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Which is the molecular target of the immunosuppressant drug Rapamycin? | The molecular target of Rapamycin is mTOR | The molecular target of rapamycin (mTOR), which is a member of the
phosphoinositide 3-kinase related kinase (PIKK) family and a central modulator
of cell growth, is a prime strategic target for anti-cancer therapeutic
development. mTOR plays a critical role in transducing proliferative signals
mediated through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)
signaling pathway, principally by activating downstream protein kinases that are
required for both ribosomal biosynthesis and translation of key mRNAs of
proteins required for G(1) to S phase traverse. By targeting mTOR, the
immunsuppressant and antiproliferative agent rapamycin (RAP) inhibits signals
required for cell cycle progression, cell growth, and proliferation. RAP, a
complex macrolide and highly potent fungicide, immunosuppressant, and
anti-cancer agent, is a highly specific inhibitor of mTOR. In essence, RAP gains
function by binding to the immunophilin FK506 binding protein 12 (FKBP12) and
the resultant complex inhibits the activity of mTOR. Since mTOR activates both
the 40S ribosomal protein S6 kinase ((p)70(s6k)) and the eukaryotic initiation
factor 4E-binding protein-1 (4E-BP1), RAP blocks activation of these downstream
signaling elements, which results in cell cycle arrest in the G1 arrest. RAP
also prevents cyclin-dependent kinase (cdk) activation, inhibits retinoblastoma
protein ((p)Rb) phosphorylation, and accelerates the turnover of cyclin D1 that
leads to a deficienciy of active cdk4/cyclin D1 complexes, all of which
potentially contribute to the prominent inhibitory effects of RAP at the G(1)/S
phase transition. Both RAP and several RAP analogs with more favorable
pharmaceutical properties have demonstrated prominent growth inhibitory effects
against a broad range of human cancers in both preclinical and early clinical
evaluations. This review will summarize the principal mechanisms of action of
RAP and RAP derivatives and their potential utility of these agents as
anti-cancer therapeutics. The preliminary results of early clinical evaluations
with RAP analogs and the unique developmental challenges that lie ahead will
also be discussed. Endocrine therapy is the most effective systemic treatment for patients with
hormone-receptor-positive (HR(+)) breast cancer. Unfortunately, efficacy is
often limited by the onset of resistance, which is almost inevitable for
patients with advanced disease. Several patterns of endocrine resistance are
recognizable clinically, including: A) tumors that are inherently insensitive to
all attempts at estrogen receptor (ER) targeting despite expression of ER
(pan-endocrine therapy resistance); B) tumors that are estrogen dependent but
resistant to one or more specific endocrine therapies (agent-selective
resistance); and C) tumors that initially respond but subsequently progress
(acquired resistance). Current insights into the molecular basis for these
resistance patterns are rudimentary, but are most clearly illuminated by
investigations that focus on the crosstalk between the ErbB or HER peptide
growth factor family and the ER. The data are sufficiently compelling to be
addressed by ongoing clinical trials that examine combinations of endocrine
agents and either trastuzumab (Herceptin; Genentech, Inc.; South San Francisco,
CA) or ErbB-specific tyrosine kinase (TK) inhibitors. Preliminary data from a
small "proof of concept" phase II study of letrozole (Femara; Novartis
Pharmaceuticals Corporation; East Hanover, NJ) and trastuzumab demonstrated
durable responses despite tamoxifen (Nolvadex; AstraZeneca Pharmaceuticals;
Wilmington, DE) resistance. Efficacy was variable, however, despite the
selection of patients on the basis of ER and ErbB-2 coexpression. Complicating
matters further, resistance often occurs in the absence of any evidence for ErbB
TK family member expression. In the absence of a clear target, common downstream
signal transduction proteins that are known to intersect with the ER pathway can
be inhibited to address resistance, including G proteins with
farnesyltransferase inhibitors and molecular target of rapamycin (mTOR) with
rapamycin analogues. With a number of phase III clinical trials now under way,
major advances in the endocrine treatment of advanced disease are possible. PURPOSE OF REVIEW: The molecular target of rapamycin, which is a member of the
phosphoinositide 3-kinase related kinase family and a central modulator of cell
growth, is a unique and prime strategic target for anticancer therapeutic
development.
RECENT FINDINGS: The molecular target of rapamycin plays a critical role in
transducing proliferative signals mediated through the phosphatidylinositol 3
kinase and protein kinase B signaling pathways, principally by activating
downstream protein kinases that are required for both ribosomal biosynthesis and
translation of mRNAs of proteins that are essential for G1 to S phase traverse.
By targeting the molecular target of rapamycin with high potency and
specificity, the immunosuppressant and antiproliferative agent rapamycin
inhibits signals required for cell cycle progression, cell growth, and
proliferation. Both rapamycin and several rapamycin analogs with more favorable
pharmaceutical properties have demonstrated impressive growth inhibitory effects
against a broad range of human cancers in both preclinical and early clinical
evaluations.
SUMMARY: This review discusses recent findings regarding the principal
mechanisms of action of the rapamycins, the potential utility of these agents as
anticancer therapeutics, clinical results to date, and developmental challenges
that lie ahead. Farnesyl protein transferase inhibitors (FTIs) have demonstrated clinical
activity in certain solid tumors and hematological maligcies. Little is known
about mechanisms of resistance to these agents. To provide a basis for better
understanding FTI resistance, the colorectal carcinoma cell line HCT 116 was
selected by stepwise exposure to increasing
4-(2-(4-(8-chloro-3,10-dibromo-6,11-dihydro-5H-benzo-(5,6)-cyclohepta(1,2-b)-pyridin-11(R)-yl)-1-piperidinyl)-2-oxo-ethyl)-1-piperidinecarboxamide
(SCH66336) concentrations. The resulting line, HCT 116R, was 100-fold resistant
to SCH66336 and other FTIs, including methyl
{N-[2-phenyl-4-N[2(R)-amino-3-mercaptopropylamino] benzoyl]}-methionate
(FTI-277), but was less than 2-fold resistant to the standard agents
gemcitabine, cisplatin, and paclitaxel. Accumulation of the unfarnesylated forms
of prelamin A and HDJ-2, two substrates that reflect farnesyl transferase
inhibition, was similar in FTI-treated parental and HCT 116R cells, indicating
that alterations in drug uptake or inhibition of farnesyl protein transferase is
not the mechanism of resistance. Changes in signal-transduction pathways that
might account for this resistance were examined by immunoblotting and confirmed
pharmacologically. There was no difference in activation of the
mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated
kinase pathway or sensitivity to the MEK1/2 inhibitor 2'-amino-3'-methoxyflavone
(PD98059) in HCT 116R cells. In contrast, increased phosphorylation of the
molecular target of rapamycin (mTOR) and its downstream target p70 S6 kinase and
increased levels of Akt1 and Akt2 were demonstrated in HCT 116R cells. Further
experiments demonstrated that the mTOR inhibitor rapamycin selectively
sensitized HCT 116R cells to SCH66336 but not to gemcitabine, cisplatin, or
paclitaxel. These findings provide evidence that alterations in the
phosphatidylinositol-3 kinase/Akt pathway can contribute to FTI resistance and
suggest a potential strategy for overcoming this resistance. The story of rapamycin is a pharmaceutical fairytale. Discovered as an
antifungal activity in a soil sample collected on Easter Island, this
macrocyclic lactone and its derivatives are now billion dollar drugs, used in,
and being evaluated for, a number of clinical applications. Taking advantage of
its antifungal property, the molecular Target Of Rapamycin, TOR, was first
described in the budding yeast Saccharomyces cerevisiae. TORs encode large,
Ser/Thr protein kinases that reside in two distinct, structurally and
functionally conserved, multi-protein complexes. In yeast, these complexes
coordinate many different aspects of cell growth. TOR complex 1, TORC1, promotes
protein synthesis and other anabolic processes, while inhibiting macroautophagy
and other catabolic and stress-response processes. TORC2 primarily regulates
cell polarity, although additional readouts of this complex are beginning to be
characterized. TORC1 appears to be activated by nutrient cues and inhibited by
stresses and rapamycin; however, detailed mechanisms are not known. In contrast,
TORC2 is insensitive to rapamycin and physiological regulators of this complex
have yet to be defined. Given the unsurpassed resources available to yeast
researchers, this simple eukaryote continues to contribute to our understanding
of eukaryotic cell growth in general and TOR function in particular. The molecular target of rapamycin (mTOR) is central to a complex intracellular
signaling pathway and is involved in diverse processes including cell growth and
proliferation, angiogenesis, autophagy, and metabolism. Although sirolimus
(rapamycin), the oldest inhibitor of mTOR, was discovered more than 30 years
ago, renewed interest in this pathway is evident by the numerous rapalogs
recently developed. These newer agents borrow from the structure of sirolimus
and, although there are some pharmacokinetic differences, they appear to differ
little in terms of pharmacodynamic effects and overall tolerability. Given the
multitude of potential applications for this class of agents and the decrease in
cost that can be expected upon the expiration of sirolimus patents, renewed
focus on this agent is warranted. Cutaneous maligt melanoma is the most aggressive form of skin cancer with an
extremely poor survival rate for the patients diagnosed with locally invasive
and metastatic disease states. Intensive research has led in last few years to
an improvement of the early detection and curative treatment of primary
cutaneous melanomas that are confined to the skin by tumor surgical resection.
However, locally advanced and disseminated melanomas are generally resistant to
conventional treatments, including ionizing radiation, systemic chemotherapy,
immunotherapy and/or adjuvant stem cell-based therapies, and result in the death
of patients. The rapid progression of primary melanomas to locally invasive
and/or metastatic disease states remains a major obstacle for an early effective
diagnosis and a curative therapeutic intervention for melanoma patients.
Importantly, recent advances in the melanoma research have led to the
identification of different gene products that are often implicated in the
maligt transformation of melanocytic cells into melanoma cells, including
melanoma stem/progenitor cells, during melanoma initiation and progression to
locally advanced and metastatic disease states. The frequent deregulated genes
products encompass the oncogenic B-RafV600E and N-RasQ61R mutants, different
receptor tyrosine kinases and developmental pathways such as epidermal growth
factor receptor (EGFR), stem cell-like factor (SCF) receptor KIT, hedgehog,
Wnt/β-catenin, Notch, stromal cell-derived factor-1 (SDF-1)/CXC chemokine
receptor-4 (CXCR4) and vascular endothelial growth factor (VEGF)/VEGFR receptor.
These growth factors can cooperate to activate distinct tumorigenic downstream
signaling elements and epithelial-mesenchymal transition (EMT)-associated
molecules, including phosphatidylinositol 3'-kinase (PI3K)/Akt/ molecular target
of rapamycin (mTOR), nuclear factor-kappaB (NF-κB), macrophage inhibitory
cytokine-1 (MIC-1), vimentin, snail and twist. Of therapeutic relevance, these
deregulated signal transduction components constitute new potential biomarkers
and therapeutic targets of great clinical interest for improving the efficacy of
current diagnostic and prognostic methods and management of patients diagnosed
with locally advanced, metastatic and/or relapsed melanomas. The molecular target of rapamycin (mTOR) is up-regulated in glioblastoma (GBM)
and this is associated with the rate of cell growth, stem cell proliferation and
disease relapse. Rapamycin is a powerful mTOR inhibitor and strong autophagy
inducer. Previous studies analyzed the effects of rapamycin in GBM cell lines.
However, to our knowledge, no experiment was carried out to evaluate the effects
of rapamycin neither in primary cells derived from GBM patients nor in vivo in
brain GBM xenograft. These data are critical to get a deeper insight into the
effects of such adjuvant therapy in GBM patients. In the present study, various
doses of rapamycin were tested in primary cell cultures from GBM patients. These
effects were compared with that obtained by the same doses of rapamycin in GBM
cell lines (U87Mg). The effects of rapamycin were also evaluated in vivo, in
brain tumors developed from mouse xenografts. Rapamycin, starting at the dose of
10nm inhibited cell growth both in U87Mg cell line and primary cell cultures
derived from various GBM patients. When administered in vivo to brain xenografts
in nude mice rapamycin almost doubled the survival time of mice and inhibited by
more than 95% of tumor volume. Recent studies in the field of cancer stem cells have revealed that the
alterations in key gene products involved in the epithelial-mesenchymal
transition (EMT) program, altered metabolic pathways such as enhanced
glycolysis, lipogenesis and/or autophagy and treatment resistance may occur in
cancer stem/progenitor cells and their progenies during cancer progression.
Particularly, the sustained activation of diverse developmental cascades such as
hedgehog, epidermal growth factor receptor (EGFR), Wnt/β-catenin, Notch,
transforming growth factor-β (TGF-β)/TGF-βR receptors and/or stromal
cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) can play critical
functions for high self-renewal potential, survival, invasion and metastases of
cancer stem/progenitor cells and their progenies. It has also been observed that
cancer cells may be reprogrammed to re-express different pluripotency-associated
stem cell-like markers such as Myc, Oct-3/4, Nanog and Sox-2 along the EMT
process and under stressful and hypoxic conditions. Moreover, the enhanced
expression and/or activities of some drug resistance-associated molecules such
as Bcl-2, Akt/molecular target of rapamycin (mTOR), nuclear factor-kappaB
(NF-κB), hypoxia-inducible factors (HIFs), macrophage inhibitory cytokine-1
(MIC-1) and ATP-binding cassette (ABC) multidrug transporters frequently occur
in cancer cells during cancer progression and metastases. These molecular events
may cooperate for the survival and acquisition of a more aggressive and
migratory behavior by cancer stem/progenitor cells and their progenies during
cancer transition to metastatic and recurrent disease states. Of therapeutic
interest, these altered gene products may also be exploited as molecular
biomarkers and therapeutic targets to develop novel multitargeted strategies for
improving current cancer therapies and preventing disease relapse. |
Is the protein KCNQ2 associated with idiopathic epilepsy? | Yes, sequence variations of the KCNQ2 gene may contribute to the etiology of idiopathic epilepsy | Mutations in the voltage gated potassium channel gene KCNQ2 and the homologous
gene KCNQ3 have been found to cause a rare monogenic subtype of idiopathic
generalized epilepsy, the benign familial neonatal convulsions. Recently, the
heteromeric KCNQ2/KCNQ3 channel was found to contribute to the native M-current,
one of the most important regulators of neuronal excitability. By performing a
systematic mutation scan of the coding region and an association study involving
a frequent Thr752Asn substitution polymorphism, we, therefore, investigated
whether allelic variation of the KCNQ2 gene confers susceptibility to common
subtypes of idiopathic generalized epilepsy. Our results do not provide evidence
that allelic variation of the KCNQ2 gene contributes a common and relevant
effect to the pathogenesis of common subtypes of idiopathic generalized
epilepsy. Several potassium channel genes have been implicated in epilepsy. We have
investigated three such genes, KCNJ3, KCNJ6 and KCNQ2, by association studies
using a broad sample of idiopathic generalised epilepsy (IGE) unselected by
syndrome. One of the two single nucleotide polymorphisms (SNPs) examined in one
of the inward rectifying potassium channel genes, KCNJ3, was associated with IGE
by genotype (P=0.0097), while its association by allele was of borderline
significance (P=0.051). Analysis of the different clinical subgroups within the
IGE sample showed more significant association with the presence of absence
seizures (P=0.0041) and which is still significant after correction for multiple
testing. Neither SNP in the other rectifying potassium channel gene, KCNJ6, was
associated with IGE or any subgroup. None of the three SNPs in the voltage-gated
potassium channel gene, KCNQ2, was associated with IGE. However, one SNP was
associated with epilepsy with generalised tonic clonic seizures only (P=0.016),
as was an SNP approximately 56 kb distant in the closely linked nicotinic
acetylcholine gene CHRNA4 (P=0.014). These two SNPs were not in linkage
disequilibrium with each other, suggesting that if they are not true
associations they have independently occurred by chance. Neither association
remains significant after correcting for multiple testing. KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current
(I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or
KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare
autosomal-domit idiopathic epilepsy of the newborn. In the present study, we
have investigated, by means of electrophysiological, biochemical, and
immunocytochemical techniques in transiently transfected cells, the consequences
prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this
frameshift mutation caused the substitution of the last 163 amino acids of the
KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The
2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric
expression of KCNQ2 subunits, dramatically reduced the steady-state cellular
levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane.
Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent
faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20
microm) at least partially reversed such enhanced degradation. Co-expression
with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and
led to their expression on the plasma membrane. Finally, co-expression of KCNQ2
2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+
currents having pharmacological and biophysical properties of heteromeric
channels. Collectively, the present results suggest that mutation-induced
reduced stability of KCNQ2 subunits may cause epilepsy in neonates. The idiopathic generalized epilepsies (IGEs) are considered to be primarily
genetic in origin. They encompass a number of rare mendelian or monogenic
epilepsies and more common forms which are familial but manifest as complex,
non-mendelian traits. Recent advances have demonstrated that many monogenic IGEs
are ion channelopathies. These include benign familial neonatal convulsions due
to mutations in KCNQ2 or KCNQ3, generalized epilepsy with febrile seizures plus
due to mutations in SCN1A, SCN2A, SCN1B, and GABRG2, autosomal-domit juvenile
myoclonic epilepsy (JME) due to a mutation in GABRA1 and mutations in CLCN2
associated with several IGE sub-types. There has also been progress in
understanding the non-mendelian IGEs. A haplotype in the Malic Enzyme 2 gene,
ME2, increases the risk for IGE in the homozygous state. Five missense mutations
have been identified in EFHC1 in 6 of 44 families with JME. Rare sequence
variants have been identified in CACNA1H in sporadic patients with childhood
absence epilepsy in the Chinese Han population. These advances should lead to
new approaches to diagnosis and treatment. OBJECTIVE: Benign familial infantile convulsions (BFIC) is a form of idiopathic
epileptic syndrome characterized by onset of afebrile seizures between 3 and 12
months of life, Spontaneous remission after several weeks or months, and
autosomal domit mode of inheritance. Previous linkage analysis in western
countries defined three susceptible loci on chromosomes 19q12.0-13.1, 16p12-q12,
and 2q23-31, but studies performed in several Chinese families with BFIC got
negative results of these previously reported loci. The authors investigated the
relation of voltage-gated potassium channel gene KCNQ2 to BFIC in a Chinese
family and thus to understand the molecular pathogenesis of BFIC.
METHODS: A four-generation Chinese BFIC family was investigated. All the
affected 17 members had similar pattern of seizures starting from 2 to 6 months
of age. In 15 of them, the seizures disappeared spontaneously within the first
year of life. The phenotype extended beyond infancy only in two patients. Blood
sample was collected from the 41 family members and 75 unassociated normal
individuals. Polymerase chain reaction (PCR)-DNA direct sequencing was performed
to screen all exons and their flanking introns of KCNQ2 gene for mutation
analysis. Polymerase chain reaction-single strand conformation polymorphism
(PCR-SSCP) was used to ascertain the co-segregation of genotype and phenotype
and to exclude polymorphism.
RESULTS: PCR amplification and subsequent direct sequencing of KCNQ2 from the
DNA of proband revealed a heterozygous guanine to thymine nucleotide exchange
(G812T) in exon 5, leading to the substitution of glycine by valine at amino
acid position 271 (G271V) of the predicted protein. The same mutation with a
comparable localization has been previously described for KCNQ3 in benign
familial neonatal convulsions (BFNC). The glycine at this position (G271) is
located in pore region of KCNQ2 protein and is evolutionarily highly conserved.
The same SSCP variant as that of the proband was shown in the rest of the
affected members of this family but not in the unaffected members enrolled in
the study of this family and all the 75 unrelated normal individuals.
CONCLUSION: Previously reported mutations of KCNQ2 were mainly identified in
BFNC family in which at least one individual had an onset of seizures during the
first week of life, a hallmark of the BFNC disorder. The results of the present
study suggest the possibility that KCNQ2 mutation exist in patients with BFIC
diagnosis. G812T of KCNQ2 gene is a novel mutation found in BFIC and functional
expression of KCNQ2 G812T is required for understanding the mechanism of BFIC
and other idiopathic epilepsy. Benign familial neonatal convulsions (BFNC) is a rare monogenic subtype of
idiopathic epilepsy exhibiting autosomal domit mode of inheritance. The
disease is caused by mutations in the two homologous genes KCNQ2 and KCNQ3 that
encode the subunits of the voltage-gated potassium channel. Most KCNQ2 mutations
are found in the pore region and the cytoplasmic C domain. These mutations are
either deletions/insertions that result in frameshift or truncation of the
protein product, splice-site variants or missense mutations. This study reveals
a novel missense mutation (N258S) in the KCNQ2 gene between the S5 domain and
the pore of the potassium channel in two BFNC patients in a Turkish family. The
absence of the mutation both in the healthy members of the family and in a
control group, and the lack of any other change in the KCNQ2 gene of the
patients indicate that N258S substitution is a pathogenic mutation leading to
epileptic seizures in this family. OBJECTIVE: To explore the involvement of M-type potassium channels KCNQ2, Q3,
and Q5 in the pathogenesis of common idiopathic epilepsies.
METHODS: Sequence analysis of the KCNQ2, Q3, and Q5 coding regions was performed
in a screening sample consisting of 58 nuclear families with rolandic epilepsy.
Subsequently, an association study was conducted for all discovered variants in
a case-control sample comprising 459 German patients with idiopathic generalized
epilepsy (IGE) and 462 population controls.
RESULTS: An in-frame deletion of codon 116 in KCNQ2 (p.Lys116del) and a missense
mutation in KCNQ3 (p.Glu299Lys) were detected in two index cases exhibiting
rolandic epilepsy and benign neonatal convulsions. Both mutations resulted in
reduced potassium current amplitude in Xenopus oocytes. Mutation analysis of
families with rolandic epilepsy without neonatal seizures discovered three novel
missense variations (KCNQ2 p.Ile592Met, KCNQ3 p.Ala381Val, KCNQ3 p.Pro574Ser).
The KCNQ2 p.Ile592Met variant displayed a significant reduction of potassium
current amplitude in Xenopus oocytes and was present only once in 552 controls.
Both missense variants identified in KCNQ3 (p.Ala381Val and p.Pro574Ser) were
present in all affected family members and did not occur in controls, but did
not show obvious functional abnormalities. The KCNQ3 missense variant
p.Pro574Ser was also detected in 8 of 455 IGE patients but not in 454 controls
(p = 0.008). In KCNQ2, a silent single nucleotide polymorphism (rs1801545) was
found overrepresented in both epilepsy samples (IGE, p = 0.004).
CONCLUSION: Sequence variations of the KCNQ2 and KCNQ3 genes may contribute to
the etiology of common idiopathic epilepsy syndromes. BACKGROUND: The underlying genetic abnormalities of rare familial idiopathic
epilepsy have been identified, such as mutation in KCNQ2, a K(+) channel gene.
Yet, few genetic abnormalities have been reported for commoner epilepsy, i.e.,
sporadic idiopathic epilepsy, which share a phenotype similar to those of
familial epilepsy.
OBJECTIVE: To search for the genetic cause of seizures in a girl with the
diagnosis of non-familial benign neonatal convulsions, and define the
consequence of the genetic abnormality identified.
METHODS: Genetic abnormality was explored within candidate genes for benign
familial neonatal and infantile convulsions, such as KCNQ2, 3, 5, KCNE2, SCN1A
and SCN2A. The electrophysiological properties of the channels harboring the
identified mutation were examined. Western blotting and immunostaining were
employed to characterize the expression and intracellular localization of the
mutant channel molecules.
RESULTS: A novel heterozygous mutation (c.910-2delTTC or TTT, Phe304del) of
KCNQ2 was identified in the patient. The mutation was de novo verified by
parentage analysis. The mutation was associated with impaired functions of KCNQ
K(+) channel. The mutant channels were expressed on the cell surface.
CONCLUSION: The mutant Phe304del of KCNQ2 leads to null function of the KCNQ
K(+) channel but the mutation does not alter proper channel sorting onto the
cell membrane. Our findings indicate that the genes responsible for rare
inherited forms of idiopathic epilepsy could be also involved in sporadic forms
of idiopathic epilepsy and expand our notion of the involvement of molecular
mechanisms in the more common forms of idiopathic epilepsy. Mutations in the SCN1A gene are found in up to 80% of individuals with severe
myoclonic epilepsy of infancy (SMEI), and mutations in KCNQ2 and KCNQ3 were
identified in benign familial neonatal convulsions (BFNC) as well as in single
families with Rolandic epilepsy (RE) and idiopathic generalized epilepsies
(IGE). This paper summarizes recent findings concerning sodium (SCN1A) and
potassium channel (KCNQ2 and KCNQ3) dysfunctions in the pathogenesis of rare and
common idiopathic epilepsies (IE). SMEI, severe idiopathic generalized epilepsy
of infancy (SIGEI), and myoclonic-astatic epilepsy (MAE) are rare IE. Because of
some semeiologic overlap, a comparative analysis of the SCN1A gene performed in
20 patients with MAE and in 18 with SIGEI. This revealed mutations in three
subjects with SIGEI only. Since BFNC are over-represented in families with RE, a
mutational analysis was performed in 58 families with RE with and without BNFC.
This revealed functionally relevant mutations in two index cases with BNFC, and
three missense mutations (one resulting in a significantly reduced potassium
current amplitude) in three patients with RE, but without BNFC. One KCNQ3
missense variant was also detected in eight out of 455 IGE patients but not in
454 controls, and a silent KCNQ2-SNP was found over-represented in both epilepsy
samples. These findings confirm that mutations in the SCN1A gene are mainly
involved in the pathogenesis of SMEI, rarely in that of SIGEI, and are commonly
not found in patients with MAE. They also demonstrate that sequence variations
of the KCNQ2 and KCNQ3 genes may contribute to the etiology of common IE
syndromes. Potassium channel subunits encoded by several genes of the KCNQ family underlie
the M-current. Specifically, KCNQ2 and KCNQ3 play a major role at most neuronal
sites. Mutations in KCNQ2 or KCNQ3 that reduce the M-current are responsible for
benign familial neonatal seizures, a rare autosomal domit idiopathic epilepsy
of the newborn. The aim of this study was to investigate a single family with
benign familial neonatal seizures for mutations in KCNQ genes and to analyze the
association of mutation type with disease prognosis. A family in which members
in several generations had signs and symptoms compatible with a diagnosis of
benign familial neonatal seizures had DNA testing with single-stranded
conformation polymorphism analysis for various mutations known to cause benign
familial neonatal seizures. A novel KCNQ2 mutation c.63-66delGGTG (p.K21fsX40),
causing a framework shift and early chain termination, was identified in the
affected family members. In all cases, there was complete remission of the
seizures after the neonatal period. This KCNQ2 mutation has implications for
diagnosis and prognosis of familial neonatal seizures. Its presence suggests a
benign disease with good prognosis and its identification can spare patients and
physicians the need for extensive investigations or prolonged therapy. Benign familial neonatal convulsions is an autosomal-domit idiopathic form of
epilepsy primarily caused by gene mutations of the voltage-gated
Kv7.2/KCNQ2/M-channel that exert only partial domit-negative effects.
However, the mechanism underlying the incomplete domice of channel mutations,
which cause epilepsy in infancy, remains unknown. Using mutagenesis and
biochemistry combined with electrophysiology, we identified a novel degradation
signal derived from distal C-terminal frameshift mutations, which impairs
channel function. This degradation signal, transferable to non-channel CD4, can
lead to accelerated degradation of mutant proteins through ubiquitin-independent
proteasome machinery but does not affect mRNA quantity and protein trafficking.
Functional dissection of this signal has revealed a key five-amino acid (RCXRG)
motif critical for degradation. Taken together, our findings reveal a mechanism
by which proteins that carry this signal are subject to degradation, leading to
M-current dysfunction, which causes epilepsy. |
PBT2 has been tested for which disorder? | PBT2 has been tested for treatment of Alzheimer's disease. | Alzheimer's disease is the most common form of dementia in the elderly, and it
is characterized by elevated brain iron levels and accumulation of copper and
zinc in cerebral beta-amyloid deposits (e.g., senile plaques). Both ionic zinc
and copper are able to accelerate the aggregation of Abeta, the principle
component of beta-amyloid deposits. Copper (and iron) can also promote the
neurotoxic redox activity of Abeta and induce oxidative cross-linking of the
peptide into stable oligomers. Recent reports have documented the release of
Abeta together with ionic zinc and copper in cortical glutamatergic synapses
after excitation. This, in turn, leads to the formation of Abeta oligomers,
which, in turn, modulates long-term potentiation by controlling synaptic levels
of the NMDA receptor. The excessive accumulation of Abeta oligomers in the
synaptic cleft would then be predicted to adversely affect synaptic
neurotransmission. Based on these findings, we have proposed the "Metal
Hypothesis of Alzheimer's Disease," which stipulates that the neuropathogenic
effects of Abeta in Alzheimer's disease are promoted by (and possibly even
dependent on) Abeta-metal interactions. Increasingly sophisticated
pharmaceutical approaches are now being implemented to attenuate abnormal
Abeta-metal interactions without causing systemic disturbance of essential
metals. Small molecules targeting Abeta-metal interactions (e.g., PBT2) are
currently advancing through clinical trials and show increasing promise as
disease-modifying agents for Alzheimer's disease based on the "metal
hypothesis." BACKGROUND: PBT2 is a metal-protein attenuating compound (MPAC) that affects the
Cu2(+)-mediated and Zn2(+)-mediated toxic oligomerisation of Abeta seen in
Alzheimer's disease (AD). Strong preclinical efficacy data and the completion of
early, clinical safety studies have preceded this phase IIa study, the aim of
which was to assess the effects of PBT2 on safety, efficacy, and biomarkers of
AD.
METHODS: Between December 6, 2006, and September 21, 2007, community-dwelling
patients over age 55 years were recruited to this 12-week, double-blind,
randomised trial of PBT2. Patients were randomly allocated to receive 50 mg
PBT2, 250 mg PBT2, or placebo. Inclusion criteria were early AD (mini-mental
state examination [MMSE] score between 20 and 26 points or Alzheimer's disease
assessment scale-cognitive subscale (ADAS-cog) score between 10 and 25 points),
taking a stable dose of acetylcholinesterase inhibitor (donepezil, galantamine,
or rivastigmine) for at least 4 months, a modified Hachinski score of 4 points
or less, and CT or MRI results that were consistent with AD. The principal
outcomes were safety and tolerability. Secondary outcomes were plasma and CSF
biomarkers and cognition. Analysis was intention to treat. The trial is
registered with ClinicalTrials.gov, number NCT00471211.
FINDINGS: 78 patients were randomly assigned (29 to placebo, 20 to PBT2 50 mg,
and 29 to PBT2 250 mg) and 74 (95%) completed the study. 42 (54%) patients had
at least one treatment emergent adverse event (10 [50%] on PBT2 50 mg, 18 [62%]
on PBT2 250 mg, and 14 [48%] on placebo). No serious adverse events were
reported by patients on PBT2. Patients treated with PBT2 250 mg had a
dose-dependent (p=0.023) and significant reduction in CSF Abeta(42)
concentration compared with those treated with placebo (difference in least
squares mean change from baseline was -56.0 pg/mL, 95% CI -101.5 to -11.0;
p=0.006). PBT2 had no effect on plasma biomarkers of AD or serum Zn(2+) and
Cu(2+) concentrations. Cognition testing included ADAS-cog, MMSE, and a
neuropsychological test battery (NTB). Of these tests, two executive function
component tests of the NTB showed significant improvement over placebo in the
PBT2 250 mg group: category fluency test (2.8 words, 0.1 to 5.4; p=0.041) and
trail making part B (-48.0 s, -83.0 to -13.0; p=0.009).
INTERPRETATION: The safety profile is favourable for the ongoing development of
PBT2. The effect on putative biomarkers for AD in CSF but not in plasma is
suggestive of a central effect of the drug on Abeta metabolism. Cognitive
efficacy was restricted to two measures of executive function. Future trials
that are larger and longer will establish if the effects of PBT2 on biomarkers
and cognition that are reported here translate into clinical effectiveness. The recent report of positive results from a Phase IIa clinical trial of PBT2, a
novel drug that targets amyloid-beta-metal interactions, underscores the value
of abnormal transition metal metabolism as a potential therapeutic target in
Alzheimer's disease. The Metals Hypothesis of Alzheimer's disease is based upon
observations of the precipitation of amyloid-beta by zinc and its radicalization
by copper. Both metals are markedly enriched in plaques. The Hypothesis involves
the perturbance of these endogenous brain metals, and it does not consider
toxicological exposure part of pathogenesis. Recent descriptions of the release
of ionic zinc and copper in the cortical glutamatergic synapse, modulating the
response of the NMDA receptor, may explain the vulnerability of amyloid-beta to
abnormal interaction with these metal ions in the synaptic region leading to
aggregation and fostering toxicity. Increasingly sophisticated medicinal
chemistry approaches are being tested which correct the abnormalities without
causing systemic disturbance of these essential minerals. PBT2, clioquinol and
related compounds are ionophores rather than chelators. PBT2 is a once per day,
orally bioavailable, second generation 8-OH quinoline derivative of clioquinol.
It has performed very satisfactorily in toxicology and Phase I clinical trials
and is advancing as a disease-modifying candidate drug for Alzheimer's disease. The prevention and treatment of cognitive impairment in the elderly has assumed
increasing importance in an aging population. This article presents a
qualitative review of recent research on experimental interventions for the
prevention and treatment of mild cognitive impairment and Alzheimer's disease in
elderly subjects. Interventions addressed range from lifestyle measures to
pharmacological treatments. Epidemiological studies suggest that dietary
measures, physical exercise, and mental activity may reduce the risk of
cognitive impairment and Alzheimer's disease in elderly subjects. Statins may
protect against incident dementia, and lithium may convey similar benefits to
bipolar patients. Ginkgo appears ineffective as a primary preventive measure.
Donepezil but not Vitamin E may benefit persons with mild cognitive impairment.
Experimental treatments potentially useful for Alzheimer's disease include
dimebon, PBT2 and etanercept; the safety and efficacy of the Alzheimer's vaccine
remains to be proven, and growth hormone secretagogue and tarenflurbil are
likely ineffective. Herbal treatments merit study in elderly subjects with
cognitive syndromes. Clioquinol (CQ), a once popular antibiotic, was used to inhibit the growth of
microorganisms. Recently, CQ and its analog PBT2 have shown encouraging effects
in the animal and clinical trials for Alzheimer's disease (AD). However, the
mechanism by which this class of molecules works remains controversial. In this
work, we used the yeast Saccharomyces cerevisiae as a model to study how CQ
affects molecular and cellular functions and particularly, copper, iron, and
zinc homeostasis. We observed a CQ-induced inhibition of yeast growth, which
could be slightly relieved by supplementation of copper or iron. Microarray
results indicated that yeast cells treated with CQ sense a general deficiency in
metals, despite elevated total cellular contents of copper and iron. Consistent
with this, reduced activities of some metal-sensitive enzymes were observed.
Intriguingly, CQ can increase the SOD1 activity, likely through Ccs1's
accessibility to CQ-bound copper ions. Further studies revealed that CQ
sequestrates copper and iron at the cellular membrane, likely the plasma
membrane, resulting overall metal accumulation but cytosolic metal depletion.
CQ's effects on metal-sensitive metalloenzymes were also verified in mammalian
cell line SH-SY5Y. Together, our results revealed that CQ can regulate metal
homeostasis by binding metal ions, resulting the cell sensing a state of
deficiency of bioavailable metal ions while simultaneously increasing available
metals to SOD1 (via Ccs1) and possibly some other metalloproteins that can
access CQ-bound metals. We hope this regulation of metal homeostasis may be
helpful in explaining the therapeutic effects of CQ used in disease treatment. Impaired metal ion homeostasis causes synaptic dysfunction and treatments for
Alzheimer's disease (AD) that target metal ions have therefore been developed.
The leading compound in this class of therapeutic, PBT2, improved cognition in a
clinical trial with AD patients. The aim of the present study was to examine the
cellular mechanism of action for PBT2. We show PBT2 induces inhibitory
phosphorylation of the α- and β-isoforms of glycogen synthase kinase 3 and that
this activity is dependent on PBT2 translocating extracellular Zn and Cu into
cells. This activity is supported when Aβ:Zn aggregates are the source of
extracellular Zn and adding PBT2 to Aβ:Zn preparations promotes Aβ degradation
by matrix metalloprotease 2. PBT2-induced glycogen synthase kinase 3
phosphorylation appears to involve inhibition of the phosphatase calcineurin.
Consistent with this, PBT2 increased phosphorylation of other calcineurin
substrates, including cAMP response element binding protein and
Ca²⁺/calmodulin-dependent protein kinase. These data demonstrate PBT2 can
decrease Aβ levels by sequestering the Zn that promotes extracellular formation
of protease resistant Aβ:Zn aggregates, and that subsequent intracellular
translocation of the Zn by PBT2 induces cellular responses with synapto-trophic
potential. Intracellular translocation of Zn and Cu via the metal chaperone
activity of PBT2 may be an important mechanism by which PBT2 improves cognitive
function in people with AD. BACKGROUND: Alzheimer's dementia (AD) may be caused by the formation of
extracellular senile plaques comprised of beta-amyloid (Aß). In vitro and mouse
model studies have demonstrated that metal protein attenuating compounds (MPACs)
promote the solubilisation and clearance of Aß.
OBJECTIVES: To evaluate the efficacy of metal protein attenuating compounds
(MPACs) for the treatment of cognitive impairment due to Alzheimer's dementia.
SEARCH METHODS: We searched ALOIS, the Cochrane Dementia and Cognitive
Improvement Group Specialized Register, on 29 July 2010 using the terms:
Clioquinol OR PBT1 OR PBT2 OR "metal protein" OR MPACS OR MPAC.
SELECTION CRITERIA: Randomised double-blind trials in which treatment with an
MPAC was administered to participants with Alzheimer's dementia in a parallel
group comparison with placebo were included.
DATA COLLECTION AND ANALYSIS: Three review authors (RM, LJ, ELS) independently
assessed the quality of trials according to the Cochrane Handbook for Systematic
Reviews of Interventions.The primary outcome measure of interest was cognitive
function (as measured by psychometric tests). The secondary outcome measures of
interest were in the following areas: quality of life, functional performance,
effect on carer, biomarkers, safety and adverse effects, and death.
MAIN RESULTS: Two MPAC trials were identified. One trial compared clioquinol
(PBT1) with placebo in 36 patients and 32 had sufficient data for per protocol
analysis. There was no statistically significant difference in cognition (as
measured on the Alzheimer's Disease Assessment Scale - Cognition (ADAS-Cog))
between the active treatment and placebo groups at 36 weeks. The difference in
mean change from baseline ADAS-Cog score in the clioquinol arm compared with the
placebo arm at weeks 24 and 36 was a difference of 7.37 (95% confidence interval
(CI) 1.51 to 13.24) and 6.36 (95% CI -0.50 to 13.23), respectively.There was no
significant impact on non-cognitive symptoms or clinical global impression. One
participant in the active treatment group developed neurological symptoms
(impaired visual acuity and colour vision) which resolved on cessation of
treatment and were possibly attributable to the drug.In the second trial a
successor compound, PBT2, was compared with placebo in 78 participants with mild
Alzheimer's dementia; all were included in the intention-to-treat analysis.
There was no significant difference in the Neuropsychological Test Battery (NTB)
composite, memory or executive scores between placebo and PBT2 in the least
squares mean change from baseline at week 12. However, two executive function
component tests of the NTB showed significant improvement over placebo in the
PBT2 250 mg group from baseline to week 12: category fluency test (2.8 words,
95% CI 0.1 to 5.4; P = 0.041) and trail making part B (-48.0 s, 95% CI -83.0 to
-13.0; P = 0.009). There was no significant effect on cognition on Mini-Mental
State Examination (MMSE) or ADAS-Cog scales. PBT2 had a favourable safety
profile.
AUTHORS' CONCLUSIONS: There is an absence of evidence as to whether clioquinol
(PBT1) has any positive clinical benefit for patients with AD, or whether the
drug is safe. We have some concerns about the quality of the study methodology;
there was an imbalance in treatment and control groups after randomisation
(participants in the active treatment group had a higher mean pre-morbid IQ) and
the secondary analyses of results stratified by baseline dementia severity. The
planned phase III trial of PBT1 has been abandoned and this compound has been
withdrawn from development. The second trial of PBT2 was more rigorously
conducted and showed that after 12 weeks this compound appeared to be safe and
well tolerated in people with mild Alzheimer's dementia. Larger trials are now
required to demonstrate cognitive efficacy. Currently, therapeutics that modify Alzheimer's disease (AD)are not available.
Increasing age is the primary risk factor for AD and due to an aging global
population the urgent need for effective therapeutics increases every year. This
Account presents the development of an AD treatment strategy that incorporates
diverse compounds with a common characteristic: the ability to redistribute
metal ions within the brain. Central to cognitive decline in AD is the amyloid-β
peptide (Aβ) that accumulates in the AD brain. A range of therapeutic strategies
have been developed based on the premise that decreasing the brain Aβ burden
will attenuate the severity of the disease symptoms. Unfortunately these
treatments have failed to show any positive outcomes in large-scale clinical
trials, raising many questions regarding whether therapeutics for AD can rely
solely on decreasing Aβ levels. An alternate strategy is to target the
interaction between Aβ and metal ions using compounds with the potential to
redistribute metal ions within the brain. The original rationale for this
strategy came from studies showing that metal ions promote Aβ toxicity and
aggregation. In initial studies using the prototype metal-chelating compound
clioquinol (CQ), CQ prevented Aβ toxicity in vitro, out-competed Aβ for metal
ions without affecting the activity of metal-dependent enzymes, and attenuated
the rate of cognitive decline in AD subjects in a small phase II clinical trial.
All these outcomes were consistent with the original hypothesized mechanism of
action for CQ where prevention or reversal of the extracellular Aβ-metal
interactions could prevent Aβ toxicity. Soon after the completion of these
studies, a new body of work began to suggest that this hypothesized mechanism of
action for CQ was simplistic and that other factors were also important for the
positive therapeutic outcomes. Perhaps most significantly, it was shown that
after CQ sequesters metal ions the neutral CQ-metal complex crosses cell
membranes to increase intracellular levels of the metals, thereby initiating
protective cell signaling cascades. The activity of CQ therefore appeared to be
two-fold: it prevented toxic interactions between Aβ and metal ions outside the
cell, and it redistributed the metal ions into the cell to promote healthy cell
function. To determine the significance of redistributing metal ions into the
cell, glyoxalbis(N(4)-methylthiosemicarbazonato)Cu(II) [Cu(II)(gtsm)] was tested
in models of AD. Cu(II)(gtsm) delivers Cu into cells, but, unlike CQ, it cannot
out-compete Aβ for metal ions. When tested in AD model mice, the Cu(II)(gtsm)
treatment restored cognitive function back to levels expected for cognitively
healthy mice. The most advanced compound from this therapeutic strategy, PBT2,
can sequester metal ions from Aβ and redistribute them into the cell like CQ.
PBT2 improved cognition in a phase II clinical trial with AD patients, and
further clinical testing is currently underway. |
What is the basic secondary structure of the variable domains of a typical antibody? | The variable domains of heavy and light chains of antibodies consist of two β-sheets. The first one is composed of four strands, A, B, E and D, and the second one is composed of six strands, named A', G, F, C, C' and C''. The antigen binding site is formed by the inter-strand links BC, C′C″ and FG from each domain. | 1. The temperature function of the myeloma IgG(K) IVA, Bence-Jones protein
(K-type) IVA and its fragments (Fab(t), Fc'(t), VL and CL) was studied by
thermal perturbation difference spectroscopy and circular dichroism. 2. The IgG
and Bence-Jones protein studied were found to be capable of a fully reversible
structural changes at temperatures between 25 and 35 degrees C. The changes
occurring at the higher temperature are accompanied by the screening of the
significant part of exposed tyrosine residues. The transition is not accompanied
by an appreciable change in the main IgG secondary structure-beta-pleated sheet,
according to the CD data. 3. It was found that the temperature-dependent changes
of IgG occur in its Fab fragments, the changes of Bence-Jones protein occur in
its variable part (VL domains). 4. The temperature changes in the interval 25-35
degrees C are explained by the flexibility of amino acid side chains composed
hypervariable loops delineated the "sides" of cavity between variable domains. Antibody heavy chain variable domains (VH) lacking their light chain (VL)
partner are prime candidates for the design of minimum-size immunoreagents. To
obtain structural information about isolated VH domains, a human VH was labelled
with 15N or doubly labelled with both 15N and 13C and was studied by
heteronuclear nuclear magnetic resoce spectroscopy. Most (90%) of the 1H and
15N main-chain signals were assigned through two-dimensional TOCSY and NOESY
experiments on the unlabelled VH and three-dimensional heteronuclear multiple
quantum correlation TOCSY and NOESY experiments on the 15N-labelled VH. Four
short stretches of the polypeptide chain could only be assigned on the basis of
three-dimensional HNCA and HN(CO)CA experiments on the 13C-/15N-labelled
protein. Long-range interstrand backbone NOEs suggest the presence of two
adjacent beta-sheets formed by altogether nine antiparallel beta-strands. 3JNHC
alpha H coupling constants and the location of slowly exchanging backbone amides
support this interpretation. The secondary structure of the isolated VH is
identical to that of heavy chain variable domains in intact antibodies, where VH
domains are packed against a VL domain. The backbone assignments of the VH made
it possible to locate its Protein A binding site. Chemical shift movements after
complexing with the IgG binding fragment of Protein A indicate binding through
one of the two beta-sheets of the VH. This beta-sheet is solvent exposed in
intact antibodies. The Protein A binding site obviously differs from that on the
Fc portion of immunoglobulins and is unique to members of the human VHIII gene
subgroup. The solution structure of the isolated antibody heavy chain variable domain
(VH)-P8 was determined by NMR spectroscopy. The VH had previously been modified
(camelised) at three positions in its former antibody light chain variable
domain (VL) interface to reduce hydrophobicity by mimicking camelid heavy chains
naturally devoid of light chains. The architecture of two pleated beta-sheets
and the conformation of the H1 and H2 loops in VH-P8 are very similar to those
in non-camelised, VL-associated VH domains. Major differences concern the H3
loop, which no longer points towards the now absent VL, and three residues in
the former VL interface. The side-chains of Val37 and Trp103 are buried and the
Arg38 side-chain exposed in VH-P8. In non-camelised, VL-associated VH domains
the side-chains of Val37 and Trp103 are in contact with the VL while the Arg38
side-chain is buried within the VH. Reorientation of Trp103 is due to the local
structure in the beta-bulge of strand G. Reorientation of Val37 and Arg38 is
caused by a disruption of regular beta-structure in strand C opposite the
beta-bulge in strand C'. These changes, combined with the more hydrophilic
side-chains of the camelised residues, reduce hydrophobicity and prevent
non-specific binding of camelised VH domains, which proved critical for their
use as small recognition units. The VH-P8 structure also indicates structural
reasons for two other mutations specific for light-chain-lacking camel
immunoglobins. Absence of the VH-typical Arg94/Asp101 salt bridge at the base of
the H3 loop in VH-P8 may explain why a positively charged residue at position 94
is not conserved in camels. Reorientation of Val37 suggests a function of the
camel-specific phenylalanine residue at this position in the hydrophobic core of
light-chain-lacking camel heavy chains. BACKGROUND: Antibodies are prototypes of multimeric proteins and consist of
structurally similar domains. The two variable domains of an antibody (VH and
VL) interact through a large hydrophobic interface and can be expressed as
covalently linked single-chain Fv (scFv) fragments. The in vitro folding of scFv
fragments after long-term denaturation in guanidinium chloride is known to be
slow. In order to delineate the nature of the rate-limiting step, the folding of
the scFv fragment of an antibody after short-term denaturation has been
investigated.
RESULTS: Secondary structure formation, measured by H/D-exchange protection, of
a mutant scFv fragment of an antibody after short incubation in 6 M guanidinium
chloride was shown to be multiphasic. NMR analysis shows that an intermediate
with significant proton protection is observed within the dead time of the
manual mixing experiments. Subsequently, the folding reaction proceeds via a
biphasic reaction and mass spectrometry analyses of the exchange experiments
confirm the existence of two parallel pathways. In the presence of cyclophilin,
however, the faster of the two phases vanishes (when followed by intrinsic
tryptophan fluorescence), while the slower phase is not significantly enhanced
by equimolar cyclophilin.
CONCLUSIONS: The formation of an early intermediate, which shows amide-proton
exchange protection, is independent of proline isomerization. Subsequently, a
proline cis-trans isomerization reaction in the rapidly formed intermediate,
producing 'non-native' isomers, competes with the fast formation of native
species. Interface formation in a folding intermediate of the scFv fragment is
proposed to prevent the back-isomerization of these prolines from being
efficiently catalyzed by cyclophilin. Light chain, or AL, amyloidosis is a pathological condition arising from
systemic extracellular deposition of monoclonal immunoglobulin light chain
variable domains in the form of insoluble amyloid fibrils, especially in the
kidneys. Substantial evidence suggests that amyloid fibril formation from native
proteins occurs via a conformational change leading to a partially folded
intermediate conformation, whose subsequent association is a key step in
fibrillation. In the present investigation, we have examined the properties of a
recombit amyloidogenic light chain variable domain, SMA, to determine whether
partially folded intermediates can be detected and correlated with aggregation.
The results from spectroscopic and hydrodynamic measurements, including far- and
near-UV circular dichroism, FTIR, NMR, and intrinsic tryptophan fluorescence and
small-angle X-ray scattering, reveal the build-up of two partially folded
intermediate conformational states as the pH is decreased (low pH destabilized
the protein and accelerated the kinetics of aggregation). A relatively
nativelike intermediate, I(N), was observed between pH 4 and 6, with little loss
of secondary structure, but with significant tertiary structure changes and
enhanced ANS binding, indicating exposed hydrophobic surfaces. At pH below 3, we
observed a relatively unfolded, but compact, intermediate, I(U), which was
characterized by decreased tertiary and secondary structure. The I(U)
intermediate readily forms amyloid fibrils, whereas I(N) preferentially leads to
amorphous aggregates. Except at pH 2, where negligible amorphous aggregate is
formed, the amorphous aggregates formed significantly more rapidly than the
fibrils. This is the first indication that different partially folded
intermediates may be responsible for different aggregation pathways (amorphous
and fibrillar). The data support the hypothesis that amyloid fibril formation
involves the ordered self-assembly of partially folded species that are critical
soluble precursors of fibrils. BACKGROUND: Distantly related proteins adopt and retain similar structural
scaffolds despite length variations that could be as much as two-fold in some
protein superfamilies. In this paper, we describe an analysis of indel regions
that accommodate length variations amongst related proteins. We have developed
an algorithm CUSP, to examine multi-membered PASS2 superfamily alignments to
identify indel regions in an automated manner. Further, we have used the method
to characterize the length, structural type and biochemical features of indels
in related protein domains.
RESULTS: CUSP, examines protein domain structural alignments to distinguish
regions of conserved structure common to related proteins from structurally
unconserved regions that vary in length and type of structure. On a
non-redundant dataset of 353 domain superfamily alignments from PASS2, we find
that 'length- deviant' protein superfamilies show > 30% length variation from
their average domain length. 60% of additional lengths that occur in indels are
short-length structures (< 5 residues) while 6% of indels are > 15 residues in
length. Structural types in indels also show class-specific trends.
CONCLUSION: The extent of length variation varies across different superfamilies
and indels show class-specific trends for preferred lengths and structural
types. Such indels of different lengths even within a single protein domain
superfamily could have structural and functional consequences that drive their
selection, underlying their importance in similarity detection and computational
modelling. The availability of systematic algorithms, like CUSP, should enable
decision making in a domain superfamily-specific manner. |
How does long-range epigenetic silencing (LRES) occur? | Long Range Epigenetic Silencing (LRES) is a mechanism of gene inactivation that affects multiple contiguous CpG islands and has been described in different human cancer types. | Genetic and epigenetic alterations have been identified that lead to
transcriptional deregulation in cancers. Genetic mechanisms may affect single
genes or regions containing several neighboring genes, as has been shown for DNA
copy number changes. It was recently reported that epigenetic suppression of
gene expression can also extend to a whole region; this is known as long-range
epigenetic silencing. Various techniques are available for identifying regional
genetic alterations, but no large-scale analysis has yet been carried out to
obtain an overview of regional epigenetic alterations. We carried out an
exhaustive search for regions susceptible to such mechanisms using a combination
of transcriptome correlation map analysis and array CGH data for a series of
bladder carcinomas. We validated one candidate region experimentally,
demonstrating histone methylation leading to the loss of expression of
neighboring genes without DNA methylation. Despite the completion of the Human Genome Project, we are still far from
understanding the molecular events underlying epigenetic change in cancer.
Cancer is a disease of the DNA with both genetic and epigenetic changes
contributing to changes in gene expression. Epigenetics involves the interplay
between DNA methylation, histone modifications and expression of non-coding RNAs
in the regulation of gene transcription. We now know that tumour suppressor
genes, with CpG island-associated promoters, are commonly hypermethylated and
silenced in cancer, but we do not understood what triggers this process or when
it occurs during carcinogenesis. Epigenetic gene silencing has always been
envisaged as a local event silencing discrete genes, but recent data now
indicates that large regions of chromosomes can be co-coordinately suppressed; a
process termed long range epigenetic silencing (LRES). LRES can span megabases
of DNA and involves broad heterochromatin formation accompanied by
hypermethylation of clusters of contiguous CpG islands within the region. It is
not clear if LRES is initiated by one critical gene target that spreads and
conscripts innocent bystanders, analogous to large genetic deletions or if
coordinate silencing of multiple genes is important in carcinogenesis? Over the
next decade with the exciting new genomic approaches to epigenome analysis and
the initiation of a Human Epigenome Project, we will understand more about the
interplay between DNA methylation and chromatin modifications and the expression
of non-coding RNAs, promising a new range of molecular diagnostic cancer markers
and molecular targets for cancer epigenetic therapy. The CpG island methylator phenotype (CIMP), characterized by an exceptionally
high frequency of methylation of discrete CpG islands, is observed in 18% to 25%
of sporadic colorectal cancers. Another hypermethylation pattern found in
colorectal cancers, termed long-range epigenetic silencing, is associated with
DNA/histone methylation in three distinct gene clusters at chromosome 2q14.2,
showing that DNA hypermethylation can span larger chromosomal domains and lead
to the silencing of flanking, unmethylated genes. We investigated whether these
two phenotypes are interrelated in colorectal cancers. The CIMP status of 148
sporadic colorectal cancers was determined by methylation-specific PCR. We
determined the BRAF V600E mutation by mutant allele-specific PCR amplification.
The methylation status of the MLH1 gene and of three CpG islands (EN1, SCTR, and
INHBB), corresponding to three distinct clusters along 2q14.2, was determined by
methylation-specific PCR. The average number of sites showing methylation in
CIMP+ tumors was 2.21, compared with 1.22 for CIMP- individuals, and this
difference was highly significant (P = 3.6 x 10(-8), Mann-Whitney test).
Moreover, all CIMP+ tumors showed hypermethylation of at least one of these
loci, in contrast to CIMP- tumors, where 18 (16%) samples remained unmethylated.
The mean number of simultaneously hypermethylated CpG islands at 2q14.2 differs
significantly between CIMP- and CIMP+ tumors, suggesting varying effects of
domain silencing in this region. Given that the number of hypermethylated loci
at 2q14.2 likely affects the range of silenced flanking genes, high frequency of
simultaneous hypermethylation of three CpG islands (EN1, SCTR, and INHBB) may
have potential influence on specific characteristics of CIMP+ colorectal
cancers. Wilms' tumour (WT) is a pediatric tumor of the kidney that arises via failure of
the fetal developmental program. The absence of identifiable mutations in the
majority of WTs suggests the frequent involvement of epigenetic aberrations in
WT. We therefore conducted a genome-wide analysis of promoter hypermethylation
in WTs and identified hypermethylation at chromosome 5q31 spanning 800 kilobases
(kb) and more than 50 genes. The methylated genes all belong to alpha-, beta-,
and gamma-protocadherin (PCDH) gene clusters (Human Genome Organization
nomenclature PCDHA@, PCDHB@, and PCDHG@, respectively). This demonstrates that
long-range epigenetic silencing (LRES) occurs in developmental tumors as well as
in adult tumors. Bisulfite polymerase chain reaction analysis showed that PCDH
hypermethylation is a frequent event found in all Wilms' tumor subtypes.
Hypermethylation is concordant with reduced PCDH expression in tumors. WT
precursor lesions showed no PCDH hypermethylation, suggesting that de novo PCDH
hypermethylation occurs during maligt progression. Discrete boundaries of the
PCDH domain are delimited by abrupt changes in histone modifications;
unmethylated genes flanking the LRES are associated with permissive marks which
are absent from methylated genes within the domain. Silenced genes are marked
with non-permissive histone 3 lysine 9 dimethylation. Expression analysis of
embryonic murine kidney and differentiating rat metanephric mesenchymal cells
demonstrates that Pcdh expression is developmentally regulated and that Pcdhg@
genes are expressed in blastemal cells. Importantly, we show that PCDHs
negatively regulate canonical Wnt signalling, as short-interfering RNA-induced
reduction of PCDHG@ encoded proteins leads to elevated beta-catenin protein,
increased beta-catenin/T-cell factor (TCF) reporter activity, and induction of
Wnt target genes. Conversely, over-expression of PCDHs suppresses
beta-catenin/TCF-reporter activity and also inhibits colony formation and growth
of cancer cells in soft agar. Thus PCDHs are candidate tumor suppressors that
modulate regulatory pathways critical in development and disease, such as
canonical Wnt signaling. Aberrant promoter DNA-hypermethylation and repressive chromatin constitutes a
frequent mechanism of gene inactivation in cancer. There is great interest in
dissecting the mechanisms underlying this abnormal silencing. Studies have shown
changes in the nuclear organization of chromatin in tumor cells as well as the
association of aberrant methylation with long-range silencing of neighboring
genes. Furthermore, certain tumors show a high incidence of promoter methylation
termed as the CpG island methylator phenotype. Here, we have analyzed the role
of nuclear chromatin architecture for genes in hypermethylated inactive versus
nonmethylated active states and its relation with long-range silencing and CpG
island methylator phenotype. Using combined immunostaining for active/repressive
chromatin marks and fluorescence in situ hybridization in colorectal cancer cell
lines, we show that aberrant silencing of these genes occurs without requirement
for their being positioned at heterochromatic domains. Importantly,
hypermethylation, even when associated with long-range epigenetic silencing of
neighboring genes, occurs independent of their euchromatic or heterochromatic
location. Together, these results indicate that, in cancer, extensive changes
around promoter chromatin of individual genes or gene clusters could potentially
occur locally without preference for nuclear position and/or causing
repositioning. These findings have important implications for understanding
relationships between nuclear organization and gene expression patterns in
cancer. DNA methylation and chromatin remodeling are frequently implicated in the
silencing of genes involved in carcinogenesis. Long Range Epigenetic Silencing
(LRES) is a mechanism of gene inactivation that affects multiple contiguous CpG
islands and has been described in different human cancer types. However, it is
unknown whether there is a coordinated regulation of the genes embedded in these
regions in normal cells and in early stages of tumor progression. To better
characterize the molecular events associated with the regulation and remodeling
of these regions we analyzed two regions undergoing LRES in human colon cancer
in the mouse model. We demonstrate that LRES also occurs in murine cancer in
vivo and mimics the molecular features of the human phenomenon, namely,
downregulation of gene expression, acquisition of inactive histone marks, and
DNA hypermethylation of specific CpG islands. The genes embedded in these
regions showed a dynamic and autonomous regulation during mouse intestinal cell
differentiation, indicating that, in the framework considered here, the
coordinated regulation in LRES is restricted to cancer. Unexpectedly, benign
adenomas in Apc(Min/+) mice showed overexpression of most of the genes affected
by LRES in cancer, which suggests that the repressive remodeling of the region
is a late event. Chromatin immunoprecipitation analysis of the transcriptional
insulator CTCF in mouse colon cancer cells revealed disrupted chromatin domain
boundaries as compared with normal cells. Maligt regression of cancer cells
by in vitro differentiation resulted in partial reversion of LRES and gain of
CTCF binding. We conclude that genes in LRES regions are plastically regulated
in cell differentiation and hyperproliferation, but are constrained to a
coordinated repression by abolishing boundaries and the autonomous regulation of
chromatin domains in cancer cells. |
What is the mode of inheritance of Acromicric dysplasia? | Acromicric dysplasia has an autosomal dominant mode of inheritance | Acromicric dysplasia is a rare bone dysplasia characterised by short stature,
short hands and feet, normal intelligence, mild facial dysmorphism, and
characteristic x ray abnormalities of the hands. Only a very small number of
children with this condition have been reported so far. Here we report on a
series of 22 patients including 10 boys and 12 girls with acromicric dysplasia.
Length was normal at birth and height fell progressively off the centiles
postnatally. The mean adult height was 130 cm (133 cm in males, 129 cm in
females). The hands, feet, and limbs were short and OFC was normal. Intelligence
was normal and mild dysmorphic features were noted. Other occasional features
included well developed muscles, a hoarse voice, generalised joint limitation in
some patients, frequent ear, tracheal, and respiratory complication, and spine
abnormalities. Long term follow up showed that facial dysmorphism was less
obvious in adults and that carpal tunnel syndrome was frequent in older
patients. Apart from short metacarpals and phalanges, internal notch of the
second metacarpal, external notch of the fifth metacarpal, and internal notch of
the femoral heads, there were no major x ray abnormalities. No major
complications, such as cardiac disease or major orthopaedic problems, occurred
in the course of the disease. The condition appeared to be sporadic in 16 cases
but the observation of vertical transmission in three families was consistent
with an autosomal domit mode of inheritance. |
Can RNAPolII function as an RNA-dependent RNA-polymerase? | RNA polymerase II (Pol II) is a well-characterized DNA-dependent RNA polymerase, which has also been reported to have RNA-dependent RNA polymerase (RdRP) activity. Pol II can use a homopolymeric RNA template, can extend RNA by several nucleotides in the absence of DNA, and has been implicated in the replication of the RNA genomes of hepatitis delta virus (HDV) and plant viroids. The RdRP activity of Pol II provides a missing link in molecular evolution, because it suggests that Pol II evolved from an ancient replicase that duplicated RNA genomes. | Chimeric proteins joining the histone methyltransferase MLL with various fusion
partners trigger distinctive lymphoid and myeloid leukemias. Here, we
immunopurified proteins associated with ENL, a protein commonly fused to MLL.
Identification of these ENL-associated proteins (EAPs) by mass spectrometry
revealed enzymes with a known role in transcriptional elongation (RNA polymerase
II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation
factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase
DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and
polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was
further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and
colocalization experiments. Purified EAP showed a histone H3 lysine 79-specific
methylase activity, displayed a robust RNAPolII CTD kinase function, and
counteracted the effect of the pTEFb inhibitor
5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished
genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on
elongation, and inhibited transient transcriptional reporter activity. According
to structure-function data, DOT1L recruitment was important for transformation
by the MLL-ENL fusion derivative. These results suggest a function of ENL in
histone modification and transcriptional elongation. RNA polymerase (Pol) II catalyses DNA-dependent RNA synthesis during gene
transcription. There is, however, evidence that Pol II also possesses
RNA-dependent RNA polymerase (RdRP) activity. Pol II can use a homopolymeric RNA
template, can extend RNA by several nucleotides in the absence of DNA, and has
been implicated in the replication of the RNA genomes of hepatitis delta virus
(HDV) and plant viroids. Here we show the intrinsic RdRP activity of Pol II with
only pure polymerase, an RNA template-product scaffold and nucleoside
triphosphates (NTPs). Crystallography reveals the template-product duplex in the
site occupied by the DNA-RNA hybrid during transcription. RdRP activity resides
at the active site used during transcription, but it is slower and less
processive than DNA-dependent activity. RdRP activity is also obtained with part
of the HDV antigenome. The complex of transcription factor IIS (TFIIS) with Pol
II can cleave one HDV strand, create a reactive stem-loop in the hybrid site,
and extend the new RNA 3' end. Short RNA stem-loops with a 5' extension suffice
for activity, but their growth to a critical length apparently impairs
processivity. The RdRP activity of Pol II provides a missing link in molecular
evolution, because it suggests that Pol II evolved from an ancient replicase
that duplicated RNA genomes. RNA polymerase II (Pol II) is a well-characterized DNA-dependent RNA polymerase,
which has also been reported to have RNA-dependent RNA polymerase (RdRP)
activity. Natural cellular RNA substrates of mammalian Pol II, however, have not
been identified and the cellular function of the Pol II RdRP activity is
unknown. We found that Pol II can use a non-coding RNA, B2 RNA, as both a
substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt
on its 3'-end in an internally templated reaction. The RNA product resulting
from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a
factor present in nuclear extracts. Treatment of cells with α-amanitin or
actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA.
Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to
control the stability of a cellular RNA by extending its 3'-end. |
Which are the best treatment options to treat Helicobacter pylori? | The best treatment options for eradication of Helicobacter pylori involve triple or quadruple drugs therapy with different types of antibiotics.
Bismuth may be also an additional option. Proton pump inhibitors are also included in treatment.
The more effective drug list includes: amoxicillin, claritromycin, metronidazole rifabutin.
Also chitosan microspheres with Eudragit L100 have been tested. | OBJECTIF : Évaluer l’efficacité d’une thérapie de rattrapage faisant appel à de
la rifabutine, de l’amoxicilline et un inhibiteur de la pompe à protons (IPP)
pour éradiquer l’Helicobacter pylori chez des patients qui n’ont pas réagi à au
moins une cure de trithérapie à base d’IPP.
MÉTHODOLOGIE : La présente étude était une série de cas monocentrique auprès de
16 patients consécutifs qui avaient reçu au moins une cure de traitement
d’éradication standard. L’évaluation avant le traitement incluait une endoscopie
et des biopsies afin de procéder à l’histologie et à la culture de l’infection à
H pylori. Le traitement se composait d’un régime d’une semaine contet un IPP
deux fois par jour, 1 g d’amoxicilline (A) deux fois par jour et 300 g de
rifabutine (R) une fois par jour (IPP-AR). L’évaluation après le traitement
était constituée d’une reprise de l’endoscopie et d’une biopsie afin de procéder
à l’histologie et à la culture ou d’un test respiratoire à l’urée validé,
effectué au moins quatre semaines après la fin du traitement. La susceptibilité
au métronidazole, à la clarithromycine et à l’A avant le traitement ont été
évaluées au moyen d’un test d’epsilomètre validé.
RÉSULTATS : Des 16 patients, quatre avaient déjà reçu une cure de trithérapie,
dix en avaient reçu deux et deux, plus de deux. Le taux de réussite global de
l’IPP-AR s’élevait à 63 % (dix cas sur 16). La résistance à l’A correspondait à
0 % (zéro cas sur 13), au métronidazole, de 77 % (dix cas sur 13), à la
clarithromycine, de 70 % (sept cas sur dix), et à la fois au métronidazole et à
la clarithromycine, de 60 % (six cas sur dix). Il n’y avait pas de corrélation
entre les profils de résistance et le taux de guérison.
CONCLUSIONS : Un régime posologique contet de la R comme l’IPP-AR est un
option viable en guise de thérapie de rattrapage de l’infection à H pylori. Insufficient gastric mucosa drug concentration and short contact time were the
main reason for the lack of eradication efficacy of Helicobacter pylori for
peptic ulcer patients. Novel multi-core chitosan microspheres were prepared for
stomach-specific delivery of hydrophilic antibiotics for the treatment of peptic
ulcer. Chitosan microspheres with multiple Eudragit L100 cores were easily
prepared by a new emulsification/coagulation encapsulating method. Swelling
behaviors, surface amino groups and mucin absorption ability were investigated
and the formulation that showed best mucoadhesive potential was adopted. The
multi-core chitosan microspheres exhibited good mucoadhesiveness as well as
controlled release manner for incorporated antibiotics in acidic environment.
The release rate could be easily modulated with accumulative release ranging
from 47.3 to 79.3% in 6 h. Accordingly, the multi-core chitosan microspheres
could serve as a satisfactory vehicle for stomach-specific delivery of
hydrophilic antibiotics. Management of Helicobacter pylori infection is evolving. New data concern the
role of the bacterium in various clinical conditions, the indications of H.
pylori testing, diagnosis procedures and eradication treatment regimens. H.
pylori should be sought and eradicated before starting NSAIDs treatment, in
aspirin users with a history of gastroduodenal ulcer, and in patients presenting
with chronic dyspepsia, high risk factors of gastric cancer, unexplained iron
deficiency anaemia, vitamin B12 deficiency, or immune (idiopathic)
thrombocytopenic purpura. The Urea Breath Test remains the best test to diagnose
H. pylori infection, whereas serology is the only test, which is not affected by
local changes in the stomach. Molecular tests can be used to detect H. pylori
and clarithromycin and/or fluoroquinolone resistance in gastric biopsies without
necessitating culture. In regions of high clarithromycin resistance, such as
France, sequential treatment or bismuth-containing quadruple therapies are
replacing standard triple therapies for the first-line empirical treatment. The treatment of Helicobacter pylori infection is in a state of flux as
traditional therapies fail and new therapies do not achieve the 90% eradication
rates desired by clinicians. Triple therapy, which has been the mainstay of
treatment in many countries over the last decade, now has suboptimal results in
many parts of the world. A number of new treatments have been described with
variable success in different parts of the world. In this article, the
fundamentals of treatment for H. pylori treatment are reviewed and new treatment
algorithms are proposed for regions of the world where triple therapy is
failing. Sequential therapy and quadruple therapy (either bismuth-based or
non-bismuth-based) are the best current options to replace initial treatment
with triple therapy. When initial treatment fails, salvage treatments using
rifabutin and levofloxacin are the best options. With knowledge of local
resistance patterns and with meticulous confirmation of eradication with
retreatment, most H. pylori infections can be successfully eradicated. |
List sclerostin interaction partners. | alkaline phosphatase
carbonic anhydrase
gremlin-1
fetuin A
midkine
annexin A1
annexin A2
collagen α1
casein kinase II
secreted frizzled related protein 4
Phex
asporin
follistatin
erbB-3
LRP5
noggin | The secreted glycoprotein, sclerostin alters bone formation. To gain insights
into the mechanism of action of sclerostin, we examined the interactions of
sclerostin with bone proteins using a sclerostin affinity capture technique.
Proteins from decalcified rat bone were captured on a sclerostin-maltose binding
protein (MBP) amylose column, or on a MBP amylose column. The columns were
extensively washed with low ionic strength buffer, and bound proteins were
eluted with buffer containing 1M sodium chloride. Eluted proteins were separated
by denaturing sodium-dodecyl sulfate gel electrophoresis and were identified by
mass spectrometry. Several previously unidentified full-length
sclerostin-interacting proteins such as alkaline phosphatase, carbonic
anhydrase, gremlin-1, fetuin A, midkine, annexin A1 and A2, and collagen α1,
which have established roles in bone formation or resorption processes, were
bound to the sclerostin-MBP amylose resin but not to the MBP amylose resin.
Other full-length sclerostin-interacting proteins such as casein kinase II and
secreted frizzled related protein 4 that modulate Wnt signaling were identified.
Several peptides derived from proteins such as Phex, asporin and follistatin
that regulate bone metabolism also bound sclerostin. Sclerostin interacts with
multiple proteins that alter bone formation and resorption and is likely to
function by altering several biologically relevant pathways in bone. |
Under which conditions does AMPK phosphorylate TSC2? | The AMP-activated serine/threonine protein kinase (AMPK) is a sensor of cellular energy status found in all eukaryotes, and it is activated under conditions of low intracellular ATP following stresses such as nutrient deprivation or hypoxia. | Germline mutations in LKB1, TSC2, or PTEN tumor suppressor genes result in
hamartomatous syndromes with shared tumor biological features. The recent
observations of LKB1-mediated activation of AMP-activated protein kinase (AMPK)
and AMPK inhibition of mTOR through TSC2 prompted us to examine the biochemical
and biological relationship between LKB1 and mTOR regulation. Here, we report
that LKB1 is required for repression of mTOR under low ATP conditions in
cultured cells in an AMPK- and TSC2-dependent manner, and that Lkb1 null MEFs
and the hamartomatous gastrointestinal polyps from Lkb1 mutant mice show
elevated signaling downstream of mTOR. These findings position aberrant mTOR
activation at the nexus of these germline neoplastic conditions and suggest the
use of mTOR inhibitors in the treatment of Peutz-Jeghers syndrome. Target of Rapamycin (TOR), a giant protein kinase expressed by all eucaryotic
cells, controls cell size in response to nutrient signals. In metazoans, cell
and organismal growth is controlled by nutrients and the insulin/insulin-like
growth factor (IGF) system, and the understanding of how these inputs
coordinately regulate TOR signaling has advanced greatly in the past 5 years. In
single-cell eucaryotes and Caenorhabditis elegans, TOR is a domit regulator
of overall mRNA translation, whereas in higher metazoans, TOR controls the
expression of a smaller fraction of mRNAs that is especially important to cell
growth. TOR signals through two physically distinct multiprotein complexes, and
the control of cell growth is mediated primarily by TOR complex 1 (TORC1), which
contains the polypeptides raptor and LST8. Raptor is the substrate binding
element of TORC1, and the ability of raptor to properly present substrates, such
as the translational regulators 4E-BP and p70 S6 kinase, to the TOR catalytic
domain is essential for their TOR-catalysed phosphorylation, and is inhibited by
the Rapamycin/FKBP-12 complex. The domit proximal regulator of TORC1
signaling and kinase activity is the ras-like small GTPase Rheb. Rheb binds
directly to the mTOR catalytic domain, and Rheb-GTP enables TORC1 to attain an
active configuration. Insulin/IGF enhances Rheb GTP charging through the ability
of activated Akt to inhibit the Rheb-GTPase-activating function of the tuberous
sclerosis heterodimer (TSC1/TSC2). Conversely, energy depletion reduces Rheb-GTP
charging through the ability of the adenosine monophosphate-activated protein
kinase to phosphorylate TSC2 and stimulate its Rheb-GTPase activating function,
as well as by HIFalpha-mediated transcriptional responses that act upstream of
the TSC1/2 complex. Amino-acid depletion inhibits TORC1 acting predomitly
downstream of the TSC complex, by interfering with the ability of Rheb to bind
to mTOR. The components of the insulin/IGF pathway to TORC1 are now well
established, whereas the elements mediating the more ancient and functionally
domit input of amino acids remain largely unknown. AMPK is a highly conserved sensor of cellular energy status that is activated
under conditions of low intracellular ATP. AMPK responds to energy stress by
suppressing cell growth and biosynthetic processes, in part through its
inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK
phosphorylation of the TSC2 tumor suppressor contributes to suppression of
mTORC1; however, TSC2-deficient cells remain responsive to energy stress. Using
a proteomic and bioinformatics approach, we sought to identify additional
substrates of AMPK that mediate its effects on growth control. We report here
that AMPK directly phosphorylates the mTOR binding partner raptor on two
well-conserved serine residues, and this phosphorylation induces 14-3-3 binding
to raptor. The phosphorylation of raptor by AMPK is required for the inhibition
of mTORC1 and cell-cycle arrest induced by energy stress. These findings uncover
a conserved effector of AMPK that mediates its role as a metabolic checkpoint
coordinating cell growth with energy status. AMP-activated protein kinase (AMPK) performs a pivotal function in energy
homeostasis via the monitoring of intracellular energy status. Once activated
under the various metabolic stress conditions, AMPK regulates a multitude of
metabolic pathways to balance cellular energy. In addition, AMPK also induces
cell cycle arrest or apoptosis through several tumor suppressors including LKB1,
TSC2, and p53. LKB1 is a direct upstream kinase of AMPK, while TSC2 and p53 are
direct substrates of AMPK. Therefore, it is expected that activators of AMPK
signal pathway might be useful for treatment or prevention of cancer. In the
present study, we report that cryptotanshinone, a natural compound isolated from
Salvia miltiorrhiza, robustly activated AMPK signaling pathway, including LKB1,
p53, TSC2, thereby leading to suppression of mTORC1 in a number of
LKB1-expressing cancer cells including HepG2 human hepatoma, but not in
LKB1-deficient cancer cells. Cryptotanshinone induced HepG2 cell cycle arrest at
the G1 phase in an AMPK-dependent manner, and a portion of cells underwent
apoptosis as a result of long-term treatment. It also induced autophagic HepG2
cell death in an AMPK-dependent manner. Cryptotanshinone significantly
attenuated tumor growth in an HCT116 cancer xenograft in vivo model, with a
substantial activation of AMPK signal pathways. Collectively, we demonstrate for
the first time that cryptotanshinone harbors the therapeutic potential for the
treatment of cancer through AMPK activation. |
What imaging modalities have been listed as method of choice to diagnose CSF leak? | CT cisternography in the investigation of cerebrospinal fluid rhinorrhoea. CTC is an accurate, well-tolerated procedure and should be regarded as the method of choice for investigation of this condition.
...unenhanced (three-dimensional constructive interference in steady state (3D-CISS)...In conclusion, 3D-CISS is a non-invasive and reliable technique, and should be the first-choice method to localise CSF leak. | A case of intracranial hypotension with spontaneous cerebrospinal fluid (CSF)
leak was reported. A Tc-99m diethyltriaminepentacetic acid radionuclide
cisternography (RNC) showed the accumulation of radioactivity in the area of the
subarachnoid space, the poor migration of the isotope over the convexities, and
the early appearance of kidney and bladder activity. To localize the site of CSF
leak, RNC will be the choice, and when the time comes, RNC will work well in the
location of the leak. The aim of this prospective study was to evaluate the value of unenhanced
(three-dimensional constructive interference in steady state (3D-CISS)) and
contrast-enhanced MR cisternography (CE-MRC) in detecting the localisation of
cerebrospinal fluid (CSF) leak in patients with rhinorrhoea. 17 patients with
active or suspected CSF rhinorrhoea were included in the study. 3D-CISS
sequences in coronal and sagittal planes and fat-suppressed T1-weighted
spin-echo sequences in three planes before and after intrathecal contrast media
administration were obtained. Images were obtained of the cribriform plate and
sphenoid sinus. In addition, high-resolution CT (HRCT) was performed in order to
evaluate the bony elements. The leak was present in 9/17 patients with 3D-CISS
and 10/17 patients with CE-MRC. The leak from the cribriform plate to the nasal
cavity in six patients and from the sphenoid sinus in four patients was nicely
shown by CE-MRC. Eight of those patients were surgically treated, but
spontaneous regression of the symptoms in two precluded any intervention. The
leak localisations shown with CE-MRC were fully compatible with surgical
results. The sensitivities of HRCT, 3D-CISS and CE-MRC for showing CSF leakage
were 88%, 76% and 100%, respectively. In conclusion, 3D-CISS is a non-invasive
and reliable technique, and should be the first-choice method to localise CSF
leak. CE-MRC is helpful in conditions when there is no leak or in complicated
cases with a positive beta2-transferrin measurement. |
Which are currently available software tools for detecting rare codon clusters in coding sequences? | Rare codon clusters (RCCs) correspond to regions along mRNA sequences where among the possible choices of synonymous codons those with lower usage are observed. Due to the fact that relative codon frequencies have been shown to correlate with their cognate tRNA frequencies, RCCs indicate possible translational attenuation sites. A few tools specific for this task have been described in the literature, namely: LaTcOm, %MinMax, PAUSE, Sherlocc, Sliding Window (RiboTempo) | The PAUSE software has been developed as a new tool to study translational
control over protein targeting. This makes it possible to correlate the position
of clusters of rare codons in a gene, predicted to cause a translational pause,
with the position of hydrophobic stretches in the encoded protein, predicted to
span a membrane or to act as a cleavable signal for targeting to the secretory
pathway. Furthermore, this software gathers these correlations over whole sets
of genes. The PAUSE software is described here, and its use is illustrated on a
set of membrane proteins from the fungus Emericella nidulans. Preferential
distances of about 45 codons and of about 70 codons between putative
transmembrane domains and predicted translational pauses were observed. Given
that approximately 30 residues are required to span the large ribosomal subunit,
the predicted pauses would therefore occur when the hydrophobic domain starts
protruding from the ribosome ('+45 pause'), or fully protrudes as a hairpin
('+70 pause'). Thus, these specific pauses might reflect a translational control
over membrane protein targeting or early recognition ('+45 pause'), and over
insertion or folding ('+70 pause'). Most amino acids are encoded by more than one codon. These synonymous codons are
not used with equal frequency: in every organism, some codons are used more
commonly, while others are more rare. Though the encoded protein sequence is
identical, selective pressures favor more common codons for enhanced translation
speed and fidelity. However, rare codons persist, presumably due to neutral
drift. Here, we determine whether other, unknown factors, beyond neutral drift,
affect the selection and/or distribution of rare codons. We have developed a
novel algorithm that evaluates the relative rareness of a nucleotide sequence
used to produce a given protein sequence. We show that rare codons, rather than
being randomly scattered across genes, often occur in large clusters. These
clusters occur in numerous eukaryotic and prokaryotic genomes, and are not
confined to unusual or rarely expressed genes: many highly expressed genes,
including genes for ribosomal proteins, contain rare codon clusters. A rare
codon cluster can impede ribosome translation of the rare codon sequence. These
results indicate additional selective pressures govern the use of synonymous
codons, and specifically that local pauses in translation can be beneficial for
protein biogenesis. We present LaTcOm, a new web tool, which offers several alternative methods for
'rare codon cluster' (RCC) identification from a single and simple graphical
user interface. In the current version, three RCC detection schemes are
implemented: the recently described %MinMax algorithm and a simplified sliding
window approach, along with a novel modification of a linear-time algorithm for
the detection of maximally scoring subsequences tailored to the RCC detection
problem. Among a number of user tunable parameters, several codon-based scales
relevant for RCC detection are available, including tRNA abundance values from
Escherichia coli and several codon usage tables from a selection of genomes.
Furthermore, useful scale transformations may be performed upon user request
(e.g. linear, sigmoid). Users may choose to visualize RCC positions within the
submitted sequences either with graphical representations or in textual form for
further processing.
AVAILABILITY: LaTcOm is freely available online at the URL
http://troodos.biol.ucy.ac.cy/latcom.html. MOTIVATION: An increasing amount of evidence from experimental and computational
analysis suggests that rare codon clusters are functionally important for
protein activity. Most of the studies on rare codon clusters were performed on a
limited number of proteins or protein families. In the present study, we present
the Sherlocc program and how it can be used for large scale protein family
analysis of evolutionarily conserved rare codon clusters and their relation to
protein function and structure. This large-scale analysis was performed using
the whole Pfam database covering over 70% of the known protein sequence
universe. Our program Sherlocc, detects statistically relevant conserved rare
codon clusters and produces a user-friendly HTML output.
RESULTS: Statistically significant rare codon clusters were detected in a
multitude of Pfam protein families. The most statistically significant rare
codon clusters were predomitly identified in N-terminal Pfam families. Many
of the longest rare codon clusters are found in membrane-related proteins which
are required to interact with other proteins as part of their function, for
example in targeting or insertion. We identified some cases where rare codon
clusters can play a regulating role in the folding of catalytically important
domains. Our results support the existence of a widespread functional role for
rare codon clusters across species. Finally, we developed an online filter-based
search interface that provides access to Sherlocc results for all Pfam families.
AVAILABILITY: The Sherlocc program and search interface are open access and are
available at http://bcb.med.usherbrooke.ca |
How many tissue kallikrein genes are present in the human genome? | Tissue kallikreins (KLKs) are a group of closely related serine proteinases that are represented by multigene families in the human genome. The human tissue kallikrein gene family consists of 15 genes, denoted KLK1–KLK15, tandemly arranged on chromosomal locus 19q13.4. | The cDNA for the trypsin-like serine protease gene (TLSP, HGMW-approved symbol
PRSS20) has been recently identified. TLSP is expressed in brain and skin
tissues but little else is known about this new serine protease gene. In this
paper, we describe the complete genomic organization and precise mapping of the
TLSP gene. This gene spans 5.3 kb of genomic sequence on chromosome 19q13.3-q13.
4. The gene consists of six exons, the first of which is untranslated. All
splice junctions follow the GT/AG rule, and the intron phases are identical to
those of other kallikrein-like genes, including zyme (PRSS9), NES1 (PRSSL1), and
neuropsin (PRSS19). Fine-mapping of the area indicates that TLSP lies downstream
from the PSA, zyme, neuropsin, and NES1 genes. Significant sequence homologies
were found between TLSP and other human kallikreins. Furthermore, there is
conservation of the catalytic triad (histidine, aspartic acid, serine) and of
the number of coding exons (five; the same in all members of the kallikrein gene
family). We thus suggest that TLSP is a new member of the human kallikrein gene
family. TLSP is expressed in many tissues including cerebellum, prostate,
salivary glands, stomach, lung, thymus, small intestine, spleen, liver, and
uterus. TLSP expression appears to be regulated by steroid hormones in the
breast carcinoma cell line BT-474. The glandular kallikrein family is composed of structurally related serine
proteases. Studies show that the mouse family encompasses at least 14 highly
conserved functional genes, but of these only the tissue kallikarein has a human
ortholog. In man, the tissue kallikrein display high sequence similarity with
prostate specific antigen and human glandular kallikrein 2, suggesting that they
evolved after the separation of primates and rodents. A phylogenetic study of
the genes encoding glandular kallikreins in species evolutionarily located
between rodents and man may reveal interesting details on how the gene family
evolved, which in turn could yield information about the function of the
proteins. Therefore, we have initiated a study of the glandular kallikreins of
the cotton-top tamarin (Saguinus oedipus), a New World Monkey. Here, we report
the cloning and nucleotide sequence of one of these, the tissue kallikrein gene.
The gene of 4.4 kb is composed of five exons, and the structure is 90% similar
to that of the orthologous human gene. It gives rise to a polypeptide of 261
amino acids, including a signal peptide of 17 residues, a pro-piece of 7
residues, and the mature protein of 237 residues with an estimated molecular
mass of 26.3 kD. The similarity to the human prostate specific antigen and human
glandular kallikrein 2 genes is 73% and 72%, respectively, including introns and
flanking regions. The lower similarity to these genes compared with the human
tissue kallikrein gene indicates that they, or a progenitor to them, arose in
primates prior to the separation of New and Old World monkeys. Genomic Southern
blots also show that the cotton-top tamarin genome encompasses at least one more
glandular kallikrein gene. Minisatellites are repetitive sequences of DNA that are present throughout the
genome. Although the origin and function of these minisatellites is still
unknown, they found clinical applications as markers of many diseases, including
cancer. Also, they are useful tools for DNA fingerprinting and linkage analysis.
Kallikreins are serine proteases that appear to be involved in many diseases
including brain disorders and maligcy. We have recently characterized the
human kallikrein gene locus on chromosome 19q13.4, which includes 15 kallikrein
genes. In this study, we examined the kallikrein locus ( approximately 300 Kb)
for all known repeat elements. About 50% of this genomic area is occupied by
different repeat elements. We also identified unique minisatellite elements that
are restricted to chromosome 19q13. Ten clusters of these minisatellites are
distributed along the locus on either DNA strand. The clusters are located in
the promoters and enhancers of genes, in introns, and in untranslated regions of
the mRNA. Analysis of these elements indicates that they are polymorphic, thus
they can be useful in linkage analysis and DNA fingerprinting. Our preliminary
results indicate also that the distribution of the different alleles of these
minisatellites might be associated with maligcy. Kallikreins are proteolytic enzymes that constitute a subfamily of serine
proteases. Novel kallikrein genes were cloned recently, and it was shown that
the human kallikrein family contains 15 genes tandemly aligned on chromosomal
locus 19q13.3-q13.4. Based on their altered expression in tumor cells,
kallikreins may be involved in the pathogenesis and/or progression of cancer.
Evidence is presented that certain kallikreins may be exploited as diagnostic
cancer biomarkers. Although the function(s) of novel kallikreins is currently
unknown, increasing evidence suggests that kallikreins may participate in
regulatory enzymatic cascade(s). Elucidation of the function of novel
kallikreins largely depends on the availability of active recombit proteins.
Here, the zymogen for kallikrein 13 was overexpressed in Pichia pastoris and
biochemically characterized. It was shown that the kallikrein 13 zymogen
displays intrinsic catalytic activity leading to autoactivation. A clipped form
of kallikrein 13 was identified, indicating autocatalytic cleavage at the
internal bond R114-S115. Mature kallikrein 13 displays trypsin-like activity
with restricted specificity on synthetic and protein substrates. Combinatorial
P1-Lys libraries of tetrapeptide fluorogenic substrates were synthesized and
used for the profiling of the P2 specificity of selected kallikreins.
Interestingly, it was shown that human kallikrein 13, similarly to PSA, could
specifically cleave human plasminogen to generate angiostatin-like fragments,
suggesting that specific kallikreins may have antiangiogenic actions. An
understanding of the physiology of human kallikreins is emerging with potential
clinical applications. Recent evidence suggests that many members of the human kallikrein (KLK) gene
family are differentially regulated in ovarian cancer and have potential as
diagnostic and/or prognostic markers. We used the serial analysis of gene
expression and expressed sequence tag databases of the Cancer Genome Anatomy
Project to perform in silico analyses of the expression pattern of the 15 human
KLK genes in normal and cancerous ovarian tissues and cell lines. We found that
seven KLK genes (KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, and KLK14) are
up-regulated in ovarian cancer. Probing 2 normal and 10 ovarian cancer serial
analysis of gene expression libraries with gene-specific tags for each KLK
indicated that whereas no expression was detected in any normal libraries (with
the exception of KLK10 and KLK11), these KLKs were found to be expressed with
moderate densities (103-408 tags per million) in 40-60% of the ovarian cancer
libraries analyzed. These data were verified by screening the expressed sequence
tag databases, where 78 of 79 mRNA clones isolated for these genes were from
ovarian cancer libraries. X-profiler comparison of the pools of normal and
cancerous ovaries identified a significant difference in expression levels for
six of the seven KLKs. We experimentally verified the overexpression of six KLK
proteins in cancer versus normal or benign tissues with highly sensitive and
specific immunofluorometric assays. A statistically significant stepwise
increase in protein levels was found among normal, benign, and cancerous ovarian
tissues. The expression of five KLKs showed a strong degree of correlation at
the protein level, suggesting the existence of a common mechanism or pathway
that controls the expression of this group of adjacent genes during ovarian
cancer progression. Tissue kallikreins are a group of serine proteases that are found in many organs
and biologic fluids. Tissue kallikrein genes (KLKs) are found on chromosome
19q13.3-4 as a gene cluster encoding 15 different serine proteases. In skin, two
tissue kallikrein proteins, hK5 and hK7, are expressed in the stratum corneum
and are known to be involved in desquamation of corneocytes. The possible
involvement of other kallikrein proteins has not been clarified, however, nor
has the significance of each member in the serine protease activity of skin been
delineated. In the study described here, we examined expression and localization
of KLK mRNA in normal human skin by means of RT-PCR and in situ hybridization.
Quantitative RT-PCR analysis showed abundant expression of KLK1 and KLK11 mRNA,
moderate expression of KLK4, KLK5, KLK6, KLK7, and KLK13 mRNA, and low
expression of KLK8 mRNA in normal human skin. For KLK4, KLK8, and KLK13 mRNA,
splice variants were identified to be their major mRNA species. Two variants for
KLK13 mRNA were novel. The amount of the serine protease inhibitor Kazal-type 5
(SPINK5) mRNA was comparable to KLK1 and KLK11 mRNA. In situ hybridization
revealed intense expression of all KLK mRNA studied except KLK12 mRNA in the
stratum granulosum of normal epidermis, where SPINK5 mRNA coexisted. Excluding
KLK13 mRNA, they are also expressed in hair sheath, eccrine sweat glands, and
sebaceous glands. Coexpression of various KLK and SPINK5 mRNA suggests that
their proteins are the candidates to balance and maintain serine protease
activities in both the skin and appendages. Recent evidence suggests that many members of the human kallikrein gene family
are differentially regulated in breast cancer and other endocrine-related
maligcies. In this study, we utilised the serial analysis of gene expression
(SAGE) and expressed sequence tag (EST) databases of the Cancer Genome Anatomy
Project (CGAP) to perform in silico analyses of the expression pattern of the 15
human kallikrein genes in normal and cancerous breast tissues and cell lines
using different analytical tools such as Virtual Northern blotting, Digital
Differential Display and X-profiler. Our results indicate that at least four
kallikrein genes (KLK5, 6, 8, 10) are downregulated in breast cancer. Probing
eight normal and 24 breast cancer SAGE libraries with gene-specific tags for
each of the above kallikreins indicated moderate-to-high expression densities in
normal breast (27-319 tags per million; tpm, in two to five out of eight
libraries), compared to no or low expression (0 - 34 tpm in zero to two
libraries out of 24) in breast cancer. These data were verified by screening the
EST databases, where all mRNA clones isolated for these genes, except for one in
each, were from normal breast libraries, with no clones detected from breast
cancer tissues or cell lines (with the exception of KLK8). X-profiler comparison
of two pools of normal and breast cancer libraries further verified the presence
of significant downregulation of expression levels of 4 of the kallikreins genes
(KLK5, 6, 10, 12). We experimentally verified the downregulation of these four
kallikreins (KLK5, 6, 8, 10 and 12) by RT - PCR analysis. Human kallikreins are a cluster of 15 serine protease genes located in the
chromosomal band 19q13.4, a non-randomly rearranged region in many solid tumors,
including pancreatic cancer. We utilized the SAGE and EST databases of the
Cancer Genome Anatomy Project to perform in-silico analysis of kallikrein gene
expression in normal and cancerous pancreatic and colon tissues and cell lines
using virtual Northern blotting (VNB), digital differential display (DDD) and
X-profiler. At least two kallikreins, KLK6 and KLK10, are significantly
up-regulated in pancreatic cancer. We probed 2 normal and 6 pancreatic cancer
SAGE libraries with gene-specific tags for each of these kallikreins. KLK6 was
found to be expressed in 5/6 cancer libraries and showed the most marked
(5-fold) increase in average expression levels in cancer vs. normal. These data
were verified by screening the EST databases, where all mRNA clones isolated
were from cancerous libraries, with no clones detected in normal pancreatic
tissues or cell lines. X-profiler comparison of two pools of normal and
cancerous pancreatic libraries further verified the significant increase of KLK6
expression levels in pancreatic cancer. DDD data showed a 13-fold increase in
KLK10 expression in pancreatic cancer. Three kallikrein genes, KLK6, 8 and 10
are overexpressed in colon cancer compared to normal colon, while one
kallikrein, KLK1, is down-regulated. While no expression of KLK6 was detected in
normal colon, KLK6-specific tags were detectable in 2 cancer libraries. Similar
results were obtained by EST screening; no KLK6 clones were detected in any of
the 28 normal libraries examined, while 10 KLK6 EST clones were found in colon
adenocarcinoma. KLK10 was not detectable in normal colon. Gene-specific tags
were, however, detectable with high density in colon cancer and 7 EST clones
were found to be expressed in colon Adenocarcinoma. Human tissue kallikreins (hKs), which are encoded by the largest contiguous
cluster of protease genes in the human genome, are secreted serine proteases
with diverse expression patterns and physiological roles. Although primarily
known for their clinical applicability as cancer biomarkers, recent evidence
implicates hKs in many cancer-related processes, including cell-growth
regulation, angiogenesis, invasion and metastasis. They have been shown to
promote or inhibit neoplastic progression, acting individually and/or in
cascades with other hKs and proteases, and might represent attractive targets
for therapeutic intervention. Seminal fluid proteins show striking effects on reproduction, involving
manipulation of female behavior and physiology, mechanisms of sperm competition,
and pathogen defense. Strong adaptive pressures are expected for such
manifestations of sexual selection and host defense, but the extent of positive
selection in seminal fluid proteins from divergent taxa is unknown. We
identified adaptive evolution in primate seminal proteins using genomic
resources in a tissue-specific study. We found extensive signatures of positive
selection when comparing 161 human seminal fluid proteins and 2,858
prostate-expressed genes to those in chimpanzee. Seven of eight outstanding
genes yielded statistically significant evidence of positive selection when
analyzed in divergent primates. Functional clues were gained through divergent
analysis, including several cases of species-specific loss of function in
copulatory plug genes, and statistically significant spatial clustering of
positively selected sites near the active site of kallikrein 2. This study
reveals previously unidentified positive selection in seven primate seminal
proteins, and when considered with findings in Drosophila, indicates that
extensive positive selection is found in seminal fluid across divergent
taxonomic groups. Human tissue kallikreins (hKs) are attracting increased attention owing to their
association with various forms of cancer and other diseases. Human tissue
kallikrein genes represent the largest contiguous group of proteases within the
human genome. There are many areas of kallikrein research that need to be
further explored, including their tissue expression patterns, their regulation,
identification of specific substrates, their participation in proteolytic
cascades, and their clinical applicability as cancer biomarkers and therapeutic
targets. In this review, we briefly describe the current status of kallikrein
research and identify future avenues that will enhance our understanding of
their function and involvement in human diseases. The tissue kallikrein gene family consists of 15 genes tandemly arranged on
human chromosome 19q13.4. Most kallikrein genes are characterized by aberrant
expression patterns in various human cancers, a feature that makes them ideal
cancer biomarkers. In the present study, we investigated the effect of the
epigenetic drug compound 5-aza-2'-deoxycytidine on the expression of
downregulated kallikrein genes in prostate, breast, and ovarian cancer cell
lines. Reactivation of multiple kallikrein genes was observed, although some of
these genes do not contain CpG islands in their genomic sequence. Epigenetic
regulation provides a new mechanism for the pharmacological modulation of
kallikreins in human cancers with putative therapeutic implications. The human tissue kallikrein family of serine proteases (hK1-hK15 encoded by the
genes KLK1-KLK15) is involved in several cancer-related processes. Accumulating
evidence suggests that certain tissue kallikreins are part of an enzymatic
cascade pathway that is activated in ovarian cancer and other maligt
diseases. In the present study, OV-MZ-6 ovarian cancer cells were stably
co-transfected with plasmids expressing hK4, hK5, hK6, and hK7. These cells
displayed similar proliferative capacity as the vector-transfected control cells
(which do not express any of the four tissue kallikreins), but showed
significantly increased invasive behavior in an in vitro Matrigel invasion assay
(p<0.01; Mann-Whitney U-test). For in vivo analysis, the cancer cells were
inoculated into the peritoneum of nude mice. Simultaneous expression of hK4,
hK5, hK6, and hK7 resulted in a remarkable 92% mean increase in tumor burden
compared to the vector-control cell line. Five out of 14 mice in the 'tissue
kallikrein overexpressing' group displayed a tumor/situs ratio greater than
0.198, while this weight limit was not exceeded at all in the vector control
group consisting of 13 mice (p=0.017; chi2 test). Our results strongly support
the view that tumor-associated overexpression of tissue kallikreins contributes
to ovarian cancer progression. Kallikrein gene families have been identified previously in genomes of the
human, the mouse, and the rat, and individual kallikrein-like genes have been
found in many more species. This study presents the in silico identification of
kallikrein gene families in the recently sequenced genomes of four additional
mammalian species, the chimpanzee, the dog, the pig, and the opossum.
Phylogenies were constructed with gene sequences from all seven mammalian
families, using Bayesian analysis, which clarified the evolutionary
relationships between these genes. Individual gene sequences, as well as
concatenated constructs of multiple sequences, were used. Fifteen kallikrein
genes were located in the chimpanzee (Pan troglodytes) genome, while only 14
were identified in the canine (Canis familiaris) genome as no orthologue to
human KLK3 was found. Thirteen genes were identified from the pig (Sus scrofa)
genome, which lacked homologues to KLK2 and KLK3, and 11 genes, orthologous to
human KLK5 through KLK15, were found in the opossum (Monodelphis domestica)
genome. No kallikrein genes were identified from the available genome sequences
of the chicken (Gallus gallus) or African clawed frog (Xenopus tropicalis).
Within the family of kallikreins several subfamilies were suggested by
phylogenetic analysis. One consisted of KLK4, KLK5, and KLK14; another of KLK9,
KLK11, and KLK15; a third of KLK10 and KLK12; a fourth of KLK6 and KLK13; and
finally one of KLK8 and the classical kallikreins (KLK1, KLK2, and KLK3). Kallikreins belong to a family of serine proteases that are widespread
throughout living organisms, expressed in diverse tissue-specific patterns, and
known to have highly diverse physiological functions. The 15 human and 24 mouse
kallikreins have been implicated in pathophysiology of brain, kidney, and
respiratory and reproductive systems and often are used as cancer biomarkers. To
better elucidate the structure and evolutionary origin of this important gene
family in the pig, we have constructed a contiguous BAC clone-derived physical
map of the porcine kallikrein gene region and have fully sequenced a BAC clone
containing 13 kallikrein genes, 11 of which are novel. Radiation hybrid mapping
assigns this kallikrein-gene-rich region to porcine chromosome 6. Phylogenetic
and percent identity plot-based analyses revealed strong structure and order
conservation of kallikreins among four mammalian species. Reverse
transcriptase-polymerase chain reaction-based expression analysis of porcine
kallikreins showed a complex expression pattern across different tissues with
the thymus being the only tissue expressing all 13 kallikrein genes. [The
sequence data described in this paper has been submitted to GenBank under
Accession No. AC149292]. microRNAs (miRNAs) are a recently discovered class of small non-coding RNAs that
regulate gene expression. Rapidly accumulating evidence has revealed that miRNAs
are associated with cancer. The human tissue kallikrein gene family is the
largest contiguous family of proteases in the human genome, containing 15 genes.
Many kallikreins have been reported as potential tumor markers. In this review,
recent bioinformatics and experimental evidence is presented indicating that
kallikreins are potential miRNA targets. The available experimental approaches
to investigate these interactions and the potential diagnostic and therapeutic
applications are also discussed. miRNAs represent a possible regulatory
mechanism for controlling kallikrein expression at the post-transcriptional
level. Many miRNAs were predicted to target kallikreins and a single miRNA can
target more than one kallikrein. Recent evidence suggests that miRNAs can also
exert 'quantitative' control of kallikreins by utilizing multiple targeting
sites in the kallikrein mRNA. More research is needed to experimentally verify
the in silico predictions and to investigate the possible role in tumor
initiation and/or progression. The 15 members of the kallikrein-related serine peptidase (KLK) family have
diverse tissue-specific expression profiles and putative proteolytic functions.
The kallikrein family is also emerging as a rich source of disease biomarkers
with KLK3, commonly known as prostate-specific antigen, being the current serum
biomarker for prostate cancer. The kallikrein locus is also notable because it
is extraordinarily responsive to steroids and other hormones. Indeed, at least
14 functional hormone response elements have been identified in the kallikrein
locus. A more comprehensive understanding of the transcriptional regulation of
kallikreins may help the field make more informed hypotheses about the
physiological functions of kallikreins and their effectiveness as biomarkers. In
this review, we describe the organization of the kallikrein locus and the
structure of kallikrein genes and proteins. We also focus on the transcriptional
regulation of kallikreins by androgens, progestins, glucocorticoids,
mineralocorticoids, estrogens, and other hormones in animal models and human
prostate, breast, and reproductive tract tissues. The interaction of the
androgen receptor with androgen response elements in the promoter and enhancer
of KLK2 and KLK3 is also summarized in detail. There is evidence that all
kallikreins are regulated by multiple nuclear receptors. Yet, apart from KLK2
and KLK3, it is not clear whether all kallikreins are direct transcriptional
targets. Therefore, we argue that gaining more detailed information about the
mechanisms that regulate kallikrein expression should be a priority of future
studies and that the kallikrein locus will continue to be an important model in
the era of genome-wide analyses. BACKGROUND: Kallikrein-related peptidases (KLKs) are a family of serine
proteases that have been shown to be dysregulated in several maligcies
including ovarian cancer. The control of kallikrein genes and their
physiological function in cancer is not well understood. We hypothesized that
microRNAs (miRNAs) represent a novel mechanism for post-transcriptional control
of KLK expression in cancer.
METHODS: We first analysed miRNA expression in ovarian cancer in silico. A total
of 98 miRNAs were reported to have altered expression in ovarian cancer. Three
of these miRNAs were predicted to target KLK10. We experimentally verified the
predicted miR-KLK10 interaction using two independent techniques, a luciferase
assay with a construct containing the KLK10 3' untranslated region (UTR),
pMIR-KLK10, and measuring KLK10 protein levels after transfection with miRNA.
RESULTS: When we co-transfected cells with pMIR-KLK10 and either let-7f,
miR-224, or mR-516a, we saw decreased luciferase signal, suggesting that these
miRNAs can target KLK10. We then examined the effect of these three miRNAs on
KLK10 protein expression and cell growth. Transfection of all miRNAs, let-7f,
miR-224, and miR-516a led to a decrease in protein expression and cellular
growth. This effect was shown to be dose dependent. The KLK10 protein levels
were partially restored by co-transfecting let-7f and its inhibitor. In
addition, there was a slight decrease in KLK10 mRNA expression after
transfection with let-7f.
CONCLUSION: Our results confirm that KLKs can be targeted by more than one
miRNA. Increased expression of certain miRNAs in ovarian cancer can lead to
decreased KLK protein expression and subsequently have a negative effect on cell
proliferation. This dose-dependent effect suggests that a 'tweaking' or
'fine-tuning' mechanism exists in which the expression of one KLK can be
controlled by multiple miRNAs. These data together suggest that miRNA may be
used as potential therapeutic options and further studies are required. Early diagnosis of cancer and early detection of relapse following surgery are
critical for the effective treatment of the disease and for a positive clinical
outcome. Identification of novel diagnostic, prognostic and predictive
biomarkers will contribute utmost to clinical decision-making. The human tissue
kallikrein and kallikrein-related peptidases (KLKs), encoded by the largest
contiguous cluster of protease genes in the human genome, are secreted serine
proteases with diverse expression patterns and physiological roles. The aberrant
expression of KLKs in various maligcies as well as their involvement in many
cancer-related processes, such as cell growth regulation, angiogenesis,
invasion, and metastasis, has prompted scientists to investigate their potential
as cancer biomarkers. Expression of distinct KLKs is associated with
clinicopathological parameters of cancer patients. Moreover, several KLKs
possess significant favorable or unfavorable prognostic value in various
maligcies, with prostate-specific antigen (PSA) being the most widely used
biomarker in clinical practice, today. KLKs are also considered as very
promising biomarkers for cancer personalized medicine, especially for prediction
and monitoring of patients' response to chemotherapy, therefore opening up new
horizons towards effective patient monitoring post-treatment. This review
describes the current status of KLKs as tumor biomarkers. |
Does the Oncotype DX test work with paraffin embedded tissues? | Yes, the Oncotype DX test works with paraffin embedded tissue. | BACKGROUND: Oncotype DX is a clinically validated, high-complexity, multianalyte
reverse transcription-PCR genomic test that predicts the likelihood of breast
cancer recurrence in early-stage, node-negative, estrogen receptor-positive
breast cancer. The Recurrence Score (RS) provides a more accurate, reproducible
measure of breast cancer aggressiveness and therapeutic responsiveness than
standard measures. Individualized patient management requires strict performance
criteria for clinical laboratory tests. We therefore investigated the analytical
performance of the assay.
METHODS: Assays used a pooled RNA sample from fixed paraffin-embedded tissues to
evaluate the analytical performance of a 21-gene panel with respect to
amplification efficiency, precision, linearity, and dynamic range, as well as
limits of detection and quantification. Performance variables were estimated
from assays carried out with sample dilutions. In addition, individual patient
samples were used to test the optimized assay for reproducibility and sources of
imprecision.
RESULTS: Assay results defined acceptable operational performance ranges,
including an estimated maximum deviation from linearity of <1 cycle threshold
(C(T)) units over a > or =2000-fold range of RNA concentrations, with a mean
quantification bias of 0.3% and CVs of 3.2%-5.7%. An analysis of study design
showed that assay imprecision contributed by instrument, operator, reagent, and
day-to-day baseline variation was low, with SDs of <0.5 C(T).
CONCLUSION: The analytical and operational performance specifications defined
for the Oncotype DX assay allow the reporting of quantitative RS values for
individual patients with an SD within 2 RS units on a 100-unit scale. Novel genetic profiling tests of breast cancer tissue have been shown to be
prognostic for overall survival and predictive of local and distant rates of
recurrence in breast cancer patients. One of these tests, Oncotype DXtrade mark,
is a diagnostic test comprised of a 21-gene assay applied to paraffin-embedded
breast cancer tissue, which allows physicians to predict subgroups of
hormone-receptor-positive, node-negative patients who may benefit from hormonal
therapy alone or require adjuvant chemotherapy to attain the best survival
outcome. The results of the assay are converted to a recurrence score (0-100)
that has been found to be predictive of 10- and 15-year local and distant
recurrence in node-negative, estrogen-receptor-positive breast cancer patients.
Previous studies have shown that patients with high recurrence scores benefit
from adjuvant chemotherapy, whereas patients with low recurrence scores do not.
To evaluate the ability to guide treatment decisions in the group with a
mid-range recurrence score, the North American Cooperative Groups developed the
Trial Assessing IndiviuaLized Options for Treatment for breast cancer, a
randomized trial of chemotherapy followed by hormonal therapy versus hormonal
therapy alone on invasive disease-free survival-ductal carcinoma in situ
(IDFS-DCIS) survival in women with node-negative, estrogen-receptor-positive
breast cancer with a recurrence score of 11-25. The study was initiated in May
2006 and approximately 4500 patients will be randomized. This article describes
the rationale, methodology, statistical ana-lysis and implications of the
results on clinical practice. In February 2010, the Medical Advisory Secretariat (MAS) began work on
evidence-based reviews of published literature surrounding three pharmacogenomic
tests. This project came about when Cancer Care Ontario (CCO) asked MAS to
provide evidence-based analyses on the effectiveness and cost-effectiveness of
three oncology pharmacogenomic tests currently in use in Ontario.Evidence-based
analyses have been prepared for each of these technologies. These have been
completed in conjunction with internal and external stakeholders, including a
Provincial Expert Panel on Pharmacogenomics (PEPP). Within the PEPP, subgroup
committees were developed for each disease area. For each technology, an
economic analysis was also completed by the Toronto Health Economics and
Technology Assessment Collaborative (THETA) and is summarized within the
reports.THE FOLLOWING REPORTS CAN BE PUBLICLY ACCESSED AT THE MAS WEBSITE AT:
www.health.gov.on.ca/mas or at
www.health.gov.on.ca/english/providers/program/mas/mas_about.htmlGENE EXPRESSION
PROFILING FOR GUIDING ADJUVANT CHEMOTHERAPY DECISIONS IN WOMEN WITH EARLY BREAST
CANCER: An Evidence-Based and Economic AnalysisEpidermal Growth Factor Receptor
Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine
Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung
Cancer: An Evidence-Based and Ecopnomic AnalysisK-RAS testing in Treatment
Decisions for Advanced Colorectal Cancer: an Evidence-Based and Economic
Analysis
OBJECTIVE: To review and synthesize the available evidence regarding the
laboratory performance, prognostic value, and predictive value of Oncotype-DX
for the target population.
CLINICAL NEED: CONDITION AND TARGET POPULATION The target population of this
review is women with newly diagnosed early stage (stage I-IIIa) invasive breast
cancer that is estrogen-receptor (ER) positive and/or progesterone-receptor (PR)
positive. Much of this review, however, is relevant for women with early stage
(I and II) invasive breast cancer that is specifically ER positive, lymph node
(LN) negative and human epidermal growth factor receptor 2 (HER-2/neu) negative.
This refined population represents an estimated incident population of 3,315 new
breast cancers in Ontario (according to 2007 data). Currently it is estimated
that only 15% of these women will develop a distant metastasis at 10 years;
however, a far great proportion currently receive adjuvant chemotherapy,
suggesting that more women are being treated with chemotherapy than can benefit.
There is therefore a need to develop better prognostic and predictive tools to
improve the selection of women that may benefit from adjuvant chemotherapy.
TECHNOLOGY OF CONCERN: The Oncotype-DX Breast Cancer Assay (Genomic Health,
Redwood City, CA) quantifies gene expression for 21 genes in breast cancer
tissue by performing reverse transcription polymerase chain reaction (RT-PCR) on
formalin-fixed paraffin-embedded (FFPE) tumour blocks that are obtained during
initial surgery (lumpectomy, mastectomy, or core biopsy) of women with early
breast cancer that is newly diagnosed. The panel of 21 genes include genes
associated with tumour proliferation and invasion, as well as other genes
related to HER-2/neu expression, ER expression, and progesterone receptor (PR)
expression.
RESEARCH QUESTIONS: What is the laboratory performance of Oncotype-DX?How
reliable is Oncotype-DX (i.e., how repeatable and reproducible is
Oncotype-DX)?How often does Oncotype-DX fail to give a useable result?What is
the prognostic value of Oncotype-DX?Is Oncotype-DX recurrence score associated
with the risk of distant recurrence or death due to any cause in women with
early breast cancer receiving tamoxifen?What is the predictive value of
Oncotype-DX?Does Oncoytpe-DX recurrence score predict significant benefit in
terms of improvements in 10-year distant recurrence or death due to any cause
for women receiving tamoxifen plus chemotherapy in comparison to women receiving
tamoxifen alone?How does Oncotype-DX compare to other known predictors of risk
such as Adjuvant! Online?How does Oncotype-DX impact patient quality of life and
clinical/patient decision-making?
SEARCH STRATEGY: A literature search was performed on March 19(th), 2010 using
OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, the
Cumulative Index to Nursing & Allied Health Literature (CINAHL), the Cochrane
Library, and the International Agency for Health Technology Assessment (INAHTA)
for studies published from January 1(st), 2006 to March 19(th), 2010. A starting
search date of January 1(st), 2006 was because a comprehensive systematic review
of Oncotype-DX was identified in preliminary literature searching. This
systematic review, by Marchionni et al. (2008), included literature up to
January 1(st), 2007. All studies identified in the review by Marchionni et al.
as well as those identified in updated literature searching were used to form
the evidentiary base of this review. The quality of the overall body of evidence
was identified as high, moderate, low or very low according to GRADE
methodology.
INCLUSION CRITERIA: Any observational trial, controlled clinical trial,
randomized controlled trial (RCT), meta-analysis or systematic review that
reported on the laboratory performance, prognostic value and/or predictive value
of Oncotype-DX testing, or other outcome relevant to the Key Questions, specific
to the target population was included.
EXCLUSION CRITERIA: Studies that did not report original data or original data
analysis,Studies published in a language other than English,Studies reported
only in abstract or as poster presentations (such publications were not sought
nor included in this review since the MAS does not generally consider evidence
that is not subject to peer review nor does the MAS consider evidence that lacks
detailed description of methodology).
OUTCOMES OF INTEREST: Outcomes of interest varied depending on the Key Question.
For the Key Questions of prognostic and predictive value (Key Questions #2 and
#3), the prospectively defined primary outcome was risk of 10-year distant
recurrence. The prospectively defined secondary outcome was 10-year death due to
any cause (i.e., overall survival). All additional outcomes such as risk of
locoregional recurrence or disease-free survival (DFS) were not prospectively
determined for this review but were reported as presented in included trials;
these outcomes are referenced as tertiary outcomes in this review. Outcomes for
other Key Questions (i.e., Key Questions #1, #4 and #5) were not prospectively
defined due to the variability in endpoints relevant for these questions.
SUMMARY OF FINDINGS: A total of 26 studies were included. Of these 26 studies,
only five studies were relevant to the primary questions of this review (Key
Questions #2 and #3). The following conclusions were drawn from the entire body
of evidence: There is a lack of external validation to support the reliability
of Oncotype-DX; however, the current available evidence derived from internal
industry validation studies suggests that Oncotype-DX is reliable (i.e.,
Oncotype-DX is repeatable and reproducible).Current available evidence suggests
a moderate failure rate of Oncotype-DX testing; however, the failure rate
observed across clinical trials included in this review is likely inflated; the
current Ontario experience suggests an acceptably lower rate of test failure.In
women with newly diagnosed early breast cancer (stage I-II) that is
estrogen-receptor positive and/or progesterone-receptor positive and lymph-node
negative:There is low quality evidence that Oncotype-DX has prognostic value in
women who are being treated with adjuvant tamoxifen or anastrozole (the latter
for postmenopausal women only),There is very low quality evidence that
Oncotype-DX can predict which women will benefit from adjuvant CMF/MF
chemotherapy in women being treated with adjuvant tamoxifen.In postmenopausal
women with newly diagnosed early breast cancer that is estrogen-receptor
positive and/or progesterone-receptor positive and lymph-node positive:There is
low quality evidence that Oncotype-DX has limited prognostic value in women who
are being treated with adjuvant tamoxifen or anastrozole,There is very low
quality evidence that Oncotype-DX has limited predictive value for predicting
which women will benefit from adjuvant CAF chemotherapy in women who are being
treated with adjuvant tamoxifen.There are methodological and statistical
limitations that affect both the generalizability of the current available
evidence, as well as the magnitude and statistical strength of the observed
effect sizes; in particular:Of the major predictive trials, Oncotype-DX scores
were only produced for a small subset of women (<40% of the original randomized
population) potentially disabling the effects of treatment randomization and
opening the possibility of selection bias;Data is not specific to
HER-2/neu-negative women;There were limitations with multivariate statistical
analyses.Additional trials of observational design may provide further
validation of the prognostic and predictive value of Oncotype-DX; however, it is
unlikely that prospective or randomized data will become available in the near
future due to ethical, time and resource considerations.There is currently
insufficient evidence investigating how Oncoytpe-DX compares to other known
prognostic estimators of risk, such as Adjuvant! Online, and there is
insufficient evidence investigating how Oncotype-DX would impact
clinician/patient decision-making in a setting generalizable to Ontario. |
Where does CTCF colocalize with cohesin? | Cohesin subunits associate with viral and cellular CTCF sites involved in complex gene regulation and chromatin organization. Cohesin cobinds across the genome with transcription factors independently of CTCF, plays a functional role in estrogen-regulated transcription, and may help to mediate tissue-specific transcriptional responses via long-range chromosomal interactions.
Numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. Cohesins colocalize with CTCF at two additional imprinted loci, the Dlk1-Dio3 and the Kcnq1/Kcnq1ot1 loci. | Cohesins, which mediate sister chromatin cohesion, and CTCF, which functions at
chromatin boundaries, play key roles in the structural and functional
organization of chromosomes. We examined the binding of these two factors on the
Kaposi's sarcoma-associated herpesvirus (KSHV) episome during latent infection
and found a striking colocalization within the control region of the major
latency transcript responsible for expressing LANA (ORF73), vCyclin (ORF72),
vFLIP (ORF71), and vmiRNAs. Deletion of the CTCF-binding site from the viral
genome disrupted cohesin binding, and crippled colony formation in 293 cells.
Clonal instability correlated with elevated expression of lytic cycle gene
products, notably the neighbouring promoter for K14 and vGPCR (ORF74). siRNA
depletion of RAD21 from latently infected cells caused an increase in K14 and
ORF74, and lytic inducers caused a rapid dissociation of RAD21 from the viral
genome. RAD21 and SMC1 also associate with the cellular CTCF sites at mammalian
c-myc promoter and H19/Igf2 imprinting control region. We conclude that cohesin
subunits associate with viral and cellular CTCF sites involved in complex gene
regulation and chromatin organization. The cohesin protein complex holds sister chromatids in dividing cells together
and is essential for chromosome segregation. Recently, cohesin has been
implicated in mediating transcriptional insulation, via its interactions with
CTCF. Here, we show in different cell types that cohesin functionally behaves as
a tissue-specific transcriptional regulator, independent of CTCF binding. By
performing matched genome-wide binding assays (ChIP-seq) in human breast cancer
cells (MCF-7), we discovered thousands of genomic sites that share cohesin and
estrogen receptor alpha (ER) yet lack CTCF binding. By use of human
hepatocellular carcinoma cells (HepG2), we found that liver-specific
transcription factors colocalize with cohesin independently of CTCF at
liver-specific targets that are distinct from those found in breast cancer
cells. Furthermore, estrogen-regulated genes are preferentially bound by both ER
and cohesin, and functionally, the silencing of cohesin caused aberrant re-entry
of breast cancer cells into cell cycle after hormone treatment. We combined
chromosomal interaction data in MCF-7 cells with our cohesin binding data to
show that cohesin is highly enriched at ER-bound regions that capture
inter-chromosomal loop anchors. Together, our data show that cohesin cobinds
across the genome with transcription factors independently of CTCF, plays a
functional role in estrogen-regulated transcription, and may help to mediate
tissue-specific transcriptional responses via long-range chromosomal
interactions. The human CCCTC-binding factor, CTCF, regulates transcription of the
double-stranded DNA genomes of herpesviruses. The architectural complex cohesin
and RNA Polymerase II also contribute to this organization. We profiled the
occupancy of CTCF, cohesin, and RNA Polymerase II on the episomal genome of the
Epstein-Barr virus in a cell culture model of latent infection. CTCF colocalizes
with cohesin but not RNA Polymerase II. CTCF and cohesin bind specific sequences
throughout the genome that are found not just proximal to the regulatory
elements of latent genes, but also near lytic genes. In addition to tracking
with known transcripts, RNA Polymerase II appears at two unotated positions,
one of which lies within the latent origin of replication. The widespread
occupancy profile of each protein reveals binding near or at a myriad of
regulatory elements and suggests context-dependent functions. The cohesin complex holds sister chromatids together and is essential for
chromosome segregation. Recently, cohesins have been implicated in
transcriptional regulation and insulation through genome-wide colocalization
with the insulator protein CTCF, including involvement at the imprinted H19/Igf2
locus. CTCF binds to multiple imprinted loci and is required for proper
imprinted expression at the H19/Igf2 locus. Here we report that cohesins
colocalize with CTCF at two additional imprinted loci, the Dlk1-Dio3 and the
Kcnq1/Kcnq1ot1 loci. Similar to the H19/Igf2 locus, CTCF and cohesins
preferentially bind to the Gtl2 differentially methylated region (DMR) on the
unmethylated maternal allele. To determine the functional importance of the
binding of CTCF and cohesins at the three imprinted loci, CTCF and cohesins were
depleted in mouse embryonic fibroblast cells. The monoallelic expression of
imprinted genes at these three loci was maintained. However, mRNA levels for
these genes were typically increased; for H19 and Igf2 the increased level of
expression was independent of the CTCF-binding sites in the imprinting control
region. Results of these experiments demonstrate an unappreciated role for CTCF
and cohesins in the repression of imprinted genes in somatic cells. RNA polymerase III (Pol III) transcription is regulated by modifications of the
chromatin. DNA methylation and post-translational modifications of histones,
such as acetylation, phosphorylation and methylation have been linked to Pol III
transcriptional activity. In addition to being regulated by modifications of DNA
and histones, Pol III genes and its transcription factors have been implicated
in the organization of nuclear chromatin in several organisms. In yeast, the
ability of the Pol III transcription system to contribute to nuclear
organization seems to be dependent on direct interactions of Pol III genes
and/or its transcription factors TFIIIC and TFIIIB with the structural
maintece of chromatin (SMC) protein-containing complexes cohesin and
condensin. In human cells, Pol III genes and transcription factors have also
been shown to colocalize with cohesin and the transcription regulator and genome
organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been
identified in yeast and humans that are bound by partial Pol III machineries
(extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC
as well as Pol III genes possess the ability to act as boundary elements that
restrict spreading of heterochromatin. |
Name triad of Wernicke encephalopathy. | Wernicke's encephalopathy is a triad of ophthalmoplegia, ataxia and confusion seen in alcoholics with dietary vitamin B1 (thiamine) deficiency. | Records of the emergency medical admissions to a large teaching hospital over a
one year period were examined for evidence of Wernicke's encephalopathy or
Korsakoff's syndrome. It was found that only 0.4% of the population studied had
the classical triad of Wernicke's encephalopathy, namely confusion,
ophthalmoplegia, and ataxia. If two of these three criteria are allowed in the
absence of other causes then 2.2% of the population had this limited Wernicke's
encephalopathy or Korsakoff's syndrome. It is concluded that the diagnosis of
Wernicke's encephalopathy should not rely on the presence of all three criteria;
any two of the three in the absence of other causes will suffice for the
diagnosis. Wernicke's encephalopathy and Korsakoff's psychosis represent a continuum of the
same pathologic process. The etiology is an absolute deficiency of thiamine
rather than a direct toxic effect of alcohol. The triad of Wernicke's
encephalopathy--global confusional state, ophthalmoplegia and nystagmus, and
ataxia--is occasionally seen in chronic alcoholics and is often attenuated by
immediate thiamine treatment. The triad of Korsakoff's psychosis--memory loss,
learning deficits and confabulation--may be seen in either the acute or the
long-term care setting. Achalasia is an incurable neuromuscular disorder of the esophagus, resulting
from destruction of the esophageal myenteric plexus. This leads to aperistalsis
and failure of the lower esophageal sphincter to relax after swallowing.
Symptoms of achalasia are gradual in onset and include dysphagia, regurgitation,
and weight loss. Severe malnutrition can ensue. Wernicke's encephalopathy (WE)
is a serious, potentially fatal, neurologic disorder caused by thiamine
deficiency (vitamin B(1)), classically described as presenting with a triad of
ocular abnormalities, ataxia, and confusion. The incidence is uncertain, and
many cases likely go unrecognized. It is usually diagnosed in the alcoholic
population. We describe its onset after the successful surgical treatment of
achalasia. Wernicke encephalopathy is caused by thiamine deficiency in the central nervous
system, and is defined by the triad of confusional symptoms, ocular alterations
and ataxia. Some other factors may also predispose alcoholic patients to this
deficiency. We report two patients with hyperglicaemia and ketoacidosis due to
diabetes mellitus decompensation and chronic alcoholism who developed Wernicke
encephalopathy before their hospital admission. The outcome was successful after
intravenous thiamine administration and insulinotherapy. The presence of
Wernicke encephalopathy in alcoholics with diabetic ketoacidosis, suggests that
metabolic decompensation is essential in the onset of the disease. Wernicke's encephalopathy is a metabolic disorder caused by deficiency of
thiamine (vitamin B1) seen in alcoholics and even in nonalcoholic patients,
classically presenting with a triad of ataxia, ophthalmoplegia, and altered
mental status. Typical findings in magnetic resoce imaging are represented by
symmetric signal alterations in medial thalami, mamillary bodies, tectal plate,
and periaqueductal area and atypical findings involve lesions in cerebellum,
midline vermis, red nuclei, dentate, caudate, cranial nerve nuclei, splenium and
cerebral cortex. We report here a case of nonalcoholic starvation induced
atypical WE showing symmetrical lesions in substantia nigra in addition to the
classical neuroradiological findings. Wernicke's encephalopathy is an acute neurological syndrome due to thiamine
deficiency, which is characterized by a typical triad of mental status changes,
oculomotor dysfunction and ataxia. Despite the fact that Wernicke's
encephalopathy, in developed countries, is frequently associated with chronic
alcoholism, there have been a number of published cases associating this
encephalopathy with parenteral feeding without vitamin supplementation.
Diagnosis is primarily a clinical one, and can be supported by laboratory tests
and imaging studies; treatment should start as soon as possible, for the
morbidity and mortality (almost 20%) associated with this syndrome is high.
Thiamine supplementation, along with other vitamins, is recommended for patients
in risk of developing this syndrome. We present a descriptive, retrospective study of initial symptoms, comorbidity,
and alcohol withdrawal in 73 alcoholic patients with subsequent Korsakoff
syndrome. In 25/73 (35%) of the patients the classic triad of Wernicke's
encephalopathy with ocular symptoms, ataxia and confusion, was found. In at
least 6/35 (17%) of the initial deliria (95% confidence interval: 10-25%) we
observed no other underlying causes, thus excluding other somatic causes,
medication, (recent) alcohol withdrawal, or intoxication. We suggest that these
deliria may have been representing Wernicke's encephalopathy. A high frequency
(15%) of diabetics may reflect a contributing factor of diabetes mellitus in the
evolution of the Wernicke-Korsakoff syndrome. Introduction. Wernicke's encephalopathy is a well-described syndrome
characterized by the classic triad of confusion, ataxia, and ophthalmoplegia.
Wernicke's encephalopathy results from thiamine (vitamin B1) deficiency. Common
causes include alcoholism and gastric disorders. Wernicke's has been described
in patients with acquired immune deficiency syndrome (AIDS); however, given
these patients' immunosuppressed state, the diagnosis of Wernicke's
encephalopathy is not apparent. Case Presentation. A 31-year-old previously
healthy male presented to the ER complaining of progressive dyspnea. Workup
revealed HIV/AIDS and PCP pneumonia. He was treated and improved. On day 14 he
became confused and developed nystagmus and ataxia. Considering his
immunocompromised state, infectious and neoplastic etiologies topped the
differential diagnosis. CT head was negative. Lumbar puncture was unremarkable.
Brain MRI revealed increased T2 signal in the medial thalamus bilaterally.
Intravenous thiamine was administered resulting in resolution of symptoms.
Discussion. The classic triad of Wernicke's encephalopathy occurs in 10% of
cases. When immunosuppressed patients develop acute neurologic symptoms
infectious or neoplastic etiologies must be excluded. However, given the
relative safety of thiamine supplementation, there should be a low threshold for
initiating therapy in order to reverse the symptoms and prevent progression to
Korsakoff dementia, which is permanent. BACKGROUND: Wernicke encephalopathy is caused by thiamine (vitamin B1)
deficiency. It is generally considered to be a disease of adult alcoholics.
However, it is known to occur in the pediatric population and in non-alcoholic
conditions.
DATA SOURCES: We searched PubMed with the key words Wernicke, thiamine,
pediatric, children and adolescents and selected publications that were deemed
appropriate.
RESULTS: The global prevalence rates of hunger, poverty and resultant nutrient
deprivation have decreased in the 21st century. However, several scenarios which
may predispose to Wernicke encephalopathy may be increasingly prevalent in
children and adolescents such as maligcies, intensive care unit stays and
surgical procedures for the treatment of obesity. Other predisposing conditions
include magnesium deficiency and defects in the SLC19A3 gene causing thiamine
transporter-2 deficiency. The classic triad consists of encephalopathy,
oculomotor dysfunction and gait ataxia but is not seen in a majority of
patients. Treatment should be instituted immediately when the diagnosis is
suspected clinically without waiting for laboratory confirmation. Common
magnetic resoce findings include symmetric T2 hyperintensities in dorsal
medial thalamus, mammillary bodies, periaqueductal gray matter, and tectal
plate.
CONCLUSIONS: Wernicke encephalopathy is a medical emergency. Delay in its
recognition and treatment may lead to significant morbidity, irreversible
neurological damage or even death. This article aims to raise the awareness of
this condition among pediatricians. |
Is the PTPN22 gene a biomarker for Rheumatoid Arthritis? | Most association studies have indeed confirmed an association between mutations at the PTPN22 gene and rheumatoid arthritis | Several multiple, large-scale, genetic studies on autoimmune-disease-associated
SNPs have been reported recently: peptidylarginine deiminase type 4 (PADI4) in
rheumatoid arthritis (RA); solute carrier family 22 members 4 and 5 (SLC22A4 and
5) in RA and Crohn's disease (CD); programmed cell death 1 (PDCD1) in systemic
lupus erythematosus (SLE), type 1 diabetes mellitus (T1D), and RA; and protein
tyrosine phosphatase nonreceptor type 22 (PTPN22) in T1D, RA, and SLE. Because
these reports on association were not always evaluated in multiple ethnic groups
and because ethnic difference in allele frequency of the variants has been also
reported, we investigated allele frequencies of nine SNPs in four
autoimmune-disease-associated loci in Caucasian, African-descent, and Japanese
populations. Although SNPs in PADI4 had similar allele frequency among three
groups [maximal difference 11%; (P >0.05)], the other three loci revealed
statistically significant allele frequency differences (maximal difference 39%
(P <0.00001), 13% (P <0.00001), and 8% (P <0.00001) in SLC22A4, PDCD1, and
PTPN22, respectively). Of note, three SNPs in the three loci that had allele
frequency more than 8% in the Caucasian population were either not polymorphic
at all or extremely rare in the Japanese population. Our data suggest that
ethnic variations of polymorphisms should be evaluated in detail, and
differences should be incorporated into investigations of susceptibility
variants for common diseases. The challenges in identifying genetic polymorphisms that influence the
susceptibility to rheumatoid arthritis are the same as those faced in most
complex diseases; genetic and phenotypic heterogeneity, an unknown number of
loci presumed to have small genetic effects, non-genetic modifying effects that
have yet to be fully characterised and a history of unconfirmed genetic
associations. Despite the difficulties, the chronic nature of the disease,
incomplete efficacy of existing therapies and resultant heavy healthcare burden
for the developed world in managing patients with this condition, mean that an
understanding of the genetic basis of disease susceptibility, severity and
response to therapy is keenly sought. Many linkage and association studies have
been carried out and in this article the results of linkage studies are
summarised. Recently a number of convincing candidate genes have begun to emerge
and an update has been provided for three of these: PTPN22, CTLA-4 and MIF. Rheumatoid arthritis (RA) is a multifactorial disease due to a combination of
genetic and environmental factors. Identification of the genetic factors
involved in the pathogenesis of RA should open up avenues for developing radical
treatment strategies directed at the cause of the disease. The Association de
Recherche sur la Polyarthrite (ARP) supports research in this field, in which
our group has been involved since 1993. Thanks to this support, considerable
progress has been made. Several combinations of susceptibility alleles of
various genes are probably involved in the development of RA. Although HLA-DRB1
is the main RA gene, it accounts for only part of the familial risk for RA.
HLA-DRB1 alleles are neither necessary nor sufficient to cause the development
of RA in a given individual. Several genome scans conducted in populations from
France, Japan, North America and UK have confirmed the role of the HLA region
and suggested several other susceptibility loci. Association studies support a
role for several genes, including TNFR2, PADI4, SLC22A4, RUNX1, and PTPN22.
However, the imperfect matching of cases and controls requires that confirmation
of these results be obtained. To confirm that a gene confers susceptibility to
RA, the association must be replicated in several independent studies and, more
importantly, evidence of genetic linkage must be obtained in family studies. The
identification of genetic factors conferring susceptibility to RA will open up
new avenues toward radical treatments for RA and may help to optimize the
diagnostic, prognostic, and pharmacogenetic management of today's patients with
RA. OBJECTIVE: Analyses of families with multiple autoimmune disorders have revealed
a functional polymorphism, 620W, in the intracellular tyrosine phosphatase gene
PTPN22 as a predisposing factor for type 1 diabetes, seropositive rheumatoid
arthritis, systemic lupus erythematosus, and Hashimoto thyroiditis, and the
presence of the PTPN22 protein appears to herald the development of
autoantibodies in these disorders. This study therefore examined whether the
functionally relevant PTPN22 polymorphism is associated with Wegener's
granulomatosis (WG).
METHODS: A population-based study was performed for the PTPN22 polymorphism in
199 patients with WG and in 399 healthy individuals. The R620W variation was
investigated by simple restriction fragment-length polymorphism analysis.
RESULTS: The PTPN22 620W allele frequency was significantly increased in
antineutrophil cytoplasmic antibody (ANCA)-positive WG patients compared with
healthy controls (P < 0.001). The association was particularly striking in
patients with kidney, lung, eye, and peripheral nervous system involvement
(i.e., those with generalized WG).
CONCLUSION: The PTPN22 620W allele appears to be involved in the pathogenesis of
WG, and ANCA positivity seems to be the hallmark. OBJECTIVES: Anti-citrullinated peptide antibodies (ACPA) and the C1858T missense
single-nucleotide polymorphism (SNP) in the PTPN22 gene are both associated with
the development of rheumatoid arthritis (RA). We investigated whether the
combination of these two biomarkers yielded better test characteristics to
predict progression from undifferentiated arthritis (UA) to RA compared with
ACPA alone.
METHODS: A total of 394 individuals with UA from a Dutch population-based
inception cohort were included in this study. At baseline, ACPA were measured
and the PTPN22 C1858T and HLA-DRB1 genotypes determined. Progression to RA was
monitored at 1 yr after entry into the cohort.
RESULTS: A priori, UA patients had a 35% (95% CI 30-40%) risk of developing RA,
which increased to 66% (95% CI 57-75%) in patients who were ACPA-positive. There
was an additional, although non-significant (P = 0.34), increase in RA risk to
76% (95% CI 57-90%) when patients were positive for both ACPA and the PTPN22
1858T-allele. The area under the receiver operator characteristic curve
increased from 0.68 for ACPA-status alone to 0.70 for the combination of
ACPA-status and the PTPN22 C1858T polymorphism. In logistic regression analysis,
ACPA predicted RA-development independent of PTPN22, while the PTPN22
polymorphism had no independent effect. In HLA-DRB1 shared epitope positive,
ACPA-positive UA patients, ACPA-levels were significantly increased in PTPN22
1858T allele carriers compared with non-1858T carriers.
CONCLUSIONS: In this Dutch cohort of UA-patients, the PTPN22 1858T allele does
not markedly improve individual decision-making to predict RA-development over
ACPA alone, but it is associated with higher ACPA-levels. OBJECTIVE: To analyse the relationship between the presence of auto-antibodies
[rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP)],
HLA-DRB1 alleles and PTPN22 1858 C/T polymorphism and test the value of their
combination as susceptibility markers for rheumatoid arthritis (RA).
METHODS: Patients with early arthritis were included. At entry in the cohort or
during follow-up, 191 patients fulfilled the criteria for RA and 184 individuals
suffered from other arthropathies. RF was measured by nephelometry and anti-CCP
antibody by enzyme-linked immunosorbent assay. HLA class II alleles were
determined by polymerase chain reaction. Samples were genotyped for PTPN22
1858C/T variants using a TaqMan 5'-allele discrimination assay.
RESULTS: The presence of shared epitope (SE) alleles was strongly associated
with anti-CCP and RF-positive RA [P = 7.05 x 10(-10), odds ratio (OR) 4.57, 95%
confidence interval (CI) 2.76-7.57 and P = 1.68 x 10(-6), OR 2.99, 95% CI
1.89-4.74, respectively). The combination of the PTPN22 1858T variant and
anti-CCP antibodies gave a high specificity for the disease, and was
significantly associated with RA (P = 8.86 x 10(-5), OR 10.05, 95% CI
1.88-53.73).
CONCLUSION: The combination of the T variant of the 1858 polymorphism of the
PTPN22 gene in combination with the presence of anti-CCP antibodies,
preferentially in a SE-positive individual, is associated with the development
of RA. OBJECTIVE: To examine the role of the variants of the PTPN22 and HLA-DRB1 genes
as predictors of mortality in inflammatory polyarthritis (IP) and rheumatoid
arthritis (RA).
METHODS: Patients were recruited from a primary care-based inception cohort of
patients with IP and were followed up prospectively. For patients who died, the
cause and date of death was obtained. Cox proportional hazards regression models
were used to assess the association of the HLA-DRB1 (including the shared
epitope [SE]) and PTPN22 genes with the risk of death from all causes and from
cardiovascular disease (CVD) and to assess the interactions between SE, smoking,
and anti-cyclic citrullinated peptide (anti-CCP) status, adjusted by age at
symptom onset and sex.
RESULTS: DNA samples were available from 1,022 IP patients. During followup, 751
of them (74%) satisfied the American College of Rheumatology 1987 criteria for
RA, and 242 of them (24%) died. Carriage of 2 copies of SE alleles predicted
death from all causes (hazard ratio [HR] 1.57 [95% confidence interval (95% CI)
1.1-2.2]) and from CVD (HR 1.68 [95% CI 1.1-2.7]). This effect was most marked
for individuals with the HLA-DRB1*01/*04 combination. An interaction of smoking,
SE alleles, and anti-CCP antibodies was observed and was associated with the
greatest risk of death from CVD (HR 7.81 [95% CI 2.6-23.2]). No association of
the PTPN22 gene with mortality was detected.
CONCLUSION: SE alleles, particularly compound heterozygotes, are associated with
death from all causes and from CVD, independently of autoantibody status.
However, the combination of SE, smoking, and anti-CCP antibodies is associated
with a high risk of premature death in patients with IP and RA, which raises the
possibility of a targeted strategy to prevent CVD in these patients. OBJECTIVE: The disease association of the common 1858C>T Arg620Trp (rs2476601)
nonsynonymous single nucleotide polymorphism (SNP) of protein tyrosine
phosphatase; nonreceptor type 22 (PTPN22) on chromosome 1p13 has been confirmed
in type 1 diabetes and also in other autoimmune diseases, including rheumatoid
arthritis and Graves' disease. Some studies have reported additional associated
SNPs independent of rs2476601/Trp(620), suggesting that it may not be the sole
causal variant in the region and that the relative risk of rs2476601/Trp(620) is
greater in lower risk by HLA class II genotypes than in the highest risk class
II risk category.
RESEARCH DESIGN AND METHODS: We resequenced PTPN22 and used these and other data
to provide >150 SNPs to evaluate the association of the PTPN22 gene and its
flanking chromosome region with type 1 diabetes in a minimum of 2,000 case
subjects and 2,400 control subjects.
RESULTS: Due to linkage disequilibrium, we were unable to distinguish between
rs2476601/Trp(620) (P = 2.11 x10(-87)) and rs6679677 (P = 3.21 x10(-87)), an
intergenic SNP between the genes putative homeodomain transcription factor 1 and
round spermatid basic protein 1. None of the previously reported
disease-associated SNPs proved to be independent of rs2476601/Trp(620). We did
not detect any interaction with age at diagnosis or sex. However, we found that
rs2476601/Trp(620) has a higher relative risk in type 1 diabetic case subjects
carrying lower risk HLA class II genotypes than in those carrying higher risk
ones (P = 1.36 x 10(-4) in a test of interaction).
CONCLUSIONS: In our datasets, there was no evidence for allelic heterogeneity at
the PTPN22 locus in type 1 diabetes, indicating that the SNP rs2476601/Trp(620)
remains the best candidate in this chromosome region in European populations.
The heterogeneity of rs2476601/Trp(620) disease risk by HLA class II genotype is
consistent with previous studies, and the joint effect of the two loci is still
greater in the high-risk group. OBJECTIVE: To evaluate the predictive values for disease progression of various
antibodies against citrullinated peptide proteins (ACPA) and their relation to
PTPN22 1858C/T polymorphism and HLA-DRB1 alleles in early rheumatoid arthritis
(RA).
METHODS: The ACPA, e.g., antibodies against mutated citrullinated vimentin
(MCV), cyclic citrullinated peptides (CCP) type 2 and 3 (both of IgG isotype)
and 3.1 (of both IgG and IgA isotypes), were analyzed at baseline in patients
with early RA (n = 210) and in population controls (n = 102) using an enzyme
immunoassay. A receiver-operating characteristic curve was constructed for each
antibody. Disease activity [swollen and tender joints, visual analog scale for
global health, and erythrocyte sedimentation rate (ESR)] was evaluated at
baseline and regularly for 24 months. Radiographs of hands and feet were graded
using the Larsen score.
RESULTS: Patients with anti-MCV antibodies had significantly less reduction in
Disease Activity Score (DAS28) over time (p < 0.01), and significantly increased
area under the curve (AUC) for DAS28 (p < 0.05), ESR (p < 0.01), C-reactive
protein (p < 0.01), and swollen joint count (p = 0.057) compared to those
without. Corresponding differences were not found in patients with anti-CCP2,
CCP3, and CCP3.1 antibodies. Radiological progression (p < 0.0001-0.01) and
radiological outcome (p < 0.0001-0.01) at 24 months were significantly predicted
by all ACPA after baseline adjustments. PTPN22 T variant and HLA-DRB1 alleles
were not related to radiological progression or inflammatory activity over time.
CONCLUSION: Anti-MCV antibodies are associated with a more severe RA disease, as
measured by DAS28, ESR, and swollen joint count over time, compared with
anti-CCP2, CCP3, and CCP3.1 antibodies. Radiological progression was predicted
equally by all 4 autoantibodies. OBJECTIVES: To evaluate the predictive value of TNFRII 196R, PTPN22 1858T and
HLA-shared epitope (SE) alleles, RFs and anti-citrullinated protein antibodies
(ACPAs) for RA diagnosis in a cohort of patients with very early arthritis.
METHODS: We followed up 284 patients who had swelling of at least two joints
that had persisted for longer than 4 weeks but had been evolving for <6 months.
At 2 yrs, patients were classified as having RA or non-RA rheumatic diseases
according to the ACR criteria. Patients were genotyped with respect to TNFRII
196M/R and PTPN22 1858C/T polymorphisms and HLA-SE. The presence of IgA, IgG and
IgM RF isotypes and ACPA was sought in sera collected at disease onset.
RESULTS: HLA-SE alleles alone, concomitant presence of TNFRII 196R and PTPN22
1858T alleles, IgA, IgG and IgM RF alone and ACPA were found to be significantly
associated with RA diagnosis. Using logistic regression analysis, the
concomitant presence of RF and ACPA at disease onset was the best association to
predict RA diagnosis. In patients (n = 34) who did not fulfil the ACR criteria
for RA at inclusion but who progressed to ACR positivity, the study of the
genetic risk markers did not contribute to predict RA diagnosis at 2 yrs.
CONCLUSIONS: PTPN22 1858T, TNFRII 196R and HLA-SE alleles do not improve the
predictive value of RF and ACPA for RA diagnosis in our cohort, and do not
contribute to an earlier diagnosis in undifferentiated patients initially
negative for RF and ACPA. Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease affecting
both joints and extra-articular tissues. Although some genetic risk factors for
RA are well-established, most notably HLA-DRB1 and PTPN22, these markers do not
fully account for the observed heritability. To identify additional
susceptibility loci, we carried out a multi-tiered, case-control association
study, genotyping 25,966 putative functional SNPs in 475 white North American RA
patients and 475 matched controls. Significant markers were genotyped in two
additional, independent, white case-control sample sets (661 cases/1322 controls
from North America and 596 cases/705 controls from The Netherlands) identifying
a SNP, rs1953126, on chromosome 9q33.2 that was significantly associated with RA
(OR(common) = 1.28, trend P(comb) = 1.45E-06). Through a comprehensive
fine-scale-mapping SNP-selection procedure, 137 additional SNPs in a 668 kb
region from MEGF9 to STOM on 9q33.2 were chosen for follow-up genotyping in a
staged-approach. Significant single marker results (P(comb)<0.01) spanned a
large 525 kb region from FBXW2 to GSN. However, a variety of analyses identified
SNPs in a 70 kb region extending from the third intron of PHF19 across TRAF1
into the TRAF1-C5 intergenic region, but excluding the C5 coding region, as the
most interesting (trend P(comb): 1.45E-06 --> 5.41E-09). The observed
association patterns for these SNPs had heightened statistical significance and
a higher degree of consistency across sample sets. In addition, the allele
frequencies for these SNPs displayed reduced variability between control groups
when compared to other SNPs. Lastly, in combination with the other two known
genetic risk factors, HLA-DRB1 and PTPN22, the variants reported here generate
more than a 45-fold RA-risk differential. OBJECTIVES: To analyse the distribution of single nucleotide polymorphisms
(SNPs) in the 5'-regulatory region of the DNASE2 gene, in patients with
rheumatoid arthritis (RA) and healthy controls.
METHODS: A total of 906 patients with RA and 878 healthy controls were
genotyped. All subjects were of German Caucasian origin. Genotyping was
performed by real-time polymerase chain reaction technology, using a TaqMan
5'-allele discrimination assay.
RESULTS: In the initial analysis of unrelated case-control samples, three DNASE2
SNP alleles in the 5'-regulatory region were significantly more frequent in
patients with RA than in healthy controls. The strongest association was found
for the -1066G allele (33.5% vs 27.2%, p = 0.007, odds ratio (OR) = 1.34).
Homozygosity for this allele (genotype GG) resulted in an additional increase in
disease susceptibility (12.5% vs 6.2%, OR = 2.17). The association was
replicated in a second case-control series of 483 patients with RA from two
German multicentre studies and 474 controls. The association of DNASE2 -1066 GG
homozygosity with RA was limited to rheumatoid factor-positive disease, but was
not influenced by the presence of anti-cyclic citrullinated peptide or
antinuclear antibodies. Similarly, the presence or absence of the HLA-DRB1
shared epitope or the RA-associated PTPN22 allele had no influence on this
association.
CONCLUSIONS: The association of SNPs in the 5'-regulatory region of the DNA
degrading enzyme DNASE2 with RA implies a role for this enzyme in the
pathogenesis of this autoimmune disease. The C1858T allele of the PTPN22 gene has been reported to confer risk for RA;
but in some reports, the effect was restricted to RF- and/or
anti-CCP-seropositive patients. Hungarian RA patients and matched controls were
genotyped. The 1858T allele showed an increased prevalence in RA patients
compared to controls. The 1858T allele represents a risk factor in the whole RA
population (P = 0.001); an association was found both in RF-seropositive (P =
0.001) and anti-CCP-seropositive patients (P = 0.001), and in subjects with the
combination of these factors (P = 0.002). In TT homozygotes, the estimated
susceptibility to RA was more than double (OR = 5.04) of that seen in TC
heterozygotes (OR = 1.89); the same gene dosage effect was observed in all
seropositive RA subgroups. Our data show that the Hungarian RA patients belong
to the populations in which the 1858T allele represents a susceptibility factor
both in the RF- and/or anti-CCP-seropositive subjects, and the association
exhibit a gene dosage dependency. A new age has begun in the genetics of rheumatoid arthritis (RA), as genome-wide
association studies scanning the human genome have been put into practical use.
Among the RA-susceptibility genes identified by genetic studies, HLA-DRB1 gene
appears to represent the most major determit of genetic predisposition to RA.
However, inconsistent results of the contributions of non-HLA susceptibility
genes have been described, with the exception of a few genes repeatedly
associated with RA-susceptibility, such as PTPN22 gene in populations of
European ancestry and PADI4 gene in populations of Asian ancestry, revealing the
presence of genetic heterogeneity in RA. We review herein recent advances in the
genetics of RA and discuss the underlying differences among populations of
European and Asian ancestries, taking as examples our previous findings for
RA-susceptibility genes in the Japanese population: PADI4; FCRL3; and CD244. OBJECTIVE: Recent advances have led to novel identification of genetic
polymorphisms that are associated with susceptibility to rheumatoid arthritis
(RA). Currently, 5 loci (HLA, PTPN22, TRAF1/C5, TNFAIP3, and STAT4) have been
consistently reported, whereas others have been observed less systematically.
The aim of the present study was to independently replicate 3 recently described
RA susceptibility loci, STAT4, IL2/IL21, and CTLA4, in a large Dutch
case-control cohort, and to perform a meta-analysis of all published studies to
date and investigate the relevance of the findings in clinically well-defined
subgroups of RA patients with or without autoantibodies.
METHODS: The STAT4, IL2/IL21, and CTLA4 gene polymorphisms (rs7574865,
rs6822844, and rs3087243, respectively) were genotyped in 877 RA patients and
866 healthy individuals. A meta-analysis of all published studies of disease
association with these polymorphisms was performed using the Mantel-Haenszel
fixed-effects method.
RESULTS: An association of STAT4, IL2/IL21, and CTLA4 with RA was detected in
Dutch patients (odds ratio [OR] 1.19 [P=0.031], OR 0.84 [P=0.051], and OR 0.87
[P=0.041], respectively). Results from the meta-analysis confirmed an
association of all 3 polymorphisms with RA in Caucasians (OR 1.24
[P=1.66x10(-11)], OR 0.78 [P=5.6x10(-5)], and OR 0.91 [P=1.8x10(-3)],
respectively). The meta-analysis also revealed that STAT4 predisposed to disease
development equally in patients with autoantibodies and those without
autoantibodies, and that CTLA4 enhanced the development of anti-citrullinated
protein antibody (ACPA)-positive RA as compared with ACPA-negative RA.
CONCLUSION: Our results replicate and firmly establish the association of STAT4
and CTLA4 with RA and provide highly suggestive evidence for IL2/IL21 loci as a
risk factor for RA. Given the strong statistical power of our meta-analysis to
confirm a true-positive association, these findings provide considerable support
for the involvement of CTLA4 in distinct subsets of RA patients. INTRODUCTION: Both genetic and environmental factors contribute to rheumatoid
arthritis (RA), a common and complex autoimmune disease. As well as the major
susceptibility gene HLA-DRB1, recent genome-wide and candidate-gene studies
reported additional evidence for association of single nucleotide polymorphism
(SNP) markers in the PTPN22, STAT4, OLIG3/TNFAIP3 and TRAF1/C5 loci with RA.
This study was initiated to investigate the association between defined genetic
markers and RA in a Slovak population. In contrast to recent studies, we
included intensively-characterized osteoarthritis (OA) patients as controls.
METHODS: We used material of 520 RA and 303 OA samples in a case-control
setting. Six SNPs were genotyped using TaqMan assays. HLA-DRB1 alleles were
determined by employing site-specific polymerase chain reaction (PCR)
amplification.
RESULTS: No statistically significant association of TRAF1/C5 SNPs rs3761847 and
rs10818488 with RA was detected. However, we were able to replicate the
association signals between RA and HLA-DRB1 alleles, STAT4 (rs7574865), PTPN22
(rs2476601) and OLIG3/TNFAIP3 (rs10499194 and rs6920220). The strongest signal
was detected for HLA-DRB1*04 with an allelic P = 1.2*10-13 (OR = 2.92, 95%
confidence interval (CI) = 2.18 - 3.91). Additionally, SNPs rs7574865STAT4 (P =
9.2*10-6; OR = 1.71, 95% CI = 1.35 - 2.18) and rs2476601PTPN22 (P = 9.5*10-4; OR
= 1.67, 95% CI = 1.23 - 2.26) were associated with susceptibility to RA, whereas
after permutation testing OLIG3/TNFAIP3 SNPs rs10499194 and rs6920220 missed our
criteria for significance (Pcorr = 0.114 and Pcorr = 0.180, respectively).
CONCLUSIONS: In our Slovak population, HLA-DRB1 alleles as well as SNPs in STAT4
and PTPN22 genes showed a strong association with RA. Both genetic and environmental factors affect susceptibility, severity and
probably drugs' efficacy in patients with rheumatoid arthritis (RA). After
initial completion of the Human Genome Project on the 16th February 2001,
significant progress has been made in identifying other than HLA genome regions
linked to the increased RA susceptibility. As an effect several new genes have
been recognized as an HLA-independent genetic risk factors of RA. PTPN22 gene
polymorphism, C5/TRAF1 genes region polymorphism and TNFAIP3-OLIG3 genes region
polymorphism(s) are among newly identified and already confirmed genetic risk
factors, whereas STAT 4, CTLA4, PADI4 and IRF5 genes polymorphisms are listed
among probable RA development genetic risk factors. OBJECTIVE: To describe a large, multicenter prospective cohort study of
first-degree relatives (FDRs) of probands with rheumatoid arthritis (RA), and
outline the use of such a study in investigating the natural history of RA
development.
METHODS: A total of 1,058 FDRs, none of whom met the American College of
Rheumatology criteria for RA, were enrolled in a prospective study investigating
genetic and environmental influences on the development of RA-related
autoimmunity. Demographic, epidemiologic, genetic, autoantibody, and physical
examination data from the initial study enrollment visit were described for
these FDRs, and the relationship was examined between genetic factors,
autoantibodies, inflammation, and joint disease.
RESULTS: Fifty-five percent of the FDRs had > or =1 copy of the shared epitope,
20% had > or =1 copy of the PTPN22 polymorphism, and approximately 16% were
positive for rheumatoid factor (RF; including isotypes) and/or anti-cyclic
citrullinated peptide antibody. IgM-RF positivity is associated with > or =1
tender joint on examination (odds ratio [OR] 2.50, 95% confidence interval [95%
CI] 1.27-4.89; P < 0.01) and elevated C-reactive protein (CRP) levels (OR 5.31,
95% CI 1.45-19.52; P = 0.01).
CONCLUSION: FDRs without RA demonstrate high prevalences of genetic risk factors
and RA-related autoantibodies. Additionally, an RF association with tender
joints and elevated CRP levels suggests that autoantibodies are a valid
intermediate marker of RA-related autoimmunity in this cohort. This prospective
FDR cohort will be a valuable resource for evaluating the relationship between
genetic and epidemiologic factors and the development of RA-related
autoimmunity. PURPOSE OF REVIEW: To update progress made between December 2008 and November
2009 on the role of the rheumatoid arthritis (RA)-shared epitope in the cause
and pathogenesis of RA.
RECENT FINDINGS: New evidence has been recently presented to suggest that
noninherited human leukocyte antigens (HLAs) originating through pregcy or
exposure to maternal antigens in utero could contribute to RA development in
shared epitope-negative women. An interaction between smoking and shared
epitope-coding non-*04 HLA-DRB1 alleles (particularly HLA-DRB1*01 and
HLA-DRB1*10) was formally established for the first time. Progress has been made
in determining the relative contributions and the interaction of the shared
epitope, PTPN22 and smoking in conferring the risk of anticitrullinated protein
antibodies-positive and negative RA. The autoantigen that anticitrullinated
protein antibodies recognize in a significant number of RA patients has been
identified as citrullinated alpha-enolase and the importance of genetic factors
in anticitrullinated protein antibodies-negative RA has been highlighted.
Additionally, associations of RA risk with several new genetic markers have been
reported. Among them: two new major histocompatibility complex, non-DRB1 loci, a
polymorphism marker in major histocompatibility complex class I
polypeptide-related sequence A, an allele of the Fcgamma receptor, a
polymorphism marker in the beta2-adrenergic receptor and a low-inducible allele
of the cytochrome P450 subtype 1A2.
SUMMARY: Although the mechanistic basis of shared epitope-RA association remains
an enigma, observations made during the last year shed new light on the
conditions in which the shared epitope - alone or in combination with other
genes or environmental factors - affects the risk of RA and the phenotype of the
disease. INTRODUCTION: This study investigated five confirmed rheumatoid arthritis (RA)
susceptibility genes/loci (HLA-DRB1, PTPN22, STAT4, OLIG3/TNFAIP3 and TRAF1/C5)
for association with susceptibility and severity in an inception cohort.
METHODS: The magnitude of association for each genotype was assessed in 1,046 RA
subjects from the Yorkshire Early RA cohort and in 5,968 healthy UK controls.
Additional exploratory subanalyses were undertaken in subgroups defined by
autoantibody status (rheumatoid factor and anti-cyclic citrullinated peptide) or
disease severity (baseline articular erosions, Health Assessment Questionnaire
(HAQ) score and swollen joint count (SJC)).
RESULTS: In the total RA inception cohort, the HLA-DRB1 shared epitope
(per-allele odds ratio (OR) = 2.1, trend P < 0.0001), PTPN22 (per-allele OR =
1.5, trend P < 0.0001), OLIG3/TNFAIP3 locus (per-allele OR = 1.2, trend P =
0.009) and TRAF1/C5 locus (per-allele OR = 1.1, trend P = 0.04) were associated
with RA. The magnitude of association for these loci was increased in those
patients who were autoantibody-positive. PTPN22 was associated with
autoantibody-negative RA (per-allele OR = 1.3, trend P = 0.04). There was no
evidence of association between these five genetic loci and baseline erosions or
SJC in the total RA cohort, after adjustment for symptom duration. TRAF1/C5 was
significantly associated with baseline HAQ, however, following adjustment for
symptom duration (P trend = 0.03).
CONCLUSIONS: These findings support the mounting evidence that different genetic
loci are associated with autoantibody-positive and autoantibody-negative RA,
possibly suggesting that many of the genes identified to date are associated
with autoantibody production. Additional studies with a specific focus on
autoantibody-negative RA will be needed to identify the genes predisposing to
this RA subgroup. The TRAF1/C5 locus in particular warrants further
investigation in RA as a potential disease severity locus. Both genetic and environmental factors contribute to rheumatoid arthritis (RA)
as well as osteoarthritis (OA). For RA, most of the known genetic markers are
linked with genes from immunological pathways. Recent genome-wide association
studies (GWAS) on RA identified known and novel susceptibility genes like
HLA-DRB1, PTPN22, STAT4, TRAF1/C5, OLIG3/TNFAIP3, CD40, CCL21, MMEL1-TNFRSF14,
CDK6, PRKCQ, IL2RB, and KIF5A-PIP4K2C. These association signals explain more
than 50% of the genetic influence on RA. In contrast, less GWAS data for OA
exist. Most OA susceptibility genes arose from classical candidate gene analyses
and were not replicated in all study samples. Neuroendocrine factors are
hypothesized to play an important role both in RA and OA etiology. Here, we
discuss these findings and present an outlook for genetic association studies
after GWAS. BACKGROUND: Genetic factors have a substantial role in determining development
of rheumatoid arthritis (RA), and are likely to account for 50-60% of disease
susceptibility. Genome-wide association studies have identified non-human
leucocyte antigen RA susceptibility loci which associate with RA with
low-to-moderate risk.
OBJECTIVES: To investigate recently identified RA susceptibility markers using
cohorts from six European countries, and perform a meta-analysis including
previously published results.
METHODS: 3311 DNA samples were collected from patients from six countries (UK,
Germany, France, Greece, Sweden and Denmark). Genotype data or DNA samples for
3709 controls were collected from four countries (not Sweden or Denmark).
Eighteen single nucleotide polymorphisms (SNPs) were genotyped using Sequenom
MassArray technology. Samples with a >95% success rate and only those SNPs with
a genotype success rate of >95% were included in the analysis. Scandinavian
patient data were pooled and previously published Swedish control data were
accessed as a comparison group. Meta-analysis was used to combine results from
this study with all previously published data.
RESULTS: After quality control, 3209 patients and 3692 controls were included in
the study. Eight markers (ie, rs1160542 (AFF3), rs1678542 (KIF5A), rs2476601
(PTPN22), rs3087243 (CTLA4), rs4810485 (CD40), rs5029937 (6q23), rs10760130
(TRAF1/C5) and rs7574865 (STAT4)) were significantly associated with RA by
meta-analysis. All 18 markers were associated with RA when previously published
studies were incorporated in the analysis. Data from this study increased the
significance for association with RA and nine markers.
CONCLUSIONS: In a large European RA cohort further evidence for the association
of 18 markers with RA development has been obtained. This study aimed at examining the association of the single nucleotide
polymorphism (SNP) in the protein tyrosine phosphatase gene (PTPN22) with the
risk of rheumatoid arthritis (RA) in a Chinese population. A total of 200 RA
patients and age and gender-matched healthy controls were recruited. Their
genotypes and allelic frequency were determined by the TaqMan-MGB probe-based
polymerase chain reaction (PCR). The frequencies of the CC genotype and C allele
in RA patient group were significantly higher than that of controls (P < 0.01 or
P < 0.05) with an odds ratio of 1.67, respectively. These data suggest, the CC
genotype and C allele of the -1123G > C in the PTPN22 gene are associated with
an increased risk for RA in Chinese population. Therefore, the CC genotype and C
allele of the -1123G > C in the PTPN22 gene may be used as a genetic marker for
the predisposition of RA in Chinese. Whole-genome association studies in rheumatoid arthritis have identified
single-nucleotide polymorphisms (SNPs) predisposing to disease with moderate
risk. We aimed to investigate the role of these markers in predicting
methotrexate (MTX) response, measured by continuation on MTX monotherapy in
patients with recent onset inflammatory polyarthritis (IP). In all, 19 SNPs were
genotyped in 736 patients treated with MTX following registration, or not more
than 3 months before registration, to the Norfolk Arthritis Register. The
association of SNPs with MTX continuation by year 1 and by year 2 was
investigated using Cox proportional hazard regression models. A SNP within the
OLIG3/TNFAIP3 locus (rs6920220) was associated with being less likely to
maintain MTX monotherapy at year 1, hazards ratio (HR) 1.73 (1.18, 2.52) and
year 2, HR 1.49 (1.11, 2.00); correlating with an increased in adverse events.
Weak evidence for an effect at the PTPN22 locus was also observed. These
findings require replication in other large datasets. Recent results from genetic and treatment studies have shed new light on chronic
inflammatory and autoimmune diseases such as rheumatoid arthritis (RA). In
particular, genome-wide association studies (GWAS) have provided supportive
evidence that RA is a disease with a strong genetic background. Interestingly, a
series of candidate genes have been identified outside of the classical major
histocompatibility (MHC) locus, which had long been regarded as the major
contributor to the pathogenesis of this disease. Among these genes, PTPN22 plays
an outstanding role. CD40, STAT4, PRM1, and TNFAIP3 also seem to be of
relevance. Interestingly, there is a significant overlap between RA
susceptibility genes and those of other autoimmune diseases such as systemic
lupus erythematosus (SLE) and type 1 diabetes, which suggests common pathogenic
mechanisms. Genetic analyses may not only provide new insights into the
pathogenesis of RA, but may also open new avenues for therapeutic approaches,
because overactive immune-signaling pathways might specifically be addressed by
biologic therapies. However, the predictive value of many of the recent findings
of large-scale genetic analyses in identifying new genetic polymorphisms remains
low. We describe the current knowledge about the role of non-MHC genes in the
pathogenesis of rheumatoid arthritis. Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory, multi-factorial
disease sustained by environmental and genetic factors. These seem to be
necessary but not sufficient in the disease development, nonetheless they can be
responsible of different clinical pictures and response to therapy, and they can
represent potential therapeutic targets. Several genes have been indicated so
far in the pathogenesis of RA. The most important region is the Human Leukocyte
Antigen (HLA) that contributes to approximately half of the genetic
susceptibility for RA. The association seems to be stronger or specific for
anti-citrullinated protein antibodies positive disease. Several alleles in the
epitope-recognition part of the HLA molecule that show the highest association
with RA susceptibility, also share a common string of amminoacid residues (the
so-called shared-epitope hypothesis). Other variants in potentially pathogenic
genes located in non-MHC regions have been implicated by recently performed
genome wide analysis studies. These genes include PTPN22, TRAF1-C5, PADI4,
STAT4. Other polymorphisms seem to be responsible for more aggressive disease
phenotype such as those located at TNF, IL-1, IL-6, IL-4, IL-5, OPN, PRF1.
However, still nowadays, the genetic background of RA remains to be clearly
depicted, and the efforts in the post-genomic era can bring to an estimation of
the real likelihood of the genetic effect on RA. Finally, the discovery of new
genes associated with the disease can be relevant in finding potential
biomarkers, potentially useful in disease diagnosis and treatment. BACKGROUND: The genome-wide association study era has made great progress in
identifying susceptibility genes and genetic loci for rheumatoid arthritis (RA)
in populations of White European ancestry. However, few studies have tried to
dissect disease aetiopathogenesis in other ethnic populations.
OBJECTIVE: To investigate these associations in the Han Chinese population.
METHODS: Haplotypes from the HapMap database Chinese population were used to
select tag-single-nucleotide polymorphisms (SNPs) (r(2)=0.8) across 19 distinct
RA genomic regions. A two phase case-control association study was performed,
with 169 SNPs genotyped in phase I (n=571 cases, n=880 controls), and 64 SNPs
achieving p<0.2 in the first phase being genotyped in phase II (n=464 cases,
n=822 controls). Association statistics were calculated using permutation tests
both unadjusted and adjusted for the number of markers studied.
RESULTS: Robust association was detected for MMEL1 and CTLA4, and modest
association was identified for another six loci: PADI4, STAT4, PRDM1, CDK6,
TRAF1-C5 and KIF5A-PIP4K2C. All three markers genotyped in MMEL1 demonstrated
association, with peak signal for rs3890745 (p=2.6 × 10(-5) unadjusted, p=0.003
adjusted, OR=0.79). For CTLA4, significance was detected for three of five
variants showing association, with peak association for marker rs12992492 (p=4.3
× 10(-5) unadjusted, p=0.0021 adjusted, OR=0.77). Lack of association of common
variants in PTPN22 with RA in Han Chinese was confirmed.
CONCLUSION: This study identifies MMEL1 and CTLA4 as RA susceptibility genes,
provides suggestive evidence of association for a further six loci in the Han
Chinese population and confirms lack of PTPN22 association in Asian populations.
It also confirms the value of multiethnic population studies to help dissect
disease aetiopathogenesis. PTPN22 is a tyrosine phosphatase and functions as a damper of TCR signals. A
C-to-T single nucleotide polymorphism (SNP) located at position 1858 of human
PTPN22 cDNA and converting an arginine (R620) to tryptophan (W620) confers the
highest risk of rheumatoid arthritis among non-HLA genetic variations that are
known to be associated with this disease. The effect of the R-to-W conversion on
the phosphatase activity of PTPN22 protein and the impact of the minor T allele
of the C1858T SNP on the activation of T cells has remained controversial. In
addition, how the overall activity of PTPN22 is regulated and how the R-to-W
conversion contributes to rheumatoid arthritis is still poorly understood. Here
we report the identification of an alternative splice form of human PTPN22,
namely PTPN22.6. It lacks the nearly entire phosphatase domain and can function
as a domit negative isoform of the full length PTPN22. Although conversion of
R620 to W620 in the context of PTPN22.1 attenuated T cell activation, expression
of the tryptophan variant of PTPN22.6 reciprocally led to hyperactivation of
human T cells. More importantly, the level of PTPN22.6 in peripheral blood
correlates with disease activity of rheumatoid arthritis. Our data depict a
model that can reconcile the conflicting observations on the functional impact
of the C1858T SNP and also suggest that PTPN22.6 is a novel biomarker of
rheumatoid arthritis. OBJECTIVE: The TRAF1 genetic region conferring susceptibility to rheumatoid
arthritis (RA) has been reported to associate with radiological damage. We aimed
to test RA genetic susceptibility markers for association with a continuous
measure of radiological damage over time using longitudinal modeling techniques.
METHODS: Sixty-seven RA susceptibility variants were genotyped in 474 patients
in the Early Rheumatoid Arthritis Study (ERAS) using Sequenom MassArray
technology. Correlation between genetic markers and Larsen score was assessed
longitudinally using zero-inflated negative binomial regression to include
repeat measurements in the same individual at different timepoints. Genetic
markers associated with radiological damage in ERAS were tested using the same
modeling techniques on previously published data from the Norfolk Arthritis
Register (NOAR).
RESULTS: The single marker associated longitudinally with Larsen score in ERAS
(p = 0.02) and in NOAR (p = 0.04) was rs2900180 at the TRAF1 locus. Analysis of
individual timepoints in ERAS showed that rs2900180 displays its effect
primarily on the extent of Larsen score early in the disease course. Combined
longitudinal analysis of the 2 cohorts suggests further association of several
loci with Larsen score (KIF5A, PTPN22, AFF3, TAGAP) and therefore a significant
accumulation of RA severity markers among RA susceptibility markers (p = 0.016).
CONCLUSION: The marker rs2900180 is associated with the extent of radiological
damage in the ERAS cohort. This represents the second independent study
correlating rs2900180 at the TRAF1 locus with radiological severity in RA.
Replication in a large dataset is required to establish the role of other RA
susceptibility loci in disease severity. |
Is marijuana use associated with increased risk for stroke? | Yes, the use of marijuana is associated with increased risk for ischemic stroke, especially in young adults. The mechanisms underlying such association remain largely unclear, but increased vascular reactivity and increased cerebrovascular resistance were implicated. | OBJECTIVE AND METHOD: This paper reviews acute and chronic effects of drugs of
abuse on cerebral blood flow (CBF) and metabolism and their clinical
significance. The most important source of information for the review is human
research reports published in refereed journals. A few animal studies, book
chapters, and abstracts that are especially relevant are also included.
RESULTS: In humans, ethanol in small doses produces cerebral vasodilation;
higher doses induce cerebral vasoconstriction. Chronic alcoholism is associated
with reduced CBF and cerebral metabolism. Sedatives and antianxiety drugs lead
to global reduction in CBF and cerebral metabolism. Caffeine, even in small
doses, is a potent cerebral vasoconstrictor. Cerebral vasodilation is seen
immediately after cigarette smoking, but chronic smokers show global reduction
in CBF. Changes in CBF after marijuana smoking are variable; both increases and
decreases are seen. Chronic marijuana smoking, however, seems to reduce CBF.
Most inhalants and solvents are vasodilators; chronic abuse is accompanied by a
decrease in CBF. A number of drugs of abuse, including ethanol, amphetamines,
cocaine, nicotine, and caffeine-phenylpropanolamine combinations, increase the
risk for stroke. Reduction in CBF associated with chronic use of ethanol,
nicotine, inhalants, and solvents is at least partially reversible upon
abstinence.
CONCLUSIONS: Topics for future research include regional brain function, which
mediates drug-induced mood changes (euphoria); CBF concomitants of psychological
and physiological characteristics that increase addiction potential; changes in
CBF that accompany withdrawal syndromes; mechanisms responsible for drug-induced
stroke; and effects of functional and organic complications on CBF. A 22-year-old man with a five-year history of drug and alcohol abuse presented
with a left hemiparesis preceded by three transient ischaemic attacks, two of
which occurred whilst smoking cannabis. Substance abuse was the only
identifiable risk factor for cerebrovascular disease. We have recorded blood flow velocity in the anterior and middle cerebral
arteries by transcranial Doppler sonography in abstinent marijuana abusers (n =
16) and control subjects (n = 19) to assess the effects of prolonged marijuana
use of the cerebrovascular system. The pulsatility index, a measure of
cerebrovascular resistance, and systolic velocity were significantly (p < 0.005)
increased in marijuana abusers compared to the control subjects. These findings
suggest that cerebral perfusion observed in 18-30 year old marijuana abusers is
comparable to that of normal 60 year-olds. Thus, chronic abuse of marijuana
might be a risk factor for stroke. Factor V Leiden is a well-recognized etiology of venous thrombosis, but reports
of stroke in patients with this mutation are few. Marijuana smoking has rarely
been associated with thrombosis of cerebral and renal arteries and may be due to
a direct toxic effect on the endothelium. Reported here is the case of a
previously healthy young man who smoked marijuana on a daily basis and had an
occipital lobe stroke; he was found to be heterozygous for factor V Leiden. This
case suggests that marijuana smoking may increase the risk of arterial
thrombosis in otherwise healthy individuals who are heterozygous for factor V
Leiden. When admitted in an emergency unit, young patients often present acute
neurological effects of smoked marijuana. Other chronic adverse effects of
marijuana are probably underestimated: postural syncope, arteritis, chronic
bronchitis, amnesia. Marijuana may trigger a myocardial infarction and have a
vasospastic effect. Marijuana has impairing effects on driving ability. Smoked
marijuana is a potential respiratory tract carcinogen. INTRODUCTION: Drug use is a well-kown risk factor for cerebrovascular disease in
young people. Cannabis is the most widely consumed among the illicit drugs
worldwide, but it has only exceptionally been associated to cerebrovascular
disease.
CLINICAL CASE: We here describe 2 young patients (26 and 29 years, respectively)
who suffered from ischemic stroke in temporal relation with cannabis
consumption.
CONCLUSIONS: The review of the literature on this topic reveals another 18
patients with stroke in association to cannabis use. They all were young people
with ischemic stroke. Although a causal relationship is difficult to establish
due to the widespread use of cannabis, this drug may play an etiologic role in
ischemic stroke. Reversible cerebral vasoconstriction syndrome (RCVS) is characterized by the
association of severe headaches with or without additional neurological symptoms
and a 'string and beads' appearance on cerebral arteries, which resolves
spontaneously in 1-3 months. We present the clinical, neuroimaging and outcome
data of 67 consecutive patients prospectively diagnosed over 3 years in our
institution with an angiographically confirmed RCVS. There were 43 females and
24 males with a mean age of 42 years (19-70). RCVS was spontaneous in 37% of
patients and secondary in the 63% others, to postpartum in 5 and to exposure to
various vasoactive substances in 37, mainly cannabis, selective
serotonin-recapture inhibitors and nasal decongestants. The main pattern of
presentation (94% of patients) was multiple thunderclap headaches recurring over
a mean period of 1 week. In 51 patients (76%), headaches resumed the clinical
presentation. Various complications were observed, with different time courses.
Cortical subarachnoid haemorrhage (cSAH) (22%), intracerebral haemorrhage (6%),
seizures (3%) and reversible posterior leukoencephalopathy (9%) were early
complications, occurring mainly within the first week. Ischaemic events,
including TIAs (16%) and cerebral infarction (4%), occurred significantly later
than haemorrhagic events, mainly during the second week. Significant sex
differences were observed: women were older, had more frequent single-drug
exposure and a higher rate of stroke and cSAH. Sixty-one patients were treated
by nimodipine: 36% had recurrent headaches, 7% TIAs and one multiple infarcts.
The different time courses of thunderclap headaches, vasoconstriction and
strokes suggest that the responsible vasospastic disorder starts distally and
progresses towards medium sized and large arteries. No relapse was observed
during the 16 +/- 12.4 months of follow-up. Our data suggest that RCVS is more
frequent than previously thought, is more often secondary particularly to
vasoactive substances, and should be considered in patients with recurrent
thunderclap headaches, cSAH or cryptogenic strokes with severe headaches. OBJECTIVES: To report risk factors, aetiology and neuroimaging features among a
large series of young Australian patients who were admitted to hospital for a
first-ever occurrence of ischaemic stroke; to analyse the effect of age, sex and
ethnicity on the presence of risk factors; and to compare Australian and
overseas data.
DESIGN, SETTING AND PATIENTS: Retrospective evaluation of data for all patients
aged from 15 to 50 years who were admitted to a public hospital in Adelaide,
South Australia, from January 2006 to June 2010 with a primary diagnosis of
ischaemic stroke.
RESULTS: Among 326 patients (184 males), the most frequent stroke risk factors
overall were dyslipidaemia (187), smoking (161), hypertension (105) and obesity
(92). Fifty-one patients used illicit drugs, mostly comprising marijuana and
amphetamines. The most frequent stroke aetiologies overall were cardioembolism
(85), arterial dissection (49), and small-vessel occlusion (31). Cardioembolism
was highly prevalent among our study population compared with patients in other
countries. Neuroimaging showed that more patients in our study had strokes that
involved both vascular territories concurrently (9%) compared with patients in
other countries.
CONCLUSIONS: Risk factors, aetiology and features of ischaemic stroke among
young people in Adelaide differ significantly from published data for young
patients around the world. Patients in Adelaide are more likely to be obese, to
be misusing marijuana and amphetamines, to suffer a cardioembolic event and to
have a stroke that concurrently affects both the anterior and posterior cerebral
circulation. |
How many and which are the different isoforms for the ryanodine receptor? | Generally, three ryanodine receptor isoforms (RyR1-RyR3) are known. RyR1, expressed in skeletal muscle; RyR2, expressed in cardiac muscle; and RyR3, expressed in various cells. RyR3 is preferentially expressed in the brain especially in the hippocampus and striatum. | The rapid cooling (RC) response in muscle is an increase in cytoplasmic Ca2+
concentration ([Ca2+]i) that is probably caused by Ca2+ release from the
sarcoplasmic reticulum (SR). However, the molecular bases of this response have
not been completely elucidated. Three different isoforms of the SR Ca2+ release
channels, or ryanodine receptors (RyRs), have been isolated (RyR1, RyR2, and
RyR3). In the current investigation, the RC response was studied in RyR-null
muscle cells (1B5) before and after transduction with HSV-1 virions containing
the cDNAs encoding for RyR1, RyR2, or RyR3. Cells were loaded with fluo 4-AM to
monitor changes in [Ca2+]i and perfused with either cold ( approximately 0
degrees C), room temperature (RT), or RT buffer containing 40 mM caffeine.
Control cells showed no significant response to cold or caffeine, whereas robust
Ca2+ transients were recorded in response to both RC and caffeine in transduced
cells expressing any one of the three RyR isoforms. Our data demonstrate
directly that RyRs are responsible for the RC response and that all three
isoforms respond in a similar manner. Ca2+ release from RyRs is likely caused by
a RC-induced conformational change of the channel from the closed to the open
state. Ryanodine receptor (RyR) is a Ca(2+) channel that mediates Ca(2+) release from
intracellular stores. Altered Ca(2+) homeostasis in skeletal muscle which
usually occurs as a result of point mutations in type 1 RyR1 (RyR1) is a key
molecular event triggering maligt hyperthermia (MH). There are three RyR
isoforms, and we herein show, for the first time, that human dendritic cells
(DCs) preferentially express RyR1 mRNA among them. The RyR activator,
4-chloro-m-cresol (4CmC), induced Ca(2+) release in DCs, and this response was
eliminated by dantrolene, an inhibitor of the RyR1, and was unaffected by
xestospongin C, a selective inhibitor of IP(3) receptor. Activation of RyR1
reduced LPS-induced IL-10 production, promoted the expression of HLA-DR and
CD86, and thereby exhibited an improved capacity to stimulate allogeneic T
cells. These findings demonstrate that RyR1-mediated calcium signaling modifies
diverse DC responses and suggest the feasibility of using DC preparations for
the diagnosis of MH. AIM: Functional evidence suggests the presence of two types of intracellular
Ca(2+) channels responsible for the release of Ca(2+) from Ca(2+)-stores, i.e.
inositol-1,4,5-trisphosphate (IP(3)R) and ryanodine receptors (RyR), in rat
colonic epithelium. Generally, three ryanodine receptor isoforms (RyR1-RyR3) are
known; however, the type of RyR at this epithelium is unknown and was the focus
of the present study.
METHODS: RyRs were characterized by molecular biological and immunohistochemical
methods in the rat colon.
RESULTS: A transcript of RyR1 was found in mRNA from colonic crypts. In
contrast, RyR2 and RyR3 were found in their corresponding reference tissues, but
not in the cDNA from colonic crypts suggesting a predomit expression of the
RyR1 isoform in this epithelium. In order to characterize the subcellular
localization of RyR1, immunohistochemical experiments were performed. They
showed that RyR1 is present in the lamina epithelialis mucosae and smooth muscle
cells and is distributed equally along the whole crypt axis with no difference
between surface and crypt cells. A double staining with IP(3)R3, the domit
cytoplasmic isoform of IP3Rs in this epithelium, revealed that there is only
little colocalization of the two receptor subtypes within the epithelial cells.
Furthermore, the epithelium is equipped with the enzyme CD38 responsible for the
production of cyclic adenosine diphosphate ribose, the physiological agonist of
RyR. RyRs are known to be activated by changes in the redox state. The oxidant,
monochloramine evoked a ruthenium red-sensitive Ca(2+) release all over the
crypt axis. This release was unaffected by prior stimulation of IP(3) receptors
with ATP (and vice versa).
CONCLUSION: The present data suggest a functional separation of IP(3)- and
ryanodine receptor-carrying Ca(2+) stores in the colonic epithelium. In excitable cells such as skeletal and cardiac myocytes excitation-contraction
coupling is an important intermediate step between initiation of the action
potential and induction of contraction. This process is predomitly controlled
by Ca(2+) release from the sarcoplasmic reticulum via the ryanodine receptor.
This very large protein (MW 560 kDa) exists as a homotetramer (~2.2 MDa) and is
expressed in three isoforms: RyR1, expressed in skeletal muscle; RyR2, expressed
in cardiac muscle; and RyR3, expressed in various cells at lower levels than the
other isoforms. Release of Ca(2+) via RyR2 is induced by Ca(2+) influx through
L-type Ca(2+) channels and is modulated by multiple factors, including
phosphorylation of RyR2 protein by protein kinase A, calmodulin kinase II and
FKBP12.6, and stimulation via the beta-adrenergic receptor signaling pathway.
Hyperphosphorylation of RyR2 induces Ca(2+) leak during diastole, which can
cause fatal arrhythmias and lead to heart failure. This makes RyR2 an important
therapeutic target. Although there are few commercially available drugs that
inhibit Ca(2+) leak from RyR2, K201 (JTV-519), a benzothiazepine derivative, has
emerged as a new ryanodine receptor-selective agent that prevents atrial
fibrillation, ventricular arrhythmias, heart failure and exercise-induced sudden
cardiac death. In this review, we discuss recent advances in our understanding
of the basic structure and function of ryanodine receptors, their involvement in
heart disease, and the development of drugs to prevent ryanodine receptor
malfunction and recent patents. Skeletal (RyR1) and cardiac muscle (RyR2) isoforms of ryanodine receptor calcium
channels are inhibited by millimollar Ca(2+), but the affinity of RyR2 for
inhibitory Ca(2+) is ~10 times lower than that of RyR1. Previous studies
demonstrated that the C-terminal quarter of RyR has critical domain(s) for
Ca(2+) inactivation. To obtain further insights into the molecular basis of
regulation of RyRs by Ca(2+), we constructed and expressed 18 RyR1-RyR2 chimeras
in HEK293 cells and determined the Ca(2+) activation and inactivation affinities
of these channels using the [(3)H]ryanodine binding assay. Replacing two
distinct regions of RyR1 with corresponding RyR2 sequences reduced the affinity
for Ca(2+) inactivation. The first region (RyR2 amino acids 4020-4250) contains
two EF-hand Ca(2+) binding motifs (EF1, amino acids 4036-4047; EF2, amino acids
4071-4082), and the second region includes the putative second transmembrane
segment (S2). A RyR1-backbone chimera containing only EF2 from RyR2 had a modest
(not significant) change in Ca(2+) inactivation, whereas another chimera channel
carrying only EF1 from RyR2 had a significantly reduced level of Ca(2+)
inactivation. The results suggest that EF1 is a more critical determit for
RyR inactivation by Ca(2+). In addition, activities of the chimera carrying RyR2
EF-hands were suppressed at 10-100 μM Ca(2+), and the suppression was relieved
by 1 mM Mg(2+). The same effects have been observed with wild-type RyR2. A
mutant RyR1 carrying both regions replaced with RyR2 sequences (amino acids
4020-4250 and 4560-4618) showed a Ca(2+) inactivation affinity comparable to
that of RyR2, indicating that these regions are sufficient to confer RyR2-type
Ca(2+)-dependent inactivation on RyR1. |
Which compound is a specific inhibitor for Nox1 and Nox4? | GKT136901 is a specific inhibitor of Nox1 and Nox4. | The functional significance and regulation of NAD(P)H oxidase (Nox) isoforms by
angiotensin II (Ang II) and endothelin-1 (ET-1) in vascular smooth muscle cells
(VSMCs) from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats
(SHR) was studied. Expression of Nox1, Nox2, and Nox4 (gene and protein) and
NAD(P)H oxidase activity were increased in SHR. Basal NAD(P)H oxidase activity
was blocked by GKT136901 (Nox1/4 inhibitor) and by Nox1 siRNA in WKY cells and
by siNOX1 and siNOX2 in SHR. Whereas Ang II increased expression of all Noxes in
WKY, only Nox1 was influenced in SHR. Ang II-induced NAD(P)H activity was
inhibited by siNOX1 in WKY and by siNOX1 and siNOX2 in SHR. ET-1 upregulated Nox
expression only in WKY and increased NAD(P)H oxidase activity, an effect
inhibited by siNOX1 and siNOX2. Nox1 co-localized with Nox2 but not with Nox4,
implicating association between Nox1 and Nox2 but not between Nox1 and Nox4.
These data highlight the complexity of Nox biology in VSMCs, emphasising that
more than one Nox member, alone or in association, may be involved in NAD(P)H
oxidase-mediated •O(2)(-) production. Nox1 regulation by Ang II, but not by
ET-1, may be important in •O(2)(-) formation in VSMCs from SHR. Author information:
(1)Diabetic Complications Division, Juvenile Diabetes Research Foundation
Danielle Alberti Memorial Centre for Diabetic Complications, Baker IDI Heart &
Diabetes Institute, Melbourne, Victoria, Australia; Department of Medicine,
Monash University, Melbourne, Victoria, Australia;
(2)Diabetic Complications Division, Juvenile Diabetes Research Foundation
Danielle Alberti Memorial Centre for Diabetic Complications, Baker IDI Heart &
Diabetes Institute, Melbourne, Victoria, Australia;
(3)Human Epigenetics Laboratory, Baker IDI Heart & Diabetes Institute,
Melbourne, Victoria, Australia;
(4)Diabetic Complications Division, Juvenile Diabetes Research Foundation
Danielle Alberti Memorial Centre for Diabetic Complications, Baker IDI Heart &
Diabetes Institute, Melbourne, Victoria, Australia; Department of Nephrology and
Hypertension, Kawasaki Medical School, Kurashiki, Japan;
(5)Department of Pharmacology, Cardiovascular Research Institute Maastricht,
Faculty of Medicine, Health & Life Science, Maastricht University, Maastricht,
The Netherlands;
(6)Genkyotex SA, Geneva, Switzerland;
(7)Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; and Institute of
Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United
Kingdom.
(8)Diabetic Complications Division, Juvenile Diabetes Research Foundation
Danielle Alberti Memorial Centre for Diabetic Complications, Baker IDI Heart &
Diabetes Institute, Melbourne, Victoria, Australia; Department of Medicine,
Monash University, Melbourne, Victoria, Australia;
[email protected]. |
Which molecule is targeted by the drug Gevokizumab? | Gevokizumab is an allosteric anti-IL-1β monoclonal antibody. | Interleukin-1β (IL-1β) is a potent mediator of inflammatory responses and plays
a role in the differentiation of a number of lymphoid cells. In several
inflammatory and autoimmune diseases, serum levels of IL-1β are elevated and
correlate with disease development and severity. The central role of the IL-1
pathway in several diseases has been validated by inhibitors currently in
clinical development or approved by the FDA. However, the need to effectively
modulate IL-1β-mediated local inflammation with the systemic delivery of an
efficacious, safe and convenient drug still exists. To meet these challenges, we
developed XOMA 052 (gevokizumab), a potent anti-IL-1β neutralizing antibody that
was designed in silico and humanized using Human Engineering™ technology. XOMA
052 has a 300 femtomolar binding affinity for human IL-1β and an in vitro
potency in the low picomolar range. XOMA 052 binds to a unique IL-1β epitope
where residues critical for binding have been identified. We have previously
reported that XOMA 052 is efficacious in vivo in a diet-induced obesity mouse
model thought to be driven by low levels of chronic inflammation. We report here
that XOMA 052 also reduces acute inflammation in vivo, neutralizing the effect
of exogenously administered human IL-1β and blocking peritonitis in a mouse
model of acute gout. Based on its high potency, novel mechanism of action, long
half-life, and high affinity, XOMA 052 provides a new strategy for the treatment
of a number of inflammatory, autoimmune and metabolic diseases in which the role
of IL-1β is central to pathogenesis. The inflammatory cytokine IL-1β has an essential role in the innate immune
response. High levels of IL-1β have been implicated in the development of many
diseases, including type 1 and 2 diabetes (T1D and T2D), rheumatoid arthritis
(RA) and cardiovascular disease. XOMA is developing gevokizumab (XOMA-052), an
IgG2 humanized mAb against human IL-1β, for the potential treatment of these
diseases. Gevokizumab has a high affinity for IL-1β and a long t1/2, which would
allow for once-monthly dosing and offer a considerable advantage for patients
over agents requiring more frequent dosing. Data from preclinical studies and
clinical trials suggest that gevokizumab is a potentially effective and
well-tolerated treatment for the indicated diseases. At the time of publication,
phase II clinical trials were ongoing in patients with T1D, T2D and RA, with the
T2D trials assessing key cardiovascular markers. Following promising data from a
recent pilot trial, XOMA was also planning a phase I/II trial of gevokizumab for
the potential treatment of uveitis in patients with the vasculitic inflammatory
disorder Behçet's disease and the autoinflammatory conditions familial cold
autoinflammatory syndrome and Muckle-Wells syndrome. OBJECTIVE: Uveitis and retinal vasculitis are sight-threatening manifestations
of Behçet's disease with limited treatment options. This pilot study aimed to
evaluate the safety, pharmacokinetics and clinical activity of XOMA 052
(gevokizumab), a recombit humanised anti-interleukin 1β antibody, in Behçet's
disease patients with uveitis.
METHODS: Patients with acute posterior or panuveitis, and/or retinal vasculitis,
resistant to azathioprine and/or ciclosporin, and receiving 10 mg/day or less of
prednisolone, were enrolled into the 98-day study. Immunosuppressive agents were
discontinued at baseline. Patients received a single infusion of XOMA 052 (0.3
mg/kg). The safety and uveitis status and pharmacokinetics of XOMA 052 were
evaluated.
RESULTS: Seven patients enrolled and completed the study. No treatment-related
adverse event was observed. XOMA 052 treatment was associated with rapid and
durable clinical response in all patients. Complete resolution of intraocular
inflammation was achieved in 4-21 days (median 14 days), with a median duration
of response of 49 days (range 21-97 days); one patient remained exacerbation
free throughout the study.
CONCLUSIONS: Well tolerated, XOMA 052 resulted in a rapid onset and sustained
reduction in intraocular inflammation in patients with resistant uveitis and
retinal vasculitis. Moreover, the effect was observed despite discontinuation of
immunosuppressive agents and without the need to increase corticosteroid
dosages. OBJECTIVE: Metabolic activation of the innate immune system governed by
interleukin (IL)-1β contributes to β-cell failure in type 2 diabetes.
Gevokizumab is a novel, human-engineered monoclonal anti-IL-1β antibody. We
evaluated the safety and biological activity of gevokizumab in patients with
type 2 diabetes.
RESEARCH DESIGN AND METHODS: In a placebo-controlled, dose-escalation study, a
total of 98 patients were randomly assigned to placebo (17 subjects) or
gevokizumab (81 subjects) at increasing doses and dosing schedules. The primary
objective of the study was to evaluate the safety profile of gevokizumab in type
2 diabetes. The secondary objectives were to assess pharmacokinetics for
different dose levels, routes of administration, and regimens and to assess
biological activity.
RESULTS: The study drug was well tolerated with no serious adverse events. There
was one hypoglycemic event whereupon concomitant insulin treatment had to be
reduced. Clearance of gevokizumab was consistent with that for a human IgG(2),
with a half-life of 22 days. In the combined intermediate-dose group (single
doses of 0.03 and 0.1 mg/kg), the mean placebo-corrected decrease in glycated
hemoglobin was 0.11, 0.44, and 0.85% after 1, 2 (P = 0.017), and 3 (P = 0.049)
months, respectively, along with enhanced C-peptide secretion, increased insulin
sensitivity, and a reduction in C-reactive protein and spontaneous and inducible
cytokines.
CONCLUSIONS: This novel IL-1β-neutralizing antibody improved glycemia, possibly
via restored insulin production and action, and reduced inflammation in patients
with type 2 diabetes. This therapeutic agent may be able to be used on a
once-every-month or longer schedule. Behçet's disease (BD) is a multisystem inflammatory disorder of uncertain
origin, although it remains defined within the spectrum of systemic
immune-mediated vasculitic disorders and also represents a spectrum of putative
autoimmune disease. Major symptoms include oral aphthous ulcers, genital
ulcerations, skin lesions, and ocular lesions. Despite afflicting many systems,
ocular complications of BD are some of the more devastating for the patient and
their quality of life. Eye involvement, which affects 60-80 % of BD patients, is
characterized in its more severe form by posterior or panuveitis including
occlusive retinal vasculitis. While pathogenesis of BD remains complex,
association with Class I MHC (HLA-B*51) predisposing to inflammation with
engagement of the innate-immune system (neutrophils, NK cells), and perpetuated
by the adaptive T cell responses against infectious- and/or auto-antigens.
Despite the choice of conventional immunosuppressive therapies available, only
recently with the advent of biologic therapy has visual prognosis and outcomes
been substantially and favorably altered. For example, both interferon-α (IFN-α)
and tumour necrosis factor (TNF)-α antagonists deliver promising results and for
the first time improve prognosis. With IFN-α therapy, durable remissions of
uveitis can be achieved and lead to drug-free remission. Similarly, anti-TNF
therapy with infliximab is reported to be rapidly effective in inducing and
maintaining remission. Most recently, rising evidence reports on the use of
adalimumab, etanercept, and golimumab, while use of anti-interleukin (IL)-1
agents (anakinra, canakinumab, gevokizumab), IL-6 blockers (tocilizumab), and
rituximab (depleting anti-CD20 antibody) is also increasing. The aim of this
review is to provide evidence for the role of conventional therapies combined
with evidence for advantages and disadvantages of biologic therapies in the
treatment of ocular BD. Although randomized controlled trials remain sparse,
evidence remains strong and enticing that biologic agents are invaluable for the
treatment of sight-threatening Behçet's uveitis and makes it an exciting time
for Behçet's specialists worldwide. Interleukin-1β (IL-1β) is a proinflammatory cytokine that is implicated in many
autoinflammatory disorders, but is also important in defense against pathogens.
Thus, there is a need to safely and effectively modulate IL-1β activity to
reduce pathology while maintaining function. Gevokizumab is a potent anti-IL-1β
antibody being developed as a treatment for diseases in which IL-1β has been
associated with pathogenesis. Previous data indicated that gevokizumab
negatively modulates IL-1β signaling through an allosteric mechanism. Because
IL-1β signaling is a complex, dynamic process involving multiple components, it
is important to understand the kinetics of IL-1β signaling and the impact of
gevokizumab on this process. In the present study, we measured the impact of
gevokizumab on the IL-1β system using Schild analysis and surface plasmon
resoce studies, both of which demonstrated that gevokizumab decreases the
binding affinity of IL-1β for the IL-1 receptor type I (IL-1RI) signaling
receptor, but not the IL-1 counter-regulatory decoy receptor (IL-1 receptor type
II). Gevokizumab inhibits both the binding of IL-1β to IL-1RI and the subsequent
recruitment of IL-1 accessory protein primarily by reducing the association
rates of these interactions. Based on this information and recently published
structural data, we propose that gevokizumab decreases the association rate for
binding of IL-1β to its receptor by altering the electrostatic surface potential
of IL-1β, thus reducing the contribution of electrostatic steering to the rapid
association rate. These data indicate, therefore, that gevokizumab is a unique
inhibitor of IL-1β signaling that may offer an alternative to current therapies
for IL-1β-associated autoinflammatory diseases. INTRODUCTION: Insulin is the cornerstone of type 1 diabetes mellitus (T1DM)
therapy. However, it cannot achieve a delay in the onset or evolution of this
condition, while cardiovascular morbidity remains an unquestionable threat.
AREAS COVERED: In this review, the authors discuss gevokizumab (XOMA 052), a
recombit monoclonal antibody that can neutralize human IL-1β by binding to
it. This is relevant, because this IL has been associated with β-cell toxicity
in both diabetes types. Moreover, gevokizumab presents two major advantages: it
spares IL-1α and it exhibits favorable pharmacokinetic properties. Gevokizumab
has already proven its safety and efficacy in improving glycemic control, β cell
function and inflammation markers in clinical trials in diabetic patients.
EXPERT OPINION: Despite the very promising characteristics of gevokizumab,
important questions remain to be answered. One important question is what to
expect from a combination of this agent with insulin and if there is a subset of
patients that might respond more favorably to treatment. We also need to know at
what stage in the natural history of T1DM could gevokizumab be most efficacious,
as well as its potential effects on cardiovascular outcomes. OBJECTIVE: Excessive neointima formation often occurs after arterial injury.
Interleukin-1β (IL-1β) is a potent pleiotropic cytokine that has been shown to
regulate neointimal proliferation. We investigated the effects of the IL-1β
modulator gevokizumab in a rat carotid denudation model.
METHODS: Sprague-Dawley rats were subjected to balloon denudation of the right
carotid artery and were then randomized to receive a single subcutaneous
infusion immediately after balloon injury of saline (control group, n = 13) or
gevokizumab (gevokizumab groups, n = 15 in each group: 1, 10 and 50 mg/kg). We
evaluated the treatment effects on carotid intima-media thickness (IMT) using
ultrasonography, on endothelial regrowth using Evans Blue staining and on
inflammatory response using histology. We also assessed the effects of IL-1β and
gevokizumab on human umbilical vein endothelial cells (HUVEC) and rat smooth
muscle cells.
RESULTS: We found that carotid IMT, in the proximal part of the denuded artery
at day 28, was decreased by gevokizumab 1 mg/kg compared with controls.
Neointima area and the intima/media area ratio were both reduced in the
gevokizumab 1 mg/kg-treated group. Gevokizumab at the 1 mg/kg dose also improved
endothelial regrowth. No effect was observed with gevokizumab 10 or 50 mg/kg.
Gevokizumab also decreased the inflammatory effect of IL-1β in in vitro cell
experiments and protected HUVECs from IL-1β's deleterious effects on cell
migration, apoptosis and proliferation.
CONCLUSION: A single administration of gevokizumab 1 mg/kg improves endothelial
regrowth and reduces neointima formation in rats following carotid denudation,
at least in part through its beneficial effects on endothelial cells. |
Which protein phosphatases have been found to dephosphorylate phospholamban? | The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)-associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70-80% of the SR-associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated Ca2+/calmodulin-dependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity. | The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum
(SR)-associated phospholamban were studied in cardiac muscle extracts and in a
Triton fraction prepared by detergent extraction of myofibrils, the latter
fraction containing 70-80% of the SR-associated proteins present in the tissue.
At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1)
accounted for approximately 70% of the total phospholamban phosphatase activity
in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent
protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated
Ca2+/calmodulin-dependent protein kinase). Protein phosphatase 2A (PP2A) and
protein phosphatase 2C (PP2C) accounted for the remainder of the activity. A
major form of cardiac PP1, present in comparable amounts in both the extract and
Triton fraction, was similar, if not identical, to skeletal muscle protein
phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit
complexed to a G subunit of approximately 160 kDa, responsible for targeting PP1
to both the SR and glycogen particles of skeletal muscle. This conclusion was
based on immunoblotting experiments using antibody to the G subunit, ability to
bind to glycogen and the release of PP1 activity from glycogen upon incubation
with PK-A and MgATP. PP1 accounted for approximately 90% of the phospholamban
(Ser-16 or Thr-17) phosphatase activity in the material sedimented by
centrifugation at 45,000 x g, a fraction prepared from cardiac extracts which is
enriched in SR membranes. The G subunit in this fraction could be solubilised by
Triton X-100, but not with 0.5 M NaCl or digestion with alpha-amylase,
indicating that it is bound to membranes and not to glycogen. By analogy with
the situation in skeletal muscle, the PK-A catalysed phosphorylation of the G
subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from
dephosphorylating SR-bound substrates and represent one of the mechanisms by
which adrenalin increases the phosphorylation of cardiac phospholamban (Ser-16
and Thr-17) in vivo. Hearts left in situ post mortem lose 85-95% of their PP1
activity within 20-30 min. This remarkable disappearance of PP1 may partly
explain why the importance of this enzyme in cardiac muscle metabolism has not
been recognized previously. The phosphorylation of rat cardiac microsomal proteins was investigated with
special attention to the effects of okadaic acid (an inhibitor of protein
phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic
AMP-dependent protein kinase (protein kinase A). The results showed that okadaic
acid (5 microM) modestly but reproducibly augmented the protein kinase
A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect
on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes
contained three other substrates (M(r) 23, 19 and 17 kDa) that were
phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The
protein kinase A-catalyzed phosphorylation of these three substrates was
markedly (2-3 fold) increased by 5 microM okadaic acid. Calmodulin was found to
antagonize the action of okadaic acid on such phosphorylation. Protein kinase A
inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation
of microsomal polypeptides. Unexpectedly, inhibitor 2 was also found to markedly
decrease protein kinase A-catalyzed phosphorylation of phospholamban as well
these other microsomal substrates. These results are consistent with the views
that protein phosphatase 1 is capable of dephosphorylating membrane-associated
phospholamban when it is phosphorylated by protein kinase A, but not by
calcium/calmodulin kinase, and that under certain conditions,
calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is
also able to dephosphorylate PLN phosphorylated by protein kinase A.
Additionally, the observations show that protein phosphatase 1 is extremely
active against the three protein kinase A substrates (M(r) 23, 19 and 17 kDa)
that were present in the isolated microsomes and whose state of phosphorylation
was particularly affected in the presence of dimethylsulfoxide. Protein
phosphatase 2B is also capable of dephosphorylating these three substrates. |
What is the most prominent sequence consensus for the polyadenylation site? | Functional polyadenylation [poly(A)] sites consist of two sequence elements, the AAUAAA and G/U box signals, that closely flank the site of mRNA 3'-end formation. The canonical polyadenylation signal sequence AATAAA | Functional polyadenylation [poly(A)] sites consist of two sequence elements, the
AAUAAA and G/U box signals, that closely flank the site of mRNA 3'-end
formation. In agreement with previous results, random sequence insertions
between the AAUAAA and G/U box signals were observed to inhibit poly(A) site
function. However, sequence insertions of similar size that were predicted to
form RNA stem-loop structures were found to have little effect on the efficiency
of polyadenylation and instead induced a 3' shift in the site of polyadenylation
that was equal to the length of the inserted stem-loop. The in vivo utilization
of a poly(A) site bearing an internal RNA stem-loop structure was inhibited by
mutations that destabilized the predicted stem but was restored by compensatory
mutations. These results strongly support the hypothesis that the appropriate
spacing of the AAUAAA and G/U box signals is critical for poly(A) site function.
Sequence insertions that are able to form RNA secondary structures that maintain
the correct spacing of these two RNA target sequences are well tolerated,
whereas sequence insertions that disturb this spacing inhibit poly(A) site
recognition. It is proposed that the effect of sequence insertions on poly(A)
site function may be sufficiently predictable to allow the development of an
assay for in vivo RNA secondary structure that uses poly(A) site selection as a
readout. A compilation of the pre-mRNA ends of the genes of nuclear encoded mitochondrial
proteins resulted in a consensus sequence of the type (T/A)NTTNNNNNTTTNAATAAA.
Nucleotide positions +8, +13, +14, +16 and +17 downstream of the AATAAA sequence
show also a predomice of nucleotide T. This consensus sequence suggests the
importance of the immediate surroundings of the cannonical polyadenylation
signal sequence AATAAA on the efficiency of the cleavage and polyadenylation of
this specific group of pre-mRNAs. Several cDNAs encoding H-protein, a constituent of the glycine cleavage system,
were cloned from chicken liver cDNA libraries with an antibody raised against
rat H-protein or with a nick-translated cDNA of an immunoreactive clone. The
structure of the H-protein cDNA consisting of 910 base pairs was determined
using clones with an apparent overlap in the nucleotide sequence. The cDNA
encodes the precursor form of H-protein that is comprised of 39 amino acid
residues for a mitochondrial presequence and 125 amino acid residues for the
mature protein, following a 5' untranslated region of 13 base pairs. There are
two genuine consensus sequences for the cleavage/polyadenylation of the
precursor H-protein mRNA in the 3' untranslated region of the cDNA sequence. We
showed by comparison with the delta-aminolevulinate synthase gene that only one
copy of the H-protein cDNA occurs in the haploid genome of the chicken.
Nevertheless, two types of H-protein mRNAs, which differ by the length of their
3' untranslated region, are produced in liver. The chicken H-protein gene
extends over 8 kilobase pairs on the genome and includes 5 exons that encode the
entire cDNA sequence. Two AATAAA motifs are coded in the last exon of this gene,
suggesting that the differently size H-protein mRNAs are produced by the
alternative use of these motifs. We have determined the nucleotide sequence of the murine immunoglobulin gamma 2a
membrane 3' untranslated region (1413 nucleotides) and approximately 679
nucleotides of downstream sequence. Two AATAAA hexanucleotide sequences are
present in the 2092 nucleotide interval. The first one functions as the major
polyA signal, directing cleavage and polyadenylation at a site 20 nucleotides
downstream. Within 41 nucleotides downstream of the major membrane polyA signal
are two sequences with 75% homology to the consensus sequence,
(C/T)GTGTT(C/T)(C/T), identified by McLauchlan et al. [Nucl. Acids Res. 13,
1347-1365 (1985)]. An 80% homology match to the Berget consensus sequence,
CA(C/T)TG, begins five nucleotides 3' of the major polyA site (used 20 times
more than the second, downstream polyA site) [Berget Nature 309, 179-182
(1984)]. The second AATAAA, located 73 nucleotides 3' of the first, directs
cleavage and polyadenylation 18 nucleotides downstream at a minor polyA site.
One match with 75% homology to the McLauchlan consensus sequence begins 17
nucleotides 3' of the second (weaker) polyA site. No matches to the Berget
consensus sequence are located near this second, weaker polyA site. A single U leads to G transversion in the 3' consensus sequence AAUAAA of the
adenovirus early region 1A gene was constructed and the effect of this mutation
on processing of the 3' end of the nuclear early region 1A RNAs was analysed.
The results demonstrate that the intact AAUAAA is not required for RNA
polyadenylation but is required for the cleavage step preceding polyadenylation
to occur efficiently. The great majority of viral mRNAs in mouse C127 cells transformed by bovine
papillomavirus type 1 (BPV) have a common 3' end at the early polyadenylation
site which is 23 nucleotides (nt) downstream of a canonical poly(A) consensus
signal. Twenty percent of BPV mRNA from productively infected cells bypasses the
early polyadenylation site and uses the late polyadenylation site approximately
3,000 nt downstream. To inactivate the BPV early polyadenylation site, the early
poly(A) consensus signal was mutated from AAUAAA to UGUAAA. Surprisingly, this
mutation did not result in significant read-through expression of downstream
RNA. Rather, RNA mapping and cDNA cloning experiments demonstrate that virtually
all of the mutant RNA is cleaved and polyadenylated at heterogeneous sites
approximately 100 nt upstream of the wild-type early polyadenylation site. In
addition, cells transformed by wild-type BPV harbor a small population of mRNAs
with 3' ends located in this upstream region. These experiments demonstrate that
inactivation of the major poly(A) signal induces preferential use of otherwise
very minor upstream poly(A) sites. Mutational analysis suggests that
polyadenylation at the minor sites is controlled, at least in part, by UAUAUA,
an unusual variant of the poly(A) consensus signal approximately 25 nt upstream
of the minor polyadenylation sites. These experiments indicate that inactivation
of the major early polyadenylation signal is not sufficient to induce expression
of the BPV late genes in transformed mouse cells. |
Does fibronectin constitute a serum biomarker for Duchenne muscular dystrophy? | Compared to age-matched controls, fibronectin levels in DMD patients were found to be significantly increased, whereas in patients with Becker muscular dystrophy, Bethlem myopathy, or myasthenia gravis were close to the control levels. Additionally, progressive elevation in fibronectin levels was observed in longitudinal samples from DMD patients followed up for a period of 6 months up to 4 years. Therefore, fibronectin is a serum biomarker for Duchenne muscular dystrophy. | PURPOSE: To identify and validate serum biomarkers for the progression of
Duchenne muscular dystrophy (DMD) using a MS-based bottom-up pipeline.
EXPERIMENTAL DESIGN: We used a bottom-up proteomics approach, including a
protein concentration equalization step, different proteolytic digestions, and
MS detection schemes, to identify candidate biomarkers in serum samples from
control subjects and DMD patients. Fibronectin was chosen for follow-up based on
the differences in peptide spectral counts and sequence coverage observed
between the DMD and control groups. Subsequently, fibronectin levels were
determined with ELISA in 68 DMD patients, 38 milder Becker muscular dystrophy
patients, 33 patients with other neuromuscular disorders, and 15 age-matched
adult and child controls.
RESULTS: There was a significant increase in fibronectin levels in DMD patients
compared to age-matched controls. Fibronectin levels in patients with Becker
muscular dystrophy, Bethlem myopathy, or myasthenia gravis were comparable to
control levels. Progressive elevation in fibronectin levels was observed in
longitudinal samples from 22 DMD patients followed up for a period of 6 months
up to 4 years.
CONCLUSION AND CLINICAL RELEVANCE: This study suggests that serum fibronectin
levels may constitute a promising biomarker to monitor disease progression in
DMD patients. |
What colonoscopy findings have been reported in autism | Endoscopy trials have demonstrated a higher prevalence of nonspecific colitis, lymphoid hyperplasia and focally enhanced gastritis compared with controls. | BACKGROUND: Intestinal mucosal pathology, characterized by ileo-colonic lymphoid
nodular hyperplasia (LNH) and mild acute and chronic inflammation of the
colorectum, small bowel and stomach, has been reported in children with autistic
spectrum disorder (ASD).
AIM: To assess ileo-colonic LNH in ASD and control children and to test the
hypothesis that there is an association between ileo-colonic LNH and ASD in
children.
PATIENTS AND METHODS: One hundred and forty-eight consecutive children with ASD
(median age 6 years; range 2-16; 127 male) with gastrointestinal symptoms were
investigated by ileo-colonoscopy. Macroscopic and histological features were
scored and compared with 30 developmentally normal (non-inflammatory bowel
disease, non-coeliac disease) controls (median age 7 years; range 1-11; 25 male)
showing mild non-specific colitis in 16 cases (13 male) and normal colonic
histology in 14 cases (12 male). Seventy-four ASD children and 23 controls also
underwent upper gastrointestinal endoscopy. The influence on ileal LNH of
dietary restriction, age at colonoscopy, and co-existent LNH elsewhere in the
intestine, was examined.
RESULTS: The prevalence of LNH was significantly greater in ASD children
compared with controls in the ileum (129/144 (90%) vs. 8/27 (30%), P < 0.0001)
and colon (88/148 (59%) vs. 7/30 (23%), P = 0.0003), whether or not controls had
co-existent colonic inflammation. The severity of ileal LNH was significantly
greater in ASD children compared with controls, with moderate to severe ileal
LNH present in 98 of 144 (68%) ASD children versus 4 of 27 (15%) controls (P <
0.0001). Severe ileal LNH was associated with co-existent colonic LNH in ASD
children (P = 0.01). The presence and severity of ileal LNH was not influenced
by either diet or age at colonoscopy (P = 0.2). Isolated ileal LNH without
evidence of pathology elsewhere in the intestine was a rare event, occurring in
less than 3% of children overall. On histopathological examination, hyperplastic
lymphoid follicles are significantly more prevalent in the ileum of ASD children
(84/138; 61%) compared with controls (2/23; 9%, P = 0.0001).
CONCLUSION: Ileo-colonic LNH is a characteristic pathological finding in
children with ASD and gastrointestinal symptoms, and is associated with mucosal
inflammation. Differences in age at colonoscopy and diet do not account for
these changes. The data support the hypothesis that LNH is a significant
pathological finding in ASD children. Les troubles du spectre autistique font référence à des syndromes de gravité
diverse, caractérisés par une perturbation des interactions sociales, des
retards sur le plan de la communication, ainsi que des comportements et des
intérêts limités et répétés. La prévalence des troubles du spectre autistique
est en hausse, tandis que leur étiologie reste imprécise et fort probablement
multifactorielle. Plusieurs rapports ont avancé l’hypothèse d’un lien entre
l’autisme et certains symptômes gastro-intestinaux chroniques. Des études
endoscopiques ont notamment montré une prévalence plus élevée de colite non
spécifique, d’hyperplasie lymphoïde et de gastrite en foyers chez des sujets
malades comparativement à des témoins. Parmi les mécanismes potentiellement en
cause, on retrouve : réponse immunitaire aberrante à certaines protéines
alimentaires, perméabilité intestinale anormale et microflore intestinale
inappropriée. On décrit ici le cas de deux patients atteints de troubles du
spectre autistique qui présentaient des symptômes intestinaux chroniques et des
anomalies endoscopiques, le tout est suivi d’une revue sur ce sujet controversé. |
What is a disordered protein? | Intrinsically disordered proteins lack stable tertiary and/or secondary structures under physiological conditions in vitro. Intrinsically disordered proteins undergo significant conformational transitions to well folded forms only on binding to partner. | Intrinsically disordered proteins lack stable tertiary and/or secondary
structures under physiological conditions in vitro. Intrinsically disordered
proteins undergo significant conformational transitions to well folded forms
only on binding to partner. Molecular dynamics simulations are used to research
the mechanism of folding for intrinsically disordered protein upon partner
binding. Room-temperature MD simulations suggest that the intrinsically
disordered proteins have nonspecific and specific interactions with the partner.
Kinetic analysis of high-temperature MD simulations shows that bound and
apo-states unfold via a two-state process, respectively. Φ-value analysis can
identify the key residues of intrinsically disordered proteins.
Kolmogorov-Smirnov (KS) P test analysis illustrates that the specific
recognition between intrinsically disordered protein and partner might follow
induced-fit mechanism. Furthermore, these methods can be widely used for the
research of the binding induced folding for intrinsically disordered proteins. The intrinsically disordered protein (IDP) stathmin plays an important
regulatory role in cytoskeletal maintece through its helical binding to
tubulin and microtubules. However, it lacks a stable fold in the absence of its
binding partner. Although stathmin has been a focus of research over the past
two decades, the solution-phase conformational dynamics of this IDP are poorly
understood. It has been reported that stathmin is purely monomeric in solution
and that it bears a short helical region of persistent foldedness, which may act
to nucleate helical folding in the C-terminal direction. Here we report a
comprehensive study of the structural equilibria local to this region in
stathmin that contradicts these two claims. Using the technique of electron
paramagnetic resoce (EPR) spectroscopy on spin-labeled stathmin mutants in
the solution-phase and when immobilized on Sepharose solid support, we show that
all sites in the helical nucleation region of stathmin exhibit multiple spectral
components that correspond to dynamic states of differing mobilities and
stabilities. Importantly, a state with relatively low mobility dominates each
spectrum with an average population greater than 50%, which we suggest
corresponds to an oligomerized state of the protein. This is in contrast to a
less populated, more mobile state, which likely represents a helically folded
monomeric state of stathmin, and a highly mobile state, which we propose is the
random coil conformer of the protein. Our interpretation of the EPR data is
confirmed by further characterization of the protein using the techniques of
native and SDS PAGE, gel filtration chromatography, and multiangle and dynamic
light scattering, all of which show the presence of oligomeric stathmin in
solution. Collectively, these data suggest that stathmin exists in a diverse
equilibrium of states throughout the purported helical nucleation region and
that this IDP exhibits a propensity toward oligomerization. BACKGROUND: Analyzing the amino acid sequence of an intrinsically disordered
protein (IDP) in an evolutionary context can yield novel insights on the
functional role of disordered regions and sequence element(s). However, in the
case of many IDPs, the lack of evolutionary conservation of the primary sequence
can hamper the study of functionality, because the conservation of their
disorder profile and ensuing function(s) may not appear in a traditional
analysis of the evolutionary history of the protein.
RESULTS: Here we present DisCons (Disorder Conservation), a novel pipelined tool
that combines the quantification of sequence- and disorder conservation to
classify disordered residue positions. According to this scheme, the most
interesting categories (for functional purposes) are constrained disordered
residues and flexible disordered residues. The former residues show conservation
of both the sequence and the property of disorder and are associated mainly with
specific binding functionalities (e.g., short, linear motifs, SLiMs), whereas
the latter class correspond to segments where disorder as a feature is important
for function as opposed to the identity of the underlying sequence (e.g.,
entropic chains and linkers). DisCons therefore helps with elucidating the
function(s) arising from the disordered state by analyzing individual proteins
as well as large-scale proteomics datasets.
CONCLUSIONS: DisCons is an openly accessible sequence analysis tool that
identifies and highlights structurally disordered segments of proteins where the
conformational flexibility is conserved across homologs, and therefore
potentially functional. The tool is freely available both as a web application
and as stand-alone source code hosted at http://pedb.vib.be/discons . P granules and other RNA/protein bodies are membrane-less organelles that may
assemble by intracellular phase separation, similar to the condensation of water
vapor into droplets. However, the molecular driving forces and the nature of the
condensed phases remain poorly understood. Here, we show that the Caenorhabditis
elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates
into P granule-like droplets in vitro. We adapt a microrheology technique to
precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets,
revealing purely viscous properties highly tunable by salt and RNA
concentration. RNA decreases viscosity and increases molecular dynamics within
the droplet. Single molecule FRET assays suggest that this RNA fluidization
results from highly dynamic RNA-protein interactions that emerge close to the
droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine
rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and
sufficient for both phase separation and RNA-protein interactions. In vivo, RNAi
knockdown of LAF-1 results in the dissolution of P granules in the early embryo,
with an apparent submicromolar phase boundary comparable to that measured in
vitro. Together, these findings demonstrate that LAF-1 is important for
promoting P granule assembly and provide insight into the mechanism by which
IDP-driven molecular interactions give rise to liquid phase organelles with
tunable properties. Intrinsically disordered proteins (IDPs) are ubiquitously involved in cellular
processes and often implicated in human pathological conditions. The critical
biological roles of these proteins, despite not adopting a well-defined fold,
encouraged structural biologists to revisit their views on the protein
structure-function paradigm. Unfortunately, investigating the characteristics
and describing the structural behavior of IDPs is far from trivial, and
inferring the function(s) of a disordered protein region remains a major
challenge. Computational methods have proven particularly relevant for studying
IDPs: on the sequence level their dependence on distinct characteristics
determined by the local amino acid context makes sequence-based prediction
algorithms viable and reliable tools for large scale analyses, while on the
structure level the in silico integration of fundamentally different
experimental data types is essential to describe the behavior of a flexible
protein chain. Here, we offer an overview of the latest developments and
computational techniques that aim to uncover how protein function is connected
to intrinsic disorder. We show here that chicken gizzard caldesmon (CaD) and its C-terminal domain
(residues 636-771, CaD136) are intrinsically disordered proteins. The
computational and experimental analyses of the wild type CaD136 and series of
its single tryptophan mutants (W674A, W707A, and W737A) and a double tryptophan
mutant (W674A/W707A) suggested that although the interaction of CaD136 with
calmodulin (CaM) can be driven by the non-specific electrostatic attraction
between these oppositely charged molecules, the specificity of CaD136-CaM
binding is likely to be determined by the specific packing of important CaD136
tryptophan residues at the CaD136-CaM interface. It is suggested that this
interaction can be described as the "buttons on a charged string" model, where
the electrostatic attraction between the intrinsically disordered CaD136 and the
CaM is solidified in a "snapping buttons" manner by specific packing of the
CaD136 "pliable buttons" (which are the short segments of fluctuating local
structure condensed around the tryptophan residues) at the CaD136-CaM interface.
Our data also show that all three "buttons" are important for binding, since
mutation of any of the tryptophans affects CaD136-CaM binding and since CaD136
remains CaM-buttoned even when two of the three tryptophans are mutated to
alanines. |
What is the Drosophila melanogaster Groucho protein? | Groucho proteins are abundant and broadly expressed nuclear factors that lack intrinsic DNA-binding activity but can interact with a variety of DNA-binding proteins. The recruitment of Groucho to specific gene regulatory sequences results in transcriptional repression.
Groucho (Gro) is a Drosophila melanogaster transcriptional corepressor. | The proteins termed TLE in humans, Grg in mice and Groucho in Drosophila
constitute a family of transcriptional corepressors. In mammalians there are
five different genes encoding an even larger number of proteins. Interactions
between these TLE/Grg proteins and an array of transcription factors has been
described. But is there any specificity? This review tries to make a case for a
non-redundant function of individual TLE/Grg proteins. The specificity may be
brought about by a tightly controlled temporo-spatial expression pattern,
post-translational modifications, and subtle structural differences leading to
distinct preferences for interacting transcription factors. A confirmation of
this concept will ultimately need to come from genetic experiments. The Groucho/Tle family of corepressor proteins has important roles in
development and in adult tissue in both Protostomes and Deuterostomes. In
Drosophila, a single member of this family has been identified. Unlike in
Protostomes, most Deutrostomes contain more than two full-length Tle genes. In
this study, I analyse the genomic organization and phylogenetic relationship
between the long and short forms of the Groucho/Tle family members in Chordata.
The genomic location and sequence similarities suggest that Aes/Grg5 and
Tle6/Grg6 arose from duplication of the Tle2 gene; each evolved independently
and acquired new functions as negative regulators of the other Tle proteins.
Based on these data, a model for Groucho/Tle gene evolution is proposed. Groucho proteins are abundant and broadly expressed nuclear factors that lack
intrinsic DNA-binding activity but can interact with a variety of DNA-binding
proteins. The recruitment of Groucho to specific gene regulatory sequences
results in transcriptional repression. In both invertebrates and vertebrates,
Groucho family members act as important regulators of several signaling
mechanisms, including the Notch, Wingless/Wnt and Dpp/BMP/TGF-beta signaling
pathways. Recent studies of embryonic development in several species point to an
important role for Groucho in the regulation of multiple patterning and
differentiation events. Moreover, a deregulated expression of human Groucho
family members is correlated with several neoplastic conditions. Here we focus
on the functions of Groucho proteins during body patterning and their
implication in tumorigenesis. The Groucho (Gro)/transducin-like enhancer of split family of transcriptional
corepressors are implicated in many signalling pathways that are important in
development and disease, including those mediated by Notch, Wnt and Hedgehog.
Here, we describe a genetic screen in Drosophila that yielded 50 new gro
alleles, including the first protein-null allele, and has two mutations in the
conserved Q oligomerization domain that have been proposed to have an essential
role in corepressor activity. One of these latter mutations, encoding an
amino-terminal protein truncation that lacks part of the Q domain, abolishes
oligomerization in vitro and renders the protein unstable in vivo. Nevertheless,
the mutation is not a null: maternal mutant embryos have intermediate
segmentation phenotypes and relatively normal terminal patterning suggesting
that the mutant protein retains partial corepressor activity. Our results show
that homo-oligomerization of Gro is not obligatory for its action in vivo, and
that Gro represses transcription through more than one molecular mechanism. The Drosophila Groucho (Gro) protein was the founding member of the family of
transcriptional co-repressor proteins that now includes the transducin-like
enhancer of split (TLE) and Grorelated gene (Grg) proteins in vertebrates. Gro
family proteins do not bind DNA directly, but are recruited by a diverse profile
of transcription factors, including members of the Hes, Runx, Nkx, LEF1/Tcf,
Pax, Six and c-Myc families. The primary structure of Gro proteins includes five
identifiable regions, of which the most highly conserved are the amino-terminal
glutamine-rich Q domain and the carboxy-terminal WD-repeat domain. The Q domain
contains two coiled-coil motifs that facilitate oligomerization into tetramers
and binding to some transcription factors. The WD domain folds to form a
beta-propeller, which mediates protein-protein interactions. Many transcription
factors interact with the WD domain via a short peptide motif that falls into
either of two classes: WRPW and related tetrapeptides; and the 'eh1' motif
(FxIxxIL). Gro family proteins are broadly expressed during development and in
the adult. They have essential functions in many developmental pathways
(including Notch and Wnt signaling) and are implicated in the pathogenesis of
some cancers. The molecular mechanisms through which Gro proteins act to repress
transcription are not yet well understood. It is becoming clear that Gro
proteins have different modes of action in vivo dependent on biological context
and these include direct and indirect modification of chromatin structure at
target genes. BACKGROUND: Transcriptional co-repressors of the Groucho/transducin-like
Enhancer of split (Gro/TLE) family regulate the expression of a variety of genes
and are involved in numerous developmental processes in both invertebrate and
vertebrate species. More specifically, Gro/TLE1 participates in mechanisms that
inhibit/delay the differentiation of cerebral cortex neural progenitor cells
into neurons during mammalian forebrain development. The anti-neurogenic
function of Gro/TLE1 depends on the formation of protein complexes with specific
DNA-binding transcription factors that engage Gro/TLE1 through WRP(W/Y)
sequences. Interaction with those transcription partners results in Gro/TLE1
recruitment to selected DNA sites and causes increased Gro/TLE1 phosphorylation.
The physiological significance of the latter event, termed "cofactor-activated
phosphorylation," had not been determined. Therefore, this study aimed at
clarifying the role of cofactor-activated phosphorylation in the anti-neurogenic
function of Gro/TLE1.
METHODS AND PRINCIPAL FINDINGS: A combination of site-directed mutagenesis, mass
spectrometry, biochemistry, primary cell culture, and immunocytochemical assays
was utilized to characterize point mutations of Ser-286, a residue that is
phosphorylated in vivo and is located within the serine/proline-rich (SP) domain
of Gro/TLE1. Mutation of Ser-286 to alanine or glutamic acid does not perturb
the interaction of Gro/TLE1 with DNA-binding partners, including the basic
helix-loop-helix transcription factor Hes1, a prototypical anti-neurogenic
WRP(W/Y) motif protein. Ser-286 mutations do not prevent the recruitment of
Gro/TLE1 to DNA, but they impair cofactor-activated phosphorylation and weaken
the interaction of Gro/TLE1 with chromatin. These effects are correlated with an
impairment of the anti-neurogenic activity of Gro/TLE1. Similar results were
obtained when mutations of Ser-289 and Ser-298, which are also located within
the SP domain of Gro/TLE1, were analyzed.
CONCLUSION: Based on the positive correlation between Gro/TLE1
cofactor-activated phosphorylation and ability to inhibit cortical neuron
differentiation, we propose that hyperphosphorylation induced by cofactor
binding plays a positive role in the regulation of Gro/TLE1 anti-neurogenic
activity. Groucho (Gro) is a Drosophila melanogaster transcriptional corepressor that
directly interacts with the histone deacetylase Rpd3. Although previous studies
suggest that this interaction is required for repression of Gro-responsive
reporters in cultured cells, the in vivo significance of this interaction and
the mechanism by which it leads to repression remain largely unexplored. In this
study, we show that Gro is partially dependent on Rpd3 for repression,
supporting the idea that Rpd3-mediated repression is one mode of Gro-mediated
repression. We demonstrate that Gro colocalizes with Rpd3 to the chromatin of a
target gene and that this is accompanied by the deacetylation of specific
lysines within the N-terminal tails of histones H3 and H4. Gro overexpression
leads to wing patterning defects and ectopic repression in the wing disc of
transcription directed by the vestigial quadrant enhancer. These effects are
reversed by the histone deacetylase inhibitors TSA and HC-Toxin and by the
reduction of Rpd3 gene dosage. Furthermore, repression of the vestigial quadrant
enhancer is accompanied by a Gro-mediated increase in nucleosome density, an
effect that is reversed by histone deacetylase inhibitors. We propose a model in
which Gro-mediated histone deacetylation results in increased nucleosome density
leading to transcriptional repression. Ephrin signaling is involved in various morphogenetic events, such as axon
guidance, hindbrain segmentation, and angiogenesis. We conducted a yeast
two-hybrid screen using the intracellular domain (ICD) of EphrinB1 to gain
biochemical insightinto the function of the EphrinB1 ICD. We identified the
transcriptional co-repressor xTLE1/Groucho as an EphrinB1 interacting protein.
Whole-mount in situ hybridization of Xenopus embryos confirmed the
co-localization of EphrinB1 and a Xenopus counterpart to TLE1, xTLE4, during
various stages of development. The EphrinB1/xTLE4 interaction was confirmed by
co-immunoprecipitation experiments. Further characterization of the interaction
revealed that the carboxy-terminal PDZ binding motif of EphrinB1 and the SP
domain of xTLE4 are required for binding. Additionally, phosphorylation of
EphrinB1 by a constitutively activated fibroblast growth factor receptor
resulted in loss of the interaction, suggesting that the interaction is
modulated by tyrosine phosphorylation of the EphrinB1 ICD. The caudal homeobox (cdx) gene family is critical for specification of caudal
body formation and erythropoiesis. In zebrafish, cdx4 expression is controlled
by the Wnt pathway, but the molecular mechanism of this regulation is not fully
understood. Here, we provide evidence that Tcf3 suppresses cdx4 expression
through direct binding to multiple sites in the cdx4 gene regulatory region.
Tcf3 requires corepressor molecules such as Groucho (Gro)/TLE and HDAC1 for
activity. Using zebrafish embryos and cultured mammalian cells, we show that the
transcription factor E4f1 derepresses cdx4 by dissociating corepressor proteins
from Tcf3 without inhibiting its binding to cis-regulatory sites in the DNA.
Further, the E3 ubiquitin ligase Lnx2b, acting as a scaffold protein
irrespective of its enzymatic activity, counteracts the effects of E4f1. We
propose that the modulation of Tcf3 repressor function by E4f1 assures precise
and robust regulation of cdx4 expression in the caudal domain of the embryo. Drosophila Groucho (Gro) is the founding member of a family of metazoan
corepressors. Gro mediates repression through interactions with a myriad of
DNA-binding repressor proteins to direct the silencing of genes involved in many
developmental processes, including neurogenesis and patterning of the main body
axis, as well as receptor tyrosine kinase/Ras/MAPK, Notch, Wingless (Wg)/Wnt,
and Decapentaplegic (Dpp) signaling. Gro mediates repression by multiple
molecular mechanisms, depending on the regulatory context. Because Gro is a
broadly expressed nuclear factor, whereas its repressor partners display
restricted temporal and spatial distribution, it was presumed that this
corepressor played permissive rather than instructive roles in development.
However, a wide range of studies demonstrates that this is not the case. Gro can
sense and integrate many cellular inputs to modulate the expression of variety
of genes, making it a versatile corepressor with crucial instructive roles in
development and signaling. Groucho (Gro) is a Drosophila corepressor required by numerous DNA-binding
repressors, many of which are distributed in gradients and provide positional
information during development. Gro contains well-conserved domains at its N-
and C-termini, and a poorly conserved central region that includes the GP, CcN,
and SP domains. All lethal point mutations in gro map to the conserved regions,
leading to speculation that the unconserved central domains are dispensable.
However, our sequence analysis suggests that the central domains are disordered
leading us to suspect that the lack of lethal mutations in this region reflects
a lack of order rather than an absence of essential functions. In support of
this conclusion, genomic rescue experiments with Gro deletion variants
demonstrate that the GP and CcN domains are required for viability.
Misexpression assays using these same deletion variants show that the SP domain
prevents unrestrained and promiscuous repression by Gro, while the GP and CcN
domains are indispensable for repression. Deletion of the GP domain leads to
loss of nuclear import, while deletion of the CcN domain leads to complete loss
of repression. Changes in Gro activity levels reset the threshold concentrations
at which graded repressors silence target gene expression. We conclude that
co-regulators such as Gro are not simply permissive components of the repression
machinery, but cooperate with graded DNA-binding factors in setting borders of
gene expression. We suspect that disorder in the Gro central domains may provide
the flexibility that allows this region to mediate multiple interactions
required for repression. |
Which enzyme is inhibited by Imetelstat? | Imetelstat works by inhibiting telomerase. | Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to
be responsible for the maintece and recurrence of cancer and metastasis.
Telomerase is constitutively active in both bulk tumor cell and CSC populations
but has only limited expression in normal tissues. Thus, inhibition of
telomerase has been shown to be a viable approach in controlling cancer growth
in nonclinical studies and is currently in phase II clinical trials. In this
study, we investigated the effects of imetelstat (GRN163L), a potent telomerase
inhibitor, on both the bulk cancer cells and putative CSCs. When breast and
pancreatic cancer cell lines were treated with imetelstat in vitro, telomerase
activity in the bulk tumor cells and CSC subpopulations were inhibited.
Additionally, imetelstat treatment reduced the CSC fractions present in the
breast and pancreatic cell lines. In vitro treatment with imetelstat, but not
control oligonucleotides, also reduced the proliferation and self-renewal
potential of MCF7 mammospheres and resulted in cell death after <4 weeks of
treatment. In vitro treatment of PANC1 cells showed reduced tumor engraftment in
nude mice, concomitant with a reduction in the CSC levels. Differences between
telomerase activity expression levels or telomere length of CSCs and bulk tumor
cells in these cell lines did not correlate with the increased sensitivity of
CSCs to imetelstat, suggesting a mechanism of action independent of telomere
shortening for the effects of imetelstat on the CSC subpopulations. Our results
suggest that imetelstat-mediated depletion of CSCs may offer an alternative
mechanism by which telomerase inhibition may be exploited for cancer therapy. Telomerase is a cellular ribonucleoprotein reverse transcriptase that plays a
crucial role in telomere maintece. This enzyme is expressed in approximately
90% of human tumors, but not in the majority of normal somatic cells. imetelstat
sodium (GRN163L), is a 13-mer oligonucleotide N3'→P5' thio-phosphoramidate lipid
conjugate, which represents the latest generation of telomerase inhibitors
targeting the template region of the human functional telomerase RNA (hTR)
subunit. In preclinical trials, this compound has been found to inhibit
telomerase activity in multiple cancer cell lines, as well as in vivo xenograft
mouse models. Currently, GRN163L is being investigated in several clinical
trials, including a phase II human non‑small cell lung cancer clinical trial, in
a maintece setting following standard doublet chemotherapy. In addition to
the inhibition of telomerase activity in cancer cell lines, GRN163L causes
morphological cell rounding changes, independent of hTR expression or telomere
length. This leads to the loss of cell adhesion properties; however, the
mechanism underlying this effect is not yet fully understood. In the present
study, we observed that GRN163L treatment leads to the loss of adhesion in A549
lung cancer cells, due to decreased E-cadherin expression, leading to the
disruption of the cytoskeleton through the alteration of actin, tubulin and
intermediate filament organization. Consequently, the less adherent cancer cells
initially cease to proliferate and are arrested in the G1 phase of the cell
cycle, accompanied by decreased matrix metalloproteinase-2 (MMP-2) expression.
These effects of GRN163L are independent of its telomerase catalytic activity
and may increase the therapeutic efficacy of GRN163L by decreasing the adhesion,
proliferation and metastatic potential of cancer cells in vivo. Telomerase is required for the unlimited lifespan of cancer cells. The vast
majority of pancreatic adenocarcinomas overexpress telomerase activity and
blocking telomerase could limit their lifespan. GRN163L (Imetelstat) is a
lipid-conjugated N3'→P5' thio-phosphoramidate oligonucleotide that blocks the
template region of telomerase. The aim of this study was to define the effects
of long-term GRN163L exposure on the maintece of telomeres and lifespan of
pancreatic cancer cells. Telomere size, telomerase activity, and telomerase
inhibition response to GRN163L were measured in a panel of 10 pancreatic cancer
cell lines. The cell lines exhibited large differences in levels of telomerase
activity (46-fold variation), but most lines had very short telomeres (2-3 kb in
size). GRN163L inhibited telomerase in all 10 pancreatic cancer cell lines, with
IC50 ranging from 50 nM to 200 nM. Continuous GRN163L exposure of CAPAN1
(IC50 = 75 nM) and CD18 cells (IC50 = 204 nM) resulted in an initial rapid
shortening of the telomeres followed by the maintece of extremely short but
stable telomeres. Continuous exposure to the drug eventually led to crisis and
to a complete loss of viability after 47 (CAPAN1) and 69 (CD18) doublings.
Crisis In these cells was accompanied by activation of a DNA damage response
(γ-H2AX) and evidence of both senescence (SA-β-galactosidase activity) and
apoptosis (sub-G1 DNA content, PARP cleavage). Removal of the drug after
long-term GRN163L exposure led to a reactivation of telomerase and re-elongation
of telomeres in the third week of cultivation without GRN163L. These findings
show that the lifespan of pancreatic cancer cells can be limited by continuous
telomerase inhibition. These results should facilitate the design of future
clinical trials of GRN163L in patients with pancreatic cancer. Pediatric ependymomas are highly recurrent tumors resistant to conventional
chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless
replication, has been found to be critically important for the maintece of
tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the
tumor from which they are identified, and are drivers of recurrence in numerous
cancers. In this study, telomerase enzymatic activity was directly measured and
inhibited to assess the therapeutic potential of targeting telomerase.
Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle
assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas
to determine the prevalence and prognostic potential of telomerase activity or
alternative lengthening of telomeres (ALT) as telomere maintece mechanisms,
respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate
the effect of telomerase inhibition on proliferation and tumorigenicity in
established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and
xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas
were found to rely on telomerase activity to maintain telomeres, while no
ependymomas showed evidence of ALT. Children with telomerase-active tumors had
reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and
overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with
tumors lacking telomerase activity. Imetelstat inhibited proliferation and
self-renewal by shortening telomeres and inducing senescence in vitro. In vivo,
Imetelstat significantly reduced subcutaneous xenograft growth by 40 %
(p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma
TICs in an orthotopic xenograft model. Telomerase inhibition represents a
promising therapeutic approach for telomerase-active pediatric ependymomas found
to characterize high-risk ependymomas. Novel treatment approaches are desperately needed for maligt rhabdoid tumor
(MRT). Telomerase is an attractive therapeutic target because it is specific to
cancer and critical for cancer cell immortality. We evaluated the effect of the
telomerase inhibitor imetelstat in preclinical models of MRT. Three MRT cell
lines, BT-12, G401, and RT-peri, were treated with the telomerase inhibitor
imetelstat. The effects of imetelstat on telomere length, DNA damage response,
and cell proliferation were assessed. The efficacy of imetelstat in vivo was
evaluated in subcutaneous xenografts derived from each of the cell lines.
Treatment with imetelstat resulted in inhibition of telomerase activity, marked
telomere shortening, and activation of the DNA damage response pathway, as
measured by formation of γ-H2AX nuclear foci, phosphorylation of ATM, and
phosphorylation of TP53. Imetelstat-treated G401 cells underwent complete growth
arrest after 16 passages. The other two cell lines exhibited growth inhibition.
Imetelstat resulted in 40-50% growth inhibition compared to placebo-treated
controls in all three xenograft models. The activity of imetelstat as a single
agent suggests that further studies of telomerase inhibitors in combination with
other agents may be warranted. Cancer stem cells (CSCs) are thought to be responsible for tumor progression,
metastasis, and recurrence. HER2 overexpression is associated with increased
CSCs, which may explain the aggressive phenotype and increased likelihood of
recurrence for HER2(+) breast cancers. Telomerase is reactivated in tumor cells,
including CSCs, but has limited activity in normal tissues, providing potential
for telomerase inhibition in anti-cancer therapy. The purpose of this study was
to investigate the effects of a telomerase antagonistic oligonucleotide,
imetelstat (GRN163L), on CSC and non-CSC populations of HER2(+) breast cancer
cell lines. The effects of imetelstat on CSC populations of HER2(+) breast
cancer cells were measured by ALDH activity and CD44/24 expression by flow
cytometry as well as mammosphere assays for functionality. Combination studies
in vitro and in vivo were utilized to test for synergism between imetelstat and
trastuzumab. Imetelstat inhibited telomerase activity in both subpopulations.
Moreover, imetelstat alone and in combination with trastuzumab reduced the CSC
fraction and inhibited CSC functional ability, as shown by decreased mammosphere
counts and invasive potential. Tumor growth rate was slower in
combination-treated mice compared to either drug alone. Additionally, there was
a trend toward decreased CSC marker expression in imetelstat-treated xenograft
cells compared to vehicle control. Furthermore, the observed decrease in CSC
marker expression occurred prior to and after telomere shortening, suggesting
that imetelstat acts on the CSC subpopulation in telomere length-dependent and
-independent mechanisms. Our study suggests addition of imetelstat to
trastuzumab may enhance the effects of HER2 inhibition therapy, especially in
the CSC population. |
What is the catalytic mechanism of DNA (cytosine-5) methyltransferases? | The catalytic mechanism of the DNA (Cytosine-5-)-methyltransferase involves nucleophilic attack of the C6 of the substrate cytosine by the single conserved cysteine of the enzyme, followed by C5 nucleophilic replacement of the methyl group of the cofactor S-adenosyl-L-methionine (AdoMet) to produce 5-methyl-6-Cys-81-S-5,6-dihydrocytosine. It has been also demonstrated that Phe and Glu, which are found in the catalytic motifs I and II of the enzyme are important for AdoMet binding and catalysis. | 2'-Deoxyoligonucleotides with 5-fluorocytosine residues incorporated at specific
positions of the nucleotide sequence are tools of great potential in the study
of the catalytic mechanism by which DNA cytosine methyltransferases methylate
the 5-position of DNA cytosine residues in specific sequence contexts. Chemical
synthesis of such oligonucleotides is described. Two alternative approaches have
been developed, one of which proceeds via a fully protected phosphoramidite of
5-fluoro-4-methylmercapto-2'-deoxyuridine 2, the other via a fully protected
phosphoramidite of 5-fluoro-2'-deoxycytidine 3. Either building block can be
used in automated oligonucleotide synthesis applying standard elongation cycles
and deprotection procedures exclusively. The methylmercapto function of 2 is
replaced by an amino group in the final ammonia treatment used for cleavage from
support and base deprotection. Kinetic and catalytic properties of the DNA (cytosine-5)-methyltransferase HhaI
are described. With poly(dG-dC) as substrate, the reaction proceeds by an
equilibrium (or processive) ordered Bi-Bi mechanism in which DNA binds to the
enzyme first, followed by S-adenosylmethionine (AdoMet). After methyl transfer,
S-adenosylhomocysteine (AdoHcy) dissociates followed by methylated DNA. AdoHcy
is a potent competitive inhibitor with respect to AdoMet (Ki = 2.0 microM) and
its generation during reactions results in non-linear kinetics. AdoMet and
AdoHcy significantly interact with only the substrate enzyme-DNA complex; they
do not bind to free enzyme and bind poorly to the methylated enzyme-DNA complex.
In the absence of AdoMet, HhaI methylase catalyzes exchange of the 5-H of
substrate cytosines for protons of water at about 7-fold the rate of
methylation. The 5-H exchange reaction is inhibited by AdoMet or AdoHcy. In the
enzyme-DNA-AdoHcy complex, AdoHcy also suppresses dissociation of DNA and
reassociation of the enzyme with other substrate sequences. Our studies reveal
that the catalytic mechanism of DNA (cytosine-5)-methyltransferases involves
attack of the C6 of substrate cytosines by an enzyme nucleophile and formation
of a transient covalent adduct. Based on precedents of other enzymes which
catalyze similar reactions and the susceptibility of HhaI to inactivation by
N-ethylmaleimide, we propose that the sulfhydryl group of a cysteine residue is
the nucleophilic catalyst. Furthermore, we propose that Cys-81 is the
active-site catalyst in HhaI. This residue is found in a Pro-Cys doublet which
is conserved in all DNA (cytosine-5)-methyltransferases whose sequences have
been determined to date and is found in related enzymes. Finally, we discuss the
possibility that covalent adducts between C6 of pyrimidines and nucleophiles of
proteins may be important general components of protein-nucleic acid
interactions. A DNA methyltransferase was partially purified from bovine thymus heavy cells.
The enzyme has Mr 130 000, and introduces methyl groups from
S-adenosylmethionine into the 5 position of cytosines in DNA. Sequence
specificity analysis revealed that about 60% of the total methylation occurred
in the 5'd(C-G)3' doublet. Single-stranded and hemi-methylated DNAs were
methylated at an elevated rate by the enzyme. The kinetic analysis showed that
the reaction obeys a random sequential mechanism. These results suggest that the
enzyme serves primarily as a maintece DNA methyltransferase. Previous X-ray crystallographic studies have revealed that the catalytic domain
of a DNA methyltransferase (Mtase) generating C5-methylcytosine bears a striking
structural similarity to that of a Mtase generating N6-methyladenine. Guided by
this common structure, we performed a multiple sequence alignment of 42
amino-Mtases (N6-adenine and N4-cytosine). This comparison revealed nine
conserved motifs, corresponding to the motifs I to VIII and X previously defined
in C5-cytosine Mtases. The amino and C5-cytosine Mtases thus appear to be more
closely related than has been appreciated. The amino Mtases could be divided
into three groups, based on the sequential order of motifs, and this variation
in order may explain why only two motifs were previously recognized in the amino
Mtases. The Mtases grouped in this way show several other group-specific
properties, including differences in amino acid sequence, molecular mass and DNA
sequence specificity. Surprisingly, the N4-cytosine and N6-adenine Mtases do not
form separate groups. These results have implications for the catalytic
mechanisms, evolution and diversification of this family of enzymes.
Furthermore, a comparative analysis of the S-adenosyl-L-methionine and
adenine/cytosine binding pockets suggests that, structurally and functionally,
they are remarkably similar to one another. All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It
has been proposed that this cysteine initiates catalysis by attacking the C6 of
cytosine and thereby activating the normally inert C5 position. We show here
that substitutions of this cysteine in the E. coli methylase M. EcoRII with
either serine or tryptophan results in a complete loss of ability to transfer
methyl groups to DNA. Interestingly, mutants with either serine or glycine
substitution bind tightly to substrate DNA. These mutants resemble the wild-type
enzyme in that their binding to substrate is not eliminated by the presence of
non-specific DNA in the reaction, it is sensitive to methylation status of the
substrate and is stimulated by an analog of the methyl donor. Hence the
conserved cysteine is not essential for the specific stable binding of the
enzyme to its substrate. However, substitution of the cysteine with the bulkier
tryptophan does reduce DNA binding. We also report here a novel procedure for
the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA
substrate for M. EcoRII in which the target cytosine is replaced by
5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms
an irreversible complex with the enzyme. As expected, this modified substrate
does not form irreversible complexes with the mutants. The product of the dcm gene is the only DNA cytosine-C5 methyltransferase of
Escherichia coli K-12; it catalyses transfer of a methyl group from S-adenosyl
methionine (SAM) to the C-5 position of the inner cytosine residue of the
cognate sequence CCA/TGG. Sequence-specific, covalent crosslinking of the enzyme
to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine is
demonstrated. This reaction is abolished if serine replaces the cysteine at
residue #177 of the enzyme. These results lend strong support to a catalytic
mechanism in which an enzyme sulfhydryl group undergoes Michael addition to the
C5-C6 double bond, thus activating position C-5 of the substrate DNA cytosine
residue for electrophilic attack by the methyl donor SAM. The enzyme is capable
of self-methylation in a DNA-independent reaction requiring SAM and the presence
of cysteine at position #177. We have determined the structure of Pvu II methyltransferase (M. Pvu II)
complexed with S -adenosyl-L-methionine (AdoMet) by multiwavelength anomalous
diffraction, using a crystal of the selenomethionine-substituted protein. M. Pvu
II catalyzes transfer of the methyl group from AdoMet to the exocyclic amino
(N4) nitrogen of the central cytosine in its recognition sequence 5'-CAGCTG-3'.
The protein is dominated by an open alpha/beta-sheet structure with a prominent
V-shaped cleft: AdoMet and catalytic amino acids are located at the bottom of
this cleft. The size and the basic nature of the cleft are consistent with
duplex DNA binding. The target (methylatable) cytosine, if flipped out of the
double helical DNA as seen for DNA methyltransferases that generate
5-methylcytosine, would fit into the concave active site next to the AdoMet.
This M. Pvu IIalpha/beta-sheet structure is very similar to those of M. Hha I (a
cytosine C5 methyltransferase) and M. Taq I (an adenine N6 methyltransferase),
consistent with a model predicting that DNA methyltransferases share a common
structural fold while having the major functional regions permuted into three
distinct linear orders. The main feature of the common fold is a seven-stranded
beta-sheet (6 7 5 4 1 2 3) formed by five parallel beta-strands and an
antiparallel beta-hairpin. The beta-sheet is flanked by six parallel
alpha-helices, three on each side. The AdoMet binding site is located at the
C-terminal ends of strands beta1 and beta2 and the active site is at the
C-terminal ends of strands beta4 and beta5 and the N-terminal end of strand
beta7. The AdoMet-protein interactions are almost identical among M. Pvu II, M.
Hha I and M. Taq I, as well as in an RNA methyltransferase and at least one
small molecule methyltransferase. The structural similarity among the active
sites of M. Pvu II, M. Taq I and M. Hha I reveals that catalytic amino acids
essential for cytosine N4 and adenine N6 methylation coincide spatially with
those for cytosine C5 methylation, suggesting a mechanism for amino methylation. Most prokaryotic (cytosine-5)-DNA methyltransferases increase the frequency of
deamination at the cytosine targeted for methylation in vitro in the absence of
the cofactor S-adenosylmethionine (AdoMet) or the reaction product
S-adenosylhomocysteine (AdoHcy). We show here that, under the same in vitro
conditions, the prokaryotic methyltransferase, M.MspI (from Moraxella sp.),
causes very few cytosine deaminations, suggesting a mechanism in which M.MspI
may avoid enzyme-mediated cytosine deamination. Two analogues of AdoMet,
sinefungin and 5'-amino-5'-deoxyadenosine, greatly increased the frequency of
cytosine deamination mediated by M.MspI presumably by introducing a
proton-donating amino group into the catalytic centre, thus facilitating the
formation of an unstable enzyme-dihydrocytosine intermediate and hydrolytic
deamination. Interestingly, two naturally occurring analogues, adenosine and
5'-methylthio-5'-deoxyadenosine, which do not contain a proton-donating amino
group, also weakly increased the deamination frequency by M.MspI, even in the
presence of AdoMet or AdoHcy. These analogues may trigger a conformational
change in the enzyme without completely inhibiting the access of solvent water
to the catalytic centre, thus allowing hydrolytic deamination of the
enzyme-dihydrocytosine intermediate. Under normal physiological conditions the
enzymes M.HpaII (from Haemophilus parainfluenzae), M. HhaI (from Haemophilus
hemolytica) and M.MspI all increased the in vivo deamination frequency at the
target cytosines with comparable efficiency. The enzymes that transfer a methyl group to C5 of cytosine within specific
sequences (C5 Mtases) deaminate the target cytosine to uracil if the methyl
donor S-adenosylmethionine (SAM) is omitted from the reaction. Recently, it was
shown that cytosine deamination caused by C5 Mtases M.HpaII, M.SssI and M.MspI
is enhanced in the presence of several analogues of SAM, and a mechanism for
this analogue-promoted deamination was proposed. According to this mechanism,
the analogues protonate C5 of the target cytosine, creating a dihydrocytosine
intermediate that is susceptible to deamination. We show here that one of these
analogues, 5'-aminoadenosine (AA), enhances cytosine deamination by the Mtase M.
EcoRII, but it does so without enhancing protonation of C5. Further, we show
that uracil is an intermediate in the mutational pathway and propose an
alternate mechanism for the analogue-promoted deamination. The new mechanism
involves a facilitated water attack at C4 but does not require attack at C6 by
the enzyme. The latter feature of the mechanism was tested by using M.EcoRII
mutants defective in the nucleophilic attack at C6 in the deamination assay. We
find that although these proteins are defective in methyl transfer and cytosine
deamination, they cause cytosine deaminations in the presence of AA in the
reaction. Our results point to a possible connection between the catalytic
mechanism of C5 Mtases and of enzymes that transfer methyl groups to N(4) of
cytosine. Further, they provide an unusual example where a coenzyme activates an
otherwise "dead" enzyme to perform catalysis by a new reaction pathway. Co-transfections of reporter plasmids and plasmids encoding the catalytic domain
of the murine Dnmt3a DNA methyltransferase lead to inhibition of reporter gene
expression. As Dnmt3a mutants with C-->A and E-->A exchanges in the conserved
PCQ and ENV motifs in the catalytic center of the enzyme also cause repression,
we checked for their catalytic activity in vitro. Surprisingly, the activity of
the cysteine variant and of the corresponding full-length Dnmt3a variant is only
two to sixfold reduced with respect to wild-type Dnmt3a. In contrast, enzyme
variants carrying E-->A, E-->D or E-->Q exchanges of the ENV glutamate are
catalytically almost inactive, demonstrating that this residue has a central
function in catalysis. Since the glutamic acid residue contacts the flipped
base, its main function could be to hold the target base at a position that
supports methyl group transfer. Whereas wild-type Dnmt3a and the ENV variants
form covalent complexes with 5-fluorocytidine modified DNA, the PCN variant does
not. Therefore, covalent complex formation is not essential in the reaction
mechanism of Dnmt3a. We propose that correct positioning of the flipped base and
the cofactor and binding to the transition state of methyl group transfer are
the most important roles of the Dnmt3a enzyme in the catalytic cycle of methyl
group transfer. On the basis of amino acid sequence alignments and structural data of related
enzymes, we have performed a mutational analysis of 14 amino acid residues in
the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase.
The target residues are located within the ten conserved amino acid sequence
motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA
recognition domain of the enzyme (TRD). Mutant proteins were purified and tested
for their catalytic properties and their abilities to bind DNA and AdoMet. We
prepared a structural model of Dnmt3a to interpret our results. We demonstrate
that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and
catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity
under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating
that the contact of Asp96 to AdoMet is not required for catalysis. R130A
(following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both
located in front of motif X) showed reduced DNA binding. R130A displayed a
strong reduction in catalytic activity and a complete change in flanking
sequence preferences, indicating that Arg130 has an important role in the DNA
interaction of Dnmt3a. R292A also displayed reduced activity and changes in the
flanking sequence preferences, indicating a potential role in DNA contacts
farther away from the CG target site. N167A (motif VI) and R202A (motif VIII)
have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167
might contribute to the positioning of residues from motif VI, according to
structural data Arg202 has a role in catalysis of cytosine-C5
methyltransferases. The R295A variant was catalytically inactive most likely
because of destabilization of the hinge sub-domain of the protein. DNA damage caused by the binding of the tumorigen 7R,8S-diol 9S,10R-epoxide
(B[a]PDE), a metabolite of bezo[a]pyrene, to guanine in CpG dinucleotide
sequences could affect DNA methylation and, thus, represent a potential
epigenetic mechanism of chemical carcinogenesis. In this work, we investigated
the impact of stereoisomeric (+)- and (-)-trans-anti-B[a]P-N(2)-dG adducts (B(+)
and B(-)) on DNA methylation by prokaryotic DNA methyltransferases M.SssI and
M.HhaI. These two methyltransferases recognize CpG and GCGC sequences,
respectively, and transfer a methyl group to the C5 atom of cytosine (C). A
series of 18-mer unmethylated or hemimethylated oligodeoxynucleotide duplexes
containing trans-anti-B[a]P-N(2)-dG adducts was generated. The B(+) or B(-)
residues were introduced either 5' or 3' adjacent or opposite to the target
2'-deoxycytidines. The B[a]PDE lesions practically produced no effect on M.SssI
binding to DNA but reduced M.HhaI binding by 1-2 orders of magnitude. In most
cases, the benzo[a]pyrenyl residues decreased the methylation efficiency of
hemimethylated and unmethylated DNA by M.SssI and M.HhaI. An absence of the
methylation of hemimethylated duplexes was observed when either the (+)- or the
(-)-trans-anti-B[a]P-N(2)-dG adduct was positioned 5' to the target dC. The
effects observed may be related to the minor groove conformation of the bulky
benzo[a]pyrenyl residue and to a perturbation of the normal contacts of the
methyltransferase catalytic loop with the B[a]PDE-modified DNA. Our results
indicate that a trans-anti-B[a]P-N(2)-dG lesion flanking a target dC in the CpG
dinucleotide sequence on its 5'-side has a greater adverse impact on methylation
than the same lesion when it is 3' adjacent or opposite to the target dC. Arg165 forms part of a previously identified base flipping motif in the
bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala
has no detectable effect on either DNA or AdoMet affinity, yet causes the base
flipping and restacking transitions to be decreased approximately 16 and
190-fold respectively, thus confirming the importance of this motif. However,
these kinetic changes cannot account for the mutant's observed 10(5)-fold
decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure
(2.79 A resolution) shows the target cytosine to be positioned approximately 30
degrees into the major groove, which is consistent with a major groove pathway
for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to
approximately +171 degrees, from a range of -95 degrees to -120 degrees in B
DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for
maintaining the cytosine positioned for nucleophilic attack by Cys81. The
cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in
contrast to the previously reported C3'-endo (North conformation) described for
the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex.
We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 A)
to better determine the sugar pucker. This new structure is similar to the
original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is
O4'-endo (East conformation), intermediate between the South and North
conformers. In summary, Arg165 plays significant roles in base flipping,
cytosine positioning, and catalysis. Furthermore, the previously proposed
M.HhaI-mediated changes in sugar pucker may not be an important contributor to
the base flipping mechanism. These results provide insights into the base
flipping and catalytic mechanisms for bacterial and eukaryotic DNA
methyltransferases. Although their amino acid sequences and structure closely resemble DNA
methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to
function as RNA methyltransferases transferring a methyl group to the C5
position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA
isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium
discoideum. RNA extracted from wild type D. melanogaster was methylated to a
lower degree, but in the case of Dictyostelium, there was no difference in the
methylation of RNA isolated from wild-type and Dnmt2 knock-out strains.
Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of
DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA
methyltransferase-like mechanism, because similar residues from motifs IV, VI,
and VIII are involved in catalysis as identified in DNA methyltransferases. In
addition, exchange of C292, which is located in a CFT motif conserved among
Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2
represents the first example of an RNA methyltransferase using a DNA
methyltransferase type of mechanism. Maintece of genomic methylation patterns is mediated primarily by DNA
methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1
composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase
domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically
binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker
between the DNA and the active site of DNMT1, preventing de novo methylation. In
addition, a loop projecting from BAH2 interacts with the target recognition
domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted
position and preventing it from inserting into the DNA major groove. Our studies
identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides
are occluded from the active site to ensure that only hemimethylated CpG
dinucleotides undergo methylation. DNA cytosine methyltransferases regulate the expression of the genome through
the precise epigenetic marking of certain cytosines with a methyl group, and
aberrant methylation is a hallmark of human diseases including cancer. Targeting
these enzymes for drug design is currently a high priority. We have utilized ab
initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD)
simulations to investigate extensively the reaction mechanism of the
representative DNA methyltransferase HhaI (M.HhaI) from prokaryotes, whose
overall mechanism is shared with the mammalian enzymes. We obtain for the first
time full free energy profiles for the complete reaction, together with reaction
dynamics in atomistic detail. Our results show an energetically preferred
mechanism in which nucleophilic attack of cytosine C5 on the
S-adenosyl-L-methionine (AdoMet) methyl group is concerted with formation of the
Michael adduct between a conserved Cys in the active site with cytosine C6.
Spontaneous and reversible proton transfer between a conserved Glu in the active
site and cytosine N3 at the transition state was observed in our simulations,
revealing the chemical participation of this Glu residue in the catalytic
mechanism. Subsequently, the β-elimination of the C5 proton utilizes as base an
OH(-) derived from a conserved crystal water that is part of a proton wire water
channel, and this syn β-elimination reaction is the rate-limiting step. Design
of novel cytosine methylation inhibitors would be advanced by our structural and
thermodynamic characterization of the reaction mechanism. |
Are mutations in the STXBP1 gene associated with epilepsy? | Yes,mutations in STXBP1 gene, encoding the syntaxin binding protein 1, have been recently described in Ohtahara syndrome, or early infantile epileptic encephalopathy with suppression-burst pattern, and in other early-onset epileptic encephalopathies. | We sequenced genes coding for components of the SNARE complex (STX1A, VAMP2,
SNAP25) and their regulatory proteins (STXBP1/Munc18-1, SYT1), which are
essential for neurotransmission, in 95 patients with idiopathic mental
retardation. We identified de novo mutations in STXBP1 (nonsense, p.R388X;
splicing, c.169+1G>A) in two patients with severe mental retardation and
nonsyndromic epilepsy. Reverse transcriptase polymerase chain reaction and
sequencing showed that the splicing mutation creates a stop codon downstream of
exon-3. No de novo or deleterious mutations in STXBP1 were found in 190 control
subjects, or in 142 autistic patients. These results suggest that STXBP1
disruption is associated with autosomal domit mental retardation and
nonsyndromic epilepsy. A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of
four individuals (group A) with early-onset West syndrome, severe
hypomyelination, poor visual attention, and developmental delay. Although
haploinsufficiency of STXBP1 was involved in early infantile epileptic
encephalopathy in a previous different cohort study (group B), no mutations of
STXBP1 were found in two of the remaining three subjects of group A (one was
unavailable). We assumed that another gene within the deletion might contribute
to the phenotype of group A. SPTAN1 encoding alpha-II spectrin, which is
essential for proper myelination in zebrafish, turned out to be deleted. In two
subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found
at the initial nucleation site of the alpha/beta spectrin heterodimer. SPTAN1
was further screened in six unrelated individuals with WS and hypomyelination,
but no mutations were found. Recombit mutant (mut) and wild-type (WT)
alpha-II spectrin could assemble heterodimers with beta-II spectrin, but
alpha-II (mut)/beta-II spectrin heterodimers were thermolabile compared with the
alpha-II (WT)/beta-II heterodimers. Transient expression in mouse cortical
neurons revealed aggregation of alpha-II (mut)/beta-II and alpha-II
(mut)/beta-III spectrin heterodimers, which was also observed in lymphoblastoid
cells from two subjects with in-frame mutations. Clustering of ankyrinG and
voltage-gated sodium channels at axon initial segment (AIS) was disturbed in
relation to the aggregates, together with an elevated action potential
threshold. These findings suggest that pathological aggregation of alpha/beta
spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations
were involved in pathogenesis of infantile epilepsy. The ARX gene is involved in the development of GABAergic interneurons in the
forebrain. Loss-of-function mutations, such as nonsense or frameshifts mutation,
of ARX cause a group of brain malformations, such as hydranencephaly,
lissencephaly, and agenesis of the corpus callosum, while expansion mutations of
the polyalanine tracts of ARX, supposed to be gain-of-function mutations, result
in a non-malformation group, such as non-syndromic mental retardation, mental
retardation with dystonia, West syndrome, and Ohtahara syndrome. A variety of
phenotypes caused by pleiotropic mutations of the ARX gene are considered to
share a common pathological mechanism connected with the structural and
functional disturbance of interneurons, designated as 'interneuronopathies'. We
identified the second gene responsible for Ohtahara syndrome, STXBP1, which is
essential for synaptic vesicle release. Molecular studies of the diseases will
reveal the relationships between the structure and function of the brain. It is
indispensable to clarify the etiology of hereditary diseases and identify new
approaches to treatment. OBJECTIVES: Heterozygous mutations in STXBP1, encoding the syntaxin binding
protein 1, have recently been identified in Ohtahara syndrome, an epileptic
encephalopathy with very early onset. In order to explore the phenotypic
spectrum associated with STXBP1 mutations, we analyzed a cohort of patients with
unexplained early-onset epileptic encephalopathies.
METHODS: We collected and clinically characterized 106 patients with early-onset
epileptic encephalopathies. Mutation analysis of the STXBP1 gene was done using
sequence analysis of the exon and intron-exon boundaries and multiplex
amplification quantification to detect copy number variations.
RESULTS: We identified 4 truncating mutations and 2 microdeletions partially
affecting STXBP1 in 6 of the 106 patients. All mutations are predicted to
abolish STXBP1 function and 5 mutations were proven to occur de novo. None of
the mutation-carrying patients had Ohtahara syndrome. One patient was diagnosed
with West syndrome at disease onset, while the initial phenotype of 5 further
patients did not fit into a specific recognized epilepsy syndrome. Three of
these patients later evolved to West syndrome. All patients had severe to
profound mental retardation, and ataxia or dyskinetic movements were present in
5 patients.
CONCLUSION: This study shows that mutations in STXBP1 are not limited to
patients with Ohtahara syndrome, but are also present in 10% (5/49) of patients
with an early-onset epileptic encephalopathy that does not fit into either
Ohtahara or West syndrome and rarely in typical West syndrome. STXBP1 mutational
analysis should be considered in the diagnostic evaluation of this challenging
group of patients. PURPOSE: De novo STXBP1 mutations have been found in individuals with early
infantile epileptic encephalopathy with suppression-burst pattern (EIEE). Our
aim was to delineate the clinical spectrum of subjects with STXBP1 mutations,
and to examine their biologic aspects.
METHODS: STXBP1 was analyzed in 29 and 54 cases of cryptogenic EIEE and West
syndrome, respectively, as a second cohort. RNA splicing was analyzed in
lymphoblastoid cells from a subject harboring a c.663 + 5G>A mutation.
Expression of STXBP1 protein with missense mutations was examined in
neuroblastoma2A cells.
RESULTS: A total of seven novel STXBP1 mutations were found in nine EIEE cases,
but not in West syndrome. The mutations include two frameshift mutations, three
nonsense mutations, a splicing mutation, and a recurrent missense mutation in
three unrelated cases. Including our previous data, 10 of 14 individuals (71%)
with STXBP1 aberrations had the onset of spasms after 1 month, suggesting
relatively later onset of epileptic spasms. Nonsense-mediated mRNA decay
associated with abnormal splicing was demonstrated. Transient expression
revealed that STXBP1 proteins with missense mutations resulted in degradation in
neuroblastoma2A cells.
DISCUSSION: Collectively, STXBP1 aberrations can account for about one-third
individuals with EIEE (14 of 43). These genetic and biologic data clearly showed
that haploinsufficiency of STXBP1 is the important cause for cryptogenic EIEE. PURPOSE: Pyridoxine-dependent epilepsy (PDE) is characterized by
therapy-resistant seizures (TRS) responding to intravenous (IV) pyridoxine. PDE
can be identified by increased urinary alpha-aminoadipic semialdehyde (α-AASA)
concentrations and mutations in the ALDH7A1 (antiquitin) gene. Prompt
recognition of PDE is important for treatment and prognosis of seizures. We
aimed to determine whether immediate electroencephalography (EEG) alterations by
pyridoxine-IV can identify PDE in neonates with TRS.
METHODS: In 10 neonates with TRS, we compared online EEG alterations by
pyridoxine-IV between PDE (n = 6) and non-PDE (n = 4). EEG segments were
visually and digitally analyzed for average background amplitude and total power
and relative power (background activity magnitude per frequency band and
contribution of the frequency band to the spectrum).
RESULTS: In 3 of 10 neonates with TRS (2 of 6 PDE and 1 of 4 non-PDE neonates),
pyridoxine-IV caused flattening of the EEG amplitude and attenuation of
epileptic activity. Quantitative EEG alterations by pyridoxine-IV consisted of
(1) decreased central amplitude, p < 0.05 [PDE: median -30% (range -78% to -3%);
non-PDE: -20% (range -45% to -12%)]; (2) unaltered relative power; (3) decreased
total power, p < 0.05 [PDE: -31% (-77% to -1%); -27% (-73% to -13%); -35% (-56%
to -8%) and non-PDE: -16% (-43% to -5%); -28% (-29% to -17%); -26% (-54% to
-8%), in delta-, theta- and beta-frequency bands, respectively]; and (4) similar
EEG responses in PDE and non-PDE.
DISCUSSION: In neonates with TRS, pyridoxine-IV induces nonspecific EEG
responses that neither identify nor exclude PDE. These data suggest that
neonates with TRS should receive pyridoxine until PDE is fully excluded by
metabolic and/or DNA analysis. Ohtahara syndrome (OS) is one of the most severe and earliest forms of epilepsy.
We have recently identified that the de novo mutations of STXBP1 are important
causes for OS. Here we report a paternal somatic mosaicism of an STXBP1
mutation. The affected daughter had onset of spasms at 1 month of age, and
interictal electroencephalogram showed suppression-burst pattern, leading to the
diagnosis of OS. She had a heterozygous c.902+5G>A mutation of STXBP1, which
affects donor splicing of exon 10, resulting in 138-bp insertion of intron 10
sequences in the transcript. The mutant transcript had a premature stop codon,
and was degraded by nonsense-mediated mRNA decay in lymphoblastoid cells derived
from the patient. High-resolution melting analysis of clinically unaffected
parental DNAs suggested that the father was somatic mosaic for the mutation,
which was also suggested by sequencing. Cloning of PCR products amplified with
the paternal DNA samples extracted from blood, saliva, buccal cells, and nails
suggested that 5.3%, 8.7%, 11.9%, and 16.9% of alleles harbored the mutation,
respectively. This is a first report of somatic mosaicism of an STXBP1 mutation,
which has implications in genetic counseling of OS. Human epilepsy is a common and heterogeneous condition in which genetics play an
important etiological role. We begin by reviewing the past history of epilepsy
genetics, a field that has traditionally included studies of pedigrees with
epilepsy caused by defects in ion channels and neurotransmitters. We highlight
important recent discoveries that have expanded the field beyond the realm of
channels and neurotransmitters and that have challenged the notion that single
genes produce single disorders. Finally, we project toward an exciting future
for epilepsy genetics as large-scale collaborative phenotyping studies come face
to face with new technologies in genomic medicine. PURPOSE: Domit mutations in the STXBP1 gene are a recently identified cause
of infantile epileptic encephalopathy without metabolic and structural brain
anomalies. To date, 25 patients with heterozygous mutation or deletion of STXBP1
have been reported. A diagnosis of early infantile epileptic encephalopathy with
suppression-burst (Ohtahara syndrome) was made in most of them, with infantile
spasms and nonsyndromic infantile epileptic encephalopathy being the diagnosis
in other patients. Although the phenotypic spectrum of STXBP1-related
encephalopathy is emerging with evidence suggesting the relatively frequent
involvement of this gene in infantile epileptic encephalopathies, accurate
clinical descriptions of patients are still necessary to delineate this entity.
METHODS: The sequence of the STXPB1 gene was analyzed in 29 patients with early
onset syndromic or nonsyndromic infantile epileptic encephalopathy without brain
magnetic resoce imaging (MRI) anomalies and with normal chromosomal and
metabolic checkup. Another patient with a complex phenotype was analyzed by
comparative genomic hybridization (CGH) array.
KEY FINDINGS: From the studied series, 2 of 29 patients were found to carry a de
novo heterozygous mutation in STXBP1. One patient carried the recurrent
p.Arg406His mutation and the other an insertion of 10 bases leading to a
premature termination codon. CGH array experiment detected a deletion of 3-3.5
Mbp in the third patient with infantile epileptic encephalopathy and nail
malformations. All three had infantile spasms associated with partial seizures
that responded to antiepileptic drug therapy. Intellectual abilities were
severely impaired in all of them. Generalized tremor was the main neurologic
striking feature in the three patients, with one of them further displaying
unilateral akinetic-hypertonic syndrome.
SIGNIFICANCE: Mutations in STXBP1 are relatively frequent in patients with
infantile epileptic encephalopathies. STXBP1-related encephalopathy may present
as drug-responsive infantile spasms with focal/lateralized discharges.
Generalized tremor appearing after the first year of life may be a clue to the
diagnosis in some patients. PURPOSE: STXBP1 (MUNC18-1) mutations have been associated with various types of
epilepsies, mostly beginning early in life. To refine the phenotype associated
with STXBP1 aberrations in early onset epileptic syndromes, we studied this gene
in a cohort of patients with early onset epileptic encephalopathy.
METHODS: STXBP1 was screened in a multicenter cohort of 52 patients with early
onset epilepsy (first seizure observed before the age of 3 months), no cortical
malformation on brain magnetic resoce imaging (MRI), and negative metabolic
screening. Three groups of patients could be distinguished in this cohort: (1)
Ohtahara syndromes (n = 38); (2) early myoclonic encephalopathies (n = 7); and
(3) early onset epileptic encephalopathies that did not match any familiar
syndrome (n = 7). None of the patients displayed any cortical malformation on
brain MRI and all were screened through multiple video-electroencephalography
(EEG) recordings for a time period spanning from birth to their sixth postnatal
month. Subsequently, patients had standard EEG or video-EEG recordings.
KEY FINDINGS: We found five novel STXBP1 mutations in patients for whom
video-EEG recordings could be sampled from the beginning of the disease. All
patients with a mutation displayed Ohtahara syndrome, since most early seizures
could be classified as epileptic spasms and since the silent EEG periods were on
average shorter than bursts. However, each patient in addition displayed a
particular clinical and EEG feature: In two patients, early seizures were
clonic, with very early EEG studies exhibiting relatively low amplitude bursts
of activity before progressing into a typical suppression-burst pattern, whereas
the three other patients displayed epileptic spasms associated with typical
suppression-burst patterns starting from the early recordings. Epilepsy
dramatically improved after 6 months and finally disappeared before the end of
the first year of life for four patients; the remaining one patient had few
seizures until 18 months of age. In parallel, EEG paroxysmal abnormalities
disappeared in three patients and decreased in two, giving place to continuous
activity with fast rhythms. Each patient displayed frequent nonepileptic
movement disorders that could easily be mistaken for epileptic seizures. These
movements could be observed as early as the neonatal period and, unlike
seizures, persisted during all the follow-up period.
SIGNIFICANCE: We confirm that STXBP1 is a major gene to screen in cases of
Ohtahara syndrome, since it is mutated in >10% of the Ohtahara patients within
our cohort. This gene should particularly be tested in the case of a surprising
evolution of the patient condition if epileptic seizures and EEG paroxysmal
activity disappear and are replaced by fast rhythms after the end of the first
postnatal year. Ohtahara syndrome or Early Infantile Epileptic Encephalopathy (EIEE) with
Suppression-Burst, is the most severe and the earliest developing age-related
epileptic encephalopathy. Clinically, the syndrome is characterized by early
onset tonic spasms associated with a severe and continuous pattern of burst
activity. It is a debilitating and early progressive neurological disorder,
resulting in intractable seizures and severe mental retardation. Specific
mutations in at least four genes (whose protein products are essential in lower
brain's neuronal and interneuronal functions, including mitochondrial
respiratory chains have been identified in unrelated individuals with EIEE and
include: (a) the ARX (aristaless-related) homeobox gene at Xp22.13 (EIEE-1
variant); (b) the CDKL5 (SYK9) gene at Xp22 (EIEE-2 variant); (c) the SLC25A22
(GC1) gene at 11p15.5 (EIEE-3 variant); and (d) the Stxbp1 (MUNC18-1) gene at
9q34-1 (EIEE-4 variant). A yet unresolved issue involves the relationship
between early myoclonic encephalopathy (EME-ErbB4 mutations) versus the EIEE
spectrum of disorders. Mutations of the syntaxin binding protein 1 (STXBP1) have been associated with
severe infantile epileptic encephalopathies (Ohtahara syndrome and West
syndrome), but also with moderate to severe cognitive impairment and
nonsyndromic epilepsy. We have studied a white infant who presented with focal
seizures at age 2 weeks. Brain imaging was unremarkable. The
electroencephalograph (EEG) demonstrated normal background frequency content but
with multifocal sharp waves and no evidence of the typical patterns associated
with Ohtahara or West syndrome. Therapy with levetiracetam and oxcarbazepine
effectively managed the seizure episodes. Investigation of genes associated with
infantile forms of epilepsy such as SCN1A, SCN1B, and ARX were negative, but we
identified a novel single-nucleotide duplication mutation, c.931dupT
(p.S311FfsX3), in exon 11 of the STXBP1 gene. This previously unreported STXBP1
mutation in a subject with neonatal-onset focal seizures broadens the spectrum
of clinically relevant human disorders caused by STXBP1 mutations. PURPOSE: Epilepsies have a highly heterogeneous background with a strong genetic
contribution. The variety of unspecific and overlapping syndromic and
nonsyndromic phenotypes often hampers a clear clinical diagnosis and prevents
straightforward genetic testing. Knowing the genetic basis of a patient's
epilepsy can be valuable not only for diagnosis but also for guiding treatment
and estimating recurrence risks.
METHODS: To overcome these diagnostic restrictions, we composed a panel of genes
for Next Generation Sequencing containing the most relevant epilepsy genes and
covering the most relevant epilepsy phenotypes known so far. With this method,
265 genes were analyzed per patient in a single step. We evaluated this panel on
a pilot cohort of 33 index patients with concise epilepsy phenotypes or with a
severe but unspecific seizure disorder covering both sporadic and familial
cases.
KEY FINDINGS: We identified presumed disease-causing mutations in 16 of 33
patients comprising sequence alterations in frequently as well as in less
commonly affected genes. The detected aberrations encompassed known and unknown
point mutations (SCN1A p.R222X, p. E289V, p.379R, p.R393H; SCN2A p.V208E; STXBP1
p.R122X; KCNJ10 p.L68P, p.I129V; KCTD7 p.L108M; KCNQ3 p.P574S; ARHGEF9 p.R290H;
SMS p.F58L; TPP1 p.Q278R, p.Q422H; MFSD8 p.T294K), a putative splice site
mutation (SCN1A c.693A> p.T/P231P) and small deletions (SCN1A p.F1330Lfs3X [1
bp]; MFSD8 p.A138Dfs10X [7 bp]). All mutations have been confirmed by
conventional Sanger sequencing and, where possible, validated by parental
testing and segregation analysis. In three patients with either Dravet syndrome
or myoclonic epilepsy, we detected SCN1A mutations (p.R222X, p.P231P, p.R393H),
even though other laboratories had previously excluded aberrations of this gene
by Sanger sequencing or high-resolution melting analysis.
SIGNIFICANCE: We have developed a fast and cost-efficient diagnostic screening
method to analyze the genetic basis of epilepsies. We were able to detect
mutations in patients with clear and with unspecific epilepsy phenotypes, to
uncover the genetic basis of many so far unresolved cases with epilepsy
including mutation detection in cases in which previous conventional methods
yielded falsely negative results. Our approach thus proved to be a powerful
diagnostic tool that may contribute to collecting information on both common and
unknown epileptic disorders and in delineating associated phenotypes of less
frequently mutated genes. PURPOSE: Ohtahara syndrome (OS) is one of the most severe and earliest forms of
epilepsy. STXBP1 and ARX mutations have been reported in patients with OS. In
this study, we aimed to identify new genes involved in OS by copy number
analysis and whole exome sequencing.
METHODS: Copy number analysis and whole exome sequencing were performed in 34
and 12 patients with OS, respectively. Fluorescence in situ hybridization,
quantitative polymerase chain reaction (PCR), and breakpoint-specific and
reverse-transcriptase PCR analyses were performed to characterize a deletion.
Immunoblotting using lymphoblastoid cells was done to examine expression of CASK
protein.
KEY FINDINGS: Genomic microarray analysis revealed a 111-kb deletion involving
exon 2 of CASK at Xp11.4 in a male patient. The deletion was inherited from his
mother, who was somatic mosaic for the deletion. Sequencing of the mutant
transcript expressed in lymphoblastoid cell lines derived from the patient
confirmed the deletion of exon 2 in the mutant transcript with a premature stop
codon. Whole exome sequencing identified another male patient who was harboring
a c.1A>G mutation in CASK, which occurred de novo. Both patients showed severe
cerebellar hypoplasia along with other congenital anomalies such as
micrognathia, a high arched palate, and finger anomalies. No CASK protein was
detected by immunoblotting in lymphoblastoid cells derived from two patients.
SIGNIFICANCE: The detected mutations are highly likely to cause the loss of
function of the CASK protein in male individuals. CASK mutations have been
reported in patients with intellectual disability with microcephaly and
pontocerebellar hypoplasia or congenital nystagmus, and those with FG syndrome.
Our data expand the clinical spectrum of CASK mutations to include OS with
cerebellar hypoplasia and congenital anomalies at the most severe end. PURPOSE: A number of genes in the 9q34.11 region may be haploinsufficient.
However, studies analyzing genotype-phenotype correlations of deletions
encompassing multiple dosage-sensitive genes in the region are lacking.
METHODS: We mapped breakpoints of 10 patients with 9q34.11 deletions using
high-resolution 9q34-specific array comparative genomic hybridization (CGH) to
determine deletion size and gene content.
RESULTS: The 9q34.11 deletions range in size from 67 kb to 2.8 Mb. Six patients
exhibit intellectual disability and share a common deleted region including
STXBP1; four manifest variable epilepsy. In five subjects, deletions include
SPTAN1, previously associated with early infantile epileptic encephalopathy,
infantile spasms, intellectual disability, and hypomyelination. In four
patients, the deletion includes endoglin (ENG), causative of hereditary
hemorrhagic telangiectasia. Finally, in four patients, deletions involve TOR1A,
of which molecular defects lead to early-onset primary dystonia. Ninety-four
other RefSeq genes also map to the genomic intervals investigated.
CONCLUSION: STXBP1 haploinsufficiency results in progressive encephalopathy
characterized by intellectual disability and may be accompanied by epilepsy,
movement disorders, and autism. We propose that 9q34.11 genomic deletions
involving ENG, TOR1A, STXBP1, and SPTAN1 are responsible for multisystemic
vascular dysplasia, early-onset primary dystonia, epilepsy, and intellectual
disability, therefore revealing cis-genetic effects leading to complex
phenotypes. Mutations in STXBP1 have been identified in a subset of patients with early
onset epileptic encephalopathy (EE), but the full phenotypic spectrum remains to
be delineated. Therefore, we screened a cohort of 160 patients with an
unexplained EE, including patients with early myoclonic encephalopathy (EME),
Ohtahara syndrome, West syndrome, nonsyndromic EE with onset in the first year,
and Lennox-Gastaut syndrome (LGS). We found six de novo mutations in six
patients presenting as Ohtahara syndrome (2/6, 33%), West syndrome (1/65, 2%),
and nonsyndromic early onset EE (3/64, 5%). No mutations were found in LGS or
EME. Only two of four mutation carriers with neonatal seizures had Ohtahara
syndrome. Epileptic spasms were present in five of six patients. One patient
with normal magnetic resoce imaging (MRI) but focal seizures underwent
epilepsy surgery and seizure frequency dropped drastically. Neuropathology
showed a focal cortical dysplasia type 1a. There is a need for additional
neuropathologic studies to explore whether STXBP1 mutations can lead to
structural brain abnormalities. Early-onset epileptic encephalopathies (EOEEs) are characterised by epileptic
seizures beginning in the first months of life, abnormal background EEG
activity, and are associated with severe developmental delay and poor prognosis.
Mutations and deletions in the STXBP1 gene are associated with Ohtahara
syndrome, also known as "early infantile epileptic encephalopathy". We report an
infant affected by EOEE with a 9q34.11 deletion that encompassed the genes
STXBP1 and SPTAN1. The infant presented with neonatal encephalopathy without
epileptic seizures and an EEG pattern varying from highly discontinuous to
suppression-burst. This was followed by West syndrome at 2 months with atypical
hypsarrhythmia and spasms, easily controlled by therapy. Our findings suggest
that molecular analysis of STXBP1 should be considered for newborns affected by
neonatal encephalopathy associated with a peculiar EEG pattern, even in the
absence of neonatal epileptic seizures. Mutations in STXBP1 gene, encoding the syntaxin binding protein 1, have been
recently described in Ohtahara syndrome, or early infantile epileptic
encephalopathy with suppression-burst pattern, and in other early-onset
epileptic encephalopathies. A 3-year-old boy affected by epileptic
encephalopathy started at 8 months of age is described. Focal epilepsy was
characterized by drug resistance seizures with multifocal interictal and ictal
electroencephalographic (EEG) features and variable EEG focus. Direct sequencing
of the STXBP1 gene showed a novel de novo mutation (c.751G>A), leading to a
p.Ala251Thr substitution. Based on reported data, treatment with vigabatrin was
attempted and patient became immediately seizure free for 4 months. The present
case further expands the clinical spectrum of "STXBP1-related encephalopathy"
suggesting molecular analysis of STXBP1 in early onset epileptic
encephalopathies of unknown etiology (with onset within the first year of life).
In addition, the case provides valuable suggestions on seizures treatment in
STXBP1 mutated subjects. Author information:
(1)Department of Neuropediatrics, Centre de Reference des Epilepsies Rares,
Hopital Necker Enfants Malades, Paris Descartes University, Paris, France;
Inserm U663, University Paris Descartes, PRES Sorbonne Paris Cité, Paris
F-75005; CEA, Neurospin, 91190 Gif/Yvette, France.
(2)Department of Genetics, Inserm U781, Hopital Necker Enfants Malades, Paris
Descartes University, Paris, France.
(3)APHM, CINAPSE, Pediatric Neurology Department, Timone Children Hospital,
Marseille, France.
(4)Department of Pediatric Neurology, Hôpital Erasme, Bruxelles, Belgium.
(5)Department of Neuropediatrics, Centre de Reference des Epilepsies Rares,
Hopital Necker Enfants Malades, Paris Descartes University, Paris, France.
(6)Department of Pediatric Radiology, Hopital Necker Enfants Malades, APHP,
Paris, Descartes University, Paris, France.
(7)Department of Neuropediatrics, Centre de Reference des Epilepsies Rares,
Hopital Necker Enfants Malades, Paris Descartes University, Paris, France.
Electronic address: [email protected]. BACKGROUND: Ohtahara syndrome is a severe condition with early onset of
recurrent unprovoked seizures associated with abnormal electroencephalography
and global developmental delay. Folinic acid-responsive seizures are treatable
causes of Ohtahara syndrome, which is thought to be due to recessive mutations
in the ALDH7A1 gene, resulting in deficiency of antiquitin.
METHOD: Here we report a girl with Ohtahara syndrome who exhibited transient
folinic acid responsiveness but without evidence of antiquitin dysfunction.
RESULTS: She was later found to have a known missense mutation (c.1439 C > T,
p.P480 L) in exon 16 of the STXBP1 gene.
CONCLUSION: For infants presenting with Ohtahara syndrome with responsiveness to
folinic acid and negative antiquitin deficiency analyses, genetic testing for
other possible causative genes such as STXBP1 mutation is recommended. |
What is the mechanism of action of geldanamycin? | Geldanamycin is an ansamycin antibiotic which holds the ability to bind heat-shock protein 90. This interaction can lead to the disruption of heat-shock protein 90-containing multimolecular complexes. Additionally, it can induce inhibition or even degradation of partner proteins dissociated from the 90 kDa chaperone and, eventually, cause apoptosis in a variety of cell types. | The mineralocorticoid receptor (MR) forms oligomers with the heat-shock protein
90 (Hsp90) -based heterocomplex, which contains tetratricopeptide repeat (TPR)
domain immunophilins (IMMs). Here we investigated the unknown biological role of
IMMs in the MR.Hsp90 complex. Upon hormone binding, FKBP52 was greatly recruited
to MR.Hsp90 complexes along with dynein motors, whereas FKBP51 was dissociated.
Importantly, the Hsp90 inhibitor geldanamycin impaired the retrograde transport
of MR, suggesting that the Hsp90.IMM.dynein molecular machinery is required for
MR movement. To elucidate the mechanism of action of MR, the synthetic ligand
11,19-oxidoprogesterone was used as a tool. This steroid showed equivalent
agonistic potency to natural agonists and was able to potentiate their
mineralocorticoid action. Importantly, aldosterone binding recruited greater
amounts of FKBP52 and dynein than 11,19-oxidoprogesterone binding to MR.
Interestingly, 11,19-oxidoprogesterone binding also favored the selective
recruitment of the IMM-like Ser/Thr phosphatase PP5. Each hormone/MR complex
yielded different proteolytic peptide patterns, suggesting that MR acquires
different conformations upon steroid binding. Also, hormone/MR complexes showed
different nuclear translocation rates and subnuclear redistribution. All these
observations may be related to the selective swapping of associated factors. We
conclude that (a) the Hsp90.FKBP52.dyenin complex may be responsible for the
retrotransport of MR; (b) a differential recruitment of TPR proteins such as
FKBP51, FKBP52, and PP5 takes place during the early steps of hormone-dependent
activation of the receptor; (c) importantly, this swapping of TPR proteins
depends on the nature of the ligand; and (d) inasmuch as FKBP51 also showed an
inhibitory effect on MR-dependent transcription, it should be dissociated from
the MR.Hsp90 complex to positively regulate the mineralocorticoid effect. The development of new anticancer agents derived from natural resources requires
a rapid identification of their molecular mechanism of action. To make this step
short, we have initiated the proteomic profiling of HeLa cells treated with
anticancer drugs representing a wide spectrum of mechanisms of action using
two-dimensional difference gel electrophoresis (2D-DIGE). Unique proteome
patterns were observed in HeLa cells treated with the HSP90 inhibitor
geldanamycin, and were similar to the patterns induced by radicicol, a
structurally different HSP90 inhibitor. On the other hand, etoposide and
ICRF-193, compounds claimed to be topoisomerase II inhibitors, showed different
proteomic profiles, which reflect their different biological activities as
revealed by cell-cycle analysis. Thus far, combined data from 19 compounds have
allowed their successful classification by cluster analysis according to the
mechanism of action. Molecular chaperones are proteins that assist the folding, unfolding, and
remodeling of other proteins. In eukaryotes, heat shock protein 90 (Hsp90)
proteins are essential ATP-dependent molecular chaperones that remodel and
activate hundreds of client proteins with the assistance of cochaperones. In
Escherichia coli, the activity of the Hsp90 homolog, HtpG, has remained elusive.
To explore the mechanism of action of E. coli Hsp90, we used in vitro protein
reactivation assays. We found that E. coli Hsp90 promotes reactivation of
heat-inactivated luciferase in a reaction that requires the prokaryotic Hsp70
chaperone system, known as the DnaK system. An Hsp90 ATPase inhibitor,
geldanamycin, inhibits luciferase reactivation demonstrating the importance of
the ATP-dependent chaperone activity of E. coli Hsp90 during client protein
remodeling. Reactivation also depends upon the ATP-dependent chaperone activity
of the DnaK system. Our results suggest that the DnaK system acts first on the
client protein, and then E. coli Hsp90 and the DnaK system collaborate
synergistically to complete remodeling of the client protein. Results indicate
that E. coli Hsp90 and DnaK interact in vivo and in vitro, providing additional
evidence to suggest that E. coli Hsp90 and the DnaK system function together. BACKGROUND: The rs2736100 single nucleotide polymorphism (SNP) is located in the
intron 2 of human telomerase reverse transcriptase (hTERT) gene. Recent
genome-wide association studies (GWAS) have consistently supported the strong
association between this SNP and risk for multiple cancers. Given the important
role of the hTERT gene and this SNP in cancer biology, we hypothesize that
rs2736100 may also confer susceptibility to anti-cancer drug sensitivity. In
this study we aim to investigate the correlation between the rs2736100 genotype
and the responsiveness to anti-cancer agents in the NCI-60 cancer cell panel.
METHODS AND MATERIALS: The hTERT rs2736100 was genotyped in the NCI-60 cancer
cell lines. The relative telomere length (RTL) of each cell line was quantified
using real-time PCR. The genotype was then correlated with publically available
drug sensitivity data of two agents with telomerase-inhibition activity:
Geldanamycin (HSP90 inhibitor) and RHPS4/BRACO19 (G-quadruplex stabilizer) as
well as additional 110 commonly used agents with established mechanism of
action. The association between rs2736100 and mutation status of TP53 gene was
also tested.
RESULTS: The C allele of the SNP was significantly correlated with increased
sensitivity to RHPS4/BRACO19 with an additive effect (r = -0.35, p = 0.009) but
not with Geldanamycin. The same allele was also significantly associated with
sensitivity to antimitotic agents compared to other agents (p = 0.003). The
highest correlation was observed between the SNP and paclitaxel (r = -0.36, p =
0.005). The telomere length was neither associated with rs2736100 nor with
sensitivity to anti-cancer agents. The C allele of rs2736100 was significantly
associated with increased mutation rate in TP53 gene (p = 0.004).
CONCLUSION: Our data suggested that the cancer risk allele of hTERT rs2736100
polymorphism may also affect the cancer cell response to both TERT inhibitor and
anti-mitotic agents, which might be attributed to the elevated
telomerase-independent activity of hTERT, as well as the increased risk for TP53
gene mutagenesis conferred by the polymorphism. Detailed mechanisms need to be
further investigated. |
Which is the most widely used anti-TNF drug? | Etanercept is the most widely used anti-TNF drug. | OBJECTIVES: Small studies have shown an improvement in disease activity in
patients with RA who have switched between anti-TNF therapies for reasons of
inefficacy. However, it is not clear whether switching improves longer term
outcomes, such as disability. This analysis compares changes in HAQ scores 1 yr
following lack of response to a first anti-TNF based on subsequent treatment
during that year.
METHODS: Analysis was limited to RA patients with inefficacy to a first anti-TNF
based on (i) clinician opinion and/or (ii) disease activity score in 28 joints
and had an HAQ measured at time of non-response and 12 months later. Patients
were classified into three groups based on treatment during the next 12 months:
(i) continued anti-TNF despite non-response; (ii) stopped anti-TNF with no
further biologics; and (iii) switched to a second anti-TNF. Mean improvement in
HAQ was compared among the groups using multivariable linear regression models.
RESULTS: As of July 2006, 868 patients met the inclusion for this analysis. Four
hundred and seventy-nine patients stopped anti-TNF of whom 331 switched to a
second anti-TNF. Three hundred and eighty-nine continued treatment. Patients who
continued and those who switched had improvements in HAQ over the 12 months,
unlike patients who discontinued all biologic therapy. The best improvement was
seen in those who switched [adjusted mean improvement in HAQ 0.15 (95% CI 0.26,
0.05)].
CONCLUSION: There is a significant improvement in HAQ in patients who switch to
a second anti-TNF, providing an effective next choice of therapy for some
patients who fail to respond to their first anti-TNF. BACKGROUND: The risk of tuberculosis (TB) in patients with rheumatoid arthritis
(RA) is thought to be increased following anti-tumour necrosis factor (anti-TNF)
therapy, with a proposed differential risk between the anti-TNF drugs etanercept
(ETA), infliximab (INF) and adalimumab (ADA).
OBJECTIVE: To compare directly the risk between drugs, to explore time to event,
site of infection and the role of ethnicity.
METHODS: Data from the British Society for Rheumatology Biologics Register
(BSRBR), a national prospective observational study, were used to compare TB
rates in 10 712 anti-TNF treated patients (3913 ETA, 3295 INF, 3504 ADA) and
3232 patients with active RA treated with traditional disease-modifying
antirheumatic drugs.
RESULTS: To April 2008, 40 cases of TB were reported, all in the anti-TNF
cohort. The rate of TB was higher for the monoclonal antibodies ADA (144
events/100,000 person-years) and INF (136/100,000 person-years) than for ETA
(39/100,000 person-years). After adjustment, the incidence rate ratio compared
with ETA-treated patients was 3.1 (95% CI 1.0 to 9.5) for INF and 4.2 (1.4 to
12.4) for ADA. The median time to event was lowest for INF (5.5 months) compared
with ETA (13.4 months) and ADA (18.5 months). 13/40 cases occurred after
stopping treatment. 25/40 (62%) cases were extrapulmonary, of which 11 were
disseminated. Patients of non-white ethnicity had a sixfold increased risk of TB
compared with white patients treated with anti-TNF therapy.
CONCLUSION: The rate of TB in patients with RA treated with anti-TNF therapy was
three- to fourfold higher in patients receiving INF and ADA than in those
receiving ETA. BACKGROUND: Anti-tumour necrosis factor (TNF) therapy may be associated with
opportunistic infections (OIs).
OBJECTIVE: To describe the spectrum of non-tuberculosis OIs associated with
anti-TNF therapy and identify their risk factors.
METHODS: A 3-year national French registry (RATIO) collected all cases of OI in
patients receiving anti-TNF treatment for any indication in France. A
case-control study was performed with three controls treated with anti-TNF
agents per case, matched for gender and underlying inflammatory disease.
RESULTS: 45 cases were collected of non-TB OIs in 43 patients receiving
infliximab (n=29), adalimumab (n=10) or etanercept (n=4) for rheumatoid
arthritis (n=26), spondyloarthritides (n=3), inflammatory colitis (n=8),
psoriasis (n=1) or other conditions (n=5). One-third (33%) of OIs were bacterial
(4 listeriosis, 4 nocardiosis, 4 atypical mycobacteriosis, 3 non-typhoid
salmonellosis), 40% were viral (8 severe herpes zoster, 3 varicella, 3 extensive
herpes simplex, 4 disseminated cytomegalovirus infections), 22% were fungal (5
pneumocystosis, 3 invasive aspergillosis, 2 cryptococcosis) and 4% were
parasitic (2 leishmaniasis). Ten patients (23%) required admission to the
intensive care unit, and four patients (9%) died. Risk factors for OIs were
treatment with infliximab (OR=17.6 (95% CI 4.3 - 72.9); p<0.0001)or adalimumab
(OR=10.0 (2.3 to 44.4); p=0.002) versus etanercept, and oral steroid use >10
mg/day or intravenous boluses during the previous year (OR=6.3 (2.0 to 20.0);
p=0.002).
CONCLUSION: Various and severe OIs, especially those with intracellular
micro-organisms, may develop in patients receiving anti-TNF treatment.
Monoclonal anti-TNF antibody rather than soluble TNF receptor therapy and
steroid use >10 mg/day are independently associated with OI. |
What is the association between number of pregnancies and rheumatoid arthritis | Greater parity significantly reduced the odds of RA. A larger number of pregnancies and late menopause show a protective effect, delaying the onset of the disease. | PIP: A case control study of rheumatoid arthritis and oral contraceptives (OC)
or postmenopausal hormones was begun in women aged 20 in 2 hospitals in Athens,
with same hospital, age-matched controls. Cases have diagnosed definite or
classical rheumatoid arthritis and are being treated in rheumatology clinics. To
date 135 cases and 104 controls have been interviewed. 5 cases (3.7%) and 5
controls (4.8% used OCs, with a mean duration of 2 years for both, giving a
relative risk of 0.76. 8 more cases and 6 controls were found who used OC for
regulation of their menstrual cycle. 1.5% of cases and 3.8% of controls had used
post-menopausal hormones. Univariate analysis showed that the cases had later
menarche and fewer pregcies and live births. Multiple regression analysis did
not find OC or postmenopausal hormones significantly linked with rheumatoid
arthritis. Number of pregcies and children, however, were significantly
higher in controls. Because of the small numbers and low power of the study,
more subjects are being collected. OBJECTIVE: To examine reproductive history and rheumatoid arthritis (RA) risk in
a highly predisposed population of North American Natives (NAN) with unique
fertility characteristics.
METHODS: The effect of pregcy on the risk of RA was examined by comparing
women enrolled in 2 studies: a study of RA in NAN patients and their unaffected
relatives; and NAN patients with RA and unrelated healthy NAN controls enrolled
in a study of autoimmunity. All participants completed questionnaires detailing
their reproductive history.
RESULTS: Patients with RA (n = 168) and controls (n = 400) were similar overall
in age, education, shared epitope frequency, number of pregcies, age at first
pregcy, smoking, and breastfeeding history. In multivariate analysis, for
women who had ≥ 6 births the OR for developing RA was 0.43 (95% CI 0.21-0.87)
compared with women who had 1-2 births (p = 0.046); for women who gave birth for
the first time after age 20 the OR for developing RA was 0.33 (95% CI 0.16-0.66)
compared with women whose first birth occurred at age ≤ 17 (p = 0.001). The
highest risk of developing RA was in the first postpartum year (OR 3.8; 95% CI
1.45-9.93) compared with subsequent years (p = 0.004).
CONCLUSION: In this unique population, greater parity significantly reduced the
odds of RA; an early age at first birth increased the odds, and the postpartum
period was confirmed as high risk for RA onset. The protective effect of
repeated exposure to the ameliorating hormonal and immunological changes of
pregcy may counterbalance the effect of early exposure to the postpartum
reversal of these changes. |
Is mitofusin 2 a receptor for parkin? | Yes, Mfn2 functions as a mitochondrial receptor for Parkin. | Mitochondrial dysfunction is a common characteristic of all neurodegenerative
diseases. However, the cause of this dysfunction remains a mystery. Here, we
discuss the potential role of mitochondrial fission and fusion in the onset and
progression of neurodegenerative diseases. Specifically, we propose that an
imbalance in mitochondrial fission and fusion may underlie both familial and
sporadic neurodegenerative disorders. There is substantial evidence that links
disruption of the mitochondrial fission and fusion equilibrium, resulting in
abnormally long or short mitochondria, to neurodegeneration. First, hereditary
mutations in the mitochondrial fusion GTPases optic atrophy-1 and mitofusin-2
cause neuropathies in humans. In addition, recent findings report increased
mitochondrial fission in Parkinson's disease (PD) models and induction of
mitochondrial fission by two proteins, PTEN-induced kinase 1 and parkin, which
are mutant in familial forms of PD. Furthermore, mutant huntingtin, the
disease-causing protein in Huntington's disease, alters mitochondrial morphology
and dynamics. Rotenone, a pesticide and inducer of PD symptoms, and amyloid-beta
peptide, which is causally linked to Alzheimer's disease, initiate mitochondrial
fission. Finally, mitochondrial fission is an early event in ischemic stroke and
diabetic neuropathies. In sum, a growing body of research suggests that a better
understanding of mitochondrial fission and fusion and the regulatory factors
involved may lead to improved treatments and cures for neurodegenerative
diseases. Mitochondrial dysfunction and perturbed degradation of proteins have been
implicated in Parkinson's disease (PD) pathogenesis. Mutations in the Parkin and
PINK1 genes are a cause of familial PD. PINK1 is a putative kinase associated
with mitochondria, and loss of PINK1 expression leads to mitochondrial
dysfunction, which increases with time. Parkin is suggested to be downstream of
PINK1 and also mediates the removal of damaged mitochondria by macroautophagy
(mitophagy). We investigated whether mitochondrial dysfunction in dopaminergic
SH-SY5Y cells following decreased PINK1 expression by RNAi may in part be due to
the inhibition of mitophagy. Reduced flux through the macroautophagy pathway was
found to be coincident with the inhibition of ATP synthesis following 12 days of
PINK1 silencing. Overexpression of parkin in these cells restored both
autophagic flux and ATP synthesis. Overexpression and RNAi studies also
indicated that PINK1 and parkin were required for mitophagy following
CCCP-induced mitochondrial damage. The ubiquitination of several mitochondrial
proteins, including mitofusin 1 and mitofusin 2, were detected within 3 h of
CCCP treatment. These post-translational modifications were reduced following
the silencing of parkin or PINK1. The ubiquitination of mitochondrial proteins
appears to identify mitochondria for degradation and facilitate mitophagy. PINK1
and parkin are thus required for the removal of damaged mitochondria in
dopaminergic cells, and inhibition of this pathway may lead to the accumulation
of defective mitochondria which may contribute to PD pathogenesis. Parkinson disease is characterized by the accumulation of aggregated α-synuclein
as the major component of the Lewy bodies. α-Synuclein accumulation in turn
leads to compensatory effects that may include the up-regulation of autophagy.
Another common feature of Parkinson disease (PD) is mitochondrial dysfunction.
Here, we provide evidence that the overactivation of autophagy may be a link
that connects the intracellular accumulation of α-synuclein with mitochondrial
dysfunction. We found that the activation of macroautophagy in primary cortical
neurons that overexpress mutant A53T α-synuclein leads to massive mitochondrial
destruction and loss, which is associated with a bioenergetic deficit and
neuronal degeneration. No mitochondrial removal or net loss was observed when we
suppressed the targeting of mitochondria to autophagosomes by silencing Parkin,
overexpressing wild-type Mitofusin 2 and domit negative Dynamin-related
protein 1 or blocking autophagy by silencing autophagy-related genes. The
inhibition of targeting mitochondria to autophagosomes or autophagy was also
partially protective against mutant A53T α-synuclein-induced neuronal cell
death. These data suggest that overactivated mitochondrial removal could be one
of the contributing factors that leads to the mitochondrial loss observed in PD
models. BACKGROUND: Mutations in parkin have been associated with autosomal recessive
early-onset Parkinson's disease (PD). Here, we report on unusual phenotypic
variability within a family with mutations in parkin.
METHODS: The proband and her parents were clinically assessed. Mutation analysis
was performed using genomic DNA and complementary DNA. The protein expression of
Parkin and Mitofusin 2 was examined by western blotting.
RESULTS: The proband was a compound heterozygote with no detectable Parkin and
presented with early-onset PD. The father, a single heterozygote with reduced
expression of Parkin, had mild loss of arm swing. The mother, who had only very
mild rigidity, was unexpectedly found to be a homozygote with no Parkin
expression. The proband, but not the parents, met the Queen Square Brain Bank
criteria for PD. Parkin-dependent ubiquitination of Mitofusin 2 was impaired in
the mother and the proband.
CONCLUSION: We report the first case of a homozygous mutation carrier in parkin
who had no functional protein and only mild signs of parkinsonism in her seventh
decade, whereas her daughter developed typical early-onset PD. This family
demonstrates phenotypic variability in parkin-related parkinsonism. © 2012
Movement Disorder Society. Senescent and damaged mitochondria undergo selective mitophagic elimination
through mechanisms requiring two Parkinson's disease factors, the mitochondrial
kinase PINK1 (PTEN-induced putative kinase protein 1; PTEN is phosphatase and
tensin homolog) and the cytosolic ubiquitin ligase Parkin. The nature of the
PINK-Parkin interaction and the identity of key factors directing Parkin to
damaged mitochondria are unknown. We show that the mitochondrial outer membrane
guanosine triphosphatase mitofusin (Mfn) 2 mediates Parkin recruitment to
damaged mitochondria. Parkin bound to Mfn2 in a PINK1-dependent manner; PINK1
phosphorylated Mfn2 and promoted its Parkin-mediated ubiqitination. Ablation of
Mfn2 in mouse cardiac myocytes prevented depolarization-induced translocation of
Parkin to the mitochondria and suppressed mitophagy. Accumulation of
morphologically and functionally abnormal mitochondria induced respiratory
dysfunction in Mfn2-deficient mouse embryonic fibroblasts and cardiomyocytes and
in Parkin-deficient Drosophila heart tubes, causing dilated cardiomyopathy.
Thus, Mfn2 functions as a mitochondrial receptor for Parkin and is required for
quality control of cardiac mitochondria. |
What are the main benefits of pharmacophore models? | As researchers continue to search for new targets of therapeutic interest, transmembrane and G-protein coupled receptors are of ever-increasing importance. However, crystal structures for these targets may be impossible to resolve, posing great challenges in rational drug design. Structure-based virtual screening is not an option when the active site geometry is unknown, but assaying an entire library for hits is an inefficient and expensive proposition.
Pharmacophore modeling solves this problem by determining the spatial arrangement of chemical features that confer drug activity toward a target receptor. Having established the chemical space occupied by active ligands, pharmacophore modeling software allows researchers to create 3-D structure-activity relationships, screen databases, and generate hits without the benefit of a receptor structure. | The release of arachidonic acid, a precursor in the production of prostaglandins
and leukotrienes, is achieved by activity of the cytosolic phospholipase A(2)α
(cPLA(2)α). Signaling mediated by this class of bioactive lipids, which are
collectively referred to as eicosanoids, has numerous effects in physiological
and pathological processes. Herein, we report the development of a ligand-based
pharmacophore model and pharmacophore-based virtual screening of the National
Cancer Institute (NCI) database, leading to the identification of
4-(hexadecyloxy)-3-(2-(hydroxyimino)-3-oxobutanamido)benzoic acid (NSC 119957)
as cPLA(2)α inhibitor in cell-free and cell-based in vitro assays. Febrifugine and its derivatives are effective against Plasmodium falciparum.
Using PHASE algorithm, a five-point pharmacophore model with two hydrogen bond
acceptor (A), one positively ionizable (P) and two aromatic rings (R), was
developed to derive a predictive ligand-based statistically significant
3D-quantitative structure-activity relationship (QSAR) model (r(2) = 0.972, SD =
0.3, F = 173.4, Q(2) = 0.712, RMSE = 0.3, Person-R = 0.94, and r(2) pred = 0.8)
to explicate the structural attributes crucial for antimalarial activity. The
developed pharmacophore model and 3D QSAR model can be a substantial tool for
virtual screening and related antimalarial drug discovery research. Tamoxifen has been shown to be active in vitro against Leishmania and effective
in the treatment for leishmaniasis in murine models. Through the screening of a
compound library of estrogen receptor modulator analogs, we identified the major
characteristics required for antileishmanial activity. To overcome the
difficulties presented by tamoxifen's propensity for E/Z isomerization, we used
the 2-arylbenzothiophene compound BTP as a more stable alternative. Directed
screening of a small compound library based on BTP led to active compounds
against Leishmania. Subsequent structure-activity data for the synthetic
2-arylbenzothiophenes evaluated in this study indicate that optimal
antileishmanial potency is dependent on the presence of two basic side chains.
In addition, the primary structural features required for estrogen receptor
binding, the phenols, are not required for inhibiting parasitic growth.
Significantly, the most active antileishmanial benzothiophenes lack the
pharmacophore for estrogen receptor activity and therefore address potential
concerns about the undesirable effects of using selective estrogen receptor
modulators in women and children with leishmaniasis. Three compounds selected
from the screening have shown consistent activity against all species and stages
of Leishmania in vitro although improvements in selectivity are needed. These
compounds represent viable starting points for further optimization as
antileishmanial agents. A series of novel diastereoisomeric σ ligands 3 was designed, synthesized and
pharmacologically evaluated. The highly rigid [4.3.3]propellane scaffold was
used to fix the three dimensional orientation of the pharmacophoric moieties
required for σ affinity. The syn,syn-configured aminocarbamate syn,syn-3a
reveals the most promising σ₁ affinity (Ki = 77 nM) and selectivity over the σ₂
subtype (21-fold). The σ₂ affinity of all four diastereomers 3 was in the low
micromolar range. Analysis of the distance between the hydrophobic regions
(phenyl moieties) of the diastereomers 3 led to the longest range of distances
(10.3-15.2 Å) for the most potent σ₁ ligand syn,syn-3a, which is in good
agreement with pharmacophore models. Among the different mammalian isoforms of Carbonic Anhydrase, the hCA VII is
mainly expressed in the brain where it is involved in several neurological
diseases. Thereby hCA VII has been validated as an attractive target for the
discovery of selective inhibitors for the treatment of epilepsy and neurological
pain. To identify new chemical entities as carbonic anhydrase inhibitors (CAIs)
targeting hCA VII, we used a structure-based approach. By means of LigandScout
software we built pharmacophore models from crystal structures of two well-known
CAIs in complex with hCA VII. A merged pharmacophore hypothesis has been
obtained. Subsequently, a focused library of compounds was screened against
pharmacophore model and the most interesting hits were docked into the crystal
structure of hCA VII. As a result, we identified new compounds displaying
significant CA inhibitory effects in the omolar range. Tankyrases (TNKS1 and TNKS2) are proteins in the poly ADP-ribose polymerase
(PARP) family. They have been shown to directly bind to axin proteins, which
negatively regulate the Wnt pathway by promoting β-catenin degradation.
Inhibition of tankyrases may offer a novel approach to the treatment of
APC-mutant colorectal cancer. Hit compound 8 was identified as an inhibitor of
tankyrases through a combination of substructure searching of the Amgen compound
collection based on a minimal binding pharmacophore hypothesis and
high-throughput screening. Herein we report the structure- and property-based
optimization of compound 8 leading to the identification of more potent and
selective tankyrase inhibitors 22 and 49 with improved pharmacokinetic
properties in rodents, which are well suited as tool compounds for further in
vivo validation studies. |
How Flaviviridae family of viruses infects vertebrates? | A wide range of about 500 different viruses is transmitted by arthropods such as ticks, mosquitoes and sandflies. These arboviruses multiply in the arthropod vector, and for each virus there is a natural cycle involving vertebrates (various birds or mammals) and arthropods. The virus enters the arthropod when the latter takes a blood meal from the infected vertebrate, and passes through the gut wall to reach the salivary gland where replication takes place. Once this has occurred, 1–2 weeks after ingesting the virus, the arthropod becomes infectious, and can transmit the virus to another vertebrate during a blood meal. Certain arboviruses that infect ticks are also transmitted directly from adult tick to egg (transovarial transmission), so that future generations of ticks are infected without the need for a vertebrate host. | At least 27 alphaviruses and 68 flaviviruses have been recognized, approximately
one-third of which are medically important human pathogens. They vary widely in
their basic ecology; each virus occupies a distinct ecologic niche, often with
restricted geographic and biologic distribution. As shown in Tables 54-1 and
54-2, alphaviruses and flaviviruses can cause various syndromes, ranging from
benign febrile illnesses to severe systemic diseases with hemorrhagic
manifestations or major organ involvement. The neurotropic alphaviruses and
flaviviruses can produce severe destructive central nervous system disease with
serious sequelae. Several alphaviruses (chikungunya, Mayaro, and Ross River)
cause painful arthritis that persists for weeks or months after the initial
febrile illness. Yellow fever virus has unique hepatotropic properties that
cause a clinically and pathologically distinct form of hepatitis with a
hemorrhagic diathesis. The dengue viruses, which cause more human illness than
all other members of their family, may produce a serious, sometimes fatal,
immunopathologic disease in which shock and hemorrhage occur. Hepatitis C virus
(Chapter 70) may be a flavivirus. Alphavirus is one of the two genera in the
family Togaviridae; the other genus (Rubivirus) has rubella virus (Chapter 55)
as its only member. Flavivirus, once classified in the Togaviridae, now
constitutes one of three genera in the family Flaviviridae; the other two genera
are Pestivirus and “Hepatitis C-like viruses”. Pestivirus includes animal
pathogens (bovine viral diarrhea and hog cholera viruses) that are of
considerable economic importance, but contains no known human pathogens.
Hepatitis C virus is described in Chapter 70. All alphaviruses and flaviviruses
that cause disease in humans are arthropod-borne viruses (arboviruses). In the
original classification scheme based on antigenic relationships, alphaviruses
and flaviviruses were termed group A and group B arboviruses, respectively. Most
alphaviruses and flaviviruses survive in nature by replicating alternately in a
vertebrate host and a hematophagous arthropod (mosquitoes or, for some
flaviviruses, ticks). Arthropod vectors acquire the viral infection by biting a
viremic host, and after an extrinsic incubation period during which the virus
replicates in the vector's tissues, they transmit virus through salivary
secretions to another vertebrate host. Virus replicates in the vertebrate host,
causing viremia and sometimes illness. The ability to infect and replicate in
both vertebrate and arthropod cells is an essential quality of alphaviruses and
flaviviruses. The principal vertebrate hosts for most are various species of
wild mammals or birds. The natural zoonotic cycles that maintain the virus do
not usually involve humans. However, a few viruses (yellow fever virus, dengue
virus types 1, 2, 3 and 4 and chikungunya virus) can be transmitted in a
human-mosquito-human cycle. As a result of being pathogenic for humans and
capable of transmission in heavily populated areas, these viruses can cause
widespread and serious epidemics. Because of their high transmission potential,
these viruses are major public health problems in many tropical and subtropical
regions of the world where appropriate mosquito vectors are present. Because
some of these agents are dangerous human pathogens and are highly infectious,
special containment and safety precautions in the laboratory are required. The focal distribution of tick-borne encephalitis virus (TBEV; Flaviviridae,
Flavivirus) appears to depend mainly on cofeeding transmission between infected
Ixodes ricinus L. nymphs and uninfected larvae. To better understand the role of
cofeeding ticks in the transmission of TBEV, we investigated tick infestation of
rodents and the influence of microclimate on the seasonality of questing I.
ricinus ticks. A 3-yr study was carried out at four sites, including two
confirmed TBEV foci. Free-living ticks and rodents were collected monthly, and
microclimatic data were recorded. A decrease in questing nymph density was
observed in 2007, associated with low relative humidity and high temperatures in
spring. One site, Thun, did not show this decrease, probably because of
microclimatic conditions in spring that favored the questing nymph population.
During the same year, the proportion of rodents carrying cofeeding ticks was
lower at sites where the questing nymph density decreased, although the
proportion of infested hosts was similar among years. TBEV was detected in 0.1%
of questing ticks, and in 8.6 and 50.0% of larval ticks feeding on two rodents.
TBEV was detected at all but one site, where the proportion of hosts with
cofeeding ticks was the lowest. The proportion of hosts with cofeeding ticks
seemed to be one of the factors that distinguished a TBEV focus from a non-TBEV
focus. The enzootic cycle of TBEV might be disrupted when dry and hot springs
occur during consecutive years. TO THE EDITOR: West Nile virus (WNV) is a member of the genus Flavivirus within
the Japanese encephalitis antigenic complex. The enzootic virus cycle involves
transmission between avian hosts and ornithophilic mosquitoes, whereas humans
and horses are considered dead-end hosts. Given the recent increase of WNV
infection in humans and horses in Europe, concern has been raised regarding
public and animal health. West Nile virus (WNV) (Flaviviridae: Flavivirus) is transmitted from mosquitoes
to birds, but can cause fatal encephalitis in infected humans. Since its
introduction into North America in New York in 1999, it has spread throughout
the western hemisphere. Multiple outbreaks have also occurred in Europe over the
last 20 years. This review highlights recent efforts to understand how host
pressures impact viral population genetics, genotypic and phenotypic changes
which have occurred in the WNV genome as it adapts to this novel environment,
and molecular epidemiology of WNV worldwide. Future research directions are also
discussed. BACKGROUND: Knowledge of spatial patterns of dengue virus (DENV) infection is
important for understanding transmission dynamics and guiding effective disease
prevention strategies. Because movement of infected humans and mosquito vectors
plays a role in the spread and persistence of virus, spatial dimensions of
transmission can range from small household foci to large community clusters.
Current understanding is limited because past analyses emphasized clinically
apparent illness and did not account for the potentially large proportion of
inapparent infections. In this study we analyzed both clinically apparent and
overall infections to determine the extent of clustering among human DENV
infections.
METHODOLOGY/PRINCIPAL FINDINGS: We conducted spatial analyses at global and
local scales, using acute case and seroconversion data from a prospective
longitudinal cohort in Iquitos, Peru, from 1999-2003. Our study began during a
period of interepidemic DENV-1 and DENV-2 transmission and transitioned to
epidemic DENV-3 transmission. Infection status was determined by seroconversion
based on plaque neutralization testing of sequential blood samples taken at
approximately six-month intervals, with date of infection assigned as the
middate between paired samples. Each year was divided into three distinct
seasonal periods of DENV transmission. Spatial heterogeneity was detected in
baseline seroprevalence for DENV-1 and DENV-2. Cumulative DENV-3 seroprevalence
calculated by trimester from 2001-2003 was spatially similar to preexisting
DENV-1 and DENV-2 seroprevalence. Global clustering (case-control Ripley's K
statistic) appeared at radii of ∼200-800 m. Local analyses (Kuldorf spatial scan
statistic) identified eight DENV-1 and 15 DENV-3 clusters from 1999-2003. The
number of seroconversions per cluster ranged from 3-34 with radii from zero (a
single household) to 750 m; 65% of clusters had radii >100 m. No clustering was
detected among clinically apparent infections.
CONCLUSIONS/SIGNIFICANCE: Seroprevalence of previously circulating DENV
serotypes can be a predictor of transmission risk for a different invading
serotype and, thus, identify targets for strategically placed surveillance and
intervention. Seroprevalence of a specific serotype is also important, but does
not preclude other contributing factors, such as mosquito density, in
determining where transmission of that virus will occur. Regardless of the
epidemiological context or virus serotype, human movement appears to be an
important factor in defining the spatial dimensions of DENV transmission and,
thus, should be considered in the design and evaluation of surveillance and
intervention strategies. A total of 54,673 mosquitoes were collected at 11 sites located near the
China-Myanmar border in the western part of Yun Province during July and
August 2007. There were 29 species in 4 genera identified from the collections,
including 12 species of Culex, 12 species of Anopheles, 3 species of Aedes, and
2 species of Armigeres. Culex tritaeniorhynchus Giles (67.9%, 37,119/54,673) and
Anopheles sinensis Wiedemann (25.9%, 14,170/54,673) were the most abundant
species in this investigation. Virus was isolated using BHK-21 and C6/36 cells
from 22 of 510 mosquito pools. Isolates included Japanese encephalitis virus
(JEV) and Getah virus (GETV), which were identified by serological and molecular
methods. Twenty JEV strains were isolated from Cx. tritaeniorhynchus (15
isolates), An. sinensis (3 isolates), and Armigeres subalbatus Coquillett (2
isolates), and 2 GETV strains were isolated from Culex pseudovishnui Colless and
Cx. tritaeniorhynchus. This study suggests that Ar. subalbatus is a potentially
important local vector because of the high JEV infection ratio found in this
species. Enzootic JEV transmission persists in this area and therefore,
surveillance for human disease caused by JEV and GETV should be conducted in the
region. After the acute infection period, birds persistently infected with West Nile
virus (family Flaviviridae, genus Flavivirus, WNV) occasionally shed virus into
the bloodstream, but these virions normally are inactivated by neutralizing
antibody. The current work tested the hypothesis that these host neutralizing
antibodies protect mosquito vectors from WNV infection and reevaluated the
minimum WNV infectious dose necessary to infect Culex tarsalis Coquillett. To
determine whether host antibodies protect mosquitoes from infection, Cx.
tarsalis and Culex stigmatosoma Dyar were fed bloodmeals containing avian blood,
WNV, and sera with or without WNV-specific neutralizing antibodies. When viral
particles were completely bound by antibody, mosquitoes were protected from
infection; however, when incompletely bound, WNV titers as low as 10(2.3)
plaque-forming units (pfu)/ml resulted in 5% infection. These data indicated
that avian antibodies were protective to mosquito vectors and were not
dissociated during digestion. Because recrudescent viremias may not attain the
same magnitude as initial acute viremias, Cx. tarsalis vector competence was
reevaluated focusing on the fate of low-titered bloodmeals. Females were
evaluated for vector competence after ingesting bloodmeals containing 10(2.2),
10(3.4), 10(4.5), 10(5.5), or 10(6.5) WNV pfu/ml. Infection increased with
bloodmeal titer, with 1% of the mosquitoes ingesting 10(3.4) pfu/ml and 45% of
the mosquitoes ingesting 10(6.5) pfu/ml developing disseminated infections. The
incomplete neutralization of recrudescent virus may be sufficient to infect a
low proportion of competent blood-feeding Culex mosquitoes and perhaps allow
persistently infected birds to provide a mechanism for arbovirus overwintering. West Nile virus (WNV) is a zoonotic arthropod-borne pathogen with continued
geographical expansion in Europe. We present and evaluate data on the temporal,
spatial and bird species focus of the WNV surveillance programme in dead wild
birds in Great Britain (2002-2009). During this period all bird samples tested
negative for WNV. Eighty-two per cent of the 2072 submissions occurred during
the peak period of vector activity with 53% tested during April-July before
human and equine infection would be expected. Samples were received from every
county, but there was significant geographical clustering (nearest neighbour
index=0·23, P<0·001). Over 240 species were represented, with surveillance more
likely to detect WNV in resident bird species (92% of submissions) than migrants
(8%). Evidence indicates that widespread avian mortality is not generally a
reported feature of WNV in Europe and hence additional activities other than
dead bird surveillance may maximize the ability to detect WNV circulation before
the onset of human and equine infections. Yellow fever virus (YFV) is historically one of the most important viruses to
affect human populations. Despite the existence of highly effective vaccines for
over 70 years, yellow fever remains a significant and re-emerging cause of
morbidity and mortality in endemic and high-risk regions of South America and
Africa. The virus may be maintained in sylvatic enzootic/epizootic, transitional
and urban epidemic transmission cycles with geographic variation in terms of
levels of genetic diversity, the nature of transmission cycles and patterns of
outbreak activity. In this review we consider evolutionary and ecological
factors underlying YFV emergence, maintece and spread, geographic
distribution and patterns of epizootic/epidemic activity. Powassan virus (POWV) is a rare tick-borne agent of encephalitis in North
America. Historically, confirmed cases occurred mainly in the northeastern
United States. Since 2008, confirmed cases in Minnesota and Wisconsin have
increased. We report a fatal case of POWV encephalitis in Minnesota. POWV
infection should be suspected in tick-exposed patients with viral encephalitis. BACKGROUND: Hepatitis C virus infection (HCV) is not infrequent among
haemodialysis patients. Most published reports suggest that patient-to-patient
spread, either directly or indirectly, is the most common mode of transmission
in renal units.
AIM: To investigate the source of an outbreak, and the route of transmission, of
acute HCV infection in two Scottish patients occurring within eight weeks of
receiving haemodialysis in the same unit while on holiday in Majorca.
METHODS: This was an international epidemiological and molecular investigation
of HCV infection among a cohort of haemodialysis patients from nine countries.
FINDINGS: No further HCV-positive infections were observed among residents and
holidaymakers receiving haemodialysis at the unit in Majorca. Molecular
investigations confirmed that a Spanish healthcare worker (HCW) was the source
of infection for the two Scottish patients. The investigators were unable to
determine the route of transmission.
CONCLUSIONS: This outbreak is the first reported case of HCW-to-patient
transmission of HCV in a renal unit, and the third reported case of transmission
involving a HCW who had not performed invasive procedures. The issue of whether
renal units are an exceptional case with regards to the risk of transmission
associated with non-invasive procedures should be considered, in conjunction
with the need to improve surveillance of blood-borne virus transmissions in
renal units in the UK and abroad. Dengue and West Nile viruses are enveloped RNA viruses that belong to genus
Flavivirus (family Flaviviridae) and are considered important mosquito-borne
viral pathogenic agents worldwide. A potential target for intervention
strategies is the virus cell entry mechanism. Previous studies of flavivirus
entry have focused on the effects of biochemical and molecular inhibitors on
viral entry leading to controversial conclusions suggesting that the process is
dependent upon endocytosis and low pH mediated membrane fusion. In this study we
analyzed the early events in the infection process by means of electron
microscopy and immuno-gold labeling of viral particles during cell entry, and
used as a new approach for infecting cells with viruses obtained directly from
mosquitoes. The results show that Dengue and West Nile viruses may infect cells
by a mechanism that involves direct penetration of the host cell plasma membrane
as proposed for alphaviruses. OBJECTIVE: To simulate the probability of HCV transmission from an HCV
seropositive index patient to susceptible household contacts through non-sexual
exposures.
METHODS: A modified Reed-Frost stochastic simulation model was used to assess
the probability of HCV transmission from an HCV seropositive index patient to
susceptible household contacts through non-sexual exposures. This mathematical
model does not require the specification of infection onset times for
individual, nor is it necessary to identify the chains of household infections.
Therefore, this model can be used with serologic data on detected asymptomatic
infections. The HCV serological data on 341 non-sexual household contacts of 86
HCV seropositive index patients were used in this simulation study. The
frequency distribution of HCV infection of susceptibles for each household size
of 4-8 initial susceptibles was calculated. A maximum likelihood procedure was
used to estimate the non-sexual household transmission parameter for HCV
infection for the range of household sizes studied and was used in 1000
stochastic iterations. The goodness-of-fit test was carried out to compare the
observed proportions of households where HCV transmission occurred to one or
more initial susceptible with mean expected simulated proportions of such
households with varying sizes ranging from 4 to 8 initial susceptibles.
RESULTS: The maximum likelihood estimates (90% probability interval (PI)) of
binomial probability of HCV transmission within households with varying number
of initial susceptible non-sexual household contacts ranged from 0.248 (90%PI:
0.031, 0.560) to 0.164 (90%PI: 0.011, 0.440) for household size of 4 and 8
respectively. The χ(2) goodness-of-fit test of observed and mean expected
simulated proportions of households wherein at least one of the susceptibles was
infected revealed good fit for households of all sizes examined (P ≥ 0.96). In a
household, the probability of HCV transmission from the index HCV seropositive
patient to susceptible via non-sexual contacts tended to decrease linearly as
the household size increased from four to seven.
CONCLUSION: Intra-household HCV transmission through non-sexual contacts may
have substantial impact on HCV transmission and needs to be considered in an HCV
control program. Dengue is a mosquito-borne disease caused by four closely related dengue virus
(genus Flavivirus)serotypes (DENV-1–4). The clinical outcomes vary from mild
febrile illness to life-threatening haemorrhagic manifestations. DENVs are
endemic in the tropics and subtropics globally and currently no specific
treatment or vaccines are available. In Venezuela, the American-Asian genotype
of DENV-2 is the most prevalent and has been associated with severe disease
outcomes.We aimed to follow-up the molecular epidemiology of DENV-2 in Venezuela
to investigate if the evolution of the virus has remained the same throughout
time or if the same dynamics documented in Brazil (hyperendemic co-circulation)
also occurred. The results show that whereas the epidemiology of DENV in several
endemic areas is characterized by serotype replacements through time, in
Venezuela the American-Asian genotype DENV-2 has evolved into several genetic
lineages and has remained in hyperendemic co-circulation with the other
serotypes. BACKGROUND: Hepatitis C virus (HCV) transmission among people who inject drugs
remains a challenging public health problem. We investigated the risk of HCV
transmission by analyzing the direct association of HCV with filters, water to
dilute drugs, and water containers.
METHODS: Experiments were designed to replicate practices by people who inject
drugs and include routinely used injection equipment. HCV stability in water was
assessed by inoculation of bottled water with HCV. Viral association with
containers was investigated by filling the containers with water, inoculating
the water with HCV, emptying the water, and refilling the container with fresh
water. Transmission risk associated with drug preparation filters was determined
after drawing virus through a filter and incubating the filter to release
infectious particles.
RESULTS: HCV can survive for up to 3 weeks in bottled water. Water containers
present a risk for HCV transmission, as infectious virions remained associated
with water containers after washing. Physical properties of the water containers
determined the degree of HCV contamination after containers were refilled with
water. HCV was also associated with filter material, in which around 10% of the
viral inoculum was detectable.
CONCLUSIONS: This study demonstrates the potential risk of HCV transmission
among injection drug users who share water, filters, and water containers and
will help to define public health interventions to reduce HCV transmission. BACKGROUND: The genus Flavivirus currently consists of approximately 80
single-strand positive-sense RNA viruses. These replicate in a range of hosts
including myriad vertebrate, insect, and tick species. As a consequence of this
broad host range, the majority of flaviviruses can be propagated in most
vertebrate and insect cell cultures. This ability to infect arthropods and
vertebrates usually is essential for maintece of these viruses in nature. But
recently, there has been the discovery of a number of flaviviruses that infect
mosquitoes but not vertebrates. It remains largely unknown why certain
flaviviruses infect vertebrates and mosquitoes while others infect mosquitoes or
vertebrates exclusively.
METHODS: Here, we initiated in vitro host range studies of Rabensburg virus
(RABV), an intermediate between the mosquito-specific and horizontally
transmitted flaviviruses, to provide information on the factor(s) that underlie
the varying host range of flaviviruses. RABV is an intermediate between the
mosquito-specific and horizontally transmitted flaviviruses because it does not
infect mammalian or avian cell cultures, house sparrows, or chickens, but it
does share genetic characteristics with the Japanese Encephalitis serogroup of
flaviviruses.
RESULTS: In vitro growth kinetic assays revealed the complete abrogation of RABV
growth on Vero and E6 cells incubated at temperatures 35°C and higher, but
surprisingly RABV infected, replicated efficiently, and displayed overt
cytopathic effects (CPE) on Vero and E6 cell cultures incubated below 35°C. In
contrast, RABV was fully viable, replicated efficiently, and displayed overt CPE
on C6/36 cells incubated at 28°C or 37°C, thus implicating temperature as an
important factor limiting the host range of RABV.
CONCLUSIONS: These data are critical for further study to more fully identify
the determits that mediate the evolution of biological transmission among
flaviviruses. It also will be useful for studies that look to provide a
comprehensive molecular definition of flavivirus-host cell interactions. And it
will provide a cadre of information to design wet lab experiments to investigate
the genetic changes that facilitate host switching, which may lead to new
vertebrate pathogens or transmission pathways. PURPOSE OF REVIEW: Increasing evidence has emerged for permucosal transmission
of hepatitis C amongst HIV-infected MSM.
RECENT FINDINGS: A rising incidence of acute hepatitis C virus (HCV) in
HIV-infected MSM has been observed since 2000 in Europe, Australia, USA and
Asia. Transmission appears to occur through the permucosal rather than the more
usual parenteral route. Although often multifactorial, permucosal risk factors
can be classified as behavioural (sexual practices and mucosally administered
drugs) and biological (HIV and sexually transmitted infections). This review
will describe the epidemiology of HCV infection in this cohort. Current and
future treatment strategies will also be outlined in the context of novel,
orally bioavailable, directly acting antiviral therapies.
SUMMARY: An improved understanding of HCV epidemiology will allow implementation
of more effective public health interventions to limit onward transmission of
HCV. BACKGROUND: Dengue is the most important vector-borne disease in many different
parts of the world and is expanding into other areas of the globe without
hindrance. The morbidity and mortality due to dengue complications are
increasing globally at an alarming rate. Although transmission of the dengue
virus has been documented in well-characterized areas of Pakistan, its incidence
in Khyber Pakhtunkhawa has not been characterized. To address this issue we
aimed to determine the seroprevalence of dengue (IgM and IgG) antibodies and the
disease symptoms in the population of Khyber Pakhtunkhawa, and to investigate
the incidence of dengue fever in different seasons and in urban as well as in
rural areas.
METHODS: From August to October 2011, data of suspected dengue patients were
collected from different primary, secondary, and tertiary collection centers
situated in Khyber Pakhtunkhawa in order to determine the actual seroprevalence
of dengue antibodies (IgM and IgG) in Khyber Pakhtunkhawa.
RESULTS: A total 612 subjects with a suspected infection were enrolled in our
study. Of the 612 suspected cases, 319 were found positive for dengue IgG, IgM,
or both IgG and IgM. The overall weighted prevalence of dengue-specific
antibodies (IgM and/or IgG) was 52.12%. Overall, of the 52.12%, 31.86% (95%
confidence interval (CI) 28.17-35.55) were positive for dengue IgM and 20.26%
(95% CI 17.03-23.39) were positive for dengue IgG. Only 23 (3.75%) samples
showed both IgG and IgM antibodies. A higher prevalence of IgM (39.35%, 95% CI
32.84-45.86) and IgG (22.42%, 95% CI 16.86-27.98) antibodies was found in the
age group 21-30 years as compared to the children age group (≤10 years) and the
oldest age group (≥51 years). The mean age of the febrile cohort was 53.16 ±
44.22 years, ranging from 4 to 85 years. Age group was not statistically
associated with IgM (p=0.64) or IgG (p=0.49) positivity. A higher seroprevalence
of IgM (37.24%, 95%CI 32.84-45.86) was observed in males as compared to females
(IgM 17.88%, 95% CI 11.11-24.65) while higher seroprevalnce of IgG (22.76%, 95%
CI 15.35-30.17) was seen in females as compared to males (IgG 17.58%, 95% CI
14.21-20.95). Gender was not significantly associated with IgM (p=0.06) or IgG
(p=0.53) positivity. Dengue IgM (35.38%, 95% CI 38.61-62.91) and IgG (50.76%,
95% CI 38.61-62.91) were higher in patients who had a history of travel to a
dengue endemic area as compared to those who did not (IgM 33%, 95% CI
29.06-36.94, and IgG 15%, 95% CI 12.01-17.99). History of travel to an endemic
area was significantly associated with IgM (p=0.023) and IgG (p=0.041)
positivity. A higher incidence of IgM (41.13%, 95% CI 35.55-46.71) and IgG
(27.42%, 95% CI 22.36-32.48) was observed in urban areas than in rural areas
(IgM 23%, 95% CI 18.34- 27.66, and IgG 13.41%, 95% CI 9.63-17.19). IgM
(p=0.0005) and IgG (p=0.0007) positivity was significantly associated with area
of residence. Symptoms including fever (p=0.007), headache (p=0.001), Skin rash
(0.005), joint pain (0.004) and Fatigue were significantly linked to dengue
fever. IgM and IgG antibodies were more frequently seen in the post-monsoon
season (68.33%) than in the monsoon period (31.68%). The death ratio in the
overall weighted prevalence was 2.19%.
CONCLUSION: The results of the present cohort study of febrile subjects show
that young people and males are more susceptible to dengue fever. Dengue
infection was most prominent in the post-monsoon season, in urban areas, and in
patients with a history of travel to an endemic locality. Furthermore seven
deaths were found in our cohort study. West Nile virus (WNV), a mosquito-borne flavivirus in the Japanese encephalitis
antigenic group, has caused sporadic outbreaks in humans, horses and birds
throughout many of the warmer regions of Europe for at least 20 years.
Occasional cases of West Nile encephalitis have also been associated with
infected blood transfusions and organ donations. Currently, WNV appears to be
expanding its geographical range in Europe and causing increasing numbers of
epidemics/outbreaks associated with human morbidity and mortality. This brief
review reports on the current epidemic situation regarding WNV in Europe,
highlighting the clinical, diagnostic and preventive measures available for
controlling this apparently emerging human pathogen. West Nile virus (WNV), an arbovirus maintained in a bird-mosquito enzootic
cycle, can infect other vertebrates including humans. WNV was first reported in
the US in 1999 where, to date, three genotypes belonging to WNV lineage I have
been described (NY99, WN02, SW/WN03). We report here the WNV sequences obtained
from two birds, one mosquito, and 29 selected human samples acquired during the
US epidemics from 2006-2011 and our examination of the evolutionary dynamics in
the open-reading frame of WNV isolates reported from 1999-2011.
Maximum-likelihood and Bayesian methods were used to perform the phylogenetic
analyses and selection pressure analyses were conducted with the HyPhy package.
Phylogenetic analysis identified human WNV isolates within the main WNV
genotypes that have circulated in the US. Within genotype SW/WN03, we have
identified a cluster with strains derived from blood donors and birds from Idaho
and North Dakota collected during 2006-2007, termed here MW/WN06. Using
different codon-based and branch-site selection models, we detected a number of
codons subjected to positive pressure in WNV genes. The mean nucleotide
substitution rate for WNV isolates obtained from humans was calculated to be
5.06×10(-4) substitutions/site/year (s/s/y). The Bayesian skyline plot shows
that after a period of high genetic variability following the introduction of
WNV into the US, the WNV population appears to have reached genetic stability.
The establishment of WNV in the US represents a unique opportunity to understand
how an arbovirus adapts and evolves in a naïve environment. We describe a novel,
well-supported cluster of WNV formed by strains collected from humans and birds
from Idaho and North Dakota. Adequate genetic surveillance is essential to
public health since new mutants could potentially affect viral pathogenesis,
decrease performance of diagnostic assays, and negatively impact the efficacy of
vaccines and the development of specific therapies. Birds, particularly passerines, can be parasitized by Ixodid ticks, which may be
infected with tick-borne pathogens, like Borrelia spp., Babesia spp., Anaplasma,
Rickettsia/Coxiella, and tick-borne encephalitis virus. The prevalence of ticks
on birds varies over years, season, locality and different bird species. The
prevalence of ticks on different species depends mainly on the degree of feeding
on the ground. In Europe, the Turdus spp., especially the blackbird, Turdus
merula, appears to be most important for harboring ticks. Birds can easily cross
barriers, like fences, mountains, glaciers, desserts and oceans, which would
stop mammals, and they can move much faster than the wingless hosts. Birds can
potentially transport tick-borne pathogens by transporting infected ticks, by
being infected with tick-borne pathogens and transmit the pathogens to the
ticks, and possibly act as hosts for transfer of pathogens between ticks through
co-feeding. Knowledge of the bird migration routes and of the spatial
distribution of tick species and tick-borne pathogens is crucial for
understanding the possible impact of birds as spreaders of ticks and tick-borne
pathogens. Successful colonization of new tick species or introduction of new
tick-borne pathogens will depend on suitable climate, vegetation and hosts.
Although it has never been demonstrated that a new tick species, or a new tick
pathogen, actually has been established in a new locality after being seeded
there by birds, evidence strongly suggests that this could occur. Mosquito feeding behavior determines the degree of vector-host contact and may
have a serious impact on the risk of pathogen transmission, including that of
the West Nile virus (WNV). To measure the role of Culex mosquitoes as WNV
vectors, host-seeking females were collected using animal-baited traps
containing live birds (quail) or mammals (rabbits) and CO2-baited Center for
Disease Control and Prevention traps placed in several wetland areas in the
Czech Republic. Culex pipiens (L.) and Culex modestus (F.) were the most
frequently collected species. Although Cx. modestus did not distinguish between
baits, Cx. pipiens was collected significantly more frequently in bird-baited
traps. Based on mitochondrial DNA analysis of bloodmeals from engorged females
collected by CO2-baited traps situated within reed beds, a diverse group of
birds were the predomit hosts (93.7%), followed by mammals (4.2%) including
humans, and amphibians (2.1%). Among birds, Anseriformes were fed upon most
frequently by Cx. modestus, whereas Cx. pipiens fed most frequently on
Passeriformes. To measure the infection risk and confirm the distribution of
mosquito species in various biotopes, transects of CO2-baited CDC traps were
operated from wetland reed beds into upland vegetated areas. Even though both
Culex species occurred in all biotopes sampled and frequently dispersed hundreds
of meters away from fishpond shore vegetation, the spatial distribution of Cx.
modestus was significantly associated with reed beds at wetlands. The first
detection of WNV (subtype RabV) in Cx. modestus in Bohemia and confirmation of
WNV presence in Cx. pipiens in Moravia together with observed feeding behavior
supports the presumed role of both Culex species in the avian-to-avian enzootic
WNV cycle and in avian-to-mammal transmission in the Czech Republic. West Nile virus (WNV) is a neurotropic flavivirus that cycles between mosquitoes
and birds but that can also infect humans, horses, and other vertebrate animals.
In most humans, WNV infection remains subclinical. However, 20%-40% of those
infected may develop WNV disease, with symptoms ranging from fever to
meningoencephalitis. A large variety of WNV strains have been described
worldwide. Based on their genetic differences, they have been classified into
eight lineages; the pathogenic strains belong to lineages 1 and 2. Ten years
ago, Beasley et al. (2002) found that dramatic differences exist in the
virulence and neuroinvasion properties of lineage 1 and lineage 2 WNV strains.
Further insights on how WNV interacts with its hosts have recently been gained;
the virus acts either at the periphery or on the central nervous system (CNS),
and these observed differences could help explain the differential virulence and
neurovirulence of WNV strains. This review aims to summarize the current state
of knowledge on factors that trigger WNV dissemination and CNS invasion as well
as on the inflammatory response and CNS damage induced by WNV. Moreover, we will
discuss how WNV strains differentially interact with the innate immune system
and CNS cells, thus influencing WNV pathogenesis. |
What was the aim of the HAMLET clinical trial? | The aim of the HAMLET (Hemicraniectomy After Middle Cerebral Artery Infarction With Life-Threatening Edema Trial) clinical trial was to compare the efficacy of decompressive surgery to improve functional outcome with that of conservative treatment in patients with space-occupying supratentorial infarction. | Patients with a hemispheric infarct and massive space-occupying brain oedema
have a poor prognosis. Despite intensive conservative treatment, the case
fatality rate may be as high as 80%, and most survivors are left severely
disabled. Non-randomised studies suggest that decompressive surgery
substantially reduces mortality and improves the functional outcome of
survivors. The 'Hemicraniectomy after middle cerebral artery infarction with
life-threatening edema trial' (HAMLET) is a newly-conceived randomised
multi-centre clinical trial that compares the efficacy of decompressive surgery
to improve functional outcome with that of conservative treatment in patients
with space-occupying supratentorial infarction. BACKGROUND: Patients with a hemispheric infarct and massive space-occupying
brain oedema have a poor prognosis. Despite maximal conservative treatment, the
case fatality rate may be as high as 80%, and most survivors are left severely
disabled. Non-randomised studies suggest that decompressive surgery reduces
mortality substantially and improves functional outcome of survivors. This study
is designed to compare the efficacy of decompressive surgery to improve
functional outcome with that of conservative treatment in patients with
space-occupying supratentorial infarction
METHODS: The study design is that of a multi-centre, randomised clinical trial,
which will include 112 patients aged between 18 and 60 years with a large
hemispheric infarct with space-occupying oedema that leads to a decrease in
consciousness. Patients will be randomised to receive either decompressive
surgery in combination with medical treatment or best medical treatment alone.
Randomisation will be stratified for the intended mode of conservative treatment
(intensive care or stroke unit care). The primary outcome measure will be
functional outcome, as determined by the score on the modified Rankin Scale, at
one year. BACKGROUND: Maligt infarction of the middle cerebral artery (MCA) is
associated with an 80% mortality rate. Non-randomised studies have suggested
that decompressive surgery reduces this mortality without increasing the number
of severely disabled survivors. To obtain sufficient data as soon as possible to
reliably estimate the effects of decompressive surgery, results from three
European randomised controlled trials (DECIMAL, DESTINY, HAMLET) were pooled.
The trials were ongoing when the pooled analysis was planned.
METHODS: Individual data for patients aged between 18 years and 60 years, with
space-occupying MCA infarction, included in one of the three trials, and treated
within 48 h after stroke onset were pooled for analysis. The protocol was
designed prospectively when the trials were still recruiting patients and
outcomes were defined without knowledge of the results of the individual trials.
The primary outcome measure was the score on the modified Rankin scale (mRS) at
1 year dichotomised between favourable (0-4) and unfavourable (5 and death)
outcome. Secondary outcome measures included case fatality rate at 1 year and a
dichotomisation of the mRS between 0-3 and 4 to death. Data analysis was done by
an independent data monitoring committee.
FINDINGS: 93 patients were included in the pooled analysis. More patients in the
decompressive-surgery group than in the control group had an mRS<or=4 (75%vs
24%; pooled absolute risk reduction 51% [95% CI 34-69]), an mRS<or=3 (43%vs 21%;
23% [5-41]), and survived (78%vs 29%; 50% [33-67]), indicating numbers needed to
treat of two for survival with mRS<or=4, four for survival with mRS<or=3, and
two for survival irrespective of functional outcome. The effect of surgery was
highly consistent across the three trials.
INTERPRETATION: In patients with maligt MCA infarction, decompressive surgery
undertaken within 48 h of stroke onset reduces mortality and increases the
number of patients with a favourable functional outcome. The decision to perform
decompressive surgery should, however, be made on an individual basis in every
patient. BACKGROUND: Patients with space-occupying hemispheric infarctions have a poor
prognosis, with case fatality rates of up to 80%. In a pooled analysis of
randomised trials, surgical decompression within 48 h of stroke onset reduced
case fatality and improved functional outcome; however, the effect of surgery
after longer intervals is unknown. The aim of HAMLET was to assess the effect of
decompressive surgery within 4 days of the onset of symptoms in patients with
space-occupying hemispheric infarction.
METHODS: Patients with space-occupying hemispheric infarction were randomly
assigned within 4 days of stroke onset to surgical decompression or best medical
treatment. The primary outcome measure was the modified Rankin scale (mRS) score
at 1 year, which was dichotomised between good (0-3) and poor (4-6) outcome.
Other outcome measures were the dichotomy of mRS score between 4 and 5, case
fatality, quality of life, and symptoms of depression. Analysis was by intention
to treat. This trial is registered, ISRCTN94237756.
FINDINGS: Between November, 2002, and October, 2007, 64 patients were included;
32 were randomly assigned to surgical decompression and 32 to best medical
treatment. Surgical decompression had no effect on the primary outcome measure
(absolute risk reduction [ARR] 0%, 95% CI -21 to 21) but did reduce case
fatality (ARR 38%, 15 to 60). In a meta-analysis of patients in DECIMAL
(DEcompressive Craniectomy In MALigt middle cerebral artery infarction),
DESTINY (DEcompressive Surgery for the Treatment of maligt INfarction of the
middle cerebral arterY), and HAMLET who were randomised within 48 h of stroke
onset, surgical decompression reduced poor outcome (ARR 16%, -0.1 to 33) and
case fatality (ARR 50%, 34 to 66).
INTERPRETATION: Surgical decompression reduces case fatality and poor outcome in
patients with space-occupying infarctions who are treated within 48 h of stroke
onset. There is no evidence that this operation improves functional outcome when
it is delayed for up to 96 h after stroke onset. The decision to perform the
operation should depend on the emphasis patients and relatives attribute to
survival and dependency. BACKGROUND AND PURPOSE: We assessed whether the effects of surgical
decompression for space-occupying hemispheric infarction, observed at 1 year,
are sustained at 3 years.
METHODS: Patients with space-occupying hemispheric infarction, who were enrolled
in the Hemicraniectomy After Middle cerebral artery infarction with
Life-threatening Edema Trial within 4 days after stroke onset, were followed up
at 3 years. Outcome measures included functional outcome (modified Rankin
Scale), death, quality of life, and place of residence. Poor functional outcome
was defined as modified Rankin Scale >3.
RESULTS: Of 64 included patients, 32 were randomized to decompressive surgery
and 32 to best medical treatment. Just as at 1 year, surgery had no effect on
the risk of poor functional outcome at 3 years (absolute risk reduction, 1%; 95%
confidence interval, -21 to 22), but it reduced case fatality (absolute risk
reduction, 37%; 95% confidence interval, 14-60). Sixteen surgically treated
patients and 8 controls lived at home (absolute risk reduction, 27%; 95%
confidence interval, 4-50). Quality of life improved between 1 and 3 years in
patients treated with surgery.
CONCLUSIONS: In patients with space-occupying hemispheric infarction, the
effects of decompressive surgery on case fatality and functional outcome
observed at 1 year are sustained at 3 years.
CLINICAL TRIAL REGISTRATION URL: http://www.controlled-trials.com. Unique
identifier: ISRCTN94237756. BACKGROUND AND PURPOSE: Surgical decompression reduces mortality and increases
the probability of a favorable functional outcome after space-occupying
hemispheric infarction. Its cost-effectiveness is uncertain.
METHODS: We assessed clinical outcomes, costs, and cost-effectiveness for the
first 3 years in patients who were randomized to surgical decompression or best
medical treatment within 48 hours after symptom onset in the Hemicraniectomy
After Middle Cerebral Artery Infarction With Life-Threatening Edema Trial
(HAMLET). Data on medical consumption were derived from case record files,
hospital charts, and general practitioners. We calculated costs per
quality-adjusted life year (QALY). Uncertainty was assessed with bootstrapping.
A Markov model was constructed to estimate costs and health outcomes after 3
years.
RESULTS: Of 39 patients enrolled within 48 hours, 21 were randomized to surgical
decompression. After 3 years, 5 surgical (24%) and 14 medical patients (78%) had
died. In the first 3 years after enrollment, operated patients had more QALYs
than medically treated patients (mean difference, 1.0 QALY [95% confidence
interval, 0.6-1.4]), but at higher costs (mean difference, €127,000 [95%
confidence interval, 73,100-181,000]), indicating incremental costs of €127,000
per QALY gained. Ninety-eight percent of incremental cost-effectiveness ratios
replicated by bootstrapping were >€80,000 per QALY gained. Markov modeling
suggested costs of ≈€60,000 per QALY gained for a patient's lifetime.
CONCLUSIONS: Surgical decompression for space-occupying infarction results in an
increase in QALYs, but at very high costs.
CLINICAL TRIAL REGISTRATION URL: http://www.controlled-trials.com. Unique
identifier: ISRCTN94237756. |
Can we use prodrug amifostine to protect healthy cell during chemotherapy? | Effective radiotherapy for patients with cancer should include maximal tumor cell killing with minimal injury to normal tissue. However, current radiation doses that can be delivered without causing severe damage to surrounding normal tissues are often insufficient to eradicate a tumor. Recently, a number of agents have been developed to protect normal tissue from the harmful effects of antitumor therapies. The aminothiol amifostine (Ethyol; Alza Pharmaceuticals, Palo Alto, CA/US Bioscience, West Conshohocken, PA) has been the subject of extensive research as a prospective protector. While this drug has been approved for use to reduce toxicities associated with cisplatin, several studies also have demonstrated that amifostine protects normal tissues from both acute and late radiation damage without protecting the tumor. Consequently, higher radiation doses could be used with less than or equal risk to surrounding normal tissues. | PURPOSE: Based on preclinical and clinical studies that suggested amifostine may
potentiate the effects of cytotoxic drugs, we conducted a phase II trial of
amifostine, cisplatin, and vinblastine (ACV) in patients with metastatic
non-small-cell lung cancer (NSCLC).
PATIENTS AND METHODS: Twenty-five patients with metastatic NSCLC received
amifostine (740 or 910 mg/m2) before 120 mg/m2 of cisplatin on day 1, plus
weekly 5 mg/m2 of vinblastine without amifostine. Cycles were repeated every 4
weeks. Patients were required to have good performance status, no prior
chemotherapy or biologic therapy, adequate organ function, and measurable
disease.
RESULTS: Sixteen of 25 assessable patients had an objective response documented
by computed tomographic (CT) scan (64%; 95% confidence interval, 45% to 85%).
With a median duration of follow-up of 19.2 months, the estimated median
survival is 17 months and 1-year survival is 64% (+/- 10%). Toxicities included
grades 3 and 4 neutropenia (8% and 92%, respectively) and nausea and vomiting
(32% and 4%, respectively). Reversible grade 3 nephrotoxicity occurred in 12% of
patients, although only one of 13 patients (7%) who received > or = four cycles
of therapy had > or = 40% reduction in creatinine clearance. Grade 3 neuropathy
was observed in seven patients at cumulative cisplatin doses that ranged from
324 to 660 mg/m2; grade 3 ototoxicity occurred in three patients at cumulative
cisplatin doses that ranged from 390 to 450 mg/m2. Four patients (16%) required
early stopping of an amifostine infusion due to hypotension.
CONCLUSION: ACV appears to be a highly active regimen in metastatic NSCLC. Acute
toxicities were generally reversible and the data suggest that amifostine may
protect against long-term renal insufficiency from cumulative doses of
cisplatin. Although the sample size of this trial is small, the results are
significantly encouraging to warrant confirmation in randomized
multiinstitutional trials. Preclinical studies demonstrate that amifostine has the potential to selectively
protect normal tissues from the harmful effects of radiation without
significantly protecting neoplastic tissue. The potential value of such an agent
includes reducing treatment-related toxicity and the opportunity for radiation
dose escalation in the curative treatment of cancer. An increasing number of
human clinical trials have been conducted that define the toxicity profile and
efficacy of radioprotection by amifostine when used during fractionated
radiation therapy. These trials demonstrate that amifostine is safe and
practical to administer in the outpatient setting during fractionated radiation
therapy. These studies also illustrate the challenge of accurately evaluating
the end point of radioprotection in the clinical setting. This article reviews
the recent clinical literature on amifostine, the evidence for normal tissue
protection, and the lack of tumor protection by this agent, and suggests
possible avenues for future investigation and application of this agent in the
field of radiation oncology. Originally developed against the effects of ionizing radiations, amifostine is
an organic thiophosphate compound shown able to selectively protect normal
tissues against cytotoxic agents in cellular and animal models, without
protecting tumor tissues. Amifostine is a prodrug which is dephosphorylated into
its active metabolite, a free thiol derivative, by membrane alkaline phosphatase
of the target issue. This unique metabolism supports its cellular selectivity
and its preferential uptake by normal tissues. In phase II clinical trials, a
decreased toxicity has been demonstrated in patients given alkylating agents;
however, reduction of the response has not been observed. On the basis of these
results, a prospective, randomized, phase III study has been conducted in
patients with ovarian carcinoma receiving a combination of cisplatinum and
cyclophosphamide. A significant decrease in hematologic, renal and neurologic
toxicity was observed in the amifostine-treated patients compared with the
control group, and response rates did not significantly differ between the two
groups. Insufficient or emerging data are only available for other applications,
including either in vitro manipulation of hematopoietic grafts or in vivo
treatment of non-Hodgkin's lymphoma, head and neck carcinoma, non-small cell
lung cancer and radioprotection. No data are yet available in regard to the
potential protective effects of amifostine against mutagenicity and
cancerogenicity of both chemo- and radiotherapy. The mechanism of action, pharmacokinetics, clinical efficacy, adverse effects,
and dosage and administration of amifostine are reviewed. Amifostine is a
prodrug converted by alkaline phosphatase to the active sulfhydryl compound
WR-1065. WR-1065 protects normal cells by scavenging free radicals, donating
hydrogen ions to free radicals, depleting oxygen, and binding to active
derivatives of antineoplastic agents. The immediate conversion of amifostine to
WR-1065, its small volume of distribution, and the limited amount of drug and
metabolite recovered in the urine suggest that amifostine is rapidly
dephosphorylated and enters cells as its active metabolite. The selectivity of
amifostine for normal tissue is hypothesized to be a results of the decreased
vascularity of tumors, decreased activity of alkaline phosphatase in tumor
cells, and pH dependence of WR-1065 uptake. In clinical studies, amifostine
decreased the frequency of cisplatin-induced nephrotoxicity, ototoxicity,
neurotoxicity, and myelosuppression. Amifostine has demonstrated an ability to
decrease the hematologic toxicity of cyclophosphamide, carboplatin, mitomycin,
and antineoplastic drug combinations. Amifostine has FDA-approved labeling for
use in reducing cumulative renal toxicity in patients receiving repeat doses of
cisplatin for advanced ovarian cancer and non-small-cell lung cancer. The
recommended dose in adults is 910 mg/m2 administered as a 15-minute infusion 30
minutes before the start of chemotherapy. The major adverse effects of
amifostine include hypotension and emesis. The benefits of amifostine must be
weighted against its potential adverse effects, and the drug's impact on the
efficacy of antineoplastics should be further investigated. Amifostine has shown
promise in protecting non-maligt cells from the toxic effects of
antineoplastics, apparently without compromising toxicity against cancer cells. The objectives of this study were to evaluate the protective effects of
amifostine against paclitaxel-induced toxicity to normal and maligt human
tissues. Haematopoietic progenitor colony assays were used to establish the
number of CFU-GEMM and BFU-E colonies after incubation with WR-1065 alone,
Amifostine alone, paclitaxel (2.5 or 5 microM) +/- WR-1065 or amifostine. MTT
and alkaline elution assays evaluated the in vitro growth inhibitory and DNA
damaging effects, respectively, of paclitaxel with or without amifostine against
normal human fibroblasts and human non-small cell lung cancer (NSCLC) cells.
This combination was also evaluated in vivo using severe combined immune
deficient (scid) mouse models of early (non-palpable tumours) and advanced
(palpable tumours) human ovarian cancer. Human 2780 ovarian cancer cells were
inoculated subcutaneously while paclitaxel and amifostine were administered
intraperitoneally. A brief exposure (15 min) to amifostine not only protected
human haematopoietic progenitor colonies from paclitaxel toxicity, but
stimulated the growth of CFU-GEMM and BFU-E beyond control values. Amifostine
protected normal human lung fibroblasts from paclitaxel-induced cytotoxicity and
DNA single-strand breaks. However, paclitaxel cytotoxicity and DNA single-strand
breaks were actually enhanced by pretreatment with amifostine in the NSCLC
model. Importantly, amifostine did not interfere with paclitaxel antitumour
activity even with prolonged exposure (24.5 h) of the lung cancer cells to high
concentrations (1.2 mM) in vitro or following five repetitive high doses (200
mg/kg) given to scid mice with human ovarian cancer xenografts. Indeed, under
certain circumstances, amifostine resulted in sensitisation of tumour cells to
paclitaxel. Our results confirm previous reports of the ability of amifostine to
protect normal tissues from the toxic effects of chemotherapy drugs and now
extend these observations to paclitaxel. Amifostine (WR-2721, Ethyol), S-2[3-aminopropylamino]-ethyl-phosphorothioic
acid, was selected as a clinically usable radioprotector from more than 4,400
compounds in the 1950s. A considerable amount of preclinical work suggested that
amifostine, or its activated thiol WR-1065, protected normal cells effectively
against the adverse effects of irradiation and several anticancer drugs without
exhibiting tumor protection. In non-randomized and randomized trials in
maligt melanoma, colorectal cancer, head and neck cancer, non-small cell lung
cancer, and epithelial ovarian carcinoma, amifostine significantly reduced the
hematological and non-hematological toxicity of DNA-damaging agents such as
alkylators, platinum compounds, or mitomycin C. In more recent studies, the drug
also protected patients from side effects produced by taxanes or topoisomerase I
inhibitors and is thus likely to allow higher cytostatic doses to be
administered. Currently, there is no evidence that amifostine compromises the
antineoplastic effect of the drugs studied. Otherwise, W/R-2721 may even improve
the therapeutic efficacy of agents like cisplatin, carboplatin, or paclitaxel.
Moreover, amifostine appears to produce growth-factor like properties resulting
in growth-promoting effects on primitive blood progenitor cells ex vivo.
Amifostine offers a rational approach to protect patients against
chemotherapy-specific and often dose-limiting effects and is thus likely to
improve therapeutic outcome significantly. Future studies should be focused on
both new indications like childhood cancer, myelodysplastic syndromes,
dose-intensified or high- dose chemotherapy, and multimodality approaches and
optimization of amifostine dosage in order to reduce dose-limiting side effects.
Then, the drug may play a major role in more specific and individualized
oncologic strategies. BACKGROUND AND OBJECTIVE: Amifostine is an inorganic thiophosphate
cytoprotective agent known chemically as ethanethiol,
2-[(3-aminopropyl)amino]dihydrogen phosphate. It is a pro-drug of free
thiol that may act as a scavenger of free radicals generated in tissues exposed
to cytotoxic drugs, and binds to reactive metabolites of such drugs. Amifostine
was originally developed as a radioprotective agent in a classified nuclear
warfare project. Following declassification of the project it was evaluated as a
cytoprotective agent against toxicity of the alkylating drugs and cisplatin. In
fact, pretreatment with amifostine was well tolerated and reduced the cumulative
hematologic, renal and neurological toxicity associated with cisplatin,
cyclophosphamide and vinblastine therapy of advanced and metastatic solid
tumors. The objective of this review is to focus the importance of amifostine as
a myeloprotective and cytoprotective drug during treatment with
chemotherapeutics, presenting the most recent results, and to discuss the
application of amifostine in the therapy of myelodysplastic syndromes.
EVIDENCE AND INFORMATION SOURCES: The material analyzed in this study includes
data published or under publication by the authors as full papers or clinical
protocols. Articles and abstracts published in Journals covered by Medline
constitute the other source of information.
STATE OF THE ART AND PERSPECTIVES: Amifostine, formerly known as WR-2721, is an
organic thiophosphate that was developed to protect normal tissues selectively
against the toxicities of chemotherapy and radiation. Amifostine is a pro-drug
that is dephosphorylated at the tissue site to its active metabolite by alkaline
phosphatase. Differences in the alkaline phosphatase concentrations of normal
versus tumor tissues can result in greater conversion of amifostine in normal
tissues. Once inside the cell the free thiol provides an alternative target to
DNA and RNA for the reactive molecules of alkylating or platinum agents and acts
as a potent scavenger of the oxygen free radicals induced by ionizing radiation
and some chemotherapies. Preclinical animal studies demonstrated that the
administration of amifostine protected against a variety of chemotherapy-related
toxicities including cisplatin-induced nephrotoxicity, cisplatin-induced
neurotoxicity, cyclophosphamide- and bleomycin-induced pulmonary toxicity, and
the cytotoxicities (including cardiotoxicity) induced by doxorubicin and related
chemotherapeutic agents. Amifostine was shown to protect a variety of animal
species from lethal doses of radiation. Studies in tumor-bearing animals
demonstrated that the administration of amifostine results in cytoprotection
without loss of antitumor activity. Multiple phase I studies were carried out
with amifostine in combination with chemotherapy for various neoplasms.
Appropriate doses of amifostine resulted to be 740-910 mg/m(2) in a single dose
regimen, and 340 mg/m(2) in a multiple dose regimen. Amifostine afforded not
only hematologic protection, but also other organ protection from cytotoxic
agents such as nephrotoxicity, mucositis and peripheral neuropathy from
cisplatin. Many studies have been performed to investigate cytoprotective
efficacy of amifostine. In brief, amifostine gives hematologic protection from
cyclophosphamide, carboplatin, mitomycin C, fotemustine and radiotherapy; renal
and peripheral nerve protection from cisplatin; mucosa, skin, and salivary gland
from radiotherapy. In phase I/II studies these properties have been confirmed,
together with a generally good tolerability of the drug, hypotension being the
most common side effect. It has been observed that amifostine possibly enhances
the anti-tumor effect of carboplatin, nitrogen mustard, melphalan, and cisplatin
combined with 5-FU or vinblastine. For all these characteristics, amifostine is
at present broadly used as supportive treatment during chemotherapy, in
lymphomas and solid tumors, and its spec Amifostine is a protective agent of normal tissue from adverse effects of
radiochemotherapy. It is the prodrug that is dephosphorylated by alkaline
phosphatase on plasma membrane into the active form named WR-1065. More than 90
per cent of the drug is cleared from plasma in 6 minutes and the peak tissue
concentration is 10-30 minutes after intravenous administration. Amifostine has
the selective property to protect normal tissue but not cancer cells by mainly
scavenging free radicals induced by radiation and chemocytotoxic agents. Both
preclinical and clinical studies of this drug provide the significant protection
of hematopoietic progentitors from a broad range of cytotoxic agents such as
cyclophosphamide, cisplatin, vinblastine, carboplatin, mitomycin-C, fotemustine,
doxorubicin, daunorubicin and radiation as well. Moreover, this drug can protect
other normal organs or tissues including kidney, salivary gland, liver, heart,
lung and small intestine. Amifostine is quite safe, the two major side effects
are vomiting and hypotension, and the minor effects are flushing, sneezing,
dizziness, chills, metallic taste etc. The drug was approved by the FDA of
U.S.A. for use as a cytoprotectant in cyclophosphamide and cisplatin treatment
for advanced ovarian cancer and non small cell lung cancer. Experimental studies have shown that vinorelbine is a powerful radiosensitizer
in vitro. To date, no reports on clinical activity of the single agent
vinorelbine as radiosensitizer have been published. The aim of the present phase
I study was to determine the maximum tolerated dose (MTD) of vinorelbine
administered daily concurrently with thoracic radiotherapy, with or without
amifostine support, in the treatment of locally advanced non small cell lung
cancer. In vitro studies have shown that vinorelbine can potentiate the
antitumor effects of radiation therapy. Amifostine is a sulphydril compound that
has shown to protect normal tissues from chemotherapy and radiotherapy-induced
toxicities. Radiotherapy lasted 6 weeks and the total dose was 55 Gy. The daily
fraction was 1.8 Gy, administered 5 days a week for 5 weeks and increased to 2.0
Gy during the sixth and last week. Concurrent vinorelbine was administered daily
with a planned escalation of the dose from 4, to 5 and 6 mg/m(2). Fourteen
patients were enrolled in the study. The first dose of vinorelbine was 4 mg/m(2)
and it showed to be feasible without dose-limiting toxicity (DLT). Instead, the
second dose level of 5 mg/m(2) was unfeasible because three out of six patients
had DLT (grade 4 neutropenia, treatment interruption longer than 2 weeks for
prolonged grade 2 neutropenia and treatment interruption longer than 2 weeks for
prolonged grade 3 esophagitis together with grade 4 dyspnea). At that time, the
study continued adding amifostine to vinorelbine in order to increase its MTD.
Amifostine was administered by means of subcutaneous injection 15 min before
each radiotherapy fraction at the fixed dose of 300 mg/m(2). However, 5 mg/m(2)
of vinorelbine were considered unfeasible even with amifostine support because
three out of five patients showed DLT (grade 4 neutropenia, febrile grade 4
neutropenia and grade 3 liver toxicity). Among 14 patients enrolled in the
study, eight completed the planned treatment because six patients experienced
DLT, which determined treatment interruption. Overall, four partial and two
complete responses were observed. Two partial and one complete response were
observed in those three patients who had been treated with the first dose of
vinorelbine. In conclusion, our data show that the MTD of daily vinorelbine is 4
mg/m(2). Therefore, this is the recommended dose of daily vinorelbine to be
administered with concurrent thoracic radiotherapy in a phase II trial. Finally,
amifostine administered subcutaneously failed to increase the MTD of daily
vinorelbine. Amifostine (Ethyoltrade mark, Alza Pharmaceuticals) is an inorganic
thiophosphate cytoprotective agent known chemically as ethanethiol, 2-[3-
aminopropyl)amino]dihydrogen phosphate. It is a prodrug of free thiol (WR-1065)
that may act as a scavenger of free radicals generated in tissues exposed to
cytotoxic drugs and binds to reactive metabolites of such drugs. Amifostine was
originally developed as a radioprotective agent in a classified nuclear warfare
project. Following declassification of the project it was evaluated as a
cytoprotective agent against toxicity of the alkylating drugs and cisplatin.
Differences in the alkaline phosphatase concentration of normal versus tumour
tissues can result in greater conversion of amifostine in normal tissues. Inside
the cell, WR-1065 provides an alternative target to DNA and RNA for the reactive
molecules of alkylating or platinum agents and acts as a potent scavenger of the
oxygen free radicals induced by ionizing radiation and some chemotherapy agents.
Preclinical animal studies have demonstrated that the administration of
amifostine protects against a variety of chemotherapy-related toxicities
including cisplatin-induced nephrotoxicity, cisplatin-induced neurotoxicity,
cyclophosphamide- and bleomycin-induced pulmonary toxicity and the
cytotoxicities (including cardiotoxicity) induced by doxorubicin and related
chemotherapeutic agents. Amifostine has been shown to protect a variety of
animal species from lethal doses of radiation. Amifostine gives haematological
protection from cyclophosphamide, carboplatin, mitomycin C, fotemustine and
radiotherapy; renal and peripheral nerve protection from cisplatin; mucosa, skin
and salivary gland protection from radiotherapy. Multiple Phase I studies were
carried out with amifostine in combination with chemotherapy for various
neoplasms. Appropriate doses of amifostine were found to be 740 - 910 mg/m(2) in
single-dose regimens and 340 mg/m(2) in multiple-dose regimens. In
radioprotection, doses are generally 200 - 350 mg/m(2). For all these
characteristics, amifostine has been recently approved and suggested in ASCO
clinical practice guidelines as a radioprotector for head and neck cancer
treatment and supportive agent during cisplatin-based chemotherapy, in lymphomas
and solid tumours. Moreover, its spectrum of possible applications is enlarging.
As data have been provided indicating that amifostine stimulates haematopoiesis,
it has been employed with intriguing results in the treatment of myelodysplastic
syndromes (MDS). Amifostine has shown to selectively protect normal tissues against cytotoxic and
mutagenic effects of several anti-neoplastic drugs, such as alkylating agents,
organoplatinum compounds, anthracyclines, taxanes, and ionising radiation. This
cytoprotection is broad-spectrum and selective, without loss of therapeutic
efficacy. In this study we have treated 31 patients affected with inoperable or
metastatic breast cancer, not previously submitted to chemotherapy for advanced
disease, with amifostine 910 mg/m(2) followed by doxorubicin 75 mg/m(2). The
overall response rate was 52% with a median response duration of 13 months
(range 6-53+) and a median overall survival of 21 months (range 3-59+). With
regard to toxicity, 14 patients (45%) experienced transient g4 neutropenia which
was febrile only in one case (3%). Grade 3-4 thrombocytopenia was not recorded.
Nausea and vomiting occurred in 14% of cycles. Grade 3 mucositis was observed in
only 1 patient, whereas 2 patients (6%) developed an asymptomatic drop of left
ventricular ejection fraction (LVEF) >10% below basal value. In conclusion, this
study suggests that amifostine can reduce doxorubicin related toxicity, thus
improving the patients' quality of life and the efficacy/toxicity ratio of this
drug. Amifostine (WR-2721) is an inorganic thiophosphate-cytoprotective agent
developed to selectively protect normal tissues against the toxicity of
chemotherapy and radiation. We have previously shown that amifostine protects
both chicken embryo chorioallantoic membrane (CAM) vessels and cells from the
effects of X-rays. In the present work, we studied the effect of amifostine on
angiogenesis in vivo, using the CAM model. Amifostine decreased the number of
CAM vessels in a dose-dependent manner, without being toxic for the tissue. It
also decreased the mRNA levels of both vascular endothelial growth factor (VEGF)
isoforms VEGF(165) and VEGF(190), 6 and up to 48 h after its application onto
the CAM. Similarly, it decreased the mRNA levels of inducible nitric-oxide
synthase, 24 and 48 h after drug application. Furthermore, amifostine decreased
the deposited amounts of laminin and collagen I 24 h after its application,
without affecting the expression of the corresponding genes. The protein amounts
and activity of matrix metalloproteinase-2 were not affected, whereas the
expression of the corresponding gene was decreased up to 48 h after drug
application. Finally, the activity of plasmin was increased 6 h after amifostine
application and remained increased at later time points. These findings suggest
that amifostine alters the expression of several molecules implicated in the
angiogenesis process and affects the composition of the extracellular matrix in
a way that leads to inhibition of angiogenesis. Such an antiangiogenic action of
amifostine, together with its radioprotective effects, further supports its use
in combination with radiotherapy for increased therapeutic efficacy. Amifostine is a phosphorylated aminothiol that not only protects hematopoietic
progenitor cells from chemotherapy and radiotherapy, but also stimulates normal
hematopoiesis. The effect of amifostine on the in vitro growth of hematopoietic
progenitors derived from B-cell chronic lymphocytic leukemia(B-CLL) was
investigated. The colony-forming units (CFU)-granulocyte macrophage (CFU-GM),
the burst-forming units-erythroid (BFU-E) and the CFU-granulocyte erythroid
macrophage megakaryocytes (CFU-GEMM) increased 38, 20 and 100%, respectively,
after the incubation with amifostine. There was no statistical difference in the
in vitro progenitor growth of patients grouped according to their disease stage,
bone marrow lymphocytic infiltration or therapy. Our data indicate that apart
from cytoprotection the parallel use of amifostine and chemotherapy in patients
with B-CLL could enhance bone marrow recovery. Amifostine (AMF) has been shown to protect some normal tissues from acute
effects of radiation therapy +/- chemotherapy. We enrolled 62 patients in a
randomized study investigating the efficacy of AMF: 31 had concurrent
chemoradiation for non-small cell lung cancer and 31 had the same treatment +
AMF. AMF reduced the frequency and severity of esophagitis, pneumonitis, and
neutropenic fever. We have tried to identify patients who get more benefit from
AMF by checking their DNA repair capability of normal cells. It was hypothesized
that DNA repair capacity from patients' lymphocytes damaged by bleomycin could
predict their normal tissue sensitivity to chemoradiation and could be protected
by AMF. Forty-six of the 62 patients provided pretreatment blood for assessment
of mutagen sensitivity (MS) using a peripheral lymphocyte assay that infers DNA
repair capacity from cellular damage remaining after in vitro mutagenic exposure
and recovery. Bleomycin-induced chromosome breaks in 50 metaphases were counted
and expressed as the mean number of breaks per cell. Patients with an average of
more than one break/cell were deemed to exhibit the MS phenotype. Data analysis
used Pearson's chi-square and Kaplan-Meier survival function estimates with
Strata 8.2 statistical software. The Log-rank test was used to assess the
equality of survival function using a P value of .05. Twelve patients (10 AMF, 2
control) exhibited the MS phenotype. The remaining 34 patients (13 AMF, 21
control) were considered to have normal DNA repair. There were no significant
differences in overall survival, disease specific survival, or local control by
MS. Those with high MS had shorter distant metastasis-free survival compared
with low MS patients ( P = .029). There were no differences in severe
esophagitis or neutropenic fever by MS. Both high and low MS patients from the
control group developed severe lung fibrosis compared with five of 21 who had
AMF ( P = .025). The incidence of grade 3/4 lung fibrosis was two of 10 with AMF
compared with two of two in the control group ( P = .025) with higher MS. Higher
MS was associated with shorter distant metastasis-free survival and more
frequent grade 3/4 lung fibrosis. AMF reduced the incidence of grade 3/4 lung
fibrosis among higher MS. These data suggest that MS might help identify
subgroups of patients who could receive more benefit from AMF with respect to
lung damage. The underlying primary damage to the seminiferous epithelium caused by
chemotherapeutic regimens at childhood is largely unknown. The present
investigation was designed to identify acute cytotoxic events in the testis
caused by a single dose of doxorubicin. Male rats at 6, 16, and 24 days of age
were injected with doxorubicin (3 mg/kg, i.p.) or vehicle (saline) alone and 24
and 48 hours later, the germ cell types and apoptotic cells in the seminiferous
epithelium were examined. As indicated by microscopy and terminal
deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, an
8-fold increase in the number of apoptotic germ cells in the testes of 6-day-old
rats was observed 48 hours after doxorubicin treatment. Spermatogonia migrating
to the basement membrane were the primary cell type undergoing this induced
apoptosis. A single dose of amifostine (200 mg/kg) administered i.p. 15 minutes
before injection of doxorubicin provided no protection against this enhanced
apoptosis. Under the same conditions, testicular levels of p53 and activated
caspase 8 were elevated, whereas the level of murine double minute-2 was
lowered. In contrast, doxorubicin treatment did not result in any significant
change in the physiologic, stage-specific germ cell apoptosis occurring in the
testes of 16- and 24-day-old rats. These observations suggest that the
initiation phase of spermatogenesis is highly sensitive to doxorubicin-induced
apoptosis. Gonocytes and early spermatogonia are the cell types that are
vulnerable to this p53-trigged apoptosis, which results in a decrease in the
size of the pool of germ-line stem cells. Amifostine fails to protect the germ
cells against this cytotoxic insult. After several decades of preclinical and clinical research, the first approved
radioprotective drug, amifostine, is being used in clinical practice. Amifostine
has been shown to specifically protect normal tissues from damage caused by
radiation and chemotherapy. An inactive prodrug, amifostine is converted to an
active thiol by dephosphorylation by alkaline phosphatase in the normal
endothelium. The hypovascularity and acidity of the tumor environment and the
differential expression of alkaline phosphatase in normal and neoplastic tissues
contribute to its cytoprotective selectivity. The cytoprotective mechanism of
amifostine is complicated, involving free-radical scavenging, DNA protection and
repair acceleration, and induction of cellular hypoxia. The U.S. Food and Drug
Administration has approved the i.v. use of amifostine to reduce the cumulative
renal toxicity associated with repeated administration of cisplatin in patients
with advanced ovarian cancer and to reduce the incidence of moderate to severe
xerostomia in patients undergoing postoperative radiation treatment for head and
neck cancer, where the radiation port includes a substantial portion of the
parotid glands. Nonetheless, amifostine has potential applications in many other
oncologic settings. Novel schedules and routes of administration are under
investigation and may further simplify the use of amifostine, reduce any
undesired effects, and considerably broaden its applications. This review
summarizes the clinical experience with amifostine and provides insight into
future clinical directions. Following treatment with bleomycin- and cisplatin-containing chemotherapy,
testicular cancer patients frequently develop vascular complications, which may
result from damage to endothelial cells. Understanding bleomycin- and
cisplatin-induced endothelial alterations may help to develop strategies to
prevent or reduce vascular toxicity. The effects of bleomycin and cisplatin on
proliferation and apoptosis of the human dermal microvascular endothelial cell
line HMEC-1 were determined. In addition, modulation of drug-induced
cytotoxicity by the free radical scavenger amifostine, the low molecular weight
heparin dalteparin, the iron-chelator dexrazoxane, the HMG-CoA reductase
inhibitor rosuvastatin and the PPAR agonist troglitazone was tested.
Furthermore, the effects of bleomycin and cisplatin on endothelial activation
measured by the expression of the intercellular adhesion molecule-1 (ICAM-1) and
on two main proteins involved in fibrinolysis, tissue-type plasminogen activator
(tPA) and plasminogen activator inhibitor type 1 (PAI-1), were measured.
Decreased endothelial cell survival induced by bleomycin and cisplatin coincided
with the induction of apoptosis. Only troglitazone was able to protect the
endothelial cells from both bleomycin- and cisplatin-induced cytotoxicity. At
high concentrations, amifostine and dexrazoxane also protected HMEC-1 from
drug-induced cytotoxicity. However, due to the required high (toxic)
concentrations of both modulators no absolute cell survival benefit could be
achieved. Both bleomycin and cisplatin induced up-regulation of ICAM-1, tPA and
PAI-1. Summarizing, bleomycin and cisplatin induce alterations in the function
of endothelial cells regarding proliferation, inflammation and fibrinolysis in
vitro. Strategies aimed at these functions should be developed in order to
ameliorate or prevent cytostatic agent-induced vascular damage. Cyclophosphamide (CYP), a commonly prescribed chemotherapy drug, has multiple
adverse side effects including alteration of taste. The effects on taste are a
cause of concern for patients as changes in taste are often associated with loss
of appetite, malnutrition, poor recovery and reduced quality of life. Amifostine
is a cytoprotective agent that was previously shown to be effective in
preventing chemotherapy-induced mucositis and nephrotoxicity. Here we determined
its ability to protect against chemotherapy-induced damage to taste buds using a
mouse model of CYP injury. We conducted detection threshold tests to measure
changes in sucrose taste sensitivity and found that administration of amifostine
30 mins prior to CYP injection protected against CYP-induced loss in taste
sensitivity. Morphological studies showed that pre-treatment with amifostine
prevented CYP-induced reduction in the number of fungiform taste papillae and
increased the number of taste buds. Immunohistochemical assays for markers of
the cell cycle showed that amifostine administration prevented CYP-induced
inhibition of cell proliferation and also protected against loss of mature taste
cells after CYP exposure. Our results indicate that treatment of cancer patients
with amifostine prior to chemotherapy may improve their sensitivity for taste
stimuli and protect the taste system from the detrimental effects of
chemotherapy. |
Which is the main difference between Alu and B1 repeats? | B1 is a murine homolog of the human SINE Alu. B1 (Alu-equivalent) is a murine short interspersed element whose amplification probably involved an RNA intermediate. The modern B1 elements are similar to the left Alu monomer, but with a 9 bp deletion and a 29 bp duplication. | The Alu sequence family comprises the major dispersed repeat sequences of rodent
and primate genomes, numbering greater than 300,000 copies in the human haploid
genome. The function of these elements is unknown. The sequences can be
transcribed by RNA polymerase III and represent a substantial fraction of total
heterogeneous nuclear RNA. Alu sequences can be found both in the flanking
regions and within the transcription units of several well-characterized genes.
Here we show that some members of the mouse B1 Alu sequence family encode a
small cytoplasmic RNA. The mouse B1 sequence is congruent to 130 nucleotides
long and shows homology with the monomeric units of the dimeric 300-nucleotide
primate sequence. By means of microinjection studies in the Xenopus laevis
oocyte, we have elucidated a novel pathway leading to the appearance of a
processed B1-type Alu RNA species in the cytoplasm. The abundance of this small
Alu RNA differs between various mouse tissues, suggesting a role in
tissue-specific gene expression. A complex study on various evolutionary peculiarities of the mammalia dispersed
Alu repeats (Alu repeats of primates and B1 of rodents) has been carried out by
phylogenetic analysis. A phylogenetic tree, containing the 7SL RNA genes and the
Alu repeats of primates and rodents has been constructed. It has been shown that
the branch of the phyletic line leading to the Alu repeats of primates and B1 of
rodents from the 7SL RNA genes occurred after the divergence of the 7SL RNA
genes of amphibia and mammalia, but before the divergence of the 7SL RNA genes
of primates and rodents (250.10 years ago). A statistically reliable slowing
down in the evolutionary rates of one of two monomers for the human Alu repeats
has been proved. It may be caused by the functional load of the corresponding
monomer in connection with the presence of a definit regulatory site in it. B1 and Alu are sequence-homologous interspersed elements of unknown function
that have expanded in the genomes of mice and humans, respectively. A minority
of B1 and Alu sequences are expressed as small cytoplasmic RNAs. These RNAs have
conserved a secondary structure motif also present in signal recognition
particle (SRP) RNA despite substantial sequence divergence, whereas random B1
and Alu sequences have not. This RNA structure has also been conserved by the
source sequences that gave rise to successive transpositions during B1 and Alu
evolution. In the present work small cytoplasmic B1 and Alu RNAs synthesized in
vitro were found to bind a cellular protein by mobility shift and UV
cross-linking analyses. The mouse and human proteins demonstrate the same
specificity to a panel of competitor RNAs. Results using mutated B1 RNA indicate
that a single strand loop in the conserved Alu motif is essential for binding.
Previous work by Strub et al. (Stub, K., Moss, J. B., and Walter, P. (1991) Mol.
Cell. Biol. 11, 3949-3959) demonstrated that the Alu-specific protein SRP 9/14
does not footprint to this region of SRP RNA. This observation coupled with the
failure of anti-SRP/9 antibodies to identify SRP 9/14 in the B1 RNA-protein
complex as well as the apparent mass and other characteristics of the protein
described here suggest that it is a novel B1-Alu RNA-binding protein.
Conservation of primary and secondary structure by B1 and Alu small cytoplasmic
RNAs as well as features of their specific expression and ability to interact
with the conserved binding protein indicate that these RNAs are more homologous
than previously appreciated. The question of the origin of the B1 family of rodents is addressed. The modern
B1 elements are similar to the left Alu monomer, but with a 9 bp deletion and a
29 bp duplication. Search of databases for B1 elements that do not exhibit those
modern features revealed sequence fragments that are very similar to the free
left Alu monomers (FLAMs) described in the primate genomes. In addition, the
analysis reveals elements that have 10 bp or 7 bp deletion in place of the 9 bp
deletion but without the 29 bp tandem duplication. The elements described define
families of proto B1 elements (referred as PB1, PB1D10 and PB1D7) that appeared
before the first modern B1 element. A phylogenetic reconstruction suggest that
the origin of Alu and B1 families took place before the divergence between the
primate and the rodent lineages and that each family has followed different
evolutionary routes since this radiation. We have determined sequences of PCR-amplified B1 elements from hamster and rat
(Myomorpha), chipmunk (Sciuromorpha), and guinea pig (Caviomorpha). Between
three and six B1 subfamilies were found in these species. In the phylogenetic
analysis B1 sequences of hamster, mouse, and rat clustered separately from those
of chipmunk and those of guinea pig. This is consistent with an independent
evolution of B1 elements in separate rodent lineages. We exclude the possibility
of convergent mutations to explain certain diagnostic characters within the
modern B1 quasi-dimers and view these elements as mosaic structures assembling
preexisting mutations. Furthermore, the presence of Alu-like structural motifs
supports the hypothesis of the monophyletic origin of Alu and B1 repeats, i.e.,
from a common 7SL RNA-derived retroposing monomeric element. Efficient synthesis of many small abundant RNAs is achieved by the proficient
recycling of RNA polymerase (pol) III and stable transcription complexes.
Cellular Alu and related retroposons represent unusual pol III genes that are
normally repressed but are activated by viral infection and other conditions.
The core sequences of these elements contain pol III promoters but must rely on
fortuitous downstream oligo(dT) tracts for terminator function. We show that a
B1-Alu gene differs markedly from a classical pol III gene (tRNAiMet) in
terminator sequence requirements. B1-Alu genes that differ only in terminator
sequence context direct differential RNA 3' end formation. These genes are
assembled into stable transcription complexes but differ in their ability to be
recycled in the presence of the La transcription termination factor. La binds to
the nascent RNA 3' UUUOH end motif that is generated by transcriptional
termination within the pol III termination signal, oligo(dT). We found that the
recycling efficiency of the B1-Alu genes is correlated with the ability of La to
access the 3' end of the nascent transcript and protect it from 3'-5'
exonucleolytic processing. These results illuminate a relationship between RNA
3' end formation and transcription termination, and La-mediated reinitiation by
pol III. Alus and B1s are short interspersed repeat elements (SINEs) derived from the 7SL
RNA gene. Alus and B1s exist in the cytoplasm as non-coding RNA indicating that
they are actively transcribed, but their function, if any, is unknown.
Transcription of individual SINEs is a prerequisite for retroposition, but it is
also possible that individual Alu and B1 elements have some cellular functions.
Previous studies suggest that transcription of Alu elements depends on the
presence of an RNA polymerase-III bipartite promoter and the poly-A tail.
Sequencing of small RNAs has demonstrated that the members of the Y and S
subfamily are expressed. We analyzed almost one million Alu sequences longer
than 200 nucleotides for the presence of RNA polymerase-III bipartite promoter
sequences. More than half contained a promoter indicating some potential for
expression. We searched 7.7 million human EST sequences in dbEST for the
presence of Alu non-coding RNAs and found evidence for the expression of 452.
Analysis of mouse spermatogenic dbEST libraries revealed an apparent
relationship between the level of differentiation and the level of B1-related
sequences in the EST library. |
Is there a relationship between thyroid hormone altered metabolism and coronary artery disease? | The major part of the studies and metaanalysis data show that hypothyroidism, both primary and secondary forms, is associated with higher incidence and severity of coronary artery disease. | The activity of the pituitary hormones (ACTH, STH, TTH, FSH, LH), the adrenal
hormones (cortisol, aldosterone), the kidney hormone (renin), and the thyroid
hormones (thyroxine tri-iodthyronine), the thyroxine binding capacity of blood
proteins and the activity of the hormones of the pancreas (insulin) and the sex
glands (testosterone, estradiol) were studied in 26 males suffering from
ischemic heart disease verified by means of selective coronarography and in 20
healthy males with no atherosclerosis of the coronary arteries of the heart.
Patients with ischemic heart disease were found to be marked by increased
activity in the blood of ACTH, TTH, cortisol, aldosterone, insulin, and
estradiol and reduced concentration of STH, thyroxine, and testosterone. These
shifts in the activity of the hypothalamo-hypophyseal system and in its
subordinate hormonal systems play an important role in the origin of the
atherosclerotic process and assosiated ischemic heart disease. OBJECTIVE: To study the effect of adequacy of thyroid hormone replacement
therapy on coronary atherosclerosis.
DESIGN: Retrospective cohort study of elderly hypothyroid patients with
coexisting coronary artery disease. The association between the adequacy of
thyroid replacement and the progression of angiographic coronary artery disease
was investigated. Fisher's exact test was used for statistical analysis.
SETTING: Coronary angiographies were performed at the Cardiac Catheterization
Laboratory of the Victoria General Hospital, Halifax, Nova Scotia, the only
tertiary referral centre for Nova Scotia and Prince Edward Island. Information
about the past and current thyroid status of the subjects was collected from
their family physicians.
PATIENTS: Of 4103 patients admitted for coronary angiography during 1992 and
1993, 25 were on thyroid replacement therapy to treat hypothyroidism. Ten
patients who underwent more than one coronary arteriography were selected (seven
females and three males, mean age 65 +/- 10 years).
RESULTS: Of five patients inadequately treated for hypothyroidism, all
demonstrated angiographic evidence of coronary atherosclerosis progression.
However, five of seven who were treated adequately did not show atherosclerosis
progression (P = 0.02, OR = 0.72, 95% CI 1.36 to infinity). Decreasing or
maintaining the dose of L-thyroxine at 100 micrograms or less resulted in
coronary atherosclerosis progression in six of six patients, whereas five of six
patients taking fixed or increasing doses of L-thyroxine 150 micrograms or
higher were spared from disease progression (P = 0.015, OR = 0.41, 95% CI 2.4 to
infinity).
CONCLUSIONS: Angiographic coronary artery disease progression may be prevented
by adequate thyroid replacement in hypothyroidism. With the help of modern,
sensitive thyroid stimulating hormone assays higher doses of L-thyroxine may be
safer and more effective in the atherosclerosis management of this patient
population. Thyroid hormones can protect against atherosclerosis, presumably due
to their metabolic affects on plaque progression. To determine whether thyroid hormones, triiodothyronine (T3) and thyroxine (T4),
have any direct, nongenomic effects on vascular smooth muscle cells, we
evaluated the effects of these hormones on rat coronary arteries. Bolus
injection of T3 or T4 elicited a transient, dose-dependent decrease in coronary
perfusion pressure (CPP), as well as an increase in arterial vasodilation.
Vasodilation occurred immediately after injection, peaked at 15 seconds, and
lasted 80 seconds. Reverse T3 had no effect on CPP or vasodilation. The rapidity
of these effects suggests that they are not mediated by the T3-nuclear receptor,
but are direct, nongenomic effects of thyroid hormones. Our results also suggest
that thyroid hormones may play a role in preventing myocardial ischemia by
inducing coronary artery vasodilation. OBJECTIVE: To assess the potential benefits and risks associated with
levothyroxine (LT4) therapy in patients with subclinical hypothyroidism (SH) and
concomitant coronary artery disease (CAD).
METHODS: We enrolled 33 patients (4 male and 29 female subjects) with SH and CAD
in this study. The study cohort consisted of 2 groups: 19 patients who were
randomly assigned to receive LT4 therapy, titrated to maintain a normal serum
thyrotropin level (main group), and 14 patients who did not receive any LT4
replacement therapy (control group). Variables of the lipid profile and left
ventricular diastolic function were measured and 24-hour electrocardiographic
monitoring was performed before randomization and at 6-month follow-up. Medical
therapy for the CAD remained unchanged throughout the 6-month study period.
RESULTS: In the main group, no statistically significant differences were found
in the lipids, variables of left ventricular diastolic function, and heart rate
pattern between the hypothyroid and euthyroid states. Individual analysis
revealed, however, that LT4 therapy was beneficial in terms of lipid
abnormalities in those patients with lower body mass index, shorter history of
CAD, and higher cholesterol levels at baseline. In the control group, we noted
statistically significant prolongation of early filling deceleration time after
6 months, which indicated less flexibility of the left ventricular myocardium
and diastolic myocardial dysfunction with long-term SH. In reference to adverse
effects of LT4 therapy, 5 of the 19 patients had an increased rate of
ventricular premature beats. These 5 patients were significantly older and
initially had more supraventricular and ventricular premature beats than the
rest of the main group. No ST depressions were recorded during LT4 therapy.
CONCLUSION: In patients with SH and CAD, LT4 therapy can be beneficial in
diminishing lipid abnormalities in those with lower body mass index, briefer
duration of CAD, and higher levels of cholesterol at baseline. Patients in our
study who experienced adverse effects of LT4 treatment were older and had more
supraventricular premature beats at baseline in comparison with the other
patients. BACKGROUND: Increased rates of depression are reported in coronary artery
disease (CAD). In heart disease, depression increases disability, reduces
quality of life, and increases mortality.
HYPOTHESIS: The study was undertaken to examine the relationship between
depression and thyroid axis function in patients with CAD.
METHODS: In all, 73 patients with CAD, consecutively admitted to a cardiac
rehabilitation hospital, were assessed for depression using the Hospital Anxiety
and Depression scale (HADS). Blood was drawn for assessment of thyroid axis
hormones and the N-amino terminal fragment of the pro-B-type natriuretic peptide
(NT-pro BNP).
RESULTS: The patients with CAD with depressive symptoms had a higher prevalence
of cardiac failure (p = 0.04), higher NT-pro BNP concentrations (p = 0.02), and
lower free triiodothyronine (T3) concentrations (p = 0.04) than patients with
CAD but without depressive symptoms. They also showed a strong trend (p = 0.058)
toward a higher incidence of the low T3 syndrome. Higher NT-pro BNP
concentrations were related to lower total T3 concentrations (r = -0.294, p =
0.011) and to higher reverse T3 concentrations (r = 0.353, p = 0.002). In men,
higher scores of depression were related to lower total T3 concentration (r =
-0.289, p = 0.034) and to higher NT-pro BNP concentration (r = 0.380, p =
0.005).
CONCLUSION: These findings suggest that symptoms of depression in patients with
CAD are associated with changes in thyroid axis function and with cardiac
impairment, especially in men. BACKGROUND: In the previous recent reports, subclinical hypothyroidism is an
independent risk factor for acute myocardial infarction in elderly women. It was
not established whether a normal range of thyroid stimulating hormone (TSH)
influences the presence of coronary atherosclerosis. We postulated that the
level of TSH is risk factor of coronary atherosclerosis in angina patients who
have normal thyroid function.
METHODS: We studied 344 angina patients (62.5+/-9.72 years, male 50%) who
underwent elective coronary angiography. TSH, free thyroxine, serum lipid levels
and high-sensitivity C-reactive protein levels were measured and compared to the
severity of coronary artery disease.
RESULTS: In patients with high level of TSH (> or =2.1 microIU/mL), age
(p=0.016), the levels of serum creatinine (p=0.004) and Gensini's score
(p=0.016) were significantly higher than those in patients with low TSH levels.
The incidence of multi-vessel disease was higher in patients with high TSH level
(p=0.026). TSH level showed a significant correlation with age (r=0.109,
p=0.044) and Gensini's score (r=0.117, p=0.045). The multivariate analysis
revealed that age (OR 2.39, p=0.001), diabetes (OR 3.74, p=0.001), creatinine
(OR 2.06, p=0.008), and smoking (OR 1.85, p=0.045) were independent predictors
for significant coronary artery disease, but TSH level did not predict coronary
artery stenosis.
CONCLUSIONS: Although the high level of serum TSH is associated with
multi-vessel disease, it was not the determit of coronary artery disease in
patients with normal range of thyroid function. BACKGROUND: Previous studies have suggested that sub-clinical thyroid states may
have detrimental effects on the coronary heart disease (CHD). Whether
subclinical thyroid dysfunction is a risk factor for the above is controversial.
METHODS: A systemic search of the literature using Pubmed, Medline and Ovid
online tool was performed to identify relevant studies. Amongst the clinical
studies, crossectional study and studies with follow-up period ranging between 4
and 20 yr were identified (Walsh JP, Bremner AP, Bulsara MK, et al. Subclinical
thyroid dysfunction as a risk factor for cardiovascular disease. Arch Intern Med
2005 Nov 28;165 (21):2467-72.; Rodondi N, Newman AB, Vittinghoff E, et al.
Subclinical hypothyroidism and the risk of heart failure, other cardiovascular
events, and death. Arch Intern Med 2005 Nov 28; 165 (21):2460-6.; Rotterdam
study, Imaizumi M, Akahoshi M, Ichimaru S, et al. Risk for coronary heart
disease and all-cause mortality in subclinical hypothyroidism. J Clin Endocrinol
Metab 2004 Jul; 89 (7):3365-70.; Capolla et al.; Parle JV, Maisonneuve P,
Sheppard MC, Boyle P, Franklyn JA. Prediction of all-cause and cardiovascular
mortality in elderly people from one low serum thyrotropin result: a 10-year
cohort study. Lancet 2001 Sep 15; 358 (9285):861-5).
RESULTS: Sub-clinical hypothyroidism: The pooled estimate of the relative risk
of CHD revealed significant difference both at baseline [RR with 95% CI: 1.533
(1.312-1.791), P<0.05] and at follow-up [RR with 95% CI: 1.188 (1.024-1.379),
P<0.05]. The relative risk of all-cause mortality at follow-up revealed no
significant difference. However, the relative risk of death from cardiovascular
causes at follow-up was significantly higher [RR with 95% CI: 1.278
(1.023-1.597), P<0.05]. Sub-clinical hyperthyroidism: The pooled estimate of the
relative risk of CHD revealed no significant difference both at baseline [RR
with 95% CI: 1.156 (0.709-1.883)] and at follow-up [RR with 95% CI: 1.207
(0.780-1.870)].The relative risk of death from cardiovascular causes at
follow-up was also not significantly higher.
CONCLUSION: The present meta-analysis indicates that sub-clinical hypothyroidism
is associated with both, a significant risk of CHD at baseline and at follow-up.
In addition, mortality from cardiovascular causes is significantly higher at
follow-up. Sub-clinical hyperthyroidism is not associated with CHD or mortality
from cardiovascular causes. Altered cardiac function in thyroid disease is well recognized and has been
extensively investigated, vascular function has however been less well studied
in those with thyroid dysfunction. Thyroid hormones, thyroxine (T(4)) and
triiodothyronine (T(3)) are important regulators of cardiac function and
cardiovascular hemodynamics. The cardiovascular system responds to minimal but
persistent changes in circulating thyroid hormone levels producing changes in
vascular reactivity and endothelial function. The detection of endothelial
dysfunction and/or arterial stiffness allows early identification of individuals
at risk as these occur in both patients with risk factors for coronary artery
disease and in those with established disease. This may allow treatment to be
targeted at high risk individuals with the aim of slowing the progression of
vascular disease. The various methods used to assess arterial function are
reviewed and the changes demonstrated in human and animal models of thyroid
dysfunction. Thyroid hormone has many effects on the heart and cardiovascular system.
Thyrotoxicosis is associated with increased cardiovascular morbidity and
mortality, primarily due to heart failure and thromboembolism. However, the
relationship between thyroid hormone excess and the cardiac complications of
angina pectoris and myocardial infarction remains largely speculative. Moreover,
few studies have been reported on the effect of thyroid hormone levels within
normal range on coronary artery disease (CAD). Therefore we examined the
association of thyroid function with coronary artery diseases in euthyroid
angina patients. Total 192 subjects (mean age; 60.8 yrs) were enrolled in which
coronary angiograms were performed due to chest pain. We measured free thyroxine
(FT(4)), thyroid stimulating hormone (TSH), serum lipid levels and
high-sensitivity C-reactive protein (hsCRP) levels and analyzed their
association with the presence of CAD. Serum FT(4) levels were higher in patients
with CAD compared with the patients without CAD (1.31 +/- 0.30 vs 1.20 +/- 0.23,
p = 0.006), and high FT(4) level was associated with the presence of
multi-vessel disease. Multivariate analysis showed that age (odds ratio (OR)
1.04; 95% confidence interval (CI) 1.01-1.07, p = 0.007), hypertension (OR 2.04;
95% CI 1.06-3.90, p = 0.036) and FT(4) (OR 4.23; 95% CI 1.12-15.99, p = 0.033),
were the determits for CAD. The relative risk (RR) for CAD in highest tertile
of FT(4) showed increased risk compared with the lowest tertile (RR 1.98; 95% CI
0.98-3.99, p<0.001). Our study showed that FT(4) levels were associated with the
presence and the severity of CAD. Also, this study suggests that elevated serum
FT(4) levels even within normal range could be a risk factor for CAD. Further
studies will be necessary to confirm the relationship of thyroid function and
CAD. BACKGROUND: Overt thyroid dysfunction, hypothyroidism in particular, may lead to
coronary artery disease (CAD). Whether more subtle anomalies of thyroid hormone
metabolism influence the progression of CAD remains a matter of speculation.
HYPOTHESIS: The occurrence of CAD and long-term prognosis in patients without a
history of either primary thyroid disease, myocardial infarction, or chronic
heart failure is related to serum levels of biologically active free
triiodothyronine (fT3).
METHODS: The cohort consisted of 1047 clinically and biochemically euthyroid
patients (median age 65.6 y and 69% male) who underwent coronary angiography in
our institute for suspected CAD.
RESULTS: Lower fT3 levels were predictive of both single-vessel (p = 0.012) and
multivessel (p = 0.009) CAD. Through a multivariate logistic regression
analysis, fT3 was still linked to the presence of CAD (hazard ratio [HR]: 0.48,
95% confidence interval [CI]: 0.34-0.68, p < 0.001). After a mean follow-up of
31 months, the survival rate was 95% and total mortality (log-rank 6.75, p =
0.009), as well as cardiac mortality (log-rank 8.26, p = 0.004), was greater
among patients with low T3 (fT3 < 2.10 pg/mL) syndrome. At subsequent
multivariate Cox regression analysis, the association between low T3 syndrome
and survival was maintained (total mortality HR: 1.80, 95% CI: 1.05-3.10, p =
0.034; cardiac mortality HR: 2.58, 95% CI: 1.13-5.93, p = 0.025).
CONCLUSIONS: In this selected population, fT3 levels were inversely correlated
to the presence of CAD and low T3 syndrome conferred an adverse prognosis, even
after adjusting for traditional coronary risk factors. Dyslipidemia is a common finding in patients with thyroid disease, explained by
the adverse effects of thyroid hormones in almost all steps of lipid metabolism.
Not only overt but also subclinical hypo- and hyperthyroidism, through different
mechanisms, are associated with lipid alterations, mainly concerning total and
LDL cholesterol and less often HDL cholesterol, triglycerides, lipoprotein (a),
apolipoprotein A1, and apolipoprotein B. In addition to quantitative,
qualitative alterations of lipids have been also reported, including atherogenic
and oxidized LDL and HDL particles. In thyroid disease, dyslipidemia coexists
with various metabolic abnormalities and induce insulin resistance and oxidative
stress via a vice-vicious cycle. The above associations in combination with the
thyroid hormone induced hemodynamic alterations, might explain the increased
risk of coronary artery disease, cerebral ischemia risk, and angina pectoris in
older, and possibly ischemic stroke in younger patients with overt or
subclinical hyperthyroidism. OBJECTIVE: In a mortality follow-up of the HUNT Study, serum TSH within the
reference range was positively associated with the risk of coronary death in
women. We now aimed to confirm the association of high serum TSH with the risk
of coronary heart disease, using hospital-based diagnoses of myocardial
infarction.
DESIGN: Prospective population-based study with linkage to hospital information
on myocardial infarction and to the national Cause of Death Registry.
PARTICIPANTS: A total of 26, 707 people without previously known thyroid or
cardiovascular disease or diabetes at baseline.
MEASUREMENTS: Hazard ratios (HR) of coronary death and HRs of hospitalization
with a first-time acute myocardial infarction, by baseline thyroid function.
RESULTS: During 12, years of follow-up, 960 (3·6%) participants had been
hospitalized with first-time myocardial infarction and 558 (2·1%) had died from
coronary heart disease. High TSH within the reference range was associated with
increased risk of coronary death in women (P(trend) 0·005), but not in men. The
risk of coronary death was also increased among women with subclinical
hypothyroidism or subclinical hyperthyroidism, compared to women with TSH of
0·50-1·4 mU/l. However, thyroid function was not associated with the risk of
being hospitalized with myocardial infarction.
CONCLUSIONS: High serum TSH was associated with increased mortality from
coronary heart disease in women, but we found no association of thyroid function
with the risk of being hospitalized with myocardial infarction. Thus, the
morbidity finding does not confirm the suggestion that low thyroid function
within the clinically normal range is associated with increased risk of coronary
heart disease. OBJECTIVE: The aim of this study is to investigate the relationship between
serum thyroid hormone levels that are within the normal range and the presence
and severity of coronary artery disease (CAD) in patients referred for coronary
angiography.
METHODS: In this observational study, we enrolled 119 consecutive patients (77
men, mean age 60.7±13.8 years) who underwent coronary angiography. Blood samples
were tested for thyroid stimulating hormone (TSH), free triiodothyronine (FT3)
and free thyroxine (FT4) concentrations. Additionally, risk factors, clinical
characteristics and angiographic results were obtained. The patients were
separated into two groups according to the Gensini score as those with mild or
severe atherosclerosis. Statistical analysis was performed using the Chi-square,
Mann-Whitney U, correlation and logistic regression tests, and ROC analysis.
RESULTS: FT3 levels were significantly lower in subjects with CAD (4.0±0.7 vs.
4.6±0.6 pmol/L; p<0.001). Moreover, lower FT3 levels were found in patients with
severe atherosclerosis (3.9±0.7 vs. 4.5±0.6 pmol/L; p<0.001). Logistic
regression analysis demonstrated that the lower FT3 levels were associated with
the presence (OR =0.266, 95% CI: 0.097-0.731, p=0.01) and severity (OR=0.238,
95% CI:0.083-0.685, p=0.008) of CAD. In the ROC analysis, a level of FT3 ≤4.2
pmol/L was found to predict the presence of CAD with 69% sensitivity and 71%
specificity (AUC:0.744, 95% CI:0.653-0.834, p<0.001); and the severity of CAD
with 75% sensitivity and 67% specificity (AUC:0.733, 95% CI:0.642-0.824,
p<0.001).
CONCLUSIONS: FT3 levels within the normal range were inversely correlated with
the presence and severity of CAD. Moreover, lower FT3 concentrations were
correlated with the Gensini score and independently predicted the presence and
severity of CAD. Thus, the FT3 levels may be used as the indicator of increased
risk for CAD. The thyroid and the cardiovascular system are closely related, both in
physiological and pathological conditions. The adverse consequences on the heart
of overt thyroid disease are well-known and even subclinical forms of both
hyperthyroidism and hypothyroidism are associated with increased cardiovascular
mortality. In recent years, attention has shifted towards milder forms of
thyroid disease, such as the so-called "low T3 syndrome", which is characterized
by an isolated reduction in circulating levels of the biologically active form
of thyroid hormone, triiodothyronine (T3). Furthermore, variations of T3 within
the physiological range have been linked to coronary artery disease, one of the
leading causes of morbidity and mortality worldwide. The present manuscript
provides an overview of thyroid physiology and pathophysiology, with a
particular focus on cardiovascular disease in patients with milder forms of
thyroid dysfunction. Studies on the relationship between thyroid stimulating hormone (TSH) within the
reference range and coronary artery disease (CAD) have produced conflicting
results. Furthermore, the effect of age on this relationship has never been
explored. The aim of this study was to investigate the association between TSH
levels and CAD among euthyroid subjects and whether age influenced this
relationship. A total of 318 subjects who underwent coronary angiography were
included. Serum TSH, T3, T4, lipid, blood glucose and creatinine levels were
measured and compared between the groups with and without CAD. Age-stratified
analysis and multivariate logistic regression analysis were performed. Levels of
TSH, T3 and T4 did not differ significantly between CAD (n=196) and non-CAD
group (n=122) (TSH: 1.77 ± 0.99 vs 1.89 ± 0.98 mIU/L, T3: 1.45 ± 0.36 vs 1.51 ±
0.35 nmol/L, T4: 100.06 ± 20.49 vs 103.95 ± 24.06 nmol/L, respectively) when
comparisons were performed among all subjects. A significant between-group
difference in levels of TSH was observed among subjects less than or equal to 65
years old (CAD group: n=121, non-CAD group: n=106), with higher TSH levels in
CAD group (2.03 ± 0.94 vs 1.75 ± 0.97 mIU/L, adjusted p=0.024). Multivariate
logistic regression analysis revealed that elevated level of TSH was an
independent predictor for CAD (odds ratio: 1.512, p=0.011). No significant
between-group difference in TSH levels was observed among subjects older than 65
years (CAD group: n=75, non-CAD group: n=16). The results showed that higher
levels of TSH within the reference range were independently associated with the
presence of CAD only among subjects less than or equal to 65 years old,
suggesting age might influence the relationship. |
What is the frequency of mutations induced spontaneously through Ethylnitrosourea (ENU) mutagenesis? | Theoretical considerations and empirical analysis suggest that the per-base mutation frequency for a fractionated-dose treatment protocol is on the order of 1 sequence change per 10(5) bp. | In order to maximize the mutagenic effectiveness of N-ethyl-N-nitrosourea in
mouse stem-cell spermatogonia, advantage was taken of the fact that these cells
can accumulate mutations from repeated doses given over relatively long time
periods. Repeated doses (100 mg/kg) of ethylnitrosourea injected
intraperitoneally into male mice at weekly intervals were found to allow
adequate survival and fertility with total dosages of 300 and 400 mg/kg. The
specific-locus mutation frequencies at these dosages were, respectively, 1.8 and
2.2 times that obtained with the maximal practicable single dose of 250 mg/kg.
The mutation frequency induced by a 400 mg/kg dosage of ethylnitrosourea is 12
times the maximal mutation frequency achievable with a single exposure to x-rays
and 36 times that reported for procarbazine, the most effective chemical mutagen
previously known for mouse stem-cell spermatogonia. Ethylnitrosourea is already
the mutagen of choice in deliberate attempts to create mouse models for human
disease and in any experiments in which a maximal mutation rate is desired.
Repeated-dose regimens similar to the ones reported here should increase the
efficiency of such studies. The ability to generate mutations is a prerequisite to functional genetic
analysis. Despite a long history of using mice as a model system for genetic
analysis, the scientific community has not generated a comprehensive collection
of multiple alleles for most mouse genes. The chemical mutagen of choice for
mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly
causes base substitutions in DNA, and therefore allows for recovery of complete
and partial loss-, as well as gain-, of-function alleles . Specific locus tests
designed to detect recessive mutations showed that ENU is the most efficient
mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes. In
fact, several genome-wide and region-specific screens based on phenotypes have
been carried out. The anticipation of the completion of the human and mouse
genome projects, however, now emphasizes genotype-driven genetics--from sequence
to mutants. To take advantage of the mutagenicity of ENU and its ability to
create allelic series of mutations, we have developed a complementary approach
to generating mutations using mouse embryonic stem (ES) cells. We show that a
high mutation frequency can be achieved and that modulating DNA-repair
activities can enhance this frequency. The treated cells retain germline
competency, thereby rendering this approach applicable for efficient generation
of an allelic series of mutations pivotal to a fine-tuned dissection of
biological pathways. Treating mice with ethylnitrosourea (ENU) is an efficient means for mutagenizing
spermatogonial cells, and this treatment has been proven effective in a variety
of screens for both domit and recessive mutations. However, a significant
problem for this technology is that the efficiency of mutagenesis is assessed
most often by the empiric determination of a per-locus mutation frequency by
using the specific locus test, which is expensive, time-consuming, and
logistically difficult. To approach this question more directly and more
efficiently, one can utilize methods of PCR-based mutation detection for the
characterization of progeny of mutagenized mice. Since this analysis can be done
after a single generation of breeding, it is useful as a rapid means for the
assessment of the efficiency of mutagen treatment. Furthermore, it is readily
imaginable that this strategy can be applied for the general determination of
gene function in a systematic manner. Theoretical considerations and empirical
analysis suggest that the per-base mutation frequency for a fractionated-dose
treatment protocol is on the order of 1 sequence change per 10(5) bp. OBJECTIVE: To explore the molecular spectra and mechanism of human hypoxanthine
guanine phosphoribosyl transferase (HPRT) gene mutation induced by
ethyluitrosourea (ENU).
METHODS: Single cell cloning culture, two-way screening, multiple PCR
amplification and electrophoresis technique were used.
RESULTS: With dose of ENU increasing, cell plating efficiency reduced (in the
group with 100-200 micro g/ml doses, P < 0.01) and mutation frequency increased
(in the group with 12.5-200.0 micro g/ml doses, P < 0.05) significantly. There
was no all exons deletion in spontaneous mutations, and only 7.7% of them were
detected as single exon deletion. But, deletion was found in 79.7% of
ENU-induced mutations (62.5%-89.4%, P < 0.01), and deletion mutations in all
nine exons of HPRT gene. Most of ENU-induced mutations were chain deletion with
multiple exons (88.1%).
CONCLUSIONS: The spectra in spontaneous mutations differed completely from
ENU-induced ones. ENU was liable to cause bigger changes in genetic structure,
which suggested a stronger ENU's mutagenesis. A perceived disadvantage of transgenic rodent mutation assays is that
spontaneous mutant frequencies are high compared to those of endogenous genes
and may consequently reduce sensitivity to induced mutation. We have previously
argued that unrepaired G:T mismatches from spontaneous deamination of
5-methylcytosine at CpG sites could be converted to apparent in vivo mutations
in the bacterial recovery systems because of rapid, random, mismatch repair in
Escherichia coli. In this study, we have measured mutation frequencies in spleen
of male mice induced by N-ethyl-N-nitrosourea (ENU) using the PhiX174 transgene,
which is not subject to mismatch repair in E.coli, using single-burst analysis,
a unique method to identify in vivo mutation. In order to compare our results to
those using the lacI and cII transgenes, we converted all mutant frequencies to
base pair substitution (bps) mutation frequencies per nucleotide based on mutant
spectra from this study and published literature. We found this frequency in
control spleen to be similar for lacI (3.8 +/- 0.7 x 10(-8)) and PhiX174 (3.1
+/- 1.2 x 10(-8)) at 6 weeks of age. We found a strong age dependence for
spontaneous lacI mutation that extrapolated to a value at conception (1.8 +/-
0.9 x 10(-8)) that was not significantly different from the human germ line bps
mutation frequency per nucleotide of 1.7 +/- 0.2 x 10(-8). These two transgenes
provided similar mutational responses to 40 mg/kg ENU, 7- to 9-fold. In
contrast, the cII target gene in the same tissue produces both spontaneous and
induced mutation frequencies approximately 10 times higher, for unknown reasons.
We conclude that the spontaneous mutant frequencies measured by the lacI and
PhiX174 transgenes in this moderately dividing tissue accurately measure in vivo
mutation frequencies at early ages. For these two transgenes, seemingly high
mutant frequencies may reflect the expected accumulation of somatic mutation
with age. |
What is the molecular pathogenesis of Spinal Muscular Atrophy? | Spinal muscular atrophy is an autosomal recessive neurodegenerative disease characterised by degeneration of spinal cord motor neurons, atrophy of skeletal muscles, and generalised weakness. It is caused by homozygous disruption of the survival motor neuron 1 (SMN1) gene by deletion, conversion, or mutation, resulting in downregulated biogenesis of uridine-rich small nuclear RNAs (U snRNAs), which are major components of the splicing machinery. | Spinal muscular atrophy is an autosomal recessive neurodegenerative disease
characterised by degeneration of spinal cord motor neurons, atrophy of skeletal
muscles, and generalised weakness. It is caused by homozygous disruption of the
survival motor neuron 1 (SMN1) gene by deletion, conversion, or mutation.
Although no medical treatment is available, investigations have elucidated
possible mechanisms underlying the molecular pathogenesis of the disease.
Treatment strategies have been developed to use the unique genomic structure of
the SMN1 gene region. Several candidate treatment agents have been identified
and are in various stages of development. These and other advances in medical
technology have changed the standard of care for patients with spinal muscular
atrophy. In this Seminar, we provide a comprehensive review that integrates
clinical manifestations, molecular pathogenesis, diagnostic strategy,
therapeutic development, and evidence from clinical trials. Disappearance of TAR-DNA-binding protein 43 kDa (TDP-43) from the nucleus
contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS), but the
nuclear function of TDP-43 is not yet fully understood. TDP-43 associates with
nuclear bodies including Gemini of coiled bodies (GEMs). GEMs contribute to the
biogenesis of uridine-rich small nuclear RNA (U snRNA), a component of splicing
machinery. The number of GEMs and a subset of U snRNAs decrease in spinal
muscular atrophy, a lower motor neuron disease, suggesting that alteration of U
snRNAs may also underlie the molecular pathogenesis of ALS. Here, we
investigated the number of GEMs and U11/12-type small nuclear ribonucleoproteins
(snRNP) by immunohistochemistry and the level of U snRNAs using real-time
quantitative RT-PCR in ALS tissues. GEMs decreased in both TDP-43-depleted HeLa
cells and spinal motor neurons in ALS patients. Levels of several U snRNAs
decreased in TDP-43-depleted SH-SY5Y and U87-MG cells. The level of U12 snRNA
was decreased in tissues affected by ALS (spinal cord, motor cortex and
thalamus) but not in tissues unaffected by ALS (cerebellum, kidney and muscle).
Immunohistochemical analysis revealed the decrease in U11/12-type snRNP in
spinal motor neurons of ALS patients. These findings suggest that loss of TDP-43
function decreases the number of GEMs, which is followed by a disturbance of
pre-mRNA splicing by the U11/U12 spliceosome in tissues affected by ALS. |
Which drugs affect insulin resistance in obesity? | Enistein treatment could help reduce insulin resistance
ACE inhibitor drugs may improve insulin resistance | Visceral obesity is frequently associated with muscle insulin resistance. Rats
fed a high-fat diet rapidly develop obesity and insulin resistance.
Dehydroepiandrosterone (DHEA) has been reported to protect against the
development of obesity. This study tested the hypothesis that DHEA protects
against the increase in visceral fat and the development of muscle insulin
resistance induced by a high-fat diet in rats. Feeding rats a diet providing 50%
of the energy as fat for 4 wk resulted in a twofold greater visceral fat mass
and a 50% lower rate of maximally insulin-stimulated muscle 2-deoxyglucose
(2-DG) uptake compared with controls. Rats fed the high-fat diet plus 0.3% DHEA
were largely protected against the increase in visceral fat (+ 11.3 g in high
fat vs. + 2.9 g in high fat plus DHEA, compared with controls) and against the
decrease in insulin-stimulated muscle 2-DG uptake (0.94 +/- 0.15 mumol.ml-1.20
min-1, controls; 0.46 +/- 0.06 mumol.ml-1.20 min-1, high-fat diet; 0.78 +/- 0.07
mumol.ml-1.20 min-1, high fat + DHEA). DHEA did not affect food intake. These
results show that DHEA has a protective effect against accumulation of visceral
fat and development of muscle insulin resistance in rats fed a high-fat diet. BACKGROUND: Insulin resistance, which can lead to a number of diseases including
type 2 diabetes and coronary heart disease, is believed to have evolved as an
adaptation to periodic starvation. The "thrifty gene" and "thrifty phenotype"
hypotheses constitute the domit paradigm for over four decades. With an
increasing understanding of the diverse effects of impairment of the insulin
signaling pathway, the existing hypotheses are proving inadequate.
PRESENTATION OF THE HYPOTHESIS: We propose a hypothesis that insulin resistance
is a socio-ecological adaptation that mediates two phenotypic transitions, (i) a
transition in reproductive strategy from "r" (large number of offspring with
little investment in each) to "K" (smaller number of offspring with more
investment in each) and (ii) a transition from "stronger to smarter" or "soldier
to diplomat" i.e. from relatively more muscle dependent to brain dependent
lifestyle. A common switch could have evolved for the two transitions since the
appropriate environmental conditions for the two transitions are highly
overlapping and interacting.
TESTING THE HYPOTHESIS: Gestational insulin resistance diverts more energy
through the placenta, resulting in increased investment per offspring. On the
other hand, insulin resistance is associated with reduced ovulation. The insulin
signaling pathway is also related to longevity. Insulin resistance diverts more
nutrients to the brain as compared to muscle. Also, hyperinsulinemia has direct
positive effects on cognitive functions of the brain. The hypothesis gets
support from known patterns in human clinical data and recent research on the
molecular interactions in the insulin signaling pathway. Further we state many
predictions of the hypothesis that can be tested experimentally or
epidemiologically.
IMPLICATIONS OF THE HYPOTHESIS: The hypothesis can bring about a significant
change in the line of treatment as well as public health policies for the
control of metabolic syndrome. BACKGROUND: There is a great deal of controversy surrounding the relationship
between alcohol consumption and insulin resistance. This association may be
further confounded by the presence of obesity. We aimed to clarify whether
regular alcohol consumption improves insulin resistance in healthy Japanese men
and whether obesity affects this relationship.
METHODS: We examined 1029 men (ages 24 to 87 y) who had undergone medical
checkups. They were divided into non-obese (body mass index (BMI) <25 kg/m(2))
or obese subjects (BMI > or =25 kg/m(2)) and further classified into non-regular
drinkers (NRD), moderate drinkers (MD; 1-6 days/week), and daily drinkers (DD; 7
days/week). The homeostasis model assessment of insulin resistance (HOMA-IR) and
other cardiac risk factors were compared between the groups.
RESULTS: In both non-obese and obese men, alcohol consumption decreased HOMA-IR
in a dose-dependent manner, although HOMA-IR was about 2 times greater in obese
men compared to non-obese men in any category (p<0.001). Stepwise logistic
regression analysis revealed that alcohol consumption was the independent
negative risk factor for HOMA-IR [OR, 0.576 (95% C.I. 0.402-0.824), p=0.003]
after adjusting for age, BMI, systolic blood pressure, smoking status,
LDL-cholesterol, HDL-cholesterol, and liver dysfunction.
CONCLUSIONS: Regular alcohol consumption improves insulin resistance in healthy
Japanese men independent of obesity. OBJECTIVES: The aim of this study was to investigate serum leptin, adiponectin
and paraoxonase1 levels in adult females receiving pharmacotherapy for various
psychiatric disorders.
METHODS: The study group consisted of 32 obese females (mean age 40.53 +/- 11.00
years, mean body mass index 35.44 +/- 5.33 kg/m(2)) who were receiving treatment
for psychiatric disorders, and the control group included 22 obese females (mean
age 35.95 +/- 9.16 years, mean body mass index 30.78 +/- 3.33 kg/m(2)) who were
free of psychiatric disorders. Analyses were performed using a bioelectrical
impedance device. Fasting blood samples were obtained for complete blood count
and various biochemical tests, including determination of leptin, adiponectin
and paraoxonase1 activity.
RESULTS: Body mass index, waist and hip circumference, body fat percentage,
fasting blood glucose, insulin, glycosylated hemoglobin, homeostasis model
assesement of insulin resistance, alanine transaminase, aspartate tarnsaminase,
and leptin levels were significantly higher in the study group than in controls.
Although body weight was positively correlated with leptin levels in both
groups, body weight was negatively correlated with adiponectin levels in the
control group and positively correlated with adiponectin levels in the study
group. In the study group, body mass index and hip circumference correlated
positively with leptin levels, hip circumference correlated positively with
adiponectin levels, and waist to hip ratio correlated positively with
paraoxonase levels. In the control group, body mass index as well as waist and
hip circumferences were positively correlated with leptin levels. Weight, body
mass index, and hip circumference were also negatively correlated with the
adiponectin/leptin ratio in the control group.
CONCLUSION: This study indicates a higher risk for obesity-related disorders,
particularly metabolic syndrome, diabetes and cardiovascular disease, in
patients treated with psychiatric drugs. OBJECTIVE: Chronically elevated free fatty acids contribute to insulin
resistance and pancreatic β-cell failure. Among numerous potential factors, the
involvement of endoplasmic reticulum (ER) stress has been postulated to play a
mechanistic role. Here we examined the efficacy of the chemical chaperone,
sodium phenylbutyrate (PBA), a drug with known capacity to reduce ER stress in
animal models and in vitro, on lipid-induced insulin resistance and β-cell
dysfunction in humans.
RESEARCH DESIGN AND METHODS: Eight overweight or obese nondiabetic men underwent
four studies each, in random order, 4 to 6 weeks apart. Two studies were
preceded by 2 weeks of oral PBA (7.5 g/day), followed by a 48-h i.v. infusion of
intralipid/heparin or saline, and two studies were preceded by placebo
treatment, followed by similar infusions. Insulin secretion rates (ISRs) and
sensitivity (S(I)) were assessed after the 48-h infusions by hyperglycemic and
hyperinsulinemic-euglycemic clamps, respectively.
RESULTS: Lipid infusion reduced S(I), which was significantly ameliorated by
pretreatment with PBA. Absolute ISR was not affected by any treatment; however,
PBA partially ameliorated the lipid-induced reduction in the disposition index
(DI = ISR × S(I)), indicating that PBA prevented lipid-induced β-cell
dysfunction.
CONCLUSIONS: These results suggest that PBA may provide benefits in humans by
ameliorating the insulin resistance and β-cell dysfunction induced by prolonged
elevation of free fatty acids. Obesity is associated with metabolic derangements such as insulin resistance,
inflammation and hypercoagulobility which can all be understood as consequences
of adipose tissue dysfunction. The potential role for adipose tissue derived
cytokines and adipokines in the development of vascular disease and diabetes may
produce a clinical need to influence adipose tissue function. Various
pharmacological and non-pharmacological interventions affect plasma cytokine and
adipokine levels. The effects of these interventions depend on weight loss per
se, changes in fat distribution without weight loss and/or direct effects on
adipose tissue inflammation.Weight loss, as a result of diet, pharmacology and
surgery, positively influences plasma adipokines and systemic inflammation.
Several classes of drugs influence systemic inflammation directly through their
anti-inflammatory actions. PPAR-γ agonism positively influences adipose tissue
inflammation in several classes of intervention such as the thiazolidinediones
and perhaps salicylates, CB1-antagonists and angiotensin II receptor blockers.
Furthermore, within drug classes there are differential effects of individual
pharmacologic agents on adipose tissue function.It can be concluded that several
commonly used pharmacological and non-pharmacological interventions have
unintended influences on adipose tissue function. Improving adipose tissue
function may contribute to reducing the risk of vascular diseases and the
development of type 2 diabetes. BACKGROUND: Pomegranate seed oil has been shown to protect against diet induced
obesity and insulin resistance.
OBJECTIVE: To characterize the metabolic effects of punicic acid on high fat
diet induced obesity and insulin resistance.
DESIGN: High-fat diet or high-fat diet with 1% Pomegranate seed oil (PUA) was
fed for 12 weeks to induce obesity and insulin resistance. We assessed body
weight and composition (pSABRE DEXA-scan), energy expenditure (Columbus
Instruments) and insulin sensitivity at the end of the 12 weeks.
RESULTS: PSO intake resulted in a lower body weight, 30.5±2.9 vs 33.8±3.2 g PSO
vs HFD respectively, p=0.02, without affecting food intake or energy
expenditure. The lower body weight was fully explained by a decreased body fat
mass, 3.3±2.3 vs 6.7±2.7 g for PSO and HFD fed mice, respectively, p=0.02.
Insulin clamps showed that PSO did not affect liver insulin sensitivity but
clearly improved peripheral insulin sensitivity, 164±52% vs 92±24% for PSO and
HFD fed mice respectively, p=0.01.
CONCLUSIONS: We conclude that dietary PSO ameliorates high-fat diet induced
obesity and insulin resistance in mice, independent of changes in food intake or
energy expenditure. Obesity is a significant health problem worldwide and is associated with a
number of co-morbidities including type 2 diabetes mellitus, hypertension,
dyslipidemia, obstructive sleep apnea, and cardiovascular disease. A number of
different pathophysiologic mechanisms including increased inflammation,
oxidative stress, and insulin resistance have been associated with initiation
and progression of atherosclerotic disease in obese individuals. Lifestyle
modifications have provided modest results in weight reduction and the focus of
interest has now shifted towards drug development to treat severely obese
individuals with a body mass index (BMI) >30 kg/m(2) or those with a BMI >27
kg/m(2) who have additional co-morbidities. Different regimens focusing on
dietary absorption or acting centrally to control hunger and food intake have
been developed. However, their weight loss effect is, in most cases, modest and
this effect is lost once the medication is discontinued. In addition, long-term
use of these drugs is limited by significant side effects and lack of long-term
safety and efficacy data. Orlistat is the only US FDA-approved medication for
long-term use. A number of new medications are currently under investigation in
phase III trials with promising preliminary results. This review comments on
available anti-obesity pharmacologic regimens, their weight-loss benefit, and
their impact on cardiovascular risk factors. Obesity is a chronic medical condition that is expected to become an indirect
but leading cause of mortality and morbidity. Obesity results in type 2 diabetes
mellitus, insulin resistance, hypertension, dyslipidemia, coronary heart
disease. These factors contribute to cardiovascular disease that is a leading
cause of death. Therefore, the approach to obesity therapy should be designed to
reduce cardiovascular disease risk and mortality. Diet and lifestyle changes
remain the cornerstones of therapy for obesity, but the resultant weight loss is
often small. For more effective weight loss, individuals have shown to benefit
from anti-obesity medications. Anti-Obesity therapy is considered for
individuals with a body mass index greater than 30 kg/m2 or ranging from 25 to
30 kg/m2, or individuals with co-morbid conditions. Recent anti-obese
medications affect biological mechanisms that suppress appetite and absorb
nutrients to regulate body weight. In this review, we discuss the FDA approved
anti-obesity drugs and recent patents which include phentermine/topiramate,
pramlintide, lorcaserin, AOD9604, oleoyl-estrone, trk-beta antagonists and
melanin concentrating hormone that can reduce adiposity at the molecular level. AIM: Ezetimibe selectively blocks intestinal cholesterol absorption by
inhibiting Niemann-Pick C1-like 1 (NPC1L1) and reducing LDL cholesterol (LDL-C).
In animals, ezetimibe reversed diet-induced obesity, liver steatosis, and
insulin resistance. In humans, its potential effects on liver steatosis and
insulin resistance have been suggested. We investigated the effects of ezetimibe
on postprandial hyperlipidaemia and hyperglycaemia in obese subjects with
dyslipidaemia in a double-blind randomized crossover trial.
METHODS: Twenty obese men with hypertriglyceridaemia were assigned randomly to
an ezetimibe- or a placebo-precedence-treated group. Subjects in the ezetimibe
group were treated with ezetimibe (10 mg/day) for the first 4 weeks, followed by
a 4-week interval and then treated with placebo for another 4 weeks. The placebo
group received these treatments in reverse order. Subjects were requested to
fast for at least 12 hours and then received a standard meal. Blood samples were
collected at 0, 30, 60, 120, 240, 360 and 480 minutes after the meal on Days 0,
28, 56 and 84 and were used to measure the lipid and glucose metabolism markers.
RESULTS: Ezetimibe significantly decreased the postprandial serum triglyceride
excursion (p=0.01) and fasting serum LDL-C, remt-like particles(RLP) and
ApoB48 levels (p<0.05). Postprandial glucose excursion, serum insulin levels,
serum glucose-dependent insulinotropic polypeptide (GIP) and active
glucagon-like peptide-1 (GLP-1) were not significantly affected by ezetimibe
treatment.
CONCLUSION: Ezetimibe restored the postprandial dysregulation of lipid but did
not affect glucose metabolism in a double-blind randomized crossover trial. OBJECTIVE: Long-term treatment with topiramate reduces body weight and improves
insulin sensitivity in obese humans. Our aim was to evaluate the effect of
topiramate treatment for 4 weeks on insulin sensitivity and secretion,
independent of weight loss.
DESIGN: Randomized, double-blind, crossover, placebo-controlled study.
METHODS: Thirteen obese (BMI 36.6 ± 1.3 kg/m(2) (mean ± s.e.m.)),
insulin-resistant (homeostasis model of assessment-insulin resistance 2.0 ± 0.2)
women received topiramate (T, maximum dose of 75 mg) and placebo (P) for 4
weeks, separated by a 4-week washout period. Insulin sensitivity and β-cell
function were assessed using a two-step hyperinsulinemic euglycemic clamp with
stable isotopes and a hyperglycemic clamp.
RESULTS: Hepatic and peripheral insulin sensitivities were not affected by
topiramate treatment (glucose disposal rate (step 1 (insulin infusion rate 10
MU/M(2) per min) T: 17.5 ± 0.8 vs P: 18.5 ± 1.0 μmol/kg(LBM) per min, t=1.016,
P=0.33; step 2 (insulin infusion rate 40 mU/m(2) per min) T: 27.9 ± 3.2 vs P:
28.8 ± 1.9 μmol/kg(LBM) per min, t=0.418, P=0.68)). Subjects lost a small amount
of weight during the topiramate period (T: -1.0 ± 0.2 vs P: -0.1 ± 0.2 kg,
t=2842, P=0.15). There were no changes in body fat mass, blood pressure, and
fasting glucose. β-Cell function was not affected by topiramate as evidenced by
an unaltered area under the curve of early (0-10 min; T: 1929.6 ± 265.7 vs P:
2024.7 ± 333.6 pmol/l, t=-0.357, P=0.73) and late (80-120 min; T: 28,017.7 ±
5029.9 vs P: 31,567.7 ± 5376.2 pmol/l, t=-1.481, P=0.16) phase insulin levels
during hyperglycemia. The use of topiramate was associated with significant side
effects such as paresthesia, nausea, dizziness, and concentration problems.
CONCLUSIONS: Low-dose topiramate treatment for 4 weeks, relative to placebo, had
no significant effect on insulin sensitivity in overweight/obese adult females
without established diabetes. Obesity/metabolic syndrome are common risk factors for overactive bladder. This
study aimed to investigate the functional and molecular changes of detrusor
smooth muscle (DSM) in high-fat insulin resistant obese mice, focusing on the
role of protein kinase C (PKC) and Ca(v)1.2 in causing bladder dysfunction. Male
C57BL/6 mice were fed with high-fat diet for 10 weeks. In vitro functional
responses and cystometry, as well as PKC and Ca(v)1.2 expression in bladder were
evaluated. Obese mice exhibited higher body weight, epididymal fat mass, fasting
glucose and insulin resistance. Carbachol (0.001-100 µM), α,β-methylene ATP
(1-10 µM), KCl (1-300 mM), extracellular Ca(2+) (0.01-100 mM) and
phorbol-12,13-dibutyrate (PDBu; 0.001-3 µM) all produced greater DSM
contractions in obese mice, which were fully reversed by the Ca(v)1.2 blocker
amlodipine. Cystometry evidenced augmented frequency, non-void contractions and
post-void pressure in obese mice that were also prevented by amlodipine.
Metformin treatment improved the insulin sensitivity, and normalized the in
vitro bladder hypercontractility and cystometric dysfunction in obese mice. The
PKC inhibitor GF109203X (1 µM) also reduced the carbachol induced contractions.
PKC protein expression was markedly higher in bladder tissues from obese mice,
which was normalized by metformin treatment. The Ca(v)1.2 channel protein
expression was not modified in any experimental group. Our findings show that
Ca(v)1.2 blockade and improvement of insulin sensitization restores the enhanced
PKC protein expression in bladder tissues and normalizes the overactive
detrusor. It is likely that insulin resistance importantly contributes for the
pathophysiology of this urological disorder in obese mice. The rapidly increasing number of people with obesity and type 2 diabetes is one
of the most serious problems of the contemporary world. Until recently, it was
thought that the main cause of this phenomenon is the change of lifestyle and
dietary habits. According to recent reports, the gut microbiota may also play an
important role in the "epidemic" of obesity and diabetes. Changes in its
composition have been observed in people suffering from these diseases. In
addition, the fact that the intestinal microbiota may affect body weight,
insulin sensitivity or sugar and lipid metabolism has led to the hypothesis that
these changes may contribute to the pathogenesis of obesity and diabetes.
Scientists, using antibacterial drugs, pro- and prebiotics, are trying to modify
the intestinal flora and thus affect its interaction with the host. The results
are very promising, lead to further analysis and indicate gut microbiota as a
potential therapeutic target for obesity and diabetes treatment. OBJECTIVE: The metabolic effects of an aloe vera gel complex (Aloe QDM complex)
on people with prediabetes or early diabetes mellitus (DM) are unknown. The goal
of this study was to determine the effects of Aloe QDM complex on body weight,
body fat mass (BFM), fasting blood glucose (FBG), fasting serum insulin, and
Homeostasis Model of Assessment - Insulin Resistance (HOMA-IR) in obese
individuals with prediabetes or early DM who were not on diabetes medications.
METHODS: Participants (n = 136) were randomly assigned to an intervention or a
control group and evaluated at baseline and at 4 and 8 wk.
RESULTS: The study lost six participants in the control group and eight in the
intervention group. At 8 wk, body weight (P = 0.02) and BFM (P = 0.03) were
significantly lower in the intervention group. At 4 wk, serum insulin level (P =
0.04) and HOMA-IR (P = 0.047) were lower in the intervention group; they also
were lower at 8 wk but with borderline significance (P = 0.09; P = 0.08,
respectively). At 8 wk, FBG tended to decrease in the intervention group (P =
0.02), but the between-group difference was not significant (P = 0.16).
CONCLUSION: In obese individuals with prediabetes or early untreated DM, Aloe
QDM complex reduced body weight, BFM, and insulin resistance. Metabolic syndrome is defined as cluster of independent risk factors of coronary
heart disease and type 2 diabetes mellitus including prediabetic glucose
metabolism disorders associated with insulin resistance as impaired fasting
glucose, impaired glucose tolerance and/ or borderline increasing of
glycosylated haemoglobin; central obesity, atherogenic dyslipidaemia with
increasing of triglyceride levels and decreasing of high density lipoprotein
levels and hypertension. In diagnosis of prediabetic states there are used
fasting glycaemia, 2 hours glycaemia during oral glucose tolerant test and HbA1c
level, which importance in diagnostic is discussed. In DM2 prevention there is
important mainly physical activity at least 30 min daily. In the case of
pharmacotherapy there was confirmed efficiency of metformin, which could be used
in states with high risk of DM2 conversion and some antihypertensive drugs,
mainly sartans. In the case of treatment of dyslipidaemia by statins there is
moderate increased risk of DM2 in prediabetic states, but cardiovascular benefit
from treatment some times exceeds this risk. OBJECTIVE: The aim of this study was to evaluate the effects of using ACE
inhibitors on insulin resistance, glucose metabolism, body fat composition, and
lipid profile in children over 10 years of age with obesity-associated metabolic
syndrome (MS).
METHODS: A total of 53 children with MS, who had been followed for at least one
year were included in the study. The sample was divided into two groups: Group
1-30 obese children (13 female, 17 male) who were not using an ACE inhibitor and
Group 2-23 obese children (13 female, 10 male) who were using an ACE inhibitor.
Anthropometric and laboratory data obtained at baseline and at the 3rd, 6th, and
12th months of follow-up were compared in the two groups.
RESULTS: Comparison of the data in the two groups at 3rd, 6th, and 12th months
revealed no statistically significant differences in terms of weight standard
deviation score (SDS), body mass index SDS, weight for height percentile, body
fat percentage, and very low-density lipoprotein (VLDL)values. However, there
were statistically significant differences in mean glucose and insulin levels,
homeostasis model assessment for insulin resistance, LDL and high-density
lipoprotein values, and highly significant differences in mean triglyceride
values.
CONCLUSIONS: The positive effects of ACE inhibitor drugs, particularly on
hypertriglyceridemia and insulin resistance, might bring them forth as
first-line drugs in the treatment of obese and hypertensive children.
Randomized, controlled, double-blind, and long-term studies are needed for a
definitive conclusion. |
Elaborate on the link between conserved noncoding elements (CNEs) and fractality. | Well-developed fractality is revealed for the chromosomal distribution of different classes of CNEs in the human genome by employing the scaling of block entropy and box-counting. This is characteristic of elements that are either extremely conserved between species or are of ancient origin, i.e. conserved between distant organisms across evolution. There are also power-law-like distributions, i.e. linearity in double logarithmic scale, in the inter-CNE distances, a feature which is connected with fractality and self-similarity. | Conserved, ultraconserved and other classes of constrained elements
(collectively referred as CNEs here), identified by comparative genomics in a
wide variety of genomes, are non-randomly distributed across chromosomes. These
elements are defined using various degrees of conservation between organisms and
several thresholds of minimal length. We here investigate the chromosomal
distribution of CNEs by studying the statistical properties of distances between
consecutive CNEs. We find widespread power-law-like distributions, i.e.
linearity in double logarithmic scale, in the inter-CNE distances, a feature
which is connected with fractality and self-similarity. Given that CNEs are
often found to be spatially associated with genes, especially with those that
regulate developmental processes, we verify by appropriate gene masking that a
power-law-like pattern emerges irrespectively of whether elements found close or
inside genes are excluded or not. An evolutionary model is put forward for the
understanding of these findings that includes segmental or whole genome
duplication events and eliminations (loss) of most of the duplicated CNEs.
Simulations reproduce the main features of the observed size distributions.
Power-law-like patterns in the genomic distributions of CNEs are in accordance
with current knowledge about their evolutionary history in several genomes. Conserved non-coding elements (CNEs) are defined using various degrees of
sequence identity and thresholds of minimal length. Their conservation
frequently exceeds the one observed for protein-coding sequences. We explored
the chromosomal distribution of different classes of CNEs in the human genome.
We employed two methodologies: the scaling of block entropy and box-counting,
with the aim to assess fractal characteristics of different CNE datasets. Both
approaches converged to the conclusion that well-developed fractality is
characteristic of elements that are either extremely conserved between species
or are of ancient origin, i.e. conserved between distant organisms across
evolution. Given that CNEs are often clustered around genes, we verified by
appropriate gene masking that fractal-like patterns emerge even when elements
found in proximity or inside genes are excluded. An evolutionary scenario is
proposed, involving genomic events that might account for fractal distribution
of CNEs in the human genome as indicated through numerical simulations. |
Which is the primary protein component of Lewy bodies? | The primary protein component of Lewy bodies are fibrils composed of alpha-synuclein. | Lewy bodies and Lewy neurites are the defining neuropathological characteristics
of Parkinson's disease and dementia with Lewy bodies. They are made of abnormal
filamentous assemblies of unknown composition. We show here that Lewy bodies and
Lewy neurites from Parkinson's disease and dementia with Lewy bodies are stained
strongly by antibodies directed against amino-terminal and carboxyl-terminal
sequences of alpha-synuclein, showing the presence of full-length or close to
full-length alpha-synuclein. The number of alpha-synuclein-stained structures
exceeded that immunoreactive for ubiquitin, which is currently the most
sensitive marker of Lewy bodies and Lewy neurites. Staining for alpha-synuclein
thus will replace staining for ubiquitin as the preferred method for detecting
Lewy bodies and Lewy neurites. We have isolated Lewy body filaments by a method
used for the extraction of paired helical filaments from Alzheimer's disease
brain. By immunoelectron microscopy, extracted filaments were labeled strongly
by anti-alpha-synuclein antibodies. The morphologies of the 5- to 10-nm
filaments and their staining characteristics suggest that extended
alpha-synuclein molecules run parallel to the filament axis and that the
filaments are polar structures. These findings indicate that alpha-synuclein
forms the major filamentous component of Lewy bodies and Lewy neurites. Alpha-synuclein forms the major component of Lewy bodies and Lewy neurites, the
defining neuropathological characteristics of Parkinson's disease and dementia
with Lewy bodies. Here we show that alpha-synuclein is also the major component
of the filamentous inclusions of multiple system atrophy which comprises several
neurodegenerative diseases with a shared filamentous pathology in nerve cells
and glial cells. These findings provide an unexpected link between multiple
system atrophy and Lewy body disorders and establish that
alpha-synucleinopathies constitute a major class of human neurodegenerative
disorder. Alpha-synuclein is believed to play an important role in Parkinson's disease
(PD). Mutations in the alpha-synuclein gene are responsible for familial forms
of PD and alpha-synuclein protein is a major component of Lewy bodies in
patients with sporadic PD. Synphilin-1 is a novel protein that we have
previously found to associate in vivo with alpha-synuclein. We now show that
synphilin-1 is present in Lewy bodies of patients with PD. Our data suggest that
synphilin-1 could play a role in Lewy body formation and the pathogenesis of PD. Lewy bodies, neuropathological hallmarks of Parkinson's disease and dementia
with Lewy bodies, comprise alpha-synuclein filaments and other less defined
proteins. Characterization of Lewy body proteins that interact with
alpha-synuclein may provide insight into the mechanism of Lewy body formation.
Double immunofluorescence labeling and confocal microscopy revealed
approximately 80% of cortical Lewy bodies contained microtubule-associated
protein 1B (MAP-1B) that overlapped with alpha-synuclein. Lewy bodies were
isolated using an immunomagnetic technique from brain tissue of patients dying
with dementia with Lewy bodies. Lewy body proteins were resolved by
polyacrylamide gel electrophoresis. Immunoblotting confirmed the presence of
MAP-1B and alpha-synuclein in purified Lewy bodies. Direct binding studies
revealed a high affinity interaction (IC(50) approximately 20 nm) between MAP-1B
and alpha-synuclein. The MAP-1B-binding sites were mapped to the last 45 amino
acids of the alpha-synuclein C terminus. MAP-1B also bound in vitro assembled
alpha-synuclein fibrils. Thus, MAP-1B may be involved in the pathogenesis of
Lewy bodies via its interaction with monomeric and fibrillar alpha-synuclein. Parkinson's disease (PD) is a neurodegenerative disorder characterized by the
appearance of intracytoplasmic inclusions called Lewy bodies (LB) in dopamine
neurons in the substantia nigra and the progressive loss of these neurons.
Recently, mutations in the alpha-synuclein gene have been identified in
early-onset familial PD, and alpha-synuclein has been shown to be a major
component of LB in all patients. Yet, the pathophysiological function of
alpha-synuclein remains unknown. In this report, we have investigated the toxic
effects of adenovirus-mediated alpha-synuclein overexpression on dopamine
neurons in rat primary mesencephalic cultures and in a rat dopaminergic cell
line - the large T-antigen immortalized, mesencephalon-derived 1RB3AN27 (N27).
Adenovirus-transduced cultures showed high-level expression of alpha-synuclein
within the cells. Overexpression of human mutant alpha-synuclein (Ala(53)Thr)
selectively induced apoptotic programmed cell death of primary dopamine neurons
as well as N27 cells. The mutant protein also potentiated the neurotoxicity of
6-hydroxydopamine (6-OHDA). By contrast, overexpression of wild-type human
alpha-synuclein was not directly neurotoxic but did increase cell death after
6-OHDA. Overexpression of wild-type rat alpha-synuclein had no effect on
dopamine cell survival or 6-OHDA neurotoxicity. These results indicate that
overexpression of human mutant alpha-synuclein directly leads to dopamine neuron
death, and overexpression of either human mutant or human wild-type
alpha-synuclein renders dopamine neurons more vulnerable to neurotoxic insults. alpha-Synuclein and ubiquitin are two Lewy body protein components that may play
antagonistic roles in the pathogenesis of Lewy bodies. We examined the
relationship between alpha-synuclein, ubiquitin, and lipids in Lewy bodies of
fixed brain sections or isolated from cortical tissues of dementia with Lewy
bodies. Lewy bodies exhibited a range of labeling patterns for alpha-synuclein
and ubiquitin, from a homogeneous pattern in which alpha-synuclein and ubiquitin
were evenly distributed and overlapped across the inclusion body to a concentric
pattern in which alpha-synuclein and ubiquitin were partially segregated, with
alpha-synuclein labeling concentrated in the peripheral domain and ubiquitin in
the central domain of the Lewy body. Lipids represented a significant component
in both homogeneous and concentric Lewy bodies. These results suggest that Lewy
bodies are heterogeneous in their subregional composition. The segregation of
alpha-synuclein to Lewy body peripheral domain is consistent with the hypothesis
that alpha-synuclein is continually deposited onto Lewy bodies. It is increasingly clear that the normal protein alpha-synuclein is in some
manner closely associated with presynaptic components of select neuronal types
within the adult human central nervous system (CNS) and, in addition, that in
its pathologically altered state alpha-synuclein aggregates selectively in the
form of filamentous inclusion bodies during certain progressive
neurodegenerative disorders, such as familial and sporadic Parkinson's disease.
By having the antibody AFshp raised specifically to alpha-synuclein to label
Parkinson disease-specific Lewy bodies and Lewy neurites as well as synaptic
boutons containing the unaltered protein, an initial attempt is made to map the
overall distribution pattern and describe the staining behavior of the
immunoreactive punctae in select regions of the prosencephalon. Neocortical
immunolabeling is most prominent in the prodigious, but incompletely myelinated,
association fields and faintest in the heavily myelinated primary motor and
primary sensory fields, with the premotor and first order sensory association
areas occupying an intermediate position. Of the thalamic grays evaluated, those
containing powerfully myelinated fiber tracts (e.g. centrum medianum, habenular
complex) show the weakest immunolabeling, whereas, less sturdily myelinated
structures are highly immunoreactive. The fact that the immunostaining spectrum
for normal alpha-synuclein is so broad, together with the fact that some
thalamic sites actually are immunonegative leads to the following conclusions
(1) alpha-synuclein, although present in the synaptic boutons of many nerve
cells in the adult human CNS, is by no means ubiquitous there, and (2) neuronal
types lacking the normal protein cannot generate the Parkinson's
disease-specific filamentous pathology. Alpha-synuclein accumulates in the brains of sporadic Parkinson's disease
patients as a major component of Lewy bodies, and mutations in alpha-synuclein
are associated with familial forms of Parkinson's disease. The pathogenic
mechanisms that precede and promote the aggregation of alpha-synuclein into Lewy
bodies in neurons remain to be determined. Here, we constructed a series of
alpha-synuclein-enhanced green fluorescent protein (alpha-synucleinEGFP,
SynEGFP) fusion proteins to address whether the Parkinson's disease-associated
mutations alter the subcellular distribution of alpha-synuclein, and to use as a
tool for experimental manipulations to induce aggregate formation. When
transfected into mouse cultured primary neurons, the 49-kDa alpha-synucleinEGFP
fusion proteins are partially truncated to a approximately 27-kDa form. This
non-fluorescent carboxy-terminally modified fusion protein spontaneously forms
inclusions in the neuronal cytoplasm. A marked increase in the accumulation of
inclusions is detected following treatment with each of three proteasome
inhibitors, n-acetyl-leu-leu-norleucinal, lactacystin and MG132. Interestingly,
Ala30Pro alpha-synucleinEGFP does not form the cytoplasmic inclusions that are
characteristic of wild-type and Ala53Thr alpha-synucleinEGFP, supporting the
idea that the Ala30Pro alpha-synuclein protein conformation differs from
wild-type alpha-synuclein. Similar inclusions are formed if alpha-synuclein
carboxy-terminus is modified by the addition of a V5/6xHistidine epitope tag. By
contrast, overexpression of unmodified alpha-synuclein does not lead to
aggregate formation. Furthermore, synphilin-1, an alpha-synuclein interacting
protein also found in Lewy bodies, colocalizes with the carboxy-terminally
truncated alpha-synuclein fusion protein in discrete cytoplasmic inclusions.Our
finding that manipulations of the carboxy-terminus of alpha-synuclein lead to
inclusion formation may provide a model for studies of the pathogenic mechanisms
of alpha-synuclein aggregation in Lewy bodies. Mutations in the alpha-synuclein gene have been linked to rare cases of familial
Parkinson's disease (PD). Alpha-synuclein is a major component of Lewy bodies
(LB), a pathological hallmark of PD. Transgenic mice and Drosophila expressing
either wild-type or mutant human alpha-synuclein develop motor deficits, LB-like
inclusions in some neurons, and neuronal degeneration. However, the relationship
between abnormal aggregates of alpha-synuclein and human dopamine (DA) neuron
degeneration remains unclear. In this report, we have investigated the influence
of alpha-synuclein expression on DA neurons in primary culture of embryonic
human mesencephalon. Two days after culture, human DA cells were transduced with
wild-type or mutant human (Ala(53)Thr) alpha-synuclein adenoviruses and
maintained for 5 days. Overexpression of mutant and wild-type human
alpha-synuclein resulted in 49% (P<0.01) and 27% (P<0.05) loss of DA neurons,
respectively, while not affecting viability of other cells in the culture.
Overexpression of rat alpha-synuclein or GFP (green fluorescent protein) had no
effect on DA neuron survival. Cytoplasmic inclusions of alpha-synuclein were
detected immunohistochemically in DA cells transduced with mutant human
alpha-synuclein, but not wild-type alpha-synuclein. These results show that
overexpression of human alpha-synuclein, particularly the mutant form, can cause
human DA neuron death, suggesting that alpha-synuclein may have a primary role
in the pathogenesis of PD. Lewy bodies, the characteristic pathological lesion of substantia nigra neurons
in Parkinson's disease (PD), are frequently observed to accompany the amyloid
plaque and neurofibrillary tangle pathology of Alzheimer's disease (AD). However
the typical anatomic distribution of Lewy bodies in AD is distinct from PD. The
most common site of occurrence is the amygdala, where Lewy bodies are observed
in approximately 60% of both sporadic and familial AD. Other common sites of
occurrence include the periamygdaloid and entorhinal cortex, while neocortical
and brainstem areas develop Lewy bodies in a lower percentage of cases. In
contrast, dementia with Lewy bodies (DLB), defined by widespread neocortical and
brainstem Lewy bodies but frequently accompanied by variable levels of AD-type
pathology, represents the other end of a spectrum of pathology associated with
dementia. The observation of Lewy bodies in familial AD cases suggests that like
neurofibrillary tangles, the formation of Lewy bodies can be induced by the
pathological state caused by Abeta-amyloid overproduction. The role of Lewy body
formation in the dysfunction and degeneration of neurons remains unclear. The
protein alpha-synuclein appears to be an important structural component of Lewy
bodies, an observation spurred by the discovery of point mutations in the
alpha-synuclein gene linked to rare cases of autosomal domit PD. Further
investigation of alpha-synuclein and its relationship to pathological conditions
promoting Lewy body formation in AD, PD, and DLB may yield further insight into
pathogenesis of these diseases. Parkinson's disease (PD) is characterized by the progressive loss of substantia
nigra dopaminergic neurons and the presence of cytoplasmic inclusions named Lewy
bodies. Two missense mutations of the alpha-synuclein (alpha-syn; A30P and A53T)
have been described in several families with an autosomal domit form of PD.
alpha-Syn also constitutes one of the main components of Lewy bodies in sporadic
cases of PD. To develop an animal model of PD, lentiviral vectors expressing
different human or rat forms of alpha-syn were injected into the substantia
nigra of rats. In contrast to transgenic mice models, a selective loss of nigral
dopaminergic neurons associated with a dopaminergic denervation of the striatum
was observed in animals expressing either wild-type or mutant forms of human
alpha-syn. This neuronal degeneration correlates with the appearance of abundant
alpha-syn-positive inclusions and extensive neuritic pathology detected with
both alpha-syn and silver staining. Lentiviral-mediated expression of wild-type
or mutated forms of human alpha-syn recapitulates the essential
neuropathological features of PD. Rat alpha-syn similarly leads to protein
aggregation but without cell loss, suggesting that inclusions are not the
primary cause of cell degeneration in PD. Viral-mediated genetic models may
contribute to elucidate the mechanism of alpha-syn-induced cell death and allow
the screening of candidate therapeutic molecules. Mutations of the alpha-synuclein gene have shown to be relevant in some rare
families with autosomal domit Parkinson's disease (PD). Furthermore,
alpha-synuclein protein is a major component of the Lewy bodies also in sporadic
PD patients. Increased levels of wildtype alpha-synuclein in the cell leads to
increased intracellular hydrogen peroxide levels and causes death of
dopaminergic neurons in rat primary culture. Subsequently, oxidative stress has
been directly linked with alpha-synuclein aggregation in vitro. This raises the
question whether increased alpha-synuclein expression might be linked to higher
susceptibility to PD and whether alpha-synuclein promoter polymorphisms are
associated with PD. Here, two polymorphisms (-116C>G and -668T>C) of the
alpha-synuclein promoter defining four haplotypes have been characterized in 315
German PD patients. The influence of the four haplotypes on gene expression was
studied by CAT reporter gene assays in neuronal SK-N-AS cells. The -668C/-116G
haplotype revealed significant higher CAT expression than the -668T/-116G or the
-668T/-116C haplotype, respectively. Although the -668C/-116G haplotype was more
common in PD patients, this difference was not significant. The major protein constituent of Lewy bodies (LBs), the pathological hallmark of
Parkinson disease and dementia with Lewy bodies, is considered to be
alpha-synuclein, but other proteins, in particular the microtubule-associated
protein tau, have been implicated in the pathogenesis of LBs. Tau is the major
structural component of neurofibrillary tangles (NFTs). Both direct
immunochemical studies of partially purified LBs and indirect
immunohistochemical studies have suggested that LBs may contain tau, but most of
these studies were based upon a single tau antibody, and immunologic
cross-reactivity was not completely excluded. To gain insight into the relation
between tau and alpha-synuclein in LBs, double immunostaining was performed in
Lewy body cases with a rabbit polyclonal antibody to alpha-synuclein and a panel
of monoclonal antibodies to phospho- and nonphospho-tau epitopes (Alz50, CP9,
CP13, PG5, TG3, PHFI) that spanned the length of the tau molecule.
Tau-immunoreactive LBs were present in the medulla in 80% of the cases,
irrespective of Braak stage. All tau antibodies recognized at least some LBs,
arguing against nonspecific antibody cross-reactivity. In most lesions the tau
immunostaining was present at the periphery of the LB. The phospho-tau antibody,
TG3, detected more LBs than any of the other tau antibodies. The proportion of
LBs with tau immunoreactivity was greatest in neurons vulnerable to NETs, such
as those in the locus ceruleus and basal nucleus of Meynert, and least in
neurons resistant to NFTs, such as the dorsal motor nucleus of the vagus in the
medulla. The present results suggest that tau may coaggregate with
alpha-synuclein in LBs, especially in neuronal populations vulnerable to both
NFTs and LBs. Synucleinopathies comprise a diverse group of neurodegenerative proteinopathies
that share common pathological lesions composed of aggregates of conformational
and posttranslational modifications of alpha-synuclein in selected populations
of neurons and glia. Abnormal filamentous aggregates of misfolded
alpha-synuclein protein are the major components of Lewy bodies, dystrophic
(Lewy) neurites, and the Papp-Lantos filaments in oligodendroglia and neurons in
multiple system atrophy linked to degeneration of affected brain regions. The
synucleinopathies include (1) Lewy body disorders and dementia with Lewy bodies,
(2) multiple system atrophy (MSA), and (3) Hallervorden-Spatz disease. (1) The
pathological diagnosis of Lewy body disorders and dementia with Lewy bodies is
established by validated consensus criteria based on semiquantitative assessment
of subcortical and cortical Lewy bodies as their common hallmarks. They are
accompanied by subcortical multisystem degeneration with neuronal loss and
gliosis with or without Alzheimer pathologic state. Lewy bodies also occur in
numerous other disorders, including pure autonomic failure, neuroaxonal
dystrophies, and various amyloidoses and tauopathies. (2) Multiple system
atrophy, a sporadic, adult-onset degenerative movement disorder of unknown
cause, is characterized by alpha-synuclein-positive glial cytoplasmic and rare
neuronal inclusions throughout the central nervous system associated with
striatonigral degeneration, olivopontocerebellar atrophy, and involvement of
medullar and spinal autonomic nuclei. (3) In neurodegeneration with brain iron
accumulation type I, or Hallervorden-Spatz disease, alpha-synuclein is present
in axonal spheroids and glial and neuronal inclusions. While the identity of the
major components of Lewy bodies suggests that a pathway leading from normal
soluble to abnormal misfolded filamentous proteins is central for their
pathogenesis, regardless of the primary disorder, there are conformational
differences in alpha-synuclein between neuronal and glial aggregates, showing
nonuniform mapping for its epitopes. Despite several cellular and transgenic
models, it is not clear whether inclusion body formation is an
adaptive/neuroprotective or a pathogenic reaction/process generated in response
to different, mostly undetermined, functional triggers linked to
neurodegeneration. The protein alpha-synuclein (ASYN) is thought to be involved in the development
of dementia with Lewy bodies (DLB). Overexpression of ASYN has been linked to
cellular toxicity and human disease, and in experimental models, chaperones such
as heat shock proteins (HSPs) are protective against ASYN toxicity. We have
assessed the abundance of mRNA for ASYN and chaperones and the abundance and
solubility of the encoded proteins in temporal cortex from sporadic human DLB.
We found a reduction of ASYN mRNA in DLB (44.9% of control). The abundance of
the Triton-soluble fraction (bioavailable protein) was not altered, but there
was an increase of the Triton-insoluble component (likely representing
aggregates). We evaluated 3 chaperones: HSP70, HSP90, and HDJ1. HSP70 mRNA was
increased in DLB, whereas the mRNAs for HSP90 and HDJ1 were unchanged. HSP70
accumulated in the Triton-soluble fraction, whereas HSP90 and HDJ1 proteins
accumulated in the Triton-insoluble fraction. These observations suggest that
sporadic DLB is not associated with overexpression of ASYN. Rather, the
persistence of normal soluble ASYN protein levels, despite the reduction of its
mRNA, suggests a primary defect in clearance of the protein. However, this
reduced clearance cannot be attributed to a failure of chaperone expression,
because their mRNA is unchanged or increased in the DLB brain. The natively disordered protein alpha-synuclein is the primary component of Lewy
bodies, the cellular hallmark of Parkinson's disease. Most studies of this
protein are performed in dilute solution, but its biologically relevant role is
performed in the crowded environment inside cells. We addressed the effects of
macromolecular crowding on alpha-synuclein by combining NMR data acquired in
living Escherichia coli with in vitro NMR data. The crowded environment in the
E.coli periplasm prevents a conformational change that is detected at 35 degrees
C in dilute solution. This change is associated with an increase in hydrodynamic
radius and the formation of secondary structure in the N-terminal 100 amino acid
residues. By preventing this temperature-induced conformational change, crowding
in the E.coli periplasm stabilizes the disordered monomer. We obtain the same
stabilization in vitro upon crowding alpha-synuclein with 300 g/l of bovine
serum albumin, indicating that crowding alone is sufficient to stabilize the
disordered, monomeric protein. Two disease-associated variants (A30P and A53T)
behave in the same way in both dilute solution and in the E.coli periplasm.
These data reveal the importance of approaching the effects of macromolecular
crowding on a case-by-case basis. Additionally, our work shows that discrete
structured protein conformations may not be achieved by alpha-synuclein inside
cells, implicating the commonly overlooked aspect of macromolecular crowding as
a possible factor in the etiology of Parkinson's disease. Natively disordered proteins are a growing class of anomalies to the
structure-function paradigm. The natively disordered protein alpha-synuclein is
the primary component of Lewy bodies, the cellular hallmark of Parkinson's
disease. We noticed a dramatic difference in dilute solution 1H-15N
Heteronuclear Single Quantum Coherence (HSQC) spectra of wild-type
alpha-synuclein and two disease-related mutants (A30P and A53T), with spectra
collected at 35 degrees C showing fewer cross-peaks than spectra acquired at 10
degrees C. Here, we show the change to be the result of a reversible
conformational exchange linked to an increase in hydrodynamic radius and
secondary structure as the temperature is raised. Combined with analytical
ultracentrifugation data showing alpha-synuclein to be monomeric at both
temperatures, we conclude that the poor quality of the 1H-15N HSQC spectra
obtained at 35 degrees C is due to conformational fluctuations that occur on the
proton chemical shift time scale. Using a truncated variant of alpha-synuclein,
we show the conformational exchange occurs in the first 100 amino acids of the
protein. Our data illustrate a key difference between globular and natively
disordered proteins. The properties of globular proteins change little with
solution conditions until they denature cooperatively, but the properties of
natively disordered proteins can vary dramatically with solution conditions. The protein alpha-synuclein (AS) is the primary fibrillar component of Lewy
bodies, the pathological hallmark of Parkinson's disease. Wild-type human AS and
the three mutant forms linked to Parkinson's disease (A53T, A30P, and E46K) all
form fibrils through a nucleation-dependent pathway; however, the biophysical
details of these fibrillation events are not yet well understood. Atomic-level
structural insight is required in order to elucidate the potential role of AS
fibrils in Parkinson's disease. Here we show that low temperature acquisition of
magic-angle spinning NMR spectra of wild type AS fibrils-greatly enhances
spectral sensitivity, enabling the detection of a substantially larger number of
spin systems. At 0 +/- 3 degrees C sample temperature, cross polarization (CP)
experiments yield weak signals. Lower temperature spectra (-40 +/- 3 degrees C)
demonstrated several times greater signal intensity, an effect further amplified
in 3D 15N-13C-13C experiments, which are required to perform backbone
assignments on this sample. Thus 3D experiments enabled assignments of most
amino acids in the rigid part of the fibril (approximately residues 64 to 94),
as well as tentative site-specific assignments for T22, V26, A27, Y39, G41, S42,
H50, V52, A53, T54, V55, V63, A107, I112, and S129. Most of these signals were
not observed in 2D or 3D spectra at 0 +/- 3 degrees C. Spectra acquired at low
temperatures therefore permitted more complete chemical shift assignments.
Observation of the majority of residues in AS fibrils represents an important
step towards solving the 3D structure. The manifestation of Lewy bodies (LB) in the brain is a hallmark of Parkinson's
disease. Here, we present a comprehensive analysis of protein elements in Lewy
bodies by comparative mass spectrometry. Cortical LB inclusions were enriched by
sucrose gradient centrifugation from postmortem brains, and a negative control
sample was prepared from specimen without LB pathology. Whereas approximately
550 proteins were identified in the LB-enriched sample by mass spectrometry,
quantitative comparison with the control sample revealed that approximately 40
proteins were co-enriched with alpha-synuclein, the major component in Lewy
bodies. As expected, the list of proteins included previously reported
constituents, such as those involved in protein folding, membrane trafficking
and oxidative stress. More interestingly, we discovered in the LB-enriched
sample several kinases (MAPKK1/MEK1, protein kinase C, and doublecortin-like
kinase), a novel deubiquitinating enzyme (otubain 1), and numerous ubiquitin
ligases (KPC and SCF). The proteomic studies provide enzyme candidates to
investigate the regulation of alpha-synuclein and/or other LB proteins, which
may contribute to the formation of Lewy bodies and the toxicity of
alpha-synuclein in the related neurodegenerative disorders. Many neurodegenerative diseases are associated with the aggregation of misfolded
proteins into amyloid oligomers or fibrils that are deposited as pathological
lesions within areas of the brain. An attractive therapeutic strategy for
preventing or ameliorating amyloid formation is to identify agents that inhibit
the onset or propagation of protein aggregation. Here we demonstrate how
solid-state nuclear magnetic resoce (ssNMR) may be used to identify key
residues within amyloidogenic protein sequences that may be targeted to inhibit
the aggregation of the host protein. For alpha-synuclein, the major protein
component of Lewy bodies associated with Parkinson's disease, we have used a
combination of ssNMR and biochemical data to identify the key region for
self-aggregation of the protein as residues 77-82 (VAQKTV). We used our new
structural information to design a peptide derived from residues 77 to 82 of
alpha-synuclein with an N-methyl group at the C-terminal residue, which was able
to disrupt the aggregation of alpha-synuclein. Thus, we have shown how
structural data obtained from ssNMR can guide the design of modified peptides
for use as amyloid inhibitors, as a primary step toward developing therapeutic
compounds for prevention and/or treatment of amyloid diseases. Alpha-synuclein (alphaS) is the primary component of Lewy bodies, the
pathological hallmark of Parkinson's Disease. Aggregation of alphaS is thought
to proceed from a primarily disordered state with nascent secondary structure
through intermediate conformations to oligomeric forms and finally to mature
amyloid fibrils. Low pH conditions lead to conformational changes associated
with increased alphaS fibril formation. Here we characterize these structural
and dynamic changes using solution state NMR measurements of secondary chemical
shifts, relaxation parameters, residual dipolar couplings, and paramagnetic
relaxation enhancement. We find that the neutralization of negatively charged
side-chains eliminates electrostatic repulsion in the C-terminal tail of alphaS
and leads to a collapse of this region at low pH. Hydrophobic contacts between
the compact C-terminal tail and the NAC (non-amyloid-beta component) region are
maintained and may lead to the formation of a globular domain. Transient
long-range contacts between the C-terminus of the protein and regions N-terminal
to the NAC region are also preserved. Thus, the release of long-range contacts
does not play a role in the increased aggregation of alphaS at low pH, which we
instead attribute to the increased hydrophobicity of the protein. α-Synuclein is the major protein component of Lewy bodies--the pathological
hallmark of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Its
accumulation into intracellular aggregates is implicated in the process of Lewy
body formation. However, its roles in both normal function, and disease, remain
controversial. Using a novel model of chronic oxidative stress in cultured
dopaminergic and cortical neurons, we report that endogenous α-synuclein is
upregulated in response to low dose toxicity. This response is conserved between
subpopulations of cortical and dopaminergic neurons, and confers relative
resistance to apoptosis following secondary insult. Additional acute oxidative
stress leads to intracellular accumulation of α-synuclein. These punctate
deposits colocalize with ubiquitin, which is central to proteosome-mediated
protein degeneration, and is the second major component of Lewy bodies. The
current results imply that differential levels of α-synuclein expression may
influence neuronal vulnerability in chronic neurodegenerative diseases. They
further support a 'two hit' hypothesis for Lewy body formation, whereby mild
stress causes a protective upregulation of α-synuclein. However, such increased
levels of α-synuclein may drive its accumulation, following additional toxic
insult. Finally, these results support a common mechanism for degeneration of
dopaminergic and cortical neurons, affected in PD, and DLB, respectively. Parkinson's disease is the most common neurodegenerative movement disorder.
α-Synuclein is a small synaptic protein that has been linked to familial
Parkinson's disease (PD) and is also the primary component of Lewy bodies, the
hallmark neuropathology found in the brain of sporadic and familial PD patients.
The function of α-synuclein is currently unknown, although it has been
implicated in the regulation of synaptic vesicle localization or fusion.
Recently, overexpression of α-synuclein was shown to cause cytoplasmic vesicle
accumulation in a yeast model of α-synuclein toxicity, but the exact role
α-synuclein played in mediating this vesicle aggregation is unclear. Here, we
show that α-synuclein induces aggregation of many yeast Rab GTPase proteins,
that α-synuclein aggregation is enhanced in yeast mutants that produce high
levels of acidic phospholipids, and that α-synuclein colocalizes with yeast
membranes that are enriched for phosphatidic acid. Significantly, we demonstrate
that α-synuclein expression induces vulnerability to perturbations of Ypt6 and
other proteins involved in retrograde endosome-Golgi transport, linking a
specific trafficking defect to α-synuclein phospholipid binding. These data
suggest new pathogenic mechanisms for α-synuclein neurotoxicity. Parkinson's disease (PD) is characterized as a neurodegenerative movement
disorder presenting with rigidity, resting tremor, disturbances in balance and
slowness in movement. An important pathologic feature of PD is the presence of
Lewy bodies. The primary structural component of Lewy bodies are fibrils
composed primarily of alpha-synuclein, a highly conserved 140 amino acid protein
that is predomitly expressed in neurons and which may play a role in synaptic
plasticity and neurotransmission. Numerous studies suggest the aggregation and
modification of alpha-synuclein as a key step leading to Lewy body formation and
neuronal cell loss associated with PD. Because of the central role of
alpha-synuclein in PD, it represents a novel drug target for the possible
treatment of this disease. In this review, an overview of the role of
alpha-synuclein in PD will be discussed with an emphasis on recent studies
utilizing an immunization approach against alpha-synuclein as a possible
treatment option for this debilitating disease. Fibrillar α-synuclein (α-Syn) is the principal component of Lewy bodies, which
are evident in individuals affected by Parkinson disease (PD). This
neuropathologic form of α-Syn plays a central role in PD progression as it has
been shown to propagate between neurons. Tools that interfere with α-Syn
assembly or change the physicochemical properties of the fibrils have potential
therapeutic properties as they may be sufficient to interfere with and/or halt
cell-to-cell transmission and the systematic spread of α-Syn assemblies within
the central nervous system. Vertebrate molecular chaperones from the
constitutive/heat-inducible heat shock protein 70 (Hsc/p70) family have been
shown to hinder the assembly of soluble α-Syn into fibrils and to bind to the
fibrils and very significantly reduce their toxicity. To understand how Hsc70
family members sequester soluble α-Syn, we set up experiments to identify the
molecular chaperone-α-Syn surface interfaces. We cross-linked human Hsc70 and
its yeast homologue Ssa1p and α-Syn using a chemical cross-linker and mapped the
Hsc70- and Ssa1p-α-Syn interface. We show that the client binding domain of
Hsc70 and Ssa1p binds two regions within α-Syn similar to a tweezer, with the
first spanning residues 10-45 and the second spanning residues 97-102. Our
findings define what is necessary and sufficient for engineering Hsc70- and
Ssa1p-derived polypeptide with minichaperone properties with a potential as
therapeutic agents in Parkinson disease through their ability to affect α-Syn
assembly and/or toxicity. α-Synuclein is an abundant presynaptic protein and a primary component of Lewy
bodies in Parkinson disease. Although its pathogenic role remains unclear, in
healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and
dissociation. An α-synuclein binding assay was used to screen for vesicle
proteins involved in α-synuclein membrane interactions and showed that
antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI
abrogated α-synuclein membrane binding. Biochemical analyses, including density
gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein
interacts with membrane-associated GTP-bound Rab3a but not to cytosolic
GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the
expression of a GTPase-deficient Rab3a mutant, by a domit-negative GDP
dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by
Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a
dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling
also increased α-synuclein sequestration on intracellular membranes. Our results
suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic
vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls
Rab3a membrane dissociation also regulates α-synuclein dissociation during
synaptic activity. Alpha-synuclein (α-Syn) is the principal protein component of Lewy bodies, a
pathological hallmark of Parkinson's disease (PD). This protein may regulate
protein phosphatase 2A (PP2A) activity, although the molecular mechanisms for
α-Syn-mediated regulation of PP2A and the potential neuroprotective actions of
PP2A against PD-associated pathology remain largely unexplored. We found that
α-Syn gene overexpression in SK-N-SH cells and primary neurons led to PP2A/C
phosphorylation at Y307, a known target of Src kinase, and consequent
phosphatase inhibition. In addition, phospho-activated Src (p-Y416 Src, pSrc)
was higher in SK-N-SH cells and primary neurons overexpressing α-Syn. Thus,
α-Syn may promote Src activation and PP2A inactivation, leading to
hyperphosphorylation of proteins. Immunoprecipitation revealed higher
calmodulin/Src complex formation in α-Syn-overexpressing cells and α-Syn
transgenic mice. A TUNEL apoptosis assay and an MTT cell viability assay
demonstrated that the PP2A activator C2-ceramide protected neurons against
α-Syn-induced cell injury. Buffering the Ca(2+) elevations induced by α-Syn
overexpression ameliorated the cytotoxicity of α-Syn. Our findings define a
potential molecular mechanism for α-Syn-mediated regulation of PP2A through
formation of the calmodulin/Src complex, activation of Src, and Src-mediated
phospho-inhibition of PP2A. Overexpression of α-Syn may lead to
neurodegeneration in PD in part by suppressing the endogenous neuroprotective
activity of PP2A. Aggregation of α-synuclein (αSyn), the primary protein component in Lewy body
inclusions of patients with Parkinson's disease, arises when the normally
soluble intrinsically disordered protein converts to amyloid fibrils. In this
work, we provide a mechanistic view of the role of N-terminal acetylation on
fibrillation by first establishing a quantitative relationship between monomer
secondary structural propensity and fibril assembly kinetics, and secondly by
demonstrating in the N-terminal acetylated form of the early onset A53T
mutation, that N-terminal transient helices formed and/or inhibited by
N-terminal acetylation modulate the fibril assembly rates. Using NMR chemical
shifts and fluorescence experiments, we report that secondary structural
propensity in residues 5-8, 14-31, and 50-57 are highly correlated to fibril
growth rate. A four-way comparison of secondary structure propensity and fibril
growth rates of N-terminally acetylated A53T and WT αSyn with non-acetylated
A53T and WT αSyn present novel mechanistic insight into the role of N-terminal
acetylation in amyloid fibril formation. We show that N-terminal acetylation
inhibits the formation of the "fibrillation promoting" transient helix at
residues 14-31 resulting from the A53T mutation in the non-acetylated variant
and supports the formation of the "fibrillation inhibiting" transient helix in
residues 1-12 thereby resulting in slower fibrillation rates relative to the
previously studied non-acetylated A53T variant. Our results highlight the
critical interplay of the region-specific transient secondary structure of the
N-terminal region with fibrillation, and the inhibitory role of the N-terminal
acetyl group in fibril formation. BACKGROUND: ATP13A2 (PARK9) loss of function mutations are a genetic cause of an
early-onset form of Parkinson's disease (PD), with in vitro studies showing that
ATP13A2 deficits lead to lysosomal and mitochondrial dysfunction and α-synuclein
accumulation, while elevated ATP13A2 expression reduces α-synuclein toxicity.
The three human brain tissue studies assessing changes in ATP13A2 expression in
PD produced divergent results; mRNA is increased while protein levels were
observed to be either increased or decreased. This apparent conflict in protein
levels might have arisen from examining Lewy body disease cases with coexisting
Alzheimer-type pathologies.To assess whether ATP13A2 levels in Lewy body disease
are modified by Alzheimer-type β-amyloid deposition, we evaluated cases of pure
PD and pure dementia with Lewy bodies (DLB) for changes in ATP13A2, α-synuclein
and β-amyloid protein levels in cortical regions with and without Lewy bodies.
RESULTS: In all Lewy body disease cases, we identified decreased ATP13A2 protein
levels that correlated with increases in both α-synuclein and β-amyloid. Partial
colocalization was observed between ATP13A2 and α-synuclein in Lewy bodies,
whereas ATP13A2 did not colocalize with pathological β-amyloid deposition.
CONCLUSIONS: Our data show that patients with Lewy body diseases have an overall
deficit in ATP13A2 protein levels, with the remaining protein being more
insoluble and partially redistributing towards Lewy bodies. This supports the
concept that increasing ATP13A2 levels may offer potential therapeutic benefits
to patients with Lewy body diseases. Parkinson's disease (PD) is the second most common neurodegenerative disease. A
key pathological feature of PD is Lewy bodies, of which the major protein
component is α-synuclein (α-syn). Human genetic studies have shown that
mutations (A53T, A30P, E46K) and multiplication of the α-syn gene are linked to
familial PD. Mice overexpressing the human A53T mutant α-syn gene develop severe
movement disorders. However, the molecular mechanisms of α-syn toxicity are not
well understood. Recently, mitochondrial dysfunction has been linked with
multiple neurodegenerative diseases including Parkinson's disease. Here we
investigated whether mitochondrial motility, dynamics and respiratory function
are affected in primary neurons from a mouse model expressing the human A53T
mutation. We found that mitochondrial motility was selectively inhibited in A53T
neurons while transport of other organelles was not affected. In addition, A53T
expressing neurons showed impairment in mitochondrial membrane potential and
mitochondrial respiratory function. Furthermore, we found that rapamycin, an
autophagy inducer, rescued the decreased mitochondrial mobility. Taken together,
these data demonstrate that A53T α-syn impairs mitochondrial function and
dynamics and the deficit of mitochondrial transport is reversible, providing
further understanding of the disease pathogenesis and a potential therapeutic
strategy for PD. α-Synuclein is the major pathological component of synucleinopathies including
Parkinson's disease and dementia with Lewy bodies. Recent studies have
demonstrated that α-synuclein also plays important roles in the release of
synaptic vesicles and synaptic membrane recycling in healthy neurons. However,
the precise relationship between the pathogenicity and physiological functions
of α-synuclein remains to be elucidated. To address this issue, we investigated
the subcellular localization of α-synuclein in normal and pathological
conditions using primary mouse hippocampal neuronal cultures. While some neurons
expressed high levels of α-synuclein in presynaptic boutons and cell bodies,
other neurons either did not or only very weakly expressed the protein. These
α-synuclein-negative cells were identified as inhibitory neurons by
immunostaining with specific antibodies against glutamic acid decarboxylase
(GAD), parvalbumin, and somatostatin. In contrast, α-synuclein-positive synapses
were colocalized with the excitatory synapse marker vesicular glutamate
transporter-1. This expression profile of α-synuclein was conserved in the
hippocampus in vivo. In addition, we found that while presynaptic α-synuclein
colocalizes with synapsin, a marker of presynaptic vesicles, it is not essential
for activity-dependent membrane recycling induced by high potassium treatment.
Exogenous supply of preformed fibrils generated by recombit α-synuclein was
shown to promote the formation of Lewy body (LB) -like intracellular aggregates
involving endogenous α-synuclein. GAD-positive neurons did not form LB-like
aggregates following treatment with preformed fibrils, however, exogenous
expression of human α-synuclein allowed intracellular aggregate formation in
these cells. These results suggest the presence of a different mechanism for
regulation of the expression of α-synuclein between excitatory and inhibitory
neurons. Furthermore, α-synuclein expression levels may determine the efficiency
of intracellular aggregate formation in different neuronal subtypes. Alpha-synuclein (α-syn) is the main protein component of Lewy bodies (LBs), that
together with nigrostriatal dopamine neuron loss constitute typical pathological
hallmarks of Parkinson's disease (PD). Glutamate N-methyl-d-aspartate receptor
(NMDAR) abnormalities, peculiarly involving NR2B-containing NMDAR, have been
observed in the brain of PD patients and in several experimental models of the
disease. Recent findings, indicating that α-syn can modulate NMDAR trafficking
and function, suggest that this protein may be a pivotal regulator of NMDAR
activity. Prompted by these evidences, we used fluorescence immunocytochemistry,
western blotting and ratiometric Ca(2+) measurements to investigate whether wild
type (wt) or C-terminally truncated α-syn can specifically modulate
NR2B-containing NMDAR levels, subcellular trafficking and function. In addition,
we evaluated whether the exposure of primary cortical neurons to increasing
concentrations of rotenone could differentially regulate NR2B levels and cell
viability in the presence or in the absence of α-syn. Our results indicate that
both wt and C-terminally truncated α-syn negatively modulate NR2B-containing
NMDAR levels, membrane translocation and function. Moreover, we found that
absence of α-syn abolishes the rotenone-dependent decrease of NR2B levels and
reduces neuronal vulnerability in primary cortical neurons. These findings
suggest that α-syn can modulate neuronal resilience by regulating
NR2B-containing NMDAR, whose specific alterations could connect α-syn pathology
to neuronal degeneration in PD. α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal
pathology of Parkinson's disease, as it is the major fibrillar component of Lewy
bodies, the inclusions that are characteristic of the disease. Oligomeric
structures, common to many neurodegenerative disease-related proteins, may in
fact be the primary toxic species, while the amyloid fibrils exist either as a
less toxic dead-end species or even as a beneficial mechanism for clearing
damaged proteins. To alter the progression of the aggregation and gain insights
into the prefibrillar structures, we determined the effect of heme on αSyn
oligomerization by several different techniques, including native
(nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence,
transmission electron microscopy, atomic force microscopy, circular dichroism,
and membrane permeation using a calcein release assay. During aggregation, heme
is able to bind the αSyn in a specific fashion, stabilizing distinct oligomeric
conformations and promoting the formation of αSyn into annular structures,
thereby delaying and/or inhibiting the fibrillation process. These results
indicate that heme may play a regulatory role in the progression of Parkinson's
disease; in addition, they provide insights into how the aggregation process may
be altered, which may be applicable to the understanding of many
neurodegenerative diseases. OBJECTIVE: Dementia with Lewy bodies is an α-synucleinopathy characterized by
neocortical Lewy-related pathology (LRP). We carried out a genome-wide
association study (GWAS) on neocortical LRP in a population-based sample of
subjects aged 85 or over.
METHODS: LRP was analyzed in 304 subjects in the Vantaa 85+ sample from Southern
Finland. The GWAS included 41 cases with midbrain, hippocampal, and neocortical
LRP and 177 controls without midbrain and hippocampal LRP. The Medical Research
Council Cognitive Function and Ageing Study (CFAS) material was used for
replication (51 cases and 131 controls).
RESULTS: By analyzing 327,010 markers the top signal was obtained at the
HLA-DPA1/DPB1 locus (P = 1.29 × 10(-7)); five other loci on chromosomes 15q14,
2p21, 2q31, 18p11, and 5q23 were associated with neocortical LRP at P < 10(-5).
Two loci were marked by multiple markers, 2p21 (P = 3.9 × 10(-6), upstream of
the SPTBN1 gene), and HLA-DPA1/DPB1; these were tested in the CFAS material.
Single marker (P = 0.0035) and haplotype (P = 0.04) associations on 2p21 were
replicated in CFAS, whereas HLA-DPA1/DPB1 association was not. Bioinformatic
analyses suggest functional effects for the HLA-DPA1/DPB1 markers as well as the
15q14 marker rs8037309.
INTERPRETATION: We identified suggestive novel risk factors for neocortical LRP.
SPTBN1 is the candidate on 2p21, it encodes beta-spectrin, an α-synuclein
binding protein and a component of Lewy bodies. The HLA-DPA1/DPB1 association
suggests a role for antigen presentation or alternatively, cis-regulatory
effects, one of the regulated neighboring genes identified here (vacuolar
protein sorting 52) plays a role in vesicular trafficking and has been shown to
interact with α-synuclein in a yeast model. BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease with
characteristics and symptoms that are well defined. Nevertheless, its aetiology
remains unknown. PD is characterized by the presence of Lewy bodies inside
neurons. α-Synuclein (α-syn) is a soluble protein present in the pre-synaptic
terminal of neurons. Evidence suggests that α-syn has a fundamental role in PD
pathogenesis, given that it is an important component of Lewy bodies localized
in the dopaminergic neurons of PD patients.
METHODS: In the present study, we investigated the influence of wild type (WT)
and A30P α-syn overexpression on neuroblastoma SH-SY5Y toxicity induced by the
conditioned medium (CM) from primary cultures of glia challenged with
lipopolysaccharide (LPS) from Escherichia coli.
RESULTS: We observed that SH-SY5Y cells transduced with α-syn (WT or A30P) and
treated with CM from LPS-activated glia cells show evidence of cell death, which
is not reverted by NF-κB inhibition by sodium salicylate or by blockage of P50
(NF-κB subunit). Furthermore, the expression of A30P α-syn in neuroblastoma
SH-SY5Y decreases the cell death triggered by the CM of activated glia versus WT
α-syn or control group. This effect of A30P α-syn may be due to the low
MAPK42/44 phosphorylation. This finding is substantiated by MEK1 inhibition by
PD98059, decreasing LDH release by CM in SH-SY5Y cells.
CONCLUSION: Our results suggest that SH-SY5Y cells transduced with α-syn (WT or
A30P) and treated with CM from LPS-activated glia cells show cell death, which
is not reverted by NF-κB blockage. Additionally, the expression of A30P α-syn on
neuroblastoma SH-SY5Y leads to decreased cell death triggered by the CM of
activated glia, when compared to WT α-syn or control group. The mechanism
underlying this process remains to be completely elucidated, but the present
data suggest that MAPK42/44 phosphorylation plays an important role in this
process.
PROSPERO: CRD42015020829. Neuronal loss in specific brain regions and neurons with intracellular
inclusions termed Lewy bodies are the pathologic hallmark in both Parkinson's
disease (PD) and dementia with Lewy bodies (DLB). Lewy bodies comprise of
aggregated intracellular vesicles and proteins and α-synuclein is reported to be
a major protein component. Using human brain tissue from control, PD and DLB and
light and confocal immunohistochemistry with antibodies to superoxide dismutase
2 as a marker for mitochondria, α-synuclein for Lewy bodies and βIII Tubulin for
microtubules we have examined the relationship between Lewy bodies and
mitochondrial loss. We have shown microtubule regression and mitochondrial and
nuclear degradation in neurons with developing Lewy bodies. In PD, multiple Lewy
bodies were often observed with α-synuclein interacting with DNA to cause marked
nuclear degradation. In DLB, the mitochondria are drawn into the Lewy body and
the mitochondrial integrity is lost. This work suggests that Lewy bodies are
cytotoxic. In DLB, we suggest that microtubule regression and mitochondrial loss
results in decreased cellular energy and axonal transport that leads to cell
death. In PD, α-synuclein aggregations are associated with intact mitochondria
but interacts with and causes nuclear degradation which may be the major cause
of cell death. |
Are microtubules marked by glutamylation? | Yes, glutamylation is the most prevalent tubulin posttranslational modification and marks stable microtubules. | In most eukaryotic cells, tubulin is subjected to posttranslational
glutamylation, a conserved modification of unclear function. The glutamyl side
chains form as branches of the primary sequence glutamic acids in two
biochemically distinct steps: initiation and elongation. The length of the
glutamyl side chain is spatially controlled and microtubule type specific. Here,
we probe the significance of the glutamyl side chain length regulation in vivo
by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena.
Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the
tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the
same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic
microtubules and destabilized axonemal microtubules. Our observations suggest
that the cellular outcomes of glutamylation are mediated by spatially restricted
tubulin interactors of diverse nature. Glutamylation, the most prevalent tubulin posttranslational modification, marks
stable microtubules and regulates recruitment and activity of microtubule-
interacting proteins. Nine enzymes of the tubulin tyrosine ligase-like (TTLL)
family catalyze glutamylation. TTLL7, the most abundant neuronal glutamylase,
adds glutamates preferentially to the β-tubulin tail. Coupled with ensemble and
single-molecule biochemistry, our hybrid X-ray and cryo-electron microscopy
structure of TTLL7 bound to the microtubule delineates a tripartite microtubule
recognition strategy. The enzyme uses its core to engage the disordered anionic
tails of α- and β-tubulin, and a flexible cationic domain to bind the
microtubule and position itself for β-tail modification. Furthermore, we
demonstrate that all single-chain TTLLs with known glutamylase activity utilize
a cationic microtubule-binding domain analogous to that of TTLL7. Therefore, our
work reveals the combined use of folded and intrinsically disordered substrate
recognition elements as the molecular basis for specificity among the enzymes
primarily responsible for chemically diversifying cellular microtubules. Enzymes of the tubulin tyrosine ligase-like (TTLL) family posttranslationally
modify and thereby mark microtubules by glutamylation, generating specific
recognition sites for microtubule-interacting proteins. Garnham et al. report
the first structure of a TTLL protein alone and in complex with microtubules,
elucidating their mechanism of action. Microtubules have important functions ranging from maintece of cell
morphology to subcellular transport, cellular signaling, cell migration, and
formation of cell polarity. At the organismal level, microtubules are crucial
for various biological processes, such as viral entry, inflammation, immunity,
learning and memory in mammals. Microtubules are subject to various covalent
modifications. One such modification is tubulin acetylation, which is associated
with stable microtubules and conserved from protists to humans. In the past
three decades, this reversible modification has been studied extensively. In
mammals, its level is mainly governed by opposing actions of α-tubulin
acetyltransferase 1 (ATAT1) and histone deacetylase 6 (HDAC6). Knockout studies
of the mouse enzymes have yielded new insights into biological functions of
tubulin acetylation. Abnormal levels of this modification are linked to
neurological disorders, cancer, heart diseases and other pathological
conditions, thereby yielding important therapeutic implications. This review
summarizes related studies and concludes that tubulin acetylation is important
for regulating microtubule architecture and maintaining microtubule integrity.
Together with detyrosination, glutamylation and other modifications, tubulin
acetylation may form a unique 'language' to regulate microtubule structure and
function. |
What is known about the Kub5-Hera/RPRD1B interactome? | Ku70-binding protein 5 (Kub5)-Hera (K-H)/RPRD1B maintains genetic integrity by concomitantly minimizing persistent R-loops and promoting repair of DNA double strand breaks (DSBs). Thus, the Kub5-Hera/RPRD1B interactome plays a novel role in preserving genetic stability by regulating DNA mismatch repair. | Ku70-binding protein 5 (Kub5)-Hera (K-H)/RPRD1B maintains genetic integrity by
concomitantly minimizing persistent R-loops and promoting repair of DNA double
strand breaks (DSBs). We used tandem affinity purification-mass spectrometry,
co-immunoprecipitation and gel-filtration chromatography to define higher-order
protein complexes containing K-H scaffolding protein to gain insight into its
cellular functions. We confirmed known protein partners (Ku70, RNA Pol II,
p15RS) and discovered several novel associated proteins that function in RNA
metabolism (Topoisomerase 1 and RNA helicases), DNA repair/replication processes
(PARP1, MSH2, Ku, DNA-PKcs, MCM proteins, PCNA and DNA Pol δ) and in protein
metabolic processes, including translation. Notably, this approach directed us
to investigate an unpredicted involvement of K-H in DNA mismatch repair (MMR)
where K-H depletion led to concomitant MMR deficiency and compromised global
microsatellite stability. Mechanistically, MMR deficiency in K-H-depleted cells
was a consequence of reduced stability of the core MMR proteins (MLH1 and PMS2)
caused by elevated basal caspase-dependent proteolysis. Pan-caspase inhibitor
treatment restored MMR protein loss. These findings represent a novel mechanism
to acquire MMR deficiency/microsatellite alterations. A significant proportion
of colon, endometrial and ovarian cancers exhibit k-h expression/copy number
loss and may have severe mutator phenotypes with enhanced maligcies that are
currently overlooked based on sporadic MSI+ screening. |
Willis-Ekbom disease is also known as? | Restless legs syndrome (RLS), also known as Willis-Ekbom disease (WED), is a common movement disorder characterized by an uncontrollable urge to move because of uncomfortable, sometimes painful sensations in the legs with a diurnal variation and a release with movement. | Restless legs syndrome (RLS), also known as Willis-Ekbom disease, is a
sensory-motor neurological disorder with a circadian component. RLS is
characterized by uncomfortable sensations in the extremities, generally at night
or during sleep, which often leads to an uncontrollable urge to move them for
relief. Recently, genomic studies identified single-nucleotide polymorphisms in
BTBD9, along with three other genes, as being associated with a higher risk of
RLS. Little is known about the function of BTBD9 or its potential role in the
pathophysiology of RLS. We therefore examined a line of Btbd9 mutant mice we
recently generated for phenotypes similar to symptoms found in RLS patients. We
observed that the Btbd9 mutant mice had motor restlessness, sensory alterations
likely limited to the rest phase, and decreased sleep and increased wake times
during the rest phase. Additionally, the Btbd9 mutant mice had altered serum
iron levels and monoamine neurotransmitter systems. Furthermore, the sensory
alterations in the Btbd9 mutant mice were relieved using ropinirole, a
dopaminergic agonist widely used for RLS treatment. These results, taken
together, suggest that the Btbd9 mutant mice model several characteristics
similar to RLS and would therefore be the first genotypic mouse model of RLS.
Furthermore, our data provide further evidence that BTBD9 is involved in RLS,
and future studies of the Btbd9 mutant mice will help shine light on its role in
the pathophysiology of RLS. Finally, our data argue for the utility of Btbd9
mutant mice to discover and screen novel therapeutics for RLS. BACKGROUND: Since the publication of the first European Federation of
Neurological Societies (EFNS) guidelines in 2005 on the management of restless
legs syndrome (RLS; also known as Willis-Ekbom disease), there have been major
therapeutic advances in the field. Furthermore, the management of RLS is now a
part of routine neurological practice in Europe. New drugs have also become
available, and further randomized controlled trials have been undertaken. These
guidelines were undertaken by the EFNS in collaboration with the European
Neurological Society and the European Sleep Research Society.
OBJECTIVES: To provide an evidence-based update of new treatments published
since 2005 for the management of RLS.
METHODS: First, we determined what the objectives of management of primary and
secondary RLS should be. We developed the search strategy and conducted a review
of the scientific literature up to 31 December 2011 (print and electronic
publications) for the drug classes and interventions employed in RLS treatment.
Previous guidelines were consulted. All trials were analysed according to class
of evidence, and recommendations made according to the 2004 EFNS criteria for
rating.
RECOMMENDATIONS: Level A recommendations can be made for rotigotine, ropinirole,
pramipexole, gabapentin enacarbil, gabapentin and pregabalin, which are all
considered effective for the short-term treatment for RLS. However, for the
long-term treatment for RLS, rotigotine is considered effective, gabapentin
enacarbil is probably effective, and ropinirole, pramipexole and gabapentin are
considered possibly effective. Cabergoline has according to our criteria a level
A recommendation, but the taskforce cannot recommend this drug because of its
serious adverse events. Many patients with restless legs syndrome (Willis-Ekbom disease) complain of
burning sensations in their feet associated with the desire to move, such that
they seek cooler environments. This pilot study aimed to characterise the
microvascular skin changes in 12 patients with restless legs syndrome compared
with 12 age- and sex-matched controls. Patients with moderate or severe restless
legs syndrome and controls underwent detailed thermovascular assessment in a
controlled temperature room at three different stages (normothermic phase 23 °C,
hot phase 30 °C, cold phase 18 °C). Microvascular activity was recorded during
all phases by bilateral great toe laser-Doppler flowmetry and also by whole-body
thermography. Patient and control measurements were compared. The study protocol
was well tolerated. Parameters extracted from the laser-Doppler flowmetry
measurements were used to model a logistic function using binary logistic
regression. This demonstrated a statistically significant difference between
patients with restless legs syndrome and healthy controls (P < 0.001). Visual
inspection of the body thermography image sequences showed increased lower limb
movement in patients with restless legs syndrome patients compared with
controls. Thermography analysis also showed significant differences between foot
temperatures in patients with restless legs syndrome compared with controls
during the hot phase (P = 0.011). Notably, patients with restless legs syndrome
had more uniform foot temperatures, whereas controls had a wider variability in
surface temperature across the feet. This novel study demonstrates impaired
microvascular circulation in patients with restless legs syndrome in comparison
to matched controls and a potential mechanism for the sensation of burning feet.
The protocol also provides an experimental paradigm to test therapeutic
interventions for the future. A Task Force was established by the International Restless Legs Syndrome Study
Group (IRLSSG) to develop evidence-based and consensus-based recommendations for
the long-term pharmacologic treatment of restless legs syndrome/Willis-Ekbom
disease (RLS/WED). The Task Force reviewed the results of all studies of RLS/WED
treatments with durations of 6 months or longer presented at meetings over the
past 2 years, posted on Web sites of pharmaceutical companies, or published in
peer-reviewed journals, asking the questions, "What is the efficacy of this
treatment in patients with RLS/WED?" and "What is the safety of this treatment
in patients with RLS/WED?" The Task Force developed guidelines based on their
review of 61 papers meeting inclusion criteria, and using a modified
evidence-grading scheme. Pregabalin has been established as effective for up to
1 year in treating RLS/WED (Level A evidence). Pramipexole, ropinirole, and
rotigotine have been established as effective for up to 6 months in treating
RLS/WED (Level A). The following drugs have been established as probably
effective (Level B) in treating RLS/WED for durations ranging from 1 to 5 years:
gabapentin enacarbil, pramipexole, and ropinirole (1 year); levodopa (2 years);
and rotigotine (5 years). Because of associated safety concerns, pergolide and
cabergoline should not be used in the treatment of RLS/WED unless the benefits
clearly outweigh the risks. Other pharmacologic therapies have insufficient
evidence to support their long-term use in treating RLS/WED. The IRLSSG Task
Force also developed consensus-based strategies for the prevention and treatment
of complications (such as augmentation, loss of efficacy, excessive daytime
sleepiness, and impulse control disorders) that may develop with the long-term
pharmacologic treatment of RLS/WED. The use of either a dopamine-receptor
agonist or α2δ calcium-channel ligand is recommended as the first-line treatment
of RLS/WED for most patients, with the choice of agent dependent on the
patient's severity of RLS/WED symptoms, cognitive status, history, and comorbid
conditions. Restless legs syndrome (RLS)/Willis-Ekbom disease (WED) is a common disorder,
occurring at least twice a week and causing at least moderate distress in 1.5%
to 2.7% of the population. It is important for primary care physicians to be
familiar with this disorder and its management. Much has changed in its
management since our previous algorithm was published in 2004, including the
availability of several new drugs. This revised algorithm was written by members
of the Medical Advisory Board of the Willis-Ekbom Disease Syndrome Foundation
based on scientific evidence and expert opinion. It considers the management of
RLS/WED under intermittent RLS/WED, chronic persistent RLS/WED, and refractory
RLS/WED. Nonpharmacological approaches, including mental alerting activities,
avoiding substances or medications that may exacerbate RLS, and the role of iron
supplementation, are outlined. Chronic persistent RLS/WED should be treated with
either a nonergot dopamine agonist or a calcium channel α-2-δ ligand. We discuss
the available drugs, the factors determining which to use, and their adverse
effects. We define refractory RLS/WED and describe management approaches,
including combination therapy and the use of high-potency opioids. There has been no previous side-by-side comparison of the diagnostic criteria
for restless legs syndrome (RLS) (Willis-Ekbom disease) and growing pains. In
our review, we explore this comparison emphasizing overlaps and disconnects,
summarize recent literature exploring the relationship between the 2 entities,
and make suggestions for future research. There is considerable overlap in the
diagnostic criteria for childhood RLS and growing pains. The literature also
indicates that RLS and growing pains more commonly occur together than one would
expect based on chance alone, and the family histories of RLS and growing pains
often are overlapping. Leg rubbing to obtain relief from leg discomfort is
common to both disorders, though walking to obtain relief seems unique to RLS.
Childhood RLS also has been reported to be painful in up to 45% of cases. The
development of standard diagnostic criteria is necessary to move forward in the
field of growing pains research. A quantitative and validated rating scale for
growing pains severity already exists. Because of the clinical and genetic
similarity between RLS and growing pains, studies that parallel those previously
performed in RLS patients are recommended for growing pains patients. For
example, a genome wide association study in growing pains patients of all
possible genes with particular attention to those identified as related to RLS
and a therapeutic trial of medications known to be effective in RLS would be
welcome. Abnormalities in vitamin D metabolism also may be common to both
disorders. STUDY OBJECTIVES: Recent genome-wide association studies (GWAS) for Caucasians
identified several allelic variants associated with increased risk of developing
restless legs syndrome (RLS), also known as Willis-Ekbom disease. Although the
pathogenic mechanisms of RLS are not entirely understood, it is becoming
increasingly evident that many diseases such as RLS can be attributed to an
epistasis. The study objectives were to evaluate whether the associations of RLS
with all loci determined in previous GWAS for Caucasians can be replicated
significantly for the Korean population and to elucidate whether an epistasis
plays a role in the pathogenesis of RLS.
DESIGN SETTING AND PARTICIPANTS: DNA from 320 patients with RLS and 320 age- and
sex-matched controls were genotyped for variants in the RLS loci.
MEASUREMENTS AND RESULTS: A significant association was found for rs3923809 and
rs9296249 in BTBD9 (P < 0.0001 and P = 0.001, respectively); the odds ratio (OR)
for rs3923809 was 1.61 (P < 0.0001) to 1.88 (P < 0.0001) and the OR for
rs9296249 was 1.44 (P = 0.001) to 1.73 (P = 0.002), according to the model of
inheritance. The OR for the interaction between rs3923809 in BTBD9 and rs4626664
in PTPRD was 2.05 (P < 0.0001) in the additive model, 1.80 (P = 0.002) in the
domit model and 2.47 (P = 0.004) in the recessive model. There was no
significant association between genotypes of all tested single nucleotide
polymorphisms and the mean value of serum iron parameters.
CONCLUSIONS: Our results suggest that the role of BTBD9 in the pathogenesis of
restless legs syndrome is more universal across populations than previously
reported and more efforts should be focused on the role of epistasis in the
genetic architecture of restless legs syndrome. OBJECTIVES: Both restless legs syndrome ([RLS], also known as Willis-Ekbom
Disease [WED]) and depression are common during pregcy. However, no prior
studies have assessed if pregt women with RLS have an elevated risk of
depression during and/or after pregcy.
METHODS: 1,428 women who were pregt in gestational week 16-17 were asked to
participate in a longitudinal survey. They were followed by web-based
questionnaires in gestational week 17 and 32, and 6 weeks after delivery. Data
were also retrieved from prenatal and birth records. Two different sets of
criteria were used to examine the prevalence of RLS in the cohort (International
Restless Legs Syndrome Society Group standard criteria and the later developed
CH-RLSQ11 questionnaire). The latter questionnaire attempts to exclude those
with common "mimics" of RLS.
RESULTS: Adjusted odds ratio for depression in gestational week 17, 32, and
postpartum week 6 in relation to pre-pregcy RLS onset and moderate to severe
symptom severity were 4.74 (2.30 - 9.76), 3.67 (1.85 - 7.28), and 2.58 (1.28 -
5.21), respectively. No significant associations were seen in pregt women
with de novo RLS during pregcy. When using the standard diagnostic RLS
criteria and frequency of symptoms more than 2-3 days per week, the prevalence
of RLS was 12.3%. With the CH-RLSQ11 questionnaire and the same threshold for
frequency of symptoms the prevalence was 6.5%.
CONCLUSION: Women with RLS onset before pregcy with moderate or severe
symptoms had an increased risk of both antenatal and postnatal depression. The
self-reported prevalence of RLS during pregcy is lower when a questionnaire
dealing with "mimics" is used. Restless leg syndrome (RLS), also known as Willis-Ekbom disease, is a condition
that includes sensations such as crawling, tingling, or aching in the limbs and
creates an urge to move. The prevalence is estimated at 3% to 15% of the
population and may present as primary RLS or secondary RLS. Secondary RLS may be
a result of some medications, iron deficiency, or conditions such as
neuropathies, or it may be related to pregcy. The guidelines for diagnosis,
which is usually made on clinical presentation, are discussed in the article.
Medication use is not always necessary in the management of RLS. Multiple
options are available and are reviewed within the article. Since 2011, two
medications have been approved for the treatment of RLS, and these are discussed
in detail. Neupro (rotigotine) is a dopamine agonist available as a patch that
has been approved for the treatment of RLS as well as Parkinson disease. One of
the major issues in treating RLS with dopamine agonists is augmentation, meaning
symptoms occur earlier in the day due to medication use. This rate of
augmentation with use of rotigotine is significantly lower than other dopamine
agonists. Horizant (gabapentin enacarbil) is the only nondopaminergic medication
approved for the treatment of RLS. Bioavailability is greater in gabapentin
enacarbil as compared to gabapentin. Augmentation has not been associated with
gabapentin or gabapentin enacarbil. Neupro (rotigotine) and Horizant (gabapentin
enacarbil) provide additional treatment options for patients with RLS who are in
need of medications. Consideration of each individual patient is necessary when
determining if medication is needed and in choosing the appropriate agent. OBJECTIVE: Restless legs syndrome (RLS), also known as Willis-Ekbom disease, is
a sensorimotor disorder that can result in considerable sleep disruption. This
narrative review provides an overview of RLS diagnosis and reports epidemiologic
evidence for an association between RLS and mood disorders. Possible links
between RLS, sleep disturbances, and mood disorders are considered, and
theoretical pathophysiologic pathways are discussed. Finally, pharmacologic
therapies for RLS are summarized.
DATA SOURCES: A PubMed search was performed using the search term restless legs
syndrome in combination with affective/anxiety, antidepressants, anxiety/anxiety
disorder, attention deficit hyperactivity disorder, depression/depressive
disorder, mood/mood disorder, neuropsychiatric, panic/panic disorder,
psychiatric disorder, and psychosis. English-language articles published between
January 1993 and May 2013 were retrieved. Additional studies were identified
from the reference lists of relevant publications.
STUDY SELECTION: 173 publications were retrieved. Articles related to the
association between idiopathic RLS and depression, anxiety, and mood disorders
were reviewed. In total, 32 epidemiologic studies were identified. These studies
were reviewed in detail and ranked according to quality.
DATA EXTRACTION: Data were extracted on the basis of relevance to the topic.
Epidemiologic studies were assessed using 3 parameters: methodology, data
quality, and generalizability of the results. Each factor was scored from 1
(high quality) to 4 (low quality), giving a total score of between 3 and 12 for
each study.
RESULTS AND CONCLUSIONS: RLS and mood disorders are frequently comorbid.
Recognition and appropriate treatment of comorbid RLS are particularly important
in patients with psychiatric disorders, as RLS is a common medical reason for
insomnia, and antidepressant use may exacerbate sensory symptoms. OBJECTIVE: Restless legs syndrome, now called Willis-Ekbom Disease (RLS/WED), is
a sensorimotor-related sleep disorder. Little is known of the effect of RLS/WED
on motor function. The current study investigated upper limb function in RLS/WED
patients. We hypothesised that RLS/WED patients exhibit subtle changes in tremor
amplitude but normal dexterity and movement speed and rhythmicity compared to
healthy controls.
METHODS: RLS/WED patients (n=17, 59 ± 7 years) with moderate disease and healthy
controls (n=17, 58 ± 6 years) completed screening tests and five tasks including
object manipulation, maximal pinch grip, flexion and extension of the index
finger (tremor assessment), maximal finger tapping (movement speed and
rhythmicity assessment), and the grooved pegboard test. Force, acceleration,
and/or first dorsal interosseus EMG were recorded during four of the tasks.
RESULTS: Task performance did not differ between groups. Learning was evident on
tasks with repeated trials and the magnitude of learning did not differ between
groups.
CONCLUSIONS: Hand function, tremor, and task learning were unaffected in RLS/WED
patients. Patients manipulated objects in a normal manner and exhibited normal
movement speed, rhythmicity, and tremor.
SIGNIFICANCE: Further research is needed to assess other types of movement in
RLS/WED patients to gain insight into the motor circuitry affected and the
underlying pathophysiology. Restless legs syndrome (RLS), also known as Willis-Ekbom Disease (WED), is a
sensorimotor disorder for which the exact pathophysiology remains unclear. Brain
iron insufficiency and altered dopaminergic function appear to play important
roles in the etiology of the disorder. This concept is based partly on extensive
research studies using cerebrospinal fluid (CSF), autopsy material, and brain
imaging indicating reduced regional brain iron and on the clinical efficacy of
dopamine receptor agonists for alleviating RLS symptoms. Finding causal
relations, linking low brain iron to altered dopaminergic function in RLS, has
required however the use of animal models. These models have provided insights
into how alterations in brain iron homeostasis and dopaminergic system may be
involved in RLS. The results of animal models of RLS and biochemical,
postmortem, and imaging studies in patients with the disease suggest that
disruptions in brain iron trafficking lead to disturbances in striatal dopamine
neurotransmission for at least some patients with RLS. This review examines the
data supporting an iron deficiency-dopamine metabolic theory of RLS by relating
the results from animal model investigations of the influence of brain iron
deficiency on dopaminergic systems to data from clinical studies in patients
with RLS. Author information:
(1)From the Department of Preventive Medicine and Biometrics (A.I.S.), Uniformed
Services University, Bethesda; National Institute on Aging (A.I.S., M.G.,
L.J.L.), Laboratory of Epidemiology and Population Sciences, Bethesda, MD;
Veterans Affairs Pacific Islands Health Care System (G.W.R.), Honolulu; Pacific
Health Research & Education Institute (G.W.R.), Honolulu, HI; Icelandic Heart
Association (S. Sigurdsson, V.G.), Kopavogur; School of Health Sciences (L.S.G.)
and Faculty of Medicine (V.G.), University of Iceland, Reykjavik; Department of
Neurology (S. Sveinbjörnsdóttir), Broomfield Hospital, UK; and Department of
Physical Medicine and Rehabilitation (A.K.W.), University of Pittsburgh, PA.
[email protected].
(2)From the Department of Preventive Medicine and Biometrics (A.I.S.), Uniformed
Services University, Bethesda; National Institute on Aging (A.I.S., M.G.,
L.J.L.), Laboratory of Epidemiology and Population Sciences, Bethesda, MD;
Veterans Affairs Pacific Islands Health Care System (G.W.R.), Honolulu; Pacific
Health Research & Education Institute (G.W.R.), Honolulu, HI; Icelandic Heart
Association (S. Sigurdsson, V.G.), Kopavogur; School of Health Sciences (L.S.G.)
and Faculty of Medicine (V.G.), University of Iceland, Reykjavik; Department of
Neurology (S. Sveinbjörnsdóttir), Broomfield Hospital, UK; and Department of
Physical Medicine and Rehabilitation (A.K.W.), University of Pittsburgh, PA. Restless legs syndrome (RLS), recently renamed Willis-Ekbom disease (WED), is a
common movement disorder. It is characterised by the need to move mainly the
legs due to uncomfortable, sometimes painful sensations in the legs, which have
a diurnal variation and a release with movement. Management is complex. First,
centres should establish the severity of RLS using a simple 10-item RLS severity
rating scale (IRLS). They should also exclude secondary causes, in particular
ensuring normal iron levels. Mild cases can be managed by lifestyle changes, but
patients with a IRLS score above 15 usually require pharmacological treatment.
Dopaminergic therapies remain the mainstay of medical therapies, with recent
evidence suggesting opioids may be particularly effective. This article focuses
on the different treatment strategies in RLS, their associated complications and
ways to manage them. BACKGROUND: Reported prevalence of restless legs syndrome (RLS), also known as
Willis-Ekbom disease (WED), varies from country to country, and methodologic
inconsistencies limit comparison of data. Impact of RLS on quality of life and
health has been studied primarily in industrialized countries, particularly
Europe and the United States. Many studies have relied exclusively on
self-report of symptoms or have assessed only medical populations. Recently,
interest has emerged on the impact of WED in rural, underserved populations
globally.
METHODS: In a population-based survey conducted in rural Ecuador, we assessed
the relationship of psychological distress to WED, evaluated with the Depression
Anxiety Stress Scales-21. WED was diagnosed through a 2-phase method in which
all residents were screened with the International Restless Legs Syndrome Study
Group (IRLSSG) questionnaire and all suspected cases were subsequently confirmed
through expert medical examination. WED severity was assessed with the IRLSSG
rating scale.
RESULTS: Of 665 persons (mean [SD] age, 59.5 [12.6] years; women, 386 [58%]), 76
had depression, 93 had anxiety, and 60 reported stress. Forty persons (6%) had
WED, with 15 (38%) having severe disease. In a regression model adjusted for age
and sex, the prevalence of depression, anxiety, and stress was about 3 times
greater among persons with WED than the general population.
CONCLUSIONS: Although cross-sectional data cannot establish causation, this
study shows the large behavioral health burden associated with WED in an
untreated, rural population. OBJECTIVES: Willis-Ekbom disease/restless legs syndrome (WED/RLS) is the most
common sleep-related movement disorder in pregcy. We designed a prospective
longitudinal study to investigate the correlates of WED/RLS during and after
pregcy.
DESIGN: A total of 138 pregt women with WED/RLS and a control group of 251
age-matched pregt women were enrolled prospectively. A questionnaire was
administered during a face-to-face interview at first evaluation during
pregcy and three months after delivery.
RESULTS: Among all women in the first trimester, 15.6% were diagnosed with
WED/RLS, whereas 32.8% of those in the second trimester and 38.8% of those in
the third trimester were diagnosed with WED/RLS (p = 0.032). In regression
analysis, later gestational age [p < 0.001; odds ratio (OR) 1.054] and previous
history of WED/RLS (p = 0.001; OR 2.795) were positively correlated with the
presence of WED/RLS, while ferritin levels (p = 0.001; OR 0.956) were negatively
correlated with the presence of WED/RLS. Ferritin levels were also negatively
correlated with the International RLS Study Group severity index (p = 0.041).
Forty-eight patients (34.8%) experienced WED/RLS symptomatology after delivery.
The ferritin levels were lower, and the mean number of pregcies was higher,
in women with residual WED/RLS (p = 0.008).
CONCLUSION: Our survey showed that WED/RLS was more common in the second and
third trimesters. Emergence of WED/RLS during the second trimester was strongly
associated with residual WED/RLS. Lower ferritin levels were associated with
both WED/RLS in pregcy and residual WED/RLS after delivery. A higher number
of pregcies were also associated with a greater likelihood of having residual
WED/RLS after delivery. BACKGROUND: Restless Legs Syndrome (RLS) or Willis-Ekbom Disease (WED) is highly
prevalent, but patients and healthcare providers alike know little about it.
Furthermore, controversy persists as to the best way of diagnosing this
nosological entity.
OBJECTIVE: To verify whether the term used to refer to this disease entity
(Restless Legs Syndrome or Willis-Ekbom Disease) affects the prevalence of
self-diagnosed RLS/WED in a sample of newly graduated physicians.
METHODS: Newly graduated physicians were asked to self-evaluate for the presence
of RLS/WED. Briefly, participants were allocated randomly across two groups. One
was asked to self-assess for RLS, while the other was asked to self-assess for
WED. The evaluation form given to one group asked 'Do you have Restless Legs
Syndrome?' whereas the form given to participants in the other group asked 'Do
you have Willis-Ekbom Disease?'. Both forms also contained the four criteria for
diagnosing RLS proposed by the International Restless Legs Syndrome Study Group
(IRLSSG) and instructions for self-diagnosis according to these criteria.
RESULTS: The study sample comprised 1413 newly graduated physicians. Of the 708
participants who were given the form that used the term RLS, 87 (12.28%)
diagnosed themselves with the condition. Conversely, of 705 physicians given the
form with the term WED, 13 (1.84%) diagnosed themselves with the condition
(p <0.0001).
CONCLUSION: A greater proportion of newly graduated physicians diagnosed
themselves with RLS/WED when presented with the term Restless Legs Syndrome than
when presented with the term Willis-Ekbom Disease. This suggests that the term
Restless Legs Syndrome may not be the most appropriate term to denote this
nosological entity. There is no consensus about mechanisms underlying restless legs syndrome (RLS),
also known as Willis-Ekbom disease (WED). Cortical excitability may be abnormal
in RLS. Transcranial magnetic stimulation (TMS) can provide insight about
cortical excitability. We reviewed studies about measures of excitability to TMS
in RLS. Original studies published between January 1999 and January 2015 were
searched in PubMed, Scopus, and Web of Science databases. Inclusion criteria
were as follows: original studies involving primary RLS in patients from both
sexes and ages between 18 and 85 years; TMS protocols clearly described; and
they were written in English, in peer-reviewed journals. Fifteen manuscripts
were identified. TMS protocols were heterogeneous across studies. Resting motor
threshold, active motor threshold, and amplitudes of motor-evoked potentials
were typically reported to be normal in RLS. A reduction in short-interval
intracortical inhibition (SICI) was the most consistent finding, whereas
conflicting results were described in regard to short-interval intracortical
facilitation and the contralateral silent period. Decreased SICI can be reversed
by treatment with dopaminergic agonists. Plasticity in the motor cortex and
sensorimotor integration may be disrupted. TMS may become a useful biomarker of
responsiveness to drug treatment in RLS. The field can benefit from increases in
homogeneity and sizes of samples, as well as from decrease in methodological
variability across studies. BACKGROUND: Willis-Ekbom disease (WED), also called restless legs syndrome
(RLS), is a neurologic sensorimotor disease that may be associated with
cardiovascular disease. Given high morbidity and mortality rates of
cardiovascular disease worldwide, we assessed the relation between WED/RLS and
cardiovascular health risks in a native South American population. We
prospectively analyzed data from The Atahualpa Project of Ecuadorian adults aged
40 years and older. Physicians interviewed consented persons on the health
behavior and health factors of the American Heart Association (AHA) for ideal
cardiovascular health in adults and underwent fasting laboratory blood
collection and blood pressure evaluation. Certified neurologists conducted
face-to-face interviews using the International Restless Legs Syndrome Study
Group (IRLSSG) field instrument. Persons testing positive for WED/RLS and
age-and sex-matched controls underwent confirmatory physical examinations
conducted by a neurologist and a sleep specialist to whom IRLSSG designation was
blinded.
FINDINGS: Of 665 persons, 94 (14 %) tested positive in IRLSSG; 40 (6 %) had a
diagnosis of WED/RLS after neurologic examination and interview. Patients with
WED/RLS were younger (53.5 vs 59.9 years, P = .001), without significant
differences in sex ratios. Among AHA risk factors, only obesity was
significantly more prevalent among patients with WED/RLS (42.5 % vs 23.5 %,
P = .01). However, after adjustment for confounders, body mass index was not
significantly associated with WED/RLS.
CONCLUSIONS: In adult Amerindians, although obesity and body mass index were
associated with WED/RLS on univariate analyses, the association was not present
after adjustment for confounders. No other significant associations were found
between WED/RLS and AHA cardiovascular metrics. Restless Legs Syndrome/Willis-Ekbom Disease (RLS/WED) is a common condition
characterized by an irresistible urge to move the legs, concomitant with an
unpleasant sensation in the lower limbs, which is typically relieved by
movement. Symptoms occur predomitly at rest and prevail in the afternoon or
evening. Treatment of patients with RLS/WED is indicated for those patients who
suffer from clinically relevant symptoms. The management of mild forms of
RLS/WED is mainly based on dopamine agonists (DA) therapy (including pramipexole
and ropinirole) and α-2-δ calcium-channel ligand. Nevertheless, with passing of
time, symptoms tend to become more severe and the patient can eventually develop
pharmacoresistance. Furthermore, long-term treatment with dopaminergic agents
may be complicated by the development of augmentation, which is defined by an
increase in the severity and frequency of RLS/WED symptoms despite adequate
treatment. Here, we discuss which are the best therapeutic options when RLS/WED
becomes intractable, with a focus on advantages and side effects of the
available medications. Prevention strategies include managing lifestyle changes
and a good sleep hygiene. Different drug options are available. Switching to
longer-acting dopaminergic agents may be a possibility if the patient is
well-tolerating DA treatment. An association with α-2-δ calcium-channel ligand
is another first-line approach. In refractory RLS/WED, opioids such as
oxycodone-naloxone have demonstrated good efficacy. Other pharmacological
approaches include IV iron, benzodiazepines such as clonazepam, and
antiepileptic drugs, with different level of evidence of efficacy. Therefore,
the final decision regarding the agent to use in treating severe RLS/WED
symptoms should be tailored to the patient, taking into account the
symptomatology, comorbidities, the availability of treatment and the history of
the disease. BACKGROUND: Willis-Ekbom disease/restless legs syndrome (WED/RLS) seems to be a
frequent cause of intractable chronic insomnia (ICI) but is under-recognized in
children/adolescents with neurodevelopmental conditions (NDCs), as many patients
do not have the ability to express the underlying "urge-to-move". In light of
this, we aim to develop a protocol for behavioral observations supporting the
diagnosis of WED/RLS.
METHODS: We investigated 26 pediatric patients (age 1-16 years, median 8) with
NDCs, ICI and evidence of familial WED/RLS employing (1) "emplotted narratives"
for description of the various "urge-to-move" presentations and (2)
self-description and "behavioral observations" during a "suggested clinical
immobilization test" (SCIT).
RESULTS: Parental narratives reflected typical WED/RLS-related "urge-to-move"
symptoms during day-, bed-, and nighttime in all patients. Fifteen out of 26
patients could describe the "urge-to-move" during the SCIT. Ten out of 26
patients, unable to describe their symptoms due to cognitive disabilities,
showed patterns of "relieving-movements" upon observation. Sensory processing
abnormalities were reported in all patients, with tactile sensitivities (26/26)
(including shifted pain threshold) as the most common sensory domain.
CONCLUSION: "Emplotted narratives" and structured "behavioral observations"
support recognition of familial WED/RLS associated movement patterns and provide
a useful tool for the diagnosis of WED/RLS in children with NDCs in a clinical
office setting. Restless legs syndrome (RLS), also known as Willis-Ekbom disease (WED), is a
common movement disorder characterised by an uncontrollable urge to move because
of uncomfortable, sometimes painful sensations in the legs with a diurnal
variation and a release with movement. The pathophysiology is only partially
known and a genetic component together with dopaminergic and brain iron
dysregulation plays an important role. Secondary causes for RLS need to be
excluded. Treatment depends on the severity and frequency of RLS symptoms,
comprises non-pharmacological (eg lifestyle changes) and pharmacological
interventions (eg dopaminergic medication, alpha-2-delta calcium channel
ligands, opioids) and relieves symptoms only. Augmentation is the main
complication of long-term dopaminergic treatment of RLS. This article will
provide a clinically useful overview of RLS with provision of diagnostic
criteria, differential diagnoses, possible investigations and different
treatment strategies with their associated complications. INTRODUCTION: Restless legs syndrome (RLS), also known as Willis-Ekbom disease,
is characterised by abnormal sensations in the legs as well as dysaesthesia.
Although the aetiology of RLS has not yet been determined, it may be associated
with systemic inflammation. The neutrophil-to-lymphocyte ratio (NLR) is a new
and simple marker indicating systemic inflammation. The present study aimed to
investigate the relationship between systemic inflammation and RLS through the
use of the NLR.
METHODS: A total of 75 newly diagnosed patients with RLS and 56 healthy control
subjects were included in the study. Baseline NLR was calculated by dividing the
absolute neutrophil count by the absolute lymphocyte count. The NLRs of the two
groups were compared.
RESULTS: There were no significant differences in gender and age between the two
groups. The NLR was 1.96 ± 0.66 in the patient group and 1.67 ± 0.68 in the
control group (p = 0.005). Receiver operating characteristic analysis was
performed to determine the cut-off value of NLR to predict RLS. The NLR was
predictive at 1.58 with a 64% sensitivity and 50% specificity (95% confidence
interval 0.55-0.74, area under curve 0.648 ± 0.05). The NLR was found to be
statistically higher in patients with RLS and may be used to predict RLS.
CONCLUSION: The aetiology of RLS remains undetermined. The present study showed
that systemic inflammation may play a role in RLS. However, RLS could also be
associated with systemic inflammatory diseases. This relationship is supported
by high NLR values, which are related to chronic systemic inflammation. |
List two most common symptoms of Aagenaes syndrome. | Aagenaes syndrome, also called lymphoedema cholestasis syndrome 1, is characterized by neonatal intrahepatic cholestasis, often lessening and becoming intermittent with age and severe chronic lymphoedema, mainly affecting the lower extremities. | Hereditary intrahepatic cholestasis with lymph oedema is now a well defined
autosomal recessive inherited syndrome. More than 75% of the known cases (about
40) are Norwegian, and most of these came from a few communities in the
south-western part of Norway. Cholestasis is present prior to or shortly after
birth. With modern treatment the cholestasis usually improves considerably
during the first two years of life, but periods of recurrent cholestasis occur
later. In some cases, lymph oedema is present at birth, but usually comes to
light during childhood. Lymph oedema needs continuous treatment. As a rule, the
prognosis for the liver disease is good, but cirrhosis has developed in about
15% of the Norwegian cases. As for the pathogenesis of the cholestasis, the
hypothesis is that the cause is an anomaly of the lymph function. We report a mother and daughter with features of Aagenaes syndrome. Unlike most
previous cases, there is no Norwegian ancestry and the pedigree favours domit
rather than recessive inheritance. Patients with cholestasis-lymphedema syndrome (CLS) suffer severe neonatal
cholestasis that usually lessens during early childhood and becomes episodic;
they also develop chronic severe lymphedema. The genetic cause of CLS is
unknown. We performed a genome screen, using DNA from eight Norwegian patients
with CLS and from seven unaffected relatives, all from an extended pedigree.
Regions potentially shared identical by descent in patients were further
characterized in a larger set of Norwegian patients. The patients manifest
extensive allele and haplotype sharing over the 6.6-cM D15S979-D15S652 region:
30 (83.3%) of 36 chromosomes of affected individuals carry a six-marker
haplotype not found on any of the 32 nontransmitted parental chromosomes. All
Norwegian patients with CLS are likely homozygous for the same disease mutation,
inherited from a shared ancestor. The combination of neonatal intrahepatic cholestasis and lymphoedema in feet and
legs is a specific syndrome named after the Norwegian paediatrician Øystein
Aagenaes, who described the syndrome in 1968. The condition is autosomal
recessively inherited and the gene is located to 15q, but not yet identified.
The condition is particularly frequent in the southern most part of Norway and
the gene frequency is estimated to be about 3%. The development of small
lymphoid vessels is probably deficient around the small biliary tracts and in
general. Aagenaes' syndrome is found in patients from other parts of Europe and
the US, but more than half of the cases are of Norwegian origin. This case describes the clinical course and treatment of a 17-year-old male
patient with advanced hepatocellular carcinoma (HCC) arising in a non-cirrhotic
liver. The disease was thought to be caused by a congenital cholestatic syndrome
associated with intermittent oedema in childhood, resembling the rare Aagenaes
syndrome. Treatment choices in advanced HCC arising in adolescence are
discussed. Aagenaes syndrome/lymphedema cholestasis syndrome 1 (LCS1) is a rare genetic
disorder characterized by neonatal cholestasis and lymphedema. The aim was to
assess dental care and oral health in adults with LCS1. Fifteen (9M, 6F)
individuals diagnosed with LCS1, aged 19-59 years participated. The study
evaluated salivary secretion rate, dental radiographs, intraoral photos and
included a questionnaire. Eight (53%) had regular dental checkups. Three had
received subsidized dental care. Seven (47%) had two or more subjective symptoms
of xerostomia. Three (20%) had a decreased stimulated salivary secretion rate
below 0.7 mL/minute. Seven (47%) had dentin caries. Marginal periodontitis was
found in all six patients above 35 years of age, but not before that age.
Thirteen (87%) had tooth discoloration, which was extensive in three (20%).
CONCLUSION: Several patients with LCS1 have problems with periodontitis and
tooth discoloration. Frequent dental checkups are therefore recommended. BACKGROUND: The characterizations of primary lymphedemas in different hereditary
diseases are often published as case reports. In this study, 17 out of 20
Norweigian adult patients with lymphedema cholestasis syndrome 1 (LCS1)/Aagenaes
syndrome were examined. The patients exhibited lymphedema and sporadic
cholestasis. Individual clinical variations are described.
METHODS AND RESULTS: Lymphedema was classified from Grade I to IV by clinical
examinations and ultrasound B-mode scanning. To support the clinical findings,
direct segmental multifrequency bioelectrical impedance analysis (DSM-BIA) was
included and was compared to healthy matched controls. The lymphedema was
similar to other hereditary lymphedemas, with more pronounced fluid retention in
the lower extremities. It was generally more extensive, as it also included
lymphedema in the arms, face, and trunk. Limited tissue fibrosis was observed,
even after long-standing lymphedema.
CONCLUSIONS: Approximately one-third of the patients had severe forms of
lymphedema in the limbs (grades III and IV) and their conditions required close
followup. A more frequent use of compression in the upper extremities is
advised. Aagenaes syndrome, also called lymphoedema cholestasis syndrome 1 (LSC1), is
characterized by neonatal intrahepatic cholestasis, often lessening and becoming
intermittent with age and severe chronic lymphoedema, mainly affecting the lower
extremities. The condition is autosomal recessively inherited, and the gene is
located on chromosome 15q. The locus, LCS1, was mapped to a 6.6 cM region on
chromosome 15. Angioid streaks are visible irregular crack-like dehiscences in
bruch's membrane that are associated with atrophic degeneration of the overlying
retinal pigment epithelium. Angioid streaks have been described to be associated
with pseudoxanthoma elasticum, paget's disease, sickle-cell anaemia, acromegaly,
Ehlers-Danlos syndrome, and diabetes mellitus, but also appear in patients
without any systemic diseases. Patients with angioid streaks are generally
asymptomatic, unless the lesions extend towards the foveola or develop
complications such as traumatic bruch's membrane rupture or macular choroidal
neovascularization. |
Which are the side effects during tacrine administration in patients with Alzheimer's Disease? | The side effects during tacrine administration in patients with Alzheimer's Disease are:
1) Hepatotoxicity
2) Gastrointestinal (diarrhea, anorexia, dyspepsia, abdominal pain, nausea, vomiting)
3) Mitochondrial impairement | OBJECTIVE: To see whether combined treatment with oral tacrine
(tetrahydroaminoacridine; THA) and lecithin improves the symptoms of patients
with Alzheimer's disease.
DESIGN: Multicentre double blind, placebo controlled, random order crossover
trial with individual determination of maximum tolerated dosage and four month
follow up.
SETTING: Outpatient departments at six university neurological centres.
PATIENTS: 67 Outpatients (24 men, 43 women) aged 53-81 (mean 66 (SD 7.3))
selected according to the following criteria: probable Alzheimer's disease as
defined by the National Institute of Neurological and Communicative Disorders
and Stroke and the Alzheimer's Disease and Related Disorders Association;
absence of mood disorder; mini mental state score lower than 26; availability of
a close relative able to complete questionnaires; and informed consent of the
patient or his or her closest relative, or both.
INTERVENTIONS: Mean of 114 mg tacrine or placebo daily plus 1200 mg lecithin
daily given in three divided doses for one four week active treatment period and
one four week control period without washout at crossover.
MAIN OUTCOME MEASURES: Cognitive state as assessed by Folstein's mini mental
state rating scale, behavioural state as assessed by the Stockton geriatric
rating scale, and overall state as assessed with a visual analogue scale rated
by both the relative and the physician.
RESULTS: Compared with placebo tacrine did not improve either the mini mental
state score (mean 14.9 (SD 7.3) v 14.8 (7.3)) or the Stockton geriatric score
(28.2 (15.7) v 28.7 (17.8)), but a slight and statistically significant
improvement occurred in the physician's score on the visual analogue scale (6.3
(10.2) v 11.6 (17.9)). Seven patients dropped out. Six patients were excluded
because of acute hepatitis and one withdrew for personal reasons not related to
treatment. Two other patients developed acute hepatitis at the end of the eight
week crossover trial and another during the follow up study. Twenty patients
complained of gastrointestinal side effects.
CONCLUSIONS: Neither short term nor long term treatment with oral tacrine at
dosages lower than 125 mg/day improves the symptoms of Alzheimer's disease.
Moreover, these dosages may induce hepatitis (nine of 67 patients in this
series). A clinical comparison of tacrine (THA) and placebo was performed in 15 Alzheimer
patients using a double blind crossover technique over 4 plus 4 weeks with one
drug-free week in between. Treatment results, as evaluated by clinical rating
scales and neuropsychological tests, were mostly negative. Side effects were
few, except for elevation liver enzymes which occurred in one third of the
patients. CSF levels of the monoamine metabolites HVA and 5-HIAA increased on
tacrine as evidence for activation of dopamine and serotonin pathways through
cholinergic receptors. Pharmacokinetic investigations showed that the oral
bioavailability of tacrine was low and greatly varying between subjects.
Patients with high bioavailability of the drug tended to improve more, and also
to have more liver enzyme elevations, than those with low bioavailability. A gel
preparation for rectal administration was manufactured for comparison of plasma
levels attained during one week's treatment with levels attained with oral
capsules. Preliminary results indicate that the dose of tacrine can be reduced
to 50 per cent when administered rectally, probably as by this route the rapid
first-pass metabolism of the drug in the liver is diminished. A clinical trial
of tacrine via the rectal route would be justified as this could decrease the
number of patients with liver side effects and increase the number of patients
improving on the treatment. Up to one-third of patients with mild to moderate Alzheimer's disease may show
improvement in cognitive function with tacrine, a centrally-acting,
noncompetitive inhibitor of acetylcholinesterase. Candidates for tacrine must
have a diagnosis of probable Alzheimer's dementia based on NINCDS-ADRDA or
DSM-IV criteria and should have no history of liver disease. For patients
receiving the drug, do follow-up cognitive testing with a sensitive measure such
as the Alzheimer's Disease Assessment Scale. The most common adverse effects
associated with tacrine therapy are elevated liver transaminases and
gastrointestinal effects. Weekly blood tests are necessary to monitor liver
function. A reliable caregiver is essential to ensure compliance with frequent
dosing and weekly blood testing. In a previous pharmacokinetic study in Alzheimer patients great inter-individual
variation and low oral bioavailability of the cholinesterase inhibitor tacrine
(tetrahydroaminoacridine, THA) were found. In the present investigation oral and
rectal administration of tacrine were compared with the aim to find a route for
improved bioavailability through diminished first-pass metabolism in the liver.
Eight patients suffering from Alzheimer's dementia were given tacrine by oral
(25 and 50 mg b.i.d.) and rectal (12.5 and 25 mg b.i.d.) routes for 1 week with
4-6 weeks washout in between. Drug hydroxylation capacity in the patients was
determined using the debrisoquine test. Levels of tacrine in plasma and
cerebrospinal fluid (CSF) were determined and the cognitive performance was
examined by the Mini-Mental State Examination (MMSE) and the Alzheimer Deficit
Assessment Scale (ADAS). Tacrine was well tolerated in all but one patient, a
slow hydroxylator, who developed an aplastic anemia. MMSE and ADAS scores did
not significantly change, except for word recall which was improved on tacrine
when given by the rectal route. Pharmacokinetic analysis of the two
administration routes revealed that the drug dose may be reduced by almost 50%
when given rectally compared to orally. Concentrations of tacrine in the CSF
were significantly lower and correlated linearly with the concentrations in
plasma. OBJECTIVE: To review the pharmacology, biopharmaceutics/pharmacokinetics,
clinical efficacy, adverse effects, and therapeutic considerations regarding the
use of tacrine in patients with Alzheimer's disease.
DATA SOURCES: Data from the scientific and professional literature were
analyzed, interpreted, and summarized. Citations were obtained by performing a
MEDLINE search using the following indexing terms: tacrine,
tetrahydroaminoacridine, and Alzheimer's drug therapy. Data also were obtained
from a summary of the New Drug Application (Summary Basis of Approval of Cognex)
and from the approved product labeling.
STUDY SELECTION: Studies in Alzheimer's disease have been plagued by
methodologic inconsistencies and deficiencies. Only efficacy studies subsequent
to the Food and Drug Administration's (FDA) issuance of recommendations for
studies in Alzheimer's disease (1991) were used. Therefore, only double-blind,
placebo-controlled, parallel design studies of at least three-month's duration
using the Alzheimer's Disease Assessment Scale-Cognitive Subscale and the
Clinical Interview-Based Impression of Change as efficacy parameters were
included.
DATA EXTRACTION: Trials were assessed according to the criteria listed above.
Results were evaluated on the basis of both completed patients and last
observation carried forward models.
DATA SYNTHESIS: Tacrine is a cholinesterase inhibitor that increases the
availability of acetylcholine in muscarinic neurons. It has a mean
bioavailability after oral administration of about 17 percent and an elimination
half-life of approximately three hours. Although drug interactions are poorly
studied, tacrine is metabolized by isoenzyme P-450IA2 and may interact with
other drugs metabolized by this isoenzyme. Tacrine has been shown to have
efficacy in mildly to moderately impaired Alzheimer's patients on both
psychometric testing and a clinician's structured interview. Although
efficacious, its effects are not dramatic, and it does not affect the ultimate
course of the disease. Adverse effects are frequent, and significantly elevated
hepatic transaminase concentrations may occur in approximately 25 percent of
patients.
CONCLUSIONS: Tacrine is the first drug approved by the FDA for treatment of
Alzheimer's disease. Although it may improve psychometric test scores in mild to
moderately impaired patients, it is not a panacea and does not affect the course
of the disease. Patients must be monitored closely for elevated transaminase,
cholinergic adverse effects, and interactions with drugs metabolized through
P-450IA2. OBJECTIVE: To characterize the hepatic effects of tacrine treatment in patients
with Alzheimer's disease.
DESIGN: Controlled trials of tacrine therapy consisting of two blinded,
parallel-group trials; three blinded, enrichment-design trials; and their
respective open-label extensions.
SETTING: Multicenter clinical trials in the United States, France, and Canada.
PATIENTS: A total of 2446 men and women at least 50 years of age with a
diagnosis of probable Alzheimer's disease of mild to moderate severity and in
good health without significant hepatic, cardiovascular, or renal disease.
INTERVENTION: Administration of tacrine vs placebo, with weekly measurement of
serum hepatic enzymes.
MAIN OUTCOME MEASURES: Incidence, maximum severity, and timing of event for
serum alanine aminotransferase (ALT) elevation.
RESULTS: Among the 2446 patients who received tacrine in clinical trials, ALT
levels greater than the upper limit of normal (ULN) occurred on at least one
occasion in 1203 patients (49%), ALT levels greater than three times the ULN
occurred in 621 patients (25%), and ALT levels greater than 20 times the ULN
occurred in 40 patients (2%). The elevated ALT levels were generally
asymptomatic and occurred more frequently in women than men. The mean time from
initiation of tacrine treatment to first ALT level greater than three times the
ULN was 50 days, and 90% of all initial ALT levels greater than three times the
ULN occurred during the first 12 weeks of treatment. Of 145 patients who
discontinued tacrine treatment because of an ALT level greater than three times
the ULN and were rechallenged, 127 (88%) were able to resume long-term therapy
with the drug. In all instances, discontinuing tacrine completely reversed
elevations in ALT levels, and no deaths related to hepatotoxicity occurred.
CONCLUSIONS: These data suggest that the potential for serious hepatic toxicity
can be reduced through careful monitoring of ALT levels in patients who may
benefit from tacrine therapy. BACKGROUND: Therapy to enhance cholinergic function in the brain is under
evaluation for the treatment of Alzheimer's disease. Tetrahydroaminoacridine
(tacrine) has recently received a product licence in the United States of
America for the treatment of Alzheimer's disease, and the licence application in
the United Kingdom will shortly be reviewed. It is therefore possible that this
drug will become available for use in the UK in due course. There will then be a
need for screening procedures for a large number of elderly patients to decide
whether or not they have dementia and, if so, whether it is the result of
Alzheimer's disease and is suitable for treatment with the new drug.
METHOD: A total of 246 patients aged 75 years or over in two general practices
in Bristol were assessed to investigate the potential workload such screening
would engender. Three different assessment schedules for the diagnosis of
dementia were compared--the mini-mental state examination, the Kew test, and the
abbreviated mental test score.
RESULTS: None of the assessment schedules was found to be particularly onerous,
with median times for administration of five, three and two minutes,
respectively. A score of 23 or less on the mini-mental state examination was
taken as the main cut-off point for further evaluation. Sixty six patients
obtained this score--in 25 the low score reflected factors other than dementia,
and 11 others declined further assessment. Of the remaining 30 patients only
four had probable Alzheimer's disease at an appropriate level of severity for
treatment, and lived with a carer who could ensure compliance and monitor side
effects. Two of these patients were receiving conflicting medical treatment and
a third declined therapy, leaving only one person for whom treatment could be
prescribed.
CONCLUSION: It seems likely that of those medically suitable for treatment, it
may not be possible to prescribe tacrine for an appreciable proportion.
Nevertheless, all potential patients should be screened as the procedures
involved are not onerous and at least some of those found suitable for treatment
are likely to benefit from this new approach. Previous work has shown that cholinomimetic drugs induce "vacuous" or
non-directed jaw movements in rats. In the present study, five experiments were
conducted to provide a pharmacological, anatomical and behavioral
characterization of tacrine-induced vacuous jaw movements. In the first
experiment, tacrine produced vacuous chewing in a dose-related manner in a range
of 1.25 mg/kg to 1.0 mg/kg. This effect was reduced, also in a dose-related
manner, by the co-administration of the muscarinic antagonist scopolamine in a
range of 0.125 to 1.0 mg/kg, but not by N-methylscopolamine. The fourth
experiment examined the effect of scopolamine (2.5 to 10.0 micrograms) injected
into the ventrolateral striatum on vacuous jaw movements induced by 5.0 mg/kg
tacrine. Intrastriatal injections of scopolamine completely blocked
tacrine-induced jaw movements. The fifth experiment utilized a slow-motion
videotaping system to analyze the temporal characteristics of vacuous chewing
induced by 5.0 mg/kg tacrine. The vast majority of the movements occurred in
rapid "bursts," and analysis of interresponse times (i.e., the time between each
jaw movement) showed that most of the jaw movements occurred within a local
frequency range of 3 to 7 Hz. Thus, tacrine-induced jaw movements are reduced by
antimuscarinic treatment, and most of these movements occur in the parkinsonian
tremor frequency range. Tremulous jaw movements induced by tacrine in rats
appear to share some characteristics with Parkinsonian tremor. OBJECTIVE: Tacrine is extensively metabolized by cytochrome P4501A2 (CYP1A2).
Fluvoxamine, a potent CYP1A2 inhibitor, may be coadministered with tacrine. The
aim of this study was to examine the influence of fluvoxamine administration on
the disposition kinetics of single-dose tacrine administration.
METHODS: Thirteen healthy volunteers participated in this double-blind,
randomized crossover study, which compared the effects of fluvoxamine (100
mg/day during 6 days) and placebo on the pharmacokinetics of a single oral dose
of tacrine (40 mg).
RESULTS: Fluvoxamine caused a significant increase in tacrine area under the
plasma concentration versus time curve (AUC): arythmetic mean, 27 (95%
confidence interval [CI], 19 to 38) ng.hr/ml versus 224 (95% CI, 166 to 302) ng.
hr/ml. Fluvoxamine caused a decrease in the apparent oral clearance of tacrine
from 1683 +/- 802 to 200 +/- 106 L/hr (mean +/- SD), which was explained by a
decrease in its nonrenal clearance. Five subjects had gastrointestinal side
effects during fluvoxamine administration. Fluvoxamine administration was
associated with significant increases in the plasma AUC values of three
monohydroxylated tacrine metabolites and in the total urinary recovery
measurements of tacrine and its metabolites (9.1% +/- 4.6% versus 24.0% +/- 2.6%
of recovery). These results may be attributable to fluvoxamine-dependent
inhibition of CYP1A/, which is responsible of the biotransformation of tacrine
into its monohydroxylated metabolites and further into dihydroxylated and
reactive metabolites.
CONCLUSION: Fluvoxamine inhibits the metabolism of tacrine. CYP1A2 may be the
target of this inhibition. Fluvoxamine may modulate the hepatotoxicity of
tacrine, depending on the relative contribution of tacrine and its reactive
metabolites to this toxicity. Hepatotoxicity limits the clinical utility of the cholinesterase inhibitor
tacrine as a palliative therapy for Alzheimer's disease. The present studies
examined the effects of E2020, a selective acetylcholinesterase inhibitor not
associated with liver toxicity in man, on cognitive performance in rhesus
monkeys using tasks employed previously to evaluate tacrine and other
cholinomimetic agents. The ability of E2020 to prevent the induction of a
cognitive impairment by the muscarinic receptor antagonist scopolamine was
assessed using an automated spatial delayed response task. Coadministration of
E2020 (0.5-1.75 mg/kg) caused a dose-dependent reversal of the scopolamine (0.03
mg/kg) induced impairment observed after retention intervals of 10 and 20 s. At
the highest dose of E2020 examined (1.75 mg/kg), choice accuracy approached
normal control levels. In this dose range, E2020 was well tolerated, but at the
higher dose of 2 mg/kg, cholinergic side-effects were apparent. The effect of
E2020 on choice accuracy in a visual recognition task was also assessed as this
task does not require the use of scopolamine to disrupt performance and
beneficial effects of cholinomimetics can therefore be detected at lower doses
than in the spatial memory paradigm. In this task, administration of E2020
increased choice accuracy from 59 +/- 1% correct to up to 71 +/- 2% at doses of
0.03 and 0.05 mg/kg. No observable adverse effects were induced by E2020 in this
dose range. The ability of E2020 to improve performance in these cognitive tasks
resembles the profile of other cholinesterase inhibitors, including tacrine,
that also improve cognitive function in Alzheimer's disease patients. Because of
its more favourable clinical safety profile, E2020 may provide a significantly
improved palliative therapy for dementia. BACKGROUND: Tacrine is one of the first drugs to be widely marketed for the loss
of memory and intellectual decline in Alzheimer's disease. The alleged success
of tacrine in the treatment of these symptoms has been heralded as confirmation
of the cholinergic theory of Alzheimer's disease. However, the efficacy of
tacrine for symptoms of dementia remains controversial. This is reflected by the
low rate of prescription of tacrine in countries where it is approved and the
lack of approval by several regulatory authorities in Europe and elsewhere. The
uncertainty about the efficacy of tacrine is due to the difficulties in
interpretation of the results from the clinical trials. The reasons for this are
the small effects of tacrine compared to placebo for all outcomes; the high
incidence of adverse events; the lack of benefit observed in several trials; the
use of cross-over designs and their associated methodological problems in a
disease like dementia; the use of different measurement scales to assess outcome
in different trials; and the problem of high dropout rates.
OBJECTIVES: To determine the clinical efficacy of tacrine for the symptoms of
Alzheimer's disease.
SEARCH STRATEGY: The Cochrane Dementia Group Register of Clinical Trials was
searched using the terms 'tacrine', 'tetrahydroaminoacridine' and 'THA' (see the
Group's search strategy for full details).
SELECTION CRITERIA: All unconfounded, double-blind, randomized trials in which
treatment with tacrine was administered for more than a day and compared to
placebo in patients with dementia of the Alzheimer's type.
DATA COLLECTION AND ANALYSIS: Data were extracted independently by two
reviewers, pooled if appropriate and possible, and the pooled odds ratios
(95%CI) or the average differences (95%CI) were estimated. Where possible,
intention-to-treat data were used.
MAIN RESULTS: This review produced no clear results. The results were compatible
with tacrine producing improvement, no change or even harm for those with
Alzheimer's disease. It was not possible to use many of the published results in
a combined analysis. For measures of overall clinical improvement, the
intention-to-treat analyses failed to detect any difference between tacrine and
placebo (OR 0.87; 95%CI 0.61 - 1.23). Behavioural disturbance, as measured by
the Alzheimer's Disease Assessment Scale-noncognitive, failed to detect any
difference between tacrine and placebo (SMD -0.04; 95%CI -0.52 - 0.43). For
cognition function, the effect of tacrine was not statistically significantly
different from placebo for the MiniMental State Examination score (0-30; high
=good) (SMD 0.14; 95%CI -0.02 - 0.30) and was barely statistically significantly
in favour of treatment for the Alzheimer's Disease Assessment Scale-cognitive
scale (SMD -0.22; 95%CI -0.32 - -0.13). Adverse events were not reported in a
systematic way in the different trials, making formal comparison difficult.
Raised serum liver enzymes was the major reason for withdrawal. The odds ratio
for withdrawal due to an adverse event was significantly different from one, the
control group experienced fewer events (OR 5.7; 95%CI 4.1-7.9). Gastrointestinal
side effects (diarrhoea, anorexia, dyspepsia and abdominal pain) were the other
major cause of adverse events and for withdrawal, and the odds ratio for
withdrawal was also significantly different from one in favour of the control
group (OR 3.8; 95%CI 2.8-5.1). No deaths were reported in any of the studies
during the trial period, up to six months.
REVIEWER'S CONCLUSIONS: This review provides no convincing evidence that tacrine
is a useful treatment for the symptoms of Alzheimer's disease. However, as so
few trials presented data in a format suitable for pooling, the results of this
review may be modified when further data from all relevant trials are included.
There is an urgent need for the independent evaluation of the data already
existing in the trials but not accessible through published or grouped data. A BACKGROUND: Tacrine is one of the first drugs to be widely marketed for the loss
of memory and intellectual decline in Alzheimer's disease, often accompanied by
abnormal behaviour and physical decline. The alleged success of tacrine in the
treatment of these symptoms has been heralded as confirmation of the cholinergic
theory of Alzheimer's disease. The efficacy of tacrine for symptoms of dementia
remains controversial. This is reflected by the low rate of prescription of
tacrine in countries where it is approved and the lack of approval by several
regulatory authorities in Europe and elsewhere. The uncertainty about the
efficacy of tacrine is due to the difficulties in interpretation of the results
from the clinical trials. The reasons for this are the small effects of tacrine
compared to placebo for all outcomes; the high incidence of adverse events; the
lack of benefit observed in several trials; the use of cross-over designs and
their associated methodological problems in a disease like dementia; the use of
different measurement scales to assess outcome in different trials; and the
problem of high dropout rates.
OBJECTIVES: To determine the clinical efficacy of tacrine for the symptoms of
Alzheimer's disease.
SEARCH STRATEGY: The Cochrane Dementia Group Register of Clinical Trials was
searched using the terms 'tacrine', 'tetrahydroaminoacridine' and 'THA' (see the
Group's search strategy for full details).
SELECTION CRITERIA: All unconfounded, double-blind, randomized trials in which
treatment with tacrine was administered for more than a day and compared to
placebo in patients with dementia of the Alzheimer's type.
DATA COLLECTION AND ANALYSIS: Data were extracted independently by two
reviewers, pooled if appropriate and possible, and the pooled odds ratios
(95%CI) or the average differences (95%CI) were estimated. Where possible,
intention-to-treat data were used.
MAIN RESULTS: This review produced no clear results. The results were compatible
with tacrine producing improvement, no change or even harm for those with
Alzheimer's disease. It was not possible to use many of the published results in
a combined analysis. For measures of overall clinical improvement, the
intention-to-treat analyses failed to detect any difference between tacrine and
placebo (OR 0.87; 95%CI 0.61 - 1.23). Behavioural disturbance, as measured by
the Alzheimer's Disease Assessment Scale-noncognitive, failed to detect any
difference between tacrine and placebo (SMD -0.04; 95%CI -0.52 - 0.43). For
cognition function, the effect of tacrine was not statistically significantly
different from placebo for the MiniMental State Examination score (0-30; high
=good) (SMD 0.14; 95%CI -0.02 - 0.30) and was barely statistically significantly
in favour of treatment for the Alzheimer's Disease Assessment Scale-cognitive
scale (SMD -0.22; 95%CI -0.32 - -0.13). Adverse events were not reported in a
systematic way in the different trials, making formal comparison difficult.
Raised serum liver enzymes was the major reason for withdrawal. The odds ratio
for withdrawal due to an adverse event was significantly different from one, the
control group experienced fewer events (OR 5.7; 95%CI 4.1-7.9). Gastrointestinal
side effects (diarrhoea, anorexia, dyspepsia and abdominal pain) were the other
major cause of adverse events and for withdrawal, and the odds ratio for
withdrawal was also significantly different from one in favour of the control
group (OR 3.8; 95%CI 2.8-5.1). No deaths were reported in any of the studies
during the trial period, up to six months.
AUTHORS' CONCLUSIONS: This review provides no convincing evidence that tacrine
is a useful treatment for the symptoms of Alzheimer's disease. However, as so
few trials presented data in a format suitable for pooling, the results of this
review may be modified when further data from all relevant trials are included.
There is an urgent need for the independent evaluation of the data already
existing in the trials but not accessible through published or grouped data. An
independent meta-analysis of the individual-patient data is required. The
results and conclusions of this update are unaltered by further searching as the
additional studies do not add any further valid/eligible data. In the treatment of Alzheimer's disease tacrine, a cholinesterase inhibitor, is
not the drug of choice due to its low oral bioavailability, extensive hepatic
first-pass effect, rapid clearance from the systemic circulation, pronounced
hepatotoxicity, and the availability of drugs better than tacrine in the same
pharmacological class. Hence, the aim of this investigation was to ascertain the
possibility of direct nose-to-brain delivery of tacrine to improve
bioavailability, to avoid the first-pass effect and to minimize hepatotoxicity.
Tacrine solution (TS) in propylene glycol was radiolabelled with (99m)Tc
(technetium) and administered in BALB/c mice intranasally (i.n.) and
intravenously (i.v.). Drug concentrations in blood and brain were determined at
predetermined time intervals post dosing. Drug targeting efficiency (DTE %) and
the brain drug direct transport percentage (DTP %) were calculated to evaluate
the brain targeting efficiency. Brain scintigraphy imaging in rabbits was
performed to ascertain the uptake of the drug into the brain. Tacrine solution
was effectively labelled with (99m)Tc and was found to be stable and suitable
for in-vivo studies. Following intranasal administration tacrine was delivered
quickly (T(max) 60 min) to the brain compared with intravenous administration
(T(max) 120 min). The brain/blood ratios of the drug were found to be higher for
[(99m)Tc]TS(i.n.) compared with [(99m)Tc]TS(i.v.) at all time points. The DTE
(207.23%) and DTP (51.75%) following intranasal administration suggested that
part of tacrine was directly transported to brain from the nasal cavity. Rabbit
brain scintigraphy imaging showed higher uptake of the drug into the brain
following intranasal administration compared with intravenous administration.
The results showed that tacrine could be directly transported into the brain
from the nasal cavity and intranasal administration resulted in higher
bioavailability of drug with reduced distribution into non-targeted tissues.
This selective localization of tacrine in the brain may be helpful in reducing
dose, frequency of dosing and dose-dependent side effects, and may prove an
interesting new approach in delivery of the drug to the brain for the treatment
of Alzheimer's disease. The aim of this review is to describe side effects of five antidementives which
are approved by the United States Food and Drug Administration (FDA); four
acetylcholinesterase inhibitors and one glutamate - or N-metyl-D-aspartat
receptor antagonist - memantine. The antidementives are well tolerated and
undesired effects are rare; except hepatotoxicity of tacrine and
gastrointestinal side effects of donepezil, rivastigmine, galantamin and tacrine
that result from acetylcholinesterase inhibition. Nausea, diarrhea, vomiting,
and weight loss are the most common side effects of the acetylcholinesterase
inhibitors. Significant cholinergic side effects can occur in patients receiving
higher doses; often they are related to the rate of initial titration of
medication. Memantine is the first noncholinesterase inhibitor indicated for
Alzheimer's disease. The side effects which may occur during the treatment with
memantine are constipation, dizziness, headache and confusion. These effects if
appears are mild end transient. La cognition, atteinte au cours de la maladie d'Alzheimer, répond maintet à
plusieurs médicaments. Les anticholinestérasiques ciblent le déficit en
acetylcholine. Au cours de la maladie d'Alzheimer légère à modérée, ils
procurent tous un bénéfice significatif comparés au placebo sur l'échelle
ADASCog (Alzheimer's Disease Assessment Schedule-Cognitive Section, échelle de
mesure de la performance cognitive). Les effets secondaires, dans 5 % à 15 % des
cas, incluent nausées, vomissements, diarrhée, anorexie et vertiges. La tacrine,
le principal anticholinestérasique, provoquait souvent une élévation des enzymes
hépatiques et a été retirée du marché ; en dose unique quotidienne, le donépézil
respecte le foie et améliore les mesures globales de détérioration dans la
démence sévère ; la rivastigmine est indiquée en cas de comorbidité avec la
maladie vasculaire ; enfin, la galantamine agit sur les récepteurs cérébraux
nicotiniques de l'acétyleholine qui potential isent la réponse à
l'acétyleholine. Parmi les autres traitements on peut noter l'antagoniste du
récepteur du N-méthyl-D-aspartate (NMDA), la mémantine, autorisée en Europe dans
le traitement de la maladie d'Alzheimer modérée à sévère ; elle agit sur un
système différent de neuromédiateurs, présent dans 70 % des neurones, protecteur
contre l'activation glutamatergique pathologique et respectant ou même
rétablissant l'activation glutamatergique physiologique. L'arsenal thérapeutique
du praticien n'a jamais été plus vaste dans la maladie d'Alzheimer. Tacrine is an acetylcholinesterase (AChE) inhibitor used as a cognitive enhancer
in the treatment of Alzheimer's disease (AD). However, its low therapeutic
efficiency and a high incidence of side effects have limited its clinical use.
In this study, the molecular mechanisms underlying the impact on brain activity
of tacrine and two novel tacrine analogues (T1, T2) were approached by focusing
on three aspects: (i) their effects on brain cholinesterase activity; (ii)
perturbations on electron transport chain enzymes activities of non-synaptic
brain mitochondria; and (iii) the role of mitochondrial lipidome changes induced
by these compounds on mitochondrial bioenergetics. Brain effects were evaluated
18 h after the administration of a single dose (75.6 μmol/kg) of tacrine or
tacrine analogues. The three compounds promoted a significant reduction in brain
AChE and butyrylcholinesterase (BuChE) activities. Additionally, tacrine was
shown to be more efficient in brain AChE inhibition than T2 tacrine analogue and
less active than T1 tacrine analogue, whereas BuChE inhibition followed the
order: T1 > T2 > tacrine. The studies using non-synaptic brain mitochondria show
that all the compounds studied disturbed brain mitochondrial bioenergetics
mainly via the inhibition of complex I activity. Furthermore, the activity of
complex IV is also affected by tacrine and T1 treatments while FoF(1) -ATPase is
only affected by tacrine. Therefore, the compounds' toxicity as regards brain
mitochondria, which follows the order: tacrine >> T1 > T2, does not correlate
with their ability to inhibit brain cholinesterase enzymes. Lipidomics
approaches show that phosphatidylethanolamine (PE) is the most abundant
phospholipids (PL) class in non-synaptic brain mitochondria and cardiolipin (CL)
present the greatest diversity of molecular species. Tacrine induced significant
perturbations in the mitochondrial PL profile, which were detected by means of
changes in the relative abundance of phosphatidylcholine (PC), PE,
phosphatidylinositol (PI) and CL and by the presence of oxidized
phosphatidylserines. Additionally, in both the T1 and T2 groups, the lipid
content and molecular composition of brain mitochondria PL are perturbed to a
lesser extent than in the tacrine group. Abnormalities in CL content and the
amount of oxidized phosphatidylserines were associated with significant
reductions in mitochondrial enzymes activities, mainly complex I. These results
indicate that tacrine and its analogues impair mitochondrial function and
bioenergetics, thus compromising the activity of brain cells. BACKGROUND: Alzheimer's disease is the commonest cause of dementia affecting
older people. One of the therapeutic strategies aimed at ameliorating the
clinical manifestations of Alzheimer's disease is to enhance cholinergic
neurotransmission in the brain by the use of cholinesterase inhibitors to delay
the breakdown of acetylcholine released into synaptic clefts. Tacrine, the first
of the cholinesterase inhibitors to undergo extensive trials for this purpose,
was associated with significant adverse effects including hepatotoxicity. Other
cholinesterase inhibitors, including rivastigmine, with superior properties in
terms of specificity of action and lower risk of adverse effects have since been
introduced. Rivastigmine has received approval for use in 60 countries including
all member states of the European Union and the USA.
OBJECTIVES: To determine the clinical efficacy and safety of rivastigmine for
patients with dementia of Alzheimer's type.
SEARCH METHODS: We searched ALOIS, the Cochrane Dementia and Cognitive
Improvement Group Specialized Register, on 2 March 2015 using the terms:
Rivastigmine OR exelon OR ENA OR "SDZ ENA 713". ALOIS contains records of
clinical trials identified from monthly searches of a number of major healthcare
databases (Cochrane Library, MEDLINE, EMBASE, PsycINFO, CINAHL, LILACS),
numerous trial registries and grey literature sources.
SELECTION CRITERIA: We included all unconfounded, double-blind, randomised,
controlled trials in which treatment with rivastigmine was administered to
patients with dementia of the Alzheimer's type for 12 weeks or more and its
effects compared with those of placebo in a parallel group of patients, or where
two formulations of rivastigmine were compared.
DATA COLLECTION AND ANALYSIS: One review author (JSB) applied the study
selection criteria, assessed the quality of studies and extracted data.
MAIN RESULTS: A total of 13 trials met the inclusion criteria of the review. The
trials had a duration of between 12 and 52 weeks. The older trials tested a
capsule form with a dose of up to 12 mg/day. Trials reported since 2007 have
tested continuous dose transdermal patch formulations delivering 4.6, 9.5 and
17.7 mg/day.Our main analysis compared the safety and efficacy of rivastigmine 6
to 12 mg/day orally or 9.5 mg/day transdermally with placebo.Seven trials
contributed data from 3450 patients to this analysis. Data from another two
studies were not included because of a lack of information and methodological
concerns. All the included trials were multicentre trials and recruited patients
with mild to moderate Alzheimer's disease with a mean age of about 75 years. All
had low risk of bias for randomisation and allocation but the risk of bias due
to attrition was unclear in four studies, low in one study and high in two
studies.After 26 weeks of treatment rivastigmine compared to placebo was
associated with better outcomes for cognitive function measured with the
Alzheimer's Disease Assessment Scale-Cognitive (ADAS-Cog) score (mean difference
(MD) -1.79; 95% confidence interval (CI) -2.21 to -1.37, n = 3232, 6 studies)
and the Mini-Mental State Examination (MMSE) score (MD 0.74; 95% CI 0.52 to
0.97, n = 3205, 6 studies), activities of daily living (SMD 0.20; 95% CI 0.13 to
0.27, n = 3230, 6 studies) and clinician rated global impression of changes,
with a smaller proportion of patients treated with rivastigmine experiencing no
change or a deterioration (OR 0.68; 95% CI 0.58 to 0.80, n = 3338, 7
studies).Three studies reported behavioural change, and there were no
differences compared to placebo (standardised mean difference (SMD) -0.04; 95%
CI -0.14 to 0.06, n = 1529, 3 studies). Only one study measured the impact on
caregivers using the Neuropsychiatric Inventory-Caregiver Distress (NPI-D) scale
and this found no difference between the groups (MD 0.10; 95% CI -0.91 to 1.11,
n = 529, 1 study). Overall, participants who received rivastigmine were about
twice as likely to withdraw from the trials (odds ratio (OR) 2.01, 95% CI 1.71
to 2.37, n = 3569, 7 studies) or to experience an adverse event during the
trials (OR 2.16, 95% CI 1.82 to 2.57, n = 3587, 7 studies).
AUTHORS' CONCLUSIONS: Rivastigmine (6 to 12 mg daily orally or 9.5 mg daily
transdermally) appears to be beneficial for people with mild to moderate
Alzheimer's disease. In comparisons with placebo, better outcomes were observed
for rate of decline of cognitive function and activities of daily living,
although the effects were small and of uncertain clinical importance. There was
also a benefit from rivastigmine on the outcome of clinician's global
assessment. There were no differences between the rivastigmine group and placebo
group in behavioural change or impact on carers. At these doses the transdermal
patch may have fewer side effects than the capsules but has comparable efficacy.
The quality of evidence is only moderate for all of the outcomes reviewed
because of a risk of bias due to dropouts. All the studies with usable data were
industry funded or sponsored. This review has not examined economic data. |
Is synapsin a phosphoprotein? | Yes, synapsin is an evolutionarily conserved presynaptic phosphoprotein. | Synapsins as a family of presynaptic terminal phosphoprotein participates in
neuronal development, but their role in the synaptic plasticity of visual cortex
is unclear. In this study, the impact of monocular deprivation (MD) on dynamic
changes of isoform-specific protein expression and site 1 phosphorylation of
synapsins in visual cortex of the postnatal mice were observed by using the
technique of Western blot analysis. The results showed that the total (T-)
protein levels of synapsins including the isoform of Ia/b, IIa/b and IIIa were
about 21-26% of adult level in visual cortex of mice at postnatal 7 days (P7),
and then the T-synapsin Ia/b and IIb could quickly reach adult level at P35.
However, the T-synapsin IIa and IIIa increased more slowly (71-74% at P35), and
then kept increasing in the visual cortex of mice at P60. Unlike to the changes
of T-synapsins, the level of phosphorylated (P-) synapsin Ia/b (not IIa/b and
IIIa) at site 1 increased with development to the highest level at P21, and then
decreased rapidly to a low level in visual cortex of mice at P35-60. In
addition, we found that the levels of P-synapsin Ia/b increased significantly in
left visual cortex of P28 and P35 (not P21 and P42) mice with 1-week MD of right
eye; and no significant changes of T-synapsins were observed in both left and
right sides of visual cortex in P21-42 mice with MD treatment. These results
suggested that the isoform-specific protein expression and site-1
phosphorylation of synapsins might play a different role in the synaptic
plasticity of visual cortex, and MD delays the dynamic changes of phosphorylated
synapsin Ia/b at site-1 in contralateral visual cortex of juvenile mice. Synapsin III (SynIII) is a phosphoprotein that is highly expressed at early
stages of neuronal development. Whereas in vitro evidence suggests a role for
SynIII in neuronal differentiation, in vivo evidence is lacking. Here, we
demonstrate that in vivo downregulation of SynIII expression affects neuronal
migration and orientation. By contrast, SynIII overexpression affects neuronal
migration, but not orientation. We identify a cyclin-dependent kinase-5 (CDK5)
phosphorylation site on SynIII and use phosphomutant rescue experiments to
demonstrate its role in SynIII function. Finally, we show that SynIII
phosphorylation at the CDK5 site is induced by activation of the semaphorin-3A
(Sema3A) pathway, which is implicated in migration and orientation of cortical
pyramidal neurons (PNs) and is known to activate CDK5. Thus, fine-tuning
of SynIII expression and phosphorylation by CDK5 activation through Sema3A
activity is essential for proper neuronal migration and orientation. The main neuropathological features of Parkinson's disease are dopaminergic
nigrostriatal neuron degeneration, and intraneuronal and intraneuritic
proteinaceous inclusions named Lewy bodies and Lewy neurites, respectively,
which mainly contain α-synuclein (α-syn, also known as SNCA). The neuronal
phosphoprotein synapsin III (also known as SYN3), is a pivotal regulator of
dopamine neuron synaptic function. Here, we show that α-syn interacts with and
modulates synapsin III. The absence of α-syn causes a selective increase and
redistribution of synapsin III, and changes the organization of synaptic vesicle
pools in dopamine neurons. In α-syn-null mice, the alterations of synapsin III
induce an increased locomotor response to the stimulation of synapsin-dependent
dopamine overflow, despite this, these mice show decreased basal and
depolarization-dependent striatal dopamine release. Of note, synapsin III seems
to be involved in α-syn aggregation, which also coaxes its increase and
redistribution. Furthermore, synapsin III accumulates in the caudate and putamen
of individuals with Parkinson's disease. These findings support a reciprocal
modulatory interaction of α-syn and synapsin III in the regulation of dopamine
neuron synaptic function. Adverse life events can induce two kinds of memory with opposite valence,
dependent on timing: "negative" memories for stimuli preceding them and
"positive" memories for stimuli experienced at the moment of "relief." Such
punishment memory and relief memory are found in insects, rats, and man. For
example, fruit flies (Drosophila melanogaster) avoid an odor after odor-shock
training ("forward conditioning" of the odor), whereas after shock-odor training
("backward conditioning" of the odor) they approach it. Do these
timing-dependent associative processes share molecular determits? We focus on
the role of Synapsin, a conserved presynaptic phosphoprotein regulating the
balance between the reserve pool and the readily releasable pool of synaptic
vesicles. We find that a lack of Synapsin leaves task-relevant sensory and motor
faculties unaffected. In contrast, both punishment memory and relief memory
scores are reduced. These defects reflect a true lessening of associative memory
strength, as distortions in nonassociative processing (e.g., susceptibility to
handling, adaptation, habituation, sensitization), discrimination ability, and
changes in the time course of coincidence detection can be ruled out as
alternative explanations. Reductions in punishment- and relief-memory strength
are also observed upon an RNAi-mediated knock-down of Synapsin, and are rescued
both by acutely restoring Synapsin and by locally restoring it in the mushroom
bodies of mutant flies. Thus, both punishment memory and relief memory require
the Synapsin protein and in this sense share genetic and molecular determits.
We note that corresponding molecular commonalities between punishment memory and
relief memory in humans would constrain pharmacological attempts to selectively
interfere with excessive associative punishment memories, e.g., after traumatic
experiences. Synapsin III (SynIII) is a neuron-specific phosphoprotein that plays a unique
role in neuronal development. SynIII is phosphorylated by cAMP-dependent protein
kinase (PKA) at a highly conserved phosphorylation site and by cyclin-dependent
kinase-5 (Cdk5) at a newly described site. Although SynIII is known to be
involved in axon elongation in vitro, the role of its phosphorylation by PKA and
Cdk5 in the modulation of this process is unknown. We expressed either wild-type
(WT) or phosphorylation-site mutants of SynIII in primary SynIII knock-out (KO)
mouse neurons at early stages of in vitro development. Whereas the neurite
elongation phenotype of SynIII KO neurons was fully rescued by the expression of
WT SynIII, the expression of nonphosphorylatable and pseudo-phosphorylated PKA
mutants was ineffective. Also, the nonphosphorylatable Cdk5 mutant was unable to
rescue the neurite elongation phenotype of SynIII KO neurons. By contrast, the
pseudo-phosphorylated mutant rescued the delay in neuronal maturation and axonal
elongation, revealing a Cdk5-dependent regulation of SynIII function.
Interestingly, SynIII KO neurons also exhibited decreased survival that was
fully rescued by the expression of WT SynIII, but not by its phosphorylation
mutants, and was associated with increased activated caspase3 and altered
tropomyosin receptor kinase B isoform expression. These results indicate that
PKA and Cdk5 phosphorylation is required for the physiological action of SynIII
on axon specification and neurite outgrowth and that the expression of a
functional SynIII is crucial for cell survival. Significance statement: Synapsin
III is an atypical member of the synapsin family of synaptic vesicle-associated
phosphoproteins that is precociously expressed in neurons and is downregulated
afterward. Although experimental evidence suggests a specific role for Synapsin
III in neuronal development, the molecular mechanisms are still largely unknown.
We found that Synapsin III plays a central role in early stages of neuronal
development involving neuronal survival, polarization, and neuritic growth and
that these effects are dependent on phosphorylation by cAMP-dependent protein
kinase and cyclin-dependent protein kinase-5. These results explain the recently
described neurodevelopmental defects in the migration and orientation of
Synapsin III-depleted cortical neurons and support the potential association of
Synapsin III with neurodevelopmental disorders such as schizophrenia. Synapsin II is a member of the neuronal phosphoprotein family. These
phosphoproteins are evolutionarily conserved across many organisms and are
important in a variety of synaptic functions, including synaptogenesis and the
regulation of neurotransmitter release. A number of genome-wide scans,
meta-analyses, and genetic susceptibility studies have implicated the synapsin
II gene (3p25) in the etiology of schizophrenia (SZ) and other psychiatric
disorders. Further studies have found a reduction of synapsin II mRNA and
protein in the prefrontal cortex in post-mortem samples from schizophrenic
patients. Disruptions in the expression of this gene may cause synaptic
dysfunction, which can result in neurotransmitter imbalances, likely
contributing to the pathogenesis of SZ. SZ is a costly, debilitating psychiatric
illness affecting approximately 1.1% of the world's population, amounting to 51
million people today. The disorder is characterized by positive (hallucinations,
paranoia), negative (social withdrawal, lack of motivation), and cognitive
(memory impairments, attention deficits) symptoms. This review provides a
comprehensive summary of the structure, function, and involvement of the
synapsin family, specifically synapsin II, in the pathophysiology of SZ and
possible target for therapeutic intervention/implications. Many endocytic proteins accumulate in the reserve pool of synaptic vesicles
(SVs) in synapses and relocalize to the endocytic periactive zone during
neurotransmitter release. Currently little is known about their functions
outside the periactive zone. Here we show that in the Drosophila neuromuscular
junction (NMJ), the endocytic scaffolding protein Dap160 colocalizes during the
SV cycle and forms a functional complex with the SV-associated phosphoprotein
synapsin, previously implicated in SV clustering. This direct interaction is
strongly enhanced under phosphorylation-promoting conditions and is essential
for proper localization of synapsin at NMJs. In a dap160 rescue mutant lacking
the interaction between Dap160 and synapsin, perturbed reclustering of SVs
during synaptic activity is observed. Our data indicate that in addition to the
function in endocytosis, Dap160 is a component of a network of protein-protein
interactions that serves for clustering of SVs in conjunction with synapsin.
During the SV cycle, Dap160 interacts with synapsin dispersed from SVs and helps
direct synapsin back to vesicles. The proteins function in synergy to achieve
efficient clustering of SVs in the reserve pool.
SIGNIFICANCE STATEMENT: We provide the first evidence for the function of the
SH3 domain interaction in synaptic vesicle (SV) organization at the synaptic
active zone. Using Drosophila neuromuscular junction as a model synapse, we
describe the molecular mechanism that enables the protein implicated in SV
clustering, synapsin, to return to the pool of vesicles during neurotransmitter
release. We also identify the endocytic scaffolding complex that includes Dap160
as a regulator of the events linking exocytosis and endocytosis in synapses. Synapsin is an evolutionarily conserved presynaptic phosphoprotein. It is
encoded by only one gene in the Drosophila genome and is expressed throughout
the nervous system. It regulates the balance between reserve and releasable
vesicles, is required to maintain transmission upon heavy demand, and is
essential for proper memory function at the behavioral level. Task-relevant
sensorimotor functions, however, remain intact in the absence of Synapsin. Using
an odor-sugar reward associative learning paradigm in larval Drosophila, we show
that memory scores in mutants lacking Synapsin (syn(97)) are lower than in
wild-type animals only when more salient, higher concentrations of odor or of
the sugar reward are used. Furthermore, we show that Synapsin is selectively
required for larval short-term memory. Thus, without Synapsin Drosophila larvae
can learn and remember, but Synapsin is required to form memories that match in
strength to event salience-in particular to a high saliency of odors, of
rewards, or the salient recency of an event. We further show that the residual
memory scores upon a lack of Synapsin are not further decreased by an additional
lack of the Sap47 protein. In combination with mass spectrometry data showing an
up-regulated phosphorylation of Synapsin in the larval nervous system upon a
lack of Sap47, this is suggestive of a functional interdependence of Synapsin
and Sap47. |
Is Thalidomide currently a marketed drug? | Several mechanisms for the teratogenic action of thalidomide are currently under review, but this mode of action of the drug still remains unclear and we review evidence-based hypotheses for the teratogenicity of thalidomide. Thalidomide is considered the drug of choice for the treatment of ENL, but for other conditions, it is recommended only when resistance to the currently available form of therapy is encountered. THE USE OF A DRUG WITH A TEMPORARY MARKETING AUTHORISATION: Thalidomide is currently available in France for nominative or cohort use with a temporary marketing authorisation (TMA). Examples of the basis for such regulation are drawn from historical situations (thalidomide, benoxaprofen) as well as currently marketed drugs (arylpropionic acids, disopyramide, indacrinone). In 1998 the US Food and Drug Administration approved thalidomide exclusively for the treatment of ENL, and strict conditions were stipulated for its use in order to prevent teratogenic adverse effects. The US Food and Drug Administration (FDA) has approved Thalomid (thalidomide) capsules for the acute treatment of the cutaneous manifestations of moderate to severe ENL. The revival of thalidomide began shortly after the drug was withdrawn from the market because of its teratogenic properties. | Thalidomide is now available as an investigational drug in the USA. A synthetic
derivative of glutamic acid, it was marketed in Europe in 1957 as a sedative but
withdrawn four years later after being associated with severe human
teratogenicity (PF D'Arcy and JP Griffin, Adverse Drug React Toxicol Rev, 13:65,
1994). The drug has since been found effective for several different
indications. The Titanic has become a metaphor for the disastrous consequences of an
unqualified belief in the safety and invincibility of new technology. Similarly,
the thalidomide tragedy stands for all of the "monsters" that can be
inadvertently or negligently created by modern medicine. Thalidomide, once
banned, has returned to the center of controversy with the Food and Drug
Administration's (FDA's) announcement that thalidomide will be placed on the
market for the treatment of erythema nodosum leprosum, a severe dermatological
complication of Hansen's disease. Although this indication is very restricted,
thalidomide will be available for off-label uses once it is on the market. New
laws regarding abortion and a new technology, ultrasound, make reasonable the
approval of thalidomide for patients who suffer from serious conditions it can
alleviate. In addition, the FDA and the manufacturer have proposed the most
stringent postmarketing monitoring ever used for a prescription drug, including
counseling, contraception, and ultrasonography in the event of pregcy. The
Titanic/thalidomide lesson for the FDA and public health is that rules and
guidelines alone are not sufficient to guarantee safety. Continuous vigilance
will be required to ensure that all reasonable postmarketing monitoring steps
are actually taken to avoid predictable and preventable teratogenic disasters. The various physician, patient, and pharmacy requirements for participation in
the System for Thalidomide Education and Prescribing Safety (S.T.E.P.S.) program
and procedures that institutions may implement in order to comply with these
requirements are described. In 1998, FDA approved the marketing of thalidomide
(Thalomid, Celgene). Because of the drug's known teratogenic effects, FDA
tightly controls the distribution of thalidomide in the United States. To comply
with FDA requirements, Celgene developed the S.T.E.P.S. oversight program, which
includes registration of thalidomide prescribers and pharmacies that dispense
thalidomide, extensive patient education about the risks associated with
thalidomide, and a registry of all patients receiving thalidomide. The
S.T.E.P.S. program is considered part of the product label. The pharmacy
requirements of the program were developed with a focus on a retail pharmacy
practice model, which does not adequately reflect current hospital practice. The
pharmacy department of the National Institutes of Health Clinical Center
developed a model that adapts the S.T.E.P.S. program requirements to inpatient
and outpatient institutional pharmacy practice. Procedures for registering
patients and prescribers and dispensing thalidomide in the hospital setting were
developed; the procedures were designed to meet the needs of both the inpatient
and outpatient pharmacies and to comply with the requirements of the S.T.E.P.S.
program. BACKGROUND: The use of thalidomide during the 1950s resulted in teratogenic
effects in thousands of infants. Although thalidomide is currently approved for
the treatment of a complication of leprosy, it is commercially available to
treat other diseases through a controlled distribution system. This article
presents a summary of a scientific conference organized to assess clinical
research on thalidomide, its new clinical applications, and the social and
ethical implications for its use.
METHODS: Summaries of 10 presentations and two panel discussions were developed
from the authors's, oral presentations, conference slides, responses to
questions, and supporting literature.
RESULTS: Thalidomide shows promise in treating several diseases, including
HIV/AIDS, rheumatoid arthritis, Crohn's disease, and multiple myeloma. The
STEPStrade mark (System for Thalidomide Education and Prescribing Safety)
Program has been developed by Celgene, the commercial manufacturer of
thalidomide, to ensure compliance with prescription and usage protocols. A
surveillance system is also in place to monitor and report compliance patterns.
CONCLUSIONS: Despite the tragic past associated with thalidomide, the drug shows
promise as a treatment for many clinical disorders. The challenge is to answer
lingering questions of risks and benefits through clinical trials and discovery,
to monitor participation and compliance with protocols developed to avoid use of
the drug during pregcy, and to continue to search for safer and more
effective treatment options. Thalidomide was synthesized in 1954 in erstwhile West Germany and marketed as a
sedative in over 46 countries until the early 1960s. Owing to serious
teratogenic effects, the drug was withdrawn from the market in 1961. A chance
observation suggested the utility of thalidomide in erythema nodosum leprosum
(ENL). After many controlled and uncontrolled trials were published, the World
Health Organization recommended its use in ENL. The Food and Drug
Administration, USA approved it for use in ENL in July 1998. Only established
and well-defined studies conducted to substantiate the efficacy of thalidomide
have been included in this review. Thalidomide is considered the drug of choice
for the treatment of ENL, but for other conditions, it is recommended only when
resistance to the currently available form of therapy is encountered. Once the
anti-inflammatory, immuno-modulatory, anti-TNF-alpha and anti-angiogenic
properties of thalidomide were discovered, it was also tried in AIDS and related
wasting, apthous ulcers, microsporidiosis and Kaposi's sarcoma. Thalidomide has
no clinical place as an immunosuppressant in solid organ transplantation.
However, it has a therapeutic role in graft-verus-host-disease. Among the
dermatological conditions, thalidomide has been found to be effective in
systemic lupus erythematosus, discoid lupus erythematosus, actinic prurigo and
prurigo nodularis. Used correctly, it is a safe and effective medicine (except
for its teratogenic potential and delayed neuropathy) in a variety of disease
conditions. The revival of thalidomide began shortly after the drug was withdrawn from the
market because of its teratogenic properties. Therapeutic effects of thalidomide
were found accidentally in leprosy patients with erythema nodosum leprosum
(ENL). Subsequent research widened the understanding of the activity of
thalidomide, and with improved methodology and the augmented background
knowledge of immunology it was possible to interpret the properties of
thalidomide more coherently. Effects on tumour necrosis factor-alpha (TNFalpha)
release play an important role in the ability of thalidomide to affect the
immune system. Alteration of synthesis and release of cytokines such as
interleukin (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 and interferon-gamma is
involved in the complex mechanisms of thalidomide. Thalidomide targets
leucocytes, endothelial cells and keratinocytes, affecting them in a different
manner and at different cellular levels. Changes in the density of adhesion
molecules alter leucocyte extravasation and the inflammatory response in the
tissue involved. Several mechanisms for the teratogenic action of thalidomide
are currently under review, but this mode of action of the drug still remains
unclear and we review evidence-based hypotheses for the teratogenicity of
thalidomide. Thalidomide shows significant clinical impact in several diseases
such as ENL in lepromatous leprosy, chronic graft-versus-host disease, systemic
lupus erythematosus, sarcoidosis, aphthous lesions in HIV infection, wasting
syndrome in chronic illness, inflammatory bowel disease, multiple myeloma and
some solid tumours. In 1998 the US Food and Drug Administration approved
thalidomide exclusively for the treatment of ENL, and strict conditions were
stipulated for its use in order to prevent teratogenic adverse effects. However,
despite the promising findings of thalidomide at the molecular level, namely its
anti-TNFalpha properties and its intercalation with DNA, and activity in
clinical trials, there is still a great need for more intensive research. BACKGROUND: Thalidomide is an anti-inflammatory and anti-angiogenic drug
currently used for the treatment of several diseases, including erythema nodosum
leprosum, which occurs in patients with lepromatous leprosy. In this research,
we use DNA microarray analysis to identify the impact of thalidomide on gene
expression responses in human cells after lipopolysaccharide (LPS) stimulation.
We employed a two-stage framework. Initially, we identified 1584 altered genes
in response to LPS. Modulation of this set of genes was then analyzed in the LPS
stimulated cells treated with thalidomide.
RESULTS: We identified 64 genes with altered expression induced by thalidomide
using the rank product method. In addition, the lists of up-regulated and
down-regulated genes were investigated by means of bioinformatics functional
analysis, which allowed for the identification of biological processes affected
by thalidomide. Confirmatory analysis was done in five of the identified genes
using real time PCR.
CONCLUSIONS: The results showed some genes that can further our understanding of
the biological mechanisms in the action of thalidomide. Of the five genes
evaluated with real time PCR, three were down regulated and two were up
regulated confirming the initial results of the microarray analysis. BACKGROUND: Thalidomide could relieve clinical symptoms and intestinal mucosal
lesions effectively in children with refractory inflammatory bowel disease (IBD)
from the pre-clinical study. This study aimed to observe the therapeutic effect
of thalidomide by the established animal model of IBD model of
2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in Sprague-Dawley
(SD) rats and to investigate the possible mechanism of action.
METHODS: A total of 82 SD rats of about 4-5 weeks were randomly divided into
three groups: the control group (25 rats), TNBS-treated group (29 rats), and
thalidomide treatment group (28 rats). Daily activities were recorded. At least
eight rats from each group were killed on the 4th, 7th, and 14th days.
Morphological and histological changes in the colon were individually assessed.
Serum was collected and the levels of TNF-α and interleukins (IL-1β and IL-10)
were assayed by ELISA method. The expression of colonic mucosal nuclear factor
(NF)-κB was assayed with the immunohistochemical method.
RESULTS: (1) In the control group, diarrhea and rectal bleeding recovered
rapidly and no death was recorded. In the TNBS-treated group, diarrhea and
rectal bleeding persisted for a longer time. The mortality rate was 10.34%
during the observation period. In the thalidomide treatment group, diarrhea and
rectal bleeding persisted for a significantly shorter time than the TNBS-treated
group (P < 0.01). The rats of this group also exhibited faster weight gain on
day 7 compared with the TNBS-treated group but still lower than that of the
control group. The mortality rate of the thalidomide treatment group was 3.57%.
(2) Macroscopic and microscopic scores of the thalidomide-treated group were
significantly lower than those of the TNBS model group on the 14th day (P <
0.01). These results suggested faster and better colonic recovery in the
thalidomide-treated group. (3) NF-κB expression in the colonic mucosa of the
control group was lower than in the others, mainly distributed in the cytoplasm.
A large amount of intra-nuclear and cytoplasm staining was observed (more
prominently intra-nuclear) in the TNBS model group and the thalidomide treatment
group. On the 7th and 14th days, intra-nuclear NF-κB-containing cells in the
thalidomide treatment group were still significantly lower than those in the
TNBS model group (P < 0.01). (4) In the control group, the cellular inflammatory
factors (TNF-α, IL-1β, and IL-10) were expressed at a low level while in the
other two groups they were already expressed at a significantly higher level on
day 4. On day 7 the expressions of TNF-α and IL-1β in the thalidomide treatment
group were lower than in the TNBS model group. On day 14, the expressions of
TNF-α and IL-1β in the thalidomide treatment group were significantly lower than
in the TNBS model group (P < 0.05). On day 4, the IL-10 levels of the
thalidomide treatment group became significantly elevated. The levels gradually
decreased but still remained at a higher level. In the TNBS model group, the
IL-10 expression peaked later than in the thalidomide treatment group.
CONCLUSIONS: Thalidomide was effective in the management of TNBS-induced colitis
in young rats. This may be due to the suppression and down-regulation of NF-κB
and the expression of the downstream inflammatory mediators (TNF-α and IL-1β).
There is also indication that the expression of the anti-inflammatory cytokine
(IL-10) is concomitantly up-regulated as well. INTRODUCTION: Polyneuropathy, organomegaly, endocrinopathy, M-protein and skin
changes (POEMS) syndrome is a fatal systemic disorder associated with plasma
cell dyscrasia and the overproduction of the vascular endothelial growth factor
(VEGF). Recently, the prognosis of POEMS was substantially improved by
introduction of therapeutic intervention for myeloma. However, no randomised
clinical trial has been performed because of the rarity and severity of the
disease.
METHODS AND ANALYSIS: The Japanese POEMS syndrome with Thalidomide (J-POST)
Trial is a phase II/III multicentre, double-blinded, randomised, controlled
trial that aims to evaluate the efficacy and safety of a 24-week treatment with
thalidomide in POEMS syndrome, with an additional 48-week open-label safety
study. Adults with POEMS syndrome who have no indication for transplantation are
assessed for eligibility at 12 tertiary neurology centres in Japan. Patients who
satisfy the eligibility criteria are randomised (1:1) to receive thalidomide
(100-300 mg daily) plus dexamethasone (12 mg/m(2) on days 1-4 of a 28-day cycle)
or placebo plus dexamethasone. Both treatments were administered for 24 weeks
(six cycles; randomised comparative study period). Patients who complete the
randomised study period or show subacute deterioration during the randomised
period participate in the subsequent 48-week open-label safety study (long-term
safety period). The primary end point of the study is the reduction rate of
serum VEGF levels at 24 weeks.
ETHICS AND DISSEMINATION: The protocol was approved by the Institutional Review
Board of each hospital. The trial was notified and registered at the
Pharmaceutical and Medical Devices Agency, Japan (No. 22-1716). The J-POST Trial
is currently ongoing and is due to finish in August 2015. The findings of this
trial will be disseminated through peer-reviewed publications and conference
presentations and will also be disseminated to participants.
TRIAL REGISTRATION NUMBER: UMIN000004179 and JMA-IIA00046. OBJECTIVE: To examine direct cost to patients associated with oral oncolytics
for the management of multiple myeloma (MM) both before and after ficial
assistance, and assess the effect on adherence.
METHODS: In this retrospective study, pharmacy claims were analyzed for those
patients with a diagnosis of MM who received thalidomide, lenalidomide, or
pomalidomide from a large specialty pharmacy in the US between January 1, 2011,
and December 31, 2013. Average direct cost to patients, per prescription, was
analyzed both before and after ficial assistance. Adherence was assessed
through an analysis of medication possession ratio (MPR) for those patients who
filled a prescription ≥2 times throughout the 3-year time period.
RESULTS: A total of 77,821 prescriptions for thalidomide, lenalidomide, and
pomalidomide were filled by 6731 unique patients between January 1, 2011, and,
December 31, 2013. The average direct cost to patients, per prescription, for
any of these three agents was $227.23 prior to ficial assistance and $80.11
after ficial assistance, representing an average patient savings of $147.14
per prescription. Prior to ficial assistance, the average direct cost to
patients was ≤$50 for 57.6% of all prescriptions. After ficial assistance,
86.2% of patients had a direct cost of ≤$50 per prescription. Adherence, as
assessed by MPR, did not vary significantly based on direct cost to the patient.
LIMITATIONS: This study included patients receiving therapy from a single
specialty pharmacy for a single indication. There may be patients included in
the analysis who received prescriptions from other pharmacies prior to or after
the prescriptions available for analysis. Most of the prescriptions included in
the analysis were for lenalidomide.
CONCLUSIONS: This retrospective study demonstrated that the specialty pharmacy
helped patients significantly reduce their direct cost expenditures by securing
funding and co-pay assistance. |
Which enzymes synthesize catecholamines in adrenal glands? | The enzymes that synthesize catecholamines in adrenal glands are:
1) Tyrosine Hydroxylase (TH)
2) Aromatic L-amino acid decarboxylase (AAAD)
3) Dopamine β-hydroxylase (DBH)
4) Phenylethanolamine N-methyltransferase (PNMT) | Male Wistar specific-pathogen-free rats aged 2, 7, 17, 30, 60, 120, 200, 360 and
600 days, all killed in experiment on the same day, were examined. The body
weight significantly increased until the 200th day, the weight of adrenals until
the 120th day and the adrenal protein content until the 30th day of life. The
adrenaline content of the adrenals increased continuously during the 600 days
studied. Adrenal noradrenaline content increased rapidly over the first 17 days,
remained at a stable level until the 120th day, and rose to a higher level after
200 days. The activity of adrenal catecholamine-synthesizing enzymes also
increased with age: tyrosine hydroxylase gradually increased until the 360th
day, dopamine-beta-hydroxylase and phenylethanolamine-N-methyl transferase until
the 200th day. Our results demonstrate that, in the rat, during development
there is a gradual increase of adrenal weight, adrenaline content, tyrosine
hydroxylase and phenylethanolamine-N-methyl transferase activity until
maturation (120th day), whereas the adrenal noradrenaline content reaches the
adult values earlier, around the 17th day. During aging, adrenal catecholamines
significantly increase when compared to young-adult rats (120-day-old), probably
due to the elevated activity of the adrenal catecholamine-synthesizing enzymes.
The increased adrenal catecholamine levels in old animals might be connected
with a higher incidence of cardiovascular diseases in aged. Tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT)
activities were assayed in adrenal glands of the following groups of the Alaskan
red-backed vole (Clethrionomys rutilus dawsoni): 1) laboratory reared at 20
degrees C and 2) exposed to 5 degrees C for 1, 3, 7, and 28 days; 3) wild,
summer acclimatized; 4) wild, fall acclimatized; and 5) wild, winter
acclimatized. TH activity in laboratory-acclimated voles exposed to 5 degrees C
was increased by 2 times after 3 days and remained elevated after 28 days. PNMT
activity in these same voles was increased after 7 days and also remained
elevated after 28 days of cold exposure. In wild-acclimatized voles TH activity
and PNMT activity in summer were equivalent to levels in 28-day cold-acclimated
laboratory voles. In fall, TH activity was increased to 2.5 times the summer
value. It decreased by midwinter, but remained elevated above the summer level.
In contrast, PNMT activity appeared unchanged from summer through fall and
winter. Pregt summer voles had markedly increased TH activity. Adrenal
norepinephrine and epinephrine did not change significantly with cold
acclimation or seasonal acclimatization. Thus, acclimatization of wild voles to
fall and winter conditions involved aquisition of a greater capacity to
synthesize adrenal catecholamines than that produced by exposing
laboratory-reared voles to an extended period of cold. Rats on calcium-deficient diets developed hypocalcemia, hyperparathyroidism and
hypertension and showed an increase in plasma catecholamines. Adrenal gland
catecholamines were decreased while tyrosine hydroxylase (TH) and dopamine
beta-hydroxylase (DBH) were found to be increased, as compared to controls. In
contrast, no significant differences were found between controls and
parathyroidectomized rats in plasma catecholamines, and catecholamines, TH and
DBH of the adrenal gland. These findings seem to indicate that the genesis of
hypertension in rats on a low calcium diet is secondary to hyperparathyroidism
caused by a low calcium diet. Furthermore, some relation between catecholamines
and parathyroid hormone seems to exist in the regulation of blood pressure in
rats. Adrenal medullary cells in adult primates contain catecholamines and several
neuropeptides. Among these peptides are several products of the three opiate
precursor proteins: proenkephalin, prodynorphin, and proopiomelanocortin. We
used immunocytochemistry to study the ontogeny of leu-enkephalin and the
catecholamine-synthesizing enzymes dopamine beta-hydroxylase and
phenylethanolamine N-methyltransferase in adjacent sections of 14 fetal rhesus
and 31 fetal human adrenal glands. The adrenal medulla of a 24-week-old human
fetus as well as medullas of 11 134- to 172-day-old rhesus fetuses were
immunopositive with all 3 antisera employed. Furthermore, in thin serial
sections of these glands, dopamine beta-hydroxylase, phenylethanolamine
N-methyltransferase, and leu-enkephalin appeared to be colocalized in the same
cells of the adrenal medulla. Twenty-six adrenals from fetuses 15-26 weeks
stained lightly with one or more of the antisera. Dopamine beta-hydroxylase
could be detected at 15 weeks, followed by leu-enkephalin and phenylethanolamine
N-methyltransferase at 18-19 weeks. The role of the enkephalins during fetal
life or in the adaptation to extrauterine life is not yet clear. In adults,
enkephalins are cosecreted with catecholamines in response to stress. Our
results suggest that the fetal adrenal may be capable of cosecretion of
catecholamines and enkephalins, at least by the end of the second trimester of
gestation. 1. We sought to determine if catecholamine biosynthetic enzymes of spontaneously
hypertensive rats (SHR) differed from those of normotensive Wistar--Kyoto (WKY)
and Sprague--Dawley (SD) control rats before birth. 2. By immunocytochemical and
biochemical methods we compared strains for the time of appearance and
maturation of the enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase
(DBH) and phenylethanolamine-N-methyltransferase (PNMT) in sympathetic ganglia
and adrenals. 3. The time of appearance of enzymes was identical in all three
strains: TH and DBH first appeared in sympathetic ganglia on embryonic day 11
(E11) and in adrenal medulla on E16. PNMT, restricted to adrenal medulla,
appeared later on E18. 4. The activity of adrenal TH prenatally on E18 and E21
and at day of birth (P1) in SHR was approximately two fold that in WKY or SD
rats. In contrast PNMT was lower in SHR but only on E18. 5. Thus, although the
timing of the first expression of adrenergic phenotypes is similar in SHR and
normotensive controls, the differences in TH activity in adrenals suggest an
enhanced biosynthetic capacity for catecholamines in this strain before birth.
6. We conclude that SHR differ from normotensive rats from the first expression
of some of the genes controlling catecholamine biosynthesis. Pheochromocytomas synthesize and release catecholamines, which subsequently are
related to various clinical manifestations of the disease. However,
pheochromocytomas are not innervated and the catecholamine release and synthesis
are not initiated by neural impulses. It is still unknown how catecholamine
synthesis is regulated in pheochromocytomas. As a first step toward
understanding the molecular mechanisms by which catecholamine synthesis is
controlled in the tumor, we measured the levels of mRNA coding for the
catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) and catecholamines
in 6 pheochromocytomas and 2 normal adrenal glands. The TH mRNA level was
overexpressed and the catecholamine contents were high in 4 out of 6
pheochromocytomas. There was a close correlation between the TH mRNA level and
the catecholamines content in the tumors. We also examined the gene expression
of the messengers of other catecholamine synthesizing enzymes, dopamine
beta-hydroxylase (DBH) and aromatic 1-amino acid decarboxylase (AADC) in
pheochromocytomas. The expression of these genes was in parallel with that of TH
mRNA in the tumors. These findings indicate that catecholamine overproduction in
pheochromocytomas is mediated by the overexpression of genes coding for
catecholamines synthesizing enzymes, TH, DBH, and AADC. The expression of catecholamine-synthesizing enzymes in the adrenal medulla is
upregulated in parallel by stress and pharmacological treatments. In this study
we examined whether a neuropeptide and its processing enzyme are regulated in
parallel with catecholamine enzyme genes after drug treatment. Because the main
effect of stress on the adrenal medulla is via splanchnic nerve stimulation of
nicotinic receptors, we used nicotine to stimulate the medulla and visualized
expression of catecholamine enzyme genes, the medullary peptide neuropeptide Y
(NPY), and the neuropeptide-processing enzyme peptidylglycine alpha-amidating
monooxygenase (PAM) by in situ hybridization quantified by image analysis of
autoradiographic images. Rats received a single injection of nicotine (0, 1, or
5 mg/kg sc). Six hours later, rats were transcardially perfused. Free-floating
adrenal gland sections were hybridized with 35S-labeled cDNA probes for tyrosine
hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine
N-methyltransferase (PNMT), PAM, and NPY. Nicotine treatment upregulated the
expression of TH, PNMT, and NPY genes in a dose-dependent fashion. Small but
nonsignificant increases were observed in DBH and PAM mRNA levels. These results
suggest that common transcriptional activation mechanisms may upregulate both
catecholamine and neuropeptide synthesis in the adrenal medulla after nicotinic
stimulation. OBJECTIVE: To understand the molecular mechanisms by which catecholamine
synthesis is controlled in pheochromocytomas--tumors that synthesize and release
catecholamines, which are related to various clinical manifestations of the
condition.
METHODS: We measured the concentrations of mRNA coding for the
catecholamine-synthesizing enzymes tyrosine hydroxylase, aromatic L-amino acid
decarboxylase (AADC), dopamine beta-hydroxylase (DBH) and phenylethanolamine
N-methyl transferase (PNMT) and for the catecholamine contents in 12
pheochromocytomas and 12 normal adrenal medullas.
RESULTS: The mean content of total catecholamine and the beta-actin mRNA
expression in the pheochromocytomas were almost the same as those in the normal
adrenal medullas. However, the tyrosine hydroxylase, AADC and DBH mRNA
concentrations in the pheochromocytomas were greater than those of the normal
adrenal medullas. Conversely, the PNMT mRNA concentration in the
pheochromocytomas was lower than that in the normal adrenal medullas. These
differences are responsible for the difference in the proportions of
catecholamines between pheochromocytomas and normal adrenal medullas. The
constitutive expression of the catecholamine-synthesizing enzyme mRNAs varied in
magnitude among the pheochromocytomas, and the tyrosine hydroxylase mRNA
expressions correlated with the contents of total catecholamine in the tumors
(r=0.964, P<0.0001).
CONCLUSIONS: These findings indicate that catecholamine production in
pheochromocytomas is primarily controlled by the level of gene expression. The present study showed the acetylcholinesterase (AChE) activity, and
neurofilament protein (NFP), catecholamine-synthesizing enzymes, dopamine
beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT)
immunoreactivities in the mouse adrenal gland during postnatal development. From
birth to postnatal-1-day, AChE activity was weakly and diffusely found in some
medullary cells and in very few nerve fibers whereas strong NFP immunoreactivity
was seen in a few ganglion cells and in remarkably numerous nerve fibers in the
medulla. Almost all meduallary cells were reactive for both DBH and PNMT during
this period. From postnatal-2- or -3-day to postnatal-1-week, strong AChE
activity was observed in a few large ganglion cells, but the reaction was weak
in clusters of chromaffin cells, and the number of strong AChE-active nerve
fibers in the medulla was rapidly increased. From postnatal-2-day onwards, the
number of NFP-immunoreactive nerve fibers in the medulla were remarkably
numerous. Numerous chromaffin cells were reactive for both DBH and PNMT whereas
some chromaffin cells were reactive for only DBH from postnatal-2-day onwards.
These results suggest that drastic changes such as an increase of acetylcholine
in the nerve fibers, differentiation of noradrenaline and adrenaline cells of
the medulla may occur during this period. From postnatal-2-week to
postnatal-3-week, weak AChE activity was seen in the clusters of several
chromaffin cells and a few ganglion cells, and the number of AChE-active nerve
fibers in the medulla was gradually increased. From postnatal-4-week to
postnatal-8-week (adult), the distribution and frequency of AChE activity in the
adrenal gland were similar to those at postnatal-3-week. In the adult, AChE
activity was weakly seen in the clusters of several chromaffin cells showing
noradrenaline fluorescence in the adrenal medulla. The noradrenaline cells were
contacted by denser AChE-reactive nerve fibers than adrenaline cells. These
results suggest that the development of cholinergic nervous system in the mouse
adrenal medulla may be completed by postnatal-3-week. The mechanisms underlying the onset of obesity are complex and not completely
understood. An imbalance of autonomic nervous system has been proposed to be a
major cause of great fat deposits accumulation in hypothalamic obesity models.
In this work we therefore investigated the adrenal chromaffin cells in
monosodium glutamate (MSG)-treated obese female mice. Newborn mice were injected
daily with MSG (4 mg/g body weight) or saline (controls) during the first five
days of life and studied at 90 days of age. The adrenal catecholamine content
was 56.0% lower in the obese group when compared to lean controls (P < 0.0001).
Using isolated adrenal medulla we observed no difference in basal catecholamine
secretion percentile between obese and lean animals. However, the percentile of
catecholamine secretion stimulated by high K+ concentration was lower in the
obese group. There was a decrease in the tyrosine hydroxylase enzyme expression
(57.3%, P < 0.004) in adrenal glands of obese mice. Interestingly, the
expression of dopamine beta-hydroxylase was also reduced (47.0%, P < 0.005).
Phenylethanolamine N-methyltransferase expression was not affected. Our results
show that in the MSG model, obesity status is associated with a defective
adrenal chromaffin cell function. We conclude that in MSG obesity the low total
catecholamine content is directly related to a decrease of key
catecholamine-synthesizing enzymes, which by its turn may lead to a defective
catecholamine secretion. The hypothalamic suprachiasmatic nuclei (SCN) comprise the main site in the
brain involved in the control of the homeostatic mechanism which respond to
environmental daily light changes. The sympathetic nervous system and
hypothalamic releasing or inhibiting factors mediate the SCN control of a number
of peripheral organs and tissues. In this work we analyzed the involvement of
two environmental light conditions, constant light (LL) and constant dark (DD)
for 20 days, on the expression of mRNAs for catecholamines biosynthetic enzymes
and neuropeptide Y (NPY) genes in rat superior cervical ganglia (SCG) and
adrenal gland. The results of Northern blot analysis show that LL exposure
reduces mRNA levels for tyrosine hydroxylase (TH) the rate limiting
catecholamine biosynthetic enzyme and also of dopamine beta-hydroxylase (DBH) as
well as for NPY in SCG to about half the levels in control animals. In contrast,
exposure of the rats to DD did not elicit any change in the SCG. In the adrenal
gland, both, LL and DD conditions increased the TH, DBH as well as
phenylethanolamine N-methyltransferase (PNMT) mRNA levels. Under the same
conditions, adrenal NPY mRNA levels were decreased by either LL or DD. The
results show, for the first time, that prolonged changes in environmental light
can alter the gene expression of catecholamine biosynthetic enzymes and of NPY.
There was differential response in SCG and adrenal gland. Social stressors, like other stressors, are powerful activators of the
sympathoadrenomedullary system. Differential housing (single vs. group) and
social defeat of rats is known to alter the activity of
catecholamine-synthesizing enzymes in the medulla. The present studies examined
the effect of 70 days of triad (3 rats per large cage) and individual housing of
male rats on adrenal mRNA levels of tyrosine hydroxylase (TH),
dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase
(PNMT) and on TH protein levels. Behavioral ratings carried out at the triad
formation indicated that domit rats exhibited mostly offensive aggressive
behaviors. By contrast, subordinate rats expressed primarily defensive
behaviors, while the subdomit rats displayed intermediate levels of these
behaviors. Overall, compared with single housing, triad housing resulted in
lower gene expression for TH, DBH and PNMT and lower TH protein in the adrenals.
Within triads, gene expression for these enzymes and TH protein concentration
were higher in subordinate compared with domit and subdomit rats. The
domit rats tended to have the lowest gene expression of these enzymes. These
data indicate that in rodents, individual housing and a subject's social rank
have a differential impact on the regulation of catecholamine biosynthesis
already during the process of gene expression of catecholamine biosynthetic
enzymes in the adrenals. The present study investigated the cellular localization of 3 catecholamine
biosynthetic enzymes, tyrosine hydroxylase (TH), dopamine beta-hydroxylase
(DBH), and phenylethanolamine N-methyltransferase (PNMT) to identify and analyze
the localization of norepinephrine (NE) and epinephrine (E) cells in the adrenal
gland in the chicken using peroxidase-antiperoxidase immunohistochemical
techniques. Tyrosine hydroxylase immunoreactivity (IR) was observed in almost
all adrenal medullary cells of the adult chicken. Dopamine beta-hydroxylase IR
coincided with that of TH. Many medullary cells also exhibited PNMT IR, but
PNMT-immunonegative cells were also observed. Tyrosine hydroxylase IR was
localized in the E- and NE-containing cells, but PNMT IR was localized only in
the E-containing cells. Approximately 69% of medullary cells were E-containing,
and the remaining were NE-containing cells. The ratio of E- and NE-containing
cells between the subcapsular and central zone was statistically significant (P
< 0.01). Although cortical cells of the adrenal gland did not show TH-, DBH-, or
PNMT-positive reactions, ganglia close to the adrenal gland showed TH, DBH, and
PNMT immunoreactivities. These findings indicated the cellular localization of 3
catecholamine-biosynthesizing enzymes in chicken adrenal medulla and suggest
that the majority of medullary cell are E-containing cells. The ratio of E cells
to NE cells varies among the 3 zones in the adrenal glands of the chicken. The effects of training are dependent on complex, adaptive changes which are
induced by acute physical exercise at different levels. In particular, evidence
shows that the hypothalamus-pituitary-adrenocortical axis, as well as the
sympatho-adrenomedullary system, is mainly involved in mediating the
physiological effects of physical exercise. The aim of the present study was to
investigate, through a morphological and biochemical approach, the effects of
training on the adrenal gland of mice, following two different protocols
consisting of either low- or high-intensity training. Mice were run daily on a
motorised treadmill for 8 weeks, at a velocity corresponding to 60%
(low-intensity exercise) or 90% (high-intensity exercise) of the maximal running
velocity previously determined by an incremental exercise test. We found that
physical exercise produced an increase in the adrenal gland size compared with
the control (sedentary) mice. The increase was 31.04% for mice that underwent
high-intensity exercise and 10.08% for mice that underwent low intensity
exercise, and this appeared to be the result of an increase in the area of both
the adrenal cortex and adrenal medulla. Morphological analysis of the adrenal
cortex showed that both types of exercise produced an increase in cytoplasmic
vacuoles in steroidogenic cells, appearing more abundant after high-intensity
exercise. No change was found in the reticulate zone. In the adrenal medulla,
despite the absence of morphological changes, immunohistochemistry for tyrosine
hydroxylase, dopamine β-hydroxylase and phenyl-ethanolamine-N-methyltransferase
demonstrated an increased immunopositivity for these cathecolamine-synthesizing
enzymes after intense exercise. These results were confirmed by immunoblot
accompanied by densitometric analysis. PURPOSE: The effect of an AA deficiency on catecholamine biosynthesis in adult
mice in vivo is unknown. Therefore, we quantified catecholamine and the
expression of catecholamine synthetic enzymes in the adrenal glands of
senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice
placed in an AA-deficient state.
METHODS: At 30 days of age, mice were divided into the following 4 groups: AA
(-) SMP30/GNL KO, AA (+) SMP30/GNL KO, AA (-) wild type (WT), and AA (+) WT. The
AA (+) groups were given water containing 1.5 g/L AA, whereas the AA (-) groups
received water without AA until the experiment ended. In addition, all mice were
fed an AA-depleted diet. Catecholamine levels were measured by a liquid
chromatographic method. Tyrosine hydroxylase, dopa decarboxylase, dopamine
β-hydroxylase, and phenylethanolamine N-methyltransferase mRNA expression levels
were measured with the quantitative real-time polymerase chain reaction (qPCR).
Tyrosine hydroxylase and dopamine β-hydroxylase protein levels were quantified
by Western blot analysis.
RESULTS: In the adrenals of AA (-) SMP30/GNL KO mice, noradrenaline and
adrenaline levels decreased significantly compared to other three groups of
mice, although there were no significant differences in dopamine β-hydroxylase
or phenylethanolamine N-methyltransferase mRNA content. Moreover, there was no
significant difference in their dopamine β-hydroxylase protein levels. On the
other hand, AA depletion did not affect dopamine levels in adrenal glands of
mice.
CONCLUSION: An AA deficiency decreases the noradrenaline and adrenaline levels
in adrenal glands of mice in vivo. Chronic isolation of adult animals represents a form of psychological stress
that produces sympatho-adrenomedullar activation. Exercise training acts as an
important modulator of sympatho-adrenomedullary system. This study aimed to
investigate physical exercise-related changes in gene expression of
catecholamine biosynthetic enzymes (tyrosine hydroxylase, dopamine-ß-hydroxylase
and phenylethanolamine N-methyltransferase) and cyclic adenosine monophosphate
response element-binding (CREB) in the adrenal medulla, concentrations of
catecholamines and corticosterone (CORT) in the plasma and the weight of adrenal
glands of chronically psychosocially stressed adult rats exposed daily to 20 min
treadmill running for 12 weeks. Also, we examined how additional acute
immobilization stress changes the mentioned parameters. Treadmill running did
not result in modulation of gene expression of catecholamine synthesizing
enzymes and it decreased the level of CREB mRNA in the adrenal medulla of
chronically psychosocially stressed adult rats. The potentially negative
physiological adaptations after treadmill running were recorded as increased
concentrations of catecholamines and decreased morning CORT concentration in the
plasma, as well as the adrenal gland hypertrophy of chronically psychosocially
stressed rats. The additional acute immobilization stress increases gene
expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well
as catecholamines and CORT levels in the plasma. Treadmill exercise does not
change the activity of sympatho-adrenomedullary system of chronically
psychosocially stressed rats. High levels of circulating catecholamines have been established as fundamental
pathophysiological elements of heart failure (HF). However, it is unclear
whether the increased gene expression of catecholamine-synthesis enzymes in the
adrenal glands contributes to these hormone abnormalities in large animal HF
models. We analyzed the mRNA levels of catecholamine-synthesizing enzymes:
tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AAAD),
dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT)
in adrenal glands of 18 pigs with chronic systolic non-ischaemic HF
(tachycardia-induced cardiomyopathy due to right ventricle pacing) and 6
sham-operated controls. Pigs with severe HF demonstrated an increased expression
of TH and DBH (but neither AAAD nor PNMT) as compared to animals with milder HF
and controls (P<0.05 in all cases). The increased adrenal mRNA expression of TH
and DBH was accompanied by a reduced left ventricle ejection fraction (LVEF)
(P<0.001) and an elevated plasma B-type natriuretic peptide (BNP) (P<0.01), the
other indices reflecting HF severity. There was a positive relationship between
the increased adrenal mRNA expression of TH and DBH, and the high levels of
circulating adrenaline and noradrenaline (all P<0.05). The association with
noradrenaline remained significant also when adjusted for LVEF and plasma BNP,
suggesting a significant contribution of adrenals to the circulating pool of
catecholamines in subjects with systolic HF. |
How many cysteines have alpha-defensins? | Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions. | Defensins are small, multifunctional cationic peptides. They typically contain
six conserved cysteines whose three intramolecular disulfides stabilize a
largely β-sheet structure. This review of human α-defensins begins by describing
their evolution, including their likely relationship to the Big Defensins of
invertebrates, and their kinship to the β-defensin peptides of many if not all
vertebrates, and the θ-defensins found in certain non-human primates. We provide
a short history of the search for leukocyte-derived microbicidal molecules,
emphasizing the roles played by luck (good), preconceived notions (mostly bad),
and proper timing (essential). The antimicrobial, antiviral, antitoxic, and
binding properties of human α-defensins are summarized. The structural features
of α-defensins are described extensively and their functional contributions are
assessed. The properties of HD6, an enigmatic Paneth cell α-defensin, are
contrasted with those of the four myeloid α-defensins (HNP1-4) and of HD5, the
other α-defensin of human Paneth cells. The review ends with a decalogue that
may assist researchers or students interested in α-defensins and related aspects
of neutrophil function. Antimicrobial peptides have been widely identified from amphibian skins except
salamanders. A novel antimicrobial peptide (CFBD) was isolated and characterized
from skin secretions of the salamander, Cynops fudingensis. The cDNA encoding
CFBD precursor was cloned from the skin cDNA library of C. fudingensis. The
precursor was composed of three domains: signal peptide of 17 residues, mature
peptide of 41 residues and intervening propeptide of 3 residues. There are six
cysteines in the sequence of mature CFBD peptide, which possibly form three
disulfide-bridges. CFBD showed antimicrobial activities against Staphylococcus
aureus, Bacillus subtilis, Candida albicans and Escherichia coli. This peptide
could be classified into family of β-defensin based on its sequence similarity
with β-defensins from other vertebrates. Evolution analysis indicated that CFBD
was close to fish β-defensin. As far as we know, CFBD is the first β-defensin
antimicrobial peptide from salamanders. Defensins are endogenous peptides with cysteine-rich antimicrobial ability that
contribute to host defence against bacterial, fungal and viral infections. There
are three subfamilies of defensins in primates: α, β and θ-defensins.
α-defensins are most present in neutrophils and Paneth cells; β-defensins are
involved in protecting the skin and the mucous membranes of the respiratory,
genitourinary and gastrointestinal tracts; and θ-defensins are physically
distinguished as the only known fully-cyclic peptides of animal origin, which
are first isolated from rhesus macaques. All three kinds of defensins have six
conserved cysteines, three intramolecular disulfide bonds, a net positive
charge, and β-sheet regions. α and θ-defensins are closely related, comparative
amino acid sequences showed that the difference between them is that θ-defensins
have an additional stop codon limits the initial defensin domain peptides to 12
residues. Humans, chimpanzees and gorillas do not produce θ-defensin peptides
due to a premature stop codon present in the signal sequence of all θ-defensin
pseudogenes. By using comprehensive computational searches, here we report the
discovery of complete repertoires of the α and θ-defensin gene family in ten
primate species. Consistent with previous studies, our phylogenetic analyses
showed all primate θ-defensins evident formed one distinct clusters evolved from
α-defensins. β-defensins are ancestors of both α and θ-defensins. Human has two
copies of DEFA1 and DEFT1P, and two extra DEFA3 and DEFA10P genes compared with
gorilla. As different primates inhabit in quite different ecological niches, the
production of species-specific α and θ-defensins and these highly evolved
θ-defensins in old world monkeys would presumably allow them to better respond
to the specific microbial challenges that they face. Human alpha defensins are a class of antimicrobial peptides with additional
antiviral activity. Such antimicrobial peptides constitute a major part of
mammalian innate immunity. Alpha defensins contain six cysteines, which form
three well defined disulfide bridges under oxidizing conditions. Residues
C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the
peptide. The major tissue in which HD5 is expressed is the crypt of the small
intestine, an anaerobic niche that should allow for substantial pools of both
oxidized and (partly) reduced HD5. We used ion mobility coupled to mass
spectrometry to track the structural changes in HD5 upon disulfide bond
reduction. We found evidence of stepwise unfolding of HD5 with sequential
reduction of the three disulfide bonds. Alkylation of free cysteines followed by
tandem mass spectrometry of the corresponding partially reduced states revealed
a domit pathway of reductive unfolding. The majority of HD5 unfolds by
initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional
evidence for a minor pathway that starts with reduction of C3-C31, followed by
C5-C20 and C10-C30. Our results provide insight into the pathway and
conformational landscape of disulfide bond reduction in HD5. |
Could BRCA gene test used for breast and ovarian cancer risk? | Yes, BRCA gene test could be used for breast and ovarian cancer risk, as female BRCA1 and BRCA2 mutations are significantly associated with risk of developing breast and ovarian cancers. | Early-onset breast cancer characterize genetic predisposition to cancer in
women. BRCA-1 gene was identified as one of the predisposition genes of
breast/ovarian cancer. About 90% of the reported mutations in the hereditary
breast and ovarian cancer gene, BRCA-1, result in truncated proteins. The aim of
our study is to detect rapidly BRCA-1 mutations by the protein truncation test
(PTT) in Tunisian women with early breast cancer. Population and methods. We
underwent molecular analysis in families with more than: (a) a women under 40
years-old with breast cancer, uni or bilateral; (b) two 1st degree relatives
women under 50 years-old with beast cancer. Sixteen women from 12 families were
studied by PTT to screen mutations in exon 11 which encodes 61% of BRCA-1.
RESULTS: PTT analysis of exon 11 revealed a normal and truncated protein in one
patient between 16 from 12 families.
CONCLUSION: BRCA-1 gene seems to contribute to at least 1/16 or 6.25% in women
with hereditary predisposition to breast/ovarian cancer in Tunisia. PTT promises
to be a valuable technique in detecting BRCA-1 mutations in our country. BACKGROUND: Clinically significant mutations of BRCA1 and BRCA2 genes are
associated with increased susceptibility for breast and ovarian cancer. Although
these mutations are uncommon, public interest in testing for them is growing.
PURPOSE: To determine benefits and harms of screening for inherited breast and
ovarian cancer susceptibility in the general population of women without cancer
presenting for primary health care in the United States.
DATA SOURCES: MEDLINE (1966 to 1 October 2004), Cochrane Library databases,
reference lists, reviews, Web sites, and experts.
STUDY SELECTION: Eligibility was determined by inclusion criteria specific to
key questions about risk assessment, genetic counseling, mutation testing,
prevention interventions, and potential adverse effects.
DATA EXTRACTION: After review of studies, data were extracted, entered into
evidence tables, and summarized by using descriptive or statistical methods.
Study quality was rated by using predefined criteria.
DATA SYNTHESIS: Tools assessing risks for mutations and referral guidelines have
been developed; their accuracy, effectiveness, and adverse effects in primary
care settings are unknown. Risk assessment, genetic counseling, and mutation
testing did not cause adverse psychological outcomes, and counseling improved
distress and risk perception in the highly selected populations studied.
Intensive cancer screening studies are inconclusive. Chemoprevention trials
indicate risk reduction for breast cancer in women with varying levels of risk,
as well as increased adverse effects. Observational studies of prophylactic
surgeries report reduced risks for breast and ovarian cancer in mutation
carriers.
LIMITATIONS: No data describe the range of risk associated with BRCA mutations,
genetic heterogeneity, and moderating factors; studies conducted in highly
selected populations contain biases; and information on adverse effects is
incomplete.
CONCLUSIONS: A primary care approach to screening for inherited breast and
ovarian cancer susceptibility has not been evaluated, and evidence is lacking to
determine benefits and harms for the general population. Women from site-specific hereditary breast cancer families who carry a BRCA1 or
BRCA2 mutation are at increased risk for ovarian cancer. It is less clear,
however, whether individuals from hereditary breast cancer families who do not
carry such a mutation are also at increased ovarian cancer risk. To determine
whether women from BRCA mutation-negative hereditary breast cancer families are
at increased risk for ovarian cancer, 199 probands from BRCA mutation-negative,
site-specific breast cancer kindreds who consented to prospective follow-up at
the time of genetic testing were identified. The incidence of new breast and
ovarian cancers in probands and their families since receipt of their genetic
test results was determined by questionnaire. The expected number of cancers and
standardized incidence ratios (SIRs) were determined from age-specific cancer
incidence rates from the Surveillance, Epidemiology, and End Results (SEER)
program by using the method of Byar. All statistical tests were two-sided.
During 2534 women-years of follow-up in 165 kindreds, 19 new cases of breast
cancer were diagnosed, whereas only 6.07 were expected (SIR = 3.13, 95%
confidence interval [CI] = 1.88 to 4.89; P < .001), and one case of ovarian
cancer was diagnosed, whereas only 0.66 was expected (SIR = 1.52, 95% CI = 0.02
to 8.46; P = .48). These results suggest that women from BRCA mutation-negative,
site-specific breast cancer families are not at increased risk for ovarian
cancer. BRCA1 and BRCA2 genes are responsible for 5-10% of breast and ovarian cancer
cases. However, the vast majority of ovarian and breast cancer cases do not
display the hereditary form of the disease. Estrogen-metabolizing genes may also
contribute to the predisposition of breast or ovarian cancer. Polymorphic
variants of the estrogen-metabolizing gene, CYP17, have been associated with the
risk of hormone-related cancers. In this study we investigated the CYP17
polymorphisms in ovarian cancer patients harboring mutations in the BRCA1 and
BRCA2 genes, patients displaying familial characteristics but not carrying
mutations and patients with sporadic ovarian cancer. Association between the
allele frequencies, the CYP17 genotype and tumor characteristics or clinical
parameters was evaluated. Our data suggest evidence for an association between
ovarian cancer risk and the CYP17 genotype in the subgroup of patients with
familial disease in whom no mutations in the BRCA genes are found. Although
there were no statistically significant differences in the genotype distribution
between the control group and the subgroup of patients with BRCA mutations, the
frequency of the CYP17 A2 allele was significantly higher in the subgroup of
patients without BRCA mutations. We found a four- to eightfold higher risk in
ovarian cancer patients with family history but without BRCA mutations. Our data
indicate that the CYP17 A2 allele polymorphism may confer an increased risk and
can provide a biomarker for ovarian cancer patients in whom no mutations in the
BRCA genes are observed. PURPOSE: Familial ovarian cancer is most often associated with hereditary breast
and ovarian cancer, implicating mutations in the BRCA1 and BRCA2 genes.
Hereditary nonpolyposis colorectal cancer, another common syndrome, is also
associated with ovarian cancer and is caused by DNA mismatch repair genes. We
sought to identify the role of hereditary nonpolyposis colorectal cancer in
women with family histories of ovarian cancer.
METHODS: The likelihood of a genetic syndrome in 226 oophorectomized women in
the Gilda Radner Familial Ovarian Cancer Registry was determined by pedigree
analysis using clinical criteria and by calculating the probability of a
mutation in genes responsible for hereditary breast and ovarian cancer and
hereditary nonpolyposis colorectal cancer using available risk models.
RESULTS: Some 86% had a BRCA gene mutation likelihood of 7.8% or higher,
warranting consideration of hereditary breast and ovarian cancer. Of the 32
women below this threshold, 4 (12.5%) had family histories that met criteria for
clinical diagnosis of hereditary nonpolyposis colorectal cancer. In addition, 16
women (7%) with a BRCA mutation likelihood greater than 7.8% met clinical
criteria for hereditary nonpolyposis colorectal cancer or warranted its
inclusion in the differential diagnosis. Among all study respondents, 9% had
family histories warranting consideration of hereditary nonpolyposis colorectal
cancer.
CONCLUSION: Hereditary nonpolyposis colorectal cancer should be considered in
the differential diagnosis of women with family histories of ovarian cancer. BACKGROUND: Women who are at increased risk for breast and ovarian cancers,
especially BRCA1 and BRCA2 mutation carriers, face a myriad of risk-reduction
options, including increased surveillance, chemoprevention, prophylactic
oophorectomy, and prophylactic mastectomy. However, little is known about which
clinical, demographic, or cancer-related factors are associated with
risk-reduction interventions.
METHODS: The authors conducted a retrospective review of records for 554 women
who had undergone testing at The University of Texas M. D. Anderson Cancer
Center between 2000 and 2006 for deleterious BRCA1 and BRCA2 gene mutations.
Data were collected on the risk-reduction interventions these women adopted
after they underwent genetic testing. These data were tested for associations
with demographic and clinical characteristics.
RESULTS: Among the 554 women who underwent genetic testing for BRCA mutation, 78
were found to have a deleterious mutation in the BRCA1 gene, and 54 had a
mutation in the BRCA 2 gene. Of the 554 women, 85 underwent prophylactic
mastectomy, 30 prophylactic oophorectomy, and 52 both surgeries; 387 women opted
for surveillance. Women who had BRCA mutations, a history of breast cancer or
ductal carcinoma in situ (DCIS), or previous breast biopsies were more likely to
have prophylactic surgery. Women with a family history of ovarian cancer were
more likely to undergo prophylactic oophorectomy. Women with a personal history
of ovarian cancer or advanced breast cancer were more likely to undergo
surveillance only. Women with breast cancer who had had a total mastectomy as
part of their prior breast cancer treatment underwent prophylactic mastectomy
more frequently than women who either had breast-conserving surgery or no
history of breast cancer. In multivariate analysis, only positive BRCA mutation
carrier status was associated with having had prophylactic surgery. In addition,
breast cancer history was significantly associated with prophylactic mastectomy.
CONCLUSIONS: Women who were BRCA carriers, women who had a history of breast
cancer, DCIS, or breast biopsy, or had a family history of ovarian cancer were
more likely to have undergone surgery for cancer risk reduction. Women with
ovarian cancer or advanced breast cancer were more likely to have undergone
surveillance. CONTEXT: An autosomal domit pattern of hereditary breast cancer may be masked
by small family size or transmission through males given sex-limited expression.
OBJECTIVE: To determine if BRCA gene mutations are more prevalent among single
cases of early onset breast cancer in families with limited vs adequate family
structure than would be predicted by currently available probability models.
DESIGN, SETTING, AND PARTICIPANTS: A total of 1543 women seen at US high-risk
clinics for genetic cancer risk assessment and BRCA gene testing were enrolled
in a prospective registry study between April 1997 and February 2007. Three
hundred six of these women had breast cancer before age 50 years and no first-
or second-degree relatives with breast or ovarian cancers.
MAIN OUTCOME MEASURE: The main outcome measure was whether family structure,
assessed from multigenerational pedigrees, predicts BRCA gene mutation status.
Limited family structure was defined as fewer than 2 first- or second-degree
female relatives surviving beyond age 45 years in either lineage. Family
structure effect and mutation probability by the Couch, Myriad, and BRCAPRO
models were assessed with stepwise multiple logistic regression. Model
sensitivity and specificity were determined and receiver operating
characteristic curves were generated.
RESULTS: Family structure was limited in 153 cases (50%). BRCA gene mutations
were detected in 13.7% of participants with limited vs 5.2% with adequate family
structure. Family structure was a significant predictor of mutation status (odds
ratio, 2.8; 95% confidence interval, 1.19-6.73; P = .02). Although none of the
models performed well, receiver operating characteristic analysis indicated that
modification of BRCAPRO output by a corrective probability index accounting for
family structure was the most accurate BRCA gene mutation status predictor (area
under the curve, 0.72; 95% confidence interval, 0.63-0.81; P<.001) for single
cases of breast cancer.
CONCLUSIONS: Family structure can affect the accuracy of mutation probability
models. Genetic testing guidelines may need to be more inclusive for single
cases of breast cancer when the family structure is limited and probability
models need to be recreated using limited family history as an actual variable. Approximately 5% to 10% of breast and ovarian cancers are related to an
inherited gene mutation. Of these cases, 84% of hereditary breast cancer and
more than 90% of hereditary ovarian cancer are caused by mutations in the BRCA1
or BRCA2 genes. Family histories of cancer are an essential tool in identifying
features of and individuals at risk for hereditary breast-ovarian cancer
syndrome. The risk to carry an identifiable BRCA gene mutation can be assessed
by trained healthcare providers using various pre-test risk models. Individuals
who carry a BRCA gene mutation have increased lifetime risks of developing
hereditary breast and ovarian cancer syndrome-related cancers. Genetic testing
for the BRCA gene mutations is offered in accordance with American Society of
Clinical Oncology guidelines. In accordance with guidelines, patients are
informed of the risks, benefits, and limitations of testing prior to electing to
undergo the testing process. Upon receipt of results, healthcare providers offer
the patient with appropriate medical management recommendations. Persons exhibiting mutations in two tumor suppressor genes, BRCA1 and BRCA2,
have a greatly increased risk of developing breast and/or ovarian cancer. The
incidence of BRCA gene mutation is very high in Ashkenazi Jewish women of
European descent, and many issues can arise, particularly for observant Orthodox
women, because of their genetic status. Their obligations under the Jewish code
of ethics, referred to as Jewish law, with respect to the acceptability of
various risk-reducing strategies, may be poorly understood. In this article the
moral direction that Jewish law gives to women regarding testing,
confidentiality, and other issues is explored. The intent is to broaden nurses'
knowledge of how a particular religious tradition could impact on decision
making around genetics testing, with the aim of enhancing their understanding of
culturally sensitive ethical care. BACKGROUND: The American Cancer Society (ACS) guidelines for screening with
breast magnetic resoce imaging (MRI) recommend MRI for women who have a
lifetime risk > or = 20% of developing breast cancer. Genetic testing for breast
cancer gene (BRCA) mutations is offered to women who have a risk > or = 10% of
carrying a mutation. The objectives of the current study were 1) to identify the
number of women in a breast cancer screening population who had > or = 20%
lifetime breast cancer risk and, thus, were candidates for screening MRI; and 2)
to determine the number of women who had > or = 10% risk of BRCA mutation yet
had <20% lifetime risk of breast cancer and, thus, may not have been identified
as candidates for MRI screening.
METHODS: From 2003 to 2005, women who underwent screening mammography completed
a self-administered questionnaire regarding breast cancer risk factors. For each
patient, the lifetime breast cancer risk and the risk of BRCA mutation was
determined by using the computerized BRCAPRO breast cancer risk-assessment
model.
RESULTS: Of 18,190 women, 78 (0.43%) had > or = 20% lifetime risk of breast
cancer, all of whom had > or = 10% risk of carrying a BRCA mutation. An
additional 374 women (2.06%) had <20% lifetime breast cancer risk but > or = 10%
risk of mutation. Overall, there were 183 (1%) predicted mutation carriers, 27
women (0.15%) who had > or = 20% lifetime risk of breast cancer, and 62 women
(0.34%) who had > or = 10% risk of mutation but <20% lifetime breast cancer
risk.
CONCLUSIONS: The ACS guidelines for breast MRI screening may systematically
exclude MRI screening for many women who have a substantial risk for BRCA
mutation. The current results demonstrated a need for greater awareness of
breast cancer risk factors in the screening mammography population, so that
high-risk women can be identified and given access to genetic testing and
counseling regarding all risk-reducing interventions. PURPOSE/OBJECTIVES: To investigate cancer surveillance behaviors of women at
risk for hereditary breast and ovarian cancer (HBOC) who presented for clinical
BRCA cancer susceptibility testing, specifically to describe cancer surveillance
behaviors and reasons for not engaging in behaviors, compare surveillance
behaviors with existing surveillance guidelines, and evaluate associations of
cancer surveillance behaviors with BRCA results.
DESIGN: Cross-sectional, descriptive.
SETTING: Genetic risk-assessment programs in a National Cancer
Institute-designated comprehensive cancer center and a community cancer center,
both in the southwestern region of the United States.
SAMPLE: Purposive sample of 107 at-risk women.
METHODS: Self-report survey.
MAIN RESEARCH VARIABLES: Breast and ovarian cancer surveillance behaviors and
BRCA test results.
FINDINGS: Ninety percent of participants had a personal history of breast
cancer; 84% had a negative BRCA result. About 60% of participants engaged in at
least the minimum recommended breast cancer surveillance behaviors, but 70% had
suboptimal ovarian cancer surveillance behaviors. Lack of physician
recommendation was the most frequently reported reason for not having
surveillance procedures. BRCA results were not associated with the breast cancer
surveillance categories and the ovarian cancer surveillance recommendations.
CONCLUSIONS: Although most participants were not carriers of a mutation, the
presence of other risk factors for breast and ovarian cancer dictates continued
cancer surveillance. At-risk women may not be informed adequately about cancer
surveillance.
IMPLICATIONS FOR NURSING: Healthcare providers should be aware of changing
breast and ovarian cancer surveillance recommendations and counsel their at-risk
patients accordingly. Discovery of mutations in the breast and ovarian cancer susceptibility genes
BRCA1 and BRCA2 can have emotional consequences for both the tested individual
and his or her relatives. This secondary analysis study investigated how BRCA
testing impacts family dynamics and relationships. For the original study, a
grounded theory inquiry, participants were recruited from a hereditary
breast/ovarian cancer syndrome support website and open-ended interviews were
performed asking about individual and family experiences after BRCA testing. All
12 participants whose interviews were included in the secondary analysis had a
BRCA mutation. For the secondary analysis, thematic analysis was conducted and
revealed three main themes characterizing the effect of BRCA testing on family
relationships: 1. That the first in the family to have testing or seek genetic
counseling takes on a special family role that can be difficult for them; 2.
That discussions in the family often change; and 3. That individuals may feel
more or less connected to certain family members. These changes seemed to relate
to family cancer history, relationships, coping strategies, communication
patterns, and mutation status. Genetic counselors might find it useful to
explore these issues in order to prepare clients before BRCA testing and to
support them through shifts in family dynamics after disclosure of results. PURPOSE: Women with ovarian cancer have a 10% probability of carrying a BRCA
mutation. If a mutation is identified, unaffected family members can undergo
genetic testing and cancer risk-reducing strategies. We estimated the net health
benefits and cost-effectiveness of different criteria for BRCA mutation testing
in women with ovarian cancer, and the downstream benefits for their first-degree
relatives (FDRs).
METHODS: We developed a Markov Monte Carlo simulation model to compare four
criteria for BRCA testing in women with ovarian cancer: no testing (reference);
only if personal history of breast cancer, family history of breast/ovarian
cancer, or Ashkenazi Jewish ancestry; only if invasive serous cancer; any
invasive nonmucinous epithelial cancer. Net health benefit was life expectancy
for FDRs and primary outcome was the incremental cost-effectiveness ratio
(ICER). The model estimated the number of future breast and ovarian cancer cases
in FDRs.
RESULTS: BRCA testing based on personal/family history and ancestry could
prevent future cases in FDRs with an ICER of $32,018 per year of life (LY)
gained compared with the reference strategy. BRCA testing based on serous or any
nonmucinous epithelial ovarian cancer could prevent more cancer cases, but at
ICERs of $128,465 and $148,363 per LY gained, respectively.
CONCLUSION: BRCA testing of women with ovarian cancer based on personal/family
history of cancer or Ashkenazi Jewish ancestry is a cost-effective strategy to
prevent future breast and ovarian cancers among FDRs. More inclusive testing
strategies prevent additional cancer cases but at significant cost. PURPOSE: Germline mutations in BRCA genes are associated with breast and ovarian
cancer susceptibility. Because infertility is associated with breast and ovarian
cancer risks, we hypothesized that the mutations in the BRCA gene may be
associated with low response to fertility treatments.
METHODS: We performed ovarian stimulation in 126 women with breast cancer by
using letrozole and gonadotropins for the purpose of fertility preservation by
embryo or oocyte cryopreservation. As surrogates of ovarian reserve, the oocyte
yield and the incidence of low response were compared with ovarian stimulation
according to BRCA mutation status.
RESULTS: Of the 82 women who met the inclusion criteria, 47 women (57%) had
undergone BRCA testing, and 14 had a mutation in BRCA genes, of which two were
of clinically undetermined significance. In BRCA mutation-positive patients, low
ovarian response rate was significantly higher compared with BRCA
mutation-negative patients (33.3 v 3.3%; P = .014) and with BRCA-untested women
(2.9%; P = .012). All BRCA mutation-positive low responders had BRCA1 mutations,
but low response was not encountered in women who were only BRCA2 mutation
positive. Compared with controls, BRCA1 mutation- but not BRCA2
mutation-positive women produced lower numbers of eggs (7.4 [95% CI, 3.1 to
17.7] v 12.4 [95% CI, 10.8 to 14.2]; P = .025) and had as many as 38.3 times the
odds ratio of low response (95% CI, 4.1 to 353.4; P = .001).
CONCLUSION: BRCA1 mutations are associated with occult primary ovarian
insufficiency. This finding may, at least in part, explain the link between
infertility and breast/ovarian cancer risks. A substantial proportion of Ashkenazi Jewish (AJ) breast and ovarian cancer
families carry one of three founder mutations in BRCA1 (185delAG, 5382InsC) and
BRCA2 (6174delT). Non-founder mutations are identified in another 2-4% of such
families. The extent to which major genomic rearrangements in BRCA contribute to
breast and ovarian cancer in the Ashkenazim is not well understood. We
identified AJ individuals with breast and/or ovarian cancer undergoing
hereditary breast/ovarian cancer risk assessment since 2006 without evidence of
a deleterious mutation on BRCA gene sequencing who were screened for major gene
rearrangements in BRCA1 and BRCA2. For each proband, the pre-test probability of
identifying a deleterious BRCA mutation was estimated using the Myriad II model.
We identified 108 affected individuals who underwent large rearrangement testing
(80 breast cancer, 19 ovarian cancer, nine both breast and ovarian cancer). The
mean estimated AJ specific pre-test probability of a deleterious mutation in
BRCA1 and BRCA2 was 24.7% (range: 4.4-88.9%). No genomic rearrangements were
identified in either the entire group or in the 26 subjects with pre-test
mutation prevalence estimates exceeding 30%. Major gene rearrangements involving
the BRCA1 and BRCA2 genes appear to contribute little to the burden of inherited
predisposition to breast and ovarian cancer in the Ashkenazim. This paper discusses the presentation I held at the symposium on genetics during
the 4th European Breast Cancer Conference held in Hamburg in March
2004.Primarily, the goals and working methods of the advocacy group specialised
in Hereditary Breast/Ovarian Cancer of the Dutch Breast Cancer Patient
Organisation known as BorstkankerVereniging Nederland (BVN) are explained.
Furthermore, some specific individual problems that mutation carriers might
encounter before and after BRCA1/2 susceptibility testing are discussed. These
include: dilemmas in choosing preventive interventions, dealing with the
psychological impact of knowing you are a mutation carrier, dealing with the
social implications of being genetically at risk, an example of insurance
discrimination. In addition, some controversial social and ethical issues that
are currently under debate are highlighted, such as the issue of the European
patenting of the breast cancer susceptibility genes BRCA1 and BRCA2. Since this
topic could also become relevant for other gene-related diseases, society as a
whole has to consider the ethical and social implications related to the
patenting of human genes in general. Another ethical area of debate is the
controversial issue of prenatal BRCA testing and the choice of pregcy
termination.Finally, the Working Party pleads for the international co-operation
and exchange of data and experience among professionals as well as patients. It
appears that professionals in different European countries tend to advise on
different risk management strategies and treatments and as such, the Working
Party strongly advocates the international standardisation of risk management
and treatment of mutation carriers. In this respect, specific attention should
be given to a group that has had a non-informative or negative BRCA test result,
because this group is still considered to be at high risk to develop the
disease. The aim of the present study is to analyze the relationship of the incidence of
mutations in the two major genes BRCA1 and BRCA2 conferring risk of breast
cancer (BC) and ovarian cancer (OC) with the cancer burden in families and with
the presence and age of onset of BC/OC. We included 704 index patients (IP) and
668 family members of the IP who tested positive for BRCA1/BRCA2 who were
studied in the Program of Genetic Counselling in Cancer of the Valencia
Community (Spain). We found 129 IPs with deleterious mutations (18.3%), 59 in
BRCA1 and 70 in BRCA2, detecting 396 mutations in this kindred. The incidence of
mutations and their distribution between BRCA1 and BRCA2 showed a significantly
uneven incidence among the family groups (P < 0.001). We found 179 tumors in the
396 mutation carriers (45%) and detected only 11 cancers among the 272
non-mutation carriers (P < 0.001). No differences in the tumor prevalence or the
age of onset of cancer between the genes among the mutation carriers were found.
The mutation carriers showed a 50% probability of having BC/OC at a median age
of 49 years (95% CI 46-52 years) and 78% at the age of 70 years (95% CI:
71-85%). In conclusion the family burden of BC and OC is strongly associated
with the incidence of BRCAs mutations and could foretell which of the two BRCAs
genes is more likely to have mutations. Mutation carriers have a 50% risk of
having BC/OC by the age of 50 years. The responsibility for informing at-risk relatives of the availability of
genetic testing for breast/ovarian cancer gene (BRCA1 or BRCA2) mutations
currently falls on the probands. This study explored the support needs of
individuals from families with identified BRCA1 or BRCA2 mutations when
communicating about genetic risk and genetic testing with at-risk family
members. Thirty-nine semi-structured telephone interviews were conducted with
individuals from families with identified BRCA mutations. Interview responses
were cross-tabulated by sample characteristics using the qualitative research
analysis software NVivo8. The development of educational materials, which
individuals could use when communicating the risks of carrying a BRCA gene
mutation with their relatives, was identified as a specific need. Many
participants expressed a preference for a staged approach, where relatives are
notified of their increased risk and the availability of genetic testing risk
either face-to-face or via a letter, with additional educational sources,
including brief written information or access to a website, made available for
those wishing to access more in-depth information. This research identified a
need for the development of educational/informational resources to support
individuals with identified breast/ovarian cancer mutations to communicate with
their at-risk relatives about genetic risk and genetic testing availability. BACKGROUND: Every year, 60,000 women in Germany are found to have breast cancer,
and 9000 to have ovarian cancer. Familial clustering of carcinoma is seen in
about 20% of cases.
METHODS: We selectively review relevant articles published up to December 2010
that were retrieved by a search in PubMed, and we also discuss findings from the
experience of the German Consortium for Hereditary Breast and Ovarian Cancer.
RESULTS: High risk is conferred by the highly penetrant BRCA1 and BRCA2 genes as
well as by other genes such as RAD51C. Genes for breast cancer that were
originally designated as moderately penetrant display higher penetrance than
previously thought in families with a hereditary predisposition. The role these
genes play in DNA repair is thought to explain why tumors associated with them
are sensitive to platin derivatives and PARP inhibitors. In carriers of BRCA1
and BRCA2, prophylactic bilateral mastectomy and adnexectomy significantly
lowers the incidence of breast and ovarian cancer. Moreover, prophylactic
adnexectomy also lowers the breast-and-ovarian-cancer-specific mortality, as
well as the overall mortality. If a woman bearing a mutation develops cancer in
one breast, her risk of developing cancer in the other breast depends on the
particular gene that is mutated and on her age at the onset of disease.
CONCLUSION: About half of all monogenically determined carcinomas of the breast
and ovary are due to a mutation in one or the other of the highly penetrant BRCA
genes (BRCA1 and BRCA2). Women carrying a mutated gene have an 80% to 90% chance
of developing breast cancer and a 20% to 50% chance of developing ovarian
cancer. Other predisposing genes for breast and ovarian cancer have been
identified. Clinicians should develop and implement evidence-based treatments on
the basis of these new findings. BACKGROUND: Women who are diagnosed with a deleterious mutation in either breast
cancer (BRCA) gene have a high risk of developing breast and ovarian cancers at
young ages. In this study, the authors assessed age at diagnosis in 2
generations of families with known mutations to investigate for earlier onset in
subsequent generations.
METHODS: Of the 132 BRCA-positive women with breast cancer who participated in a
high-risk protocol at The University of Texas MD Anderson Cancer Center (Gen 2),
106 women could be paired with a family member in the previous generation (Gen
1) who was diagnosed with a BRCA-related cancer (either breast cancer or ovarian
cancer). Age at diagnosis, location of the mutation, and year of birth were
recorded. A previously published parametric anticipation model was applied in
these genetically predisposed families.
RESULTS: The median age of cancer diagnosis was 42 years (range, 28-55 years) in
Gen 2 and 48 years (range, 30-72 years) in Gen 1 (P < .001). [corrected]. In the
parametric model, the estimated change in the expected age at onset for the
entire cohort was 7.9 years (P < .0001). Statistically significant earlier ages
at diagnosis also were observed within subgroups of BRCA1 and BRCA2 mutations,
maternal inheritance, paternal inheritance, breast cancer only, and breast
cancer-identified and ovarian cancer-identified families.
CONCLUSIONS: Breast and ovarian cancers in BRCA mutation carriers appeared to be
diagnosed at an earlier age in later generations. The authors concluded that
patients who are younger at the onset of BRCA-related cancers should continue to
be tracked to offer appropriate screening modalities at appropriate ages. BRCAPRO and Myriad II are widely used models for predicting BRCA1/2 mutation
probability before genetic testing. However, the accuracy of these models in
Koreans is not known. This study was performed to evaluate the accuracy of the
BRCAPRO and Myriad II models. Two hundred thirty-six women with breast cancer
who underwent comprehensive BRCA1/2 genetic testing at our hospital between 2003
and 2010 were included in this study. We evaluated the performance of each model
by comparing the numbers of observed versus predicted mutation carriers. We
calculated sensitivity, specificity, and predictive values at 10 % estimated
probability. Forty-six individuals were identified to carry a deleterious BRCA
mutation. The prevalence of BRCA mutation (19.5 %) was significantly higher than
that predicted by BRCAPRO (9.0 %, p = 0.001) and Myriad (5.6 %, p < 0.001). In
familial breast cancer patients, BRCA mutation rate (observed 22.7 %) was
underestimated by both BRCAPRO (expected 11.4 %, p = 0.006) and Myriad II
(expected 6.4 %, p < 0.001). Subgroup analyses showed that both models
underestimated the risk of BRCA mutation in patients with a family history of
breast cancer (probands' age at breast cancer diagnosis >50 years), with only
one relative with breast cancer, and with non-familial early-onset breast cancer
or bilateral breast cancer. Using a 10 % cut-off, the sensitivities were 47.8 %
(BRCAPRO) and 50.0 % (Myriad), and positive predictive values were 44.9 %
(BRCAPRO) and 43.4 % (Myriad). Both BRCAPRO and Myriad II underestimated the
risk of BRCA1/2 mutation in Koreans. Our findings suggest that these models are
less sensitive in Korean women, and therefore a new BRCA mutation prediction
model based on Korean data is needed for proper genetic counseling. BACKGROUND: BRCA1 and BRCA2 germline mutations predispose heterozygous carriers
to hereditary breast/ovarian cancer. However, unclassified variants (UVs)
(variants with unknown clinical significance) and missense polymorphisms in
BRCA1 and BRCA2 genes pose a problem in genetic counseling, as their impact on
risk of breast and ovarian cancer is still unclear. The objective of our study
was to identify UVs and missense polymorphisms in Algerian breast/ovarian cancer
patients and relatives tested previously for BRCA1 and BRCA2 genes germline
mutations analysis.
METHODS: We analyzed 101 DNA samples from 79 breast/ovarian cancer families. The
approach used is based on BRCA1 and BRCA2 sequence variants screening by SSCP or
High-Resolution Melting (HRM) curve analysis followed by direct sequencing. In
silico analyses have been performed using different bioinformatics programs to
individualize genetics variations that can disrupt the BRCA1 and BRCA2 genes
function.
RESULTS: Among 80 UVs and polymorphisms detected in BRCA1/2 genes (33 BRCA1 and
47 BRCA2), 31 were new UVs (10 BRCA1 and 21 BRCA2), 7 were rare UVs (4 BRCA1 and
3 BRCA2) and 42 were polymorphic variants (19 BRCA1 and 23 BRCA2). Moreover, 8
new missense UVs identified in this study: two BRCA1 (c.4066C>A/p.Gln1356Lys,
c.4901G>T/p.Arg1634Met) located respectively in exons 11 and 16, and six BRCA2
(c.1099G>A/p.Asp367Asn, c.2636C>A/p.Ser879Tyr, c.3868T>A/p.Cys1290Ser,
c.5428G>T/p.Val1810Phe, c.6346C>G/p.His2116Asp and c.9256G>A/p.Gly3086Arg)
located respectively in exons 10, 11 and 24, show a damaging PSIC score yielded
by PolyPhen2 program and could be pathogenic. In addition, 5 new BRCA} missense
UVs out of six that were found to be damaging by PolyPhen2 program, also were
deleterious according to SIFT program. The rare BRCA1 UV c.5332G>A/p.Asp1778Asn
was found here for the first time in co-occurrence in trans with the deleterious
BRCA1 mutation c.798_799delTT/p.Ser267LysfsX19 in young breast cancer patient.
Moreover, 10 new identified intronic variants with unknown clinical significance
(3 BRCA1 and 7 BRCA2) in the present study, could be considered as benign,
because GeneSplicer, SpliceSiteFinder and MaxEntScan prediction programs show no
splice site alteration for these variants. Several missense polymorphisms of
BRCA1 c.2612C>T/p.Pro871Leu, c.3548A>G/p.Lys1183Arg, c.4837A>G/p.Ser1613Gly and
BRCA2 c.865A>C/p.Asn289His, c.1114A>C/p.Asn372His, c.2971A>G/p.Asn991Asp,
c.7150C>A/p.Gly2384Lys have been identified with high frequency in patients who
were tested negative for BRCA1 and BRCA2 mutations. These missense polymorphisms
could have a role as susceptibility breast cancer markers in Algerian
breast/ovarian cancer families where pathological BRCA1 and BRCA2 mutations were
not present.
CONCLUSIONS: For the first time, UVs and missense polymorphisms in BRCA1 and
BRCA2 genes have been identified in Algerian breast/ovarian cancer families.
Evaluation of breast/ovarian cancer risk induced by the eight new missense UVs
and common polymorphisms detected in our present work is on going in a larger
study. Germline mutations in the BRCA1 and BRCA2 genes highly predispose to breast and
ovarian cancers and are responsible for a substantial proportion of familial
breast and ovarian cancers. No female individuals from families from Morocco
affected by breast cancer with mutations of these genes have previously been
reported, and clinicians in Morocco are unaccustomed to dealing with healthy
female individuals carrying mutations in the BRCA genes. This study aimed to
report the initial experience of a group of Moroccan investigators carrying out
predictive genetic testing to detect a known familial mutation in healthy
Moroccan females with a high risk of developing breast cancer and to introduce
supervision of these asymptomatic female carriers as a new approach in the
prevention and early diagnosis of breast and ovarian cancers in Morocco.
Presymptomatic diagnosis was carried out using DNA genetic testing in 5 healthy
Moroccan female individuals from three families with an elevated risk of
developing breast cancer. These are the first Moroccan families reported to be
affected by breast cancers associated with BRCA mutations. Presymptomatic
diagnosis was carried out for breast cancer in 5 female individuals from three
Moroccan families with BRCA mutations. Two of the families are the first
reported incidence of the founder mutation Ashkenazi BRCA1-185_186delAG in
Moroccan patients. The third family carried the known BRCA2 mutation
c.5073dupA/p.trp1692metfsX3. We tested the presence of these mutations in 5
asymptomatic healthy females from the three families. Two sisters from family 1
carried the BRCA1-185_186delAG mutation, whereas the third female individual
from family 2 carried the c.5073dupA/p.trp1692metfsX3 mutation. However, one
healthy female individual and her mother from family 3 did not carry the
familial mutation of the BRCA1 gene. This study found BRCA mutations in three
asymptomatic subjects, suggesting that this is the first step towards the
development of persistent medical monitoring of females from families with a
history of breast and ovarian cancers. Consequently, it is crucial for
oncologists in Morocco to initiate the supervision of healthy female individuals
with genetic defects which may lead to hereditary cancers. OBJECTIVE: Women who carry a BRCA1 or BRCA2 gene mutation face a risk of
developing breast or ovarian cancer at an earlier age than women without such a
mutation. Relatively little is known about the psychosocial
consequences-especially regarding marriage and childbearing-in young women who
test positive for one of these mutations.
METHODS: In 2006, participants were recruited from Web sites for women with
breast cancer or BRCA gene mutations. Forty-four women ages 18 to 39 from 22
states and Canada who had had genetic testing and were found to carry a BRCA
mutation were interviewed by phone or e-mail. A qualitative, grounded theory
analysis was performed on the data, focusing on the participants' being young
and having had genetic testing for the BRCA mutation. The findings reported here
focus on three characteristics of the participants-whether or not they were
married, had children, or had a breast cancer diagnosis-and how those
characteristics were affected by the women's knowledge of their genetic risk.
RESULTS: Among the 13 unmarried participants, issues of when to disclose
information about their genetic risk in intimate relationships were discussed.
Many of the 24 participants who had children reported "staying alive" for their
children as a primary goal; the childless women reported an urgency to have
children. Of the 21 who had a breast cancer diagnosis, the youngest was 24 years
old, and several said knowledge of their genetic risk influenced their decision
to have the unaffected breast removed prophylactically.
CONCLUSIONS: A sense of being different and not understood was expressed in
these interviews. These findings suggest that nurses should be aware of
psychosocial issues, especially those surrounding marriage and childbearing, in
their interactions with young women who carry a BRCA1 or BRCA2 gene mutation. PURPOSE: We investigated the relationship between BRCA mutations and the
distribution of familial cancers other than breast or ovary in high-risk breast
cancer patients.
METHODS: PATIENTS WITH BREAST CANCER WHO HAD AT LEAST ONE OF THE FOLLOWING RISK
FACTORS WERE ENROLLED: reported family history of breast or ovarian cancer; 40
years of age or younger age at diagnosis; bilateral breast cancer; or male
gender. Genetic testing for BRCA mutation and questionnaires about personal and
family histories of maligcies were performed.
RESULTS: Among the 238 eligible patients, 49 (20.6%) patients had BRCA1/2
mutations, which were more frequent in patients with multiple risk factors
(p<0.0001). There were 271 members of 156 (65.5%) families who had histories of
other primary cancer. The distribution of the families was 119 (63.0%) and 37
(75.5%) in the BRCA-negative and positive group, respectively (p=0.0996).
Multiple familial cancers occurred in 70 families, which were significantly more
frequent in BRCA-positive families (p=0.0034). By ordinal logistic regression,
the occurrence of multiple familial cancers was associated with BRCA mutations
(p=0.0045), not with other risk factors. The most common site of disease was the
stomach, which is the most common in nationwide. And the proportional incidence
of pancreatic cancer (6.8%) was significantly higher than that of nationwide
cancer statistics (2.4%, p=0.0137).
CONCLUSION: BRCA mutations in high-risk breast cancer patients were associated
with multiple risk factors and multiple family members with other primary
cancers. Genetic counseling based on accurate information should be provided to
families with BRCA mutation carriers. OBJECTIVE: Female BRCA (breast cancer gene)-1 and BRCA-2 mutations are
significantly associated with risk of developing breast and ovarian cancers, in
turn, associated with female infertility. BRCA-1 mutations have also been
associated with occult primary ovarian insufficiency (OPOI), as have different
mutations of the FMR1 gene. We, therefore, hypothesized that FMR1 genotypes may
be associated with menarcheal and menopausal ages of BRCA mutation carriers.
PATIENTS: We compared the FMR1 genotype and sub-genotype distribution in 99
BRCA1/2 positive women and in 182 healthy women without a known history of
familial breast and ovarian cancer and searched for associations with age at
menarche and menopause. T-test was used to assess differences in menarcheal and
menopause ages, with times of menarche and menopause as continuous variables.
RESULTS: Women with BRCA1/2 mutations showed significantly different FMR1
genotype and sub-genotype distributions when compared with the control group
(p<0.001). This result remained stable in a sub-group analysis of Caucasian
BRCA1/2 carriers and healthy controls (p<0.001). In addition, BRCA1/2 carriers
indicated a trend toward shorter reproductive lifespan (p=0.18).
CONCLUSIONS: Our data confirm the previously reported highly skewed distribution
of FMR1 genotypes and sub-genotypes toward a high preponderance of low FMR1
alleles in BRCA1/2 carriers. We could demonstrate that BRCA-1 mutations are
associated with an earlier onset of menopause compared to BRCA-2 carriers,
although the distribution of the het-norm/low genotype is similar in both
groups. Our findings suggest that there may be other factors beside the genotype
that has an influence on menarche and especially menopause age in BRCA mutation
carriers. BACKGROUND: Mutations in BRCA1/2 genes confer ovarian, alongside breast, cancer
risk. We examined the risk of developing ovarian cancer in BRCA1/2-positive
families and if this risk is extended to BRCA negative families.
PATIENTS AND METHODS: A prospective study involving women seen at a single
family history clinic in Manchester, UK. Patients were excluded if they had
ovarian cancer or oophorectomy prior to clinic. Follow-up was censored at the
latest date of: 31/12/2010; ovarian cancer diagnosis; oophorectomy; or death. We
used person-years at risk to assess ovarian cancer rates in the study
population, subdivided by genetic status (BRCA1, BRCA2, BRCA negative, BRCA
untested) compared with the general population.
RESULTS: We studied 8005 women from 895 families. Women from BRCA2 mutation
families showed a 17-fold increased risk of invasive ovarian cancer (relative
risk (RR) 16.67; 95% CI 5.41 to 38.89). This risk increased to 50-fold in women
from families with BRCA1 mutations (RR 50.00; 95% CI 26.62 to 85.50). No
association was found for women in families tested negative for BRCA1/2, where
there was 1 observed invasive ovarian cancer in 1613 women when 2.74 were
expected (RR 0.37; 95% CI 0.01 to 2.03). There was no association with ovarian
cancer in families untested for BRCA1/2 (RR 0.99; 95% CI 0.45 to 1.88).
DISCUSSION: This study showed no increased risk of ovarian cancer in families
that tested negative for BRCA1/2 or were untested. These data help counselling
women from BRCA1/2 negative families with breast cancer that their risk of
invasive ovarian cancer is not higher than the general population. Mutations in BRCA genes elevate risk for breast and ovarian cancer. These
mutations are population specific. As there are no data on BRCA mutation
screening on larger number of probands in Serbia to date, aim of this study was
to determine types and frequencies of BRCA mutations in individuals from
high-risk families from Serbia, as well as to determine which BRCA mutations may
be considered as founder for Serbian population. We analyzed 94 probands and
detected 9 frameshift mutations in 12 individuals, 1 benign BRCA2 nonsense
mutation and numerous missense and synonymous mutations in both genes. Frequency
of frameshift mutations is 12.77%. In addition to two novel mutations detected
in our population we reported previously, we detected another novel
mutation--c.7283delT in BRCA2 exon 14. None of the detected deleterious
mutations may be considered as founder mutations for Serbian population, as each
of them was found in no more than two high-risk families. This mutation
diversity is most probably due to high migration rate in history of this part of
Europe. Interpretation of genetic testing results with missense mutations of
unknown clinical importance is very challenging and should be approached with
caution, using all available data sources for results' interpretation. Background. Inherited BRCA gene mutations convey a high risk for breast and
ovarian cancer, but current guidelines limit BRCA mutation testing to women with
early-onset cancer and relatives of mutation-positive cases. Benefits and risks
of providing this information directly to consumers are unknown. Methods. To
assess and quantify emotional and behavioral reactions of consumers to their
23andMe Personal Genome Service(®) report of three BRCA mutations that are
common in Ashkenazi Jews, we invited all 136 BRCA1 and BRCA2 mutation-positive
individuals in the 23andMe customer database who had chosen to view their BRCA
reports to participate in this IRB-approved study. We also invited 160
mutation-negative customers who were matched for age, sex and ancestry.
Semi-structured phone interviews were completed for 32 mutation carriers, 16
women and 16 men, and 31 non-carriers. Questions addressed personal and family
history of cancer, decision and timing of viewing the BRCA report, recollection
of the result, emotional responses, perception of personal cancer risk,
information sharing, and actions taken or planned. Results. Eleven women and 14
men had received the unexpected result that they are carriers of a BRCA1
185delAG or 5382insC, or BRCA2 6174delT mutation. None of them reported extreme
anxiety and four experienced moderate anxiety that was transitory. Remarkably,
five women and six men described their response as neutral. Most carrier women
sought medical advice and four underwent risk-reducing procedures after
confirmatory mutation testing. Male carriers realized that their test results
implied genetic risk for female relatives, and several of them felt considerably
burdened by this fact. Sharing mutation information with family members led to
screening of at least 30 relatives and identification of 13 additional carriers.
Non-carriers did not report inappropriate actions, such as foregoing cancer
screening. All but one of the 32 mutation-positive participants appreciated
learning their BRCA mutation status. Conclusions. Direct access to BRCA mutation
tests, considered a model for high-risk actionable genetic tests of proven
clinical utility, provided clear benefits to participants. The unexpected
information demonstrated a cascade effect as relatives of newly identified
carriers also sought testing and more mutation carriers were identified. Given
the absence of evidence for serious emotional distress or inappropriate actions
in this subset of mutation-positive customers who agreed to be interviewed for
this study, broader screening of Ashkenazi Jewish women for these three BRCA
mutations should be considered. BRCA1 and BRCA2 are two major genes associated with familial breast and ovarian
cancer susceptibility. In Poland standard BRCA gene test is usually limited to
Polish founder BRCA1 mutations: 5382insC, C61G and 4153delA. To date, just a few
single large genomic rearrangements (LGRs) of BRCA1 gene have been reported in
Poland. Here we report the first comprehensive analysis of large mutations in
BRCA1 and BRCA2 genes in this country. We screened LGRs in BRCA1 and BRCA2 genes
by multiplex ligation-dependent probe amplification in 200 unrelated patients
with strong family history of breast/ovarian cancers and negative for BRCA1
Polish founder mutations. We identified three different LGRs in BRCA1 gene:
exons 13-19 deletion, exon 17 deletion and exon 22 deletion. No LGR was detected
in BRCA2 genes. Overall, large rearrangements accounted for 3.7 % of all BRCA1
mutation positive families in our population and 1.5 % in high-risk families
negative for Polish founder mutation. Dr Wera Hofmann is an expert in biochemistry and has over 12 years of human
genetic diagnostics experience. Until 2006, she supervised a diagnostic unit for
BRCA gene testing at the Interdisciplinary Center for Hereditary Breast Cancer
(Max Delbrück Center, Berlin, Germany). She has also been the Managing Director
of the Professional Association of German Human Geneticists, BVDH, which is a
trade association. In 2008, Hofmann became a Medical Director at LifeCodexx
(Konstanz, Germany), where she has worked on the development of a noninvasive
prenatal diagnostic test that detects chromosomal aneuploidies in fetuses. Author information:
(1)Human Genetics Group, Spanish National Cancer Centre (CNIO), Madrid, Spain;
Biomedical Network on Rare Diseases (CIBERER), Madrid, Spain.
(2)Cancer Epidemiology Centre, Cancer Council Victoria, Melbourne, Australia.
(3)Centre for Cancer Genetic Epidemiology, Department of Public Health and
Primary Care, University of Cambridge, Cambridge, United Kingdom.
(4)Human Genetics Group, Spanish National Cancer Centre (CNIO), Madrid, Spain.
(5)Genotyping Unit (CeGen), Spanish National Cancer Centre (CNIO), Madrid,
Spain.
(6)IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy.
(7)Genetic Counseling Unit, Hereditary Cancer Program, IDIBELL-Catalan Institute
of Oncology, Barcelona, Spain.
(8)Molecular Oncology Laboratory, Hospital Clinico San Carlos, IdISSC, Madrid,
Spain.
(9)Institute of Biology and Molecular Genetics, Universidad de Valladolid
(IBGM-UVA), Valladolid, Spain.
(10)Oncogenetics Laboratory, University Hospital Vall d'Hebron, Vall d'Hebron
Institute of Oncology (VHIO), Vall d'Hebron Institut de Recerca (VHIR), and
Universitat Autonoma de Barcelona, Barcelona, Spain.
(11)Oncology Service, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.
(12)Molecular Diagnostics Laboratory IRRP, National Centre for Scientific
Research Demokritos Aghia Paraskevi Attikis, Athens, Greece.
(13)Molecular Genetics Laboratory (Department of Biochemistry), Cruces Hospital
Barakaldo, Bizkaia, Spain.
(14)Medical Oncology Service, Hospital Clínico Lozano Blesa, San Juan Bosco,
Zaragoza, Spain.
(15)Cancer Genomics Laboratory, Centre Hospitalier Universitaire de Québec and
Laval University, Quebec City, Canada.
(16)Department of Oncology, Lund University, Lund, Sweden.
(17)Department of Oncology, Karolinska University Hospital, Stockholm, Sweden.
(18)Department of Clinical Genetics, Karolinska University Hospital, Stockholm,
Sweden.
(19)Department of Oncology, Lund University Hospital, Lund, Sweden.
(20)Department of Clinical Genetics, Lund University Hospital, Lund, Sweden.
(21)Center for Clinical Cancer Genetics and Global Health, University of Chicago
Medical Center, Chicago, Illinois, United States of America.
(22)Departments of Medicine, Epidemiology, and Biostatistics, University of
California, San Francisco, San Francisco, California, United States of America.
(23)Abramson Cancer Center and Department of Medicine, The University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of
America.
(24)Abramson Cancer Center and Center for Clinical Epidemiology and
Biostatistics, The University of Pennsylvania Perelman School of Medicine,
Philadelphia, Pennsylvania, United States of America.
(25)University of Texas MD Anderson Cancer Center, Houston, Texas, United States
of America.
(26)Women's Cancer Program at the Samuel Oschin Comprehensive Cancer Institute,
Cedars-Sinai Medical Center, Los Angeles, California, United States of America.
(27)Department of Epidemiology, Cancer Prevention Institute of California,
Fremont, California, United States of America.
(28)Department of Health Research & Policy, Stanford University School of
Medicine, Stanford, California, United States of America.
(29)Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of
America.
(30)Genetic Epidemiology Laboratory, Department of Pathology, University of
Melbourne, Parkville, Australia.
(31)Centre for Molecular, Environmental, Genetic and Analytic Epidemiology,
University of Melbourne, Melbourne, Victoria, Australia.
(32)Department of Epidemiology, Columbia University, New York, New York, United
States of America.
(33)Department of Oncological Sciences, Huntsman Cancer Institute, University of
Utah School of Medicine, Salt Lake City, Utah, United States of America.
(34)Vilnius University Hospital Santariskiu Clinics, Hematology, oncology and
transfusion medicine center, Department of Molecular and Regenerative Medicine,
Vilnius, Lithuania.
(35)Department of Genetics, University of Pretoria, Pretoria, South Africa.
(36)Department of Population Sciences, Beckman Research Institute of City of
Hope, Duarte, California, United States of America.
(37)Center for Genomic Medicine, Rigshospitalet, University of Copenhagen,
Copenhagen, Denmark.
(38)Department of Oncology, Rigshospitalet, University of Copenhagen,
Copenhagen, Denmark.
(39)Department of Clinical Genetics, Rigshospitalet, University of Copenhagen,
Copenhagen, Denmark.
(40)Clinical Cancer Genetics, City of Hope, Duarte, California, United States of
America.
(41)Unit of Medical Genetics, Department of Preventive and Predictive Medicine,
Fondazione IRCCS Istituto Nazionale Tumori (INT), Milan, Italy.
(42)Division of Cancer Prevention and Genetics, Istituto Europeo di Oncologia,
Milan, Italy.
(43)IFOM, Fondazione Istituto FIRC di Oncologia Molecolare and Cogentech Cancer
Genetic Test Laboratory, Milan, Italy.
(44)Division of Experimental Oncology 1, Centro di Riferimento Oncologico,
IRCCS, Aviano, Italy.
(45)Unit of Hereditary Cancer, Department of Epidemiology, Prevention and
Special Functions, IRCCS AOU San Martino - IST Istituto Nazionale per la Ricerca
sul Cancro, Genoa, Italy.
(46)Unit of Medical Genetics, Department of Biomedical, Experimental and
Clinical Sciences, University of Florence, Florence, Italy.
(47)Department of Molecular Medicine, "Sapienza" University, Rome, Italy.
(48)UO Anatomia Patologica, Ospedale di Circolo-Università dell'Insubria,
Varese, Italy.
(49)Unit of Molecular bases of genetic risk and genetic testing, Department of
Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazionale Tumori
(INT), Milan, Italy.
(50)Dana-Farber Cancer Institute, Boston, Massachusetts, United States of
America.
(51)Genetic Medicine, Manchester Academic Health Sciences Centre, Central
Manchester University Hospitals NHS Foundation Trust, Manchester, United
Kingdom.
(52)South East Thames Regional Genetics Service, Guy's Hospital London, United
Kingdom.
(53)Oncogenetics Team, The Institute of Cancer Research and Royal Marsden NHS
Foundation Trust, London, United Kingdom.
(54)Yorkshire Regional Genetics Service, Leeds, United Kingdom.
(55)Ferguson-Smith Centre for Clinical Genetics, Yorkhill Hospitals, Glasgow,
United Kingdom.
(56)West Midlands Regional Genetics Service, Birmingham Women's Hospital
Healthcare NHS Trust, Edgbaston, Birmingham, United Kingdom.
(57)Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton,
United Kingdom.
(58)Sheffield Clinical Genetics Service, Sheffield Children's Hospital,
Sheffield, United Kingdom.
(59)Clinical Genetics Department, St Georges Hospital, University of London,
London, United Kingdom.
(60)Department of Clinical Genetics, Royal Devon & Exeter Hospital, Exeter,
United Kingdom.
(61)Department of Clinical Genetics, East Anglian Regional Genetics Service,
Addenbrookes Hospital, Cambridge, United Kingdom.
(62)Institute of Human Genetics, Centre for Life, Newcastle Upon Tyne Hospitals
NHS Trust, Newcastle upon Tyne, United Kingdom.
(63)South East of Scotland Regional Genetics Service, Western General Hospital,
Edinburgh, United Kingdom.
(64)North East Thames Regional Genetics Service, Great Ormond Street Hospital
for Children NHS Trust, London, United Kingdom.
(65)Oxford Regional Genetics Service, Churchill Hospital, Oxford, United
Kingdom.
(66)Northern Ireland Regional Genetics Centre, Belfast City Hospital, Belfast,
United Kingdom.
(67)South West Regional Genetics Service, Bristol, United Kingdom.
(68)Academic Unit of Clinical and Molecular Oncology, Trinity College Dublin and
St James's Hospital, Dublin, Eire.
(69)Cheshire & Merseyside Clinical Genetics Service, Liverpool Women's NHS
Foundation Trust, Liverpool, United Kingdom.
(70)Department of Pathology and Laboratory Medicine, University of Kansas
Medical Center, Kansas City, Kansas, United States of America.
(71)Centre of Familial Breast and Ovarian Cancer and Centre for Integrated
Oncology (CIO), University Hospital of Cologne, Cologne, Germany.
(72)Institute for Medical Informatics, Statistics and Epidemiology, University
of Leipzig, Leipzig, Germany.
(73)Department of Gynaecology and Obstetrics, Division of Tumor Genetics,
Klinikum rechts der Isar, Technical University Munich, Munich, Germany.
(74)Department of Gynecology and Obstetrics, University Medical Center
Schleswig-Holstein, Campus Kiel, Kiel, Germany.
(75)Institute of Human Genetics, University Medical Center Schleswig-Holstein,
Campus Kiel, Kiel, Germany.
(76)Department of Gynaecology and Obstetrics, University Hospital Düsseldorf,
Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
(77)Institute of Human Genetics, Department of Human Genetics, University
Hospital Heidelberg, Heidelberg, Germany.
(78)Department of Gynaecology and Obstetrics, University Hospital Ulm, Ulm,
Germany.
(79)Institute of Cell and Molecular Pathology, Hannover Medical School,
Hannover, Germany.
(80)Institute of Human Genetics, University of Münster, Münster, Germany.
(81)Department of Gynaecology and Obstetrics, University Hospital Carl Gustav
Carus, Technical University Dresden, Dresden, Germany.
(82)Institute of Human Genetics, Campus Virchov Klinikum, Charite Berlin,
Berlin, Germany.
(83)Centre of Familial Breast and Ovarian Cancer, Department of Medical
Genetics, Institute of Human Genetics, University Würzburg, Würzburg, Germany.
(84)Institut Curie, Department of Tumour Biology, Paris, France; Institut Curie,
INSERM U830, Paris, France; Université Paris Descartes, Sorbonne Paris Cité,
Paris, France.
(85)Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Hospices
Civils de Lyon - Centre Léon Bérard, Lyon, France; INSERM U1052, CNRS UMR5286,
Université Lyon 1, Centre de Recherche en Cancérologie de Lyon, Lyon, France.
(86)INSERM U1052, CNRS UMR5286, Université Lyon 1, Centre de Recherche en
Cancérologie de Lyon, Lyon, France.
(87)Center for Medical Genetics, Ghent University, Ghent, Belgium.
(88)Gynecologic Oncology Group Statistical and Data Center, Roswell Park Cancer
Institute, Buffalo, New York, United States of America.
(89)Prince of Wales Hospital. Sydney, Australia.
(90)Ohio State University, Columbus Cancer Council, Columbus, Ohio, United
States of America.
(91)Division of Gynecologic Oncology, NorthShore University HealthSystem,
Evanston, Illinois, United States of America.
(92)Division of Gynecologic Oncology, NorthShore University HealthSystem,
Chicago, Illinois, United States of America.
(93)For Tufts Medical Center, Boston, Massachusetts, United States of America.
(94)Yale University School of Medicine, New Haven, Connecticut, United States of
America.
(95)Department of Obstetrics and Gynecology, University of Helsinki and Helsinki
University Central Hospital, Helsinki, Finland.
(96)Department of Epidemiology, Netherlands Cancer Institute, Amsterdam, The
Netherlands.
(97)Department of Clinical Genetics, Academic Medical Center, Amsterdam, The
Netherlands.
(98)Family Cancer Clinic, Netherlands Cancer Institute, Amsterdam, The
Netherlands.
(99)Department of Clinical Genetics, VU University Medical Centre, Amsterdam,
The Netherlands.
(100)University of Groningen, University Medical Center Groningen, Department of
Genetics, Groningen, The Netherlands.
(101)Department of Clinical Genetics, Leiden University Medical Center Leiden,
Leiden, The Netherlands.
(102)Department of Clinical Genetics and GROW, School for Oncology and
Developmental Biology, MUMC, Maastricht, The Netherlands.
(103)Department of Human Genetics, Radboud University Nijmegen Medical Centre,
Nijmegen, The Netherlands.
(104)Department of Clinical Genetics, Family Cancer Clinic, Erasmus University
Medical Center, Rotterdam, The Netherlands.
(105)Department of Pathology, Family Cancer Clinic, Erasmus University Medical
Center, Rotterdam, The Netherlands.
(106)Department of Medical Genetics, University Medical Center Utrecht, Utrecht,
The Netherlands.
(107)Department of Human Genetics & Department of Pathology, Leiden University
Medical Center, Leiden, The Netherlands.
(108)The Hereditary Breast and Ovarian Cancer Research Group, Netherlands Cancer
Institute, Amsterdam, The Netherlands.
(109)Department of Molecular Genetics, National Institute of Oncology, Budapest,
Hungary.
(110)Molecular Diagnostic Unit, Hereditary Cancer Program, IDIBELL-Catalan
Institute of Oncology, Barcelona, Spain.
(111)Department of Genetics and Pathology, Pomeranian Medical University,
Szczecin, Poland.
(112)Department of Genetics and Pathology, Pomeranian Medical University,
Szczecin, Poland; Postgraduate School of Molecular Medicine, Warsaw Medical
University, Warsaw, Poland.
(113)Department of Oncology, Landspitali University Hospital and BMC, Faculty of
Medicine, University of Iceland, Reykjavik Iceland.
(114)Laboratoire de Diagnostic Génétique et Service d'Onco-hématologie, Hopitaux
Universitaire de Strasbourg, CHRU Nouvel Hôpital Civil, Strasbourg, France.
(115)Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV -
IRCCS, Padua, Italy.
(116)Department of Genetics, Portuguese Oncology Institute, Porto, and
Biomedical Sciences Institute (ICBAS), Porto University, Porto, Portugal.
(117)Department of Genetics and Computational Biology, Queensland Institute of
Medical Research, Brisbane, Australia.
(118)Kathleen Cuningham Consortium for Research into Familial Breast Cancer,
Peter MacCallum Cancer Center, Melbourne, Australia.
(119)Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota,
United States of America.
(120)Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester,
Minnesota, United States of America.
(121)Center for Individualized Medicine, Mayo Clinic, Scottsdale, Arizona,
United States of America.
(122)Center for Translational Cancer Research, Department of Biological
Sciences, University of Delaware, Newark, Delaware, United States of America.
(123)Clinical Genetics Service, Memorial Sloan-Kettering Cancer Center, New
York, New York, United States of America; Cancer Biology and Genetics Program,
Memorial Sloan-Kettering Cancer Center, New York, New York, United States of
America.
(124)Clinical Genetics Service, Memorial Sloan-Kettering Cancer Center, New
York, New York, United States of America.
(125)Diagnostic Molecular Genetics Laboratory, Memorial Sloan-Kettering Cancer
Center, New York, New York, United States of America.
(126)Department of OB/GYN and Comprehensive Cancer Center, Medical University of
Vienna, Vienna, Austria.
(127)Department of Cancer Epidemiology, Moffitt Cancer Center, Tampa, Florida,
United States of America.
(128)Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics,
National Cancer Institute, National Institutes of Health, Rockville, Maryland,
United States of America.
(129)Clalit National Israeli Cancer Control Center, Haifa, Israel.
(130)Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto,
Ontario, Canada, and Cancer Care Ontario, Departments of Molecular Genetics and
Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario,
Canada.
(131)Department of Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, Ontario, Canada; Laboratory Medicine Program, University Health
Network, Toronto, Ontario, Canada.
(132)Ontario Cancer Genetics Network: Samuel Lunenfeld Research Institute, Mount
Sinai Hospital, Toronto, Ontario, Canada.
(133)Division of Human Cancer Genetics, Departments of Internal Medicine and
Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer
Center, The Ohio State University, Columbus, Ohio, United States of America.
(134)Department of Clinical Genetics, Vejle Hospital, Vejle, Denmark.
(135)Section of Molecular Diagnostics, Department of Clinical Biochemistry,
Aalborg University Hospital, Aalborg, Denmark.
(136)Department of Clinical Genetics, Aarhus University Hospital, Aarhus,
Denmark.
(137)Department of Clinical Genetics, Odense University Hospital, Odense,
Denmark.
(138)Sheba Medical Center, Tel Aviv, Israel.
(139)Canada Research Chair in Oncogenetics, Cancer Genomics Laboratory, Centre
Hospitalier Universitaire de Québec and Laval University, Quebec City, Canada.
(140)Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota,
United States of America; Department of Laboratory Medicine and Pathology, Mayo
Clinic, Rochester, Minnesota, United States of America.
(141)Human Genetics Group, Spanish National Cancer Centre (CNIO), Madrid, Spain;
Biomedical Network on Rare Diseases (CIBERER), Madrid, Spain; Genotyping Unit
(CeGen), Spanish National Cancer Centre (CNIO), Madrid, Spain. PURPOSE: Little is known about how women with hereditary breast and/or ovarian
cancer who test positive for a BRCA gene manage the impact of a positive test
result on their everyday lives and in the longer term. This study defined the
experience and needs of women with hereditary breast and ovarian cancer and a
positive BRCA test over time.
METHODS: A grounded theory approach was taken using qualitative interviews (n =
49) and reflective diaries. Data collected from December 2006 until March 2010
was analysed using the constant comparative technique to trace the development
of how women manage their concerns of inherited cancer.
RESULTS: A four stage substantive theory of maximising survival was generated
that defines the experience of women and how they resolve their main concerns.
The process of maximising survival begins prior to genetic testing in women from
high risk families as they expect to get a cancer diagnosis at some time. Women
with cancer felt they had experienced the worst with a cancer diagnosis and
altruistically tested for the sake of their children but a positive test result
temporarily shifted their focus to decision-making around their personal health
needs.
CONCLUSION: This study adds to clinical practice through raising awareness and
adding insights into how women cope with living with inherited cancer risk and
the personal and familial ramifications that ensue from it. A clear
multi-professional structured care pathway for women from genetic testing result
disclosure to undergoing risk-reducing surgery and/or surveillance should be
developed. The assessment of BRCA1 and BRCA2 coding sequences to identify pathogenic
mutations associated with inherited breast/ovarian cancer syndrome has provided
a method to identify high-risk individuals, allowing them to seek preventative
treatments and strategies. However, the current test is expensive, and cannot
differentiate between pathogenic variants and those that may be benign. Focusing
only on one of the two BRCA partners, we have developed a biological assay for
haploinsufficiency of BRCA1. Using a series of EBV-transformed cell lines, we
explored gene expression patterns in cells that were BRCA1 wildtype compared to
those that carried (heterozygous) BRCA1 pathogenic mutations. We identified a
subset of 43 genes whose combined expression pattern is a sensitive predictor of
BRCA1 status. The gene set was disproportionately made up of genes involved in
cellular differentiation, lending credence to the hypothesis that single copy
loss of BRCA1 function may impact differentiation, rendering cells more
susceptible to undergoing maligt processes. BACKGROUND: Frequent recurrent mutations in the breast and ovarian cancer
susceptibility (BRCA) genes BRCA1 and BRCA2 among Hispanics, including a large
rearrangement Mexican founder mutation (BRCA1 exon 9-12 deletion [ex9-12del]),
suggest that an ancestry-informed BRCA-testing strategy could reduce disparities
and promote cancer prevention by enabling economic screening for hereditary
breast and ovarian cancer in Mexico.
METHODS: In a multistage approach, 188 patients with cancer who were unselected
for family cancer history (92 with ovarian cancer and 96 with breast cancer)
were screened for BRCA mutations using a Hispanic mutation panel (HISPANEL) of
115 recurrent mutations in a multiplex assay (114 were screened on a mass
spectroscopy platform, and a polymerase chain reaction assay was used to screen
for the BRCA1 ex9-12del mutation). This was followed by sequencing of all BRCA
exons and adjacent intronic regions and a BRCA1 multiplex ligation-dependent
probe amplification assay (MLPA) for HISPANEL-negative patients. BRCA mutation
prevalence was calculated and correlated with histology and tumor receptor
status, and HISPANEL sensitivity was estimated.
RESULTS: BRCA mutations were detected in 26 of 92 patients (28%) with ovarian
cancer, in 14 of 96 patients (15%) with breast cancer overall, and in 9 of 33
patients (27%) who had tumors that were negative for estrogen receptor,
progesterone receptor, and human epithelial growth factor 2 (triple-negative
breast cancer). Most patients with breast cancer were diagnosed with locally
advanced disease. The Mexican founder mutation (BRCA1 ex9-12del) accounted for
35% of BRCA-associated ovarian cancers and 29% of BRCA-associated breast
cancers. At 2% of the sequencing and MLPA cost, HISPANEL detected 68% of all
BRCA mutations.
CONCLUSIONS: In this study, a remarkably high prevalence of BRCA mutations was
observed among patients with ovarian cancer and breast cancer who were not
selected for family history, and the BRCA1 ex9-12del mutation explained 33% of
the total. The remarkable frequency of BRCA1 ex9-12del in Mexico City supports a
nearby origin of this Mexican founder mutation and may constitute a regional
public health problem. The HISPANEL mutation panel presents a translational
opportunity for cost-effective genetic testing to enable breast and ovarian
cancer prevention. HBOC is the most common and well-described hereditary breast cancer syndrome and
is often at the center of professional recommendations, accreditation standards,
and insurance company coverage policies. A person’s BRCA gene mutation status
may alter their decisions about surgical treatment, eligibility for a clinical
trial, and their approach to cancer risk reduction and screening. The potential
for knowledge gained from undergoing BRCA gene testing is great, but there are
limitations and pitfalls of which patients should be aware before providing
informed consent, including the possibility of uncertain or uninformative
results, potential for psychological distress, and effect on family members. As
such, it is important for clinicians across the health care spectrum to be able
to appropriately identify patients at risk of having HBOC, understand the effect
that this diagnosis has on their patients with and without cancer, and be able
to identify resources to support their patients throughout genetic testing
process. OBJECTIVE: The aim of our study was to determine the rate of participation in
genetic testing, to determine the reasons for non-participation and to identify
the factors affecting participation in BRCA genetic testing for high-risk
patients.
METHODS: This study was performed through a retrospective review of 804
individuals who underwent genetic counseling for BRCA1/2 gene mutations at Seoul
National University Bundang Hospital between July 2003 and September 2012.
RESULTS: In total, 728 (90.5%) individuals underwent BRCA1/2 mutation screening
after the initial genetic counseling; 88.2% of 647 probands and 100% of 157
family members were screened. In multivariate analysis, family history of breast
cancer and younger age were independent variables affecting participation in
genetic testing. Of the 132 people who initially declined genetic testing, 58
(43.9%) postponed the decision, 30 (22.7%) needed time to discuss the issue with
family members, 22 (16.7%) did not want to know if they had a BRCA1/2 mutation,
and 22 (16.7%) declined the test because of ficial problems. When analyzing
refusal of testing according to the time period before and after the
implementation of national health insurance coverage for BRCA1/2 genetic
testing, the critical reason given for refusal was different. After insurance
coverage, refusal for ficial reason was decreased from 61.1 to 9.6%.
CONCLUSIONS: A family history of breast cancer and a younger age were important
factors associated with participation in genetic testing. National health
insurance decreased the proportion of individuals who did not participate in
testing owing to a ficial reason. In genetic counseling, we have to
understand these issues and consider several factors that may influence an
individual's decision to be tested. Author information:
(1)Abramson Cancer Center, Perelman School of Medicine at the University of
Pennsylvania, Philadelphia2Center for Clinical Epidemiology and Biostatistics,
Perelman School of Medicine at the University of Pennsylvania, Philadelphia.
(2)Center for Clinical Epidemiology and Biostatistics, Perelman School of
Medicine at the University of Pennsylvania, Philadelphia.
(3)Centre de Recherche en Cancérologie de Lyon, UMR Inserm, Centre Léon Bérard,
Lyon, France.
(4)Department of Genetics and Computational Biology, Queensland Institute of
Medical Research, Brisbane, Australia.
(5)Centre for Cancer Genetic Epidemiology, Department of Public Health and
Primary Care, University of Cambridge, Cambridge, United Kingdom.
(6)Abramson Cancer Center, Perelman School of Medicine at the University of
Pennsylvania, Philadelphia6Department of Medicine, Perelman School of Medicine
at the University of Pennsylvania, Philadelphia.
(7)Sheba Medical Center, Tel Aviv, Israel.
(8)Susanne Levy Gertner Oncogenetics Unit, Sheba Medical Center, Tel Hashomer,
Israel.
(9)Oncology Institute, Sheba Medical Center, Tel Hashomer, Israel.
(10)Oncology Institute, Rivkah Ziv Medical Center Zefat, Israel.
(11)Department of Oncology, Lund University, Lund, Sweden12Department of
Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
(12)Division of Clinical Genetics, Department of Clinical and Experimental
Medicine, Linköping University, Linköping, Sweden.
(13)Department of Oncology, Sahlgrenska University Hospital, Gothenburg, Sweden.
(14)Department of Oncology, Lund University, Lund, Sweden.
(15)Department of Clinical Genetics, Karolinska University Hospital, Stockholm,
Sweden.
(16)Department of Radiation Sciences, Oncology, Umeå University, Umeå, Sweden.
(17)Center for Clinical Cancer Genetics and Global Health, University of Chicago
Medical Center, Chicago, Illinois.
(18)UCLA Schools of Medicine and Public Health, Division of Cancer Prevention
and Control Research, Jonsson Comprehensive Cancer Center, Los Angeles,
California.
(19)Department of Medicine and Genetics, University of California, San
Francisco.
(20)Cancer Risk Program, Helen Diller Family Cancer Center, University of
California, San Francisco.
(21)Department of Gynecologic Oncology, Roswell Park Cancer Institute, Buffalo,
New York.
(22)Department of Preventive Medicine, Keck School of Medicine, University of
Southern California, Los Angeles.
(23)University of Texas MD Anderson Cancer Center, Houston.
(24)Division of Cancer Medicine, Peter MacCallum Cancer Centre, East Melbourne,
Victoria, Australia 25Sir Peter MacCallum Department of Oncology, University of
Melbourne, Melbourne, Australia.
(25)Women's Cancer Program at the Samuel Oschin Comprehensive Cancer Institute,
Cedars-Sinai Medical Center, Los Angeles, California.
(26)Department of Pathology and Laboratory Medicine, University of Kansas
Medical Center, Kansas City.
(27)Fox Chase Cancer Center, Philadelphia, Pennsylvania.
(28)Department of Health Research and Policy, Stanford University School of
Medicine, Stanford, California.
(29)Department of Epidemiology, Cancer Prevention Institute of California,
Fremont.
(30)Dana-Farber Cancer Institute, Boston, Massachusetts.
(31)Department of Epidemiology, Columbia University, New York, New York.
(32)Departments of Pediatrics and Medicine, Columbia University, New York, New
York.
(33)Department of Dermatology, University of Utah School of Medicine, Salt Lake
City.
(34)Department of Oncological Sciences, Huntsman Cancer Institute, University of
Utah School of Medicine, Salt Lake City.
(35)Vilnius University Hospital Santariskiu Clinics, Hematology, Oncology, and
Transfusion Medicine Center, Department of Molecular and Regenerative Medicine,
State Research Institute Innovative Medicine Center, Vilnius, Lithuania.
(36)Latvian Biomedical Research and Study Centre, Riga, Latvia.
(37)Department of Medical Oncology, Beth Israel Deaconess Medical Center,
Boston, Massachusetts.
(38)Department of Genetics, University of Pretoria, Pretoria, South Africa.
(39)Department of Population Sciences, Beckman Research Institute of City of
Hope, Duarte, California.
(40)Departments of Oncology or Clinical Genetics, Rigshospitalet, Copenhagen
University Hospital, Copenhagen, Denmark.
(41)Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital,
Copenhagen, Denmark.
(42)Oncology Service, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.
(43)Human Genetics Group, Spanish National Cancer Centre (CNIO), and Biomedical
Network on Rare Diseases (CIBERER), Madrid, Spain.
(44)Human Genetics Group and Genotyping Unit, Spanish National Cancer Centre
(CNIO), and Biomedical Network on Rare Diseases (CIBERER), Madrid, Spain.
(45)Hospital clinico Universitario "Lozano Blesa," Instituto de investigación
sanitaria de Aragón (IIS), Zaragoza, Spain.
(46)Molecular Genetics Laboratory (Department of Genetics), Cruces University
Hospital Barakaldo, Bizkaia, Spain.
(47)Institute of Biology and Molecular Genetics. Universidad de Valladolid
(IBGM-UVA), Valladolid, Spain.
(48)Clinical Cancer Genetics, City of Hope Clinical Cancer Genetics Community
Research Network, Duarte, California.
(49)Unit of Genetic Counselling, Medical Oncology Department, Istituto Nazionale
Tumori Regina Elena, Rome, Italy.
(50)Department of Medical Science, University of Turin, and AO Città della
Salute e della Scienza, Turin, Italy.
(51)Unit of Medical Genetics, Department of Preventive and Predictive Medicine,
Fondazione IRCCS Istituto Nazionale dei Tumori (INT), Milan, Italy.
(52)Division of Cancer Prevention and Genetics, Istituto Europeo di Oncologia,
Milan, Italy.
(53)AO Città della Salute e della Scienza, Turin, Italy.
(54)Department of Molecular Medicine, University La Sapienza, Rome, Italy.
(55)Department of Experimental Oncology, Istituto Europeo di Oncologia, Milan,
Italy57Cogentech Cancer Genetic Test Laboratory, Milan, Italy.
(56)Institute of Medical Genetics, Catholic University, "A. Gemelli" Hospital,
Rome, Italy.
(57)Unit of Molecular Bases of Genetic Risk and Genetic Testing, Department of
Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazionale Tumori
(INT), Milan, Italy60IFOM, Fondazione Istituto FIRC di Oncologia Molecolare,
Milan, Italy.
(58)Cancer Bioimmunotherapy Unit, Centro di Riferimento Oncologico, IRCCSCRO
Aviano National Cancer Institute, Aviano (PN), Italy.
(59)Cogentech Cancer Genetic Test Laboratory, Milan, Italy60IFOM, Fondazione
Istituto FIRC di Oncologia Molecolare, Milan, Italy.
(60)Unit of Hereditary Cancer, IRCCS AOU San Martino-IST Istituto Nazionale per
la Ricerca sul Cancro, Genoa, Italy.
(61)Molecular Diagnostics Laboratory, IRRP, National Centre for Scientific
Research "Demokritos" Aghia Paraskevi Attikis, Athens, Greece.
(62)Instituto de Genética Humana, Pontificia Universidad Javeriana, Bogotá,
Colombia65Molecular Genetics of Breast Cancer, Deutsches Krebsforschungszentrum
(DKFZ), Heidelberg, Germany.
(63)Molecular Genetics of Breast Cancer, Deutsches Krebsforschungszentrum
(DKFZ), Heidelberg, Germany 66Department of Basic Sciences, Shaukat Khanum
Memorial Cancer Hospital and Research Centre (SKMCH & RC), Lahore, Pakistan.
(64)Molecular Genetics of Breast Cancer, Deutsches Krebsforschungszentrum
(DKFZ), Heidelberg, Germany.
(65)Genetic Medicine, Manchester Academic Health Sciences Centre, Central
Manchester University Hospitals, NHS Foundation Trust, Manchester, United
Kingdom.
(66)Oncogenetics Team, Institute of Cancer Research and Royal Marsden, NHS
Foundation Trust, London, United Kingdom.
(67)Ferguson-Smith Centre for Clinical Genetics, Yorkhill Hospitals, Glasgow,
United Kingdom.
(68)Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton,
United Kingdom.
(69)West Midlands Regional Genetics Service, Birmingham Women's Hospital
Healthcare NHS Trust, Edgbaston, Birmingham, United Kingdom.
(70)Sheffield Clinical Genetics Service, Sheffield Children's Hospital,
Sheffield, United Kingdom.
(71)Department of Clinical Genetics, Royal Devon and Exeter Hospital, Exeter,
United Kingdom.
(72)Clinical Genetics Department, St Georges Hospital, University of London,
United Kingdom.
(73)Northern Ireland Regional Genetics Centre, Belfast City Hospital, Belfast,
United Kingdom.
(74)Oxford Regional Genetics Service, Churchill Hospital, Oxford, United
Kingdom.
(75)South East of Scotland Regional Genetics Service, Western General Hospital,
Edinburgh, United Kingdom.
(76)Academic Unit of Clinical and Molecular Oncology, Trinity College Dublin and
St James's Hospital, Dublin, Eire.
(77)South East Thames Regional Genetics Service, Guy's Hospital London, United
Kingdom.
(78)Yorkshire Regional Genetics Service, Leeds, United Kingdom.
(79)South West Regional Genetics Service, Bristol, United Kingdom.
(80)Department of Hematology and Oncology, University of Kansas Medical Center,
Kansas City.
(81)Center for Hereditary Breast and Ovarian Cancer, Center for Integrated
Oncology (CIO), and Center for Molecular Medicine Cologne (CMMC), Medical
Faculty, University of Cologne and University Hospital Cologne, Cologne,
Germany.
(82)Institute for Medical Informatics, Statistics and Epidemiology, University
of Leipzig, Leipzig, Germany.
(83)Department of Gynaecology and Obstetrics, Division of Tumor Genetics,
Klinikum rechts der Isar, Technical University Munich, Munich, Germany.
(84)Department of Gynecology and Obstetrics, University Medical Center
Schleswig-Holstein, Campus Kiel, Germany.
(85)Institute of Human Genetics, University Medical Center Schleswig-Holstein,
Campus Kiel, Germany.
(86)Department of Gynaecology and Obstetrics, University Hospital Düsseldorf,
Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
(87)Institute of Human Genetics, Department of Human Genetics, University
Hospital Heidelberg, Heidelberg, Germany.
(88)Department of Gynaecology and Obstetrics, University Hospital Ulm, Ulm,
Germany.
(89)Institute of Cell and Molecular Pathology, Hannover Medical School,
Hannover, Germany.
(90)Department of Gynaecology and Obstetrics, University Hospital Carl Gustav
Carus, Technical University Dresden, Dresden, Germany.
(91)Institute of Human Genetics, Technical University Dresden, Dresden, Germany.
(92)Institute of Human Genetics, Campus Virchov Klinikum, Charite Berlin,
Berlin, Germany.
(93)Centre of Familial Breast and Ovarian Cancer, Department of Medical
Genetics, Institute of Human Genetics, University of Würzburg, Würzburg,
Germany.
(94)Institute of Human Genetics, University of Regensburg, Regensburg, Germany.
(95)Institut Curie, Department of Tumour Biology, Paris, France98Institut Curie,
INSERM U830, Paris, France99Université Paris Descartes, Sorbonne Paris Cité,
France.
(96)Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Hospices
Civils de Lyon-Centre Léon Bérard, Lyon, France101INSERM U1052, CNRS UMR5286,
Université Lyon 1, Centre de Recherche en Cancérologie de Lyon, Lyon, France.
(97)INSERM U1052, CNRS UMR5286, Université Lyon 1, Centre de Recherche en
Cancérologie de Lyon, Lyon, France.
(98)Institut Curie, Department of Tumour Biology, Paris, France99Université
Paris Descartes, Sorbonne Paris Cité, France.
(99)Institut Curie, Department of Tumour Biology, Paris, France.
(100)Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Hospices
Civils de Lyon-Centre Léon Bérard, Lyon, France.
(101)Université Lyon 1, CNRS UMR5558, Lyon, France103Unité de Prévention et
d'Epidémiologie Génétique, Centre Léon Bérard, Lyon, France.
(102)Département Oncologie Génétique, Prévention et Dépistage, INSERM CIC-P9502,
Institut Paoli-Calmettes/Université d'Aix-Marseille II, Marseille, France.
(103)Laboratoire d'Oncologie Moléculaire Humaine, Centre Oscar Lambret, Lille,
France.
(104)Unité d'Oncogénétique, CLCC Paul Strauss, Strasbourg, France.
(105)Service de Génétique Biologique-Histologie-Biologie du Développement et de
la Reproduction, Centre Hospitalier Universitaire de Besançon, Besançon, France.
(106)Service de Génétique, Centre Hospitalier Universitaire Bretonneau, Tours,
France.
(107)Oncogénétique Clinique, Hôpital René Huguenin/Institut Curie, Saint-Cloud,
France.
(108)Laboratoire d'Oncogénétique, Hôpital René Huguenin/Institut Curie,
Saint-Cloud, France.
(109)Lombardi Comprehensive Cancer Center, Georgetown University, Washington,
DC.
(110)Center for Medical Genetics, Ghent University, Ghent, Belgium.
(111)GOG Statistical and Data Center, Buffalo, New York.
(112)Evanston Hospital, Evanston, Illinois.
(113)Tufts University, Medford, Massachusetts.
(114)University of North Carolina, Chapel Hill.
(115)New York University, New York, New York.
(116)Ohio State, Good Samaritan Hospital, Cincinnati.
(117)Yale University School of Medicine, New Haven, Connecticut.
(118)Molecular Oncology Laboratory, Hospital Clinico San Carlos, IdISSC, Madrid,
Spain.
(119)Department of Obstetrics and Gynecology, University of Helsinki and
Helsinki University Central Hospital, Helsinki, Finland.
(120)Department of Clinical Genetics, Helsinki University Central Hospital,
Helsinki, Finland.
(121)Department of Genetics, University Medical Center, Groningen University,
Groningen, The Netherlands.
(122)Family Cancer Clinic, Netherlands Cancer Institute, Amsterdam, The
Netherlands.
(123)Department of Clinical Genetics, Family Cancer Clinic, Erasmus University
Medical Center, Rotterdam, The Netherlands.
(124)Department of Medical Oncology, Family Cancer Clinic, Erasmus University
Medical Center, Rotterdam, The Netherlands.
(125)Department of Clinical Genetics, VU University Medical Centre, Amsterdam,
The Netherlands.
(126)Department of Human Genetics and Department of Clinical Genetics, Leiden
University Medical Center, Leiden, The Netherlands.
(127)Department of Clinical Genetics and GROW, School for Oncology and
Developmental Biology, MUMC, Maastricht, The Netherlands.
(128)Department of Human Genetics, Radboud University Nijmegen Medical Centre,
Nijmegen, The Netherlands.
(129)Department of Medical Genetics, University Medical Center Utrecht, Utrecht,
The Netherlands.
(130)Department of Clinical Genetics, Academic Medical Center, Amsterdam, The
Netherlands.
(131)Department of Human Genetics and Department of Pathology, Leiden University
Medical Center, Leiden, The Netherlands.
(132)Hong Kong Hereditary Breast Cancer Family Registry, Hong Kong135Cancer
Genetics Center, Hong Kong Sanatorium and Hospital, Hong Kong136Department of
Surgery, University of Hong Kong, Hong Kong.
(133)Department of Molecular Genetics, National Institute of Oncology, Budapest,
Hungary.
(134)Oncogenetics Laboratory, Vall d'Hebron Institute of Oncology (VHIO),
Universitat Autonoma de Barcelona, Barcelona, Spain139University Hospital of
Vall d'Hebron, Barcelona, Spain.
(135)Molecular Diagnostic Unit, Hereditary Cancer Program, IDIBELL-Catalan
Institute of Oncology, Barcelona, Spain.
(136)Genetic Counseling Unit, Hereditary Cancer Program, IDIBGI-Catalan
Institute of Oncology, Girona, Spain.
(137)Genetic Counseling Unit, Hereditary Cancer Program, IDIBELL-Catalan
Institute of Oncology, Barcelona, Spain.
(138)Department of Genetics and Pathology, Pomeranian Medical University,
Szczecin, Poland.
(139)Department of Genetics and Pathology, Pomeranian Medical University,
Szczecin, Poland144Postgraduate School of Molecular Medicine, Warsaw Medical
University, Warsaw, Poland.
(140)Department of Surgical Gynecology and Gynecological Oncology of Adults and
Adolescents, Pomeranian Medical University, Szczecin, Poland.
(141)Department of Pathology, Landspitali University Hospital, Reykjavík,
Iceland147BMC, Faculty of Medicine, University of Iceland, Reykjavik, Iceland.
(142)Canada Research Chair in Oncogenetics, Cancer Genomics Laboratory, Centre
Hospitalier Universitaire de Québec Research Center, Quebec City, Quebec,
Canada149Laval University, Quebec City, Quebec, Canada.
(143)Medical Genetics Division, Centre Hospitalier Universitaire de Québec,
Quebec City, Quebec, Canada151Immunology and Molecular Oncology Unit, Veneto
Institute of Oncology, IOV-IRCCS, Padua, Italy.
(144)Immunology and Molecular Oncology Unit, Veneto Institute of Oncology,
IOV-IRCCS, Padua, Italy.
(145)Department of Genetics, Portuguese Oncology Institute, Porto, Portugal.
(146)Department of Genetics, Portuguese Oncology Institute, Porto,
Portugal153Biomedical Sciences Institute (ICBAS), University of Porto, Portugal.
(147)Department of Surgery, Soonchunhyang University and Hospital, Seoul, Korea.
(148)Department of Preventive Medicine, Seoul National University College of
Medicine and Cancer Research Institute, Seoul National University, Seoul, Korea.
(149)Department of Surgery, Daerim St Mary's Hospital, Seoul, Korea.
(150)Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester,
Minnesota159Department of Health Sciences Research, Mayo Clinic, Rochester,
Minnesota.
(151)Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota.
(152)Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester,
Minnesota.
(153)Program in Cancer Genetics, Departments of Human Genetics and Oncology,
McGill University, Montreal, Quebec, Canada161Department of Medical Genetics,
University of Cambridge, Cambridge, United Kingdom.
(154)Department of Cancer Epidemiology and Genetics, Masaryk Memorial Cancer
Institute and MF MU, Brno, Czech Republic.
(155)Clinical Genetics Service, Cancer Biology and Genetics Program, Memorial
Sloan-Kettering Cancer Center, New York, New York.
(156)Clinical Genetics Service, Memorial Sloan-Kettering Cancer Center, New
York, New York.
(157)Department of OB/GYN and Comprehensive Cancer Center, Medical University of
Vienna, Vienna, Austria.
(158)Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics,
National Cancer Institute, National Institutes of Health, Rockville, Maryland.
(159)N. N. Petrov Institute of Oncology, St Petersburg, Russia.
(160)Divison of Human Cancer Genetics, Departments of Internal Medicine and
Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer
Center, Ohio State University, Columbus.
(161)Divison of Human Genetics, Department of Internal Medicine, Comprehensive
Cancer Center, Ohio State University, Columbus.
(162)Department of Clinical Genetics, Vejle Hospital, Vejle, Denmark.
(163)Section of Molecular Diagnostics, Department of Clinical Biochemistry,
Aalborg University Hospital, Aalborg, Denmark.
(164)Department of Clinical Genetics, Aarhus University Hospital, Aarhus N,
Denmark.
(165)Department of Clinical Genetics, Odense University Hospital, Odense C,
Denmark.
(166)Section of Genetic Oncology, Department of Oncology, University of Pisa and
University Hospital of Pisa, Pisa, Italy.
(167)Cancer Research Initiatives Foundation, Sime Darby Medical Centre, Subang
Jaya, Malaysia176Department of Surgery, Faculty of Medicine, University Malaya
Cancer Research Institute, University Malaya, Kuala Lumpur, Malaysia.
(168)NorthShore University HealthSystem, Department of Medicine, Evanston,
Illinois.
(169)Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital and University
of Toronto, Toronto, Ontario, Canada. The Korean Hereditary Breast Cancer (KOHBRA) study was established to evaluate
the prevalence and spectrum of BRCA1/2 mutations in Korean breast cancer
patients at risk for hereditary breast and ovarian cancer. A total of 2953
subjects (2403 index patients and 550 family members of affected carriers) from
36 centers participated in this study between May 2007 and December 2013. All
participants received genetic counseling and BRCA genetic testing. In total, 378
mutation carriers among 2403 index patients were identified. The prevalence of
BRCA mutations in specific subgroups was as follows: 22.3 % (274/1228) for
breast cancer patients with a family history of breast/ovarian cancers, 8.8 %
(39/441) for patients with early-onset (<35 years) breast cancer without a
family history, 16.3 % (34/209) for patients with bilateral breast cancer, 4.8 %
(1/21) for male patients with breast cancer, and 37.5 % (3/8) for patients with
both breast and ovarian cancer. From an analysis of the mutation spectrum, 63
BRCA1 and 90 BRCA2 different mutations, including 44 novel mutations, were
identified. The c.7480 (p.Arg2494Ter) mutation in BRCA2 (10.1 %) was the most
commonly identified in this cohort. The KOHBRA study is the largest cohort to
identify BRCA mutation carriers in Asia. The results suggest that the prevalence
of BRCA mutations in familial breast cancer patients is similar to that among
Western cohorts. However, some single risk factors without family histories
(early-onset breast cancer, male breast cancer, or multiple organ cancers) may
limit the utility of BRCA gene testing in the Korean population. Germline BRCA gene mutations are reportedly associated with hereditary breast
and ovarian cancers. Identification of BRCA mutations greatly improves the
preventive strategies and management of breast cancer. Sanger sequencing has
been the gold standard in identifying these mutations. However, 4-28% of
inherited BRCA mutations may be due to large genomic rearrangements (LGRs),
which could be missed by using Sanger sequencing alone. Our aim is to evaluate
the pick-up rate of LGRs in our cohort. A total of 1,236 clinically high-risk
patients with breast and/or ovarian cancers were recruited through The Hong Kong
Hereditary Breast Cancer Family Registry from 2007 to 2014. Full gene sequencing
(either Sanger or next generation sequencing) and multiplex ligation-dependent
probe amplification (MLPA) were performed. We identified 120 deleterious BRCA
mutations: 57 (4.61%) were in BRCA1 and 63 (5.10%) were in BRCA2. LGRs accounted
for 6.67% (8 of 120) of all BRCA mutations, whereas 8.77 % (5 of 57) were BRCA1
mutations and 4.76% (3 of 63) were BRCA2 mutations. Through this integrated
approach, both small nucleotide variations and LGRs could be detected. We
suggest that MLPA should be incorporated into the standard practice for genetic
testing to avoid false-negative results, which would greatly affect the
management of these high-risk families. OBJECTIVES: To investigate and analyze the BRCA mutations in Korean ovarian
cancer patients with or without family history and to find founder mutations in
this group.
METHODS/MATERIALS: One hundred two patients who underwent a staging operation
for pathologically proven epithelial cancer between January 2013 and December
2014 were enrolled. Thirty-two patients declined to analyze BRCA1/2 gene
alterations after genetic counseling and pedigree analysis. Lymphocyte specimens
from peripheral blood were assessed for BRCA1/2 by direct sequencing.
RESULTS: BRCA genetic test results of 70 patients were available. Eighteen
BRCA1/2 mutations and 17 unclassified variations (UVs) were found. Five of the
BRCA1/2 mutations and 4 of the UVs were not reported in the Breast Cancer
Information Core database. One BRCA2 UV (8665_8667delGGA) was strongly
suspicious to be a deleterious mutation. BRCA1/2 mutations were identified in 11
(61.1%) of 18 patients with a family history and in 7 (13.5%) of 52 patients
without a family history.Candidates for founder mutations in Korean ovarian
cancer patients were assessed among 39 BRCA1/2 mutations from the present study
and from literature reviews. The analysis showed that 1041_1043delAGCinsT (n =
4; 10.2%) and 3746insA (n = 4; 10.2%) were possible BRCA1 founder mutations.
Only one of the BRCA2 mutations (5804_5807delTTAA) was repeated twice (n = 2;
5.1%).
CONCLUSIONS: The prevalence of BRCA1/2 mutations in Korean ovarian cancer
patients irrespective of the family history was significantly higher than
previously reported. Possible founder mutations in Korean ovarian cancer
patients were identified. BACKGROUND: PALB2 (Partner and Localizer of BRCA2) was identified as a
moderate-risk gene in breast and pancreatic cancers. Recently, it was reported
that PALB2 carriers have a high risk of developing breast cancer, with the
cumulative risk of 34 % by the age of 70.
PATIENTS AND METHODS: Peripheral blood samples from 155 patients at risk for
hereditary breast and/or ovarian cancer were tested for BRCA1/2 and PALB2 by
targeted sequencing using a next-generation sequencer. Of these 155, 146 met
NCCN criteria and the remaining 9 did not.
RESULTS: BRCA1/2 analysis was performed on 155 patients, for whom the results
were reported previously (Hirotsu Y et al. Mol Genet Genomic Med,
doi:10.1002/mgg3.157, 2015). Eleven patients were identified to have deleterious
BRCA mutations (Hirotsu Y et al. Mol Genet Genomic Med, doi:10.1002/mgg3.157,
2015). However, none of the 155 patients were found to have deleterious PALB2
germline mutations. Missense mutations [variants of uncertain significance
(VUS)] of PALB2 were found in 12 cases. In silico analyses by SIFT (Sorting
Intolerant Form Tolerant) and PolyPhen2 (Polymorphism Phenotyping version 2)
indicated that 2 of 12 VUS were deleterious and probably damaging.
CONCLUSIONS: This is the first report on PALB2 mutations in Japan, revealing two
missense mutations as "deleterious and probably damaging" by in silico analyses,
but no PALB2 premature truncation mutations were identified. The sample size is
relatively small and a larger cohort study is needed in Japan. BACKGROUND: The PALB2 (FANCN) gene was identified as a component of endogenous
BRCA2 complex that encodes a DNA repair protein participating along with BRCA1
and BRCA 2 proteins in DNA double-strand break repair. Hereditary PALB2
mutations are associated with an increased risk of breast and pancreatic cancers
in heterozygotes. Breast cancer risk for PALB2 mutation carriers has recently
been estimated at 33-58% depending on family history of breast cancer;
pancreatic cancer risk in carriers of PALB2 mutations has not been precisely
quantified, yet.
MATERIALS AND RESULTS: Results of a study identifying PALB2 mutations in
high-risk, BRCA1/2-negative, breast and/or ovarian cancer patients in the Czech
Republic indicate that the frequency of hereditary PALB2 mutations in our
population is quite high. Interestingly, almost 20% of all recognized mutations
comprised large genomic rearrangements. The highest proportion of PALB2
mutations (comparable with the number of mutations reported for BRCA2) was found
in a subgroup of hereditary breast cancer patients (5.5%). Frequency of
mutations in an independent group of Czech unselected pancreatic cancer patients
was approximately 1.3%.
CONCLUSION: Considering the frequency of pathogenic, hereditary PALB2 mutations
in our population, their phenotypic similarity to BRCA2, and expected risk of
breast cancer associated with PALB2 mutations, its screen-ing (including large
genomic rearrangements) should be encouraged in patients from hereditary breast
cancer families. The follow-up of pathogenic PALB2 mutation carriers should be
similar to that in BRCA2 mutation carriers, enabling early diagnosis,
prevention, and possible targeted therapy. Preventive surgical interventions for
the carriers could be considered in case of strong family cancer history and
evident segregation of a pathogenic mutation with a tumor phenotype. Additional
analysis of various cancer patient populations and further meta-analyses will be
necessary for accurate assessment of PALB2 gene penetrance and its significance
for the risk of pancreatic and other cancers. Technological advances in DNA sequencing have made gene testing fast and more
affordable. Evidence of effectiveness and cost-effectiveness of genetic service
models is essential for the successful translation of sequencing improvements
for patient benefit, but remain sparse in the genetics literature. In
particular, there is a lack of detailed cost data related to genetic services. A
detailed micro-costing of 28 possible pathways relating to breast and/or ovarian
cancer and BRCA testing was carried out by defining service activities and
establishing associated costs. These data were combined with patient-level data
from a Royal Marsden Cancer Genetics Service audit over a 6-month period during
which BRCA testing was offered to individuals at ≥10 % risk of having a
mutation, in line with current NICE guidance. The average cost across all
patient pathways was £2227.39 (range £376.51 to £13,553.10). The average cost
per pathway for an affected person was £1897.75 compared to £2410.53 for an
unaffected person. Of the women seen in the Cancer Genetics Service during the
audit, 38 % were affected with breast and/or ovarian cancer, and 62 % were
unaffected but concerned about their family history. The most efficient service
strategy is to identify unaffected relatives from an affected individual with an
identified BRCA mutation. Implementation of this strategy would require more
comprehensive testing of all eligible cancer patients, which could be achieved
by integrating BRCA testing into oncology services. Such integration would be
also more time-efficient and deliver greater equity of access to BRCA testing
than the standard service model. BACKGROUND: Breast cancer associated (BRCA) genes are critical for DNA repair.
Mutations in BRCA1 and BRCA2 (BRCAm) result in loss of these repair mechanisms
and potential carcinogenesis. Germline BRCAm are common in ovarian carcinomas,
particularly in platinum-sensitive disease. The increased prevalence of BRCAm in
platinum-sensitive disease is likely due to enhanced responsiveness to platinum
chemotherapy from homologous recombination repair deficiency. The purpose of
this study was to explore BRCA testing, treatment patterns and survival in
platinum-sensitive recurrent (PSR) ovarian cancer.
METHODS: This was an observational cohort analysis of PSR ovarian cancer treated
at the Huntsman Cancer Institute from 1995 to 2012. Germline BRCA status was
ascertained through chart review and categorized as BRCAm (BRCA1/2 positive),
BRCAwt (BRCA wild type or variant of uncertain significance), and untested.
Treatment patterns and survival were assessed from recurrence until death or
last follow-up. The Kaplan-Meier method was used to evaluate survival from
recurrence by BRCA status. Logistic regression and COX proportional hazard model
was used to estimate predictors of BRCA testing and survival, respectively.
RESULTS: Of the 168 PSR patients, 15 (9 %) were BRCAm, 25 (15 %) were BRCAwt,
and 128 (76 %) were untested. Median age at PSR was 56 years for BRCAm and
BRCAwt (p = 0.90) and 63 years for those untested (p = 0.033 vs BRCAm). Overall
survival was similar between BRCAm and BRCAwt (median 50.4 vs 67.5 months, p =
0.86) and was 24.9 months in untested patients. Significant predictors for the
likelihood of BRCA testing were age (OR = 0.93, 95 % CI 0.89, 0.97, p = 0.002),
family history of breast or ovarian cancer (OR = 8.33, 95 % CI: 3.08, 22.59, p <
0.001), and cancer diagnosis year (OR = 10.02, 95 % CI: 3.22, 31.21, p < 0.001).
BRCA-tested patients had a lower risk of death versus untested (HR 0.35, 95 % CI
0.17, 0.68, p = 0.001).
CONCLUSIONS: BRCAwt patients had similar outcomes to BRCAm patients, potentially
owing to similar age at diagnosis, representing a BRCA testing channeling bias.
Younger patients, those with a family history of breast or ovarian cancer, and
those diagnosed more recently were more likely to be BRCA tested. BRCA tested
patients had a lower risk of death. Since the 2013 Supreme Court ruling on BRCA1/BRCA2 patenting, hereditary cancer
gene panels now include BRCA1 and BRCA2, making these panels an option for
first-tier testing. However, questions remain about the clinical utility and
implications of these panels for medical management with inclusion of genes of
unknown to moderate penetrance. To better understand how use of these panels
affected our practice, we reviewed patients who underwent testing in our clinic
from July 1, 2013 through May 23, 2014. Indications for testing included
personal and/or family history of breast and/or ovarian cancer. A total of 136
patients underwent panel testing via a single commercial laboratory; 12 (8.8 %)
patients were positive for a pathogenic or likely pathogenic mutation (four
BRCA2 mutations, two TP53 mutations, one CDH1 mutation, two ATM mutations, and
one patient each with a CHEK2, NBN, or PALB2 mutation). Of these positive
patients, 100 % met the National Comprehensive Cancer Network (NCCN) guidelines
for Hereditary Breast and Ovarian Cancer genetic testing (2.2014). Mutations in
seven of twelve (58 %) patients led to changes in medical management; three of
seven (43 %) had a non-BRCA1 or BRCA2 gene mutation. Our findings suggest that
there is clinical utility of panels that include genes of unknown to moderate
penetrance. BACKGROUND: Mutations in the BRCA1 and BRCA2 genes are associated with increased
risk of breast, ovarian, and several other cancers. The purpose of the present
study was to evaluate the incidence of cancer in first- and second-degree
relatives of BRCA mutation carriers compared with the general population.
MATERIALS AND METHODS: A total of 1,086 pedigrees of BRCA mutation carriers was
obtained from a prospectively maintained, internal review board-approved study
of persons referred for clinical genetic counseling at the University of Texas
MD Anderson Cancer Center. We identified 9,032 first- and second-degree
relatives from 784 pedigrees that had demonstrated a clear indication of
parental origin of mutation. Standardized incidence ratios (SIRs) were used to
compare the observed incidence of 20 primary cancer sites to the expected
incidence of each cancer based on the calculated risk estimates according to
each subject's age, sex, and ethnicity.
RESULTS: BRCA1 families had increased SIRs for breast and ovarian cancer (p <
.001) and decreased SIRs for kidney, lung, prostate, and thyroid cancer and
non-Hodgkin's lymphoma (p < .001). BRCA2 families had increased SIRs for breast,
ovarian, and pancreatic cancer (p < .001) and decreased SIRs for kidney, lung,
thyroid, and uterine cancer and non-Hodgkin's lymphoma (p < .0025). Analysis of
only first-degree relatives (n = 4,099) identified no decreased SIRs and agreed
with the increased SIRs observed in the overall study population.
CONCLUSION: We have confirmed previous reports of an association between breast,
ovarian, and pancreatic cancers with BRCA mutations. Additional research to
quantify the relative risks of these cancers for BRCA mutation carriers can help
tailor recommendations for risk reduction and enhance genetic counseling.
IMPLICATIONS FOR PRACTICE: BRCA gene mutations have been well described to carry
an increased risk of both breast and ovarian cancer. However, the implications
and risks of other cancers continues to be investigated. Evaluating the risks
for other cancers further is key in identifying and managing risk reduction
strategies. OBJECTIVE: The presence of deleterious mutations in breast cancer (BRCA)-1 or
BRCA-2 gene has a decisive influence on the development of various types of
neoplasms, such as breast, ovarian, tubal, and peritoneal cancers. Primary
peritoneal cancer is an aggressive maligcy which, due to the absence of a
specific screening test, cannot be diagnosed in its early stages. As a
risk-reducing option, prophylactic bilateral salpingo-oophorectomy and
mastectomy are often proposed in BRCA gene carriers. The effectiveness of a
preventive surgical treatment is, however, unclear in the development of
peritoneal cancer.
MATERIAL AND METHODS: An extensive electronic search was performed in PubMed,
Scopus, and Cochrane databases.
RESULTS: The total number of patients who underwent prophylactic bilateral
salpingo-oophorectomy was 1,830, of whom 28 presented with peritoneal cancer
(1.53%). The age of the included patients ranged from 48 to 61 years. BRCA-1 was
present in 9 out of 28 patients and BRCA-2 in 2 patients, while the type of BRCA
was unclear in 17 patients. Salpingo-oophorectomy was performed in 23 out of 28
patients, while oophorectomy was carried out in 5 patients. The interval from
initial risk-reducing surgical treatment to the presentation of peritoneal
cancer ranged from 12 to 84 months.
CONCLUSION: Modification of the follow-up guidelines and increase in healthcare
providers' awareness may reduce the risk of peritoneal cancer. During the past years, several empirical and statistical models have been
developed to discriminate between carriers and non-carriers of germline
BRCA1/BRCA2 (breast cancer 1, early onset/breast cancer 2, early onset)
mutations in families with hereditary breast or ovarian cancer. Among these, the
BRCAPRO or CaGene model is commonly used during genetic counseling, and plays a
central role in the identification of potential carriers of BRCA1/2 mutations.
We compared performance and clinical applicability of BRCAPRO version 5.1 vs
version 6.0 in order to assess diagnostic accuracy of updated version. The study
was carried out on 517 pedigrees of patients with familial history of breast or
ovarian cancer, 150 of which were submitted to BRCA1/2 mutation screening,
according to BRCAPRO evaluation or to criteria based on familial history. In our
study, CaGene 5.1 was more sensitive than CaGene 6.0, although the latter showed
a higher specificity. Both BRCAPRO versions better discriminate BRCA1 than BRCA2
mutations. This study evidenced similar performances in the two BRCAPRO versions
even if the CaGene 6.0 has underestimated the genetic risk prediction in some
BRCA mutation-positive families. Genetic counselors should recognize this
limitation and during genetic counseling would be advisable to use a set of
criteria in order to improve mutation carrier prediction. |
What is Desomorphine? | Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin Desomorphine is the semi-synthetic opioid claimed to be the main component of krokodil | In order to summarize current knowledge about the drug "Krokodil" a systematic
review including a literature search of the databases PubMed, Embase, Scopus,
and Google was conducted in December 2011. According to information acquired,
"Krokodil" is a mixture of several substances and was first reported to have
been used in Russia in 2003. The core agent of "Krokodil" is desomorphine, an
opioid-analogue that can be easily and cheaply manufactured by oneself.
Self-production results in a contaminated suspension that is injected
intravenously. Due to its pharmacologic features, desomorphine shows a high
potential to cause dependence. Against the background of first possible cases of
"Krokodil" use in Western Europe, it appears advisable to provide information
regarding the fatal consequences of "Krokodil." A systematic review was conducted to identify the available data for the term
Krokodil, which is a jargon expression for an allegedly new drug. Krokodil seems
to be a mixture of several substances and was first used in Russia in 2003, with
a tremendous increase in the number of addicted individuals since then. The
psychoactive core agent of Krokodil is desomorphine, an opioid-analogon that can
be manufactured by boiling tablets containing codeine and other ingredients. The
procedure results in a suspension that is used intravenously and regularly
causes complications such as abscess, thrombophlebitis, and gangrene. conduct needs assessments and treatment compliance evaluations in MMT and
Suboxone Substitution State Programs in Georgia (Republic of). 506 patients (2
females) were surveyed (92% on Methadone, 8% on Suboxone) from 6 Tbilisi and 4
regional State Programs in 2011 November. Mean age - 40±8,56 (22-65) year; 254
(51.4%) were in treatment for 1-3 year. Evaluation was carried out on the base
of structured self-questionnaire that covers demographics, drug use history,
general drug use trends, psychotherapeutic sessions' acceptance and open label
question regarding treatment challenges and satisfaction. 305 (60.3%) attended
individual and 57 (11.3%) group psychotherapy sessions with 50.79% attending
once/month or rare. The main reason given for therapy non-attendance - no needs
for it (29.48%); the main drugs before admission - heroin (80.04%),
buprenorphine (53.49%); Main drugs used in Georgia nowadays - desomorphine
("crocodile"), alcohol and marihuana. Commonly used drugs by program patients
(136 positive answers) - alcohol-13.62%, marihuana-10.39%, pregabalin - 8.17%,
opioids- 6.62% (mostly-"crocodile"), home-made stimulants-6.23%, sedatives
-5.45%. 55.4% are extremely satisfied with treatment, 82.4% - with program
staff. Patients' main wishes- free of charge programs (46.4%) and provide
take-home doses (22.07%). Methadone and Suboxone ST are being well accepted in
Georgia and appear to be reducing illegal opioid use. However, the
psychotherapeutic sessions' attendance is very low. "Krokodil" is the street name for the semi-synthetic opioid derivative
desomorphine. Although an old drug, it re-staged on "drug arena" during the last
decade causing detrimental effects to its users. Despite the fact that Russia
and other former Soviet Republics were the initial plagued countries, "krokodil"
arrived in Europe and United States lately, as a substitute of the relative
expensive, and in many cases unavailable, heroin. It can be easily manufactured
in home-environment from codeine and causes significant health problems, even
deaths worldwide. The aim of this review is to summarize the current knowledge
about this drug, concerning its chemistry, synthesis, pharmacology and
toxicology. Published or reported "krokodil" related cases, fatalities or
intoxications, as well as self reports from drug users are reviewed. The
existing analytical methodologies for the determination of desomorphine in
biological samples as well as its legal status are also presented. BACKGROUND: Krokodil is an informal term for a cheap injectable illicit drug
domestically prepared from codeine-containing medication (CCM). The method of
krokodil preparation may produce desomorphine as well as toxic reactants that
cause extensive tissue necrosis. The first confirmed report of krokodil use in
Russia took place in 2004. In 2012, reports of krokodil-related injection
injuries began to appear beyond Russia in Western Europe and the United States.
OBJECTIVE: This exploratory study had two main objectives: (1) to determine if
Internet search patterns could detect regularities in behavioral responses to
Russian CCM policy at the population level, and (2) to determine if
complementary data sources could explain the regularities we observed.
METHODS: First, we obtained krokodil-related search pattern data for each Russia
subregion (oblast) between 2011 and 2012. Second, we analyzed several
complementary data sources included krokodil-related court cases, and related
search terms on both Google and Yandex to evaluate the characteristics of terms
accompanying krokodil-related search queries.
RESULTS: In the 6 months preceding CCM sales restrictions, 21 of Russia's 83
oblasts had search rates higher than the national average (mean) of 16.67
searches per 100,000 population for terms associated with krokodil. In the 6
months following restrictions, mean national searches dropped to 9.65 per
100,000. Further, the number of oblasts recording a higher than average search
rate dropped from 30 to 16. Second, we found krokodil-related court appearances
were moderately positively correlated (Spearman correlation=.506, P≤.001) with
behaviors consistent with an interest in the production and use of krokodil
across Russia. Finally, Google Trends and Google and Yandex related terms
suggested consistent public interest in the production and use of krokodil as
well as for CCM as analgesic medication during the date range covered by this
study.
CONCLUSIONS: Illicit drug use data are generally regarded as difficult to obtain
through traditional survey methods. Our analysis suggests it is plausible that
Yandex search behavior served as a proxy for patterns of krokodil production and
use during the date range we investigated. More generally, this study
demonstrates the application of novel methods recently used by policy makers to
both monitor illicit drug use and influence drug policy decision making. Author information:
(1)Department of Legal Medicine and Forensic Sciences, Faculty of Medicine,
University of Porto, Porto, Portugal; REQUIMTE, Laboratory of Toxicology,
Department of Biological Sciences, Faculty of Pharmacy, University of Porto,
Porto, Portugal; EPSJV-Polythecnical School of Health Joaquim Venâncio, Oswaldo
Cruz Foundation, Rio de Janeiro, Brazil. Electronic address:
[email protected].
(2)DoA-CUNI-Addiction Research Centre, Utrecht, The Netherlands; Department of
Addictology, 1st Faculty of Medicine, Charles University in Prague and General
University Hospital in Prague, Prague, Czech Republic.
(3)Center of Medical Chemistry (CEQUIMED-UP), Faculty of Pharmacy, University of
Porto, Porto, Portugal; Interdisciplinary Center of Marine and Environmental
Investigation (CIIMAR/CIMAR) , Porto, Portugal.
(4)Department of Analytical Chemistry, Chemistry Institute, Fluminense Federal
University, Niterói, Brazil.
(5)REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences,
Faculty of Pharmacy, University of Porto, Porto, Portugal.
(6)Department of Legal Medicine and Forensic Sciences, Faculty of Medicine,
University of Porto, Porto, Portugal; REQUIMTE, Laboratory of Toxicology,
Department of Biological Sciences, Faculty of Pharmacy, University of Porto,
Porto, Portugal; IINFACTS-Institute of Research and Advanced Training in Health
Sciences and Technologies, Department of Sciences, Advanced Institute of Health
Sciences-North (ISCS-N), CESPU, CRL, Gandra, Portugal. Electronic address:
[email protected]. Author information:
(1)UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences,
Faculty of Pharmacy, University of Porto, Porto, Portugal; Department of Legal
Medicine and Forensic Sciences, Faculty of Medicine, University of Porto, Porto,
Portugal; EPSJV - Polytechnic School of Health Joaquim Venâncio, Oswaldo Cruz
Foundation, Rio de Janeiro, Brazil. Electronic address: [email protected].
(2)REQUIMTE, Department of Chemical Sciences, Laboratory of Applied Chemistry,
Faculty of Pharmacy, University of Porto, Porto, Portugal.
(3)Center of Medical Chemistry (CEQUIMED-UP), Faculty of Pharmacy, University of
Porto, Porto, Portugal; Interdisciplinary Center of Marine and Environmental
Investigation (CIIMAR/CIMAR), Porto, Portugal.
(4)CVO - Addiction Research Centre, Utrecht, Netherlands; Department of
Addictology, 1st Faculty of Medicine, Charles University in Prague and General
University Hospital in Prague, Czech Republic.
(5)Department of Legal Medicine and Forensic Sciences, Faculty of Medicine,
University of Porto, Porto, Portugal.
(6)Center of Medical Chemistry (CEQUIMED-UP), Faculty of Pharmacy, University of
Porto, Porto, Portugal.
(7)Department of Analytical Chemistry, Chemistry Institute, Fluminense Federal
University, Niterói, Brazil.
(8)UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences,
Faculty of Pharmacy, University of Porto, Porto, Portugal.
(9)UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences,
Faculty of Pharmacy, University of Porto, Porto, Portugal; Department of Legal
Medicine and Forensic Sciences, Faculty of Medicine, University of Porto, Porto,
Portugal; IINFACTS - Institute of Research and Advanced Training in Health
Sciences and Technologies, Department of Sciences, University Institute of
Health Sciences (IUCS), CESPU, CRL, Gandra, Portugal. Electronic address:
[email protected]. Injecting drug user size estimation studies carried out in 2009, 2012 and 2015
revealed growing trends of drug abuse in Georgia:estimated number of people who
inject drugs (PWID) have been increased from 40000 and 45000 to 50000. Since
Soviet period the most popular injective narcotics have been opioids: home-made
opium, heroine, buprenorphine and home-made desomorphine ("Krokodile") replacing
each other on the black market. Self-made desomorphine typically contains big
amounts of different toxic substances and causes significant somatic disorders,
especially skin, bone, blood infections, liver and kidney failure; is highly
addictive, associates with frequent injections that enhance injecting-related
harm, including the risk of HIV transmission, in comparison with typical
opioids. The aim of the study was to determine the effectiveness of opioid
substitution treatment (OST) on depression and anxiety in opioid dependent
clients with history of different opioid substance use. 104 opioid drug users
undergoing OST with intensive psychological counseling have been divided in 5
groups according to the principal opioid drug that was abused during past 6
months before starting treatment: heroine, desomorphine, illicit methadone
injectors, illicit buprenorphine injectors, and multiple drug abusers consuming
opioids as primary drugs. Level of depression (Beck Depression Inventory),
anxiety (Spielberger Anxiety Inventory) as well as clinical symptoms, risky
behavior, quality of life (WHO), and other data were measured before starting
and after 3, 9, 15, 21 months of treatment. The illegal use of
psychotropic-narcotics was checked through random urine-testing 1-2 times per
patient per month. In all five groups remarkable decrease of depression and
anxiety was observed in comparison with the starting data. Before inclusion
desomorphine and poly-drug users had the highest scores of depression and
anxiety while buprenorphine users manifested the lowest rate. Improvement of
figures was observed in all groups in three month period that have been
continuing during 21 month of treatment process. Study revealed normalization of
scores forbothstatesin groups of heroine, desomorphine, methadone and
buprenorphine users. The highest scores of depression and anxiety were observed
in the group of poly-drug abusers andwhile depression rate hesitated in the
range of clear "no-depression", anxiety index still remained close to the
clinical important threshold after 21 month of treatment. Urine-testingon
psychotropic-narcotic substances indicated remarkable decrease of illegal drug
abuse in all studied groups in three month and although abuse of benzodiazepine
drugs was highest in desomorphine and poly-drug abusers, the difference between
groups was not statistically significant. Although some disparities have been
observed in dynamics of subjects with different spectrum of initial opioid
substance use, including homemade desomorphine, there is no significant
difference between groups and OST effectively supports to improve depression and
anxiety indices, and dramatically decreases use of illegal psychotropic-narcotic
drugs during treatment. However poly-drug users seems to be the most resistant
to achieve stabilization and require more treatment time and targeted
interventions. |
Which two drugs are included in the Harvoni pill? | Harvoni contains 400 mg sofosbuvir and 90 mg ledipasvir. It used for treatment of hepatitis C virus infection. | Ledipasvir/sofosbuvir (Harvoni) for hepatitis C virus genotype 1 infection;
dulaglutide (Trulicity) for glycemic control in type-2 diabetes;
netupitant/palonosetron (Akynzeo) for prevention of nausea and vomiting related
to chemotherapy; and naloxegol (Movantik) for opioid-induced constipation in
patients with chronic noncancer pain. The single-tablet regimen of the hepatitis C virus (HCV) NS5A inhibitor
ledipasvir and the HCV NS5B polymerase inhibitor sofosbuvir
(ledipasvir/sofosbuvir; Harvoni(®)) was recently approved in the US and the EU.
The phase III ION trials included treatment-naive (ION-1 and -3) or
treatment-experienced (ION-2) patients with chronic HCV genotype 1 infection
(≈20 % of patients in ION-1 and -2 had cirrhosis, whereas no patient in ION-3
had cirrhosis). A sustained virological response 12 weeks' post-treatment
(SVR12) was seen in 99 % of treatment-naive patients receiving
ledipasvir/sofosbuvir for 12 weeks in ION-1, with no additional benefit
conferred by the addition of ribavirin or extending the treatment duration to 24
weeks. Moreover, in ION-3, an 8-week regimen achieved an SVR12 rate of 94 %
overall and 97 % in the subgroup of patients with a baseline HCV RNA level of <6
million IU/mL. SVR12 rates of 94 and 99 % were seen in treatment-experienced
patients who received ledipasvir/sofosbuvir for 12 and 24 weeks in ION-2. Data
also support the use of ledipasvir/sofosbuvir in chronic HCV genotype 4
infection, in HCV and HIV co-infection and, in combination with ribavirin, in
patients with chronic HCV genotype 1 or 4 infection who have decompensated
cirrhosis or are liver transplant recipients and in chronic HCV genotype 3
infection. Oral ledipasvir/sofosbuvir was generally well tolerated. In
conclusion, ledipasvir/sofosbuvir is an important new single-tablet regimen that
represents a significant advance in the treatment of chronic hepatitis C. PURPOSE: Public discourse regarding the hepatitis C virus (HCV) drug Sovaldi®
(sofosbuvir) has become inflamed, generating much heat but little light
concerning the clinical, health economic, and quality-of-life merits of
Sovaldi®. The purpose of this article is to provide a factual basis for
evaluating the claims regarding the benefits of Sovaldi® relative to its costs.
METHODS: A comprehensive review was conducted of news stories highlighted in the
daily updates of the electronic newsletters BIO SmartBrief, FiercePharma,
FierceBiotech and BioCentury Extra published from November 1, 2013, through
December 31, 2014, on the topics of the HCV market, Sovaldi®, and other HCV
therapeutics. Also reviewed were recent practice guidelines on the management of
HCV infections, prescribing information on all HCV drugs approved by the US Food
and Drug Administration, and health technology assessments of Sovaldi® and
Harvoni(TM) (sofosbuvir/ledipasvir).
FINDINGS: Sovaldi® and Harvoni(TM) have provided significant improvements in the
treatment of HCV, with all-oral regimens and cure rates exceeding 90% in some
populations of patients with HCV. Sovaldi® prevents significant health care
resource utilization in patients who would otherwise develop cirrhosis and
require a liver transplant; however, only a small proportion of patients with
HCV develop cirrhosis, and fewer require liver transplants. Because it is not
possible to identify those patients whose HCV will progress to severe liver
disease, it would be necessary to treat a large number of patients with HCV to
prevent disease progression in this subpopulation, resulting in a considerable
loss to health plans even over a 20-year horizon. The claim that treating all
patients with HCV with Sovaldi® would cost nearly as much as the current total
US expenditure on all prescription drugs, while factually correct, is not a
realistic scenario. Many patients with HCV will continue to go undiagnosed. In
addition, the medical expense for those who are treated will be spread out over
many years. However, the unexpectedly large, up-front cost of covering these
drugs has had a major impact on health plan budgets, resulting in losses for
some plans.
IMPLICATIONS: Sovaldi® represents an enormous advance in the care of some
populations of HCV-infected patients, but also a major cost burden to health
plans. As the first of a number of anticipated, paradigm-changing drugs to treat
medical conditions affecting large patient populations, Sovaldi® should act as a
wake-up call for all health care stakeholders to engage in a meaningful,
fact-based discussion about managing the cost of innovative new drugs to balance
the needs of drug manufacturers, health plans, providers, and, above all,
patients. Ledipasvir/Sofosbuvir (harvoni): improving options for hepatitis C virus
infection. The availability of direct-acting antiviral (DAA) therapy has launched a new era
in the management of chronic hepatitis C. Sofosbuvir, a uridine nucleotide
analog that inhibits the hepatitis C RNA-dependent RNA polymerase, is the
backbone of chronic hepatitis C therapy. Acting at the catalytic site of the
polymerase, sofosbuvir is highly potent in suppressing viral replication and has
a high genetic barrier to resistance. Sofosbuvir is effective across all
hepatitis C genotypes, and is a mainstay of interferon-free combination therapy.
In Phase II and III studies, genotype 1 patients who took sofosbuvir in
combination with another DAA such as the NS3-4A protease inhibitor, simeprevir,
or the NS5A replication complex inhibitors, ledipasvir or daclatasvir, achieved
a sustained virologic response rate of over 90%. Harvoni(®), a combination
tablet of sofosbuvir and ledipasvir, dosed once daily is recommended for 24
weeks for treatment-experienced genotype 1 patients with cirrhosis, but 12 weeks
of therapy is sufficient for all other populations. While genotype 2 (12 weeks
or 16 weeks) and treatment-naïve genotype 3 patients (24 weeks) have excellent
response rates with sofosbuvir and ribavirin, treatment-experienced cirrhotic
genotype 3 patients may need the addition of another DAA such as daclatasvir.
Sofosbuvir is efficacious in special populations such as HIV-hepatitis C
virus-coinfected patients and liver transplant recipients and has already made a
profound impact in these groups. Since it is renally eliminated, patients with
advanced kidney disease or on dialysis must await dosing recommendations.
Sofosbuvir-based regimens appear to be well tolerated with headache and fatigue
being the most common side effects. The opportunity to cure patients with
hepatitis C with sofosbuvir combination therapy is likely to change the future
for our patients, particularly if the emphasis shifts to identifying those
patients unaware that they are infected and providing affordable access to
treatment. Nucleotide compounds like sofosbuvir, acyclovir, and tenofovir have proven to be
amongst the most potent orally available antiviral treatments. These drugs
exhibit high efficacy and a wide therapeutic index, with demonstrated utility in
a number of chronic viral infections. The approval of Sovaldi™, brand name for
sofosbuvir, by the U.S. Food and Drug Administration heralded improvements in
chronic hepatitis C virus (HCV) treatment. Sofosbuvir was originally discovered
by Pharmasset Corporation and named PSI-7977. It was subsequently acquired and
advanced through phase 3 development by Gilead Sciences, Inc. In Sofosbuvir both
a unique pharmacology and a high specificity for the HCV ribonucleic acid
polymerase are present in a molecule that is well tolerated and highly
efficacious. Phase 2 and 3 clinical trials have consistently demonstrated
durable and high rates of sustained virologic response (SVR), curing patients in
excess of 80% in all genotypes and >90% in treatment-naïve subjects being
administered combination therapy with other agents. Harvoni(®) is the
combination of sofosbuvir and the NS5A inhibitor ledipasvir in a fixed-dose oral
tablet, and it has demonstrated high SVR rates in patients infected with HCV
genotype 1, without the need for exogenous interferon and/or ribavirin. Here, we
discuss the discovery, development, pharmacologic characterization, and results
from the phase 3 trials of sofosbuvir. Hepatitis C is a chronic disease, for
which most patients have been undiagnosed, are unwilling to start treatment, or
are ineligible for treatment because of the high toxicity and low efficacy of
interferon and ribavirin-based therapy. Clinical studies with sofosbuvir have
demonstrated significant improvement over the prior standard of care, thus
ushering in a new paradigm of HCV treatment and an update of treatment
guidelines. Treatment for chronic hepatitis C depends on the hepatitis C virus (HCV)
genotype and the patient's clinical characteristics. A fixed-dose combination of
ledipasvir + sofosbuvir has been authorised in the European Union for adults
with HCV genotype 1 (HCV-1), HCV-3 or HCV-4 infection. Ledipasvir targets the
HCV protein NS5A, while sofosbuvir inhibits the HCV RNA polymerase NS5B. The
ledipasvir+ sofosbuvircombination has not been compared directly with other
antiviral drugs. No information is available on its ability to prevent hepatic
complications, even in patients with cirrhosis. In four trials including over
1800 treatment-naive patients infected with HCV-1, a 12-week course of
ledipasvir + sofosbuviryielded a sustained virological response in nearly every
case. This is better than that reported with peginterferon alfa-based protocols.
In four trials including more than 900 HCV-1-infected patients in whom
treatments including peginterferon alfa had failed, a 24-week course of
ledipasvir+ sofosbuvir yielded a sustained virological response in nearly every
case, which is far better than reported with peginterferon alfa + ribavirin +
protease inhibitor combinations, based on indirect comparison. In these trials,
a 24-week course of the ledipasvir + sofosbuvir combination was effective in
almost all patients with compensated cirrhosis. The same treatment also showed
major efficacy in a non-comparative trial in 337 HCV-1-infected patients with
decompensated cirrhosis or who had undergone liver transplantation. In mid-2015,
very few data are available on the ledipasvir + sofosbuvir combination in
HCV-1-infected patients in whom sofosbuvir combination therapy has failed, or in
patients with HCV-3 or HCV-4 infection. Comparative data on the adverse effects
of the ledipasvir + sofosbuvir combination are mainly based on a double-blind,
placebo-controlled trial in 155 patients. Overall, serious adverse effects were
infrequent in this and other trials. The main adverse effects appear to be
headache, fatigue, sleep disorders, irritability and lipase elevations.
Hypertension, muscle disorders and dyspnoea are other plausible adverse effects.
Bradycardia and cardiac conduction disorders have been reported with concomitant
use of sofosbuvir and amiodarone, an antiarrhythmic drug. In practice, in
mid-2015, when drug therapy is warranted for chronic hepatitis C due to HCV
genotype 1, the ledipasvir + sofosbuvir combination is a first-choice treatment
because of its virological efficacy, despite its poorly documented adverse
effects. These important outstanding questions call for rigorous
pharmacovigilance on the part of all healthcare professionals. It is too early
to recommend the ledipasvir + sofosbuvir combination for patients infected with
other HCV genotypes. The exorbitant price imposed by Gilead endangers public
healthcare systems and undermines access to high-quality care. Ledipasvir/sofosbuvir (Harvoni®), a fixed-dose combination tablet of an NS5A
inhibitor ledipasvir and an NS5B polymerase inhibitor sofosbuvir, is approved in
the US, European Union, Canada, and other regions for the treatment of chronic
hepatitis C virus infection in adults. Following absorption, ledipasvir reaches
maximum plasma concentrations (T max) 4-4.5 h post-dose and is eliminated with a
terminal half-life (t 1/2) of 47 h. Sofosbuvir undergoes intracellular
activation to an active triphosphate GS-461203 (not detected in plasma) and
ultimately to GS-331007, a predomit circulating metabolite, which is the
primary analyte of interest in clinical pharmacology studies. Sofosbuvir is
rapidly absorbed and eliminated from plasma (T max: 0.8-1 h; t 1/2: 0.5 h). The
peak plasma concentrations for GS-331007 are achieved between 3.5 and 4 h
post-dose; the elimination t 1/2 for GS-331007 is 27 h. Ledipasvir/sofosbuvir
exhibits a favorable clinical pharmacology profile; it can be administered once
daily without regard to food and does not require dose modification in hepatitis
C virus-infected patients with any degree of hepatic impairment or mild to
moderate renal impairment. The pharmacokinetic profiles of ledipasvir,
sofosbuvir, and GS-331007 (predomit circulating metabolite of sofosbuvir) are
not significantly affected by demographic variables;
pharmacokinetic/pharmacodynamic analyses reveal no exposure-response
relationships for efficacy or safety. The review summarizes the clinical
pharmacokinetics, pharmacodynamics, and pharmacokinetic/pharmacodynamic analyses
for ledipasvir/sofosbuvir. Author information:
(1)Second Liver Cirrhosis Diagnosis and Treatment Center, 302 Hospital, Beijing,
China.
(2)Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases,
Peking University People's Hospital, Peking University Hepatology Institute,
Beijing, China.
(3)Division of Gastroenterology & Hepatology, Humanity & Health Medical Centre,
Hong Kong, Hong Kong SAR, China.
(4)The Translational Hepatology Institue, Beijing You'an Hospital, Capital
University of Medicine, Beijing, China.
(5)Department of Infectious Diseases, Shengjing Hospital, China Medical
University, Shenyang, China.
(6)Department of Infectious diseases, Shanghai Ruijin Hospital, Jiaotong
University, School of Medicine, Shanghai, China.
(7)Key Laboratory of Medical Molecular Virology, Huashan Hospital, Fudan
University, Shanghai, China Institute of Biomedical Sciences, Shanghai Medical
College, Fudan University, Shanghai, China.
(8)Department of Gastroenterology, Shanghai First People's Hospital, Shanghai
Jiao Tong University School of Medicine, Shanghai, China.
(9)Department of Gastroenterology, Xinhua Hospital, Shanghai Jiaotong University
School of Medicine, Shanghai, China.
(10)Liver Disease Center, Beijing Ditan Hospital, Capital University of
Medicine, Beijing, China.
(11)Department of Infectious Diseases, Center for Liver Diseases, Peking
University First Hospital, Beijing, China.
(12)Institute for Viral Hepatitis, Second Affiliated Hospital, Chongqing
University of Medical Sciences, Chongqing, China.
(13)Department of Infectious Disease, Xijing Hospital, Fourth Military Medical
University, Xi'an, China.
(14)Department of Infection, Sichuan Provincial People's Hospital, Chengdu,
Sichuan, China.
(15)Center of Diagnosis and Treatment for Infectious Diseases of Chinese PLA,
Tangdu Hospital, Fourth Military Medical University, Xi'an, China.
(16)Shanghai Liver Diseases Research Center, Nanjing Military Command, Shanghai,
China.
(17)People's Hospital, Shanghai Jiaotong University, School of Medicine,
Shanghai, China.
(18)Department of Infectious Diseases, He Provincal People's Hospital,
Zhengzhou University, Zhengzhou, China.
(19)Department of Infectious Disease, Xinjiang Medical University First
Affiliated Hospital, Urumqi, China.
(20)Department of Digestive Diseases, Xijing Hospital, Fourth Military Medical
University, Xi'an, China.
(21)Liver Disease Center for Combined Traditional Chinese Medicine and Western
Medicine, 302 Hospital, Beijing, China.
(22)State Key Laboratory of Organ Failure Research, Guangdong Provincial Key
Laboratory of Viral Hepatitis Research, Department of Infectious Diseases,
Nanfang Hospital, Southern Medical University, Guangzhou, China.
(23)Liver Disease Center, Beijing Friendship Hospital, Capital Medical
University, Beijing, China.
(24)Department of Microbiology and Center of Infectious Disease, School of Basic
Medical Sciences, Peking University Health Science Center, Beijing, China. A new validated bioanalytical method based on LC tandem MS has been developed
for the simultaneous extraction and determination of sofosbuvir and ledipasvir
in human plasma using antiviral daclatasvir as an internal standard (IS).
Liquid-liquid extraction of samples was used for the purification and
preconcentration of the analytes from a human plasma matrix. Good and consistent
recoveries were obtained, with average extraction recoveries of 91.61 and 88.93%
for sofosbuvir and ledipasvir, respectively. The chromatographic separation of
the three analytes was achieved within only 2.8 min by an isocratic mobile phase
consisting of 10 mM ammonium acetate, which was then adjusted to pH 4.0 by
acetic acid-acetonitrile-0.1% methanolic formic acid (12 + 25 + 63, v/v/v)
flowing through a C18 Zorbax eclipse plus column (5 μm, 100 × 4.6 mm; Agilent).
Multiple reaction monitoring transitions were measured in positive ion mode for
sofosbuvir, ledipasvir, and daclatasvir (IS). A detailed validation of the
method was performed and the standard curves were found to be linear in the
range of 0.5 to 2500 and 5 to 2100 ng/mL for sofosbuvir and ledipasvir,
respectively, applying weighted (1/X(2)) linear regression. The developed method
was applied to the analysis of the two drugs after a single oral administration
of Harvoni 400/90 mg film-coated tablets containing 400 mg sofosbuvir and 90 mg
ledipasvir to four healthy volunteers. After the introductions of sofosbuvir (Sovaldi) and ledipasvir plus sofosbuvir
(Harvoni) for the treatment of hepatitis C, employers have become very sensitive
to new, and especially unforeseen, factors that significantly raise healthcare
costs. With the recent launch of the proprotein convertase subtilisin/kexin type
9 (PCSK9) inhibitors, self-insured and fully insured employers have been seeking
information on this drug class and its potential for off-label use, which could
amount to up to $23 billion in healthcare expenditures, according to a report
from Prime Therapeutics. Based on their approved indications, 0.4% of commercial
members may be eligible to use PCSK9 inhibitors, at a cost of $3.29 per member
per month. Corporate employers are evaluating their options to manage the new
expense associated with the novel PCSK9 inhibitors. |
What is the result of Mff overexpression in mitochondria? | The Drp1 receptor Mff is a major regulator of mitochondrial fission, and its overexpression results in increased fission. | The cytoplasmic dynamin-related guanosine triphosphatase Drp1 is recruited to
mitochondria and mediates mitochondrial fission. Although the mitochondrial
outer membrane (MOM) protein Fis1 is thought to be a Drp1 receptor, this has not
been confirmed. To analyze the mechanism of Drp1 recruitment, we manipulated the
expression of mitochondrial fission and fusion proteins and demonstrated that
(a) mitochondrial fission factor (Mff) knockdown released the Drp1 foci from the
MOM accompanied by network extension, whereas Mff overexpression stimulated
mitochondrial recruitment of Drp1 accompanied by mitochondrial fission; (b)
Mff-dependent mitochondrial fission proceeded independent of Fis1; (c) a Mff
mutant with the plasma membrane-targeted CAAX motif directed Drp1 to the target
membrane; (d) Mff and Drp1 physically interacted in vitro and in vivo; (e)
exogenous stimuli-induced mitochondrial fission and apoptosis were compromised
by knockdown of Drp1 and Mff but not Fis1; and (f) conditional knockout of Fis1
in colon carcinoma cells revealed that it is dispensable for mitochondrial
fission. Thus, Mff functions as an essential factor in mitochondrial recruitment
of Drp1. elongation, constriction, and fission. Translocation of dynamin-like protein 1
(DLP1), a member of the large GTPase family, from the cytosol to peroxisomes is
a prerequisite for membrane fission; however, the molecular machinery for
peroxisomal targeting of DLP1 remains unclear. This study investigated whether
mitochondrial fission factor (Mff), which targets DLP1 to mitochondria, may also
recruit DLP1 to peroxisomes. Results show that endogenous Mff is localized to
peroxisomes, especially at the membrane-constricted regions of elongated
peroxisomes, in addition to mitochondria. Knockdown of MFF abrogates the fission
stage of peroxisomal division and is associated with failure to recruit DLP1 to
peroxisomes, while ectopic expression of MFF increases the peroxisomal targeting
of DLP1. Co-expression of MFF and PEX11β, the latter being a key player in
peroxisomal elongation, increases peroxisome abundance. Overexpression of MFF
also increases the interaction between DLP1 and Pex11pβ, which knockdown of MFF,
but not Fis1, abolishes. Moreover, results show that Pex11pβ interacts with Mff
in a DLP1-dependent manner. In conclusion, Mff contributes to the peroxisomal
targeting of DLP1 and plays a key role in the fission of the peroxisomal
membrane by acting in concert with Pex11pβ and DLP1. |
Has Glucose-6-phosphate dehydrogenase (G6PD) deficiency an X-linked inheritance? | Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest red cell enzymopathy in humans and has an X-linked inheritance. | Severe red cell glucose-6-phosphate dehydrogenase (G-6-PD) deficiency has been
found in an 'aboriginal' Finnish family. 2 male and 9 female carriers of the
variant G-6-PD were studied. The genetic pattern is consistent with x-linked
recessive inheritance and the defect is associated with drug (primaquine)
induced haemolysis. This was demonstrated by enzyme deficient red cell
(51Cr-labelled) survival studies on a normal volunteer recipient. In addition,
one of the hemizygotes studied had a slight chronic nonspherocytic haemolytic
disorder. The partially purified enzyme had many of the characteristics of
G-6-PD Mediterranean. The occurrence of this G-6-PD Mediterranean type variant
in the Finnish population, which differs greatly from Mediterranean ethnic
groups, as well as the association of slight chronic haemolysis with severe
G-6-PD deficiency is discussed. Wallaroos (Macropus robustus robustus), which have the G6PD-F electrophoretic
phenotype, crossed with euros (M.r.erubescens), of G6PD-S phenotype, produced F1
animals which had only the maternal G6PD type regardless of the direction of the
cross. When F1 hybrids were backcrossed to wallaroos or euros, backcross progeny
of either perental phenotype resulted. Sex-linked inheritance of allelic G6PD
genes is shown to occur in wallaroos, euros and red kangaroos (M. rufus). Dose
compensation for X chromosomes at the G6PD locus in kangaroow is achieved by
inactivation of the allele of male parental origin. Five hundred members belonging to the Bania community of Punjab were screened
for erythrocytic glucose-6-phosphate dehydrogenase (G-6-PD) deficiency. The
incidence of enzyme deficiency in males was 2.84 per cent and in females 2.75
per cent, with an overall incidence of 2.80 per cent. No correlation between age
and G-6-PD deficiency was found. The mean values for haemoglobin and haematocrit
did not differ significantly in the normal and deficient subjects. Study of the
deficiency pattern amongst family members of the enzyme deficient subjects
confirmed the X-linked inheritance of G-6-PD deficiency. The human X-linked gene encoding glucose 6-phosphate dehydrogenase (G6PD) is
highly polymorphic; more than 300 G6PD variants have been identified. G6PD
deficiency in different geographical areas appears to have arisen through
independent mutational events, but within the same population it may also be
heterogeneous. One example is the island of Sardinia, where careful clinical and
biochemical studies have identified four different G6PD variants. We cloned and
sequenced the four G6PD variants from Sardinia and found that only two mutations
are responsible for G6PD deficiency in this area: one mutation is the cause of
the G6PD Seattle-like phenotype, a milder form of G6PD deficiency; the other
mutation is responsible for all forms of very severe G6PD deficiency in Sardinia
and, possibly, in the Mediterranean. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is transmitted as an
X-linked recessive disorder, and thus female infants are expected to be only
rarely affected. Review of the records of 1,478 jaundiced newborn infants (728
boys and 750 girls) screened for G6PD deficiency at the Foothills Provincial
Hospital in Calgary showed 41 (5.6%) boys and 17 (2.2%) girls with this
disorder. In view of the unexpected and unexplained high frequency of G6PD
deficiency in female infants, I recommend that screening for this disorder be
done in selected jaundiced infants regardless of sex. This is a report about a 9 year old turkish boy suffering from recurrent
episodes of high fever caused by Plasmodium vivax-infection (Malaria tertiana),
12 months after returning from his malarious homeland. After a 3-day course of
Chloroquin, we administrated Primaquin to eliminate residual extraerythrocyte
forms of Plasmodium vivax. On the 7th day of treatment acute haemolysis
developped. This was caused by Glucose-6-Phosphate-Dehydrogenase-Deficiency,
which could be demonstrated by a red-cell-enzyme analysis. The investigation of
the patient's whole family showed the typical recessive X-linked inheritance of
this enzyme-defect. Frequency and clinical manifestations of this defect are
discussed. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a well-characterized
X-linked inherited disorder in humans but has not been reported in horses. We
describe a persistent hemolytic anemia and hyperbilirubinemia due to a severe
G6PD deficiency in an American Saddlebred colt. Other abnormalities in the
colt's erythrocytes as compared with those of healthy horses (n = 22-35)
included increased activities of hexokinase and pyruvate kinase, decreased
concentrations of reduced glutathione and reduced nicotinamide adenine
dinucleotide phosphate (NADP), and increased concentration of oxidized NADP.
Morphologic abnormalities included eccentrocytosis, pyknocytosis, anisocytosis,
macrocytosis, and increased number of Howell-Jolly bodies. Scanning and
transmission electron microscopic examinations revealed that eccentrocytes had
contracted to spherical regions and thin collapsed regions. Eccentrocytes were
more electron dense than were normal erythrocytes when examined by transmission
electron microscopy. When exposed to acetylphenylhydrazine, erythrocytes from
the G6PD-deficient colt produced more and smaller Heinz bodies than did
erythrocytes from normal horses. Abnormalities in the colt's dam included
presence of eccentrocytes and pyknocytes; her average erythrocyte G6PD activity
was slightly below the range of reference values. G6PD deficiency is the most common enzymopathy in the world. The highest
frequency values are found in tropical Africa, in the Middle East, in some areas
of the Mediterranean, in tropical and sub-tropical Asia and in Oceania. This
genetic defect shows sex linked inheritance and a marked heterogeneity. At least
400 abnormal variants with different biochemical characteristics and about 100
diverse mutations have been identified. In most cases the phenotypic expression
is a marked decrease in erythrocyte G6PD activity. The most common clinical
consequences are neonatal jaundice and sporadic haemolytic crises caused by a
number of drugs, by infections or by ingestion of fava beans. A few cases of
chronic non-spherocytic haemolytic anaemia associated with rare molecular
variants have been reported. Early diagnosis, education and epidemiologic
surveillance have been proved to be cornerstones in the prevention of the
haemolytic disease. Therefore they should be taken into account in the national
health programmes, especially in the countries with high prevalence rates. Glucose 6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a
housekeeping X-linked gene whose main function is to produce NADPH, a key
electron donor in the defense against oxidizing agents and in reductive
biosynthetic reactions. Inherited G6PD deficiency is associated with either
episodic hemolytic anemia (triggered by fava beans or other agents) or life-long
hemolytic anemia. We show here that an evolutionary analysis is a key to
understanding the biology of a housekeeping gene. From the alignment of the
amino acid (aa) sequence of 52 glucose 6-phosphate dehydrogenase (G6PD) species
from 42 different organisms, we found a striking correlation between the aa
replacements that cause G6PD deficiency in humans and the sequence conservation
of G6PD: two-thirds of such replacements are in highly and moderately conserved
(50-99%) aa; relatively few are in fully conserved aa (where they might be
lethal) or in poorly conserved aa, where presumably they simply would not cause
G6PD deficiency. This is consistent with the notion that all human mutants have
residual enzyme activity and that null mutations are lethal at some stage of
development. Comparing the distribution of mutations in a human housekeeping
gene with evolutionary conservation is a useful tool for pinpointing amino acid
residues important for the stability or the function of the corresponding
protein. In view of the current explosive increase in full genome sequencing
projects, this tool will become rapidly available for numerous other genes. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest red cell
enzymopathy in humans and has an X-linked inheritance. It has been reported from
India more than 30 years ago and the prevalence varies from 0-27% in different
caste, ethnic and linguistic groups. The major clinical manifestations are drug
induced hemolytic anemia, neonatal jaundice and chronic non-spherocytic
hemolytic anemia. Individuals with G6PD deficiency have a selective advantage
against falciparum malaria. Thirteen biochemically characterized variants have
been reported from India. At the molecular level, G6PD Mediterranean is the most
common deficient variant in the caste groups whereas, G6PD Orissa is more
prevalent among the tribal of India. The third common variant seen in India is
G6PD Kerala-Kalyan. BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) is important in the control
of oxidant stress in erythrocytes, the host cells for Plasmodium falciparum.
Mutations in this enzyme produce X-linked deficiency states associated with
protection against malaria, notably in Africa where the A- form of G6PD
deficiency is widespread. Some reports have proposed that heterozygous females
with mosaic populations of normal and deficient erythrocytes (due to random X
chromosome inactivation) have malaria resistance similar to or greater than
hemizygous males with populations of uniformly deficient erythrocytes. These
proposals are paradoxical, and they are not consistent with currently
hypothesized mechanisms of protection.
METHODS AND FINDINGS: We conducted large case-control studies of the A- form of
G6PD deficiency in cases of severe or uncomplicated malaria among two ethnic
populations of rural Mali, West Africa, where malaria is hyperendemic. Our
results indicate that the uniform state of G6PD deficiency in hemizygous male
children conferred significant protection against severe, life-threatening
malaria, and that it may have likewise protected homozygous female children. No
such protection was evident from the mosaic state of G6PD deficiency in
heterozygous females. We also found no significant differences in the parasite
densities of males and females with differences in G6PD status. Pooled odds
ratios from meta-analysis of our data and data from a previous study confirmed
highly significant protection against severe malaria in hemizygous males but not
in heterozygous females. Among the different forms of severe malaria, protection
was principally evident against cerebral malaria, the most frequent form of
life-threatening malaria in these studies.
CONCLUSIONS: The A- form of G6PD deficiency in Africa is under strong natural
selection from the preferential protection it provides to hemizygous males
against life-threatening malaria. Little or no such protection is present among
heterozygous females. Although these conclusions are consistent with data from
at least one previous study, they have not heretofore been realized to our
knowledge, and they therefore give fresh perspectives on malaria protection by
G6PD deficiency as an X-linked trait. Glucose 6-phosphate dehydrogenase (G6PD) is the first enzyme of the pentose
phosphate pathway. It exists in over 250 variants which are divided broadly into
five classes on the basis of residual enzyme activity and clinical
manifestations. The variants with reduced activity result in G6PD deficiency.
This is inherited as an X-linked recessive disorder and occurs at a much higher
frequency in the male than in the female. Most G6PD deficient individuals show
no clinical abnormality under normal conditions, but acute hemolytic crisis may
occur. Several nonhemolytic abnormalities occur as well in G6PD deficients at a
higher frequency than in nondeficients. Several nonhemolytic abnormalities occur
as well in G6PD deficients at a higher frequency than in nondeficients. The
genetic, pathophysiologic, and therapeutic aspects of G6PD deficiency are
presented; and the possibility of genetic counselling, care in blood banks, and
benefits of education the G6PD deficients are discussed. BACKGROUND: Primaquine is a key drug for malaria elimination. In addition to
being the only drug active against the dormant relapsing forms of Plasmodium
vivax, primaquine is the sole effective treatment of infectious P. falciparum
gametocytes, and may interrupt transmission and help contain the spread of
artemisinin resistance. However, primaquine can trigger haemolysis in patients
with a deficiency in glucose-6-phosphate dehydrogenase (G6PDd). Poor information
is available about the distribution of individuals at risk of primaquine-induced
haemolysis. We present a continuous evidence-based prevalence map of G6PDd and
estimates of affected populations, together with a national index of relative
haemolytic risk.
METHODS AND FINDINGS: Representative community surveys of phenotypic G6PDd
prevalence were identified for 1,734 spatially unique sites. These surveys
formed the evidence-base for a Bayesian geostatistical model adapted to the
gene's X-linked inheritance, which predicted a G6PDd allele frequency map across
malaria endemic countries (MECs) and generated population-weighted estimates of
affected populations. Highest median prevalence (peaking at 32.5%) was predicted
across sub-Saharan Africa and the Arabian Peninsula. Although G6PDd prevalence
was generally lower across central and southeast Asia, rarely exceeding 20%, the
majority of G6PDd individuals (67.5% median estimate) were from Asian countries.
We estimated a G6PDd allele frequency of 8.0% (interquartile range: 7.4-8.8)
across MECs, and 5.3% (4.4-6.7) within malaria-eliminating countries. The
reliability of the map is contingent on the underlying data informing the model;
population heterogeneity can only be represented by the available surveys, and
important weaknesses exist in the map across data-sparse regions. Uncertainty
metrics are used to quantify some aspects of these limitations in the map.
Finally, we assembled a database of G6PDd variant occurrences to inform a
national-level index of relative G6PDd haemolytic risk. Asian countries, where
variants were most severe, had the highest relative risks from G6PDd.
CONCLUSIONS: G6PDd is widespread and spatially heterogeneous across most MECs
where primaquine would be valuable for malaria control and elimination. The maps
and population estimates presented here reflect potential risk of
primaquine-associated harm. In the absence of non-toxic alternatives to
primaquine, these results represent additional evidence to help inform safe use
of this valuable, yet dangerous, component of the malaria-elimination toolkit.
Please see later in the article for the Editors' Summary. Glucose-6-Phosphate Dehydrogenase (G6PD) gene is located at the X-chromosome at
Xq28 and the disease is recessively inherited predomitly in males. More than
400 variants have been proposed based on clinical and enzymatic studies. The aim
of the current study was to identify C563T mutation in G6PD-deficient newborns
and to correlate the enzyme residual activity with the presence of the mutation.
Some 1189 full-term neonates aged 3-5 days old were tested for G6PD activity in
dried blood spots from Guthrie cards using a commercial kit. DNA extraction from
Guthrie cards and mutation identification among the deficient samples were
performed with current techniques. A total of 92 (7.7%) newborns were
G6PD-deficient. In 46 (50%), the mutation C563T was identified. The residual
activity in C563T hemizygote males (n = 28) was statistically significantly
lower (1.23 ± 0.93 U/g Hb) than that in non-C563T G6PD-deficient males (n = 25)
(4.01 ± 1.20 U/g Hb, p < 0.0001) and in controls (13.6 ± 2.9 U/g Hb, p <
0.0001). In C563T heterozygote females, the estimated enzyme activity was lower
than that determined in non-C563T females. Male C563T hemizygotes suffer from
G6PD deficiency and severe neonatal jaundice. G6PD activity showed statistically
significant correlation with total bilirubin blood levels. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked disease
responsible for moderate to severe hemolytic anaemia. Despite being the most
common erythrocyte enzyme disorder, it is often overlooked in the regular
diagnostic parlance. A 40-year-old male patient admitted to the casualty with an
acutely exacerbated diabetic ketoacidosis, showed features of hemolytic anaemia
on peripheral smear examination. Crucially, the spherocytes and bite cells
suggested a possibility of G6PD deficiency. This was substantiated by an
increased reticulocyte count (6.8%) and a reduced quantitative G6PD enzyme assay
(7.2%). There was no significant family or prior medical/ drug history.
Interestingly, the hemolytic features were evidenced when blood glucose levels
were returning to normal values. The insulin mediated NADPH loss may have
resulted in an increased erythrocyte oxidant sensitivity and a loss of
sulfhydryl group availability; causing hemolysis to manifest. G6PD deficiency is
conventionally affiliated with drug induced oxidative stress. But an association
with a diabetes mellitus is seldom reported. This case is being presented as it
highlights the lesser known complication of diabetic crisis such as hemolysis
secondary to a G6PD deficiency. BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) is a metabolic enzyme
involved in the pentose phosphate pathway, its especially important in red blood
cell metabolism. Glucose-6-phosphate dehydrogenase deficiency is an X-linked
recessive hereditary disease characterised by abnormally low levels of G6PD.
About 400 million people worldwide have a deficiency of this enzyme. The
remarkable geographic correlation of G6PD deficiency distribution with
historical endemicity patterns of malaria has led to suggestions that the two
could be linked. Some studies have concluded that G6PD deficiency confers
resistance to malaria.
OBJECTIVE: To determine the prevalence of G6PD deficiency, and determine its
relationship with prevalence and incidence of P. falciparum infection among
children in Uganda.
METHODS: This was longitudinal study involving 245 children, 135 were actively
followed up for 12 months. G6PD status was assessed for using PCR-RFLP method. A
thick smear was done to determine presence of plasmodium trophozoites and
parasite densities.
RESULTS: A total of 245 children between 6 months and 9 years were recruited. Of
these 46.5% were males. Overall prevalence for the X-linked G6PD A- mutation
was; 79.59% wild type, 12.65% heterozygous and 7.76% homozygous or hemizygous.
Among the males 14% were hemizygous. At baseline, 40.8% had asymptomatic P
falciparum infection. There was no statistically significant difference in
prevalence and incidence rates of malaria infection among the different G6PD
genotypes with prevalence among heterozygous, homozygous, and wild type being
29%, 42.6% and 43% respectively (p = 0.11) and incidence among heterozygous and
wild type being 0.56 and 0.52 episodes/year (p = 0.5). The heterozygous G6PD A-
females had a lower parasite density compared to the wild type (2505 vs 941
parasites/μL; P = 0.024).
CONCLUSIONS: This study showed that 20.41% of the population in this part of
Uganda carry the G6PD A-mutation, within the range of 15-32% seen in other parts
of Africa. P. falciparum infection incidence and prevalence rates are similar
among the G6PD genotypes though, once infected, P. falciparum parasite densities
are lowest among G6PD A- heterozygous females. This suggests differences in P.
falciparum infection rates and severity of disease could be mediated by
differences in parasite densities among the different G6PD genotypes. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked incompletely
domit enzyme deficiency that results from G6PD gene mutations. Women
heterozygous for G6PD mutations exhibit variation in the loss of enzyme activity
but the cause of this phenotypic variation is unclear. We determined DNA
methylation and X-inactivation patterns in 71 G6PD-deficient female
heterozygotes and 68 G6PD non-deficient controls with the same missense
mutations (G6PD Canton c.1376G>T or Kaiping c.1388G>A) to correlate determits
with variable phenotypes. Specific CpG methylations within the G6PD promoter
were significantly higher in G6PD-deficient heterozygotes than in controls.
Preferential X-inactivation of the G6PD wild-type allele was determined in
heterozygotes. The incidence of preferential X-inactivation was 86.2% in the
deficient heterozygote group and 31.7% in the non-deficient heterozygote group.
A significant negative correlation was observed between X-inactivation ratios of
the wild-type allele and G6PD/6-phosphogluconate dehydrogenase (6PGD) ratios in
heterozygous G6PD Canton (r=-0.657, p<0.001) or Kaiping (r=-0.668, p<0.001).
Multivariate logistic regression indicated that heterozygotes with
hypermethylation of specific CpG sites in the G6PD promoter and preferential
X-inactivation of the wild-type allele were at risk of enzyme deficiency. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive
genetic defect that can cause hemolytic crisis. However, this disease affects
both males and females. In Turkey, the frequency of this enzyme deficiency was
reported to vary, from 0.25 to 18%, by the geographical area. Its prevalence in
the northern Black Sea region of Turkey is unknown. The aims of this study were
to assess the prevalence of G6PD deficiency in the northern region Turkey in
children and adults with hyperbilirubinemia and hemolytic anemia. This report
included a total of 976 G6PD enzyme results that were analyzed between May 2005
and January 2014. G6PD deficiency was detected in 5.0% of all patients. G6PD
deficiency was significantly less frequent in females (1.9%, 6/323) than in
males (6.6%, 43/653). G6PD deficiency was detected in 3.7% of infants with
hyperbilirubinemia, 9.2% of children, and 4.5% of adults with hemolytic anemia.
In both the newborn group and the group of children, G6PD deficiency was
significantly more frequent in males. In the combined group of children (groups
I and II), the proportion of males was 74% and 67% in all groups (P = .0008). In
conclusion, in northern region of Turkey, G6PD deficiency is an important cause
of neonatal hyperbilirubinemia and hemolytic crisis in children and adults. This
study suggests that most pediatricians thought that G6PD deficiency is
exclusively a male disease. For this reason, some female patients may have been
undiagnosed. Glucose-6-phosphate dehydrogenase deficiency (G6PD) is the most common enzyme
pathology in humans; it is X-linked inherited and causes neonatal
hyperbilirubinaemia, chronic nonspherocytic haemolytic anaemia and drug-induced
acute haemolytic anaemia. G6PD deficiency has scarcely been studied in the
northern region of Mexico, which is important because of the genetic
heterogeneity described in Mexican population. Therefore, samples from the
northern Mexico were biochemically screened for G6PD deficiency, and PCR-RFLPs,
and DNA sequencing used to identify mutations in positive samples. The frequency
of G6PD deficiency in the population was 0.95% (n = 1993); the mutations in 86%
of these samples were G6PD A(-202A/376G), G6PDA(-376G/968C) and G6PD
Santamaria(376G/542T). Contrary to previous reports, we demonstrated that G6PD
deficiency distribution is relatively homogenous throughout the country (P =
0.48336), and the unique exception with high frequency of G6PD deficiency does
not involve a coastal population (Chihuahua: 2.4%). Analysis of eight
polymorphic sites showed only 10 haplotypes. In one individual we identified a
new G6PD mutation named Mexico DF(193A>G) (rs199474830), which probably results
in a damaging functional effect, according to PolyPhen analysis. Proteomic
impact of the mutation is also described. INTRODUCTION: Glucose-6-phosphate dehydrogenase deficiency (G6PD) is an X-linked
genetic disorder with a relatively high frequency in malaria-endemic regions. It
is an obstacle to malaria elimination, as primaquine administered in the
treatment of malaria can cause hemolysis in G6PD-deficient individuals. This
study presents information on the prevalence of G6PD deficiency in Sistan and
Balouchetsan province, which hosts more than 90% of Plasmodium vivax malaria
cases in Iran. This type of information is needed for a successful malaria
elimination program.
METHODOLOGY: A total of 526 students were randomly recruited through schools
located in southeast Iran. Information was collected by interviewing the
students using a structured questionnaire. Blood samples taken on filter papers
were examined for G6PD deficiency using the fluorescent spot test.
RESULTS: Overall, 72.8% (383/526) of the subjects showed normal G6PD enzyme
function. Mild and severe G6PD deficiency was observed in 14.8% (78) and 12.2%
(64) of subjects, respectively. A total 193/261 males (73.9%) and 190/265 (72%)
females had normal enzyme activity. Mild G6PD deficiency was observed in 10.8%
(28) and 18.9% (50) of male and female subjects, respectively. However, in
comparison with females, a greater proportion of males showed severe enzyme
deficiency (15.3% versus 9.1%). All these differences were statistically
significant (p < 0.006).
CONCLUSIONS: G6PD deficiency is highly prevalent in southeast Iran.
G6PD-deficient individuals are susceptible to potentially severe and
life-threatening hemolytic reactions after primaquine treatment. In order to
achieve malaria elimination goals in the province, G6PD testing needs to be made
routinely available within the health system. It is believed that the tribal people, who constitute 8.6 per cent of the total
population (2011 census of India), are the original inhabitants of India.
Glucose-6-phosphate-dehydrogenase (G6PD) deficiency is an X-linked genetic
defect, affecting around 400 million people worldwide and is characterized by
considerable biochemical and molecular heterogeneity. Deficiency of this enzyme
is highly polymorphic in those areas where malaria is/has been endemic. G6PD
deficiency was reported from India more than 50 years ago. t0 he prevalence
varies from 2.3 to 27.0 per cent with an overall prevalence of 7.7 per cent in
different tribal groups. Since the tribal populations live in remote areas where
malaria is/has been endemic, irrational use of antimalarial drugs could result
in an increased number of cases with drug induced haemolysis. Therefore, before
giving antimalarial therapy, routine screening for G6PD deficiency should be
undertaken in those tribal communities where its prevalence is high. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked hereditary
disease that predisposes red blood cells to oxidative damage. G6PD deficiency is
particularly prevalent in historically malaria-endemic areas. Use of primaquine
for malaria treatment may result in severe hemolysis in G6PD deficient patients.
In this study, we systematically evaluated the prevalence of G6PD deficiency in
the Kachin (Jingpo) ethnic group along the China-Myanmar border and determined
the underlying G6PD genotypes. We surveyed G6PD deficiency in 1770 adult
individuals (671 males and 1099 females) of the Kachin ethnicity using a G6PD
fluorescent spot test. The overall prevalence of G6PD deficiency in the study
population was 29.6% (523/1770), among which 27.9% and 30.6% were males and
females, respectively. From these G6PD deficient samples, 198 unrelated
individuals (147 females and 51 males) were selected for genotyping at 11 known
G6PD single nucleotide polymorphisms (SNPs) in Southeast Asia (ten in exons and
one in intron 11) using a multiplex SNaPshot assay. Mutations with known
association to a deficient phenotype were detected in 43.9% (87/198) of cases,
intronic and synonymous mutations were detected alone in 34.8% (69/198) cases
and no mutation were found in 21.2% (42/198) cases. Five non-synonymous
mutations, Mahidol 487G>A, Kaiping 1388G>A, Canton 1376G>T, Chinese 4 392G>T,
and Viangchan 871G>A were detected. Of the 87 cases with known deficient
mutations, the Mahidol variant was the most common (89.7%; 78/87), followed by
the Kaiping (8.0%; 7/87) and the Viangchan (2.2%; 2/87) variants. The Canton and
Chinese 4 variants were found in 1.1% of these 87 cases. Among them, two females
carried the Mahidol/Viangchan and Mahidol/Kaiping double mutations,
respectively. Interestingly, the silent SNPs 1311C>T and IVS11nt93T>C both
occurred in the same 95 subjects with frequencies at 56.4% and 23.5% in tested
females and males, respectively (P<0.05). It is noteworthy that 24 subjects
carrying the Mahidol mutation and two carrying the Kaiping mutation also carried
the 1311C>T/IVS11nt93T>C SNPs. Further studies are needed to determine the
enzyme levels of the G6PD deficient people and presence of additional G6PD
mutations in the study population. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive
hemolytic anemia caused by a mutation in the G6PD gene on Xq28. Herein, we
describe a Korean boy with G6PD deficiency resulting from a novel mutation in
G6PD. A 20-month-old boy with hemolytic anemia was referred for molecular
diagnosis. He had no relevant family history. The G6PD activity was severely
decreased at 0.2 U/g Hb (severe deficiency). Direct sequencing analyses on the
G6PD gene revealed that he was hemizygous for a novel missense variant,
c.1187C>G (p.Pro396Arg), in exon 10 of G6PD. Family study involving his parents
revealed the de novo occurrence of the mutation. This is the first report of
genetically confirmed G6PD deficiency in Korea. OBJECTIVE: The enzyme glucose-6-phosphate dehydrogenase (G6PD) catalyses the
first step in the pentose phosphate pathway, producing nicotinamide adenine
dinucleotide phosphate (NADPH). NADPH plays a crucial role in preventing
oxidative damage to proteins and other molecules in cells, mostly red blood
cells. G6PD deficiency has an x-linked pattern of inheritance in which
hemizygous males are deficient, while females may or may not be deficient
depending on the number of affected alleles. We report two novel DNA variants in
the G6PD gene detected in two male probands with chronic nonspherocytic
hemolytic anemia (CNSHA), who were referred for hematological evaluation.
METHOD: Probands and their relatives underwent clinical, biochemical, and
molecular assessment.
RESULTS: Two novel DNA variants, c.995C>T and c.1226C>A, were found in this
study. At the protein level, they produce the substitution of Ser332Phe and
Pro409Gln, respectively. These DNA variants were analyzed in the female
relatives of probands for genetic counseling.
CONCLUSIONS: The novel DNA variants were classified as class I based on the
clinical, biochemical, and molecular evaluations performed. |
What is FFI, fatal familial insomnia | Familial fatal insomnia (FFI) is a prion disease caused by a mutation (D178N-129M haplotype) in the Prion Protein gene (PRNP). FFI is manifested by sleep disturbances with insomnia, autonomic disorders and spontaneous and evoked myoclonus, among other symptoms. FFI is considered to be a rare disease. | Fatal familial insomnia (FFI), a condition characterized by inability to sleep,
dysautonomia, motor disturbances, and selective thalamic atrophy is a prion
disease linked to a GAC----AAC mutation at codon 178 of the prion gene. These
data were obtained from one kindred. We now report a second kindred affected by
FFI and carrying the same mutation. The finding of the same disease phenotype
and genotype in a second family further validates FFI as a distinct disease
entity and a phenotype of the GAC----AAC mutation at codon 178 of the prion
gene. Fatal familial insomnia (FFI) is a unique hereditary prion disease with
characteristic disturbances of sleep. We studied the serotonergic system in 8
FFI-affected subjects by immunohistochemistry for the serotonin-synthesizing
enzyme, tryptophan hydroxylase (TH). Quantification of neurons in median raphe
nuclei showed no total neuronal loss in FFI but a substantial increase of TH+
neurons (approximately 62%) in FFI subjects compared with controls. Our data
indicate an alteration of the serotonergic system that might represent the
functional substrate of some typical symptoms of FFI. Fatal familial insomnia (FFI) is a rare hereditary human prion disease with
unique clinical features including progressive sleep impairment and autonomic
dysfunction. The serotonergic system is considered to be involved in the
regulation of the sleep-wake cycle. In this study we demonstrate a reduced
availability of serotonin transporters of 57% and 73% respectively in a
thalamus-hypothalamus region of two FFI patients examined with beta-CIT SPECT as
compared to age-expected control values. BACKGROUND: Fatal familial insomnia (FFI) is an autosomal domit disease
linked to a mutation in the prion protein gene. Fatal familial insomnia is
characterized by sleep disturbance and loss of neurons, with gliosis in the
thalamic nuclei.
OBJECTIVE: To describe the clinical, neurophysiological, radiological, and
neuropathological data in a Chinese family with FFI.
SETTING: Tertiary referral university hospital setting.
PATIENTS: Patient 1 was a 36-year-old man who presented with insomnia and
myoclonus. In the subsequent 9 months, he developed ataxia and dementia,
followed by death. Patient 2 was the aunt of patient 1, and presented at the age
of 47 years with insomnia, myoclonus, and dementia; her condition declined
during a 12-month period. Genetic analysis was performed, followed by
neuropathological and biochemical analysis of the disease-associated form of the
prion protein PrPSc on the postmortem brain specimen.
RESULTS: Molecular analysis demonstrated an aspartic acid to asparagine mutation
at codon 178 and homozygosity for methionine at codon 129. Both patients showed
severe neuronal loss and prominent gliosis in the thalamus and brainstem
involvement, with evidence of astrogliosis in the inferior olivary nucleus.
Patient 1 also had neuronal loss and astrogliosis in the region of the superior
colliculus and in the periaqueductal region. PrPSc was detected on Western blot
analysis, and had a wide distribution. The strongest signals were present in the
amygdala, hypothalamus, caudate, parahippocampal gyrus, periaqueductal gray
matter, and mediodorsal thalamus.
CONCLUSIONS: To our knowledge, this is the first report of FFI in a family of
Chinese descent. This supports the worldwide distribution of FFI, and despite
differences in genetic background, the clinical and pathological findings are
similar to those found in white patients with FFI. BACKGROUND: Sporadic fatal insomnia (sFI) and fatal familial insomnia (FFI) are
rare human prion diseases.
CASE PRESENTATION: We report a case of a 33-year-old female who died of a prion
disease for whom the diagnosis of sFI or FFI was not considered clinically.
Following death of this patient, an interview with a close family member
indicated the patient's illness included a major change in her sleep pattern,
corroborating the reported autopsy diagnosis of sFI. Genetic tests identified no
prion protein (PrP) gene mutation, but neuropathological examination and
molecular study showed protease-resistant PrP (PrPres) in several brain regions
and severe atrophy of the anterior-ventral and medial-dorsal thalamic nuclei
similar to that described in FFI.
CONCLUSIONS: In patients with suspected prion disease, a characteristic change
in sleep pattern can be an important clinical clue for identifying sFI or FFI;
polysomnography (PSG), genetic analysis, and nuclear imaging may aid in
diagnosis. OBJECTIVE: Fatal familial insomnia (FFI) is an autosomal domit prion disease
characterized clinically by inattention, sleep loss, dysautonomia, and motor
signs. This study is aimed to investigate clinical and familial characteristics
of ten Chinese Patients with FFI.
METHODS: We identified ten FFI cases from the surveillance network for
Creutafeldt-Jakob disease (CJD) in China. Final diagnosis of FFI cases was made
in accordance with the WHO criteria for CJD. The main clinical features and
family histories of these ten FFI cases were analyzed.
RESULTS: The median age of ten cases at onset was 38 years (from 19 to 55). The
foremost symptoms seemed to be various, including sleep disturbances, vision
disorder, dizziness and anorexia. Sleep disturbances appeared in all cases and
lasted in the whole clinical courses. Progressive sympathetic symptoms, memory
loss, movement disturbances, myoclonus and hypertension were also frequently
observed. The median duration of the disease was 9.5 months. EEG and MRI did not
figure out special abnormality. 14-3-3 protein in CSF was positive in five out
of eight tested patients. Clear family histories were identified in 8 patients.
CONCLUSION: The data from our study confirm that the Chinese FFI cases have
similar clinical characteristics as that of the Caucasian cases. Compared with
other genetic CJD associated mutations, the genetic frequencies of D178N in PRNP
are apparently high among the Chinese cases. Human prion diseases are fatal neurodegenerative disorders that are
characterized by spongiform changes, astrogliosis, and the accumulation of an
abnormal prion protein (PrP(Sc)). Approximately 10%-15% of human prion diseases
are familial variants that are caused by pathogenic mutations in the prion
protein gene (PRNP). Point mutations or the insertions of one or more copies of
a 24 bp repeat are associated with familial human prion diseases including
familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker
syndrome, and fatal familial insomnia. These mutations vary significantly in
frequency between countries. Here, we compare the frequency of PRNP mutations
between European countries and East Asians. Associations between single
nucleotide polymorphisms (SNPs) of several candidate genes including PRNP and
CJD have been reported. The SNP of PRNP at codon 129 has been shown to be
associated with sporadic, iatrogenic, and variant CJD. The SNPs of several genes
other than PRNP have been showed contradictory results. Case-control studies and
genome-wide association studies have also been performed to identify candidate
genes correlated with variant and/or sporadic CJD. This review provides a
general overview of the genetic mutations and polymorphisms that have been
analyzed in association with human prion diseases to date. BACKGROUND AND METHODS: Fatal familial insomnia (FFI) is a rare genetic disease
characterized by intractable insomnia, dysautonomia, and dementia. Herein we
describe a patient with FFI. In order to study brain glucose hypometabolism in
the patient, we used statistical parametric mapping (SPM) analysis of
[(18)F]-fluorodeoxyglucose positron emission tomography (FDG-PET).
CASE REPORT: The patient was a 34-year-old Korean man. He presented with
intractable insomnia, rapidly progressive dementia and autonomic disturbances. A
comprehensive clinical investigation was conducted, including brain MRI,
electroencephalography, polysomnography, neuropsychological tests, FDG-PET and
genomic tests. SPM analysis was performed using 7 healthy controls. Direct
sequencing of the PRNP gene identified a heterozygous p. Asp179Asn mutation
homozygous for methionine at codon 129 and for glutamate at codon 219. The
results of the SPM analysis showed marked hypometabolism in the deep cerebral
nuclei (including the bilateral thalami, caudate nuclei, and hypothalamus),
association cortices (including the frontal, lateral temporal, inferior parietal
lobule and posterior cingulate gyri), and midbrain.
CONCLUSIONS: This is the first Korean report of FFI, in which the family showed
male phenotypic predomice. The patient's SPM analysis demonstrated brain
hypometabolism in the midbrain and the hypothalamus, as well as the thalami,
caudate nuclei, and multiple cortical regions. These results contribute further
to the overall understanding of the pathophysiology of FFI. Fatal familial insomnia is a rare disease caused by a D178N mutation in
combination with methionine (Met) at codon 129 in the mutated allele of PRNP
(D178N-129M haplotype). FFI is manifested by sleep disturbances with insomnia,
autonomic disorders and spontaneous and evoked myoclonus, among other symptoms.
This study describes new neuropathological and biochemical observations in a
series of eight patients with FFI. The mediodorsal and anterior nuclei of the
thalamus have severe neuronal loss and marked astrocytic gliosis in every case,
whereas the entorhinal cortex is variably affected. Spongiform degeneration only
occurs in the entorhinal cortex. Synaptic and fine granular proteinase K
digestion (PrPres) immunoreactivity is found in the entorhinal cortex but not in
the thalamus. Interleukin 6, interleukin 10 receptor alpha subunit, colony
stimulating factor 3 receptor and toll-like receptor 7 mRNA expression increases
in the thalamus in FFI. PrPc levels are significantly decreased in the thalamus,
entorhinal cortex and cerebellum in FFI. This is accompanied by a particular
PrPc and PrPres band profile. Altered PrP solubility consistent with
significantly reduced PrP levels in the cytoplasmic fraction and increased PrP
levels in the insoluble fraction are identified in FFI cases. Amyloid-like
deposits are only seen in the entorhinal cortex. The RT-QuIC assay reveals that
all the FFI samples of the entorhinal cortex are positive, whereas the thalamus
is positive only in three cases and the cerebellum in two cases. The present
findings unveil particular neuropathological and neuroinflammatory profiles in
FFI and novel characteristics of natural prion protein in FFI, altered PrPres
and Scrapie PrP (abnormal and pathogenic PrP) patterns and region-dependent
putative capacity of PrP seeding. Although prion diseases are generally thought to present as rapidly progressive
dementias with survival of only a few months, the phenotypic spectrum for
genetic prion diseases (gPrDs) is much broader. The majority have a rapid
decline with short survival, but many patients with gPrDs present as slowly
progressive ataxic or parkinsonian disorders with progression over a few to
several years. A few very rare mutations even present as neuropsychiatric
disorders, sometimes with systemic symptoms such as gastrointestinal disorders
and neuropathy, progressing over years to decades. gPrDs are caused by mutations
in the prion protein gene (PRNP), and have been historically classified based on
their clinicopathological features as genetic Jakob-Creutzfeldt disease (gJCD),
Gerstmann-Sträussler-Scheinker (GSS), or Fatal Familial Insomnia (FFI).
Mutations in PRNP can be missense, nonsense, and octapeptide repeat insertions
or a deletion, and present with diverse clinical features, sensitivities of
ancillary testing, and neuropathological findings. We present the UCSF gPrD
cohort, including 129 symptomatic patients referred to and/or seen at UCSF
between 2001 and 2016, and compare the clinical features of the gPrDs from 22
mutations identified in our cohort with data from the literature, as well as
perform a literature review on most other mutations not represented in our
cohort. E200K is the most common mutation worldwide, is associated with gJCD,
and was the most common in the UCSF cohort. Among the GSS-associated mutations,
P102L is the most commonly reported and was also the most common at UCSF. We
also had several octapeptide repeat insertions (OPRI), a rare nonsense mutation
(Q160X), and three novel mutations (K194E, E200G, and A224V) in our UCSF cohort.
© 2016 Wiley Periodicals, Inc. |
Which mutated genes are associated with the Tourette's syndrome? | A mutation in histidine decarboxylase (Hdc) gene as well as mutations in the SLITRK1 (Slit and Trk-like 1) gene have been implicated as rare genetic causes of Tourette's syndrome. | Tourette syndrome is a neurologic disorder characterized by both motor and vocal
tics. Recently, two variants, including a single-base deletion resulting in a
truncated protein and a 3'-untranslated-region variant altering a binding site
for micro-RNA in the Slit and Trk-like 1 gene, were found to be a genetic cause
of Tourette syndrome. The Slit and Trk-like 1 family was identified as neuronal
transmembrane proteins that control neurite outgrowth. This study aimed to
determine whether mutations in the gene can be found in Taiwanese patients with
Tourette syndrome. In total, 160 patients were included. All children underwent
peripheral blood sampling for genotype analyses. We sequenced the whole Slit and
Trk-like 1 gene, including the promoter, the 3'-untranslated region, the
5'-untranslated region, and the whole coding region. We found that none of the
160 samples revealed any mutation in the whole gene sequence. In addition, there
was only one polymorphism, c.3225 T>C, detected in 10 individuals. We conclude
that in rare variants, it may be difficult to establish an association with
disorder. Therefore, genetic screening in the Slit and Trk-like 1 gene for the
recently identified mutations does not appear to be of utility in the diagnosis
of Tourette syndrome. Mutations in the gene SLITRK1 (Slit and Trk-like 1) have been reported in
patients with Tourette's disorder (TD). We sequenced the entire SLITRK1 gene
including the coding region the 5' and 3' untranslated region in 92 Austrian
patients with TD. No nucleotide changes within the protein-coding region were
identified. One patient was found to carry a variant within the 3' untranslated
region (3383g>a), which was absent in 192 control individuals and which
segregated in two additional family members with tic symptoms. In conclusion,
our results provide no evidence for SLITRK1 playing a major role in TD. Obsessive compulsive disorder (OCD) is a syndrome characterized by recurrent and
intrusive thoughts and ritualistic behaviors or mental acts that a person feels
compelled to perform. Twin studies, family studies, and segregation analyses
provide compelling evidence that OCD has a strong genetic component. The SLITRK1
gene encodes a developmentally regulated stimulator of neurite outgrowth and
previous studies have implicated rare variants in this gene in disorders in the
OC spectrum, specifically Tourette syndrome (TS) and trichotillomania (TTM). The
objective of the current study was to evaluate rare genetic variation in SLITRK1
in risk for OCD and to functionally characterize associated coding variants. We
sequenced SLITRK1 coding exons in 381 individuals with OCD as well as in 356
control samples and identified three novel variants in seven individuals. We
found that the combined mutation load in OCD relative to controls was
significant (p = 0.036). We identified a missense N400I change in an individual
with OCD, which was not found in more than 1000 control samples (P<0.05). In
addition, we showed the the N400I variant failed to enhance neurite outgrowth in
primary neuronal cultures, in contrast to wildtype SLITRK1, which enhanced
neurite outgrowth in this assay. These important functional differences in the
N400I variant, as compared to the wildtype SLITRK1 sequence, may contribute to
OCD and OC spectrum symptoms. A synonymous L63L change identified in an
individual with OCD and an additional missense change, T418S, was found in four
individuals with OCD and in one individual without an OCD spectrum disorder.
Examination of additional samples will help assess the role of rare SLITRK1
variation in OCD and in related psychiatric illness. Author information:
(1)Department of Psychiatry, Yale University School of Medicine.
(2)Department of Child Study Center, Yale University School of Medicine.
(3)Department of Diagnostic Radiology, Yale University School of Medicine.
(4)Department of Laboratory Medicine, Yale University School of Medicine.
(5)Department of Genetics, Yale University School of Medicine.
(6)Department of Program on Neurogenetics, Yale University School of Medicine.
(7)Neuroscience Research Unit, Pfizer, Inc., Cambridge, MA.
(8)Dept. of Biochem., Faculty of Science, Chulalongkorn Univ., Bangkok,
Thailand.
(9)Nathan S. Kline Institute for Psychiatric Research.
(10)New York University Dept of Child and Adolescent Psychiatry.
(11)Tohoku University, Graduate School of Engineering, Sendai, Japan.
(12)Department of Pediatrics, Yale University School of Medicine.
(13)Department of Psychology, Yale University School of Medicine.
(14)John B. Pierce Laboratory, New Haven, CT.
(15)Integrated Neuroscience Research Program; New Haven, CT 06520.
(#)Contributed equally PURPOSE OF REVIEW: This update summarizes progress in understanding Tourette
syndrome clinical characteristics, etiology, and treatment over the past year.
RECENT FINDINGS: Premonitory sensory phenomena were found to have important
impacts on Tourette syndrome quality of life. A rare genetic form of Tourette
syndrome due to L-histidine-decarboxylase mutation, with similar features in
human and rodent, has inspired new research on functional anatomy of Tourette
syndrome. In response to new data, treatment guidelines have been revised to
include behavioral therapy as first-line treatment. Novel dopamine receptor
antagonists aripiprazole and ecopipam have shown potential efficacy - as well as
tolerability concerns. Recent work has suggested efficacy and tolerability of
topiramate and fluphenazine, but more rigorous studies are needed to further
understand their role in Tourette syndrome management. Recent consensus
guidelines explain when deep brain stimulation can be considered for severe
refractory cases under a multidisciplinary team.
SUMMARY: More research is needed to identify better tolerated treatments for, to
understand pathophysiology or functional anatomy of, and to predict or influence
longitudinal outcome of Tourette syndrome. Tic disorders produce substantial morbidity, but their pathophysiology remains
poorly understood. Convergent evidence suggests that dysregulation of the
cortico-basal ganglia circuitry is central to the pathogenesis of tics. Tourette
syndrome (TS), the most severe end of the continuum of tic disorders, is
substantially genetic, but causative mutations have been elusive. We recently
described a mouse model, the histidine decarboxylase (Hdc) knockout mouse, that
recapitulates a rare, highly penetrant mutation found in a single family; these
mice exhibit TS-like phenomenology. These animals have a global deficit in brain
histamine and a consequent dysregulation of DA in the basal ganglia. Histamine
modulation of DA effects is increasingly appreciated, but the mechanisms
underlying this modulation remain unclear; the consequences of modest DA
elevation in the context of profound HA deficiency are difficult to predict, but
understanding them in the Hdc knockout mouse may provide generalizable insights
into the pathophysiology of TS. Here we characterized signaling pathways in
striatal cells in this model system, at baseline and after amphetamine
challenge. In vivo microdialysis confirms elevated DA in Hdc-KO mice. We find
dephosphorylation of Akt and its target GSK3β and activation of the MAPK
signaling cascade and its target rpS6; these are characteristic of the effects
of DA on D2- and D1-expressing striatal neurons, respectively. Strikingly, there
is no alteration in mTOR signaling, which can be regulated by DA in both cell
types. These cellular effects help elucidate striatal signaling abnormalities in
a uniquely validated mouse model of TS and move towards the identification of
new potential therapeutic targets for tic disorders. |
Which enzyme is inhibited by niraparib? | Niraparib is a Poly(ADP-ribose) Polymerase (PARP) Inhibitor. It is used for ovarian cancer treatment. | Small-molecule inhibitors of PARP are thought to mediate their antitumor effects
as catalytic inhibitors that block repair of DNA single-strand breaks (SSB).
However, the mechanism of action of PARP inhibitors with regard to their effects
in cancer cells is not fully understood. In this study, we show that PARP
inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA
complexes were more cytotoxic than unrepaired SSBs caused by PARP inactivation,
arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on
DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors
with niraparib (MK-4827) > olaparib (AZD-2281) >> veliparib (ABT-888), a pattern
not correlated with the catalytic inhibitory properties for each drug. We also
analyzed repair pathways for PARP-DNA complexes using 30 genetically altered
avian DT40 cell lines with preestablished deletions in specific DNA repair
genes. This analysis revealed that, in addition to homologous recombination,
postreplication repair, the Fanconi anemia pathway, polymerase β, and FEN1 are
critical for repairing trapped PARP-DNA complexes. In summary, our study
provides a new mechanistic foundation for the rational application of PARP
inhibitors in cancer therapy. BACKGROUND: Poly(ADP-ribose) polymerase (PARP) is implicated in DNA repair and
transcription regulation. Niraparib (MK4827) is an oral potent, selective PARP-1
and PARP-2 inhibitor that induces synthetic lethality in preclinical tumour
models with loss of BRCA and PTEN function. We investigated the safety,
tolerability, maximum tolerated dose, pharmacokinetic and pharmacodynamic
profiles, and preliminary antitumour activity of niraparib.
METHODS: In a phase 1 dose-escalation study, we enrolled patients with advanced
solid tumours at one site in the UK and two sites in the USA. Eligible patients
were aged at least 18 years; had a life expectancy of at least 12 weeks; had an
Eastern Cooperative Oncology Group performance status of 2 or less; had
assessable disease; were not suitable to receive any established treatments; had
adequate organ function; and had discontinued any previous anticancer treatments
at least 4 weeks previously. In part A, cohorts of three to six patients,
enriched for BRCA1 and BRCA2 mutation carriers, received niraparib daily at ten
escalating doses from 30 mg to 400 mg in a 21-day cycle to establish the maximum
tolerated dose. Dose expansion at the maximum tolerated dose was pursued in 15
patients to confirm tolerability. In part B, we further investigated the maximum
tolerated dose in patients with sporadic platinum-resistant high-grade serous
ovarian cancer and sporadic prostate cancer. We obtained blood, circulating
tumour cells, and optional paired tumour biopsies for pharmacokinetic and
pharmacodynamic assessments. Toxic effects were assessed by common toxicity
criteria and tumour responses ascribed by Response Evaluation Criteria in Solid
Tumors (RECIST). Circulating tumour cells and archival tumour tissue in prostate
patients were analysed for exploratory putative predictive biomarkers, such as
loss of PTEN expression and ETS rearrangements. This trial is registered with
ClinicalTrials.gov, NCT00749502.
FINDINGS: Between Sept 15, 2008, and Jan 14, 2011, we enrolled 100 patients: 60
in part A and 40 in part B. 300 mg/day was established as the maximum tolerated
dose. Dose-limiting toxic effects reported in the first cycle were grade 3
fatigue (one patient given 30 mg/day), grade 3 pneumonitis (one given 60
mg/day), and grade 4 thrombocytopenia (two given 400 mg/day). Common
treatment-related toxic effects were anaemia (48 patients [48%]), nausea (42
[42%]), fatigue (42 [42%]), thrombocytopenia (35 [35%]), anorexia (26 [26%]),
neutropenia (24 [24%]), constipation (23 [23%]), and vomiting (20 [20%]), and
were predomitly grade 1 or 2. Pharmacokinetics were dose proportional and the
mean terminal elimination half-life was 36·4 h (range 32·8-46·0).
Pharmacodynamic analyses confirmed PARP inhibition exceeded 50% at doses greater
than 80 mg/day and antitumour activity was documented beyond doses of 60 mg/day.
Eight (40% [95% CI 19-64]) of 20 BRCA1 or BRCA2 mutation carriers with ovarian
cancer had RECIST partial responses, as did two (50% [7-93]) of four mutation
carriers with breast cancer. Antitumour activity was also reported in sporadic
high-grade serous ovarian cancer, non-small-cell lung cancer, and prostate
cancer. We recorded no correlation between loss of PTEN expression or ETS
rearrangements and measures of antitumour activity in patients with prostate
cancer.
INTERPRETATION: A recommended phase 2 dose of 300 mg/day niraparib is well
tolerated. Niraparib should be further assessed in inherited and sporadic
cancers with homologous recombination DNA repair defects and to target
PARP-mediated transcription in cancer.
FUNDING: Merck Sharp and Dohme. Excision repair cross-complementation group 1 (ERCC1) is a DNA repair enzyme
that is frequently defective in non-small cell lung cancer (NSCLC). Although low
ERCC1 expression correlates with platinum sensitivity, the clinical
effectiveness of platinum therapy is limited, highlighting the need for
alternative treatment strategies. To discover new mechanism-based therapeutic
strategies for ERCC1-defective tumours, we performed high-throughput drug
screens in an isogenic NSCLC model of ERCC1 deficiency and dissected the
mechanism underlying ERCC1-selective effects by studying molecular biomarkers of
tumour cell response. The high-throughput screens identified multiple clinical
poly (ADP-ribose) polymerase 1 and 2 (PARP1/2) inhibitors, such as olaparib
(AZD-2281), niraparib (MK-4827) and BMN 673, as being selective for ERCC1
deficiency. We observed that ERCC1-deficient cells displayed a significant delay
in double-strand break repair associated with a profound and prolonged G₂/M
arrest following PARP1/2 inhibitor treatment. Importantly, we found that ERCC1
isoform 202, which has recently been shown to mediate platinum sensitivity, also
modulated PARP1/2 sensitivity. A PARP1/2 inhibitor-synthetic lethal siRNA screen
revealed that ERCC1 deficiency was epistatic with homologous recombination
deficiency. However, ERCC1-deficient cells did not display a defect in RAD51
foci formation, suggesting that ERCC1 might be required to process PARP1/2
inhibitor-induced DNA lesions before DNA strand invasion. PARP1 silencing
restored PARP1/2 inhibitor resistance in ERCC1-deficient cells but had no effect
in ERCC1-proficient cells, supporting the hypothesis that PARP1 might be
required for the ERCC1 selectivity of PARP1/2 inhibitors. This study suggests
that PARP1/2 inhibitors as a monotherapy could represent a novel therapeutic
strategy for NSCLC patients with ERCC1-deficient tumours. The aim of this study was to assess niraparib (MK-4827), a novel
poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize
human tumor cells. Human tumor cells derived from lung, breast and prostate
cancers were tested for radiosensitization by niraparib using clonogenic
survival assays. Both p53 wild-type and p53-defective lines were included. The
ability of niraparib to alter the repair of radiation-induced DNA double strand
breaks (DSBs) was determined using detection of γ-H2AX foci and RAD51 foci.
Clonogenic survival analyses indicated that micromolar concentrations of
niraparib radiosensitized tumor cell lines derived from lung, breast, and
prostate cancers independently of their p53 status but not cell lines derived
from normal tissues. Niraparib also sensitized tumor cells to H2O2 and converted
H2O2-induced single strand breaks (SSBs) into DSBs during DNA replication. These
results indicate that human tumor cells are significantly radiosensitized by the
potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The
mechanism of this effect appears to involve a conversion of sublethal SSBs into
lethal DSBs during DNA replication due to the inhibition of base excision repair
by the drug. Taken together, our findings strongly support the clinical
evaluation of niraparib in combination with radiation. Recent findings indicate that a major mechanism by which poly(ADP-ribose)
polymerase (PARP) inhibitors kill cancer cells is by trapping PARP1 and PARP2 to
the sites of DNA damage. The PARP enzyme-inhibitor complex "locks" onto damaged
DNA and prevents DNA repair, replication, and transcription, leading to cell
death. Several clinical-stage PARP inhibitors, including veliparib, rucaparib,
olaparib, niraparib, and talazoparib, have been evaluated for their
PARP-trapping activity. Although they display similar capacity to inhibit PARP
catalytic activity, their relative abilities to trap PARP differ by several
orders of magnitude, with the ability to trap PARP closely correlating with each
drug's ability to kill cancer cells. In this article, we review the available
data on molecular interactions between these clinical-stage PARP inhibitors and
PARP proteins, and discuss how their biologic differences might be explained by
the trapping mechanism. We also discuss how to use the PARP-trapping mechanism
to guide the development of PARP inhibitors as a new class of cancer therapy,
both for single-agent and combination treatments. Poly(ADP-ribose) polymerases (PARPs) are involved in DNA repair following damage
by endogenous or exogenous processes. It has become clear over the past decade
that inhibition of PARP in the context of defects in other DNA repair mechanisms
provide a tumor specific way to kill cancer cells. We describe the rationale for
this approach and the design and discovery of niraparib, a potent PARP-1/2
inhibitor with good cell based activity, selectivity for cancer over normal
cells, and oral bioavailability. Niraparib was characterized in a number of
preclinical models before moving to phase I clinical trials, where it showed
excellent human pharmacokinetics suitable for once a day oral dosing, achieved
its pharmacodynamic target for PARP inhibition, and had promising activity in
cancer patients. It is currently being tested in phase 3 clinical trials as
maintece therapy in ovarian cancer and as a treatment for breast cancer. Poly(ADP-ribose) polymerases (PARP1, -2, and -3) play important roles in DNA
damage repair. As such, a number of PARP inhibitors are undergoing clinical
development as anticancer therapies, particularly in tumors with DNA repair
deficits and in combination with DNA-damaging agents. Preclinical evidence
indicates that PARP inhibitors potentiate the cytotoxicity of DNA alkylating
agents. It has been proposed that a major mechanism underlying this activity is
the allosteric trapping of PARP1 at DNA single-strand breaks during base
excision repair; however, direct evidence of allostery has not been reported.
Here the data reveal that veliparib, olaparib, niraparib, and talazoparib
(BMN-673) potentiate the cytotoxicity of alkylating agents. Consistent with
this, all four drugs possess PARP1 trapping activity. Using biochemical and
cellular approaches, we directly probe the trapping mechanism for an allosteric
component. These studies indicate that trapping is due to catalytic inhibition
and not allostery. The potency of PARP inhibitors with respect to trapping and
catalytic inhibition is linearly correlated in biochemical systems but is
nonlinear in cells. High-content imaging of γH2Ax levels suggests that this is
attributable to differential potentiation of DNA damage in cells. Trapping
potency is inversely correlated with tolerability when PARP inhibitors are
combined with temozolomide in mouse xenograft studies. As a result, PARP
inhibitors with dramatically different trapping potencies elicit comparable in
vivo efficacy at maximum tolerated doses. Finally, the impact of trapping on
tolerability and efficacy is likely to be context specific.
IMPLICATIONS: Understanding the context-specific relationships of trapping and
catalytic inhibition with both tolerability and efficacy will aid in determining
the suitability of a PARP inhibitor for inclusion in a particular clinical
regimen. Poly(ADP-ribose) polymerases(PARP) synthesize the ADP-ribose polymers onto
proteins and play a role in DNA repair. PARP inhibitors block the repair of
single-strand breaks, which in turn gives rise to double-strand breaks during
DNA replication. Thus, PARP inhibitors elicit synthetic lethality in cancer with
BRCA1/2 loss-of-function mutations that hamper homologous recombination repair
of double-strand breaks. Olaparib, the first-in-class PARP inhibitor, was
approved for treatment of BRCA-mutated ovarian cancer in Europe and the United
States in 2014. Other PARP inhibitors under clinical trials include rucaparib,
niraparib, veliparib, and the "PARP-trapping" BMN-673. BRCA1/2 sequencing is an
FDA-approved companion diagnostics, which predicts the cancer vulnerability to
PARP inhibition. Together, synthetic lethal PARP inhibition is a novel promising
strategy for cancer intervention even in cases without prominent driver
oncogenes. Pediatric high-grade astrocytomas (pHGA) and diffuse intrinsic pontine gliomas
(DIPG) are devastating maligcies for which no effective therapies exist. We
investigated the therapeutic potential of PARP1 inhibition in preclinical models
of pHGA and DIPG. PARP1 levels were characterized in pHGA and DIPG patient
samples and tumor-derived cell lines. The effects of PARP inhibitors veliparib,
olaparib, and niraparib as monotherapy or as radiosensitizers on cell viability,
DNA damage, and PARP1 activity were evaluated in a panel of pHGA and DIPG cell
lines. Survival benefit of niraparib was examined in an orthotopic xenograft
model of pHGA. About 85% of pHGAs and 76% of DIPG tissue microarray samples
expressed PARP1. Six of 8 primary cell lines highly expressed PARP1.
Interestingly, across multiple cell lines, some PARP1 protein expression was
required for response to PARP inhibition; however, there was no correlation
between protein level or PARP1 activity and sensitivity to PARP inhibitors.
Niraparib was the most effective at reducing cell viability and proliferation
(MTT and Ki67). Niraparib induced DNA damage (γH2AX foci) and induced growth
arrest. Pretreatment of pHGA cells with a sublethal dose of niraparib (1 μmol/L)
before 2 Gy of ionizing radiation (IR) decreased the rate of DNA damage repair,
colony growth, and relative cell number. Niraparib (50 mg/kg) inhibited PARP1
activity in vivo and extended survival of mice with orthotopic pHGA xenografts,
when administered before IR (20 Gy, fractionated), relative to control mice (40
vs. 25 days). Our data provide in vitro and in vivo evidence that niraparib may
be an effective radiosensitizer for pHGA and DIPG. Inhibition of Poly(ADP-ribose) Polymerase1 (PARP1) impairs DNA damage repair,
and early generation PARP1/2 inhibitors (olaparib, niraparib, etc.) have
demonstrated clinical proof of concept for cancer treatment. Here, we describe
the development of the novel PARP inhibitor E7449, a potent PARP1/2 inhibitor
that also inhibits PARP5a/5b, otherwise known as tankyrase1 and 2 (TNKS1 and 2),
important regulators of canonical Wnt/β-catenin signaling. E7449 inhibits PARP
enzymatic activity and additionally traps PARP1 onto damaged DNA; a mechanism
previously shown to augment cytotoxicity. Cells deficient in DNA repair pathways
beyond homologous recombination were sensitive to E7449 treatment. Chemotherapy
was potentiated by E7449 and single agent had significant antitumor activity in
BRCA-deficient xenografts. Additionally, E7449 inhibited Wnt/β-catenin signaling
in colon cancer cell lines, likely through TNKS inhibition. Consistent with this
possibility, E7449 stabilized axin and TNKS proteins resulting in β-catenin
de-stabilization and significantly altered expression of Wnt target genes.
Notably, hair growth mediated by Wnt signaling was inhibited by E7449. A
pharmacodynamic effect of E7449 on Wnt target genes was observed in tumors,
although E7449 lacked single agent antitumor activity in vivo, a finding typical
for selective TNKS inhibitors. E7449 antitumor activity was increased through
combination with MEK inhibition. Particularly noteworthy was the lack of
toxicity, most significantly the lack of intestinal toxicity reported for other
TNKS inhibitors. E7449 represents a novel dual PARP1/2 and TNKS1/2 inhibitor
which has the advantage of targeting Wnt/β-catenin signaling addicted tumors.
E7449 is currently in early clinical development. PARP-family ADP-ribosyltransferases (PARPs) and sirtuin deacetylases all use
NAD(+) as cosubstrate for ADP-ribosyl transfer. PARP inhibitors are important
research tools and several are being evaluated in cancer treatment. With the
exception of a few tankyrase inhibitors, all current PARP inhibitors mimic the
nicotinamide moiety in NAD(+) and block the nicotinamide binding pocket. We
report here that while the activities of the four human sirtuin isoforms SIRT1,
SIRT2, SIRT3 and SIRT6 are blocked by sirtuin inhibitor Ex527 in vitro, they are
unaffected by the seven clinical and commonly used PARP inhibitors niraparib,
olaparib, rucaparib, talazoparib, veliparib, PJ34, and XAV939. These findings
indicate that PARP inhibitors containing planar nicotinamide mimetics do not
bind to sirtuin cofactor sites. In conclusion, a simple commercially available
assay can be used to rule out interference of novel PARP inhibitors with sirtuin
NAD(+) binding. For several years, a major obstacle in the systemic treatment of ovarian cancer
has been the lack of a therapeutic strategy tailored to specific biomarkers
present in the individual patient's tumour. However, considerable progress has
been made recently through the development of drugs targeting cells deficient in
the key mechanism of double-strand DNA repair, known as homologous recombination
(HRD). These drugs, inhibitors of the enzyme poly (ADP) ribose polymerase
(PARP), selectively kill HRD cells through a process known as tumour-selective
synthetic lethality. Olaparib is the first such agent, now approved for the
treatment of ovarian cancer associated with mutations in the BRCA 1/2 genes,
since these are characterised by cells with HRD. Importantly, another group of
patients with tumours bearing a similar repair deficiency but without BRCA
mutations may also be susceptible to PARP inhibition and efforts to develop an
HRD assay are therefore a priority so that these patients can be identified as
PARPi candidates. In addition, combination strategies are an area of intense
research; these include combinations with antiangiogenic agents and with
inhibitors of the P13K/AKT pathway and others are likely to merit assessment
since resistance to PARP inhibitors will certainly emerge as the next challenge.
While olaparib is the first PARP inhibitor to receive approval for ovarian
cancer treatment, others including rucaparib and niraparib are clearly effective
in this disease and, within the next year or two, the results of ongoing
randomised trials will clarify their respective roles. PARP inhibitors are
generally well tolerated; regulatory approval at present supports their use as a
maintece therapy (in Europe) and as treatment for advanced recurrent disease
(in the United States), but it is likely that these indications will extend as
the results of ongoing trials become available. Ten years have elapsed between
the first pre-clinical publications and the regulatory approval of PARP
inhibitors and the next 10 years promise to be even more productive. BACKGROUND: Slow progress in improving the outcome of ovarian cancer with
chemotherapy over the last decade has stimulated research into molecularly
targeted therapy. Poly(ADP-ribose) polymerase (PARP) inhibitors target DNA
repair and are specifically active in cells that have impaired repair of DNA by
the homologous recombination (HR) pathway. Cells with mutated BRCA function have
HR deficiency (HRD), which is also present in a significant proportion of
non-BRCA-mutated ovarian cancer.
DESIGN: In the last decade, olaparib, the first and most-investigated oral PARP
inhibitor, has undergone phase I-III trials as a single agent, in comparison
with and in addition to chemotherapy, and as a maintece therapy following
chemotherapy.
RESULTS: The greatest benefit to-date has been in the maintece setting,
prolonging the progression-free survival of high-grade serous ovarian cancer
with a BRCA1/2 mutation. In this group of patients, olaparib has received
approval as maintece following chemotherapy from the EMA, and accelerated
approval as a single agent in women who have had three or more lines of therapy.
Olaparib can be given for a prolonged period with few significant side-effects
in most patients. Similar trials with other PARP inhibitors (rucaparib,
niraparib and veliparib) are in progress and include non-BRCA-mutated ovarian
cancer. Second-generation studies are exploring the combination of PARP
inhibitors with anti-angiogenic drugs.
CONCLUSIONS: PARP inhibitors represent a step change in the management of
ovarian cancer. BRCA mutations are the first genotypic predictive markers in
ovarian cancer and can be used to select patients who will most likely benefit
from PARP inhibitors. BRCA testing is now becoming a routine part of the
evaluation of women with ovarian cancer, and tests for HRD are being used to
evaluate PARP inhibitors in an extended population of non-BRCA-mutated ovarian
cancer. OBJECTIVE: Poly(ADP-ribose) polymerase (PARP) inhibitors have yielded
encouraging responses in high-grade serous ovarian carcinomas (HGSOCs), but the
optimal treatment setting remains unknown. We assessed the effect of niraparib
on HGSOC patient-derived xenograft (PDX) models as well as the relationship
between certain markers of homologous recombination (HR) status, including
BRCA1/2 mutations and formation of RAD51 foci after DNA damage, and response of
these PDXs to niraparib in vivo.
METHODS: Massively parallel sequencing was performed on HGSOCs to identify
mutations contributing to HR deficiency. HR pathway integrity was assessed using
fluorescence microscopy-based RAD51 focus formation assays. Effects of niraparib
(MK-4827) on treatment-naïve PDX tumor growth as monotherapy, in combination
with carboplatin/paclitaxel, and as maintece therapy were assessed by
transabdominal ultrasound. Niraparib responses were correlated with changes in
levels of poly(ADP-ribose), PARP1, and repair proteins by western blotting.
RESULTS: Five PDX models were evaluated in vivo. Tumor regressions were induced
by single-agent niraparib in one of two PDX models with deleterious BRCA2
mutations and in a PDX with RAD51C promoter methylation. Diminished formation of
RAD51 foci failed to predict response, but Artemis loss was associated with
resistance. Niraparib generally failed to enhance responses to
carboplatin/paclitaxel chemotherapy, but maintece niraparib therapy delayed
progression in a BRCA2-deficient PDX.
CONCLUSIONS: Mutations in HR genes are neither necessary nor sufficient to
predict response to niraparib. Assessment of repair status through multiple
complementary assays is needed to guide PARP inhibitor therapy, design future
clinical trials and identify ovarian cancer patients most likely to benefit from
PARP inhibition. Recent advances in our understanding of the molecular biology of epithelial
ovarian cancer have led to the development of a number of targeted therapies,
including poly-ADP-ribose polymerase (PARP) inhibitors. PARP inhibitors are a
novel class of therapeutic agents that target tumors with deficiencies in the
homologous recombination DNA repair pathway. Early studies have shown
significant efficacy for PARP inhibitors in patients with germline BRCA1/2
mutations. It has become evident that BRCA wild-type patients with other defects
in the homologous recombination repair pathway benefit from this therapeutic
approach. Importantly, companion homologous recombination deficiency scores are
being developed to help guide the selection of patients most likely to gain
clinical benefit from PARP inhibition. Olaparib, the first and most extensively
investigated PARP inhibitor, is now licensed in Europe for maintece treatment
of patients with platinum-sensitive relapsed BRCA-mutated (germline or somatic)
high-grade serous ovarian cancer who have responded to platinum-based
chemotherapy. In the United States, olaparib is licensed for treatment of
patients with germline BRCA-mutated ovarian cancer who have received 3 or more
lines of chemotherapy. There are a number of other PARP inhibitors in late phase
clinical development in ovarian cancer including rucaparib, niraparib,
veliparib, and talazoparib. This review will focus on the current evidence for
PARP inhibitors in ovarian cancer and discuss ongoing clinical trials and future
research directions in this rapidly evolving area. Author information:
(1)From the Nordic Society of Gynecological Oncology and
Rigshospitalet-Copenhagen University Hospital, Copenhagen (M.R.M.), Odense
University Hospital (J.H.) and European Network for Gynacological Oncological
Trial and Research Unit of General Practice, Institute of Public Health,
University of Southern Denmark, Odense (R.D.C.) - all in Denmark; University of
Arizona and Creighton University-Phoenix, Phoenix (B.J.M.), and Arizona Oncology
Associates, Tuscon (B.J.M., J.B.) - all in Arizona; Princess Margaret
Consortium, Princess Margaret Cancer Centre, University Health Network,
University of Toronto, Toronto (A.M.O.), British Columbia Cancer Agency,
Vancouver (A.V.T.), and McGill University-McGill University Health Centre,
Montreal (L.G.) - all in Canada; Arbeitsgemeinschaft Gynäkologische Onkologie
(AGO) and the University of Munich, Munich (S.M.), and Kliniken Essen Mitte,
Essen (A.B.) - both in Germany; Grupo Español de Investigación en Cáncer de
Ovario (GEICO) and Hospital Universitario La Paz (A.R.), and GEICO and M.D.
Anderson Cancer Center Madrid (A.G.-M.), Madrid; French Investigator Group for
Ovarian and Breast Cancer (GINECO) and Institut du Cancer de Montpellier,
Montpellier (M.F.), and GINECO and Centre Antoine Lacassagne, Nice (P.F.) - both
in France; National Cancer Research Institute and UCL Cancer Institute,
University College London, London (J.A.L.); Multicenter Italian Trials in
Ovarian Cancer/Mario Negri Gynecologic Oncology Group, Fondazione Istituto di
Ricovero e Cura a Carattere Scientifico, Istituto Nazionale dei Tumori, Milan
(D.L.); Belgium and Luxembourg Gynecological Oncology Group and University of
Leuven, Leuven, Belgium (I.V.); Kaplan Medical Center, Rehovot, Israel
(N.E.B.-B.); AGO-Austria and Medical University Innsbruck, Innsbruck, Austria
(C.M.); Central and Eastern European Gynecologic Oncology Group and Uniwersytet
Medyczny w Poziu, Poz, Poland (R.M.); Stanford Comprehensive Cancer
Institute, Stanford (J.S.B.), and Cedars-Sinai Medical Center, West Hollywood
(B.J.R.) - both in California; Oslo University Hospital, Radiumhospitalet, Oslo
(A.D.); Northside Hospital, Atlanta (B.B.); Universitetssjukhuset, Linköping,
Sweden (P.R.); and Veristat, Southborough (J.P.B.), Tesaro, Waltham (S.A.), and
Dana-Farber Cancer Institute, Boston (U.A.M.) - all in Massachusetts. Results from a phase III trial indicate that maintece therapy with the PARP
inhibitor niraparib is more effective than placebo in slowing the progression of
recurrent platinum-sensitive ovarian cancer. Improved progression-free survival
was seen regardless of the presence or absence of germline BRCA mutations, or of
homologous recombination deficiency; however, patients who had these mutations
or defective DNA repair did better. |
Which peak calling algorithm employs mixture model clustering under the hood? | JAMM (Joint Analysis of NGS replicates via Mixture Model clustering) is a peak finder that can integrate information from biological replicates, determine enrichment site widths accurately and resolve neighboring narrow peaks. JAMM is a universal peak finder that is applicable to different types of datasets. | MOTIVATION: Although peak finding in next-generation sequencing (NGS) datasets
has been addressed extensively, there is no consensus on how to analyze and
process biological replicates. Furthermore, most peak finders do not focus on
accurate determination of enrichment site widths and are not widely applicable
to different types of datasets.
RESULTS: We developed JAMM (Joint Analysis of NGS replicates via Mixture Model
clustering): a peak finder that can integrate information from biological
replicates, determine enrichment site widths accurately and resolve neighboring
narrow peaks. JAMM is a universal peak finder that is applicable to different
types of datasets. We show that JAMM is among the best performing peak finders
in terms of site detection accuracy and in terms of accurate determination of
enrichment sites widths. In addition, JAMM's replicate integration improves peak
spatial resolution, sorting and peak finding accuracy.
AVAILABILITY AND IMPLEMENTATION: JAMM is available for free and can run on Linux
machines through the command line: http://code.google.com/p/jamm-peak-finder. |
Data from which major epigenome projects are contained in the DeepBlue epigenomic data server? | The DeepBlue Epigenomic Data Server contains data from four major epigenome projects, namely ENCODE, ROADMAP, BLUEPRINT and DEEP. | Large amounts of epigenomic data are generated under the umbrella of the
International Human Epigenome Consortium, which aims to establish 1000 reference
epigenomes within the next few years. These data have the potential to unravel
the complexity of epigenomic regulation. However, their effective use is
hindered by the lack of flexible and easy-to-use methods for data retrieval.
Extracting region sets of interest is a cumbersome task that involves several
manual steps: identifying the relevant experiments, downloading the
corresponding data files and filtering the region sets of interest. Here we
present the DeepBlue Epigenomic Data Server, which streamlines epigenomic data
analysis as well as software development. DeepBlue provides a comprehensive
programmatic interface for finding, selecting, filtering, summarizing and
downloading region sets. It contains data from four major epigenome projects,
namely ENCODE, ROADMAP, BLUEPRINT and DEEP. DeepBlue comes with a user manual,
examples and a well-documented application programming interface (API). The
latter is accessed via the XML-RPC protocol supported by many programming
languages. To demonstrate usage of the API and to enable convenient data
retrieval for non-programmers, we offer an optional web interface. DeepBlue can
be openly accessed at http://deepblue.mpi-inf.mpg.de. |
Which diseases that can be treated using the focused ultrasound thalamotomy. | Focused ultrasound thalamotomy is used for treatment of Parkinson disease, essential tremor, obsessive-compulsive disorder and chronic neuropathic pain. | OBJECT: Recent technological developments open the field of therapeutic
application of focused ultrasound to the brain through the intact cranium. The
goal of this study was to apply the new transcranial magnetic resoce
imaging-guided focused ultrasound (tcMRgFUS) technology to perform noninvasive
central lateral thalamotomies (CLTs) as a treatment for chronic neuropathic
pain.
METHODS: In 12 patients suffering from chronic therapy-resistant neuropathic
pain, tcMRgFUS CLT was proposed. In 11 patients, precisely localized thermal
ablations of 3-4 mm in diameter were produced in the posterior part of the
central lateral thalamic nucleus at peak temperatures between 51 ° C and 64 ° C
with the aid of real-time patient monitoring and MR imaging and MR thermometry
guidance. The treated neuropathic pain syndromes had peripheral (5 patients) or
central (6 patients) origins and covered all body parts (face, arm, leg, trunk,
and hemibody).
RESULTS: Patients experienced mean pain relief of 49% at the 3-month follow-up
(9 patients) and 57% at the 1-year follow-up (8 patients). Mean improvement
according to the visual analog scale amounted to 42% at 3 months and 41% at 1
year. Six patients experienced immediate and persisting somatosensory
improvements. Somatosensory and vestibular clinical manifestations were always
observed during sonication time because of ultrasound-based neuronal activation
and/or initial therapeutic effects. Quantitative electroencephalography (EEG)
showed a significant reduction in EEG spectral overactivities. Thermal ablation
sites showed sharply delineated ellipsoidal thermolesions surrounded by
short-lived vasogenic edema. Lesion reconstructions (18 lesions in 9 patients)
demonstrated targeting precision within a millimeter for all 3 coordinates.
There was 1 complication, a bleed in the target with ischemia in the motor
thalamus, which led to the introduction of 2 safety measures, that is, the
detection of a potential cavitation by a cavitation detector and the maintece
of sonication temperatures below 60 ° C.
CONCLUSIONS: The authors assert that tcMRgFUS represents a noninvasive, precise,
and radiation-free neurosurgical technique for the treatment of neuropathic
pain. The procedure avoids mechanical brain tissue shift and eliminates the risk
of infection. The possibility of applying sonication thermal spots free from
trajectory restrictions should allow one to optimize target coverage. The
real-time continuous MR imaging and MR thermometry monitoring of targeting
accuracy and thermal effects are major factors in optimizing precision, safety,
and efficacy in an outpatient context. BACKGROUND: Essential tremor is the most common movement disorder and is often
refractory to medical treatment. Surgical therapies, using lesioning and deep
brain stimulation in the thalamus, have been used to treat essential tremor that
is disabling and resistant to medication. Although often effective, these
treatments have risks associated with an open neurosurgical procedure. MR-guided
focused ultrasound has been developed as a non-invasive means of generating
precisely placed focal lesions. We examined its application to the management of
essential tremor.
METHODS: Our study was done in Toronto, Canada, between May, 2012, and January,
2013. Four patients with chronic and medication-resistant essential tremor were
treated with MR-guided focused ultrasound to ablate tremor-mediating areas of
the thalamus. Patients underwent tremor evaluation and neuroimaging at baseline
and 1 month and 3 months after surgery. Outcome measures included tremor
severity in the treated arm, as measured by the clinical rating scale for
tremor, and treatment-related adverse events.
FINDINGS: Patients showed immediate and sustained improvements in tremor in the
domit hand. Mean reduction in tremor score of the treated hand was 89·4% at 1
month and 81·3% at 3 months. This reduction was accompanied by functional
benefits and improvements in writing and motor tasks. One patient had
postoperative paraesthesias which persisted at 3 months. Another patient
developed a deep vein thrombosis, potentially related to the length of the
procedure.
INTERPRETATION: MR-guided focused ultrasound might be a safe and effective
approach to generation of focal intracranial lesions for the management of
disabling, medication-resistant essential tremor. If larger trials validate the
safety and ascertain the efficacy and durability of this new approach, it might
change the way that patients with essential tremor and potentially other
disorders are treated.
FUNDING: Focused Ultrasound Foundation. BACKGROUND: Recent advances have enabled delivery of high-intensity focused
ultrasound through the intact human cranium with magnetic resoce imaging
(MRI) guidance. This preliminary study investigates the use of transcranial
MRI-guided focused ultrasound thalamotomy for the treatment of essential tremor.
METHODS: From February 2011 through December 2011, in an open-label,
uncontrolled study, we used transcranial MRI-guided focused ultrasound to target
the unilateral ventral intermediate nucleus of the thalamus in 15 patients with
severe, medication-refractory essential tremor. We recorded all safety data and
measured the effectiveness of tremor suppression using the Clinical Rating Scale
for Tremor to calculate the total score (ranging from 0 to 160), hand subscore
(primary outcome, ranging from 0 to 32), and disability subscore (ranging from 0
to 32), with higher scores indicating worse tremor. We assessed the patients'
perceptions of treatment efficacy with the Quality of Life in Essential Tremor
Questionnaire (ranging from 0 to 100%, with higher scores indicating greater
perceived disability).
RESULTS: Thermal ablation of the thalamic target occurred in all patients.
Adverse effects of the procedure included transient sensory, cerebellar, motor,
and speech abnormalities, with persistent paresthesias in four patients. Scores
for hand tremor improved from 20.4 at baseline to 5.2 at 12 months (P=0.001).
Total tremor scores improved from 54.9 to 24.3 (P=0.001). Disability scores
improved from 18.2 to 2.8 (P=0.001). Quality-of-life scores improved from 37% to
11% (P=0.001).
CONCLUSIONS: In this pilot study, essential tremor improved in 15 patients
treated with MRI-guided focused ultrasound thalamotomy. Large, randomized,
controlled trials will be required to assess the procedure's efficacy and
safety. (Funded by the Focused Ultrasound Surgery Foundation; ClinicalTrials.gov
number, NCT01304758.). Neurosurgical procedures are indispensable in management of various types of
movement disorders (MD). Stereotactic operations that have been well established
include deep brain stimulation for tremor, dystonia, and Parkinsonian symptoms.
Recently the actual role of stereotactic ablative procedures such as thalamotomy
and pallidotomy has been re-explored, and Vo thalamotomy shows long-term
improvement of task specific focal dystonia like writer's cramp and musician's
dystonia. A new less invasive treatment of tremor using MR guided focused
ultrasound has started and is promising. Intrathecal administration of baclofen
is also an established treatment for severe spasticity, but other ablative
procedures such as peripheral neurotomy and dorsal rhizotomy are also important
in spasticity treatment. It seems that most neurologists are unfamiliar, at
least in Japan, with such neurosurgical procedures. However, neurologists
involved in management of MD should understand the important roles of
neurosurgical management of intractable MD and should refer such patients to
appropriate neurosurgeons before permanent contracture and deformity develop. OBJECT: The authors report different MRI patterns in patients with essential
tremor (ET) or obsessive-compulsive disorder (OCD) after transcranial MR-guided
focused ultrasound (MRgFUS) and discuss possible causes of occasional MRgFUS
failure.
METHODS: Between March 2012 and August 2013, MRgFUS was used to perform
unilateral thalamotomy in 11 ET patients and bilateral anterior limb capsulotomy
in 6 OCD patients; in all patients symptoms were refractory to drug therapy.
Sequential MR images were obtained in patients across a 6-month follow-up
period.
RESULTS: For OCD patients, lesion size slowly increased and peaked 1 week after
treatment, after which lesion size gradually decreased. For ET patients, lesions
were visible immediately after treatment and markedly reduced in size as time
passed. In 3 ET patients and 1 OCD patient, there was no or little temperature
rise (i.e., < 52°C) during MRgFUS. Successful and failed patient groups showed
differences in their ratio of cortical-to-bone marrow thickness (i.e., skull
density).
CONCLUSIONS: The authors found different MRI pattern evolution after MRgFUS for
white matter and gray matter. Their results suggest that skull characteristics,
such as low skull density, should be evaluated prior to MRgFUS to successfully
achieve thermal rise. Transcranial magnetic resoce imaging-guided high-intensity focused ultrasound
(MRgHIFU) is gaining attention as a potent substitute for surgical intervention
in the treatment of neurologic disorders. To discern the neurophysiologic
correlates of its therapeutic effects, we applied MRgHIFU to an intractable
neurologic disorder, essential tremor, while measuring magnetoencephalogram mu
rhythms from the motor cortex. Focused ultrasound sonication destroyed tissues
by focusing a high-energy beam on the ventralis intermedius nucleus of the
thalamus. The post-treatment effectiveness was also evaluated using the clinical
rating scale for tremors. Thalamic MRgHIFU had substantial therapeutic effects
on patients, based on MRgHIFU-mediated improvements in movement control and
significant changes in brain mu rhythms. Ultrasonic thalamotomy may reduce
hyper-excitable activity in the motor cortex, resulting in normalized behavioral
activity after sonication treatment. Thus, non-invasive and spatially accurate
MRgHIFU technology can serve as a potent therapeutic tool with broad clinical
applications. BACKGROUND: Radiofrequency (RF) subthalamotomies have been proposed since the
1960s to treat patients suffering from Parkinson's disease (PD). Recently, the
magnetic resoce (MR)-guided focused ultrasound technology (MRgFUS) offers the
possibility to perform subthalamic thermocoagulations with reduced risks and
optimized accuracy. We describe here the initial results of the MRgFUS
pallidothalamic tractotomy (PTT), an anatomical and physiological update of the
earlier subthalamotomies.
METHODS: Thirteen consecutive patients suffering from chronic (mean disease
duration 9.7 years) and therapy-resistant PD were treated unilaterally with an
MRgFUS PTT. Primary relief assessment indicators were the score reduction of the
Unified Parkinson Disease Rating Scale (UPDRS) and the patient estimation of
global symptom relief (GSR) taken at 3 months follow-up. Final temperatures at
target were between 52°C and 59°C. The MR examinations were performed before the
treatment, 2 days and 3 months after it. The accuracy of the targeting was
calculated on 2 days post-treatment MR pictures for each PTT lesion.
RESULTS: The first four patients received a PTT using the lesional parameters
applied for thalamotomies. They experienced clear-cut recurrences at 3 months
(mean UPDRS relief 7.6%, mean GSR 22.5%), and their MR showed no sign of thermal
lesion in T2-weighted (T2w) images. As a consequence, the treatment protocol was
adapted for the following nine patients by applying repetition of the final
temperatures 4 to 5 times. That produced thermocoagulations of larger volumes
(172 mm(3) against 83 mm(3) for the first four patients), which remained visible
at 3 months on T2w images. These nine patients enjoyed a mean UPDRS reduction of
60.9% and a GSR of 56.7%, very close to the results obtained with radiofrequency
lesioning. The targeting accuracy for the whole patient group was 0.5, 0.5, and
0.6 mm for the anteroposterior (AP), mediolateral (ML), and dorsoventral (DV)
dimensions, respectively.
CONCLUSIONS: This study demonstrated the feasibility, safety, and accuracy of
the MRgFUS PTT. To obtain similar results as the ones of RF PTT, it was
necessary to integrate the fact that white matter, in this case, the
pallidothalamic tract, requires repeated thermal exposition to achieve full
lesioning and thus full therapeutic effect. While no real breakthrough in the medical treatment of Essential Tremor (ET) has
recently emerged, surgical field is expanding exponentially. Purpose of this
review is to examine the recent and future developments of the surgical
treatments for ET. Technological advances are shaping the present and the future
application of deep brain stimulation (DBS) in ET. New electrode configurations
as well as new implantable pulse generators are now available. Application of
closed-loop or adaptive stimulation in clinical practice will allow DBS to
deliver stimulation in a truly physiological way to restore aberrant
neurological circuits on demand, thus avoiding side effects, tolerance and also
saving the battery life. Besides DBS and standard thalamotomy, novel surgical
approaches for ET are on the horizon. The development of MRI-guided focused
ultrasound technique has been the new frontier of deep brain lesional therapies.
Although the benefit of motor cortex stimulation is yet to be defined, this
minimally invasive approach remains intriguing. Although the advances of
surgical treatments along the clinical and technological directions described in
this review will certainly contribute to a successful management of ET patients,
future studies need to consider critical issues such as the heterogeneity of ET
and the development of tolerance. Background. Thalamotomy is effective in alleviating tremor in Parkinson's
disease (PD). Methods. Seven PD patients, mean age 59.4 ± 9.8 years (range,
46-74) with a mean disease duration of 5.4 ± 2.8 years (range, 2-10) suffering
from severe refractory tremor, underwent ventral intermediate nucleus
thalamotomy using MRI guided focused ultrasound (MRgFUS), an innovative
technology that enables noninvasive surgery. Results. Tremor stopped in the
contralateral upper extremity in all patients immediately following treatment.
Total UPDRS decreased from 37.4 ± 12.2 to 18.8 ± 11.1 (p = 0.007) and PDQ-39
decreased from 42.3 ± 16.4 to 21.6 ± 10.8 (p = 0.008) following MRgFUS. These
effects were sustained (mean follow-up 7.3 months). Adverse events during MRgFUS
included headache (n = 3), dizziness (n = 2), vertigo (n = 4), and lip
paresthesia (n = 1) and following MRgFUS were hypogeusia (n = 1), unsteady
feeling when walking (n = 1, resolved), and disturbance when walking tandem (n =
1, resolved). Conclusions. Thalamotomy using MRgFUS is safe and effective in PD
patients. Large randomized studies are needed to assess prolonged efficacy and
safety. Progressively less invasive neurosurgical approaches for the treatment of
movement disorders have evolved, beginning with open craniotomy for placement of
lesions within pyramidal structures followed by refined stereotactic ablation of
extrapyramidal targets that encouraged nondestructive electrode stimulation of
deep brain structures. A noninvasive approach using transcranial high-energy
focused ultrasound has emerged for the treatment of intractable tremor. The
ability to target discreet intracranial sites millimeters in size through the
intact skull using focused acoustic energy marks an important milestone in
movement disorders surgery. This article describes the evolution of magnetic
resoce-guided focused ultrasound for ventrolateral thalamotomy for tremor. BACKGROUND: Thalamic deep brain stimulation (DBS) has largely replaced
radiofrequency thalamotomy as the treatment of choice for disabling,
medication-refractory essential tremor. Recently, the development of
transcranial, high-intensity focused ultrasound has renewed interest in thalamic
lesioning. The purpose of this study is to compare functional outcomes and
quality of life in essential tremor patients treated with either bilateral Vim
DBS or unilateral procedures (focused ultrasound or DBS). We hypothesized that
all three would effectively treat the domit hand and positively impact
functional outcomes and quality of life as measured with the Clinical Rating
Scale for Tremor and the Quality of Life in Essential Tremor Questionnaire.
METHODS: This is a retrospective study of medication-refractory essential tremor
patients treated at the University of Virginia with bilateral Vim DBS (n = 57),
unilateral Vim DBS (n = 13), or unilateral focused ultrasound Vim thalamotomy
(n = 15). Tremor was rated for all patients before and after treatment, using
the Clinical Rating Scale for Tremor and Quality of Life in Essential Tremor
Questionnaire.
RESULTS: Patients undergoing bilateral DBS treatment had more baseline tremor
and worse quality of life scores. Patients had significant improvements in
tremor symptoms and quality of life with all three treatments. Both DBS
procedures improved axial tremor. No difference was seen in the degree of
improvement in upper extremity tremor score, disability, or overall quality of
life between bilateral and either unilateral procedure.
CONCLUSIONS: Bilateral thalamic DBS improves overall tremor more than unilateral
DBS or focused ultrasound treatment; however, unilateral treatments are equally
effective in treating contralateral hand tremor. Despite the greater overall
tremor reduction with bilateral DBS, there is no difference in disability or
quality of life comparing bilateral versus unilateral treatments. BACKGROUND: Already in the late 1960s and early 1970s, targeting of the
"posterior subthalamic area (PSA)" was explored by different functional
neurosurgical groups applying the radiofrequency (RF) technique to treat
patients suffering from essential tremor (ET). Recent advances in magnetic
resoce (MR)-guided focused ultrasound (MRgFUS) technology offer the
possibility to perform thermocoagulation of the cerebellothalamic fiber tract in
the PSA without brain penetration, allowing a strong reduction of the
procedure-related risks and increased accuracy. We describe here the first
results of the MRgFUS cerebellothalamic tractotomy (CTT).
METHODS: Twenty-one consecutive patients suffering from chronic (mean disease
duration 29.9 years), therapy-resistant ET were treated with MRgFUS CTT. Three
patients received bilateral treatment with a 1-year interval. Primary relief
assessment indicators were the Essential Tremor Rating Scale (Fahn, Tolosa, and
Marin) (ETRS) taken at follow-up (3 months to 2 years) with accent on the hand
function subscores (HF16 for treated hand and HF32 for both hands) and
handwriting. The evolution of seven patients with HF32 above 28 points over 32
(group 1) differentiated itself from the others' (group 2) and was analyzed
separately. Global tremor relief estimations were provided by the patients.
Lesion reconstruction and measurement of targeting accuracy were done on 2-day
post-treatment MR pictures for each CTT lesion.
RESULTS: The mean ETRS score for all patients was 57.6 ± 13.2 at baseline and
25.8 ± 17.6 at 1 year (n = 10). The HF16 score reduction was 92 % in group 2 at
3 months and stayed stable at 1 year (90 %). Group 1 showed only an improvement
of 41 % at 3 months and 40 % at 1 year. Nevertheless, two patients of group 1
treated bilaterally had an HF16 score reduction of 75 and 88 % for the domit
hand at 1 year after the second side. The mean patient estimation of global
tremor relief after CTT was 92 % at 2 days and 77 % at 1-year follow-up.
CONCLUSIONS: CTT with MRgFUS was shown to be an effective and safe approach for
patients with therapy-refractory essential tremor, combining neurological
function sparing with precise targeting and the possibility to treat patients
bilaterally. 1. BACKGROUND: Uncontrolled pilot studies have suggested the efficacy of focused
ultrasound thalamotomy with magnetic resoce imaging (MRI) guidance for the
treatment of essential tremor.
METHODS: We enrolled patients with moderate-to-severe essential tremor that had
not responded to at least two trials of medical therapy and randomly assigned
them in a 3:1 ratio to undergo unilateral focused ultrasound thalamotomy or a
sham procedure. The Clinical Rating Scale for Tremor and the Quality of Life in
Essential Tremor Questionnaire were administered at baseline and at 1, 3, 6, and
12 months. Tremor assessments were videotaped and rated by an independent group
of neurologists who were unaware of the treatment assignments. The primary
outcome was the between-group difference in the change from baseline to 3 months
in hand tremor, rated on a 32-point scale (with higher scores indicating more
severe tremor). After 3 months, patients in the sham-procedure group could cross
over to active treatment (the open-label extension cohort).
RESULTS: Seventy-six patients were included in the analysis. Hand-tremor scores
improved more after focused ultrasound thalamotomy (from 18.1 points at baseline
to 9.6 at 3 months) than after the sham procedure (from 16.0 to 15.8 points);
the between-group difference in the mean change was 8.3 points (95% confidence
interval [CI], 5.9 to 10.7; P<0.001). The improvement in the thalamotomy group
was maintained at 12 months (change from baseline, 7.2 points; 95% CI, 6.1 to
8.3). Secondary outcome measures assessing disability and quality of life also
improved with active treatment (the blinded thalamotomy cohort)as compared with
the sham procedure (P<0.001 for both comparisons). Adverse events in the
thalamotomy group included gait disturbance in 36% of patients and paresthesias
or numbness in 38%; these adverse events persisted at 12 months in 9% and 14% of
patients, respectively.
CONCLUSIONS: MRI-guided focused ultrasound thalamotomy reduced hand tremor in
patients with essential tremor. Side effects included sensory and gait
disturbances. (Funded by InSightec and others; ClinicalTrials.gov number,
NCT01827904.). Thalamotomy at the ventralis intermedius nucleus has been an effective treatment
method for essential tremor, but how the brain network changes immediately
responding to this deliberate lesion and then reorganizes afterwards are not
clear. Taking advantage of a non-cranium-opening MRI-guided focused ultrasound
ablation technique, we investigated functional network changes due to a focal
lesion. To classify the diverse time courses of those network changes with
respect to symptom-related long-lasting treatment effects and symptom-unrelated
transient effects, we applied graph-theoretic analyses to longitudinal
resting-state functional magnetic resoce imaging data before and 1 day,
7 days, and 3 months after thalamotomy with essential tremor. We found reduced
average connections among the motor-related areas, reduced connectivity between
substantia nigra and external globus pallidum and reduced total connection in
the thalamus after thalamotomy, which are all associated with clinical rating
scales. The average connectivity among whole brain regions and inter-hemispheric
network asymmetry show symptom-unrelated transient increases, indicating
temporary reconfiguration of the whole brain network. In summary, thalamotomy
regulates interactions over the motor network via symptom-related connectivity
changes but accompanies transient, symptom-unrelated diaschisis in the global
brain network. This study suggests the significance of longitudinal network
analysis, combined with minimal-invasive treatment techniques, in understanding
time-dependent diaschisis in the brain network due to a focal lesion. |
Is the toxin produced by Clostridium botulinum always deadly? | There are numerous examples where children and animals survived infection with clostridium botulinum. | Botulism is a rare but potentially fatal disease caused by toxins produced by
Clostridium botulinum. We report a case of botulism in a 38-year-old man after
eating canned "garlic in chilli-oil". The patient was treated with antiserum.
The diagnosis was confirmed by detection of botulinum B toxin by a bio-assay and
growth of Clostridium botulinum from the food left-overs. In the last fifty years, Clostridium botulinum has become notorious for its
ability to produce the deadly botulinum neurotoxins. While botulinum toxin A,
better known as Botox, is universally recognised by the public as a cosmetic
enhancement tool, the botulinum neurotoxins are commonly used off-label for many
medical conditions in ophthalmology, neurology and dermatology. The versatility
of these botulinum toxins has made Clostridium botulinum one of the most widely
known bacterial pathogens in medical history. This article outlines the
discovery of botulinum toxins through to their present day applications in
medicine. BACKGROUND: Benign masseter muscle hypertrophy is an uncommon clinical
phenomenon of uncertain aetiology which is characterised by a soft swelling near
the angle of the mandible. The swelling may on occasion be associated with
facial pain and can be prominent enough to be considered cosmetically
disfiguring. Varying degrees of success have been reported for some of the
treatment options for masseter hypertrophy, which range from simple
pharmacotherapy to more invasive surgical reduction. Injection of botulinum
toxin type A into the masseter muscle is generally considered a less invasive
modality and has been advocated for cosmetic sculpting of the lower face.
Botulinum toxin type A is a powerful neurotoxin which is produced by the
anaerobic organism Clostridium botulinum and when injected into a muscle causes
interference with the neurotransmitter mechanism producing selective paralysis
and subsequent atrophy of the muscle.
OBJECTIVES: To assess the effects of botulinum toxin type A in the management of
benign bilateral masseter hypertrophy.
SEARCH STRATEGY: We searched the following databases in August 2008: the
Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library
2008, issue 3); MEDLINE (via PubMed) (1950 to August 2008); EMBASE (via
embase.com) (1980 to August 2008); and LILACS via BIREME. We searched two
bibliographic databases of regional journals which may be expected to contain
relevant trials (IndMED and Iranmedex) using free text terms appropriate for
this review.
SELECTION CRITERIA: Randomised controlled clinical trials (RCTs) and controlled
clinical trials (CCTs) comparing intra-masseteric injections of botulinum toxin
versus placebo administered for cosmetic facial sculpting in individuals of any
age with bilateral benign masseter hypertrophy, which had been self-evaluated
and confirmed by clinical and radiological examination. We excluded participants
with unilateral or compensatory contralateral masseter hypertrophy resulting
from head and neck radiotherapy.
DATA COLLECTION AND ANALYSIS: Two review authors conducted screening of studies
in duplicate and independently, and although no eligible trials were identified,
the two authors had planned to extract data independently and assess trial
quality using standard Cochrane Collaboration methodologies.
MAIN RESULTS: We retrieved 167 references to studies, none of which matched the
inclusion criteria for this review and all of which were excluded.
AUTHORS' CONCLUSIONS: We were unable to identify any randomised controlled
trials on the efficacy of intra-masseteric injections of botulinum toxin for
people with bilateral benign masseter hypertrophy. The absence of high level
evidence for the effectiveness of this intervention emphasises the need for
well-designed, adequately powered, randomised controlled clinical trials (RCTs)
and controlled clinical trials (CCTs). BACKGROUND AND OBJECTIVES: Botulinum toxin (BTX) is one of the most potent
bacterial toxins known and its effectiveness in the treatment of some pain
syndromes is well known. However, the efficacy of some of its indications is
still in the process of being confirmed. The objective of this study was to
review the history, pharmacological properties, and clinical applications of BTX
in the treatment of pain of different origins.
CONTENTS: Botulinum toxin is produced by fermentation of Clostridium botulinum,
a Gram-positive, anaerobic bacterium. Commercially, BTX comes in two
presentations, types A and B. Botulinum toxin, a neurotoxin with high affinity
for cholinergic synapses, blocks the release of acetylcholine by nerve endings
without interfering with neuronal conduction of electrical signals or synthesis
and storage of acetylcholine. It has been proven that BTX can selectively weaken
painful muscles, interrupting the spasm-pain cycle. Several studies have
demonstrated the efficacy and safety of BTX-A in the treatment of tension
headaches, migraines, chronic lumbar pain, and myofascial pain.
CONCLUSIONS: Botulinum toxin type A is well tolerated in the treatment of
chronic pain disorders in which pharmacotherapy regimens can cause side effects.
The reduction in the consumption of analgesics and length of action of 3 to 4
months per dose represent other advantages of its use. However, further studies
are necessary to establish the efficacy of BTX-A in chronic pain disorders and
its exact mechanism of action, as well as its potential in multifactorial
treatments. BACKGROUND: On 7 and 11 July 2007, health officials in Texas and Indiana,
respectively, reported 4 possible cases of type A foodborne botulism to the US
Centers for Disease Control and Prevention. Foodborne botulism is a rare and
sometimes fatal illness caused by consuming foods containing botulinum
neurotoxin.
METHODS: Investigators reviewed patients' medical charts and food histories.
Clinical specimens and food samples were tested for botulinum toxin and
neurotoxin-producing Clostridium species. Investigators conducted inspections of
the cannery that produced the implicated product.
RESULTS: Eight confirmed outbreak associated cases were identified from Indiana
(n = 2), Texas (n = 3), and Ohio (n = 3). Botulinum toxin type A was identified
in leftover chili sauce consumed by the Indiana patients and 1 of the Ohio
patients. Cannery inspectors found violations of federal canned-food regulations
that could have led to survival of Clostridium botulinum spores during
sterilization. The company recalled 39 million cans of chili. Following the
outbreak, the US Food and Drug Administration inspected other canneries with
similar canning systems and issued warnings to the industry about the danger of
C. botulinum and the importance of compliance with canned food manufacturing
regulations.
CONCLUSIONS: Commercially produced hot dog chili sauce caused these cases of
type A botulism. This is the first US foodborne botulism outbreak involving a
commercial cannery in >30 years. Sharing of epidemiologic and laboratory
findings allowed for the rapid identification of implicated food items and swift
removal of potentially deadly products from the market by US food regulatory
authorities. BACKGROUND: Infantile botulism is the result of ingestion of Clostridium
botulinum spores, and is the most common form of infection with botulism in the
United States. Ninety percent of cases occur in infants <6 months old. The
infants typically present with vague symptoms such as hypotonia and poor
feeding. This article reports an infant with confirmed infantile botulism that
presented to the Emergency Department (ED) with complaints of decreased feeding
and absence of bowel movements for >1 week.
OBJECTIVES: Review a case of infantile botulism, its diagnosis, and treatment.
CASE REPORT: A 4-month-old healthy Caucasian male presented to the ED with a
6-day history of decreased feeding after referral from the pediatrician. He had
not had a bowel movement for 9 days, and his parents were also concerned about
increasing weakness, as he was no longer able to hold his head up on his own. In
the ED, he was minimally interactive. His vital signs were within normal limits,
and he had hypoactive bowel sounds and decreased tone throughout. He was
admitted to the Children's Hospital and eventually transferred to the Pediatric
Intensive Care Unit requiring intubation and mechanical ventilation. The
botulism immunoglobulin was administered, and a diagnosis was confirmed with
positive botulinum toxin in the stool samples. Full recovery was made by the
infant.
CONCLUSION: Awareness of the symptoms of botulism and a high degree of clinical
suspicion is needed to make a prompt diagnosis. We present the first case of abnormal neuroimaging in a case of infant botulism.
The clinical findings of the patient with constipation, bulbar weakness, and
descending, symmetric motor weakness are consistent with the classic findings of
infant botulism. Magnetic resoce imaging (MRI), however, revealed restricted
diffusion in the brain and enhancement of the cervical nerve roots.
Traditionally, normal neuroimaging was used to help differentiate infant
botulism from other causes of weakness in infants. Abnormal neuroimaging is seen
in other causes of weakness in an infant including metabolic disorders and
hypoxic-ischemic injury, but these diagnoses did not fit the clinical findings
in this case. The explanation for the MRI abnormalities in the brain and
cervical nerve roots is unclear as botulinum toxin acts at presynaptic nerve
terminals and does not cross the blood-brain barrier. Possible explanations for
the findings include inflammation from the botulinum toxin at the synapse,
alterations in sensory signaling and retrograde transport of the botulinum
toxin. The patient was treated with human botulism immune globulin and had rapid
recovery in weakness. A stool sample from the patient was positive for Type A
Clostridium botulinum toxin eventually confirming the diagnosis of infant
botulism. The findings in this case support use of human botulism immune
globulin when the clinical findings are consistent with infant botulism despite
the presence of MRI abnormalities in the brain and cervical nerve roots. |
Is ocrelizumab effective for treatment of multiple sclerosis? | Yes, ocrelizumab is effective for primary progressive form of multiple sclerosis. | Biogen Idec Inc, Genentech Inc, Roche Holding AG and Chugai Pharmaceutical Co
Ltd are developing ocrelizumab, a humanized mAb against CD20, for the potential
treatment of inflammatory disorders and B-cell maligcies. Ocrelizumab is
undergoing phase III clinical trials for rheumatoid arthritis and lupus
nephritis, and phase II trials for multiple sclerosis and hematological cancer.
Previously, ocrelizumab was also being developed for the treatment of systemic
lupus erythematosus (SLE) and neuromyelitis optica; however, development for SLE
has been discontinued. No development has been reported for neuromyelitis optica
and as of January 2007, this indication had been removed from the company
pipeline. Disease modifying therapy (DMT) first became available for relapsing-remitting
multiple sclerosis (RRMS) fifteen years ago with the development of the
moderately effective injectable agents interferon (IFN)-beta and glatiramer
acetate (GA). The subsequent licensure of mitoxantrone (MX) and natalizumab (NZ)
has allowed for better control of refractory disease at the expense of
potentially life-threatening side effects in a minority of patients. This
dichotomy between DMT potency and safety also characterizes the next generation
of DMTs. Five oral medications (fingolimod, cladribine, teriflunomide,
laquinimod and fumarate) are at various stages of phase III trials and it is
anticipated that at least some of these will be on the market within the next
year. The development of oral agents would be a tremendous advance with respect
to convenience and it is anticipated that this would dramatically increase the
number of patients on therapy. In parallel with oral therapies, powerful
immunosuppressive monoclonal antibodies (alemtuzumab, rituximab/ocrelizumab,
daclizumab) are also being evaluated. Enthusiasm for the next generation of
therapies is tempered by safety concerns. Serious and occasionally fatal
complications have occurred with the emerging monoclonal therapies and rigorous
patient selection will be required for these agents. Moreover, some of the oral
DMTs that are most eagerly awaited by patients have also been associated with
serious side-effects in the trials to date. It is unclear how oral agents will
be incorporated into future treatment algorithms given the need to weigh the
ease of oral administration against the relative inconvenience but long-term
safety of current first-line injectable therapies. BACKGROUND: B lymphocytes are implicated in the pathogenesis of multiple
sclerosis. We aimed to assess efficacy and safety of two dose regimens of the
humanised anti-CD20 monoclonal antibody ocrelizumab in patients with
relapsing-remitting multiple sclerosis.
METHODS: We did a multicentre, randomised, parallel, double-blind,
placebo-controlled study involving 79 centres in 20 countries. Patients aged
18-55 years with relapsing-remitting multiple sclerosis were randomly assigned
(1:1:1:1) via an interactive voice response system to receive either placebo,
low-dose (600 mg) or high-dose (2000 mg) ocrelizumab in two doses on days 1 and
15, or intramuscular interferon beta-1a (30 μg) once a week. The randomisation
list was not disclosed to the study centres, monitors, project statisticians or
to the project team at Roche. All groups were double blinded to group
assignment, except the interferon beta-1a group who were rater masked. At week
24, patients in the initial placebo, 600 mg ocrelizumab, and interferon beta-1a
groups received ocrelizumab 600 mg; the 2000 mg group received 1000 mg. Our
primary endpoint was the total number of gadolinium-enhancing lesions (GEL) and
T1-weighted MRI at weeks 12, 16, 20, and 24. Analyses were done on an
intention-to-treat basis. This trial is registered with ClinicalTrials.gov,
number NCT00676715.
FINDINGS: 218 (99%) of the 220 randomised patients received at least one dose of
ocrelizumab, 204 (93%) completed 24 weeks of the study and 196 (89%) completed
48 weeks. In the intention-to-treat population of 218 patients, at week 24, the
number of gadolinium-enhancing lesions was 89% (95% CI 68-97; p<0·0001) lower in
the 600 mg ocrelizumab group than in the placebo group, and 96% (89-99;
p<0·0001) lower in the 2000 mg group. In exploratory analyses, both 600 mg and
2000 mg ocrelizumab groups were better than interferon beta-1a for GEL
reduction. We noted serious adverse events in two of 54 (4%; 95% CI 3·0-4·4)
patients in the placebo group, one of 55 (2%; 1·3-2·3) in the 600 mg ocrelizumab
group, three of 55 (5%; 4·6-6·3) in the 2000 mg group, and two of 54 (4%;
3·0-4·4) in the interferon beta-1a group.
INTERPRETATION: The similarly pronounced effects of B-cell depletion with both
ocrelizumab doses on MRI and relapse-related outcomes support a role for B-cells
in disease pathogenesis and warrant further assessment in large, long-term
trials.
FUNDING: F Hoffmann-La Roche Ltd, Biogen Idec Inc. Recent years have broadened the spectrum of therapeutic strategies and specific
agents for treatment of multiple sclerosis (MS). While immune-modulating drugs
remain the first-line agents for MS predomitly due to their benign safety
profile, our growing understanding of key processes in initiation and
progression of MS has pioneered development of new agents with specific targets.
One concept of these novel drugs is to hamper migration of immune cells towards
the affected central nervous system (CNS). The first oral drug approved for MS
therapy, fingolimod inhibits egress of lymphocytes from lymph nodes; the
monoclonal antibody natalizumab prevents inflammatory CNS infiltration by
blocking required adhesion molecules. The second concept is to deplete T cells
and/or B cells from the peripheral circulation using highly specific monoclonal
antibodies such as alemtuzumab (anti-CD52) or rituximab/ocrelizumab (anti-CD20).
All of these novel, highly effective agents are a substantial improvement in our
therapeutic armamentarium; however, they have in common to potentially lower the
abundance of immune cells within the CNS, thereby collaterally affecting immune
surveillance within this well-controlled compartment. In this review, we aim to
critically evaluate the risk/benefit ratio of therapeutic strategies in
treatment of MS with a specific focus on infectious neurological side effects. Drug development for multiple sclerosis (MS), as with any other neurological
disease, faces numerous challenges, with many drugs failing at various stages of
development. The disease-modifying therapies (DMTs) first introduced for MS are
only moderately effective, but given the lack of competition, they have been
widely accepted in clinical practice. Although safety and efficacy continue to
be the two main metrics by which drugs will be judged, the newer agents in the
market also face challenges of a more comparative nature-are they more
efficacious than the currently available drugs on the market? Are they safer or
better tolerated? Do they offer any practical advantages over current
treatments? Fingolimod represented a milestone following its approval as an oral
drug for MS in 2010, offering patients a far more convenient administration
route. However, association with cardiovascular complications has led to a more
cautious approach in its initial prescribing, now requiring cardiac monitoring
for the first 6 h as well as subsequent monitoring of blood pressure and for
macular oedema. Natalizumab, amongst licensed drugs, represents the current
benchmark for efficacy. The risk of progressive multifocal leukoencephalopathy
during natalizumab treatment is now more quantifiable. Other monoclonal
antibodies are in various phases of development. Marketing authorisation for
alemtuzumab has been filed, and whilst trial data suggest that its efficacy
outperforms both licensed drugs and others in development, there is a
significant risk of secondary autoimmunity. Its once-yearly administration,
however, seems particularly advantageous. Rituximab is unlikely to be developed
further as its license will expire, but ocrelizumab, another monoclonal antibody
directly targeting B cells, is currently in phase 2 development and looks
promising. Daclizumab is also moderately efficacious but may struggle to
establish itself given its monthly subcutaneous dosing. There are new oral drugs
in development, and it is likely that BG-12 will be licensed this year. This has
been licensed for psoriasis so there are good safety data in humans that may
also hold true in MS; however, its three times daily dosage will probably impact
on patient compliance. Laquinimod has lower efficacy than BG-12 but appears safe
and could find a place as a first-line agent. Teriflunomide has just been
licensed by the US FDA and may challenge the current injectable first-line
therapies as it has a similar efficacy but the advantage of being taken orally.
However, risk of teratogenicity may caution against its use in some women of
child-bearing potential. This review will examine drugs that have been recently
approved as well as those that are in late phase 2 or 3 development as treatment
for relapsing MS, highlighting their mechanism of action as well as the clinical
trial and safety data before discussing their potential for success in an
increasingly florid and complex DMT armamentarium. This article reviews and discusses the approved and emerging therapies for
multiple sclerosis (MS). MS is a chronic and disabling immune-mediated disease
of the central nervous system (CNS) that affects mainly young adults. MS imposes
a huge economic burden on healthcare systems and the society. Although the last
20 years have brought a continuous expansion in therapeutic options, there are
still unmet needs in MS management. Available MS drugs have varying degrees of
efficacy in reducing relapse risk. The long-term term effects of these
treatments are incompletely known. New therapies, along with variations of
currently available treatments, may prove more effective and tolerable than the
available drugs. Treatments for MS differ with respect to the mode of
administration, tolerability and likelihood of treatment adherence, side
effects, and risk of major toxicity. The armamentarium of approved
disease-modifying therapies in MS and those in development include: (1) the
first approved, moderately effective, injectable interferon-β and glatiramer
acetate; (2) oral drugs (fingolimod, laquinimod, teriflunomide, dimethyl
fumarate); (3) monoclonal antibodies (rituximab, ocrelizumab, ofatumumab,
daclizumab, alemtuzumab); and (4) immunosuppressive agents (e.g. mitoxantrone).
The place of each drug in the therapeutic algorithm is dependent on its specific
risk-benefit profile. Patients' clinical and paraclinical phenotypes and
biomarker profile may help to elucidate disease subtypes and response to therapy
in the future, thus allowing treatment individualization. In multiple sclerosis (MS), B cell-depleting therapy using monoclonal anti-CD20
Abs, including rituximab (RTX) and ocrelizumab, effectively reduces disease
activity. Based on indirect evidence, it is generally believed that elimination
of the Ag-presenting capabilities and Ag nonspecific immune functions of B cells
underlie the therapeutic efficacy. However, a small subset of T lymphocytes (T
cells) was shown to also express CD20, but controversy prevails surrounding the
true existence of this T cell subpopulation. Using single-cell imaging flow
cytometry and expression profiling of sorted lymphocyte subsets, we
unequivocally demonstrate the existence of CD3(+)CD20(dim) T cells. We show that
in MS patients, increased levels of CD3(+)CD20(dim) T cells are effectively
depleted by RTX. The pathological relevance of this T cell subset in MS remains
to be determined. However, given their potential proinflammatory functionality,
depletion of CD20-expressing T cells may also contribute to the therapeutic
effect of RTX and other mAbs targeting CD20. Until the mid 1990s, with the appearance of interferon beta and glatiramer
acetate, there was no treatment for multiple sclerosis (MS). However, due to
their moderate therapeutic potential in some patients, a broad search was
continued to find new and more effective treatment strategies, largely
concentrated on monoclonal antibodies (MOAB). Natalizumab, the first MOAB for
the treatment of MS, was approved at the end of 2004, representing a major
advance in the field of neuroimmunology. Today, there is broad experience with
natalizumab and other MOAB (alemtuzumab, daclizumab, rituximab, ocrelizumab,
ofatumumab and anti-lingo-1) that are pending commercialization or are under
phase II or III of development with promising results. The present review
analyzes the efficacy and safety results of all these drugs. PURPOSE OF REVIEW: The purpose of this study is to highlight the pathological
features and clinical aspects of progressive multiple sclerosis (PMS) and also
the results of clinical trial experience to date and review ongoing clinical
trials and prospective new treatment options. This study will explain the
challenges of clinical trial design in PMS.
RECENT FINDINGS: Multiple sclerosis (MS) has been identified as a chronic immune
mediated disease, and the progressive phase of the disease appears to have
significant neurodegenerative mechanisms. The classification of the course of
PMS has been reorganized into categories of active vs. inactive inflammatory
disease and the presence vs. absence of gradual disease progression. This
differentiation allows clearer conceptualization of PMS and possibly even more
efficient recruitment of PMS patients into clinical trials. Clinical trial
experience to date in PMS has been negative with anti-inflammatory medications
used in relapsing MS. Simvastatin was recently tested in a phase II trial and
showed a 43% reduction of annualized atrophy progression in secondary
progressive MS. Ongoing PMS trials are currently being conducted with the
phosphodiesterase inhibitor ibudilast, S1P modulator siponimod and anti-B-cell
therapy ocrelizumab. Several efforts for development of outcome measures in PMS
are ongoing.
SUMMARY: PMS represents a significant challenge, as the pathogenesis of the
disease is not well understood, no validated outcome metrics have been
established and clinical trial experience to date has been disappointing.
Advances in the understanding of the disease and lessons learned in previous
clinical trials are paving the way for successful development of
disease-modifying agents for this disease. PURPOSE OF REVIEW: To summarize mechanisms of action, efficacy, and safety of
novel and imminently emerging disease-modifying treatments (DMTs) intended to be
used in relapsing-remitting multiple sclerosis (RRMS).
RECENT FINDINGS: Novel and imminently emerging DMTs for the treatment of RRMS
include alemtuzumab, daclizumab, ocrelizumab, pegylated interferon-β-1a, and
three times weekly glatiramer acetate. These DMTs have substantially different
mechanisms of action, efficacy, and safety and tolerability profiles, which are
summarized concisely in this article.
SUMMARY: The treatment landscape of RRMS is evolving rapidly as the available
treatment options have doubled in recent years, and a number of novel DMTs will
likely become available in the near future. Choosing the optimal DMT for
patients is becoming an increasingly complex process, and the care of patients
with MS will likely require regular input from neurologists subspecializing in
the care of patients with MS. As the use of novel DMTs with unknown long-term
safety profiles increases, postmarketing surveillance and vigilance with regards
to safety monitoring will be essential to confirm the safety and clinical
efficacy of these DMTs for patients with RRMS. BACKGROUND: Multiple sclerosis (MS) is the most common cause of nontraumatic
neurological disability in young adults. There is great need for developing
effective treatments to arrest the disease. As of today, there is no cure for MS
but several agents mitigating its effects are available. The era of
disease-modifying therapy began with the use of interferon beta and glatiramer
acetate in the 1990s. Given the injectable nature and the limited efficacy of
these agents, efforts are ongoing to develop new treatments.
SUMMARY OF REVIEW: We provide an overview of the ongoing developments in MS
therapy. After considering the clinical features and measures of drug efficacy
in MS clinical trials, we report the phase-III clinical trials results of: (1) 3
oral agents approved within the last 5 years, fingolimod (Gilenya),
dimethylfumarate (Tecfidera), and teriflunomide (Aubagio); (2) the oral agent
laquinimod; and (3) the monoclonal antibodies daclizumab, ocrelizumab, and
alemtuzumab. We will then briefly mention remyelinating and neuroprotective
agents that are in very early studies. We will end with a possible approach to
different clinical scenarios to guide the choice of disease-modifying therapy in
patients.
CONCLUSIONS: The newer agents offer the convenience of oral administration (for
the oral agents) and potentially higher efficacy, but their long-term safety
profile remains unknown. All available agents attack only 1 aspect of MS, that
is, inflammatory demyelination. Arresting or reversing the progression of
disability will be feasible only with agents affecting remyelination and
neuroprotection, still in relatively early research. B cells play a central role in the pathogenesis in multiple sclerosis (MS),
being involved in the activation of proinflammatory T cells, secretion of
proinflammatory cytokines, and production of autoantibodies directed against
myelin. Hence, the usage of B-cell-depleting monoclonal antibodies as therapy
for autoimmune diseases including MS lay near at hand. Rituximab was the first
therapeutic B-cell-depleting chimeric monoclonal antibody to be used
successfully in MS. Ocrelizumab, a second-generation humanized anti-CD20
antibody, was explored in a large phase II, randomized, placebo-controlled
multicentre trial in patients with relapsing-remitting disease. Compared with
placebo, two doses of ocrelizumab (600 and 2000 mg on days 1 and 15) showed a
pronounced effect on disease activity seen in magnetic resoce imaging (MRI)
as gadolinium-enhanced lesions (89% and 96% relative reduction, both p < 0.001)
and also had a significant effect on relapses. In exploratory analyses, both
doses of ocrelizumab had better effect on gadolinium-enhanced lesions than
interferon beta-1a intramuscularly that was used as a reference arm. Adverse
effects were mainly infusion-related reactions, in particular during the first
infusion. Serious infections occurred at similar rates in ocrelizumab and
placebo-treated patients, and no opportunistic infections were reported.
However, progressive multifocal leukoencephalopathy (PML) has been reported in
patients treated with anti-CD20 monoclonal antibodies for other indications.
Other anti-CD20 monoclonal antibodies have been tested as treatments for MS,
including ofatumumab that has shown beneficial results in placebo-controlled
phase II trials in patients with relapsing-remitting MS. Ocrelizumab is now in
phase III development for the treatment of relapsing-remitting MS, as well as
primary progressive MS, and the results of ongoing clinical trials are eagerly
awaited and will determine the place of ocrelizumab in the armamentarium of MS
therapies. Publisher: TITLE: Revision de las novedades del XXXI Congreso ECTRIMS 2015,
presentadas en la VIII Reunion Post-ECTRIMS.
Reconocidos especialistas nacionales en esclerosis multiple (EM) se han reunido,
por octavo año consecutivo, para exponer lo mas novedoso que se presento en la
ultima edicion del congreso ECTRIMS 2015 y que recoge esta revision. En esta
edicion ha destacado la nueva clasificacion de los fenotipos de la EM. Tambien
se revisaron los criterios diagnosticos del espectro de la neuromielitis optica
y los problemas en el diagnostico diferencial derivados de la falta de
definicion del espectro radiologico. La microbiota adquiere protagonismo como
posible factor determite de la enfermedad, junto con factores extrinsecos
como el tabaco, la ingesta de sal o el deficit de vitamina D. Los avances en
inmunomodulacion impulsan el progreso en el tratamiento de la EM. El ocrelizumab
es el primer tratamiento con resultados positivos en las formas primariamente
progresivas, y el tocilizumab, un farmaco para la artritis reumatoide, destaca
como candidato potencial para el tratamiento de la neuromielitis optica. Ciertos
antibioticos y vitaminas tambien podrian tener un papel en el tratamiento de la
EM. En esta edicion se presto especial atencion a la terapia personalizada.
Actualmente disponemos de 11 farmacos aprobados en Europa. Se necesitan
algoritmos terapeuticos que nos ayuden a elegir el mejor tratamiento para cada
paciente. Asimismo, necesitamos poder identificar en los estadios precoces de la
enfermedad el riesgo de desarrollar discapacidad, para diseñar estrategias
terapeuticas, para lo que se precisan biomarcadores moleculares y otras
herramientas pronosticas. Los problemas aun existentes en la tecnologia del
software en resocia magnetica dificultan su traslacion a la practica clinica
diaria. INTRODUCTION: Despite recent advances in pharmacological management, multiple
sclerosis (MS), an autoimmune disease of the central nervous system, remains a
leading cause of disability. In relapsing-remitting (RR)MS, neurologists most
commonly utilize immunomodulatory or immunosuppressive agents to benefit their
patients. With the introduction of humanized monoclonal antibodies (mAbs)
ablation of distinct immune populations has become possible. Depletion of B
cells by anti-CD20 mAbs has repeatedly proven to be a very rapid and effective
means to diminish disease activity in RRMS.
AREAS COVERED: We discuss the biological rationale, development, and recent
clinical study results of the second generation anti-CD20 mAb ocrelizumab.
Expert commentary: The topline results of two phase-III randomized clinical
trials demonstrate superiority of ocrelizumab over interferon beta in RRMS
patients with regards to clinical and paraclinical outcome parameters. The short
term adverse events profile appears favorable. However, long-term effects of
repeated B cell depletion are currently unknown. The therapeutic utility of the anti-CD20 monoclonal antibodies (mAbs) is
currently being evaluated in multiple sclerosis (MS) in line with the better
understanding of the role of B lymphocytes in MS pathogenesis. Area covered: We
conducted a literature search using Medline/Pub Med database of basic research
and available controlled trials about anti-CD20 mAbs in MS. Additionally,
ongoing studies were identified in the ClinicalTrials.gov database. B cells
exert multiple inflammatory and regulatory functions playing an important role
in MS pathogenesis as is demonstrated by the production of autoantibodies,
infiltration of B cells in MS lesions and the formation of ectopic B cell
follicle-like structures in meninges, among others. B-cell depletion by
anti-CD20 mAbs has been shown to have an impact on these pathogenic mechanisms.
The efficacy of three of them, rituximab, ocrelizumab and ofatumumab in MS has
been confirmed by placebo-controlled clinical trials demonstrating a significant
reduction of the annualized relapsing rate (ARR), new gadolinium-enhancing (GdE)
and T2 lesions. There have been no significant safety problems so far but the
overall benefit to risk profile is still to be determined. Expert commentary:
After recent good results of these agents in MS therapy, questions related to
maintece therapy, markers of response and control of B cells values remain
uswered. Multiple sclerosis (MS) is a chronic inflammatory condition of the central
nervous system with heterogeneous features. Primary progressive (PP) MS is a
rare disease subtype characterized by continuous disability worsening from
onset. No disease-modifying therapy is currently approved for PP MS due to the
negative or inconsistent results of clinical trials conducted on a wide range of
interventions, which are reviewed in the present paper. Areas covered: The
features and results of randomized trials of disease-modifying treatments for PP
MS are discussed, including immunosuppressants, immunomodulators, monoclonal
antibodies, and putative neuroprotective agents. Expert commentary: The recent
encouraging results of the ocrelizumab trial in PP MS, the first to reach the
primary disability endpoint, indicate B cells as a promising therapeutic target
to prevent disease progression. Other emerging treatment strategies include cell
metabolism modulation and inflammatory pathways inhibition, which are being
investigated in several ongoing phase II and III placebo-controlled trials.
Future PP MS trials will need to systematically include efficacy endpoints other
than physical disability alone, such as cognition, quality of life, advanced MRI
measures and molecular biomarkers. |
Elaborate on the role of CARMEN in cardiac specification. | CARMEN, (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA, is a human super enhancer-associated long noncoding RNA controlling cardiac specification, differentiation and homeostasis. CARMEN exhibits RNA-dependent enhancing activity and is upstream of the cardiac mesoderm-specifying gene regulatory network. CARMEN interacts with SUZ12 and EZH2, two components of the polycomb repressive complex 2 (PRC2). CARMEN expression is activated during pathological remodeling in the mouse and human hearts, and is necessary for maintaining cardiac identity in differentiated cardiomyocytes. | Long noncoding RNAs (lncRNAs) are emerging as important regulators of
developmental pathways. However, their roles in human cardiac precursor cell
(CPC) remain unexplored. To characterize the long noncoding transcriptome during
human CPC cardiac differentiation, we profiled the lncRNA transcriptome in CPCs
isolated from the human fetal heart and identified 570 lncRNAs that were
modulated during cardiac differentiation. Many of these were associated with
active cardiac enhancer and super enhancers (SE) with their expression being
correlated with proximal cardiac genes. One of the most upregulated lncRNAs was
a SE-associated lncRNA that was named CARMEN, (CAR)diac (M)esoderm
(E)nhancer-associated (N)oncoding RNA. CARMEN exhibits RNA-dependent enhancing
activity and is upstream of the cardiac mesoderm-specifying gene regulatory
network. Interestingly, CARMEN interacts with SUZ12 and EZH2, two components of
the polycomb repressive complex 2 (PRC2). We demonstrate that CARMEN knockdown
inhibits cardiac specification and differentiation in cardiac precursor cells
independently of MIR-143 and -145 expression, two microRNAs located proximal to
the enhancer sequences. Importantly, CARMEN expression was activated during
pathological remodeling in the mouse and human hearts, and was necessary for
maintaining cardiac identity in differentiated cardiomyocytes. This study
demonstrates therefore that CARMEN is a crucial regulator of cardiac cell
differentiation and homeostasis. |
Is there a role of regorafenib for sarcoma treatment? | Yes, there is evidence to suggest that regorafenib can be effective for sarcoma treatment. Clinical trials are under-way. | INTRODUCTION: Adult sarcomas are rare tumors characterized, in general, by their
poor prognosis and the paucity of effective treatments. However, the deeper
understanding of their underlying molecular pathology, signaling pathways and
key effectors has permitted the development of a number of drugs able to inhibit
important processes in sarcoma pathogenesis. Some of these novel compounds have
been assessed in clinical trials with successful results.
AREAS COVERED: The latest reported trials are comprehensively reviewed. Thus,
the Phase III studies with pazopanib, regorafenib, muramyl tripeptide (MTP) and
ridaforolimus are extensively discussed as well as the biological rationale for
the use of these compounds. In addition, the most promising drugs that still are
in earlier stages of development such as CDK4 and MDM2 inhibitors, cediranib,
eribulin and crizotinib are also discussed.
EXPERT OPINION: It is crucial for the correct identification of active drugs in
sarcomas that new clinical trials are focused on specific subtypes and/or
molecular alterations. The results of these studies should improve the prognosis
of the patients affected by sarcoma in forthcoming years. BACKGROUND: Increasing studies implicate cancer stem cells (CSCs) as the source
of resistance and relapse following conventional cytotoxic therapies. Few
studies have examined the response of CSCs to targeted therapies, such as
tyrosine kinase inhibitors (TKIs). We hypothesized that TKIs would have
differential effects on CSC populations depending on their mechanism of action
(anti-proliferative vs. anti-angiogenic).
METHODS: We exposed human sarcoma cell lines to sorafenib, regorafenib, and
pazopanib and assessed cell viability and expression of CSC markers (ALDH, CD24,
CD44, and CD133). We evaluated survival and CSC phenotype in mice harboring
sarcoma metastases after TKI therapy. We exposed dissociated primary sarcoma
tumors to sorafenib, regorafenib, and pazopanib, and we used tissue microarray
(TMA) and primary sarcoma samples to evaluate the frequency and intensity of CSC
markers after neoadjuvant therapy with sorafenib and pazopanib. Parametric and
non-parametric statistical analyses were performed as appropriate.
RESULTS: After functionally validating the CSC phenotype of ALDHbright sarcoma
cells, we observed that sorafenib and regorafenib were cytotoxic to sarcoma cell
lines (P < 0.05), with a corresponding 1.4 - 2.8 fold increase in ALDHbright
cells from baseline (P < 0.05). In contrast, we observed negligible effects on
viability and CSC sub-populations with pazopanib. At low doses, there was
progressive CSC enrichment in vitro after longer term exposure to sorafenib
although the anti-proliferative effects were attenuated. In vivo, sorafenib
improved median survival by 11 days (P < 0.05), but enriched ALDHbright cells
2.5 - 2.8 fold (P < 0.05). Analysis of primary human sarcoma samples revealed
direct cytotoxicity following exposure to sorafenib and regorafenib with a
corresponding increase in ALDHbright cells (P < 0.05). Again, negligible effects
from pazopanib were observed. TMA analysis of archived specimens from sarcoma
patients treated with sorafenib demonstrated significant enrichment for
ALDHbright cells in the post-treatment resection specimen (P < 0.05), whereas
clinical specimens obtained longitudinally from a patient treated with pazopanib
showed no enrichment for ALDHbright cells (P > 0.05).
CONCLUSIONS: Anti-proliferative TKIs appear to enrich for sarcoma CSCs while
anti-angiogenic TKIs do not. The rational selection of targeted therapies for
sarcoma patients may benefit from an awareness of the differential impact of
TKIs on CSC populations. BACKGROUND: Angiogenesis, among other signaling pathways, plays a key-role in
sarcoma biology. Regorafenib (RE) has recently been shown to be effective in
imatinib and sunitinib-refractory GIST in a phase III trial.
METHODS/DESIGN: We are conducting an international trial (France, Austria and
Germany) consisting in 4 parallel double-blind placebo-controlled randomized
(1/1) phase II trials to assess the activity and safety of RE in
doxorubicin-refractory STS (ClinicalTrials.gov: NCT01900743). Each phase II
trial is dedicated to one of the 4 following histological subgroups:
liposarcoma, leiomyosarcoma, synovial sarcoma and other sarcoma. Within each
randomized trial the following stratification factors will be applied: countries
and prior exposure to pazopanib. Key-eligibility criteria are: measurable
disease, age ≥18, not > 3 previous systemic treatment lines for metastatic
disease, metastatic disease not amenable to surgical resection. The primary
endpoint is progression-free survival (PFS) according to central radiological
review. Secondary endpoints are: Toxicity (NCI-CTC AE V4.0); time to
progression; Growth modulation index in pts receiving RE after randomization; 3
and 6 months PFS-Rates, best response rate and overall survival. Each phase II
trial will be separately analyzed. In 3 trials, statistical assumptions are:
PFS0 = 1.6 & PFS1 = 4.6 months; 1-sided α = 0.1; β = 0.05 with a total sample
size of 192 pts. To take into account the rarity of synovial sarcoma, the
statistical assumptions are: PFS0 = 1.6 & PFS1 = 4.6 months; 1-sided α = 0.1;
β = 0.2 Tumor assessment is done monthly during the 4 first months, and every 3
months thereafter. After central radiological confirmation of tumor progression,
an optional open-label option is offered to eligible patients.
DISCUSSION: The design of this trial allows an assessment of regorafenib
activity over placebo in four sarcoma strata and might provide evidence for
launching a phase III trial. This study includes both integrative and
exploratory translational research program. The study is enrolling since June
2013 (TRIAL REGISTRATION NUMBER: EudraCT N°: 2012-005743-24, on the 15(th)
February 2012). We report a response to pazopanib in a 69-year-old man with heavily pre-treated
metastatic extraosseous Ewing sarcoma in addition to molecular profiling of his
tumor. To our knowledge, this case is the earliest to demonstrate activity of an
oral multi-targeted kinase inhibitor in Ewing sarcoma. This case provides
rationale for adding a Ewing sarcoma arm to SARC024, a phase II study of
regorafenib, another multi-targeted kinase inhibitor, in patients with
liposarcoma, osteosarcoma and Ewing and Ewing-like sarcomas (NCT02048371). This
national multi-institutional study is ongoing. Soft tissue sarcomas are rare tumors that represent a major challenge due to
varying clinical presentations and often interdisciplinary treatment concepts.
Gold standard for the treatment of localized resectable soft tissue sarcomas is
complete surgical removal. In metastatic soft tissue sarcoma, systemic therapy
is the treatment of choice. The most active drugs are anthracyclines and
ifosfamide. Combination chemotherapy has improved both response rate and
progression-free survival at the cost of increased toxicity. Imatinib at a dose
of 400 mg/day is the gold standard for patients with advanced or metastatic
gastrointestinal stromal tumors (GIST). In patients with a mutation in KIT
exon 9, 800 mg/day is the recommended dose. In imatinib refractory or intolerant
patients, sunitinib is recommended. Regorafenib has been approved for third-line
therapy. |
Which drug was tested in the TEMSO Trial for multiple sclerosis? | Teriflunomide was evaluated in the Teriflunomide Multiple Sclerosis Oral (TEMSO) trial. | Teriflunomide, being developed as a potential oral treatment for multiple
sclerosis (MS) by sanofi-aventis, is the active metabolite of the rheumatoid
arthritis drug leflunomide. Both teriflunomide and leflunomide are inhibitors of
the mitochondrial enzyme dihydroorotate dehydrogenase, which is critically
involved in pyrimidine synthesis. The production of activated T-cells largely
depends on de novo pyrimidine synthesis, and thus pyrimidine depletion is
thought to result in the inhibition of immune cell proliferation. Therapeutic
efficacy of teriflunomide has been demonstrated in vivo in an experimental
autoimmune encephalomyelitis model of MS using Dark Agouti rats. In a phase II,
randomized, double-blind, placebo-controlled clinical trial of patients with
relapsing-remitting MS, treatment with teriflunomide reduced the number of
active lesions in the brain and preliminary evidence indicated a slowing in the
development of disability. Recently reported data from the phase III TEMSO
clinical trial support these initial findings. Compared with current therapies,
teriflunomide has the advantage of oral administration. Thus, if good efficacy
is demonstrated, teriflunomide may have a role to play in the future treatment
of MS. Conflict of interest statement: Conflicts of interest: Aaron E Miller has
received research support from Acorda Therapeutics, Biogen Idec, Genentech,
Genzyme, sanofi-aventis, Novartis, Roche and Teva, and consulting fees from
Acorda Therapeutics, Avanir, Biogen Idec, BioMarin, Chelsea Therapeutics,
Daiichi-Sankyo, EMD Serono, GlaxoSmithKline, La-Ser, Merck Serono, Novartis,
Nuron Biotech, ONO, and sanofi-aventis. Paul O’Connor has received consulting
fees and/or research support for MS trials from Actelion, Bayer, Biogen Idec,
BioMS, Cognosci, Daiichi Sankyo, EMD Serono, Genentech, Genmab, Novartis, Roche,
sanofi-aventis, Teva, and Warburg Pincus. Jerry S Wolinsky, within the past two
years, has had consulting agreements with, or served as a speaker for, Astellas,
Bayer HealthCare, Celgene, Consortium of MS Clinics, Eli Lilly, Medscape CME,
Novartis, PRIME, sanofi-aventis, Serono Symposia International Foundation, Teva
and Teva Neurosciences, the National MS Society; has received royalties from
Millipore (Chemicon International Corporation); and has research or contractual
support from Clayton Foundation for Research, National Institutes of Health, and
sanofi-aventis. Christian Confavreux has received consulting fees from Biogen
Dompé, Biogen Idec, Gemacbio, Genzyme Corporation, Hertie Foundation, Novartis,
sanofi-aventis, Teva Pharma and UCB Pharma; lecture fees from Bayer Schering,
Biogen Idec, Merck Serono, Novartis, sanofi-aventis, and Teva Pharma; research
support from Bayer Schering, Biogen Idec, Merck Serono, Novartis,
sanofi-aventis, and Teva Pharma; and fees for membership of Company Advisory
Boards from Biogen Idec, Genzyme, Novartis, sanofi-sventis, Teva Pharma, and UCB
Pharma. Ludwig Kappos has received research support from Actelion, Advancell,
Allozyne, BaroFold, Bayer Health Care Pharmaceuticals, Bayer Schering Pharma,
Bayhill, Biogen Idec, BioMarin, CLC Behring, Elan, Genmab, Genmark, GeNeuro SA,
Glaxo-SmithKline, Lilly, Merck Serono, MediciNova, Novartis, Novonordisk,
Peptimmune, sanofi-aventis, Santhera, Roche, Teva, UCB, and Wyeth, and from the
Swiss MS Society, the Swiss National Research Foundation, the European Union,
and the Gianni Rubatto, Novartis, and Roche Research Foundations. Tomas P Olsson
has received consulting fees and/or research support for MS trials from Biogen
Idec, Merck Serono, and sanofi-aventis, and for participation in scientific
advisory boards and/or speaking activities from Merck Serono, Biogen Idec, and
sanofi-aventis. Philippe Truffinet and Lin Wang are employees of sanofi-aventis.
Laura D’Castro is an employee of Fishawack Communications Ltd, which has been
contracted to provide editorial services for sanofi-aventis. Giancarlo Comi has
received, in the past year, consulting fees for participating on advisory boards
from Novartis, Teva Pharmaceutical Ind. Ltd, sanofi-aventis, Merck Serono,
Actelion and Bayer Schering, and lecture fess from Novartis, Teva Pharmaceutical
Ind. Ltd, sanofi-aventis, Merck Serono, Biogen Dompè, Bayer Schering, and Serono
Symposia International Foundation. Mark S Freedman has received research or
educational grant support from Bayer Healthcare and Genzyme; he has received
honoraria or consultation fees from Bayer Healthcare, Biogen Idec, EMD Canada,
Novartis, sanofi-aventis, Teva Canada Innovation, and is a member of Company
Advisory Board/Board of Directors/or other similar group for: Bayer Healthcare,
Biogen Idec, Merck Serono, Novartis, sanofi-aventis, and Celgene. Teriflunomide is a new active drug which has recently been approved as a
first-line treatment of relapsing forms of MS in the US, Australia, Argentina,
and the European Union. It is characterized by a once-daily oral application and
a well-established long-term safety profile. The main therapeutic effect is
considered to be mediated via the inhibition of the de novo synthesis of
pyrimidine in proliferating immune cells. Two phase III clinical trials (TEMSO,
TOWER) tested teriflunomide in patients with relapsing forms of MS: efficacy was
shown, with positive effects on relapse rates and disease progression for 14
mg/day. Overall, the safety profile in these studies was favorable. In patients
treated with teriflunomide, the regular monitoring of blood cell counts and
liver enzymes is required. Teriflunomide must not be used during pregcy. In
this article, we review recent phase II and phase III clinical trial data, and
discuss the potential of teriflunomide for the treatment of relapsing forms of
MS. PURPOSE: The purpose was to summarize US prescribing information for
teriflunomide in the treatment of patients with relapsing forms of multiple
sclerosis (RMS), with reference to clinical efficacy and safety outcomes.
METHODS: In September 2012, the US Food and Drug Administration granted approval
for the use of teriflunomide, 14 mg and 7 mg once daily, to treat RMS on the
basis of the results of a Phase II study and the Phase III TEMSO (Teriflunomide
Multiple Sclerosis Oral) trial. After recent updates to the prescribing
information (October 2014), key findings from these and 2 other Phase III
clinical trials, TOWER (Teriflunomide Oral in People With Relapsing Multiple
Sclerosis) and TOPIC (Oral Teriflunomide for Patients with a First Clinical
Episode Suggestive of Multiple Sclerosis), and practical considerations for
physicians are summarized.
FINDINGS: Teriflunomide, 14 mg and 7 mg, significantly reduced mean number of
unique active lesions on magnetic resoce imaging (MRI; P < 0.05 for both
doses) in the Phase II study. In the TEMSO and TOWER studies, the 14-mg dose of
teriflunomide significantly reduced annualized relapse rate (31% and 36%
relative risk reduction compared with placebo, respectively; both P < 0.001) and
risk of disability progression sustained for 12 weeks (hazard ratio vs placebo
0.70 and 0.69, respectively; both P < 0.05). The 7-mg dose significantly
(P < 0.02) reduced annualized relapse rate in both studies, although the
reduction in risk of disability progression was not statistically significant.
Teriflunomide treatment was also associated with significant efficacy on MRI
measures of disease activity in TEMSO; both doses significantly reduced total
lesion volume and number of gadolinium-enhancing T1 lesions. TOPIC evaluated
patients with a first clinical event consistent with acute demyelination and
brain MRI lesions characteristic of multiple sclerosis. More patients were free
of relapse in the teriflunomide 14-mg and 7-mg groups than in the placebo group
(P < 0.05 for both comparisons). In safety data pooled from the 4 studies,
adverse events occurring in ≥2% of patients and ≥2% higher than in the placebo
group were headache, alanine aminotransferase increase, diarrhea, alopecia (hair
thinning), nausea, paresthesia, arthralgia, neutropenia, and hypertension.
Routine monitoring procedures before and on treatment are recommended to assess
potential safety issues. Women of childbearing potential must use effective
contraception and, in the event of pregcy, undergo an accelerated elimination
procedure to reduce plasma concentrations of teriflunomide.
IMPLICATIONS: Clinical evidence suggests that teriflunomide is an effective
therapeutic choice for patients with RMS, both as an initial treatment and as an
alternative for patients who may have experienced intolerance or inadequate
response to a previous or current disease-modifying therapy. Multiple sclerosis (MS) is the autoimmune, demyelinating, neurodegenerative
disorder of the central nervous system (CNS). There are nine drugs available in
Hungary reimbursed by the National Health Insurance Fund of Hungary (OEP) to
reduce the activity of the disease, from which seven can be used as first line
therapies. We have approximately 20 years of experience with the interferon
beta-1a/1b and glatiramer-acetate products. Though in case of approximately 30%
of the patients using one of the first line drugs, the disease remains active,
that we call break-through disease. The reasons for breakthrough disease could
be the insufficient adherence and compliance, the appearance of neutralizing
antibodies or the high activity of the disease. One of the oral immunomodulating
drugs for MS, teriflunomide, was registered in Europe in 2013. Because of the
anti-proliferative and anti-inflammatory effect of teriflunomide, it can be used
for the reduction of the disease activity in the relapsing-remitting course of
MS. The effect of teriflunomide was proved in one Phase II. and four Phase III.
(TEMSO, TOWER, TENERE, TOPIC) studies. Teriflunomide 14 mg once daily was able
to demonstrate in two consecutive placebo-controlled phase 3 clinical trials
that significantly reduces the relapse rate (31.5% and 36.3%) and in both
studies significantly reduces the sustained disability progression (29.8% and
31.5%) moreover delays the appearance of the clinically definitive MS in
patients with clinically isolated syndrome (CIS). According to the TENERE study
there were no significant differences observed between teriflunomide 14 mg and
IFNβ-α a s.c. in time to failure and annualized relapse rate but the treatment
satisfaction domains of global satisfaction, side-effects and convenience were
significantly improved with teriflunomide compared with s.c. IFNβ-α. Teriflunomide is a once-daily oral agent that has been licensed in the EU since
August 2013 for the treatment of adult patients with relapsing-remitting
multiple sclerosis (RRMS). More recently (September 2014), the EU summary of
product characteristics (SmPC) was updated to include data from patients with a
first clinical demyelinating event. This review examines the EU SmPC for
teriflunomide, with reference to key clinical and safety outcomes and practical
considerations for prescribing physicians. In two phase III trials (TEMSO and
TOWER) in patients with relapsing forms of MS, teriflunomide 14 mg significantly
reduced the annualized relapse rate and the risk of confirmed disability
progression sustained for at least 12 weeks. Magnetic resoce imaging (MRI)
total lesion volume, gadolinium-enhancing lesions, and unique active lesions
were reduced with teriflunomide treatment in TEMSO. In the TOPIC study, in
patients with a first clinical demyelinating event, teriflunomide treatment
significantly reduced the time to a second clinical episode (relapse). Across
the clinical studies, teriflunomide was generally well tolerated; adverse events
reported in ≥ 10% of teriflunomide-treated patients were diarrhea, nausea,
increased alanine aminotransferase, and alopecia. Data from the clinical
development program support the use of teriflunomide in a broad spectrum of
patients with RRMS. Three oral disease-modifying drugs-fingolimod, teriflunomide, and dimethyl
fumarate (DMF)-are available for treatment of relapsing forms of multiple
sclerosis (MS). All three agents were approved in the last decade, primarily on
the basis of a moderate to substantial reduction in the occurrence of MS
relapses and central nervous system lesion formation detected by MRI. In the
trials leading to approval, the first oral disease-modifying drug, fingolimod,
reduced the annualized relapse rate (ARR) from 0.40 in placebo-treated patients
to 0.18 (FREEDOMS) and from 0.33 in patients treated with interferon β1a
intramuscularly to 0.16 (TRANSFORMS). Teriflunomide, approved on the basis of
the two placebo-controlled trials TEMSO and TOWER, demonstrated a reduction in
the ARR from 0.54 to 0.37 and from 0.50 to 0.32 respectively. The latest oral MS
medication, approved in 2014, is DMF, which had been used in a different
formulation for treatment of psoriasis for decades. In the 2-year DEFINE study,
the proportion of patients with a relapse was reduced to 27 %, compared with 46
% in placebo arm, whereas in the CONFIRM trial, the ARR was reduced from 0.40
(placebo) to 0.22 in the DMF-treated group of patients. In this review, we will
elucidate the mechanisms of action of these three medications and compare their
efficacy, safety, and tolerability as a practical guideline for their use. We
will further discuss effects other than relapse reduction these small molecules
may exert, including potential activities within the central nervous system, and
briefly summarize emerging data on new oral MS drugs in clinical development. Dimethyl fumarate (DMF), fingolimod, and teriflunomide are oral
disease-modifying therapies (DMTs) indicated for the treatment of
relapsing-remitting multiple sclerosis. Despite well-established limitations of
cross-trial comparisons, DMTs are still frequently compared in terms of relative
reductions in specific endpoints, most commonly annualized relapse rate.
Consideration of absolute risk reduction and number needed to treat (NNT)
provides an alternative approach to assess the magnitude of treatment effect and
can provide valuable additional information on therapeutic gain. Using data from
pivotal studies of DMF (DEFINE, NCT00420212; CONFIRM, NCT00451451), fingolimod
(FREEDOMS, NCT00289978; FREEDOMS II, NCT00355134), and teriflunomide (TEMSO,
NCT00134563; TOWER, NCT00751881), we calculated NNTs to prevent any relapse,
more severe relapses (such as those leading to hospitalization or requiring
intravenous corticosteroids), and disability worsening. Higher relative
reductions were reported for DMF and fingolimod vs placebo on overall relapse
and relapses requiring intravenous corticosteroids in both individual and pooled
studies (pooled data unavailable for fingolimod). However, NNTs for each outcome
were similar for DMF and teriflunomide, with marginally lower NNTs observed with
fingolimod. By contrast, for relapses requiring hospitalization, relative
reductions were higher and NNTs were substantially lower for teriflunomide
compared with DMF. For fingolimod, there were inconsistent outcomes between the
two studies for relapses requiring hospitalization; thus, comparative
conclusions against DMF or teriflunomide cannot be clearly established. The risk
of disability worsening was significantly reduced in both teriflunomide studies,
but only in a single study for DMF (DEFINE) and fingolimod (FREEDOMS). NNTs to
prevent one patient from experiencing disability worsening were similar in
DEFINE, FREEDOMS, and TEMSO and TOWER but were higher in CONFIRM and FREEDOMS
II. This NNT analysis demonstrates broadly comparable effects for DMF,
fingolimod, and teriflunomide across key clinical outcomes. These observations
are clinically relevant and may help to inform treatment decisions by providing
additional information on therapeutic gain beyond informal assessments of
relative reductions alone. Teriflunomide, a once-daily, oral disease-modifying therapy, has demonstrated
efficacy in patients with relapsing forms of multiple sclerosis (MS) and
patients with a first clinical episode suggestive of MS. As the only
disease-modifying therapy with positive disability results in two Phase III
trials, teriflunomide significantly slowed disability in patients with relapsing
forms of MS. We highlight data from the Phase II study and the TEMSO, TOWER,
TOPIC and TENERE teriflunomide studies. TEMSO MRI outcomes have been supported
with Structural Image Evaluation Using Normalization of Atrophy analyses. We
present data from long-term extensions of the Phase II study, TEMSO and TOWER,
as well as results from patients who switched from other disease-modifying
therapies to teriflunomide, patient-reported outcomes and supplementary measures
of response. |
What is the mechanism of action of peginesatide? | Peginesatide (Omontys) is synthetic, PEGylated, peptide-based erythropoiesis-stimulating agent that is designed to specifically stimulate the erythropoietin receptor. It was recalled because of serious side-effects including cases of death. | Erythropoiesis stimulating agents (ESAs) are effective drugs that correct anemia
in patients with chronic kidney disease (CKD). Recombit human erythropoietin
(EPO), the first ESA that became available more than 20 years ago, is similar to
the naturally occurring molecule. In subsequent years, pharmacological research
focused on the development of new agents with improved characteristics, with the
creation of high molecular weight ESAs having been the first approach. In more
recent years, new agents have been developed, including peginesatide (Hematide;
Affymax Inc/Takeda Pharmaceutical Co Ltd), which is a dimeric peptide with a
chemical structure unrelated to EPO that is being evaluated in phase III
clinical trials. In addition, the clinical development of two inhibitors of
hypoxia-inducible transcription factor has been resumed recently, while other
approaches, such as gene therapy and EPO fusion proteins, and the inhibition of
GATA and hematopoietic cell phosphatase remain far from being applicable in
clinical practice. New iron compounds, which are becoming increasingly
available, will facilitate an integrated approach to anemia management using
both iron and/or ESAs, according to the clinical needs of patients. This review
discusses new therapeutic options (already available or still under development)
for the treatment of CKD-associated anemia, including ESAs and intravenous iron
molecules. Erythropoiesis stimulating agents (ESAs) have revolutionized the management of
anemia of chronic kidney disease (CKD). Peginesatide is an investigational
pegylated, peptide-based, once-monthly ESA for increasing and maintaining
hemoglobin (Hb). In phase 2 studies, peginesatide increases and maintains target
Hb levels in patients with CKD, both those on hemodialysis and those not on
hemodialysis; phase 3 trials have recently been completed. This article
discusses unmet needs in the management of anemia of CKD, presents peginesatide
attributes, reviews the results of select peginesatide clinical studies, and
discusses the potential value of peginesatide as an alternative anemia
management option. Peginesatide is a PEGylated, investigational, peptide-based
erythropoiesis-stimulating agent (ESA) that was designed and engineered to
stimulate specifically the erythropoietin receptor dimer that governs
erythropoiesis. Clinical use of peginesatide is anticipated to result in chronic
dosing in chronic kidney disease (CKD) patients, and the nonclinical data to
support development should include an evaluation of carcinogenic potential
evaluation. Peginesatide was not mutagenic or clastogenic in a standard
genotoxicity battery of tests. Doses for a rasH2 transgenic mouse
carcinogenicity assay were defined in a 28-day study in the wild-type
littermates of the rasH2 transgenic mouse strain, using intravenous doses of
1-25 mg/kg on days 1 and 22. The findings were consistent with exaggerated
pharmacology, including polycythemia, with associated increases in hemoglobin
level and extramedullary hematopoiesis and bone marrow hypercellularity. Erythropoietin (EPO) and other erythropoiesis-stimulating agents possess a high
misuse potential in elite sport due to their ability to increase the oxygen
transport capacity, which plays a vital role in enhancing endurance performance.
Currently, a new generation of EPO-mimetic peptides is under development from
which peginesatide (also referred to as Hematide™), a pegylated homodimeric
peptide of approximately 45 kDa with no structural relationship to
erythropoietin, is the most advanced drug candidate undergoing phase-III
clinical trials. Since preventive doping research aims at the development of
detection methods before a drug receives clinical approval, a selective and
sensitive assay has to be established knowing that conventional doping control
assays for EPO will not succeed in detecting peginesatide. Thus, a pegylated
EPO-mimetic peptide simulating the structure and properties of the lead drug
candidate peginesatide was synthesised as a model compound for this new class of
emerging drugs and characterised by means of gel electrophoresis,
matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry, as well
as liquid chromatography/electrospray ionisation tandem mass spectrometry
(LC/ESI-MS/MS) after proteolytic digestion. Based on these results, a mass
spectrometric detection method of the product in plasma was developed targeting
a pentapeptide fragment after protein precipitation and subtilisin digestion.
Its fitness for purpose was evaluated by the determination of selected method
characteristics focusing particularly on specificity, recovery (ca. 60%), and
limit of detection (1 ng/mL). Since the 1990's, cheating athletes have abused substances to increase their
oxygen transport capabilities; among these substances, recombit EPO is the
most well known. Currently, other investigational pharmaceutical products are
able to produce an effect similar to EPO but without having chemical structures
related to EPO; these are the synthetic erythropoiesis stimulating agents
(ESAs). Peginesatide (also known as Hematide™) is being developed by Affymax and
Takeda and, if approved by regulatory authorities, could soon be released on the
international market. To detect potential athletic abuse of this product and
deter athletes who consider cheating, we initiated a collaboration to implement
a detection test for anti-doping purposes. Peginesatide is a synthetic,
PEGylated, investigational, peptide-based erythropoiesis-stimulating agent that
is designed and engineered to stimulate specifically the erythropoietin receptor
dimer that governs erythropoiesis. It is undetectable using current anti-doping
tests due to its lack of sequence homology to EPO. To detect and deter potential
abuse of peginesatide, we initiated an industry/antidoping laboratory
collaboration to develop and validate screening and confirmation assays so that
they would be available before peginesatide reaches the market. We describe a
screening ELISA and a confirmation assay consisting of immune-purification
followed by separation with SDS-PAGE and revelation with Western double
blotting. Both assays can detect 0.5 ng/mL concentrations of peginesatide in
blood samples, enabling detection for several days after administration of a
physiologically relevant dose. This initial report describes experimental
characterization of these assays, including testing with a blinded set of
samples from a clinical study conducted in healthy volunteers. BACKGROUND AND OBJECTIVES: Peginesatide is a synthetic, PEGylated,
investigational, peptide-based erythropoiesis-stimulating agent. We report the
first assessment of its efficacy and safety in correcting renal anemia in a
population of 139 nondialysis chronic kidney disease patients.
DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Chronic kidney disease patients
who were not on dialysis and not receiving treatment with
erythropoiesis-stimulating agents in the 12 weeks before study drug
administration were sequentially assigned to one of 10 cohorts; cohorts differed
in starting peginesatide dose (different body weight-based or absolute doses),
route of administration (intravenous or subcutaneous), and frequency of
administration (every 4 or 2 weeks).
RESULTS: Across all cohorts, 96% of patients achieved a hemoglobin response. A
dose-response relationship was evident for hemoglobin increase. Comparable
subcutaneous and intravenous peginesatide doses produced similar hemoglobin
responses. Rapid rates of hemoglobin rise and hemoglobin excursions >13 g/dl
tended to occur more frequently with every-2-weeks dosing than they did with
every-4-weeks dosing. The range of final median doses in the every-4-weeks
dosing groups was 0.019 to 0.043 mg/kg. Across all cohorts, 20% of patients
reported serious adverse events (one patient had a possibly drug-related serious
event) and 81% reported adverse events (11.5% reported possibly drug-related
events); these events were consistent with those routinely observed in this
patient population.
CONCLUSIONS: This study suggests that peginesatide administered every 4 weeks
can increase and maintain hemoglobin in nondialysis chronic kidney disease
patients. Additional long-term data in larger groups of patients are required to
further elucidate the efficacy and safety of this peptide-based
erythropoiesis-stimulating agent. The pharmacokinetics (PK) (absorption, distribution, metabolism, excretion) of
peginesatide, a synthetic, PEGylated, investigational, peptide-based
erythropoiesis-stimulating agent (ESA), was evaluated in rats. The PK profile
was evaluated at 0.1-5 mg·kg(-1) IV using unlabeled or [(14)C]-labeled
peginesatide. Mass balance, tissue distribution and metabolism were evaluated
following IV administration of 5 mg·kg(-1) [(14)C]-peginesatide, with tissue
distribution also evaluated by quantitative whole-body autoradiography (QWBA)
following an IV dose of 17 mg·kg(-1) [(14)C]-peginesatide. Plasma clearance was
slow and elimination was biphasic with unchanged peginesatide representing >90%
of the total radioactivity of the total radioactive exposure. Slow uptake of the
radiolabeled compound from the vascular compartment into the tissues was
observed. Biodistribution to bone marrow and extramedullary hematopoietic sites,
and to highly vascularized lymphatic and excretory tissues occurred. A
predomit degradation event to occur in vivo was the loss of one PEG chain
from the branched PEG moiety to generate mono-PEG. Renal excretion was the
primary mechanism (41%) of elimination, with parent molecule (67%) the major
moiety excreted. In conclusion, elimination of [(14)C]-peginesatide-derived
radioactivity was extended, retention preferentially occurred at sites of
erythropoiesis (bone marrow), and urinary excretion was the primary elimination
route. Recombit human erythropoietin (epoetin) has been available for the treatment
of renal anemia for more than 20 years, and within the last decade two
molecularly engineered analogues darbepoetin alfa and pegylated epoetin beta
were introduced as longer-acting erythropoiesis-stimulating agents. Recently,
newer strategies for correcting anemia have been explored, some of which remain
in the laboratory while others are translating across into clinical trials.
Peginesatide has completed phase 3 clinical trials for the treatment of anemia
associated with chronic kidney disease; this molecule is immunologically
distinct from the erythropoietic proteins, with no cross-reactivity with
anti-erythropoietin antibodies. HIF (hypoxia inducible factor) stabilization
involves the pharmacologic inhibition of prolyl hydroxylation of HIF-α (the
major transcription factor controlling erythropoietin gene expression), thereby
preventing its degradation in the proteasome. Hepcidin is the master regulator
of iron metabolism, and this peptide is upregulated in inflammatory conditions,
including uremia; its antagonism has been shown to cause amelioration of
inflammatory anemia in animal models. For the time being,
erythropoiesis-stimulating agent therapy remains the mainstay of anemia
management in chronic kidney disease, but it is possible that one or more of the
strategies discussed in this review may have a future role in the treatment of
this condition. As recently reported, dried blood spot (DBS) analysis is an advantageous
technique for doping control purposes due to the minimal invasive sample
collection, the simple and economic manner, as well as the low susceptibility to
manipulation. Its general applicability to the sports drug testing arena has
been shown for analytes of various substance classes, all of which comprise
exclusively low molecular mass compounds. The aim of the present study was to
investigate whether the technique of DBS analysis is applicable also to
(pegylated) peptides with relevance for doping controls. As target analyte,
peginesatide (Omontys, Hematide), a recently approved pegylated
erythropoietin-mimetic peptide of approximately 45 kDa, tested for the treatment
of anaemia in patients with renal failure, was chosen, which has been prohibited
in elite sports due to its assumed endurance enhancing effects. Therefore, a
detection method for peginesatide employing DBS was developed based on
extraction, proteolytic digestion and cation-exchange purification followed by
liquid chromatography-tandem mass spectrometry analysis. Eventually, the assay
was validated for qualitative purposes and proved to be specific, sensitive
(limit of detection, 10 ng/mL) and precise (relative standard deviations below
18%), demonstrating the general suitability of DBS analysis in sports drug
testing also for (pegylated) peptides. Peginesatide is a synthetic, dimeric peptide that is covalently linked to
polyethylene glycol (PEG). The amino acid sequence of peginesatide is unrelated
to that of erythropoietin (EPO) and is not immunologically cross-reactive with
EPO. Peginesatide binds to and activates the human EPO receptor, stimulating the
proliferation and differentiation of human red cell precursors in vitro in a
manner similar to other EPO-stimulating agents (ESAs). In Phase II and III
studies in dialysis and predialysis patients, peginesatide administered once
monthly was as effective as epoetin alfa given thrice weekly (dialysis patients)
or darbepoetin given once weekly (nondialysis patients), in correcting anemia of
chronic kidney disease as well as maintaining hemoglobin within the desired
target range. In the dialysis population, the reported side-effect profile of
peginesatide was comparable to that known with other marketed ESAs. In the
nondialysis studies, compared with those treated with darbepoetin, patients
treated with peginesatide experienced a higher adverse-effect profile.
Peginesatide is likely to be licensed for treatment of renal anemia in dialysis
patients and not in nondialysis patients. Despite this limitation, peginesatide
is likely to prove valuable in treating dialysis patients because of its
infrequent mode of administration, thereby allowing for a reduced number of
injections, with associated better compliance, reduced cold storage requirement,
and improved stock accountability. PEGylated therapeutic proteins can elicit
immunological response to the PEG moiety of the therapeutic complex. Only
long-term experience and post-marketing surveillance will address whether this
immunological response will have any impact on the clinical efficacy or safety
of peginesatide in clinical practice. Anemia is a major complication in patients with chronic kidney disease, as the
damaged kidney is unable to produce enough erythropoietin. Peginesatide
(formerly known as Hematide™) is a synthetic, peptide-based
erythropoiesis-stimulating agent linked to polyethylene glycol. Based on
extensive preclinical and clinical data substantiating the efficacy and safety
of this agent, it was approved in the U.S. in March 2012 for the treatment of
anemia due to chronic kidney disease in adult patients on dialysis. Peginesatide
(Omontys®) was launched in the U.S. in April 2012. Erythropoiesis-stimulating agents (ESAs) have frequently been confessed to be
illicitly used in elite sports due to their endurance enhancing effects.
Recently, peginesatide, the first representative of a new generation of ESAs,
referred to as Erythropoietin (EPO)-mimetic peptides, obtained approval in the
USA under the trade name Omontys(®) for the treatment of anaemic patients.
Lacking sequence homology with EPO, it consists of a pegylated homodimeric
peptide of approximately 45 kDa, and thus, specific approaches for the
determination of peginesatide in blood were developed as conventional detection
assays for EPO do not allow for the analysis of the EPO-mimetic peptides.
However, as urine specimens are the most frequently provided doping control
samples and pharmacokinetic studies conducted in rats and monkeys revealed the
excretion of the pegylated peptide into urine, a detection method for
peginesatide in urine would be desirable. A mass spectrometric assay in human
urine was developed consisting of protein precipitation with acetonitrile
followed by proteolytic digestion after the removal of the acetonitrile fraction
under reduced pressure. Purification and concentration of the resulting
proteotypic target peptide was accomplished by means of solid-phase extraction
on strong cation-exchange resin prior to liquid chromatographic-tandem mass
spectrometric analysis. Method validation was performed for qualitative purposes
and demonstrated specificity, precision, linearity as well as sufficient
sensitivity (limit of detection: 0.5 ng/ml) while proof-of-concept for the
applicability of the assay for the determination of peginesatide in authentic
urine samples was obtained by analyzing animal in vivo specimens collected after
a single i.v. administration of peginesatide over a period of 4 days. Erythropoietin (EPO) and its recombit analogues are suspected to be illicitly
administered to horses for performance enhancing purposes and, consequently,
prohibited in equine sports. Recently, a new erythropoiesis-stimulating agent,
peginesatide (Omontys, formerly referred to as Hematide), belonging to the
upcoming class of EPO-mimetic peptides, received approval for the treatment of
anaemia in humans with chronic kidney disease on dialysis. As the pegylated
dimeric peptide of approximately 45 kDa without sequence homology to EPO is not
detectable by conventional EPO detection assays, specific methods are bound to
be established for horse sports drug testing. Thus, by fortifying equine serum
with peginesatide, an approach consisting of a proteolytic digestion with
subtilisin after protein precipitation was developed, eventually targeting a
proteotypic and xenobiotic pentapeptide which is easily accessible to liquid
chromatography- tandem mass spectrometry analysis. The method was validated for
qualitative purposes and demonstrated to be specific, precise (relative standard
deviations below 14%), sensitive (limit of detection 10 ng mL(-1)) and linear.
Being simple, cost-effective and readily transferable to other doping control
laboratories, a mass spectrometric assay for the detection of therapeutic
concentrations of peginesatide in equine serum is, in terms of preventive doping
research, applicable to routine analysis shortly after approval of the drug. The erythropoiesis-stimulating agents (ESAs) erythropoietin and darbepoetin
prevent transfusions among chemotherapy-associated anemia patients. Clinical
trials, meta-analyses, and guidelines identify mortality, tumor progression, and
venous thromboembolism (VTE) risks with ESA administration in this setting.
Product labels advise against administering ESAs with potentially curative
chemotherapy (United States) or to conduct risk-benefit assessments
(Europe/Canada). Since 2007, fewer chemotherapy-associated anemia patients in
the United States and Europe receive ESAs. ESAs and the erythropoietin receptor
agonist peginesatide prevent transfusions among chronic kidney disease (CKD)
patients; clinical trials, guidelines, and meta-analyses demonstrate myocardial
infarction, stroke, VTE, or mortality risks with ESAs targeting high hemoglobin
levels. U.S. labels recommend administering ESAs or peginesatide at doses
sufficient to prevent transfusions among dialysis CKD patients. For dialysis CKD
patients, Canadian and European labels recommend targeting hemoglobin levels of
10 to 12 g/dL and 11 to 12 g/dL, respectively, with ESAs. ESA utilization for
dialysis CKD patients has decreased in the United States. BACKGROUND AND OBJECTIVES: Peginesatide (Omontys) is a novel, synthetic,
PEGylated, peptide-based erythropoiesis-stimulating agent (ESA) that is designed
to specifically stimulate the erythropoietin receptor. This study evaluated
maintece of hemoglobin levels in patients after conversion from darbepoetin
alfa to once-monthly peginesatide.
DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This open-label, multicenter
study included 101 CKD patients, 52 of whom were receiving dialysis. The
duration of the study was 24 weeks. The primary endpoint was the mean change in
hemoglobin from baseline to the evaluation period (weeks 19-24). The study was
conducted during the period from September 22, 2008 to December 24, 2009.
RESULTS: The mean change among hemodialysis patients was -0.42 g/dl (95%
confidence interval, -0.65 to -0.19) and the mean change among CKD nondialysis
patients was 0.49 g/dl (95% confidence interval, 0.26-0.71). The percentages of
patients who maintained hemoglobin levels within ±1.0 g/dl of baseline values
were as follows: 80.0% for hemodialysis and 68.1% for nondialysis, and73.3% for
hemodialysis and 68.1% for nondialysis within the target range of 10.0-12.0
g/dl. Few patients received red blood cell transfusions (hemodialysis, 5.8%;
nondialysis, 2.0%). Seventy-nine patients experienced adverse events, the
majority of which were mild or moderate in severity. There were 40 serious
adverse events and 2 deaths reported.
CONCLUSIONS: In this study, once-monthly peginesatide resulted in a slight
decrease in mean hemoglobin levels in individuals on hemodialysis and a small
increase in individuals with CKD who were not on dialysis. Erythropoiesis-stimulating agents (ESAs) have become a hallmark of anaemia
therapy in patients with chronic kidney disease (CKD). Although different ESAs
are available for the treatment of renal anaemia, each nephrologist should
select a single ESA for an individual patient. Epoetin alfa and epoetin beta
have been used 1-3 times weekly but extended-interval dosing up to every 4 weeks
is also effective in a substantial majority of CKD patients. However, the
epoetin dose necessary to achieve or maintain target haemoglobin (Hb) levels
increases substantially as the dosing interval increases. Subcutaneous
administration of short-acting ESAs is more effective than the intravenous route
of administration. Darbepoetin alfa and the continuous erythropoietin receptor
activator (CERA) have been developed as a treatment for anaemia with extended
administration intervals (every 2 weeks and every 4 weeks, respectively). Dose
requirements for these long-acting ESAs are independent of the route of
administration. Patents of short-acting ESAs have expired, which has opened the
field for biosimilars. Epoetin biosimilars approved by the European Medicines
Agency (EMA) or the US Food and Drug Administration (FDA) have been shown to
have a comparable efficacy and safety profile to their originators. An alarming
increase in pure red cell aplasia (PRCA) in Thailand with follow-on epoetins
manufactured in Asia (but also those manufactured in Latin America) indicates
that stringent country-specific approval and pharmacovigilance protocols for
ESAs manufactured in non-North American and non-EU European countries are
urgently needed. Two PRCA cases occurring with subcutaneous HX575 (one certain,
one likely) indicate that chances of inducing a more immunogenic product are
unpredictable, even with a biosimilar epoetin approved under the EMA biosimilar
approval pathway. Phase III clinical trials with peginesatide, a pegylated
synthetic peptide-based ESA without any homology to erythropoietin raised safety
concerns in non-dialysis CKD patients but not in dialysis patients. BACKGROUND: Peginesatide, a synthetic peptide-based erythropoiesis-stimulating
agent (ESA), is a potential therapy for anemia in patients with advanced chronic
kidney disease.
METHODS: We conducted two randomized, controlled, open-label studies (EMERALD 1
and EMERALD 2) involving patients undergoing hemodialysis. Cardiovascular safety
was evaluated by analysis of an adjudicated composite safety end point--death
from any cause, stroke, myocardial infarction, or serious adverse events of
congestive heart failure, unstable angina, or arrhythmia--with the use of pooled
data from the two EMERALD studies and two studies involving patients not
undergoing dialysis. In the EMERALD studies, 1608 patients received peginesatide
once monthly or continued to receive epoetin one to three times a week, with the
doses adjusted as necessary to maintain a hemoglobin level between 10.0 and 12.0
g per deciliter for 52 weeks or more. The primary efficacy end point was the
mean change from the baseline hemoglobin level to the mean level during the
evaluation period; noninferiority was established if the lower limit of the
two-sided 95% confidence interval was -1.0 g per deciliter or higher in the
comparison of peginesatide with epoetin. The aim of evaluating the composite
safety end point in the pooled cohort was to exclude a hazard ratio with
peginesatide relative to the comparator ESA of more than 1.3.
RESULTS: In an analysis involving 693 patients from EMERALD 1 and 725 from
EMERALD 2, peginesatide was noninferior to epoetin in maintaining hemoglobin
levels (mean between-group difference, -0.15 g per deciliter; 95% confidence
interval [CI], -0.30 to -0.01 in EMERALD 1; and 0.10 g per deciliter; 95% CI,
-0.05 to 0.26 in EMERALD 2). The hazard ratio for the composite safety end point
was 1.06 (95% CI, 0.89 to 1.26) with peginesatide relative to the comparator ESA
in the four pooled studies (2591 patients) and 0.95 (95% CI, 0.77 to 1.17) in
the EMERALD studies. The proportions of patients with adverse and serious
adverse events were similar in the treatment groups in the EMERALD studies. The
cardiovascular safety of peginesatide was similar to that of the comparator ESA
in the pooled cohort.
CONCLUSIONS: Peginesatide, administered monthly, was as effective as epoetin,
administered one to three times per week, in maintaining hemoglobin levels in
patients undergoing hemodialysis. (Funded by Affymax and Takeda Pharmaceutical;
ClinicalTrials.gov numbers, NCT00597753 [EMERALD 1], NCT00597584 [EMERALD 2],
NCT00598273 [PEARL 1], and NCT00598442 [PEARL 2].). BACKGROUND: Peginesatide is a peptide-based erythropoiesis-stimulating agent
(ESA) that may have therapeutic potential for anemia in patients with advanced
chronic kidney disease. We evaluated the safety and efficacy of peginesatide, as
compared with another ESA, darbepoetin, in 983 such patients who were not
undergoing dialysis.
METHODS: In two randomized, controlled, open-label studies (PEARL 1 and 2),
patients received peginesatide once a month, at a starting dose of 0.025 mg or
0.04 mg per kilogram of body weight, or darbepoetin once every 2 weeks, at a
starting dose of 0.75 μg per kilogram. Doses of both drugs were adjusted to
achieve and maintain hemoglobin levels between 11.0 and 12.0 g per deciliter for
52 weeks or more. The primary efficacy end point was the mean change from the
baseline hemoglobin level to the mean level during the evaluation period;
noninferiority was established if the lower limit of the two-sided 97.5%
confidence interval was -1.0 g per deciliter or higher. Cardiovascular safety
was evaluated on the basis of an adjudicated composite end point.
RESULTS: In both studies and at both starting doses, peginesatide was
noninferior to darbepoetin in increasing and maintaining hemoglobin levels. The
mean differences in the hemoglobin level with peginesatide as compared with
darbepoetin in PEARL 1 were 0.03 g per deciliter (97.5% confidence interval
[CI], -0.19 to 0.26) for the lower starting dose of peginesatide and 0.26 g per
deciliter (97.5% CI, 0.04 to 0.48) for the higher starting dose, and in PEARL 2
they were 0.14 g per deciliter (97.5% CI, -0.09 to 0.36) and 0.31 g per
deciliter (97.5% CI, 0.08 to 0.54), respectively. The hazard ratio for the
cardiovascular safety end point was 1.32 (95% CI, 0.97 to 1.81) for peginesatide
relative to darbepoetin, with higher incidences of death, unstable angina, and
arrhythmia with peginesatide.
CONCLUSIONS: The efficacy of peginesatide (administered monthly) was similar to
that of darbepoetin (administered every 2 weeks) in increasing and maintaining
hemoglobin levels. However, cardiovascular events and mortality were increased
with peginesatide in patients with chronic kidney disease who were not
undergoing dialysis. (Funded by Affymax and Takeda Pharmaceutical;
ClinicalTrials.gov numbers, NCT00598273 [PEARL 1], NCT00598442 [PEARL 2],
NCT00597753 [EMERALD 1], and NCT00597584 [EMERALD 2].). INTRODUCTION: Erythropoiesis-stimulating agents (ESAs) are the mainstay of
treatment in anemic chronic kidney disease (CKD) patients. A tailored ESA
therapy should combine maximal efficacy and safety with greatest convenience in
dosing. Peginesatide, recently approved in the US for once-monthly dosing in
adult patients on dialysis, is a promising novel PEGylated
erythropoietin-mimetic peptide for the treatment of renal disease-induced
anemia.
AREAS COVERED: Published animal and human studies that evaluated the
pharmacodynamics, pharmacokinetics, clinical efficacy and safety of peginesatide
were critically analyzed.
EXPERT OPINION: Peginesatide has a well-studied pharmacological and
immunological profile, and latest published data favor the use of peginesatide
in place of epoetin in dialysis patients. A more detailed evaluation of its
safety profile particularly in trials with CKD patients not requiring dialysis
is urgently needed, as peginesatide could be a perfect treatment solution for
these patients. In addition, clinical long-term data and results from
supplemental studies, e.g., with the PEGylated continuous erythropoietin
receptor activator as comparator, should briefly follow. The fate of
peginesatide on the highly competitive ESA market is currently not predictable
and depends on safety and efficacy results of upcoming trials as well as finally
on market and price policy. Any scientific innovation needs to translate into a significant benefit.
Peginesatide is noninferior to other erythropoiesis stimulating agents (ESAs) in
terms of efficacy, and it shares the advantages of other long-acting ESAs:
delayed administration frequency and no changes in dose needs according to the
administration route. The molecular structure of peginesatide does not require
the use of recombit DNA technology during the manufacturing process, making
its synthesis simpler and likely economically cheaper. During clinical
development, its safety profile seemed to be safe, excepting the potential
increase in the risk of safety end-point events in nondialysis CKD patients.
However postmarketing serious hypersensitivity reactions have completely changed
the scenario and urgently needs in-depth clarification. This promising drug
seems to have prematurely finished its prospects. BACKGROUND: In non-dialysis patients (ND-CKD), C.E.R.A. has been extensively
investigated in ESA-naïve subjects but no data are available on its efficacy
after switch from other ESA.
METHODS: In this prospective, multicenter, open-label study lasting 24 weeks,
ND-CKD patients (n = 157) receiving ESA were converted to C.E.R.A. at doses
lower than recommended. Primary outcome was the prevalence of Hb target (11-12.5
g/dl).
RESULTS: Age was 73 ± 13 years and GFR was 26.2 ± 9.4 ml/min/1.73 m(2); male
gender, diabetes and prior cardiovascular disease were 49, 33 and 19%,
respectively. Doses of darbepoetin (25 ± 16 µg/week, n = 124) and epoetin (5,702
± 3,190 IU/week, n = 33) were switched to low dose C.E.R.A. (87 ± 17 µg/month).
During the study, prevalence of Hb target increased from 60% to 68% at week-24,
while that of Hb < 11 g/dl declined from 32% to 16% (p < 0.001). Hb increased
from 11.3 ± 0.8 at baseline to 11.7 ± 0.9 g/dl at week-24 (p = 0.01) without
changes in C.E.R.A. dose. Significant predictors of Hb increase were low BMI,
low Hb and longer dosing intervals before switch. These factors also predicted
the risk of Hb overshooting (Hb > 12.5 g/dl) occurring in 57 patients.
CONCLUSIONS: In ND-CKD, conversion from other ESAs to C.E.R.A. is associated
with a better anemia control induced by a greater Hb increase in patients
previously treated with ESAs at extended dosing interval. This parameter should
be considered when switching to long-acting ESA for its potential impact on the
risk of overshooting. Anemia in chronic kidney disease is a prevalent and expensive problem in the
United States, and it is well documented that anemia worsens as glomerular
filtration rates decline. The complications of severe anemia in this patient
population contribute significantly to their overall morbidity with increased
cardiovascular complications, decreased quality of life, and increased
dependence on transfusions to maintain adequate hemoglobin levels.
Erythropoietin-stimulating agents (ESAs) have revolutionized the treatment of
anemia in this population, but there has been a great deal of controversy
surrounding the quest for the ideal hemoglobin target. In addition, there are
economic and practice management implications where anemia treatment is
concerned, with ongoing refinement of Centers for Medicare and Medicaid
Services-bundled payments. One of the newest additions to the arsenal used to
fight anemia in end-stage renal disease patients is peginesatide (Omontys), a
synthetic, PEGylated, peptide-based ESA that acts by stimulating the
erythropoietin receptor. The role of peginesatide in the future treatment of
anemia in chronic kidney disease remains uncertain, with new safety concerns
being brought to attention as it emerges on the market, prompting a national
recall. ♦
BACKGROUND: Peginesatide is a novel, synthetic, peptide-based pegylated
erythropoiesis-stimulating agent that is designed specifically to stimulate the
erythropoietin receptor. The purpose of the present study was to assess, for the
first time, the efficacy and safety of peginesatide in chronic kidney disease
(CKD) patients receiving peritoneal dialysis (PD) and previously on epoetin
treatment. ♦
METHODS: In this open-label multicenter study, 59 PD patients with CKD were
converted from epoetin (alfa or beta) to once-monthly peginesatide. Doses were
titrated to maintain hemoglobin levels between 10 g/dL and 12 g/dL during the 25
weeks of the study. The primary endpoint was change from baseline in mean
hemoglobin values during the evaluation period (weeks 20 - 25). ♦
RESULTS: The mean hemoglobin value during the evaluation period was 11.3 ± 1.07
g/dL, and the mean change from baseline was 0.10 ± 1.15 g/dL (95% confidence
limits: -0.24, 0.44 g/dL). During the evaluation period, most patients
maintained hemoglobin levels between 10 g/dL and 12 g/dL (63.0%) and within ±1.0
g/dL of baseline (60.9%). The median weekly epoetin dose at baseline was 96.0
U/kg, and the median starting peginesatide dose was 0.047 mg/kg. Forty-three
patients (72.9%) completed the study. Six patients (10.2%) received red blood
cell transfusions. The observed adverse event profile was consistent with
underlying conditions in the PD patient population. The most common adverse
event was peritonitis (20.3%), a complication commonly associated with PD. Four
deaths occurred during the study (2 related to septic shock, and 1 each to
myocardial ischemia and myasthenia gravis). ♦
CONCLUSIONS: In this study, once-monthly peginesatide maintained hemoglobin
levels in PD patients after conversion from epoetin. PURPOSE: Peginesatide, a long-acting erythropoiesis-stimulating agent, was
recalled in February 2013 following reports of serious and sometimes fatal
hypersensitivity reactions in dialysis patients who received a first dose. We
assessed the relative risks of mortality and morbidity in peginesatide-treated
and matched epoetin alfa-treated patients.
METHODS: From standardized extracts of paid Medicare claims in 2012 and 2013, we
identified dialysis patients treated with peginesatide or epoetin between 1 July
2012 and 28 February 2013. For each peginesatide-treated patient, we identified
with propensity score matching two epoetin-treated control patients. Patients
were followed for up to 2 days after the first peginesatide dose or the referent
epoetin dose for death or hospitalization as a result of cardiovascular
morbidity or symptoms (composite event), all-cause hospitalization, and
emergency room care.
RESULTS: We identified 15 633 peginesatide-treated patients and 31 266 matched
epoetin-treated controls. On the day of dose administration, 19 composite events
occurred with peginesatide (incidence, 0.12%) and 14 with epoetin (0.04%); the
hazard ratio was 2.7 (95% confidence interval, 1.4-5.4). With follow-up for 1
and 2 subsequent days, hazard ratios were 1.6 (1.0-2.4) and 1.5 (1.1-2.0),
respectively. Corresponding hazard ratios were larger among hemodialysis
patients with neither intravenous antibiotic nor intravenous iron exposure on
the day of dose administration. Hazard ratios for all-cause hospitalization and
emergency room care exceeded 1 on and after the day of dose administration.
CONCLUSIONS: Relative to administration of epoetin alfa, first administration of
peginesatide in dialysis patients was acutely associated with higher risk of
death or hospitalization as a result of cardiovascular morbidity or symptoms. The development of a new class of erythropoietin mimetic agents (EMA) for
treating anemic conditions has been initiated with the discovery of
oligopeptides capable of dimerizing the erythropoietin (EPO) receptor and thus
stimulating erythropoiesis. The most promising amino acid sequences have been
mounted on various different polymeric structures or carrier molecules to obtain
highly active EPO-like drugs exhibiting beneficial and desirable pharmacokinetic
profiles. Concomitant with creating new therapeutic options, erythropoietin
mimetic peptide (EMP)-based drug candidates represent means to artificially
enhance endurance performance and necessitate coverage by sports drug testing
methods. Therefore, the aim of the present study was to develop a strategy for
the comprehensive detection of EMPs in doping controls, which can be used
complementary to existing protocols. Three model EMPs were used to provide
proof-of-concept data. Following EPO receptor-facilitated purification of target
analytes from human urine, the common presence of the cysteine-flanked core
structure of EMPs was exploited to generate diagnostic peptides with the aid of
a nonenzymatic cleavage procedure. Sensitive detection was accomplished by
targeted-SIM/data-dependent MS(2) analysis. Method characterization was
conducted for the EMP-based drug peginesatide concerning specificity, linearity,
precision, recovery, stability, ion suppression/enhancement, and limit of
detection (LOD, 0.25 ng/mL). Additionally, first data for the identification of
the erythropoietin mimetic peptides EMP1 and BB68 were generated, demonstrating
the multi-analyte testing capability of the presented approach. Erythropoiesis-stimulating agents (ESAs) that exert long-acting antianemia
effects have been developed recently, but their mechanisms are poorly
understood. Analyses reveal unique erythropoietin receptor (EPOR)-binding
properties for one such ESA, the synthetic EPOR agonist peginesatide. Compared
with recombit human EPO and darbepoietin, peginesatide exhibited a slow on
rate, but sustained EPOR residency and resistant displacement. In EPO-dependent
human erythroid progenitor UT7epo cells, culture in peginesatide unexpectedly
upmodulated endogenous cell surface EPOR levels with parallel increases in
full-length EPOR-68K levels. These unique properties are suggested to contribute
to the durable activity of this (and perhaps additional) dimeric peptide
hematopoietic growth factor receptor agonist. The erythropoietin-mimetic peptide (EMP) peginesatide belongs to the group of
erythropoiesis-stimulating agents (ESAs) that are prohibited when misused in
sports. Peginesatide is a synthetic pegylated homodimer of two cyclic 21-amino
acid chains. It was approved for the treatment of anaemic patients with chronic
kidney disease in the USA in 2012, but recalled in 2013 due to prevalent cases
of acute severe anaphylactoid reactions and associated fatalities (0.02%). The
drug was considered obsolete for athletes and part of the anti-doping scene lost
sight of it. However, recent research indicates that the adverse events were not
caused by the drug substance, but by the drug product formulated in multi-use
vials. These vials contained comparably high levels of subvisible particles.
Phenol was identified as a critical component of the drug formulation, which
caused the release of histamine from mast cells. Tricky athletes might consider
peginesatide a pharmacologically safe ESA in an appropriate formulation. In
addition, other EMPs may get a second wind for therapy including misuse in
sports. Therefore, it is very important to proceed in developing
electrophoretic, immunological, and mass spectroscopic methods for detecting
peginesatide and other EMPs in human urine and blood samples. Copyright © 2016
John Wiley & Sons, Ltd. INTRODUCTION: The introduction of recombit human erythropoietin
revolutionized the management of anemia in patients with chronic kidney disease
(CKD). In order to circumvent costly recombit DNA technology, synthetic
chemistry techniques were used to manufacture peginesatide, a synthetic peptide
that bore no resemblance to previous erythropoiesis-stimulating agents (ESAs),
and yet was capable of stimulating erythropoiesis. Compared with other ESAs,
peginesatide was deemed to have advantages related to immunogenicity,
administration schedule, and cost. Marketing approval was restricted to CKD
patients on dialysis because cardiovascular events were more common with
peginesatide than with darbepoetin in non-dialysis CKD patients. Unfortunately,
unexplained serious adverse drug reactions (sADR) led to quick withdrawal of
peginesatide from the market.
AREAS COVERED: This review describes the efficacy and safety of peginesatide in
pre-approval clinical trials, sADRs after marketing approval, and lessons
learned during its short life-span.
EXPERT OPINION: The case of peginesatide illustrates the difficulties in
detecting rare sADRs in trials with limited patient populations and the need for
improved pharmacovigilance after marketing approval. However, the need for
simpler drug production methods as a result of non-dependence on recombit DNA
techniques and mammalian cell lines remains. Lessons learned during the
scientific development of peginesatide can be used in developing other drugs. |
Describe mechanism of action of Eteplirsen? | Eteplirsen (Exondys 51) is an antisense oligonucleotide designed to induce exon 51 skipping. Eteplirsen is approved for the treatment of Duchenne muscular dystrophy (DMD) in patients with a confirmed mutation of the DMD gene amenable to exon 51 skipping. | We previously conducted a proof of principle; dose escalation study in Duchenne
muscular dystrophy (DMD) patients using the morpholino splice-switching
oligonucleotide AVI-4658 (eteplirsen) that induces skipping of dystrophin exon
51 in patients with relevant deletions, restores the open reading frame and
induces dystrophin protein expression after intramuscular (i.m.) injection. We
now show that this dystrophin expression was accompanied by an elevated
expression of α-sarcoglycan, β-dystroglycan (BDG) and--in relevant
cases--neuronal nitric oxide synthase (nNOS) at the sarcolemma, each of which is
a component of a different subcomplex of the dystrophin-associated glycoprotein
complex (DAPC). As expected, nNOS expression was relocalized to the sarcolemma
in Duchenne patients in whom the dystrophin deletion left the nNOS-binding
domain (exons 42-45) intact, whereas this did not occur in patients with
deletions that involved this domain. Our results indicate that the novel
internally deleted and shorter dystrophin induced by skipping exon 51 in
patients with amenable deletions, can also restore the dystrophin-associated
complex, further suggesting preserved functionality of the newly translated
dystrophin. Restoration of the open reading frame of the DMD gene and dystrophin protein
production in Duchenne muscular dystrophy (DMD) can be achieved by exon skipping
using antisense oligomers (AOs) targeted to splicing elements. Several such
RNA-based gene therapy approaches are in clinical development in which all
studies to date have assessed AO efficacy by semiquantitative nested
reverse-transcription polymerase chain reaction (RT-PCR). Precise evaluation of
dystrophin protein levels is complex and hindered by the large size and low
abundance of dystrophin; thus an accurate and standardized measurement of DMD
exon skipping at the RNA level remains important to assess and compare patient
responses in DMD exon skipping clinical trials. Here we describe the development
of a Taqman quantitative (q)RT-PCR assay to quantify exon skipping and highlight
its use to determine the levels of exon skipping in DMD patients treated
intramuscularly with a morpholino AO to skip exon 51, eteplirsen (AVI-4658). The
muscle biopsies of these patients were previously thoroughly characterized,
providing a valuable benchmark for the evaluation of novel methodology. We
demonstrate that levels of dystrophin protein restoration, and thus patient
response, correlate accurately with the RNA level. Furthermore, this sensitive
assay detects revertant exon 51 skipped fibers in untreated biopsies, providing
an important baseline to precisely quantify treatment success. This study
represents the first quantitative assessment of exon skipping in a clinical
trial setting. We present a standardized and reproducible method to assess
patient response that will complement protein studies in future preclinical and
clinical exon skipping-based gene therapy studies for DMD. OBJECTIVE: In prior open-label studies, eteplirsen, a phosphorodiamidate
morpholino oligomer, enabled dystrophin production in Duchenne muscular
dystrophy (DMD) with genetic mutations amenable to skipping exon 51. The present
study used a double-blind placebo-controlled protocol to test eteplirsen's
ability to induce dystrophin production and improve distance walked on the
6-minute walk test (6MWT).
METHODS: DMD boys aged 7 to 13 years, with confirmed deletions correctable by
skipping exon 51 and ability to walk 200 to 400 m on 6 MWT, were randomized to
weekly intravenous infusions of 30 or 50 mg/kg/wk eteplirsen or placebo for 24
weeks (n = 4/group). Placebo patients switched to 30 or 50 mg/kg eteplirsen
(n=2/group) at week 25; treatment was open label thereafter. All patients had
muscle biopsies at baseline and week 48. Efficacy included dystrophin-positive
fibers and distance walked on the 6MWT.
RESULTS: At week 24, the 30 mg/kg eteplirsen patients were biopsied, and
percentage of dystrophin-positive fibers was increased to 23% of normal; no
increases were detected in placebo-treated patients (p≤0.002). Even greater
increases occurred at week 48 (52% and 43% in the 30 and 50 mg/kg cohorts,
respectively), suggesting that dystrophin increases with longer treatment.
Restoration of functional dystrophin was confirmed by detection of sarcoglycans
and neuronal nitric oxide synthase at the sarcolemma. Ambulation-evaluable
eteplirsen-treated patients experienced a 67.3 m benefit compared to
placebo/delayed patients (p≤0.001).
INTERPRETATION: Eteplirsen restored dystrophin in the 30 and 50 mg/kg/wk
cohorts, and in subsequently treated, placebo-controlled subjects. Duration,
more than dose, accounted for dystrophin production, also resulting in
ambulation stability. No severe adverse events were encountered. PURPOSE OF REVIEW: The most encouraging recent advances regarding
pharmacological agents for treating Duchenne muscular dystrophy (DMD) are
summarized. Emphasis is given to compounds acting downstream of dystrophin, the
protein lacking in DMD, on cellular pathways leading to pathological
consequences. The author highlights the progress that may have the greatest
potential for clinical use in DMD.
RECENT FINDINGS: Modifying the transcripts of the mutated gene by exon skipping
has led to expression of shortened dystrophins in DMD patients. Currently, the
most promising potential drugs are the exon-skipping agents eteplirsen and
drisapersen. Biglycan and SMTC1100 upregulate utrophin. The steroid receptor
modulating compounds VBP15 and tamoxifen, and specific antioxidants appear
promising agents for symptomatic therapy.
SUMMARY: The past 18 months have seen a strong increase in the number of
exciting reports on novel therapeutic agents for DMD. Exon-skipping agents have
been fine-tuned to improve tissue delivery and stability. Impressive discoveries
regarding pathogenic events in cellular signalling have revealed targets that
were unknown in the context of DMD, thus enabling approaches that limit
inflammation, fibrosis and necrosis. The targets are nuclear hormone receptors,
NADPH-oxidases and Ca channels. Inhibition of NF-KB, transforming growth
factor-alpha (TGF-α) and transforming growth factor-beta (TGF-β)/myostatin
production or action are also promising routes in counteracting the complex
pathogenesis of DMD. Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in
the dystrophin gene that disrupt the open reading frame, while in frame
mutations result in Becker muscular dystrophy (BMD). Ullrich congenital muscular
dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific
muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring
the outcome of therapeutic interventions and disease progression. We quantified
miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD
and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but
unchanged in UCMD compared to controls. Milder DMD patients had higher levels of
dystromirs than more severely affected patients. Patients with low forced vital
capacity (FVC) values, indicating respiratory muscle weakness, had low levels of
serum miR-1 and miR-133b. There was no significant difference in the level of
the dystromirs in BMD compared to controls. We also assessed the effect of
dystrophin restoration on the expression of the five dystromirs in serum of DMD
patients treated systemically for 12 weeks with antisense oligomer eteplirsen
that induces skipping of exon 51 in the dystrophin gene. The dystromirs were
also analysed in muscle biopsies of DMD patients included in a single dose
intramuscular eteplirsen clinical trial. Our analysis detected a trend towards
normalization of these miRNA between the pre- and post-treatment samples of the
systemic trial, which however failed to reach statistical significance. This
could possibly be due to the small number of patients and the short duration of
these clinical trials. Although longer term studies are needed to clarify the
relationship between dystrophin restoration following therapeutic intervention
and the level of circulating miRNAs, our results indicate that miR-1 and miR-133
can be considered as exploratory biomarkers for monitoring the progression of
muscle weakness and indirectly the remaining muscle mass in DMD. Eteplirsen (Exondys 51) is an antisense oligonucleotide designed to induce exon
51 skipping that is developed by Sarepta Therapeutics. Intravenous eteplirsen
has received accelerated approval from the US FDA for the treatment of Duchenne
muscular dystrophy (DMD) in patients with a confirmed mutation of the DMD gene
amenable to exon 51 skipping. Eteplirsen has orphan drug designation in the USA
and EU, and rare paediatric disease designation in the USA for use in DMD. In
the phase III PROMOVI trial, eteplirsen significantly increased dystrophin
levels from baseline in muscle tissues of 12 evaluable patients with DMD after
48 weeks of treatment. This finding is supported by data from phase II trials.
Long-term treatment with eteplirsen was associated with a decrease in the rate
of decline in ambulation and pulmonary function in an open-label extension of a
phase II trial. Eteplirsen was generally well tolerated in clinical trials. This
article summarizes the milestones in the development of eteplirsen leading to
this first approval for DMD. BACKGROUND: Phosphorodiamidate morpholino oligomers (PMOs) are a class of exon
skipping drugs including eteplirsen, which has shown considerable promise for
treatment of the degenerative neuromuscular disease, Duchenne musculardystrophy
(DMD).
OBJECTIVE: Toxicity studies in non-human primates (NHPs) of 12 weeks duration
with two new PMOs for DMD, SRP-4045 and SRP-4053, along with results from a
chronic study in NHPs of 39 weeks duration for eteplirsen, are described here.
METHODS: PMOs were administered once-weekly by bolus intravenous (IV) injections
to male NHPs. Endpoints evaluated included plasma exposures, clinical
observations, body weight/food consumption, eye exams, electrocardiograms, male
reproductive hormones/endpoints, complement alternative pathway, clinical
pathology, urinalysis, and macroscopic/light microscopic pathology.
RESULTS: Findings in these studies were limited to the kidneys, with a common
presentation of tubular basophilia, vacuolation, and/or minimal degeneration
that was considered non-adverse. No necrosis, glomerular lesions, or effects on
renal function tests such as serum creatinine or urea nitrogen were observed,
suggesting that PMO-related kidney findings are not likely to develop into frank
nephrotoxicity. There were no adverse effects on other potential target organs
after repeated IV injections at the highest dose levels tested, 320 mg/kg.
CONCLUSIONS: Nonclinical results in NHPs for these three PMOs, together with the
excellent clinical safety established for eteplirsen to date, suggest that
once-weekly IV administration of PMOs for lifetime durations at therapeutic
doses will be well tolerated by patients with DMD. Exon skipping is a therapeutic approach for Duchenne muscular dystrophy (DMD)
that has been in development for close to two decades. This approach uses
antisense oligonucleotides (AONs) to modulate pre-mRNA splicing of dystrophin
transcripts to restore the disrupted DMD reading frame. The approach has moved
from in vitro proof of concept studies to the clinical trial phase and marketing
authorization applications with regulators. The first AON (eteplirsen) has
recently received accelerated approval by the Food and Drug Administration in
the US. Areas covered: In this review the authors explain the antisense-mediated
exon skipping approach, outline how it needs be tailored for different DMD
mutation types and describe the challenges and opportunities for each mutation
type. The authors summarize the clinical development of antisense-mediated exon
51 skipping, and discuss methods to improve efficiency. Finally, the authors
provide their opinion on current developments and identify topics for future
prioritization. Expert opinion: Exon skipping development has been a learning
experience for all those involved. Aside from an approved therapy, its
development has yielded side benefits including the development of tools for
clinical trials and has increased collaboration between academics, patients,
industry and regulators. |
Which genes of the marmoset genome exhibit rapid sequence evolution? | Both protein-coding and microRNA genes related to reproduction exhibit evidence of rapid sequence evolution in the marmoset genome. | Collaborators: Worley KC, Warren WC, Rogers J, Locke D, Muzny DM, Mardis ER,
Weinstock GM, Tardif SD, Aagaard KM, Archidiacono N, Rayan NA, Batzer MA, Beal
K, Brejova B, Capozzi O, Capuano SB, Casola C, Chandrabose MM, Cree A, Dao MD,
de Jong PJ, Del Rosario RC, Delehaunty KD, Dinh HH, Eichler EE, Fitzgerald S,
Flicek P, Fontenot CC, Fowler RG, Fronick C, Fulton LA, Fulton RS, Gabisi RA,
Gerlach D, Graves TA, Gunaratne PH, Hahn MW, Haig D, Han Y, Harris RA, Herrero
J, Hillier LW, Hubley R, Hughes JF, Hume J, Jhangiani SN, Jorde LB, Joshi V,
Karakor E, Konkel MK, Kosiol C, Kovar CL, Kriventseva EV, Lee SL, Lewis LR, Liu
YS, Lopez J, Lopez-Otin C, Lorente-Galdos B, Mansfield KG, Marques-Bonet T, Minx
P, Misceo D, Moncrieff JS, Morgan MB, Nazareth LV, Newsham I, Nguyen NB, Okwuonu
GO, Prabhakar S, Perales L, Pu LL, Puente XS, Quesada V, Ranck MC, Raney BJ,
Raveendran M, Deiros DR, Rocchi M, Rodriguez D, Ross C, Ruffier M, Ruiz SJ,
Sajjadian S, Santibanez J, Schrider DR, Searle S, Skaletsky H, Soibam B, Smit
AF, Tennakoon JB, Tomaska L, Ullmer B, Vejnar CE, Ventura M, Vilella AJ, Vinar
T, Vogel JH, Walker JA, Wang Q, Warner CM, Wildman DE, Witherspoon DJ, Wright
RA, Wu Y, Xiao W, Xing J, Zdobnov EM, Zhu B, Gibbs RA, Wilson RK. |
What is the importance of Janus Kinases in dermatology? | Janus Kinase (JAK) is active in many skin diseases and recent evidence show that inhibitors of JAK kinase could be used to treat vitiligo, psoriasis, lupus, alopecia areata and other inflammatory skin diseases. | Many cytokines function through interaction with receptors of the cytokine
receptor superfamily. Although lacking catalytic domains, cytokine receptors
couple ligand binding to induction of protein tyrosine phosphorylation. Recent
studies have shown that one or more of the Janus kinase family members (Jaks)
associate with cytokine receptors and are tyrosine phosphorylated and activated
following ligand binding. Here we describe a new Jak family kinase, Jak-3, and
demonstrate that Jak-3, and to a lesser extent Jak-1, are tyrosine
phosphorylated and Jak-3 is activated in the responses to interleukin-2 and
interleukin-4 in T cells and myeloid cells. Jak-3 activation requires the
serine-rich, membrane-proximal domain of the interleukin-2 receptor beta-chain,
but does not require the acidic domain that is required for association and
activation of Src family kinases. Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase essential for signaling
via cytokine receptors that comprise the common gamma-chain (gammac), i.e., the
receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Jak3 is preferentially
expressed in hemopoietic cells and is up-regulated upon cell differentiation and
activation. Despite the importance of Jak3 in lymphoid development and immune
function, the mechanisms that govern its expression have not been defined. To
gain insight into this issue, we set out to characterize the Jak3 promoter. The
5'-untranslated region of the Jak3 gene is interrupted by a 3515-bp intron.
Upstream of this intron and the transcription initiation site, we identified an
approximately 1-kb segment that exhibited lymphoid-specific promoter activity
and was responsive to TCR signals. Truncation of this fragment revealed that
core promoter activity resided in a 267-bp fragment that contains putative Sp-1,
AP-1, Ets, Stat, and other binding sites. Mutation of the AP-1 sites
significantly diminished, whereas mutation of the Ets sites abolished, the
inducibility of the promoter construct. Chromatin immunoprecipitation assays
showed that histone acetylation correlates with mRNA expression and that Ets-1/2
binds this region. Thus, transcription factors that bind these sites, especially
Ets family members, are likely to be important regulators of Jak3 expression. Cytokines are key mediators of the development and homeostasis of haematopoietic
cells, critical for host defense, but also for the development of autoimmune and
inflammatory diseases such as psoriasis or rheumatoid arthritis (RA). Blocking
cytokines activity by interfering with the ligand-receptor association has been
successfully employed to treat several immune disorders. A subgroup of cytokines
signals through receptors requiring the association with a family of cytoplasmic
protein tyrosine kinases known as Janus kinases (Jaks). Jaks have recently
gained significant attention as therapeutic targets in inflammation and
autoimmunity, and several Jak inhibitory small molecules have been developed.
The first two Jak inhibitors, tofacitinib and ruxolitinib, have been approved
for the treatment of RA and primary myelofibrosis, respectively. Efficacy and
safety data suggest that some of these oral Jak inhibitors as well as their
topical formulations may soon enter the daily clinical practice for treating
patients with psoriasis, lupus erythematosus or other inflammatory skin
diseases. While biologics typically target one single cytokine, these new
immunomodulators can inhibit signals from multiple cytokines intra-cellularly
and therefore could be useful when other therapies are ineffective. Thus, Jak
inhibitors may replace some traditional immunosuppressive agents and help
patients not responding to previous therapies. Many emerging studies have implicated the Janus kinase/signal transducer and
activator of transcription (JAK-STAT) cytokine signalling mechanism in disease
pathogenesis. This signalling pathway is involved in haematopoiesis and immune
development. Mutations in genes regulating JAK-STAT signalling can cause common
inflammatory disorders and myeloproliferative disorders. JAK and STAT inhibitors
are new management tools for disorders such as myelofibrosis and rheumatoid
arthritis. Evidence suggests that the cytokine components of the JAK-STAT
pathways play a crucial role in common skin disorders, including psoriasis and
atopic dermatitis. We present an overview for the clinical dermatologist of the
significance of these signalling pathways in various skin disorders, and
introduce the potential application of JAK and STAT inhibition as a new
therapeutic tool in dermatology. BACKGROUND: Plaque psoriasis is a chronic and often debilitating skin disorder
and proinflammatory cytokines are known to play a key role in the disease
process.
OBJECTIVES: To evaluate the safety and efficacy of baricitinib, an oral Janus
kinase (JAK) 1/JAK2 inhibitor, in patients with moderate-to-severe psoriasis in
a randomized, double-blind, placebo-controlled, dose-ranging phase 2b study.
METHODS: Patients were randomized (n = 271) to receive placebo or oral
baricitinib at 2, 4, 8 or 10 mg once daily for 12 weeks (Part A). Dose
adjustment for 12 additional weeks was based on percentage improvement in the
Psoriasis Area and Severity Index (PASI) score. The primary end point was
Psoriasis Area and Severity Index (PASI) 75% (PASI-75) at 12 weeks for North
American patients (n = 238); secondary end points were safety and efficacy
measures in the entire population.
RESULTS: At week 12, more North American patients in the 8-mg (43%) and 10-mg
(54%) baricitinib groups than in placebo group (17%; P < 0·05) achieved PASI-75.
All baricitinib-treated groups had greater mean changes from baseline in their
PASI scores (P < 0·05) at 12 weeks and (except 2 mg) had higher rates of PASI-50
than the placebo group; statistically significant PASI-90 responses were
achieved in the 8-mg and 10-mg groups at 8 and 12 weeks. More than 81% of
PASI-75 responders maintained their scores through 24 weeks. During Part A,
study discontinuations due to adverse events (AEs) were 0%, 0%, 2·8%, 6·3% and
5·8% and treatment-emergent AE rates were 44%, 50%, 47%, 58% and 64% for placebo
and 2-, 4-, 8- and 10-mg baricitinib groups, respectively. No opportunistic
infections were observed in any treatment group. Dose-dependent changes in
laboratory values were observed.
CONCLUSIONS: Patients with moderate-to-severe psoriasis treated with baricitinib
for 12 weeks achieved significant improvements in PASI-75. BACKGROUND: Tofacitinib is an oral Janus kinase inhibitor being investigated for
psoriasis.
OBJECTIVES: We sought to report longer-term tofacitinib efficacy and safety in
patients with moderate to severe psoriasis.
METHODS: Data from 2 identical phase-III studies, Oral-treatment Psoriasis Trial
Pivotal 1 and 2, were pooled with data from these patients in an ongoing
open-label long-term extension study. Patients (n = 1861) were randomized 2:2:1
to tofacitinib 5 mg, 10 mg, or placebo twice daily (BID). At week 16, placebo
patients were rerandomized to tofacitinib. Pivotal study participants could
enroll into the long-term extension where they received tofacitinib at 10 mg BID
for 3 months, after which dosing could be 5 or 10 mg BID.
RESULTS: At week 28, the proportions of patients randomized to tofacitinib 5 and
10 mg BID achieving 75% or greater reduction in Psoriasis Area and Severity
Index score from baseline were 55.6% and 68.8%, and achieving Physician Global
Assessment of clear or almost clear were 54.7% and 65.9%. Efficacy was
maintained in most patients through 24 months. Serious adverse events and
discontinuations because of adverse events were reported in less than 11% of
patients over 33 months of tofacitinib exposure.
LIMITATIONS: There was no dose comparison beyond week 52.
CONCLUSIONS: Oral tofacitinib demonstrated sustained efficacy in patients with
psoriasis through 2 years, with 10 mg BID providing greater efficacy than 5 mg
BID. No unexpected safety findings were observed. BACKGROUND: Tofacitinib is an oral Janus kinase inhibitor that improves clinical
measures of psoriasis.
OBJECTIVE: We sought to assess patient-reported outcomes in tofacitinib-treated
patients with moderate to severe plaque psoriasis over 52 weeks.
METHODS: In 2 identical, phase III studies (Oral treatment for Psoriasis Trial
Pivotal 1 [NCT01276639], n = 901, and Pivotal 2 [NCT01309737], n = 960),
patients were randomized 2:2:1 to receive 5 or 10 mg of tofacitinib or placebo,
twice daily. At week 16, placebo-treated patients were re-randomized to
tofacitinib. Dermatology Life Quality Index score, Itch Severity Item score,
Patient Global Assessment score, and patient satisfaction were assessed.
RESULTS: Baseline Dermatology Life Quality Index score indicated substantial
health-related quality of life impairment. At week 16, a greater proportion of
patients achieved Dermatology Life Quality Index score of 1 or less (no effect
of psoriasis on health-related quality of life) with tofacitinib 5 and 10 mg
twice daily versus placebo (Oral treatment for Psoriasis Trial Pivotal 1/2:
26.7%/28.6% and 40.2%/48.2% vs 4.6%/6.0%, respectively; P < .0001); improvements
were maintained through week 52. Similar patterns were observed with Patient
Global Assessment. Improvements in itch were particularly rapid, observed 1 day
after treatment initiation for both tofacitinib doses versus placebo (P < .05).
At week 16, more patients were satisfied with tofacitinib versus placebo
(P < .0001).
LIMITATIONS: Clinical nonresponders discontinued at week 28.
CONCLUSIONS: Tofacitinib demonstrated improvement in health-related quality of
life and patient-reported symptoms that persisted over 52 weeks. |
What is the incidence of beta-thalassemia in Greek population? | The incidence of beta-thalassemia trait is 8% in Greek population. | A program for the detection of thalassemias and other hemoglobinopathies in
high-risk populations is described. This program, based on two screening tests,
was applied to the Hellenic Army recruits and was found to work well. Red cell
one-point osmotic fragility was used for the detection of thalassemic samples
and hemoglobin electrophoresis for screening of other hemoglobinopathies.
Samples with decreased red cell osmotic fragility and/or abnormal
electrophoretic pattern were submitted for further detailed investigation.
Following this program, 64,814 recruits, representing 0.651% of the total Greek
population and 9.917% of the 20-year-old Greek male population, were tested.
beta-Thalassemia was found with an average incidence of 5.476% and
alpha-Thalassemia with an incidence of 0.201%. Hemoglobinopathy Lepore was
detected in 51 samples (0.079%) and hemoglobinopathy-S in 352 samples (0.543%). Hemoglobinopathies are very common in Greece, the incidence of beta-thalassemia
trait being 8% and that of sickle cell trait ranging from 1 to 32% in various
districts. In Greek populations, sickle cell disease (SCD) is mainly represented
by S-beta thalassemia. |
Is exon skipping correlated with exon circularization? | Yes. Circularization of exons is widespread and correlates with exon skipping, a feature that adds considerably to the regulatory complexity of the human transcriptome. | Circular RNAs are found in a wide range of organisms and it has been proposed
that they perform disparate functions. However, how RNA circularization is
connected to alternative splicing remains largely unexplored. Here, we
stimulated primary human endothelial cells with tumor necrosis factor α or tumor
growth factor β, purified RNA, generated >2.4 billion RNA-seq reads, and used a
custom pipeline to characterize circular RNAs derived from coding exons. We find
that circularization of exons is widespread and correlates with exon skipping, a
feature that adds considerably to the regulatory complexity of the human
transcriptome. |
What is the target of daratumumab? | Daratumumab is a fully human anti-CD38 IgG1-κ monoclonal antibody. It is approved for treatment of multiple myeloma. | BACKGROUND: In our efforts to develop novel effective treatment regimens for
multiple myeloma we evaluated the potential benefits of combining the
immunomodulatory drug lenalidomide with daratumumab. Daratumumab is a novel
human CD38 monoclonal antibody which kills CD38+ multiple myeloma cells via
antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity
and apoptosis.
DESIGN AND METHODS: To explore the effect of lenalidomide combined with
daratumumab, we first carried out standard antibody-dependent cell-mediated
cytotoxicity and complement-dependent cytotoxicity assays in which the CD38+
multiple myeloma cell line UM-9 and primary multiple myeloma cells isolated from
patients were used as target cells. We also tested the effect of lenalidomide on
daratumumab-dependent cell-mediated-cytotoxicity and complement-dependent
cytotoxicity of multiple myeloma cells directly in the bone marrow mononuclear
cells of multiple myeloma patients. Finally, we determined the
daratumumab-dependent cell-mediated cytotoxicity using peripheral blood
mononuclear cells of multiple myeloma patients receiving lenalidomide treatment.
RESULTS: Daratumumab-dependent cell-mediated cytotoxicity of purified primary
multiple myeloma cells, as well as of the UM-9 cell line, was significantly
augmented by lenalidomide pre-treatment of the effector cells derived from
peripheral blood mononuclear cells from healthy individuals. More importantly,
we demonstrated a clear synergy between lenalidomide and daratumumab-induced
antibody-dependent cell-mediated cytotoxicity directly in the bone marrow
mononuclear cells of multiple myeloma patients, indicating that lenalidomide can
also potentiate the daratumumab-dependent lysis of myeloma cells by activating
the autologous effector cells within the natural environment of maligt cells.
Finally, daratumumab-dependent cell-mediated cytotoxicity was significantly
up-regulated in peripheral blood mononuclear cells derived from 3 multiple
myeloma patients during lenalidomide treatment.
CONCLUSIONS: Our results indicate that powerful and complementary effects may be
achieved by combining lenalidomide and daratumumab in the clinical management of
multiple myeloma. CD38, a type II transmembrane glycoprotein highly expressed in hematological
maligcies including multiple myeloma (MM), represents a promising target for
mAb-based immunotherapy. In this study, we describe the cytotoxic mechanisms of
action of daratumumab, a novel, high-affinity, therapeutic human mAb against a
unique CD38 epitope. Daratumumab induced potent Ab-dependent cellular
cytotoxicity in CD38-expressing lymphoma- and MM-derived cell lines as well as
in patient MM cells, both with autologous and allogeneic effector cells.
Daratumumab stood out from other CD38 mAbs in its strong ability to induce
complement-dependent cytotoxicity in patient MM cells. Importantly,
daratumumab-induced Ab-dependent cellular cytotoxicity and complement-dependent
cytotoxicity were not affected by the presence of bone marrow stromal cells,
indicating that daratumumab can effectively kill MM tumor cells in a
tumor-preserving bone marrow microenvironment. In vivo, daratumumab was highly
active and interrupted xenograft tumor growth at low dosing. Collectively, our
results show the versatility of daratumumab to effectively kill CD38-expressing
tumor cells, including patient MM cells, via diverse cytotoxic mechanisms. These
findings support clinical development of daratumumab for the treatment of
CD38-positive MM tumors. Multiple myeloma (MM) has been mostly incurable due to its highly complex and
heterogeneous molecular abnormalities and the support from myeloma
microenvironment factors. A therapeutic strategy which effectively targets
relevant and specific molecule to myeloma cells, and which is potent in
overcoming tumor microenvironment-mediated drug resistance needs to be
developed. One of the promising fields is the development of immunotherapy using
monoclonal antibodies (MoAbs) against myeloma-specific antigens. This review
focuses on the basic and clinical aspects of two emerging and promising novel
MoAbs for MM, elotuzumab which targets CS1 and daratumumab which targets CD38.
Both antigens are relatively specific to myeloma cells and expressed in more
than 90% of MM patients, and mediate adhesion of myeloma cells to bone marrow
stromal cells. We also discuss the unique characteristics of the two MoAbs by
comparing with other MoAbs being developed for MM. Conflict of interest statement: Conflicts of interest:EMO: Consultancy: Onyx;
Bristol Myers Squibb; Array Pharmaceuticals. Research Funding: Celgene; Onyx;
Pharmamar; Array Pharmaceuticals. PGR. Consultancy: Celgene; Millennium Takeda;
Johnson & Johnson; Novartis; Bristol Myer Squibb. Research Funding: Celgene and
Millenium. SVR: No conflicts to disclose. AP: Consultancy & Honoraria: Amgen;
Bristol Myers Squibb; Celgene; Janssen-Cilag; Millennium; ONYX. MVM:
Consultancy: Janssen-Cilag; Celgene; Millennium. RO: Consultancy: Abbott
Laboratories; Centocor Ortho Biotech; Cephalon; Millennium; Novartis; Onyx.
Research Funding: Celgene; Johnson and Johnson; Millennium; Onyx. SK:
Consultancy: Millennium; Celgene; Onyx. Research Funding: Celgene; Millennium;
Novartis; Celphalon; Sanofi; Onyx. SU: Consultancy: Celgene. Honoraria: Celgene;
Onyx. Research Funding: Celgene; Onyx; Millennium. DR: Honoraria: Amgen.
Research Funding: Eli Lilly. RN: Consultancy: Onyx; Millennium; Celgene.
Honoraria: Onyx; Millennium; Celgene. Research Funding: Onyx; Millennium;
Celgene. HE: Consultancy: Celgene; Janssen. Honoraria: Celgene; Janssen.
Research Funding: Celgene; Janssen. KCA: Consultancy: Gilead; Sanofi-Aventis;
Onyx; Celgene. Stock Ownership; Acetylon; Oncoprep. MAD: Consultancy: Celgene;
Ortho Biotech. Honoraria: Celgene; Ortho Biotech. Research Funding: Celgene. HA:
Honoraria: Celgene; Janssen; Onyx. UHM: Honoraria: Celgene; Janssen-Cilag. IT:
No conflicts to disclose. GM: Consultancy: Millennium-Takeda; Neotype.
Honoraria: Millennium-Takeda; Pfizer. RS: No conflicts to disclose. PM:
Consultancy: Celgene; Janssen. Honoraria: Celgene; Janssen. PLB: Honoraria:
Onyx. CSC: No conflicts to disclose. JJL: Honoraria: Celgene. Research Funding:
Celgene; Janssen-Cilag. JS; Research Funding: Janssen-Cilag; Celgene; Onyx. AR:
Consultancy: Celgene. Research Funding: Celgene; Bristol Myers Squibb;
Millennium; Astra Zeneca; Onyx. JM: Research Funding: Celgene; Onyx; Sanofi. SZ:
Research Funding: Celgene; Janssen-Cilag; Millennium. SL: Consultancy: Celgene;
Millennium; Novartis; Bristol Myers Squibb; Onyx; Janssen-Cilag. RC:
Consultancy: Millennium. Research Funding: Millennium; Prothena Biotech. WJC:
Honoraria: Janssen; Celgene; Novartis. Research Funding: Celgene; Roche. PM:
Consultancy: Celgene; Janssen; Millennium. Honoraria: Celgene; Janssen. PS:
Research Funding: Janssen-Cilag; Celgene; Onyx. HL: Honoraria: Celgene; Mundi
Pharma; Janssen-Cilag. Research Funding: Celgene; Mundi-Pharma; Janssen-Cilag.
BD: Honoraria: Celgene Corporation; Onyx Pharmaceutical; Millennium
Pharmaceutical, The Takeda Company. JFSM: Consultancy & Honoraria:
Janssen-Cilag; Millennium; Celgene; Onyx; Novartis; Bristol Myers Squibb Multiple myeloma (MM) remains incurable despite important recent advances in
treatment due to its inherent resistance, characterized by highly complex and
heterogeneous molecular abnormalities, as well as the support from myeloma bone
marrow (BM) microenvironment. A novel therapeutic strategy that effectively
targets specific molecules on myeloma cells and also potentially overcomes tumor
microenvironment-mediated drug resistance and the downstream effects of genetic
instability is thus urgently needed. Over the last 2 years, an anti-CD38
monoclonal antibody daratumumab (DARA) has emerged as a breakthrough targeted
therapy for patients with MM. Early-stage clinical trials have found DARA to be
safe and to have encouraging clinical activity as a single agent and in
combination with lenalidomide in heavily pretreated, relapsed patients in whom
other novel agents (such as bortezomib, thalidomide and lenalidomide) as well as
stem cell transplant has already failed. DARA may, therefore, be the first mAb
with significant anti-MM activity both as a monotherapy and in combination. It
is currently being further evaluated both alone and in combination with
conventional and novel anti-MM agents as part of prospective clinical trials.
This review discusses the preclinical and clinical development of DARA, its
pathophysiological basis, and its prospects for future use in MM. The diagnosis and treatment of multiple myeloma (MM) are progressing
continuously. This article aims at summarizing the current status in the
diagnosis and treatment of MM, emphasizing a clinical point of view. Prognostic
factors can be determined by clinical parameters, molecular analyses and patient
characteristics (e.g. age and comorbidities). The international staging system
(ISS) and cytogenetics, such as the high-risk aberrations 17p deletion,
translocation (4;14) and insertion 1q21 > 2 copies, are key factors in risk
stratification of MM patients. Induction therapy based on novel agents, namely
bortezomib, followed by subsequent high-dose melphalan and autologous stem cell
transplantation is considered the standard of care for younger, newly diagnosed
MM patients (≤ 70 years). Transplant-ineligible patients should receive
thalidomide or bortezomib-based chemotherapy. The combination of bortezomib,
melphalan and prednisone (VMP) was shown to significantly improve overall
survival (OS) compared to melphalan and prednisone (MP, 56.4 vs. 43.1 months,
p = < 0.01). Recent results suggest that lenalidomide-based therapy not
incorporating alkylating agents might be a competitive alternative with a
favorable toxicity profile for transplant-ineligible patients. Maintece
therapies are of increasing clinical significance in MM as they have the ability
to prolong overall survival; however, thalidomide maintece therapy should not
be used in MM patients with high-risk cytogenetics as it shortens OS. Refractory
or relapsed MM treatment continues to improve with the development of second and
third generation immunomodulatory agents and proteasome inhibitors. For example,
pomalidomide and dexamethasone vs. high-dose dexamethasone significantly
improved OS (12.7 vs. 8.1 months, p = 0.03). Novel therapy strategies include
targeted and stroma-directed approaches. Antibodies targeting CS-1 (elotuzumab)
and CD38 (daratumumab) in particular are currently undergoing advanced clinical
phase II/III trials. Multiple myeloma is the second most common hematologic maligcy in the US.
Treatments utilizing alkylating agents, corticosteroids, proteasome inhibitors,
and immunomodulatory drugs have resulted in significant survival benefits,
however, despite the advances, relapse is inevitable. Decreased depth and
duration of response obtained with each successive relapse of disease is typical
of the disease course, thereby highlighting a continuing need for new treatment
options. With the introduction of monoclonal antibodies for multiple myeloma,
new options for treatment in the relapsed setting are on the horizon. Among the
new immunologic agents is daratumumab (DARA), a humanized antibody to CD38 with
potent multifaceted antitumor activity. Phase I and II clinical trials have
demonstrated significant reduction in serum M-protein and bone marrow plasma
cell percentage in refractory patients, with an acceptable toxicity profile.
Moreover, ex vivo studies have shown that DARA may be particularly useful in
combination with currently used anti-myeloma agents. With a recent breakthrough
drug designation by the US Food and Drug Administration, DARA shows promise as
mono- and combination therapy for the treatment of relapsed/refractory multiple
myeloma. Multiple myeloma (MM) remains mostly incurable despite the recent progress in
the treatment strategy. One of novel fields for anti-MM therapeutic strategy is
the development of immunotherapy using monoclonal antibodies (MoAbs) against
myeloma-specific antigens. This article focuses on the basic and clinical
aspects of several emerging and promising novel MoAbs for MM, such as elotuzumab
which targets CS1 and daratumumab which targets CD38. Both antigens are highly
expressed in more than 90% of MM patients, and the clinical trials have shown
promising anti-MM effects, especially in combination with immunomodulatory agent
lenalidomide. We also discuss the characteristics and the results of clinical
trials of other MoAbs, such as tabalumab against B cell activating factor or
dacetuzumab against CD40, being developed for MM. Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical
development for the treatment of multiple myeloma (MM). The potential for IgG1
antibodies to induce macrophage-mediated phagocytosis, in combination with the
known presence of macrophages in the tumor microenvironment in MM and other
hematological tumors, led us to investigate the contribution of
antibody-dependent, macrophage-mediated phagocytosis to DARA's mechanism of
action. Live cell imaging revealed that DARA efficiently induced
macrophage-mediated phagocytosis, in which individual macrophages rapidly and
sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse
and human macrophages was also observed in an in vitro flow cytometry assay,
using a range of MM and Burkitt's lymphoma cell lines. Phagocytosis contributed
to DARA's anti-tumor activity in vivo, in both a subcutaneous and an intravenous
leukemic xenograft mouse model. Finally, DARA was shown to induce
macrophage-mediated phagocytosis of MM cells isolated from 11 of 12 MM patients
that showed variable levels of CD38 expression. In summary, we demonstrate that
phagocytosis is a fast, potent and clinically relevant mechanism of action that
may contribute to the therapeutic activity of DARA in multiple myeloma and
potentially other hematological tumors. BACKGROUND: Daratumumab (DARA), a promising novel therapy for multiple myeloma,
is an IgG1κ monoclonal antibody that recognizes CD38 on myeloma cells. During
routine compatibility testing, we observed that the plasma of five of five
DARA-treated patients demonstrated a positive antibody screen and panreactivity
on red blood cell (RBC) panel testing. We hypothesized that the observed
panreactivity reflected DARA binding to CD38 on reagent RBCs, and we
investigated methods to prevent this binding.
STUDY DESIGN AND METHODS: DARA binding to CD38+ or CD38- HL60 cells was assessed
by flow cytometry. To remove cell surface CD38, cells were incubated with
dithiothreitol (DTT) or trypsin. Soluble CD38 or anti-DARA was used to
neutralize DARA in solution. Routine blood bank serologic methods were used to
test samples from DARA-treated patients and normal plasma samples spiked with
DARA and/or alloantibodies.
RESULTS: Normal plasma samples spiked with DARA (0.1-10 µg/mL) and incubated
with reagent RBCs recapitulated the interference observed with samples from
DARA-treated patients. Flow cytometry experiments confirmed DARA binding to
CD38+ HL60 cells, but not to CD38- controls. DTT treatment of CD38+ HL60 cells
reduced DARA binding by 92% by denaturing cell surface CD38. Treating
DARA-containing plasma with soluble CD38 or anti-DARA idiotype also inhibited
DARA binding.
CONCLUSION: DARA causes panreactivity in vitro by binding to CD38 on reagent
RBCs. Treating reagent RBCs with DTT is a robust method to negate the DARA
interference, enabling the safe provision of blood to DARA-treated patients.
Because DTT denatures Kell antigens, K- units are provided to these patients. No standard chemotherapy regimens have been defined yet for extranodal natural
killer/T cell lymphoma (ENKTL), and the prognosis of patients with advanced or
relapsed disease is very poor. Daratumumab, an investigated anti-cancer drug
targeting CD38, has been of great interest in the treatment of CD38-expressing
maligcies, especially multiple myeloma. In this study, we reviewed the
clinical data of 94 patients with ENKTL, investigated the expression of CD38,
and analyzed the prognostic value of CD38 expression. Forty-seven patients had
weak expression of CD38, and the other 47 patients had strong expression. The
complete response (CR) rate was significantly higher in patients who were
treated with asparaginase-based therapy (83.8 vs. 59.6 %, p = 0.025). There was
a trend towards higher CR rate in CD38 weak expression group (78.7 vs. 59.6 %,
p = 0.074). At a median follow-up time of 42 months, the 2-year and 5-year
progression-free survival (PFS) rates were 53.0 and 39.0 %, respectively, and
the 2-year and 5-year overall survival (OS) rates were 68.0 and 58.0 %,
respectively. In multivariate survival analysis including CD38 expression
status, International Prognostic Index (IPI) score, local tumor invasion, and
chemotherapy regimens, it was found that strong expression of CD38 and
non-asparaginase-based chemoregimens were independent adverse prognostic factors
for PFS (p = 0.009 and 0.027, respectively), while local tumor invasion and
higher IPI score were independent adverse prognostic factors for OS (p = 0.002
and 0.035, respectively). In subgroup analysis, strong expression of CD38
significantly correlated with inferior survival outcomes in patients without
local tumor invasion (p = 0.011) or with stage I-II disease (p = 0.008). In
conclusion, we firstly found that the majority of ENKTL cases were CD38
positive, with half had strong expression of CD38, which significantly
correlated with poor outcomes, indicating the potential role of CD38 as a
therapy target for ENKTL. Author information:
(1)Jerome Lipper Multiple Myeloma Center, Division of Hematologic Maligcy,
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical
School, Boston, Massachusetts.
(2)Jerome Lipper Multiple Myeloma Center, Division of Hematologic Maligcy,
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical
School, Boston, Massachusetts. [email protected]. Monoclonal antibodies (mAb) have had tremendous success in treating a variety of
cancers over the past twenty years. Yet despite their widespread clinical use,
which includes treatments for haematological maligcies, there are still no
approved mAb therapies for multiple myeloma (MM). This is likely to change
within the next few years with a number of mAb therapies being assessed in late
stage clinical trials, most notably, the anti-CS-1 mAb, elotuzumab, and the
anti-CD38 mAb, daratumumab, which are currently being evaluated in Phase III
clinical trials for MM. In this review, we will discuss the preclinical and
clinical development of MDX-1097, a Phase II candidate which targets cell
membrane-associated kappa immunoglobulin free light chains expressed on the
surface of MM cells. Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against
multiple myeloma (MM) cells, including ADCC (antibody-dependent cellular
cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked
heterogeneity of response to daratumumab therapy in MM, we investigated
determits of the sensitivity of MM cells toward daratumumab-mediated ADCC and
CDC. In bone marrow samples from 144 MM patients, we observed no difference in
daratumumab-mediated lysis between newly diagnosed or relapsed/refractory
patients. However, we discovered, next to an expected effect of effector
(natural killer cells/monocytes) to target (MM cells) ratio on ADCC, a
significant association between CD38 expression and daratumumab-mediated ADCC
(127 patients), as well as CDC (56 patients). Similarly, experiments with
isogenic MM cell lines expressing different levels of CD38 revealed that the
level of CD38 expression is an important determit of daratumumab-mediated
ADCC and CDC. Importantly, all-trans retinoic acid (ATRA) increased CD38
expression levels but also reduced expression of the complement-inhibitory
proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted
in a significant enhancement of the activity of daratumumab in vitro and in a
humanized MM mouse model as well. Our results provide the preclinical rationale
for further evaluation of daratumumab combined with ATRA in MM patients. BACKGROUND: Monoclonal antibodies (MoAbs) are increasingly integrated in the
standard of care. The notion that therapeutic MoAbs can interfere with clinical
laboratory tests is an emerging concern that requires immediate recognition and
the development of appropriate solutions. Here, we describe that treatment of
multiple myeloma patients with daratumumab, a novel anti-CD38 MoAb, resulted in
false-positive indirect antiglobulin tests (IATs) for all patients for 2 to 6
months after infusion. This precluded the correct identification of irregular
blood group antibodies for patients requiring blood transfusion.
STUDY DESIGN AND METHODS: The IAT was performed using three- and 11-donor-cell
panels. Interference of daratumumab and three other anti-CD38 MoAbs was studied
using fresh-frozen plasma spiked with different MoAb concentrations.
Additionally it was tested whether two potentially neutralizing agents,
anti-idiotype antibody and recombit soluble CD38 (sCD38) extracellular
domain, were able to inhibit the interference.
RESULTS: The CD38 MoAbs caused agglutination in the IAT in a dose-dependent
manner. Addition of an excess of anti-idiotype antibodies or sCD38 protein to
the test abrogated CD38 MoAb interference and successfully restored irregular
antibody screening and identification.
DISCUSSION: CD38 MoAb therapy causes false-positive results in the IAT. The
reliability of the test could be restored by adding a neutralizing agent against
the CD38 MoAb to the patient's plasma. This study emphasizes that during drug
development, targeted therapeutics should be investigated for potential
interference with laboratory tests. Clinical laboratories should be informed
when patients receive MoAb treatments and matched laboratory tests to prevent
interference should be employed. Despite the recent major advancement in therapy for multiple myeloma, it remains
an incurable disease. There remains an unmet need for novel therapies that
target different mechanisms of action. Immunotherapy with monoclonal antibodies
is a promising area of development and will expand our therapeutic armamentarium
in the fight against myeloma. Daratumumab is a novel, high-affinity, therapeutic
human monoclonal antibody against unique CD38 epitope with broad-spectrum
killing activity. It has a favorable safety profile as monotherapy in patients
with relapsed/refractory myeloma and also demonstrates significant single-agent
activity. Abundant preclinical data supports its use in combination therapy and
clinical studies on various exciting combinations are underway. This review
focuses on the CD38 antigen and its targeting with daratumumab and provides an
update on the results of recent clinical studies involving daratumumab. Monoclonal antibodies (mAbs) are currently the most investigated therapeutic
compounds in oncology, but there is no monoclonal antibody approved in the
treatment of multiple myeloma (MM). Nevertheless several really promising
molecules are under investigation in phase III clinical trials. Domitly
daratumumab (anti-CD38) and elotuzumab (anti-CS1) showed extraordinary
effectiveness in phase I/II trials. The toxicity was acceptable which is
important for their addition to standard anti-myeloma agents like proteasome
inhibitors or immunomodulatory drugs. Monoclonal antibodies such as denosumab
(anti-RANKL) or BHQ880 (anti-DKK-1) are investigated also in the management of
myeloma bone disease. This review is focused on the most promising mAbs, their
mechanisms of action and the rationale of use. Practically all available results
have been described. If the ongoing trials confirm the efficacy and safety of
mAbs, they would become an important part of MM treatment that would be
translated in the further improvement of therapeutic outcomes. Immunotherapeutic strategies are emerging as promising therapeutic approaches in
multiple myeloma (MM), with several monoclonal antibodies in advanced stages of
clinical development. Of these agents, CD38-targeting antibodies have marked
single agent activity in extensively pretreated MM, and preliminary results from
studies with relapsed/refractory patients have shown enhanced therapeutic
efficacy when daratumumab and isatuximab are combined with other agents.
Furthermore, although elotuzumab (anti-SLAMF7) has no single agent activity in
advanced MM, randomized trials in relapsed/refractory MM have demonstrated
significantly improved progression-free survival when elotuzumab is added to
lenalidomide-dexamethasone or bortezomib-dexamethasone. Importantly, there has
been no significant additive toxicity when these monoclonal antibodies are
combined with other anti-MM agents, other than infusion-related reactions
specific to the therapeutic antibody. Prevention and management of infusion
reactions is important to avoid drug discontinuation, which may in turn lead to
reduced efficacy of anti-MM therapy. Therapeutic antibodies interfere with
several laboratory tests. First, interference of therapeutic antibodies with
immunofixation and serum protein electrophoresis assays may lead to
underestimation of complete response. Strategies to mitigate interference, based
on shifting the therapeutic antibody band, are in development. Furthermore,
daratumumab, and probably also other CD38-targeting antibodies, interfere with
blood compatibility testing and thereby complicate the safe release of blood
products. Neutralization of the therapeutic CD38 antibody or CD38 denaturation
on reagent red blood cells mitigates daratumumab interference with transfusion
laboratory serologic tests. Finally, therapeutic antibodies may complicate flow
cytometric evaluation of normal and neoplastic plasma cells, since the
therapeutic antibody can affect the availability of the epitope for binding of
commercially available diagnostic antibodies. In the last few weeks, the FDA approved three new therapies for multiple
myeloma: ixazomib, the first oral proteasome inhibitor; and daratumumab and
elotuzumab, two monoclonal antibodies that target CD38 and SLAMF7, respectively. BACKGROUND: New treatment options are needed for patients with multiple myeloma
that is refractory to proteasome inhibitors and immunomodulatory drugs. We
assessed daratumumab, a novel CD38-targeted monoclonal antibody, in patients
with refractory multiple myeloma.
METHODS: In this open-label, multicentre, phase 2 trial done in Canada, Spain,
and the USA, patients (age ≥18 years) with multiple myeloma who were previously
treated with at least three lines of therapy (including proteasome inhibitors
and immunomodulatory drugs), or were refractory to both proteasome inhibitors
and immunomodulatory drugs, were randomly allocated in a 1:1 ratio to receive
intravenous daratumumab 8 mg/kg or 16 mg/kg in part 1 stage 1 of the study, to
decide the dose for further assessment in part 2. Patients received 8 mg/kg
every 4 weeks, or 16 mg/kg per week for 8 weeks (cycles 1 and 2), then every 2
weeks for 16 weeks (cycles 3-6), and then every 4 weeks thereafter (cycle 7 and
higher). The allocation schedule was computer-generated and randomisation, with
permuted blocks, was done centrally with an interactive web response system. In
part 1 stage 2 and part 2, patients received 16 mg/kg dosed as in part 1 stage
1. The primary endpoint was overall response rate (partial response [PR] + very
good PR + complete response [CR] + stringent CR). All patients who received at
least one dose of daratumumab were included in the analysis. The trial is
registered with ClinicalTrials.gov, number NCT01985126.
FINDINGS: The study is ongoing. In part 1 stage 1 of the study, 18 patients were
randomly allocated to the 8 mg/kg group and 16 to the 16 mg/kg group. Findings
are reported for the 106 patients who received daratumumab 16 mg/kg in parts 1
and 2. Patients received a median of five previous lines of therapy (range
2-14). 85 (80%) patients had previously received autologous stem cell
transplantation, 101 (95%) were refractory to the most recent proteasome
inhibitors and immunomodulatory drugs used, and 103 (97%) were refractory to the
last line of therapy. Overall responses were noted in 31 patients (29.2%, 95% CI
20.8-38.9)-three (2.8%, 0.6-8.0) had a stringent CR, ten (9.4%, 4.6-16.7) had a
very good PR, and 18 (17.0%, 10.4-25.5) had a PR. The median time to first
response was 1.0 month (range 0.9-5.6). Median duration of response was 7.4
months (95% CI 5.5-not estimable) and progression-free survival was 3.7 months
(95% CI 2.8-4.6). The 12-month overall survival was 64.8% (95% CI 51.2-75.5)
and, at a subsequent cutoff, median overall survival was 17.5 months (95% CI
13.7-not estimable). Daratumumab was well tolerated; fatigue (42 [40%] patients)
and anaemia (35 [33%]) of any grade were the most common adverse events. No
drug-related adverse events led to treatment discontinuation.
INTERPRETATION: Daratumumab monotherapy showed encouraging efficacy in heavily
pretreated and refractory patients with multiple myeloma, with a favourable
safety profile in this population of patients.
FUNDING: Janssen Research & Development. Daratumumab is a fully human anti-CD38 IgG1-κ monoclonal antibody (mAb)
currently being evaluated in several Phase 2 and 3 clinical studies for the
treatment of multiple myeloma (MM). In this clinical case study we demonstrate
that daratumumab can be detected as an individual monoclonal band in serum
immunofixation electrophoresis (IFE). M-protein follow-up by IFE is part of the
International Myeloma Working Group (IMWG) criteria to assess treatment
response. Therefore, it is crucial that the daratumumab band is not confused
with the endogenous M-protein of the patient during IFE interpretation.
Moreover, a significant number of IgG-κ M-proteins co-migrate with daratumumab.
Co-migration introduces a bias in the M-protein quantification since
pharmacokinetic studies show that daratumumab peak plasma concentrations reach
up to 1 g/L. More importantly, co-migration can mask clearance of the M-protein
by IFE which is necessary for classification of complete response by IMWG
criteria (negative serum IFE). For optimal M-protein monitoring the laboratory
specialist needs to be informed when patients receive daratumumab, and it is
essential that the laboratory specialist is aware that a slow migrating band in
the γ-region in those patients may be derived from the daratumumab. A
daratumumab specific IFE reflex assay (DIRA) has been developed and can be
utilized to abrogate interference. The here described mAb interference is not
limited to daratumumab, and as therapeutic antibodies gain approval and enter
into common clinical practice, laboratory specialists will need additional
processes to characterize IFE interference and distinguish endogenous M-protein
from therapeutic antibodies. INTRODUCTION: Monoclonal antibodies mark the beginning of a new era in the
context of multiple myeloma (MM) treatment. Numerous antibodies have been tested
or are currently in development for patients with MM, in order to improve
tolerability and quality of life.
AREAS COVERED: This manuscript reviews emerging antibodies for the treatment of
MM i.e. elotuzumab, daratumumab, MOR03087, isatuximab, bevacizumab, cetuximab,
siltuximab, tocilizumab, elsilimomab, azintrel, rituximab, tositumomab,
milatuzumab, lucatumumab, dacetuzumab, figitumumab, dalotuzumab, AVE1642,
tabalumab, pembrolizumab, pidilizumab, nivolumab.
EXPERT OPINION: Amongst these antibodies, elotuzumab which targets SLAMF-7 and
daratumumab which targets CD38, have been recently approved by FDA for patients
with relapsed/refractory MM. Both agents are well tolerated. Multiple clinical
trials incorporating these monoclonal antibodies in MM treatment are currently
ongoing. Of special interest are the anticipated results of phase III clinical
trials with elotuzumab [NCT0189164; NCT01335399; NCT02495922] and daratumumab
[NCT02252172; NCT02195479] in newly diagnosed MM patients. Moreover, of great
interest are the awaited data on pembrolizumabin combination with pomalidomide
and dexamethasone in refractory/relapsed MM patients [NCT02576977] and in
combination with lenalidomide and dexamethasone in newly diagnosed MM patients.
It seems that the incorporation of monoclonal antibodies will change the
landscape of myeloma therapy in the near future. Daratumumab targets CD38-expressing myeloma cells through a variety of
immune-mediated mechanisms (complement-dependent cytotoxicity,
antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular
phagocytosis) and direct apoptosis with crosslinking. These mechanisms may also
target nonplasma cells that express CD38, which prompted evaluation of
daratumumab's effects on CD38-positive immune subpopulations. Peripheral blood
(PB) and bone marrow (BM) from patients with relapsed/refractory myeloma from 2
daratumumab monotherapy studies were analyzed before and during therapy and at
relapse. Regulatory B cells and myeloid-derived suppressor cells, previously
shown to express CD38, were evaluated for immunosuppressive activity and
daratumumab sensitivity in the myeloma setting. A novel subpopulation of
regulatory T cells (Tregs) expressing CD38 was identified. These Tregs were more
immunosuppressive in vitro than CD38-negative Tregs and were reduced in
daratumumab-treated patients. In parallel, daratumumab induced robust increases
in helper and cytotoxic T-cell absolute counts. In PB and BM, daratumumab
induced significant increases in CD8(+):CD4(+) and CD8(+):Treg ratios, and
increased memory T cells while decreasing naïve T cells. The majority of
patients demonstrated these broad T-cell changes, although patients with a
partial response or better showed greater maximum effector and helper T-cell
increases, elevated antiviral and alloreactive functional responses, and
significantly greater increases in T-cell clonality as measured by T-cell
receptor (TCR) sequencing. Increased TCR clonality positively correlated with
increased CD8(+) PB T-cell counts. Depletion of CD38(+) immunosuppressive cells,
which is associated with an increase in T-helper cells, cytotoxic T cells,
T-cell functional response, and TCR clonality, represents possible additional
mechanisms of action for daratumumab and deserves further exploration. BACKGROUND: Daratumumab, a human IgGκ monoclonal antibody that targets CD38,
induces direct and indirect antimyeloma activity and has shown substantial
efficacy as monotherapy in heavily pretreated patients with multiple myeloma, as
well as in combination with bortezomib in patients with newly diagnosed multiple
myeloma.
METHODS: In this phase 3 trial, we randomly assigned 498 patients with relapsed
or relapsed and refractory multiple myeloma to receive bortezomib (1.3 mg per
square meter of body-surface area) and dexamethasone (20 mg) alone (control
group) or in combination with daratumumab (16 mg per kilogram of body weight)
(daratumumab group). The primary end point was progression-free survival.
RESULTS: A prespecified interim analysis showed that the rate of
progression-free survival was significantly higher in the daratumumab group than
in the control group; the 12-month rate of progression-free survival was 60.7%
in the daratumumab group versus 26.9% in the control group. After a median
follow-up period of 7.4 months, the median progression-free survival was not
reached in the daratumumab group and was 7.2 months in the control group (hazard
ratio for progression or death with daratumumab vs. control, 0.39; 95%
confidence interval, 0.28 to 0.53; P<0.001). The rate of overall response was
higher in the daratumumab group than in the control group (82.9% vs. 63.2%,
P<0.001), as were the rates of very good partial response or better (59.2% vs.
29.1%, P<0.001) and complete response or better (19.2% vs. 9.0%, P=0.001). Three
of the most common grade 3 or 4 adverse events reported in the daratumumab group
and the control group were thrombocytopenia (45.3% and 32.9%, respectively),
anemia (14.4% and 16.0%, respectively), and neutropenia (12.8% and 4.2%,
respectively). Infusion-related reactions that were associated with daratumumab
treatment were reported in 45.3% of the patients in the daratumumab group; these
reactions were mostly grade 1 or 2 (grade 3 in 8.6% of the patients), and in
98.2% of these patients, they occurred during the first infusion.
CONCLUSIONS: Among patients with relapsed or relapsed and refractory multiple
myeloma, daratumumab in combination with bortezomib and dexamethasone resulted
in significantly longer progression-free survival than bortezomib and
dexamethasone alone and was associated with infusion-related reactions and
higher rates of thrombocytopenia and neutropenia than bortezomib and
dexamethasone alone. (Funded by Janssen Research and Development;
ClinicalTrials.gov number, NCT02136134.). Author information:
(1)Janssen Research & Development, LLC, Raritan, New Jersey, USA.
(2)Janssen Research & Development, LLC, Spring House, Pennsylvania, USA.
(3)Department of Hematology and Medical Oncology, Winship Cancer Institute,
Emory University, Atlanta, Georgia, USA.
(4)VU University Medical Center, Amsterdam, The Netherlands.
(5)Division of Hematology/Oncology, Lineberger Comprehensive Cancer Center,
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
(6)Vejle Hospital and University of Southern Denmark, Vejle, Denmark.
(7)Janssen Research & Development, Beerse, Belgium. |
Which gene-defect causes the Vel-blood type? | A cohort of 70 Vel- individuals was found to be uniformly homozygous for a 17 nucleotide deletion in the coding sequence of SMIM | Here, we report the biochemical and genetic basis of the Vel blood group
antigen, which has been a vexing mystery for decades, especially as anti-Vel
regularly causes severe haemolytic transfusion reactions. The protein carrying
the Vel blood group antigen was biochemically purified from red blood cell
membranes. Mass spectrometry-based de novo peptide sequencing identified this
protein to be small integral membrane protein 1 (SMIM1), a previously
uncharacterized single-pass membrane protein. Expression of SMIM1 cDNA in Vel-
cultured cells generated anti-Vel cell surface reactivity, confirming that SMIM1
encoded the Vel blood group antigen. A cohort of 70 Vel- individuals was found
to be uniformly homozygous for a 17 nucleotide deletion in the coding sequence
of SMIM1. The genetic homogeneity of the Vel- blood type, likely having a common
origin, facilitated the development of two highly specific DNA-based tests for
rapid Vel genotyping, which can be easily integrated into blood group genotyping
platforms. These results answer a 60-year-old riddle and provide tools of
immediate assistance to all clinicians involved in the care of Vel- patients. |
Is NADPH oxidase 5 expressed in rodents? | No, NADPH oxidase 5 is not expressed in rodents, because the gene is absent. | BACKGROUND: NADPH-oxidases (Nox) and the related Dual oxidases (Duox) play
varied biological and pathological roles via regulated generation of reactive
oxygen species (ROS). Members of the Nox/Duox family have been identified in a
wide variety of organisms, including mammals, nematodes, fruit fly, green
plants, fungi, and slime molds; however, little is known about the molecular
evolutionary history of these enzymes.
RESULTS: We assembled and analyzed the deduced amino acid sequences of 101
Nox/Duox orthologs from 25 species, including vertebrates, urochordates,
echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In
contrast to ROS defense enzymes, such as superoxide dismutase and catalase that
are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared
later in evolution. Molecular taxonomy revealed seven distinct subfamilies of
Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies
diverged early and are the most widely distributed in biology. Subunit-regulated
Noxes represent a second major subdivision, and appeared first in fungi and
amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in
gravity perception, emerged the most recently, corresponding to full-time
adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus
possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4
first appeared somewhat later in urochordates. Comparison of evolutionary
substitution rates demonstrates that Nox2, the regulatory subunits p47phox and
p67phox, and Duox are more stringently conserved in vertebrates than other Noxes
and Nox regulatory subunits. Amino acid sequence comparisons identified key
catalytic or regulatory regions, as 68 residues were highly conserved among all
Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in
variants of X-linked chronic granulomatous disease. In addition to canonical
motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were
identified as important for Nox activity, as verified by mutational analysis.
The presence of these non-canonical, but highly conserved regions suggests that
all Nox/Duox may possess a common biological function remained in a long history
of Nox/Duox evolution.
CONCLUSION: This report provides the first comprehensive analysis of the
evolution and conserved functions of Nox and Duox family members, including
identification of conserved amino acid residues. These results provide a guide
for future structure-function studies and for understanding the evolution of
biological functions of these enzymes. NADPH oxidase (Nox) enzymes are a significant source of reactive oxygen species,
which contribute to glomerular podocyte dysfunction. Although studies have
implicated Nox1, -2, and -4 in several glomerulopathies, including diabetic
nephropathy, little is known regarding the role of Nox5 in this context. We
examined Nox5 expression and regulation in kidney biopsies from diabetic
patients, cultured human podocytes, and a novel mouse model. Nox5 expression
increased in human diabetic glomeruli compared with nondiabetic glomeruli.
Stimulation with angiotensin II upregulated Nox5 expression in human podocyte
cultures and increased reactive oxygen species generation. siRNA-mediated Nox5
knockdown inhibited angiotensin II-stimulated production of reactive oxygen
species and altered podocyte cytoskeletal dynamics, resulting in an Rac-mediated
motile phenotype. Because the Nox5 gene is absent in rodents, we generated
transgenic mice expressing human Nox5 in a podocyte-specific manner
(Nox5(pod+)). Nox5(pod+) mice exhibited early onset albuminuria, podocyte foot
process effacement, and elevated systolic BP. Subjecting Nox5(pod+) mice to
streptozotocin-induced diabetes further exacerbated these changes. Our data show
that renal Nox5 is upregulated in human diabetic nephropathy and may alter
filtration barrier function and systolic BP through the production of reactive
oxygen species. These findings provide the first evidence that podocyte Nox5 has
an important role in impaired renal function and hypertension. NADPH oxidases are the major sources of reactive oxygen species in
cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5) gene is
present in humans but not rodents. Because Nox isoforms in renal proximal
tubules (RPTs) are involved in the pathogenesis of hypertension, we tested the
hypothesis that NOX5 is differentially expressed in RPT cells from normotensive
(NT) and hypertensive subjects (HT). We found that NOX5 mRNA, total NOX5
protein, and apical membrane NOX5 protein were 4.2±0.7-fold, 5.2±0.7-fold, and
2.8±0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was
4.5±0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2±0.2-fold
greater in HT than NT (P=<0.001, n=6-14/group). Ionomycin increased total NOX
and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA), effects that were
abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or
superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase
activity (-45.1±3.2% vs. mock-siRNA, n=6-8) in HT. D1-like receptor stimulation
decreased NADPH oxidase activity to a greater extent in NT (-32.5±1.8%) than HT
(-14.8±1.8). In contrast to the marked increase in expression and activity of
NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to
NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the
increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT,
relative to NT, was much less than those observed with NOX5. Thus, we
demonstrate, for the first time, that NOX5 is expressed in human RPT cells and
to greater extent than the other Nox isoforms in HT than NT. We suggest that the
increased expression of NOX5, which may be responsible for the increased
oxidative stress in RPT cells in human essential hypertension, is caused, in
part, by a defective renal dopaminergic system. |
List selective estrogen receptor degraders. | Selective estrogen receptor degraders (SERD) are fulvestrant, RAD1901 and ARN-810. Fulvestrant is the only SERD approved for the treatment of breast cancer. | It has become apparent of late that even in tamoxifen and/or aromatase resistant
breast cancers, ERα remains a bona fide therapeutic target. Not surprisingly,
therefore, there has been considerable interest in developing Selective ER
Degraders (SERDs), compounds that target the receptor for degradation.
Currently, ICI 182,780 (ICI, fulvestrant) is the only SERD approved for the
treatment of breast cancer. However, the poor pharmaceutical properties of this
injectable drug and its lack of superiority over second line aromatase
inhibitors in late stage breast cancer have negatively impacted its clinical
use. These findings have provided the impetus to develop second generation,
orally bioavailable SERDs with which quantitative turnover of ERα in tumors can
be achieved. Interestingly however, the contribution of SERD activity to
fulvestrant efficacy is unclear, making it difficult to define the
characteristics desired of the next generation of ER antagonists. It is of
significance therefore, that we have determined that the antagonist activity of
ICI and its ability to induce ERα degradation are not coupled processes.
Specifically, our results indicate that it is the ability of ICI to interact
with ERα and to (a) competitively displace estradiol and (b) induce a
conformational change in ER incompatible with transcriptional activation that
are likely to be the most important pharmacological characteristics of this
drug. Collectively, these data argue for a renewed emphasis on the development
of high affinity, orally bioavailable pure antagonists and suggest that SERD
activity though proven effective may not be required for ERα antagonism in
breast cancer. Conjugated estrogen/bazedoxifene (CE/BZA) therapy represents a new,
progestin-free treatment in the management of postmenopausal health. CE/BZA
pairs CE with the selective estrogen receptor modulator, BZA. The rationale for
the development of CE/BZA was that BZA, acting primarily as a selective estrogen
receptor degrader in uterine and breast tissue, would sufficiently inhibit the
proliferative effects of CE on the endometrium. The absence of a progestin would
reduce the incidence of uterine bleeding, breast pain and increased breast
density associated with progestin-containing hormone therapy. CE/BZA has been
evaluated in five multicenter, randomized, double-blind, placebo-controlled, and
active-controlled Phase III trials known as the SMART trials. CE/BZA has been
shown to maintain the established benefits of estrogen therapy for treatment of
vasomotor symptoms and prevention of a loss in bone mineral density (bone mass),
while minimizing certain estrogenic effects, particularly in the uterine
endometrium and breast. PURPOSE: Estrogen receptor (ER) targeting is key in management of
receptor-positive breast cancer. Currently, there are no methods to optimize
anti-ER therapy dosing. This study assesses the use of 16α-(18)F-fluoroestradiol
((18)F-FES) PET for fulvestrant dose optimization in a preclinical ER(+) breast
cancer model.
EXPERIMENTAL DESIGN: In vitro, (18)F-FES retention was compared with ERα protein
expression (ELISA) and ESR1 mRNA transcription (qPCR) in MCF7 cells (ER(+))
after treatment with different fulvestrant doses. MCF7 xenografts were grown in
ovariectomized nude mice and assigned to vehicle, low- (0.05 mg), medium- (0.5
mg), or high-dose (5 mg) fulvestrant treatment groups (5-7 per group). Two and 3
days after fulvestrant treatment, PET/CT was performed using (18)F-FES and
(18)F-FDG, respectively. ER expression was assessed by immunohistochemistry,
ELISA, and qPCR on xenografts. Tumor proliferation was assessed using Ki67
immunohistochemistry.
RESULTS: In vitro, we observed a parallel graded reduction in (18)F-FES uptake
and ER expression with increased fulvestrant doses, despite enhancement of ER
mRNA transcription. In xenografts, ER expression significantly decreased with
increased fulvestrant dose, despite similar mRNA expression and Ki67 staining
among the treatment groups. We observed a significant dose-dependent reduction
of (18)F-FES PET mean standardized uptake value (SUV(mean)) with fulvestrant
treatment but no significant difference among the treatment groups in (18)F-FDG
PET SUV(mean).
CONCLUSIONS: We demonstrated that (18)F-FES uptake mirrors the dose-dependent
changes in functional ER expression with fulvestrant resulting in ER degradation
and/or blockade; these precede changes in tumor metabolism and proliferation.
Quantitative (18)F-FES PET may be useful for tracking early efficacy of ER
blockade/degradation and guiding ER-targeted therapy dosing in patients with
breast cancer. Approximately 80% of breast cancers are estrogen receptor alpha (ER-α) positive,
and although women typically initially respond well to antihormonal therapies
such as tamoxifen and aromatase inhibitors, resistance often emerges. Although a
variety of resistance mechanism may be at play in this state, there is evidence
that in many cases the ER still plays a central role, including mutations in the
ER leading to constitutively active receptor. Fulvestrant is a steroid-based,
selective estrogen receptor degrader (SERD) that both antagonizes and degrades
ER-α and is active in patients who have progressed on antihormonal agents.
However, fulvestrant suffers from poor pharmaceutical properties and must be
administered by intramuscular injections that limit the total amount of drug
that can be administered and hence lead to the potential for incomplete receptor
blockade. We describe the identification and characterization of a series of
small-molecule, orally bioavailable SERDs which are potent antagonists and
degraders of ER-α and in which the ER-α degrading properties were prospectively
optimized. The lead compound 11l (GDC-0810 or ARN-810) demonstrates robust
activity in models of tamoxifen-sensitive and tamoxifen-resistant breast cancer,
and is currently in clinical trials in women with locally advanced or metastatic
estrogen receptor-positive breast cancer. Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains a
first-line treatment for estrogen receptor 1 (ESR1) positive breast cancer.
However, tumor resistance limits the duration of response. The clinical efficacy
of fulvestrant, a selective ER degrader (SERD) that triggers receptor
degradation, has confirmed that ESR1 often remains engaged in endocrine therapy
resistant cancers. Recently developed, selective ER modulators (SERMs)/SERD
hybrids (SSHs) that facilitate ESR1 degradation in breast cancer cells and
reproductive tissues have been advanced as an alternative treatment for advanced
breast cancer, particularly in the metastatic setting. RAD1901 is one SSH
currently being evaluated clinically that is unique among ESR1 modulators in
that it readily enters the brain, a common site of breast cancer metastasis. In
this study, RAD1901 inhibited estrogen activation of ESR1 in vitro and in vivo,
inhibited estrogen-dependent breast cancer cell proliferation and xenograft
tumor growth, and mediated dose-dependent downregulation of ESR1 protein.
However, doses of RAD1901 insufficient to induce ESR1 degradation were shown to
result in the activation of ESR1 target genes and in the stimulation of
xenograft tumor growth. RAD1901 is an SSH that exhibits complex pharmacology in
breast cancer models, having dose-dependent agonist/antagonist activity
displayed in a tissue-selective manner. It remains unclear how this unique
pharmacology will impact the utility of RAD1901 for breast cancer treatment.
However, being the only SERD currently known to access the brain, RAD1901 merits
evaluation as a targeted therapy for the treatment of breast cancer brain
metastases. Agents that inhibit estrogen production, such as aromatase inhibitors or those
that directly block estrogen receptor (ER) activity, such as selective estrogen
receptor modulators and selective estrogen receptor degraders, are routinely
used in the treatment of ER-positive breast cancers. However, although initial
treatment with these agents is often successful, many women eventually relapse
with drug-resistant breast cancers. To overcome some of the challenges
associated with current endocrine therapies and to combat the development of
resistance, there is a need for more durable and more effective ER-targeted
therapies. Here we describe and characterize a novel, orally bioavailable
small-molecule selective estrogen receptor degrader, RAD1901, and evaluate its
therapeutic potential for the treatment of breast cancer. RAD1901 selectively
binds to and degrades the ER and is a potent antagonist of ER-positive breast
cancer cell proliferation. Importantly, RAD1901 produced a robust and profound
inhibition of tumor growth in MCF-7 xenograft models. In an intracranial MCF-7
model, RAD1901-treated animals survived longer than those treated with either
control or fulvestrant, suggesting the potential benefit of RAD1901 in the
treatment of ER-positive breast cancer that has metastasized to the brain.
Finally, RAD1901 preserved ovariectomy-induced bone loss and prevented the
uterotropic effects of E2, suggesting that it may act selectively as an agonist
in bone but as an antagonist in breast and uterine tissues. RAD1901 is currently
under clinical study in postmenopausal women with ER-positive advanced breast
cancer. Traditional menopausal hormone therapy containing estrogens/progestin has been
associated with an increased risk of breast cancer, and estrogen exposure is
known to promote growth and proliferation of a majority of breast cancers.
Therefore, it is important for clinicians to consider the breast safety profile
of any hormone-based therapy used in postmenopausal women. This review provides
an overview of the breast safety and tolerability profiles of currently marketed
selective estrogen receptor modulators, antiestrogens, and the first tissue
selective estrogen complex combining conjugated estrogens with the selective
estrogen receptor modulator bazedoxifene in postmenopausal women. Selective
estrogen receptor modulators and antiestrogens act as estrogen receptor
antagonists in the breast. Tamoxifen, toremifene, and the selective estrogen
receptor degrader fulvestrant are used to treat breast cancer, and tamoxifen and
raloxifene protect against breast cancer in high-risk women. Postmenopausal
women using selective estrogen receptor modulators for prevention or treatment
of osteoporosis (raloxifene, bazedoxifene) can be reassured that these hormonal
treatments do not adversely affect their risk of breast cancer and may, in the
case of raloxifene, even be protective. There are limited data on breast cancer
in women who use ospemifene for dyspareunia. Conjugated estrogens/bazedoxifene
use for up to two years did not increase mammographic breast density or breast
pain/tenderness, and there was no evidence of an increased risk of breast
cancer, suggesting that conjugated estrogens/bazedoxifene has an improved breast
safety profile compared with traditional menopausal hormone therapies. Future
research will continue to focus on development of selective estrogen receptor
modulators and selective estrogen receptor modulator combinations capable of
achieving the ideal balance of estrogen receptor agonist and antagonist effects. Author information:
(1)Department of Pathology and Laboratory Medicine, Hollings Cancer Center, 86
Jonathan Lucas St, Charleston, SC 29425, USA; Department of Cancer Biology,
Wayne State University School of Medicine, 540 E Canfield St, Detroit, MI 48201,
USA. Electronic address: [email protected].
(2)Department of Pathology and Laboratory Medicine, Hollings Cancer Center, 86
Jonathan Lucas St, Charleston, SC 29425, USA. Electronic address:
[email protected].
(3)Department of Pathology and Laboratory Medicine, Hollings Cancer Center, 86
Jonathan Lucas St, Charleston, SC 29425, USA. Electronic address:
[email protected].
(4)Department of Pathology and Laboratory Medicine, Hollings Cancer Center, 86
Jonathan Lucas St, Charleston, SC 29425, USA. Electronic address:
[email protected].
(5)Department of Pathology and Laboratory Medicine, Hollings Cancer Center, 86
Jonathan Lucas St, Charleston, SC 29425, USA. Electronic address:
[email protected].
(6)Department of Pathology and Laboratory Medicine, Hollings Cancer Center, 86
Jonathan Lucas St, Charleston, SC 29425, USA. Electronic address:
[email protected].
(7)Department of Cancer Biology, Wayne State University School of Medicine, 540
E Canfield St, Detroit, MI 48201, USA. Electronic address: [email protected].
(8)Department of Pathology and Laboratory Medicine, Hollings Cancer Center, 86
Jonathan Lucas St, Charleston, SC 29425, USA. Electronic address:
[email protected].
(9)Department of Medicine and Public Health, Medical University of South
Carolina, 171 Ashley Ave, Charleston, SC 29425, USA. Electronic address:
[email protected].
(10)Department of Pathology and Laboratory Medicine, Hollings Cancer Center, 86
Jonathan Lucas St, Charleston, SC 29425, USA. Electronic address:
[email protected]. For women with hormone receptor-positive advanced breast cancer, endocrine
therapies, including the selective estrogen receptor modulator tamoxifen, the
aromatase inhibitors anastrozole, letrozole, and exemestane, and the selective
estrogen receptor degrader fulvestrant, are recommended in clinical guidelines.
The addition of targeted agents such as everolimus or palbociclib to aromatase
inhibitors are also recommended as treatment options. Chemotherapy remains an
option, although clinical guidelines have recommended these agents be reserved
for patients with immediately life-threatening disease or if resistance to
endocrine therapy is known or suspected. The present review has consolidated the
tolerability profiles of the agents approved for use in the treatment of hormone
receptor-positive advanced or metastatic breast cancer based on phase III
registration trial data. Endocrine therapies are generally well tolerated,
although the addition of targeted therapies to aromatase inhibitors or
fulvestrant appears to increase the proportion of patients experiencing adverse
events, and palbociclib and chemotherapy appear to be more closely associated
with serious adverse events, including neutropenia. The purpose of this study was to address the role of ESR1 hormone-binding
mutations in breast cancer. Soft agar anchorage-independent growth assay,
Western blot, ERE reporter transactivation assay, proximity ligation assay
(PLA), coimmunoprecipitation assay, silencing assay, digital droplet PCR
(ddPCR), Kaplan-Meier analysis, and statistical analysis. It is now generally
accepted that estrogen receptor (ESR1) mutations occur frequently in metastatic
breast cancers; however, we do not yet know how to best treat these patients. We
have modeled the three most frequent hormone-binding ESR1 (HBD-ESR1) mutations
(Y537N, Y537S, and D538G) using stable lentiviral transduction in human breast
cancer cell lines. Effects on growth were examined in response to hormonal and
targeted agents, and mutation-specific changes were studied using microarray and
Western blot analysis. We determined that the HBD-ESR1 mutations alter
anti-proliferative effects to tamoxifen (Tam), due to cell-intrinsic changes in
activation of the insulin-like growth factor receptor (IGF1R) signaling pathway
and levels of PIK3R1/PIK3R3. The selective estrogen receptor degrader,
fulvestrant, significantly reduced the anchorage-independent growth of ESR1
mutant-expressing cells, while combination treatments with the mTOR inhibitor
everolimus, or an inhibitor blocking IGF1R, and the insulin receptor
significantly enhanced anti-proliferative responses. Using digital drop (dd)
PCR, we identified mutations at high frequencies ranging from 12 % for Y537N,
5 % for Y537S, and 2 % for D538G in archived primary breast tumors from women
treated with adjuvant mono-tamoxifen therapy. The HBD-ESR1 mutations were not
associated with recurrence-free or overall survival in response in this patient
cohort and suggest that knowledge of other cell-intrinsic factors in combination
with ESR1 mutation status will be needed determine anti-proliferative responses
to Tam. |
What is the role of ZNF335 in microcephaly? | Znf335 null mice are embryonically lethal, and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that ZNF335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. | ZNF335 was first reported in 2012 as a causative gene for microcephaly. Because
only 1 consanguineous pedigree has ever been reported, the key clinical features
associated with ZNF335 mutations remain unknown. In this article, we describe
another family harboring ZNF335 mutations. The female proband was the first
child of nonconsanguineous Japanese parents. At birth, microcephaly was absent;
her head circumference was 32.0 cm (-0.6 SD). At 3 months, microcephaly was
noted, (head circumference, 34.0 cm [-4.6 SD]). Brain MRI showed invisible basal
ganglia, cerebral atrophy, brainstem hypoplasia, and cerebellar atrophy. At 33
months, (head circumference, 41.0 cm [-5.1 SD]), she had severe psychomotor
retardation. After obtaining informed consent from her parents, we performed
exome sequencing in the proband and identified 1 novel and 1 known mutation in
ZNF335, namely, c.1399T>C (p.C467R) and c.1505A>G (p.Y502C), respectively. The
mutations were individually transmitted by her parents, indicating that the
proband was compound heterozygous for the mutations. Her brain imaging findings,
including invisible basal ganglia, were similar to those observed in the
previous case with ZNF335 mutations. We speculate that invisible basal ganglia
may be the key feature of ZNF335 mutations. For infants presenting with both
microcephaly and invisible basal ganglia, ZNF335 mutations should be considered
as a differential diagnosis. |
What molecule is targeted by Avelumab? | Avelumab is a monoclonal antibody that binds programmed death-ligand 1 (PD-L1). | Several anti-PD-1/PD-L1 monoclonal antibodies (mAb) are currently providing
evidence of clinical benefit in subsets of cancer patients. The mode of action
of these mAbs is to inhibit PD-1 on immune cells interacting with PD-L1 on tumor
cells. These mAbs are either designed or engineered to eliminate
antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been
implicated as an important mechanism in several highly effective mAb-mediated
cancer therapies. A fully human anti-PD-L1 mAb would potentially be able to
block PD-1/PD-L1 interactions and also mediate the ADCC lysis of tumor cells.
MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 mAb. The
studies reported here demonstrate (i) the ability of avelumab to lyse a range of
human tumor cells in the presence of PBMC or NK effectors; (ii) IFNγ can enhance
tumor cell PD-L1 expression and, in some cases, enhance ADCC tumor cell lysis;
(iii) purified NK cells are potent effectors for avelumab; (iv) similar levels
of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as
effectors from either healthy donors or cancer patients; (v) very low levels of
avelumab-mediated lysis are seen using whole PBMCs as targets; this finding
complements results seen in analyses of PBMC subsets of patients receiving
avelumab; and (vi) the addition of IL12 to NK cells greatly enhances
avelumab-mediated ADCC. These studies thus provide an additional mode of action
for an anti-PD-L1 mAb and support the rationale for further studies to enhance
avelumab-mediated ADCC activity. In non-small cell lung cancer (NSCLC), the first immune checkpoint inhibitor to
be approved by the US Food and Drug Administration was nivolumab, based on a
survival advantage over docetaxel in recurrent squamous NSCLC, a
difficult-to-treat histology. In addition, several other immune checkpoint
inhibitors are also in late-stage development. Most of these agents inhibit the
programmed cell death protein 1 (PD-1) pathway, targeting either the PD-1
receptor or its ligand, programmed cell death ligand 1 (PD-L1). In addition to
nivolumab, pembrolizumab is a PD-1 inhibitor under investigation in NSCLC, and
atezolizumab (MPDL3280A), durvalumab (MEDI4736), and avelumab (MSB0010718C) are
PD-L1 inhibitors under investigation. The cytotoxic T-lymphocyte-associated
antigen 4 (CTLA-4) immune checkpoint inhibitors ipilimumab and tremelimumab are
also under investigation in NSCLC, largely as part of combination approaches
rather than as monotherapy. PD-L1 expression as a potential biomarker to select
patients most likely to respond to inhibitors of the PD-1 pathway has been
widely studied. Gastric cancer (GC) is a major world-wide health problem. It is the third
leading cause of death from cancer. The treatment of advanced GC by chemotherapy
has limited efficacy. The addition of some targeted therapies like trastuzumab
and ramucirumab have added a modest benefit, but only in human epidermal growth
factor receptor 2 (ERBB2 or HER2)-positive patients and in the second-line
setting, respectively. The development of new and effective therapeutic
strategies must consider the genetic complexity and heterogeneity of GC;
prognostic and predictive biomarkers should be identified for clinical
implementation. Immune deregulation has been associated with some GC subtypes,
especially those that are associated with virus infection and those with a high
mutational rate. Different mechanisms to prevent immunologic escape have been
characterized during the last years; in particular the PD-1/PD-L1 inhibitors
pembrolizumab, avelumab, durvalumab and atezolizumab have shown early sign of
efficacy. Therefore, immunotherapeutic strategies may provide new opportunities
for GC patients. This review will discuss (1) the main characteristics of GC
treatment, (2) the immune response in GC, and (3) the current status of
immune-related strategies in clinical development in GC patients, focusing on
immune checkpoints therapies. Bacillus Calmette-Guerin (BCG) is the standard of care for intravesical therapy
for carcinoma in situ and non-muscle invasive, nonmetastatic human urothelial
carcinoma. Although the responsiveness to this immunotherapeutic is believed to
be linked with (i) a high number of somatic mutations and (ii) a large number of
tumor-infiltrating lymphocytes, recent findings of the roles that inhibitory
immune receptors and their ligands play in tumor evasion may provide insights
into the limitations of the effectiveness of BCG and offer new targets for
immune-based therapy. In this study, an aggressive, bioluminescent orthotopic
bladder cancer model, MB49 tumor cells transfected with luciferase (MB49(luc)),
was used to study the antitumor effects of avelumab, an antibody to PD-L1.
MB49(luc) murine tumor cells form multifocal tumors on the mucosal wall of the
bladder reminiscent of non-muscle invasive, nonmetastatic urothelial carcinomas.
MB49(luc) bladder tumors are highly positive for the expression of PD-L1, and
avelumab administration induced significant (P < 0.05) antitumor effects. These
antitumor effects were more dependent on the presence of CD4 than CD8 T cells,
as determined by in vivo immune cell depletions. The findings suggest that in
this bladder tumor model, interruption of the immune-suppressive PD-1/PD-L1
complex releases a local adaptive immune response that, in turn, reduces tumor
growth. This bladder tumor model can be used to further identify host antitumor
immune mechanisms and evaluate combinations of immune-based therapies for
carcinoma in situ and non-muscle invasive, nonmetastatic urothelial carcinoma,
to provide the rationale for subsequent clinical studies. Cancer Immunol Res;
4(5); 452-62. ©2016 AACR. Chordoma, a rare bone tumor derived from the notochord, has been shown to be
resistant to conventional therapies. Checkpoint inhibition has shown great
promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb
avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1
capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of
PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential
therapy for chordoma. We examined 4 chordoma cell lines, first for expression of
PD-L1, and in vitro for ADCC killing using NK cells and avelumab. PD-L1
expression was markedly upregulated by IFN-γ in all 4 chordoma cell lines, which
significantly increased sensitivity to ADCC. Brachyury is a transcription factor
that is uniformly expressed in chordoma. Clinical trials are ongoing in which
chordoma patients are treated with brachyury-specific vaccines. Co-incubating
chordoma cells with brachyury-specific CD8+ T cells resulted in significant
upregulation of PD-L1 on the tumor cells, mediated by the CD8+ T cells' IFN-γ
production, and increased sensitivity of chordoma cells to avelumab-mediated
ADCC. Residential cancer stem cell subpopulations of chordoma cells were also
killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell
populations. These findings suggest that as a monotherapy for chordoma, avelumab
may enable endogenous NK cells, while in combination with T-cell immunotherapy,
such as a vaccine, avelumab may enhance NK-cell killing of chordoma cells via
ADCC. A new approach to the management of non-small-cell lung cancer (NSCLC) has
recently emerged that works by manipulating the immune checkpoint controlled by
programmed death receptor 1 (PD-1) and its ligand programmed death ligand 1
(PD-L1). Several drugs targeting PD-1 (pembrolizumab and nivolumab) or PD-L1
(atezolizumab, durvalumab, and avelumab) have been approved or are in the late
stages of development. Inevitably, the introduction of these drugs will put
pressure on healthcare systems, and there is a need to stratify patients to
identify those who are most likely to benefit from such treatment. There is
evidence that responsiveness to PD-1 inhibitors may be predicted by expression
of PD-L1 on neoplastic cells. Hence, there is considerable interest in using
PD-L1 immunohistochemical staining to guide the use of PD-1-targeted treatments
in patients with NSCLC. This article reviews the current knowledge about PD-L1
testing, and identifies current research requirements. Key factors to consider
include the source and timing of sample collection, pre-analytical steps (sample
tracking, fixation, tissue processing, sectioning, and tissue prioritization),
analytical decisions (choice of biomarker assay/kit and automated staining
platform, with verification of standardized assays or validation of
laboratory-devised techniques, internal and external quality assurance, and
audit), and reporting and interpretation of the results. This review addresses
the need for integration of PD-L1 immunohistochemistry with other tests as part
of locally agreed pathways and protocols. There remain areas of uncertainty, and
guidance should be updated regularly as new information becomes available. Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being
investigated for the treatment of infectious diseases and cancer, with the aim
of enhancing the function of an impaired immune system. Avelumab (MSB0010718C)
is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which
differs from other checkpoint-blocking antibodies in its ability to mediate
antibody-dependent cell-mediated cytotoxicity. These studies were conducted to
define whether avelumab could enhance the detection of antigen-specific immune
response in in vitro assays. Peripheral blood mononuclear cells from 17 healthy
donors were stimulated in vitro, with and without avelumab, with peptide pools
encoding for cytomegalovirus, Epstein-Barr virus, influenza and tetanus toxin or
the negative peptide control encoding for human leukocyte antigen. These studies
show for the first time that the addition of avelumab to an antigen-specific IVS
assay (a) increased the frequency of activated antigen-specific CD8(+) T
lymphocytes, and did so to a greater extent than that seen with commercially
available PD-L1-blocking antibodies, (b) reduced CD4(+) T-cell proliferation and
(c) induced a switch in the production of Th2 to Th1 cytokines. Moreover, there
was an inverse correlation between the enhancement of CD8(+) T-cell activation
and reduction in CD4(+) T-cell proliferation induced by avelumab. These findings
provide the rationale for the use of avelumab anti-PD-L1 in in vitro assays to
monitor patient immune responses to immunotherapies. INTRODUCTION: The functional aspects of programmed death 1 (PD-1) and PD ligand
1 (PD-L1) immune checkpoints in maligt mesothelioma have not been studied.
METHODS: Tumor samples from 65 patients with mesothelioma were evaluated for
PD-L1 expression by immunohistochemistry, and its prognostic significance was
examined. Maligt effusions from patients with pleural and peritoneal
mesothelioma were evaluated for PD-1-positive and PD-L1-positive infiltrating
lymphocytes and their role in inducing PD-L1 expression in tumor cells.
Antibody-dependent cellular cytotoxicity (ADCC) of avelumab, a fully humanized
immunoglobulin G1 anti PD-L1 antibody against primary mesothelioma cell lines,
was evaluated in presence of autologous and allogeneic natural killer cells.
RESULTS: Of 65 pleural and peritoneal mesothelioma tumors examined, 41 (63%)
were PD-L1-positive, which was associated with slightly inferior overall
survival compared to patients with PD-L1-negative tumors (median 23.0 versus
33.3 months, p = 0.35). The frequency of PD-L1 expression was similar in
patients with pleural and peritoneal mesothelioma, with 62% and 64% of samples
testing positive, respectively. In nine mesothelioma effusion samples evaluated,
the fraction of cells expressing PD-L1 ranged from 12% to 83%. In seven patients
with paired maligt effusion and peripheral blood mononuclear cell (PBMC)
samples, PD-L1 expression was significantly higher on CD3-positive T cells
present in maligt effusions as compared with PBMCs (p = 0.016). In addition,
the numbers of CD14-positive PD-1-positive cells were increased in maligt
effusions compared with PBMCs (p = 0.031). The lymphocytes present in maligt
effusions recognized autologous tumor cells and induced interferon-γ-mediated
PD-L1 expression on the tumor cell surface. Of the three primary mesothelioma
cell lines tested, two were susceptible to avelumab-mediated ADCC in the
presence of autologous natural killer cells.
CONCLUSIONS: Most pleural as well as peritoneal mesotheliomas express PD-L1.
Maligt effusions in this disease are characterized by the presence of tumor
cells and CD3-positive T cells that highly express PD-L1. In addition,
mesothelioma tumor cells are susceptible to ADCC by the anti-PD-L1 antibody
avelumab. BACKGROUND: Merkel cell carcinoma is a rare, aggressive skin cancer with poor
prognosis in patients with advanced disease. Current standard care uses various
cytotoxic chemotherapy regimens, but responses are seldom durable. Tumour
oncogenesis is linked to Merkel cell polyomavirus integration and
ultraviolet-radiation-induced mutations, providing rationale for treatment with
immunotherapy antibodies that target the PD-L1/PD-1 pathway. We assessed
treatment with avelumab, an anti-PD-L1 monoclonal antibody, in patients with
stage IV Merkel cell carcinoma that had progressed after cytotoxic chemotherapy.
METHODS: In this multicentre, international, prospective, single-group,
open-label, phase 2 trial, patients with stage IV chemotherapy-refractory,
histologically confirmed Merkel cell carcinoma (aged ≥18 years) were enrolled
from 35 cancer treatment centres and academic hospitals in North America,
Europe, Australia, and Asia. Key eligibility criteria were an ECOG performance
status of 0 or 1, measurable disease by Response Evaluation Criteria in Solid
Tumors (RECIST) version 1.1, adequate haematological, hepatic, and renal
function, and immune-competent status (patients with HIV, immunosuppression,
haematological maligcies, and previous organ transplantation were excluded).
Patient selection was not based on PD-L1 expression or Merkel cell polyomavirus
status. Collection of biopsy material or use of archival tissue for these
assessments was mandatory. Avelumab was given intravenously at a dose of 10
mg/kg every 2 weeks. The primary endpoint was confirmed objective response
(complete response or partial response) assessed according to RECIST version 1.1
by an independent review committee. Safety and clinical activity were assessed
in all patients who received at least one dose of study drug (the modified
intention-to-treat population). This trial is registered with ClinicalTrials.gov
as NCT02155647.
FINDINGS: Between July 25, 2014, and Sept 3, 2015, 88 patients were enrolled and
received at least one dose of avelumab. Patients were followed up for a median
of 10·4 months (IQR 8·6-13·1). The proportion of patients who achieved an
objective response was 28 (31·8% [95·9% CI 21·9-43·1]) of 88 patients, including
eight complete responses and 20 partial responses. Responses were ongoing in 23
(82%) of 28 patients at the time of analysis. Five grade 3 treatment-related
adverse events occurred in four (5%) patients: lymphopenia in two patients,
blood creatine phosphokinase increase in one patient, aminotransferase increase
in one patient, and blood cholesterol increase in one patient; there were no
treatment-related grade 4 adverse events or treatment-related deaths. Serious
treatment-related adverse events were reported in five patients (6%):
enterocolitis, infusion-related reaction, aminotransferases increased,
chondrocalcinosis, synovitis, and interstitial nephritis (n=1 each).
INTERPRETATION: Avelumab was associated with durable responses, most of which
are still ongoing, and was well tolerated; hence, avelumab represents a new
therapeutic option for advanced Merkel cell carcinoma.
FUNDING: Merck KGaA, Darmstadt, Germany. Near Infrared-Photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment
that employs an antibody-photo-absorber conjugate (APC). Programmed cell death
protein-1 ligand (PD-L1) is emerging as a molecular target. Here, we describe
the efficacy of NIR-PIT, using fully human IgG1 anti-PD-L1 monoclonal antibody
(mAb), avelumab, conjugated to the photo-absorber, IR700DX, in a PD-L1
expressing H441 cell line, papillary adenocarcinoma of lung. Avelumab-IR700
showed specific binding and cell-specific killing was observed after exposure of
the cells to NIR in vitro. In the in vivo study, avelumab-IR700 showed high
tumor accumulation and high tumor-background ratio. Tumor-bearing mice were
separated into 4 groups: (1) no treatment; (2) 100 μg of avelumab-IR700 i.v.;
(3) NIR light exposure only, NIR light was administered; (4) 100 μg of
avelumab-IR700 i.v., NIR light was administered. Tumor growth was significantly
inhibited by NIR-PIT treatment compared with the other groups (p < 0.001), and
significantly prolonged survival was achieved (p < 0.01 vs other groups). In
conclusion, the anti-PD-L1 antibody, avelumab, is suitable as an APC for
NIR-PIT. Furthermore, NIR-PIT with avelumab-IR700 is a promising candidate of
the treatment of PD-L1-expressing tumors that could be readily translated to
humans. Author information:
(1)CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of
Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
(2)National Institute for Viral Disease Control and Prevention, Chinese Center
for Disease Control and Prevention (China CDC), Beijing, 102206, China.
(3)College of Laboratory Medicine and Life Sciences, Wenzhou Medical University,
Wenzhou, 325035, China.
(4)College of Life Sciences, University of Chinese Academy of Sciences, Beijing,
100049, China.
(5)Research Network of Immunity and Health (RNIH), Beijing Institutes of Life
Science, Chinese Academy of Sciences, Beijing, 100101, China.
(6)ImmuFucell Biotechnology Co.Ltd., Beijing, 100102, China.
(7)CAS Key Laboratory of Microbial Physiological and Metabolic Engineering,
Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
(8)CAS Key Laboratory of Bio-medical Diagnostics, Suzhou Institute of Biomedical
Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu,
215163, China. [email protected].
(9)CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of
Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
[email protected].
(10)National Institute for Viral Disease Control and Prevention, Chinese Center
for Disease Control and Prevention (China CDC), Beijing, 102206, China.
[email protected].
(11)Research Network of Immunity and Health (RNIH), Beijing Institutes of Life
Science, Chinese Academy of Sciences, Beijing, 100101, China. [email protected].
(12)Savaid Medical School, University of Chinese Academy of Sciences, Beijing,
100049, China. [email protected]. |
What are the exonic splice enhancers? | In mammals there is a bias in amino acid usage near splice sites that is explained, in large part, by the high density of exonic splicing enhancers (ESEs) in these regions. Exonic splicing enhancers (ESEs) activate pre-mRNA splicing by promoting the use of the flanking splice sites. Some of these variants may have an effect on pre-mRNA splicing, either by altering degenerate positions of splice site sequences or by affecting intronic or exonic splicing regulatory sequences such as exonic splicing enhancers (ESEs). For inherited disease, the main mechanism responsible for the splicing defect is splice site loss, whereas for cancer the predominant mechanism of splicing disruption is predicted to be exon skipping via loss of exonic splicing enhancers or gain of exonic splicing silencer elements. FELINES was shown to be useful for characterizing branchsites, polypyrimidine tracts and 5' and 3' splice sites in the intron databases and exonic splicing enhancers (ESEs) in S.pombe exons. Unexpectedly, a fully experimental dataset identifies motifs that commonly behave opposite to the consensus, for example, being enriched in exon cores where splice-associated mutations are rare.Prior analyses that used the RESCUE-ESE set of hexamers captured the properties of consensus exonic splice enhancers. | |
What are the birth defects associated with Zika-virus infection? | Although the full spectrum of adverse reproductive outcomes caused by Zika virus infection is not yet determined, a distinctive phenotype-the congenital Zika syndrome-has emerged. Zika virus infection during pregnancy is a cause of microcephaly and other severe birth defects. | Zika virus is a mosquito-borne flavivirus discovered in Africa in 1947. Most
persons with Zika virus infection are asymptomatic; symptoms when present are
generally mild and include fever, maculopapular rash, arthralgia, and
conjunctivitis. Since early 2015, Zika virus has spread rapidly through the
Americas, with local transmission identified in 31 countries and territories as
of February 29, 2016, including several US territories. All age groups are
susceptible to Zika virus infection, including children. Maternal-fetal
transmission of Zika virus has been documented; evidence suggests that
congenital Zika virus infection is associated with microcephaly and other
adverse pregcy and infant outcomes. Perinatal transmission has been reported
in 2 cases; 1 was asymptomatic, and the other had thrombocytopenia and a rash.
Based on limited information, Zika virus infection in children is mild, similar
to that in adults. The long-term sequelae of congenital, perinatal, and
pediatric Zika virus infection are largely unknown. No vaccine to prevent Zika
virus infection is available, and treatment is supportive. The primary means of
preventing Zika virus infection is prevention of mosquito bites in areas with
local Zika virus transmission. Given the possibility of limited local
transmission of Zika virus in the continental United States and frequent travel
from affected countries to the United States, US pediatric health care providers
need to be familiar with Zika virus infection. This article reviews the Zika
virus, its epidemiologic characteristics, clinical presentation, laboratory
testing, treatment, and prevention to assist providers in the evaluation and
management of children with possible Zika virus infection. Zika virus is endemic in several parts of the world. February 1, 2016 Zika virus
was declared a public health emergency by the WHO. This declaration is mainly
due to a convincing association between Zika virus infection during pregcy
and birth defects, like microcephaly, among some of the newborns. Imported cases
of Zika virus infection to North America, Europe and Denmark have been
described. The infection in itself is mild and self-limiting. The available
diagnostic methods are under development, validation and evaluation. In Denmark,
some promising diagnostics are available at Statens Serum Institut. In January 2016, the World Health Organization warned that Zika virus is
"spreading explosively" in the Americas and that up to 4 million infections
could be present worldwide within a year. Soon thereafter, some politicians and
authors publicly advocated for quarantine of travelers returning from regions
where mosquitoes carrying Zika virus are prevalent. The public health tool of
quarantine can be used to prevent the spread of infection by restricting the
movement of persons who have been exposed to a deadly disease that can be
transmitted from person to person before symptom onset. With 80% of Zika virus
infections being asymptomatic, no rapid test being available to detect the
virus, and primary transmission being via the bites of certain mosquitoes,
application of quarantine in this setting is not scientifically sound or
practically feasible. Rather, public health interventions should focus on
preventing bites from infected mosquitoes, counseling pregt women on the
risks of fetal microcephaly and other birth defects, and identifying patients
with signs and symptoms of Guillain-Barré syndrome. As was seen in the Ebola
virus disease outbreak of 2014, non-evidence-based factors can influence policy
decisions. Public health experts must ensure that policy makers are informed
that quarantine is not a scientifically sound approach for the control of Zika
virus. (Disaster Med Public Health Preparedness. 2016;0:1-3). Prior to 2015, Zika Virus (ZIKV) outbreaks had occurred in areas of Africa,
Southeast Asia, and the Pacific Islands. Although a causal relationship between
Zika infection during pregcy and microcephaly is strongly suspected, such a
connection has not yet been scientifically proven. In May 2015, the outbreak of
ZIKV infection in Brazil led to reports of syndrome and pregt women giving
birth to babies with birth defects and poor pregcy outcomes; the Pan American
Health Organization (PAHO) issued an alert regarding the first confirmed ZIKV
infection in Brazil. Currently, ZIKV outbreaks are ongoing and it will be
difficult to predict how the virus will spread over time. ZIKV is transmitted to
humans primarily through the bite of infected mosquitos, Aedes aegypti and Aedes
albopictus. These mosquitoes are the principle vectors of dengue, and ZIKV
disease generally is reported to include symptoms associated with acute febrile
illnesses that clinically resembles dengue fever. The laboratory diagnosis can
be performed by using reverse-transcriptase polymerase chain reaction (RT-PCR)
on serum, viral nucleic acid and virus-specific immunoglobulin M. There is
currently no vaccine and antiviral treatment available for ZIKV infection, and
the only way to prevent congenital ZIKV infection is to prevent maternal
infection. In February 2016, the Taiwan Centers for Disease Control (Taiwan CDC)
activated ZIKV as a Category V Notifiable Infectious Disease similar to Ebola
virus disease and MERS. Zika virus (ZIKV), a mosquito-borne flavivirus, belongs to the Flaviviridae
family, genus Flavivirus. ZIKV was initially isolated in 1947 from a sentinel
monkey in the Zika forest, Uganda. Little clinical importance was attributed to
ZIKV, once only few symptomatic cases were reported in some African and
Southeast Asiatic countries. This situation changed in 2007, when a large
outbreak was registered on the Yap Island, Micronesia, caused by the Asian ZIKV
lineage. Between 2013 and 2014, ZIKV spread explosively and caused many
outbreaks in different islands of the Southern Pacific Ocean and in 2015
autochthonous transmission was reported in Brazil. Currently, Brazil is the
country with the highest number of ZIKV-positive cases in Latin America.
Moreover, for the first time after the discovery of ZIKV, the Brazilian
scientists are studying the possibility for the virus to cause severe congenital
infection related to microcephaly and serious birth defects due to the
time-spatial coincidence of the alarming increase of newborns with microcephaly
and the Brazilian ZIKV epidemic. The present review summarizes recent
information for ZIKV epidemiology, clinical picture, transmission, diagnosis and
the consequences of this emerging virus in Brazil. Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (family
Flaviviridae) and was first described in 1947 in Uganda following blood analyses
of sentinel Rhesus monkeys. Until the twentieth century, the African and Asian
lineages of the virus did not cause meaningful infections in humans. However, in
2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy
epidemic on the Yap Island in Micronesia. Patients experienced fever, skin rash,
arthralgia and conjunctivitis. From 2013 to 2015, the Asian lineage of the virus
caused further massive outbreaks in New Caledonia and French Polynesia. In 2013,
ZIKV reached Brazil, later spreading to other countries in South and Central
America. In Brazil, the virus has been linked to congenital malformations,
including microcephaly and other severe neurological diseases, such as
Guillain-Barré syndrome. Despite clinical evidence, direct experimental proof
showing that the Brazilian ZIKV (ZIKV(BR)) strain causes birth defects remains
absent. Here we demonstrate that ZIKV(BR) infects fetuses, causing intrauterine
growth restriction, including signs of microcephaly, in mice. Moreover, the
virus infects human cortical progenitor cells, leading to an increase in cell
death. We also report that the infection of human brain organoids results in a
reduction of proliferative zones and disrupted cortical layers. These results
indicate that ZIKV(BR) crosses the placenta and causes microcephaly by targeting
cortical progenitor cells, inducing cell death by apoptosis and autophagy, and
impairing neurodevelopment. Our data reinforce the growing body of evidence
linking the ZIKV(BR) outbreak to the alarming number of cases of congenital
brain malformations. Our model can be used to determine the efficiency of
therapeutic approaches to counteracting the harmful impact of ZIKV(BR) in human
neurodevelopment. BACKGROUND: The single-stranded RNA Flavivirus, Zika virus (ZIKV), has recently
re-emerged and spread rapidly across the western hemisphere's equatorial
countries, primarily through Aedes mosquito transmission. While symptoms in
adult infections appear to be self-limiting and mild, severe birth defects, such
as microcephaly, have been linked to infection during early pregcy. Recently,
Tang et al. (Cell Stem Cell 2016, doi: 10.1016/j.stem.2016.02.016) demonstrated
that ZIKV efficiently infects induced pluripotent stem cell (iPSC) derived human
neural progenitor cells (hNPCs), resulting in cell cycle abnormalities and
apoptosis. Consequently, hNPCs are a suggested ZIKV target.
METHODS: We analyzed the transcriptomic sequencing (RNA-seq) data (GEO:
GSE78711) of ZIKV (Strain: MR766) infected hNPCs. For comparison to the
ZIKV-infected hNPCs, the expression data from hNPCs infected with human
cytomegalovirus (CMV) (Strain: AD169) was used (GEO: GSE35295). Utilizing a
combination of Gene Ontology, database of human diseases, and pathway analysis,
we generated a putative systemic model of infection supported by known molecular
pathways of other highly related viruses.
RESULTS: We analyzed RNA-sequencing data for transcript expression alterations
in ZIKV-infected hNPCs, and then compared them to expression patterns of
iPSC-derived hNPCs infected with CMV, a virus that can also induce severe
congenital neurological defects in developing fetuses. We demonstrate for the
first time that many of cellular pathways correlate with clinical pathologies
following ZIKV infection such as microcephaly, congenital nervous system
disorders and epilepsy. Furthermore, ZIKV activates several inflammatory signals
within infected hNPCs that are implicated in innate and acquired immune
responses, while CMV-infected hNPCs showed limited representation of these
pathways. Moreover, several genes related to pathogen responses are
significantly upregulated upon ZIKV infection, but not perturbed in CMV-infected
hNPCs.
CONCLUSION: The presented study is the first to report enrichment of numerous
pro-inflammatory pathways in ZIKV-infected hNPCs, indicating that hNPCs are
capable of signaling through canonical pro-inflammatory pathways following viral
infection. By defining gene expression profiles, new factors in the pathogenesis
of ZIKV were identified which could help develop new therapeutic strategies. Author information:
(1)Laboratory of Viral Diseases, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
(2)Laboratory of Viral Diseases, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic
address: [email protected]. IMPORTANCE: Zika virus infection can be prenatally passed from a pregt woman
to her fetus. There is sufficient evidence to conclude that intrauterine Zika
virus infection is a cause of microcephaly and serious brain anomalies, but the
full spectrum of anomalies has not been delineated. To inform pediatric
clinicians who may be called on to evaluate and treat affected infants and
children, we review the most recent evidence to better characterize congenital
Zika syndrome.
OBSERVATIONS: We reviewed published reports of congenital anomalies occurring in
fetuses or infants with presumed or laboratory-confirmed intrauterine Zika virus
infection. We conducted a comprehensive search of the English literature using
Medline and EMBASE for Zika from inception through September 30, 2016.
Congenital anomalies were considered in the context of the presumed pathogenetic
mechanism related to the neurotropic properties of the virus. We conclude that
congenital Zika syndrome is a recognizable pattern of structural anomalies and
functional disabilities secondary to central and, perhaps, peripheral nervous
system damage. Although many of the components of this syndrome, such as
cognitive, sensory, and motor disabilities, are shared by other congenital
infections, there are 5 features that are rarely seen with other congenital
infections or are unique to congenital Zika virus infection: (1) severe
microcephaly with partially collapsed skull; (2) thin cerebral cortices with
subcortical calcifications; (3) macular scarring and focal pigmentary retinal
mottling; (4) congenital contractures; and (5) marked early hypertonia and
symptoms of extrapyramidal involvement.
CONCLUSIONS AND RELEVANCE: Although the full spectrum of adverse reproductive
outcomes caused by Zika virus infection is not yet determined, a distinctive
phenotype-the congenital Zika syndrome-has emerged. Recognition of this
phenotype by clinicians for infants and children can help ensure appropriate
etiologic evaluation and comprehensive clinical investigation to define the
range of anomalies in an affected infant as well as determine essential
follow-up and ongoing care. |
What is Neisseria adhesin A? | Neisseria adhesin A (NadA) is one of the antigens of Bexsero, the recently licensed multicomponent vaccine against serogroup B Neisseria meningitidis (MenB). NadA belongs to the class of oligomeric coiled-coil adhesins and is able to mediate adhesion and invasion of human epithelial cells. As a vaccine antigen, NadA has been shown to induce high levels of bactericidal antibodies. More than 89 distinct NadA allele sequences and 43 distinct peptides have been described. | Specific surface proteins of Neisseria meningitidis have been proposed to
stimulate leukocytes during tissue invasion and septic shock. In this study, we
demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the
colonization of the respiratory epithelium by hypervirulent N. meningitidis B
strains also binds to and activates human monocytes/macrophages. Expression of
NadA on the surface on Escherichia coli does not increase bacterial-monocyte
association, but a NadA-positive strain induced a significantly higher amount of
TNF-alpha and IL-8 compared with the parental NadA-negative strain, suggesting
that NadA has an intrinsic stimulatory action on these cells. Consistently,
highly pure, soluble NadA(Delta351-405), a proposed component of an
antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to
secrete a selected pattern of cytokines and chemotactic factors characterized by
high levels of IL-8, IL-6, MCP-1, and MIP-1alpha and low levels of the main
vasoactive mediators TNF-alpha and IL-1. NadA(Delta351-405) also inhibited
monocyte apoptosis and determined its differentiation into a macrophage-like
phenotype. The Oca (Oligomeric coiled-coil adhesin) family is a subgroup of the bacterial
trimeric autotransporter adhesins, which includes structurally related proteins,
such as YadA of Yersinia enterocolitica and NadA of Neisseria meningitidis. In
this study, we searched in silico for novel members of this family in bacterial
genomes and identified HadA (Haemophilus adhesin A), a trimeric autotransporter
expressed only by Haemophilus influenzae biogroup aegyptius causing Brazilian
purpuric fever (BPF), a fulmit septicemic disease of children. By comparative
genomics and sequence analysis we predicted that the hadA gene is harboured on a
mobile genetic element unique to BPF isolates. Biological analysis of HadA in
the native background was limited because this organism is not amenable to
genetic manipulation. Alternatively, we demonstrated that expression of HadA
confers to a non-invasive Escherichia coli strain the ability to adhere to human
cells and to extracellular matrix proteins and to induce in vitro bacterial
aggregation and microcolony formation. Intriguingly, HadA is predicted to lack
the typical N-terminal head domain of Oca proteins generally associated with
cellular receptor binding. We propose here a structural model of the HadA
coiled-coil stalk and show that the N-terminal region is still responsible of
the binding activity and a KGD motif plays a role. Interestingly, HadA promotes
bacterial entry into mammalian cells. Our results show a cytoskeleton
re-arrangement and an involvement of clathrin in the HadA-mediated
internalization. These data give new insights on the structure-function
relationship of oligomeric coiled-coil adhesins and suggest a potential role of
this protein in the pathogenesis of BPF. The physico-chemical characterization of NadADelta(351-405), a recombit
protein discovered by reverse vaccinology, component of a candidate vaccine
against Neisseria meningitidis serotype B is presented. Analytical methods like
mass spectrometry, electrophoresis, optical spectroscopy and SEC-MALLS have been
applied to unveil the structure of NadADelta(351-405), and to evaluate
Product-Related Substances. Moreover, analysis of the protein after intentional
denaturation has been applied in order to challenge the chosen methods and to
determine their appropriateness and specificity. All the obtained results were
inserted in a model allowing in-depth understanding of the antigen
NadADelta(351-405): it is present in solution as a homo-trimer, retaining a high
percentage of alpha-helix secondary structure, and able to reassemble from
monomeric subunits after thermal denaturation; this structural organization is
consistent with that foreseen for MenB NadA (Neisseria Adhesin A). The
analytical data sets produced during process development for clinical phases
I-III material confirm product quality and manufacturing consistency. Hypervirulent MenB causing fatal human infections frequently display the
oligomeric-coiled coil adhesin NadA, a 45-kDa intrinsic outer membrane protein
implicated in binding to and invasion of respiratory epithelial cells. A
recombit soluble mutant lacking the 10-kDa COOH terminal membrane domain
(NadA(Delta351-405)) also activates human monocytes/macrophages/DCs. As NadA is
physiologically released during sepsis as part of OMVs, in this study, we tested
the hypothesis that NadA(+) OMVs have an enhanced or modified
proinflammatory/proimmune action compared with NadA(-) OMVs. To do this we
investigated the activity of purified free NadA(Delta351-405) and of OMVs from
MenB and Escherichia coli strains, expressing or not full-length NadA.
NadA(Delta351-405) stimulated monocytes and macrophages to secrete cytokines
(IL-1beta, TNF-alpha, IL-6, IL-12p40, IL-12p70, IL-10) and chemokines (IL-8,
MIP-1alpha, MCP-1, RANTES), and full-length NadA improved MenB OMV activity,
preferentially on macrophages, and only increased cytokine release.
NadA(Delta351-405) induced the lymphocyte costimulant CD80 in monocytes and
macrophages, and NadA(+) OMVs induced a wider set of molecules supporting
antigen presentation (CD80, CD86, HLA-DR, and ICAM-1) more efficiently than
NadA(-) OMVs only in macrophages. Moreover, membrane NadA effects, unlike
NadA(Delta351-405) ones, were much less IFN-gamma-sensitive. The activity of
NadA-positive E. coli OMVs was similar to that of control OMVs. NadA in MenB
OMVs acted at adhesin concentrations approximately 10(6) times lower than those
required to stimulate cells with free NadA(Delta351-405). With the aim of studying the molecular diversity of the antigens of a new
recombit vaccine against meningococcus serogroup B, the three genes coding
for the main vaccine components GNA (Genome-derived Neisseria Antigen) 1870
(fHbp, factor H Binding Protein), GNA1994 (NadA, Neisseria adhesin A) and
GNA2132 were sequenced in a panel of 85 strains collected worldwide and selected
as representative of the serogroup B meningococcal diversity. No correlations
were found between vaccine antigen variability and serogroup, geographic area
and year of isolation. Although a relevant clustering was found with MLST clonal
complexes, each showing an almost specific antigen variant repertoire, the
prediction of the antigen assortment was not possible on the basis of MLST
alone. Therefore, classification of meningococcus on the basis of MLST only is
not sufficient to predict vaccine antigens diversity. Sequencing each gene in
the different strains will be important to evaluate antigen conservation and
assortment and to allow a future prediction of potential vaccine coverage. The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria
meningitidis B (MenB). Its recombit form NadA(Δ351-405,) devoid of the outer
membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to
stimulate monocytes, macrophages and dendritic cells. In this study we
investigated the molecular mechanism of NadA(Δ351-405) cellular effects in
monocytes. We show that NadA(Δ351-405) (against which we obtained polyclonal
antibodies in rabbits), binds to hsp90, but not to other extracellular
homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of
monocytes, in a temperature dependent way. Pre-incubation of monocytes with the
MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70
antibodies to hsp90 and hsp70 at 37°C, a condition in which specific
cell-binding occurs, but not at 0°C, a condition in which specific cell-binding
is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and
anti-hsp70 antibodies did not affected NadA(Δ351-405) cell binding in any
temperature condition, indicating that it associates to another receptor on
their plasma membrane and then laterally diffuses to encounter hsp90.
Consistently, polymixin B interfered with NadA(Δ351-405) /hsp90 association,
abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due
to NadA(Δ351-405) and inhibited adhesin-induced cytokine/chemokine secretion
without affecting monocyte-adhesin binding. Co-stimulation of monocytes with
anti-hsp90 antibodies and NadA(Δ351-405) determined a stronger but polymixin B
insensitive cell activation. This indicated that the formation of a recombit
NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can
be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of
monocytes by NadA(Δ351-405) alone or in the presence of anti-hsp90 antibodies
were both inhibited by neutralizing anti-TLR4 antibodies, but not by anti-TLR2
antibodies. We propose that hsp90-dependent recruitment into an hsp90/hsp70/TLR4
transducing signal complex is necessary for the immune-stimulating activity of
NadA(Δ351-405) anti-MenB vaccine candidate. Serogroup B meningococcal (MenB) disease remains a serious public health problem
for which a cross-protective vaccine effective against a wide range of MenB
isolates has not been available. Novartis Vaccines has developed a vaccine for
the prevention of MenB disease that contains four antigenic components: factor H
binding protein (fHbp), neisserial adhesin A (NadA), Neisseria heparin binding
antigen (NHBA) and outer membrane vesicles from a New Zealand epidemic strain
(which provides PorA). This vaccine has been submitted for regulatory review in
Europe so it is timely to review the design of the vaccine, results to date in
clinical studies and the potential strain coverage provided by the vaccine. It
is also critical to discuss the key issues for the long-term success of the
vaccine which include strain coverage, potential persistence of protection,
potential effects on carriage of MenB strains, potential for escape mutants and
cost effectiveness. The surface-exposed NadA adhesin produced by a subset of capsular serogroup B
strains of Neisseria meningitidis is currently being considered as a vaccine
candidate to prevent invasive disease caused by a hypervirulent lineage of
meningococci. Levels of NadA are known to be controlled by both transcriptional
regulatory factors and a component of human saliva, 4-hydroxyphenylacetic acid.
Herein, we confirmed the capacity of a DNA-binding protein termed FarR to
negatively control nadA expression. We also found that a known transcriptional
regulator of farR in N. gonorrhoeae termed MtrR can have a negative regulatory
impact on farR and nadA expression, especially when over-expressed.
MtrR-mediated repression of nadA was found to be direct, and its binding to a
target DNA sequence containing the nadA promoter influenced formation and/or
stability of FarR::nadA complexes. The complexity of the multi-layered
regulation of nadA uncovered during this investigation suggests that N.
meningitidis modulates NadA adhesin protein levels for the purpose of
interacting with host cells yet avoiding antibody directed against surface
exposed epitopes. The poor immunogenicity of the meningococcal serogroup B (MenB) capsule has led
to the development of vaccines targeting subcapsular antigens, in particular the
immunodomit and diverse outer membrane porin, PorA. These vaccines are
largely strain specific; however, they offer limited protection against the
diverse MenB-associated diseases observed in many industrialized nations. To
broaden the scope of its protection, the multicomponent vaccine (4CMenB)
incorporates a PorA-containing outer membrane vesicle (OMV) alongside relatively
conserved recombit protein components, including factor H-binding protein
(fHbp), Neisseria adhesin A (NadA), and neisserial heparin-binding antigen
(NHBA). The expression of PorA is unique to meningococci (Neisseria
meningitidis); however, many subcapsular antigens are shared with nonpathogenic
members of the genus Neisseria that also inhabit the nasopharynx. These
organisms may elicit cross-protective immunity against meningococci and/or
occupy a niche that might otherwise accommodate pathogens. The potential for
4CMenB responses to impact such species (and vice versa) was investigated by
determining the genetic distribution of the primary 4CMenB antigens among
diverse members of the common childhood commensal, Neisseria lactamica. All the
isolates possessed nhba but were devoid of fhbp and nadA. The nhba alleles were
mainly distinct from but closely related to those observed among a
representative panel of invasive MenB isolates from the same broad geographic
region. We made similar findings for the immunogenic typing antigen, FetA, which
constitutes a major part of the 4CMenB OMV. Thus, 4CMenB vaccine responses may
impact or be impacted by nasopharyngeal carriage of commensal neisseriae. This
highlights an area for further research and surveillance should the vaccine be
routinely implemented. BACKGROUND: Serogroup B meningococcal (MenB) isolates currently account for
approximately 90% of invasive meningococcal disease (IMD) in Greece with ST-162
clonal complex predominating. The potential of a multicomponent meningococcal B
vaccine (4CMenB) recently licensed in Europe was investigated in order to find
whether the aforementioned vaccine will cover the MenB strains circulating in
Greece. A panel of 148 serogroup B invasive meningococcal strains was
characterized by multilocus sequence typing (MLST) and PorA subtyping. Vaccine
components were typed by sequencing for factor H-binding protein (fHbp),
Neisserial Heparin Binding Antigen (NHBA) and Neisseria adhesin A (NadA). Their
expression was explored by Meningococcal Antigen Typing System (MATS).
RESULTS: Global strain coverage predicted by MATS was 89.2% (95% CI 63.5%-98.6%)
with 44.6%, 38.5% and 6.1% of strains covered by one, two and three vaccine
antigens respectively. NHBA was the antigen responsible for the highest coverage
(78.4%), followed by fHbp (52.7%), PorA (8.1%) and NadA (0.7%). The coverage of
the major genotypes did not differ significantly. The most prevalent MLST
genotype was the ST-162 clonal complex , accounting for 44.6% of the strains in
the panel and with a predicted coverage of 86.4%, mainly due to NHBA and fHbp.
CONCLUSIONS: 4CMenB has the potential to protect against a significant
proportion of Greek invasive MenB strains. Neisseria adhesin A (NadA), involved in the adhesion and invasion of Neisseria
meningitidis into host tissues, is one of the major components of Bexsero, a
novel multicomponent vaccine licensed for protection against meningococcal
serogroup B in Europe, Australia, and Canada. NadA has been identified in
approximately 30% of clinical isolates and in a much lower proportion of carrier
isolates. Three protein variants were originally identified in invasive
meningococci and named NadA-1, NadA-2, and NadA-3, whereas most carrier isolates
either lacked the gene or harbored a different variant, NadA-4. Further analysis
of isolates belonging to the sequence type 213 (ST-213) clonal complex
identified NadA-5, which was structurally similar to NadA-4, but more distantly
related to NadA-1, -2, and -3. At the time of this writing, more than 89
distinct nadA allele sequences and 43 distinct peptides have been described.
Here, we present a revised nomenclature system, taking into account the complete
data set, which is compatible with previous classification schemes and is
expandable. The main features of this new scheme include (i) the grouping of the
previously named NadA-2 and NadA-3 variants into a single NadA-2/3 variant, (ii)
the grouping of the previously assigned NadA-4 and NadA-5 variants into a single
NadA-4/5 variant, (iii) the introduction of an additional variant (NadA-6), and
(iv) the classification of the variants into two main groups, named groups I and
II. To facilitate querying of the sequences and submission of new allele
sequences, the nucleotide and amino acid sequences are available at
http://pubmlst.org/neisseria/NadA/. Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein
thought to assist in the adhesion of the bacterium to host cells. We have
previously shown that NadA also promotes bacterial internalization in a
heterologous expression system. Here we have used the soluble recombit NadA
(rNadA) lacking the membrane anchor region to characterize its internalization
route in Chang epithelial cells. Added to the culture medium, rNadA internalizes
through a PI3K-dependent endocytosis process not mediated by the canonical
clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling
pathway previously described for MHC-I. The intracellular pool of rNadA reaches
a steady state level within one hour of incubation and colocalizes in endocytic
vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90.
Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and
FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly
suggesting that the extracellular secreted pool of the chaperone is involved in
rNadA intracellular trafficking. A significant number of intracellular vesicles
containing rNadA recruit Rab11, a small GTPase associated to recycling
endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell
treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA,
abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR
colocalization. Collectively, these results are consistent with a model whereby
rNadA internalizes into human epithelial cells hijacking the recycling endosome
pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11
associated and Hsp90-regulated mechanism. The present study addresses for the
first time a meningoccoccal adhesin mechanism of endocytosis and suggests a
possible entry pathway engaged by N. meningitidis in primary infection of human
epithelial cells. A new vaccine (the 4CMenB 4-component protein vaccine [Bexsero], which includes
PorA, factor H-binding protein [fHbp], neisserial heparin-binding antigen
[NHBA], and Neisseria adhesin A [NadA]) against serogroup B meningococci has
recently been approved for use in people older than age 2 months in Europe,
Australia, and Canada. Preapproval clinical efficacy studies are not feasible
for invasive meningococcal disease because its incidence is low/very low, and
the serum bactericidal antibody (SBA) titer (or the human SBA [hSBA] titer when
human complement is used in the assay) has been used as a surrogate marker of
protection. However, the hSBA assay cannot be used on a large scale, and
therefore, a meningococcal antigen typing system (MATS) was developed. MATS
combines conventional PorA genotyping with an enzyme-linked immunosorbent assay
(ELISA) that quantifies both the expression and the cross-reactivity of
antigenic variants. The assay has been used to evaluate the potential of the
4CMenB meningococcal group B vaccine to cover group B strains in several
countries. Some recent data suggest that MATS is a conservative predictor of
strain coverage. We used pooled sera from adolescents and infants to test by the
hSBA assay 10 meningococcal group B strains isolated in Spain that were negative
for the 3 antigens (n = 9) or that had very low levels of the 3 antigens (n = 1)
by MATS. We found that all strains were killed by sera from adolescents and that
5 of the 10 strains were also killed, although at a low titer, by sera from
infants. Our data confirm that MATS underestimates vaccine coverage. BACKGROUND: For decades, a broadly effective vaccine against serogroup B
Neisseria meningitidis (MenB) has remained elusive. Recently, a four-component
recombit vaccine (4CMenB) has been developed and is now approved in Europe,
Canada, Australia and some Latin American countries. This phase III, randomized
study evaluated the lot consistency, early immune responses and the safety
profile of 4CMenB in 11 to 17-year-old adolescents in Australia and Canada
(NCT01423084).
METHODS: In total, 344 adolescents received two doses of one of 2 lots of
4CMenB, 1-month apart. Immunogenicity was assessed before, 2-weeks and 1-month
following the second vaccination. Serum bactericidal activity using human
complement (hSBA) was measured against three reference strains 44/76-SL, 5/99
and NZ98/254, selected to express one of the vaccine antigens; Neisseria adhesin
A (NadA), factor H binding protein (fHbp) and porin A (PorA) containing outer
membrane vesicle (OMV), respectively. Responses to the Neisseria heparin binding
antigen (NHBA) were assessed with enzyme linked immunosorbent assay (ELISA).
Local and systemic reactions were recorded for 7 days following each
vaccination; unsolicited adverse events were monitored throughout the study.
RESULTS: Immunological equivalence of the two lots of 4CMenB was established at
1-month. At baseline, ≤7% of participants had hSBA titers ≥5 to all three
reference strains. Two weeks following the second dose of 4CMenB, all
participants had hSBA titers ≥5 against fHbp and NadA compared with 84-96%
against the PorA reference strains. At 1-month, corresponding proportions were
99%, 100% and 70-79%, respectively. Both lots were generally well tolerated and
had similar adverse event profiles.
CONCLUSIONS: Two doses of 4CMenB had an acceptable safety profile and induced a
robust immune response in adolescents. Peak antibody responses were observed at
14 days following vaccination. While a substantial non-uniform antigen-dependent
early decline in antibody titers was seen thereafter, a significant percentage
of participants continued to maintain protective hSBA titers at 1-month. Neisseria adhesin A (NadA) is one of the antigens of Bexsero, the recently
licensed multicomponent vaccine against serogroup B Neisseria meningitidis
(MenB). NadA belongs to the class of oligomeric coiled-coil adhesins and is able
to mediate adhesion and invasion of human epithelial cells. As a vaccine
antigen, NadA has been shown to induce high levels of bactericidal antibodies;
however, the domains important for protective response are still unknown. In
order to further investigate its immunogenic properties, we have characterized
the murine IgG1 mAb (6E3) that was able to recognize the 2 main antigenic
variants of NadA on the surface of MenB strains. The epitope targeted by mAb 6E3
was mapped by hydrogen-deuterium exchange mass spectrometry and shown to be
located on the coiled-coil stalk region of NadA (aa 206-249). Although no serum
bactericidal activity was observed for murine IgG1 mAb 6E3, functional activity
was restored when using chimeric antibodies in which the variable regions of the
murine mAb 6E3 were fused to human IgG3 constant regions, thus confirming the
protective nature of the mAb 6E3 epitope. The use of chimeric antibody molecules
will enable future investigations of complement-mediated antibody functionality
independently of the Fc-mediated differences in complement activation. BACKGROUND: A novel meningococcal multicomponent vaccine, 4CMenB (Bexsero®), has
been approved in Europe, Canada, Australia and US. The potential impact of
4CMenB on strain coverage is being estimated by using Meningococcal Antigen
Typing System (MATS), an ELISA assay which measures vaccine antigen expression
and diversity in each strain. Here we show the genetic characterization and the
4CMenB potential coverage of Spanish invasive strains (collected during one
epidemiological year) compared to other European countries and discuss the
potential reasons for the lower estimate of coverage in Spain.
MATERIAL AND METHODS: A panel of 300 strains, a representative sample of all
serogroup B Neisseria meningitidis notified cases in Spain from 2009 to 2010,
was characterized by multilocus sequence typing (MLST) and FetA variable region
determination. 4CMenB vaccine antigens, PorA, factor H binding protein (fHbp),
Neisseria Heparin Binding Antigen (NHBA) and Neisserial adhesin A (NadA) were
molecularly typed by sequencing. PorA coverage was assigned to strain with VR2 =
4. The levels of expression and cross-reactivity of fHbp, NHBA and NadA were
analyzed using MATS ELISA.
FINDINGS: Global estimated strain coverage by MATS was 68.67% (95% CI:
47.77-84.59%), with 51.33%, 15.33% and 2% of strains covered by one, two and
three vaccine antigens, respectively. The predicted strain coverage by
individual antigens was: 42% NHBA, 36.33% fHbp, 8.33% PorA and 1.33% NadA.
Coverage within the most prevalent clonal complexes (cc) was 70.37% for cc 269,
30.19% for cc 213 and 95.83% for cc 32.
CONCLUSIONS: Clonal complexes (cc) distribution accounts for variations in
strain coverage, so that country-by-country investigations of strain coverage
and cc prevalence are important. Because the cc distribution could also vary
over time, which in turn could lead to changes in strain coverage, continuous
detailed surveillance and monitoring of vaccine antigens expression is needed in
those countries where the multicomponent vaccine is introduced. This is really
important in countries like Spain where most of the strains are predicted to be
covered by only one vaccine antigen and the chance for escape mutants to emerge
with vaccine use is higher. Based on the observed data, cc213 should receive
special attention as it is associated with low predicted strain coverage, and
has recently emerged in Spain. In the last decade, the incidence of invasive meningococcal disease (IMD) in
Croatia remained stable at approximately 1 case per 100 000 inhabitants,
affecting mainly children aged ≤5 years. We report the molecular
characterization of meningococci causing IMD occurring from June 2009 to January
2014 in Croatia. Genomic DNA from 50 clinical isolates was analysed for
serogroup, multilocus sequence typing and allele type of the two outer membrane
protein genes, porA and the iron-regulated fetA. Furthermore, 22 of them were
characterized by using whole-genome sequencing to define the meningococcal
vaccine four-component meningococcal serogroup B (4CMenB) antigen genes factor
H-binding protein (fHbp), Neisseria heparin-binding antigen (nhba) and Neisseria
adhesin A (nadA) and the antimicrobial target resistance genes for penicillin
(penicillin binding protein 2, penA), ciprofloxacin (DNA gyrase subunit A, gyrA)
and rifampicin (β-subunit of RNA polymerase, rpoB). The Etest was used to
phenotypically determine the antimicrobial susceptibility of isolated
meningococci. The main serogroup/clonal complex combinations were MenB cc41/44,
MenC/cc11, MenW/cc174 and MenY/cc23. PorA P1.7-2, FetA F5-5 and F1-5 were the
most represented through the serogroups. Meningococci with decreased
susceptibility to penicillin (38.9 %) and one strain resistant to ciprofloxacin
were identified. Forty-two percent of MenB showed the presence of at least one
of the 4CMenB vaccine antigens (fHbp, NHBA, NadA and PorA). Our findings
highlight the genetic variability of meningococci causing IMD in Croatia,
especially for the serogroup B. Molecular-based characterization of meningococci
is crucial to enhance IMD surveillance and to better plan national immunization
programmes. |
Which R package is used for the detection of chromosomal abnormalities from microarray data? | CAFE is an R package for the detection of gross chromosomal abnormalities from gene expression microarray data. | SUMMARY: The current methods available to detect chromosomal abnormalities from
DNA microarray expression data are cumbersome and inflexible. CAFE has been
developed to alleviate these issues. It is implemented as an R package that
analyzes Affymetrix *.CEL files and comes with flexible plotting functions,
easing visualization of chromosomal abnormalities.
AVAILABILITY AND IMPLEMENTATION: CAFE is available from
https://bitbucket.org/cob87icW6z/cafe/ as both source and compiled packages for
Linux and Windows. It is released under the GPL version 3 license. CAFE will
also be freely available from Bioconductor.
CONTACT: [email protected] or [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. |
List all the available databases of super enhancers | dbSUPER and SEA are the available databases of super enhancers. | |
Which are the consequences of the hyperphosphorylated tau in Alzheimers' Disease? | The consequences of the hyperphosphorylated tau in Alzheimers' Disease is:
1) The formation of neurofibrillary tangles (NFTs)
2) Impaired glutamate metabolism
3) Decreased tau affinity for microtubules binding
4) Dendritic and axonal instability
5) Synaptic degeneration
6) Neuronal loss. | Microtubule-associated protein tau is abnormally hyperphosphorylated and
aggregated in affected neurons of Alzheimer disease brain. This
hyperphosphorylated tau can be dephosphorylated at some of the abnormal
phosphorylated sites by purified protein phosphatase-1, 2A, and 2B in vitro. In
the present study, we have developed an assay to measure protein phosphatase
activity toward tau-1 sites (Ser199/Ser202) using the hyperphosphorylated tau
isolated from Alzheimer disease brain as substrate. Using this assay, we have
identified that in normal brain, protein phosphatase-2A and 2B and, to a lesser
extent, 1 are involved in the dephosphorylation of tau. The Km values of
dephosphorylation of the hyperphosphorylated tau by protein phosphatase-2A and
2B are similar. The tau phosphatase activity is decreased by approximately 30%
in brain of Alzheimer disease patients compared with those of age-matched
controls. These findings suggest that a defect of protein phosphatase could be
the cause of the abnormal hyperphosphorylation of tau in Alzheimer disease. Adult human nerve cells contain tau protein, a phosphorylated
microtubule-associated protein, that is hyperphosphorylated in the fetus and in
patients with Alzheimer's disease. Hyperphosphorylation, which diminishes the
microtubule-binding capacity of tau, destabilizes microtubules and may enhance
the formation of paired helical filaments that constitute neurofibrillary
tangles in Alzheimer's disease. Here, we use phosphorylation-dependent anti-tau
antibodies to detect specific epitopes that characterize hyperphosphorylated
tau. Our demonstration of intracellular tangles containing full-length tau that
are not immunolabeled by these antibodies suggests that hyperphosphorylation of
tau is not obligatory in the formation of neurofibrillary tangles in Alzheimer's
disease. Pick bodies and ballooned cells of Pick's disease and the neurofibrillary
lesions of Alzheimer's disease are characterized by the presence of
hyperphosphorylated microtubule-associated protein tau. Little is known about
the mechanisms underlying tau hyperphosphorylation in Pick's disease and the
distribution of abnormal tau in affected neurons. We have used a panel of
phosphorylation-dependent (AT270, AT8, AT180, 12E8, PHF-1, AT10 and Tau-1) and
phosphorylation-independent anti-tau antibodies (N-tau 5 and 134) to stain brain
tissue sections from subjects with Pick's disease and Alzheimer's disease. These
antibodies labeled Pick bodies and neurofibrillary lesions in a similar way,
with the exception of antibody 12E8, which stained a subset of neurofibrillary
tangles, but no Pick bodies. Moreover, abundant AT8- and PHF-1-positive neuritic
profiles were observed in cortical areas rich in Pick bodies, even in the
complete absence of neurofibrillary lesions. Unlike the Gallyas-positive
neuropil threads of Alzheimer's disease, which were of variable diameter and
covered by spiny appendages, neuritic profiles of Pick's disease showed a
regular diameter, appeared smooth and were Gallyas-negative. In contrast to
Alzheimer's disease, dendritic branches of neurons containing Pick bodies were
not labeled by anti-tau antibodies. In the hippocampus, numerous tau-positive
axon terminals were found along dendrites of the polymorphic layer of the
dentate gyrus. Our results indicate that tau proteins in Pick's disease and
Alzheimer's disease share similar phosphorylated residues, with the exception of
serine 262, which is phosphorylated in Alzheimer tangles but not in Pick bodies
or neuritic profiles. Furthermore, we show that hyperphosphorylated tau
segregates to different neuronal compartments in the two diseases, with a
somatoaxonal distribution in Pick's disease and a somatodendritic distribution
in Alzheimer's disease. Hyperphosphorylated tau, the major component of the paired helical filaments of
Alzheimer's disease, was found to accumulate in the brains of mice in which the
calcineurin A alpha gene was disrupted [calcineurin A alpha knockout (CNA alpha
-/-)]. The hyperphosphorylation involved several sites on tau, especially the
Ser396 and/or Ser404 recognized by the PHF-1 monoclonal antibody. The increase
in phosphorylated tau content occurred primarily in the mossy fibers of the CNA
alpha -/- hippocampus, which contained the highest level of calcineurin in
brains of wild-type mice. The CNA alpha -/- mossy fibers also contained less
neurofilament protein than normal, although the overall level of neurofilament
phosphorylation was unchanged. In the electron microscope, the mossy fibers of
CNA alpha -/- mice exhibited abnormalities in their cytoskeleton and a lower
neurofilament/microtubule ratio than those of wild-type animals. These findings
indicate that hyperphosphorylated tau can accumulate in vivo as a result of
reduced calcineurin activity and is accompanied by cytoskeletal changes that are
likely to have functional consequences on the affected neurons. The CNA alpha
-/- mice were found in a separate study to have deficits in learning and memory
that may result in part from the cytoskeletal changes in the hippocampus. Paired helical filaments (PHFs) are the structural constituents of
neurofibrillary tangles in Alzheimer's disease and are composed of
hyperphosphorylated forms of the microtubule-associated protein tau (PHF-tau).
Pathological hyperphosphorylation of tau is believed to be an important
contributor to the destabilisation of microtubules and their subsequent
disappearance from tangle-bearing neurons in Alzheimer's disease, making
elucidation of the mechanisms that regulate tau phosphorylation an important
research goal. Thus, it is essential to identify, preferably by direct
sequencing, all of the sites in PHF-tau that are phosphorylated, a task that is
incomplete because of the difficulty to date of purifying insoluble PHF-tau to
homogeneity and in sufficient quantities for structural analysis. Here we
describe the solubilisation of PHF-tau followed by its purification by Mono Q
chromatography and reversed-phase HPLC. Phosphopeptides from proteolytically
digested PHF-tau were sequenced by oelectrospray mass spectrometry. We
identified 22 phosphorylation sites in PHF-tau, including five sites not
previously identified. The combination of our new data with previous reports
shows that PHF-tau can be phosphorylated on at least 25 different sites. Abnormal tau-immunoreactive filaments are a hallmark of tauopathies, including
Alzheimer's disease (AD). A higher phosphorylation ("hyperphosphorylation")
state of tau protein may represent a critical event. To determine the potential
role of tau hyperphosphorylation in these disorders, mutated tau proteins were
produced where serine/threonine residues known to be highly phosphorylated in
tau filaments isolated from AD patients were substituted for glutamate to
simulate a paired helical filament (PHF)-like tau hyperphosphorylation. We
demonstrate that, like hyperphosphorylation, glutamate substitutions induce
compact structure elements and SDS-resistant conformational domains in tau
protein. Hyperphosphorylation-mimicking glutamate-mutated tau proteins display a
complete functional loss in its ability to promote microtubule nucleation which
can partially be overcome by addition of the osmolyte trimethylamine N-oxide
(TMAO), which is similar to phosphorylated tau. In addition, glutamate-mutated
tau proteins fail to interact with the domit brain protein phosphatase 2A
isoform ABalphaC, and exhibit a reduced ability to assemble into filaments.
Interestingly, wild-type tau and phosphorylation-mimicking tau similarly bind to
microtubules when added alone, but the mutated tau is almost completely
displaced from the microtubule surface by equimolar concentrations of wild-type
tau. The data indicate that glutamate-mutated tau proteins provide a useful
model for analyzing the functional consequences of tau hyperphosphorylation.
They suggest that several mechanisms contribute to the abnormal tau accumulation
observed during tauopathies, in particular a selective displacement of
hyperphosphorylated tau from microtubules, a functional loss in promoting
microtubule nucleation, and a failure to interact with phosphatases. Treatment of cultured neurons with beta-amyloid (Abeta) evokes multiple
consequences, including calcium influx, production of reactive oxygen species
(ROS), hyperphosphorylation of tau. Which of these events is the major cause of
Abeta-induced neurodegeneration has been the subject of controversy. We
undertook to determine whether or not the accumulation of hyperphosphorylated
tau mediated neurodegeneration. Murine cortical neurons demonstrated increased
phospho-tau immunoreactivity between 2-8 hr after treatment of murine cortical
neurons with Abeta_25-35. Cultures underwent overall neurodegeneration between
8-16 hr as ascertained by phase-contrast microscopy, a commercial "live/dead"
assay and externalization of phosphatidyl serine. Unexpectedly, however, the
healthiest-appearing neurons in Abeta-treated cultures contained relatively more
phospho-tau immunoreactivity, while obviously degenerating neurons contained
less; degenerating neurons often contained less phospho-tau immunoreactivity
than did non-Abeta-treated control neurons. By contrast, accumulation of
reactive oxygen species, previously demonstrated to mediate Abeta-induced
neurodegeneration, was most prominent within visibly-degenerating neurons. These
studies do not address the long-term consequences of PHF formation; however,
they indicate that tau hyperphosphorylation, although a consequence of Abeta
treatment, does not directly contribute to acute degeneration of cultured
neurons. Two members of the family of low-density lipoprotein receptors (i.e., very
low-density lipoprotein [VLDL] receptor and apolipoprotein E [apoE] type 2
receptor) are expressed in brain, where they bind and transduce reelin, a
secreted glycoprotein that shares structural analogies with extracellular matrix
proteins. In the developing fetal brain, reelin-signal transduction is critical
for the correct positioning of neurons and the formation of appropriate synaptic
connections, whereas in the mature brain, reelin participates in the mediation
of experience-dependent synaptic plasticity. An important "downstream"
consequence of the reelin-signal transduction cascade is inhibition of the
phosphorylation of tau, a protein that regulates microtubule assembly and
stability. Importantly, hyperphosphorylated tau comprises the paired helical
filament, whose pathological deposition as neurofibrillary tangles is implicated
in Alzheimer's disease; hyperphosphorylated tau is also implicated in the
pathogenesis of other neurodegenerative disorders. Isoforms of apoE may affect
the binding of reelin to its cell surface receptors and, thereby, influence tau
phosphorylation, whereas insulin, insulin-like growth factor-1, and the lithium
ion have actions within the cell at the level of the specific tyrosine kinases
involved in the phosphorylation of tau. These data support the exploration of
pharmacotherapeutic interventions designed to prevent or reduce the burden of
hyperphosphorylated tau. Impaired reelin-signal transduction due to an actual
deficiency of reelin expression may occur in at least some patients with
psychotic disorders, especially schizophrenia; conceivably, hyperphosphorylation
of tau would result from deficient transduction of reelin in schizophrenia.
Schizophrenia has been conceptualized as a neurodevelopmental disorder of
impaired synaptic "connectivity", whose consequence does not become fully
apparent until late adolescence or early adulthood. In summary,
hyperphosphorylation of tau may be an underlying point of pathological
convergence for several neuropsychiatric disorders, and prevention of tau
hyperphosphorylation may be an important therapeutic target. Hyperphosphorylated tau has long been proposed as the key molecule disrupting
normal neuronal microtubule dynamics and leading to neurofibrillary degeneration
in Alzheimer disease. Here we provide a direct evidence of hyperphosphorylated
tau-induced disruption of microtubule network. Using Nocodozole-treated and
detergent-extracted cells, we created a neuronal environment in mouse embryonic
fibroblasts, 3T3 cells, by replacing their cytoplasm with adult rat brain
cytosol. By recreating neuronal microtubule network in these cells, we were able
to follow the effects of hyperphosphorylated tau on microtubule dynamics in real
time. Whereas recombit human brain tau promoted assembly and bundling of
microtubules, abnormally hyperphosphorylated tau isolated from Alzheimer disease
brain cytosol (AD P-tau) inhibited the assembly and disrupted preformed
microtubule network by sequestering normal brain tau and MAP2. This breakdown of
the microtubule network was reversed by treatment of the extracted cells with
protein phosphatase-2A. This study, for the first time, provides direct
mechanistic insights into the molecular basis of both axonal and dendritic
neurodegeneration seen in Alzheimer disease. Most individuals with Down syndrome show early onset of Alzheimer disease (AD),
resulting from the extra copy of chromosome 21. Located on this chromosome is a
gene that encodes the dual specificity tyrosine phosphorylation-regulated kinase
1A (DYRK1A). One of the pathological hallmarks in AD is the presence of
neurofibrillary tangles (NFTs), which are insoluble deposits that consist of
abnormally hyperphosphorylated Tau. Previously it was reported that Tau at the
Thr-212 residue was phosphorylated by Dyrk1A in vitro. To determine the
physiological significance of this phosphorylation, an analysis was made of the
amount of phospho-Thr-212-Tau (pT212) in the brains of transgenic mice that
overexpress the human DYRK1A protein (DYRK1A TG mice) that we recently
generated. A significant increase in the amount of pT212 was found in the brains
of DYRK1A transgenic mice when compared with age-matched littermate controls. We
further examined whether Dyrk1A phosphorylates other Tau residues that are
implicated in NFTs. We found that Dyrk1A also phosphorylates Tau at Ser-202 and
Ser-404 in vitro. Phosphorylation by Dyrk1A strongly inhibited the ability of
Tau to promote microtubule assembly. Following this, using mammalian cells and
DYRK1A TG mouse brains, it was demonstrated that the amounts of
phospho-Ser-202-Tau and phospho-Ser-404-Tau are enhanced when DYRK1A amounts are
high. These results provide the first in vivo evidence for a physiological role
of DYRK1A in the hyperphosphorylation of Tau and suggest that the extra copy of
the DYRK1A gene contributes to the early onset of AD. It has been established for a long time that brain glucose metabolism is
impaired in Alzheimer's disease. Recent studies have demonstrated that impaired
brain glucose metabolism precedes the appearance of clinical symptoms, implying
its active role in the development of Alzheimer's disease. However, the
molecular mechanism by which this impairment contributes to the disease is not
known. In this study, we demonstrated that protein O-GlcNAcylation, a common
post-translational modification of nucleocytoplasmic proteins with
beta-N-acetyl-glucosamine and a process regulated by glucose metabolism, was
markedly decreased in Alzheimer's disease cerebrum. More importantly, the
decrease in O-GlcNAc correlated negatively with phosphorylation at most
phosphorylation sites of tau protein, which is known to play a crucial role in
the neurofibrillary degeneration of Alzheimer's disease. We also found that
hyperphosphorylated tau contained 4-fold less O-GlcNAc than
non-hyperphosphorylated tau, demonstrating for the first time an inverse
relationship between O-GlcNAcylation and phosphorylation of tau in the human
brain. Downregulation of O-GlcNAcylation by knockdown of O-GlcNAc transferase
with small hairpin RNA led to increased phosphorylation of tau in HEK-293 cells.
Inhibition of the hexosamine biosynthesis pathway in rat brain resulted in
decreased O-GlcNAcylation and increased phosphorylation of tau, which resembled
changes of O-GlcNAcylation and phosphorylation of tau in rodent brains with
decreased glucose metabolism induced by fasting, but not those in rat brains
when protein phosphatase 2A was inhibited. Comparison of tau phosphorylation
patterns under various conditions suggests that abnormal tau
hyperphosphorylation in Alzheimer's disease brain may result from downregulation
of both O-GlcNAcylation and protein phosphatase 2A. These findings suggest that
impaired brain glucose metabolism leads to abnormal hyperphosphorylation of tau
and neurofibrillary degeneration via downregulation of tau O-GlcNAcylation in
Alzheimer's disease. Thus, restoration of brain tau O-GlcNAcylation and protein
phosphatase 2A activity may offer promising therapeutic targets for treating
Alzheimer's disease. The microtubule-associated protein tau, in a hyperphosphorylated form,
aggregates into insoluble paired-helical filaments (PHFs) in Alzheimer's disease
(AD) and other tauopathies. In AD, there is approximately 8 mol of phosphate per
mole of tau distributed among approximately 30 PHF phosphorylation sites as
compared to 2-3 mol of phosphate per mole in normal brain. In AD, kinases such
as glycogen synthase kinase-3beta (GSK-3beta) are believed to be involved in the
generation of hyperphosphorylated tau. However, the functional consequences of
hyperphosphorylation on the microtubule binding and polymerization of tau are
not well understood. To address this question, we have generated
pseudohyperphosphorylation mutants consisting of six and seven sites in the
proline-rich region and carboxy terminus of tau by amino acid substitution. In
addition, several single, double, and triple pseudophosphorylation mutants were
also generated. Pseudophosphorylation of tau decreases its affinity for
microtubules, and pseudohyperphosphorylated forms of tau do not have
significantly decreased levels of microtubule binding as compared to single and
double sites. Three pseudohyperphosphorylated forms of tau with altered sodium
dodecyl sulfate-polyacrylamide gel electrophoresis migration have a greater
effect on its inducer-mediated polymerization, slowing the rate of nucleation
and elongation. On the basis of the observations that pseudohyperphosphorylated
tau has decreased affinity for microtubules and reduced inducer-initiated rates
of nucleation and polymerization, we propose that this combination could be the
cause of the increased cytotoxicity of hyperphosphorylated tau in Alzheimer's
disease and also explain the potentially beneficial role of tau polymerization
and NFT formation. Alzheimer's disease (AD) is a neurodegenerative disorder characterized
pathologically by progressive neuronal loss, extracellular plaques containing
the amyloid-β (Aβ) peptides, and neurofibrillary tangles composed of
hyperphosphorylated tau proteins. Aβ is thought to act upstream of tau,
affecting its phosphorylation and therefore aggregation state. One of the major
risk factors for AD is traumatic brain injury (TBI). Acute intra-axonal Aβ and
diffuse extracellular plaques occur in ∼30% of human subjects after severe TBI.
Intra-axonal accumulations of tau but not tangle-like pathologies have also been
found in these patients. Whether and how these acute accumulations contribute to
subsequent AD development is not known, and the interaction between Aβ and tau
in the setting of TBI has not been investigated. Here, we report that controlled
cortical impact TBI in 3xTg-AD mice resulted in intra-axonal Aβ accumulations
and increased phospho-tau immunoreactivity at 24 h and up to 7 d after TBI.
Given these findings, we investigated the relationship between Aβ and tau
pathologies after trauma in this model by systemic treatment of Compound E to
inhibit γ-secretase activity, a proteolytic process required for Aβ production.
Compound E treatment successfully blocked posttraumatic Aβ accumulation in these
injured mice at both time points. However, tau pathology was not affected. Our
data support a causal role for TBI in acceleration of AD-related pathologies and
suggest that TBI may independently affect Aβ and tau abnormalities. Future
studies will be required to assess the behavioral and long-term
neurodegenerative consequences of these pathologies. The major defining pathological hallmarks of Alzheimer's disease (AD) are the
accumulations of Aβ in senile plaques and hyperphosphorylated tau in
neurofibrillary tangles and neuropil threads. Recent studies indicate that
rather than these insoluble lesions, the soluble Aβ oligomers and
hyperphosphorylated tau are the toxic agents of AD pathology. Such pathological
protein species are accompanied by cytoskeletal changes, mitochondrial
dysfunction, Ca(2+) dysregulation, and oxidative stress. In this review, we
discuss how the binding of Aβ to various integrins, defects in downstream focal
adhesion signaling, and activation of cofilin can impact mitochondrial
dysfunction, cytoskeletal changes, and tau pathology induced by Aβ oligomers.
Such pathological consequences can also feedback to further activate cofilin to
promote cofilin pathology. We also suggest that the mechanism of Aβ generation
by the endocytosis of APP is mechanistically linked with perturbations in
integrin-based focal adhesion signaling, as APP, LRP, and β-integrins are
physically associated with each other. The pathological hallmarks of Alzheimer's disease (AD)--widespread synaptic and
neuronal loss and the pathological accumulation of amyloid-beta peptide (Aβ) in
senile plaques, as well as hyperphosphorylated tau in neurofibrillary
tangles--have been known for many decades, but the links between AD pathology
and dementia and effective therapeutic strategies remain elusive. Transgenic
mice have been developed based on rare familial forms of AD and frontotemporal
dementia, allowing investigators to test in detail the structural, functional,
and behavioral consequences of AD-associated pathology. Here, we review work on
transgenic AD models that investigate the degeneration of dendritic spine
structure, synaptic function, and cognition. Together, these data support a
model of AD pathogenesis in which soluble Aβ initiates synaptic dysfunction and
loss, as well as pathological changes in tau, which contribute to both synaptic
and neuronal loss. These changes in synapse structure and function as well as
frank synapse and neuronal loss contribute to the neural system dysfunction
which causes cognitive deficits. Understanding the underpinnings of dementia in
AD will be essential to develop and evaluate therapeutic approaches for this
widespread and devastating disease. We used a nontransgenic cellular tauopathy model in which individual giant
neurons in the lamprey CNS (ABCs) overexpress human tau isoforms cell
autonomously to characterize the still poorly understood consequences of
disease-associated tau processing in situ. In this model, tau colocalizes with
endogenous microtubules and is nontoxic when expressed at low levels, but is
misprocessed by a toxicity-associated alternative pathway when expressed above
levels that saturate dendritic microtubules, causing abnormally phosphorylated,
vesicle-associated tau to accumulate in ABC distal dendrites. This causes
localized microtubule loss and eventually dendritic degeneration, which is
preceded by tau secretion to the extracellular space. This sequence is
reiterated at successively more proximal dendritic locations over time,
suggesting that tau-induced dendritic degeneration is driven by distal dendritic
accumulation of hyperphosphorylated, vesicle-associated tau perpetuated by
localized microtubule loss. The implications for the diagnosis and treatment of
human disease are discussed. In neurodegenerative diseases including Alzheimer's disease and frontotemporal
dementia, the protein tau is hyperphosphorylated and eventually aggregates to
develop neurofibrillary tangles. Here, the consequences of tau
hyperphosphorylation on both neuronal and astrocytic metabolism and amino-acid
neurotransmitter homeostasis were assessed in transgenic mice expressing the
pathogenic mutation P301L in the human tau gene (pR5 mice) compared with
nontransgenic littermate controls. Mice were injected with the neuronal and
astrocytic substrate [1-(13)C]glucose and the astrocytic substrate
[1,2-(13)C]acetate. Hippocampus and cerebral cortex extracts were analyzed using
(1)H and (13)C nuclear magnetic resoce spectroscopy, gas chromatography-mass
spectrometry and high-performance liquid chromatography. The glutamate level was
reduced in the hippocampus of pR5 mice, accompanied by reduced incorporation of
(13)C label derived from [1-(13)C]glucose in glutamate. In the cerebral cortex,
glucose utilization as well as turnover of glutamate, glutamine, and GABA, were
increased. This was accompanied by a relative increase in production of
glutamate via the pyruvate carboxylation pathway in cortex. Overall, we revealed
that astrocytes as well as glutamatergic and GABAergic neurons in the cortex of
pR5 mice were in a hypermetabolic state, whereas in the hippocampus, where
expression levels of mutant human tau are the highest, glutamate homeostasis was
impaired. Neurofibrillary pathology of abnormally hyperphosphorylated Tau is a key lesion
of Alzheimer disease and other tauopathies, and its density in the brain
directly correlates with dementia. The phosphorylation of Tau is regulated by
protein phosphatase 2A, which in turn is regulated by inhibitor 2, I2(PP2A). In
acidic conditions such as generated by brain ischemia and hypoxia, especially in
association with hyperglycemia as in diabetes, I2(PP2A) is cleaved by
asparaginyl endopeptidase at Asn-175 into the N-terminal fragment (I2NTF) and
the C-terminal fragment (I2CTF). Both I2NTF and I2CTF are known to bind to the
catalytic subunit of protein phosphatase 2A and inhibit its activity. Here we
show that the level of activated asparaginyl endopeptidase is significantly
increased, and this enzyme and I2(PP2A) translocate, respectively, from neuronal
lysosomes and nucleus to the cytoplasm where they interact and are associated
with hyperphosphorylated Tau in Alzheimer disease brain. Asparaginyl
endopeptidase from Alzheimer disease brain could cleave GST-I2(PP2A), except
when I2(PP2A) was mutated at the cleavage site Asn-175 to Gln. Finally, an
induction of acidosis by treatment with kainic acid or pH 6.0 medium activated
asparaginyl endopeptidase and consequently produced the cleavage of I2(PP2A),
inhibition of protein phosphatase 2A, and hyperphosphorylation of Tau, and the
knockdown of asparaginyl endopeptidase with siRNA abolished this pathway in
SH-SY5Y cells. These findings suggest the involvement of brain acidosis in the
etiopathogenesis of Alzheimer disease, and asparaginyl
endopeptidase-I2(PP2A)-protein phosphatase 2A-Tau hyperphosphorylation pathway
as a therapeutic target. Traumatic brain injury (TBI) survivors frequently suffer from life-long deficits
in cognitive functions and a reduced quality of life. Axonal injury, observed in
many severe TBI patients, results in accumulation of amyloid precursor protein
(APP). Post-injury enzymatic cleavage of APP can generate amyloid-β (Aβ)
peptides, a hallmark finding in Alzheimer's disease (AD). At autopsy, brains of
AD and a subset of TBI victims display some similarities including accumulation
of Aβ peptides and neurofibrillary tangles of hyperphosphorylated tau proteins.
Most epidemiological evidence suggests a link between TBI and AD, implying that
TBI has neurodegenerative sequelae. Aβ peptides and tau may be used as
biomarkers in interstitial fluid (ISF) using cerebral microdialysis and/or
cerebrospinal fluid (CSF) following clinical TBI. In the present review, the
available clinical and experimental literature on Aβ peptides and tau as
potential biomarkers following TBI is comprehensively analyzed. Elevated CSF and
ISF tau protein levels have been observed following severe TBI and suggested to
correlate with clinical outcome. Although Aβ peptides are produced by normal
neuronal metabolism, high levels of long and/or fibrillary Aβ peptides may be
neurotoxic. Increased CSF and/or ISF Aβ levels post-injury may be related to
neuronal activity and/or the presence of axonal injury. The heterogeneity of
animal models, clinical cohorts, analytical techniques, and the complexity of
TBI in the available studies make the clinical value of tau and Aβ as biomarkers
uncertain at present. Additionally, the link between early post-injury changes
in tau and Aβ peptides and the future risk of developing AD remains unclear.
Future studies using methods such as rapid biomarker sampling combined with
enhanced analytical techniques and/or novel pharmacological tools could provide
additional information on the importance of Aβ peptides and tau protein in both
the acute pathophysiology and long-term consequences of TBI. Neurofibrillary tangles (NFTs), a marker of neuronal alterations in Alzheimer's
disease (AD) and other tauopathies, are comprised of aggregates of
hyperphosphorylated tau protein. We recently studied the formation of NFTs in
the entorhinal cortex (EC) and their subsequent propagation through neural
circuits in the rTgTauEC mouse model (de Calignon et al., 2012). We now examine
the consequences of suppressing transgene expression with doxycycline on the
NFT-associated pathological features of neuronal system deafferentation, NFT
progression and propagation, and neuronal loss. At 21 months of age we observe
that EC axonal lesions are associated with an abnormal sprouting response of
acetylcholinesterase (AChE)-positive fibers, a phenotype reminiscent of human
AD. At 24 months, NFTs progress, tau inclusions propagate to the dentate gyrus,
and neuronal loss is evident. Suppression of the transgene expression from 18 to
24 months led to reversal of AChE sprouting, resolution of Gallyas-positive and
Alz50-positive NFTs, and abrogation of progressive neuronal loss. These data
suggest that propagation of NFTs, as well as some of the neural system
consequences of NFTs, can be reversed in an animal model of NFT-associated
toxicity, providing proof in principle that these lesions can be halted, even in
established disease. The formation of neurofibrillary tangles from the assembly of
hyperphosphorylated tau leads to dendritic and axonal instability, synaptic
degeneration, and neuronal loss. To understand the early physiological
consequences of aberrant tau expression, we characterized the physiology of CA1
pyramidal neurons in rTg4510 female mice and non-transgenic (wt) littermate
controls. We studied mice at the age of 10-12 weeks where only minimal
hyperphosphorylated pretangle tau was present, and 22-24 weeks old mice with
significant neurofibrillary tangle pathology. Our electrophysiological analysis
included input-output relation, paired-pulse facilitation, and whole cell
patch-clamp recordings of neurons to measure action potential threshold and
action potential properties, chord-conductance, and characterization of AMPA
receptor mediated synaptic transmission. We found that the input-output relation
in field (excitatory postsynaptic potentials, EPSP) and whole cell recordings
(excitatory postsynaptic currents, EPSC) were impaired in rTg4510 mice compared
to wt controls at both ages. We measured a diminished tail current charge after
depolarizing voltage input in rTg4510 mice compared to wt in both young and aged
mice. Additionally, mini-EPSC properties (peak and decay time) were essentially
similar between genotypes and age groups investigated. Surprisingly, in the
22-24 week old group, the mini-EPSC frequency was significantly increased
(interevent interval 0.8 ± 0.1 in wt compared to 0.3 ± 0.1 in rTg4510 mice).
These data indicate that the developmentally regulated expression of human P301L
tau in CA1 pyramidal neurons coincide with changes in neuronal excitability but
also that significant presynaptic changes occur late during the progression of
tau pathology in this mouse model. Aggregates of hyperphosphorylated tau protein are found in a group of diseases
called tauopathies, which includes Alzheimer's disease. The causes and
consequences of tau hyperphosphorylation are routinely investigated in
laboratory animals. Mice are the models of choice as they are easily amenable to
transgenic technology; consequently, their tau phosphorylation levels are
frequently monitored by Western blotting using a panel of monoclonal/polyclonal
anti-tau antibodies. Given that mouse secondary antibodies can recognize
endogenous mouse immunoglobulins (Igs) and the possible lack of specificity with
some polyclonal antibodies, non-specific signals are commonly observed. Here, we
characterized the profiles of commonly used anti-tau antibodies in four
different mouse models: non-transgenic mice, tau knock-out (TKO) mice, 3xTg-AD
mice, and hypothermic mice, the latter a positive control for tau
hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type
1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3), type 2,
demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5), and type 3,
with no non-specific signal (DA9, PHF-1, Tau1, Tau46). For polyclonal anti-tau
antibodies, some displayed non-specificity (pS262, pS409) while others did not
(pS199, pT205, pS396, pS404, pS422, A0024). With monoclonal antibodies, most of
the interfering signal was due to endogenous Igs and could be eliminated by
different techniques: i) using secondary antibodies designed to bind only
non-denatured Igs, ii) preparation of a heat-stable fraction, iii) clearing Igs
from the homogenates, and iv) using secondary antibodies that only bind the
light chain of Igs. All of these techniques removed the non-specific signal;
however, the first and the last methods were easier and more reliable. Overall,
our study demonstrates a high risk of artefactual signal when performing Western
blotting with routinely used anti-tau antibodies, and proposes several solutions
to avoid non-specific results. We strongly recommend the use of negative (i.e.,
TKO) and positive (i.e., hypothermic) controls in all experiments. Alzheimer's disease (AD) is characterized by the presence of amyloid plaques
mainly consisting of hydrophobic β-amyloid peptide (Aβ) aggregates and
neurofibrillary tangles (NFTs) composed principally of hyperphosphorylated tau.
Aβ oligomers have been described as the earliest effectors to negatively affect
synaptic structure and plasticity in the affected brains, and cellular prion
protein (PrP(C)) has been proposed as receptor for these oligomers. The most
widely accepted theory holds that the toxic effects of Aβ are upstream of change
in tau, a neuronal microtubule-associated protein that promotes the
polymerization and stabilization of microtubules. However, tau is considered
decisive for the progression of neurodegeneration, and, indeed, tau pathology
correlates well with clinical symptoms such as dementia. Different pathways can
lead to abnormal phosphorylation, and, as a consequence, tau aggregates into
paired helical filaments (PHF) and later on into NFTs. Reported data suggest a
regulatory tendency of PrP(C) expression in the development of AD, and a
putative relationship between PrP(C) and tau processing is emerging. However,
the role of tau/PrP(C) interaction in AD is poorly understood. In this study, we
show increased susceptibility to Aβ-derived diffusible ligands (ADDLs) in
neuronal primary cultures from PrP(C) knockout mice, compared to wild-type,
which correlates with increased tau expression. Moreover, we found increased
PrP(C) expression that paralleled with tau at early ages in an AD murine model
and in early Braak stages of AD in affected individuals. Taken together, these
results suggest a protective role for PrP(C) in AD by downregulating tau
expression, and they point to this protein as being crucial in the molecular
events that lead to neurodegeneration in AD. Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are
found in numerous tauopathies including Alzheimer's disease. Increasing evidence
suggests that tau pathology can be transmitted from cell-to-cell; however the
mechanisms involved in the initiation of tau fibrillization and spreading of
disease linked to progression of tau pathology are poorly understood. We show
here that intracerebral injections of preformed synthetic tau fibrils into the
hippocampus or frontal cortex of young tau transgenic mice expressing mutant
human P301L tau induces tau hyperphosphorylation and aggregation around the site
of injection, as well as a time-dependent propagation of tau pathology to
interconnected brain areas distant from the injection site. Furthermore, we show
that the tau pathology as a consequence of injection of tau preformed fibrils
into the hippocampus induces selective loss of CA1 neurons. Together, our data
confirm previous studies on the seeded induction and the spreading of tau
pathology in a different tau transgenic mouse model and reveals neuronal loss
associated with seeded tau pathology in tau transgenic mouse brain. These
results further validate the utility of the tau seeding model in studying
disease transmission, and provide a more complete in vivo tauopathy model with
associated neurodegeneration which can be used to investigate the mechanisms
involved in tau aggregation and spreading, as well as aid in the search for
disease modifying treatments for Alzheimer's disease and related tauopathies. The cause for the neurofibrillary tangles and plaques in Alzheimer disease
likely relates to an abnormal accumulation of their key components, which
include β-amyloid and hyperphosphorylated tau protein. We segregated Alzheimer
brain sections from people with end-stage disease into those with abundant
hyperphosphorylated tau protein and those without and compared each to normal
brains for global microRNA patterns. A significant reduced expression of several
microRNAs, including miR-512, was evident in the Alzheimer brain sections with
abundant hyperphosphorylated tau. Immunohistochemistry documented that 2 known
targets of microRNA-512, cFLIP and MCL1, were significantly over expressed and
each colocalized to neurons with the abnormal tau protein. Analysis for
apoptosis including activated caspase-3, increased caspase-4 and caspase-8,
apoptosis initiating factor, APAF-1 activity, and the TUNEL assay was negative
in the areas where neurons showed hyperphosphorylated tau. MCM2 expression, a
marker of neuroprogenitor cells, was significantly reduced in the Alzheimer
sections that contained the hyperphosphorylated tau. These results suggest that
a basic defect in Alzheimer disease may be the reduced microRNA-driven increased
expression of proteins that may alter the apoptotic/antiapoptotic balance of
neurons. This, in turn, could lead to the accumulation of key Alzheimer proteins
such as hyperphosphorylated tau that ultimately prevent normal neuronal function
and lead to disease symptomatology. |
Which is the "bonding hormone"? | Oxytocin is known as the 'bonding hormone' due its role in promoting mother-child and pair bonding. | Humans excel in cooperative exchanges between unrelated individuals. Although
this trait is fundamental to the success of our species, its evolution and
mechanisms are poorly understood. Other social mammals also build long-term
cooperative relationships between non-kin, and recent evidence shows that
oxytocin, a hormone involved in parent-offspring bonding, is likely to
facilitate non-kin as well as kin bonds. In a population of wild chimpanzees, we
measured urinary oxytocin levels following a rare cooperative event--food
sharing. Subjects showed higher urinary oxytocin levels after single
food-sharing events compared with other types of social feeding, irrespective of
previous social bond levels. Also, urinary oxytocin levels following food
sharing were higher than following grooming, another cooperative behaviour.
Therefore, food sharing in chimpanzees may play a key role in social bonding
under the influence of oxytocin. We propose that food-sharing events co-opt
neurobiological mechanisms evolved to support mother-infant bonding during
lactation bouts, and may act as facilitators of bonding and cooperation between
unrelated individuals via the oxytocinergic system across social mammals. Oxytocin is known as the 'love hormone' due its role in promoting mother-child
and pair bonding. More recent research indicates that oxytocin may have broader
pro-social effects on behavior and cognition, which points towards oxytocin's
potential as an agent to help improve social cognition and functioning in
psychiatric disorders such as schizophrenia and autism. However, new research on
oxytocin has also uncovered a 'darker side', including oxytocin's possible role
in social out-grouping and envy. Instead of a simple view of oxytocin as 'good'
or 'bad', a more accurate depiction of oxytocin's role in social processing
likely involves the presence of moderating factors. We review moderation effects
in oxytocin and their implications for psychiatry. One implication is that,
across diagnostic categories, oxytocin administration may have positive effects
for patients with social cognitive deficits but negative effects for patients
with social cognitive bias. We conclude that future intervention studies should
use methods such as signal detection to measure both deficit and bias parameters
of social cognition and to evaluate potential individual and contextual
moderators both within and between psychiatric diagnoses in order to determine
for whom oxytocin treatment may be beneficial and for whom it may actually be
harmful. The oxytocin (OT) hormone pathway is involved in numerous physiological
processes, and one of its receptor genes (OXTR) has been implicated in pair
bonding behavior in mammalian lineages. This observation is important for
understanding social monogamy in primates, which occurs in only a small subset
of taxa, including Azara's owl monkey (Aotus azarae). To examine the potential
relationship between social monogamy and OXTR variation, we sequenced its 5'
regulatory (4936bp) and coding (1167bp) regions in 25 owl monkeys from the
Argentinean Gran Chaco, and examined OXTR sequences from 1092 humans from the
1000 Genomes Project. We also assessed interspecific variation of OXTR in 25
primate and rodent species that represent a set of phylogenetically and
behaviorally disparate taxa. Our analysis revealed substantial variation in the
putative 5' regulatory region of OXTR, with marked structural differences across
primate taxa, particularly for humans and chimpanzees, which exhibited unique
patterns of large motifs of dinucleotide A+T repeats upstream of the OXTR 5'
UTR. In addition, we observed a large number of amino acid substitutions in the
OXTR CDS region among New World primate taxa that distinguish them from Old
World primates. Furthermore, primate taxa traditionally defined as socially
monogamous (e.g., gibbons, owl monkeys, titi monkeys, and saki monkeys) all
exhibited different amino acid motifs for their respective OXTR protein coding
sequences. These findings support the notion that monogamy has evolved
independently in Old World and New World primates, and that it has done so
through different molecular mechanisms, not exclusively through the oxytocin
pathway. Author information:
(1)Division of Comparative Medicine, Massachusetts Institute of Technology,
Cambridge, MA, USA.
(2)Division of Comparative Medicine, Massachusetts Institute of Technology,
Cambridge, MA, USA; College of Veterinary Medicine, Aristotle University,
Thessaloniki, Greece.
(3)Neurobiology of Social Behavior Laboratory, Department of Psychology, Boston
College, Chestnut Hill, MA, USA.
(4)Division of Comparative Medicine, Massachusetts Institute of Technology,
Cambridge, MA, USA; Biological Engineering, Massachusetts Institute of
Technology, Cambridge, MA, USA.
(5)University of Massachusetts Medical School and Memorial Medical Center,
Worcester, MA, USA.
(6)College of Veterinary Medicine, Aristotle University, Thessaloniki, Greece.
(7)Clinical Research Center, Institute for Medical Engineering & Science,
Massachusetts Institute or Technology, Cambridge, MA, USA.
(8)Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA,
USA; Civil and Environmental Engineering, Massachusetts Institute of Technology,
Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
(9)Division of Comparative Medicine, Massachusetts Institute of Technology,
Cambridge, MA, USA. Electronic address: [email protected]. |
What causes Scurvy? | Scurvy, or "Barlow's disease", is a widely described disease involving cutaneous and mucosal lesions resulting from vitamin C deficiency. | The human body is unable to synthesise Vitamin C and a diet deficient in Vitamin
C leads to scurvy. Scurvy may mimic other medical conditions, like bleeding
diasthesis or deep vein thrombosis, leading to delay in diagnosis and treatment,
thus prolonging sufferings of patients. Often, scurvy could have been diagnosed
if it is thought of and features of scurvy carefully looked for. Scurvy is
easily treated with high dose of oral vitamin C. Recurrences may occur.
Education of care providers cannot be overemphasised. We report three local
cases of scurvy to highlight the existence of the disease in our modern society. Scurvy, a disease of dietary deficiency of vitamin C, is uncommon today. Among
diseases, scurvy has a rich history and an ancient past. The Renaissance (14th
to 16th centuries) witnessed several epidemics of scurvy among sea voyagers. In
1747, James Lind, a British Naval surgeon, performed a carefully designed
clinical trial and concluded that oranges and lemons had the most antiscorbutic
effect. Eventually, with the provision of lemon juice to the sea voyagers,
scurvy became rare at sea. Infantile scurvy appeared almost as a new disease
toward the end of the 19th century. The increased incidence of infantile scurvy
during that period was attributed to the usage of heated milk and proprietary
foods. Thomas Barlow described the classic clinical and pathologic features of
infantile scurvy in 1883. Between 1907 and 1912, Holst and Frolich induced and
cured scurvy in guinea pigs by dietary modification. In 1914, Alfred Hess
established that pasteurization reduced the antiscorbutic value of milk and
recommended supplementation of fresh fruit and vegetable juices to prevent
scurvy. Such pioneering efforts led to the eradication of infantile scurvy in
the United States. A brief history of infantile scurvy is provided. BACKGROUND: Scurvy is the clinical manifestation of vitamin C deficiency. It is
historically linked to the era of great maritime expeditions. But it is
remerging in Western countries as in France.
SITUATION: Nowadays, scurvy mainly affects homeless populations of large
occidental cities and the isolated and malnourished inhabitants of developing
countries. The clinical presentations of scurvy are numerous and often
misleading and its evolution without treatment is always lethal. After years of
wanderings and research, the physiopathological mechanisms of scurvy were
finally understood, due to the will of outstanding personalities who took the
risk to brave the established superstitions in order to apply a strict medical
approach.
PERSPECTIVES: Scurvy must still be prevented in at risk-populations. Indeed a
pocket meal enriched with vitamin C is distributed to homeless people in Paris. Scurvy is a nondiscriminatory disease process resulting from a nutritional
deficiency of ascorbic acid (vitamin C). The severe vitamin deficiency produces
a breakdown in the cellular structure of the body. This case report describes a
middle-age woman with a history of edema, bruising of the lower extremities,
anemia, and severe periodontal disease. Her presentation and medical history are
classic for the signs of scurvy. Scurvy is now only uncommonly seen in developed
countries, but there are still vulnerable populations whose nutritional status
can lead to scurvy. The aim of this report is to help the clinician identify and
treat scurvy, a disease that was once feared for its high mortality but is now
easily treatable, even in cases that have progressed to multiple organ
dysfunction and failure. Ascorbic acid is a vitamin soluble in water and its deficiency in human body
causes scurvy. Its symptoms in adults are gingivitis, susceptibility of blood
vessels to damage and bleeding, changes in bones and cartilage and retarded
wound healing. Ascorbic acid is necessary in redox processes taking place in
cell. It is reversibly oxidized to dehydroascorbic acid and partially
metabolized to inactive sulphide and oxalic acid, which is expelled in urine. It
is well absorbed from the digestive system and easily reaches the tissues.
Healthy organism contains 1.5 g of ascorbic acid and daily requirement for
ascorbic acid is estimated for 30-100 mg. Ascorbic acid is not synthesized by
humans, but it is an essential dietary vitamin for the species. Ascorbic acid is
used in treatment deficiency in daily demand for vitamin C, caused by improper
diet, poor absorption or cigarette smoking. It is used in large doses in general
weakness, infectious diseases and during the recovery period. Positive results
have been obtained after therapy with vitamin C of Mollera-Barlowa disease,
Schonlein-Henoch disease, Werlhof disease, haemophilia and also in patients with
stable coronary artery disease. Vitamin C is assumed to be a basic antioxidant,
although its role in pathological conditions is controversial. However, it seems
that the complexity of the oxidant-antioxidant system makes the question of
participation of vitamin C (and other scavengers of free radicals) in
pathogenesis of diseases still open. Scurvy is a rarely seen disease resulting from a deficiency of vitamin C. We
present a case of scurvy in a 65-year-old man. The patient reported heavy
alcohol abuse over the last several years. He also reported that his diet
consisted of cheese pizzas only. On physical examination, he was noted to have
spontaneous ecchymosis of his lower extremities (denying any history of trauma);
poor dentition; and corkscrew hairs on his chest, abdomen, and legs, with
associated perifollicular petechia. Punch biopsy of his skin lesions revealed
perivascular lymphohistiocytic inflammation, with some focal perifollicular
erythrocyte extravasation. A serum ascorbic acid level was <0.12 mg/dL (normal
range, 0.20-1.9 mg/dL). A diagnosis of vitamin C deficiency was made. The
patient was successfully treated with 1 g/d vitamin C for the first 5 days,
followed by a dose of 500 mg/d. Though scurvy is rarely seen in modern times, it
is important to identify who is at risk and to recognize the clear and classic
signs and symptoms associated with scurvy. Failure to diagnose this disease can
potentially lead to expensive and unnecessary medical tests, as well as missing
a very simple treatment that can prevent infection and even death. Scurvy is caused by prolonged dietary deficiency of vitamin C, the plasma
concentration of which appears inversely related to mortality from all causes.
Its clinical importance relates principally to its role as a cofactor in a
number of enzyme reactions involved in collagen synthesis, dysfunction of which
disrupts connective tissue integrity, resulting in impaired wound healing and
capillary bleeding. In the UK, overt scurvy is diagnosed only rarely. However,
subclinical vitamin C deficiency appears quite common, one study estimated that
25% of men and 16% of women in the low income/materially deprived population had
vitamin C deficiency, with smoking a strong predictor. Because many of the early
symptoms of vitamin C deficiency (fatigue, malaise, depression and irritability)
are non-specific, the diagnostic possibility of scurvy is usually delayed until
haemorrhagic manifestations occur. The classical cutaneous features consist of
perifollicular purpura, contorted (corkscrew) hairs and follicular
hyperkeratosis, particularly affecting the legs. Large areas of purpura or
ecchymosis may occur. Swelling and bleeding of the gums is an early mucosal
symptom, and progressively severe gum disease causes loss of teeth.
Subperiosteal haemorrhage, particularly in the femur and tibia, can present as
pain, pseudoparalysis, swelling and discoloration of the legs. Haemorrhage into
joints and muscle is very uncomfortable. Diagnosis is generally made on the
basis of clinical features, corroborated by a history of dietary inadequacy, and
the subsequent rapid resolution of symptoms with the restoration of an adequate
vitamin C intake. Vitamin C participates in several physiological processes, among others, immune
stimulation, synthesis of collagen, hormones, neurotransmitters, and iron
absorption. Severe deficiency leads to scurvy, whereas a limited vitamin C
intake causes general symptoms, such as increased susceptibility to infections,
fatigue, insomnia, and weight loss. Surprisingly vitamin C deficiencies are
spread in both developing and developed countries, with the latter actually
trying to overcome this lack through dietary supplements and food fortification.
Therefore new strategies aimed to increase vitamin C in food plants would be of
interest to improve human health. Interestingly, plants are not only living
bioreactors for vitamin C production in optimal growing conditions, but also
they can increase their vitamin C content as consequence of stress conditions.
An overview of the different approaches aimed at increasing vitamin C level in
plant food is given. They include genotype selection by "classical" breeding,
bio-engineering and changes of the agronomic conditions, on the basis of the
emerging concepts that plant can enhance vitamin C synthesis as part of defense
responses. Scurvy, caused by lack of vitamin C, was a major problem for polar explorers. It
may have contributed to the general ill-health of the members of Scott's polar
party in 1912 but their deaths are more likely to have been caused by a
combination of frostbite, malnutrition and hypothermia. Some have argued that
Oates's war wound in particular suffered dehiscence caused by a lack of vitamin
C, but there is little evidence to support this. At the time, many doctors in
Britain overlooked the results of the experiments by Axel Holst and Theodor
Frølich which showed the effects of nutritional deficiencies and continued to
accept the view, championed by Sir Almroth Wright, that polar scurvy was due to
ptomaine poisoning from tainted pemmican. Because of this, any advice given to
Scott during his preparations would probably not have helped him minimise the
effect of scurvy on the members of his party. Vitamin C is an essential dietary nutrient for the biosynthesis of collagen and
a co-factor in the biosynthesis of catecholamines, L-carnitine, cholesterol,
amino acids, and some peptide hormones. The lack of vitamin C causes scurvy, a
pathological condition leading to blood vessel fragility and connective tissue
damage due to failure in producing collagen, and, finally, to death as result of
a general collapse. Vitamin C is potentially involved also in cancer and
cardiovascular diseases prevention. In addition, vitamin C effects on nervous
system and chronically ill patients have been also documented. This review
attempts to summarize recent and well established advances in vitamin C research
and its clinical implications. Since vitamin C has the potential to counteract
inflammation and subsequent oxidative damage that play a major role in the
initiation and progression of several chronic and acute diseases, it represents
a practical tool to administer for the early prevention of these pathologic
conditions. In modern times scurvy is a rarely encountered disease caused by ascorbic acid
(vitamin C) deficiency. However, sporadic cases of scurvy persist, particularly
within the pediatric population. Recent individual case reports highlight an
increased incidence of scurvy among patients with autism or developmental delay,
with isolated case reports detailing the magnetic resoce imaging (MRI)
findings of scurvy in these pediatric populations. We present the MRI findings
of scurvy in four patients with autism or developmental delay, and review the
literature on MRI findings in pediatric patients with scurvy. Despite its
rarity, the radiologist must consider scurvy in a pediatric patient with a
restricted diet presenting with arthralgia or myalgia. Scurvy is caused by prolonged severe dietary deficiency of vitamin C. Being rare
as compared to other nutritional deficiencies, it is seldom suspected and this
frequently leads to delayed recognition of this disorder. Children with abnormal
dietary habits, mental illness or physical disabilities are prone to develop
this disease. The disease spectrum of scurvy is quite varied and includes
dermatological, dental, bone and systemic manifestations. Subperiosteal
hematoma, ring epiphysis, metaphyseal white line and rarefaction zone along with
epiphyseal slips are common radiological findings. High index of suspicion,
detailed history and bilateral limb radiographs aids physician in diagnosing
this eternal masquerader. We searched Pubmed for recent literature (2009-2014)
with search terms "scurvy" "vitamin C deficiency" "ascorbic acid deficiency"
"scurvy and children" "scurvy and pediatric age group". There were a total of 36
articles relevant to pediatric scurvy in children (7 reviews and 29 case
reports) which were retrieved. The review briefly recapitulates the role of
vitamin C, the various disease manifestations and the treatment of scurvy to
create awareness of the disease which still is reported from our country,
although sporadically. The recent advances related to scurvy and its management
in pediatric age group are also incorporated. Collagen is mostly found in fibrous tissues such as tendons, ligaments and skin.
Collagen makes up approximately 30% of the proteins within the body. These are
tough and strong structures found all over the body: in bones, tendons and
ligaments. Collagen being the most abundant protein provides tensile strength
via cell matrix interactions to tissue architecture. Biomimetic materials of
collagen origin gained wide spread acceptance in clinical applications. Vitamin
C deficiency causes scurvy a serious and painful disease in which defective
collagen prevents the formation of strong connective tissue, gums deteriorate
and bleed, with loss of teeth; skin discolors, and wounds do not heal. Effective
collagens prevent the manifestation of such disorders. Polyurethanes on the
other hand are frequently used for various applications as they offered in
wide-ranging of compositions, properties and complex structures. Collagen/PU
bio-composites have potential array for biomedical applications. Considering
versatile properties of the elongated fibrils and wide industrial and biomedical
applications including biocompatibility of polyurethane, this review shed a
light on collagen based polyurethane materials with their potential applications
especially focusing the bio-medical field. Vitamin C is an important antioxidant and cofactor that is involved in the
regulation of development, function, and maintece of several cell types in
the body. Deficiencies in vitamin C can lead to conditions such as scurvy,
which, among other ailments, causes gingivia, bone pain, and impaired wound
healing. This review examines the functional importance of vitamin C as it
relates to the development and maintece of bone tissues. Analysis of several
epidemiological studies and genetic mouse models regarding the effect of vitamin
C shows a positive effect on bone health. Overall, vitamin C exerts a positive
effect on trabecular bone formation by influencing expression of bone matrix
genes in osteoblasts. Recent studies on the molecular pathway for vitamin C
actions that include direct effects of vitamin C on transcriptional regulation
of target genes by influencing the activity of transcription factors and by
epigenetic modification of key genes involved in skeletal development and
maintece are discussed. With an understanding of mechanisms involved in the
uptake and metabolism of vitamin C and knowledge of precise molecular pathways
for vitamin C actions in bone cells, it is possible that novel therapeutic
strategies can be developed or existing therapies can be modified for the
treatment of osteoporotic fractures. Scurvy is a rare disease in developed countries. Risk groups include children
with restricted diets, mainly patients who are autistic or have cerebral palsy.
Furthermore, consumption of plant-based beverages has increased in recent years,
especially in developed countries. When plant-based beverages are the exclusive
diet in the first year of life and not consumed as a supplement to formula or
breastfeeding, it can result in severe nutritional problems. We report a case of
scurvy after exclusive intake of almond beverages and almond flour from 2.5 to
11.0 months of life. The patient was referred for pathologic fractures of the
femur, irritability, and failure to thrive. He had typical radiologic signs of
scurvy, such as osteopenia, cortical thinning, Wimberger ring, Frankel line,
fracture, and periosteal reaction. Moreover, his plasmatic vitamin C level was
very low. The child was diagnosed with scurvy and was started on vitamin C
replacement therapy at a dose of 300 mg per day. Over the following 3 months,
his general condition, the pain in the legs, and the radiologic features
improved; the plasmatic vitamin C level was normalized; and the child started
walking. In summary, this case demonstrates that scurvy is a new and severe
complication of improper use of almond drinks in the first year of life.
Manufacturers should indicate that these beverages are inappropriate for infants
who consume a vitamin C-deficient diet. |
Describe clinical presentation of Ambras syndrome. | Ambras syndrome is a distinct form of congenital hypertrichosis characterized by excessive hair growth over the body and face associated with facial and occasional dental anomalies. In patients with this syndrome, the whole body is covered with fine long hair, except for areas where normally no hair grows. There is accompanying facial dysmorphism and teeth abnormalities, including retarded first and second dentition and absence of teeth. | Congenital hypertrichosis universalis is a rare autosomal domit disease. We
report the further development of a Greek girl, now aged 3 years, the first case
associated with a balanced structural chromosomal aberration. She was described
as a neonate by Sigalas et al. (1990). Her persistent generalized hypertrichosis
is most excessive on the face, ears and shoulders. Her fine silky hair is of the
vellus, not the lanugo type. The syndrome features are characterized, referring
to nine further published case reports. It is distinguished from other types of
congenital hypertrichoses, which have been described in the literature under
different synonyms. To avoid confusion in the terminology, we propose to name
this type of hypertrichosis Ambras syndrome in reference to the first documented
family with congenital hypertrichosis universalis in the 16th century. Ambras syndrome (AS) is a special form of congenital universal hypertrichosis
described for the first time by Baumeister et al. (1). This form differs from
other forms of congenital hypertrichosis in the pattern of hair distribution and
its associated anomalies. The molecular-genetic cause of AS is unknown; the
association of AS with a pericentric inversion (8) (p11.2; q22) described in the
case of Baumeister so far has been unique in the literature. This report is the
tenth with clinical signs of AS so far described in the literature and the
second with an inversion in chromosome 8 and the first with evaluation of
peripheral androgens. The new-born girl presented with abundant and dark hair on
the face and ears, on the shoulders and on the arms; the other parts of the body
were covered with fine, lightly pigmented hair. The face showed many dysmorphic
features. Chromosome analysis showed a paracentric inversion of one chromosome
8. The breakpoints were localised at q12 and q22. The parental karyotypes were
normal. Laboratory investigation showed normal plasma levels of testosterone,
androstenedione (A), 17-hydroxyprogesterone, dehydroepiandrosterone-sulphate
(DHA-S), free testosterone (FT), dihydrotestosterone (DHT) and
3alpha-androstanediol-glucuronide (3AG). Here we report a chromosomal inversion
similar to that found previously not associated with alterations in androgen
plasma levels. Ambras syndrome (AMS) is a unique form of congenital universal hypertrichosis.
The syndrome has been found in association with rearrangements of chromosome 8
in two isolated cases. One of these patients was reported to have an apparently
balanced paracentric inversion of chromosome 8, inv(8)(q12q22). Our cytogenetic
analysis on this patient showed that the rearrangement of chromosome 8 is more
complex than initially reported. We detected an insertion of the q23-q24 region
into a more proximal region of the long arm of chromosome 8 as well as a large
deletion in 8q23:46,XX,
rea(8)(8pter-->8q13::8q23.2-->8q24.1::8q13-->8q23.1::8q24.1-->8qter). Given the
large number of breakpoints and the presence of a substantial deletion, it is
surprising that the proposita did not show anomalies other than these
characteristic of Ambras syndrome. We report two siblings with congenital generalized hypertrichosis and
distinctive facial appearance consistent with the dysmorphic facial features
described in Ambras syndrome. The patients were born to non-consanguineous,
phenotypically normal parents. This is the first report of affected siblings and
could be explained by either autosomal recessive inheritance or by germline
mosaicism for an autosomal domit gene. We compared the phenotype of our
patients to descriptions of reported cases and discuss phenotypic variability. Congenital generalized hypertrichosis terminalis (CGHT) is a heterogenous group
of diseases with continuing excessive growth of terminal hair. "Ambras syndrome"
was first coined by Baumeister in 1993 to describe a case of nonsyndromic CGHT
which was erroneously analogized to the portrait paintings of Petrus Gonzales
and his children, exhibited in Ambras Castle near Innsbruck, Austria. This
family probably, a syndromic type with abnormal dentition, inherited as an
autosomal domit trait. CGHT associated with gingival hyperplasia is probably
a particular entity typified by the historical cases of Julia Pastrana and her
son. An X-linked type of CGHT has likewise been categorized as "Ambras
syndrome". Moreover, some reports have mistakenly classified "Ambras syndrome"
as an example of hypertrichosis lanuginosa. Potential gene loci identified so
far may include 8q22, 17q24.2-q24.3 and Xq24-q27.1. The designation "Ambras
syndrome" has thus been applied to various types of congenital hypertrichosis
that differ to such degree that the name "Ambras" has no specific meaning,
neither in the past nor in the future. Hence, this misleading term should now be
jettisoned. People with rare hypertrichosis syndromes became crowd-drawing money-making
phenomena in many 19th century sideshow acts. These individuals have been
referred to as dog-men, hair-men, and werewolves. In 1993, Baumister et al.
described congenital hypertrichosis lanuginose or Ambras syndrome: a distinct
form of congenital hypertrichosis characterized by excessive hair growth over
the body and face associated with facial and occasional dental anomalies. Much
is not known about this syndrome since fewer than 50 cases have been documented
worldwide. In this case report, a nine year old girl presented with excessive
hair growth throughout her body that was denser along her midline. Furthermore,
her face displayed the typical dysmorphic features characteristic of Ambras
syndrome: a round tip nose, thickened nasal cartilage, antiverted nares,
prominent philtrum with deep groove, and a trapezoid mouth. Oral examination
revealed normal oral mucosa with completely missing and unerupted decidious and
permanent teeth. Panoramic radiographs confirmed unerupted deciduous teeth.
Previous case reports have mentioned the presence of occasional dental anomalies
such as retarded first and second dentition and absence of some teeth. However,
this is the first reported case of Ambras syndrome presenting with complete
anodontia. Prior cytogenetic studies performed on persons with Ambras syndrome
have implicated a balanced pericentric inversion of chromosome 8. However, it is
likely that dental anomalies are likely a result of a different genetic
rearrangement. Further studies are needed to explore the cause of this rare
phenotype of Ambras syndrome with complete unerupted dentition. Congenital generalized hypertrichosis associated with gingival hyperplasia are
rare cases published in literature. The frequency incidence of generalized
congenital hypertrichosis is about one to billions of people. Hypertrichosis and
gingival hyperplasia are termed as Ambras syndrome (AS), which can be noticed at
birth or soon after. Here, is a rare case report of 4-year-old male child who
presented with generalized hypertrichosis with gingival fibromatosis and
dysmorphic facial features. Ambras syndrome is a rare and special form of congenital hypertrichosis,
characterized by dysmorphic facial features and familial pattern of inheritance.
It is rarely associated with gingival hyperplasia. We report such a rare entity
in a 38-year-old female patient with a history of consanguinity and positive
family history. |
Which diseases are involved in the severe cutaneous reactions (SCAR) spectrum? | The diseases that are involved in the severe cutaneous reactions (SCAR) spectrum are:
1) Stevens-Johnson syndrome (SJS)
2) Toxic epidermal necrolysis (TEN)
3) Acute generalized exanthematous pustulosis (AGEP). | A multicenter international case-control study has been designed to elucidate
the etiology of Stevens-Johnson syndrome and toxic epidermal necrolysis (TEN).
Although these diseases occur rarely, the morbidity is high and the mortality
for TEN is of the order of 30%. These serious dermatologic conditions have often
been linked to exposure to drugs. Infective and autoimmune diseases, as well as
other non-drug risk factors, have also been postulated to be of importance in
increasing the risk. The design and methods are described, with particular
attention to the unique challenges for an epidemiologic study of these
conditions. Patch testing may help to assess the culpability of a drug in an adverse
reaction. Our aim was to study patch testing in severe cutaneous adverse drug
reactions (ADRs) (Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN),
acute generalized exanthematous pustulosis (AGEP), and other cutaneous ADRs). 59
patients with cutaneous ADRs were included: 22 had SJS/TEN, 14 AGEP, and 23
other cutaneous ADRs. Patients were patch tested with the suspect drug, and with
a standard series of drugs. 2 patients among the 22 SJS/TEN cases had a relevant
positive test. 7 patients among the 14 AGEP cases had a relevant positive test.
6 patients among the 23 other cutaneous ADRs had a relevant positive test. Our
results suggest that patch testing has a weak sensitivity in SJS/TEN and is not
appropriate in these diseases. Patch testing seems more adapted to other
cutaneous ADRs, such as AGEP, in which the proportion of positive patch tests
was significantly higher (p < 0.02). Nevertheless, the difference of sensitivity
of patch testing in SJS/ TEN, AGEP or other cutaneous ADRs could be linked not
only to the clinical type of eruption, but also to the different spectrum of
culprit drugs in each type of eruption. Allopurinol, a commonly prescribed medication for gout and hyperuricemia, is a
frequent cause of severe cutaneous adverse reactions (SCAR), which include the
drug hypersensitivity syndrome, Stevens-Johnson syndrome, and toxic epidermal
necrolysis. The adverse events are unpredictable and carry significant morbidity
and mortality. To identify genetic markers for allopurinol-SCAR, we carried out
a case-control association study. We enrolled 51 patients with allopurinol-SCAR
and 228 control individuals (135 allopurinol-tolerant subjects and 93 healthy
subjects from the general population), and genotyped for 823 SNPs in genes
related to drug metabolism and immune response. The initial screen revealed
strong association between allopurinol-SCAR and SNPs in the MHC region,
including BAT3 (encoding HLA-B associated transcript 3), MSH5 (mutS homolog 5),
and MICB (MHC class I polypeptide-related sequence B) (P < 10(-7)). We then
determined the alleles of HLA loci A, B, C, and DRB1. The HLA-B*5801 allele was
present in all (100%) 51 patients with allopurinol-SCAR, but only in 20 (15%) of
135 tolerant patients [odds ratio 580.3 (95% confidence interval, 34.4-9780.9);
corrected P value = 4.7 x 10(-24)] and in 19 (20%) of 93 of healthy subjects
[393.51 (23.23-6665.26); corrected P value = 8.1 x 10(-18)]. HLA alleles A*3303,
Cw*0302, and DRB1*0301 were in linkage disequilibrium and formed an extended
haplotype with HLA-B*5801. Our results indicated that allopurinol-SCAR is
strongly associated with a genetic predisposition in Han Chinese. In particular,
HLA-B*5801 allele is an important genetic risk factor for this life-threatening
condition. Skin is the most frequent target of drug reactions that are reported, may be
because they are easily detected. Most (probably more than 90%) are related to
drug hypersensitivity, i.e. an individually tailored, unexpected effect mediated
by a drug specific activation of the immune response. The clinical presentation
of "drug eruptions" is highly variable, from the most common transient and
benign erythema that occurs 6-9 days after the introduction of a new drug in 1
to 3 % of users to the most severe forms, that fortunately affect less than
1/10,000 users. Even though there are some overlapping or unclassifiable cases,
it is important for clinicians to recognize and categorize severe cutaneous
adverse reactions/SCAR (bullous fixed drug eruptions/bFDE, acute generalized
exanthematous pustulosis/AGEP, drug reaction with eosinophilia and systemic
symptoms/DRESS, Stevens-Johnson syndrome/SJS, toxic epidermal necrolysis/TEN).
First they must suspect rapidly that an unusual eruption with high fever and
severe constitutional symptoms is caused by a medication and not by an
infection. Second they have to look for involvement of organs that differ
according to the type of reaction. Third they can determine a prognosis, the
mortality rate being virtually 0 for bFDE, 5% for AGEP, 10% for
"hypersensitivity syndrome"/DRESS and 25% for SJS or TEN. In addition if some
medications are "usual suspects" for all types (e.g. anticonvulsants), some
other are more specific of a given pattern (pristinamycine, hydroxychloroquine,
diltiazem for AGEP, minocycline for DRESS, anti-infectious sulfonamides,
allopurinol for epidermal necrolysis). The "phenotypic" diversity of the final
expression drug reactions can be explained by the engagement of a variety of
cytokines and inflammatory cells and by regulatory mechanisms. For example,
memory cytotoxic T-Cells are key effectors in both localized blisters of bFDE
and in extensive blisters of epidermal necrolysis. Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are
considered part of a spectrum of adverse cutaneous drug reactions showing severe
and extensive skin detachment. TEN and SJS are morphologically characterized by
active apoptotic keratinocyte cell death that results in the separation of the
epidermis from the dermis. TEN is a life-threatening disease with a high
mortality rate (20-30%). Although several therapies have been tried, there is no
specific outstanding of generally accepted treatment for TEN at present. The
pathogeneses of TEN and SJS have not yet been fully elucidated. We have
demonstrated that high concentrations of soluble FasL (sFasL) are detected in
TEN/SJS patients' serum samples and sFasL secreted by peripheral blood
mononuclear cells interacts with the Fas expressed on diseased keratinocytes in
TEN/SJS. Our data suggested sFasL is a prime candidate for therapeutic
intervention, whereas a few recent papers have reported sFasL levels were not
elevated in some TEN patients. An urgent review of the pathophysiology in
TEN/SJS is needed to resolve this issue and to determine more effective
treatment regimes. BACKGROUND: The severe adverse cutaneous reactions of erythema multiforme (EM),
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare
mucocutaneous diseases associated with significant morbidity and mortality. The
most common cause is antiepileptic drugs, particularly carbamazepine and
lamotrigine, as well as the barbiturates group (phenobarbital and phenytoin). In
this article, we present seven children with severe adverse cutaneous reactions
caused by barbiturates.
CASE REPORTS: The age of the affected children was between 2 and 11 years and
they all had a history of taking barbiturates. Their symptoms started 1-3 weeks
after the initiation of barbiturates, including a prodrome characterized by 2-3
days of malaise, fever, cough and anorexia, after which the skin and mucosal
lesions appeared and worsened. The skin lesions varied from rash to large
bullae, plus different forms of mucous membrane involvement. The offending drugs
(barbiturates) were stopped immediately and care was largely supportive.
CONCLUSION: As a result of the morbidity and/or mortality associated with EM,
SJS and TEN, physicians should keep in mind their differential diagnosis when
cutaneous reactions are observed in patients undergoing barbiturate therapy.
Furthermore, although TEN and SJS are life-threatening diseases, early detection
and appropriate care can lead to a decrease in the incidence of death. The
strategies described here seem to be successful and safe because, despite the
serious conditions, our patients responded well. All survived. Toxic epidermal necrolysis (TEN) and Stevens Johnson Syndrome (SJS) are severe
adverse cutaneous drug reactions that predomitly involve the skin and mucous
membranes. Both are rare, with TEN and SJS affecting approximately 1or
2/1,000,000 annually, and are considered medical emergencies as they are
potentially fatal. They are characterized by mucocutaneous tenderness and
typically hemorrhagic erosions, erythema and more or less severe epidermal
detachment presenting as blisters and areas of denuded skin. Currently, TEN and
SJS are considered to be two ends of a spectrum of severe epidermolytic adverse
cutaneous drug reactions, differing only by their extent of skin detachment.
Drugs are assumed or identified as the main cause of SJS/TEN in most cases, but
Mycoplasma pneumoniae and Herpes simplex virus infections are well documented
causes alongside rare cases in which the aetiology remains unknown. Several
drugs are at "high" risk of inducing TEN/SJS including: Allopurinol,
Trimethoprim-sulfamethoxazole and other sulfonamide-antibiotics,
aminopenicillins, cephalosporins, quinolones, carbamazepine, phenytoin,
phenobarbital and NSAID's of the oxicam-type. Genetic susceptibility to SJS and
TEN is likely as exemplified by the strong association observed in Han Chinese
between a genetic marker, the human leukocyte antigen HLA-B*1502, and SJS
induced by carbamazepine. Diagnosis relies mainly on clinical signs together
with the histological analysis of a skin biopsy showing typical full-thickness
epidermal necrolysis due to extensive keratinocyte apoptosis. Differential
diagnosis includes linear IgA dermatosis and paraneoplastic pemphigus, pemphigus
vulgaris and bullous pemphigoid, acute generalized exanthematous pustulosis
(AGEP), disseminated fixed bullous drug eruption and staphyloccocal scalded skin
syndrome (SSSS). Due to the high risk of mortality, management of patients with
SJS/TEN requires rapid diagnosis, evaluation of the prognosis using SCORTEN,
identification and interruption of the culprit drug, specialized supportive care
ideally in an intensive care unit, and consideration of immunomodulating agents
such as high-dose intravenous immunoglobulin therapy. SJS and TEN are severe and
life-threatening. The average reported mortality rate of SJS is 1-5%, and of TEN
is 25-35%; it can be even higher in elderly patients and those with a large
surface area of epidermal detachment. More than 50% of patients surviving TEN
suffer from long-term sequelae of the disease. Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are
represented by rare but life-threatening cutaneous adverse reactions to
different drugs. Previous studies have found that in a Han Chinese population
from Taiwan and other Asian Countries, a strong genetic association between
HLA-class I alleles (B*15:02, B*58:01) and SJS and TEN was induced by
carbamazepine and allopurinol, respectively. To identify genetic markers that
covered the MHC region, we carried out a case-control association enrolling 20
Caucasian patients with SJS/TEN. Our patient series included 10 cases related to
paracetamol, 7 to allopurinol and 3 to different drugs (plaquenil, itraconazol,
nabumetone). Healthy controls were represented by 115 Caucasian bone marrow or
stem cell donors. The HLA-A*, B*, C*, DRB1*, DQB1*, DQA1* and DPB1* genotyping
were determined. The frequencies of HLA-A*33:03 as well as C*03:02 and C*08:01
were significantly higher in SJS/TEN patient subgroup showing allopurinol
drug-induced severe cutaneous adverse reactions (SCAR) as compared to controls
(28.6% vs 0%, P=0.00002, Pc=0.0011; 28.6% vs 0%, P=0.00002, Pc=0.001; 28.6% vs
0%, P=0.00002, Pc=0.001, respectively). In the same subgroup the frequencies of
B*58:01, DRB1*15:02 and DRB1*13:02 alleles, although considerably higher than in
control group (42.8% vs 5.2%, P=0.003; 28.6% vs 1.7%, P=0.005; 28.6% vs 3.5%,
P=0.037, respectively), appeared no more statistically different after P
correction (Pc=0.248; Pc=0.29; Pc=1.00, respectively). In addition, in 10 of the
20 SJS/TEN patient subgroup with paracetamol-induced SCAR no statistically
significant association with HLA alleles could be found. However, in the same
SJS/TEN patient subgroup showing allopurinol drug-induced SCAR, haplotype
analysis indicated that B*58:01, DRB1*13:02 and DRB1*15:02 alleles, that in a
single allele analysis lost statistical significance after P correction, may
still confer susceptibility, because the B*58:01-DRB1*13:02 and
DRB1*15:02-DQB1*05:02 are positively associated with the disease (14.2% vs
0.43%, P= 0.00001, Pc=0.00028; 14.2% vs 0.43%, P=0.00001, Pc=0.00028,
respectively). Our results show that in contrast to SCAR-related to paracetamol,
where HLA alleles do not appear to be involved, HLA molecules behave as a strong
risk factor for SCAR-related to allopurinol even when a limited number of
patients are considered. BACKGROUND: Although the US FDA recommends screening for HLA-B*1502 allele in
most of Asian ancestry before initiating carbamazepine therapy, the HLA
associations with carbamazepine hypersensitivity in non-Chinese Asian
populations remain unclear. This study investigated the association between the
HLA class I genotype and carbamazepine-induced severe cutaneous adverse reaction
(SCAR) in Koreans.
METHODS: Twenty-four patients who had developed carbamazepine-induced SCAR (7
Stevens-Johnson syndrome (SJS), 17 drug hypersensitivity syndrome (HSS)), 50
carbamazepine-tolerant controls from the Korean Pharmacogenetic Adverse Drug
Reaction Research Network and data of 485 Korean general population from a
previously published study were recruited. HLA-A, -B, and -C genotyping was
performed by direct DNA sequence analysis.
RESULTS: Only one of the seven SJS patients was positive for the B*1502 allele,
but the frequency of B*1511 was much higher in the patients with CBZ-SJS than in
the CBZ-tolerant control patients (P=0.011, P(c)=not significant;
OR=18.0(2.3-141.2)). The frequencies of A*3101 in carbamazepine-induced HSS and
SCAR were significantly higher than those in carbamazepine-tolerant controls
(P(c)=0.011, OR=8.8(2.5-30.7) and P(c)=0.013, OR=7.3(2.3-22.5), respectively).
The frequencies of B*1511 in carbamazepine-SJS and A*3101 in
carbamazepine-HSS/SCAR were significantly higher than those in the general
population.
CONCLUSIONS: HLA-B*1502 does not seem to be an effective predictive marker for
carbamazepine-induced SCAR, while HLA-B*1511 and A*3101 was associated with
carbamazepine-induced SJS and HSS/SCAR respectively in the Korean population. BACKGROUND: Drug patch tests (PTs) can reproduce delayed hypersensitivity to
drugs and entail a moderate re-exposure of patients to offending drugs.
OBJECTIVES: To determine the value of PTs for identifying the responsible drug
in severe cutaneous adverse drug reactions (SCARs) such as acute generalized
exanthematous pustulosis (AGEP), drug reaction with eosinophilia and systemic
symptoms (DRESS) and Stevens-Johnson syndrome/toxic epidermal necrolysis
(SJS/TEN).
METHODS: In a multicentre study, PTs were conducted on patients referred for
DRESS, AGEP or SJS/TEN within 1 year of their SCAR. All drugs administered in
the 2 months prior to and the week following the onset of the SCAR were tested.
RESULTS: Among the 134 patients included (48 male, 86 female; mean age 51·7
years), positive drug PTs were obtained for 24 different drugs. These included
positive tests for 64% (46/72) of patients with DRESS, 58% (26/45) of those with
AGEP and 24% (4/17) of those with SJS/TEN, with only one relapse of AGEP. The
value of PTs depended on the type of drug and the type of SCAR (e.g.
carbamazepine was positive in 11/13 DRESS cases but none of the five SJS/TEN
cases). PTs were frequently positive for beta lactams (22 cases), pristinamycin
(11 cases) and in DRESS with pump proton inhibitors (five cases), but were
usually negative for allopurinol and salazopyrin. Of 18 patients with DRESS,
eight had virus reactivation and positive PTs. In DRESS, multiple drug
reactivity was frequent (18% of cases), with patients remaining sensitized many
years later.
CONCLUSIONS: PTs are useful and safe for identifying agents inducing SCAR. Allopurinol is the most commonly used drug for the treatment of hyperuricemia
and gout. However, allopurinol is also one of the most common causes of severe
cutaneous adverse reactions (SCARs), which include drug hypersensitivity
syndrome, Stevens–Johnson syndrome, and toxic epidermal necrolysis. A variant
allele of the human leukocyte antigen (HLA)-B, HLA-B*58:01, associates strongly
with allopurinolinduced SCAR. We have summarized the evidence from the published
literature and developed peer-reviewed guidelines for allopurinol use based on
HLA-B genotype. Severe Cutaneous Adverse Reaction (SCAR) represents the spectrum of adverse drug
reactions from erythema multiforme, Stevens - Johnson syndrome (SJS) to Toxic
Epidermal Necrolysis (TEN). A 55 year old lady presented in a toxic state with
peeling of skin, blisters on the body of seven days duration following
medications taken for fever and pulmonary tuberculosis. When referred to our
institution, she was diagnosed as TEN. Immediately the suspected medications
were stopped. The essential investigations were done including the screening for
immunosuppression, which was found to be negative. The patient was treated
symptomatically with emphasis on skilled nursing care. The patient's skin
condition improved gradually but tuberculosis progressively worsened over three
months. Thus patient was reinvestigated for seropositivity and was found to be
positive. Considering the benefit - risk ratio along with the advice of the
pulmonologist, a decision was made to give her a rechallenge test, first for
antitubercular drugs and later for antipyretics. The patient developed SJS
within two days of starting isoniazid (INH). On withdrawal of INH the patient
recovered. BACKGROUND: Aromatic anticonvulsant-induced severe cutaneous adverse drug
reactions (SCARs), including Stevens-Johnson syndrome (SJS), toxic epidermal
necrosis (TEN), and drug rash with eosinophilia and systemic symptoms (DRESS),
are fatal immune-mediated adverse drug reactions. CYP2C19, a cytochrome P450
isoform, plays a role in metabolic rate of aromatic anticonvulsant. HLA-B*1502
has also been demonstrated to be associated with carbamazepine-induced SJS-TEN.
METHODS: Forty case patients who were diagnosed with SCARs after initiation of
phenobarbital (PB), phenytoin (PHT), or carbamazepine (CBZ) for 1-8 wk and forty
control patients who received PB, PHT, or CBZ at least 2 months with no adverse
drug reactions were enrolled in the study. The genotypes of CYP2C19*1,
CYP2C19*2, and HLA-B*1502 were analyzed using allele-specific polymerase chain
reaction technique. Clinical characteristics of SCARs patients who used
different drugs were also analyzed.
RESULTS: There was no significant difference in sex, onset of symptoms,
laboratory results, treatment, and length of stay among patients with SCARs due
to PB, PHT, or CBZ. The patients with CYP2C19*2 variant had a trend to have a
likelihood to develop SCARs more than the patients with CYP2C19 wild type
(OR = 2.5, 95% CI (0.96-67.3) p = 0.06). In subgroup analysis, the patients with
CYP2C19*2 variant were at four times increased risk of SCARs from phenobarbital
more than the patients with CYP2C19 wild type (OR = 4.5, 95% CI (1.17-17.37)
p < 0.03). There was no association between the HLA-B*1502 and aromatic
anticonvulsant-induced severe cutaneous adverse reactions (SCARs).
CONCLUSION: CYP2C19*2 variant may play a role in the genetic predisposition of
SCARs from phenobarbital. BACKGROUND: The cutaneous manifestations of human enterovirus (HEV) infection
are usually limited, such as hand-foot-mouth disease. By comparison,
Stevens-Johnson syndrome (SJS) is a life-threatening severe cutaneous adverse
reaction (SCAR), mainly caused by drugs. During the HEV outbreaks in 2010-2012
in Taiwan, we identified 21 patients who developed widespread blistering
mucocutaneous reactions without any suspected drug causality.
METHODS: We screened possible pathogen(s) for detecting human herpes virus
(HHV1-HHV7), HEV, or Mycoplasma pneumoniae infections using throat swab virus
cultures, real-time PCR, DNA sequencing, immunochemistry and electron microscopy
analyses.
RESULTS: Coxsackievirus A6 (CVA6) DNA was identified in the blistering skin
lesions in 6 of 21 patients. Cytotoxic T lymphocytes and natural killer cells
expressing granulysin predomitly infiltrated into the skin lesions, sharing
the histopathological features with SJS. Intact CVA6 viral particles were
identified in the blister fluids and skin lesions by electron microscopy. The
phylogenetic analysis of the viral genome showed the CVA6 DNA sequence sharing
higher similarity (97.6%-98.1%) to CVA6 strains reported from Finland at 2008.
CONCLUSIONS: This study identifies a new variant of CVA6 as the causative agent
for severe mucocutaneous blistering reactions mimicking SCAR. An awareness of
this unusual presentation of HEV infection is needed in the epidemic area. BACKGROUND: Severe cutaneous adverse reactions (SCARs) include Stevens-Johnson
syndrome (SJS), toxic epidermal necrolysis (TEN), and drug reaction with
eosinophilia and systemic symptoms (DRESS). SJS and TEN (SJS/TEN) and DRESS are
thought to be different diseases; however, they share some clinical and
laboratory features. Although SCORTEN serves as an excellent prognostic marker
for SJS/TEN, there is still a need for development of other prognostic markers
for SCARs.
METHODS: The study population consisted of 88 SCAR patients. Clinical
characteristics and clinical manifestations were compared between SJS/TEN and
DRESS. Risk factor analyses for prolonged hospitalization were performed.
RESULTS: Of the 88 patients, 41 were SJS/TEN and 47 were DRESS. Mortality rates
of TEN and DRESS were 9.8 and 2.1%, respectively. Allopurinol and carbamazepine
were the most common causes of both SJS/TEN and DRESS (34.7 and 62.9%,
respectively). Some of the systemic presentations, such as fever and laboratory
abnormalities were common in both phenotypes. Thrombocytopenia tended to be
related to prolonged hospitalization (longer than 3 weeks) in SJS/TEN (odds
ratio, OR = 5.1, 95% confidence interval, CI 0.8-31.8, p = 0.076). In DRESS
patients, leukocytosis at presentation (OR 4.8, 95% CI 1.1-20.3, p = 0.03) was
related to prolonged hospitalization.
CONCLUSIONS: Clinical features of SCARs in a tertiary hospital in Korea were
similar to those reported previously. SJS/TEN and DRESS shared some clinical and
laboratory features. Thrombocytopenia for SJS/TEN and leukocytosis at
presentation for DRESS may be useful prognostic markers for prolonged
hospitalization. HLA-A*31:01 was reported to be associated with carbamazepine (CBZ)-induced
severe cutaneous adverse reactions (SCAR), including drug reaction with
eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome (SJS) and
toxic epidermal necrolysis (TEN). We conducted an international study using
consensus diagnosis criteria to enroll a total of 93 patients with CBZ-SCAR from
Europe or Asia. We found that HLA-A*31:01 showed a significant association with
CBZ-DRESS in Europeans (P<0.001; odds ratio (OR) (95% confidence interval
(CI))=57.6 (11.0-340)), and the strong association was also found in Chinese
(P<0.001; OR (95% CI)=23.0 (4.2-125)). However, HLA-A*31:01 had no association
with CBZ-SJS/TEN in neither Chinese nor Europeans. By comparison, HLA-B*15:02
showed a strong association with CBZ-SJS/TEN in Chinese (P<0.001, OR (95%
CI)=58.1 (17.6-192)). A meta-analysis of this and other published studies
confirmed that in all populations, HLA-A*31:01 had an extremely strong
association with CBZ-DRESS (P<0.001, a pooled OR (95% CI)=13.2 (8.4-20.8)), but
a much weaker association with CBZ-SJS/TEN (P=0.01, OR (95% CI)=3.94
(1.4-11.5)). Our data revealed that HLA-A*31:01 is a specific predictor for
CBZ-DRESS but not for CBZ-SJS/TEN. More studies are needed to investigate the
genetic determit of CBZ-SJS/TEN in Europeans. Considering the potential
clinical utility, the cost-effectiveness of the combined HLA-A*31:01 and
HLA-B*15:02 genetic test to prevent CBZ-SCAR in Chinese needs further
investigation. Different responses, in terms both of efficacy and toxicity, are commonly
observed for any drug administered to apparently homogeneous groups of patients.
It is estimated that adverse drug reactions (ADRs) cause 3-6% of all
hospitalizations, accounting for 5% to 9% of hospital admission costs. The skin
is often involved in ADRs and although most cutaneous ADRs have a favorable
course, they may present as severe adverse cutaneous drug reactions (SCARs),
such as Stevens-Johnson syndrome, toxic epidermal necrolysis, drug reaction with
eosinophilia and systemic symptoms (also referred to as drug-induced
hypersensitivity syndrome), and acute generalized exanthematous pustulosis.
SCARs are associated with significant mortality and require prompt diagnosis and
adequate treatment. Pharmacogenetics studies individual variants in the DNA
sequence associated with drug efficacy and toxicity, allowing prescription of a
drug to patients expected to benefit from it, and excluding from treatment those
who are at risk of developing ADRs. Pharmacogenetics already achieved several
important results in the prevention of SCARs, and pharmacogenetic testing is now
recommended by regulatory agencies before administration of abacavir and
carbamazepine, leading to reduced incidence of SCARs. In this review, the
pharmacogenetic associations of SCARs that have been validated in independent,
case-control association studies will be presented. By familiarizing with
principles of pharmacogenetics, dermatologists should be able to correlate
specific cutaneous ADR phenotypes to the underlying genotype, thus contributing
to better drug safety and facilitating drug discovery, development and approval. BACKGROUND: Severe cutaneous adverse reactions (SCAR) are rare but important
causes of morbidity and mortality. Stevens-Johnson syndrome (SJS), Toxic
Epidermal Necrolysis (TEN), and drug reaction with eosinophilia and systemic
symptoms (DRESS) are severe cutaneous drug reactions that can be potentially
life threatening. Our study aims to look at the epidemiology of SCAR in the
local setting in Singapore and the underlying characteristics of our patients
that may influence the drug reaction seen.
METHODS: Data was collected retrospectively from in-patient records in the
period of January 2007 to December 2011. We looked at several factors: (i)
patient demographics including age, gender, ethnicity, comorbidities, (ii)
culprit drug(s), (iii) latent period, (iv) drug reaction observed, (v) systemic
complications, (vi) length of hospital stay, (vii) treatment given, and (viii)
outcomes (mortality, morbidity).
RESULTS: We collected data from 42 patients. The mean age of our patients was
51.8 years. Twenty-nine (69%) of the patients had underlying comorbidities. The
most common culprit drug group was antibiotics. SJS was the most common SCAR
observed (54.8%), followed by acute generalized exanthematous pustolosis (AGEP;
24%), TEN (11.9%), and DRESS (2%). Sixteen patients (38.1%) had complications,
and there was one reported death. There was a weak correlation (correlation
coefficient 0.29, P value = 0.15, 95% CI = 2.07) between early steroid therapy
and the length of stay.
CONCLUSIONS: Antibiotics are the most common culprit drugs. The most common SCAR
observed in our study was SJS. Early initiation of steroids may lead to a more
favorable outcome. OBJECTIVE: Allopurinol, an antihyperuricaemic agent, is one of the common causes
of life-threatening severe cutaneous adverse reactions (SCAR), including drug
rash with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome
(SJS) and toxic epidermal necrosis (TEN). The prognostic factors for
allopurinol-related SCAR remain unclear. This study aimed to investigate the
relationship of dosing, renal function, plasma levels of oxypurinol and
granulysin (a cytotoxic protein of SJS/TEN), the disease severity and mortality
in allopurinol-SCAR.
METHODS: We prospectively enrolled 48 patients with allopurinol-SCAR (26 SJS/TEN
and 22 DRESS) and 138 allopurinol-tolerant controls from 2007 to 2012. The human
leucocyte antigen (HLA)-B*58:01 status, plasma concentrations of oxypurinol and
granulysin were determined.
RESULTS: In this cohort, HLA-B*58:01 was strongly associated with
allopurinol-SCAR (p<0.001, OR (95% CI) 109 (25 to 481)); however, the
initial/maintece dosages showed no relationship with the disease. Poor renal
function was significantly associated with the delayed clearance of plasma
oxypurinol, and increased the risk of allopurinol-SCAR (p<0.001, OR (95% CI) 8.0
(3.9 to 17)). Sustained high levels of oxypurinol after allopurinol withdrawal
correlated with the poor prognosis of allopurinol-SCAR. In particular, the
increased plasma levels of oxypurinol and granulysin linked to the high
mortality of allopurinol-SJS/TEN (p<0.01), and strongly associated with
prolonged cutaneous reactions in allopurinol-DRESS (p<0.05).
CONCLUSIONS: Impaired renal function and increased plasma levels of oxypurinol
and granulysin correlated with the poor prognosis of allopurinol-SCAR.
Allopurinol prescription is suggested to be avoided in subjects with renal
insufficiency and HLA-B*58:01 carriers. An early intervention to increase the
clearance of plasma oxypurinol may improve the prognosis of allopurinol-SCAR. Publisher: La syndrome de Stevens-Johnson (SJS) et la nécrolyse épidermique
toxique sont des maladies dans le spectre de réactions cutanées graves affectant
la peau et les muqueuses. Les médicaments antiépileptiques sont utilisés en
combinaison, et ceci peut provoquer des effets indésirables. Ici, nous
rapportons un cas rare de SJS déclenché par une combinaison de clobazam, la
lamotrigine et l’acide valproïque chez un garçon de 7 ans. En raison de l’
insuffisante maîtrise des crises, le lorazépam a été utilisé avec le clobazam.
Quatre semaines après l’ajout de clobazam, le patient a développé SJS avec une
éruption cutanée généralisée, de la fièvre. Il y avait la participation de la
foie et les reins, et une éosinophilie, une semaine après le début du
traitement. Tous les médicaments antiépileptiques ont été abandonnées, et la
méthylprednisolone intraveineuse, des antibiotiques systémiques prophylactiques,
supplément de liquide par voie intraveineuse, antipyrétique, les soins des
plaies spécial, et de soutien pour les soins médicaux ont été administrés. Il a
été libéré dans un état stable la 18ème journée. Notre cas suggère qu’une
interaction médicamenteuse entre le valproate, la lamotrigine et clobazam ait
contribué au développement de SJS. Lorsque le clobazam a été ajouté à l’acide
valproïque et la lamotrigine, la dose de lamotrigine aurait dû être diminué. AIM: The study sought to identify the magnitude and characteristic of severe
cutaneous adverse reactions (SCAR's) like Steven-Johnson syndrome (SJS) and
Toxic Epidermal Necrolysis (TEN).
MATERIALS AND METHODS: A prospective study was conducted by the Department of
Pharmacology in association with Department of Dermatology in SMHS hospital. The
study was carried out from June 2013-June 2015 on hospitalized cases of
cutaneous adverse drug reaction reporting in hospital. The SCAR's were reported
in a structured questionnaire based on adverse drug reaction (ADR) reporting
form provided by the Central Drug Standard Control Organization (CDSCO) Ministry
of Health and Family welfare, Government of India. The SCAR's were analysed for
their characteristics, causality, severity and prognosis. Causality assessment
was done by using a validated ADR probability scale of Naranjo as well as WHO
Uppsala Monitoring Center (WHO-UMC) system for standardized case causality
assessment. The management protocol were analysed for their clinical outcome
through a proper follow up period.
RESULTS: A total of 52 hospitalized cases of cutaneous adverse drug reactions
were reported during the study period. We identified a total of 15 cases (28%)
of SCAR's involving 9(17%) of SJS and 6 (12%) of TEN. SJS was seen in 2(22%)
males and 7(78%) females. TEN was seen in all females (100%) and in no male.
Drugs implicated in causing these life threatening reactions were identified as
anticonvulsant agents like carbamazepine (CBZ), phenytoin (PHT) and Lamotrigine
(LTG), oxicam NSAID, Sulfasalazine and levofloxacin. Despite higher reported
mortality rates in SJS and TEN all patients survived with 2 patients surviving
TEN suffered from long term opthalmological sequelae of the disease.
CONCLUSION: Present study suggest that drug induced cutaneous eruptions are
common ranging from common nuisance rashes to rare life threatening diseases
like SJS and TEN, SJS/TEN typically occur 1-3 weeks after initiation of therapy.
Aromatic AED's, LTG, oxicam NSAID's, sulfasalazine and levofloxacin have a
tremendous potential to trigger SCARS's. To ensure safe use of pharmaceutical
agents and better treatment outcomes post marketing voluntary reporting of
severe rare and unusual reactions remains inevitable. The clinical manifestations of drug eruptions can range from mild maculopapular
exanthema to severe cutaneous adverse drug reactions (SCAR), including
drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and
systemic symptoms, Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis
(TEN) which are rare but occasionally fatal. Some pathogens may induce skin
reactions mimicking SCAR. There are several models to explain the interaction of
human leukocyte antigen (HLA), drug and T-cell receptor (TCR): (i) the
"hapten/prohapten" theory; (ii) the "p-i concept"; (iii) the "altered peptide
repertoire"; and (iv) the "altered TCR repertoire". The checkpoints of molecular
mechanisms of SCAR include specific drug antigens interacting with the specific
HLA loci (e.g. HLA-B*15:02 for carbamazepine-induced SJS/TEN and HLA-B*58:01 for
allopurinol-induced SCAR), involvement of specific TCR, induction of
T-cell-mediated responses (e.g. granulysin, Fas ligand, perforin/granzyme B and
T-helper 1/2-associated cytokines) and cell death mechanism (e.g.
miR-18a-5p-induced apoptosis; annexin A1 and formyl peptide receptor 1-induced
necroptosis in keratinocytes). In addition to immune mechanism, metabolism has
been found to play a role in the pathogenesis of SCAR, such as recent findings
of strong association of CYP2C9*3 with phenytoin-induced SCAR and impaired renal
function with allopurinol SCAR. With a better understanding of the mechanisms,
effective therapeutics and prevention for SCAR can be improved. |
Which tool employs self organizing maps for analyzing synonymous codon usage? | INteractive Codon usage Analysis (INCA) provides an array of features useful in analysis of synonymous codon usage in whole genomes. In addition to computing codon frequencies and several usage indices, such as 'codon bias', effective Nc and CAI, the primary strength of INCA has numerous options for the interactive graphical display of calculated values, thus allowing visual detection of various trends in codon usage. Finally, INCA includes a specific unsupervised neural network algorithm, the self-organizing map, used for gene clustering according to the preferred utilization of codons. | |
What is MINDY-1? | MINDY-1 (motif interacting with Ub-containing novel DUB family) is a member of an evolutionarily conserved and structurally distinct new family of deubiquitinating enzymes. Found in all eukaryotes, MINDY-family DUBs are highly selective at cleaving K48-linked polyUb, a signal that targets proteins for degradation. MINDY-1 prefers cleaving long polyUb chains and works by trimming chains from the distal end. | Deubiquitinating enzymes (DUBs) remove ubiquitin (Ub) from Ub-conjugated
substrates to regulate the functional outcome of ubiquitylation. Here we report
the discovery of a new family of DUBs, which we have named MINDY (motif
interacting with Ub-containing novel DUB family). Found in all eukaryotes,
MINDY-family DUBs are highly selective at cleaving K48-linked polyUb, a signal
that targets proteins for degradation. We identify the catalytic activity to be
encoded within a previously unotated domain, the crystal structure of which
reveals a distinct protein fold with no homology to any of the known DUBs. The
crystal structure of MINDY-1 (also known as FAM63A) in complex with
propargylated Ub reveals conformational changes that realign the active site for
catalysis. MINDY-1 prefers cleaving long polyUb chains and works by trimming
chains from the distal end. Collectively, our results reveal a new family of
DUBs that may have specialized roles in regulating proteostasis. |
What is the benserazide's mechanism of function when co-administered with L-DOPA in patients with Parkinson's Disease? | Benserazide is a peripheral decarboxylase inhibitor. Benserazide in combination with L-DOPA constitutes a slow-release formulation of L-DOPA in patients with Parkinson's disease (PD), it reduces peaks and rapid fluctuations of L-DOPA plasma levels (possibly responsible for peak-dose dyskinesias and end-of-dose deterioration) and considered as the best available therapy. | An open cross-over study of 20 patients with Parkinson's disease performed with
two drugs containing L-dopa and a peripheral aromatic amino acid decarboxylase
inhibitor (benserazide, carbidopa) confirmed the conclusions reached in other
clinical trials that this combined treatment of Parkinson's disease is the most
effective form of drug therapy available at present. With both drugs, Madopar or
Sinemet, an optimum therapeutic result was obtained with relatively small doses
of L-dopa (the reduction in L-dopa dosage amounting to about 80%). A loss of
efficacy with both drugs, which has observed during long-term treatment of
patients with Parkinson's disease, could be avoided by switching the patients
from Sinemet to Madopar and vice versa. Determination of L-dopa in the plasma
demonstrated that with either drug similar plasma levels of L-dopa were achieved
during clinically effective treatment. Basic aspects and recent advances in the understanding of the pharmacological
mechanism of action of the clinically most used antiparkinson drugs are
reviewed. Recent human and animal biochemical investigations clearly confirm and
extend previous findings indicating that benserazide is much more potent than
carbidopa as peripheral decarboxylase inhibitor. L-DOPA in combination with
benserazide or carbidopa constitutes the best available therapy for Parkinson's
disease (PD). To reduce peaks and rapid fluctuations of L-DOPA plasma levels
(possibly responsible for peak-dose dyskinesias and end-of-dose deterioration) a
slow-release formulation of L-DOPA in combination with benserazide or with
benserazide plus catechol-O-methyltransferase inhibitors should be developed. In
parkinsonian patients under long-term L-DOPA therapy monoamine oxidase
inhibitors type B (MAO-B) e.g. (-)deprenyl and direct dopamine receptor agonists
(bromocriptine, lisuride, pergolide etc.), due to their L-DOPA-sparing effects,
alleviate in some cases L-DOPA-induced side-effects e.g. dyskinesias and on-off
phenomena. However, since (-)deprenyl, due to its metabolism to
(-)methamphetamine and (-)amphetamine, seem to have indirect sympathomimetic
activity, new selective MAO-B inhibitors devoid of indirect sympathomimetic
effects should be tested clinically to assess the functional role of pure MAO-B
inhibition in the therapy of PD. The auxiliary therapy with direct dopamine
receptor agonists of the D-2 subtype represents another valid approach which
should be further investigated in order to find novel dopamine agonists, less
expensive than bromocriptine, and strictly selective for D-2 receptor sites. In an open label study 63 patients with idiopathic Parkinson's disease suffering
from end-of-dose akinesia were switched from a treatment with a L-DOPA standard
formulation to a combined therapy of L-DOPA standard in the morning and L-DOPA
slow release (levodopa, benserazide, Madopar Depot) at the remaining single
doses. Substitution of L-DOPA standard by L-DOPA slow release took on average
2-4 weeks. Patients were subsequently treated for 6 months. Due to a lower
bioavailability of the slow release formulation--the latter is based on the
"hydrodynamically balanced system" (HBS)--, the patients remained initially on
their time schedule of drug intake but received a higher dose of L-DOPA slow
release compared to the preceding L-DOPA standard therapy. In 20 centers 37 men
and 26 women were included into the study. 27 males and 20 females completed the
6 month treatment period. Before switching, the patients received 438 +/- 213 mg
a day L-DOPA standard, after conversion, the average dose was 617 +/- 323 mg
L-DOPA slow release and 107 +/- 95 mg L-DOPA standard a day. Fluctuations during
the day and at night which were rated according to a newly developed clinical
5-point rating scale were significantly improved by the treatment regimen from
2.8 +/- 0.9 to 1.4 +/- 1.2. Additionally, parkinsonian symptoms were
significantly reduced during the ON-phase as there was a significant decrease of
the Webster rating score from 12.0 +/- 4.6 to 7.1 +/- 4.0. Quality of life as
measured by subjective ratings of the patients improved. The tolerability of the
new formulation of L-DOPA was rated to be good in 51.1% and very good in 48.9%.
The results of this open label study suggest that the combination of L-DOPA
standard in the morning and L-DOPA slow release formulation at the following
time points can be an efficient therapy in parkinsonian patients who suffer form
L-DOPA related end-of-dose motor akinesia. L-DOPA provides the most potent medication to treat Parkinson's disease, and
such systemic treatment is usually combined with a peripheral amino acid
decarboxylase inhibitor to amplify its central effectiveness. Since L-DOPA can
lose its efficacy or can lead to adverse effects with prolonged application,
current pharmacokinetic and dynamic research is aimed at improving the drug's
applicability. In a previous study, performed with in vivo microdialysis in the
anesthetized rat, we have shown that intranasal L-DOPA administration (without
prior decarboxylase inhibition) can increase extracellular dopamine levels in
the neostriatum. Using similar experimental conditions in the present
experiment, we tested the neurochemical effects of L-DOPA treatment in
combination with the peripheral amino acid decarboxylase inhibitor benserazide.
In accordance with other data, it was found that the combination of i.p.
benserazide and i.p. L-DOPA led to pronounced increases of extracellular levels
of dopamine, dihydroxyplenylacetic acid and homovanillic acid in the
neostriatum, whereas i.p. L-DOPA alone only moderately increased dopamine, but
strongly increased the metabolite levels. Furthermore, increased dopamine
levels, and weaker increases of dihydroxyplenylacetic acid and homovanillic acid
were observed after i.p. benserazide followed by intranasal L-DOPA. Finally, we
found that i.p. benserazide alone can lead to pronounced increases in
neostriatal dopamine and moderate increases of dihydroxyplenylacetic acid
levels, whereas it did not affect homovanillic acid. Thus, not only the
combination of L-DOPA (i.p. or intranasal) with the presumed peripheral L-DOPA
decarboxylase inhibitor benserazide, but also each component alone can affect
dopamine activity in the brain. Especially the findings with benserazide
treatment might be of relevance for understanding the mechanisms of current
L-DOPA therapy, since they indicate that part of the treatment's actions may
possibly be determined by central dopaminergic effects of the accompanying amino
acid decarboxylase inhibitor. (S)-(-)-3-(3-(methylsulfonyl)phenyl)-1-propylpiperidine ((-)-OSU6162) is a
phenylpiperidine derivative which exhibits low affinity to the dopamine D2
receptor in vitro. However, in vivo, positron emission tomography scanning
studies show that the compound displaces the selective dopamine D2 receptor
antagonist, raclopride. We have evaluated, in this study, the effect of
(-)-OSU6162, on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias in a
primate model of Parkinson's disease. Five
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cynomolgus monkeys
with a stable parkinsonian syndrome and reproducible dyskinesias to L-DOPA were
used in this study. The monkeys were housed in observation cages equipped with
an electronic motility monitoring system. They were injected subcutaneously
(s.c.) with L-DOPA methyl ester (125 mg per animal) plus benserazide (50 mg per
animal; L-DOPA/benserazide) alone or in combination with (-)-OSU6162 (1.0, 3.0,
6.0 or 10 mg/kg, s.c.). Subcutaneous injection of sterile saline was used as
control. L-DOPA/benserazide increased locomotion and improved parkinsonism but
also induced dyskinesias. Co-administration of (-)-OSU6162 with
L-DOPA/benserazide produced a significant reduction in L-DOPA-induced
dyskinesias. This improvement in L-DOPA-induced dyskinesias occurred mainly at
the onset of the L-DOPA/benserazide effect as reflected by an increase in the
duration of the "ON" state without dyskinesias up to 3.4 fold after (-)-OSU6162
co-administration as compared to L-DOPA/benserazide alone. The anti-dyskinetic
effect of (-)-OSU6162 was maintained during 14 days and no tolerance to this
effect was observed. Our data suggests that (-)-OSU6162 could be of significant
clinical value to reduce L-DOPA-induced dyskinesias in fluctuating advanced
Parkinson's disease patients. PURPOSE: To study the PK interaction of L-dopa/benserazide in rats.
METHODS: Male rats received a single oral dose of 80 mg/kg L-dopa or 20 mg/kg
benserazide or 80/20 mg/kg L-dopa/benserazide. Based on plasma concentrations
the kinetics of L-dopa, 3-O-methyldopa (3-OMD), benserazide, and its metabolite
Ro 04-5127 were characterized by noncompartmental analysis and a compartmental
model where total L-dopa clearance was the sum of the clearances mediated by
amino-acid-decarboxylase (AADC), catechol-O-methyltransferase and other enzymes.
In the model Ro 04-5127 inhibited competitively the L-dopa clearance by AADC.
RESULTS: The coadministration of L-dopa/benserazide resulted in a major increase
in systemic exposure to L-dopa and 3-OMD and a decrease in L-dopa clearance. The
compartmental model allowed an adequate description of the observed L-dopa and
3-OMD concentrations in the absence and presence of benserazide. It had an
advantage over noncompartmental analysis because it could describe the temporal
change of inhibition and recovery of AADC.
CONCLUSIONS: Our study is the first investigation where the kinetics of
benserazide and Ro 04-5127 have been described by a compartmental model. The
L-dopa/benserazide model allowed a mechanism-based view of the
L-dopa/benserazide interaction and supports the hypothesis that Ro 04-5127 is
the primary active metabolite of benserazide. L-3, 4-dihydroxyphenylalanine (L-dopa) is the gold standard for symptomatic
treatment of Parkinson's disease (PD), but long-term therapy is associated with
the emergence of L-dopa-induced dyskinesia (LID). In the present study, L-dopa
and benserazide were loaded by poly (lactic-co-glycolic acid) microspheres
(LBM), which can release levodopa and benserazide in a sustained manner in order
to continuous stimulate dopaminergic receptors. We investigated the role of
striatal DR1/PKA/P-tau signal transduction in the molecular event underlying LID
in the 6-OHDA-lesioned rat model of PD. We found that animals rendered
dyskinetic by L-dopa treatment, administration of LBM prevented the severity of
AIM score, as well as improvement in motor function. Moreover, we also showed
L-dopa elicits profound alterations in the activity of three LID molecular
markers, namely DR1/PKA/P-tau (ser396). These modifications are totally
prevented by LBM treatment, a similar way to achieve continuous dopaminergic
delivery (CDD). In conclusion, our experiments provided evidence that
intermittent administration of L-dopa, but not continuous delivery, and
DR1/PKA/p-tau (ser396) activation played a critical role in the molecular and
behavioural induction of LID in 6-OHDA-lesioned rats. In addition, LBM treatment
prevented the development of LID by inhibiting the expression of DR1/PKA/p-tau,
as well as PPEB mRNA in dyskintic rats. |
What is regioneR? | Statistically assessing the relation between a set of genomic regions and other genomic features is a common challenging task in genomic and epigenomic analyses. regioneR is an R package that implements a permutation test framework specifically designed to work with genomic regions. In addition to the predefined randomization and evaluation strategies, regioneR is fully customizable allowing the use of custom strategies to adapt it to specific questions. Finally, it also implements a novel function to evaluate the local specificity of the detected association. regioneR is an R package released under Artistic-2.0 License. The source code and documents are freely available through Bioconductor (http://www.bioconductor.org/packages/regioneR). | MOTIVATION: Statistically assessing the relation between a set of genomic
regions and other genomic features is a common challenging task in genomic and
epigenomic analyses. Randomization based approaches implicitly take into account
the complexity of the genome without the need of assuming an underlying
statistical model.
SUMMARY: regioneR is an R package that implements a permutation test framework
specifically designed to work with genomic regions. In addition to the
predefined randomization and evaluation strategies, regioneR is fully
customizable allowing the use of custom strategies to adapt it to specific
questions. Finally, it also implements a novel function to evaluate the local
specificity of the detected association.
AVAILABILITY AND IMPLEMENTATION: regioneR is an R package released under
Artistic-2.0 License. The source code and documents are freely available through
Bioconductor (http://www.bioconductor.org/packages/regioneR).
CONTACT: [email protected]. |
For what indications is thalidomide currently marketed? | Drug repositioning, exemplified by sildenafil and thalidomide, is a promising way to explore alternative indications for existing drugs. THE USE OF A DRUG WITH A TEMPORARY MARKETING AUTHORISATION: Thalidomide is currently available in France for nominative or cohort use with a temporary marketing authorisation (TMA). Currently, it is used for a few indications; in Brazil, where leprosy is endemic, thalidomide is used for the treatment of erythema nodosum leprosum, and recent cases of thalidomide embryopathy have been reported.We analyzed the frequency of births with phenotypes consistent with thalidomide embryopathy (TEP) and correlated this with the distribution of thalidomide and the prevalence of leprosy between 2005 and 2010 in Brazil.A total of 5,889,210 thalidomide tablets were distributed; the prevalence of limb reduction defects was 1.60 (CI95%: 1.54-1.66) and TEP was 0.11 (CI95%: 0.10-0.13) Currently it includes a group of new drugs (immunosuppressives tacrolimus mycophenolate, thalidomide, biologic therapy, probiotics, neuroinflammation blockers), new treatment techniques (cytaphereses, sequential immunosuppression, immunosuppression with high doses), and finally new indications (chemoprophylaxis). A review of the therapeutic indications for thalidomide in dermatology as well as the mechanisms of action and side-effects of this drug are presented. | Based on present publications we review indications of the therapy of dermatoses
with thalidomide as well as possible mechanisms of action and side effects of
this drug. In reactional states of leprosy the use of thalidomide is
established. Further indications are chronic cutaneous lupus erythematosus,
prurigo nodularis, and eventually recurrent aphthosis and certain
photodermatoses not responding to usual treatment. Therapeutical trials of
thalidomide in diseases in which such a treatment is only occasionally or not at
all mentioned in the literature will be reported. Concerning the mechanisms of
action emphasis is put on a possible immunosuppression by thalidomide. Among the
side effects the thalidomide neuropathy is stressed. Thalidomide is now available as an investigational drug in the USA. A synthetic
derivative of glutamic acid, it was marketed in Europe in 1957 as a sedative but
withdrawn four years later after being associated with severe human
teratogenicity (PF D'Arcy and JP Griffin, Adverse Drug React Toxicol Rev, 13:65,
1994). The drug has since been found effective for several different
indications. Thalidomide, mainly used for the treatment of leprosy, is a current teratogen in
South America, and it is reasonable to assume that at present this situation is
affecting many births in underdeveloped countries. Moreover, the potential
re-marketing of thalidomide for the treatment of a large variety of diseases may
extend the problem to the developed world. When the drug is available, the
control of its intake during early pregcy is very difficult since most
pregcies are unintended. The ongoing occurrence of thalidomide embryopathy
cases went undetected by the ECLAMC, due to several factors: (1) low
populational coverage through this monitoring system; (2) pre-existence of the
teratogen with its effects present in both baseline (expected) and monitored
(observed) materials; and (3) lack of a defined phenotype to be monitored. Thus,
if thalidomide re-enters the market throughout the world, due to the wide range
of new applications, occurrence of phocomelia alone might not be sufficient to
detect its effects. By a case-reference approach, the ECLAMC registered 34
thalidomide embryopathy cases born in South America after 1965 whose birthplaces
correspond to endemic areas for leprosy. Phocomelia was found in five of eleven
fully described cases. Thus, phocomelia alone is neither specific nor sufficient
to serve as a suitable phenotype to survey the teratogenic effects of
thalidomide. Therefore, a thalidomide-like phenotype, defined as any bilateral
upper and/or lower limb reduction defect of the preaxial and/or phocomelia
types, should be included in the routine surveillance of birth defects in all
programmes. Thalidomide was withdrawn from the market in the early sixties because of major
teratogenic effects such as reduction defects of the limbs. Since, however, it
has been found to be an effective drug in erythema nodosum leprosum. In the
United States it was decided in September 1997 to admit thalidomide to the
market for this indication, and in South America it has been available for this
indication all the time. Thalidomide is also efficacious in other major
disorders (e.g. aphtae and ulcers in aids) or its efficacy is being investigated
in clinical trials (e.g. autoimmune diseases, other complications in aids). The
American Food and Drug Administration has imposed conditions for the use of
thalidomide. Users have to sign an informed consent and to take adequate
contraceptive measures. Physicians should inform the patients and monitor side
effects. Pharmacists should record and control the use. Thalidomide is a synthetic derivative of glutamic acid with sedative-hypnotic
activity, which caused devastating teratogenic effects in the 1960s. This paper
reviews the possible mechanisms of its teratogenic effect, its new therapeutic
indications, the proposed mechanisms for its antitumor activity and, finally,
reviews published studies of its application in oncology. Current data
demonstrates that thalidomide is clinically promising in multiple myeloma,
glioblastoma multiforme and renal cell cancer. Furthermore, a beneficial effect
of the drug has been proposed in cancer-related cachexia, which merits further
investigation. Well-designed, randomized studies are warranted to establish the
possible indications of thalidomide as an antitumor compound. Thalidomide, which was developed as a nonbarbiturate sedative agent, was taken
off the market in 1961 after it was linked to a spate of major birth defects.
Gradually, thalidomide was reintroduced for the treatment of a few skin diseases
including leprous erythema nodosum, severe mucosal ulcers (e.g., associated with
HIV infection or Behçet's disease), lymphocytic skin infiltrations, cutaneous
lupus erythematosus, and chronic graft-versus-host disease. Recent reports of
original pharmacological properties including modulation of cytokine production
(mainly reduced TNF-alpha production) and inhibition of angiogenesis have led to
the suggestion that thalidomide may be useful in some inflammatory and
neoplastic conditions. Several open-label studies and case reports have
described the effects of thalidomide in Crohn's disease, rheumatoid arthritis,
ankylosing spondylarthritis, systemic sclerosis, and a few other systemic
disorders. In these indications, minor but dose-limiting side effects were
apparently common. Thalidomide analogs with better acceptability profiles are
under evaluation. The anti-angiogenic effects of thalidomide may make this
compound valuable as single-drug therapy or as an adjunct to chemotherapy in
patients with cancer, particularly those with metastases or multiple myeloma.
This possibility requires further evaluation. Thalidomide was withdrawn from the market in the early sixties because of its
teratogenic effects. Despite forty years of research, the mechanism of
thalidomide embryopathy has remained unsolved. Thalidomide has various
immunomodulatory effects. Thalidomide inhibits TNF alpha production, has T-cell
costimulatory properties and modulates the expression of cell surface molecules
on leukocytes in vivo. Thalidomide also has anti-angiogenic activity in vivo.
Angiogenesis plays an important role in the pathogenesis of both solid tumours
and hematologic maligcies such as multiple myeloma and lymphoma. In clinical
studies, thalidomide has been used as an inhibitor of angiogenesis. Erythema
nodosum leprosum is the only registered indication for the use of thalidomide in
the United States of America. Thalidomide is also effective in the treatment of
chronic graft-versus-host disease, mucocutaneous lesions in Behçet's syndrome
and HIV infections, and multiple myeloma. Thalidomide was first used in the late 1950s but it was withdrawn from the
market in the 1960s for its notorious teratogenic effects. This drug was more
recently rediscovered as a powerful immunomodulatory and antiinflammatory agent
and was approved by the FDA in 1998 for treatment of erythema nodosum leprosum.
Thalidomide has shown great promise in advanced or refractory multiple myeloma
either alone or in combination with other agents. It has also demonstrated
benefits in a wide variety of disparate conditions such as aphthous and genital
ulcers, cancer cachexia, HIV, tuberculosis and chronic graft versus host
disease. Thalidomide is being investigated for treatment of renal cell
carcinoma, and liver and thyroid cancers. Better understanding of its many
mechanisms of action has provoked great interest in its potential use for
treatment of various disorders. This review focuses on thalidomide's mechanisms
of action, biochemistry, pharmacokinetics and its use in erythema nodosum
leprosum as well as multiple myeloma, graft versus host disease, and renal cell
carcinoma. The resurgence of interest in thalidomide in the last decade has been
remarkable. Thalidomide has established its own niche market particularly for
the dermatological manifestations associated with HIV, Behçet's disease,
graft-versus-host disease and systemic lupus erythematosus. To a large extent
this has resulted from initial empirical uncontrolled studies in conditions
resistant to other drug therapies. Appropriate trials are now being published
for most of the prevalent indications. Thalidomide produces partial inhibition
of tumour necrosis factor-alpha production in vivo but recent data reveals that
it can also act as a co-stimulatory molecule for T cell activation in vitro,
resulting in increased production of interleukin-2 and interferon-gamma. Hence
in addition to monocyte inhibitory activity, thalidomide can exert a
co-stimulatory or adjuvant-like effect on T cell responses. The unraveling of
the molecular basis of thalidomide's therapeutic effects would suggest that an
expansion of the use of thalidomide and its analogues in other conditions is
highly likely. It remains imperative, however, that physicians using this
fascinating drug are familiar with its risks and adverse effects. Interferon (INF)-α was the maintece treatment of choice after autologous stem
cell transplantation in multiple myeloma in the past, but currently Thalidomide
is commonly used. In this prospective study, the implications of the various
types of maintece therapy on the patients T cell pattern and activation
status were assessed. T cells were analyzed for expression of surface molecules,
cytokine secretion, the presence of regulatory T cells, and the specific
activation against the multiple myeloma antigen HM1.24. T cells from 69 multiple
myeloma patients were analyzed: 19 patients were treated with IFN-α; 26 were
treated with Thalidomide; and 24 patients received no maintece therapy.
Specific T cell activation with an immunogenic HLA-A2(+)-restricted peptide from
the myeloma-associated antigen HM1.24 was impaired in the Thalidomide group. In
accordance with this observation, there was a trend toward a higher amount of
regulatory T cells in the Thalidomide group. Furthermore, patients treated with
IFN-α showed high rates of naive T cells, whereas a high rate of effector memory
T cells was observed in the Thalidomide group. Importantly, after cessation of
Thalidomide therapy, this effect was reversible in the CD8 compartment. In
conclusion, Thalidomide maintece therapy has profound implications on T cell
pattern and activation status, which compromise antigen specific antitumor
immunity. The use of thalidomide was never discontinued in Brazil where it is prescribed
for leprosy type 2 reaction. Babies with birth defects compatible with the
thalidomide embryopathy phenotype were born after 1965, an indication that
control on drug dispensing and use failed in the country. The article reports
data on thalidomide dispensing and clinical uses in the Federal District in
2011/12, when new rules were put into effect, and data on drug dispensing and
use obtained ten years earlier. It was found that the number of patients making
use of thalidomide declined from 819 in 2001 to 369 in 2011/12. Leprosy
accounted for over 70% of prescriptions in both time periods analyzed in this
study. In the same time interval, however, use for lupus erythematosus decreased
from 13.7 to 4.9%, while that for multiple myeloma increased from 2.9 to 20.3%
of all prescriptions. Thalidomide prescription for the remaining approved
indications was far less frequent, and so was the use for off label indications
that accounted for <1% of prescriptions in 2001 and 2011/12. Registration of
prescribing doctors, patients and dispensing units at the state department of
health, apparently rendered this control more effective and reliable. BACKGROUND: Thalidomide could relieve clinical symptoms and intestinal mucosal
lesions effectively in children with refractory inflammatory bowel disease (IBD)
from the pre-clinical study. This study aimed to observe the therapeutic effect
of thalidomide by the established animal model of IBD model of
2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in Sprague-Dawley
(SD) rats and to investigate the possible mechanism of action.
METHODS: A total of 82 SD rats of about 4-5 weeks were randomly divided into
three groups: the control group (25 rats), TNBS-treated group (29 rats), and
thalidomide treatment group (28 rats). Daily activities were recorded. At least
eight rats from each group were killed on the 4th, 7th, and 14th days.
Morphological and histological changes in the colon were individually assessed.
Serum was collected and the levels of TNF-α and interleukins (IL-1β and IL-10)
were assayed by ELISA method. The expression of colonic mucosal nuclear factor
(NF)-κB was assayed with the immunohistochemical method.
RESULTS: (1) In the control group, diarrhea and rectal bleeding recovered
rapidly and no death was recorded. In the TNBS-treated group, diarrhea and
rectal bleeding persisted for a longer time. The mortality rate was 10.34%
during the observation period. In the thalidomide treatment group, diarrhea and
rectal bleeding persisted for a significantly shorter time than the TNBS-treated
group (P < 0.01). The rats of this group also exhibited faster weight gain on
day 7 compared with the TNBS-treated group but still lower than that of the
control group. The mortality rate of the thalidomide treatment group was 3.57%.
(2) Macroscopic and microscopic scores of the thalidomide-treated group were
significantly lower than those of the TNBS model group on the 14th day (P <
0.01). These results suggested faster and better colonic recovery in the
thalidomide-treated group. (3) NF-κB expression in the colonic mucosa of the
control group was lower than in the others, mainly distributed in the cytoplasm.
A large amount of intra-nuclear and cytoplasm staining was observed (more
prominently intra-nuclear) in the TNBS model group and the thalidomide treatment
group. On the 7th and 14th days, intra-nuclear NF-κB-containing cells in the
thalidomide treatment group were still significantly lower than those in the
TNBS model group (P < 0.01). (4) In the control group, the cellular inflammatory
factors (TNF-α, IL-1β, and IL-10) were expressed at a low level while in the
other two groups they were already expressed at a significantly higher level on
day 4. On day 7 the expressions of TNF-α and IL-1β in the thalidomide treatment
group were lower than in the TNBS model group. On day 14, the expressions of
TNF-α and IL-1β in the thalidomide treatment group were significantly lower than
in the TNBS model group (P < 0.05). On day 4, the IL-10 levels of the
thalidomide treatment group became significantly elevated. The levels gradually
decreased but still remained at a higher level. In the TNBS model group, the
IL-10 expression peaked later than in the thalidomide treatment group.
CONCLUSIONS: Thalidomide was effective in the management of TNBS-induced colitis
in young rats. This may be due to the suppression and down-regulation of NF-κB
and the expression of the downstream inflammatory mediators (TNF-α and IL-1β).
There is also indication that the expression of the anti-inflammatory cytokine
(IL-10) is concomitantly up-regulated as well. INTRODUCTION: Polyneuropathy, organomegaly, endocrinopathy, M-protein and skin
changes (POEMS) syndrome is a fatal systemic disorder associated with plasma
cell dyscrasia and the overproduction of the vascular endothelial growth factor
(VEGF). Recently, the prognosis of POEMS was substantially improved by
introduction of therapeutic intervention for myeloma. However, no randomised
clinical trial has been performed because of the rarity and severity of the
disease.
METHODS AND ANALYSIS: The Japanese POEMS syndrome with Thalidomide (J-POST)
Trial is a phase II/III multicentre, double-blinded, randomised, controlled
trial that aims to evaluate the efficacy and safety of a 24-week treatment with
thalidomide in POEMS syndrome, with an additional 48-week open-label safety
study. Adults with POEMS syndrome who have no indication for transplantation are
assessed for eligibility at 12 tertiary neurology centres in Japan. Patients who
satisfy the eligibility criteria are randomised (1:1) to receive thalidomide
(100-300 mg daily) plus dexamethasone (12 mg/m(2) on days 1-4 of a 28-day cycle)
or placebo plus dexamethasone. Both treatments were administered for 24 weeks
(six cycles; randomised comparative study period). Patients who complete the
randomised study period or show subacute deterioration during the randomised
period participate in the subsequent 48-week open-label safety study (long-term
safety period). The primary end point of the study is the reduction rate of
serum VEGF levels at 24 weeks.
ETHICS AND DISSEMINATION: The protocol was approved by the Institutional Review
Board of each hospital. The trial was notified and registered at the
Pharmaceutical and Medical Devices Agency, Japan (No. 22-1716). The J-POST Trial
is currently ongoing and is due to finish in August 2015. The findings of this
trial will be disseminated through peer-reviewed publications and conference
presentations and will also be disseminated to participants.
TRIAL REGISTRATION NUMBER: UMIN000004179 and JMA-IIA00046. Author information:
(1)INAGEMP - Instituto Nacional de Genética Médica Populacional, Porto Alegre,
Brazil; Teratogen Information Service, Medical Genetics Service, Hospital de
Clínicas de Porto Alegre, Brazil; Genetics Department, Universidade Federal do
Rio Grande do Sul, Porto Alegre, Brazil; Postgraduate Program in Epidemiology,
Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul,
Brazil.
(2)INAGEMP - Instituto Nacional de Genética Médica Populacional, Porto Alegre,
Brazil; Genetics Department, Universidade Federal do Rio Grande do Sul, Porto
Alegre, Brazil.
(3)INAGEMP - Instituto Nacional de Genética Médica Populacional, Porto Alegre,
Brazil; Teratogen Information Service, Medical Genetics Service, Hospital de
Clínicas de Porto Alegre, Brazil.
(4)Programa Nacional da Hanseníase, Brasília, Brazil.
(5)Secretaria de Vigilância em Saúde, Ministério da Saúde, Brasília, Brazil.
(6)ECLAMC (Latin-American Collaborative Study of Congenital Malformations) in
IMBICE, Instituto Multidisciplinario de Biologia Celular, La Plata, Argentina;
ECLAMC in CEMIC, Centro de Educación Médica e Investigación Clínica, Buenos
Aires, Argentina.
(7)Postgraduate Program in Epidemiology, Universidade Federal do Rio Grande do
Sul, Porto Alegre, Rio Grande do Sul, Brazil.
(8)INAGEMP - Instituto Nacional de Genética Médica Populacional, Porto Alegre,
Brazil; Teratogen Information Service, Medical Genetics Service, Hospital de
Clínicas de Porto Alegre, Brazil; Genetics Department, Universidade Federal do
Rio Grande do Sul, Porto Alegre, Brazil. Electronic address:
[email protected]. OBJECTIVE: To examine direct cost to patients associated with oral oncolytics
for the management of multiple myeloma (MM) both before and after ficial
assistance, and assess the effect on adherence.
METHODS: In this retrospective study, pharmacy claims were analyzed for those
patients with a diagnosis of MM who received thalidomide, lenalidomide, or
pomalidomide from a large specialty pharmacy in the US between January 1, 2011,
and December 31, 2013. Average direct cost to patients, per prescription, was
analyzed both before and after ficial assistance. Adherence was assessed
through an analysis of medication possession ratio (MPR) for those patients who
filled a prescription ≥2 times throughout the 3-year time period.
RESULTS: A total of 77,821 prescriptions for thalidomide, lenalidomide, and
pomalidomide were filled by 6731 unique patients between January 1, 2011, and,
December 31, 2013. The average direct cost to patients, per prescription, for
any of these three agents was $227.23 prior to ficial assistance and $80.11
after ficial assistance, representing an average patient savings of $147.14
per prescription. Prior to ficial assistance, the average direct cost to
patients was ≤$50 for 57.6% of all prescriptions. After ficial assistance,
86.2% of patients had a direct cost of ≤$50 per prescription. Adherence, as
assessed by MPR, did not vary significantly based on direct cost to the patient.
LIMITATIONS: This study included patients receiving therapy from a single
specialty pharmacy for a single indication. There may be patients included in
the analysis who received prescriptions from other pharmacies prior to or after
the prescriptions available for analysis. Most of the prescriptions included in
the analysis were for lenalidomide.
CONCLUSIONS: This retrospective study demonstrated that the specialty pharmacy
helped patients significantly reduce their direct cost expenditures by securing
funding and co-pay assistance. |
What are reactive metabolites? | Reactive metabolites are generated when a small molecule, commonly a drug or hydrocarbon, is broken down in the body. Reactive metabolites can cause cancer and other diseases as well as hepatoxicty. | Certain drugs are transformed into reactive metabolites by cytochrome P-450, a
hepatic microsomal enzyme. The reactive metabolites covalently bind to
hepatocyte macromolecules, thus determining liver lesions. Induction of
microsomial enzymes increases the formation of reactive metabolites and
exaggerates hepatotoxicity of these drugs. Evidence strongly suggests that many adverse drug reactions, including
idiosyncratic drug reactions, involve reactive metabolites. Furthermore, certain
functional groups, which are readily oxidized to reactive metabolites, are
associated with a high incidence of adverse reactions. Most drugs can probably
form reactive metabolites, but a simple comparison of covalent binding in vitro
is unlikely to provide an accurate indication of the relative risk of a drug
causing an idiosyncratic reaction because it does not provide an indication of
how efficiently the metabolite is detoxified in vivo. In addition, the incidence
and nature of adverse reactions associated with a given drug is probably
determined in large measure by the location of reactive metabolite formation, as
well as the chemical reactivity of the reactive metabolite. Such factors will
determine which macromolecules the metabolites will bind to, and it is known
that covalent binding to some proteins, such as those in the leukocyte membrane,
is much more likely to lead to an immune-mediated reaction or other type of
toxicity. Some reactive metabolites, such as acyl glucuronides, circulate freely
and could lead to adverse reactions in almost any organ; however, most reactive
metabolites have a short biological half-life, and although small amounts may
escape the organ where they are formed, these metabolites are unlikely to reach
sufficient concentrations to cause toxicity in other organs. Many idiosyncratic
drug reactions involve leukocytes, especially agranulocytosis and drug-induced
lupus. We and others have demonstrated that drugs can be metabolized by
activated neutrophils and monocytes to reactive metabolites. The major reaction
appears to be reaction with leukocyte-generated hypochlorous acid. Hypochlorous
acid is quite reactive, and therefore it is likely that many other drugs will be
found that are metabolized by activated leukocytes. Some neutrophil precursors
contain myeloperoxidase and the NADPH oxidase system, and it is likely that
these cells can also oxidize drugs. Therefore, although there is no direct
evidence, it is reasonable to speculate that reactive metabolites generated by
activated leukocytes, or neutrophil precursors in the bone marrow, could be
responsible for drug-induced agranulocytosis and aplastic anemia. This could
involve direct toxicity or an immune-mediated reaction. These mechanisms are not
mutually exclusive, and it may be that both mechanisms contribute to the
toxicity, even in the same patient. In the case of drug-induced lupus, a
prevalent hypothesis for lupus involves modification of class II MHC
antigens.(ABSTRACT TRUNCATED AT 400 WORDS) Central to most hypotheses of the mechanism of idiosyncratic drug-induced blood
dyscrasias is the involvement of reactive metabolites. In view of the reactive
nature of the majority of such metabolites, it is likely that they are formed
by, or in close proximity to the blood cells affected. The major oxidative
system of neutrophils generates hypochlorous acid. We have demonstrated that the
drugs associated with the highest incidence of agranulocytosis are oxidized to
reactive metabolites by hypochlorous acid and/or activated neutrophils. There
are many mechanisms by which such reactive metabolites could induce
agranulocytosis. In the case of aminopyrine-induced agranulocytosis, most cases
appear to involve drug-dependent anti-neutrophil antibodies, and these are
likely to be induced by cell membrane antigens modified by the reactive
metabolite of aminopyrine. The target of agranulocytosis associated with many
other drugs is usually neutrophil precursors and may involve cytotoxicity or a
cell-mediated immune reaction induced by a reactive metabolite. In the case of
aplastic anaemia, there is evidence in some cases for involvement of cytotoxic T
cells, which could either be induced by metabolites generated by neutrophils, or
more likely, by reactive metabolites generated by stem cells. Olanzapine was shown to be oxidized to a reactive intermediate by HOCl, which is
the major oxidant produced by activated neutrophils. A mass spectrum obtained
using a flow system in which the reactants were fed into a mixing chamber and
the products flowed directly into a mass spectrometer revealed a reactive
intermediate at m/z 311. This is 2 mass units less than the protonated molecular
ion of parent olanzapine and suggests that the reactive intermediate is a
nitrenium ion. The reactive intermediate could be trapped with glutathione or
N-acetylcysteine to produce two conjugates. These data are analogous to results
we reported previously with the structurally related atypical antipsychotic
agent clozapine. However, the clozapine and olanzapine reactive metabolites
showed differences in their ability to cause toxicity to human neutrophils.
Toxicity to neutrophils was observed only at high concentrations of clozapine
(>50 microM) when HOCl was used to generate reactive metabolite. In contrast,
concentration-dependent toxicity (p < 0.05) was observed when neutrophils were
incubated with clozapine (0-20 microM) and H2O2 to generate clozapine reactive
metabolite. No toxicity was observed with clozapine alone (at concentrations of
> 50 microM). Similar results were observed in monocytes and HL-60 cells.
Olanzapine reactive metabolite only seemed to cause slight toxicity at the
highest concentrations tested (20 microM), even when the reactive metabolite was
generated using H2O2. Neutrophils from two patients with a history of
clozapine-induced agranulocytosis seemed to be more sensitive to the toxic
effects of the clozapine reactive metabolite; however, the numbers are too small
to draw any definite conclusions. Drug-induced adverse reactions, especially type B reactions, represent a major
clinical problem. They also impart a significant degree of uncertainty into drug
development because they are often not detected until the drug has been released
onto the market. Type B reactions are also termed idiosyncratic drug reactions
by many investigators due to their unpredictable nature and our lack of
understanding of the mechanisms involved. It is currently believed that the
majority of these reactions are immune-mediated and are caused by immunogenic
conjugates formed from the reaction of a reactive metabolite of a drug with
cellular proteins. It has been shown that most drugs associated with
idiosyncratic reactions form reactive metabolites to some degree. Covalent
binding of reactive metabolites to cellular proteins has also been shown in many
cases. However, studies to reveal the role of reactive metabolites and their
protein-adducts in the mechanism of drug-induced idiosyncratic reactions are
lacking. This review will focus on our current understanding and speculative
views on how a reactive metabolite of a drug might ultimately lead to
immune-mediated toxicity. Metabolic activation of a drug leading to reactive metabolite(s) that can
covalently modify proteins is considered an initial step that may lead to
drug-induced organ toxicities. Characterization of reactive metabolites is
critical to designing new drug candidates with an improved toxicological
profile. High performance liquid chromatography (HPLC) coupled with mass
spectrometry (MS) predominates over all analytical tools used for screening and
characterization of reactive metabolites. In this review, a brief description of
experimental approaches employed for assessing reactive metabolites is followed
by a discussion on the reactivity of acyl glucuronides and acyl coenzyme A
thioesters. Techniques for high-throughput screening and quantitation of
reactive metabolite formation are also described, along with proteomic
approaches used to identify protein targets and modification sites by reactive
metabolites. Strategies for dealing with reactive metabolites are reviewed. In
conclusion, we discuss the challenges and future needs in this field of
research. Troglitazone (TGZ) was developed for the treatment of type 2 diabetes but was
withdrawn from the market due to hepatotoxicity. The formation of reactive
metabolites has been associated with the observed hepatotoxicity. Such reactive
metabolites have been proposed to be formed via three different mechanisms. One
of the proposed mechanisms involves the oxidation of the chromane moiety of TGZ
to a reactive o-quinone methide. The two other mechanisms involve metabolic
activation of the thiazolidinedione moiety of TGZ. In the present study, it is
shown that electrochemical oxidations can be used to generate a reactive
metabolite of TGZ, which can be trapped by GSH or N-acetylcysteine. From
incubations of TGZ with rat and human liver microsomes in the presence of either
GSH or N-acetylcysteine, it was shown that similar conjugates were formed in
vitro as formed from electrochemical oxidations of TGZ. One- and two-dimensional
NMR studies of the troglitazone- S-( N-acetyl)cysteine conjugate revealed that
N-acetylcysteine was attached to a benzylic carbon in the chromane moiety,
showing that the conjugate was formed via a reaction between the o-quinone
methide of TGZ and N-acetylcysteine. From electrochemical oxidations of
rosiglitazone, pioglitazone, and ciglitazone in the presence of GSH, no GSH
conjugates could be identified. These three compounds all contain a
thiazolidinedione moiety. In conclusion, it has been shown that the primary
reactive metabolite of TGZ formed from electrochemical oxidation was the
o-quinone methide, and this metabolite was similar to what was observed to be
the primary reaction product in human and rat liver microsomes. Drug bioactivation leading to the formation of reactive species capable of
covalent binding to proteins represents an important cause of drug-induced
toxicity. Reactive metabolite detection using in vitro microsomal incubations is
a crucial step in assessing potential toxicity of pharmaceutical compounds. The
most common method for screening the formation of these unstable, electrophilic
species is by trapping them with glutathione (GSH) followed by liquid
chromatography/mass spectrometry (LC/MS) analysis. The present work describes
the use of a brominated analog of glutathione, N-(2-bromocarbobenzyloxy)-GSH
(GSH-Br), for the in vitro screening of reactive metabolites by LC/MS. This
novel trapping agent was tested with four drug compounds known to form reactive
metabolites, acetaminophen, fipexide, trimethoprim and clozapine. In vitro rat
microsomal incubations were performed with GSH and GSH-Br for each drug with
subsequent analysis by liquid chromatography/high-resolution mass spectrometry
on an electrospray time-of-flight (ESI-TOF) instrument. A generic LC/MS method
was used for data acquisition, followed by drug-specific processing of accurate
mass data based on mass defect filtering and isotope pattern matching. GSH and
GSH-Br incubations were compared to control samples using differential analysis
(Mass Profiler) software to identify adducts formed via the formation of
reactive metabolites. In all four cases, GSH-Br yielded improved results, with a
decreased false positive rate, increased sensitivity and new adducts being
identified in contrast to GSH alone. The combination of using this novel
trapping agent with powerful processing routines for filtering accurate mass
data and differential analysis represents a very reliable method for the
identification of reactive metabolites formed in microsomal incubations. Atazanavir (ATV) is an antiretroviral drug of the protease inhibitor class.
Multiple adverse effects of ATV have been reported in clinical practice, such as
jaundice, nausea, abdominal pain, and headache. The exact mechanisms of
ATV-related adverse effects are unknown. It is generally accepted that a
predomit pathway of drug-induced toxicity is through the generation of
reactive metabolites. Our current study was designed to explore reactive
metabolites of ATV. We used a metabolomic approach to profile ATV metabolism in
mice and human liver microsomes. We identified 5 known and 13 novel ATV
metabolites. Three potential reactive metabolites were detected and
characterized for the first time: one aromatic aldehyde, one α-hydroxyaldehyde,
and one hydrazine. These potential reactive metabolites were primarily generated
by CYP3A. Our results provide a clue for studies on ATV-related adverse effects
from the aspect of metabolic activation. Further studies are suggested to
illustrate the impact of these potential reactive metabolites on ATV-related
adverse effects. Reactive metabolites are estimated to be one of the main reasons behind
unexpected drug-induced toxicity, by binding covalently to cell proteins or DNA.
Due to their high reactivity and short lifespan, reactive metabolites are
analyzed after chemical trapping with nucleophilic agents such as glutathione or
cyanide. Recently, unexplained and uncharacterized methylated reaction products
were reported in a human liver microsome based reactive metabolite trapping
assay utilizing potassium cyanide as a trapping agent. Here, a similar assay was
utilized to produce mono- or dimethylated and further cyanide-trapped reaction
products from propranolol, amlodipine and ciprofloxacin, followed by
ultra-performance liquid chromatography/time-of-flight mass spectrometry
(UPLC/TOF-MS) and ultra-performance liquid chromatography/tandem mass
spectrometry (UPLC/MS/MS) experiments for their more detailed structural
elucidation. Formation of all observed cyanide-trapped products was clearly
NADPH-dependent and thus metabolism-mediated. The suggested reaction pathways
included N-methylation leading to iminium formation in primary and/or secondary
amines preceded by cytochrome P450 (CYP)-mediated reactions. As the methylation
reaction was suggested to be involved in formation of the actual reactive
iminium ion, the observed cyanide-trapped products were experimental artifacts
rather than trapped reactive metabolites. The results stress that to avoid
overestimating the formation of reactive metabolites in vitro, this methylation
phenomenon should be taken into account when interpreting the results of
cyanide-utilizing reactive metabolite trapping assays. This in turn emphasizes
the importance of identification of the observed cyano conjugates during such
studies. Yet, metabolite identification has a high importance to avoid
overestimation of in vitro metabolic clearance in the cases where this kind of
metabonate formation has a high impact in the disappearance rate of the
compound. Reactive oxygen species (ROS) are a family of molecules that are continuously
generated, transformed and consumed in all living organisms as a consequence of
aerobic life. The traditional view of these reactive oxygen metabolites is one
of oxidative stress and damage that leads to decline of tissue and organ systems
in aging and disease. However, emerging data show that ROS produced in certain
situations can also contribute to physiology and increased fitness. This
Perspective provides a focused discussion on what factors lead ROS molecules to
become signal and/or stress agents, highlighting how increasing knowledge of the
underlying chemistry of ROS can lead to advances in understanding their
disparate contributions to biology. An important facet of this emerging area at
the chemistry-biology interface is the development of new tools to study these
small molecules and their reactivity in complex biological systems. INTRODUCTION: Reactive metabolite-mediated toxicity is frequently limited to the
organ where the electrophilic metabolites are generated. Some reactive
metabolites, however, might have the ability to translocate from their site of
formation. This suggests that for these reactive metabolites, investigations
into the role of organs other than the one directly affected could be relevant
to understanding the mechanism of toxicity.
AREAS COVERED: The authors discuss the physiological and biochemical factors
that can enable reactive metabolites to cause toxicity in an organ distal from
the site of generation. Furthermore, the authors present a case study which
describes studies that demonstrate that S-(1,2-dichlorovinyl)-L-cysteine
sulfoxide (DCVCS) and N-acetyl-S-(1,2-dichlorovinyl-L-cysteine sulfoxide
(N-AcDCVCS), reactive metabolites of the known trichloroethylene metabolites
S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and
N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (N-AcDCVC), are generated in the liver
and translocate through the circulation to the kidney to cause nephrotoxicity.
EXPERT OPINION: The ability of reactive metabolites to translocate could be
important to consider when investigating mechanisms of toxicity. A mechanistic
approach, similar to the one described for DCVCS and N-AcDCVCS, could be useful
in determining the role of circulating reactive metabolites in extrahepatic
toxicity of drugs and other chemicals. If this is the case, intervention
strategies that would not otherwise be feasible might be effective for reducing
extrahepatic toxicity. The decline in approval of new drugs during the past decade has led to a close
analysis of the drug discovery process. One of the main reasons for attrition is
preclinical toxicity, frequently attributed to the generation of
protein-reactive drug metabolites. In this review, we present a critique of such
reactive metabolites and evaluate the evidence linking them to observed toxic
effects. Methodology for the characterization of reactive metabolites has
advanced greatly in recent years, and is summarized first. Next, we consider the
inhibition of key metabolic enzymes by electrophilic metabolites, as well as
unfavorable drug-drug interactions that may ensue. One important class of
protein-reactive metabolites, not linked conclusively to a toxic event, is acyl
glucuronides. Their properties are discussed in light of the safety
characteristics of carboxylic acid containing drugs. Many adverse drug reactions
(ADRs) are known collectively as idiosyncratic events, that is, not predictable
from knowledge of the pharmacology and pharmacokinetics of the parent compound.
Observed ADRs may take various forms. Specific organ injury, particularly of the
liver, is the most direct: we examine this in some detail. Moving to the
cellular level, we also consider the upregulation of induced cellular processes.
The related, but distinct, issue of hypersensitivity or allergic reactions to
drugs and their metabolites, possibly via the immune system, is considered next.
Finally, we discuss the impact of such data on the drug discovery process, both
through early detection of reactive metabolites and informed synthetic design,
which eliminates unfavorable functionality from drug candidates. Over the past decades, it has become abundantly clear that enzymes evolved to
detoxify and eliminate foreign chemicals from the body, occasionally generate
highly reactive metabolites which have toxicological implications. To decrease
the probability of late clinical failure or market withdrawal, there has been an
increased prioritization on understanding key metabolic processes that might
cause drug interactions or toxicities. Significant advances have been made in
the detection of reactive metabolites and in understanding the structure
activity relationship. It is now widely accepted that compounds with certain
functional groups such as anilines, quinones, hydrazines, thiophenes, furans,
acylpropionic acids, and alkynes have a much greater associated risk towards
formation of reactive metabolites than compounds that do not contain such
"structural alerts". Detection of reactive metabolites is usually done with in
vitro assays, which have become more sensitive with advances in mass
spectrometry. As an increasingly large number of compounds that form reactive
metabolites have been identified, much of the focus has shifted from detection
to evaluation of toxicological implication. While there is a disproportionate
number of compounds metabolized to reactive metabolites that are associated with
drug-induced hepatotoxicity and serious skin toxicities such as toxic
endothelial necrolysis and Steven's Johnson syndrome, attempts to predict
toxicity based on in vitro testing have been discouraging. In this review we
attempt to summarize the experimental options available to evaluate reactive
metabolites. Drug metabolism can result in the formation of highly reactive metabolites that
are known to play a role in toxicity resulting in a significant proportion of
attrition during drug development and clinical use. Thus, the earlier such
reactivity was detected, the better. This review summarizes our multi-year
project, together with pertinent literature, to examine a battery of in vitro
tests capable of detecting the formation of reactive metabolites. Principal
prerequisites for such tests were delineated: chemicals known/not known to cause
tissue injury and produce reactive metabolites, activation system (mainly
human-derived), small- and large-molecular targets (small-molecular trappers,
peptides, proteins), analytical techniques (mass spectrometry), and cellular
toxicity biomarkers. The current status of in vitro tools to detect reactive
intermediates is the following: 1. Small-molecular trapping agents such
glutathione or cyanide detect the production of reactive species with high
sensitivity by proper MS technique. However, it seems that also putative
"negatives" give rise to corresponding adducts. 2. Results from peptide and dG
(DNA targeting) trapper studies are generally in line with those of
small-molecular trappers, although also important differences exist. These two
trapping platforms do not overlap. 3. It is anticipated that the in vitro adduct
studies could be fully interpreted only in conjunction with toxicity biomarker
(such as the Nrf2 pathway) information from whole cells or tissues. However,
while there are tools to characterize the chemical liability and there are
correlation between individual/integrated endpoints and toxicity, there are
still severe gaps in understanding the mechanisms behind the link between
reactive metabolites and adverse effects. As an organochlorine insecticide, endosulfan has been widely banned or
restricted, but it is still largely used in many developing countries. Previous
studies have shown multiple adverse health effects of endosulfan. However, the
neurotoxicity of endosulfan has not been fully elucidated. In this study,
endosulfan isomers (α-/β-endosulfan) and their major metabolites (endosulfan
sulfate, endosulfan diol, and endosulfan lactone) were, respectively, exposed to
human neuroblastoma SH-SY5Y cells. Results showed that both α-endosulfan and
β-endosulfan caused decrease of cell viability and morphological damages in a
dose-dependent manner. Their median effective concentrations (EC50s) were
respectively 79.6 μM (α-endosulfan) and 50.37 μM (β-endosulfan) for 72 h
exposure. EC50s of α/β-endosulfan mixture were lower than that of the single
isomer. However, EC50s of its metabolites were higher than that of technical
endosulfan. Endosulfan and its metabolites caused increases of reactive oxygen
species and the lipid peroxidation, but decrease of superoxide dismutase in a
dose-dependent manner. These results indicate that α-endosulfan exhibits higher
neurotoxicity than β-endosulfan. Mixture of endosulfan isomers shows stronger
cytotoxicity than the single isomer. After endosulfan is degraded, cytotoxicity
of its metabolites decreases gradually. The neurotoxicity of endosulfan and its
metabolites is closely related to oxidative damage and antioxidative deficit. Diosbulbin B (DIOB), a furanoid, is a major constituent of herbal medicine
Dioscorea bulbifera L. Exposure to DIOB caused liver injury in humans and
experimental animals. The mechanisms of DIOB-induced hepatotoxicities remain
unknown. The present study demonstrated that DIOB induced hepatotoxicities in a
time- and dose-dependent manner in mice. H&E stained histopathologic image
showed the occurrence of necrosis in the liver obtained from the mice treated
with DIOB at dose of 200 mg/kg. Pretreatment with KTC protected the animals from
hepatotoxicities and hepatic GSH depletion induced by DIOB, increased area under
the concentration-time curve of blood DIOB, decreased urinary excretion of GSH
conjugates derived from DIOB, and increased urinary excretion of parent drug.
Pretreatment with BSO exacerbated DIOB-induced hepatotoxicities. In order to
define the role of furan moiety in DIOB-induced liver toxicities, we replaced
the furan of DIOB with a tetrahydrofuran group by chemical hydrogenation of the
furan ring of DIOB. No liver injury was observed in the animals given the same
doses of tetrahydro-DIOB. The furan moiety was essential for DIOB-induced
hepatotoxicities. The results implicate the cis-enedial reactive metabolite of
DIOB was responsible for the observed toxicities. The observed modest depletion
of hepatic GSH in DIOB-treated animals suggests the actions of one or more
reactive metabolites, and the hepatic injury observed could be due at least in
part to reactions of these metabolites with crucial biomolecules. Cytochrome
P450 3A enzymes are implicated in DIOB-induced hepatotoxicities by catalyzing
the formation of the reactive metabolite of DIOB. INTRODUCTION: A number of withdrawn drugs are known to undergo bioactivation by
a range of drug metabolizing enzymes to chemically reactive metabolites that
bind covalently to protein and DNA resulting in organ toxicity and
carcinogenesis, respectively. An important goal in drug discovery is to identify
structural sites of bioactivation within discovery molecules for providing
strategic modifications that eliminate or minimize reactive metabolite
formation, while maintaining target potency, selectivity and desired
pharmacokinetic properties leading to the development of efficacious and
nontoxic drugs.
AREAS COVERED: This review covers experimental techniques currently used to
detect reactive drug metabolites and provides recent examples where information
from mechanistic in vitro studies was successfully used to redesign candidate
drugs leading to blocked or minimized bioactivation. Reviewed techniques include
in vitro radiolabeled drug covalent binding to protein and reactive metabolite
trapping with reagents such as glutathione, cyanide, semicarbazide and DNA
bases. Case studies regarding reactive metabolite detection using a combination
of varied techniques, including liquid chromatography-tandem mass spectrometry
and NMR analyses and subsequent structural modification are discussed.
EXPERT OPINION: Information derived from state-of-art mechanistic drug
metabolism studies can be used successfully to direct medicinal chemistry
towards the synthesis of candidate drugs devoid of bioactivation liabilities,
while maintaining desired pharmacology and pharmacokinetic properties. Diclofenac is a widely prescribed NSAID, which by itself and its reactive
metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic
hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced
liver injury (DILI). In the present work, an investigation of the inhibitory
effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4'-OH-Dic)
and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide
(DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP
synthesis in rat liver mitochondria was carried out. A mechanism based
inhibition of ATP synthesis is exerted by diclofenac and its metabolites.
Phase-I metabolite (4'-OH-Dic) and Phase-II metabolites (DicGluA and DicSG)
showed potent inhibition (2-5 fold) of ATP synthesis, where as 5-OH-Dic, one of
the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The
calculated kinetic constants of mechanism based inhibition of ATP synthesis by
Dic showed maximal rate of inactivation (Kinact) of 2.64 ± 0.15 min(-1) and half
maximal rate of inactivation (KI) of 7.69 ± 2.48 μM with Kinact/KI ratio of
0.343 min(-1) μM(-1). Co-incubation of mitochondria with Dic and reduced GSH
exhibited a protective effect on Dic mediated inhibition of ATP synthesis. Our
data from this study strongly indicate that Dic as well as its metabolites could
be involved in the hepato-toxic action through inhibition of ATP synthesis. The formation of reactive metabolites through biotransformation is the suspected
cause of many adverse drug reactions. Testing for the propensity of a drug to
form reactive metabolites has increasingly become an integral part of
lead-optimization strategy in drug discovery. DNA reactivity is one undesirable
facet of a drug or its metabolites and can lead to increased risk of cancer and
reproductive toxicity. Many drugs are metabolized by cytochromes P450 in the
liver and other tissues, and these reactions can generate hard electrophiles.
These hard electrophilic reactive metabolites may react with DNA and may be
detected in standard in vitro genotoxicity assays; however, the majority of
these assays fall short due to the use of animal-derived organ extracts that
inadequately represent human metabolism. The current study describes the
development of bacterial systems that efficiently detect DNA-damaging
electrophilic reactive metabolites generated by human P450 biotransformation.
These assays use a GFP reporter system that detects DNA damage through induction
of the SOS response and a GFP reporter to control for cytotoxicity. Two human
CYP1A2-competent prototypes presented here have appropriate characteristics for
the detection of DNA-damaging reactive metabolites in a high-throughput manner.
The advantages of this approach include a short assay time (120-180 min) with
real-time measurement, sensitivity to small amounts of compound, and
adaptability to a microplate format. These systems are suitable for
high-throughput assays and can serve as prototypes for the development of future
enhanced versions. |
What is the "wearing-off" phenomenon in levodopa-treated patients with Parkinson's Disease? | Chronic administration of traditional levodopa/dopa decarboxylase inhibitor formulations to Paskinson's Disease patients is associated with the development of complications, such as wearing-off phenomenon. Wearing-off phenomenon is characterized by the predictable emergence of motor symptoms (e.g. rigidity and freezing) and nonmotor PD symptoms (e.g. anxiety and shortness of breath), before the next scheduled dose of medication. | "Wearing-off" effect, the most common form of levodopa-induced fluctuations,
seems to be related to the short plasma half-life of the drug. More sustained
plasma levodopa levels may be achieved with a new controlled-release formulation
of carbidopa/levodopa, Sinemet CR4. We studied 20 patients, 12 men and 8 women,
with Parkinson's disease complicated by "wearing-off" phenomenon. Mean age was
61.1 +/- 8.1 years, duration of symptoms 8.3 +/- 2.4 years, and the Hoehn-Yahr
stage 3.0 +/- 0.9. In a 12-week double-blind study, the average number of
tablets administered per day decreased from 5.7 +/- 1.2 to 3.8 +/- 0.7 when
Sinemet CR4 (50/200) was substituted for the standard Sinemet (25/100) (p less
than 0.001). However, this was at the expense of reducing the "on" time (without
dyskinesia) from 9.3 +/- 4.6 to 7.5 +/- 4.3 (p less than 0.05), although the
total "on" time did not significantly change. In a long-term follow-up of 18
patients, the "on" time with dyskinesia and morning dystonia significantly
increased (p less than 0.05). There was no significant change in the total daily
dosage of levodopa, but the daily number of doses and tablets significantly
decreased (p less than 0.001). Despite increased dyskinesia, most patients
preferred taking fewer tablets and have elected to continue taking Sinemet CR4
instead of standard Sinemet. Sinemet CR4 seems to offer a new and effective
strategy for the management of levodopa-related fluctuations. Pharmacokinetic and pharmacodynamic mechanisms for levodopa have been studied in
relation to the pathogenesis of the motor fluctuations which complicate advanced
Parkinson's disease. Since levodopa clearance from the general circulation was
found to be similar in patients with wearing-off or on-off phenomena and those
with a stable response to levodopa, peripheral pharmacokinetic factors are
unlikely to be involved. Efficacy half-time for levodopa, on the other hand, was
significantly reduced in patients with mainly wearing-off fluctuations in
comparison to those manifesting a stable response to oral levodopa; individuals
with predomitly on-off phenomenon had an even more extreme reduction in the
duration of the antiparkinsonian action of levodopa. Conversion from oral to
intravenous levodopa treatment immediately stabilized plasma levodopa levels in
both the wearing-off and on-off groups; motor variability also decreased,
especially in those with wearing-off phenomenon. During 11 days of continuous
intravenous levodopa therapy, additional reductions in motor fluctuations
occurred in both groups, but at a significantly faster rate in patients with
wearing-off than in those with on-off responses. These results suggest that
wearing-off phenomenon may arise as a consequence of the degeneration of
dopamine terminals due to natural disease progression with a resultant inability
to buffer variations in levodopa availability; on-off phenomenon, may reflect
additional postsynaptic dopamine receptor dysregulation, possibly in response to
the resultant, nonphysiologic fluctuations in synaptic dopamine. The response to continuous intravenous infusion of levodopa was evaluated in 23
patients with Parkinson's disease complicated by motor fluctuations. Conversion
from oral to intravenous levodopa treatment resulted in an immediate and
sustained stabilization of plasma levodopa levels in both the wearing-off and
on-off groups. Motor variability also diminished within the first 24 hours of
infusion, although to a much greater extent in patients with the wearing-off
phenomenon. Over the next 5 to 11 days of intravenous treatment, further
reductions in motor fluctuations occurred in both groups, at a substantially
faster rate in the wearing-off group than in the on-off group. The degree of
immediate stabilization of parkinsonian motor response correlated best with
disease duration, whereas the rate of subsequent improvement correlated most
closely with levodopa dose. The results support the view that the wearing-off
phenomenon may reflect the degeneration of dopamine terminals as a consequence
of natural disease progression with a resultant inability to buffer variations
in levodopa availability; the on-off phenomenon may reflect additional
postsynaptic changes, possibly occurring in response to the nonphysiological
fluctuations in synaptic dopamine, which gradually remit during continuous
levodopa administration. The contribution of the peripheral and central pharmacokinetic mechanisms of
levodopa to the pathogenesis of the motor fluctuations that complicate its
long-term administration was studied in 28 parkinsonian patients. The rate of
levodopa clearance from the general circulation, its plasma half-life, and
apparent volume of distribution were indistinguishable between patients with the
on-off or the wearing-off phenomenon and those with a stable response to
levodopa or those who had not been previously treated with levodopa. Peripheral
pharmacokinetic factors are thus unlikely to account for the development of
these response fluctuations. Conversely, the efficacy half-time of levodopa
differed markedly among the four response groups studied and may provide a
quantitative index of central mechanisms that favor the development of the
wearing-off and on-off phenomena. Although symptom duration was the best
predictor of the severity of untreated parkinsonism, levodopa dose correlated
best with response half-time. The wearing-off phenomenon may primarily reflect
the loss of buffering capacity caused by degeneration of the dopamine neurons,
while the development of the on-off phenomenon appears to require additional
postsynaptic changes, possibly at the receptor level. Wearing-off phenomenon that complicates levodopa therapy of Parkinson's disease
has been attributed to a reduction in striatal dopamine storage due to the
progressive degeneration of presynaptic dopaminergic terminals. To determine
whether postsynaptic mechanisms also contribute to these response fluctuations,
the duration of the antiparkinsonian response in parkinsonian patients grouped
by disease severity was compared following discontinuation of a steady-state
optimal-dose infusion of apomorphine. Although the plasma half-life of this
dopamine receptor agonist remained constant, its mean efficacy half-time
declined from 66 minutes in early, levodopa-naive patients to 33 minutes in
advanced, complicated parkinsonians (p < 0.005). Since the motor effects of
apomorphine do not depend on the presence of dopaminergic terminals, changes at
the postsynaptic level undoubtedly contribute to the diminished response
duration. The only slightly greater attenuation of levodopa's motor effects
observed previously under similar conditions suggests these postjunctional
alterations, possibly involving relatively plastic striatal dopaminoceptive
systems, account for most of the shortening in the duration of levodopa action
that underlie wearing-off fluctuations. The effects of tolcapone, a catechol-O-methyltransferase inhibitor, on the
bioavailability and efficacy of levodopa were evaluated in 12 patients with
Parkinson's disease (PD), 8 of whom showed signs of daily motor fluctuations
(wearing-off phenomenon). Motor disabilities were assessed in 12 patients at 7
time points before and after the chronic administration of tolcapone using the
Unified Parkinson's Disease Rating Scale (UPDRS). The UPDRS score was improved
at all points of determination. Eight patients with wearing-off phenomenon on
levodopa showed symptomatic improvement on the combination. The area under the
curve (AUC) for levodopa increased by 34% (p = 0.0059) after the administration
of tolcapone. The elimination half-life (T1/2) of levodopa was significantly
prolonged by 81% (p = 0.0001) after the treatment. The AUC of 3-O-methyldopa, a
metabolite of levodopa, was decreased by 79% (p = 0.0001) and the Cmax (maximum
concentration) was also decreased by 80%d after the administration (p = 0.0001)
of tolcapone. The combination of tolcapone and levodopa was well tolerated. Our
findings suggest that tolcapone improves the pharmacokinetics of levodopa in
plasma and motor symptoms of fluctuating PD patients. It is suggested that
tolcapone may be useful drug adjunct to levodopa in treating patients with PD
with wearing-off phenomena. We studied the new catechol-O-methyltransferase inhibitor tolcapone, 100 and 200
mg, three times daily (tid) in a randomized, double-blind, parallel-group trial
involving 202 parkinsonian patients who were experiencing the "wearing-off"
phenomenon on levodopa therapy. After 3 months, patients receiving tolcapone had
a significant decrease in mean daily levodopa dose requirement compared with
placebo-treated patients (p < 0.01). In patients treated with tolcapone 200 mg
tid, daily "off" time, measured using patient diaries, was reduced from baseline
by 3.25 hours; this reduction was significantly different from that seen in the
placebo group (p < 0.01). Moreover, the number of daily levodopa intakes was
reduced significantly in each tolcapone group compared with placebo (p < 0.01).
We found significant improvements in motor function and overall efficacy in the
tolcapone groups (p < 0.01). The most frequent adverse events were associated
with levodopa treatment. Dyskinesia developed or worsened in 18% of patients
receiving placebo, in 51% receiving tolcapone 100 mg tid, and in 64% receiving
200 mg tid, with most cases occurring within the first 30 days of the study.
Diarrhea was the most frequent nondopaminergic event, occurring in 14% on
placebo, 13% on tolcapone 100 mg tid, and 19% on 200 mg tid. Overall 18% of
patients withdrew because of adverse events: 15% on placebo, 17% on tolcapone
100 mg tid, and 22% on 200 mg tid. We conclude that tolcapone as an adjunct
offers promise for the relief of the "wearing-off" phenomenon in
levodopa-treated parkinsonian patients. BACKGROUND: More than 50% of patients with Parkinson's disease develop motor
response fluctuations (the "wearing off" phenomenon) after more than five years
of levodopa therapy. Inhibition of catechol-O-methyltransferase by tolcapone has
been shown to increase levodopa bioavailability and plasma elimination half
life, thereby prolonging the efficacy of levodopa.
OBJECTIVES: The primary objective was to evaluate the efficacy of tolcapone in
reducing "wearing off" in levodopa treated, fluctuating parkinsonian patients.
Secondary objectives included assessment of reduction in levodopa requirements,
improvement in patients' clinical status, duration of improvements, and
tolerability of tolcapone.
METHODS: In this multicentre, randomised, double blind, placebo controlled
trial, 58 patients received placebo, 60 received 100 mg tolcapone three times
daily (tid), and 59 received 200 mg tolcapone tid, in addition to
levodopa/benserazide.
RESULTS: After three months with 200 mg tolcapone tid, "off" time decreased by
26.2% of the baseline value, "on" time increased by 20.6% (P<O.01 v placebo),
and the mean total daily levodopa dose decreased by 122 mg from the baseline
dose of 676 mg (P<0.01). These responses were maintained up to nine months. With
100 mg tolcapone tid, "off" time decreased by 31.5% (P<0.05), "on" time
increased by 21.3% (P<0.01), and the mean total daily levodopa dose decreased by
109 mg from the baseline dose of 668 mg (P<0.05). With 200 mg tolcapone tid,
unified Parkinson's disease rating scale motor and total scores were
significantly reduced, and quality of life (sickness impact profile) scores were
significantly improved. Both dosages were well tolerated. Dyskinesia was the
most often reported levodopa induced adverse event. Diarrhea was the most often
reported non-dopaminergic adverse event and the most frequent reason for
withdrawal from the study: four patients in the 100 mg tolcapone tid group and
six in the 200 mg tid group withdrew because of diarrhea.
CONCLUSION: Tolcapone prolongs "on" time in fluctuating parkinsonian patients
while allowing a reduction in daily levodopa dosage, thereby improving the
efficacy of long term levodopa therapy. BACKGROUND: More than 50% of patients with Parkinson's disease develop motor
response fluctuations (the 'wearing off" phenomenon) after more than five years
of levodopa therapy. Inhibition of catechol-O-methyltransferase by tolcapone has
been shown to increase levodopa bioavailability and plasma elimination half
life, thereby prolonging the efficacy of levodopa.
OBJECTIVES: The primary objective was to evaluate the efficacy of tolcapone in
reducing "wearing off" in levodopa treated, fluctuating parkinsonian patients.
Secondary objectives included assessment of reduction in levodopa requirements,
improvement in patients' clinical status, duration of improvements, and
tolerability of tolcapone.
METHODS: In this multicentre, randomised, double blind, placebo controlled
trial, 58 patients received placebo, 60 received 100 mg tolcapone three times
daily (tid), and 59 received 200 mg tolcapone tid, in addition to
levodopa/benserazide.
RESULTS: After three months with 200 mg tolcapone tid, "off" time decreased by
26.2% of the baseline value, "on" time increased by 20.6% (p < 0.01 vs.
placebo), and the mean total daily levodopa dose decreased by 122 mg from the
baseline dose of 676 mg (p < 0.01). These responses were maintained up to nine
months. With 100 mg tolcapone tid, "off" time decreased by 31.5% (p < 0.05),
"on" time increased by 21.3% (p < 0.01), and the mean total daily levodopa dose
decreased by 109 mg from the baseline dose of 668 mg (p < 0.05). With 200 mg
tolcapone tid, unified Parkinson's disease rating scale motor and total scores
were significantly reduced, and quality of life (sickness impact profile) scores
were significantly improved. Both dosages were well tolerated. Dyskinesia was
the most often reported levodopa induced adverse event. Diarrhoea was the most
often reported non-dopaminergic adverse event and the most frequent reason for
withdrawal from the study: four patients in the 100 mg tolcapone tid group and
six in the 200 mg tid group withdrew because of diarrhoea.
CONCLUSION: Tolcapone prolongs "on" time in fluctuating parkinsonian patients
while allowing a reduction in daily levodopa dosage, thereby improving the
efficacy of long term levodopa therapy. We studied the new catechol-O-methyltransferase inhibitor tolcapone, 100 and 200
mg, three times daily (tid) in a randomized, double-blind, parallel-group trial
involving 202 parkinsonian patients who were experiencing the "wearing-off"
phenomenon on levodopa therapy. After 3 months, patients receiving tolcapone had
a significant decrease in mean daily levodopa dose requirement compared with
placebo-treated patients (p < 0.01). In patients treated with tolcapone 200 mg
tid, daily "off" time, measured using patient diaries, was reduced from baseline
by 3.25 hours; this reduction was significantly different from that seen in the
placebo group (p < 0.01). Moreover, the number of daily levodopa intakes was
reduced significantly in each tolcapone group compared with placebo (p < 0.01).
We found significant improvements in motor function and overall efficacy in the
tolcapone groups (p < 0.01). The most frequent adverse events were associated
with levodopa treatment. Dyskinesia developed or worsened in 18% of patients
receiving placebo, in 51% receiving tolcapone 100 mg tid, and in 64% receiving
200 mg tid, with most cases occurring within the first 30 days of the study.
Diarrhea was the most frequent nondopaminergic event, occurring in 14% on
placebo, 13% on tolcapone 100 mg tid, and 19% on 200 mg tid. Overall 18% of
patients withdrew because of adverse events: 15% on placebo, 17% on tolcapone
100 mg tid, and 22% on 200 mg tid. We conclude that tolcapone as an adjunct
offers promise for the relief of the "wearing-off " phenomenon in
levodopa-treated parkinsonian patients. Entacapone is a potent and specific peripheral catechol-O-methyltransferase
(COMT) inhibitor. It has been shown to improve the clinical benefits of levodopa
plus an aromatic L-amino acid decarboxylase inhibitor (AADC) when given to
patients with Parkinson's disease and end-of-dose deterioration in the response
to levodopa (the 'wearing off' phenomenon). The efficacy of entacapone is
currently being assessed in patients with stable Parkinson's disease. In 2 well
conducted trials of 6 months' duration and smaller short term studies, treatment
with entacapone (200 mg with each dose of levodopa/AADC inhibitor) was
associated with significant increases in daily 'on' time and decreases in 'off'
time. Changes in Unified Parkinson's Disease Rating Scale (UPDRS) scores
concurred with changes in 'on' and 'off' times: entacapone improved total,
activities of daily living and motor function scores, but it had no effect on
mentation scores. Entacapone also provided benefits when given with controlled
release levodopa/ AADC inhibitor or with standard levodopa/AADC inhibitor and
selegiline in small trials. Dopaminergic events, including dyskinesia and
nausea, are among the most common events with entacapone, and are related to the
drug's ability to potentiate the effects of levodopa. Diarrhoea, abdominal pain,
constipation and urine discolouration are the most common nondopaminergic
events, although the latter event is the only one to occur consistently more
frequently with entacapone than with placebo. However, adverse events of any
type infrequently led to study discontinuation.
CONCLUSIONS: The efficacy and tolerability of entacapone administered with
levodopa/AADC inhibitor have not yet been compared with those of other
strategies for the treatment of Parkinson's disease. However, once the decision
to initiate levodopa therapy has been made, studies generally support the use of
entacapone as an adjunct to levodopa in patients with Parkinson's disease and
the 'wearing off' phenomenon. We report a 73-year-old Japanese woman with familial Parkinson's disease. The
patient was well until her 67 years of the age, when she noted rest tremor in
her right hand. Soon after her gait became short stepped. She visited our clinic
on October 6, 1992 when she was 68 years old. She was alert and well oriented
without dementia. She showed masked face, small voice, small stepped gait,
retropulsion, resting tremor in her right hand, rigidity in the neck, and
bradykinesia. She was treated with 400 mg/day of levodopa-carbidopa, which
improved her symptoms, however, she developed wearing off phenomenon 3 years
after the initiation of levodopa treatment. On August 26, 1998, she developed
abdominal pain, diarrhea, and vomiting. She was admitted to another hospital,
where abdominal plain x-ray revealed an evidence of intestinal obstruction
(ileus). She was treated with nasogastric suction and intravenous fluid. Her
condition did not improve and she was transferred to our hospital on August 29,
1998. Her family history revealed no consanguineous marriage. She had two elder
brothers and three elder sisters. One of her brothers had been diagnosed as
Parkinson's disease. Her husband also suffered from Parkinson's disease,
however, her parents apparently did not have Parkinson's disease. On admission,
she appeared to be drowsy. Her blood pressure was 102/70 mmHg, body temperature
36.2 degrees C. The lungs were clear and no cardiac murmur was present. Abdomen
was flat and bowel sound was audible. No abnormal mass was palpable. Neurologic
examination revealed mild consciousness disturbance, masked face, and small
voice. No motor paralysis was noted. Muscle tone was hypotonic. No abnormal
involuntary movement was noted. Abnormal laboratory findings on admission were
as follows; WBC 11,300/microliter, amylase 1,373 IU/l, CK 446 IU/l, BUN 50
mg/dl, creatinine 1.17 mg/dl, CRP 22.7 mg/ dl, Na 134 mEq/l, K 3.1 mEq/l, and Cl
81 mEq/l. A chest x-ray film revealed pneumonic shadows in both lower lung
fields. She was treated by nasointestinal suction, intravenous fluids, and
chemotherapy for her infection. Her BP started to drop on September 2 and she
developed cardiac arrest on the same day. She was discussed in a neurological
CPC. The chief discussant arrived at the conclusion that the patient had a form
of autosomal domit familial Parkinson's disease. As parents did not have
Parkinson's disease, some of the participants raised the possibility of
autosomal recessive inheritance. But the age of onset was too late for autosomal
recessive inheritance. Majority thought that the mode of inheritance was
autosomal domit with low penetrance. alpha-Synuclein mutation causes an
autosomal domit familial Parkinson's disease, but this type is very rare in
non-Greek populations and the penetrance is high. Chromosome 2-linked autosomal
domit familial Parkinson's disease shows low penetrance. There are many other
autosomal domit forms of familial Parkinson's disease linked to yet unknown
chromosome loci. Majority thought that this patient also had a form of Lewy-body
positive autosomal domit familial Parkinson's disease of unknown chromosome
locus. Post mortem examination revealed ischemic intestinal lesion with
strangulation. This was thought to be the cause of her death. In the central
nervous system, the brain appeared to be normal by inspection. In the coronal
sections, the substantia nigra and the locus coeruleus showed marked
depigmentation. Histologic examination revealed marked neuronal loss and Lewy
body formation in the remaining neurons. Pathologic examination was consistent
with Parkinson's disease. Mutational analysis for the parkin gene was negative. The efficacy and tolerability of entacapone was investigated in a randomized,
double-blind, placebo-controlled, 3-month study of 162 patients with Parkinson's
disease (PD) treated with levodopa and a dopamine agonist and experiencing
wearing-off motor fluctuations. Patients were randomized in a 3 : 2 ratio to
entacapone 200 mg or placebo, administered with each dose of levodopa. Efficacy
was judged on the improvement of "on" and "off" time while awake (Patient Diary
and UPDRS part IV Item 39), Investigators' Global Assessment, the SF-36 Health
Survey, and changes in levodopa dosages. Patients were monitored for adverse
events, laboratory safety and vital signs throughout the study. Improvements in
"on" time as assessed using patient diary data showed a trend in favour of
entacapone, however these did not reach statistical significance. "Off" time
while awake (UPDRS part IV Item 39) showed an improvement of at least one
category in 36% of entacapone-treated patients, compared with 22% in the control
group (p = 0.0038). The proportion of patients showing an improvement at the
Investigators' Global Assessment was significantly higher (p = 0.0006) in the
entacapone-treated group of patients. Also, the proportion of patients with a
reduction in their daily levodopa dose was significantly higher (p = 0.02) in
the entacapone group (28%) compared with placebo (13%). As expected, the most
frequent adverse events were dopamine-mediated (dyskinesia: entacapone 31%
versus placebo 13%), and harmless urinary discoloration. The modest increase in
dyskinesias could be readily managed by levodopa down-adjustment, and, at study
end there was no significant difference for the UPDRS "overall dyskinesia score"
between entacapone and placebo. In conclusion, although the primary efficacy
variable did not reach statistical significance, the present results demonstrate
that entacapone provides additional antiparkinsonian benefits to levodopa
therapy and is well tolerated in levodopa-treated PD patients experiencing
wearing-off motor fluctuations despite adjunct dopamine agonist therapy. We investigated the short-term effects of a single dose of levodopa (L-dopa) on
micturition function in PD patients with wearing-off phenomenon. Eighteen PD
patients who had median Hoehn and Yahr scores of 5 during the off phase and 3
during the on phase were recruited. We carried out urodynamic studies before and
about 1 hour after the patients had taken 100 mg of L-dopa with
dopa-decarboxylase inhibitor (DCI). After taking the L-dopa/DCI, urinary urgency
and urge incontinence aggravated, whereas voiding difficulty was alleviated in
all 12 patients. When compared to the baseline assessment, urodynamic study
results after taking 100 mg of L-dopa/DCI showed aggravated detrusor
hyperreflexia; decreased maximum bladder capacity (P = 0.006); an increased
maximum Watts Factor value (P = 0.001), reflecting the detrusor power on
voiding; an increased Abrams-Griffiths number (P = 0.042), reflecting urethral
obstruction on voiding; decreased residual urine volume (P = 0.025); and
increased static urethral closure pressure (P = 0.012). One hundred milligrams
of L-dopa/DCI worsened detrusor hyperreflexia, producing worsened urinary
urgency and urge incontinence during the storage (bladder-filling) phase. It
also increased detrusor contractility much more than it did urethral obstruction
in the voiding phase, producing overall lessening of voiding difficulty and
improving voiding efficiency in our PD patients with the wearing-off phenomenon. Levodopa-treated Parkinson's disease is often complicated by the occurrence of
motor fluctuations, which can be predictable ('wearing-off') or unpredictable
('on-off'). In contrast, untreated dopa-responsive dystonia (DRD) is usually
characterized by predictable diurnal fluctuation. The pathogenesis of motor
fluctuations in treated Parkinson's disease and diurnal fluctuation in untreated
DRD is poorly understood. We have developed a mathematical model indicating that
all these fluctuations in motor function can be explained by presynaptic
mechanisms. The model is predicated upon the release of dopamine being subject
to probabilistic variations in the quantity of dopamine released by exocytosis
of vesicles. Specifically, we propose that the concentration of intravesicular
dopamine undergoes dynamic changes according to a log-normal distribution that
is associated with different probabilities of release failure. Changes in two
parameters, (i) the proportion of vesicles that undergo exocytosis per unit of
time and (ii) the proportion of dopamine subject to re-uptake from the synapse,
allowed us to model different curves of levodopa response, for the same degree
of nigrostriatal damage in Parkinson's disease. The model predicts the following
periods of levodopa clinical benefit: 4 h for stable responders, 3 h for
wearing-off fluctuators, and 1.5 h for on-off fluctuators. The model also
predicts that diurnal fluctuation in untreated DRD should occur some 8 h after
getting up in the morning. All these results fit well with clinical
observations. Additionally, we calculated the probability of obtaining a second
ON period after a single dose of levodopa in Parkinson's disease (the 'yo-yoing'
phenomenon). The model shows that the yo-yoing phenomenon depends on how fast
the curve crosses the threshold that separates ON and OFF states, which explains
why this phenomenon is virtually exclusive to patients with on-off fluctuations.
The model supports the idea that presynaptic mechanisms play a key role in both
short-duration and long-duration responses encountered in Parkinson's disease.
Dyskinesias may also be explained by the same mechanisms. Degenerative process in Parkinson's disease affects substantia nigra and other
central structures of an extrapyramidal system but it can also affect central
and peripheral autonomic centres. One of the most frequent late complications in
levodopa therapy is a wearing off phenomenon. We present a patient treated for
Parkinson's disease in whom during the period of levodopa wearing off we
observed a paroxysmal abdominalgia apart from other features of a typical
movement disorders like: increasing rigidity, gait disturbances and tremor.
Abdominalgia consisted of stomach cramps, with variable localization in epi-,
meso- and hypogastrium. Rectal tenesmus was also present. The patient was
treated with analgesics, spasmolytics and carminative drugs with no effect.
Abdominal pains regressed after an intake of the next levodopa dose. The patient
presented with other features of a gastrointestinal tract autonomic system
dysfunction like: chronic constipation, preterm satiety resulting in food intake
reduction and a decrease in body weight. There was no organic lesions of the
gastrointestinal system that could explain such disturbances. Pharmacologic
treatment modification (more frequent levodopa dosage, additional dopamine
agonist) resulted in some improvement.
CONCLUSION: It is possible that the abdominal pains could be a clinical
manifestation of a digestive tract dyskinesias, occurring during Levodopa is the most efficacious treatment in the management of Parkinson's
disease. Unfortunately, chronic use of traditional levodopa/dopa decarboxylase
inhibitor formulations is associated with the development of complications, such
as wearing-off and dyskinesia. In an attempt to avoid these complications, some
physicians delay the introduction of levodopa or employ levodopa-sparing
strategies; however, these strategies are frequently suboptimal for patients. As
most patients require the superior efficacy of levodopa during the course of
their disease, an appreciation of the changing response to levodopa over time
and an understanding of the pharmacokinetic principles underlying the
development of complications such as wearing-off is essential in the long-term
management of the patient. OBJECTIVE: To evaluate the role of the practicing pharmacist in the
identification and current treatment of the levodopa wearing-off phenomenon
experienced by patients with Parkinson's disease (PD) who are receiving chronic
levodopa therapy.
DATA SOURCES: Literature retrieval was accessed through MEDLINE (1967-June 2007)
using the terms levodopa, wearing-off, and Parkinson's disease. In addition,
reference citations from publications identified were reviewed.
STUDY SELECTION AND DATA EXTRACTION: All articles that were identified from the
data sources and written in English were evaluated.
DATA SYNTHESIS: Levodopa is the most efficacious therapeutic agent in PD;
however, the response of patients to levodopa changes over time. Eventually, the
duration of response becomes shorter and more unpredictable, and complications
emerge. One of the first complications observed with levodopa therapy is
wearing-off, which can emerge within 1-3 years of initiation of levodopa
treatment. Wearing-off is characterized by the predictable emergence of motor
and nonmotor PD symptoms before the next scheduled dose of medication. Despite
effective treatment options to tackle wearing-off, it remains underrecognized
and under treated. With early identification and optimization of treatment,
wearing-off can be managed effectively, resulting in improved quality of life
for patients with PD.
CONCLUSIONS: Owing to their training and accessibility, pharmacists play an
increasingly important role in the management of patients with PD. Pharmacists
are uniquely placed to identify wearing-off, offer timely advice, and facilitate
the optimization of treatment regimens to improve patients' quality of life and
enhance long-term outcomes. OBJECTIVE: To evaluate the effectiveness of entacapone in the management of
levodopa wearing-off in Parkinson's disease (PD) in a naturalistic, real-life
setting.
RESEARCH DESIGN AND METHODS: This prospective, open-label, observational study
included patients with idiopathic PD. Patients were eligible for inclusion if
they had been taking 3-5 doses of levodopa per day for ≥2 months and had shown
signs of levodopa wearing-off for ≥1 month. Subjects received entacapone
(recommended dose: 1 × 200 mg tablet with each levodopa dose) for 28 days.
Patients were asked to complete a wearing-off questionnaire and the
eight-question Parkinson's Disease Questionnaire Quality of Life assessment
(PDQ-8). Activities of daily living (both in the on and off states) were
assessed using the Unified Parkinson's Disease Rating Scale (UPDRS) part II.
Clinical Global Impression (CGI) of severity of PD-related symptoms was assessed
using a modified CGI tool. Patient global assessment of severity of PD symptoms
was also obtained.
RESULTS: A total of 341 patients were enrolled by 68 physicians across Canada.
At Day 28, 56.9% of the subjects indicated improvement compared to baseline on
the modified CGI of change (CGI-C); 21.4% reported no change. Improvements were
also observed on the UPDRS II and the PDQ-8. Benefit from entacapone appeared to
be relatively uniform across subgroups (e.g., number of daily levodopa doses,
use of other anti-PD medications).
STUDY LIMITATIONS: The results of this study may be biased due to factors
inherent in open-label, community-based trials (e.g., compliance). This is,
however, reflective of everyday clinical practice.
CONCLUSIONS: In this naturalistic, real-life study, the addition of entacapone
to levodopa therapy provided benefits in quality of life and activities of daily
living for a substantial proportion of PD patients experiencing wearing-off. Although levodopa is considered the gold standard for Parkinson's disease
therapy, prolonged use of this drug can result in motor complications such as a
'wearing-off' phenomenon. This outcome is seen in a significant number of
patients with Parkinson's disease taking levodopa and, in some cases, is
observed only a few hours after intake of the last dose of levodopa. Patients
experiencing the wearing-off period may present with sensory, autonomic,
psychiatric and motor fluctuations. Although infrequent, shortness of breath is
an important non-motor wearing-off symptom experienced by patients with
Parkinson's disease. In addition to being a symptom induced by wearing off,
other causes of shortness of breath include pulmonary diseases, coronary artery
disease and anxiety. Thus, it is important to identify the cause of shortness of
breath to ensure that the appropriate treatment is initiated. We report here on
a patient with Parkinson's disease who was taking levodopa and developed both
shortness of breath and hyperventilation during wearing-off periods. He
underwent extensive pulmonary and cardiac investigations that were unremarkable.
His shortness of breath was determined to be a wearing-off phenomenon and his
condition improved with the addition of a catechol-O-methyltransferase inhibitor
(entacapone). BACKGROUND: The duration of clinical control of motor symptoms of Parkinson
disease (PD) treated with levodopa/carbidopa preparations eventually starts to
shorten, a phenomenon known as end-of-dose "wearing off." The involvement of
core nonmotor symptoms of "wearing off" (depressed mood, pain/aching, anxiety,
and cloudy/slowed thinking) is not well understood.
METHODS: A post hoc analysis from a study to validate the self-rated 9-item,
Wearing-Off Questionnaire (WOQ-9), which was designed to identify motor and
nonmotor symptoms of "wearing off" in PD patients, was performed to compare the
frequency and sensitivity of motor and nonmotor symptoms of "wearing off" from
dopaminergic therapy.
RESULTS: Analysis of responses to the WOQ-9 from 216 PD patients found that
individual nonmotor symptoms were reported by 25% to 50% and motor symptoms by
55% to 80% of patients. Individual nonmotor symptoms improved following the next
dose of dopaminergic therapy in 43% to 53% of the patients who presented with
such symptoms, whereas motor symptoms improved in 48% to 66% of the cases,
suggesting both types of symptoms respond to dopaminergic therapies.
CONCLUSION: Nonmotor symptoms of PD appear sensitive to dopaminergic treatment.
These symptoms resemble those seen with depressive, anxiety, and somatoform
disorders suggesting potential shared mechanisms as well as possible treatment
implications. Long-term levodopa use is associated with the "End of Dose Wearing Off" (EODWO)
phenomenon wherein Parkinsonian symptoms return before a patient's next
scheduled dose of levodopa. Wearing off symptoms may include a variety of
autonomic, emotional, motor, psychological and sensory abnormalities. Abdominal
pain may be an important wearing off symptom as an early indicator of the
development of EODWO in Parkinson's disease (PD) patients. In this report, we
present two patients on levodopa therapy for PD who developed acute abdominal
pain as a symptom of EODWO. BACKGROUND AND PURPOSE: Wearing-off is one of the most frequent problems
encountered by levodopa-treated patients. Entacapone, a peripheral inhibitor of
catechol-O-methyltransferase (COMT), reduces this motor complication by
prolonging the effect of levodopa. We sought to understand the impact of
COMT-inhibition on movement execution in PD patients with wearing-off by
comparing functional magnetic resoce imaging (f-MRI) activation patterns
prior to and during entacapone treatment. Our hypothesis was to determine
whether changes in cortical activation are associated to COMT-inhibitor
treatment.
METHODS: Nine levodopa-treated non-demented PD patients with wearing-off were
prospectively studied in two f-MRI session, prior to and during entacapone
treatment. A group of control subjects were also studied for comparison.
RESULTS: The patients significantly improved under COMT-inhibitor treatment
based on home diaries. F-MRI results showed that at baseline the patients
presented a bilateral activation of the primary motor, controlateral premotor
cortex and supplementary motor area, as well as ipsilateral cerebellum. During
treatment with entacapone, PD patients showed reductions in the activations of
these cortical areas and a decreased activation in the ipsilateral cerebellum.
CONCLUSIONS: Our preliminary findings indicate that f-MRI is able to detect
cortical activation changes during long-term modulation of dopaminergic
treatment in PD patients with wearing-off, and thus, this technique could be
further investigated in advanced PD patients. The objective of this study was to investigate the risk factors of wearing-off
phenomenon in Parkinson's disease (PD) and propose safe dosage of levodopa to
reduce wearing-off development based on Chinese cohort. Patients with PD who had
taken levodopa (L-dopa) for at least 1 month were recruited. Wearing-off was
diagnosed based on validated Chinese version of a patient self-rated 9-question
Wearing-Off Questionnaire (WOQ-9) and clinical definition. Eleven variables
(gender, disease duration at L-dopa initiation, disease duration at assessment,
age at onset, age at assessment, H-Y stage, UPDRS III, L-dopa daily total dosage
and dosage adjusted to weight, duration of L-dopa treatment, initial drug
recipe) were included in our analysis. Univariate analysis, multivariate
logistic regression analysis and decision tree classification model(DTC) were
used to detect risk factors of wearing-off. Receiver operating characteristic
(ROC) curve and DTC were used to investigate cut-off value of L-dopa to best
predict wearing-off. Two hundred and thirty-four patients were investigated in
our study, among whom 111 developed wearing-off. Patients with wearing-off
tended to receive higher L-dopa dosage and endure longer duration of L-dopa
treatment. L-Dopa dosage as 281 mg/day and 4.2 mg/kg/day by ROC, as well as 269
mg/day and 3.2 mg/kg/day by DTC were cut-off values for wearing-off. L-Dopa
dosage and duration of L-dopa treatment were related to increased wearing-off
development. Cumulative L-dopa dosage and L-dopa daily dosage were better
predictive of wearing-off. Inadequate evidence was present for delayed L-dopa
initiation. L-Dopa daily dosage no more than 275 mg or 4.2 mg/kg was regarded as
safe. BACKGROUND AND PURPOSE: Opicapone (OPC) is a novel third generation
catechol-O-methyltransferase (COMT) inhibitor that enhances levodopa
availability. This study investigated the effects of OPC in comparison with
placebo on levodopa pharmacokinetics, tolerability and safety, COMT activity and
motor response to levodopa in Parkinson's disease (PD) patients with motor
fluctuations.
METHODS: This was a randomized, multicentre, double-blind and placebo-controlled
study in four parallel groups of PD patients treated with standard-release
100/25 mg levodopa/carbidopa or levodopa/benserazide and with motor fluctuations
(wearing-OFF phenomenon). Subjects were sequentially assigned to be
administered, once-daily, up to 28 days (maintece phase), placebo (n = 10) or
5 (n = 10), 15 (n = 10) and 30 mg (n = 10) OPC. Two levodopa tests were
performed, one at baseline and another following the maintece phase. Subjects
kept a diary to record motor fluctuations (ON/OFF periods) throughout the study.
RESULTS: In relation to placebo, levodopa exposure (AUC0-6) increased 24.7%,
53.9% and 65.6% following 5, 15 and 30 mg OPC, respectively. Maximum COMT
inhibition (Emax) ranged from 52% (5 mg OPC) to 80% (30 mg OPC). The study was
not designed to detect any significant differences in motor performance, but the
exploratory analysis performed shows improvement in various motor outcomes,
including a dose-dependent change in absolute OFF time corresponding to a
percentage decrease of 4.16% (P > 0.05), 29.55% (P > 0.05) and 32.71% (P < 0.05)
with 5, 15 and 30 mg OPC, respectively. Treatments were generally well tolerated
and safe.
CONCLUSIONS: OPC is a promising new COMT inhibitor that significantly decreased
COMT activity, increased systemic exposure to levodopa and improved motor
response. BACKGROUND AND PURPOSE: The 9-item Wearing-off Questionnaire (WOQ-9) is a useful
tool for screening of wearing-off. We performed a validation study of the
Japanese version of the WOQ-9 (JWOQ-9) using a cross-sectional design in
Japanese Parkinson's disease (PD) patients diagnosed with sporadic PD and
treated with levodopa.
METHODS: Subjects with severe dementia, uncontrolled psychiatric comorbidities,
and previous PD neurosurgery were excluded. The wearing-off phenomenon was
detected according to the JWOQ-9, and the results were compared with independent
evaluations of wearing-off conducted by PD specialists blinded to the JWOQ-9
results. To validate the JWOQ-9, a sample size of at least 70 patients with
wearing-off and 70 patients without wearing-off was required. Therefore, a total
of 180 patients (101 patients with wearing-off and 79 patients without
wearing-off) were enrolled.
RESULTS: The sensitivity, specificity, positive predictive value, and negative
predictive value of the JWOQ-9 were 94.1%, 39.2%, 66.4%, and 83.8%,
respectively. Motor symptom questions demonstrated both moderate sensitivity
(58.1-87.3%) and specificity (60.4-87.5%). In contrast, non-motor symptom
questions demonstrated fair to moderate sensitivity (51.5-64.6%), with high
specificity (80.0-94.1%). Like the original WOQ-9, the JWOQ-9 exhibits
significant value for detecting possible wearing-off.
CONCLUSIONS: The JWOQ-9 is a useful screening tool for detecting wearing-off of
both motor and non-motor symptoms. BACKGROUND: The wearing-off phenomenon in patients with Parkinson's disease (PD)
is a complication of prolonged levodopa usage. During this phenomenon, motor
symptoms such as rigidity and freezing re-emerge. This is often accompanied by
non-motor symptoms, including anxiety, the so-called wearing-off related anxiety
(WRA). Current treatment options are limited and typically focus on either the
physical or mental aspects of wearing-off. An integrated approach seems
warranted in order to optimally address the complex reciprocal interactions
between these aspects. Also, because wearing-off is eventually inescapable,
treatment needs to focus on coping, acceptance, and self-efficacy. We therefore
developed an integrated body awareness intervention, combining principles from
physical therapy with acceptance and commitment therapy to teach patients to
deal with WRA. This study will investigate whether this new intervention, named
BEWARE, is more effective than treatment as usual in increasing self-efficacy.
METHODS/DESIGN: This is a single-blinded randomized controlled trial in 36 PD
patients who experience WRA. Subjects will be recruited from the outpatient
clinic for movement disorders of the VU University Medical Center. After
providing written informed consent, patients will be randomly assigned to an
experimental (BEWARE) or treatment-as-usual (physical therapy) group. Clinical
assessments will be performed prior to the intervention, directly after the
6-week intervention period, and at 3-month naturalistic follow-up by a blinded
investigator not involved in the study. The primary outcome measure is
self-efficacy, and secondary outcomes focus on mobility, daily functioning,
anxiety, and quality of life.
DISCUSSION: Because wearing-off is an inevitable consequence of levodopa therapy
and current treatment options are insufficient, a multidisciplinary intervention
that addresses both physical and mental aspects of wearing-off in PD may foster
additional benefits for treating WRA in PD patients over mono-disciplinary care
alone.
TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02054845. Date of
registration: 30 January 2014. Entacapone is frequently used together with levodopa/carbidopa (LC) and
levodopa/benserazide (LB) in the treatment of Parkinson's disease (PD) patients
with wearing-off symptoms. It is generally assumed that the effects of
entacapone are independent of the type of decarboxylase inhibitor used, but
there is very little published data available on the efficacy of entacapone
administered with LB versus LC. We have performed a pooled analysis of three
randomized, double-blind, 6-month, phase III studies to compare the treatment
effects of entacapone (compared to placebo) in PD patients receiving LC or LB. A
total of 551 PD patients experiencing wearing-off were included in the analysis.
300 patients were on LB and 251 on LC at baseline. At 6 months, entacapone
(compared to placebo) improved mean daily OFF-time in patients on LB and LC by
0.76 (p = 0.016) and 0.95 (p = 0.011) hours, respectively. The corresponding
improvements in ON-time were 0.97 (p = 0.002) and 0.83 h (p = 0.022),
respectively. The treatment effects of entacapone both in LB and LC users were
statistically significant (p < 0.05) also in UPDRS II and III scores, except in
UPDRS II scores in patients receiving LC (p = 0.20). None of the treatment
effects of entacapone were statistically significantly different between
patients receiving LB or LC. Reported adverse events were comparable between LB
and LC users. We conclude that entacapone provided comparable benefits in PD
patients with wearing-off symptoms using either LB or LC. IMPORTANCE: Although levodopa remains the most effective oral pharmacotherapy
for Parkinson disease (PD), its use is often limited by wearing off effect and
dyskinesias. Management of such complications continues to be a significant
challenge.
OBJECTIVE: To investigate the efficacy and safety of safinamide (an oral
aminoamide derivative with dopaminergic and nondopaminergic actions) in
levodopa-treated patients with motor fluctuations.
DESIGN, SETTING, AND PARTICIPANTS: From March 5, 2009, through February 23,
2012, patients from academic PD care centers were randomized (1:1 ratio) to
receive double-blind adjunctive safinamide or placebo for 24 weeks. All patients
had idiopathic PD with "off" time (time when medication effect has worn off and
parkinsonian features, including bradykinesia and rigidity, return) of greater
than 1.5 hours per day (excluding morning akinesia). Their pharmacotherapy
included oral levodopa plus benserazide or carbidopa in a regimen that had been
stable for 4 weeks or longer. During screening, each patient's regimen was
optimized to minimize motor fluctuations. Study eligibility required that after
4 weeks of optimized treatment, the patients still have more than 1.5 hours per
day of off time. Adverse events caused the premature study discontinuation of 12
individuals (4.4%) in the safinamide group and 10 individuals (3.6%) in the
placebo group.
INTERVENTIONS: Patients took safinamide or placebo as 1 tablet daily with
breakfast. If no tolerability issues arose by day 14, the starting dose, 50 mg,
was increased to 100 mg.
MAIN OUTCOMES AND MEASURES: The prespecified primary outcome was each treatment
group's mean change from baseline to week 24 (or last "on" treatment value) in
daily "on" time (relief of parkinsonian motor features) without troublesome
dyskinesia, as assessed from diary data.
RESULTS: At 119 centers, 549 patients were randomized (mean [SD] age, 61.9 [9.0]
years; 334 male [60.8%] and 371 white [67.6%]): 274 to safinamide and 275 to
placebo. Among them, 245 (89.4%) receiving safinamide and 241 (87.6%) receiving
placebo completed the study. Mean (SD) change in daily on time without
troublesome dyskinesia was +1.42 (2.80) hours for safinamide, from a baseline of
9.30 (2.41) hours, vs +0.57 (2.47) hours for placebo, from a baseline of 9.06
(2.50) hours (least-squares mean difference, 0.96 hour; 95% CI, 0.56-1.37 hours;
P < .001, analysis of covariance). The most frequently reported adverse event
was dyskinesia (in 40 [14.6%] vs 15 [5.5%] and as a severe event in 5 [1.8%] vs
1 [0.4%]).
CONCLUSIONS AND RELEVANCE: The outcomes of this trial support safinamide as an
effective adjunct to levodopa in patients with PD and motor fluctuations to
improve on time without troublesome dyskinesia and reduce wearing off.
TRIAL REGISTRATION: clinicaltrials.gov Identifier NCT00627640. |
What are prions? | Prion diseases are protein conformation disorders and neither caused by viroid or virus but is a transmissible particle labeled a prion by Pruisner. Normal prion protein becomes infectious by a different folding, but the triggers are not known. | Prions are units of propagation of an altered state of a protein or proteins;
prions can propagate from organism to organism, through cooption of other
protein copies. Prions contain no necessary nucleic acids, and are important
both as both pathogenic agents, and as a potential force in epigenetic
phenomena. The original prions were derived from a misfolded form of the
mammalian Prion Protein PrP. Infection by these prions causes neurodegenerative
diseases. Other prions cause non-Mendelian inheritance in budding yeast, and
sometimes act as diseases of yeast. We report the bioinformatic construction of
the PrionHome, a database of >2000 prion-related sequences. The data was
collated from various public and private resources and filtered for redundancy.
The data was then processed according to a transparent classification system of
prionogenic sequences (i.e., sequences that can make prions), prionoids (i.e.,
proteins that propagate like prions between individual cells), and other
prion-related phenomena. There are eight PrionHome classifications for
sequences. The first four classifications are derived from experimental
observations: prionogenic sequences, prionoids, other prion-related phenomena,
and prion interactors. The second four classifications are derived from sequence
analysis: orthologs, paralogs, pseudogenes, and candidate-prionogenic sequences.
Database entries list: supporting information for PrionHome classifications,
prion-determit areas (where relevant), and disordered and
compositionally-biased regions. Also included are literature references for the
PrionHome classifications, transcripts and genomic coordinates, and structural
data (including comparative models made for the PrionHome from manually curated
alignments). We provide database usage examples for both vertebrate and fungal
prion contexts. Using the database data, we have performed a detailed analysis
of the compositional biases in known budding-yeast prionogenic sequences,
showing that the only abundant bias pattern is for asparagine bias with
subsidiary serine bias. We anticipate that this database will be a useful
experimental aid and reference resource. It is freely available at:
http://libaio.biol.mcgill.ca/prion. Prions are self-propagating infectious protein isoforms. A growing number of
prions have been identified in yeast, each resulting from the conversion of
soluble proteins into an insoluble amyloid form. These yeast prions have served
as a powerful model system for studying the causes and consequences of prion
aggregation. Remarkably, a number of human proteins containing prion-like
domains, defined as domains with compositional similarity to yeast prion
domains, have recently been linked to various human degenerative diseases,
including amyotrophic lateral sclerosis. This suggests that the lessons learned
from yeast prions may help in understanding these human diseases. In this
review, we examine what has been learned about the amino acid sequence basis for
prion aggregation in yeast, and how this information has been used to develop
methods to predict aggregation propensity. We then discuss how this information
is being applied to understand human disease, and the challenges involved in
applying yeast prediction methods to higher organisms. Several neurodegenerative diseases such as transmissible spongiform
encephalopathies, Alzheimer's and Parkinson's diseases are caused by the
conversion of cellular proteins to a pathogenic conformer. Despite differences
in the primary structure and subcellular localization of these proteins, which
include the prion protein, α-synuclein and amyloid precursor protein (APP),
striking similarity has been observed in their ability to seed and convert naïve
protein molecules as well as transfer between cells. This review aims to cover
what is known about the intracellular trafficking of these proteins as well as
their degradation mechanisms and highlight similarities in their movement
through the endocytic pathway that could contribute to the pathogenic conversion
and seeding of these proteins which underlies the basis of these diseases. Prions are proteins most commonly associated with fatal neurodegenerative
diseases in mammals but are also responsible for a number of harmless heritable
phenotypes in yeast. These states arise when a misfolded form of a protein
appears and, rather than be removed by cellular quality control mechanisms,
persists. The misfolded prion protein forms aggregates and is capable of
converting normally folded protein to the misfolded state through direct
interaction between the two forms. The domit mathematical model for prion
aggregate dynamics has been the nucleated polymerization model (NPM) which
considers the dynamics of only the normal protein and the aggregates. However,
for yeast prions the molecular chaperone Hsp104 is essential for prion
propagation. Further, although mammals do not express Hsp104, experimental
assays have shown Hsp104 also interacts with mammalian prion aggregates. In this
study, we generalize the NPM to account for molecular chaperones and develop
what we call the enzyme-limited nucleated polymerization model (ELNPM). We
discuss existence, uniqueness and stability of solutions to our model and
demonstrate that the NPM represents a quasi-steady-state reduction of our model.
We validate the ELNPM by demonstrating agreement with experimental results on
the yeast prion PSI(+) that could not be supported by the NPM. Finally, we
demonstrate that, in contrast to the NPM, the ELNPM permits the coexistence of
multiple prion strains. Prion diseases or transmissible spongiform encephalopathies are fatal
neurodegenerative diseases characterized by the aggregation and deposition of
the misfolded prion protein in the brain. α-synuclein (α-syn)-associated
multiple system atrophy has been recently shown to be caused by a bona fide
α-syn prion strain. Several other misfolded native proteins such as β-amyloid,
tau and TDP-43 share some aspects of prions although none of them is shown to be
transmissible in nature or in experimental animals. However, these prion-like
"prionoids" are causal to a variety of neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The
remarkable recent discovery of at least two new α-syn prion strains and their
transmissibility in transgenic mice and in vitro cell models raises a distinct
question as to whether some specific strain of other prionoids could have the
capability of disease transmission in a manner similar to prions. In this
overview, we briefly describe human and other mammalian prion diseases and
comment on certain similarities between prion and prionoid and the possibility
of prion-like transmissibility of some prionoid strains. INTRODUCTION: Prion diseases are protein conformation disorders and neither
caused by viroid or virus but is a transmissible particle labeled a prion by
Pruisner. Normal prion protein becomes infectious by a different folding, but
the triggers are not known. Based on the characteristic brain pathology, they
are grouped under spongiform encephalopathy affecting both man and animals.
Estimated prevalence is one per million. Creutzfeldt-Jakob disease (CJD)
registry from National Institute and Neurosciences (NIMHANS), Bengaluru,
reported 69 cases in 30 years.
PATIENT AND METHODS: Patients seen by our team from December 2011 to October
2015 who satisfied criteria for probable CJD were evaluated for clinical,
electrophysiological, radiological, and demographic factors. None of them
underwent histopathological examination of brain tissue or tonsils.
Cerebrospinal fluid protein 14-3-3 was not done. All of them were followed up by
telephonic inquiry for the course of the illness. All of them received
symptomatic medications with anticonvulsants, flupirtine 200 mg orally daily,
and other symptomatic medications.
RESULTS: Sporadic CJD is the most common form seen in India and is probably
under reported. males seem to be more affected, and the mean duration for the
bed bound state is 12 months. Drugs were only effective for a very brief period
in controlling myoclonus and behavior.
DISCUSSION: Sporadic CJD is one of the most common and rapidly fatal forms of
dementia in India. Cortical ribboning and periodic complexes are the most common
laboratory findings. Familial CJD is a very rare occurrence and variant CJD is
probably not prevalent.
CONCLUSION: All patients with rapidly progressive dementia should be handled
with biohazard precautions unless proved otherwise. Role of alcohol and smoking
in the transformation of PrPc to PrPsc needs to be evaluated. |
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