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Has "RNA interference" been awarded Nobel prize? | Since the first unequivocal description of RNA interference (RNAi) in 1998, it has remained one of the hottest topics under investigation, culminating in the award of a Nobel Prize to its discoverers in 2006. | Since the first unequivocal description of RNA interference (RNAi) in 1998, it
has remained one of the hottest topics under investigation, culminating in the
award of a Nobel Prize to its discoverers in 2006. Excitement over this
technique derives from the ease with which it can be used to switch-off a
specific gene in almost any organism, thereby allowing the role of that gene to
be identified. More importantly, it offers the potential to treat certain
diseases by switching-off the causative genes. Key to the RNAi pathway are the
small-interfering RNAs (siRNAs), which at 21-23 nucleotides in length are very
amenable to analogue development by chemists. However in comparison to the use
of oligonucleotides as antisense agents, an area where many chemists first
developed an interest in nucleic acids, the RNAi pathway is exceedingly complex.
The literature is also complicated by the fact that the phenomenon has been
studied in a wide range of organisms. In this tutorial review we have presented
the subject from a more chemical perspective, incorporating a glossary to give a
clear explanation of the specialist terms. However, the coverage of the biology
remains sufficiently detailed to give the reader the necessary insight that we
believe will be essential for the successful design of chemically modified
siRNA. One of the most significant achievements in biological science in the last
decade is the discovery of RNA interference (RNAi), a process within living
cells that regulates gene expression at post-transcriptional levels.
Historically, this process was described by other more generic names, such as
co-suppression and post transcriptional gene silencing. Only after the molecular
mechanism underlying these apparently unrelated processes was fully understood
did it become apparent that they all described the RNAi phenomenon. In 2006, Dr.
Andrew Fire and Dr. Craig C. Mello were awarded the Nobel Prize in Physiology or
Medicine for their work on RNAi interference. RNAi is an RNA-dependent gene
silencing process that is controlled by the RNA-induced silencing complex (RISC)
and is initiated by two types of small RNA molecules - microRNA (miRNA) and
small interfering RNA (siRNA). However, the function of microRNA appears to be
far beyond RNAi alone, including direct interaction with the gene promoter and
epigenetic regulation of the DNA methylation and histone modification. By
regulating gene expression, miRNAs are likely to be involved in diverse
biological activities, such as tumorigenesis, immune response, insulin
secretion, neurotransmitter synthesis, and circadian rhythm, to name a few.
MicroRNAs are 21-23 nucleotide single stranded RNA molecules found in eukaryotic
cells. The first miRNA, lin-4, was characterized in C. elegans in the early
1990s [1]. In the early years, the progress on microRNA research was slow and
experienced substantial growing pains. The short length and uniqueness of each
microRNA rendered many conventional hybridization based methods ineffective;
very small RNAs are difficult to reliably amplify or label without introducing
bias. In addition, hybridization-based methods for microRNA profiling relied on
probes designed to detect known microRNAs or known microRNA species previously
identified by sequencing or homology search. Recent evidence of target-directed
editing of mature microRNA (trimming and tailing by 3'-to-5' exonuclase and
terminal nucleotide transferase) [2] further highlighted the complexity of
microRNA processing and regulation mechanisms. Moreover, the wide range of
microRNA expression, from tens of thousands to just few molecules per cell,
complicated the detection of microRNAs expressed at low copy numbers. Hence,
many novel microRNAs may exist even in well-explored species. Nevertheless,
recent advances in genomic technologies and data analysis / bioinformatics
approaches have made a significant impact on microRNA research. For example,
next generation deep sequencing platforms are ideal for detecting and
quantifying both known and novel microRNA sequences with high sensitivity and
for a relatively low cost [3]. The microRNA field has experienced a major
explosion in recent years. The microRNA gene family is continuously growing with
novel members discovered in association with rapid advances in genomic
technologies, and reports on the functional characterizations of specific
microRNA genes have been dominating the recent literature. We devote this new
journal, MicroRNA, to the rapidly advancing field of microRNA research. We
dedicate our new journal to the scientists who work tirelessly on this family of
small molecules, and their immense contributions to the biological sciences.
MicroRNA publishes letters, full-length research articles, review articles, drug
and clinical trial studies and thematic issues on all aspects of microRNA
research. The scope of the journal covers all experimental microRNA research and
applied research in the fields of health and disease, including therapeutic,
biomarker, and diagnostic applications of microRNA. RNA interference is a cellular mechanism by which small molecules of double
stranded RNA modulate gene expression acting on the concentration and/or
availability of a given messenger RNA. Almost 10 years after Fire and Mello
received the Nobel Prize for the discovery of this mechanism in flat worms, RNA
interference is on the edge of becoming a new class of therapeutics. With
various phase III studies underway, the following years will determine whether
RNAi-therapeutics can rise up to the challenge and become mainstream medicines.
The present review gives a thorough overview of the current status of this
technology focusing on the path to the clinic of this new class of compounds. |
Is there any role for Pds5b in cohesion establishment? | Yes. Pds5 proteins are essential for cohesion establishment by allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and binding of Sororin. | Cohesin mediates sister chromatid cohesion and contributes to the organization
of interphase chromatin through DNA looping. In vertebrate somatic cells,
cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional
factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic
association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A
and Pds5B, but their functional specificity remains unclear. Here, we
demonstrate that Pds5 proteins are essential for cohesion establishment by
allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and
binding of Sororin. While both proteins contribute to telomere and arm cohesion,
Pds5B is specifically required for centromeric cohesion. Furthermore, reduced
accumulation of Aurora B at the inner centromere region in cells lacking Pds5B
impairs its error correction function, promoting chromosome mis-segregation and
aneuploidy. Our work supports a model in which the composition and function of
cohesin complexes differs between different chromosomal regions. |
Which factors are considered in the FUNC score for intracerebral hemorrhage? | FUNC score includes Age, Glasgow Coma Scale, ICH location, volume and pre-ICH cognitive impairment. | BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) is the most fatal and
disabling stroke subtype. Widely used tools for prediction of mortality are
fundamentally limited in that they do not account for effects of withdrawal of
care and are not designed to predict functional recovery. We developed an acute
clinical score to predict likelihood of functional independence.
METHODS: We prospectively characterized 629 consecutive patients with ICH at
hospital presentation. Predictors of functional independence (Glasgow Outcome
Score > or = 4) at 90 days were used to develop a logistic regression-based risk
stratification scale in a random subset of two thirds and validated in the
remaining one third of the cohort.
RESULTS: At 90 days, 162 (26%) patients achieved independence. Age, Glasgow Coma
Scale, ICH location, volume (all P<0.0001), and pre-ICH cognitive impairment
(P=0.005) were independently associated with Glasgow Outcome Score > or = 4. The
FUNC score was developed as a sum of individual points (0-11) based on strength
of association with outcome. In both the development and validation cohorts, the
proportion of patients who achieved Glasgow Outcome Score > or = 4 increased
steadily with FUNC score. No patient assigned a FUNC score < or = 4 achieved
functional independence, whereas > 80% with a score of 11 did. The predictive
accuracy of the FUNC score remained unchanged when restricted to ICH survivors
only, consistent with absence of confounding by early withdrawal of care.
CONCLUSIONS: FUNC score is a valid clinical assessment tool that identifies
patients with ICH who will attain functional independence and thus, can provide
guidance in clinical decision-making and patient selection for clinical trials. |
Where is the respirasome located? | Respirasomes are macromolecular assemblies of the respiratory chain complexes I, III and IV in the inner mitochondrial membrane. The 4.0 Å cryo-EM structure of one of the most intricate enzyme systems, the respirasome, in the mitochondrial inner membrane is now available. | Respirasomes are macromolecular assemblies of the respiratory chain complexes I,
III and IV in the inner mitochondrial membrane. We determined the structure of
supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å
resolution. Most protein-protein contacts between complex I, III and IV in the
membrane are mediated by supernumerary subunits. Of the two Rieske iron-sulfur
cluster domains in the complex III dimer, one is resolved, indicating that this
domain is immobile and unable to transfer electrons. The central position of the
active complex III monomer between complex I and IV in the respirasome is
optimal for accepting reduced quinone from complex I over a short diffusion
distance of 11 nm, and delivering reduced cytochrome c to complex IV. The
functional asymmetry of complex III provides strong evidence for directed
electron flow from complex I to complex IV through the active complex III
monomer in the mammalian supercomplex. |
Describe the usefulness of MiRduplexSVM. | MiRduplexSVM is a high-performing miRNA-duplex prediction and evaluation methodology. It's a method that combines a unique problem representation and an unbiased optimization protocol to learn from mirBase19.0 an accurate predictive model. It is the first model that provides precise information about all four ends of the miRNA duplex. | We address the problem of predicting the position of a miRNA duplex on a
microRNA hairpin via the development and application of a novel SVM-based
methodology. Our method combines a unique problem representation and an unbiased
optimization protocol to learn from mirBase19.0 an accurate predictive model,
termed MiRduplexSVM. This is the first model that provides precise information
about all four ends of the miRNA duplex. We show that (a) our method outperforms
four state-of-the-art tools, namely MaturePred, MiRPara, MatureBayes, MiRdup as
well as a Simple Geometric Locator when applied on the same training datasets
employed for each tool and evaluated on a common blind test set. (b) In all
comparisons, MiRduplexSVM shows superior performance, achieving up to a 60%
increase in prediction accuracy for mammalian hairpins and can generalize very
well on plant hairpins, without any special optimization. (c) The tool has a
number of important applications such as the ability to accurately predict the
miRNA or the miRNA*, given the opposite strand of a duplex. Its performance on
this task is superior to the 2nts overhang rule commonly used in computational
studies and similar to that of a comparative genomic approach, without the need
for prior knowledge or the complexity of performing multiple alignments.
Finally, it is able to evaluate novel, potential miRNAs found either
computationally or experimentally. In relation with recent confidence evaluation
methods used in miRBase, MiRduplexSVM was successful in identifying high
confidence potential miRNAs. |
What genes are drug targets for Fibrodysplasia Ossificans Progressiva (FOP)? | Recently, FOP has been associated with a specific mutation of ACVR1, the gene coding for a bone morphogenetic protein type I receptor. | Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and disabling
genetic disorder characterized by congenital malformation of the great toes and
by progressive heterotopic endochondral ossification in predictable anatomical
patterns. Although elevated levels of bone morphogenetic protein 4 (BMP4) occur
in lymphoblastoid cells and in lesional cells of patients with FOP, mutations
have not been identified in the BMP4 gene, suggesting that the mutation in FOP
may reside in a BMP4-interacting factor or in another component of the BMP4
pathway. A powerful antagonist of BMP4 is the secreted polypeptide noggin. A
recent case report described a heterozygous 42-bp deletion in the protein-coding
region of the noggin gene in a patient with FOP. In order to determine if noggin
mutations are a widespread finding in FOP, we examined 31 families with 1 or
more FOP patients. Linkage analysis with an array of highly polymorphic
microsatellite markers closely linked to the noggin gene was performed in four
classically-affected multigenerational FOP families and excluded linkage of the
noggin locus to FOP (the multipoint lod score was -2 or less throughout the
entire range of markers). We sequenced the noggin gene in affected members of
all four families, as well as in 18 patients with sporadic FOP, and failed to
detect any mutations. Single-strand conformation polymorphism (SSCP) analysis of
4 of these patients plus an additional 9 patients also failed to reveal any
mutations. Among the samples analyzed by SSCP and DNA sequencing was an
independently obtained DNA sample from the identical FOP patient previously
described with the 42-bp noggin deletion; no mutation was detected. Examination
of the DNA sequences of 20 cloned noggin PCR products, undertaken to evaluate
the possibility of a somatic mutation in the noggin gene which could be carried
by a small subset of white blood cells, also failed to detect the presence of
the reported 42-bp deletion. We conclude that mutations in the coding region of
noggin are not associated with FOP. Fibrodysplasia ossificans progressiva (FOP) is a severe, progressive disease of
the musculoskeletal system. Muscles, tendons and other connective tissues ossify
after minor trauma, and patients often become encased in a second immobile
skeleton. There is no known cure or treatment for FOP. It has been found that
lymphocytes from FOP patients elaborate excess levels of bone morphogenic
protein-4 (BMP-4). Given this, it has been suggested that allogenic bone marrow
transplantation (BMT) possibly could be a cure for FOP, and drawn attention to a
previously unappreciated case of an FOP patient who had successful BMT for
aplastic anemia with apparent short- and medium-term arresting of the FOP
disease process. However, BMT has non-trivial associated morbidity and
mortality. Here, it is noted that if B cells are found to be the lymphocytes
responsible for excess BMP-4 production in FOP, use of Rituximab, a monoclonal
anti-CD20 antibody which effectively targets B cells, could be a less permanent
and less risky treatment alternative for FOP. FOP is a disorder in which skeletal muscle is progressively replaced with bone.
FOP lymphocytes, a model system for exploring the BMP pathway in these patients,
exhibit a defect in BMPRIA internalization and increased activation of
downstream signaling, suggesting that altered BMP receptor trafficking underlies
ectopic bone formation in this disease.
INTRODUCTION: Fibrodysplasia ossificans progressiva (FOP) is a severely
disabling disorder characterized by progressive heterotopic ossification of
connective tissues. Whereas the genetic defect and pathophysiology of this
condition remain enigmatic, BMP4 mRNA and protein are overexpressed, and mRNAs
for a subset of secreted BMP antagonists are not synthesized at appropriate
levels in cultured lymphocytes from FOP patients. These data suggest involvement
of altered BMP signaling in the disease. In this study, we investigate whether
the abnormality is associated with defective BMP receptor function in
lymphocytes.
MATERIALS AND METHODS: Cell surface proteins were quantified by
fluorescence-activated cell sorting (FACS). Protein phosphorylation was assayed
by immunoprecipitation and immunoblotting. Protein synthesis and degradation
were examined by [35S]methionine labeling and pulse-chase assays. mRNA was
detected by RT-PCR.
RESULTS: FOP lymphocytes expressed 6-fold higher levels of BMP receptor type IA
(BMPRIA) on the cell surface compared with control cells and displayed a marked
reduction in ligand-stimulated internalization and degradation of BMPRIA.
Moreover, in control cells, BMP4 treatment increased BMPRIA phosphorylation,
whereas BMPRIA showed ligand-insensitive constitutive phosphorylation in FOP
cells. Our data additionally support that the p38 mitogen-activated protein
kinase (MAPK) signaling pathway is a major BMP signaling pathway in these cell
lines and that expression of inhibitor of DNA binding and differentiation 1
(ID-1), a transcriptional target of BMP signaling, is enhanced in FOP cells.
CONCLUSIONS: These data extend our previous observations of misregulated BMP4
signaling in FOP lymphocytes and show that cell surface overabundance and
constitutive phosphorylation of BMPRIA are associated with a defect in receptor
internalization. Altered BMP receptor trafficking may play a significant role in
FOP pathogenesis. A new mutation of the Noggin gene in a French Fybrodysplasia ossificans
progressiva (FOP) family: Fibrodysplasia ossificans progressiva (FOP) is a very
rare disease characterized by congenital malformation of the great toes and
progressive heterotopic ossification of the muscles. We previously located a FOP
gene in the 17q21-22 region and described several mutations of the noggin (NOG)
gene (located in 17q22) in four FOP patients, including the G91C mutation which
is transmitted domitly in a Spanish FOP family. We describe in the present
study a new mutation of the NOG gene in a French FOP family. This new mutation
is a guanine to adenine change at nucleotide 283 (283G --> A) of the NOG gene,
and is transmitted in the family (in the heterozygote form) by the affected
mother to her two affected children. At the peptide level this mutation (A95T)
substitutes an Alanine residue by a Threonine at position 95 of the Noggin
protein. The Alanine mutated residue is located just adjacent to the
myristoylation site of the protein, where all the mutations we described until
now are located. Identification of gene mutations in Mendelian disorders is often determined by
linkage analysis and positional cloning, an approach that is difficult for
fibrodysplasia ossificans progressiva (FOP) due to a low reproductive fitness
that results in a small number of multigenerational families showing inheritance
of the disease. Altered signaling pathways can be investigated as a
complementary method to identify the consequences of the mutated gene
responsible for FOP and to identify potential therapeutic targets. Candidate
signaling pathways for FOP are those that malfunctioning could account for the
malformation of the great toes during embryonic development and could explain
the postnatal progressive heterotopic endochondral ossification. Signaling
pathways that fit these criteria are the BMP signaling pathway and its
interacting pathways. A large body of data suggest that the BMP-4 signaling
pathway is dysregulated in FOP. Fibrodysplasia ossificans progressiva (FOP) is a disabling genetic condition
that leads to the formation of a second (heterotopic) skeleton, and is the most
catastrophic disorder of heterotopic ossification in humans. Throughout
childhood and early adult life, FOP progressively immobilizes all of the joints
of the normotopic skeleton, rendering movement impossible. At present, there is
no effective prevention or treatment. Recently, a recurrent mutation in the
glycine-serine activation domain of the activin receptor IA/activin-like
kinase-2, a bone morphogenetic protein type I receptor, was reported in all
sporadic and familial cases of classic FOP, making this one of the most highly
specific disease-causing mutations in the human genome. The discovery of the FOP
gene establishes a critical milestone in understanding FOP, reveals a highly
conserved druggable target in the TGF-beta/bone morphogenetic protein signaling
pathway and compels therapeutic approaches for the development of small molecule
signal transduction inhibitors for activin-like kinase-2. Effective therapies
for FOP, and possibly for a vast array of more common conditions of heterotopic
ossification, will be based on blocking activin-like kinase-2, a critical node
in the BMP signaling pathway. The study of FOP, a disabling genetic disorder of progressive heterotopic
ossification, is hampered by the lack of readily available connective tissue
progenitor cells. We isolated such cells from discarded primary teeth of
patients with FOP and controls and discovered dysregulation of BMP signaling and
rapid osteoblast differentiation in FOP cells compared with control cells.
INTRODUCTION: Fibrodysplasia ossificans progressiva (FOP), the most disabling
condition of progressive heterotopic ossification in humans, is caused by a
recurrent heterozygous missense mutation in activin receptor IA (ACVR1), a bone
morphogenetic protein (BMP) type I receptor, in all classically affected
individuals. A comprehensive understanding of FOP has been limited, in part, by
a lack of readily available connective tissue progenitor cells in which to study
the molecular pathology of this disorder.
MATERIALS AND METHODS: We derived connective tissue progenitor cells from
discarded primary teeth (SHED cells) of patients with FOP and controls and
examined BMP signaling and osteogenic differentiation in these cells.
RESULTS: SHED cells transmitted BMP signals through both the SMAD and p38
mitogen-activated protein kinase (MAPK) pathways and responded to BMP4 treatment
by inducing BMP responsive genes. FOP cells showed ligand-independent BMP
signaling and ligand-dependent hyper-responsiveness to BMP stimulation.
Furthermore, FOP cells showed more rapid differentiation to an osteogenic
phenotype than control cells.
CONCLUSIONS: This is the first study of BMP signaling and osteogenic
differentiation in connective tissue progenitor cells from patients with FOP.
Our data strongly support both basal and ligand-stimulated dysregulation of BMP
signaling consistent with in silico studies of the mutant ACVR1 receptor in this
condition. This study substantially extends our understanding of dysregulated
BMP signaling in a progenitor cell population relevant to the pathogenesis of
this catastrophic disorder of progressive ectopic ossification. Fibrodysplasia ossificans progressiva (FOP, MIM 135100) is a rare genetic
disorder characterized by congenital great toe malformations and progressive
heterotopic ossification transforming skeletal muscles and connective tissues to
bone following a well-defined anatomic pattern of progression. Recently, FOP has
been associated with a specific mutation of ACVR1, the gene coding for a bone
morphogenetic protein type I receptor. The identification of ACVR1 as the
causative gene for FOP now allows the genetic screening of FOP patients to
identify the frequency of the identified recurrent ACVR1 mutation and to
investigate genetic variability that may be associated with this severely
debilitating disease. We report the screening for mutations in the ACVR1 gene
carried out in a cohort of 17 Italian patients. Fifteen of these displayed the
previously described c.617G>A mutation, leading to the R206H substitution in the
GS domain of the ACVR1 receptor. In two patients, we found a novel mutation
c.774G>C, leading to the R258S substitution in the kinase domain of the ACVR1
receptor. In the three-dimensional model of protein structure, R258 maps in
close proximity to the GS domain, a key regulator of ACVR1 activity, where R206
is located. The GS domain is known to bind the regulatory protein FKBP12 and to
undergo multiple phosphorylation events that trigger a signaling cascade inside
the cell. The novel amino-acid substitution is predicted to influence either the
conformation/stability of the GS region or the binding affinity with FKBP12,
resulting in a less stringent inhibitory control on the ACVR1 kinase activity. Fibrodysplasia ossificans progressiva (FOP) is a rare but very severe disease,
characterised by congenital malformations of the toes and by progressive
heterotopic ossification of muscles and joints. Two genes, the noggin (NOG) gene
and the activin A type I receptor (ACVRI) gene, are involved in FOP. In this
study we have searched for the NOG and the 617G>A (ACVR1) mutations in a well
characterized series of twenty-seven French FOP patients. Five NOG mutations
(delta 42, 274G>C, 275G>A, 276G>A, and 283G>A) have been found in seven (26%) of
our FOP patients. The 617G>A mutation in the ACVR1 gene is found in fourteen
(52%) of the patients. With one exception (patient number 22), 617G>A and NOG
mutations are mutually exclusive in patients. Mutations 274G>C, 283G>A and
617G>A segregate with the trait in five different FOP families, some members of
them being partially affected by the disease. Fibrodysplasia ossificans progressiva (FOP, MIM 135100) is a rare autosomal
domit disorder characterized by postnatal progressive heterotopic
ossification of the connective tissue and congenital malformation of the big
toes. Recently, FOP has been associated with a specific mutation of ACVR1, the
gene coding for a bone morphogenetic protein type I receptor. We report the case
of a Moroccan patient with FOP carrying a rarely occurring mutation of ACVR1
gene. Fibrodysplasia ossificans progressiva (FOP) is a rare disabling disease
characterized by heterotopic ossification for which there is currently no
treatment available. FOP has been linked recently to a heterozygous R206H
mutation in the bone morphogenetic protein (BMP) type I receptor activin
receptor-like kinase 2 (ALK2). Expression of the mutant ALK2-R206H receptor
(FOP-ALK2) results in increased phosphorylation of the downstream Smad1 effector
proteins and elevated basal BMP-dependent transcriptional reporter activity,
indicating that FOP-ALK2 is constitutively active. FOP-ALK2-induced
transcriptional activity could be blocked by overexpressing either of the
inhibitory Smads, Smad6 or -7, or by treatment with the pharmacological BMP type
I receptor inhibitor dorsomorphin. However, in contrast to wild-type ALK2,
FOP-ALK2 is not inhibited by the negative regulator FKBP12. Mesenchymal cells
expressing the FOP-ALK2 receptor are more sensitive to undergoing BMP-induced
osteoblast differentiation and mineralization. In vivo bone formation was
assessed by loading human mesenchymal stem cells (hMSCs) expressing the
ALK2-R206H receptor onto calcium phosphate scaffolds and implantation in nude
mice. Compared with control cells FOP-ALK2-expressing cells induced increased
bone formation. Taken together, the R206H mutation in ALK2 confers constitutive
activity to the mutant receptor, sensitizes mesenchymal cells to BMP-induced
osteoblast differentiation, and stimulates new bone formation. We have generated
an animal model that can be used as a stepping stone for preclinical studies
aimed at inhibiting the heterotopic ossification characteristic of FOP. Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to
control development and tissue homeostasis. Mutant receptor kinase domains
escape regulation leading to severely degenerative diseases and represent an
important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a
rare but devastating disorder of extraskeletal bone formation. FOP-associated
mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and
promote leaky signaling in the absence of ligand. To establish structural
mechanisms of receptor regulation and to address the effects of FOP mutation, we
determined the crystal structure of the cytoplasmic domain of ALK2 in complex
with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical
interactions that stabilize the inactive state of the kinase, thereby
facilitating structural rearrangements that diminish FKBP12 binding and promote
the correct positioning of the glycine-serine-rich loop and αC helix for kinase
activation. The balance of these effects accounts for the comparable activity of
R206H and L196P. Kinase activation in the clinically benign mutant L196P is far
weaker than R206H but yields equivalent signals due to the stronger interaction
of FKBP12 with R206H. The presented ALK2 structure offers a valuable template
for the further design of specific inhibitors of BMP signaling. Fibrodysplasia ossificans progressiva (FOP), characterized by congenital
malformation of bones, is an autosomal domit disorder. This is a rare genetic
disorder and its worldwide prevalence is approximately 1/2,000,000. There is no
ethnic, racial, gender, or geographic predilection to FOP. It is regarded as one
of the intractable disorders, which is not only an extremely disabling disease
but also a condition of considerably shortened lifespan. Although the genetic
defects of FOP are not completely known, several clinical and animal model
studies have implicated that mutations in bone morphogenetic proteins, their
receptors, and activin receptor type IA (ACVR1) genes are associated with FOP
primarily. The noggin (NOG) gene has also been reported in some studies. In most
of the cases of FOP, the mutation was found as 'de novo' however there is
paternal age effect on mutations. Unfortunately, at present there is no
efficient treatment for FOP. The recent discoveries of genetic basis of FOP
provide a clue to the underlying pathophysiology and potential therapy. This
review article focuses on the genetic mutations in FOP, their usage as
diagnostic markers, and possible target specific drug development to treat FOP
patients. A heterozygous missense mutation in activin receptor IA/activin-like kinase-2
(ACVR1/ALK2), a bone morphogenetic protein (BMP) type I receptor, is responsible
for fibrodysplasia ossificans progressiva (FOP), the most catastrophic disorder
of skeletal metamorphosis in humans. The discovery of the FOP gene establishes a
crucial milestone in understanding FOP, reveals a highly conserved target in the
BMP signaling pathway for drug development and specifically stimulates
therapeutic approaches for the development of inhibitors for ACVR1/ALK2
signaling. Effective therapies for FOP, and possibly for more common conditions
of heterotopic ossification, will be based on interventions that selectively
block promiscuous ACVR1/ALK2 signaling, and/or themolecular triggers, responding
cells and tissue microenvironments that facilitate aberrant skeletal
metamorphosis in a permissive genetic background of increased BMP pathway
activity. Growth factor signaling pathways are tightly regulated by phosphorylation and
include many important kinase targets of interest for drug discovery. Small
molecule inhibitors of the bone morphogenetic protein (BMP) receptor kinase ALK2
(ACVR1) are needed urgently to treat the progressively debilitating
musculoskeletal disease fibrodysplasia ossificans progressiva (FOP).
Dorsomorphin analogues, first identified in zebrafish, remain the only BMP
inhibitor chemotype reported to date. By screening an assay panel of 250
recombit human kinases we identified a highly selective 2-aminopyridine-based
inhibitor K02288 with in vitro activity against ALK2 at low omolar
concentrations similar to the current lead compound LDN-193189. K02288
specifically inhibited the BMP-induced Smad pathway without affecting TGF-β
signaling and induced dorsalization of zebrafish embryos. Comparison of the
crystal structures of ALK2 with K02288 and LDN-193189 revealed additional
contacts in the K02288 complex affording improved shape complementarity and
identified the exposed phenol group for further optimization of
pharmacokinetics. The discovery of a new chemical series provides an independent
pharmacological tool to investigate BMP signaling and offers multiple
opportunities for pre-clinical development. BACKGROUND: The ACVR1 gene encodes a type I receptor for bone morphogenetic
proteins (BMPs). Mutations in the ACVR1 gene are associated with Fibrodysplasia
Ossificans Progressiva (FOP), a rare and extremely disabling disorder
characterized by congenital malformation of the great toes and progressive
heterotopic endochondral ossification in muscles and other non-skeletal tissues.
Several aspects of FOP pathophysiology are still poorly understood, including
mechanisms regulating ACVR1 expression. This work aimed to identify regulatory
elements that control ACVR1 gene transcription.
METHODS AND RESULTS: We first characterized the structure and composition of
human ACVR1 gene transcripts by identifying the transcription start site, and
then characterized a 2.9 kb upstream region. This region showed strong
activating activity when tested by reporter gene assays in transfected cells. We
identified specific elements within the 2.9 kb region that are important for
transcription factor binding using deletion constructs, co-transfection
experiments with plasmids expressing selected transcription factors,
site-directed mutagenesis of consensus binding-site sequences, and by
protein/DNA binding assays. We also characterized a GC-rich minimal promoter
region containing binding sites for the Sp1 transcription factor.
CONCLUSIONS: Our results showed that several transcription factors such as
Egr-1, Egr-2, ZBTB7A/LRF, and Hey1, regulate the ACVR1 promoter by binding to
the -762/-308 region, which is essential to confer maximal transcriptional
activity. The Sp1 transcription factor acts at the most proximal promoter
segment upstream of the transcription start site. We observed significant
differences in different cell types suggesting tissue specificity of
transcriptional regulation. These findings provide novel insights into the
molecular mechanisms that regulate expression of the ACVR1 gene and that could
be targets of new strategies for future therapeutic treatments. Diffuse intrinsic pontine gliomas (DIPGs) are highly infiltrative maligt
glial neoplasms of the ventral pons that, due to their location within the
brain, are unsuitable for surgical resection and consequently have a universally
dismal clinical outcome. The median survival time is 9-12 months, with neither
chemotherapeutic nor targeted agents showing substantial survival benefit in
clinical trials in children with these tumors. We report the identification of
recurrent activating mutations in the ACVR1 gene, which encodes a type I activin
receptor serine/threonine kinase, in 21% of DIPG samples. Strikingly, these
somatic mutations (encoding p.Arg206His, p.Arg258Gly, p.Gly328Glu, p.Gly328Val,
p.Gly328Trp and p.Gly356Asp substitutions) have not been reported previously in
cancer but are identical to mutations found in the germ line of individuals with
the congenital childhood developmental disorder fibrodysplasia ossificans
progressiva (FOP) and have been shown to constitutively activate the BMP-TGF-β
signaling pathway. These mutations represent new targets for therapeutic
intervention in this otherwise incurable disease. Author information:
(1)Department of Cell Growth and Differentiation, Center for iPS Cell Research
and Application, Kyoto University, Kyoto, 606-8507, Japan; iPS Cell-Based Drug
Discovery Group, Innovative Drug Discovery Laboratories, Sumitomo Dainippon
Pharma Co., Ltd., Osaka, 554-0022, Japan;
(2)Department of Cell Growth and Differentiation, Center for iPS Cell Research
and Application, Kyoto University, Kyoto, 606-8507, Japan;
[email protected] [email protected].
(3)Department of Cell Growth and Differentiation, Center for iPS Cell Research
and Application, Kyoto University, Kyoto, 606-8507, Japan; Department of Tissue
Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto,
606-8507, Japan; Department of Orthopaedic Surgery, Graduate School of Medical
Sciences, Nagoya City University, Nagoya, 467-8601, Japan;
(4)Omics Group, Genomic Science Laboratories, Sumitomo Dainippon Pharma Co.,
Ltd., Osaka, 554-0022, Japan;
(5)Department of Cell Growth and Differentiation, Center for iPS Cell Research
and Application, Kyoto University, Kyoto, 606-8507, Japan;
(6)Department of Cell Growth and Differentiation, Center for iPS Cell Research
and Application, Kyoto University, Kyoto, 606-8507, Japan; Department of Tissue
Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto,
606-8507, Japan; Department of Orthopaedic Surgery, Graduate School of Medicine,
Kyoto University, Kyoto, 606-8507, Japan.
(7)Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto
University, Kyoto, 606-8507, Japan.
(8)Department of Cell Growth and Differentiation, Center for iPS Cell Research
and Application, Kyoto University, Kyoto, 606-8507, Japan; Department of Tissue
Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto,
606-8507, Japan; Department of Orthopaedic Surgery, Graduate School of Medicine,
Kyoto University, Kyoto, 606-8507, Japan [email protected]
[email protected]. Fibrodysplasia ossificans progressiva (FOP) is a rare disease characterized by
progressive ossification of soft tissues, for which there is no effective
treatment. Mutations in the bone morphogenetic protein (BMP) type I receptor
activin receptor-like kinase 2 (ACVR1/ALK2) are the main cause of FOP. We
generated human induced pluripotent stem cells (hiPSCs) from FOP patients with
the ALK2 R206H mutation. The mutant ALK2 gene changed differentiation
efficiencies of hiPSCs into FOP bone-forming progenitors: endothelial cells
(ECs) and pericytes. ECs from FOP hiPSCs showed reduced expression of vascular
endothelial growth factor receptor 2 and could transform into mesenchymal cells
through endothelial-mesenchymal transition. Increased mineralization of
pericytes from FOP hiPSCs could be partly inhibited by the ALK2 kinase inhibitor
LDN-212854. Thus, differentiated FOP hiPSCs recapitulate some aspects of the
disease phenotype in vitro, and they could be instrumental in further
elucidating underlying mechanisms of FOP and development of therapeutic drug
candidates. Fibrodysplasia ossificans progressiva (FOP, MIM #135100) is a rare genetic
disorder of heterotopic endochondral ossification, resulting in transformation
of soft tissue into episodic bone formation. Currently, no effective treatment
for FOP has been established. The causative heterozygous genetic mutations have
been identified in either the intracellular glycine-serine-rich (GS) domain or
kinase domain of ALK2 (Activin-like kinase-2, also known as Activin A receptor
type I, ACVR1), a type I receptor of bone morphogenetic proteins (BMP).
Cumulative studies support that these mutations abnormally activate BMP
signaling in a ligandindependent manner by reducing the ALK2 interaction with
the negative regulator FKBP12, whereas others argue a ligand-dependent BMP
signaling activation in FOP. Nevertheless, in either the ligand-independent or
ligand-dependent model, ALK2 receptor activation is essential for heterotopic
ossification in FOP. Thus targeting ALK2 likely represents an effective
treatment for FOP. In this article, we critically review the recent progress on
therapeutic strategies, with a focus on development of small molecule ALK2
inhibitors to suppress BMP signaling for FOP treatment. Fibrodysplasia ossificans progressiva (FOP), a rare and as yet untreatable
genetic disorder of progressive extraskeletal ossification, is the most
disabling form of heterotopic ossification (HO) in humans and causes skeletal
deformities, movement impairment, and premature death. Most FOP patients carry
an activating mutation in a bone morphogenetic protein (BMP) type I receptor
gene, ACVR1(R206H) , that promotes ectopic chondrogenesis and osteogenesis and,
in turn, HO. We showed previously that the retinoic acid receptor γ (RARγ)
agonist palovarotene effectively inhibited HO in injury-induced and genetic
mouse models of the disease. Here we report that the drug additionally prevents
spontaneous HO, using a novel conditional-on knock-in mouse line carrying the
human ACVR1(R206H) mutation for classic FOP. In addition, palovarotene restored
long bone growth, maintained growth plate function, and protected growing mutant
neonates when given to lactating mothers. Importantly, palovarotene maintained
joint, limb, and body motion, providing clear evidence for its encompassing
therapeutic potential as a treatment for FOP. © 2016 American Society for Bone
and Mineral Research. |
What kind of analyses are performed with the software tool "unipept" | The Unipept web application (http://unipept.ugent.be) supports biodiversity analysis of large and complex metaproteome samples using tryptic peptide information obtained from shotgun MS/MS experiments. The application designed for metaproteomics analysis with a focus on interactive datavisualization. | The Unipept web application (http://unipept.ugent.be) supports biodiversity
analysis of large and complex metaproteome samples using tryptic peptide
information obtained from shotgun MS/MS experiments. Its underlying index
structure is designed to quickly retrieve all occurrences of a tryptic peptide
in UniProtKB records. Taxon-specificity of the tryptic peptide is successively
derived from these occurrences using a novel lowest common ancestor approach
that is robust against taxonomic misarrangements, misidentifications, and
inaccuracies. Not taking into account this identification noise would otherwise
result in drastic loss of information. Dynamic treemaps visualize the
biodiversity of metaproteome samples, which eases the exploration of samples
with highly complex compositions. The potential of Unipept to gain novel
insights into the biodiversity of a sample is evaluated by reanalyzing publicly
available metaproteome data sets taken from the bacterial phyllosphere and the
human gut. Unipept (http://unipept.ugent.be) is a web application that offers a
user-friendly way to explore the biodiversity of complex metaproteome samples by
providing interactive visualizations. In this article, the updates and changes
to Unipept since its initial release are presented. This includes the addition
of interactive sunburst and treeview visualizations to the multipeptide
analysis, the foundations of an application programming interface (API) and a
command line interface, updated data sources, and the open-sourcing of the
entire application under the MIT license. Unipept is an open source web application that is designed for metaproteomics
analysis with a focus on interactive datavisualization. It is underpinned by a
fast index built from UniProtKB and the NCBI taxonomy that enables quick
retrieval of all UniProt entries in which a given tryptic peptide occurs.
Unipept version 2.4 introduced web services that provide programmatic access to
the metaproteomics analysis features. This enables integration of Unipept
functionality in custom applications and data processing pipelines.
AVAILABILITY AND IMPLEMENTATION: The web services are freely available at
http://api.unipept.ugent.be and are open sourced under the MIT license.
CONTACT: [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. |
What is the role of gamma-secreatase complex in Alzheimer's Disease? | The gamma-secretase complex has a decisive role in the development of Alzheimer's disease, as it cleaves a precursor protein to create the amyloid beta peptide whose aggregates form the senile plaques encountered in the brains of patients. Gamma-secretase is a member of the intramembrane-cleaving proteases which process their transmembrane substrates within the bilayer. | Gamma-secretase catalyzes the proteolytic processing of a number of integral
membrane proteins, including amyloid precursor protein (APP) and Notch. The
native gamma-secretase is a heterogeneous population of large membrane protein
complexes containing presenilin 1 (PS1) or presenilin 2 (PS2), aph-1a or aph-1b,
nicastrin, and pen-2. Here we report the reconstitution of a gamma-secretase
complex in Sf9 cells by co-infection with baculoviruses carrying the PS1,
nicastrin, pen-2, and aph-1a genes. The reconstituted enzyme processes C99 and
the Notch-like substrate N160 and displays the characteristic features of
gamma-secretase in terms of sensitivity to a gamma-secretase inhibitor,
upregulation of Abeta42 production by a familial Alzheimer's disease (FAD)
mutation in the APP gene, and downregulation of Notch processing by PS1 FAD
mutations. However, the ratio of Abeta42:Abeta40 production by the reconstituted
gamma-secretase is significantly higher than that of the native enzyme from 293
cells. Unlike in mammalian cells where PS1 FAD mutations cause an increase in
Abeta42 production, PS1 FAD missense mutations in the reconstitution system
alter the cleavage sites in the C99 substrate without changing the
Abeta42:Abeta40 ratio. In addition, PS1DeltaE9 is a loss-of-function mutation in
both C99 and N160 processing. Reconstitution of gamma-secretase provides a
homogeneous system for studying the individual gamma-secretase complexes and
their roles in Abeta production, Notch processing and AD pathogenesis. These
studies may provide important insight into the development of a new generation
of selective gamma-secretase inhibitors with an improved side effect profile. Cleavage of the beta-secretase processed beta-amyloid precursor protein by
gamma-secretase leads to the extracellular release of Abeta42, the more
amyloidogenic form of the beta-amyloid peptide, which subsequently forms the
amyloid-plaques diagnostic of Alzheimer's disease. Mutations in beta-amyloid
precursor protein (APP), presenilin-1 and presenilin-2 associated with familial
Alzheimer's disease (FAD) increase release of Abeta42, suggesting that FAD may
directly result from increased gamma-secretase activity. Here, we show that
familial Alzheimer's disease mutations clustered near the sites of
gamma-secretase cleavage actually decrease gamma-secretase-mediated release of
the intracellular fragment of APP (CTFgamma). Concordantly, presenilin-1
mutations that result in Alzheimer's disease also decrease the release of
CTFgamma. Mutagenesis of the epsilon cleavage site in APP mimicked the effects
of the FAD mutations, both decreasing CTFgamma release and increasing Abeta42
production, suggesting that perturbation of this site may account for the
observed decrement in gamma-secretase-mediated proteolysis of APP. As CTFgamma
has been implicated in transcriptional activation, these data indicate that
decreased signaling and transcriptional regulation resulting from FAD mutations
in beta-amyloid precursor protein and presenilin-1 may contribute to the
pathology of Alzheimer's disease. Genetic analysis of familial Alzheimer's disease has revealed that mutations in
the gamma-secretase enzyme presenilin promote toxic Abeta secretion; however,
presenilin mutations might also influence tau hyperphosphorylation and
neurodegeneration through gamma-secretase-independent mechanisms. To address
this possibility and determine whether other components of the gamma-secretase
complex possess similar regulatory functions, we analyzed the roles of
presenilin, nicastrin, and aph-1 in a Drosophila model for tau-induced
neurodegeneration. Here, we show that presenilin and nicastrin prevent tau
toxicity by modulating the PI3K/Akt/GSK3beta phosphorylation pathway, whereas
aph-1 regulates aPKC/PAR-1 activities. Moreover, we found that these
transmembrane proteins differentially regulate the intracellular localization of
GSK3beta and aPKC at cell junctions. Inhibition of gamma-secretase activity
neither interfered with these kinase pathways nor induced aberrant tau
phosphorylation. These results establish new in vivo molecular functions for the
three components of the gamma-secretase complex and reveal a different mechanism
that might contribute to neuronal degeneration in Alzheimer's disease. Gamma-secretase is a membrane protein complex with unusual aspartyl protease
activity that cleaves a variety of type I transmembrane proteins, such as APP,
Notch and E-cadherin, within their transmembranous regions. Gamma-secretase was
first recognized because of its role in the production of Abeta peptides that
are pathogenic in Alzheimer's disease. There is overwhelming evidence
demonstrating that four components, presenilin, nicastrin, APH-1 and PEN-2, are
necessary and sufficient for gamma-secretase activity. However, based on the
findings of studies conducted on cells overexpressing these four components, the
existence of regulatory components of the gamma-secretase complex has been
postulated. Recently, an additional subunit of the gamma-secretase complex,
membrane protein CD147, has been identified through the purification and
characterization of endogenous complexes from HeLa cell membranes. Removal of
CD147 from gamma-secretase complexes increases the production of Abeta-peptides.
Elucidating the molecular mechanism by which CD147 exerts its effect on the
activity of the gamma-secretase complex will help us to further understand the
pathogenesis of Alzheimer's disease, and may allow for the development of novel
therapeutics. The biogenesis and accumulation of the beta amyloid protein (Abeta) is a key
event in the cascade of oxidative and inflammatory processes that characterises
Alzheimer's disease. The presenilins and its interacting proteins play a pivotal
role in the generation of Abeta from the amyloid precursor protein (APP). In
particular, three proteins (nicastrin, aph-1 and pen-2) interact with
presenilins to form a large multi-subunit enzymatic complex (gamma-secretase)
that cleaves APP to generate Abeta. Reconstitution studies in yeast and insect
cells have provided strong evidence that these four proteins are the major
components of the gamma-secretase enzyme. Current research is directed at
elucidating the roles that each of these protein play in the function of this
enzyme. In addition, a number of presenilin interacting proteins that are not
components of gamma-secretase play important roles in modulating Abeta
production. This review will discuss the components of the gamma-secretase
complex and the role of presenilin interacting proteins on gamma-secretase
activity. Genetic and biological studies provide evidence that the production and
deposition of amyloid-beta peptides (Abeta) contribute to the etiology of
Alzheimer's disease. beta- and gamma-secretases, which are responsible for the
generation of Abeta, are plausible molecular targets for Alzheimer's disease
treatment. gamma-Secretase is an unusual aspartic protease that cleaves the
scissile bond within the transmembrane domain. This unusual enzyme is composed
of a high molecular weight membrane protein complex containing presenilin,
nicastrin, Aph-1 and Pen-2. Drugs that regulate the production of Abeta by
inhibiting or modulating gamma-secretase activity could provide a
disease-modifying effect on Alzheimer's disease, although recent studies suggest
that gamma-secretase plays important roles in cellular signaling including
Notch. Thus, understanding the molecular mechanism whereby gamma-secretase
recognizes and cleaves its substrate is a critical issue for the development of
compounds that specifically regulate Abeta-generating gamma-secretase activity.
This review focuses on the structure and function relationship of
gamma-secretase complex and the mode of action of the gamma-secretase
inhibitors. In this review, we discuss the biology of gamma-secretase, an enigmatic enzyme
complex that is responsible for the generation of the amyloid-beta peptide that
constitutes the amyloid plaques of Alzheimer's disease. We begin with a brief
review on the processing of the amyloid precursor protein and a brief discussion
on the family of enzymes involved in regulated intramembrane proteolysis, of
which gamma-secretase is a member. We then identify the four major components of
the gamma-secretase complex - presenilin, nicastrin, Aph-1, and Pen-2 - with a
focus on the identification of each and the role that each plays in the
maturation and activity of the complex. We also discuss two new proteins that
may play a role in modulating the assembly and activity of the gamma-secretase
complex. Next, we summarize the known subcellular locations of each
gamma-secretase component and the sites of gamma-secretase activity, as defined
by the production of Abeta. Finally, we close by synthesizing all of the
included topics into an overarching model for the assembly and trafficking of
the gamma-secretase complex, which serves as a launching point for further
questions into the biology and function of gamma-secretase in Alzheimer's
disease. Senescence-accelerated mice (SAMP8) serve as a model for Alzheimer's disease
(AD) as they exhibit early loss of memory and increased amyloid precursor
protein (APP) expression. APP is a ubiquitous membrane protein that is
physiologically processed by site-specific proteolysis firstly by alpha- or
beta-secretases, releasing a large fragment called APP(S) that contains most of
the extracellular sequences of APP, a small extracellular stub, the
transmembrane region and the cytoplasmic tail of APP (;AICD'-APP intracellular
domain). These are subsequently cleaved by gamma-secretase at multiple sites in
the transmembrane region, releasing small peptides, Abeta(1-40) and Abeta(1-42),
the major components of AD-associated amyloid fibrils. gamma-secretase is a
high-molecular-mass complex composed of presenilin-1 (PS1), nicastrin, APH-1 and
Pen-2. As PS1 has been shown to play a critical role in facilitating
gamma-secretase activity, and mutations in this protein are associated with
familial AD (FAD), we have cloned it from SAMP8 mouse hippocampus and compared
its sequence with those of other species. Furthermore, changes in the expression
of PS1 with age in the hippocampal tissue of SAMP8 were studied. The results
showed that the SAMP8 PS1 cDNA sequence is identical to that of normal mice.
However, its expression in the hippocampus of SAMP8 exhibited an increase, while
CD-1 mice, a strain that does not exhibit premature memory loss, showed no
change with age. An increased amount or mutation(s) in PS1, which alters the
stoichiometric balance of the gamma-secretase complex, may be the cause of
aberrant or increased processing of APP, resulting in Abeta accumulation leading
to loss of memory. The gamma-secretase complex plays a role in Alzheimer's disease and cancer
progression. The development of clinically useful inhibitors, however, is
complicated by the role of the gamma-secretase complex in regulated
intramembrane proteolysis of Notch and other essential proteins. Different
gamma-secretase complexes containing different Presenilin or Aph1 protein
subunits are present in various tissues. Here we show that these complexes have
heterogeneous biochemical and physiological properties. Specific inactivation of
the Aph1B gamma-secretase in a mouse Alzheimer's disease model led to
improvements of Alzheimer's disease-relevant phenotypic features without any
Notch-related side effects. The Aph1B complex contributes to total
gamma-secretase activity in the human brain, and thus specific targeting of
Aph1B-containing gamma-secretase complexes may help generate less toxic
therapies for Alzheimer's disease. gamma-Secretase is critically involved in the Notch pathway and in Alzheimer's
disease. The four subunits of gamma-secretase assemble in the endoplasmic
reticulum (ER) and unassembled subunits are retained/retrieved to the ER by
specific signals. We here describe a novel ER-retention/retrieval signal in the
transmembrane domain (TMD) 4 of presenilin 1, a subunit of gamma-secretase. TMD4
also is essential for complex formation, conferring a dual role for this domain.
Likewise, TMD1 of Pen2 is bifunctional as well. It carries an
ER-retention/retrieval signal and is important for complex assembly by binding
to TMD4. The two TMDs directly interact with each other and mask their
respective ER-retention/retrieval signals, allowing surface transport of
reporter proteins. Our data suggest a model how assembly of Pen2 into the
nascent gamma-secretase complex could mask TMD-based ER-retention/retrieval
signals to allow plasma membrane transport of fully assembled gamma-secretase. Presenilins form the catalytic part of the gamma-secretases, protein complexes
that are responsible for the intramembranous cleavage of transmembrane proteins.
The presenilins are involved in several biological functions, but are best known
for their role in the generation of the beta-amyloid (Abeta) peptide in
Alzheimer's disease and are therefore thought to be important drug targets for
this disorder. Mutations in the presenilin genes cause early-onset familial
Alzheimer's disease, but mutation carriers have substantial phenotypic
heterogeneity. Recent evidence implicating presenilin mutations in
non-Alzheimer's dementias, including frontotemporal dementia and Lewy body
dementia, warrants further investigation. An increased understanding of the
diversity of the molecular cell biology of the gamma-secretase complex and the
effects of clinical mutations in the presenilin genes might help pave the way
for improved development of drugs that are designed to target gamma-secretase
enzymatic activity in Alzheimer's disease and potentially in other neurological
diseases. Amyloid-beta peptides (Abeta) generated by proteolysis of the beta-amyloid
precursor protein (APP) by beta- and gamma-secretases play an important role in
the pathogenesis of Alzheimer's disease (AD). There is mounting evidence that
the lipid matrix of neuronal cell membranes plays an important role in the
accumulation of Abeta peptides into senile plaques, one of the hallmarks of AD.
With the aim to clarify the molecular basis of the interaction between Abeta and
cellular membranes, we investigated the effects of various phospholipids (PLs)
and a PL-rich diet on Abeta production. Here we show that modulation of Abeta
production and Abeta42:40 ratio is not limited to individual fatty acids, rather
it is the composition of the PLs of the membrane bilayer, that influences the
specificity and level of the regulated intramembranous proteolysis of APP by the
gamma-secretase complex. We show that Abeta levels in the conditioned media, in
response to some of the PL supplements, is increased in the center and decreased
on either side of a graph that resembles bell-shaped distribution. This means
that the PLs have less of a tendency to produce unusually extreme effects on
Abeta production in SP-C99 transfected Cos-7 cultured cells. We proposed a
mechanism-based hypothesis to rationalize PLs' effects on Abeta production. Mutations in presenilins (PS1 and PS2) account for the vast majority of early
onset familial Alzheimer's disease cases. Beside the well investigated role of
presenilins as the catalytic unit in γ-secretase complex, their involvement in
regulation of intracellular calcium homeostasis has recently come into more
focus of Alzheimer's disease research. Here we report that the overexpression of
PS1 full-length holoprotein forms, in particular familial Alzheimer's
disease-causing forms of PS1, result in significantly attenuated calcium release
from thapsigargin- and bradykinin-sensitive stores. Interestingly, treatment of
HEK293 cells with γ-secretase inhibitors also leads to decreased amount of
calcium release from endoplasmic reticulum (ER) accompanying elevated PS1
holoprotein levels. Similarly, the knockdown of PEN-2 which is associated with
deficient PS1 endoproteolysis and accumulation of its holoprotein form also
leads to decreased ER calcium release. Notably, we detected enhanced PS1
holoprotein levels also in postmortem brains of patients carrying familial
Alzheimer's disease PS1 mutations. Taken together, the conditions in which the
amount of full length PS1 holoprotein is increased result in reduction of
calcium release from ER. Based on these results, we propose that the disturbed
ER calcium homeostasis mediated by the elevation of PS1 holoprotein levels may
be a contributing factor to the pathogenesis of Alzheimer's disease. γ-Secretase is a multisubunit membrane protein complex containing presenilin
(PS1) as a catalytic subunit. Familial Alzheimer disease (FAD) mutations within
PS1 were analyzed in yeast cells artificially expressing membrane-bound
substrate, amyloid precursor protein, or Notch fused to Gal4 transcriptional
activator. The FAD mutations, L166P and G384A (Leu-166 to Pro and Gly-384 to Ala
substitution, respectively), were loss-of-function in yeast. We identified five
amino acid substitutions that suppress the FAD mutations. The cleavage of
amyloid precursor protein or Notch was recovered by the secondary mutations. We
also found that secondary mutations alone activated the γ-secretase activity.
FAD mutants with suppressor mutations, L432M or S438P within TMD9 together with
a missense mutation in the second or sixth loops, regained γ-secretase activity
when introduced into presenilin null mouse fibroblasts. Notably, the cells with
suppressor mutants produced a decreased amount of Aβ42, which is responsible for
Alzheimer disease. These results indicate that the yeast system is useful to
screen for mutations and chemicals that modulate γ-secretase activity. |
Which syndromes are associated with heterochromia iridum? | The syndromes that are associated with heterochromia iridum are:
1) Ascher's syndrome
2) Waardenburg Syndrome type II (WS2)
3) Horner's syndrome. | BACKGROUND: Heterochromia iridis, asymmetry of iris pigmentation, has been well
described with congenital Horner syndrome. Acquired heterochromia associated
with lesions in the ocular sympathetic pathways in adulthood, however, is rare.
METHODS: Two cases are reported in which sympathectomy in adults resulted in
ipsilateral Horner syndrome with heterochromia. In each case, pharmacologic
testing with cocaine and hydroxyamphetamine was performed.
RESULTS: In both cases, sympathectomy occurred at the level of the second order
neuron, but hydroxyamphetamine testing suggested at least partial third order
neuron involvement.
CONCLUSION: Acquired heterochromia can occur in adults. The partial response to
hydroxyamphetamine in the two cases presented may reflect trans-synaptic
degeneration of the postganglionic neuron. A reduction in trophic influences on
iris melanocytes may have contributed to the observed heterochromia. A case of heterochromia iridis and Horner's syndrome is reported in a 7-year old
girl with paravertebral neurilemmoma. These clinical findings can be useful in
the early diagnosis of mediastinal tumors in the paravertebral axis. While
typically associated with neuroblastoma, these findings can be due to tumors
which are inately benign--in this case neurilemmoma. The mechanism for
heterochromia is briefly discussed. Sturge-Weber syndrome is a disorder characterized by ipsilateral cavernous
hemangioma of the face, uvea, and brain in patients who may present with an
enlarged eye, exudative retinal detachment, glaucoma, and seizures. This report
presents the clinicopathologic findings of an otherwise healthy infant with
ipsilateral arteriovenous and capillary hemangiomas of the face and uveal tract,
microphthalmos, iris heterochromia, hypotony, and absence of central nervous
system involvement. The association of an arteriovenous-capillary angioma of the
ocular adnexa and ipsilateral uveal tract is a syndrome that is distinct from
Sturge-Weber syndrome. Waardenburg's syndrome consists of lateral displacement of the inner canthi of
the eyes (dystopia canthorum), a broad nasal root and confluent eyebrows,
heterochromia iridum, a white forelock and congenital deafness. The syndrome is
inherited as a domit, but affected individuals do not necessarily have all of
the characteristics cited.Five hundred and fourteen pupils at a school for the
deaf were screened for features of this syndrome. Three cases were discovered.
Eleven other deaf children were found to have heterochromia iridum and two more
had white forelocks. The interocular dimensions of the remaining children were
recorded as standards by which to judge the presence of dystopia canthorum. The
results of chromosomal analysis in two cases with Waardenburg's syndrome were
normal.The findings provide further evidence that Waardenburg's syndrome is a
distinct entity and call in question Mackenzie's concept of a comprehensive
"first arch syndrome". Duane retraction syndrome has been reported in association with structural
abnormalities of the eye, including epibulbar dermoid, keratoconus, iris
dysplasia, heterochromia iridis, persistent fetal vasculature, cataract,
choroidal coloboma, microphthalmia, and optic nerve dysplasia. A novel
association, that of bilateral Duane syndrome with bilateral aniridia, is the
subject of this report. OBJECTIVE: Waardenburg syndrome is an autosomal-domit syndrome characterized
by dystopia canthorum, hyperplasia of the eyebrows, heterochromia irides, a
white forelock, and sensorineural hearing loss in 20% to 55% of patients. This
patient population accounts for approximately 2% of congenitally deaf children.
The purpose of this retrospective case review was to describe the outcomes for
those children with Waardenburg syndrome who have undergone cochlear
implantation.
METHODS: Pediatric cochlear implant recipients with documented evidence of
Waardenburg syndrome underwent retrospective case review. All patients received
their cochlear implants at the study institution followed by outpatient auditory
habilitation. Charts were reviewed for etiology and duration of deafness, age at
time of cochlear implantation, perioperative complications, duration of use, and
performance outcomes. Results of standard tests batteries for speech perception
and production administered as a part of the patients' auditory habilitation
were reviewed.
RESULTS: Seven patients with Waardenburg syndrome and cochlear implants were
identified. The average age at implantation was 37 months (range, 18-64 months)
and the average duration of use was 69 months (range, 12-143 months). All of
these patients are active users of their devices and perform very well after
implantation. There were no major complications in this small group of patients.
CONCLUSIONS: Children with congenital sensorineural hearing loss without other
comorbidities (e.g., developmental delay, inner ear malformations) perform well
when they receive cochlear implantation and auditory habilitation. Patients with
Waardenburg syndrome can be expected to have above-average performance after
cochlear implantation. Horner's syndrome (HS) is related to an interruption of the oculosympathetic
nerve pathway. The classic clinical findings associated with this condition are
ptosis, miosis, and enophthalmos. Heterochromia is typically described in
congenital HS, but it is an uncommon finding in acquired HS. We report a case of
post-traumatic HS associated with heterochromia. A literature review indicates
that this type of heterochromia may be related to a reduction in the number of
iris melanocytes. This mechanism may be the same in the physiological iris color
modifications in adulthood. We report a 3-year-old girl with autosomal domit inherited Waardenburg
syndrome type I showing circumscribed hypopigmentation of the skin,
heterochromia iridis, sensorineural deafness, and dental aberrations. Clinical
diagnosis was confirmed by the identification of an underlying missense mutation
(C811T) in the PAX3 gene. Early diagnosis of Waardenburg syndrome among children
with pigment anomalies enables a successful interdisciplinary medical care. A domitly inherited syndrome associated with hypopigmentation, heterochromia
irides, colobomatous eyes and bilateral hearing loss has been ascertained in
Fleckvieh cattle (German White Fleckvieh syndrome). This syndrome has been
mapped to bovine chromosome (BTA) 22 using a genome-wide association study with
the bovine high density single nucleotide polymorphism array. An R210I missense
mutation has been identified within microphthalmia-associated transcription
factor (MITF) as responsible for this syndrome. The mutation is located in the
highly conserved basic region of the protein and causes a negative-domit
effect. SOX10 and PAX3 promoter binding site mutations in MITF could be ruled
out as causative for the German White Fleckvieh syndrome. Molecular
characterization of this newly detected bovine syndrome means a large animal
model is now available for the Tietz syndrome in humans. Waardenburg Syndrome (WS) is an autosomal-domit disorder characterized by
sensorineural hearing loss and pigmentary abnormalities of the eyes, hair, and
skin. Microphthalmia-associated transcription factor (MITF) gene mutations
account for about 15% of WS type II (WS2) cases. To date, fewer than 40
different MITF gene mutations have been identified in human WS2 patients, and
few of these were of Chinese descent. In this study, we report clinical findings
and mutation identification in the MITF gene of 20 Chinese WS2 patients from 14
families. A high level of clinical variability was identified. Sensorineural
hearing loss (17/20, 85.0%) and heterochromia iridum (20/20, 100.0%) were the
most commonly observed clinical features in Chinese WS2 patients. Five affected
individuals (5/20, 25.0%) had numerous brown freckles on the face, trunk, and
limb extremities. Mutation screening of the MITF gene identified five mutations:
c.20A>G, c.332C>T, c.647_649delGAA, c.649A>G, and c.763C>T. The total mutational
frequency of the MITF gene was 21.4% (3/14), which is significantly higher than
the 15.0% observed in the fair-skinned WS2 population. Our results indicate that
MITF mutations are relatively common among Chinese WS2 patients. An 18-year-old Indian girl with upper lip deformity presented with on and off
painless swelling of her both upper eyelids for 3 years. Clinical evaluation
revealed bilateral blepharochalasis, narrowing of horizontal palpebral fissure,
decreased outer intercanthal distance, iris coloboma, cleft soft palate, bifid
uvula, sensorineural deafness and double upper lip. Clinical examination of the
thyroid, thyroid hormone assay and ultrasonography revealed normal thyroid gland
structure and function. Ascher's syndrome was diagnosed. To our knowledge, this
is the first reported case of Ascher's syndrome associated with iris coloboma,
heterochromia iridum, and narrowing of horizontal palpebral fissure and
decreased outer intercanthal distance secondary to lengthening of lateral
canthal ligament. |
What is the target of tanezumab? | Tanezumab is a humanized monoclonal antibody against nerve growth factor. | We describe the use of four complementary biosensors (Biacore 3000, Octet QK,
ProteOn XPR36, and KinExA 3000) in characterizing the kinetics of human nerve
growth factor (NGF) binding to a humanized NGF-neutralizing monoclonal antibody
(tanezumab, formerly known as RN624). Tanezumab is a clinical candidate as a
therapy for chronic pain. Our measurements were consistent with the
NGF/tanezumab binding affinity being tighter than 10 pM due to the formation of
an extremely stable complex that had an estimated half-life exceeding 100 h,
which was beyond the resolution of any of our methods. The system was
particularly challenging to study because NGF is an obligate homodimer, and we
describe various assay orientations and immobilization methods that were used to
minimize avidity in our experiments while keeping NGF in as native a state as
possible. We also explored the interactions of NGF with its natural receptors,
TrkA and P75, and how tanezumab blocks them. The Biacore blocking assay that we
designed was used to quantify the potency of tanezumab and is more precise and
reproducible than the currently available cell-based functional assays. Persistent pain represents a major health problem, and most current therapeutic
approaches are associated with unwanted effects and unsatisfactory pain relief.
Therefore, an urgent need exists to develop more effective drugs that are
directed toward new molecular targets. Nerve growth factor (NGF) is involved in
pain transduction mechanisms, playing a key role as a master switch in many
chronic and inflammatory pain states; the NGF ligand and its receptor TrkA
constitute well-validated targets for pain therapy. Tanezumab (RN-624), a
first-in-class recombit humanized mAb targeting NGF, is being developed by
Pfizer Inc for the potential treatment of pain associated with several
conditions. In preclinical studies, tanezumab, and its murine precursor
muMab-911, effectively targeted the NGF pathway in various chronic and
inflammatory pain models. Phase I and II clinical trials in osteoarthritic pain
and chronic lower back pain demonstrated good efficacy for the compound, as well
as a good safety and tolerability profile. Given that tanezumab is an antibody,
the drug demonstrates the general advantages of this class of products
(including good specificity and favorable pharmacokinetics), and also appears to
be particularly well suited for targeting the chronic and inflammatory-mediating
pain actions of NGF and its receptor system. Nerve growth factor (NGF) is an important mediator of pain and hyperalgesia and
has become a target of novel analgesic therapeutics. Tanezumab is a humanized
IgG(2) antibody that binds NGF with high affinity and specificity. In a study to
assess the toxicity and pharmacokinetic properties of tanezumab in adult, male
and female, cynomolgus monkeys following weekly intravenous administration of 1,
10, or 30 mg/kg for up to 26 weeks (followed by an 8-week recovery period),
tanezumab was well tolerated with no macroscopic or microscopic effects on those
brain, spinal cord, nerve, or ganglia sections evaluated. One fifth of
tanezumab-treated monkeys developed an antibody response to tanezumab that
prevented maintece of tanezumab exposure between dosing. In the
antibody-negative animals, accumulation of tanezumab was observed; steady state
was achieved approximately 8 weeks after the first dose of study drug, and
exposure to tanezumab was approximately dose proportional with no observed
difference between male and female animals. One monkey died during the study;
this monkey had findings suggestive of hypersensitivity reaction. The favorable
toxicity and pharmacokinetic profile of tanezumab seen in this study supports
its further evaluation for the treatment of pain in clinical practice. PURPOSE: In this randomized, double-blind, placebo controlled phase 2 study we
investigated tanezumab, a humanized monoclonal antibody that specifically
inhibits nerve growth factor as a treatment for interstitial cystitis pain.
MATERIALS AND METHODS: Patients with interstitial cystitis received a single
intravenous dose of 200 μg/kg tanezumab or placebo. Patients recorded daily pain
scores (on an 11-point numerical rating scale) 7 days before attending study
visits and completed a urinary symptom diary for 3 of those days. Patients also
completed the Interstitial Cystitis Symptom Index questionnaire and a global
response assessment. The primary end point was change in average daily numerical
rating scale pain score from baseline to week 6. Secondary end points included
global response assessment, Interstitial Cystitis Symptom Index score,
micturition and urgency episode frequency per 24 hours, and mean voided volume
per micturition. The incidence of adverse events was also assessed.
RESULTS: A total of 34 patients received tanezumab and 30 received placebo. At
week 6 tanezumab produced a significant reduction from baseline in average daily
pain score vs placebo (treatment difference [LS mean, 90% CI] was -1.4 [-2.2,
-0.5]). A significantly higher proportion of patients on tanezumab responded as
improved in the global response assessment and tanezumab also significantly
reduced urgency episode frequency vs placebo. Tanezumab had no significant
effect on Interstitial Cystitis Symptom Index score, micturition frequency or
mean voided volume per micturition. The most common adverse events were headache
(tanezumab 20.6%, placebo 16.7%) and paresthesia (tanezumab 17.6%, placebo
3.3%).
CONCLUSIONS: Tanezumab has shown preliminary efficacy in the treatment of pain
associated with interstitial cystitis. Increased nerve growth factor levels are associated with chronic pain
conditions, including chronic low back pain (LBP). This study examined safety
and analgesic efficacy of tanezumab, a humanized anti-nerve growth factor
antibody, in adults with chronic LBP. Patients received intravenous tanezumab
200 μg/kg plus oral placebo (n=88), intravenous placebo plus oral naproxen 500
mg twice a day (n=88), or intravenous placebo plus oral placebo (n=41). Primary
outcome was average LBP intensity (aLBPI) at Week 6. Secondary outcomes were
proportion of patients with ≥30% or ≥50% reduction in aLBPI, Roland-Morris
Disability Questionnaire and Brief Pain Inventory-short form scores, Patients'
Global Assessment of LBP, Patients' Global Evaluation of study medication, and
rescue medication use. Mean aLBPI change from baseline to Week 6 was greater
with tanezumab vs naproxen (P=0.004) and placebo (P<0.001). Greater proportions
of patients reported ≥30% and ≥50% reduction in aLBPI with tanezumab vs naproxen
(P≤0.013) and placebo (P<0.001), and greater improvements in Roland-Morris
Disability Questionnaire (P<0.001) and other secondary outcomes except rescue
medication use. Tanezumab was associated with adverse events (AEs) of abnormal
peripheral sensation that were generally mild and resolved before study
completion; however, there were no serious AEs. Nine patients (4 of whom were
tanezumab-treated) discontinued due to AEs. In conclusion, tanezumab resulted in
analgesic efficacy that was clinically and statistically superior to placebo and
naproxen in patients with chronic LBP. Tanezumab clinical development is on
regulatory hold due to AEs in osteoarthritis patients. Nerve growth factor (NGF) antagonism has long been proposed as a chronic pain
treatment. In 2010, the FDA suspended clinical trials using tanezumab, a
humanized monoclonal anti-NGF antibody, to treat osteoarthritis due to worsening
joint damage in 16 patients. Increased physical activity in the absence of acute
pain which normally prevents self-harm was purported as a potential cause. Such
an adverse effect is consistent with an extension of tanezumab's primary
mechanism of action by decreasing pain sensitivity below baseline levels. In
animal inflammatory pain models, NGF antagonism decreases intraepidermal nerve
fiber (IENF) density and attenuates increases in expression of
nociception-related proteins, such as calcitonin gene-related peptide (CGRP) and
substance P (SP). Little is known of the effects of NGF antagonism in
noninflamed animals and the hypoalgesia that ensues. In the current study, we
immunized rats with NGF or cytochrome C (cytC) and examined (1) nocifensive
behaviors with thermal latencies, mechanical thresholds, the hot plate test, and
the tail flick test, (2) IENF density, and (3) expression of CGRP, SP,
voltage-gated sodium channel 1.8 (Nav1.8), and glutaminase in subpopulations of
dorsal root ganglion (DRG) neurons separated by size and isolectin B4 (IB4)
labeling. Rats with high anti-NGF titers had delayed responses on the hot plate
test but no other behavioral abnormalities. Delayed hot plate responses
correlated with lower IENF density. CGRP and SP expression was decreased
principally in medium (400-800 μm(2)) and small neurons (<400 μm(2)),
respectively, regardless of IB4 labeling. Expression of Nav1.8 was only
decreased in small and medium IB4 negative neurons. NGF immunization appears to
result in a more profound antagonism of NGF than tanezumab therapy, but we
hypothesize that decreases in IENF density and nociception-related protein
expression are potential mechanisms for tanezumab-induced hypoalgesia. OBJECTIVE: To assess the efficacy and safety of tanezumab, a humanized
monoclonal antibody directed against the pain-mediating neurotrophin, nerve
growth factor, to treat pain and other symptoms of chronic prostatitis/chronic
pelvic pain syndrome in a Phase IIa, proof-of-concept clinical trial powered to
provide 2-sided 90% confidence interval around the primary endpoint.
METHODS: Patients received a single intravenous dose of tanezumab (20 mg) or
placebo. The primary efficacy endpoint was the change from baseline to week 6 in
average daily numerical rating scale pain score. The secondary endpoints
included the change from baseline to week 6 in the National Institutes of Health
Chronic Prostatitis Symptom Index and urinary symptoms. Safety was also
assessed.
RESULTS: Overall, 62 patients were randomized (30 to tanezumab and 32 to
placebo). At week 6, tanezumab marginally improved the average daily pain
(least-squares mean difference from placebo -0.47, 90% confidence interval
-1.150-0.209) and urgency episode frequency (least-squares mean difference from
placebo -1.37, 90% confidence interval -3.146-0.401). No difference was seen in
the National Institutes of Health chronic prostatitis symptom index total score
or micturition frequency at week 6. The most common adverse events were
paresthesia and arthralgia. The odds of having a ≥ 30% reduction in pain were
1.75-fold greater (90% confidence interval 0.65-4.69) for patients receiving
tanezumab versus placebo.
CONCLUSION: Tanezumab might improve symptoms for some patients with chronic
prostatitis/chronic pelvic pain syndrome. Although proof of concept was not
demonstrated in the present study, additional studies with larger populations
and stricter inclusion criteria according to patient phenotype might identify
populations in which antinerve growth factor treatment will provide clinical
benefit. Chronic pain arising from various pathological conditions such as
osteoarthritis, low back or spinal injuries, cancer, and urological chronic
pelvic pain syndromes presents significant challenges in diagnosis and
treatment. Specifically, since the underlying cause of these pain syndromes is
unknown or heterogeneous, physicians diagnose and treat patients based on the
symptoms presented. Nerve growth factor (NGF) has been recognized as an
important mediator of chronic pain in many pathological conditions, and has been
shown to be upregulated in a subset of individuals suffering from such pain
syndromes. These findings have led to the development of anti-NGF monoclonal
antibodies such as tanezumab as potentially effective therapeutics for chronic
pain. Although tanezumab has reached Phase II and III clinical trials, the
trials of anti-NGF antibodies were halted due to safety concerns. Some of these
trials of anti-NGF treatment have had statistically significant decreases in
pain, while others have yielded inconclusive results. These findings are
suggestive of, though do not prove, target (NGF) neutralization in chronic pain
syndromes. A biomarker-driven anti-NGF clinical study layout is proposed that
incorporates NGF measurements in the relevant samples before and after
treatment, in addition to collecting the pain scores. This approach might not
only confirm the mechanism of tanezumab's action in these chronic pain patients,
but should establish NGF levels as a predictive biomarker for patients who can
benefit from anti-NGF treatment, thereby creating a personalized approach to
pain treatment. Tanezumab is a humanized monoclonal antibody that specifically inhibits nerve
growth factor as a treatment for chronic pain. This phase IIB study investigated
the efficacy and safety of tanezumab for chronic low back pain vs placebo and
naproxen. Patients (N=1347) received intravenous tanezumab (5, 10, or 20mg every
8weeks), naproxen (500mg twice daily), or placebo. The primary efficacy end
point was mean change in daily average low back pain intensity (LBPI) from
baseline to week 16. Secondary end points included mean change from baseline to
week 16 in the Roland Morris Disability Questionnaire and Patient's Global
Assessment (PGA) of low back pain. Tanezumab 10 and 20mg had similar efficacy
profiles and significantly improved LBPI, Roland Morris Disability
Questionnaire, and PGA scores vs both placebo and naproxen (P⩽.05). Tanezumab
5mg provided improvement of PGA scores vs placebo (P⩽.05), and naproxen resulted
in significant improvement of LBPI vs placebo (P⩽.05). Adverse event incidence
was comparable across tanezumab doses but higher than with placebo or naproxen.
Arthralgia, pain in extremity, headache, and paresthesia were the most commonly
reported adverse events by tanezumab-treated patients. The most frequently
reported adverse events resulting in discontinuation of tanezumab treatment were
arthralgia and paresthesia; the highest frequency was observed with tanezumab
20mg (both 1.4%). Serious adverse event incidence was similar across treatments.
In conclusion, tanezumab provided significantly greater improvement in pain,
function, and global scores vs placebo and naproxen in patients with chronic low
back pain. BACKGROUND: Low back pain with or without radiculopathy is an important cause of
disability and economic expenditure. However, many patients are not meeting
optimal pain control through existing treatments. Recent studies have linked
nerve growth factor (NGF) and the pathophysiology of persistent pain. Anti-NGF
could be an alternative drug treatment for low back pain.
OBJECTIVE: Systematically review the efficacy and safety of anti-NGF in the
treatment of low back pain.
METHODS: A systematic review of the literature with no language, date or
publication status restriction, using Medline, EMBASE, Cochrane Library, and the
clinicaltrials.gov database. Additional literature was retrieved by conferring
with experts in the field or reviewing bibliographies and annals of meetings and
congresses. Search terms included "monoclonal antibodies," "nerve growth
factor," "anti-ngf," "fulranumab," "tanezumab," "sciatica," "back pain," and
"spine."
STUDY DESIGN: Inclusion criteria were observational studies with safety as an
outcome and randomized or nonrandomized controlled trials studying the efficacy
and/or the safety of anti-NGF drugs on low back pain. Exclusion criteria
included patients with autoimmune conditions or osteoporosis. Studies were
assessed independently by 2 authors regarding inclusion/exclusion criteria, risk
of bias, clinical relevance, and quality of evidence (GRADE approach).
RESULTS: 1,168 studies were retrieved. After excluding duplicates and applying
the inclusion/exclusion criteria, 4 RCTs remained (n = 2,109): 2 for tanezumab,
one for REGN475, and one for fulranumab. Only the tanezumab studies showed any
significant difference over placebo (n = 1,563) for both pain relief and
functional improvement.
CONCLUSIONS: There is very low evidence that systemically administered anti-NGF
therapy has a small positive effect compared to placebo for both pain relief
(standarized mean difference [SMD] = -0.29, 95% confidence interval [CI] -0.58
to 0.00) and functional improvement (SMD = -0.21, 95%CI -0.37 to -0.05 ) of low
back pain. There was low evidence of adverse effects (AEs) compared to placebo
and low evidence of neurological AEs than placebo (relative risk = 1.93, 95%CI
1.41 to 2.64).Tanezumab, as a specific anti-NGF treatment, showed low evidence
of a small to moderate effect for pain relief of low back pain (SMD = -0.44,
95%CI -0.81 to -0.07); and low evidence of a small effect for functional
improvement (SMD = -0.26, 95%CI -0.40 to -0.12) with systemic administration,
although not clinically significant. Tanezumab and anti-NGFs overall had,
respectively, moderate and low evidence of overall AEs and serious AEs and a
higher risk of developing neurological AEs when compared with placebo. Although
anti-NGF, specifically tanezumab, showed a low-to-moderate effect on pain relief
and functional improvement, it cannot be recommended for low back pain
treatment. Without more research on the pathophysiology of anti-NGFs and adverse
effects, its use is not safe in the overall population. However, as corroborated
by the US Food and Drug Administration, this meta-analysis underscores a role
for greater insight into anti-NGF therapy for painful conditions that are
refractory to current drugs, such as oncologic pain, chronic pancreatitis, and
phantom-limb pain. Given the pathophysiology of axial pain involving
inflammatory mediators and the adverse effects of systemic anti-NGF use,
consideration of local therapies may warrant further exploration. It is uimously accepted that there is an unmet need for pain medications that
are both effective and safe. Unfortunately, no really novel analgesics have been
approved over the past three decades. In view of both experimental and clinical
evidence of a major role for nerve growth factor (NGF) in the generation and
maintece of a wide range of pain states, drug discovery efforts focusing on
the development of anti-NGF agents have aroused particular interest. Several
humanized anti-NGF monoclonal antibodies (mAbs) have entered clinical trials as
potential analgesics. In this respect, tanezumab is at an advanced stage of
clinical development while fulranumab, fasinumab and ABT-110, previously known
as PG110, are in early phases of clinical development. This Current Opinion
article aims at describing the rationale for targeting NGF for pain, reviewing
the analgesic efficacy and safety of anti-NGF agents based on data from fully
published studies, conference abstracts, and the US Food and Drug Administration
(FDA) website, and discussing the possible future of these agents in managing
chronic pain. Anti-NGF mAbs produced significant pain relief and functional
improvement in patients with osteoarthritis of the knee and/or hip. Conversely,
studies in non-specific lower back pain generated mixed results; overall, this
condition appeared to be less responsive to anti-NGF agents than osteoarthritis.
Finally, there was no conclusive evidence of the effectiveness of anti-NGF mAbs
in some types of chronic visceral or neuropathic pain. Furthermore, these
studies raised safety concerns about anti-NGF mAbs. As a class, these drugs may
cause or worsen peripheral neuropathies. But the most problematic issue-which
prompted the FDA to place studies of these compounds on clinical hold in
2010-was rapid joint destruction leading to joint replacement surgery. The
aetiologies of these side effects have been much debated and their
pathophysiology is poorly understood. After an Arthritis Advisory Committee
meeting held in March 2012, pharmaceutical companies negotiated with the FDA on
the conditions for restarting clinical studies. Although the FDA lifted its
clinical hold, there remain many unresolved issues about the long-term efficacy
and safety of anti-NGF mAbs. While acknowledging that the future of these drugs
is unforeseeable, it appears that they may not be the safe and effective
painkillers that have been awaited for decades. INTRODUCTION: Tropomyosin receptor kinases (Trks) are a family of three similar
tyrosine kinases activated by peptide hormones of the neurotrophin family. The
nerve growth factor antibody tanezumab has provided clinical proof of concept
for inhibition of the TrkA pathway in pain. As an alternative modality,
small-molecule inhibitors of the Trks have been pursued in recent years to probe
the role of these neurotrophin pathways in pain, cancer and other indications.
AREAS COVERED: This paper reviews the patent literature between mid-2009 and
2013, claiming inhibitors of Trk family members as the primary biological
targets. Additional patents have been reviewed where Trk is not the main kinase
of interest but in which high Trk potency is observed and the chemical matter is
particularly noteworthy. Patents pre-dating this period have been reviewed
previously. Scifinder and Google were used to find relevant patents and clinical
information using Trk or Tropomyosin as the search term.
EXPERT OPINION: Considerable recent progress has been made in the identification
of selective pan Trk inhibitors with pharmacodynamic and pharmacokinetic
properties appropriate for clinical evaluation. Inhibitors of both active and
inactive conformations of the Trks as well as peripherally restricted molecules
have been identified. Furthermore, TrkA-selective allosteric inhibitors have
recently been disclosed, which enables the biology of this isoform to be probed.
The recent identification of a TrkA gene fusion in a subset of lung cancer
patients will increase further the attraction of Trk inhibition to the
pharmaceutical industry. Nerve growth factor (NGF) is indispensable during normal embryonic development
and critical for the amplification of pain signals in adults. Intervention in
NGF signaling holds promise for the alleviation of pain resulting from human
diseases such as osteoarthritis, cancer and chronic lower back disorders. We
developed a fast, high-fidelity method to convert a hybridoma-derived
NGF-targeted mouse antibody into a clinical candidate. This method, termed
Library Scanning Mutagenesis (LSM), resulted in the ultra-high affinity antibody
tanezumab, a first-in-class anti-hyperalgesic specific for an NGF epitope.
Functional and structural comparisons between tanezumab and the mouse 911
precursor antibody using neurotrophin-specific cell survival assays and X-ray
crystal structures of both Fab-antigen complexes illustrated high fidelity
retention of the NGF epitope. These results suggest the potential for wide
applicability of the LSM method for optimization of well-characterized
antibodies during humanization. OBJECTIVE: To evaluate peripheral nerve safety and clinical efficacy of
tanezumab in patients with painful osteoarthritis.
METHODS: Patients received intravenous tanezumab 5mg, tanezumab 10mg, or placebo
every 8 weeks for 24 weeks. Neurological safety was evaluated via a composite
score (nerve conduction attributes and heart rate variability with deep
breathing; Σ5NC + HRdb), intraepidermal nerve fiber (IENF) density, and clinical
assessments. Efficacy and general safety were also evaluated.
RESULTS: The study was stopped prematurely by an FDA partial clinical hold
(joint safety issues in other studies). Differences in change from baseline to
Week 24 in Σ5NC + HRdb were not significant. Tanezumab 5mg vs placebo exceeded
the prespecified clinically important difference using last observation carried
forward imputation, but not with observed data or when patients with evidence of
neuropathy at baseline were excluded. No significant differences were found in
individual nerve conduction measures. No treatment exceeded the prespecified
clinically important decrease in IENF. Tanezumab resulted in significant
improvement in pain, physical function, and Patient's Global Assessment. Safety
was similar to previous tanezumab clinical trials.
CONCLUSIONS: Tanezumab has a modulating effect on pain, does not appear to
increase neurological safety signals, and offers a potentially promising, novel
approach in treatment of pain. Tanezumab, an antibody to nerve growth factor, was administered to pregt
cynomolgus monkeys at 0, 0.5, 4, and 30 mg/kg weekly, beginning gestation day
(GD) 20 through parturition (∼GD165). Maternal tanezumab administration appeared
to increase stillbirths and infant mortality, but no consistent pattern of gross
and/or microscopic change was detected to explain the mortality. Offspring
exposed in utero were evaluated at 12 months of age using light microscopy (all
tissues), stereology (basal forebrain cholinergic and dorsal root ganglia
neurons), and morphometry (sural nerve). Light microscopy revealed decreased
number of neurons in sympathetic ganglia (superior mesenteric, cervicothoracic,
and ganglia in the thoracic sympathetic trunk). Stereologic assessment indicated
an overall decrease in dorsal root ganglion (thoracic) volume and number of
neurons in animals exposed to tanezumab 4 mg/kg (n = 9) and 30 mg/kg (n = 1). At
all tanezumab doses, the sural nerve was small due to decreases in myelinated
and unmyelinated axons. Existing axons/myelin sheaths appeared normal when
viewed with light and transmission electron microscopy. There was no indication
of tanezumab-related, active neuron/nerve fiber degeneration/necrosis in any
tissue, indicating decreased sensory/sympathetic neurons and axonal changes were
due to hypoplasia or atrophy. These changes in the sensory and sympathetic
portions of the peripheral nervous system suggest some degree of developmental
neurotoxicity, although what effect, if any, the changes had on normal function
and survival was not apparent. Overall, these changes were consistent with
published data from rodent studies. OBJECTIVE: Evaluate efficacy and safety of tanezumab, a humanized monoclonal
antibody against nerve growth factor, in neuropathic pain.
DESIGN: Two randomized controlled trials.
SUBJECTS: Patients with pain due to diabetic peripheral neuropathy (DPN) or
postherpetic neuralgia (PHN).
METHODS: In the DPN study, patients received subcutaneous tanezumab 20 mg or
placebo on Day 1 and Week 8. Evaluations included change from baseline in
average DPN pain (primary endpoint), Patient's Global Assessment of DPN, and
safety (including neuropathy assessments). Due to a partial clinical hold
limiting enrollment and treatment duration, the prespecified landmark analysis
was modified post hoc from Week 16 to Week 8. In the PHN study, patients
received intravenous tanezumab 50 μg/kg, tanezumab 200 μg/kg, or placebo on Day
1. Evaluations included change from baseline in average daily pain (primary
endpoint), Brief Pain Inventory-short form, Patient's Global Assessment of pain
from PHN, and safety.
RESULTS: Mean DPN pain reduction from baseline to Week 8 was greater with
tanezumab vs placebo (P = 0.009); differences in Patient's Global Assessment of
DPN were not significant (P > 0.05). Neither tanezumab dose resulted in
significant differences vs placebo in efficacy in PHN (P > 0.05), although
tanezumab 200 μg/kg provided some benefit. Neuropathy assessments showed no
meaningful changes.
CONCLUSIONS: Tanezumab provided effective pain reduction in DPN. In PHN, only
the highest tanezumab dose reduced pain; treatment differences were not
significant. No new safety concerns were observed despite preexisting
neuropathy. INTRODUCTION: Bladder pain syndrome (BPS)/interstitial cystitis (IC) is
associated with sensory lower urinary tract symptoms. Unfortunately, many of the
existing oral treatments are ineffective in most patients of BPS/IC, which is
the motivation for developing new drugs and therapeutic approaches. This review
covers the latest drugs that have been investigated in BPS/IC patients.
Intravesical treatments offer the opportunity to directly target the painful
bladder with less systemic side effects.
AREAS COVERED: In this review, the authors analyze the existing literature
supporting the treatment of BPS/IC with conventional drugs including heparin,
hyaluronic acid, chondroitin sulfate, and dimethylsulfoxide (DMSO). Furthermore,
investigational drugs such as tanezumab and adalimumab, capable of sequestering
nerve growth factor (NGF), and Tumor necrosis factor-α (TNF- α) are discussed.
Investigational treatments such as liposomes, botulinum toxin (BTX), liposomal
BTX, PD-0299685 (a Ca(2+) channel ɑ2δ ligand), continuous intravesical
lidocaine, and AQX-1125 (a novel SHIP1 activating compound) are also covered.
EXPERT OPINION: New investigational drugs offer promising improvements in
clinical outcomes for BPS/IC patients; however, BPS/IC is a chronic pain
disorder that is very vulnerable to a strong placebo effect. In addition, BPS/IC
is a heterogeneous disorder that can be classified into several phenotypes.
Since different phenotypes of BPS/IC respond differently to systemic and
intravesical treatments, the authors believe that new drugs developed for BPS/IC
are more likely to meet their predetermined clinical endpoints if the
inclusion/exclusion criterion is tailored to specific phenotype of BPS/IC
patients. The management of pain associated with chronic musculoskeletal conditions
represents a significant challenge for the clinician. There remains a need for
novel medications that have a significant analgesic benefit and are also safe
and well tolerated. Both pre-clinical and clinical data have provided evidence
of the role of nerve growth factor (NGF) in a multitude of pain eliciting
conditions. Therefore, the development of monoclonal antibodies to NGF for
chronic painful musculoskeletal conditions has generated interest. Areas
covered: This manuscript is a review that examines both the pharmacological
properties and clinical studies of tanezumab, the most widely studied antibody
to NGF, for management of osteoarthritis (OA) and low back pain. In addition,
the safety and tolerability profile and development history of tanezumab are
also discussed. Expert opinion: Most studies provide strong support for the
ability of tanezumab to provide clinically meaningful pain relief in individuals
with these conditions, with longer-term studies suggesting durability of effect.
The adverse event profile appears favorable, assuming the risk mitigation
strategies are effective at reducing the incidence of joint-related side
effects. Further data are being collected to define the optimal dose and dosing
strategy in both OA and chronic low back pain. |
Could plasmepsins be used as targets for developing anti-malaria drugs? | Yes, plasmepsins, which are essential Plasmodium proteases, could be highly promising anti-malarial drug targets. | Plasmodium falciparum is the major causative agent of malaria, a disease of
worldwide importance. Resistance to current drugs such as chloroquine and
mefloquine is spreading at an alarming rate, and our antimalarial armamentarium
is almost depleted. The malarial parasite encodes two homologous aspartic
proteases, plasmepsins I and II, which are essential components of its
hemoglobin-degradation pathway and are novel targets for antimalarial drug
development. We have determined the crystal structure of recombit plasmepsin
II complexed with pepstatin A. This represents the first reported crystal
structure of a protein from P. falciparum. The crystals contain molecules in two
different conformations, revealing a remarkable degree of interdomain
flexibility of the enzyme. The structure was used to design a series of
selective low molecular weight compounds that inhibit both plasmepsin II and the
growth of P. falciparum in culture. Plasmepsin II is a key enzyme in the life cycle of the Plasmodium parasites
responsible for malaria, a disease that afflicts more than 300 million
individuals annually. Since plasmepsin II inhibition leads to starvation of the
parasite, it has been acknowledged as an important target for the development of
new antimalarials. In this paper, we identify and characterize high-affinity
inhibitors of plasmepsin II based upon the allophenylnorstatine scaffold. The
best compound, KNI-727, inhibits plasmepsin II with a K(i) of 70 nM and a
22-fold selectivity with respect to the highly homologous human enzyme cathepsin
D. KNI-727 binds to plasmepsin II in a process favored both enthalpically and
entropically. At 25 degrees C, the binding enthalpy (DeltaH) is -4.4 kcal/mol
and the entropic contribution (-TDeltaS) to the Gibbs energy is -5.56 kcal/mol.
Structural stability measurements of plasmepsin II were also utilized to
characterize inhibitor binding. High-sensitivity differential scanning
calorimetry experiments performed in the absence of inhibitors indicate that, at
pH 4.0, plasmepsin II undergoes thermal denaturation at 63.3 degrees C. The
structural stability of the enzyme increases with inhibitor concentration in a
manner for which the binding energetics of the inhibitor can quantitatively
account. The effectiveness of the best compounds in killing the malaria parasite
was validated by performing cytotoxicity assays in red blood cells infected with
Plasmodium falciparum. EC50s ranging between 6 and 10 microM (3-6 microg/mL)
were obtained. These experiments demonstrate the viability of the
allophenylnorstatine scaffold in the design of powerful and selective plasmepsin
inhibitors. The malarial aspartic proteinases (plasmepsins) have been discovered in several
species of Plasmodium, including all four of the human malarial pathogens. In
P.falciparum, plasmepsins I, II, IV and HAP have been directly implicated in
hemoglobin degradation during malaria infection, and are now considered targets
for anti-malarial drug design. The plasmepsins are produced from inactive
zymogens, proplasmepsins, having unusually long N-terminal prosegments of more
than 120 amino acids. Structural and biochemical evidence suggests that the
conversion process of proplasmepsins to plasmepsins differs substantially from
the gastric and plant aspartic proteinases. Instead of blocking substrate access
to a pre-formed active site, the prosegment enforces a conformation in which
proplasmepsin cannot form a functional active site. We have determined crystal
structures of plasmepsin and proplasmepsin from P.vivax. The three-dimensional
structure of P.vivax plasmepsin is typical of the monomeric aspartic
proteinases, and the structure of P.vivax proplasmepsin is similar to that of
P.falciparum proplasmepsin II. A dramatic refolding of the mature N terminus and
a large (18 degrees ) reorientation of the N-domain between P.vivax
proplasmepsin and plasmepsin results in a severe distortion of the active site
region of the zymogen relative to that of the mature enzyme. The present
structures confirm that the mode of inactivation observed originally in
P.falciparum proplasmepsin II, i.e. an incompletely formed active site, is a
true structural feature and likely represents the general mode of inactivation
of the related proplasmepsins. Intraerythrocytic malaria parasites degrade haemoglobin to provide nutrients for
their own growth and maturation. Plasmodium aspartic proteases known as
plasmepsins play an important role on haemoglobin degradation and are being
studied as drug targets for chemotherapy of malaria. The rodent model for human
malaria, Plasmodium chabaudi, is an experimentally good model for therapy drug
design. The gene encoding an aspartic protease precursor (proplasmepsin) from
the rodent malaria parasite P. chabaudi was cloned and sequenced. A theoretical
3D structure model was constructed by comparative homology and used for
superimposition with other known models. Analysis of the P. chabaudi and
Plasmodium yoelli genomes revealed in both the presence of at least seven
plasmepsins and each one has sequence similarity to its plasmepsin counterpart
of the human malaria Plasmodium falciparum. The predicted proteins were
confirmed as plasmepsins by detection on Blocks Database of three characteristic
blocks of the eukaryotic and viral aspartic protease family. Analysis of the
proline-rich loop amino acid sequence of these plasmepsins suggests that they
constitute characteristic motifs of each plasmepsin group suggesting that these
sequence variations are related with different substrate specificities. Malaria remains a major disease of mankind, and resistance to existing
therapeutics is rapidly emerging. Limited ficial investment to develop new
therapeutics requires the careful selection of well-defined targets from the
causative parasite, Plasmodium falciparum. In these circumstances, protein
crystallography can provide valuable structural detail to facilitate both the
selection of suitable targets and the development of compounds to provide novel
drug candidates. This review summarises the current involvement of
crystallographic studies in anti-malarial drug development programmes. Protein
crystallography is increasingly central to the exploitation of a number of
potential Plasmodial targets. including the aspartic acid proteases
(plasmepsins) and cysteine proteases (falcipains) involved in haem degradation
within the parasite food vacuole. Lead compounds are being identified from
collections previously synthesised against homologous human enzymes. Plasmodium
have an unusual dependence on the glycolytic pathway relative to their human
hosts, and this is reflected in subtle structural differences identified in the
crystal structures of a number of parasite glycolytic enzymes including aldolase
and lactate dehydrogenase. Other enzymes from a range of biosynthetic pathways
have also been targeted in crystallographic studies. These include dihydrofolate
reductase, the target of existing anti-folate therapeutics, and enoyl reductase
from the fatty acid biosynthesis pathway which is already the target of
effective bacteriocides. Crystal structures of these drug-enzyme complexes not
only allow visualisation and improvement of inhibitor-protein contacts, but in
the former case have also been used to probe the molecular basis of emerging
anti-malarial drug resistance. Crystallography is similarly proving valuable as
a tool to facilitate the development of inhibitors of purine salvage, isoprenoid
synthesis and utilisation, and protein processing mechanisms. The increasing resistance of the malarial parasite to antimalarial drugs is a
major contributor to the reemergence of the disease and increases the need for
new drug targets. The two aspartic proteases, plasmepsins I and II, from
Plasmodium falciparum have recently emerged as potential targets. In an effort
to inhibit these hemoglobinases, a series of inhibitors encompassing a basic
hydroxyethylamine transition state isostere as a central fragment were prepared.
The synthesized compounds were varied in the P1' position and exhibited
biological activities in the range of 31 to >2000 nM. To try to rationalize the
results, molecular docking and 3D-QSAR analysis were used. Plasmepsin group of enzymes are key enzymes in the life cycle of malarial
parasites. As inhibition of plasmepsins leads to the parasite's death, these
enzymes can be utilized as potential drug targets. Although many drugs are
available, it has been observed that Plasmodium falciparum, the species that
causes most of the malarial infections and subsequent death, has developed
resistance against most of the drugs. Based on the cleavage sites of hemglobin,
the substrate for plasmepsins, we have designed two compounds
(p-nitrobenzoyl-leucine-beta-alanine and p-nitrobenzoyl-leucine-isonipecotic
acid), synthesized them, solved their crystal structures and studied their
inhibitory effect using experimental and theoretical (docking) methods. In this
paper, we discuss the synthesis, crystal structures and inhibitory nature of
these two compounds which have a potential to inhibit plasmepsins. Malaria is one of the major diseases in the world. Due to the rapid spread of
parasite resistance to available antimalarial drugs there is an urgent need for
new antimalarials with novel mechanisms of action. Several promising targets for
drug intervention have been revealed in recent years. This review addresses the
parasitic aspartic proteases termed plasmepsins (Plms) that are involved in the
hemoglobin catabolism that occurs during the erythrocytic stage of the malarial
parasite life cycle. Four Plasmodium species are responsible for human malaria;
P. vivax, P. ovale, P. malariae, and P. falciparum. This review focuses on
inhibitors of the haemoglobin-degrading plasmepsins of the most lethal species,
P. falciparum; Plm I, Plm II, Plm IV, and histo-aspartic protease (HAP).
Previously, Plm II has attracted the most attention. With the identification and
characterization of new plasmepsins and the results from recent plasmepsin
knockout studies, it now seems clear that in order to achieve high-antiparasitic
activities in P. falciparum-infected erythrocytes it is necessary to inhibit
several of the haemoglobin-degrading plasmepsins. Herein we summarize the
structure-activity relationships of the Plm I, II, IV, and HAP inhibitors. These
inhibitors represent all classes which, to the best of our knowledge, have been
disclosed in journal articles to date. The 3D structures of inhibitor/plasmepsin
II complexes available in the protein data bank are briefly discussed and
compared. Plasmepsins are aspartic proteases involved in the degradation of the host cell
hemoglobin that is used as a food source by the malaria parasite. Plasmepsins
are highly promising as drug targets, especially when combined with the
inhibition of falcipains that are also involved in hemoglobin catabolism. In
this review, we discuss the mechanism of plasmepsins I-IV in view of the
interest in transition state mimetics as potential compounds for lead
development. Inhibitor development against plasmepsin II as well as relevant
crystal structures are summarized in order to give an overview of the field.
Application of computational techniques, especially binding affinity prediction
by the linear interaction energy method, in the development of malarial
plasmepsin inhibitors has been highly successful and is discussed in detail.
Homology modeling and molecular docking have been useful in the current
inhibitor design project, and the combination of such methods with binding free
energy calculations is analyzed. Though different species of the genus Plasmodium may be responsible for malaria,
the variant caused by P. falciparum is often very dangerous and even fatal if
untreated. Hemoglobin degradation is one of the key metabolic processes for the
survival of the Plasmodium parasite in its host. Plasmepsins, a family of
aspartic proteases encoded by the Plasmodium genome, play a prominent role in
host hemoglobin cleavage. In this paper we demonstrate the use of virtual
screening, in particular molecular docking, employed at a very large scale to
identify novel inhibitors for plasmepsins II and IV. A large grid
infrastructure, the EGEE grid, was used to address the problem of large
computation resources required for docking hundreds of thousands of chemical
compounds on different plasmepsin targets of P. falciparum. A large compound
library of about 1 million chemical compounds was docked on 5 different targets
of plasmepsins using two different docking software, namely FlexX and AutoDock.
Several strategies were employed to analyze the results of this virtual
screening approach including docking scores, ideal binding modes, and
interactions to key residues of the protein. Three different classes of
structures with thiourea, diphenylurea, and guanidino scaffolds were identified
to be promising hits. While the identification of diphenylurea compounds is in
accordance with the literature and thus provides a sort of "positive control",
the identification of novel compounds with a guanidino scaffold proves that high
throughput docking can be effectively used to identify novel potential
inhibitors of P. falciparum plasmepsins. Thus, with the work presented here, we
do not only demonstrate the relevance of computational grids in drug discovery
but also identify several promising small molecules which have the potential to
serve as candidate inhibitors for P. falciparum plasmepsins. With the use of the
EGEE grid infrastructure for the virtual screening campaign against the malaria
causing parasite P. falciparum we have demonstrated that resource sharing on an
eScience infrastructure such as EGEE provides a new model for doing
collaborative research to fight diseases of the poor. Novel and potent inhibitors of Plasmodium falciparum plasmepsin II were
identified by post-processing the results of a docking screening with BEAR, a
recently reported procedure for the refinement and rescoring of docked ligands
in virtual screening. FRET substrate degradation assays performed on the 30 most
promising compounds resulted in 26 inhibitors with IC(50) values ranging from
4.3 nM to 1.8 microM.Herein we report the discovery of novel and potent
inhibitors of Plasmodium falciparum plasmepsin II using GRID computing
infrastructures. These compounds were identified by post-processing the results
of a large docking screen of commercially available compounds using an automated
procedure based on molecular dynamics refinement and binding free-energy
estimation using MM-PBSA and MM-GBSA. Among the best-scored compounds, four
highly populated and promising chemical classes were identified:
N-alkoxyamidines, guanidines, amides, and ureas and thioureas. Thirty hit
compounds representative of each class were selected on the basis of their
favourable binding free energies and molecular interactions with key active site
residues. These were experimentally validated using an inhibition assay based on
FRET substrate degradation. Remarkably, 26 of the 30 tested compounds proved to
be active as plasmepsin II inhibitors, with IC(50) values ranging from 4.3 nM to
1.8 microM. Plasmepsins (PMs) are essential proteases of the plasmodia parasites and are
therefore promising targets for developing drugs against malaria. We have
discovered six inhibitors of PM II by high-throughput fragment-based docking of
a diversity set of approximately 40,000 molecules, and consensus scoring with
force field energy functions. Using the common scaffold of the three most active
inhibitors (IC(50)=2-5 microM), another seven inhibitors were identified by
substructure search. Furthermore, these 13 inhibitors belong to at least three
different classes of compounds. The in silico approach was very effective since
a total of 13 active compounds were discovered by testing only 59 molecules in
an enzymatic assay. This hit rate is about one to two orders of magnitude higher
than those reported for medium- and high-throughput screening techniques in
vitro. Interestingly, one of the inhibitors identified by docking was
halofantrine, an antimalarial drug of unknown mechanism. Explicit water
molecular dynamics simulations were used to discriminate between two putative
binding modes of halofantrine in PM II. To invade its definitive host, the mosquito, the malaria parasite must cross the
midgut peritrophic matrix that is composed of chitin cross-linked by
chitin-binding proteins and then develop into an oocyst on the midgut basal
lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase
is important in midgut invasion. The mechanistic role of other ookinete-secreted
enzymes in midgut invasion has not been previously examined. De novo mass
spectrometry sequencing of a protein obtained by benzamidine affinity column of
Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the
presence of an ookinete-secreted plasmepsin, an aspartic protease previously
only known to be present in the digestive vacuole of asexual stage malaria
parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4,
was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P.
falciparum chitinase PfCHT1) are both localized on the ookinete apical surface,
and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic
and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies
significantly reduced parasite infectivity for mosquitoes. These results suggest
that plasmepsin 4, previously known only to function in the digestive vacuole of
asexual blood stage Plasmodium, plays a role in how the ookinete interacts with
the mosquito midgut interactions as it becomes an oocyst. These data are the
first to delineate a role for an aspartic protease in mediating Plasmodium
invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a
malaria transmission-blocking vaccine target. Malaria causes a worldwide annual mortality of about a million people. Rapidly
evolving drug-resistant species of the parasite have created a pressing need for
the identification of new drug targets and vaccine candidates. By developing
fractionation protocols to enrich parasites from low-parasitemia patient
samples, we have carried out the first ever proteomics analysis of clinical
isolates of early stages of Plasmodium falciparum (Pf) and P. vivax.
Patient-derived malarial parasites were directly processed and analyzed using
shotgun proteomics approach using high-sensitivity MS for protein
identification. Our study revealed about 100 parasite-coded gene products that
included many known drug targets such as Pf hypoxanthine guanine phosphoribosyl
transferase, Pf L-lactate dehydrogenase, and Plasmepsins. In addition, our study
reports the expression of several parasite proteins in clinical ring stages that
have never been reported in the ring stages of the laboratory-cultivated
parasite strain. This proof-of-principle study represents a noteworthy step
forward in our understanding of pathways elaborated by the parasite within the
malaria patient and will pave the way towards identification of new drug and
vaccine targets that can aid malaria therapy. Plasmodium vivax malaria is a globally widespread disease responsible for 50% of
human malaria cases in Central and South America, South East Asia and Indian
subcontinent. The rising severity of the disease and emerging resistance of the
parasite has emphasized the need for the search of novel therapeutic targets to
combat P. vivax malaria. Plasmepsin 4 (PM4) a food vacuole aspartic protease is
essential in parasite functions and viability such as initiating hemoglobin
digestion and processing of proteins and is being looked upon as potential drug
target. Although the plasmepsins of Plasmodium falciparum have been extensively
studied, the plasmepsins of P. vivax are not well characterized. This is the
first report detailing complete PM4 gene analysis from Indian P. vivax isolates.
Blast results of sequences of P. vivax plasmepsin 4 (PvPM4) shows 100% homology
among isolates of P. vivax collected from different geographical regions of
India. All of the seven Indian isolates did not contain intron within the coding
region. Interestingly, PvPM4 sequence analysis showed a very high degree of
homology with all other sequences of Plasmodium species available in the
genebank. Our results strongly suggest that PvPM4 are highly conserved except a
small number of amino acid substitutions that did not modify key motifs at
active site formation for the function or the structure of the enzymes.
Furthermore, our study shows that PvPM4 occupies unique phylogenetic status
within Plasmodium group and sufficiently differ from the most closely related
human aspartic protease, cathepsin D. The analysis of 3D model of PM4 showed a
typical aspartic protease structure with bi-lobed, compact and distinct peptide
binding cleft in both P. vivax and P. falciparum. In order to validate
appropriate use of PM4 as potential anti-malarial drug target, studies on
genetic and structural variations among P. vivax plasmepsins (PvPMs) from
different geographical regions are of utmost importance for drugs and vaccine
designs for anti-malarial strategies. There is a high demand for new drugs against malaria, which takes millions of
lives annually. The abuse of classical antimalarials from the late 1940's to the
early 1980's has bred resistant parasites, which led to the use of more potent
drugs that ended up by refueling the resistance cycle. An example is
chloroquine, once highly effective but now virtually useless against malaria.
Structure-based rational drug design relies on high-resolution target structures
to allow for screening of selective ligands/inhibitors. For the past two
decades, and especially after the unveiling of the Plasmodium falciparum genome
in 2002, enzymes of this lethal malaria parasite species have been increasingly
attracting the attention of Medicinal Chemists worldwide as promising drug
targets. There is particular emphasis on proteases having key roles on the
degradation of host's hemoglobin within the food vacuole of blood-stage
parasites, as these depend on such process for their survival. Among such
enzymes, Plasmepsins (aspartic proteases) and, especially, Falcipains (cysteine
proteases) are highly promising antimalarial drug targets. The present review
will focus on the computational approaches made so far towards the unraveling of
the structure, function and inhibition of Falcipains that, by virtue of their
quite specific features, are excellent targets for highly selective inhibitors. The development of efficient and selective antimalariais remains a challenge for
the pharmaceutical industry. The aspartic proteases plasmepsins, whose
inhibition leads to parasite death, are classified as targets for the design of
potent drugs. Combinatorial synthesis is currently being used to generate
inhibitor libraries for these enzymes, and together with computational
methodologies have been demonstrated capable for the selection of lead
compounds. The high structural flexibility of plasmepsins, revealed by their
X-ray structures and molecular dynamics simulations, made even more complicated
the prediction of putative binding modes, and therefore, the use of common
computational tools, like docking and free-energy calculations. In this review,
we revised the computational strategies utilized so far, for the
structure-function relationship studies concerning the plasmepsin family, with
special focus on the recent advances in the improvement of the linear
interaction estimation (LIE) method, which is one of the most successful
methodologies in the evaluation of plasmepsin-inhibitor binding affinity. Plasmepsin II (PM II) is an attractive target for anti-malaria drug discovery,
which involves in host hemoglobin degradation in the acidic food vacuole. In
this study, we demonstrated the successful use of structure-based virtual
screening to identify inhibitors of PM II from two chemical database. Five novel
non-peptide inhibitors were identified and revealed moderate inhibitory
potencies with IC50 ranged from 4.62 ± 0.39 to 9.47 ± 0.71 μM. The detailed
analysis of binding modes using docking simulations for five inhibitors showed
that the inhibitors could be stabilized by forming multiple hydrogen bonds with
catalytic residues (Asp 34 and Asp 214) and also with other key residues. Malaria is one of the most devastating infectious diseases in the developing
world. Until now, only one candidate malaria vaccine RTS,S/AS01 has shown modest
protection in phase 3 trial in African infants. Hence the treatment of malaria
still depends on the current chemotherapeutic drugs. Considering the resistance
of malaria parasites to almost all used antimalarial drugs, aiming at
multi-targets rather than a single target will be a more promising strategy.
Previous studies have shown that myricetin and fisetin exhibited in vitro
antimalarial activity against Plasmodium falciparum, but very little research
focused on the molecular mechanism for their parasiticidal activity. The
cysteine protease falcipain-2 and aspartic protease plasmepsin II have long been
considered as important antimalarial drug targets, especially combined
inhibition of these two proteases. In this study, we determined that myricetin
and fisetin are dual inhibitors of falcipain-2 and plasmepsin II, which might
account for their antimalarial properties. Overall, the dual inhibition of
falcipain-2 and plasmepsin II by myricetin and fisetin has shed light on a
possible mechanism for their antimalarial activity and provided a rationale for
further development as antimalarial drugs. To screen the active antimalarial novel artemisinin derivatives, a QSAR modeling
approach was used. QSAR model showed high correlation (r(2)= 0.83 and rCV(2)=
0.81) and indicated that Connectivity Index (order 1, standard), Connectivity
Index (order 2, standard), Dipole Moment (debye), Dipole Vector X (debye) and
LUMO Energy (eV) well correlate with activity. High binding likeness on
antimalarial target plasmepsin was detected through molecular docking. Active
artemisinin derivatives showed significant activity and indicated compliance
with standard parameters of oral bioavailability and ADMET. The active
artemisinin derivatives namely, β-Artecyclopropylmether HMCP (A3), β-
Artepipernoylether (PIP-1) (A4) and 9-(β-Dihydroartemisinoxy)methyl anthracene
(A5) were semi-synthesized and characterized based on its (1)H and (13)C NMR
spectroscopic data and later activity tested in vivo on mice infected with
multidrug resistant strain of P. yoelii nigeriensis. Predicted results were
successfully validated by in vivo experiments. The malaria parasite Plasmodium falciparum exports several hundred proteins into
the infected erythrocyte that are involved in cellular remodeling and severe
virulence. The export mechanism involves the Plasmodium export element (PEXEL),
which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV
gene is refractory to deletion, suggesting it is essential, but definitive proof
is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks
the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment
of PMV activity in P. falciparum revealed PEXEL cleavage occurs
cotranslationaly, similar to signal peptidase. Treatment of P.
falciparum-infected erythrocytes with the inhibitor caused dose-dependent
inhibition of PEXEL processing as well as protein export, including impaired
display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and
cytoadherence. The inhibitor killed parasites at the trophozoite stage and
knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of
PMV increased resistance. This provides the first direct evidence that PMV
activity is essential for protein export in Plasmodium spp. and for parasite
survival in human erythrocytes and validates PMV as an antimalarial drug target. Plasmepsins (Plms) are aspartic proteases involved in the degradation of human
hemoglobin by Plasmodium falciparum. Given that the parasite needs the resulting
amino acid building blocks for its growth and development, plasmepsins are an
important antimalarial drug target. Over the past decade, tremendous progress
has been achieved in the development of inhibitors of plasmepsin using two
strategies: structure-based drug design (SBDD) and structure-based virtual
screening (SBVS). Herein, we review the inhibitors of Plms I-IV developed by
SBDD or SBVS with a particular focus on obtaining selectivity versus the human
Asp proteases cathepsins and renin and activity in cell-based assays. By use of
SBDD, the flap pocket of Plm II has been discovered and constitutes a convenient
handle to obtain selectivity. In SBVS, activity against Plms I-IV and
selectivity versus cathepsins are not always taken into account. A combination
of SBVS, SBDD, and molecular dynamics simulations opens up opportunities for
future design cycles. Malaria is one of the major parasitic disease whose rapid spreading and
mortality rate affects all parts of the world especially several parts of Asia
as well as Africa. The emergence of multi-drug resistant strains hamper the
progress of current antimalarial therapy and displayed an urgent need for new
antimalarials by targeting novel drug targets. Until now, several promising
targets were explored in order to develop a promising Achilles hill to counter
malaria. Plasmepsin, an aspartic protease, which is involved in the hemoglobin
breakdown into smaller peptides emerged as a crucial target to develop new
chemical entities to counter malaria. Due to early crystallographic evidence,
plasmepsin II (Plm II) emerged as well explored target to develop novel
antimalarials as well as a starting point to develop inhibitors targeting some
other subtypes of plasmepsins i.e. Plm I, II, IV and V. With the advancements in
drug discovery, several computational and synthetic approaches were employed in
order to develop novel inhibitors targeting Plm II. Strategies such as fragment
based drug design, molecular dynamics simulation, double drug approach etc. were
employed in order to develop new chemical entities targeting Plm II. But
majority of Plm II inhibitors suffered from poor selectivity over cathepsin D as
well as other subtypes of plasmepsins. This review highlights an updated account
of drug discovery efforts targeting plasmepsin II from a medicinal chemistry
perspective. Malaria is an infectious disease caused by Plasmodium parasites. It results in
an annual death-toll of ~ 600,000. Resistance to all medications currently in
use exists, and novel antimalarial drugs are urgently needed. Plasmepsin V (PmV)
is an essential Plasmodium protease and a highly promising antimalarial target,
which still lacks molecular characterization and drug-like inhibitors. PmV,
cleaving the PExEl motif, is the key enzyme for PExEl-secretion, an
indispensable parasitic process for virulence and infection. Here, we describe
the accessibility of PmV catalytic pockets to inhibitors and propose a novel
strategy for PmV inhibition. We also provide molecular and structural data
suitable for future drug development. Using high-throughput platforms, we
identified a novel scaffold that interferes with PmV in-vitro at picomolar
ranges (~ 1,000-fold more active than available compounds). Via systematic
replacement of P and P' regions, we assayed the physico-chemical requirements
for PmV inhibition, achieving an unprecedented IC50 of ~20 pM. The
hydroxyethylamine moiety, the hydrogen acceptor group in P2', the lipophilic
groups upstream to P3, the arginine and other possible substitutions in position
P3 proved to be critically important elements in achieving potent inhibition.
In-silico analyses provided essential QSAR information and model validation. Our
inhibitors act 'on-target', confirmed by cellular interference of PmV function
and biochemical interaction with inhibitors. Our inhibitors are poorly
performing against parasite growth, possibly due to poor stability of their
peptidic component and trans-membrane permeability. The lowest IC50 for parasite
growth inhibition was ~ 15 μM. Analysis of inhibitor internalization revealed
important pharmacokinetic features for PExEl-based molecules. Our work disclosed
novel pursuable drug design strategies for highly efficient PmV inhibition
highlighting novel molecular elements necessary for picomolar activity against
PmV. All the presented data are discussed in respect to human aspartic proteases
and previously reported inhibitors, highlighting differences and proposing new
strategies for drug development. Author information:
(1)Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy and
Nutrition, University of Navarra, Pamplona, 31008, Spain; Institute of Tropical
Health (ISTUN), University of Navarra, Pamplona, 31008, Spain.
(2)Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy and
Nutrition, University of Navarra, Pamplona, 31008, Spain.
(3)Department of Chemistry, Vanderbilt University, Station B 351822, Nashville,
TN 37235, USA.
(4)Grupo Malaria, Universidad de Antioquía, Medellín, Colombia.
(5)Department of Biochemistry and Molecular Biology, University of Florida,
Gainesville, FL, USA.
(6)Laboratoire de Synthèse et Physicochimie de Molécules d'Intérêt Biologique
SPCMIB - UMR5068, CNRS - Université Paul Sabatier, 118, route de Narbonne,
31062, Toulouse Cedex 09, France.
(7)Department of Pharmacology and Toxicology, Faculty of Pharmacy and Nutrition,
University of Navarra, Pamplona, 31008, Spain.
(8)Universidad Nacional de Colombia, Sede Bogotá, Facultad de Ciencias,
Departamento de Farmacia (DFUNC), Grupo de investigación FaMeTra (Farmacología
de la Medicina tradicional y popular), Carrera 30 45-03, Bogotá D.C., Colombia.
(9)UMR 152 PHARMA-DEV, Université Toulouse, IRD, UPS, 31062, Toulouse, France.
(10)Department of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy and
Nutrition, University of Navarra, Pamplona, 31008, Spain; Institute of Tropical
Health (ISTUN), University of Navarra, Pamplona, 31008, Spain. Electronic
address: [email protected]. |
What is MRSA? | community-associated methicillin resistant staphylococcus aureus (ca-mrsa) has become a severe health concern because of its treatment difficulties. | We investigated the distribution of MRSA (methicillin-resistant Staphylococcus
aureus) on and around six patients with MRSA infection in our neurosurgical
ward. All patients had a disturbance of consciousness and had sputum
colonization of MRSA. Samples were obtained from 11 sites (patients' hands,
attendances' hands, floors, sidetables, bedclothes, chairs, walls, curtains,
door knobs, faucets and disposable gloves) in the patients' rooms by the wiping
method. High counts of MRSA were detected on horizontal planes such as floors,
sidetables and chairs, but MRSA was not detected on vertical planes such as
curtains and walls. The reason why MRSA was detected on the horizontal planes
was due to a fall of MRSA spread from sputum in the air. These findings indicate
that the disinfection of horizontal planes is important for preventing the
spread of MRSA. We also evaluated what disinfectant was useful for floor
disinfection and concluded that 0.5% chlorhexidine digluconate (Hibitane) and
0.5% benzalkonium chloride (Osvan) were more effective than the other
usually-used disinfectants such as alkyldiaminoethyl glycine (Tego-51). The susceptibility to arbekacin (ABK) of methicillin-resistant Staphylococcus
aureus (MRSA) was investigated to find out how it related to aac(6')/aph(2")
gene. In 49 isolates of MRSA for which MIC of ABK ranged from 0.125 to 64
micrograms/ml, the MICs of ABK for 38 strains carrying aac(6')/aph(2") gene were
widely distributed from 0.25 to 64, whereas those for 11 strains without that
gene were all < or = 0.5 microgram/ml. Residual rate of ABK activity was higher
than that of gentamicin after the reaction with each crude enzyme preparation
extracted from 3 isolates of MRSA, carrying aac(6')/aph(2") and aad(4',4")
genes. Furthermore, 97 strains of MRSA isolated at Kanagawa prefecture in Japan
in 1999 were all sensitive to ABK, although 28 strains of them carried
aac(6')/aph(2") gene. These results showed that ABK resistance was not
necessarily related to carrying aac(6')/aph(2") gene in clinical isolates of
MRSA. BACKGROUND: Asymptomatic colonization with methicillin-resistant Staphylococcus
aureus (MRSA) has been described as a risk factor for subsequent MRSA infection.
MRSA is an important nosocomial pathogen but has currently been reported in
patients without typical risk factors for nosocomial acquisition. This study was
designed to evaluate the impact of asymptomatic nares MRSA colonization on the
development of subsequent MRSA infection. The incidence of MRSA infection was
examined in patients with and patients without MRSA or methicillin-susceptible
S. aureus (MSSA) colonization at admission to the hospital and in those who
developed colonization during hospitalization.
METHODS: Patients admitted to 5 representative hospital units were prospectively
evaluated. Nares samples were obtained for culture at admission and during
hospitalization. Laboratory culture results were monitored to identify all MRSA
infections that occurred during the study period and 1 year thereafter.
RESULTS: Of the 758 patients who had cultures of nares samples performed at
admission, 3.4% were colonized with MRSA, and 21% were colonized with MSSA. A
total of 19% of patients with MRSA colonization at admission and 25% who
acquired MRSA colonization during hospitalization developed infection with MRSA,
compared with 1.5% and 2.0% of patients colonized with MSSA (P<.01) and
uncolonized (P<.01), respectively, at admission. MRSA colonization at admission
increased the risk of subsequent MRSA infection, compared with MSSA colonization
(relative risk [RR], 13; 95% confidence interval [CI], 2.7-64) or no
staphylococcal colonization (RR, 9.5; 95% CI, 3.6-25) at admission. Acquisition
of MRSA colonization also increased the risk for subsequent MRSA infection,
compared with no acquisition (RR, 12; 95% CI, 4.0-38).
CONCLUSION: MRSA colonization of nares, either present at admission to the
hospital or acquired during hospitalization, increases the risk for MRSA
infection. Identifying MRSA colonization at admission could target a high-risk
population that may benefit from interventions to decrease the risk for
subsequent MRSA infection. This article explains what methicillin-resistant Staphylococcus aureus (MRSA)
is, how it is spread and what the real challenges are in healthcare settings in
the UK. It explores the different strains of MRSA and points out the main ways
to control their spread. It is intended to be a reference source for all nurses. Screening specimens were homogenised in saline 0.9% w/v before either direct
inoculation or following enrichment in broth on three chromogenic media
(MRSA-ID, CHROMagar MRSA and MRSA Select) and ORSAB medium for the detection of
methicillin-resistant Staphylococcus aureus (MRSA). In total, 102 of 466
specimens yielded MRSA on at least one medium. After incubation for 16-18 h, the
sensitivity was 51%, 59%, 47% and 65% on MRSA-ID, CHROMagar MRSA, ORSAB and MRSA
Select, respectively, compared with 82%, 75%, 67% and 80%, respectively, after
42 h, and 93%, 95%, 79% and not tested, respectively, following broth
enrichment. There were significantly more MRSA colonies on MRSA-Select after
16-18 h than on ORSAB or MRSA ID (p 0.001 and 0.0022, respectively), whereas
there were more MRSA colonies after 42 h on MRSA-ID and MRSA-Select than on
ORSAB (p 0.0004 and 0.012, respectively). The specificity of the media for
identifying MRSA based on the colour of colonies after incubation for 16-18 h
was 100%, 99%, 99% and 100%, respectively, compared with 98%, 97%, 98% and 98%,
respectively, after 42 h, and 100%, 99%, 100% and not tested, respectively,
following broth enrichment. The speed of detection (mean time to report a
positive result) was 1.65, 1.72, 2.31 and 1.35 days, respectively. For each of
the three media tested following enrichment, the use of an enrichment broth
increased the detection rate of MRSA by 16-24%. OBJECTIVE: To determine the appropriate method to calculate the rate of
methicillin-resistant Staphylococcus aureus (MRSA) infection and colonization
(hereafter, MRSA rates) for interhospital comparisons, such that the large
number of patients who are already MRSA positive on admission is taken into
account.
DESIGN: A prospective, multicenter, hospital-based surveillance of MRSA-positive
case patients from January through December 2004.
SETTING: Data from 31 hospitals participating in the German national nosocomial
infections surveillance system (KISS) were recorded during routine surveillance
by the infection control team at each hospital.
RESULTS: Data for 4,215 MRSA-positive case patients were evaluated. From this
data, the following values were calculated. The median incidence density was
0.71 MRSA-positive case patients per 1,000 patient-days, and the median
nosocomial incidence density was 0.27 patients with nosocomial MRSA infection or
colonization per 1,000 patient-days (95% CI, 0.18-0.34). The median average
daily MRSA burden was 1.13 MRSA patient-days per 100 patient-days (95% CI,
0.86-1.51), with the average daily MRSA burden defined as the total number of
MRSA patient-days divided by the total number of patient-days times 100. The
median MRSA-days-associated nosocomial MRSA infection and colonization rate,
which describes the MRSA infection risk for other patients in hospitals housing
large numbers of MRSA-positive patients and/or many patients who were MRSA
positive on admission, was 23.1 cases of nosocomial MRSA infection and
colonization per 1,000 MRSA patient-days (95% CI, 17.4-28.6). The values were
also calculated for various MRSA screening levels.
CONCLUSIONS: The MRSA-days-associated nosocomial MRSA rate allows investigators
to assess the extent of MRSA colonization and infection at each hospital, taking
into account cases that have been imported from other hospitals, as well as from
the community. This information provides an appropriate incentive for hospitals
to introduce further infection control measures. Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA)
carrying pvl is an emerging problem worldwide. CA-MRSA tends to harbor
staphylococcal cassette chromosome mec type IV (SCCmec IV), to be
non-multiantibiotic resistant, and to have different genotypes from the local
hospital-acquired MRSA (HA-MRSA). However, in Ireland, 80% of HA-MRSA isolates
have the non-multiantibiotic-resistant genotype ST22-MRSA-IV. This study
investigated MRSA isolates from Ireland (CA-MRSA, health care-associated MRSA,
and HA-MRSA) for the carriage of pvl and determined the genotypic
characteristics of all pvl-positive isolates identified. All 1,389 MRSA isolates
were investigated by antibiogram-resistogram typing and SmaI DNA
macrorestriction analysis. pvl-positive isolates were further characterized by
multilocus sequence typing and SCCmec, agr, and toxin gene typing. Twenty-five
(1.8%) MRSA isolates belonging to six genotypes (ST30, ST8, ST22, ST80, ST5, and
ST154) harbored pvl. Nineteen of these (76%) were CA-MRSA isolates, but a
prospective study of MRSA isolates from 401 patients showed that only 6.7%
(2/30) of patients with CA-MRSA yielded pvl-positive isolates. Thus, pvl cannot
be used as a sole marker for CA-MRSA. Fifty-two percent of pvl-positive MRSA
isolates were recovered from patients with skin and soft tissue infections;
thirty-six percent were from patients of non-Irish ethnic origin, reflecting the
increasing heterogeneity of the Irish population due to immigration. All 25
pvl-positive isolates carried SCCmec IV; 14 (56%) harbored SCCmec IV.1 or IV.3,
and the remaining 11 isolates could not be subtyped. This study demonstrates
that pvl is not a reliable marker for CA-MRSA in Ireland and reveals the
emergence and importation of diverse genotypes of pvl-positive MRSA in Ireland. The aim of this study was to assess to what extent patients with
meticillin-resistant Staphylococcus aureus (MRSA) at respiratory sites shed
viable MRSA into the air of hospital rooms. We also evaluated whether the
distance from the patient could influence the level of contamination. Air
sampling was performed directly onto MRSA-selective agar in 24 hospital rooms
containing patients with MRSA colonization or infection of the respiratory
tract. Samplings were performed in duplicate at 0.5, 1 and 2-3 m from the
patients' heads. Clinical and environmental isolates were compared using
antimicrobial resistance patterns and pulsed-field gel electrophoresis. MRSA
strains were isolated from 21 out of 24 rooms, in quantities varying from
between 1 and 78 cfu/m3. In each of the 21 rooms, at least one of the
environmental isolates was identical to a clinical isolate from the patient in
that room. There was no significant difference in MRSA counts between the
distance from the patient's head and the sampler. This study demonstrates that
most patients with MRSA infection or colonisation of the respiratory tract shed
viable MRSA into the air of their room. The results emphasise the need to study
MRSA in air in more detail in order to improve infection control
recommendations. Detection of methicillin (meticillin)-resistant Staphylococcus aureus
colonization was assessed using combined nose and groin swabs in two commercial
PCR assays (the Xpert MRSA assay and the BD GeneOhm MRSA assay). Compared to
routine culture, both had similar sensitivities (87.0% versus 84.8%,
respectively) and specificities (93.8% versus 92.7%, respectively). Combined PCR
assays provide a rapid and more-complete assessment of colonization at a cost
similar to that of single-site analysis. The aim of the present study was to investigate the antibiotic susceptibility
patterns and molecular epidemiology of clinical methicillin-resistant
Staphylococcus aureus (MRSA) isolates recovered in 24 hospitals in 20 cities in
Croatia from October to December 2004. A total of 1815 consecutive S. aureus
isolates were recovered, 248 of which were MRSA. The MRSA isolates were analysed
using spa typing, multilocus sequence typing and SCCmec typing. Furthermore, the
presence of Panton-Valentine leukocidin (PVL) genes was determined as a genetic
marker for community-associated MRSA. The MRSA prevalence was 14%. Ninety-six
per cent of the MRSA isolates were resistant to ciprofloxacin, 95% to
clindamycin and azithromycin, 94% to gentamicin, and 93% to erythromycin. The
majority of the MRSA isolates (78%) was associated with the ST111-MRSA-I clone.
In addition, various other endemic MRSA clones were observed, such as the
ST247-MRSA-I (4%), the ST45-MRSA-IV (2%), the ST5-MRSA-I (2%), the
ST239-MRSA-III (2%), the ST5-MRSA-II (1%), the ST8-MRSA-IV (1%) and the
ST5-MRSA-IV (<1%) clones. Furthermore, we observed one PVL-negative ST80-MRSA-IV
isolate. Four PVL-positive MRSA isolates were found, associated with
ST8-MRSA-IV, ST80-MRSA-IV and ST80-MRSA-I. The ST111-MRSA-I clone was
predomit in Croatia. Future surveillance studies of MRSA are important to
elucidate whether changes in the clonal distribution of MRSA will occur, and if
the minor endemic MRSA clones observed in the present study will replace the
ST111-MRSA-I clone on a large scale. Rapid laboratory methods provide optimal support for active surveillance efforts
to screen for methicillin-resistant Staphylococcus aureus (MRSA). Most
laboratories struggle to determine the optimal use of resources, considering
options to balance cost, speed, and diagnostic accuracy. To assess the
performance of common methods, the first comparison of MRSASelect agar (MS) and
CHROMagar MRSA (CA), with and without broth enrichment followed by a 24-h
subculture to MS, was performed. Results were compared to those of the Xpert
MRSA assay. For direct culture methods, the agreement between MS and CA was
98.8%. At 18 h, direct MS identified 93% of all positive samples from direct
culture and 84% of those identified by the Xpert MRSA. For Trypticase soy
broth-enriched MS culture, incubated overnight and then subcultured for an
additional 24 h, the agreement with Xpert MRSA was 96%. The agreement between
direct MS and Xpert MRSA was 100% when semiquantitative culture revealed a
bacterial density of 2+ or greater; however, discrepancies between culture and
Xpert MRSA arose for MRSA bacterial densities of 1+ or less, indicating low
density as a common cause of false-negative culture results. Since 1+ or less
was established as the most common MRSA carrier state, broth enrichment or PCR
may be critical for the identification of all MRSA carriers who may be
reservoirs for transmission. In this active-surveillance convenience sample, the
use of broth enrichment followed by subculture to MS offered a low-cost but
sensitive method for MRSA screening, with performance similar to that of Xpert
MRSA PCR. The efficacy of the BD GeneOhm methicillin-resistant Staphylococcus aureus
(MRSA) assay was assessed by analyzing nasal swabs and swabs from other body
sites for the presence of MRSA in a low-prevalence area. From 681 patients with
a high risk for MRSA carriage, 1,601 specimens were collected and transported in
Amies agar. After discordant analysis, the sensitivity, specificity, positive
predictive value, and negative predictive value of the BD GeneOhm MRSA assay
were 84.3%, 99.2%, 88.4%, and 98.9%, respectively, compared to culture. There are few more compelling questions in clinical microbiology today than the
issue of whether or not to screen for the presence of methicillin-resistant
Staphylococcus aureus (MRSA), with the results being used to institute infection
control interventions aimed at preventing transmission of MRSA in health care
environments. Numerous different matters must be addressed when considering a
screening program. Who is to be screened, what method is to be employed to
detect MRSA, and what sites should be sampled? When and how often should the
screening be performed? Who is going to pay for the screening, and, finally and
perhaps most importantly, how are screening results to be communicated to health
care providers and what kind of interventions are best undertaken based on the
results? Numerous governmental agencies have mandated MRSA screening programs,
and yet several authorities in infection control organizations have questioned
the appropriateness of mandated screening. In this Point-Counterpoint feature,
Dr. Lance Peterson of Evanston Hospital (Evanston, IL) offers his perspective on
why screening for MRSA is to be encouraged. Dr. Daniel Diekema of the University
of Iowa Carver College of Medicine (Iowa City, IA) offers an opposing view. OBJECTIVE: To evaluate the long-term impact of successive interventions on rates
of methicillin-resistant Staphylococcus aureus (MRSA) colonization or infection
and MRSA bacteremia in an endemic hospital-wide situation.
DESIGN: Quasi-experimental, interrupted time-series analysis. The impact of the
interventions was analyzed by use of segmented regression. Representative MRSA
isolates were typed by use of pulsed-field gel electrophoresis.
SETTING: A 950-bed teaching hospital in Seville, Spain.
PATIENTS: All patients admitted to the hospital during the period from 1995
through 2008.
METHODS: Three successive interventions were studied: (1) contact precautions,
with no active surveillance for MRSA; (2) targeted active surveillance for MRSA
in patients and healthcare workers in specific wards, prioritized according to
clinical epidemiology data; and (3) targeted active surveillance for MRSA in
patients admitted from other medical centers.
RESULTS: Neither the preintervention rate of MRSA colonization or infection
(0.56 cases per 1,000 patient-days [95% confidence interval {CI}, 0.49-0.62
cases per 1,000 patient-days]) nor the slope for the rate of MRSA colonization
or infection changed significantly after the first intervention. The rate
decreased significantly to 0.28 cases per 1,000 patient-days (95% CI, 0.17-0.40
cases per 1,000 patient-days) after the second intervention and to 0.07 cases
per 1,000 patient-days (95% CI, 0.06-0.08 cases per 1,000 patient-days) after
the third intervention, and the rate remained at a similar level for 8 years.
The MRSA bacteremia rate decreased by 80%, whereas the rate of bacteremia due to
methicillin-susceptible S. aureus did not change. Eighty-three percent of the
MRSA isolates identified were clonally related. All MRSA isolates obtained from
healthcare workers were clonally related to those recovered from patients who
were in their care.
CONCLUSION: Our data indicate that long-term control of endemic MRSA is feasible
in tertiary care centers. The use of targeted active surveillance for MRSA in
patients and healthcare workers in specific wards (identified by means of
analysis of clinical epidemiology data) and the use of decolonization were key
to the success of the program. Universal surveillance upon patient admission is important in reducing the
transmission of methicillin-resistant Staphylococcus aureus (MRSA) and
associated disease in hospitals. High costs for the health care system in
conjunction with MRSA have promoted the development of rapid screening methods
to detect MRSA carriers. This study compared two real-time PCR methods, the BD
GeneOhm MRSA assay (BDGO) and the Xpert MRSA assay, with broth-enriched culture
to define their performance characteristics and rapidity in an area with low
MRSA prevalence. In total, 414 swabs from the nose and 389 swabs from the groin
from 425 patients were tested. Of those 425 patients, 378 had swabs from both
the nose and groin in parallel. Two hundred thirty-one and 194 patients were
randomly assigned to the BDGO group and the Xpert MRSA group, respectively. In
general, sensitivity, specificity, and negative predictive value (NPV) were high
for the BDGO (100%, 98.5%, and 100%, respectively) and the Xpert MRSA (100%,
98.2%, and 100%, respectively), irrespective of whether or not nasal and
inguinal specimens were considered alone or combined. In contrast, the positive
predictive value (PPV) was lower: before the resolution of discrepant results,
the PPVs for nasal and inguinal specimens alone and combined were 87.5%, 86.7%,
and 82.4% for the BDGO and 91.7%, 66.7%, and 92.9% for the Xpert MRSA,
respectively. After the resolution of discrepant results, PPVs were 93.8%, 93.3%
and 94.1% for the BDGO and 91.7%, 88.9% and 92.9% for the Xpert MRSA,
respectively. With the BDGO, 4 of 16 carriers were each identified by nasal or
inguinal swabs alone, whereas in the Xpert MRSA group, 4 of 13 carriers were
exclusively identified by nasal swabs and 2 of 13 were identified by inguinal
swabs alone. Both PCR methods showed no significant difference in the number of
discrepant results (odds ratio, 0.70 [P = 0.789]), but specimens from wounds and
other body sites (axilla, vagina, and throat) produced discrepancies more often
than nasal and groin specimens (odds ratios, 4.724 [P = 0.058] and 12.163 [P <
0.001], respectively). The facts that no false-negative PCR results were
detected and increased PPVs were found after the resolution of discrepant
results point to PCR as the actual gold standard. Since both sensitivity and NPV
were exceptionally high for PCR, backup cultures may, therefore, be unnecessary
in an area with low prevalence and with a preemptive isolation strategy but may
still be useful for PCR-positive specimens because of the lower PPV for both
methods and the possibility of susceptibility testing. The median time for
analysis, including extraction, hands-on time, and actual PCR was 2 h 20 min for
the Xpert MRSA versus 5 h 40 min for the BDGO. Concerning reporting time,
including administration and specimen collection, the Xpert MRSA was faster than
the BDGO (7 h 50 min versus 17 h). Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging threat to
public health, especially in correctional settings. Outbreaks have been seen in
jails and prisons in Mississippi, California, Texas, and Georgia in recent
years. Also, many correctional settings have seen an increase in MRSA infection
greater than in the general population. This article examines the lessons that
have been learned about MRSA in correctional settings and ponders what is yet to
be learned about this disease in these populations. PURPOSE: To characterize the patient demographics, clinical features, and
antibiotic susceptibility of ocular infections caused by methicillin-resistant
Staphylococcus aureus (MRSA), including community-associated (CA) and
healthcare-associated (HA) isolates.
DESIGN: Retrospective, observational study.
PARTICIPANTS: Patients (n = 519) with culture-proven S. aureus ocular infections
seen between January 1, 1999, and December 31, 2008, in Chang Gung Memorial
Hospital.
METHODS: Data collected included patient demographics and clinical information.
Antibiotic susceptibility was verified by disc diffusion method.
MAIN OUTCOME MEASURES: Proportion of MRSA in S. aureus ocular infections and the
clinical characteristics, diagnoses, and antibiotic susceptibility patterns of
CA-MRSA versus HA-MRSA ocular infections.
RESULTS: We identified 274 patients with MRSA ocular infections, which comprised
181 CA-MRSA and 93 HA-MRSA isolates. The average rate of MRSA in S. aureus
infections was 52.8% with a stable trend, whereas the annual ratio of CA-MRSA in
ocular MRSA infections averaged 66.1% and tended to increase over the 10-year
interval. Patients with ocular CA-MRSA were younger. Lid and lacrimal system
disorders were more common, but keratitis, endophthalmitis, and wound infection
were less common among CA-MRSA cases than HA-MRSA cases. Both CA-MRSA and
HA-MRSA isolates were resistant to clindamycin and erythromycin, but CA-MRSA was
more susceptible to sulfamethoxazole/trimethoprim.
CONCLUSIONS: Community-associated MRSA is an important pathogen of ocular
infections; CA-MRSA and HA-MRSA ocular infections differ demographically and
clinically, but both strains were multi-resistant in Chang Gung Memorial
Hospital, one of the biggest referral centers in Taiwan. In a country with a
high prevalence of MRSA, ophthalmologists should be aware of such epidemiologic
information. BACKGROUND: Community-associated methicillin-resistant Staphylococcus
aureus-(CA-MRSA) strains have emerged in Argentina. We investigated the clinical
and molecular evolution of community-onset MRSA infections (CO-MRSA) in children
of Córdoba, Argentina, 2005-2008. Additionally, data from 2007 were compared
with the epidemiology of these infections in other regions of the country.
METHODOLOGY/PRINCIPAL FINDINGS: Two datasets were used: i) lab-based prospective
surveillance of CA-MRSA isolates from 3 Córdoba pediatric hospitals-(CBAH1-H3)
in 2007-2008 (compared to previously published data of 2005) and ii) a sampling
of CO-MRSA from a study involving both, healthcare-associated
community-onset-(HACO) infections in children with risk-factors for
healthcare-associated infections-(HRFs), and CA-MRSA infections in patients
without HRFs detected in multiple centers of Argentina in 2007. Molecular typing
was performed on the CA-MRSA-(n: 99) isolates from the CBAH1-H3-dataset and on
the HACO-MRSA-(n: 51) and CA-MRSA-(n: 213) isolates from other regions. Between
2005-2008, the annual proportion of CA-MRSA/CA-S. aureus in Córdoba hospitals
increased from 25% to 49%, P<0.01. Total CA-MRSA infections increased 3.6
fold-(5.1 to 18.6 cases/100,000 annual-visits, P<0.0001), associated with an
important increase of invasive CA-MRSA infections-(8.5 fold). In all regions
analyzed, a single genotype prevailed in both CA-MRSA (82%) and HACO-MRSA(57%),
which showed pulsed-field-gel electrophoresis-(PFGE)-type-"I",
sequence-type-5-(ST5), SCCmec-type-IVa, spa-t311, and was positive for PVL. The
second clone, pulsotype-N/ST30/CC30/SCCmecIVc/t019/PVL(+), accounted for 11.5%
of total CA-MRSA infections. Importantly, the first 4 isolates of Argentina
belonging to South American-USA300
clone-(USA300/ST8/CC8/SCCmecIVc/t008/PVL(+)/ACME(-)) were detected. We also
demonstrated that a HA-MRSA clone-(pulsotype-C/ST100/CC5) caused 2% and 10% of
CA-MRSA and HACO-MRSA infections respectively and was associated with a SCCmec
type closely related to SCCmecIV(2B&5).
CONCLUSIONS/SIGNIFICANCE: The dissemination of epidemic MRSA clone,
ST5-IV-PVL(+) was the main cause of increasing staphylococcal community-onset
infections in Argentinean children (2003-2008), conversely to other countries.
The predomice of this clone, which has capacity to express the h-VISA
phenotype, in healthcare-associated community-onset cases suggests that it has
infiltrated into hospital-settings. We evaluated the new, fully automated molecular BD Max methicillin-resistant
Staphylococcus aureus (MRSA) assay for detection of methicillin-resistant S.
aureus in a low-prevalence (4.1%) setting. Sensitivity, specificity, and
positive and negative predictive values were 93.9%, 99.2%, 83.8%, and 99.7%,
respectively. The assay reported fewer unresolved results than the BD GeneOhm
MRSA ACP assay. A diverse collection of 261 Staphylococcus aureus strains from human, animal,
food, and environmental sources were tested for the presence and type of SCCmec
elements, antibiotic susceptibility to various antibiotics, and non-ß-lactam
antibiotic resistance genes. About 18.39% (48/261) of strains were
methicillin-resistant S. aureus (MRSA) including 29.75% (36/121) human strains
of which 29 strains were hospital-acquired MRSA (HA-MRSA) and 7 strains were
community-associated MRSA (CA-MRSA) and 19.67% (12/61) animal strains that all
were CA-MRSA strains. The percentage of CA-MRSA strains from animals was
significantly higher than that from human (p<0.01). Most of MRSA strains and a
part of methicillin-susceptible S. aureus (MSSA) strains harbored unique
combinations of non-ß-lactamase genes aac(6')/aph(2″), aph(3')-III, ant (4',4″),
ermA, ermC, mrsA, tetM, and tetK. Antibiotic resistance genes were detected more
frequently in HA-MRSA strains than in CA-MRSA strains (p<0.01). MRSA strains and
MSSA strains had 22 and 39 antibiotic profiles to 15 tested antibiotics,
respectively. The resistant proportion was higher in HA-MRSA strains than in
CA-MSSA strains for various antibiotics, as well as higher in MRSA strains than
in MSSA strains. Animal MRSA reservoirs (particularly pigs and cows) might
represent an important source of human CA-MRSA. CA-MRSA strains might acquire
more different resistance genes gradually, depending on the selective pressure
of antibiotics in different regions or environments. CA-MRSA is not yet endemic
in China, but could be prevalent in future, contributing to its acquiring more
resistance genes and huge animal sources. Infection with multidrug-resistant
MSSA strains acquired from food, animal, and human sources might also become a
significant problem for human medicine, which warrants further study. OBJECTIVES: To compare the BD GeneOhm Methicillin Resistant Staphylococcus
aureus (MRSA) Achromopeptidase (ACP) polymerase chain reaction (PCR) assay with
the culture method for the detection of MRSA colonization.
METHODS: One hundred and two patients were admitted to the Intensive Care Unit
in King Khalid Hospital, Najran, Kingdom of Saudi Arabia from July 2010 to
February 2011. Separate swabs from the nose, axilla, and groin of each patient
were processed by the culture method (sheep blood agar plate and mannitol salt
agar plate) and BD GeneOhm MRSA ACP assay.
RESULTS: Of the 287 samples, 62 (21.6%) were MRSA positive by the PCR assay and
26 (9%) were MRSA positive by the culture method. The PCR method showed 88.4%
sensitivity and 98.6% negative predictive value. The number of MRSA-PCR positive
groin specimens was nearly the same as nasal specimens. The PCR method gave
positive results in 22.5% of patients by nasal specimens, 27.5% of patients by
nasal and groin specimens, and 30.4% of patients by nasal, groin, and axilla
specimens. The PCR method detected 30.4% of patients as MRSA positive while the
culture method detected 19.6% of patients as positive for MRSA.
CONCLUSION: The BD GeneOhm MRSA ACP assay has high sensitivity and NPV and hence
is a useful screening method to exclude patients who are not colonized with
MRSA. AIM: to determine the rate of MRSA-carrier among patients, family members and
health care providers, and the association between MRSA-carrier family members
and health care providers on MRSA infection patient after orthopaedic surgery.
METHODS: this is a cross-sectional analytical study. Samples were taken
consecutively during December 2010 to December 2011, consisting of postoperative
patients infected with MRSA, attending family members, and the medical officers
with history of contact with the patient. Swab culture were taken from nasal and
axilla of all subjects. The incidence of MRSA infection, and MRSA-carrier on the
patient, family members and medical officers were presented descriptively, while
their association with MRSA infection was statistically tested using Fischer
exact test.
RESULTS: during the study period, there were 759 surgeries, with 4 (0.5%)
patients were identified to have MRSA infection. Of these four cases, 48
subjects were enrolled. The rate of MRSA-carrier among patients, family and
health care providers were 50%, 25% and 0% respectively. There were no
significant association between MRSA and the rates of MRSA-carrier on the family
member or health care providers.
CONCLUSION: the incidence of MRSA infection, MRSA-carrier patient, MRSA-carrier
health care providers, and family member carrier were 0.5%, 50%, 0%, and 25%
respectively. No significant association found between MRSA-carrier on the
family member or health care providers and MRSA infection patient. There were no
MRSA infection found on the health care provider. OBJECTIVE: To study the clinical and molecular characteristics of
methicillin-resistant Staphylococcus aureus (MRSA) infection in children.
METHOD: A total of 37 MRSA strains were isolated from hospitalized patients in
Children's Hospital of Fudan University from March 2009 to November 2011. The
clinical characteristics were investigated by a cohort study. Furthermore, the
mecA, Panton-Valentine leucocidin (PVL) genes were detected by polymerase chain
reaction (PCR), and the genotypes of SCCmec were determined by multiplex PCR.
RESULT: (1) Among the 37 MRSA isolates, infections with 21 were acquired from
hospital (HA-MRSA), and 16 isolates were acquired from community (CA-MRSA). (2)
In the study, MRSA frequently caused respiratory tract infection, and most of
the strains were isolated from intensive care unit (ICU). (3) CA-MRSA was most
frequently associated with skin and soft tissue infections (SSTI), suppurative
tonsillitis, even pneumonia and septicemia. HA-MRSA infection was more
aggressive, most frequently associated with pneumonia, septicemia, and central
nervous system (CNS) infections, such as meningitis. In children with fever
caused by HA-MRSA or CA-MRSA infection, HA-MRSA showed a longer duration of
fever, for 10.5 days. C-reactive protein (CRP) level caused by HA-MRSA (63.00
mg/L) was higher than CA-MRSA (9.50 mg/L) , and there were statistically
significant differences between the groups (t = 2.5670, P < 0.05). However,
there were no statistically significant differences between the groups in white
blood cell count (WBC) or procalcitonin (PCT) level. (4) Among 37 MRSA isolates,
the whole isolates were mecA gene positive (100%). SCCmec genotyping results
showed that the most frequent SCCmec types were type III, 17 isolates, the
others including type IV 8 isolates, type II1 isolates, nontypable 11 isolates,
type I and type V were not found in this group. Therein, among 21 HA-MRSA
isolates, SCCmec III was the most common, 15 isolates, type IV 1 isolates,
nontypable 5 isolates; among 16 CA-MRSA isolates, SCCmec type IV was the most
common, 7 isolates, type III 2 isolates, type II 1 isolate, nontypable 6
isolates. (5) Among the 37 MRSA isolates, 28 were PVL gene positive; and among
21 HA-MRSA isolates, 17 were PVL gene positive; Among 16 CA-MRSA isolates, 11
were PVL gene positive; There were no statistically significant differences
between the groups (χ(2) = 0.735, P > 0.05) .
CONCLUSION: Compared with CA-MRSA, HA-MRSA infection was more aggressive, and
induced higher C reactive protein; the domit epidemic strains of CA-MRSA was
SCCmec type IV, and HA-MRSA was SCCmec type III; the positive rate of PVL gene
was high. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is
increasing in prevalence among asymptomatic carriers and in cases of paediatric
soft-tissue infections alike. CA-MRSA may express virulence factors such as
Panton-Valentine leukocidin, which makes soft-tissue and hard-tissue infections
due to such organisms challenging to treat. We report a case of osteomyelitis of
the proximal tibia in a 10-year-old boy and discuss its management in what is to
the authors' knowledge the first case report of Panton-Valentine
leukocidin-positive CA-MRSA osteomyelitis in a child in the UK. Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a major
challenge in health care, yet the limited heterogeneity within this group
hinders molecular investigations of related outbreaks. Pulsed-field gel
electrophoresis (PFGE) has been the gold standard approach but is impractical
for many clinical laboratories and is often replaced with PCR-based methods.
Regardless, both approaches can prove problematic for identifying subclonal
outbreaks. Here, we explore the use of whole-genome sequencing for clinical
laboratory investigations of MRSA molecular epidemiology. We examine the
relationships of 44 MRSA isolates collected over a period of 3 years by using
whole-genome sequencing and two PCR-based methods, multilocus variable-number
tandem-repeat analysis (MLVA) and spa typing. We find that MLVA offers higher
resolution than spa typing, as it resolved 17 versus 12 discrete isolate groups,
respectively. In contrast, whole-genome sequencing reproducibly cataloged
genomic variants (131,424 different single nucleotide polymorphisms and indels
across the strain collection) that uniquely identified each MRSA clone,
recapitulating those groups but enabling higher-resolution phylogenetic
inferences of the epidemiological relationships. Importantly, whole-genome
sequencing detected a significant number of variants, thereby distinguishing
between groups that were considered identical by both spa typing (minimum, 1,124
polymorphisms) and MLVA (minimum, 193 polymorphisms); this suggests that these
more conventional approaches can lead to false-positive identification of
outbreaks due to inappropriate grouping of genetically distinct strains. An
analysis of the distribution of variants across the MRSA genome reveals 47
mutational hot spots (comprising ∼ 2.5% of the genome) that account for 23.5% of
the observed polymorphisms, and the use of this selected data set successfully
recapitulates most epidemiological relationships in this pathogen group. It was found in the present study that combined use of fusidic acid (FA) and
berberine chloride (BBR) offered an in vitro synergistic action against 7 of the
30 clinical methicillin-resistant Staphylococcus aureus (MRSA) strains, with a
fractional inhibitory concentration (FIC) index ranging from 0.5 to 0.19. This
synergistic effect was most pronounced on MRSA 4806, an FA-resistant isolate,
with a minimum inhibitory concentration (MIC) value of 1,024 μg/ml. The
time-kill curve experiment showed that FA plus BBR yielded a 4.2 log10 c.f.u./ml
reduction in the number of MRSA 4806 bacteria after 24-h incubation as compared
with BBR alone. Viable count analysis showed that FA plus BBR produced a 3.0
log10 c.f.u./ml decrease in biofilm formation and a 1.5 log10 c.f.u./ml decrease
in mature biofilm in viable cell density as compared with BBR alone. In
addition, phase contrast micrographs confirmed that biofilm formation was
significantly inhibited and mature biofilm was obviously destructed when FA was
used in combination with BBR. These results provide evidence that combined use
of FA and BBR may prove to be a promising clinical therapeutic strategy against
MRSA. BACKGROUND: Prior studies published in the cardiothoracic, orthopedic and
gastrointestinal surgery have identified the importance of nasal
(methicillin-resistant Staphylococcus aureus) MRSA screening and subsequent
decolonization to reduce MRSA surgical site infection (SSI). This is the first
study to date correlating nasal MRSA colonization with postoperative spinal MRSA
SSI.
OBJECTIVE: To assess the significance of nasal MRSA colonization in the setting
of MRSA SSI.
METHODS: A retrospective electronic chart review of patients from year 2011 to
June 2013 was conducted for patients with both nasal MRSA colonization within 30
days prior to spinal surgery. Patients who tested positive for MRSA were put on
contact isolation protocol. None of these patients received topical antibiotics
for decolonization of nasal MRSA.
RESULTS: A total of 519 patients were identified; 384 negative (74%), 110
MSSA-positive (21.2%), and 25 (4.8%) MRSA-positive. Culture positive surgical
site infection (SSI) was identified in 27 (5.2%) cases and was higher in
MRSA-positive group than in MRSA-negative and MSSA-positive groups (12% vs.
5.73% vs. 1.82%; p=0.01). The MRSA SSI rate was 0.96% (n=5). MRSA SSI developed
in 8% of the MRSA-positive group as compared to only in 0.61% of MRSA-negative
group, with a calculated odds ratio of 14.23 (p=0.02). In the presence of SSI,
nasal MRSA colonization was associated with MRSA-positive wound culture (66.67
vs. 12.5%; p<0.0001).
CONCLUSION: Preoperative nasal MRSA colonization is associated with
postoperative spinal MRSA SSI. Preoperative screening and subsequent
decolonization using topical antibiotics may help in decreasing the incidence of
MRSA SSI after spine surgery. Nasal MRSA+ patients undergoing spinal surgery
should be informed regarding their increased risk of developing surgical site
infection. Methicillin-resistant Staphylococcus aureus (MRSA) burden is increasing
worldwide in hospitals [healthcare-associated (HA)-MRSA] and in communities
[community-associated (CA)-MRSA]. However, the impact of CA-MRSA within
hospitals remains limited, particularly in Latin America. A countrywide
representative survey of S. aureus infections was performed in Argentina by
analyzing 591 clinical isolates from 66 hospitals in a prospective
cross-sectional, multicenter study (Nov-2009). This work involved
healthcare-onset infections-(HAHO, >48 hospitalization hours) and
community-onset (CO) infections [including both, infections (HACO) in patients
with healthcare-associated risk-factors (HRFs) and infections (CACO) in those
without HRFs]. MRSA strains were genetically typed as CA-MRSA and HA-MRSA
genotypes (CA-MRSAG and HA-MRSAG) by SCCmec- and spa-typing, PFGE, MLST and
virulence genes profile by PCR. Considering all isolates, 63% were from
CO-infections and 55% were MRSA [39% CA-MRSAG and 16% HA-MRSAG]. A significantly
higher MRSA proportion among CO- than HAHO-S. aureus infections was detected
(58% vs 49%); mainly in children (62% vs 43%). The CA-MRSAG/HA-MRSAG have
accounted for 16%/33% of HAHO-, 39%/13% of HACO- and 60.5%/0% of
CACO-infections. Regarding the epidemiological associations identified in
multivariate models for patients with healthcare-onset CA-MRSAG infections,
CA-MRSAG behave like HA-MRSAG within hospitals but children were the highest
risk group for healthcare-onset CA-MRSAG infections. Most CA-MRSAG belonged to
two major clones: PFGE-type N-ST30-SCCmecIVc-t019-PVL(+) and PFGE-type
I-ST5-IV-SCCmecIVa-t311-PVL(+) (45% each). The ST5-IV-PVL(+)/ST30-IV-PVL(+)
clones have caused 31%/33% of all infections, 20%/4% of HAHO-, 43%/23% of HACO-
and 35%/60% of CACO- infections, with significant differences by age groups
(children/adults) and geographical regions. Importantly, an isolate belonging to
USA300-0114-(ST8-SCCmecIVa-spat008-PVL(+)-ACME(+)) was detected for the first
time in Argentina. Most of HA-MRSAG (66%) were related to the Cordobes/Chilean
clone-(PFGE-type A-ST5-SCCmecI-t149) causing 18% of all infections (47% of HAHO-
and 13% of HACO-infections). Results strongly suggest that the CA-MRSA clone
ST5-IV-PVL(+) has begun to spread within hospitals, replacing the traditional
Cordobes/Chilean-HA-MRSA clone ST5-I-PVL(-), mainly in children. Importantly, a
growing MRSA reservoir in the community was associated with spreading of two
CA-MRSA clones: ST5-IV-PVL(+), mainly in children with HRFs, and ST30-IV-PVL(+)
in adults without HRFs. This is the first nationwide study in Argentina
providing information about the molecular and clinical epidemiology of CA-MRSA,
particularly within hospitals, which is essential for designing effective
control measures in this country and worldwide. BACKGROUND AND OBJECTIVE: Since the early 2000s, the incidence of
methicillin-resistant Staphylococcus aureus (MRSA) infections among the
community of people lacking known healthcare risk factors has increased. This
MRSA infection is referred to as community-associated MRSA (CA-MRSA) infection
and is distinct from hospital-associated MRSA (HA-MRSA) infection, which occurs
among people with known healthcare risk factors. Understanding the epidemiology
of CA-MRSA infections is critical; however, this has not been investigated in
detail in Japan. Our objective was to investigate the incidence of CA-MRSA
infections in a regional hospital.
PATIENTS AND METHODS: We investigated CA-MRSA isolates and infections in a rural
regional hospital by reviewing medical records of one year. Infections were
classified as CA-MRSA if no established risk factors were identified.
RESULTS: During 2008, 31 Staphylococcus aureus (S. aureus) isolates were
detected in 29 unique patients, with 1 methicillin-sensitive S. aureus (MSSA)
isolates obtained from 19 patients (66%) and MRSA obtained from 10 patients
(34%). In the 10 patients with MRSA, the number of HA-MRSA and CA-MRSA cases
were nine (32% of patients with S. aureus isolates) and one (3%), respectively.
The patient with CA-MRSA was diagnosed with cellulitis due to CA-MRSA. All nine
patients with HA-MRSA exhibited colonization.
CONCLUSION: We observed a CA-MRSA case in a regional hospital in Japan,
suggesting that incidence trends of CA-MRSA should be considered in future
research and treatment. PURPOSE: To assess whether vancomycin minimum inhibitory concentration (MIC)
creeps among clinical isolates of methicillin-resistant Staphylococcus aureus
(MRSA) in a regional hospital in China. Furthermore, to analyze the causes of
vancomycin MIC creeps and the relationship between vancomycin MICs and the
outcome among patients with MRSA infection.
MATERIALS AND METHODS: All clinical isolates of MRSA from 2006-2010 were
retrieved and tested by the broth microdilution procedure to determine their
vancomycin MIC. Meanwhile, related patient records were analyzed.
RESULTS: While all isolates were susceptive to vancomycin, the percentage of
isolates with a vancomycin MIC = 1 mg/L increased significantly from 2006
(37.0%) to 2010 (75.7%). Meanwhile, vancomycin usage density (DDDs/1000
bed-days) had increased significantly from 2006-2010. Mean linear correlation
analysis showed a statistically significant positive correlation (r = 0.905, P <
0.05) between the consumption of vancomycin and the percentage of MRSA isolates
with a vancomycin MIC = 1 mg/L. Clinical records revealed high vancomycin MIC
was associated with a higher microbiologic failure rate in MRSA bloodstream
infections.
CONCLUSIONS: The data demonstrated vancomycin MIC creep among clinical isolates
in our hospital, and the MIC creep may be caused by the increasing usage of
vancomycin. Furthermore, the analysis strongly suggested this shift of
vancomycin MIC within the susceptible range may be associated with an increasing
probability of treatment failure. BACKGROUND: We describe the clinical characteristics and epidemiology of
methicillin-resistant Staphylococcus aureus (MRSA) in children with cystic
fibrosis (CF) from the U.S. CF center with the highest MRSA prevalence.
METHODS: Medical records of children with CF were retrospectively reviewed from
1997-2009. MRSA clinical isolates from 2007-2009 were analyzed by polymerase
chain reaction and pulsed field gel electrophoresis.
RESULTS: The prevalence of MRSA was 1% in 1997 and 49% in 2009. Fifty-five
children (26%) had persistent MRSA infection. Sixty-eight percent of MRSA
isolates were hospital-associated (HA) MRSA, of which 52% were pulsed-field type
USA 100. Ninety-three percent of HA MRSA isolates were clindamycin resistant.
Twelve children acquired MRSA before 1 year of age, 83% of whom were
hospitalized prior to acquisition of MRSA. Ten of 11 sibling pairs carried
indistinguishable MRSA strains. Children with persistent MRSA were hospitalized
more often (P = .01), required inhaled medications more frequently (P = .01),
and had higher rates of Pseudomonas aeruginosa coinfection (P < .001).
CONCLUSION: MRSA prevalence in children with CF is increasing, and most children
are infected with HA MRSA. Exposure to health care facilities and
gastrointestinal surgeries may facilitate early acquisition of MRSA. Siblings
carry indistinguishable MRSA strains, indicating household transmission of MRSA.
Children with persistent MRSA had worse pulmonary morbidity. Coinfection with
MRSA and P aeruginosa is likely associated with further increased pulmonary
morbidity. BACKGROUND: Identifying and tackling the social determits of infectious
diseases has become a public health priority following the recognition that
individuals with lower socioeconomic status are disproportionately affected by
infectious diseases. In many parts of the world, epidemiologically and
genotypically defined community-associated (CA) methicillin-resistant
Staphylococcus aureus (MRSA) strains have emerged to become frequent causes of
hospital infection. The aim of this study was to use spatial models with
adjustment for area-level hospital attendance to determine the transmission
niche of genotypically defined CA- and health-care-associated (HA)-MRSA strains
across a diverse region of South East London and to explore a potential link
between MRSA carriage and markers of social and material deprivation.
METHODS AND FINDINGS: This study involved spatial analysis of cross-sectional
data linked with all MRSA isolates identified by three National Health Service
(NHS) microbiology laboratories between 1 November 2011 and 29 February 2012.
The cohort of hospital-based NHS microbiology diagnostic services serves 867,254
usual residents in the Lambeth, Southwark, and Lewisham boroughs in South East
London, United Kingdom (UK). Isolates were classified as HA- or CA-MRSA based on
whole genome sequencing. All MRSA cases identified over 4 mo within the
three-borough catchment area (n = 471) were mapped to small geographies and
linked to area-level aggregated socioeconomic and demographic data. Disease
mapping and ecological regression models were used to infer the most likely
transmission niches for each MRSA genetic classification and to describe the
spatial epidemiology of MRSA in relation to social determits. Specifically,
we aimed to identify demographic and socioeconomic population traits that
explain cross-area extra variation in HA- and CA-MRSA relative risks following
adjustment for hospital attendance data. We explored the potential for
associations with the English Indices of Deprivation 2010 (including the Index
of Multiple Deprivation and several deprivation domains and subdomains) and the
2011 England and Wales census demographic and socioeconomic indicators
(including numbers of households by deprivation dimension) and indicators of
population health. Both CA-and HA-MRSA were associated with household
deprivation (CA-MRSA relative risk [RR]: 1.72 [1.03-2.94]; HA-MRSA RR: 1.57
[1.06-2.33]), which was correlated with hospital attendance (Pearson correlation
coefficient [PCC] = 0.76). HA-MRSA was also associated with poor health (RR:
1.10 [1.01-1.19]) and residence in communal care homes (RR: 1.24 [1.12-1.37]),
whereas CA-MRSA was linked with household overcrowding (RR: 1.58 [1.04-2.41])
and wider barriers, which represent a combined score for household overcrowding,
low income, and homelessness (RR: 1.76 [1.16-2.70]). CA-MRSA was also associated
with recent immigration to the UK (RR: 1.77 [1.19-2.66]). For the area-level
variation in RR for CA-MRSA, 28.67% was attributable to the spatial arrangement
of target geographies, compared with only 0.09% for HA-MRSA. An advantage to our
study is that it provided a representative sample of usual residents receiving
care in the catchment areas. A limitation is that relationships apparent in
aggregated data analyses cannot be assumed to operate at the individual level.
CONCLUSIONS: There was no evidence of community transmission of HA-MRSA strains,
implying that HA-MRSA cases identified in the community originate from the
hospital reservoir and are maintained by frequent attendance at health care
facilities. In contrast, there was a high risk of CA-MRSA in deprived areas
linked with overcrowding, homelessness, low income, and recent immigration to
the UK, which was not explainable by health care exposure. Furthermore, areas
adjacent to these deprived areas were themselves at greater risk of CA-MRSA,
indicating community transmission of CA-MRSA. This ongoing community
transmission could lead to CA-MRSA becoming the domit strain types carried by
patients admitted to hospital, particularly if successful hospital-based MRSA
infection control programmes are maintained. These results suggest that
community infection control programmes targeting transmission of CA-MRSA will be
required to control MRSA in both the community and hospital. These
epidemiological changes will also have implications for effectiveness of
risk-factor-based hospital admission MRSA screening programmes. During the past 25 years an increase in the prevalence of methicillin-resistant
Staphylococcus aureus (HA-MRSA) was recorded worldwide. Additionally, MRSA
infections may occur outside and independent of hospitals, caused by community
associated MRSA (CA-MRSA). In Germany, we found that at least 10% of these
sporadic infections are due to livestock-associated MRSA (LA-MRSA), which is
initially associated with livestock. The majority of these MRSA cases are
attributed to clonal complex CC398. LA-MRSA CC398 colonizes the animals
asymptomatically in about half of conventional pig farms. For about 77%-86% of
humans with occupational exposure to pigs, nasal carriage has been reported; it
can be lost when exposure is interrupted. Among family members living at the
same farms, only 4%-5% are colonized. Spread beyond this group of people is less
frequent. The prevalence of LA-MRSA in livestock seems to be influenced by farm
size, farming systems, usage of disinfectants, and in-feed zinc. LA-MRSA CC398
is able to cause the same kind of infections in humans as S. aureus and MRSA in
general. It can be introduced to hospitals and cause nosocomial infections such
as postoperative surgical site infections, ventilator associated pneumonia,
septicemia, and infections after joint replacement. For this reason, screening
for MRSA colonization at hospital admittance is recommended for farmers and
veterinarians with livestock contacts. Intrahospital dissemination, typical for
HA-MRSA in the absence of sufficient hygiene, has only rarely been observed for
LA-MRSA to date. The proportion of LA-MRSA among all MRSA from nosocomial
infections is about 3% across Germany. In geographical areas with a
comparatively high density of conventional farms, LA-MRSA accounts for up to 10%
of MRSA from septicemia and 15% of MRSA from wound infections. As known from
comparative genome analysis, LA-MRSA has evolved from human-adapted
methicillin-susceptible S. aureus, and the jump to livestock was obviously
associated with several genetic changes. Reversion of the genetic changes and
readaptation to humans bears a potential health risk and requires tight
surveillance. Although most LA-MRSA (>80%) is resistant to several antibiotics,
there are still sufficient treatment options. BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause
of both hospital and community infections globally. It's important to illuminate
the differences between community-acquired MRSA (CA-MRSA) and hospital-acquired
MRSA (HA-MRSA), but there have been confusions on the definition, especially for
the MRSA isolates identified within 48 h of admission. This study aimed to
determine the molecular characteristics and virulence genes profile of CA and
HA-MRSA isolates identified less than 48 h after hospital admission in our
region.
METHODS: A total 62 MRSA isolates identified within 48 h after admission and the
clinical data were collected. Antimicrobial susceptibility test (AST) of
collected isolates were performed according to the guidelines of Clinical and
Laboratory Standards Institute (CLSI) 2015, and staphylococcal cassette
chromosome mec (SCCmec) typing, multilocus sequence typing (MLST), pulsed-field
gel electrophoresis (PFGE) and virulence gene profiling were performed to
explore the molecular diversity.
RESULTS: SCCmec III and sequence type (ST) 239 were the most prevalent SCCmec
type and ST in both CA and HA-MRSA groups. HA-MRSA group had higher prevalence
of SCCmec III (87.2 %) and ST239 (79.5 %) compared with CA-MRSA (60.9 and
43.4 %, both P < 0.001), while the frequency of SCCmec IV (26.0 %) and ST59
(21.7 %) were higher in CA-MRSA than its counterpart (P < 0.001 and P = 0.003).
MRSA-ST239-III was the predomit type in this study (61.3 %, 38/62),
especially in HA-MRSA group (76.9 %, 30/39). However, CA-MRSA strains exhibited
more diversity in genotypes in this study. Meanwhile, CA-MRSA tended to have
lower resistant percentage to non-β-lactams antibiotics but more virulence genes
carriage, especially the staphylococcal enterotoxins (SE) genes. Notably, seb
gene was only detected in CA-MRSA isolates (52.2 %), likely a significant marker
for CA-MRSA isolates. Panton-Valentine leukocidin gene (PVL) was highly detected
in both groups, while appeared no significantly different between CA-MRSA
(47.8 %) and HA-MRSA (43.6 %).
CONCLUSIONS: Our findings support a difference in the molecular epidemiology and
virulence genes profile of CA-MRSA and HA-MRSA. Furthermore, this study
indicates a possible transmission from HA-MRSA to CA-MRSA, which may cause the
overlap of the definition. Community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) has
become a severe health concern because of its treatment difficulties. Herein, we
report the synthesis and biological evaluation of two phenazine natural products
and a series of phenazines that show promising activities against MRSA with MIC
values in the low micromolar range. Basic studies revealed that these compounds
are bacteriostatic agents. The most active compound also displayed promising
IC50 values against HaCat cells. Finally, a QSAR model was developed to
understand the key structural features of the molecules. Bloodstream infections with Staphylococcus aureus are clinically significant and
are often treated with empirical methicillin resistance (MRSA,
methicillin-resistant S. aureus) coverage. However, vancomycin has associated
harms. We hypothesized that MRSA screening correlated with resistance in S.
aureus bacteremia and could help determine the requirement for empirical
vancomycin therapy. We reviewed consecutive S. aureus bacteremias over a 5-year
period at two tertiary care hospitals. MRSA colonization was evaluated in three
ways: as tested within 30 days of bacteremia (30-day criterion), as tested
within 30 days but accounting for any prior positive results (ever-positive
criterion), or as tested in known-positive patients, with patients with unknown
MRSA status being labeled negative (known-positive criterion). There were 409 S.
aureus bacteremias: 302 (73.8%) methicillin-susceptible S. aureus (MSSA) and 107
(26.2%) MRSA bacteremias. In the 167 patients with MSSA bacteremias, 7.2% had a
positive MRSA test within 30 days. Of 107 patients with MRSA bacteremia, 68 were
tested within 30 days (54 positive; 79.8%), and another 21 (19.6%) were
previously positive. The 30-day criterion provided negative predictive values
(NPV) exceeding 90% and 95% if the prevalence of MRSA in S. aureus bacteremia
was less than 33.4% and 19.2%, respectively. The same NPVs were predicted at
MRSA proportions below 39.7% and 23.8%, respectively, for the ever-positive
criterion and 34.4% and 19.9%, respectively, for the known-positive criterion.
In MRSA-colonized patients, positive predictive values exceeded 50% at low
prevalence. MRSA screening could help avoid empirical vancomycin therapy and its
complications in stable patients and settings with low-to-moderate proportions
of MRSA bacteremia. |
List peptide fragmentations methods in mass spectrometry | CID, HCD, ECD, ETD and PSD are different peptide fragmentation technologies used in mass spectrometry. | The use of liquid chromatography-electrospray ionization-tandem mass
spectrometry (LC-ESI-MS(n)) for the glycoproteomic characterization of
glycopeptides is a growing field of research. The N- and O-glycosylated peptides
(N- and O-glycopeptides) analyzed typically originate from protease-digested
glycoproteins where many of them are expected to be biomedically important.
Examples of LC-MS(2) and MS(3) fragmentation strategies used to pursue glycan
structure, peptide identity and attachment-site identification analyses of
glycopeptides are described in this review. MS(2) spectra, using the CID and HCD
fragmentation techniques of a complex biantennary N-glycopeptide and a core 1
O-glycopeptide, representing two examples of commonly studied glycopeptide
types, are presented. A few practical tips for accomplishing glycopeptide
analysis using reversed-phase LC-MS(n) shotgun proteomics settings, together
with references to the latest glycoproteomic studies, are presented. Confident characterization of the microheterogeneity of protein glycosylation
through identification of intact glycopeptides remains one of the toughest
analytical challenges for glycoproteomics. Recently proposed mass spectrometry
(MS)-based methods still have some defects such as lack of the false discovery
rate (FDR) analysis for the glycan identification and lack of sufficient
fragmentation information for the peptide identification. Here we proposed
pGlyco, a novel pipeline for the identification of intact glycopeptides by using
complementary MS techniques: 1) HCD-MS/MS followed by product-dependent
CID-MS/MS was used to provide complementary fragments to identify the glycans,
and a novel target-decoy method was developed to estimate the false discovery
rate of the glycan identification; 2) data-dependent acquisition of MS3 for some
most intense peaks of HCD-MS/MS was used to provide fragments to identify the
peptide backbones. By integrating HCD-MS/MS, CID-MS/MS and MS3, intact
glycopeptides could be confidently identified. With pGlyco, a standard
glycoprotein mixture was analyzed in the Orbitrap Fusion, and 309 non-redundant
intact glycopeptides were identified with detailed spectral information of both
glycans and peptides. Six ion fragmentation techniques that can distinguish aspartic acid from its
isomer, isoaspartic acid, were compared. MALDI post-source decay (PSD), MALDI
157 nm photodissociation, tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP)
charge tagging in PSD and photodissociation, ESI collision-induced dissociation
(CID), electron transfer dissociation (ETD), and free-radical initiated peptide
sequencing (FRIPS) with CID were applied to peptides containing either aspartic
or isoaspartic acid. Diagnostic ions, such as the y-46 and b+H2O, are present in
PSD, photodissociation, and charge tagging. c•+57 and z-57 ions are observed in
ETD and FRIPS experiments. For some molecules, aspartic and isoaspartic acid
yield ion fragments with significantly different intensities. ETD and charge
tagging appear to be most effective at distinguishing these residues. Graphical
Abstract ᅟ. |
Is avanafil indicated for treatment of erectile dysfunction? | Yes, avanafil is indicated for treatment of erectile dysfunction. | Study Type--Therapy (RCT) Level of Evidence 1b. What's known on the subject? and
What does the study add? Avanafil is a potent selective phosphodiesterase type 5
(PDE5) inhibitor newly developed for treating erectile dysfunction (ED).
Preclinical and clinical phase I studies showed that avanafil had enhanced
selectivity, faster onset of action and a favourable side-effect profile
relative to currently available PDE5 inhibitors. As the result of phase III
clinical trial for the efficacy and safety of avanafil treatment (100 and 200
mg), taken as needed over a period of 12 weeks, in Korean patients with ED,
avanafil is an effective and well-tolerated therapy for ED of broad-spectrum
aetiology and severity.
OBJECTIVE: • To evaluate the efficacy and safety of avanafil, a new potent
selective phosphodiesterase type 5 (PDE5) inhibitor, in patients with erectile
dysfunction (ED).
PATIENTS AND METHODS: • The present study was a multicentre, randomized,
double-blind, placebo-controlled, fix-dosed phase three clinical trial involving
200 patients with ED. • The subjects were treated with placebo or avanafil (100
or 200 mg) for 12 weeks and were asked to complete the International Index of
Erectile Function (IIEF), the Sexual Encounter Profile (SEP) diary, and the
Global Assessment Questionnaire (GAQ). • The primary outcome variable was the
change from baseline for IIEF erectile function domain (EFD) score. • The
secondary outcome variables were SEP Q2 and Q3, the shift to normal rate (EFD ≥
26), and response to the GAQ.
RESULTS: • Compared with placebo, patients who took 100 or 200 mg of avanafil
had significantly improved IIEF-EFD score. • There were similar results when
comparing Q2 and Q3 in the SEP diary and the GAQ. • Flushing was the most common
treatment-related adverse event. • Most adverse events were transient and mild
or moderate in severity.
CONCLUSION: • Avanafil is an effective and well-tolerated therapy for ED of
broad-spectrum aetiology and severity. PURPOSE: We investigated the in vitro inhibitory effects of avanafil, a novel,
potent inhibitor of phosphodiesterase-5, on 11 phosphodiesterases. We also
studied its potentiation of penile tumescence in dogs.
MATERIALS AND METHODS: Phosphodiesterase assay was done with the 4
phosphodiesterase-5 inhibitors avanafil, sildenafil, vardenafil and tadalafil
using 11 phosphodiesterase isozymes. In anesthetized dogs the pelvic nerve was
repeatedly stimulated to evoke tumescence. Intracavernous pressure was measured
after avanafil or sildenafil administration.
RESULTS: Avanafil specifically inhibited phosphodiesterase-5 activity at a 50%
inhibitory concentration of 5.2 nM. Avanafil showed higher selectivity
(121-fold) against phosphodiesterase-6 than sildenafil and vardenafil (16 to
21-fold) and showed excellent selectivity (greater than 10,000-fold) against
phosphodiesterase-1 compared with sildenafil (375-fold). Avanafil also had
higher selectivity against phosphodiesterase-11 than tadalafil (greater than
19,000 vs 25-fold). Avanafil also showed excellent selectivity against all other
phosphodiesterases. After intravenous administration in anesthetized dogs the
200% effective dose of avanafil and sildenafil on the penile tumescence was 37.5
and 34.6 μg/kg, respectively. After intraduodenal administration the 200%
effective dose of avanafil and sildenafil on tumescence was 151.7 and 79.0 μg/kg
at the peak time, respectively. Time to peak response with avanafil and
sildenafil was 10 and 30 minutes, respectively, indicating a more rapid onset of
avanafil.
CONCLUSIONS: Avanafil has a favorable phosphodiesterase-5 selectivity profile
compared to that of marketed phosphodiesterase-5 inhibitors. Avanafil shows
excellent in vitro and in vivo potency, and fast onset of action for penile
erection. Cumulative data suggest that avanafil has a promising pharmacological
profile for erectile dysfunction. INTRODUCTION: Phosphodiesterase type 5 (PDE5) inhibitors are indicated for the
treatment of erectile dysfunction (ED); however, they can also inhibit other PDE
isozymes, affecting their target tissues (e.g., PDE1: heart; PDE6: retina; and
PDE11: skeletal muscle), which in some cases can cause unwanted side effects and
therapy discontinuation. Data from in vitro studies showed that avanafil, a PDE5
inhibitor for the treatment of ED, exhibited strong selectivity toward PDE5 and
against all other PDE isozymes.
AIM: To review the inhibitory effects of avanafil for PDE isozymes compared with
those of sildenafil, tadalafil, and vardenafil and to discuss these results
within the context of clinical trial safety observations.
METHODS: Review of in vitro selectivity data for avanafil (published primary
data from a peer-reviewed journal and scientific congress abstracts); PubMed
search for pertinent publications on PDE5 inhibitor safety data; and review of
published articles and abstracts from avanafil phase 1, 2, and 3 clinical
trials.
MAIN OUTCOME MEASURES: A low incidence of some PDE-related adverse events may be
reflected by the high selectivity of avanafil against non-PDE5 isozymes.
RESULTS: Avanafil is highly selective toward PDE5 and against all other PDE
isozymes tested. Lower selectivity against PDE1, PDE6, and PDE11 is consistent
with results from randomized, placebo-controlled, phase 3 trials in which
musculoskeletal and hemodynamic adverse events were reported in <2% of patients
and no color vision-related abnormalities were reported with avanafil doses up
to 200 mg once daily.
CONCLUSIONS: Data suggest that avanafil may confer a safety benefit, in terms of
a lower incidence of specific adverse events, by virtue of its high specificity
to PDE5 and its overall selectivity against other PDE isozymes. OBJECTIVE: To prospectively assess the safety and effectiveness of the
investigational phosphodiesterase 5 inhibitor avanafil to treat erectile
dysfunction in men with diabetes mellitus.
PATIENTS AND METHODS: This 12-week, multicenter, double-blind,
placebo-controlled study conducted between December 15, 2008, and February 11,
2010, randomized 390 men with diabetes and erectile dysfunction 1:1:1 to receive
avanafil, 100 mg (n=129), avanafil, 200 mg (n=131), or placebo (n=130).
Coprimary end points assessed changes in the percentage of sexual attempts in
which men were able to maintain an erection of sufficient duration to have
successful intercourse (Sexual Encounter Profile [SEP] 3), percentage of sexual
attempts in which men were able to insert the penis into the partner's vagina
(SEP 2), and International Index of Erectile Function erectile function domain
score.
RESULTS: Compared with placebo, least-squares mean change from baseline to study
end in SEP 3, SEP 2, and International Index of Erectile Function erectile
function domain score were significantly improved with both avanafil, 100 mg
(P≤.002), and avanafil, 200 mg (P<.001). Additional analyses indicated that
successful intercourse could be initiated in 15 minutes or less through more
than 6 hours after avanafil dosing. Adverse events most commonly reported with
avanafil treatment were headache, nasopharyngitis, flushing, and sinus
congestion.
CONCLUSION: Avanafil was safe and effective for treating erectile dysfunction in
men with diabetes and was effective as early as 15 minutes and more than 6 hours
after dosing. The adverse events seen with avanafil were similar to those seen
with other phosphodiesterase 5 inhibitors.
TRIAL REGISTRATION: clinicaltrials.gov Identifier NCT00809471. PURPOSE: We evaluated the safety and efficacy of 100 and 200 mg avanafil for the
treatment of adult males with erectile dysfunction after bilateral nerve sparing
radical prostatectomy.
MATERIALS AND METHODS: This was a double-blind, placebo controlled, parallel
group, phase 3 study in males age 18 to 70 years with a history of erectile
dysfunction of 6 months or more after bilateral nerve sparing radical
prostatectomy. Patients were randomized to 100 or 200 mg avanafil or placebo
(taken 30 minutes before sexual activity) for 12 weeks. Primary end points
included successful vaginal insertion (Sexual Encounter Profile [SEP] question
2), successful intercourse (SEP3) and change in score on the erectile function
domain of the International Index of Erectile Function (IIEF-EF) questionnaire.
RESULTS: A total of 298 patients were randomized and 84.6% completed the study.
At baseline 16.1% were age 65 years or older and 71.5% had severe erectile
dysfunction (mean overall IIEF-EF domain score 9.2). After 12 weeks there were
significantly greater increases in SEP2 and SEP3 and change in mean IIEF-EF
domain score with 100 and 200 mg avanafil vs placebo (p <0.01). Following dosing
with avanafil 36.4% (28 of 77) of sexual attempts (SEP3) at 15 minutes or less
were successful vs 4.5% (2 of 44) for placebo (p <0.01). Avanafil was generally
well tolerated. No serious adverse events were reported and fewer than 2% of
patients discontinued the study due to an adverse event.
CONCLUSIONS: Avanafil in 100 and 200 mg doses was effective and well tolerated
in improving erectile function after prostatectomy. Results suggest a rapid
onset of action and sustained duration of effect, with all 3 primary end points
being achieved at both dose levels. AIM: Determine the long-term efficacy, safety and tolerability of avanafil, a
highly specific, rapidly absorbed phosphodiesterase type 5 inhibitor in male
patients with mild to severe erectile dysfunction (ED), with or without
diabetes.
METHODS: This was a 52-week, open-label extension of two 12-week, randomised,
placebo-controlled, phase 3 trials. Patients were assigned to avanafil 100 mg,
but could request 200 mg (for increased efficacy; '100/200-mg' group) or 50 mg
(for improved tolerability). Primary end points included percentage of sexual
attempts ending in successful vaginal penetration [Sexual Encounter Profile 2
(SEP2)] and intercourse (SEP3) and erectile function domain score per the
International Index of Erectile Function (IIEF-EF).
RESULTS: Some 712 patients enrolled; 686 were included in the intent to treat
population and contributed to the data. All primary end points showed sustained
improvement. SEP2 and SEP3 success rates improved from 44% to 83% and from 13%
to 68% (100-mg group) and from 43% to 79% and from 11% to 66% (100/200-mg
group), respectively. Mean IIEF-EF domain scores improved from 13.6 to 22.2
(100-mg group) and from 11.9 to 22.7 (100/200-mg group). Avanafil was effective
in some patients ≤ 15 min and > 6 h postdose. Sixty-five per cent (112/172) of
'nonresponders' to avanafil 100 mg responded to the 200-mg dose. The most common
(≥ 2%) treatment-emergent adverse events were headache, flushing,
nasopharyngitis and nasal congestion; < 3% of patients discontinued therapy
because of adverse events.
CONCLUSIONS: The long-term tolerability and improvement in sexual function,
coupled with rapid onset, suggest that avanafil is well suited for the on-demand
treatment of ED. OBJECTIVE: To review the pharmacology, pharmacokinetics, safety, and efficacy of
avanafil and evaluate relevant clinical trial data.
DATA SOURCES: A MEDLINE, International Pharmaceutical Abstracts,
ClinicalTrials.gov, and Google Scholar searches (1966 to July 2013) were
conducted using the key words: avanafil, erectile dysfunction, and
phosphodiesterase type 5 (PDE5) inhibitor.
STUDY SELECTION AND DATA EXTRACTION: Articles evaluating avanafil for erectile
dysfunction (ED) published in English and using human subjects were selected.
Five clinical trials were identified. References cited in identified articles
were used for additional citations.
DATA SYNTHESIS: Avanafil is a highly selective PDE5 inhibitor that is a
competitive antagonist of cyclic guanosine monophosphate. Specifically, avanafil
has a high ratio of inhibiting PDE5 as compared with other PDE subtypes allowing
for the drug to be used for ED while minimizing adverse effects. Absorption
occurs quickly following oral administration with a median Tmax of 30 to 45
minutes and a terminal elimination half-life of 5 hours. Additionally, it is
predomitly metabolized by cytochrome P450 3A4. As such, avanafil should not
be co-administered with strong cytochrome P450 3A4 inhibitors. Dosage
adjustments are not warranted based on renal function, hepatic function, age or
gender. Five clinical trials suggest that avanafil 100 and 200 mg doses are
effective in improving the Sexual Encounter Profile and the Erectile Function
Domain scores among men as part of the International Index of Erectile Function.
A network meta-analysis comparing the PDE5 inhibitors revealed avanafil was less
effective on Global Assessment Questionnaire question 1 while safety data
indicated no major differences among the different PDE5 inhibitors. The most
common adverse effects reported from the clinical trials associated with
avanafil were headache, flushing, nasal congestion, nasopharyngitis, sinusitis,
and dyspepsia.
CONCLUSIONS: Avanafil is a potent PDE5 inhibitor and is an effective treatment
option for ED. OBJECTIVE: To determine the effect of avanafil, a novel phosphodiesterase-5
inhibitor, on the treatment of erectile dysfunction associated with type 2
diabetes mellitus (T2DM).
METHODS: In 2-day-old rats, T2DM was induced by single intraperitoneal injection
of 90 mg/kg of streptozotocin (STZ; i.p.). Erectile responses were evaluated
after 10 weeks on intracavernosal injection of avanafil (1 μM) to anesthetized
rats and data expressed as intracavernosal pressure (ICP)/mean arterial pressure
and total ICP. The relaxant and contractile responses of corpus cavernosum (CC)
strips were obtained in vitro studies.
RESULTS: ICP/mean arterial pressure and total ICP responses were significantly
reduced in T2DM rats compared with controls. Avanafil partially restored
diminished ICP responses in diabetic rats. In CC strips from the diabetic group,
electrical field stimulation (1-20 Hz)-induced relaxation responses were
markedly enhanced by 45%, whereas acetylcholine (ACh; 10(-8)-10(-3))-induced
relaxation responses were diminished by 73%. In addition, phenylephrine (PE;
10(-8)-10(-3)) and electrical field stimulation (1-40 Hz)-induced contractile
responses were significantly reduced in the diabetic group compared with
controls. CC relaxant responses to sodium nitroprusside (SNP, 10(-8)-10(-3)) and
avanafil (10(-8)-10(-3)) were unaltered in both groups.
CONCLUSION: The cavernous injection of avanafil in T2DM rats resulted in partial
improvement in erectile responses. These findings suggest that intracavernosal
administration of avanafil might be beneficial for the treatment of erectile
dysfunction in patients with T2DM. Avanafil, a potent new selective phosphodiesterase type 5 (PDE5) inhibitor, has
been developed for the treatment of erectile dysfunction (ED). We carried out a
systematic review and meta-analysis to assess the efficacy and safety of this
drug for the treatment of ED. A literature review was performed to identify all
published randomized, double-blind, placebo-controlled trials of avanafil for
the treatment of ED. The search included the following databases: MEDLINE,
EMBASE and the Cochrane Controlled Trials Register. The reference lists of the
retrieved studies were also investigated. Four publications, involving a total
of 1381 patients, were used in the analysis, including four randomized
controlled trials (RCTs) that compared avanafil with a placebo. Among the
co-primary efficacy end points indicating that avanafil 100 mg was more
effective than a placebo were successful vaginal penetration (SEP2) (the odds
ratio (OR) =5.06, 95% confidence interval (CI) =3.29-7.78, P< 0.00001) and
successful intercourse (SEP3) (OR = 3.99, 95% CI = 2.80-5.67, P< 0.00001). Men
randomized to receive avanafil were less likely than those receiving the placebo
to drop out due to an adverse event (AE) (OR = 1.48, 95% CI = 0.54-4.08, P=
0.44). Specific AEs with avanafil included headache and flushing, which were
significantly less likely to occur with placebo. This meta-analysis indicates
that avanafil 100 or 200 mg is an effective and well-tolerated treatment for ED.
Compared with avanafil 100 mg, patients who take avanafil 200 mg are more likely
to experience headaches. OBJECTIVE: To compare the efficacy and safety between different dosages of
avanafil for the treatment of erectile dysfunction (ED).
METHODS: PubMed, Cochrane Library, and Embase were searched to identify
randomized controlled trials which compared avanafil with placebo, or compared
different dosages of avanafil for ED. International Index of Erectile
Function-Erectile Function domain score (IIEF-EF), Sexual Encounter Profile
Question (SEP) questions 2 and 3, and adverse events were considered as the
study outcomes. Both pairwise meta-analysis and network meta-analysis were
carried out.
RESULTS: Five studies including 2225 patients were assessed. The pairwise
meta-analysis suggested that avanafil was more effective than placebo in
improving IIEF-EF (mean difference [MD]: 4.47; 95% confidence interval [CI]:
3.51 to 5.43), SEP-2 (MD: 17.41; 95% CI: 14.03 to 20.79), and SEP-3 (MD: 20.01;
95% CI: 22.98 to 37.22), with an evident dose-response relationship. The
effectiveness was significantly different between the 50 mg and 100 mg groups,
or between the 50 mg and 200 mg groups, for all outcomes. Overall, avanafil was
associated with a significantly higher incidence of any adverse event (risk
ratio [RR]: 2.56; 95% CI: 1.66 to 3.94), serious adverse event (RR: 2.78; 95%
CI: 1.34 to 5.76), flushing (RR: 6.06; 95% CI: 3.37 to 10.88) and headache (RR:
7.54; 95% CI: 3.52 to 16.12) when compared with placebo. No significant
difference in safety was found among various dosage groups.
CONCLUSIONS: Avanafil, from 50 to 200 mg, is effective and well tolerated for
the treatment of ED, and an increase in dosage is associated with a significant
rise in effectiveness but not with significantly more adverse events. Author information:
(1)Medicinal Chemistry Research Laboratories II, Mitsubishi Tanabe Pharma
Corporation, 2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan.
(2)Pharmacology Research Laboratories II, Mitsubishi Tanabe Pharma Corporation,
2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan.
(3)Advanced Medical Research Laboratories, Mitsubishi Tanabe Pharma Corporation,
2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan.
(4)Advanced Medical Research Laboratories, Mitsubishi Tanabe Pharma Corporation,
2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan; Industry and Academia
Cooperation Research Project, Laboratory of Target and Drug Discovery, Graduate
School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8601, Japan.
(5)Medicinal Chemistry Research Laboratories II, Mitsubishi Tanabe Pharma
Corporation, 2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan. Electronic
address: [email protected]. OBJECTIVES: Avanafil is a highly selective phosfosdiesterase 5 inhibitor (PDE5
inhibitor), with rapid onset of action, approved by the Food and Drug
Administration (FDA) and the European Medicines Agency for the treatment of
erectile dysfunction (ED). It had been recently commercialized in Spain. This
article presents a detailed review of the available literature, where the
safety, tolerability and efficacy of avanafil were evaluated.
METHODS: A systematic literature search using the Medline database was
performed. The search included the terms Avanafil and erectile dysfunction. The
pivotal studies of clinical development of the drug, and also those randomized,
double-blind, placebo-controlled, well-designed studies were analyzed. We
included those studies published in English up to January 2014. Likewise,
studies of the pharmacokinetics and pharmacodynamics of the drug were also
included.
RESULTS: The avanafil pivotal studies, conducted in general population of
patients with ED, patients with Diabetes mellitus type I and II and patients
with ED secondary to nerve sparing radical prostatectomy were analyzed. In all
these studies, avanafil demonstrated a statistically significant improvement in
erectile function (IIEF), and all the coprimary outcomes (SEP2 and SEP3)
compared to placebo. Also, a good tolerance profile and few side effects
compared to placebo were evident.
CONCLUSIONS: Avanafil is a selective PDE5 inhibitors, that is rapidly absorbed
and that has a short time to peak response. It found to be effective in
randomized, double-blind, placebo-controlled trials conducted in men with
erectile dysfunction, including in patients with diabetes mellitus and after
radical prostatectomy. It was generally well tolerated across trials, with very
few patients withdrawing because of adverse effects. Similarly, avanafil had a
significantly lower rate of hemodynamic side effects compared with sildenafil. PURPOSE: We examined the therapeutic effects of avanafil 15 minutes after dosing
in men with mild to severe erectile dysfunction.
MATERIALS AND METHODS: This randomized, double-blind, placebo controlled,
12-week study (4-week run-in and 8-week treatment) randomized 145 men to
placebo, 147 to avanafil 100 mg and 148 to avanafil 200 mg on demand. The
primary efficacy variable was the per subject proportion of sexual attempts
during the treatment period in which subjects achieved erection sufficient for
vaginal penetration within approximately 15 minutes after dosing as measured by
a stopwatch. The attempt had to enable successful completion of sexual
intercourse according to SEP question 3.
RESULTS: Significantly greater mean per subject percentages of successful
intercourse attempts within approximately 15 minutes after dosing were observed
for avanafil 100 mg (mean 25.9%, LS mean ± SE 24.7% ± 2.9%) and 200 mg (mean
29.1%, LS mean 28.2% ± 2.9%) vs placebo (mean 14.9%, LS mean 13.8% ± 2.9%, p =
0.001 and <0.001, respectively). After treatment we noted a statistically
significant difference between avanafil and placebo in the average per subject
proportion of successful intercourse attempts according to SEP question 3 as
early as 10 minutes in the 200 mg group and 12 minutes in the 100 mg group.
Treatment emergent adverse events included headache, upper respiratory tract
infection and nasal congestion, and most such events were mild or moderate in
severity.
CONCLUSIONS: Avanafil was efficacious within approximately 15 minutes of dosing
compared to placebo. A statistically significant treatment difference in the
percentage of successful sexual attempts was demonstrated as early as 10 minutes
after treatment. CONTEXT: Avanafil (AVA) is used in the treatment of erectile dysfunction, but is
reported for its poor aqueous solubility. Solid lipid oparticles (SLNs) are
lipid carriers that can greatly enhance drug solubility and bioavailability.
OBJECTIVE: This work was aimed to formulate and optimize AVA SLNs with
subsequent loading into hydrogel films for AVA transdermal delivery.
MATERIALS AND METHODS: AVA SLNs were prepared utilizing homogenization followed
by ultra-sonication technique. The prepared SLNs were characterized for particle
size, charge, surface morphology and drug content. The optimized SLNs
formulation was incorporated into transdermal films prepared using HPMC and
chitosan. Hydrogel films were evaluated for ex-vivo rat skin permeation using
automated Franz diffusion cells. The permeation parameters and the release
mechanism were evaluated. The transdermal permeation of the prepared AVA SLNs
through the skin layers was studied using confocal laser scanning microscope.
RESULTS: Lipid concentration and % of oil in lipid had a pronounced effect on
particle size while, entrapment efficiency was significantly affected by lipid
concentration and % of cholesterol. The optimized AVA SLNs showed particle size
and entrapment efficiency of 86 nm and 85.01%, respectively. TEM images revealed
spherecity of the particles. High permeation parameters were observed from HPMC
films loaded with AVA SLNs. The release data were in favor of Higuchi diffusion
model. The prepared AVA SLNs were able to penetrate deeper in skin layers.
CONCLUSION: HPMC transdermal film-loaded AVA SLNs is an effective and
alternative to per-oral drug administration. |
What is "Epitranscriptome analysis"? | Modified nucleotides in messenger RNA (mRNA) have been discovered over 40 years ago, but until recently little was known about which transcripts contain them and what their function is. High-throughput sequencing approaches revealed a dynamic landscape of the 'Epitranscriptome' for many mRNA modifications in various organisms from yeast to humans.
The detection methods of RNA modifications has enabled investigation of a new layer of gene regulation - the epitranscriptome. | Biochemical modifications to mRNA, especially N6-methyladenosine (m6A) and
5-methylcytosine (m5C), have been recently shown to be associated with crucial
biological functions. Despite the intriguing advancements, little is known so
far about the dynamic landscape of RNA methylome across different cell types and
how the epitranscriptome is regulated at the system level by enzymes, i.e., RNA
methyltransferases and demethylases. To investigate this issue, a meta-analysis
of m6A MeRIP-Seq datasets collected from 10 different experimental conditions
(cell type/tissue or treatment) is performed, and the combinatorial
epitranscriptome, which consists of 42 758 m6A sites, is extracted and divided
into 3 clusters, in which the methylation sites are likely to be hyper- or
hypo-methylated simultaneously (or co-methylated), indicating the sharing of a
common methylation regulator. Four different clustering approaches are used,
including K-means, hierarchical clustering (HC), Bayesian factor regression
model (BFRM) and nonnegative matrix factorization (NMF) to unveil the
co-methylation patterns. To validate whether the patterns are corresponding to
enzymatic regulators, i.e., RNA methyltransferases or demethylases, the target
sites of a known m6A regulator, fat mass and obesity-associated protein (FTO),
are identified from an independent mouse MeRIP-Seq dataset and lifted to human.
Our study shows that 3 out of the 4 clustering approaches used can successfully
identify a group of methylation sites overlapping with FTO target sites at a
significance level of 0.05 (after multiple hypothesis adjustment), among which,
the result of NMF is the most significant (p-value 2.81×10(-06)). We defined a
new approach evaluating the consistency between two clustering results which
shows that clustering results of different methods are highly correlated
strongly indicating the existence of co-methylation patterns. Consistent with
recent studies, a number of cancer and neuronal disease-related bimolecular
functions are enriched in the identified clusters, which are biological
functions that can be regulated at the epitranscriptional level, indicating the
pharmaceutical prospect of RNA N6-methyladenosine-related studies. This result
successfully reveals the linkage between the global RNA co-methylation patterns
embedded in the epitranscriptomic data under multiple experimental conditions
and the latent enzymatic regulators, suggesting a promising direction towards a
more comprehensive understanding of the epitranscriptome. A common feature of ribonucleic acids (RNAs) is that they can undergo a variety
of chemical modifications. As nearly all of these chemical modifications result
in an increase in the mass of the canonical nucleoside, mass spectrometry has
long been a powerful approach for identifying and characterizing modified RNAs.
Over the past several years, significant advances have been made in method
development and software for interpreting tandem mass spectra resulting in
approaches that can yield qualitative and quantitative information on RNA
modifications, often at the level of sequence specificity. We discuss these
advances along with instrumentation developments that have increased our ability
to extract such information from relatively complex biological samples. With the
increasing interest in how these modifications impact the epitranscriptome, mass
spectrometry will continue to play an important role in bioanalytical
investigations revolving around RNA. The advent of high-throughput sequencing technologies coupled with new detection
methods of RNA modifications has enabled investigation of a new layer of gene
regulation - the epitranscriptome. With over 100 known RNA modifications,
understanding the repertoire of RNA modifications is a huge undertaking. This
review summarizes what is known about RNA modifications with an emphasis on
discoveries in plants. RNA ribose modifications, base methylations and
pseudouridylation are required for normal development in Arabidopsis, as
mutations in the enzymes modifying them have diverse effects on plant
development and stress responses. These modifications can regulate RNA
structure, turnover and translation. Transfer RNA and ribosomal RNA
modifications have been mapped extensively and their functions investigated in
many organisms, including plants. Recent work exploring the locations, functions
and targeting of N6 -methyladenosine (m6 A), 5-methylcytosine (m5 C),
pseudouridine (Ψ), and additional modifications in mRNAs and ncRNAs are
highlighted, as well as those previously known on tRNAs and rRNAs. Many
questions remain as to the exact mechanisms of targeting and functions of
specific modified sites and whether these modifications have distinct functions
in the different classes of RNAs. The recent discovery of reversible mRNA methylation has opened a new realm of
post-transcriptional gene regulation in eukaryotes. The identification and
functional characterization of proteins that specifically recognize RNA
N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to
accelerate mRNA metabolism and translation. N6-adenosine methylation directs
mRNAs to distinct fates by grouping them for differential processing,
translation and decay in processes such as cell differentiation, embryonic
development and stress responses. Other mRNA modifications, including
N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together
with m6A form the epitranscriptome and collectively code a new layer of
information that controls protein synthesis. |
Which infection can be prevented with Dapivirine? | Vaginal ring containing Dapivirine is used for HIV prevention in women. | The nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681
(Dapivirine) effectively prevented human immunodeficiency virus (HIV) infection
in cocultures of monocyte-derived dendritic cells and T cells, representing
primary targets in sexual transmission. Both drugs had a favorable therapeutic
index. A 24-h treatment with 1,000 nM UC-781 or 100 nM TMC120-R147681 prevented
cell-free HIV infection, whereas 10-fold-higher concentrations blocked
cell-associated HIV. Heterosexual transmission of human immunodeficiency virus (HIV) remains the
major route of infection worldwide; thus, there is an urgent need for additional
prevention strategies, particularly strategies that could be controlled by
women, such as topical microbicides. Potential microbicide candidates must be
both safe and effective. Using cellular and tissue explant models, we have
evaluated the activity of the nonnucleoside reverse transcriptase inhibitor
(NNRTI) dapivirine as a vaginal microbicide. In tissue compatibility studies,
dapivirine was well tolerated by epithelial cells, T cells, macrophages, and
cervical tissue explants. Dapivirine demonstrated potent dose-dependent
inhibitory effects against a broad panel of HIV type 1 isolates from different
clades. Furthermore, dapivirine demonstrated potent activity against a wide
range of NNRTI-resistant isolates. In human cervical explant cultures,
dapivirine was able not only to inhibit direct infection of mucosal tissue but
also to prevent the dissemination of the virus by migratory cells. Activity was
retained in the presence of semen or a cervical mucus simulant. Furthermore,
dapivirine demonstrated prolonged inhibitory effects: it was able to prevent
both localized and disseminated infection for as long as 6 days posttreatment.
The prolonged protection observed following pretreatment of genital tissue and
the lack of observable toxicity suggest that dapivirine has considerable promise
as a potential microbicide candidate. Dual segment polyurethane intravaginal rings (IVRs) were fabricated to enable
sustained release of antiretroviral agents dapivirine and tenofovir to prevent
the male to female sexual transmission of the human immunodeficiency virus. Due
to the contrasting hydrophilicity of the two drugs, dapivirine and tenofovir
were separately formulated into polymers with matching hydrophilicity via
solvent casting and hot melt extrusion. The resultant drug loaded rods were then
joined together to form dual segment IVRs. Compression testing of the IVRs
revealed that they are mechanically comparable to the widely accepted NuvaRing
IVR. Physical characterization of the individual IVR segments using wide angle
X-ray scattering and differential scanning calorimetry determined that
dapivirine and tenofovir are amorphous and crystalline within their polymeric
segments, respectively. In vitro release of tenofovir from the dual segment IVR
was sustained over 30 days while dapivirine exhibited linear release over the
time period. A 90 day accelerated stability study confirmed that dapivirine and
tenofovir are stable in the IVR formulation. Altogether, these results suggest
that multisegment polyurethane IVRs are an attractive formulation for the
sustained vaginal delivery of drugs with contrasting hydrophilicity such as
dapivirine and tenofovir. To assess the pharmacokinetics of dapivirine in plasma and dapivirine
concentrations in cervicovaginal fluids (CVF) and cervicovaginal tissues
following vaginal administration of dapivirine microbicide gel in healthy,
HIV-negative women for 10 days. A randomized, double-blind, phase I study was
conducted at a single research center in South Africa. A total of 18 women used
dapivirine gel (0.001%, 0.005%, or 0.02%) once daily on Days 1 and 10 and twice
daily on Days 2-9. Pharmacokinetics of dapivirine were assessed in plasma on
Days 1 and 10. Dapivirine concentrations were measured in CVF on Days 1 and 10
and in cervicovaginal tissues on Day 10. Safety was evaluated through laboratory
tests (hematology, clinical chemistry, and urinalysis), physical examinations,
and assessment of adverse events. Plasma concentrations of dapivirine increased
over time with gel dose and were greater on Day 10 (C(max) 31 to 471 pg/ml) than
Day 1 (C(max) 23 to 80 pg/ml). T(max) was 10-12 h on Day 1, and 9 h on Day 10.
Concentrations in CVF generally increased with dose but were highly variable
among participants. Mean peak values ranged from 4.6-8.3 × 10(6) pg/ml on Day 1
and from 2.3-20.7 × 10(6) pg/ml on Day 10 across dose groups. Dapivirine was
detectable in all tissue biopsies on Day 10 at concentrations of 1.0-356 × 10(3)
pg/mg.
CONCLUSIONS: Dapivirine was widely distributed throughout the lower genital
tract with low systemic absorption when administered in a vaginal gel
formulation for 10 consecutive days. The gel was safe and well tolerated. The HIV-1 epidemic remains unchecked despite existing technology; vaccines and
microbicides in development may help reverse the epidemic. Reverse transcriptase
inhibitors (RTIs) formulated in gels tenofovir (TFV) and IVRs (dapivirine) are
under clinical development. While TFV or similar products may prove successful
for HIV-1, alternatives to RTIs may provide additional benefits, e.g., broader
STI prevention. Biopharmaceutical agents under development as microbicides
include cyanovirin, RANTES analogues, commensals, and Mabs. Cost of
manufacturing biopharmaceuticals has been reduced and they can be formulated
into tablets, films, and IVRs for vaginal delivery. Nanotechnology offers a
novel approach to formulate microbicides potentially leading to uniform
epithelial delivery. Delivery through vaginal mucus may be possible by
controlling oparticle size and surface characteristics. Combining prevention
modalities may be the most effective means of preventing STI transmission,
importantly, codelivery of microbicides and vaccines has demonstrated. Finally,
the safety of microbicide preparations and excipients commonly used can be
assessed using a mouse/HSV-2 susceptibility model. Screening of new microbicide
candidates and formulation excipients may avoid past issues of enhancing HIV-1
transmission. This article forms part of a special supplement covering several
presentations on novel microbicide formulations from the symposium on "Recent
Trends in Microbicide Formulations" held on 25 and 26 January 2010, Arlington,
VA. Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is a potent and
promising anti-HIV molecule. It is currently being investigated for use as a
vaginal microbicide in two dosage forms, a semi-solid gel and a silicone
elastomer ring. Quick-dissolving films are promising and attractive dosage forms
that may provide an alternative platform for the vaginal delivery of microbicide
drug candidates. Vaginal films may provide advantages such as discreet use, no
product leakage during use, lack of requirement for an applicator for insertion,
rapid drug release and minimal packaging and reduced wastage. Within this study
the in vitro bioactivity of dapivirine as compared to the NNRTI UC781 was
further established and a quick dissolve film was developed for vaginal
application of dapivirine for prevention of HIV infection. The developed film
was characterized with respect to its physical and chemical attributes including
water content, mechanical strength, drug release profile, permeability,
compatibility with lactobacilli and bioactivity. The anti-HIV activity of the
formulated dapivirine film was confirmed in in vitro and ex vivo models.
Importantly the physical and chemical properties of the film as well as its
bioactivity were maintained for a period of 18 months. In conclusion, a vaginal
film containing dapivirine was developed and characterized. The film was shown
to prevent HIV-1 infection in vitro and ex vivo and have acceptable
characteristics which make this film a promising candidate for testing as
vaginal microbicide. Dapivirine, formerly known as TMC 120, is a poorly-water soluble anti-HIV drug,
currently being developed as a vaginal microbicide. The clinical use of this
drug has been limited due to its poor solubility. The aim of this study was to
design solid dispersion systems of Dapivirine to improve its solubility. Solid
dispersions were prepared by solvent and fusion methods. Dapivirine release from
the solid dispersion system was determined by conducting in-vitro dissolution
studies. The physicochemical characteristics of the drug and its formulation
were studied using Differential Scanning Calorimetry (DSC), powder X-ray
Diffraction (XRD), Fourier-transform Infrared Spectroscopy (FTIR) and Scanning
Electron Microscopy (SEM). A significant improvement in drug dissolution rate
was observed with the solid dispersion systems. XRD, SEM and DSC results
indicated the transformation of pure Dapivirine which exists in crystalline form
into an amorphous form in selected solid dispersion formulations. FTIR and HPLC
analysis confirmed the absence of drug-excipient interactions. Solid dispersion
systems can be used to improve the dissolution rate of Dapivirine. This
improvement could be attributed to the reduction or absence of drug
crystallinity, existence of drug particles in an amorphous form and improved
wettability of the drug. PURPOSE: To assess the potential of polymeric oparticles (NPs) to affect the
genital distribution and local and systemic pharmacokinetics (PK) of the
anti-HIV microbicide drug candidate dapivirine after vaginal delivery.
METHODS: Dapivirine-loaded, poly(ethylene oxide)-coated
poly(epsilon-caprolactone) (PEO-PCL) NPs were prepared by a oprecipitation
method. Genital distribution of NPs and their ability to modify the PK of
dapivirine up to 24 h was assessed after vaginal instillation in a female mouse
model. Also, the safety of NPs upon daily administration for 14 days was
assessed by histological analysis and chemokine/cytokine content in vaginal
lavages.
RESULTS: PEO-PCL NPs (180-200 nm) were rapidly eliminated after administration
but able to distribute throughout the vagina and lower uterus, and capable of
tackling mucus and penetrate the epithelial lining. Nanocarriers modified the PK
of dapivirine, with higher drug levels being recovered from vaginal lavages and
vaginal/lower uterine tissues as compared to a drug suspension. Systemic drug
exposure was reduced when NPs were used. Also, NPs were shown safe upon
administration for 14 days.
CONCLUSIONS: Dapivirine-loaded PEO-PCL NPs were able to provide likely favorable
genital drug levels, thus attesting the potential value of using this vaginal
drug delivery osystem in the context of HIV prophylaxis. The HIV-1 replication inhibitor dapivirine (DPV) is one of the most promising
drug candidates being used in topical microbicide products for prevention of
HIV-1 sexual transmission. To be able to block HIV-1 replication, DPV must have
access to the viral reverse transcriptase enzyme. The window for DPV to access
the enzyme happens during the HIV-1 cellular infection cycle. Thus, in order for
DPV to exert its anti-HIV activity, it must be present in the mucosal tissue or
cells where HIV-1 infection occurs. A dosage form containing DPV must be able to
deliver the drug to the tissue site of action. Polymeric films are solid dosage
forms that dissolve and release their payload upon contact with fluids. Films
have been used as vaginal delivery systems of topical microbicide drug
candidates including DPV. For use in topical microbicide products containing
DPV, polymeric films must prove their ability to deliver DPV to the target
tissue site of action. Ex vivo exposure studies of human ectocervical tissue to
DPV film revealed that DPV was released from the film and did diffuse into the
tissue in a concentration dependent manner indicating a process of passive
diffusion. Analysis of drug distribution in the tissue revealed that DPV
accumulated mostly at the basal layer of the epithelium infiltrating the upper
part of the stroma. Furthermore, as a combination microbicide product,
codelivery of DPV and TFV from a polymeric film resulted in a significant
increase in DPV tissue concentration [14.21 (single entity film) and 31.03 μg/g
(combination film)], whereas no impact on TFV tissue concentration was found. In
vitro release experiments showed that this observation was due to a more rapid
DPV release from the combination film as compared to the single entity film. In
conclusion, the findings of this study confirm the ability of polymeric films to
deliver DPV and TFV to human ectocervical tissue and show that codelivery of the
two agents has a significant impact on DPV tissue accumulation. These findings
support the use of polymeric films for topical microbicide products containing
DPV and/or TFV. OBJECTIVES: Combination microbicide vaginal rings may be more effective than
single microbicide rings at reducing/preventing sexual transmission of HIV.
Here, we report the pre-clinical development and macaque pharmacokinetics of
matrix-type silicone elastomer vaginal rings containing dapivirine and
darunavir.
METHODS: Macaque rings containing 25 mg dapivirine, 100 mg dapivirine, 300 mg
darunavir or 100 mg dapivirine+300 mg darunavir were manufactured and
characterized by differential scanning calorimetry. In vitro release was
assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal
fluid and blood serum concentrations for both antiretrovirals were measured
during 28 day ring use. Tissue levels were measured on day 28. Ex vivo challenge
studies were performed on vaginal fluid samples and IC50 values were calculated.
RESULTS: Darunavir caused a concentration-dependent reduction in the dapivirine
melting temperature in both solid drug mixes and in the combination ring. In
vitro release from rings was dependent on drug loading, the number of drugs
present and the release medium. In macaques, serum concentrations of both
microbicides were maintained between 10(1) and 10(2) pg/mL. Vaginal fluid levels
ranged between 10(3) and 10(4) ng/g and between 10(4) and 10(5) ng/g for
dapivirine and darunavir, respectively. Both dapivirine and darunavir showed
very similar concentrations in each tissue type; the range of drug tissue
concentrations followed the general rank order: vagina (1.8 × 10(3)-3.8 × 10(3)
ng/g) > cervix (9.4 × 10(1)-3.9 × 10(2) ng/g) > uterus (0-108 ng/g) > rectum
(0-40 ng/g). Measured IC50 values were >2 ng/mL for both compounds.
CONCLUSIONS: Based on these results, and in light of recent clinical progress of
the 25 mg dapivirine ring, a combination vaginal ring containing dapivirine and
darunavir is a viable second-generation HIV microbicide candidate. PURPOSE: To develop polymeric films containing dual combinations of anti-HIV
drug candidate tenofovir, maraviroc and dapivirine for vaginal application as
topical microbicides.
METHODS: A solvent casting method was used to manufacture the films. Solid phase
solubility was used to identify potential polymers for use in the film
formulation. Physical and chemical properties (such as water content, puncture
strength and in vitro release) and product stability were determined. The
bioactivity of the film products against HIV was assessed using the TZM-bl assay
and a cervical explant model.
RESULTS: Polymers identified from the solid phase solubility study maintained
tenofovir and maraviroc in an amorphous state and prevented drug
crystallization. Three combination film products were developed using cellulose
polymers and polyvinyl alcohol. The residual water content in all films was <10%
(w/w). All films delivered the active agents with release of >50% of film drug
content within 30 min. Stability testing confirmed that the combination film
products were stable for 12 months at ambient temperature and 6 months under
stressed conditions. Antiviral activity was confirmed in TZM-bl and cervical
explant models.
CONCLUSIONS: Polymeric films can be used as a stable dosage form for the
delivery of antiretroviral combinations as microbicides. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are widely used to treat
HIV-1-infected individuals; indeed most first-line antiretroviral therapies
typically include one NNRTI in combination with two nucleoside analogs. In 2008,
the next-generation NNRTI etravirine was approved for the treatment of
HIV-infected antiretroviral therapy-experienced individuals, including those
with prior NNRTI exposure. NNRTIs are also increasingly being included in
strategies to prevent HIV-1 infection. For example: (1) nevirapine is used to
prevent mother-to-child transmission; (2) the ASPIRE (MTN 020) study will test
whether a vaginal ring containing dapivirine can prevent HIV-1 infection in
women; (3) a microbicide gel formulation containing the urea-PETT derivative
MIV-150 is in a phase I study to evaluate safety, pharmacokinetics,
pharmacodynamics and acceptability; and (4) a long acting rilpivirine
formulation is under-development for pre-exposure prophylaxis. Given their
widespread use, particularly in resource-limited settings, as well as their low
genetic barriers to resistance, there are concerns about overlapping resistance
between the different NNRTIs. Consequently, a better understanding of the
resistance and cross-resistance profiles among the NNRTI class is important for
predicting response to treatment, and surveillance of transmitted
drug-resistance. When developing novel microbicide products for the prevention of HIV infection,
the preclinical safety program must evaluate not only the active pharmaceutical
ingredient but also the product itself. To that end, we applied several
relatively standard toxicology study methodologies to female sheep,
incorporating an assessment of the pharmacokinetics, safety, tolerability, and
local toxicity of a dapivirine-containing human vaginal ring formulation
(Dapivirine Vaginal Ring-004). We performed a 3-month general toxicology study,
a preliminary pharmacokinetic study using drug-loaded vaginal gel, and a
detailed assessment of the kinetics of dapivirine delivery to plasma, vaginal,
and rectal fluid and rectal, vaginal, and cervical tissue over 28 days of
exposure and 3 and 7 days after removal of the ring. The findings of the general
toxicology study supported the existing data from both preclinical and clinical
studies in that there were no signs of toxicity related to dapivirine. In
addition, the presence of the physical dapivirine ring did not alter local or
systemic toxicity or the pharmacokinetics of dapivirine. Pharmacokinetic studies
indicated that the dapivirine ring produced significant vaginal tissue levels of
dapivirine. However, no dapivirine was detected in cervical tissue samples using
the methods described here. Plasma and vaginal fluid levels were lower than
those in previous clinical studies, while there were detectable dapivirine
levels in the rectal tissue and fluid. All tissue and fluid levels tailed off
rapidly to undetectable levels following removal of the ring. The sheep
represents a very useful model for the assessment of the safety and
pharmacokinetics of microbicide drug delivery devices, such as the vaginal ring. BACKGROUND: Research is ongoing to develop multipurpose vaginal rings to be used
continuously for contraception and to prevent Human Immunodeficiency Virus (HIV)
infection. Contraceptive vaginal rings (CVRs) are available in a number of
countries and are most of the time used intermittently i.e. three weeks out of a
4-week cycle. Efficacy trials with a dapivirine-containing vaginal ring for HIV
prevention are ongoing and plans to develop multi-purpose vaginal rings for
prevention of both HIV and pregcy have been elaborated. In contrast with the
CVRs, multi-purpose vaginal rings will have to be used continuously. Women who
continuously use a CVR will no longer have menses. Furthermore, some safety
aspects of CVR use have never been studied in-depth in the past, such as the
impact of the vaginal ring on the vaginal microbiota, biofilm formation and
induction of inflammation. We studied acceptability and these novel aspects of
safety in Rwandan women. Although significant progress has been made over the
past decade, Rwanda still has a high unmet need for contraception (with 47%
unplanned births) and a generalized HIV epidemic, and CVRs are not yet
available.
METHODS: We will conduct an open label, single centre, randomized controlled
trial. A total of 120 HIV-negative women will be randomized to intermittent CVR
use (to allow menstruation) or continuous CVR use. Women will be followed for a
maximum of 14 weeks. In parallel, we will conduct a qualitative study using
in-depth interview and focus group discussion methodology.
DISCUSSION: In addition to evaluating the safety and acceptability of
intermittent and continuous CVR use in Rwandan women, we hope that our findings
will inform the development of future multipurpose vaginal rings, will prepare
Rwandan study populations for future clinical trials of multipurpose vaginal
rings, and will pave the way for introduction of CVRs on African markets.
TRIAL REGISTRATION: Clinicaltrials.gov NCT01796613 . Registered 14 February
2013. OBJECTIVES: The objectives of this study were to comprehensively assess mRNA
expression of 84 drug transporters in human colorectal biopsies and six
representative cell lines, and to investigate the alteration of drug transporter
gene expression after exposure to three candidate microbicidal antiretroviral
(ARV) drugs (tenofovir, darunavir and dapivirine) in the colorectal epithelium.
The outcome of the objectives informs development of optimal ARV-based
microbicidal formulations for prevention of HIV-1 infection.
METHODS: Drug transporter mRNA expression was quantified from colorectal
biopsies and cell lines by quantitative real-time PCR. Relative mRNA expression
was quantified in Caco-2 cells and colorectal explants after induction with
ARVs. Data were analysed using Pearson's product moment correlation (r),
hierarchical clustering and principal component analysis (PCA).
RESULTS: Expression of 58 of the 84 transporters was documented in colorectal
biopsies, with genes for CNT2, P-glycoprotein (P-gp) and MRP3 showing the
highest expression. No difference was noted between individual subjects when
analysed by age, gender or anatomical site (rectum or recto-sigmoid) (r =
0.95-0.99). High expression of P-gp and CNT2 proteins was confirmed by
immunohistochemical staining. Similarity between colorectal tissue and cell-line
drug transporter gene expression was variable (r = 0.64-0.84). PCA showed
distinct clustering of human colorectal biopsy samples, with the Caco-2 cells
defined as the best surrogate system. Induction of Caco-2 cell lines with ARV
drugs suggests that darunavir-based microbicides incorporating tenofovir may
result in drug-drug interactions likely to affect distribution of individual
drugs to sub-epithelial target cells.
CONCLUSIONS: These findings will help optimize complex formulations of rectal
microbicides to realize their full potential as an effective approach for
pre-exposure prophylaxis against HIV-1 infection. Collaborators: Bello ES, Chihana M, Huwa JC, Chisale P, Chithila F, Dadabhai SS,
Deg L, Debevec B, Fatch T, Goliati E, Gondwe DK, Kandonje DH, Kachale E,
Kaliwo V, Kamaliza M, Kamanga M, Chadza MK, Soko DK, Kaphiri T, Kumwenda N,
Mwenda WL, Madiwati B, Makunganya J, Malemia A, Malidadi M, Maruwo A, Mbilizi
YR, Mpofu J, Mulenga P, Mulima J, Munthali J, Mwafulirwa L, Mwakhwawa T, Njete
L, Nkanaunena K, Nyirenda M, Phingo M, Phiri G, Seyama L, Simkoza H, Singini I,
Singini A, Sitima E, Siyasiya A, Taulo F, Taulo E, Tung'ande T, Zulu F, Baluwa
C, Banda G, Banda B, Banda T, Chikopa L, Chindebvu M, Chintedza J, Chinula L,
Chitema F, Chitsulo G, Chiudzu G, Chome N, Gondwe N, Hoffman I, Kachipapa E,
Kaliati I, Kalulu A, Kamwana G, Kilembe J, Kumwenda M, Kumwenda W, Kumwenda P,
Lungu T, Lusewa C, Makala Y, Mapanje C, Mhango W, Mkandawire N, Mkochi T, Msiska
E, Msumba L, Mukatipa M, Mwese J, Ndovie M, Nkhalamba T, Nyirenda N, Sichali D,
Stambuli H, Tegha G, Tembo T, Tembo T, Atujuna M, Barnabas S, Belonje D,
Cengimbo N, Chabanga HA, Coba T, Crida D, Dallimore J, Danster N, Dikili S,
Dippenaar R, Dlulane P, Dukwe P, Dyabeni L, Fleurs L, Funda NP, Gelant J, Gogo
L, Gumede S, Hlwele N, Hobongwana T, Jeffery L, Jose M, Khaya B, Khumalo T,
Killa N, Maclachlan M, Magobiane N, Mahed F, Makhamba P, Malan G, Masters A,
Mayekiso N, Mcayiya T, Mngqebisa B, Mona N, Mthethi Z, Nandudu N, Ngayi B,
Ngqabe N, Nkomiyapi SM, Nompondwana M, Ntlantsana V, Ntshoko M, Pearce N,
Peterson N, Radebe M, Radzilani T, Rainers H, Rasmeni S, Reynolds D, Rode N,
Roux S, Scotch N, Snyder K, Temmers N, Thompson C, Tofile T, Tshongoyi N, Van
Der Vendt D, Van Niekerk C, Vogt M, Wallace M, Woolley P, Yola N, Young K,
Chetty D, Dias L, Dladla M, Gengiah T, Govender M, Harkoo I, Kamal S, Kasaval M,
Khan I, Khanyile S, Khwela C, Kubheka L, Luthuli L, Mabasa N, Madlala B, Maharaj
B, Mayisela N, Mdunge A, Mkhize W, Mngadi A, Moosa A, Naidoo K, Naidoo A,
Ndwandwe L, Ramdhani V, Richards J, Shozi C, Singh C, Zungu N, Abbai N, Arbee F,
Ashokumar A, Aungadh A, Baba L, Baboolal S, Balkaran K, Bebeza S, Bhayat H,
Bhengu B, Bhengu R, Biyela B, Boodhram R, Bulose P, Buthelezi P, Buthelezi S,
Buthelezi S, Buthelezi T, Buthelezi L, Buthelezi P, Buyeye L, Byroo S, Cebisa S,
Cele N, Chamane V, Chetty C, Chitray K, Chonco P, Choonilall A, Chunderduri K,
Cibane S, Devnarain S, Dhanpal N, Dladla N, Dlamini D, Dlamini E, Dubazane N,
Dube F, Duma C, Dwayisa Z, Faya N, Gaffoor T, Ganesh R, Ganesh S, Gappoo S, Gasa
T, Gounden N, Govender D, Govender D, Govender M, Govender N, Govender S,
Guddera V, Guga P, Gumede E, Gumede S, Harichund C, Haridutt A, Harilal J,
Hemchund I, Hlangu P, Hlela T, Hlophe M, Hurbans N, Imamdin R, Jagesur L,
Jainarain K, Jenneker M, Jokozela A, Juggernath V, Juggessar N, Kadwa R, Khaba
DP, Khalishwayo V, Khanyile L, Khanyile N, Khanyile R, Khomo M, Khoza N, Khubone
N, Khumalo N, Khumalo TK, Khuzwayo N, Kistansami G, Kumalo N, Labuschange L,
Lembethe P, Mabaso M, Mabaso S, Mabusela P, Sayed F, Maduna P, Magwaza P,
Maharaj R, Mahlangeni T, Makeka N, Makhanya N, Makhoba N, Maphumulo L, Maphumulo
N, Mavis V, Maphumulo B, Marx E, Masinga N, Mavundla SS, Mbatha T, Mbeje T,
Mbeko M, Mbhele S, Mbonambi S, Mbuyisa F, Mchunu M, Mchunu N, Mdepha P, Mdletshe
MI, Meyiwa S, Mhlaba N, Mhlongo R, Mhlongo Z, Mkhabela N, Mkhithi L, Mkhize L,
Mkhize Z, Mkhize S, Mkhize Z, Mkhize T, Mkhunyo Z, Mkhwanazi N, Mnikathi N,
Mnqonywa N, Mofokeng M, Moodley J, Moodley J, Moodley J, Moodley R, Moodley S,
Moonsamy S, Morar N, Mphili N, Mpofana I, Mshibe LC, Msomi LD, Msomi S, Msomi T,
Msweli S, Mthalane E, Mthembu B, Mthembu F, Mthembu T, Mthethwa NZ, Mthimkhulu
S, Mthlane MI, Mvelase S, Mvuyane GZ, Mzimela L, Mzimela N, Mzolo A, Naicker K,
Naicker S, Naicker V, Naidoo A, Naidoo S, Naidoo A, Naidoo I, Naidoo J, Naidoo
J, Naidoo K, Naidoo S, Naidoo S, Naidu N, Naras A, Nayager T, Ncayiya N, Ndaba
N, Ndaba S, Ndala T, Ndamase P, Ndlovu S, Ndlovu Z, Ndlovu J, Ndlovu S, Ndwandwe
DE, Ngobese KJ, Ngubane H, Ngubane M, Nhleko S, Nursayhe N, Nxumalo N, Nyawo K,
Padayachee K, Paramanund L, Perumal M, Phahlamohlaka B, Phillip J, Pillay J,
Pillay O, Pillay P, Pillay T, Pillay Y, Premrajh A, Punianathan R, Qhogwana L,
Rajpal P, Ramdass S, Ramluckan N, Ramnarain S, Rampersadh A, Ramsumair R,
Ratanjee A, Reddy M, Reddy M, Reddy N, Reddy T, Sakloo V, Saul N, Shangase S,
Shazi Z, Shembe Z, Shobede Z, Sibisi S, Sibisi T, Sibiya Z, Singh N, Sithole H,
Sithole L, Skhosana M, Soobryan D, Spooner E, Sukazi S, Teise R, Tenza T,
Tshabalala T, Tutshana B, Udith A, Vallabhjee L, Vijay A, Vilakazi H, Viriah B,
Wanda B, Woeber K, Zondi T, Zulu T, Zungu N, Zungu P, Zwane I, Boikanya R,
Bojosi P, Bothma R, Cebekhulu B, Cohen S, Delany-Moretlwe S, Diep L, Dott C,
Duba NG, Dube NM, Duma PI, Farisani LML, Gama L, Gegana NM, Govender N, Gubayo
S, Gumede N, Guness MP, Hoggan MA, Kekana E, Kgaritsi MD, Khan F, Langa T, Madi
MM, Maduno N, Maenetje PW, Magazi B, Maila ML, Makgoka KP, Makupula BS,
Manzingana NN, Maputla F, Maraba MC, Martin R, Marule ES, Maseko N, Maseko P,
Mashabane N, Mashiane TE, Mathebula FT, Mathipa M, Matikinca XJ, Mazibuko NV,
Mlangeni EG, Mlotshwa MD, Mnguni B, Modau M, Modise CL, Mokgatle KM, Mokoena ET,
Mothle AO, Mthethwa MJ, Mthalane ES, Mzolo ADN, Nage E, Naidoo AD, Naidoo MA,
Naidoo S, Ncgobo NLE, Ncube DE, Ndhlozi TW, Ngcobo NT, Ngoetjana J, Ngozo R,
Ngwenya LP, Nhlapo D, Nkangane L, Nyathi AT, Okwuolise OV, Palanee-Phillips B,
Pamacheche P, Pitso ST, Ramafalo MP, Ramalo P, Ramchuran P, Reddy K, Rees VH,
Sharif A, Sibeko SS, Sibisi S, Sibisi WP, Sizane V, Steingo J, Stuurman R,
Thobejane LP, Tjale LC, Tshabalala M, Verheyden B, Vuma AC, Aanyu D, Akello CA,
Asiimwe P, Biira FA, Balamusani D, Bazira JA, Bihabwa G, Birungi E, Bolton S,
Bukenya LE, Nabatanzi RB, Sseguya BB, Busuulwa BJ, Elbireer A, Ongom JE, Fowler
MG, Mirembe BG, Ggita J, Juuko A, Kizza DK, Kabwigu S, Kafufu B, Mukasa AK,
Kagoda J, Kakayi BC, Nabunya HK, Kamira B, Kampi R, Kamugisha A, Kamya J,
Bawulira FK, Karugaba P, Katongole F, Kemigisha D, Kiiza B, King B, Kintu K,
Sserwadda HL, Lwanga J, Musoke PM, Makooka H, Mayanja E, Mbabali MS, Mirembe D,
Motevalli MO, Mubiru MC, Namayanja PM, Mugenyi M, Najjemba MMK, Musisi M, Mutebo
J, Muwawu R, Mwebaza D, Nabisere J, Nabukeera J, Kiwanuka CVN, Najjuka S,
Nakacwa S, Nakakande J, Nakalema MG, Saava MN, Nakibuka J, Nakyanzi T, Nakyeyune
J, Kalungi SN, Namirembe C, Nampewo J, Nkalubo SN, Nankinga S, Nansamba W,
Nanyonga S, Nanziri SC, Kawuma CN, Ndawula P, Nolan M, Musisi JN, Nyanzi Z,
Wabwire DO, Ojok D, Onen F, Onyuthfua M, Osman S, Onyango CP, Rwanzogyera G,
Sakwa R, Semakula J, Kikonyogo FS, Ssenyonga M, Zalwango A, Bafana Munatsi TNS,
Banda M, Chandipwisa A, Chareka GT, Chasakara CZ, Chidemo T, Chigwanha D,
Chihota E, Chikono S, Chikonyora A, Chikuni A, Chitambo T, Chitsinde C,
Chitukuta M, Chivasa T, Dhlakama PM, Godo L, Gomani P, Goreraza R, Gwete NS,
Hlahla K, Hunidzarira P, Hwehwe TD, Kachingamire F, Katanda C, Katema H,
Kufakunesu T, Kundeya M, Kutinyu I, Madovi T, Magure TM, Makaya TC, Makoni R,
Makwenda S, Mangove LZ, Manyeruke T, Mapfunde A, Maponga CEC, Masango M, Masara
TB, Masawi M, Masuko S, Matanhire A, Matenda S, Matsa JM, Matubu AT, Matyanga
CMJ, Mhangami F, Mhizha ES, Mlingo M, Mlingo CT, Motsi TM, Mpofu TAM, Mugocha C,
Mugodhi F, Mugowe N, Muhlanga FGS, Mukaka S, Mukova S, Munaiwa O, Munjoma MW,
Mupunga M, Murefu T, Murewanhema G, Musara P, Mutero P, Mutisi F, Muvunzi T,
Muzamhindo TRL, Mwakurudza S, Mwandiwata E, Ncube S, Ncube E, Ndadziyira P,
Ndhlovu G, Ngwindi E, Nyabadza O, Nyakudya S, Nyawera EC, Rimau F, Rupondo N,
Rushwaya C, Samaneka LN, Shawatu BN, Shonhiwa R, Tauya TT, Thompson M, Vuta C,
Zinyongo M, Aridor O, Bezak L, Comer K, Conser E, DeGore M, Duran L, Foster M,
Leszczewski M, McCarthy K, Rossi L, Rullo C, Smolinski D, Browning-Roberts B,
Maddox T, Montoya D, Moutsos K, Scoville C, Calabrese K, Cokley C, Friedland I,
Mierzwa S, Souidi S, Wu C, Cheng H, Hartmann M, Katz A, Laborde N, Leslie J,
Lutnick A, Pleasants E, Shapley-Quinn MK, Johnson J, Manohar M, Seserko L,
Travers J, Austin M, Avolia H, Beamer M, Cosentino L, DeMarco A, Dezzutti C,
Hall W, Hendrickson S, Jones L, Petrina M, Rabe L, Stoner K, Hamanishi K, Eskay
K, Gordon K, Halvas E, Hardesty R, Opest A, Penrose K, Abullarade J, Anderson M,
Balkus J, Bassuk D, Borgerding J, Chapdu C, Chin C, Cianciola M, DeBoer C,
Dinnie E, Doyle M, Edwards D, Fitzpatrick J, Gandham S, Huang H, Kentop S,
Krause J, Liu K, Mohebalian I, Molitor C, Pan J, Schille J, Snapinn K, Tseng J,
Unten C, Weaver K, Wilson D, Adedeji B, Black R, Barnett R, Bupp J, Chow G,
Cleland N, Cogliano N, Csedrik J, Dieffenbach C, Erbelding E, Estep S, Freeman
L, Hinds J, Martinez A, O’Brien J, Payton M, Perez A, Piper J, Potter D,
Rancourt A, Rausch D, Russo D, Saint-Vil D, Sharma U, Siskind R, Leirman LS,
Stover K, Towers S, Veronese F, Zwerski S, Carstens H, Carter A, Derrick T,
Devlin B, Garg A, Malherbe M, Mans W, Nuttall J, Russell M, Seaton E, Solai L,
Spence P, Steytler J, van Niekerk N, Windle K, Bhengu N, Bucklew R, Chasakara C,
Chatani M, Dlamini C, Dlamini T, Gondwe D, Kampangire Z, Katana M, Luthuli V,
Mafumane O, Mchunu Z, Mthiyane T, Muganga J, Ndlovu Z, Ngani S, Ngqame S, Nkebi
JC, Nompondwana M, Samuels H, Sontshatsha B, Wright T, Zindela A, Cates W, Celum
C, Donnell D, Hughes J, McCormack S, Wang L. The 2016 Conference on Retroviruses and Opportunistic Infections (CROI)
highlighted hot spots in HIV infection. Men who have sex with men (MSM),
transgender populations, people who inject drugs, fisherfolk, migrants,
adolescents, and older adults are heavily impacted in a number of regions.
Stigma contributes to risk behaviors and HIV acquisition across populations. HIV
testing is a crucial first step in the HIV care continuum, and several large
community-based surveys are underway in Africa to increase HIV testing, linkage
to care, and uptake of antiretroviral treatment. Advances in preexposure
prophylaxis (PrEP) featured prominently at CROI 2016. Two large efficacy trials
of a vaginal ring containing the investigational drug dapivirine demonstrated
efficacy and safety in preventing HIV infections in women in Africa. Data on the
safety of long-acting injectable PrEP and several investigational PrEP drugs and
formulations were also presented. Knowledge and use of PrEP among MSM in the
United States appears to be increasing, and high uptake was seen among black MSM
when provided as part of a culturally tailored support program. The use of
broadly neutralizing antibodies for HIV prevention is a novel and promising
approach to be evaluated in efficacy trials. |
What is Bexsero? | Bexsero is a 4-component vaccine against capsular Meningococcus serogroup B (4CMenB), which has recently been licensed in Europe, Canada and Australia. | 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. Proteinaceous meningococcal B vaccines are under development, and the most
advanced, Bexsero, is currently being evaluated by the European Medicines
Agency. Approval, if granted, will be based on safety, immunogenicity, and
theoretical strain coverage established in vitro. Clinical effectiveness will
only be determined after market release. New, more effective influenza vaccines
are also being developed. A trivalent attenuated nasal influenza vaccine
(Fluenz) shows better efficacy in children than the classic trivalent seasonal
inactivated vaccine, but its use is restricted to children over 2 years of age
because of safety and efficacy considerations. The more potent trivalent (MF59)
adjuvated inactivated influenza vaccine (Fluad), licensed for adults over 65
years of age, is being evaluated through a pediatric investigation plan. This
vaccine could be useful for infants in whom unadjuvated inactivated vaccines are
poorly protective, but its safety must first be fully established. We previously demonstrated the immunogenicity and tolerability of the serogroup
B meningococcal vaccine, 4CMenB (Bexsero), in 11-17 y-olds randomized to receive
1, 2, or 3 doses at 1, 2, or 6 mo intervals. Participants in this extension
study provided an additional blood sample 18-24 mo after last vaccine dose, to
assess persistence of serum bactericidal activity with human complement (hSBA),
and to compare with age-matched 4CMenB-naïve controls. In the original study,
one month after one 4CMenB dose, 93% of subjects had seroprotective hSBA titers
(≥4) against indicator serogroup B strains for individual vaccine antigens
(fHbp, NadA and NZOMV), increasing to ~100% after two or three doses. After
18-24 mo, 62-73% of subjects given one dose had titers ≥4 against the three
antigens, significantly lower rates than after two (77-94%) or three (86-97%)
doses. Only proportions with titers ≥ 4 against NZOMV were significantly
different between the two (77%) and three (90%, p < 0.0001) dose groups. These
results confirm that two doses of 4CMenB, administered 1 to 6 mo apart, provide
good levels of bactericidal activity against serogroup B meningococci, which
were sustained at least 18-24 mo in over 64% of adolescents for all three tested
vaccine-related antigens. Effective polysaccharide(conjugate) vaccines against Neisseria meningitidis
serogroups A, C, W, and Y have been widely used, but serogroup B meningococci
remain a major cause of severe invasive meningococcal disease (IMD) worldwide,
especially in infants. Recently, a vaccine, 4CMenB (Bexsero(®)), containing
three recombit proteins, and outer membrane vesicles (OMV) derived from a
serogroup B meningococcal strain (MenB) has been licensed in Europe and
Australia and is indicated for persons aged 2 mo or older. This article
discusses what should be considered to enable a successful implementation of a
broad coverage MenB vaccine in national immunization programs. Epidemiology
data, vaccine characteristics including vaccine coverage, immunogenicity,
post-implementation surveillance and costs are relevant aspects that should be
taken into account when selecting an appropriate immunization strategy. The
potential impact on strain variation and carriage, as well as monitoring vaccine
effectiveness, and rare but potentially serious adverse events are points that
need to be included in a post-implementation surveillance plan. Neisseria meningitidis is the main cause of bacterial meningitis and sepsis in
the UK, and can potentially be lethal or cause long-term sequelae. Bexsero®
(4CMenB) is a new multi-component vaccine approved by the European Commission
for use in individuals aged ⩾2 months. A theoretical transmission model was
constructed to assess the long-term effectiveness of Bexsero compared to
standard care. The model was populated with UK-specific demographic data and
calibrated to ensure that the transmission dynamics of meningococcal disease in
the UK were adequately simulated. The model showed the best strategy to be a
routine vaccination programme at ages 2, 3, 4, 12 months and 14 years combined
with a 5-year catch-up programme in toddlers aged 12-24 months and adolescents
aged 15-18 years. This would lead to a 94% reduction in meningococcal cases or
150 000 cases and 15 000 deaths over a 100-year time-frame. Meningococcal disease is a major cause of morbidity and mortality worldwide. Its
epidemiology is currently dominated by five capsular serogroups (A, B, C, W, and
Y). While effective vaccines already exist for serogroups A, C, W and Y, except
for under clonal outbreaks, no vaccine was available against serogroup B.
Recently, a four component vaccine, Bexsero(®), designed to prevent infection
caused by this serogroup, has been approved in Europe and other Countries for
use in individuals from two months of age and older. The active components of
this vaccine are three recombit proteins identified by reverse vaccinology
combined with detergent extracted outer membrane vesicles (DOMV) prepared from a
New Zealand epidemic strain. Considering their intrinsic complexity, we
performed additional characterization of DOMVs on top of the standard quality
control testing carried out for batch release. We applied the Hi3 label-free
LC-MS(E) methodology to qualitatively and quantitatively characterize the DOMV
protein content. We first, successfully investigated the robustness and the
accuracy of the methodology for the DOMV characterization and we then applied it
to compare six DOMV production lots. Around 100 proteins were quantified from
each preparation. When classified according to their predicted cellular
localization, about 90% of the total protein amount belongs consistently to the
outer membrane compartment. Using nonparametric hypothesis testing and
complementary log-log linear regression, the quantifications of a subset of 21
proteins common to all lots and including approximately 90% (85-92%) of the
total protein amount quantified in any DOMV lot were found consistent across
lots. The relevance of these results is two-fold, showing that the Hi3
quantification methodology is robust for a broad range of proteins and
indicating that the manufacturing process currently used for the production of
the Bexsero(®) DOMV components is highly reproducible and consistent. 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/. Developing effective vaccines against Neisseria meningitidis serogroup B has
been challenging for several reasons, including the fact that the capsular
polysaccharide of N. meningitidis serogroup B is a poor antigen. Therefore,
studies have focused on developing vaccines that target capsular protein
meningococcal antigens using reverse vaccinology, a technique that predicts
likely vaccine candidates using computational analysis of the whole bacterial
genome. This has resulted in a multicomponent, recombit, meningococcal
serogroup B vaccine: 4CMenB (Bexsero(®), Novartis Vaccines & Diagnostics, NC,
USA), containing four main immunogenic components: two recombit fusion
proteins (Neisseria heparin-binding antigen-GNA1030 and factor H-binding
protein-GNA2091); recombit Neisserial adhesion A; and detergent-treated outer
membrane vesicles derived from the meningococcal NZ98/254 strain, where porin A
1.4 is the major immunodomit antigen. In this article, we summarize the
available clinical data on 4CMenB in healthy infants, adolescents and adults,
and discuss the methods available for assessing vaccine efficacy. OBJECTIVES: To assess the potential use of a protein-based meningococcal group B
(MenB) vaccine (Bexsero(®)) in addition to antibiotic chemoprophylaxis for
preventing secondary cases.
METHODS: Published studies on the risk of secondary meningococcal infections
were used to estimate the numbers needed to vaccinate (NNV) with Bexsero(®) to
prevent a secondary case in household and educational settings.
RESULTS: Most secondary cases occur within a few days of diagnosis in the index
case. Unlike conjugate vaccines, early protection offered after a single dose of
Bexsero(®) is likely to be low, particularly in young children, who are at
higher risk of secondary infection. NNV was dependent on predicted meningococcal
strain coverage, estimated onset of protection after one Bexsero(®) dose and
estimated vaccine efficacy. Even in the most favourable scenario where we assume
the vaccine is administered within 4 days of the index case and prevents 90% of
cases occurring after 14 days, the NNV for household contacts was >1000. NNV in
educational settings was much higher.
CONCLUSIONS: The estimated NNV should be taken into account when deciding policy
to recommend Bexsero(®) for close contacts of single cases in household or
educational settings. Bexsero(®) may have a protective role in clusters and
outbreaks. OBJECTIVE: To use mathematical and economic models to predict the
epidemiological and economic impact of vaccination with Bexsero, designed to
protect against group B meningococcal disease, to help inform vaccine policy in
the United Kingdom.
DESIGN: Modelling study.
SETTING: England.
POPULATION: People aged 0-99.
INTERVENTIONS: Incremental impact of introductory vaccine strategies simulated
with a transmission dynamic model of meningococcal infection and vaccination
including potential herd effects. Model parameters included recent evidence on
the vaccine characteristics, disease burden, costs of care, litigation costs,
and loss of quality of life from disease, including impacts on family and
network members. The health impact of vaccination was assessed through cases
averted and quality adjusted life years (QALYs) gained.
MAIN OUTCOME MEASURES: Cases averted and cost per QALY gained through
vaccination; programmes were deemed cost effective against a willingness to pay
of £20,000 (€25,420, $32,677) per QALY gained from an NHS and personal and
social services perspective.
RESULTS: In the short term, case reduction is greatest with routine infant
immunisation (26.3% of cases averted in the first five years). This strategy
could be cost effective at £3 (€3.8, $4.9) a vaccine dose, given several
favourable assumptions and the use of a quality of life adjustment factor. If
the vaccine can disrupt meningococcal transmission more cases are prevented in
the long term with an infant and adolescent combined programme (51.8% after 30
years), which could be cost effective at £4 a vaccine dose. Assuming the vaccine
reduces acquisition by 30%, adolescent vaccination alone is the most favourable
strategy economically, but takes more than 20 years to substantially reduce the
number of cases.
CONCLUSIONS: Routine infant vaccination is the most effective short term
strategy and could be cost effective with a low vaccine price. Critically, if
the vaccine reduces carriage acquisition in teenagers, the combination of infant
and adolescent vaccination could result in substantial long term reductions in
cases and be cost effective with competitive vaccine pricing. Invasive meningococcal infections can be life-threatening and cause severe
sequelae. Antibiotic therapy is only partially effective. Bexsero is the first
meningococcal B vaccine to be approved in the European Union. It contains four
capsular antigens from various strains of group B meningococci. Clinical trials
of this meningococcal B vaccine did not assess clinical protection. Two
immunogenicity studies in adults, one in adolescents and six in infants, are
available. They established the immunogenicity of the meningococcal B vaccine,
determined age-appropriate vaccination schedules, and verified that concomitant
administration of other vaccines did not undermine its immunogenicity. In the
absence of relevant clinical trials, an in vitro study showed that sera from
vaccinated individuals were likely to have bactericidal activity against 85% of
200 invasive meningococcal B strains isolated in France in 2007-2008. The
meningococcal B vaccine provoked local adverse effects in most vaccinees,
including local erythema, induration and pain. Fever occurred in about half of
vaccinated children. Six cases of Kawasaki syndrome have been reported in
children who received the vaccine, compared to only one case in control groups.
In practice, the harm-benefit balance of this meningococcal B vaccine justify
using it during outbreaks, provided the outbreak strain is covered by the
vaccine antigens. Vaccinees should be enrolled in studies designed to evaluate
clinical efficacy and to better determine the risk of Kawasaki syndrome. New generation vaccines are in demand to include only the key antigens
sufficient to confer protective immunity among the plethora of pathogen
molecules. In the last decade, large-scale genomics-based technologies have
emerged. Among them, the Reverse Vaccinology approach was successfully applied
to the development of an innovative vaccine against Neisseria meningitidis
serogroup B, now available on the market with the commercial name BEXSERO®
(Novartis Vaccines). The limiting step of such approaches is the number of
antigens to be tested in in vivo models. Several laboratories have been trying
to refine the original approach in order to get to the identification of the
relevant antigens straight from the genome. Here we report a new bioinformatics
tool that moves a first step in this direction. The tool has been developed by
identifying structural/functional features recurring in known bacterial
protective antigens, the so called "Protectome space," and using such
"protective signatures" for protective antigen discovery. In particular, we
applied this new approach to Staphylococcus aureus and Group B Streptococcus and
we show that not only already known protective antigens were re-discovered, but
also two new protective antigens were identified. Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is
composed of 3 main recombit proteins and an outer membrane vesicle component.
One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA),
is present as a fusion protein with the accessory protein genome-derived
neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene
encoding for GNA1030 is present and highly conserved in all Neisseria strains,
and although orthologs are present in numerous species, its biologic function is
unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a
homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly
involved in the electron transport chain and with antioxidant properties. Disc
diffusion assays on the wild-type and knockout mutant of GNA1030, in the
presence of various compounds, suggested that GNA1030 is not involved in
oxidative stress or electron chain transport per se, although it contributes to
constitutive refilling of the inner membrane with Ub8. These studies shed light
on an accessory protein present in Bexsero and reveal functional insights into
the family of related proteins. On the basis of our findings, we propose to name
the protein neisseria ubiquinone binding protein (NUbp). Recently approved in the EU, US, Australia, and Canada, 4CMenB (Bexsero(®), GSK
Vaccines) is a multi-component meningococcal B (MenB) vaccine containing 3
surface exposed recombit proteins (fHbp, NadA, and NHBA) and New Zealand
strain outer membrane vesicles (NZ OMV) containing PorA 1.4. The accepted
correlate of protection to assess response to MenB vaccines, the serum
bactericidal assay with human complement, is impractical for large panels of
strains with diverse antigenic profile and expression. Therefore, the
Meningococcal Antigen Typing System (MATS) was developed to identify MenB
strains with a high likelihood of being covered by 4CMenB. MATS is used to
assess MenB strain coverage without requiring sera, an advantage for testing
large panels of bacterial isolates. MATS provides an accurate, conservative
estimate of 4CMenB coverage. In a public-private partnership, 10 reference
laboratories around the world were established and standardized to facilitate
the timely collection and analysis of regional data. MATS has global public
health implications for informing local policy makers of the predicted effect of
the implementation of the 4CMenB vaccine. Coverage estimates are similar to or
better than other recently approved vaccines, ranging from 66% to 91%. The use
of MATS in post-vaccine implementation surveillance could provide data regarding
vaccine effectiveness in the field and duration of protection on a global scale
that will aid in the development of vaccine booster schedules, if necessary.
This MATS approach could potentially be applied rapidly to assess epidemiology
of other bacterial pathogens and coverage by other protein-based vaccines. Author information:
(1)Assistance Publique-Hôpitaux de Marseille (AP-HM), Pharmacie Usage Intérieur,
Hôpital Nord, 13015 Marseille, France.
(2)Assistance Publique-Hôpitaux de Marseille (AP-HM), Pharmacie Usage Intérieur,
Hôpital de la Conception, 13005 Marseille, France.
(3)Assistance Publique-Hôpitaux de Marseille (AP-HM), Oncopharma, 13005
Marseille, France; Aix-Marseille Université, CNRS, Institut de Chimie
Radicalaire ICR, UMR 7273, Laboratoire de Pharmaco-Chimie Radicalaire, 13005
Marseille, France.
(4)Aix-Marseille Université, CNRS, Institut de Chimie Radicalaire ICR, UMR 7273,
Laboratoire de Pharmaco-Chimie Radicalaire, 13005 Marseille, France; Assistance
Publique-Hôpitaux de Marseille (AP-HM), Service Central de la Qualité et de
l'Information Pharmaceutiques (SCQIP), 13005 Marseille, France.
(5)Aix-Marseille Université, CNRS, Institut de Chimie Radicalaire ICR, UMR 7273,
Laboratoire de Pharmaco-Chimie Radicalaire, 13005 Marseille, France; Assistance
Publique-Hôpitaux de Marseille (AP-HM), Service Central de la Qualité et de
l'Information Pharmaceutiques (SCQIP), 13005 Marseille, France. Electronic
address: [email protected]. Neisseria meningitidis still leads to deaths and severe disability in children,
adolescents and adults. Six different capsular groups of N. meningitidis cause
invasive meningococcal disease in the form of meningitis and septicaemia in
humans. Although conjugate meningococcal vaccines have been developed to provide
protection against four of the capsular groups causing most diseases in humans,
vaccines against capsular group B, which causes 85% of cases in Australia and
the United Kingdom, have only recently been developed. A capsular group B
meningococcal vaccine - 4CMenB (Bexsero) - has recently been licensed in the
European Union, Canada and Australia. In Australia, a submission for inclusion
of 4CMenB in the funded national immunization programme has recently been
rejected. The vaccine will now be introduced into the national immunization
programme in the United Kingdom following negotiation of a cost-effective price.
With the current low incidence of invasive meningococcal disease in many
regions, cost-effectiveness of a new capsular group B meningococcal vaccine is
borderline in both the United Kingdom and Australia. Cost-effectiveness of an
infant programme is determined largely by the direct protection of those
vaccinated and is driven by the higher rate of disease in this age group.
However, for an adolescent programme to be cost-effective, it must provide both
long-term protection against both disease and carriage. The potential of
vaccination to reduce the rate of severe invasive disease is a real possibility.
A dual approach using both an infant and adolescent immunization programme to
provide direct protection to those age groups at highest risk of meningococcal
disease and to optimize the potential herd immunity effects is likely to be the
most effective means of reducing invasive meningococcal disease. This commentary
aims to describe the known disease burden and consequences of meningococcal
disease, and the development and potential effectiveness of new capsular group B
meningococcal vaccines. 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. The 4-component meningococcal serogroup B vaccine 4CMenB (Bexsero) is the first
vaccine against this serogroup and has been approved by licensing authorities in
Europe, Canada and Australia. Therefore, the vaccine may enter soon nationwide
vaccine recommendation schemes. We report on a case of a 5-month-old infant who
developed prolonged upper extremity dysfunction after the second injection of
the 4CMenB vaccine in the left deltoid muscle and was concomitantly applied with
2 routine vaccinations. Myositis, periostitis, (peri-) vasculitis and axillary
inflammation were confirmed by magnetic resoce imaging. Two months after
initial initiation of an anti-inflammatory and an antibiotic treatment, symptoms
completely resolved. Administration of 3 vaccines requires clear recommendations
for the preferred injection site in infants because increased reactogenicity of
4CMenB may lead to local severe adverse events. The United Kingdom is the first country to introduce Bexsero(®) (GSK
Biologicals), a multicomponent, protein-based vaccine against meningococcal
group B (MenB), into the national infant immunisation programme. This vaccine is
like no other licensed vaccine and poses a number of implementation and
surveillance challenges in England. From 01 September 2015, UK infants were
offered a reduced two dose primary immunisation schedule at 2 and 4 months
followed by a booster at 12 months. Because of high rates of fever
post-vaccination, parents were advised to give their infants three doses of
prophylactic paracetamol, with the first dose given as soon as possible after
the primary MenB vaccination dose. Since the vaccine only protects against
73-88% of MenB strains causing invasive disease in England, clinical isolates
and PCR-positive samples will require extensive characterisation by the
Meningococcal Reference Unit (MRU) at Public Health England (PHE) in order to
monitor vaccine effectiveness and identify potential vaccine failures. PHE is
also conducting detailed clinical and epidemiological surveillance to assess the
impact of the MenB immunisation programme on the morbidity and mortality
associated with invasive meningococcal disease in infants and young children. In December 2013 Bexsero® became available in Germany for vaccination against
serogroup B meningococci (MenB). In August 2015 the German Standing Committee on
Vaccination (STIKO) endorsed a recommendation for use of this vaccine in persons
at increased risk of invasive meningococcal disease (IMD). This background paper
summarizes the evidence underlying the recommendation. Bexsero® is based on
surface protein antigens expressed by about 80% of circulating serogroup B
meningococci in Germany. The paper reviews available data on immunogenicity and
safety of Bexsero® in healthy children and adolescents; data in persons with
underlying illness and on the effectiveness in preventing clinical outcomes are
thus far unavailable.STIKO recommends MenB vaccination for the following persons
based on an individual risk assessment: (1) Persons with congenital or acquired
immune deficiency or suppression. Among these, persons with terminal complement
defects and properdin deficiency, including those under eculizumab therapy, are
at highest risk with reported invasive meningococcal disease (IMD) incidences up
10,000-fold higher than in the general population. Persons with asplenia were
estimated to have a ~ 20-30-fold increased risk of IMD, while the risk in
individuals with other immune defects such as HIV infection or
hypogammaglobulinaemia was estimated at no more than 5-10-fold higher than the
background risk. (2) Laboratory staff with a risk of exposure to N. meningitidis
aerosols, for whom an up to 271-fold increased risk for IMD has been reported.
(3) Unvaccinated household (-like) contacts of a MenB IMD index case, who have a
roughly 100-200-fold increased IMD risk in the year after the contact despite
chemoprophylaxis. Because the risk is highest in the first 3 months and full
protective immunity requires more than one dose (particularly in infants and
toddlers), MenB vaccine should be administered as soon as possible following
identification of the serogroup of the index case. Although rare, invasive meningococcal disease remains an important cause of
mortality and morbidity in children and young adults. Vaccines have been
successfully introduced to help protect against meningococcal disease caused by
serogroups A, C, W and Y, but until recently, a vaccine for serogroup B (MenB)
was not available. In many industrialised countries, MenB causes the majority of
meningococcal disease. Moreover, MenB outbreaks occur unpredictably,
particularly in high-risk populations, such as university students. In 2013,
Bexsero(®) became the first broad-coverage vaccine to be licensed for active
immunisation against MenB disease. Bexsero is now licensed in more than 35
countries worldwide for varying age groups, including the EU, Australia, Brazil,
Canada, Chile, Uruguay and the USA. Clinical recommendations for the use of
Bexsero have been published in several countries. Recommendations include use in
high-risk groups, outbreak control and routine infant immunisation. Since
initial licensure, considerable clinical experience has been gained. In Canada,
43,740 individuals received Bexsero during a vaccination programme in the
Saguenay-Lac-Saint-Jean region of Quebec, where local disease incidence was
high. In the USA, Bexsero was administered to >15,000 individuals during two
college outbreaks prior to licensure, under an Investigational New Drug
protocol. In the UK, the Joint Committee on Vaccination and Immunisation has
recommended the inclusion of Bexsero in the routine immunisation schedule for
infants. Publically funded vaccination programmes have been initiated in Italy,
and there has been widespread use of the vaccine outside of publically
reimbursed programmes. Overall, >1,000,000 doses of Bexsero have been
distributed in 19 countries worldwide since 2013. The emerging clinical
experience with Bexsero is consistent with findings from pre-licensure clinical
studies, and no new safety concerns have been identified. Additional data on
length of protection, potential impact on meningococcal carriage and
transmission and strain coverage have also been published and will be reviewed. Immunisation against meningococcal meningitis has a long history, which has
passed through several phases: the studies by Flexner, extraction of the
polysaccharide capsule, the development of monovalent and multivalent conjugate
vaccines, the outer membrane vesicle vaccines up to the development of effective
and safe vaccines for meningococcal B invasive disease through the application
of the techniques of molecular biology and reverse vaccinology. The new
available vaccines are Bexsero® and Trumenba®. Bexsero ® has been approved and
is available in Europe, the USA, Canada, Australia and Chile, and is currently
under review in Brazil for the prevention of MenB invasive disease in subjects ≥
2 months. Trumemba® is currently approved only in the USA, for use in
adolescents and young adults. At present, the greatest obstacle to the extensive
use of these vaccines in industrialised countries is the high cost and the need
administer multiple doses in infants. However, in some European countries and in
some Italian Regions, strategies (free and active call) to fight the disease
through vaccination (Bexsero®) are already in place. A century of traditional vaccinology lost the fight against meningococcus
serogroup B (MenB). However, thanks to an innovative genome-based approach, the
first broadly effective MenB vaccine, Bexsero® (GSK Vaccines), was developed and
has been licensed for use in various age groups by the European Commission and
other regulatory authorities. Genes encoding for the main meningococcus B
antigens were identified and screened in order to achieve a broadly protective
vaccine, taking into account the fact that meningococcus B has many different
subtypes whose membrane proteins may be different. Since the antigens selected
for Bexsero® are also harbored by meningococci belonging to other serogroups
there may be the potential for Bexsero® to offer a certain level of protection
against non-B serogroups. Therefore preliminary studies were carried out to
investigate the potential of the vaccine to also provide a degree of cross
protection against non-B serogroups. Here we review the potential for Bexsero®
to offer a certain level of protection against the diversity of meningococcus
type B subtypes and its potential ability to offer some cross protection from
non-B serogroups. Lastly, we describe the future perspectives in pentavalent
meningococcal vaccine (ABCWY) development which hopefully will result in a
vaccine able to help prevent Invasive Meningococcal Diseases (IMD) from the
majority of currently circulating meningococcal strains. INTRODUCTION: Despite its low incidence in France, invasive serogroup B
meningococcal disease remains a public health concern. A new vaccine against the
disease, Bexsero(®), has been licensed in the EU. We studied the epidemiological
impact and cost-effectiveness of routine vaccination using Bexsero(®) in order
to inform the decision-making process regarding its potential inclusion in the
vaccination schedule.
METHODS: A multi-generational Markov model was used. Time horizon was set to 100
years. Five vaccination strategies were evaluated: infants at 3, 5, 6 and 13
months, toddlers at 13, 15 and 27 months and adolescents at 15 years provided 2
doses one month apart. A booster dose at 15 years old and a catch-up for 15
years old subjects during the first 15 years of the programme were added to the
infant and toddler strategies. Costs per QALY gained were computed from a
restricted societal perspective including direct costs only. Herd immunity was
simulated in an alternative base-case scenario and sensitivity analyses.
RESULTS: In the base-case analysis without herd immunity and with all cohorts
vaccinated, at € 40 per vaccine dose, routine infant vaccination would provide
the lowest cost per QALY gained (€ 380,973) despite only preventing 18% of
cases. Under the assumption of herd immunity, the adolescent vaccination would
provide the lowest cost per QALY gained (€ 135,902) preventing 24% of cases.
Infant vaccination with a late booster and catch-up would prevent 51% of cases
with a cost of € 188,511 per QALY gained.
CONCLUSIONS: Given current meningococcal epidemiology in France and the
available data on the protection provided by Bexsero(®), our modelling work
showed that routine vaccination against serogroup B meningococcal disease is not
cost-effective. The European Medicines Agency has approved a multicomponent serogroup B
meningococcal vaccine (Bexsero®) for use in individuals of 2 months of age and
older. A cost-effectiveness analysis (CEA) from the societal and Italian
National Health Service perspectives was performed in order to evaluate the
impact of vaccinating Italian infants less than 1 y of age with Bexsero®, as
opposed to non-vaccination. The analysis was carried out by means of Excel
Version 2011 and the TreeAge Pro® software Version 2012. Two basal scenarios
that differed in terms of disease incidence (official and estimated data to
correct for underreporting) were considered. In the basal scenarios, we
considered a primary vaccination cycle with 4 doses (at 2, 4, 6 and 12 months of
age) and 1 booster dose at the age of 11 y, the societal perspective and no cost
for death. Sensitivity analyses were carried out in which crucial variables were
changed over probable ranges. In Italy, on the basis of official data on disease
incidence, vaccination with Bexsero® could prevent 82.97 cases and 5.61 deaths
in each birth cohort, while these figures proved to be three times higher on
considering the estimated incidence. The results of the CEA showed that the
Incremental Cost Effectiveness Ratio (ICER) per QALY was €109,762 in the basal
scenario if official data on disease incidence are considered and €26,599 if
estimated data are considered. The tornado diagram indicated that the most
influential factor on ICER was the incidence of disease. The probability of
sequelae, the cost of the vaccine and vaccine effectiveness also had an impact.
Our results suggest that vaccinating infants in Italy with Bexsero® has the
ability to significantly reduce meningococcal disease and, if the probable
underestimation of disease incidence is considered, routine vaccination is
advisable. An outbreak of Neisseria meningitidis serotype B infection occurred at a small
residential university; public health announced an organizational vaccination
program with the 4-component Meningococcal B (4CMenB) vaccine (Bexsero(TM),
Novartis/GlaxoSmithKline Inc.) several days later. Since there were limited
published data on reactogenicity of 4CMenB in persons over 17years of age, this
study sought to conduct rapid surveillance of health events in vaccinees and
controls using an online survey. Vaccine uptake was 84.7% for dose 1 (2967/3500)
and 70% (2456/3500) for dose 2; the survey response rates were 33.0% (987/2967)
and 18.7% (459/2456) in dose 1 and dose 1 recipients respectively, and 12% in
unvaccinated individuals (63/533). Most students were 20-29years of age
(vaccinees, 64.0%; controls, 74.0). A new health problem or worsening of an
existing health problem was reported by 30.0% and 30.3% of vaccine recipients
after doses 1 and 2 respectively; and by 15.9% of controls. These health
problems interfered with the ability to perform normal activities in most
vaccinees reporting these events (74.7% post dose 1; 62.6% post dose 2), and in
60% of controls. The health problems led to a health care provider visit
(including emergency room) in 12.8% and 14.4% of vaccinees post doses 1 and 2,
respectively and in 40% of controls. The most common reactions in vaccinees were
injection site reactions (20.6% post dose 1, 16.1% post dose 20 and non-specific
systemic complaints (22.6% post dose 1, 17.6% post dose 2). No hospitalizations
were reported. An online surveillance program during an emergency meningococcal
B vaccine program was successfully implemented, and detected higher rates of
health events in vaccinees compared to controls, and high rates of both
vaccinees and controls seeking medical attention. The types of adverse events
reported by young adult vaccinees were consistent with those previously. This policy statement provides recommendations for the prevention of serogroup B
meningococcal disease through the use of 2 newly licensed serogroup B
meningococcal vaccines: MenB-FHbp (Trumenba; Wyeth Pharmaceuticals, a subsidiary
of Pfizer, Philadelphia, PA) and MenB-4C (Bexsero; Novartis Vaccines, Siena,
Italy). Both vaccines are approved for use in persons 10 through 25 years of
age. MenB-FHbp is licensed as a 2- or 3-dose series, and MenB-4C is licensed as
a 2-dose series for all groups. Either vaccine is recommended for routine use in
persons 10 years and older who are at increased risk of serogroup B
meningococcal disease (category A recommendation). Persons at increased risk of
meningococcal serogroup B disease include the following: (1) persons with
persistent complement component diseases, including inherited or chronic
deficiencies in C3, C5-C9, properdin, factor D, or factor H or persons receiving
eculizumab (Soliris; Alexion Pharmaceuticals, Cheshire, CT), a monoclonal
antibody that acts as a terminal complement inhibitor by binding C5 and
inhibiting cleavage of C5 to C5A; (2) persons with anatomic or functional
asplenia, including sickle cell disease; and (3) healthy persons at increased
risk because of a serogroup B meningococcal disease outbreak. Both serogroup B
meningococcal vaccines have been shown to be safe and immunogenic and are
licensed by the US Food and Drug Administration for individuals between the ages
of 10 and 25 years. On the basis of epidemiologic and antibody persistence data,
the American Academy of Pediatrics agrees with the Advisory Committee on
Immunization Practices of the Centers for Disease Control and Prevention that
either vaccine may be administered to healthy adolescents and young adults 16
through 23 years of age (preferred ages are 16 through 18 years) to provide
short-term protection against most strains of serogroup B meningococcal disease
(category B recommendation). Neisseria meningitidis serogroup B is the most prevalent cause of invasive
meningococcal disease in Europe and members of laboratories working on
meningococci are at risk due to frequent handling. Recommendation for
anti-meningococcal vaccination among these workers has been recently updated
upon the licensure in Europe of Bexsero® vaccine. We tested the immunogenicity
and safety of this vaccine among adults laboratory staff using the recommended
schedule of 2 doses at 5 weeks interval. The vaccine was well tolerated in spite
of frequent local side effects and all participants reported at least one side
effect after each dose. Immunogenicity was evaluated 6 weeks and one year after
the second dose. All participants showed increase in their bactericidal titers
against the components of the vaccine 6 weeks after the second dose, however
titers declined significantly one year later. We assessed the cost-effectiveness of offering catch-up vaccination with Bexsero
against meningococcal disease to children too old to receive the vaccine under
the recently introduced infant programme. Offering catch-up vaccination to
increasingly older children is less economically attractive because of declining
disease burden. We estimate catch-up vaccination of 1year old children could be
cost-effective, incremental on the infant programme with a vaccine price of ⩽£8
per dose. Extending vaccination to 2year olds could only be cost-effective
(incremental on infant and 1year old catch-up) with a vaccine price of ⩽£3 per
dose and was not cost-effective in sensitivity analyses with more conservative
vaccine assumptions. Extending catch-up further to 3-4year olds was not
cost-effective. Employing the current criteria for assessing vaccines, our
models suggest that even with low vaccine prices only catch-up vaccination in
1year old children could be cost-effective, when considered incrementally on the
infant programme. |
Is there a role for gamma knife in treatment of Obsessive-Compulsive Disorder? | Yes. Gamma knife radiosurgery is being increasingly to treat refractory obsessive- compulsive disorder (OCD). It is reserved for severe, treatment-refractory disease that has not responded to multiple treatments. | Normal brain tissue is probably the most important tissue compartment in the
brain involved in adverse radiation effects (AREs). The capabilities of
computerized tomography, magnetic resoce imaging, and positron emission
tomography in repeat examinations of a treatment outcome provide a baseline by
which to monitor the AREs in vivo and to follow their sequential changes. This
article relates the AREs seen after radiosurgery to the dose/volume and specific
tissue effects established from 30 years of collected experience with
radiosurgery at the Karolinska Hospital. Intractable OCD and depression cause tremendous suffering in those affected and
in their families. The impaired ability to function of those affected imposes a
heavy burden on society as a whole. Existing data suggest that lesion procedures
offer benefit to a large proportion (ranging from about 35%-70%) of patients
with intractable OCD and depression. The literature also suggests that although
serious long-term adverse events have occurred, these are relatively infrequent
overall. Methodologic limitations of the earlier reports on any of these
procedures were described previously in this article. The major academic centers
conducting this work have since been obtaining systematic prospective data using
modern assessment tools. Nevertheless, even with improved methodologies, more
recent studies confront some remaining issues that have been difficult to
overcome fully. First, the number of patients who have received any one
procedure has been relatively small, constraining statistical power. This limits
the ability of researchers to enhance patient selection based on clinical
characteristics. This is important, because patients with intractable OCD and
depression referred for neurosurgery have high rates of comorbid Axis I
diagnoses, personality disorders, and functional impairments, which may have
value in predicting response. Other features, such as age of onset, chronicity,
and symptom subtypes, may be likewise useful. Another key factor in response may
be postoperative management, which has varied most over time but also across
patients enrolled in trials. As noted previously, randomized controlled trials
of neurosurgical treatment for intractable psychiatric illness have not been
reported, although one has been proposed for gamma knife capsulotomy in
intractable OCD [23]. The development of deep brain stimulation has also made
sham-controlled studies possible and also allows within-patient designs to be
considered. Bearing these problems in mind, the literature does provide
important guidance on a number of key points, including approaches to referral,
patient selection, and the need for long-term prospective follow-up and
postoperative management. Nevertheless, important gaps in knowledge remain in
all these areas. Research is expected to narrow these gaps in a number of ways,
including patient selection, optimizing the procedures themselves, and
understanding the mechanisms of therapeutic action. Neuroimaging studies will
play a key role in achieving these aims (see the article by Rauch in this
issue). So will cross-species translational research on the anatomy and
physiology of the pathways implicated in the pathophysiology and response to
treatment in these disorders. Future research in psychiatric neurosurgery must
proceed cautiously. A recent editorial statement of the OCD-DBS Collaborative
Group [26] recommends a minimum set of standards for any multidisciplinary teams
contemplating work in this domain. The rationale for those standards is found
throughout this issue and is especially developed in the article by Fins. The
need for safe and effective therapeutic options for people suffering with these
severe illnesses is just as clear. The experience over the last several decades
provides grounds for careful optimism that refined lesion procedures or
reversible deep brain stimulation may relieve suffering and improve the lives of
people with these devastating disorders. Serotonin reuptake inhibitors (SRIs), especially potent ones given at high doses
over long periods of time, are often effective in the treatment of
obsessive-compulsive disorder (OCD). However, a large percentage of patients do
not respond to treatment with SRIs, and those who do respond often do not fully
remit, which should be the standard goal of treatment in OCD. If a patient has
been treated for several months and has not yet responded to treatment with
several SRIs, the physician should perform a careful assessment of resistant
and/or residual clinical symptoms and any comorbid conditions to determine which
next-step treatment would be the most appropriate. One strategy for patients who
have not responded to treatment with an SRI is to switch them to a
serotonin-norepinephrine reuptake inhibitor, because some patients may respond
better to agents that target multiple systems. Another promising approach is the
augmentation of SRIs with neuroleptics. In addition, open trials have shown that
intravenous (IV) clomipramine and IV citalopram may be effective in the
treatment of resistant OCD. Novel pharmacotherapeutic treatments and
electroconvulsive therapy have been attempted, with mixed success. Recently,
researchers have been studying repetitive transcranial magnetic stimulation,
vagal nerve stimulation, and neurosurgical approaches such as gamma knife
capsulotomy and deep brain stimulation to learn if these procedures are
effective in treating treatment-resistant OCD. Repetitive transcranial magnetic
stimulation has possibilities not only as a therapy but also as an instrument
that can help researchers describe the neurocircuitries involved in OCD. More
results are needed before the effectiveness of the nonpharmacologic treatments
for OCD can be determined. Five refractory obsessive-compulsive patients were assessed using a
neuropsychological battery after a modified gamma knife capsulotomy. The
surgical technique was not associated with profound cognitive deficits. The
authors found improvements in attention, vocabulary, learning, abstract
reasoning, and memory. BACKGROUND: Obsessive compulsive disorder (OCD), in its severe form, can cause
tremendous disability for affected patients.
OBJECTIVE: To evaluate the results following bilateral radiosurgical anterior
capsulotomy for severe medically refractory OCD.
METHODS: We performed gamma knife anterior capsulotomy (GKAC) on 3 patients with
extreme, medically intractable OCD. According to our protocol, all patients were
evaluated by at least 2 psychiatrists who recommended surgery. The patient had
to request the procedure, and had to have severe OCD according to the Yale-Brown
Obsessive Compulsive Scale (YBOCS). Patient ages were 37, 55, and 40 years, and
pre-radiosurgery YBOCS scores were 34/40, 39/40, and 39/40. Bilateral lesions
were created with 2 4-mm isocenters to create an oval volume in the ventral
internal capsule at the putaminal midpoint. A maximum dose of 140 or 150 Gy was
used.
RESULTS: There was no morbidity after the procedure, and all patients returned
immediately to baseline function. All patients noted significant functional
improvements, and reduction in OCD behavior. Follow-up was at 55, 42, and 28
months. The first patient reduced her YBOCS score from 34 to 24. One patient
with compulsive skin picking and an open wound had later healing of the chronic
wound and a reduction in the YBOCS score from 39 to 8. At 28 months, the third
patient is living and working independently, and her YBOCS score is 18.
CONCLUSION: Within a strict protocol, gamma knife radiosurgery provided
improvement of OCD behavior with no adverse effects. This technique should be
evaluated further in patients with severe and disabling behavioral disorders. Radiosurgery for psychiatric disorders has been performed for more than 50
years. The use of deep brain stimulation has recently been expanded to the
investigational treatment of specific psychiatric disorders. A literature review
of past studies incorporating radiosurgical stereotactic lesions for psychiatric
disorders was performed to provide historic context and possible guidance for
current and future attempts at treating psychiatric disorders, especially by
gamma capsulotomy. The anatomic target localization, dose selection, and the
outcome of the radiosurgical procedures were reviewed, and the evolutions of
lesioning strategies were analyzed with particular emphasis on the dose
selection. Large-scale prospective studies with strict inclusion and
well-defined, objective outcome criteria are necessary for defining the role of
radiosurgery for the treatment of psychiatric disorders. Functional neurological disorders (FND) have been a challenge to treat both for
neurologists and neurosurgeons. Various ablative as well as non-ablative
techniques have been used to treat these disorders. Gamma knife radiosurgery
(GKRS) is also being practised to treat refractory obsessive- compulsive
disorder (OCD). The subsequent complications of GKRS reported have been
variable, with headache being the most common. We discuss here a rare
complication of 'late onset radiation necrosis in bilateral caudate nuclei' in a
patient after receiving GKRS three years back. This case highlights the need to
be more cautious before administering ablative procedures in patients suffering
with functional disorders. IMPORTANCE: Select cases of intractable obsessive-compulsive disorder (OCD) have
undergone neurosurgical ablation for more than half a century. However, to our
knowledge, there have been no randomized clinical trials of such procedures for
the treatment of any psychiatric disorder.
OBJECTIVE: To determine the efficacy and safety of a radiosurgery (gamma ventral
capsulotomy [GVC]) for intractable OCD.
DESIGN, SETTING, AND PARTICIPANTS: In a double-blind, placebo-controlled,
randomized clinical trial, 16 patients with intractable OCD were randomized to
active (n = 8) or sham (n = 8) GVC. Blinding was maintained for 12 months. After
unblinding, sham-group patients were offered active GVC.
INTERVENTIONS: Patients randomized to active GVC had 2 distinct isocenters on
each side irradiated at the ventral border of the anterior limb of the internal
capsule. The patients randomized to sham GVC received simulated radiosurgery
using the same equipment.
MAIN OUTCOMES AND MEASURES: Scores on the Yale-Brown Obsessive-Compulsive Scale
(Y-BOCS) and the Clinical Global Impression-Improvement (CGI-I) Scale. Response
was defined as a 35% or greater reduction in Y-BOCS severity and "improved" or
"much improved" CGI-I ratings.
RESULTS: Three of 8 patients randomized to active treatment responded at 12
months, while none of the 8 sham-GVC patients responded (absolute risk
reduction, 0.375; 95% CI, 0.04-0.71). At 12 months, OCD symptom improvement was
significantly higher in the active-GVC group than in the sham group (Y-BOCS,
P = .046; Dimensional Y-BOCS, P = .01). At 54 months, 2 additional patients in
the active group had become responders. Of the 4 sham-GVC patients who later
received active GVC, 2 responded by post-GVC month 12. The most serious adverse
event was an asymptomatic radiation-induced cyst in 1 patient.
CONCLUSIONS AND RELEVANCE: Gamma ventral capsulotomy benefitted patients with
otherwise intractable OCD and thus appears to be an alternative to deep-brain
stimulation in selected cases. Given the risks inherent in any psychiatric
neurosurgery, such procedures should be conducted at specialized centers.
TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01004302. |
What is the mechanism of action of the biguanide class of diabetes drugs? | this biguanide is an oral insulin-sensitizing agent capable of increasing insulin sensitivity and decreasing plasma fasting insulin levels. | Since impaired glucose tolerance (IGT) is a major risk factor for
non-insulin-dependent diabetes mellitus (NIDDM), some kinds of intervention
aiming to prevent or to delay the onset of NIDDM in subjects with IGT might be
considered. Besides life style modification, drug therapy which could correct
insulin deficiency and insulin resistance, might prevent progression to NIDDM.
One agent is an alpha-glucosidase inhibitor, which delays the absorption of
glucose from the intestine. The resulting decrease in postprandial hyperglycemia
and hyperinsulinemia could theoretically decrease insulin resistance in IGT
subjects and, it is hoped, prevent or delay progression to NIDDM. Metformin, an
antihyperglycemic drug of the biguanide class, may be effective in subjects with
IGT by reducing hepatic glucose output, enhancing insulin sensitivity, or
through other mechanisms such as weight loss. New insulin sensitizers, such as
troglitazone and pioglitazone, improve insulin-mediated glucose disposal by
enhancing tissue sensitivity to the actions of insulin and reversing the insulin
resistance, characteristic of NIDDM. Sulfonylureas might be another candidates
of drug intervention to IGT whose insulin secretory abilities are markedly
reduced. As far as the question, "Can NIDDM be prevented or delayed?" is
concerned, a prospective study using life style modification or above-mentioned
drugs, should be performed on long-term basis. Metformin is an oral anti-diabetic drug of the biguanide class that is commonly
used to treat type 2 diabetes mellitus. This study examined the molecular
mechanism for the action of metformin on osteoblast differentiation.
Metformin-induced mRNA expression of the osteogenic genes and small heterodimer
partner (SHP) in MC3T3E1 cells were determined by RT-PCR and real-time PCR.
Metformin increased significantly the expression of the key osteogenic genes,
such as alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP)
as well as SHP. Transient transfection assays were performed in MC3T3E1 cells to
confirm the effects of metformin on SHP, OC and Runx2 promoter activities.
Metformin increased the transcription of the SHP and OC genes, and the metformin
effect was inhibited by domit negative form of AMPK (DN-AMPK) or compound C
(an inhibitor of AMPK). The adenoviral overexpression of SHP increased
significantly the level of ALP staining and OC production. However, metformin
did not have any significant effect on osteogenic gene expression, ALP staining
and activity, and OC production in SHP null (SHP-/-) primary calvarial cells.
Moreover, upstream stimulatory factor-1 (USF-1) specifically mediated
metformin-induced SHP gene expression. In addition, metformin-induced AMPK
activation increased the level of Runx2 mRNA and protein. However, USF-1 and SHP
were not involved in metformin-induced Runx2 expression. Transient transfection
and chromatin immunoprecipitation assays confirmed that metformin-induced SHP
interacts physically and forms a complex with Runx2 on the osteocalcin gene
promoter in MC3T3E1 cells. These results suggest that metformin may stimulate
osteoblast differentiation through the transactivation of Runx2 via
AMPK/USF-1/SHP regulatory cascade in mouse calvaria-derived cells. Male infertility has been increasing over the last decades being nowadays a
pressing health problem. Diabetes mellitus (DM) can contribute directly or
indirectly to male infertility due to an abnormal spermatogenesis, which results
in a decreased sperm quality. Type 2 Diabetes mellitus (T2DM) is responsible for
the vast majority of DM cases, being frequently treated with oral antidiabetic
drugs. Metformin is the most cost-effective therapy for the treatment of T2DM.
This biguanide is an oral insulin-sensitizing agent capable of increasing
insulin sensitivity and decreasing plasma fasting insulin levels. The main
metabolic action of this drug occurs in the liver. However, it has been shown
that metformin acts on a variety of organs including the male reproductive
system. With the rising numbers of diabetic individuals among younger
populations, there is an increase in the consumption of metformin in individuals
of this age group. As a result, it is important to discuss the role of metformin
in male fertility. This review presents the most recent data available from
studies on the effects of metformin on male reproductive system. Together with
the discussion of these effects, their significance to male fertility is also
debated. The most widely prescribed oral anti-diabetic agent today in the world today is
a member of the biguanide class of drugs called metformin. Apart from its use in
diabetes, it is currently being investigated for its potential use in many
diseases such as cancer, cardiovascular diseases, Alzheimer's disease, obesity,
comorbidities of diabetes such as retinopathy, nephropathy to name a few.
Numerous in-vitro and in-vivo studies as well as clinical trials have been and
are being conducted with a vast amount of literature being published every day.
Numerous mechanisms for this drug have been proposed, but they have been unable
to explain all the actions observed clinically. It is of interest that insulin
has a stimulatory effect on cellular growth. Metformin sensitizes the insulin
action but believed to be beneficial in cancer. Like -wise metformin is shown to
have beneficial effects in opposite sets of pathological scenario looking from
insulin sensitization point of view. This requires a comprehensive review of the
disease conditions which are claimed to be affected by metformin therapy. Such a
comprehensive review is presently lacking. In this review, we begin by examining
the history of metformin before it became the most popular anti-diabetic
medication today followed by a review of its relevant molecular mechanisms and
important clinical trials in all areas where metformin has been studied and
investigated till today. We also review novel mechanistic insight in metformin
action in relation to microbiome and elaborate implications of such aspect in
various disease states. Finally, we highlight the quandaries and suggest
potential solutions which will help the researchers and physicians to channel
their research and put this drug to better use. |
Where is the proteasome located? | The proteasome can be found in perinuclear and nuclear location, as well as in cytosolic compartments, such as mitochondria and endoplasmic reticulum. Proteasome-mediated degradation of cell cycle regulatory proteins, production and loading of antigenic peptides onto HLA molecules, and transient homing of diverse virion proteins required for entry and/or egress have been also shown to be coordinated at the centrosome. | N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL), which reversibly inhibits the
proteasome in addition to other proteases, and a more specific irreversible
inhibitor of the proteasome, lactacystin, were found to cause the accumulation
of major histocompatibility complex (MHC) class I heavy chains in the cytosol of
the beta2-microglobulin-deficient cell line Daudi and the TAP-deficient cell
line .174. These cell lines, which are severely impaired in their ability to
fold MHC class I heavy chain, showed an accumulation of soluble class I heavy
chains at different rates over a period of hours in the presence of LLnL. The
accumulation of soluble class I heavy chains in the presence of either LLnL or
lactacystin was easily revealed in Daudi and .174 but almost undetectable in a
Daudi transfectant expressing beta2-microglobulin and in 45.1, the wild-type
parent of .174. The soluble class I heavy chain was also found to be devoid of
its N-linked glycan and to be located in the cytosol. When the gene for ICP47, a
herpes simplex virus protein that blocks the translocation of peptides into the
endoplasmic reticulum, was transfected into 45.1, a similar accumulation of
soluble MHC class I heavy chain was detectable. These data suggest that in cells
where the MHC class I molecule is unable to assemble properly, the misfolded
heavy chain is removed from the endoplasmic reticulum to the cytosol,
deglycosylated, and degraded by the proteasome. Proteasomes are proteolytic complexes involved in non-lysosomal degradation
which are localized in both the cytoplasm and the nucleus. The dynamics of
proteasomes in living cells is unclear, as is their targeting to proteins
destined for degradation. To investigate the intracellular distribution and
mobility of proteasomes in vivo, we generated a fusion protein of the proteasome
subunit LMP2 and the green fluorescent protein (GFP). The LMP2-GFP chimera was
quantitatively incorporated into catalytically active proteasomes. The
GFP-tagged proteasomes were located within both the cytoplasm and the nucleus.
Within these two compartments, proteasomes diffused rapidly, and bleaching
experiments demonstrated that proteasomes were transported slowly and
unidirectionally from the cytoplasm into the nucleus. During mitosis, when the
nuclear envelope has disintegrated, proteasomes diffused rapidly throughout the
dividing cell without encountering a selective barrier. Immediately after cell
division, the restored nuclear envelope formed a new barrier for the diffusing
proteasomes. Thus, proteasomes can be transported unidirectionally over the
nuclear membrane, but can also enter the nucleus upon reassembly during cell
division. Since proteasomes diffuse rapidly in the cytoplasm and nucleus, they
may perform quality control by continuous collision with intracellular proteins,
and degrading those proteins that are properly tagged or misfolded. Two new forms of proteasomes, designated as the endoplasmic reticulum (ER)
membrane-associated proteasome (ERa proteasome) and ER membrane-bound proteasome
(ERb proteasome), were purified to homogeneity from 0.0125 and 2.5% sodium
cholate extracts, respectively, of a rat liver microsomal fraction. SDS-PAGE
analysis revealed that the purified ERa and ERb proteasomes were composed of
multiple subunits similar to the cytosolic 20S proteasome. However,
electrophoretic, structural and immunochemical differences between the ERa, ERb
and cytosolic 20S proteasomes were observed on native PAGE, two-dimensional (2D)
PAGE, and immunoblot analyses. Purification of ERb from a 2.5% sodium cholate
extract of the trypsin-treated microsomal fraction yielded a trypsin-modified
form of ERb (tERb), which lacked the C2 subunit at least. On the other hand, no
ERa proteasome was obtained from the 0.0125% sodium cholate extract of the
trypsin-treated microsomes, suggesting that ERa and ERb are ER
membrane-associated and -bound proteasomes, respectively. The ERa, ERb, and
cytosolic 20S proteasomes exhibited similar specificities as to peptide
hydrolyzing activity, although differences in their activities were noted in the
presence of SDS and phospholipid. With respect to the proteolysis of protein
substrates, only the ERb proteasome cleaved beta-casein, and it also degraded
reduced and carboxymethylated lysozyme considerably faster than the cytosolic
20S and ERa proteasomes. Collectively our results suggest that the ERa and ERb
proteasomes may play roles in intracellular proteolysis distinct from that of
the cytosolic 20S proteasome. Proteasomes can exist in several different molecular forms in mammalian cells.
The core 20S proteasome, containing the proteolytic sites, binds regulatory
complexes at the ends of its cylindrical structure. Together with two 19S ATPase
regulatory complexes it forms the 26S proteasome, which is involved in
ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory
complexes (REG, PA28) which play a role in antigen processing, as do the three
variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the
present study, we have investigated the subcellular distribution of the
different forms of proteasomes using subunit specific antibodies. Both 20S
proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic
and microsomal preparations isolated from rat liver. LMP7 was enriched
approximately two-fold compared with core alpha-type proteasome subunits in the
microsomal preparations. 20S proteasomes were more abundant than 26S
proteasomes, both in liver and cultured cell lines. Interestingly, some
significant differences were observed in the distribution of different subunits
of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to
be relatively enriched in nuclear fractions from rat liver, and
immunofluorescent labelling of cultured cells with anti-p45 antibodies showed
stronger labelling in the nucleus than in the cytoplasm. The REG was found to be
localized predomitly in the cytoplasm. Three- to six-fold increases in the
level of REG were observed following gamma-interferon treatment of cultured
cells but gamma-interferon had no obvious effect on its subcellular
distribution. These results demonstrate that different regulatory complexes and
subpopulations of proteasomes have different distributions within mammalian
cells and, therefore, that the distribution is more complex than has been
reported for yeast proteasomes. The 26S proteasome is a large multisubunit complex involved in degrading both
cytoplasmic and nuclear proteins. We have investigated the subcellular
distribution of four regulatory ATPase subunits (S6 (TBP7/MS73), S6' (TBP1), S7
(MSS1), and S10b (SUG2)) together with components of 20S proteasomes in the
intersegmental muscles (ISM) of Manduca sexta during developmentally programmed
cell death (PCD). Immunogold electron microscopy shows that S6 is located in the
heterochromatic part of nuclei of ISM fibres. S6' is present in degraded
material only outside intact fibres. S7 can be detected in nuclei, cytoplasm and
also in degraded material. S10b, on the other hand, is initially found in nuclei
and subsequently in degraded cytoplasmic locations during PCD. 20S proteasomes
are present in all areas where ATPase subunits are detected, consistent with the
presence of intact 26S proteasomes. These results are discussed in terms of
heterogeneity of 26S proteasomes, 26S proteasome disassembly and the possible
role of ATPases in non-proteasome complexes in the process of PCD. Cell Death
and Differentiation (2000) 7, 1210 - 1217. Proteasomes play a major role in non-lysosomal proteolysis and also in the
processing of proteins for presentation by the MHC class I pathway. In animal
cells they exist in several distinct molecular forms which contribute to the
different functions. 26S proteasomes contain the core 20S proteasome together
with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the
ends of the 20S proteasome to form PA28-proteasome complexes and
PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome
subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We
have found differences in the subcellular distribution of the different forms of
proteasomes. The gamma-interferon inducible PA28 alpha and beta subunits are
predomitly located in the cytoplasm, while 19S regulatory complexes (present
at significant levels only in 26S complexes) are present in the nucleus as well
as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic
reticulum (ER) where they may facilitate the generation of peptides for
transport into the lumen of the ER. We have also investigated the effects of
gamma-interferon on the levels and subcellular distribution of inducible
subunits and regulator subunits. In each case gamma-interferon was found to
increase the level but not to alter the distribution. Several subunits of
proteasomes are phosphorylated including alpha subunits C8 (alpha7) and C9
(alpha3), and ATPase subunit S4 (rpt2). Our studies have shown that
gamma-interferon treatment decreases the level of phosphorylation of
proteasomes. We have investigated the role of phosphorylation of C8 by casein
kinase II by site directed mutagenesis. The results demonstrate that
phosphorylation at either one of the two sites is essential for the association
of 19S regulatory complexes and that the ability to undergo phosphorylation at
both sites gives the most efficient incorporation of C8 into the 26S proteasome. The transport of apolipoprotein B (apoB) between the endoplasmic reticulum (ER)
and Golgi was studied in puromycin-synchronized HepG2 cells, using an antibody
that could distinguish between apoB in ER and Golgi compartments. In cells with
normal ER-to-Golgi transport, both albumin and apoB colocalized throughout the
ER and appeared as intense, compact signals in Golgi. When ER-to-Golgi transport
was blocked with brefeldin A, apoB and albumin remained colocalized in the ER
network and three-dimensional constructed images showed more intense signals for
both proteins in a central, perinuclear region of the ER. When protein synthesis
was stopped in cells with brefeldin A-inhibited ER-to-Golgi transport, apoB
degradation was visualized as a homogeneous decrease in fluorescence signal
intensity throughout the ER that could be slowed with clasto-lactacystin
beta-lactone, a proteasome inhibitor. Incubation of cells with CP-10447, an
inhibitor of microsomal triglyceride transfer protein, inhibited apoB, but not
albumin, transport from ER to Golgi. Nanogold immunoelectron microscopy of
digitonin-permeabilized cells showed proteasomes in close proximity to the
cytosolic side of the ER membrane. Thus, newly synthesized apoB is localized
throughout the entire ER and degraded homogeneously, most likely by neighboring
proteasomes located on the cytosolic side of the ER membrane. Although albumin
is colocalized with apoB in the ER, as expected, it was not targeted for
ER-associated proteasomal degradation. In this work, we have investigated the role of the sperm proteasome during in
vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome
activity was measured in extract and intact sperm using a specific substrate. In
addition, sperm were treated with specific proteasome inhibitors and evaluated
during IVF, binding to the zona pellucida, and progesterone- and zona
pellucida-induced acrosome reactions. In other experiments, sperm membrane
proteins were obtained resuspending them in Triton X-114, shaking vigorously and
let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous
phase and sperm membrane proteins in the detergent phase. In both phases,
proteasome activity was measured. Labeling of cell surface sperm proteins was
carried out with the cell-impermeable NHS-LC biotin, extracted with Triton
X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were
incubated with an anti-proteasome monoclonal antibody and evaluated by indirect
immunofluorescence. The results indicate that sperm extracts as well as intact
sperm had proteasome activity; the sperm proteasome was involved in IVF,
specifically during sperm-zona pellucida binding and the acrosome reaction;
soluble sperm membrane proteins exhibited proteasome activity; biotin
experiments indicated the presence of proteasomes on the sperm surface, which
was corroborated by indirect immunofluorescence experiments. All these
observations indicate that the mouse sperm proteasome participates in the
binding to the zona pellucida and the acrosome reaction and that there is a pool
of proteasomes located on the sperm head. While misfolded and short-lived proteins are degraded in proteasomes located in
the nucleus and cytoplasm, the degradation of organelles and long-lived proteins
in the lysosome occurs by the process of autophagy. Central and necessary to the
autophagic process are two conserved ubiquitin-like conjugation machineries.
These conjugation machineries appear to be specific for autophagy and can
together with genetic and morphological data be used to trace the natural
history of autophagy. Here we discuss the origin and evolution of autophagy. Covalent conjugation of proteins with ubiquitin is one the most important
post-translational modifications because it controls intracellular protein
trafficking typically resulting in protein degradation. Frequently ubiquitinated
proteins are targeted to the proteasome for degradation in the cytosol. However,
ubiquitinated membrane bound proteins can also be targeted for endocytosis and
degradation in the lysosome. Ubiquitin-dependent degradation pathways have clear
cancer relevance due to their integral involvement in protein quality control,
regulation of immune responses, signal transduction, and cell cycle regulation.
In spite of its fundamental importance, little is known regarding how proteins
are specifically identified for ubiquitin-dependent degradation. In this article
we review a newly discovered family of viral and cellular ubiquitin ligases
called MARCH proteins. Recent studies of MARCH proteins define new paradigms
showing how ubiquitin E3 ligases determine the intracellular location and fate
of proteins. Proteasomes are large multicatalytic proteinase complexes located in the cytosol
and the nucleus of eukaryotic cells. The ubiquitin-proteasome system is
responsible for the degradation of most intracellular proteins and therefore
plays an essential regulatory role in critical cellular processes including cell
cycle progression, proliferation, differentiation, angiogenesis and apoptosis.
Besides involving in normal cellular functions and homeostasis, the alteration
of proteasomal activity contributes to the pathological states of several
clinical disorders including inflammation, neurodegeneration and cancer. It has
been reported that human cancer cells possess elevated level of proteasome
activity and are more sensitive to proteasome inhibitors than normal cells,
indicating that the inhibition of the ubiquitin-proteasome system could be used
as a novel approach for cancer therapy. In this review we summarize several
specific aspects of research for the proteasome complex, including the structure
and catalytic activities of the proteasome, properties and mechanisms of action
of various proteasome inhibitors, and finally the clinical development of
proteasome inhibitors as novel anticancer agents. Quiescent endothelial cells contain low concentrations of plasminogen activator
inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a
variety of inflammatory mediators. In this study, we provide evidence that PAI-2
interacts with proteasome and affects its activity in endothelial cells. To
ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were
coimmunoprecipitated from endothelial cells and identified with specific
antibodies. The specificity of this interaction was evidenced after (a)
transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both
proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using
specific small interfering RNA (siRNA). Subsequently, cellular distribution of
the PAI-2·proteasome complexes was established by immunogold staining and
electron microscopy analyses. As judged by confocal microscopy, both proteins
appeared in a diffuse cytosolic pattern, but they also could be found in a dense
perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting
that it bound to proteasome not as the substrate but rather as its inhibitor.
Consistently, increased PAI-2 expression (a) abrogated degradation of degron
analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b)
prevented degradation of p53, as evidenced both by confocal microscopy and
Western immunoblotting, and (c) inhibited proteasome cleavage of specific
fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced
with inflammatory stimuli, can inhibit proteasome and thus tilt the balance
favoring proapoptotic signaling. The cellular proteasome is an important molecular target in cancer therapy and
drug resistance research. Proteasome inhibitors are effective agents against
multiple myeloma and mantle cell lymphoma and display great potential as
treatment for a variety of other maligcies. The proteasome is a large
multicatalytic, proteinase complex located in the cytosol and the nucleus of
eukaryotic cells. The ubiquitin proteasome system is responsible for the
degradation of most intracellular proteins and therefore plays an essential
regulatory role in critical cellular processes including cell cycle progression,
proliferation, differentiation, angiogenesis, and apoptosis. Cancer cells are
particularly sensitive to proteasome inhibitors, indicating the utility for
inhibition of the ubiquitin-proteasome pathway as an approach for cancer
therapy. In eukaryotic cells, regulated protein degradation of intracellular proteins is
mediated largely by the ubiquitin proteasome system (UPS). UPS-mediated protein
degradation regulates virtually all crucial aspects of cellular physiology, such
as cell proliferation, cell division, cell differentiation, and cell death.
Concomitantly, the deregulation by the UPS contributes to human disorders
including cancer. Cellular regulation by UPS- mediated protein degradation is a
highly specific and selective process that depends on time (e.g. cell cycle) and
location (nucleus, mitochondria or endoplasmic reticulum). An ongoing challenge
in the protein degradation field is identification of degradation signals for
specific proteins that trigger their degradation by the proteasome. More than 25
years ago, the first degradation signal was discovered and defined as
destabilizing N-terminal amino-acid residue (or N-degron) of protein substrates.
The discovery and subsequent detailed analysis of N-degrons gave rise to the so
called N-end rule, which states that the half-life time of a protein is
determined by the identity of its N-terminal amino-acid residue. The N-end rule
pathway recognizes proteins containing N-terminal destabilizing residues and
mediates their polyubiquitination and subsequent degradation in the proteasome.
Recent investigations have revealed a role for N-terminal acetylation on the
recognition of N-degrons by the N-end rule pathway. Here we summarize these
recent findings and highlight the impact on our understanding of the N-end rule
pathway with respect to cellular physiology. |
What is clinical presentation of the Gardner-Diamond syndrome? | Psychogenic purpura, also known as Gardner-Diamond syndrome, is a rare, distinctive, localized cutaneous reaction pattern mostly affecting psychologically disturbed adult women. Repeated crops of tender, ill-defined ecchymotic lesions on the extremities and external bleeding from other sites characterize the condition. | Gardner-Diamond's syndrome, or autoerythrocyte sensitization, is a disorder of
spontaneous, painful ecchymoses whose pathogenesis is unresolved. The role of
psychopathologic factors in this entity has been emphasized in previous reports.
The patient in this study had a classical history and characteristic clinical
features and is, to my knowledge, the first man described with this disorder. Autoerythrocyte sensitization (Gardner-Diamond syndrome) causes painful
ecchymoses, and usually occurs in young women. It is rare and of unknown
etiology. The young woman in this report probably had the condition beginning at
age 14, but the diagnosis was not made until age 19, shortly after a
copper-containing intrauterine device (IUD) exacerbated her condition. The
ecchymoses disappeared when the IUD was removed, but recurred when replaced. A
non-copper IUD caused no ecchymoses. Taping a copper penny to the skin caused a
similar rash. It seems that in this woman, the Gardner-Diamond syndrome was
markedly worsened by exposure to copper. We describe the clinical presentation and course of a patient with
autoerythrocyte sensitization (Gardner-Diamond) syndrome, and review the
literature for similar cases. A 37-yr-old female presented with recurrent
episodes of painful ecchymotic bruising over the anterior aspect of both thighs.
These episodes were precipitated by emotional stress. The diagnosis was
confirmed by induction of similar lesions by intradermal injection of the
patient's own washed red blood cells and hemoglobin. The lesions did not recur
for 6 months after the cause of her emotional stress was relieved.
Autoerythrocyte sensitization (Gardner-Diamond) syndrome should be considered in
the differential diagnosis of purpura, especially in patients with psychiatric
problems. A 54-year old anorectic patient with painful bruising syndrome (Gardner-Diamond
syndrome) suffered from various gastrointestinal and psychologic complaints. The
episodes of painful bruising could be provoked following intradermal injection
of washed red blood cells within 96 h. The pathogenesis of this rare entity is
more likely due to a psychiatric alteration rather than to the immunological
mechanisms, as had been postulated two decades ago. Nevertheless, the
Gardner-Diamond-syndrome should be included in the differential diagnosis of
ecchymotic bleeding. INTRODUCTION: Painful bruising syndrome was described by Gardner and Diamond in
1955. It is marked by spontaneous bruising, without any biological abnormality,
affecting young women with pathological mental context.
EXEGESIS: We report three observations with painful bruising syndrome. In a
patient, psychotherapy induced improvement in dermatological and articular
manifestations. In other case, placebotherapy made clinical symptoms go away for
a prolonged period.
CONCLUSION: Some etiological hypotheses have been postulated for Gardner and
Diamond syndrome. However, published cases speak in favour of psychogenic
hypothesis. Somatic and psychological approach must be offered to these
patients. Autoerythrocyte sensitization syndrome (ASS) (Gardner-Diamond syndrome) is
characterized by painful ecchymotic lesions affecting mostly women with
emotional stress. Although it is widely accepted as a non-inflammatory disease,
ASS can be accompanied by some autoimmune diseases. In this case report, we
present a case with ASS associated with cutaneous vasculitis. We also briefly
discuss the possible inflammatory features of ASS. Le cas d’une adolescente qui a consulté à cause d’ecchymoses inexpliquées est
présenté. Tous les examens exécutés pour établir une étiologie organique étaient
normaux. On a posé un diagnostic de syndrome de Gardner-Diamond, un syndrome
d’ecchymoses prévisibles précédées par des douleurs et une sensation de chaleur
au foyer de l’ecchymose, souvent associé à un stress physique ou psychosocial.
Dans le présent article, les auteurs se servent de leur expérience de ce
syndrome rare pour souligner quelques considérations importantes au point de vue
éthique et pratique, en ce qui a trait aux examens à effectuer, au traitement à
amorcer et aux communications dans le cadre d’une maladie aux symptômes médicaux
inexpliqués. A review of the literature concerning psychogenic purpura is presented. The
diagnosis is usually based on typical anamnestic data, clinical presentation
(painful inflammatory skin lesions, which progressed to ecchymoses during the
next 24 h) and positive diagnostic tests with intracutaneous injections of 80%
solution of washed autologous erythrocytes. No pathological findings of blood
coagulation parameters are usually detected. Histopathological evaluations of
lesional biopsies revealed non-specific changes. Taking into account the high
frequency of psychic disorders and stress dependence of skin symptoms, therapy
with psychotropic drugs (according to indications) and psychotherapy are
pathogenetically grounded methods of treatment in psychogenic purpura, and
should be provided together with symptomatic therapy. Psychogenic purpura (Gardner-Diamond syndrome) is the occurrence and spontaneous
recurrence of painful ecchymosis following emotional stress and minor trauma.
Although the exact mechanism of this syndrome remains unknown, apart from skin
lesions, different types of hemorrhaging have been reported, such as epistaxis,
gastrointestinal bleeding, and bleeding from the ear canals and eyes. We report
a psychogenic purpura case that presented with hematuria in addition to skin
lesions. Based on the psychiatric evaluation she was diagnosed with major
depressive disorder, generalized anxiety disorder, and obsessive-compulsive
disorder. Additionally, sexual pain disorder accompanied these disorders. With
the help of antidepressant and supportive psychotherapy, the patient's
ecchymosis and bleeding disappeared. During 8 months of follow-up the symptoms
did not return. Vaginismus has not been reported in patients with psychogenic
purpura. The presence of vaginismus, which is seen more frequently in eastern
cultures and is thought to be related to sociocultural determits, suggests
that some cultural factors may be common to both psychogenic purpura and
vaginismus. The aim of this case report was to call attention to a syndrome that
is rarely seen and diagnosed, and to discuss its relationship to psychosocial
factors. This syndrome should be considered in the differential diagnosis of not
only ecchymotic lesions, but also various types of bleeding, including
hematuria. Despite the fact that its etiology and treatment are not clearly
understood, it should be noted that psychological factors play a role in this
disease and therefore, psychopharmacological and psychotherapeutic approaches
can be effective. Psychocutaneous disorders (PCDs) are conditions that are characterized by
psychiatric and skin manifestations. Classifications of PCDs and their
nomenclature are matters of debate. For the purpose of this review, we adopted
the classification that distinguishes primary dermatologic disorders with
psychiatric co-morbidity (PDDPC) from primary psychiatric disorders with
dermatologic manifestations (PPDDM). PDDPC includes the psychophysiologic
disorders such as atopic eczema, psoriasis, vitiligo, and alopecia areata. PPDDM
includes impulse control disorders, obsessive-compulsive disorders, factitious
disorder, factitious disorder by proxy, self-mutilation, delusions of
parasitosis, psychogenic purpura/Gardner-Diamond syndrome, and cutaneous sensory
disorders. Diagnosis and treatment of PCDs are challenging and require that the
underlying psychopathology be addressed. A specific PCD may have different
underlying psychopathologies and, at times, multiple overlapping
psychopathologies may coexist. Most often, both non-pharmacologic management and
psychopharmacologic treatment are necessary. The choice of psychopharmacologic
agent depends on the nature of the underlying psychopathology (e.g. anxiety,
depression, obsessive-compulsive disorder, psychosis). This article reviews the
spectrum of PPDDM in children. A10-year boy presented with spontaneous episodes of oral bleeding for the last 6
months. Detailed ENT examination showed no pathology, bleeding profile was
normal, endoscopy and dental examination also did not reveal any abnormality.
Child abuse or malingering was also ruled out. Initially the child was managed
with platelet transfusion and fresh frozen plasma and then put on follow-up
treatment with antifibrinolytics, Vitamin C but the episodes became recurrent.
Psychiatric evaluation revealed that child was suffering from depression.
Antidepressants were prescribed by the psychiatrist that not only cured the
depression with time but also the bleeding episodes which were actually related
to child's depression (Gardner-Diamond syndrome or psychogenic purpura). This is
a diagnosis by exclusion where the patients bleed due to dysregulated steroid
secretion secondary to stress; resulting in development of sensitization to RBC
membrane, and dysregulated fibrinolytic system activity. Psychogenic purpura, also known as Gardner-Diamond syndrome or autoerythrocyte
sensitization syndrome, is a rare condition characterized by spontaneous
development of painful edematous skin lesions progressing to ecchymosis over the
next 24 hours. Severe stress and emotional trauma always precede the skin
lesions. The condition is most commonly seen in women, but isolated cases have
been reported in adolescents and in males. Psychodermatologic evaluation and
dermatology and psychiatry liaison have been successful in the treatment of
these patients. This report provides an overview of psychogenic purpura and
presents the case of a 15-year-old girl. Gardner-Diamond syndrome (GDS) is an uncommon disease clinically characterized
by a wide spectrum of psycho-emotive symptoms associated with painful
ecchymoses/purpuric lesions and positivity of auto-erythrocyte sensitization
skin test. Herein, a perspective clinical and psychological observation of an
adolescent GDS is firstly reported focusing on her psychological features
long-term monitored for a 1-year period. The administration of a standardized
tools battery allowed us to define psychological features of the young patient
over time and to monitored clinical course and response to treatment. |
Where can you find the annulus of Zinn? | Annulus of Zinn is in the orbit. | PURPOSE: To report an elevation deficit, which was caused by an accessory
extraocular muscle, in a 6-year-old boy.
METHODS: Computed tomography and magnetic resoce imaging were used to confirm
an accessory, fusiform, well-defined, solid structure in the retrobulbar space.
RESULTS: A supernumerary intraconal muscle was detected between the annulus of
Zinn and the posterior part of the left globe.
CONCLUSION: This rare anomaly may represent an atavistic retractor bulbi muscle. Conventional strabismus surgery employs a conjunctival incision to gain access
to Tenon's space where a wide variety of procedures are routinely performed on
the tendon and anterior aspect of the extraocular muscles. Recently, transnasal
endoscopic surgical techniques have gained acceptance as effective means of
decompressing the medial wall and floor of the orbit in patients with
thyroid-related orbitopathy. The orbital surface of the medial rectus and
inferior rectus are exposed from the annulus of Zinn to a position close to
where the muscles penetrate Tenon's capsule. In theory, this technique also
provides the exposure necessary to locate and retrieve a "lost" medial rectus
when the usual sub-Tenon's approach fails to recover the muscle. Cadaver studies
demonstrate the feasibility of exposure and suture placement in the stump of a
lost medial rectus with passage of the suture through Tenon's capsule to
transmit the force of the muscle to the globe, provided that the lost muscle is
retrieved before severe contracture develops. Tumors of the orbital apex are difficult to approach through a standard lateral
orbitotomy exposure. The transcranial approach has been described, but it
requires an open craniotomy as well as dissection through the annulus of Zinn in
its tight superior segment to reach intraconal and inferior lateral tumors. It
is well recognized that the transcranial approach is optimal only for tumors of
the superomedial orbital apex. Our study demonstrates that by enlarging the bony
incision of a classic lateral orbitotomy to include a generous marginotomy and
removing the deep sphenoid wing up to the superior orbital fissure, good
exposure of the lateral orbital apex can be obtained. Tumors of the apex,
including those that extend slightly into the cavernous sinus, can be removed
from the cranial nerves and extraocular muscle origins in en face fashion,
providing optimal ability to identify the delicate neurovascular structures of
the orbital apex and avoid damage to them. The operating microscope is extremely
useful for bony and soft tissue dissection. We report four benign tumors of the
orbital apex removed using this approach. Two tumors encroached slightly into
the cavernous sinus. Three of four patients were told that they had inoperable
tumors. By use of the deep orbital apex approach described, all four tumors were
successfully exposed and removed. Visual and motor function was unchanged or
improved in all four patients, with the exception of one tumor that incorporated
the inferior division of the third cranial nerve; in that patient, the
transected nerve was anastomosed microscopically, and partial return of function
was noted. The transorbital ophthalmic approach to tumors of the inferolateral
orbital apex has significant potential advantages in comparison with a frontal
craniotomy approach. Transient diplopia, blepharoptosis, or both conditions are rare postoperative
complications of blepharoplasty performed with the patient under local
anesthesia. It has been hypothesized that some cases of postoperative diplopia
and blepharoptosis could be attributed to the myotoxic effects of local
anesthetics to the extraocular muscles and the levator muscle or to the
neurotoxic effects of lidocaine. In 30 cadavers, the superior division of the
oculomotor nerve was severed en bloc 1.5 cm anterior to the annulus of Zinn with
the levator palpebrae superioris (LPS) and the superior rectus muscles. These
muscles were detached from their origins, and their attachments to the scleral
and tarsal plates were divided respectively. The specimens were treated in
guanidine-hydrochloride and Alizarin Red solution, and were dissected under an
operating microscope. The nerve branches of the superior division of the
oculomotor nerve innervated the proximal third (type I) in 2 of 30 LPS muscles
(6.7%), in 8 of 30 muscles (26.7%) extended to the middle third (type II), and
reached the distal third (type III) in 20 of 30 muscles (66.7%). The terminal
branches ran through the medial third (type IIIa) in 6 of 20 type III LPS
muscles (30%), the central third (type IIIb) in 8 muscles(40%), and the lateral
third (type IIIc) in 6 muscles (30%). The oculomotor nerve ends that extend
forward to the distal third of the LPS muscle (type III) are exposed and
vulnerable to local anesthetics and may be numbed during blepharoplasty. If this
is so, postoperative blepharoptosis may be caused by transient paralysis of the
LPS muscle, and great care should be taken during the injection of local
anesthetics near the LPS. BACKGROUND: Although resection of the anterior clinoid process (ACP) is valuable
in the surgical treatment of aneurysms of the ophthalmic (C6) segment of the
internal carotid artery (ICA), quantitative assessment of this adjunct is
incomplete. Our morphometric study assesses the effectiveness of the anterior
clinoidectomy for exposure of the C6 segment of the ICA.
METHODS: Ten formalin-fixed adult cadaveric heads were dissected bilaterally and
pterional craniotomies were performed bilaterally. Measurements before and after
resection of the ACP included the length of C6 segment of the ICA on its lateral
aspect; C6 segment length on its medial aspect; and medial length of the optic
nerve from the optic chiasm to falciform ligament (before ACP resection) then to
the annulus of Zinn (after ACP resection).
FINDINGS: Height and width of the intradural ACP were 8.67 +/- 2.63 and 6.57 +/-
1.68 mm, respectively. After clinoidectomy, mean length of the lateral C6
segment of the ICA increased 60% and mean exposure of the medial C6 segment of
the ICA increased 113% (p < 0.001). Exposure of the optic nerve increased 150%
(p < 0.001) after clinoidectomy and sectioning of the falciform ligament. No
correlations were found between the lengths of the ACP and entire C6 segment, or
the ACP size and amount of the C6 segment covered by the clinoid.
CONCLUSIONS: Exposure of the C6 segment of the ICA is markedly increased by
increase of the mobility of the optic nerve with clinoidectomy and section of
the falciform ligament. Dissections of the bilateral orbits in a 45-year-old female cadaver, who had no
ocular movement disorders in her lifetime, revealed anomalous muscles linking
the superior and inferior rectus muscles. The muscles, situated between the
optic nerve and the lateral rectus muscle, originated from the annulus of Zinn
and branched off two heads; one inserted into the medial inferior side of the
superior rectus muscle and the other inserted into the central superior side of
the inferior rectus muscle. Each insertion was located on a distal site of the
myoneural junction of each rectus muscle. Histological investigations showed
that the muscles had a striated muscle structure. No definite nerve insertion
was observed in the muscles. Although this type of anomalous muscle has been
reported in a few Caucasian cases, the present study is the first report in an
Asian person. Anomalous orbital structures, which are a rare cause of
strabismus, are important in the differential diagnosis of intra-orbital
space-occupying lesions, rather than the differential diagnosis of strabismus. The microanatomy of the superior orbital fissure (SOF) was studied in 96 sides
of cadaver specimens. The SOF is a narrow bony cleft that lies at the apex of
the orbit between the greater and lesser wings of the sphenoid. Through this
fissure, many important structures enter the orbit from the middle cranial fossa
including the third, fourth, sixth cranial nerves, and the ophthalmic branch of
the fifth nerve. In addition, the superior opthalmamic vein exits the orbit to
drain into the cavernous sinus via the SOF. The fissure can be divided into
three anatomical regions by the annulus of Zinn (common annular tendon): the
lateral, central, and inferior regions. The lateral wall of the SOF can also be
divided between the upper and lower segments, and the angle between them was
measured to be 144.27 degrees +/- 20.03 degrees . Defining these regions is
useful in describing the course and placement of the nerves and vasculature in
the SOF. Managing lesions at the orbital apex requires an extensive knowledge of
the cranial base and the intracranial and extracranial relationships of the
anatomical structures coursing through the SOF. The goal of this study was to
describe the microanatomy of the SOF region in detail and to provide a reference
for surgical procedures involving the orbital apex. The sphenoid bony landmarks are important for endoscopic orientation in skull
base surgery but show a wide range of variations. We aimed to describe an
instructional model for the endoscopic parasellar anatomy in sphenoid sinuses
with ill-defined bony landmarks. Five preserved injected cadaveric heads and
four sides of dry skulls were studied endoscopically via transethmoid,
transsphenoidal approach. The parasellar region was exposed by drilling along
the maxillary nerve (V2) canal [the length of the foramen rotundum (FR) between
the middle cranial fossa and the pterygopalatine fossa]. This was achieved by
drilling in the inferior part of the lateral wall of posterior ethmoids
immediately above the sphenopalatine foramen. Cavernous V2 was traced to the
paraclival internal carotid artery (ICA). Cavernous sinus (CS) apex was exposed
by drilling a triangle bounded by V2 and its canal inferiorly, bone between FR
and superior orbital fissure (SOF) anteriorly, and ophthalmic nerve (V1)
superiorly. Drilling was continued toward the annulus of Zinn (AZ) and optic
nerve superiorly and over the intracavernous ICA posteriorly. Endoscopic
measurements between V2, SOF, AZ, and opticocarotid recess were obtained.
Endoscopic systematic orientation of parasellar anatomy is presented that can be
helpful for approaching sphenoid sinus with ill-defined bony landmarks. OBJECTIVE: Explored the causation of a case of incomitant vertical strabismus
accompanied with elevation deficit and globe retraction by surgery.
METHODS: Case report. Orbital imaging study of MRI was used to discover the
anatomic feature of extraocular muscle. By released the restrictive structure to
treat strabismus. Histopathologic inspection was used to confirm the origin of
the abnormal structure.
RESULTS: Abnormal extraocular muscle that located within the cone formed by the
four recti muscles was the causation of strabismus. It arose at the annulus of
Zinn, passing forwards between the inferior rectus muscle and lateral rectus
muscle, and insert directly on the sclera. After Released it from eyeball and
recession of inferior rectus muscle the strabismus was improved. Elevation
deficit was not improved. Histopathologic inspection confirmed that the
structure was muscular in origin.
CONCLUSION: The abnormal structure that found by MRI was the cause of elevation
deficit and globe retraction. Its histopathologic inspection confirmed the
muscular origin. The abnormal structure was an accessory extraocular muscle. For
incomitant vertical strabismus accompanied with elevation deficit and globe
retraction anomalous orbital structures maybe the causation. Orbital imaging
studies should be done to explore the origin of disease. PURPOSE: To describe a combined transcranial-orbital approach for en bloc
resection of optic nerve gliomas with preservation of the annulus of Zinn that
minimizes recurrence and prevents postoperative paralytic ptosis.
DESIGN: A retrospective, noncomparative, interventional case series.
STUDY POPULATION: All patients who underwent optic nerve glioma resections using
this technique with the authors between 1994 and 2010.
PROCEDURE: A transcranial-orbital approach is used to resect the intracranial
segment of the optic nerve glioma from 2 mm anterior to the chiasm to the
posterior extent of annulus of Zinn. The proximal transected edge of the nerve
is examined intraoperatively for tumor margin clearance. Through a superior
orbitotomy exposure, the entire retrobulbar segment of the tumor is transected
from the globe to the annulus of Zinn. A simulation of the procedure in a
cadaver and en bloc resection of the orbital apex are performed to demonstrate
the subdural plane of dissection within the annulus of Zinn.
MAIN OUTCOME MEASURES: Postoperative outcome measures include: health of the
ipsilateral globe, paralytic ptosis, postoperative complications, and tumor
recurrence.
RESULTS: Eleven patients underwent resection of optic nerve gliomas using this
technique. No patients had tumor recurrence or developed postoperative paralytic
ptosis.
CONCLUSIONS: The combined transcranial-orbital approach with preservation of the
annulus of Zinn is a safe and effective way to remove optic nerve gliomas and
ensure tumor clearance while avoiding paralytic ptosis. The annular tendon is commonly named 'annulus of Zinn', from the German
anatomist and botanist Johann Gottfried Zinn (1727-1759) who described this
structure in his Descriptio anatomica oculi humani (Anatomical Description of
the Human Eye, 1755). This structure, however, had been previously discovered
not by Zinn, but by Antonio Maria Valsalva (1666-1723) some decades before the
publication of Zinn, in his Dissertatio anatomica prima and Dissertatio
anatomica altera (First and Second Anatomical Dissertations), inside Valsalva's
Opera omnia published in 1740. We advance that this structure could be re-named
such as 'annulus of Valsalva-Zinn' because Valsalva, even making a mistake in
its functional interpretation, first described this anatomical structure.
Likewise, Valsalva, with his discovery, advanced a revolutionary idea for that
time on the usefulness of anatomy for clinic and pathology. AIMS: Our aim was to elucidate the etiology of Brown syndrome by evaluating the
trochlea position, morphologic characteristics of the extraocular muscles
including superior oblique muscle/tendon complex, and the presence of the
cranial nerves (CN) III, IV, and VI using magnetic resoce imaging (MRI) in
eight patients with unilateral congenital Brown syndrome and one patient with
bilateral congenital Brown syndrome.
METHODS: Nine consecutive patients diagnosed with congenital Brown syndrome had
a comprehensive ocular examination and MRI for the CN III, CN VI, and the
extraocular muscles. Five of the nine patients underwent additional high
resolution MRI for CN IV. The distance from the annulus of Zinn to the trochlea
was measured.
RESULTS: Normal sized CN III, IV, and VI, as well as all extraocular muscles,
could be identified bilaterally in all patients with available MRI. The distance
from the annulus of Zinn to the trochlea was the same in both eyes.
CONCLUSIONS: The findings for our patients, particularly in those who underwent
additional high resolution MRI, did not provide evidence of a lack of CN IV as a
cause of Brown syndrome. |
What is the indication for SLCO1B1 genotyping? | HMG Co-A reductase inhibitors, commonly known as statins, also display wide interindividual variability in plasma concentration, response and toxicity due in part to polymorphisms in transporter genes, including SLCO1B1 and ABCG2. The SLCO1B1*5 variant is a risk factor for statin side effects and exhibits statin-specific effects: highest with simvastatin/atorvastatin and lowest with pravastatin/rosuvastatin. | Personalized medicine is an emerging field with a goal of applying genomic
information as a predictor of disease risk as well as individualization of drug
therapy. For optimization of drug therapy, significant progress has been made in
the past decade in linking genetic variation in genes associated with drug
disposition to prediction of drug response and adverse reactions. For most drugs
in clinical use, the interplay of many factors, including genetics,
demographics, drug-drug interactions, disease states and the environment, result
in the interindividual variability observed during drug therapy. Broadly
speaking, such determits of drug response are mediated through modulation of
drug concentrations reflective of pharmacokinetic factors, as well as drug
targets, often referred to as pharmacodynamics. It is clear that for
personalized medicine to become clinically meaningful, genomic as well as
clinical and environmental influences must be considered together. We show, for
a number of drugs in clinical use, that genomics-guided treatment options not
only are becoming feasible but are also on the cusp of showing superiority in
terms of clinical outcomes as well as cost-benefit. One of the most widely
studied drugs with regard to genomics-guided dosing options is the oral
anticoagulant, warfarin. Genetic polymorphisms in the gene encoding cytochrome
P450 2C9 (CYP2C9) and those in the target gene responsible for the warfarin
anticoagulant effect, vitamin K epoxide reductase (VKORC1), account for much of
the variability in the warfarin maintece dose; however, routine genotyping in
warfarin therapy remains controversial. We will outline the importance of
understanding all of the variables that mediate warfarin response as the
prerequisite to successful utilization of genotype-guided warfarin therapy.
Similarly, HMG Co-A reductase inhibitors, commonly known as statins, also
display wide interindividual variability in plasma concentration, response and
toxicity due in part to polymorphisms in transporter genes, including SLCO1B1
and ABCG2. Genetic factors are also important considerations in treatment with
other therapeutic agents discussed, including clopidogrel and tamoxifen.
Implementation of personalized medicine-based treatment options for these and
other drugs, the pharmacokinetics or pharmacodynamics of which are impacted by
functional genetic variations, will require overcoming a number of challenges,
including cost, turnaround time, and demonstration of clinical benefit, as well
as better training of health care professionals about genomics in general, and
pharmacogenomics in particular. Statin adherence is often limited by side effects. The SLCO1B1*5 variant is a
risk factor for statin side effects and exhibits statin-specific effects:
highest with simvastatin/atorvastatin and lowest with pravastatin/rosuvastatin.
The effects of SLCO1B1*5 genotype guided statin therapy (GGST) are unknown.
Primary care patients (n = 58) who were nonadherent to statins and their
providers received SLCO1B1*5 genotyping and guided recommendations via the
electronic medical record (EMR). The primary outcome was the change in Beliefs
about Medications Questionnaire, which measured patients' perceived needs for
statins and concerns about adverse effects, measured before and after SLCO1B1*5
results. Concurrent controls (n = 59) were identified through the EMR to compare
secondary outcomes: new statin prescriptions, statin utilization, and change in
LDL-cholesterol (LDL-c). GGST patients had trends (p = 0.2) towards improved
statin necessity and concerns. The largest changes were the "need for statin to
prevent sickness" (p < 0.001) and "concern for statin to disrupt life" (p =
0.006). GGST patients had more statin prescriptions (p < 0.001), higher statin
use (p < 0.001), and greater decrease in LDL-c (p = 0.059) during follow-up. EMR
delivery of SLCO1B1*5 results and recommendations is feasible in the primary
care setting. This novel intervention may improve patients' perceptions of
statins and physician behaviors that promote higher statin adherence and lower
LDL-c. OBJECTIVE: The present study aims to investigate the correlation of
polymorphisms of SLCO1B1 gene with the toxicity during therapy with the
high-dose methotrexate (MTX) chemotherapy in childhood acute lymphoblastic
leukemia.
METHODS: We analyzed 2 polymorphisms (rs4149081 and rs11045897) in SLCO1B1 gene
in 280 Chinese pediatric B-ALL patients, using MTX plasma concentration as an
objective and quantifiable marker of toxicity. We utilized Enzyme-multiplied
immunoassay technique (EMIT) to measure the plasma concentration of MTX. The
polymerase chain reaction-allele specific (PCR-AS) method was utilized to
perform the genotyping.
RESULTS: We found there was a statistically significant association between MTX
plasma concentration and the SLCO1B1 rs11045879 CC genotype (P<0.05). We also
found the rs4149081 AA genotype was associated with high-MTX plasma
concentrations. A-C haplotype carriers have a higher risk for MTX delayed
clearance but G-T haplotype was associated with a lower risk for MTX delayed
clearance.
CONCLUSIONS: The rs4149081 AA genotype and the rs11045897 CC genotype could be
indicators for high-MTX plasma concentrations in children with ALL. Patients with familial hypercholesterolemia (FH) may be at increased risk for
statin-associated muscle symptoms because they require long-term treatment with
high-intensity statin therapy. We sought to determine (1) whether other
predisposing factors, including the well-known genetic variant associated with
statin-associated muscle symptoms-solute carrier organic anion transporter
family, member 1B1 (SLCO1B1) rs4149056-also increase the risk of
statin-associated muscle symptoms in FH patients, and (2) the natural history
and management for FH patients with statin-associated muscle symptoms.
METHODS: We queried electronic records (2004-2014) of 278 genetically screened
FH patients (113 men, 165 women; mean [SD] pretreatment low-density lipoprotein
cholesterol [LDL-C] 259 [72] mg/dL) recruited from lipid clinics in the Dallas,
TX, area from 2004 to 2014. Statin-associated muscle symptoms were defined as
muscle symptoms arising while taking a statin and interrupting therapy.
RESULTS: The risk of muscle symptoms was associated with age (odds ratio 1.6
[95% CI 1.2-2.2]), body mass index in non-African Americans (0.90 [0.83-0.97]),
and hypertension (0.4 [0.2-0.9]). Simvastatin was the most commonly used statin,
and it was the statin most associated with muscle symptoms. Among FH patients
with muscle symptoms, 41% (n = 40) reestablished statin therapy ("eventually
tolerant") and 29% (n = 28) never reestablished statin therapy ("never
tolerant"). Rosuvastatin (43%) and pravastatin (30%) were the most common
eventually tolerated statins, and eventually tolerant patients achieved lower
treated LDL-C levels (eventually tolerant 127 vs never tolerant 192 mg/dL, P <
.001). Never tolerant patients also developed muscle symptoms on nonstatins (16%
vs 50%, P = .003). SLCO1B1 rs4149056 genotyping revealed 224 wild-type patients
(TT) and 49 heterozygotes (TC). SLCO1B1 genotype was not associated with the
risk of statin-associated muscle symptoms (odds ratio 1.40 [95% CI 0.74-2.64]).
CONCLUSION: Age, not SLCO1B1 rs4149056 genotype, was the strongest risk factor
for statin-associated muscle symptoms in FH patients. After developing muscle
symptoms, many patients reestablished statin therapy and achieved significant
LDL-C reductions. Overall, 10% of all FH patients had statin-associated muscle
symptoms and never reestablished statin therapy. Such patients developed muscle
symptoms even on nonstatin lipid-lowering drugs and continued to have elevations
in LDL-C. Further insight is needed into the relationship between FH and
statin-associated muscle symptoms so all FH patients can be adequately treated. |
List available circular RNA prediction tools. | circRNA_finder, find_circ, CIRCexplorer, CIRI, and MapSplice. | CircRNAs are novel members of the non-coding RNA family. For several decades
circRNAs have been known to exist, however only recently the widespread
abundance has become appreciated. Annotation of circRNAs depends on sequencing
reads spanning the backsplice junction and therefore map as non-linear reads in
the genome. Several pipelines have been developed to specifically identify these
non-linear reads and consequently predict the landscape of circRNAs based on
deep sequencing datasets. Here, we use common RNAseq datasets to scrutinize and
compare the output from five different algorithms; circRNA_finder, find_circ,
CIRCexplorer, CIRI, and MapSplice and evaluate the levels of bona fide and false
positive circRNAs based on RNase R resistance. By this approach, we observe
surprisingly dramatic differences between the algorithms specifically regarding
the highly expressed circRNAs and the circRNAs derived from proximal splice
sites. Collectively, this study emphasizes that circRNA annotation should be
handled with care and that several algorithms should ideally be combined to
achieve reliable predictions. |
Which disease is treated with Nusinersen? | Nusinersen us used for treatment of Spinal Muscular Atrophy. | Nusinersen (ISIS-SMNRx or ISIS 396443) is an antisense oligonucleotide drug
administered intrathecally to treat spinal muscular atrophy. We summarize lumbar
puncture experience in children with spinal muscular atrophy during a phase 1
open-label study of nusinersen and its extension. During the studies, 73 lumbar
punctures were performed in 28 patients 2 to 14 years of age with type 2/3
spinal muscular atrophy. No complications occurred in 50 (68%) lumbar punctures;
in 23 (32%) procedures, adverse events were attributed to lumbar puncture. Most
common adverse events were headache (n = 9), back pain (n = 9), and post-lumbar
puncture syndrome (n = 8). In a subgroup analysis, adverse events were more
frequent in older children, children with type 3 spinal muscular atrophy, and
with a 21- or 22-gauge needle compared to a 24-gauge needle or smaller. Lumbar
punctures were successfully performed in children with spinal muscular atrophy;
lumbar puncture-related adverse event frequency was similar to that previously
reported in children. BACKGROUND: Nusinersen is a 2'-O-methoxyethyl phosphorothioate-modified
antisense drug being developed to treat spinal muscular atrophy. Nusinersen is
specifically designed to alter splicing of SMN2 pre-mRNA and thus increase the
amount of functional survival motor neuron (SMN) protein that is deficient in
patients with spinal muscular atrophy.
METHODS: This open-label, phase 2, escalating dose clinical study assessed the
safety and tolerability, pharmacokinetics, and clinical efficacy of multiple
intrathecal doses of nusinersen (6 mg and 12 mg dose equivalents) in patients
with infantile-onset spinal muscular atrophy. Eligible participants were of
either gender aged between 3 weeks and 7 months old with onset of spinal
muscular atrophy symptoms between 3 weeks and 6 months, who had SMN1 homozygous
gene deletion or mutation. Safety assessments included adverse events, physical
and neurological examinations, vital signs, clinical laboratory tests,
cerebrospinal fluid laboratory tests, and electrocardiographs. Clinical efficacy
assessments included event free survival, and change from baseline of two
assessments of motor function: the motor milestones portion of the Hammersmith
Infant Neurological Exam-Part 2 (HINE-2) and the Children's Hospital of
Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND) motor function
test, and compound motor action potentials. Autopsy tissue was analysed for
target engagement, drug concentrations, and pharmacological activity. HINE-2,
CHOP-INTEND, and compound motor action potential were compared between baseline
and last visit using the Wilcoxon signed-rank test. Age at death or permanent
ventilation was compared with natural history using the log-rank test. The study
is registered at ClinicalTrials.gov, number NCT01839656.
FINDINGS: 20 participants were enrolled between May 3, 2013, and July 9, 2014,
and assessed through to an interim analysis done on Jan 26, 2016. All
participants experienced adverse events, with 77 serious adverse events reported
in 16 participants, all considered by study investigators not related or
unlikely related to the study drug. In the 12 mg dose group, incremental
achievements of motor milestones (p<0·0001), improvements in CHOP-INTEND motor
function scores (p=0·0013), and increased compound muscle action potential
amplitude of the ulnar nerve (p=0·0103) and peroneal nerve (p<0·0001), compared
with baseline, were observed. Median age at death or permanent ventilation was
not reached and the Kaplan-Meier survival curve diverged from a published
natural history case series (p=0·0014). Analysis of autopsy tissue from patients
exposed to nusinersen showed drug uptake into motor neurons throughout the
spinal cord and neurons and other cell types in the brainstem and other brain
regions, exposure at therapeutic concentrations, and increased SMN2 mRNA exon 7
inclusion and SMN protein concentrations in the spinal cord.
INTERPRETATION: Administration of multiple intrathecal doses of nusinersen
showed acceptable safety and tolerability, pharmacology consistent with its
intended mechanism of action, and encouraging clinical efficacy. Results
informed the design of an ongoing, sham-controlled, phase 3 clinical study of
nusinersen in infantile-onset spinal muscular atrophy.
FUNDING: Ionis Pharmaceuticals, Inc and Biogen. |
Which disease the London mutation involved in? | London mutation that is the missense mutation in exon 17 of the amyloid precursor protein gene on chromosome 21 (Val717Ile) is involved in Alzheimer's Disease. | The major constituent of senile plaques in Alzheimer's disease is a 42-aa
peptide, referred to as beta-amyloid (Abeta). Abeta is generated from a family
of differentially spliced, type-1 transmembrane domain (TM)-containing proteins,
called APP, by endoproteolytic processing. The major, relatively ubiquitous
pathway of APP metabolism in cell culture involves cleavage by alpha-secretase,
which cleaves within the Abeta sequence, thus precluding Abeta formation and
deposition. An alternate secretory pathway, enriched in neurons and brain, leads
to cleavage of APP at the N terminus of the Abeta peptide by beta-secretase,
thus generating a cell-associated beta-C-terminal fragment (beta-CTF). A
pathogenic mutation at codons 670/671 in APP (APP "Swedish") leads to enhanced
cleavage at the beta-secretase scissile bond and increased Abeta formation. An
inhibitor of vacuolar ATPases, bafilomycin, selectively inhibits the action of
beta-secretase in cell culture, suggesting a requirement for an acidic
intracellular compartment for effective beta-secretase cleavage of APP. beta-CTF
is cleaved in the TM domain by gamma-secretase(s), generating both Abeta 1-40
(90%) and Abeta 1-42 (10%). Pathogenic mutations in APP at codon 717 (APP
"London") lead to an increased proportion of Abeta 1-42 being produced and
secreted. Missense mutations in PS-1, localized to chromosome 14, are pathogenic
in the majority of familial Alzheimer's pedigrees. These mutations also lead to
increased production of Abeta 1-42 over Abeta 1-40. Knockout of PS-1 in
transgenic animals leads to significant inhibition of production of both Abeta
1-40 and Abeta 1-42 in primary cultures, indicating that PS-1 expression is
important for gamma-secretase cleavages. Peptide aldehyde inhibitors that block
Abeta production by inhibiting gamma-secretase cleavage of beta-CTF have been
discovered. Mutations of presenilin 1 (PS1) causing Alzheimer's disease selectively increase
the secretion of the amyloidogenic betaA4(1-42), whereas knocking out the gene
results in decreased production of both betaA4(1-40) and (1-42) amyloid peptides
(De Strooper et al. 1998). Therefore, PS1 function is closely linked to the
gamma-secretase processing of the amyloid precursor protein (APP). Given the
ongoing controversy on the subcellular localization of PS1, it remains unclear
at what level of the secretory and endocytic pathways PS1 exerts its activity on
APP and on the APP carboxy-terminal fragments that are the direct substrates for
gamma-secretase. Therefore, we have reinvestigated the subcellular localization
of endogenously expressed PS1 in neurons in vitro and in vivo using confocal
microscopy and fine-tuned subcellular fractionation. We show that uncleaved PS1
holoprotein is recovered in the nuclear envelope fraction, whereas the cleaved
PS fragments are found mainly in post-ER membranes including the intermediate
compartment (IC). PS1 is concentrated in discrete sec23p- and
p58/ERGIC-53-positive patches, suggesting its localization in subdomains
involved in ER export. PS1 is not found to significant amounts beyond the
cis-Golgi. Surprisingly, we found that APP carboxy-terminal fragments also
coenrich in the pre-Golgi membrane fractions, consistent with the idea that
these fragments are the real substrates for gamma-secretase. Functional evidence
that PS1 exerts its effects on gamma-secretase processing of APP in the ER/IC
was obtained using a series of APP trafficking mutants. These mutants were
investigated in hippocampal neurons derived from transgenic mice expressing
PS1wt or PS1 containing clinical mutations (PS1(M146L) and PS1(L286V)) at
physiologically relevant levels. We demonstrate that the APP-London and PS1
mutations have additive effects on the increased secretion of betaA4(1-42)
relative to betaA4(1-40), indicating that both mutations operate independently.
Overall, our data clearly establish that PS1 controls gamma(42)-secretase
activity in pre-Golgi compartments. We discuss models that reconcile this
conclusion with the effects of PS1 deficiency on the generation of betaA4(1-40)
peptide in the late biosynthetic and endocytic pathways. Transgenic mice over-expressing a mutated form of the human amyloid precursor
protein (APP, 695 isoform) bearing a mutation associated with Alzheimer's
disease (V642I, so-called London mutation, hereafter APPLd2) and wild-type
controls were studied at age periods (3 and 10 months) prior to the overt
development of neuritic amyloid plaques. Both 3- and 10-month-old APPLd2 mice
had reflex eyelid responses like those of controls, but only younger mice were
able to acquire a classical conditioning of eyelid responses in a trace
paradigm. In vitro studies on hippocampal slices showed that 10-month-old APPLd2
mice also presented deficits in paired-pulse facilitation and long-term
potentiation, but presented a normal synaptic activation of CA1 pyramidal cells
by the stimulation of Schaffer collaterals. It is proposed that definite
functional changes may appear well in advance of noticeable structural
alterations in this animal model of Alzheimer's disease, and that specific
learning tasks could have a relevant diagnostic value. Alzheimer's disease (AD) is the most common cause of dementia in humans. A
pathological hallmark in the brain of an AD patient is extracellular amyloid
plaques formed by accumulated beta-amyloid protein (Abeta), a metabolic product
of amyloid precursor protein (APP). Studies have revealed a strong genetic
linkage in the early-onset familial form (<60 years old) of AD. For example,
some mutant APPs are transmitted domitly and are segregated with inheritance
of early onset AD. These mutants facilitate Abeta production. The "Swedish"
mutations (APP(SW)) and the "London" mutation (APP(LON)) are examples of these
mutants. Selective silencing of these mutant alleles holds therapeutic promise
for AD. Here we show that the expression of the mutant APPs was selectively
inhibited by RNA interference. The best selectivity was obtained when the
mismatches were centrally placed in the antisense strand of small interfering
RNAs. Introducing an additional mismatch in the antisense strand may improve the
selectivity. The addition of a G at 5' end of the antisense strand may enhance
the efficacy of gene silencing by RNA interference. Our results illustrate the
guiding principles for selection of targeted sequences to achieve
allele-specific silencing. The sequences that are effective to silence APP(SW)
and APP(LON) as identified in this study may be useful in both in vivo and in
vitro studies to investigate the pathophysiological role of APP(SW) and APP(LON)
in AD development. Although chronic stress is known to be linked with memory and other neurological
disorders, little is known about the relationship between chronic stress and the
onset or development of Alzheimer's disease (AD). In this study, we investigated
the effects of long-term stress on the onset and severity of cognitive deficits
and pathological changes in APPV717I-CT100 mice overexpressing human APP-CT100
containing the London mutation (V717I) after exposure to immobilization stress.
We found that chronic immobilization stress accelerated cognitive impairments,
as accessed by the Passive avoidance and the Social Transfer of Food Preference
(STFP) tests. Moreover, the numbers and densities of vascular and extracellular
deposits containing amyloid beta peptide (Abeta) and carboxyl-terminal fragments
of amyloid precursor protein (APP-CTFs), which are pathologic markers of AD,
were significantly elevated in stressed animals, especially in the hippocampus.
Moreover, stressed animals, also showed highly elevated levels of
neurodegeneration and tau phosphorylation and increased intraneuronal Abeta and
APP-CTFs immunoreactivities in the hippocampus and in the entorhinal and
piriform cortex. This study provides the first evidence that chronic stress
accelerates the onset and severity of cognitive deficits and that these are
highly correlated with pathological changes, which thus indicates that chronic
stress may be an important contributor to the onset and development of AD. The APP/PS1ki mouse model for Alzheimer's disease (AD) exhibits robust brain and
spinal cord axonal degeneration and hippocampal CA1 neuron loss starting at 6
months of age. It expresses human mutant APP751 with the Swedish and London
mutations together with two FAD-linked knocked-in mutations (PS1 M233T and PS1
L235P) in the murine PS1 gene. The present report covers a phenotypical analysis
of this model using either behavioral tests for working memory and motor
performance, as well as an analysis of weight development and body shape. At the
age of 6 months, a dramatic, age-dependent change in all of these properties and
characteristics was observed, accompanied by a significantly reduced ability to
perform working memory and motor tasks. The APP/PS1ki mice were smaller and
showed development of a thoracolumbar kyphosis, together with an incremental
loss of body weight. While 2-month-old APP/PS1ki mice were inconspicuous in all
of these tasks and properties, there is a massive age-related impairment in all
tested behavioral paradigms. We have previously reported robust axonal
degeneration in brain and spinal cord, as well as abundant hippocampal CA1
neuron loss starting at 6 months of age in the APP/PS1ki mouse model, which
coincides with the onset of motor and memory deficits described in the present
report. BACKGROUND/AIMS: Mutations in the amyloid precursor protein gene were the first
to be recognized as a cause of Alzheimer's disease (AD).
METHODS: We describe 2 Italian families showing the missense mutation in exon 17
of the amyloid precursor protein gene on chromosome 21 (Val717Ile), known as
London mutation.
RESULTS: In 1 family, this mutation was responsible for AD in 3 out of 7
siblings and it is also present in a fourth sibling who has only shown signs of
executive dysfunction so far. Two subjects of the other family with AD diagnosis
were carriers of the same mutation.
CONCLUSION: All AD subjects showed a cognitive profile characterized by early
impairment in long-term memory, shifting abilities and affective symptoms
beginning in the fifth decade of life. One major hallmark of Alzheimer's disease (AD) is the massive loss of synapses
that occurs at an early clinical stage of the disease. In this study, we
characterize alterations in spine density and the expression of
synapse-associated immediate early gene Arc (activity-regulated
cytoskeleton-associated protein) in the hippocampal CA1 regions of two different
amyloid precursor protein (APP) transgenic mouse lines before plaque development
and their connection to performance in hippocampus-dependent memory tests. The
density of mushroom-type spines was reduced by 34% in the basal dendrites
proximal to the soma of CA1 pyramidal neurons in 5.5-month-old Tg2576 mice,
carrying the Swedish mutation, compared with wild-type littermates. A similar
reduction of 42% was confirmed in the same region of 8-month-old APP/Lo mice,
carrying the London mutation. In this strain, the reduction extended to the
distal dendritic spines (28%), although no differences were found in apical
dendrites in either transgenic mouse line. Both transgenic mice lines presented
a significant increase in Arc protein expression in CA1 compared with controls,
suggesting rather an overactivity and increased spine turnover that was
supported by a significant decrease in number of somatostatin-immunopositive
inhibitory interneurons in the stratum oriens of CA1. Behaviorally, the
transgenic mice showed decrease freezing in the fear contextual conditioning
test and impairment in spatial memory assessed by Morris water maze test. These
data indicate that cognitive impairment in APP transgenic mice is correlated
with impairment of synaptic connectivity in hippocampal CA1, probably
attributable to loss of inhibitory interneurons and subsequent hyperactivity. In the present study, we used a new training paradigm in the intelliCage
automatic behavioral assessment system to investigate cognitive functions of the
transgenic mice harboring London mutation of the human amyloid precursor protein
(APP.V717I). Three groups of animals: 5-, 12- and 18-24-month old were subjected
to both Water Maze training and the IntelliCage-based appetitive conditioning.
The spatial memory deficit was observed in all three groups of transgenic mice
in both behavioral paradigms. However, the APP mice were capable to learn
normally when co-housed with the wild-type (WT) littermates, in contrast to
clearly impaired learning observed when the transgenic mice were housed alone.
Furthermore, in the transgenic mice kept in the Intellicage alone, the cognitive
deficit of the young animals was modulated by the circadian rhythm, namely was
prominent only during the active phase of the day. The novel approach to study
the transgenic mice cognitive abilities presented in this paper offers new
insight into cognitive dysfunctions of the Alzheimer's disease mouse model. There is pivotal evidence that tau pathology can be triggered by amyloid-β (Aβ)
pathology in experimental systems. On the other side, studies on human brain
specimen have elucidated that tau pathology may occur before amyloid pathology
is present indicating that in principle tau pathology could also trigger Aβ
aggregation. To address this question, we have crossed 5XFAD mice coexpressing
human mutant APP695 with the Swedish, Florida, and London mutations and human
mutant presenilin-1 (PS1) with the M146L and L286V mutations with the PS19 model
overexpressing human mutant tau with the P301S mutation. The resulting triple
transgenic model 5XFAD/PS19 has been characterized at 3 and 9 months of age. A
dramatic aggravation of hyperphosphorylated tau pathology together with a
dramatically increased inflammatory response and a loss of synapses and
hippocampal CA1 neurons in aged 5XFAD/PS19 mice were observed. Extracellular
amyloid deposits were unaltered. These data support the assumption of tau
pathology being downstream of amyloid pathology, suggesting that both
pathologies together trigger the severe neuron loss in the hippocampus in the
5XFAD/PS19 mouse model. Current therapies for Alzheimer's disease only treat the symptoms of the
disease. We have previously developed a novel monoclonal antibody, 2B3, which
binds to the β-secretase cleavage site in amyloid precursor protein (APP) and
reduces the production of amyloid-β (Aβ) in human cell lines. To determine
whether the antibody was likely to be effective in mouse models of amyloid
pathology in vivo, we investigated whether 2B3 could also bind to APP in mouse
primary cortical neurones. Primary cortical neurones were produced from
E15.5-17.5 C57Bl/6 wild-type and transgenic APP/V717I (London mutation) embryos.
The percentage of the neuronal population was determined by immunocytochemistry.
Cells were treated with 10 μg/ml 2B3 or an irrelevant IgG for 48 h and Aβ40
levels determined by ELISA. The population of cells was found to contain over
75% neurones and 2B3 bound effectively to these cells. No differences in Aβ40
were detected between wild-type and transgenic cells. Importantly, 2B3
significantly inhibited the production of Aβ40 by 75.15±1.37% of the media
control, whereas an irrelevant IgG only significantly reduced Aβ40 levels by
23.35±5.55% of the media control. The reduction in Aβ40 produced by 2B3 was
significantly greater than that caused by the IgG. These data indicate that 2B3
binds to APP in mouse neurones and can inhibit Aβ40, similar to our previous
findings. The antibody is probably therefore acting by steric hindrance of
β-secretase and these data suggest that it will be effective in mice in vivo and
could be an alternative potential therapy for Alzheimer's disease. Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized
by extracellular plaques containing amyloid β (Aβ)-protein and intracellular
tangles containing hyperphosphorylated Tau protein. Here, we describe the
generation of inducible pluripotent stem cell lines from patients harboring the
London familial AD (fAD) amyloid precursor protein (APP) mutation (V717I). We
examine AD-relevant phenotypes following directed differentiation to forebrain
neuronal fates vulnerable in AD. We observe that over differentiation time to
mature neuronal fates, APP expression and levels of Aβ increase dramatically. In
both immature and mature neuronal fates, the APPV717I mutation affects both β-
and γ-secretase cleavage of APP. Although the mutation lies near the γ-secretase
cleavage site in the transmembrane domain of APP, we find that β-secretase
cleavage of APP is elevated leading to generation of increased levels of both
APPsβ and Aβ. Furthermore, we find that this mutation alters the initial
cleavage site of γ-secretase, resulting in an increased generation of both Aβ42
and Aβ38. In addition to altered APP processing, an increase in levels of total
and phosphorylated Tau is observed in neurons with the APPV717I mutation. We
show that treatment with Aβ-specific antibodies early in culture reverses the
phenotype of increased total Tau levels, implicating altered Aβ production in
fAD neurons in this phenotype. These studies use human neurons to reveal
previously unrecognized effects of the most common fAD APP mutation and provide
a model system for testing therapeutic strategies in the cell types most
relevant to disease processes. BACKGROUND: Accumulation and deposition of β-amyloid peptides (Aβ) in the brain
is a central event in the pathogenesis of Alzheimer's disease (AD). Besides the
parenchymal pathology, Aβ is known to undergo active transport across the
blood-brain barrier and cerebral amyloid angiopathy (CAA) is a prominent feature
in the majority of AD. Although impaired cerebral blood flow (CBF) has been
implicated in faulty Aβ transport and clearance, and cerebral hypoperfusion can
exist in the pre-clinical phase of Alzheimer's disease (AD), it is still unclear
whether it is one of the causal factors for AD pathogenesis, or an early
consequence of a multi-factor condition that would lead to AD at late stage. To
study the potential interaction between faulty CBF and amyloid accumulation in
clinical-relevant situation, we generated a new amyloid precursor protein (APP)
knock-in allele that expresses humanized Aβ and a Dutch mutation in addition to
Swedish/London mutations and compared this line with an equivalent knock-in line
but in the absence of the Dutch mutation, both crossed onto the PS1M146V
knock-in background.
RESULTS: Introduction of the Dutch mutation results in robust CAA and
parenchymal Aβ pathology, age-dependent reduction of spatial learning and memory
deficits, and CBF reduction as detected by fMRI. Direct manipulation of CBF by
transverse aortic constriction surgery on the left common carotid artery caused
differential changes in CBF in the anterior and middle region of the cortex,
where it is reduced on the left side and increased on the right side. However
these perturbations in CBF resulted in the same effect: both significantly
exacerbate CAA and amyloid pathology.
CONCLUSIONS: Our study reveals a direct and positive link between vascular and
parenchymal Aβ; both can be modulated by CBF. The new APP knock-in mouse model
recapitulates many symptoms of AD including progressive vascular and parenchymal
Aβ pathology and behavioral deficits in the absence of APP overexpression. One of the major histopathological hallmarks of Alzheimer's disease (AD) is
cerebral deposits of extracellular β-amyloid peptides. Preclinical studies have
pointed to glucagon-like peptide 1 (GLP-1) receptors as a potential novel target
in the treatment of AD. GLP-1 receptor agonists, including exendin-4 and
liraglutide, have been shown to promote plaque-lowering and mnemonic effects of
in a number of experimental models of AD. Transgenic mouse models carrying
genetic mutations of amyloid protein precursor (APP) and presenilin-1 (PS1) are
commonly used to assess the pharmacodynamics of potential amyloidosis-lowering
and pro-cognitive compounds. In this study, effects of long-term liraglutide
treatment were therefore determined in two double APP/PS1 transgenic mouse
models of Alzheimer's disease carrying different clinical APP/PS1 mutations,
i.e. the 'London' (hAPPLon/PS1A246E) and 'Swedish' mutation variant
(hAPPSwe/PS1ΔE9) of APP, with co-expression of distinct PS1 variants.
Liraglutide was administered in 5 month-old hAPPLon/PS1A246E mice for 3 months
(100 or 500 ng/kg/day, s.c.), or 7 month-old hAPPSwe/PS1ΔE9 mice for 5 months
(500 ng/kg/day, s.c.). In both models, regional plaque load was quantified
throughout the brain using stereological methods. Vehicle-dosed hAPPSwe/PS1ΔE9
mice exhibited considerably higher cerebral plaque load than hAPPLon/PS1A246E
control mice. Compared to vehicle-dosed transgenic controls, liraglutide
treatment had no effect on the plaque levels in hAPPLon/PS1A246E and
hAPPSwe/PS1ΔE9 mice. In conclusion, long-term liraglutide treatment exhibited no
effect on cerebral plaque load in two transgenic mouse models of low- and
high-grade amyloidosis, which suggests differential sensitivity to long-term
liraglutide treatment in various transgenic mouse models mimicking distinct
pathological hallmarks of AD. |
Define lncRNA. | Long noncoding RNAs (lncRNAs) represent a newly discovered class of regulatory molecules that impact a variety of biological processes in cells and organ systems. In humans, it is estimated that there may be more than twice as many lncRNA genes than protein-coding genes. However, only a handful of lncRNAs have been analyzed in detail.
Long non-coding RNAs (lncRNAs) are emerging as key molecules in cancers, yet their potential molecular mechanisms are not well understood.
long noncoding RNAs (lncRNAs), the largest family of noncoding transcripts, have emerged as common regulators of many cellular stressors; including heat shock, metabolic deprivation and DNA damage. | OBJECTIVE: Long non-coding RNAs (lncRNAs) are emerging as key molecules in
cancers, yet their potential molecular mechanisms are not well understood. The
objective of this study is to examine the expression and functions of lncRNAs in
the development of colorectal cancer (CRC).
METHODS: LncRNA expression profiling of CRC, adenoma and normal colorectal
tissues was performed to identify tumour-related lncRNAs involved in colorectal
maligt transformation. Then, we used quantitative reverse transcription PCR
assays to measure the tumour-related lncRNA and to assess its association with
survival and response to adjuvant chemotherapy in 252 patients with CRC. The
mechanisms of CCAL function and regulation in CRC were examined using molecular
biological methods.
RESULTS: We identified colorectal cancer-associated lncRNA (CCAL) as a key
regulator of CRC progression. Patients whose tumours had high CCAL expression
had a shorter overall survival and a worse response to adjuvant chemotherapy
than patients whose tumours had low CCAL expression. CCAL promoted CRC
progression by targeting activator protein 2α (AP-2α), which in turn activated
Wnt/β-catenin pathway. CCAL induced multidrug resistance (MDR) through
activating Wnt/β-catenin signalling by suppressing AP-2α and further
upregulating MDR1/P-gp expression. In addition, we found that histone H3
methylation and deacetylases contributed to the upregulation of CCAL in CRC.
CONCLUSIONS: Our results suggest that CCAL is a crucial oncogenic regulator
involved in CRC tumorigenesis and progression. Genomic imprinting has been a great resource for studying transcriptional and
post-transcriptional-based gene regulation by long noncoding RNAs (lncRNAs). In
this article, I overview the functional role of intergenic lncRNAs (H19, IPW,
and MEG3), antisense lncRNAs (Kcnq1ot1, Airn, Nespas, Ube3a-ATS), and enhancer
lncRNAs (IG-DMR eRNAs) to understand the diverse mechanisms being employed by
them in cis and/or trans to regulate the parent-of-origin-specific expression of
target genes. Recent evidence suggests that some of the lncRNAs regulate
imprinting by promoting intra-chromosomal higher-order chromatin
compartmentalization, affecting replication timing and subnuclear positioning.
Whereas others act via transcriptional occlusion or transcriptional
collision-based mechanisms. By establishing genomic imprinting of target genes,
the lncRNAs play a critical role in important biological functions, such as
placental and embryonic growth, pluripotency maintece, cell differentiation,
and neural-related functions such as synaptic development and plasticity. An
emerging consensus from the recent evidence is that the imprinted lncRNAs
fine-tune gene expression of the protein-coding genes to maintain their dosage
in cell. Hence, lncRNAs from imprinted clusters offer insights into their mode
of action, and these mechanisms have been the basis for uncovering the mode of
action of lncRNAs in several other biological contexts. This article is part of
a Special Issue entitled: Clues to long noncoding RNA taxonomy, edited by Dr.
Tetsuro Hirose and Dr. Shinichi Nakagawa. Genomic studies have revealed that humans possess far fewer protein-encoding
genes than originally predicted. These over-estimates were drawn from the
inherent developmental and stimuli-responsive complexity found in humans and
other mammals, when compared to lower eukaryotic organisms. This left a
conceptual void in many cellular networks, as a new class of functional
molecules was necessary for "fine-tuning" the basic proteomic machinery.
Transcriptomics analyses have determined that the vast majority of the genetic
material is transcribed as noncoding RNA, suggesting that these molecules could
provide the functional diversity initially sought from proteins. Indeed, as
discussed in this review, long noncoding RNAs (lncRNAs), the largest family of
noncoding transcripts, have emerged as common regulators of many cellular
stressors; including heat shock, metabolic deprivation and DNA damage. These
stimuli, while divergent in nature, share some common stress-responsive
pathways, notably inhibition of cell proliferation. This role intrinsically
makes stress-responsive lncRNA regulators potential tumor suppressor or
proto-oncogenic genes. As the list of functional RNA molecules continues to
rapidly expand it is becoming increasingly clear that the significance and
functionality of this family may someday rival that of proteins. This article is
part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited
by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Long noncoding RNAs (lncRNAs) represent a newly discovered class of regulatory
molecules that impact a variety of biological processes in cells and organ
systems. In humans, it is estimated that there may be more than twice as many
lncRNA genes than protein-coding genes. However, only a handful of lncRNAs have
been analyzed in detail. In this review, we describe expression and functions of
lncRNAs that have been demonstrated to impact innate and adaptive immunity.
These emerging paradigms illustrate remarkably diverse mechanisms that lncRNAs
utilize to impact the transcriptional programs of immune cells required to fight
against pathogens and maintain normal health and homeostasis. Hepatocellular carcinoma (HCC) is the most prevalent and maligt type of liver
cancer. Besides the high incidence, the resistance to chemotherapy is a major
problem that leads to the high mortality of HCC. Recently, aberrant expression
of long noncoding RNAs (lncRNAs) has been considered as a primary feature of
many types of cancer. However, the genome-wide expression pattern and associated
functional implications of lncRNAs in chemo-resistant HCC cells remain unknown.
In this study, we identified 120 differentially expressed lncRNAs with 61
up-regulated and 59 down-regulated (fold change>2, p<0.05) along with 421
differentially expressed mRNAs with 228 up-regulated and 193 down-regulated
(fold change>2, p<0.05) in oxaliplatin-resistant (MHCC97H-OXA) HCC cells,
compared to parental oxaliplatin-sensitive (MHCC97H) by microarray. The
underlying pathways were related to cell death, proliferation, cellular response
to stimulus, including p53 pathway, ErbB pathway and MAPK pathway. Further, 16
lncRNAs were selected for validation of microarray results with quantitative
PCR, and a strong correlation was identified between the qPCR results and
microarray data. We demonstrated for the first time that ENST00000438347,
NR_073453 and ENST00000502804 were up-regulated in MHCC97H-OXA cells as well as
chemo-resistant HCC cancerous tissues. Moreover, the expression of
ENST00000518376 was significantly associated with the tumor size and
differentiation. Overall survival analysis showed that high expression of
ENST00000438347 and ENST00000518376 was associated with poor prognosis in HCC
patients. Taken together, our results reveal that the expression profile in
oxaliplatin-resistant HCC is significantly altered including lncRNAs. And a
series of de novo lncRNAs play important functions in HCC oxaliplatin resistance
and HCC progression. |
How many microorganisms are present in human normal gut? | Human gut microbiota is home to 10 to 100 trillions microorganisms. | Social behavior plays a pivotal role in the mental well-being of an individual.
Continuous efforts in the past have led to advancements in the area of how the
brain regulates emotion and cognition, while the understanding of human social
behavior still remains eluded. A major breakthrough in understanding the
etiology of neurological disorders is the recent insight on the role of the gut
microbiota (GM). Human GM also referred to as the "forgotten organ" is home to
10(13-14) microorganisms, which is 10 times the number of cells present in the
human body. In addition, the gut microbiome (total genome of GM) is 150 times
greater as compared to the human genome. An emerging concept gaining worldwide
focus and acceptance is that, this much big genome can potentially control human
behavior and other biological functions. Herein we hypothesize on the basis of
GM's ability to modify brain and behavior and that it can directly or indirectly
control social behavior. This review focuses on the association of GM with
various domains of social behavior like stress, cognition and anxiety. The gut microbiome comprises the collective genome of the trillions of
microorganisms residing in our gastrointestinal ecosystem. The interaction
between the host and its gut microbiome is a complex relationship whose
manipulation could prove critical to preventing or treating not only various gut
disorders, like irritable bowel syndrome (IBS) and ulcerative colitis (UC), but
also central nervous system (CNS) disorders, such as Alzheimer's and Parkinson's
diseases. The purpose of this review is to summarize what is known about the gut
microbiome, how it is connected to the development of disease and to identify
the bacterial and biochemical targets that should be the focus of future
research. Understanding the mechanisms behind the activity and proliferation of
the gut microbiome will provide us new insights that may pave the way for novel
therapeutic strategies. Type 1 diabetes (T1D) is an autoimmune disease resulting from T cell-mediated
destruction of the insulin-secreting pancreatic beta cells. During the past 50
years T1D incidence has increased dramatically in many countries accompanied by
an earlier age of onset especially in persons with lower genetic risk. These
observations have prompted investigations of dynamic environmental factors that
may contributor to risk for anti-pancreatic immunity. The gut and pancreas are
anatomically and biochemically linked through the enteroinsular axis, a system
in which gut-derived immune and metabolic signals have the potential to evoke
effects in the pancreas. The gut microbiome (i.e. the 100 trillion symbiotic
microorganisms which inhabit the mammalian gastrointestinal tract) influences
numerous aspects of host metabolism, development and immunity. Here we examine
recent evidence linking gut microbiome composition and function to pancreatic
autoimmunity. Studies in children with genetic risk factors for T1D and analyses
of the microbiome in rodent models have begun to associations between an altered
microbiome composition potentially favoring a pro-inflammatory intestinal
metabolic milieu and T1D. We discuss how environmental factors during critical
developmental windows - gestation, birth, weaning and puberty may contribute to
T1D risk. For example mode of delivery (vaginal or C-section) and exposure to
antibiotics (pre- or post-natally) are two factors that modulate the maternal
and/or offspring microbiome and can impact T1D development. Taken together,
these emerging data underscore the requirement for longitudinal studies and
mechanistic investigations in human subjects and rodent models to identify the
basis for microbiome modulation of T1D and to identify biomarkers and
therapeutics to improve the delayed onset and prevention of the disease. The microorganisms inhabiting the human gut are abundant (10(14) cells) and
diverse (approximately 500 species per individual). It is now acknowledged that
the microbiota has coevolved with its host to achieve a symbiotic relationship,
leading to physiological homeostasis. The gut microbiota ensures vital
functions, such as food digestibility, maturation of the host immune system, and
protection against pathogens. Over the last few decades, the gut microbiota has
also been associated with numerous diseases, such as inflammatory bowel disease,
irritable bowel syndrome, obesity, and metabolic diseases. In most of these
pathologies, a microbial dysbiosis has been found, indicating shifts in the
taxonomic composition of the gut microbiota and changes in its functionality.
Our understanding of the influence of the gut microbiota on human health is
still growing. Working with microorganisms residing in the gut is challenging
since most of them are anaerobic and a vast majority (approximately 75%) are
uncultivable to date. Recently, a wide range of new approaches (meta-omics) has
been developed to bypass the uncultivability and reveal the intricate mechanisms
that sustain gut microbial homeostasis. After a brief description of these
approaches (metagenomics, metatranscriptomics, metaproteomics, and
metabolomics), this review will discuss the importance of considering the gut
microbiome as a structured ecosystem and the use of meta-omics to decipher
dysfunctions of the gut microbiome in diseases. |
What is the role of 3,4-diaminobenzoic acid derivatives in the immune system? | 3,4-diaminobenzoic acid derivatives are inhibitors of the oxytocinase subfamily of M1 aminopeptidases with immune-regulating properties. Cell-based analysis indicated that the lead compounds can be effective in downregulating macrophage activation induced by lipopolysaccharide and interferon-γ as well as cross-presentation by bone marrow-derived dendritic cells. | Members of the oxytocinase subfamily of M1 aminopeptidases (ERAP1, ERAP2, and
IRAP) play important roles in both the adaptive and innate human immune
responses. Their enzymatic activity can contribute to the pathogenesis of
several major human diseases ranging from viral and parasitic infections to
autoimmunity and cancer. We have previously demonstrated that diaminobenzoic
acid derivatives show promise as selective inhibitors for this group of
aminopeptidases. In this study, we have thoroughly explored a series of
3,4-diaminobenzoic acid derivatives as inhibitors of this class of enzymes,
achieving submicromolar inhibitors for ERAP2 (IC50 = 237 nM) and IRAP (IC50 =
105 nM). Cell-based analysis indicated that the lead compounds can be effective
in downregulating macrophage activation induced by lipopolysaccharide and
interferon-γ as well as cross-presentation by bone marrow-derived dendritic
cells. Our results indicate that this class of inhibitors may be useful for the
targeted downregulation of immune responses. |
Can NADPH oxidase be inhibited by apocynin and diphenylene iodonium? | Yes, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be inhibited by apocynin or diphenylene iodonium (DPI). | Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of
the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a
cell-free system of NADPH oxidase activation consisting of neutrophil membranes
and cytosol from resting cells, supplemented with guanosine
5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes
isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate,
addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced
inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to
contain the Ph2I-sensitive component(s). In the presence of a concentration of
Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane
protein), addition of catalytic amounts of the redox mediator dichloroindophenol
(Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of
which was about half of that determined in non-inhibited oxidase. A marked
increase in the efficiency of this by-pass was achieved by addition of sodium
deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in
membranes isolated from resting neutrophils. At a higher concentration of Ph2I
(100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase
activity was only half inhibited, which indicated that, in the NADPH oxidase
complex, there are at least two Ph2I sensitive components, differing by their
sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10
nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil
membranes was modified, suggesting that the component sensitive to low
concentrations of Ph2I is the heme binding component of cytochrome b558. Higher
concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase
component of the oxidase complex. A number of membrane and cytosolic proteins
were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound
24-kDa protein, which might be the small subunit of cytochrome b558, responded
more specifically to the conditions of activation and reduction which are
required for inhibition of O2- production by Ph2I. The O2(-)-generating form of
xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a
non-heme iron flavoprotein, by Ph2I had a number of features in common with that
of the neutrophil NADPH oxidase, namely the requirement of reducing conditions
for inhibition of O2- production by Ph2I and the induction of a by-pass of
electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some
similarity in the molecular organization of the two enzymes. Diphenylene iodonium is an inhibitor of the respiratory burst-generating NADPH
oxidase of phagocytes. The effect of this compound on human monocyte-derived
macrophages and its usefulness in exploring the antimicrobial mechanisms of
phagocytes was examined. 1 microM diphenylene iodonium inhibited hydrogen
peroxide production by human macrophages and the activity of these cells against
Toxoplasma gondii. At this concentration macrophage degranulation was
unaffected. We have previously reported that N-ethylmaleimide induces apoptosis through
activation of K(+), Cl(-)-cotransport in HepG2 human hepatoblastoma cells. In
this study, we investigated the role for reactive oxygen species as a mediator
of the apoptosis induced by N-ethylmaleimide. N-ethylmaleimide induced a
significant elevation of intracellular level of reactive oxygen species.
Treatment with antioxidants (N-acetyl cysteine,
N,N'-diphenyl-p-phenylenediamine) which markedly suppressed generation of
reactive oxygen species, significantly inhibited the N-ethylmaleimide-induced
activation of K(+), Cl(-)-cotransport and apoptosis. Inhibitors of NADPH oxidase
(diphenylene iodonium, apocynin, D-(+)-neopterine) also significantly blunted
the generation of reactive oxygen species, activation of K(+), Cl(-)-cotransport
and apoptosis induced by N-ethylmaleimide. These results suggest that reactive
oxygen species generated through activation of NADPH oxidase may play a role in
the N-ethylmaleimide-induced stimulation of K(+), Cl(-)-cotransport and
apoptosis in HepG2 cells. Increasing evidence has suggested an important role for environmental toxins
such as pesticides in the pathogenesis of Parkinson's disease (PD). Chronic
exposure to rotenone, a common herbicide, reproduces features of Parkinsonism in
rats. Mechanistically, rotenone-induced dopaminergic neurodegeneration has been
associated with both its inhibition of neuronal mitochondrial complex I and the
enhancement of activated microglia. Our previous studies with NADPH oxidase
inhibitors, diphenylene iodonium and apocynin, suggested that NADPH
oxidase-derived superoxide might be a major factor in mediating the
microglia-enhanced rotenone neurotoxicity. However, because of the relatively
low specificity of these inhibitors, the exact source of superoxide induced by
rotenone remains to be further determined. In this study, using primary
mesencephalic cultures from NADPH oxidase--null (gp91phox-/-) or wild-type
(gp91phox+/+) mice, we demonstrated a critical role for microglial NADPH oxidase
in mediating microglia-enhanced rotenone neurotoxicity. In neuron--glia
cultures, dopaminergic neurons from gp91phox-/- mice were more resistant to
rotenone neurotoxicity than those from gp91phox+/+ mice. However, in
neuron-enriched cultures, the neurotoxicity of rotenone was not different
between the two types of mice. More importantly, the addition of microglia
prepared from gp91phox+/+ mice but not from gp91phox-/- mice to neuron-enriched
cultures markedly increased rotenone-induced degeneration of dopaminergic
neurons. Furthermore, apocynin attenuated rotenone neurotoxicity only in the
presence of microglia from gp91phox+/+ mice. These results indicated that the
greatly enhanced neurotoxicity of rotenone was attributed to the release of
NADPH oxidase-derived superoxide from activated microglia. This study also
suggested that microglial NADPH oxidase may be a promising target for PD
treatment. We have shown that intracellular superoxide (O(2)(*-)) production in CNS neurons
plays a key role in the pressor, bradycardic, and dipsogenic actions of Ang II
in the brain. In this study, we tested the hypothesis that a Rac1-dependent
NADPH oxidase is a key source of O(2)(*-) in Ang II-sensitive neurons and is
involved in these central Ang II-dependent effects. We performed both in vitro
and in vivo studies using adenoviral (Ad)-mediated expression of
domit-negative Rac1 (AdN17Rac1) to inhibit Ang II-stimulated Rac1 activation,
an obligatory step in NADPH oxidase activation. Ang II induced a time-dependent
increase in Rac1 activation and O(2)(*-) production in Neuro-2A cells, and this
was abolished by pretreatment with AdN17Rac1 or the NADPH oxidase inhibitors
apocynin or diphenylene iodonium. AdN17Rac1 also inhibited Ang II-induced
increases in NADPH oxidase activity in primary neurons cultured from central
cardiovascular control regions. In contrast, overexpression of wild-type Rac1
(AdwtRac1) caused more robust NADPH oxidase-dependent O(2)(*-) production to Ang
II. To extend the in vitro studies, the pressor, bradycardic, and drinking
responses to intracerebroventricularly (ICV) injected Ang II were measured in
mice that had undergone gene transfer of AdN17Rac1 or AdwtRac1 to the brain.
AdN17Rac1 abolished the increase in blood pressure, decrease in heart rate, and
drinking response induced by ICV injection of Ang II, whereas AdwtRac1 enhanced
these physiological effects. The exaggerated physiological responses in
AdwtRac1-treated mice were abolished by O(2)(*-) scavenging. These results, for
the first time, identify a Rac1-dependent NADPH oxidase as the source of central
Ang II-induced O(2)(*-) production, and implicate this oxidase in cardiovascular
diseases associated with dysregulation of brain Ang II signaling, including
hypertension. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) mediated
generation of reactive oxygen species (ROS) was originally identified as the
powerful host defense machinery against microorganism in phagocytes. But recent
reports indicated that some non-phagocytic cells also have the NADPH oxidase
activity, and the ROS produced by it may act as cell signal molecule. But as far
as today, whether the NADPH oxidase also plays similar role in phagocyte has not
been paid much attention. Utilizing the undifferentiated HL-60 promyelocytic
leukemia cells as a model, the aim of the present study was to determine whether
NADPH oxidase plays a role on ROS generation in undifferentiated HL-60, and the
ROS mediated by it was essential for cell's survival. For the first time, we
verified that the release of ROS in undifferentiated HL-60 was significantly
increased by the stimulation with Calcium ionophore or opsonized zymosan, which
are known to trigger respiration burst in phagocytes by NADPH oxidase pathway.
Diphenylene iodonium (DPI) or apocynin (APO), two inhibitors of NADPH oxidase,
significantly suppressed the increasing of ROS caused by opsonized zymosan. Cell
survival assay and fluorescence double dyeing with acridine orange and ethidium
bromide showed that DPI and APO, as well as superoxide dismutase (SOD) and
catalase (CAT) concentration-dependently decreased the viability of
undifferentiated HL-60 cells, whereas exogenous H2O2 can rescue the cells from
death obviously. Our results suggested that the ROS, generated by NADPH oxidase
play an essential role in the survival of undifferentiated HL-60 cells. Microglia are resident brain macrophages that become activated and proliferate
following brain damage or stimulation by immune mediators, such as IL-1beta or
TNF-alpha. We investigated the mechanisms by which microglial proliferation is
regulated in primary cultures of rat glia. We found that basal proliferation of
microglia was stimulated by proinflammatory cytokines IL-1beta or TNF-alpha, and
this proliferation was completely inhibited by catalase, implicating hydrogen
peroxide as a mediator of proliferation. In addition, inhibitors of NADPH
oxidase (diphenylene iodonium or apocynin) also prevented microglia
proliferation, suggesting that this may be the source of hydrogen peroxide.
IL-1beta and TNF-alpha rapidly stimulated the rate of hydrogen peroxide produced
by isolated microglia, and this was inhibited by diphenylene iodonium, implying
that the cytokines were acting directly on microglia to stimulate the NADPH
oxidase. Low concentrations of PMA or arachidonic acid (known activators of
NADPH oxidase) or xanthine/xanthine oxidase or glucose oxidase (generating
hydrogen peroxide) also increased microglia proliferation and this was blocked
by catalase, showing that NADPH oxidase activation or hydrogen peroxide was
sufficient to stimulate microglia proliferation. In contrast to microglia, the
proliferation of astrocytes was unaffected by the presence of catalase. In
conclusion, these findings indicate that microglial proliferation in response to
IL-1beta or TNF-alpha is mediated by hydrogen peroxide from NADPH oxidase. Increasing evidence indicates that advanced glycation end products (AGEs)
promote retinal alterations through oxidative stress. However, the pathways
involved in AGE-induced generation of reactive oxygen species (ROS) in retinal
cells are poorly defined. In the present study, we investigated the role of
nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase in
AGE-induced ROS intracellular generation and vascular endothelial growth factor
(VEGF) expression in bovine retinal endothelial cells (BRECs). Incubation of
BRECs with 100 microg/mL AGEs increased ROS generation and VEGF expression in
these cells. Treatment of the cells with the NADPH oxidase inhibitors, apocynin
and diphenylene iodonium, inhibited these effects. In retinal endothelial cells
exposed to AGEs, translocation of protein kinase C (PKC)-beta2 and p47phox was
observed. Inhibition of PKC by treatment of the cells with calphostin C,
GF10923X, and LY379196 totally suppressed AGE-mediated p47phox translocation and
ROS generation. Incubation of BRECs with gliclazide inhibited AGE-induced
PKC-beta2 and p47phox translocation and totally abrogated AGE-mediated ROS
generation and VEGF expression. Overall, these results demonstrate that AGEs
induce intracellular ROS generation and VEGF expression in retinal endothelial
cells through a PKC-dependent activation of NADPH oxidase. Inhibition of retinal
NADPH oxidase expression and ROS generated by this system provides a new
potential mechanism by which gliclazide may affect retinal VEGF expression and
exert a beneficial effect on diabetic retinopathy. Voltage-gated potassium channels (Kv) and thromboxane A(2) (TXA(2)) have been
involved in several forms of human and experimental pulmonary hypertension. We
have reported that the TXA(2) analog U46619, via activation of TP receptors and
PKCzeta, inhibited Kv currents in rat pulmonary artery smooth muscle cells
(PASMC), increased cytosolic calcium, and induced a contractile response in
isolated rat and piglet pulmonary arteries (PA). Herein, we have analyzed the
role of reactive oxygen species (ROS) in this signaling pathway. In rat PA,
U46619 increased dichlorofluorescein fluorescence, an indicator of intracellular
hydrogen peroxide, and this effect was prevented by the NADPH oxidase inhibitor
apocynin and by polyethyleneglycol-catalase (PEG-catalase, a membrane-permeable
form of catalase). U46619 inhibited Kv currents in native PASMC and these
effects were strongly inhibited by apocynin. The contractile responses to U46619
in isolated PA were inhibited by PEG-catalase and the NADPH oxidase inhibitors
diphenylene iodonium (DPI) and apocynin. A membrane permeable of hydrogen
peroxide, t-butyl hydroperoxide, also inhibited Kv currents and induced a
contractile response. Activation of NADPH oxidase and the subsequent production
of hydrogen peroxide are involved in the Kv channel inhibition and the
contractile response induced by TP receptor activation in rat PA. Oxidative stress is widely recognized as a key mediator of degenerative
processes in Parkinson's disease (PD). Recently, we demonstrated that the
dopaminergic toxin MPP+ initiates oxidative stress to cause caspase-3-dependent
apoptotic cell death in mesencephalic dopaminergic neuronal (N27) cells. In this
study, we determined the source of reactive oxygen species (ROS) produced during
MPP+-induced apoptotic cell death. In addition to mitochondria, plasma membrane
NADPH oxidase is considered a major producer of ROS inside the cell. Here, we
show that N27 neuronal cells express key NADPH oxidase subunits gp91phox and
p67phox. We used structurally diverse NADPH oxidase inhibitors,
aminoethyl-benzenesulfonylfluoride (AEBSF, 100-1000microM), apocynin
(100-1000microM), and diphenylene iodonium (DPI, 3-30microM), to inhibit
intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using
the ROS-sensitive dye hydroethidine revealed that AEBSF blocked 300microM
MPP+-induced ROS production for over 45min in N27 cells, in a dose-dependent
manner. Further treatment with DPI, apocynin, and SOD also blocked MPP+-induced
ROS production. In Sytox cell death assays, co-treatment with AEBSF, apocynin,
or DPI for 24h significantly suppressed MPP+-induced cytotoxic cell death.
Similarly, co-treatment with these inhibitors also significantly attenuated
MPP+-induced increases in caspase-3 enzymatic activity. Furthermore,
quantitative DNA fragmentation ELISA assays revealed that AEBSF, DPI, and
apocynin rescue N27 cells from MPP+-induced apoptotic cell death. Together,
these results indicate for the first time that intracellular ROS generated by
NAPDH oxidase are present within the mesencephalic neuronal cells, and are a key
determit of MPP+-mediated dopaminergic degeneration in in vitro models of
dopaminergic degeneration. This study supports a critical role of NADPH oxidase
in the oxidative damage in PD; targeting this enzyme may lead to novel therapies
for PD. Activated pancreatic stellate cells (PSCs) play an important role in pancreatic
fibrosis and inflammation, where oxidative stress is implicated in the
pathogenesis. NADPH oxidase might be a source of reactive oxygen species (ROS)
in the injured pancreas. This study aimed to clarify the expression and
regulation of cell functions by NADPH oxidase in PSCs. PSCs were isolated from
rat and human pancreas tissues. Expression of NADPH oxidase was assessed by
reverse transcription-PCR and immunostaining. Intracellular ROS production was
assessed using 2',7'-dichlorofluorescin diacetate. The effects of diphenylene
iodonium (DPI) and apocynin, inhibitors of NADPH oxidase, on key parameters of
PSC activation were evaluated in vitro. In vivo, DPI (at 1 mg.kg body
wt(-1).day(-1)) was administered in drinking water to 10-wk-old male Wistar
Bonn/Kobori rats for 10 wk and to rats with chronic pancreatitis induced by
dibutyltin dichloride (DBTC). PSCs expressed key components of NADPH oxidase
(p22(phox), p47(phox), NOX1, gp91(phox)/NOX2, NOX4, and NOX activator 1).
PDGF-BB, IL-1beta, and angiotensin II induced ROS production, which was
abolished by DPI and apocynin. DPI inhibited PDGF-induced proliferation,
IL-1beta-induced chemokine production, and expression of alpha-smooth muscle
actin and collagen. DPI inhibited transformation of freshly isolated cells to a
myofibroblast-like phenotype. In addition, DPI inhibited the development of
pancreatic fibrosis in Wistar Bonn/Kobori rats and in rats with DBTC-induced
chronic pancreatitis. In conclusion, PSCs express NADPH oxidase to generate ROS,
which mediates key cell functions and activation of PSCs. NADPH oxidase might be
a potential target for the treatment of pancreatic fibrosis. Histone acetylation regulated by histone acetyltransferases (HATs) and histone
deacetylases (HDACs) plays a critical role in the expression of inflammatory
genes, such as vascular cell adhesion molecule-1 (VCAM-1). Oxidative processes
have been shown to induce VCAM-1 expression. Here, we investigated the
mechanisms underlying IL-1beta-induced VCAM-1 expression in human tracheal
smooth muscle cells (HTSMCs). Our results showed that IL-1beta enhanced
HTSMCs-monocyte adhesion through up-regulation of VCAM-1, which was inhibited by
pretreatment with selective inhibitors of PKCalpha (Gö6976), c-Src (PP1), NADPH
oxidase [diphenylene iodonium (DPI) and apocynin (APO)], intracellular calcium
chelator (BAPTA/AM), PI-PLC (U73122), CaM (calmidazolium chloride), CaM kinase
II (KN62), p300 (garcinol), NF-kappaB (Bay11-7082), HDAC (trichostatin A), and
ROS scavenger [N-acetyl-L-cysteine (NAC)] or transfection with siRNAs of MyD88,
PKCalpha, Src, p47(phox), p300, and HDAC4. Moreover, IL-1beta stimulated
NF-kappaB and CaMKII phosphorylation through MyD88-dependent
PI-PLC/PKCalpha/c-Src/ROS and PI-PLC/Ca2+/CaM pathways, respectively. Activation
of NF-kappaB and CaMKII may eventually lead to the acetylation of histone
residues and phosphorylation of histone deacetylases. These findings suggested
that IL-1beta induced VCAM-1 expression via these multiple signaling pathways in
HTSMCs. Blockade of these pathways may reduce monocyte adhesion via VCAM-1
suppression and attenuation of the inflammatory responses in airway diseases. In swim-up-selected spermatozoa of 38 normozoospermic patients, capacitated
spermatozoa exhibited enhanced pentose phosphate pathway (PPP) activity and
increased expression of glucose-6-phosphate dehydrogenase (G6PD). The G6PD
inhibitor DHEA and the inhibitors of NADPH oxidase apocynin and diphenylene
iodonium (DPI) prevented both superoxide generation and capacitation in human
spermatozoa, but whereas DPI and DHEA inhibited PPP, apocynin did not influence
it, suggesting that PPP activation during capacitation is not a response to
increased oxidative stress but exerts a role by supplying reducing equivalents
to oxygen. BACKGROUND AND PURPOSE: Oxidative stress [i.e. increased levels of reactive
oxygen species (ROS)] has been suggested as a pathomechanism of different
diseases, although the disease-relevant sources of ROS remain to be identified.
One of these sources may be NADPH oxidases. However, due to increasing concerns
about the specificity of the compounds commonly used as NADPH oxidase
inhibitors, data obtained with these compounds may have to be re-interpreted.
EXPERIMENTAL APPROACH: We compared the pharmacological profiles of the commonly
used NADPH oxidase inhibitors, diphenylene iodonium (DPI), apocynin and
4-(2-amino-ethyl)-benzolsulphonyl-fluoride (AEBSF), as well as the novel
triazolo pyrimidine VAS3947. We used several assays for detecting cellular and
tissue ROS, as none of them is specific and artefact free.
KEY RESULTS: DPI abolished NADPH oxidase-mediated ROS formation, but also
inhibited other flavo-enzymes such as NO synthase (NOS) and xanthine oxidase
(XOD). Apocynin interfered with ROS detection and varied considerably in
efficacy and potency, as did AEBSF. Conversely, the novel NADPH oxidase
inhibitor, VAS3947, consistently inhibited NADPH oxidase activity in low
micromolar concentrations, and interfered neither with ROS detection nor with
XOD or eNOS activities. VAS3947 attenuated ROS formation in aortas of
spontaneously hypertensive rats (SHRs), where NOS or XOD inhibitors were without
effect.
CONCLUSIONS AND IMPLICATIONS: Our data suggest that triazolo pyrimidines such as
VAS3947 are specific NADPH oxidase inhibitors, while DPI and apocynin can no
longer be recommended. Based on the effects of VAS3947, NADPH oxidases appear to
be a major source of ROS in aortas of SHRs. Differentiation of human endometrial stromal cells into specialized decidual
cells is critical for embryo implantation and survival of the conceptus.
Initiation of this differentiation process is strictly dependent on elevated
cAMP levels, but the signal intermediates that control the expression of
decidual marker genes, such as prolactin (PRL) and IGFBP1, remain poorly
characterized. Here we show that cAMP-dependent decidualization can be
attenuated or enhanced upon treatment of primary cultures with a nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenylen iodonium)
or activator (apocynin), respectively. Time-course analysis demonstrated that
cAMP enhances endogenous reactive oxygen species production, apparent after 12 h
of stimulation, which coincides with a dramatic increase in decidual PRL and
IGFBP1 expression. Knockdown of the Rho GTPase RAC1, which disables activation
of the NADPH oxidase homologs NADPH oxidase (NOX)-1, NOX-2, and NOX-3, had no
effect on PRL or IGFBP1 expression. In contrast, silencing of NOX-4, or its
cofactor p22(PHOX), inhibited the expression of both decidual markers. Finally,
we show that the NOX-4/p22(PHOX) complex regulates the DNA-binding activity of
CCAAT/enhancer binding protein-β, a key regulator of human endometrial stromal
cell differentiation. Thus, NOX-4 activation and reactive oxygen species
signaling play an integral role in initiating the endometrial decidual response
in preparation of pregcy. Fibrotic disorders are typified by excessive connective tissue and extracellular
matrix (ECM) deposition that precludes normal healing processes in different
tissues. Angiotensin-II (Ang-II) is involved in the fibrotic response. Several
muscular dystrophies are characterized by extensive fibrosis. However, the exact
role of Ang-II in skeletal muscle fibrosis is unknown. Here we show that
myoblasts responded to Ang-II by increasing protein levels of connective tissue
growth factor (CTGF/CCN2), collagen-III and fibronectin. These Ang-II-induced
pro-fibrotic effects were mediated by AT-1 receptors. Remarkably, Ang-II induced
reactive oxygen species (ROS) via a NAD(P)H oxidase-dependent mechanism, as
shown by inhibition of ROS production via the NAD(P)H oxidase inhibitors
diphenylene iodonium (DPI) and apocynin. This increase in ROS is critical for
Ang-II-induced fibrotic effects, as indicated by the decrease in Ang-II-induced
CTGF and fibronectin levels by DPI and apocynin. We also show that
Ang-II-induced ROS production and fibrosis require PKC activity as indicated by
the generic PKC inhibitor chelerythrine. These results strongly suggest that the
fibrotic response induced by Ang-II is mediated by AT-1 receptor and requires
NAD(P)H-induced ROS in skeletal muscle cells. Phagocytes engulf pathogenic microbes, kill them and degrade their cellular
macromolecules by hydrolytic enzymes in phagolysosomes. However, such enzymes
are unable to degrade some microbial polysaccharides, and fate of such
indigestible polysaccharides in phagocytes remains uncertain. Using the
extracellular domain of Dectin-1 as β-glucan-specific probes, we succeeded in
detection of soluble and Dectin-1-reactive β-glucan discharged from mouse RAW
264.7 and human THP-1 macrophage cell lines as well as mouse peritoneal
macrophages, which had phagocytized insoluble β-glucan particles. The RAW 264.7
cell culture-supernatant containing the discharged β-glucan stimulated naïve RAW
264.7 cells, resulting in the induction of cytokine expression. Such discharge
of Dectin-1-reactive β-glucan from macrophage cells was inhibited by either
NADPH oxidase inhibitors (apocynin and diphenylene iodonium) or radical
scavengers (N-acetyl cysteine and MCI-186). Moreover, reactive oxygen species
(ROS) produced by a Cu(2+)/ascorbic acid system solubilized insoluble β-glucan
particles in vitro, and a part of the solubilized β-glucan was Dectin-1 reactive
and biologically active in macrophage activation. The soluble and biologically
active β-glucan was degraded further during prolonged exposure to ROS. These
results suggest that degraded but Dectin-1-reactive β-glucan is discharged from
macrophage cells phagocytizing insoluble β-glucan particles and stimulates not
only themselves again but also the other naïve phagocytes, leading to the
effective elimination of infecting microbes and the ultimate breakdown and
inactivation of metabolically resistant β-glucan. Recent studies have shown that oligomeric amyloid-β (oAβ) peptide can
potentially activate microglia in addition to inducing more potent neurotoxicity
compared with fibrillar Aβ (fAβ); however, its mechanisms of action remain
unclear. This study was designed to investigate the possible mechanisms involved
in the microglial activation induced by oAβ in BV-2 microglial cells. The
results showed that oAβ induced activated properties of microglia, including
higher proliferative capacity as well as increased production of reactive oxygen
species, nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β
(IL-1β). NADPH oxidase inhibitors [diphenylene iodonium (DPI) and apocynin
(4-hydroxy-3-methoxy-acetophenone)] prevented the microglial activation induced
by oAβ, suggesting that NADPH oxidase activation was involved in microglial
activation. In addition, TNF-α and IL-1β, which are massively released by
activated microglia, significantly induced the activation of microglia, thereby
resulting in the production of NO and proliferation of microglia, respectively.
These effects could be inhibited by diphenylene iodonium and apocynin,
indicating a self-cycle regulated by NADPH oxidase in microglial activation in
response to oAβ. In conclusion, microglial activation induced by oAβ is possibly
mediated by NADPH oxidase, suggesting that oAβ, which is normally considered a
neurotoxin, may also lead to indirect neuronal damage through the
pro-inflammation activation of microglia in Alzheimer's disease and that NADPH
oxidase could be a potential target to prevent oAβ-induced inflammatory
neurodegeneration. Angiotensin II (AII) plays a major role in the progression of chronic kidney
diseases. Podocytes are essential components of the ultrafiltration apparatus,
and are targets for AII signaling. AII has been shown to increase generation of
reactive oxygen species (ROS) in podocytes. Canonical transient receptor
potential-6 (TRPC6) channels stimulate Ca(2+) influx in podocytes, and have been
implicated in glomerular disease. We observed that AII increased cationic
currents in rat podocytes in an isolated glomerulus preparation in which
podocytes are still attached to the underlying capillary. This effect was
completely blocked by SKF-96365, by micromolar La(3+) , and by siRNA knockdown
of TRPC6, indicating that TRPC6 is the primary source of Ca(2+) influx mobilized
by endogenously expressed angiotensin II receptors in these cells. These
responses were also blocked by the AT1R antagonist losartan, the phospholipase C
inhibitor D-609, and by inhibition of G protein signaling. The pan-protein
kinase C inhibitor chelerythrine had no effect. Importantly, pretreating
podocytes with the ROS quencher manganese (III) tetrakis (4-benzoic acid)
porphyrin chloride (MnTBAP) eliminated AII activation of TRPC6. Significant
reductions of AII effects on podocyte TRPC6 were also observed after
pretreatment with NADPH oxidase inhibitors apocynin or diphenylene iodonium
(DPI). These data suggest that ROS production permits activation of TRPC6
channels by G protein and PLC-dependent cascades initiated by AII acting on
AT1Rs in podocytes. This pathway also provides a basis whereby two forms of
cellular stress-oxidative stress and Ca(2+) overload-converge on common pathways
relevant to disease. Author information:
(1)Department of Pathology, VU University Medical Center, Amsterdam, The
Netherlands; ICaR-VU, Institute for Cardiovascular Research, VU University
Medical Center, Amsterdam, The Netherlands.
(2)Department of Physiology, VU University Medical Center, Amsterdam, The
Netherlands; ICaR-VU, Institute for Cardiovascular Research, VU University
Medical Center, Amsterdam, The Netherlands.
(3)Department of Pathology, VU University Medical Center, Amsterdam, The
Netherlands.
(4)Department of Pharmacology and Chemical Biology, Vascular Medicine Institute,
University of Pittsburgh, Pittsburgh, PA, USA.
(5)Department of Cardiac Surgery, VU University Medical Center, Amsterdam, The
Netherlands; ICaR-VU, Institute for Cardiovascular Research, VU University
Medical Center, Amsterdam, The Netherlands.
(6)Department of Cardiology, VU University Medical Center, Amsterdam, The
Netherlands; ICaR-VU, Institute for Cardiovascular Research, VU University
Medical Center, Amsterdam, The Netherlands.
(7)Department of Pathology, VU University Medical Center, Amsterdam, The
Netherlands; Department of Cardiac Surgery, VU University Medical Center,
Amsterdam, The Netherlands; ICaR-VU, Institute for Cardiovascular Research, VU
University Medical Center, Amsterdam, The Netherlands.
(8)Department of Pathology, VU University Medical Center, Amsterdam, The
Netherlands; ICaR-VU, Institute for Cardiovascular Research, VU University
Medical Center, Amsterdam, The Netherlands. Electronic address:
[email protected]. |
Viliuisk encephalomyelitis is diagnosed in which geographical area? | Viliuisk encephalomyelitis (VE) is an endemic neurological disease in Northeast Siberia and generally considered to be a chronic encephalomyelitis of unknown origin actually spreading in the Sakha (Yakutian) Republic. | Viliuisk encephalomyelitis (VE), a progressive neurological disorder with a
fatal outcome usually in several months to 6 yrs after disease onset, is seen
only among the Iakut people of Siberia. The acute meningoencephalitic phase of
the disease is followed by progressive dementia, rigidity and spastic
tetraparesis. The disease is characterized by multiple micronecrotic foci with
marked inflammatory reactions and gliosis in the grey matter. The acute febrile
onset, with cerebrospinal fluid (CSF) pleocytosis and increased protein in the
CSF, the epidemiology and the inflammatory neuropathology suggest the disease is
infectious. Studies on household spread indicate an incubation time of up to
several years. Viliuisk encephalomyelitis was restricted to an ethically
distinct group of Iakut people of the Middle Viliui region, but in recent
decades, with migration from this region, it has been spreading into previously
unaffected Iakut populations. The occurrence of multiple VE cases in households
and introduction of the disease by migrants into new populations indicate
horizontal transmission in a setting of long intimate contact. Human T-lymphotropic virus type I (HTLV-I), the etiological agent of adult
T-cell leukemia/lymphoma, also appears to be the cause of tropical spastic
paraparesis, a chronic myelopathy reported in several different regions of the
world. The prevalence of antibodies to HTLV-I in patients with chronic
neurodegenerative disorders other than tropical spastic paraparesis and in
patients with some muscle inflammatory disorders has been investigated. IgG
antibodies to HTLV-I were measured in the sera and/or cerebrospinal fluid from
82 Guamanian patients with amyotrophic lateral sclerosis and
parkinsonism-dementia, 164 Guamanian normal controls, 10 patients with kuru from
the Eastern Highlands of Papua New Guinea, 4 patients with Viliuisk
encephalomyelitis from the Iakut region of eastern Siberia, 45 Italian patients
with multiple sclerosis, and 56 patients with polymyositis (49 from the United
States and 7 from Jamaica). As determined by enzyme-linked immunosorbent assay,
Western immunoblot, and gelatin particle agglutination techniques, serological
evidence of HTLV-I infection was found in 1 patient with amyotrophic lateral
sclerosis and 1 control subject from Guam, and in 1 patient from the United
States and all 7 Jamaican patients with polymyositis. Except for the high
seropositivity rate among the group of Jamaican patients with polymyositis, our
data indicate that HTLV-I is an unlikely causative agent in the spectrum of the
neurological diseases examined. The seropositivity of the 7 Jamaican patients
with polymyositis requires further study. Viliuisk encephalomyelitis (VE) is an unique neurological disease occurring in
the Iakut (Sakha) people of Siberia. Evolution of the disease follows one of
three broad clinical forms: subacute, slowly progressive or chronic. Death
occurs within 3 to 6 months in subacute cases and within 6 years in the slowly
progressive cases. Chronic cases lack a subacute phase but show a slowly
progressive dementia associated with bradykinesia, dysarthria and spastic
paraparesis that stabilizes late in the disease process. In subacute and slowly
progressive cases, focal necrotizing encephalomyelitis is seen at necropsy.
Chronic cases show multifocal areas of lysis with a gliotic margin,
predomitly within grey matter, lacking associated chronic inflammatory
changes seen in the other forms of the disease. Epidemiological studies are
consistent with a disease of low-grade communicability, but laboratory studies
have so far failed to reveal an infectious organism. The spectrum of
neuropathological changes are reviewed in this examination of 11 cases. Although
the aetiology of VE remains obscure, further studies are warranted since it may
represent a novel disease process. Viliuisk encephalomyelitis is an acute, often fatal, meningoencephalitis that
tends to develop into a prolonged chronically progressive panencephalitis.
Clinical, neuropathologic, and epidemiologic data argue for an infectious cause,
although multiple attempts at pathogen isolation have been unsuccessful. To
assess mechanisms of disease transmission and spread, we studied 6 multiplex
families. Secondary cases occurred among genetically related and unrelated
persons in a setting of prolonged intrahousehold contact with a patient
manifesting the disease. Transmission to unrelated persons was documented in a
densely populated region around the city of Yakutsk in which Viliuisk
encephalomyelitis had not been previously known. Initially identified in a small
Yakut-Evenk population on the Viliui River of eastern Siberia, the disease
subsequently spread through human contacts to new geographic areas, thus
characterizing Viliuisk encephalomyelitis as an emerging infectious disease. Viliuisk encephalomyelitis (VE) is an endemic neurological disease in
Northeastern Siberia and generally believed to be a chronic encephalomyelitis of
unknown origin. We investigated 17 patients with a clinical diagnosis of VE
within the Viliuiski region of Sakha (Yakutian) Republic to explore the core
clinical syndrome of chronic VE and subsequently whether VE is caused by
Borrelia burgdorferi infection. We found a chronic myelopathy as the core of the
syndrome, often following an acute phase with a meningo-radiculo-neuropathy,
suggestive of chronic neuroborreliosis. A search for inflammatory parameters in
a larger cohort in blood (39 VE patients and 41 controls) and CSF samples (10 VE
patients and 7 controls) excluded an ongoing chronic infection, but revealed
evidence for an immunological scar or a chronic inflammatory ("autoimmune")
response in the CSF. In addition, we detected signs of a previous exposure to
Borrelia burgdorferi antigens in a subset of chronic VE patients with positive
serological results using ELISA/immunoblot in 54/10% and 22/0% of VE patients
and controls, respectively (p values of 0.003/0.034; Fisher's exact test).
However, CSF analyses did not show a link between exposure or at least
immunological reaction against Borrelia and the risk of suffering from VE. Our
data provide the first evidence of the presence of Borrelia burgdorferi or
similar pathogens in Northeastern Siberia, but do not support a causative role
of these pathogens in the aetiopathogenesis of VE. Viliuisk encephalomyelitis (VE) is a unique disease occurring in the Yakut
(Sakha) population of Eastern Siberia. VE is always fatal, with some patients
dying during the acute encephalitic phase of illness; those surviving the acute
phase develop progressive dementia, rigidity and spastic quadriparesis as part
of a more prolonged pan-encephalitic syndrome. The disease is characterized
neuropathologically by multiple widespread micronecrotic foci with marked
inflammatory reactions and subsequent gliosis throughout the cerebral cortex,
basal ganglia, cerebellum and brain stem. The acute febrile onset with
cerebrospinal fluid pleocytosis and increased protein and neuropathology showing
inflammatory reactions suggest that VE is an infectious disease, but the
causative agent has not been identified. Initially detected in a small mixed
Yakut-Evenk population of the mid-Viliui region, the disease subsequently spread
south to densely populated areas around the capital city of Yakutsk. The
occurrence of secondary VE cases in households and the introduction of the
disease by migrants into new populations indicate that the disease is
horizontally transmitted in a setting of a long intra-household contact.
Although there has been a recent decline in the number of cases, increasing
travel may result in further spread of this fatal disease to susceptible
individuals in other regions of the world. BACKGROUND: Viliuisk encephalomyelitis is a disorder that starts, in most cases,
as an acute meningoencephalitis. Survivors of the acute phase develop a slowly
progressing neurologic syndrome characterized by dementia, dysarthria, and
spasticity. An epidemic of this disease has been spreading throughout the Yakut
Republic of the Russian Federation. Although clinical, neuropathologic, and
epidemiologic data suggest infectious etiology, multiple attempts at pathogen
isolation have been unsuccessful.
METHODS: Detailed clinical, pathologic, laboratory, and epidemiologic studies
have identified 414 patients with definite Viliuisk encephalomyelitis in 15 of
33 administrative regions of the Yakut Republic between 1940 and 1999. All data
are documented in a Registry.
RESULTS: The average annual Viliuisk encephalomyelitis incidence rate at the
height of the epidemic reached 8.8 per 100,000 population and affected
predomitly young adults. The initial outbreak occurred in a remote isolated
area of the middle reaches of Viliui River; the disease spread to adjacent areas
and further in the direction of more densely populated regions. The results
suggest that intensified human migration from endemic villages led to the
emergence of this disease in new communities. Recent social and demographic
changes have presumably contributed to a subsequent decline in disease
incidence.
CONCLUSIONS: Based on the largest known set of diagnostically verified Viliuisk
encephalomyelitis cases, we demonstrate how a previously little-known disease
that was endemic in a small indigenous population subsequently reached densely
populated areas and produced an epidemic involving hundreds of persons. BACKGROUND: Viliuisk encephalomyelitis (VE) is an endemic neurological disease
in Northeast Siberia and generally considered to be a chronic encephalomyelitis
of unknown origin actually spreading in the Sakha (Yakutian) Republic.
METHODOLOGY AND PRINCIPLE FINDINGS: In search for the pathophysiology and
causative agent of VE, we performed a cross-sectional study on clinical,
serological and neuroimaging data on chronic VE patients during two medical
expeditions to three villages within the Viliuiski river basin in the Republic
of Sakha in 2000 and to the capital Yakutsk in 2006. The severity of the core
clinical picture with predomit sensory ataxia, gait apraxia, lower limb
spasticity, cognitive impairment and bladder dysfunction correlated with the
degree of MRI findings showing enlargement of inner ventricular spaces as in
communicating hydrocephalus. Laboratory studies revealed transient eosinophilia
during the preceding acute meningitis-like phase, but no ongoing inflammatory
process in the CSF. We found immune reactions against Toxocara canis in the
majority of chronic VE patients but rarely in controls (P = 0.025; Fisher's
exact test). Histological analysis of subacute to subchronic VE brain samples
showed eosinophilic infiltrations with no signs of persistent Toxocara canis
infection.
CONCLUSIONS AND SIGNIFICANCE: Our data showed that pressure by the communicating
hydrocephalus as a mechanical factor is the major pathogenic mechanism in
chronic VE, most likely triggered by eosinophilic meningitis. There are no signs
for an ongoing inflammatory process in chronic VE. The past eosinophilic
reaction in VE might be caused by Toxocara ssp. infection and might therefore
represent the first hint for an initial cause leading to the development of
chronic VE. Our data provide a framework for future studies and potential
therapeutic interventions for this enigmatic epidemic neurological disease
potentially spreading in Sakha Republic. |
What are Septins? | Septins are an evolutionarily conserved family of GTP-binding proteins. They are involved in diverse processes including cytokinesis, apoptosis, infection, neurodegeneration and neoplasia. In yeast, septins assemble into a highly ordered array of filaments at the mother bud neck in Saccharomyces cerevisiae cells. Septins have been implicated in a diverse range of cancers, including gastric cancer, but the underlying mechanisms remain unclear. | The septins are a novel family of proteins that were first recognized in yeast
as proteins associated with the neck filaments. Recent work has shown that
septins are also present in other fungi, insects, and vertebrates. Despite the
apparent differences in modes of cytokinesis amongst species, septins appear to
be essential for this process in both fungal and animal cells. The septins also
appear to be involved in various other aspects of the organization of the cell
surface. Septins are a cytosolic GTP-binding protein family first characterized in yeast,
but gaining increasing recognition as critical protagonists in higher eukaryotic
cellular events. Mammalian septins have been associated with cytokinesis and
exocytosis, along with contributing to the development of neurological
disorders. Ten different septins, divided into four groups, have been identified
in mammals, and individual septins are capable of interacting with each other to
form macromolecular complexes. The present study characterizes the structural
requirements for human septin-septin interactions using a yeast two-hybrid
system. We focus on three septins that are highly expressed in platelets and
neurons, SEPT4 [previously designated H5, CDCrel-2
(cell-division-control-related-2), PNUTL2], SEPT5 (CDCrel-1, PNUTL1) and SEPT8
(KIAA0202). Each of these three septins contains a characteristic domain
structure consisting of unique N- and C-termini, and a central core domain
conserved among the family of proteins. The yeast two-hybrid system yielded data
consistent with a model where each of the three septins can interact with itself
(homotypic assembly) or with one of the other septins (heterotypic assembly).
For SEPT5 and SEPT8, the results illustrate a model whereby heterotypic septin
assembly is dependent on the conserved central core domain and homotypic
interactions require the N- and C-termini of each protein. We also characterized
a model in which the proper cellular localization of SEPT5 and SEPT8 requires
concomitant expression of both proteins. Co-transfection of SEPT5 and SEPT8
results in both proteins targeted to a vesicular-like location. Therefore the
cellular repertoire of human septins has an impact on function by targeting
septin macromolecular complexes to specific cellular locations. Septins are a family of conserved proteins that are essential for cytokinesis in
a wide range of organisms including fungi, Drosophila and mammals. In budding
yeast, where they were first discovered, they are thought to form a filamentous
ring at the bridge between the mother and bud cells. What regulates the assembly
and function of septins, however, has remained obscure. All septins share a
highly conserved domain related to those found in small GTPases, and septins
have been shown to bind and hydrolyze GTP, although the properties of this
domain and the relationship between polymerization and GTP binding/hydrolysis is
unclear. Here we show that human septin 2 is phosphorylated in vivo at Ser218 by
casein kinase II. In addition, we show that recombit septin 2 binds guanine
nucleotides with a Kd of 0.28 microm for GTPgammaS and 1.75 microm for GDP. It
has a slow exchange rate of 7 x 10(-5) s(-1) for GTPgammaS and 5 x 10(-4) s(-1)
for GDP, and an apparent kcat value of 2.7 x 10(-4) s(-1), similar to those of
the Ras superfamily of GTPases. Interestingly, the nucleotide binding affinity
appears to be altered by phosphorylation at Ser218. Finally, we show that a
single septin protein can form homotypic filaments in vitro, whether bound to
GDP or GTP. Septins are a conserved family of eukaryotic GTP-binding, filament-forming
proteins. In Saccharomyces cerevisiae, five septins (Cdc3p, Cdc10p, Cdc11p,
Cdc12p, and Shs1p) form a complex and colocalize to the incipient bud site and
as a collar of filaments at the neck of budded cells. Septins serve as a
scaffold to localize septin-associated proteins involved in diverse processes
and as a barrier to diffusion of membrane-associated proteins. Little is known
about the role of nucleotide binding in septin function. Here, we show that
Cdc3p, Cdc10p, Cdc11p, and Cdc12p all bind GTP and that P-loop and G4 motif
mutations affect nucleotide binding and result in temperature-sensitive defects
in septin localization and function. Two-hybrid, in vitro, and in vivo analyses
show that for all four septins nucleotide binding is important in septin-septin
interactions and complex formation. In the absence of complete complexes,
septins do not localize to the cortex, suggesting septin localization factors
interact only with complete complexes. When both complete and partial complexes
are present, septins localize to the cortex but do not form a collar, perhaps
because of an inability to form filaments. We find no evidence that nucleotide
binding is specifically involved in the interaction of septins with
septin-associated proteins. BACKGROUND: In Saccharomyces cerevisiae, nutrient limitation stimulates diploid
cells to undergo DNA replication and meiosis, followed by the formation of four
haploid spores. Septins are a family of proteins that assemble a ring structure
at the mother-daughter neck during vegetative growth, where they control
cytokinesis. In sporulating cells, the septin ring disassembles and septins
relocalize to the prospore membrane.
RESULTS: Here, we demonstrate that nutrient limitation triggers a change in the
localization of at least two vegetative septins (Cdc10 and Cdc11) from the bud
neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules
persists into meiosis, and they are found associated with the meiotic spindle
until the end of meiosis II. In addition, the meiosis-specific septin Spr28
displays similar behavior, suggesting that this is a common feature of septins.
Septin association to microtubules is a consequence of the nutrient limitation
signal, since it is also observed when haploid cells are incubated in
sporulation medium and when haploid or diploid cells are grown in medium
containing non-fermentable carbon sources. Moreover, during meiosis II, when the
nascent prospore membrane is formed, septins moved from the microtubules to this
membrane. Proper organization of the septins on the membrane requires the
sporulation-specific septins Spr3 and Spr28.
CONCLUSION: Nutrient limitation in S. cerevisiae triggers the sporulation
process, but it also induces the disassembly of the septin bud neck ring and
relocalization of the septin subunits to the nucleus. Septins remain associated
with microtubules during the meiotic divisions and later, during spore
morphogenesis, they are detected associated to the nascent prospore membranes
surrounding each nuclear lobe. Septin association to microtubules also occurs
during growth in non-fermentable carbon sources. The septins are filament-forming, GTP-binding proteins that are conserved from
yeast to humans. Septins assemble into higher-order structures such as rings,
bars, and gauzes with diverse functions including serving as membrane diffusion
barriers and scaffolds for cell signaling. The basis for septin filament
polymerization and the rules governing septin polymer dynamics are presently not
well understood. Pharmacological agents are essential tools in studying such
properties of the actin and microtubule cytoskeletons however there are only
limited reports of a drug specific to the septin cytoskeleton. Forchlorfenuron
(FCF) is a synthetic plant cytokinin used in agriculture which has been shown to
alter septin organization in yeast and mammalian tissue culture cells. Here we
assess cellular requirements and properties of septin-based structures induced
by FCF. Treatment of the filamentous fungus Ashbya gossypii with FCF leads to
assembly of extensive septin fibers throughout hyphae which is rapidly reversed
upon removal of the drug. These fibers do not exchange or add septin subunits
after assembly, indicating that FCF suppresses normal septin dynamics and
stabilizes the polymers. While FCF-induced septin fibers do not co-localize to
actin or microtubules, a polarized F-actin cytoskeleton is likely required for
the assembly of drug-induced septin fibers. Thus, FCF is a potent inducer of
septin polymerization and acts as a reversible stabilizer of extended septin
polymers. This drug will be a powerful tool for studying mechanisms of septin
polymerization and function, particularly in cell types where molecular analyses
are complicated by the presence of multiple isoforms and limited genetics. BACKGROUND: Septins are a highly conserved family of GTP-binding proteins
involved in multiple cellular functions, including cell division and
morphogenesis. Studies of septins in fungal cells underpin a clear correlation
between septin-based structures and fungal morphology, providing clues to
understand the molecular frame behind the varied morphologies found in fungal
world.
METHODOLOGY/PRINCIPAL FINDINGS: Ustilago maydis genome has the ability to encode
four septins. Here, using loss-of-function as well as GFP-tagged alleles of
these septin genes, we investigated the roles of septins in the morphogenesis of
this basidiomycete fungus. We described that septins in U. maydis could assemble
into at least three different structures coexisting in the same cell: bud neck
collars, band-like structures at the growing tip, and long septin fibers that
run from pole to pole near the cell cortex. We also found that in the absence of
septins, U. maydis cells lost their elongated shape, became wider at the central
region and ended up losing their polarity, pointing to an important role of
septins in the morphogenesis of this fungus. These morphological defects were
alleviated in the presence of an osmotic stabilizer suggesting that absence of
septins affected the proper formation of the cell wall, which was coherent with
a higher sensitivity of septin defective cells to drugs that affect cell wall
construction as well as exocytosis. As U. maydis is a phytopathogen, we analyzed
the role of septins in virulence and found that in spite of the described
morphological defects, septin mutants were virulent in corn plants.
CONCLUSIONS/SIGNIFICANCE: Our results indicated a major role of septins in
morphogenesis in U. maydis. However, in contrast to studies in other fungal
pathogens, in which septins were reported to be necessary during the infection
process, we found a minor role of septins during corn infection by U. maydis. Septin9 (Sept9) is a member of the filament-forming septin family of structural
proteins and is associated with a variety of cancers and with hereditary
neuralgic amyotrophy. We have generated mice with constitutive and conditional
Sept9 knockout alleles. Homozygous deletion of Sept9 results in embryonic
lethality around day 10 of gestation whereas mice homozygous for the conditional
allele develop normally. Here we report the consequences of homozygous loss of
Sept9 in immortalized murine embryonic fibroblasts. Proliferation rate was not
changed but cells without Sept9 had an altered morphology compared to normal
cells, particularly under low serum stress. Abnormal, fragmented, and multiple
nuclei were more frequent in cells without Sept9. Cell migration, as measured by
gap-filling and filter-invasion assays, was impaired, but individual cells did
not move less than wild-type cells. Sept9 knockout cells showed a reduced
resistance to hypo-osmotic stress. Stress fiber and vinculin staining at focal
adhesion points was less prominent. Long septin filaments stained for Sept7
disappeared. Instead, staining was found in short, often curved filaments and
rings. Furthermore, Sept7 was no longer localized to the mitotic spindle.
Together, these data reveal the importance of Sept9 for septin filament
formation and general cell stability. Septins comprise a conserved family of GTPases important in cytokinesis. These
proteins polymerize into filaments from rod-shaped heteromeric septin complexes.
Septins interact with one another at two interfaces (NC and G) that alternate
within the complex. Here, we show that small mutations at the N terminus greatly
enhance the formation of SEPT2 homopolymers. Taking advantage of this mutation
to examine polymer formation using SEPT2 alone, we show that both NC and G
interfaces are required for filament formation. However, co-expression of wild
type SEPT2 with SEPT2 containing mutations at either NC or G interfaces revealed
that only the NC mutant suppressed filament formation. NC mutants are able to
interact with one another at putative G interfaces, whereas G mutants fail to
interact at NC interfaces. In addition, all promiscuous septin pairwise
interactions occur at the G interface. These findings suggest that G interface
interactions must occur before NC interactions during polymer formation. Septins are a class of GTP-binding proteins conserved throughout many
eukaryotes. Individual septin subunits associate with one another and assemble
into heteromeric complexes that form filaments and higher-order structures in
vivo. The mechanisms underlying the assembly and maintece of higher-order
structures in cells remain poorly understood. Septins in several organisms have
been shown to be phosphorylated, although precisely how septin phosphorylation
may be contributing to the formation of high-order septin structures is unknown.
Four of the five septins expressed in the filamentous fungus, Ashbya gossypii,
are phosphorylated, and we demonstrate here the diverse roles of these
phosphorylation sites in septin ring formation and septin dynamics, as well as
cell morphology and viability. Intriguingly, the alteration of specific sites in
Cdc3p and Cdc11p leads to a complete loss of higher-order septin structures,
implicating septin phosphorylation as a regulator of septin structure formation.
Introducing phosphomimetic point mutations to specific sites in Cdc12p and Shs1p
causes cell lethality, highlighting the importance of normal septin modification
in overall cell function and health. In addition to discovering roles for
phosphorylation, we also present diverse functions for conserved septin domains
in the formation of septin higher-order structure. We previously showed the
requirement for the Shs1p coiled-coil domain in limiting septin ring size and
reveal here that, in contrast to Shs1p, the coiled-coil domains of Cdc11p and
Cdc12p are required for septin ring formation. Our results as a whole reveal
novel roles for septin phosphorylation and coiled-coil domains in regulating
septin structure and function. Septins are important components of the cytoskeleton that are highly conserved
in eukaryotes and play major roles in cytokinesis, patterning, and many
developmental processes. Septins form heteropolymers which assemble into
higher-order structures including rings, filaments, and gauzes. In contrast to
actin filaments and microtubules, the molecular mechanism by which septins
assemble is not well-understood. Here, we report that in the filamentous fungus
Aspergillus nidulans, four core septins form heteropolymeric complexes. AspE, a
fifth septin lacking in unicellular yeasts, interacts with only one of the core
septins, and only during multicellular growth. AspE is required for proper
localization of three of the core septins, and requires this same subset of core
septins for its own unique cortical localization. The ΔaspE mutant lacks
developmentally-specific septin higher-order structures and shows reduced spore
production and slow growth with low temperatures and osmotic stress. Our results
show that at least two distinct septin heteropolymer populations co-exist in A.
nidulans, and that while AspE is not a subunit of either heteropolymer, it is
required for assembly of septin higher-order structures found in multicellular
development. Septin proteins are conserved structural proteins that often demarcate regions
of cell division. The essential nature of the septin ring, composed of several
septin proteins, complicates investigation of the functions of the ring,
although careful analysis in the model yeast Saccharomyces cerevisiae has
elucidated the role that septins play in the cell cycle. Mutation analysis of
nonessential septins in the pathogenic fungus Candida albicans has shown that
septins also have vital roles in cell wall regulation (CWR), hyphal formation,
and pathogenesis. While mutations in nonessential septins have been useful in
establishing phenotypes, the septin defect is so slight that identifying
causative associations between septins and downstream effectors has been
difficult. In this work, we describe decreased abundance by mRNA perturbation
(DAmP) alleles of essential septins, which display a septin defect more severe
than the defect observed in deletions of nonessential septins. The septin DAmP
alleles have allowed us to genetically separate the roles of septins in hyphal
growth and CWR and to identify the cyclic AMP pathway as a pathway that likely
acts in a parallel manner with septins in hyphal morphogenesis. Septins are a family of 14 cytoskeletal proteins that dynamically form
hetero-oligomers and organize membrane microdomains for protein complexes. The
previously reported interactions with SNARE proteins suggested the involvement
of septins in exocytosis. However, the contradictory results of up- or
down-regulation of septin-5 in various cells and mouse models or septin-4 in
mice suggested either an inhibitory or a stimulatory role for these septins in
exocytosis. The involvement of the ubiquitously expressed septin-2 or general
septin polymerization in exocytosis has not been explored to date. Here, by
o-LC with tandem MS and immunoblot analyses of the septin-2 interactome in
mouse brain, we identified not only SNARE proteins but also Munc-18-1
(stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF)
(disassembles SNARE complexes after each membrane fusion event), and the
chaperones Hsc70 and synucleins (maintain functional conformation of SNARE
proteins after complex disassembly). Importantly, α-soluble NSF attachment
protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE
complex, did not interact with septin-2, indicating that septins undergo
reorganization during each exocytosis cycle. Partial depletion of septin-2 by
siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive
and stimulated exocytosis of secreted and transmembrane proteins in various cell
types. Forchlorfenuron impaired the interaction between SNAP-25 and its
chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and
inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor
neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic
reorganization of septin oligomers in exocytosis. Septins are GTP-binding proteins that form filaments and higher-order structures
on the cell cortex of eukaryotic cells and associate with actin and microtubule
cytoskeletal networks. When assembled, septins coordinate cell division and
contribute to cell polarity maintece and membrane remodeling. These functions
manifest themselves via scaffolding of cytosolic proteins and cytoskeletal
networks to specific locations on membranes and by forming diffusional barriers
that restrict lateral diffusion of proteins embedded in membranes. Notably, many
neurodegenerative diseases and cancers have been characterized as having
misregulated septins, suggesting that their functions are relevant to diverse
diseases. Despite the importance of septins, little is known about what features
of the plasma membrane influence septin recruitment and alternatively, how
septins influence plasma membrane properties. Septins have been localized to the
cell cortex at the base of cilia, the mother-bud neck of yeast, and branch
points of filamentous fungi and dendritic spines, in cleavage furrows, and in
retracting membrane protrusions in mammalian cells. These sites all possess some
degree of curvature and are likely composed of distinct lipid pools. Depending
on the context, septins may act alone or in concert with other cytoskeletal
elements to influence and sense membrane properties. The degree to which septins
react to and/or induce changes in shape and lipid composition are discussed
here. As septins are an essential player in basic biology and disease,
understanding the interplay between septins and the plasma membrane is critical
and may yield new and unexpected functions. Septins are a conserved family of GTP-binding proteins that assemble into a
highly ordered array of filaments at the mother bud neck in Saccharomyces
cerevisiae cells. Many molecular functions and mechanisms of the septins in S.
cerevisiae were already uncovered. However, structural information is only
available from modeling the crystallized subunits of the human septins into the
EM cryomicroscopy data of the yeast hetero-octameric septin rod. Octameric rods
are the building block of septin filaments in yeast. We present here the first
crystal structure of Cdc11, the terminal subunit of the octameric rod and
discuss its structure in relation to its human homologues. Size exclusion
chromatography analysis revealed that Cdc11 forms homodimers through its
C-terminal coiled coil tail. Author information:
(1)Section of Microbiology, Medical Rresearch Council Centre for Molecular
Bacteriology and Infection, Imperial College London, London SW7 2AZ, England,
UK.
(2)Section of Microbiology, Medical Rresearch Council Centre for Molecular
Bacteriology and Infection, Imperial College London, London SW7 2AZ, England, UK
[email protected]. Septins are a family of cytoskeletal GTP-binding proteins that assemble into
membrane-associated hetero-oligomers and organize scaffolds for recruitment of
cytosolic proteins or stabilization of membrane proteins. Septins have been
implicated in a diverse range of cancers, including gastric cancer, but the
underlying mechanisms remain unclear. The hypothesis tested here is that septins
contribute to cancer by stabilizing the receptor tyrosine kinase ErbB2, an
important target for cancer treatment. Septins and ErbB2 were highly
over-expressed in gastric cancer cells. Immunoprecipitation followed by MS
analysis identified ErbB2 as a septin-interacting protein. Knockdown of septin-2
or cell exposure to forchlorfenuron (FCF), a well-established inhibitor of
septin oligomerization, decreased surface and total levels of ErbB2. These
treatments had no effect on epidermal growth factor receptor (EGFR), emphasizing
the specificity and functionality of the septin-ErbB2 interaction. The level of
ubiquitylated ErbB2 at the plasma membrane was elevated in cells treated with
FCF, which was accompanied by a decrease in co-localization of ErbB2 with
septins at the membrane. Cathepsin B inhibitor, but not bafilomycin or
lactacystin, prevented FCF-induced decrease in total ErbB2 by increasing
accumulation of ubiquitylated ErbB2 in lysosomes. Therefore, septins protect
ErbB2 from ubiquitylation, endocytosis and lysosomal degradation. The
FCF-induced degradation pathway is distinct from and additive with the
degradation induced by inhibiting ErbB2 chaperone Hsp90. These results identify
septins as novel regulators of ErbB2 expression that contribute to the
remarkable stabilization of the receptor at the plasma membrane of cancer cells
and may provide a basis for the development of new ErbB2-targeting anti-cancer
therapies. Septins are highly conserved and essential eukaryotic cytoskeletal proteins that
interact with the inner plasma membrane. They are involved in essential
functions requiring cell membrane remodeling and compartmentalization, such as
cell division and dendrite morphogenesis, and have been implicated in numerous
diseases. Depending on the organisms and on the type of tissue, a specific set
of septins genes are expressed, ranging from 2 to 13. Septins self-assemble into
linear, symmetric rods that can further organize into linear filaments several
microns in length. Only a subset of human septins has been described at high
resolution by X-ray crystallography (Sirajuddin et al., 2007). Electron
microscopy (EM) has proven to be a method of choice for analyzing the molecular
organization of septins. It is possible to localize each septin subunit within
the rod complex using genetic tags, such as maltose-binding protein or green
fluorescent protein, to generate a visible label of a specific septin subunit in
EM images that are processed using single-particle EM methodology. In this
chapter we present, in detail, the methods that we have used to analyze the
molecular organization of budding yeast septins (Bertin et al., 2008). These
methods include purification of septin complexes, sample preparation for EM, and
image processing procedures. Such methods can be generalized to analyze the
organization of septins from any organism. |
What is the drug target for Simtuzumab? | These results suggest that LOXL2 could be an appealing target for treatment of scar formation after glaucoma surgery, and point to the potential therapeutic benefits of simtuzumab, a humanized monoclonal antibody derived from GS-607601. | There is worldwide epidemic of non-alcoholic fatty liver disease (NAFLD). NAFLD
is a clinical entity related to metabolic syndrome. Majority of the patients are
obese but the disease can affect non-obese individuals as well. Metabolic
factors and genetics play important roles in the pathogenesis of this disorder.
The spectrum of disorders included in NAFLD are benign macrovesicular hepatic
steatosis, non-alcoholic steatohepatitis, hepatic fibrosis, cirrhosis of liver
and hepatocellular carcinoma. Although the disease remains asymptomatic most of
the time, it can slowly progress to end stage liver disease. It will be the most
common indication of liver transplantation in the future. It is diagnosed by
abnormal liver chemistry, imaging studies and liver biopsy. As there are risks
of potential complications during liver biopsy, many patients do not opt for
liver biopsy. There are some noninvasive scoring systems to find out whether
patients have advanced hepatic fibrosis. At the present time, there are limited
treatment options which include lifestyle modification to loose weight, vitamin
E and thioglitazones. Different therapeutic agents are being investigated for
optimal management of this entity. There are some studies done on incretin based
therapies in patients with NAFLD. Other potential agents will be silent
information regulator protein Sirtuin and antifibrotic monoclonal antibody
Simtuzumab against lysyl oxidase like molecule 2. But they are still in the
investigational phase. BACKGROUND: Lysyl oxidase-like 2 (LOXL2) catalyses collagen cross-linking and is
implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The aim
of this study was to investigate the efficacy and safety of simtuzumab, a
monoclonal antibody against LOXL2, in patients with IPF.
METHODS: In this randomised, double-blind, phase 2 trial, we recruited patients
aged 45-85 years with definite IPF diagnosed prior to 3 years of screening from
183 hospitals and respiratory clinics in 14 countries. Eligible patients,
stratified by baseline forced vital capacity (FVC), serum LOXL2 (sLOXL2)
concentrations, and pirfenidone and nintedanib use, were randomly assigned (1:1)
to inject 125 mg/mL simtuzumab or placebo subcutaneously once a week. The
primary endpoints were progression-free survival, defined as time to all-cause
death or a categorical decrease from baseline in FVC % predicted, in the
intention-to-treat population, in patients with sLOXL2 concentrations in the
50th percentile or higher, and in patients with sLOXL2 concentrations in the
75th percentile or higher. Treatment duration was event-driven, and interim
analyses were planned and conducted after approximately 120 and 200
progression-free survival events, respectively, occurred. We compared treatment
groups with the stratified log-rank test. This study is registered with
ClinicalTrials.gov, number NCT01769196.
FINDINGS: Patients with IPF were recruited between Jan 31, 2013, and June 1,
2015. The intention-to-treat population included 544 randomly assigned patients
(272 patients in both groups), and the safety population included 543 randomly
assigned patients who received at least one dose of study medication. The study
was terminated when the second interim analysis met the prespecified futility
stopping criteria in the intention-to-treat population. We noted no difference
in progression-free survival between simtuzumab and placebo in the
intention-to-treat population (median progression free survival times of 12·6
months and 15·4 months for simtuzumab and placebo, respectively; stratified HR
1·13, 95% CI 0·88-1·45; p=0·329) and in patients with baseline sLOXL2 in the
50th percentile or higher (median progression-free survival 11·7 months and 14·3
months for simtuzumab and placebo, respectively; stratified HR 1·03, 95% CI
0·74-1·43; p=0·851), or in the 75th percentile or higher (median
progression-free survival 11·6 months and 16·9 months for simtuzumab and
placebo, respectively; stratified HR 1·20, 95% CI 0·72-2·00; p=0·475). The
incidence of adverse events and serious adverse events was similar between
treatment groups. The most common adverse events in both the simtuzumab and
placebo groups were dyspnoea, cough, upper respiratory tract infection, and
worsening of IPF; and the most common grade 3 or 4 adverse events were worsening
of IPF, dyspnoea, and pneumonia.
INTERPRETATION: Simtuzumab did not improve progression-free survival in a
well-defined population of patients with IPF. Our data do not support the use of
simtuzumab for patients with IPF.
FUNDING: Gilead Sciences Inc. |
What is Dravet syndrome? | Dravet syndrome is one of the most severe epilepsy syndromes of early childhood, and it comes with very high morbidity and mortality. It is likely that Dravet syndrome is underdiagnosed in adults with treatment-resistant epilepsy. | OBJECTIVE: To describe the long term efficacy and tolerance of stiripentol
associated with valproate and clobazam in an exhaustive cohort of patients with
severe myoclonic epilepsy of infancy (Dravet's syndrome), in which short term
efficacy of such a treatment has recently been demonstrated in a
placebo-controlled trial.
RESULTS: In 46 patients the frequency and the duration of seizures was
significantly reduced (p < 0.001) as well as the number of convulsive status at
a median of three-year follow-up. Ten patients were dramatically improved
(seizures significantly decreased in number [p = 0.002] and duration [p = 0.002]
and status epilepticus disappeared), 20 were moderately improved (seizures
significantly decreased in duration [p = 0.001] and status were less numerous),
four had no response and efficacy was non evaluable in 12 mainly because of
adverse events. Efficacy was better in the youngest patients. The most frequent
adverse events were loss of appetite and loss of weight. They could be so severe
in patients over 12 years of age that the stiripentol dosage could not be
increased to 50 mg kg-1 j-1.
CONCLUSION: This follow-up study shows that stiripentol added to valproate and
clobazam maintains its efficacy at long term in children with severe myoclonic
epilepsy of infancy and suggests that the tritherapy should be introduced as
early as possible in these patients in order to suppress the convulsive status
epilepticus. Dravet syndrome and genetic epilepsy with febrile seizures plus (GEFS+) can both
arise due to mutations of SCN1A, the gene encoding the alpha 1 pore-forming
subunit of the sodium channel. GEFS+ refers to a familial epilepsy syndrome
where at least two family members have phenotypes that fit within the GEFS+
spectrum. The GEFS+ spectrum comprises a range of mild to severe phenotypes
varying from classical febrile seizures to Dravet syndrome. Dravet syndrome is a
severe infantile onset epilepsy syndrome with multiple seizure types,
developmental slowing and poor outcome. More than 70% of patients with Dravet
syndrome have mutations of SCN1A; these include both truncation and missense
mutations. In contrast, only 10% of GEFS+ families have SCN1A mutations and
these comprise missense mutations. GEFS+ has also been associated with mutations
of genes encoding the sodium channel beta 1 subunit, SCN1B, and the GABA(A)
receptor gamma 2 subunit, GABRG2. The phenotypic heterogeneity that is
characteristic of GEFS+ families is likely to be due to modifier genes.
Interpretation of the significance of a SCN1A missense mutation requires a
thorough understanding of the phenotypes in the GEFS+ spectrum whereas a de novo
truncation mutation is likely to be associated with a severe phenotype. Early
recognition of Dravet syndrome is important as aggressive control of seizures
may improve developmental outcome. BACKGROUND: Dravet syndrome is a severe infantile epileptic encephalopathy
caused in approximately 80% of cases by mutations in the voltage gated sodium
channel subunit gene SCN1A. The majority of these mutations are de novo. The
parental origin of de novo mutations varies widely among genetic disorders and
the aim of this study was to determine this for Dravet syndrome.
METHODS: 91 patients with de novo SCN1A mutations and their parents were
genotyped for single nucleotide polymorphisms (SNPs) in the region surrounding
their mutation. Allele specific polymerase chain reaction (PCR) based on
informative SNPs was used to separately amplify and sequence the paternal and
maternal alleles to determine in which parental chromosome the mutation arose.
RESULTS: The parental origin of SCN1A mutations was established in 44 patients
for whom both parents were available and SNPs were informative. The mutations
were of paternal origin in 33 cases and of maternal origin in the remaining 11
cases. De novo mutation of SCN1A most commonly, but not exclusively, originates
from the paternal chromosome. The average age of parents originating mutations
did not differ from that of the general population.
CONCLUSIONS: The greater frequency of paternally derived mutations in SCN1A is
likely to be due to the greater chance of mutational events during the increased
number of mitoses which occur during spermatogenesis compared to oogenesis, and
the greater susceptibility to mutagenesis of the methylated DNA characteristic
of sperm cells. Dravet syndrome, or as it was called in the past 'severe myoclonic epilepsy in
infancy', is a drug-resistant epilepsy first described by Charlotte Dravet in
1978. Besides the well-known and well-described therapy resistance, Dravet
syndrome dramatically impacts the development and behaviour of the affected
children. As it is still not a curable disease, families need to be taught how
to cope with the disorder and will require assistance from both clinical and
non-clinical structures. At the onset of the disease, many questions arise
regarding the diagnosis of Dravet syndrome, the severity of the illness and its
deleterious effects, and the management of seizures, especially the long-lasting
status epilepticus. Once the diagnosis has been established, severe convulsions,
often unpredictable and long-lasting, are still a major worry, but developmental
and behavioural problems also rapidly become a serious concern. Later on, nearly
all parents will have a child who becomes an adult with special needs, requiring
specialised attention from professionals. INTRODUCTION: Dravet syndrome is a drug resistant epilepsy which starts in the
first year of life with febrile seizures, followed by cognitive impairment and
epilepsy with multiple seizure types. Diagnosis has been typically made at the
age of three to four years, but earlier diagnosis is now possible as clinical
features are better recognised and molecular diagnosis is available.
PATIENTS AND METHODS: We studied a series of 14 children with Dravet syndrome or
Dravet spectrum epilepsy. A screening test, developed by other authors to
distinguish the febrile seizures in Dravet syndrome from febrile seizures from
other origin, was applied to the clinical features of the seizures occurring
during the first year of life in our patients.
RESULTS: Clinical suspicion of Dravet spectrum epilepsy was possible in 100% of
children in our series. Moreover, taking into consideration only the first
seizure, 79% of patients scored sufficiently to detect Dravet syndrome.
CONCLUSIONS: Dravet syndrome can be recognised during the first year of life. It
is important that physicians are made aware of these clinical criteria capable
to distinguish febrile seizures in Dravet syndrome from febrile seizures of
other origin, and set up a protocol to collect appropriate data regarding
febrile seizures occurring in the first year of life. BACKGROUND: Dravet syndrome is a severe form of epilepsy. Majority of patients
have a mutation in SCN1A gene, which encodes a voltage-gated sodium channel. A
recent study has demonstrated that 16% of SCN1A-negative patients have a
mutation in PCDH19, the gene encoding protocadherin-19. Mutations in other genes
account for only a very small proportion of families. TSPYL4 is a novel
candidate gene within the locus 6q16.3-q22.31 identified by linkage study.
OBJECTIVE: The present study examined the mutations in epileptic Chinese
children with emphasis on Dravet syndrome.
METHODS: A hundred children with severe epilepsy were divided into Dravet
syndrome and non-Dravet syndrome groups and screened for SCN1A mutations by
direct sequencing. SCN1A-negative Dravet syndrome patients and patients with
phenotypes resembling Dravet syndrome were checked for PCDH19 and TSPYL4
mutations.
RESULTS: Eighteen patients (9 males, 9 females) were diagnosed to have Dravet
syndrome. Among them, 83% (15/18) had SCN1A mutations including truncating (7),
splice site (2) and missense mutations (6). The truncating/splice site mutations
were associated with moderate to severe degree of intellectual disability
(p<0.05). During the progression of disease, 73% (11/15) had features fitting
into the diagnostic criteria of autism spectrum disorder and 53% (8/15) had
history of vaccination-induced seizures. A novel PCDH19 p.D377N mutation was
identified in one SCN1A-negative female patient with Dravet syndrome and a known
PCDH19 p.N340S mutation in a female non-Dravet syndrome patient. The former also
inherited a TSPYL4 p.G60R variant.
CONCLUSION: A high percentage of SCN1A mutations was identified in our Chinese
cohort of Dravet syndrome patients but none in the rest of patients. We
demonstrated that truncating/splice site mutations were linked to moderate to
severe intellectual disability in these patients. A de novo PCDH19 missense
mutation together with an inherited TSPYL4 missense variant were identified in a
patient with Dravet syndrome. Dravet syndrome is an intractable epileptic syndrome beginning in the first year
of life. De novo mutations of SCN1A, which encode the Na(v)1.1 neuronal
voltage-gated sodium channel, are considered the major cause of Dravet syndrome.
In this study, we investigated genetic modifiers of this syndrome. We performed
a mutational analysis of all coding exons of CACNA1A in 48 subjects with Dravet
syndrome. To assess the effects of CACNA1A variants on the epileptic phenotypes
of Dravet syndrome, we compared clinical features in two genotype groups: 1)
subjects harboring SCN1A mutations but no CACNA1A variants (n=20) and 2)
subjects with SCN1A mutations plus CACNA1A variants (n=20). CACNA1A variants
detected in patients were studied using heterologous expression of recombit
human Ca(v)2.1 in HEK 293 cells and whole-cell patch-clamp recording. Nine
CACNA1A variants, including six novel ones, were detected in 21 of the 48
subjects (43.8%). Based on the incidence of variants in healthy controls, most
of the variants seemed to be common polymorphisms. However, the subjects
harboring SCN1A mutations and CACNA1A variants had absence seizures more
frequently than the patients with only SCN1A mutations (8/20 vs. 0/20, p=0.002).
Moreover, the former group of subjects exhibited earlier onset of seizures and
more frequent prolonged seizures before one year of age, compared to the latter
group of subjects. The electrophysiological properties of four of the five novel
Ca(v)2.1 variants exhibited biophysical changes consistent with
gain-of-function. We conclude that CACNA1A variants in some persons with Dravet
syndrome may modify the epileptic phenotypes. Dravet syndrome is a severe form of epileptic encephalopathy characterized by
early onset epileptic seizures followed by ataxia and cognitive decline.
Approximately 80% of patients with Dravet syndrome have been associated with
heterozygous mutations in SCN1A gene encoding voltage-gated sodium channel
(VGSC) α(I) subunit, whereas a homozygous mutation (p.Arg125Cys) of SCN1B gene
encoding VGSC β(I) subunit was recently described in a patient with Dravet
syndrome. To further examine the involvement of homozygous SCN1B mutations in
the etiology of Dravet syndrome, we performed mutational analyses on SCN1B in
286 patients with epileptic disorders, including 67 patients with Dravet
syndrome who have been negative for SCN1A and SCN2A mutations. In the cohort, we
found one additional homozygous mutation (p.Ile106Phe) in a patient with Dravet
syndrome. The identified homozygous SCN1B mutations indicate that SCN1B is an
etiologic candidate underlying Dravet syndrome. Dravet syndrome is an epileptic encephalopathy characterized by multiple types
of seizures. We report the first case of musicogenic reflex seizures in a
7-year-old male with a mutation in the SCN1A gene causing Dravet syndrome.
Reflex seizures have been reported in patients with Dravet syndrome provoked by
body temperature elevation, looking at visual patterns, or under intermittent
photic stimulation. The case we report widens the spectrum of reflex seizures
recorded in patients with Dravet syndrome. Cortical hyperexcitability of genetic
origin could explain the tendency of these patients to experience reflex
seizures. OBJECTIVES: To determine the prevalence of Dravet syndrome, an epileptic
encephalopathy caused by SCN1A-mutations, often with seizure onset after
vaccination, among infants reported with seizures following vaccination. To
determine differences in characteristics of reported seizures after vaccination
in children with and without SCN1A-related Dravet syndrome.
METHODS: Data were reviewed of 1,269 children with seizures following
immunization in the first two years of life, reported to the safety surveillance
system of the Dutch national immunization program between 1 January 1997 and 31
December 2006. Selective, prospective follow-up was performed of children with
clinical characteristics compatible with a diagnosis of Dravet syndrome.
RESULTS: In 21.9% (n = 279) of children, a diagnosis of Dravet syndrome could
not be excluded based on available clinical data (median age at follow-up 16
months). Additional follow-up data were obtained in 83.9% (n = 234) of these
children (median age 8.5 years). 15 (1.2% of 1,269; 95%CI:0.6 to 1.8%) children
were diagnosed with SCN1A-related Dravet syndrome. Of all reported seizures
following vaccinations in the first year of life, 2.5% (95%CI:1.3 to 3.6%) were
due to SCN1A-related Dravet syndrome, as were 5.9% of reported seizures
(95%CI:3.1 to 8.7%) after 2(nd) or 3(rd) DTP-IPV-Hib vaccination. Seizures in
children with SCN1A-related Dravet syndrome occurred more often with a body
temperature below 38.5°C (57.9% vs. 32.6%, p = 0.020) and reoccurred more often
after following vaccinations (26.7% vs. 4.0%, p = 0.003), than in children
without a diagnosis of SCN1A-related Dravet Syndrome.
CONCLUSIONS: Although Dravet syndrome is a rare genetic epilepsy syndrome, 2.5%
of reported seizures following vaccinations in the first year of life in our
cohort occurred in children with this disorder. Knowledge on the specific
characteristics of vaccination-related seizures in this syndrome might promote
early diagnosis and indirectly, public faith in vaccination safety. OBJECTIVE: Dravet syndrome is a severe form of intractable pediatric epilepsy
with a high incidence of SUDEP: Sudden Unexpected Death in epilepsy. Cardiac
arrhythmias are a proposed cause for some cases of SUDEP, yet the susceptibility
and potential mechanism of arrhythmogenesis in Dravet syndrome remain unknown.
The majority of Dravet syndrome patients have de novo mutations in SCN1A,
resulting in haploinsufficiency. We propose that, in addition to neuronal
hyperexcitability, SCN1A haploinsufficiency alters cardiac electrical function
and produces arrhythmias, providing a potential mechanism for SUDEP.
METHODS: Postnatal day 15-21 heterozygous SCN1A-R1407X knock-in mice, expressing
a human Dravet syndrome mutation, were used to investigate a possible cardiac
phenotype. A combination of single cell electrophysiology and in vivo
electrocardiogram (ECG) recordings were performed.
RESULTS: We observed a 2-fold increase in both transient and persistent Na(+)
current density in isolated Dravet syndrome ventricular myocytes that resulted
from increased activity of a tetrodotoxin-resistant Na(+) current, likely
Nav1.5. Dravet syndrome myocytes exhibited increased excitability, action
potential duration prolongation, and triggered activity. Continuous
radiotelemetric ECG recordings showed QT prolongation, ventricular ectopic foci,
idioventricular rhythms, beat-to-beat variability, ventricular fibrillation, and
focal bradycardia. Spontaneous deaths were recorded in 2 DS mice, and a third
became moribund and required euthanasia.
INTERPRETATION: These data from single cell and whole animal experiments suggest
that altered cardiac electrical function in Dravet syndrome may contribute to
the susceptibility for arrhythmogenesis and SUDEP. These mechanistic insights
may lead to critical risk assessment and intervention in human patients. BACKGROUND: Severe myoclonic epilepsy in infants (SMEI), also known as Dravet
syndrome, is a rare, refractory form of epilepsy, for whose treatment
stiripentol (STP) has been recently licensed for add-on use.
OBJECTIVES: To evaluate the efficacy and tolerability of STP and other
antiepileptic drug treatments (including ketogenic diet) as therapy for patients
with SMEI.
SEARCH METHODS: We searched the Cochrane Epilepsy Group Specialised Register (15
May 2013), the Cochrane Central Register of Controlled Trials (CENTRAL, Issue 4
of 12, The Cochrane Library, April 2013), MEDLINE (1946 to May 2013) and SCOPUS
(1823 to May 2013). The online trials registries ClinicalTrials.gov and the WHO
International Clinical Trials Registry Platform were systematically searched.
The bibliographies of any identified study were searched for further references.
We handsearched selected journals and conference proceedings. No language
restrictions were imposed.
SELECTION CRITERIA: Randomised controlled trials (RCTs) or quasi-randomised
controlled trials; double- or single-blinded or unblinded trials; and
parallel-group studies. Administration of at least one antiepileptic drug
therapy given singly (monotherapy) or in combination (add-on therapy) compared
with add-on placebo or no add-on treatment.
DATA COLLECTION AND ANALYSIS: Review authors independently selected trials for
inclusion according to predefined criteria, extracted relevant data and
evaluated the methodological quality of trials. The following outcomes were
assessed: at least 50% seizure reduction, seizure freedom, adverse effects,
proportion of dropouts and quality of life. Outcomes were assessed using a
Mantel-Haenszel meta-analysis to calculate risk ratio (RR) with 95% confidence
intervals (95% CIs).
MAIN RESULTS: No RCTs assessing drugs other than STP were found. Two RCTs
evaluating the use of STP (total of 64 children) were included. Both studies
were generally at unclear risk of bias. A significantly higher proportion of
participants had 50% or greater reduction in seizure frequency in the STP group
compared with the placebo group (22/33 vs 2/31; RR 10.40, 95% CI 2.64 to 40.87).
A significantly higher proportion of participants achieved seizure freedom in
the STP group compared with the placebo group (12/33 vs 1/31; RR 7.93, 95% CI
1.52 to 41.21). No significant difference in the proportion of dropouts was
found in the STP group compared with the placebo group (2/33 vs 8/31; RR 0.24,
95% CI 0.06 to 1.03). Only one study explicitly reported the occurrence of side
effects; higher proportions of participants were reported to experience side
effects in the STP group compared with the placebo group (100% vs 25%; RR 3.73,
95% CI 1.81 to 7.67).
AUTHORS' CONCLUSIONS: Data derived from two small RCTs indicate that STP is
significantly better than placebo with regards to 50% or greater reduction in
seizure frequency and seizure freedom. Adverse effects occurred more frequently
with STP. Further adequately powered studies with long-term follow-up should be
conducted to unequivocally establish the long-term efficacy and tolerability of
STP in the treatment of SMEI. OBJECTIVE: Dravet syndrome or severe myoclonic epilepsy of infancy (SMEI) is a
baleful epileptic encephalopathy that begins in the first year of life. This
syndrome specified by febrile seizures followed by intractable epilepsy,
disturbed psychomotor development, and ataxia. Clinical similarities between
Dravet syndrome and generalized epilepsy with febrile seizure plus (GEFS+)
includes occurrence of febrile seizures and joint molecular genetic etiology.
Shared features of these two diseases support the idea that these two disorders
represent a severity spectrum of the same illness. Nowadays, more than 60
heterozygous pattern SCN1A mutations, which many are de novo mutations, have
been detected in Dravet syndrome.
MATERIALS & METHODS: From May 2008 to August 2012, 35 patients who referred to
Pediatric Neurology Clinic of Mofid Children Hospital in Tehran were enrolled in
this study. Entrance criterion of this study was having equal or more than four
criteria for Dravet syndrome. We compared clinical features and genetic findings
of the patients diagnosed as Dravet syndrome or GEFS+.
RESULTS: 35 patients (15 girls and 20 boys) underwent genetic testing. Mean age
of them was 7.7 years (a range of 13 months to 15 years). Three criteria that
were best evident in SCN1A mutation positive patients are as follows: "Normal
development before the onset of seizures, onset of seizure before age of one
year, and psychomotor retardation after onset of seizures. Our genetic testing
showed that 1 of 3 (33.3%) patients with clinical Dravet syndrome and 3 of 20
(15%) patients that diagnosed as GEFS+, had SCN1A mutation.
CONCLUSION: In this study, normal development before seizure onset, seizures
beginning before age of one year and psychomotor retardation after age of two
years are the most significant criteria in SCN1A mutation positive patients. Dravet syndrome, a severe infantile epilepsy syndrome, is typically resistant to
anti-epileptic drugs (AED). Lamotrigine (LTG), an AED that is effective for both
focal and generalized seizures, has been reported to aggravate seizures in
Dravet syndrome. Therefore, LTG is usually avoided in Dravet syndrome. We
describe two adults and a child with Dravet syndrome in whom LTG resulted in
decreased seizure duration and frequency. This benefit was highlighted in each
patient when LTG was withdrawn after 6 to 15 years, and resulted in an increased
frequency of convulsive seizures together with longer seizure duration. A
25-year-old male required hospital admission for frequent seizures for the first
time in 7 years, 6 weeks after ceasing LTG. Reintroduction of LTG improved
seizure control, suggesting that in some patients with Dravet syndrome, LTG may
be beneficial. Dravet syndrome is a severe infantile-onset epileptic encephalopathy associated
with mutations in the sodium channel alpha-1 subunit gene SCN1A. We aimed to
describe the incidence of Dravet syndrome in the Danish population. Based on a
6-year birth cohort from 2004 to 2009, we propose an incidence of 1:22,000,
which is higher than what has been established earlier. We identified 17 cases
with SCN1A mutation-positive Dravet syndrome. Fifteen patients were found, by
conventional Sanger sequencing. Two additional patients with clinical Dravet
syndrome, but without a detectable SCN1A mutation by Sanger sequencing, were
diagnosed with a SCN1A mutation after using a targeted next-generation
sequencing gene panel. Neurological and psychiatric syndromes often have multiple disease traits, yet
it is unknown how such multi-faceted deficits arise from single mutations.
Haploinsufficiency of the voltage-gated sodium channel Nav1.1 causes Dravet
syndrome, an intractable childhood-onset epilepsy with hyperactivity, cognitive
deficit, autistic-like behaviours, and premature death. Deletion of Nav1.1
channels selectively impairs excitability of GABAergic interneurons. We studied
mice having selective deletion of Nav1.1 in parvalbumin- or
somatostatin-expressing interneurons. In brain slices, these deletions cause
increased threshold for action potential generation, impaired action potential
firing in trains, and reduced amplification of postsynaptic potentials in those
interneurons. Selective deletion of Nav1.1 in parvalbumin- or
somatostatin-expressing interneurons increases susceptibility to
thermally-induced seizures, which are strikingly prolonged when Nav1.1 is
deleted in both interneuron types. Mice with global haploinsufficiency of Nav1.1
display autistic-like behaviours, hyperactivity and cognitive impairment.
Haploinsufficiency of Nav1.1 in parvalbumin-expressing interneurons causes
autistic-like behaviours, but not hyperactivity, whereas haploinsufficiency in
somatostatin-expressing interneurons causes hyperactivity without autistic-like
behaviours. Heterozygous deletion in both interneuron types is required to
impair long-term spatial memory in context-dependent fear conditioning, without
affecting short-term spatial learning or memory. Thus, the multi-faceted
phenotypes of Dravet syndrome can be genetically dissected, revealing synergy in
causing epilepsy, premature death and deficits in long-term spatial memory, but
interneuron-specific effects on hyperactivity and autistic-like behaviours.
These results show that multiple disease traits can arise from similar
functional deficits in specific interneuron types. Dravet syndrome is one of the most severe epilepsy syndromes of early childhood,
and it comes with very high morbidity and mortality. The typical presentation is
characterized by hemiclonic or generalized clonic seizures triggered by fever
during the first year of life, followed by myoclonic, absence, focal and
generalized tonic-clonic seizures. Non-convulsive status epilepticus and
epileptic encephalopathy are common. Development is normal in the first year of
life, but most individuals eventually suffer from intellectual impairment.
Dravet syndrome is associated with mutations in the sodium channel alpha1
subunit gene (SCN1A) in 70-80% of individuals. SCN1A mutation results in
inhibition of the GABAergic inhibitory interneurons, leading to excessive
neuronal excitation. The "interneuron hypothesis" is the current most accepted
pathophysiological mechanism of Dravet syndrome. The mortality rate is increased
significantly in Dravet syndrome. Ataxia, a characteristic crouched gait and
Parkinson's symptoms may develop in some individuals. It is likely that Dravet
syndrome is underdiagnosed in adults with treatment-resistant epilepsy. Early
diagnosis is important to avoid anti-seizure medications that exacerbate
seizures. |
What makes telomerase a good drug target? | Human telomerase is absent in most normal tissues, but is abnormally activated in all major cancer cells. Telomerase enables tumor cells to maintain telomere length, allowing indefinite replicative capacity. | The activation of telomerase, which specifically occurs in neoplastic cells to
avoid telomere attrition at each cell division, is a necessary event in
tumorigenesis. The evidence that telomerase is also present in normal B cells at
different levels according to their differentiation and activation state makes
the study of telomerase activity in B cell tumors particularly interesting. This
review summarizes data concerning telomerase activity in chronic
lymphoproliferative disorders of B-cell lineage (B-CLD), making suggestions
regarding B-cell development and B-cell tumor histogenesis. The role of
telomerase activity as a potential prognostic marker, as well as a target of new
antineoplastic strategies is discussed. Telomerase is the ribonucleoprotein that enables cancer and stem cells to
maintain their telomeres, resulting in unlimited proliferative potential. The
catalytic component of telomerase in humans, hTERT, is upregulated in nearly 90%
of all cancers, making it the most widely expressed marker of maligcy. With
the exception of germ cells and stem cells, hTERT is undetectable in somatic
human tissues. Together, these properties make telomerase a leading candidate
for cancer therapy. Various therapies have been tested in tissue culture and in
mouse models utilizing genetic, pharmacological, and immunological approaches.
The purpose of this review is to critically examine the role of hTERT in cancer
immunotherapy by providing a comparison of the current experiments and a
proposal for an innovative method utilizing DNA vaccination. Telomerase is a ribonucleoprotein that maintains telomeres and is essential for
cellular immortality and tumour growth. The differential expression of
telomerase in cancer cells makes it an attractive therapeutic target. Anti-sense
oligonucleotides directed against the RNA template of hTR and small molecules
that can interact and stabilise the G-quadruplex represent promising therapeutic
strategies. Human trials investigating the potential role of the catalytic
subunit hTERT as a universal cancer vaccine have already commenced. Alternative
lengthening of telomeres (ALT) and efficacy delay remain important limitations
to anti-telomerase therapy. Prostate cancer is the leading cause of cancer-related deaths in men. Androgen
ablation is the mainstay of treatment for advanced prostate cancer. This therapy
is very effective in androgen-dependent cancer; however, these cancers
eventually become androgen independent, rendering anti-androgen therapy
ineffective. The exploration of novel modalities of treatment is therefore
essential to improve the prognosis of this neoplasia. Telomeres are specialized
heterochromatin structures that act as protective caps at the ends of
chromosomes. Telomere maintece in the majority of tumor cells is achieved by
telomerase, a reverse transcriptase enzyme that catalyzes the synthesis of
further telomeric DNA. Telomerase is detected in the majority of prostate
cancers, but not in normal or benign prostatic hyperplasia tissue. Moreover, the
human telomerase reverse transcriptase (hTERT) gene, the catalytic subunit of
telomerase, is regulated by androgens as well as by different oncogenes
including Her-2, Ras, c-Myc and Bcl-2, which seem to play an important role in
prostate cancer progression. Thus, telomerase may represent a very good
candidate for targeted therapy in prostate tumors. To inhibit telomere
maintece by telomerase, approaches that directly target either telomerase and
telomeres or the telomerase regulatory mechanisms have been used. Moreover,
strategies targeting telomerase-positive cells as a means to directly kill the
tumor cells have been tested. This review summarizes the most promising results
achieved by anti-telomerase strategy in different solid tumors. Most of the
telomerase-associated therapies described here have proved very promising for
the treatment of prostate cancer. On the basis of the good results obtained and
considering the multigenic defects of human tumors, including prostate cancer,
the combination of anti-telomerase strategies with conventional drugs and/or
molecules capable of interfering with oncogenic pathways could efficiently
improve the response of this neoplasia. The human telomerase reverse transcriptase (hTERT) is expressed in more than 85%
of tumor cells but is usually not found in normal cells, which makes hTERT as an
ideal tumor-associate antigen (TAA) to develop potential vaccine specifically
destroying cancers without impairing normal tissues in human cancer
immunotherapy. Here are reviewed the fundamental advances of studies on
immunogenicity of hTERT or its peptides and the early clinical trials using the
hTERT vaccine approach in the last decades. Telomerase plays an important role in cell proliferation and carcinogenesis and
is believed to be a good target for anti-cancer drugs. Elimination of template
function of telomerase RNA may repress the telomerase activity. A hammer-headed
ribozyme (telomerase ribozyme, teloRZ) directed against the RNA component of
human telomerase (hTR) was designed and synthesized. TeloRZ showed a specific
cleavage activity against the hTR. The cleavage efficacy reached 60%. A
eukaryotic expression plasmid containing teloRZ gene was inducted into HeLa
cells by lipofectamine, the telomerase activity in HeLa cells expressing teloRZ
decreased to one eighth of that in the control cells. The doubling time
increased significantly and the apoptosis ratio was elevated with increasing
population doublings (PDS). After 19-20 PDS 95% cells were apoptotic. To further
investigate the effect of teloRZ on tumor growth, the eukaryotic expression
plasmid containing teloRZ was injected into transplanted tumor of nude mouse.
The teloRZ effectively inhibited the telomerase activity in transplanted tumor,
promoted apoptosis of the transplanted tumor cells, and decreased the tumor size
significantly. These results indicate that teloRZ can effectively inhibit
telomerase activity and growth of tumor cells, and suggest the potential use of
this ribozyme in anti-cancer therapy. The ideal cancer treatment would specifically target cancer cells yet have
minimal or no adverse effects on normal somatic cells. Telomerase, the
ribonucleoprotein reverse transcriptase that maintains the ends of human
chromosome, is an attractive cancer therapeutic target for exactly this reason
[1]. Telomerase is expressed in more than 85% of cancer cells, making it a
nearly universal cancer marker, while the majority of normal somatic cells are
telomerase negative. Telomerase activity confers limitless replicative potential
to cancer cells, a hallmark of cancer which must be attained for the continued
growth that characterizes almost all advanced neoplasms [2]. In this review we
will summarize the role of telomeres and telomerase in cancer cells, and how
properties of telomerase are being exploited to create targeted cancer therapies
including telomerase inhibitors, telomerase-targeted immunotherapies and
telomerase-driven virotherapies. A frank and balanced assessment of the current
state of telomerase inhibitors with caveats and potential limitations will be
included. The tanshinone natural products possess a variety of pharmacological properties
including anti-bacterial, anti-inflammatory, anti-oxidant, and anti-neoplastic
activity. The molecular basis of these effects, however, remains largely
unknown. In the present study, we explored the direct effect of tanshinones on
the enzyme telomerase. Telomerase is up-regulated in the majority of cancer
cells and is essential for their survival, making it a potential anti-cancer
drug target. We found that the ortho-quinone tanshinone II-A inhibits telomerase
in a time- and DTT-dependent fashion, and the hydrogen peroxide scavenger
catalase protected telomerase from inactivation. These findings demonstrate that
ortho-quinone containing tanshinones can inhibit telomerase owing to their
ability to generate reactive oxygen species. The results also provide evidence
that telomerase is directly and negatively regulated by reactive oxygen species. The observation that the enzyme telomerase is up-regulated in 80-90% of cancer
cells isolated from primary human tumors but is absent in neighboring cells of
healthy tissue has resulted in significant efforts to validate telomerase as an
anticancer drug target and to develop effective approaches toward its
inhibition. In addition to inhibitors that target the enzymatic function of
telomerase, efforts toward immunotherapy using peptides derived from its
catalytic subunit hTERT and hTERT-promoter driven gene therapy have made
significant advances. The increased level of telomerase in cancer cells also
provides a potential platform for cancer diagnostics. Telomerase inhibition
leads to disruption of a cell's ability to maintain the very ends of the
chromosomes, which are called telomeres. Thus, the telomere itself has also
attracted attention as an anticancer drug target. In this Perspective,
interdisciplinary efforts to realize the therapeutic potential of targeting
telomere maintece with a focus on telomerase are discussed. In contrast to cancer cells, most normal human cells have no or low telomerase
levels which makes it an attractive target for anti-cancer drugs. The small
molecule sulforaphane from broccoli is known for its cancer therapeutic
potential in vitro and in vivo. In animals and humans it was found to be quickly
metabolized into 4-methylthiobutyl isothiocyanate (MTBITC, erucin) which we
recently identified as strong selective apoptosis inducer in hepatocellular
carcinoma (HCC) cells. Here, we investigated the relevance of telomerase
abrogation for cytotoxic efficacy of MTBITC against HCC. The drug was effective
against telomerase, independent from TP53 and MTBITC also blocked telomerase in
chemoresistant subpopulations. By using an orthotopic human liver cancer
xenograft model, we give first evidence that MTBITC at 50 mg/KG b.w./d
significantly decreased telomerase activity in vivo without affecting enzyme
activity of adjacent normal tissue. Upon drug exposure, telomerase decrease was
consistent with a dose-dependent switch to anti-survival, cell arrest and
apoptosis in our in vitro HCC models. Blocking telomerase by the specific
inhibitor TMPyP4 further sensitized cancer cells to MTBITC-mediated
cytotoxicity. Overexpression of hTERT, but not enzyme activity deficient
DNhTERT, protected against apoptosis; neither DNA damage nor cytostasis
induction by MTBITC was prevented by hTERT overexpression. These findings imply
that telomerase enzyme activity does not protect against MTBITC-induced DNA
damage but impacts signalling processes upstream of apoptosis execution level. Uncontrolled cell proliferation is the hallmark of cancer, and cancer cells have
typically acquired damage to genes that directly regulate their cell cycles. The
synthesis of DNA onto the end of chromosome during the replicative phase of cell
cycle by telomerase may be necessary for unlimited proliferation of cells.
Telomerase, a ribonucleoprotein enzyme is considered as a universal therapeutic
target of cancer because of its preferential expression in cancer cells and its
presence in 90 % of tumors. We studied the regulation of telomerase and
telomerase reverse transcriptase catalytic subunit (TERT) by diclofenac and
curcumin, alone and also in combination, in 1, 2-dimethylhydrazine
dihydrochloride-induced colorectal cancer in rats. The relationship of
telomerase activity with tumors suppressor proteins (p51, Rb, p21), cell cycle
machinery, and apoptosis was also studied. Telomerase is highly expressed in DMH
group and its high activity is associated with increased TERT expression.
However, telomerase is absent or is present at lower levels in normal tissue.
CDK4, CDK2, cyclin D1, and cyclin E are highly expressed in DMH as assessed by
RT-PCR, qRT-PCR, Western blot, and immunofluorescence analysis. Diclofenac and
curcumin overcome these carcinogenic effects by downregulating telomerase
activity, diminishing the expression of TERT, CDK4, CDK2, cyclin D1, and cyclin
E. The anticarcinogenic effects shown after the inhibition of telomerase
activity by diclofenac and curcumin may be associated with upregulation of tumor
suppressor proteins p51, Rb, and p21, whose activation induces the cells cycle
arrest and apoptosis. Human telomerase is absent in most normal tissues, but is abnormally activated
in all major cancer cells. Telomerase enables tumor cells to maintain telomere
length, allowing indefinite replicative capacity. Albeit not sufficient in
itself to induce neoplasia, telomerase is believed to be necessary for cancer
cells to grow without limit. Studies using an antisense oligonucleotide (ASODN)
to the RNA component of telomerase or human telomerase reverse transcriptase
(hTERT) demonstrate that telomerase in human tumor lines can be blocked in vivo.
Inhibition of hTERT led to telomere shortening and cancer cell death, validating
telomerase as a target for anticancer genetic therapy. Varieties of approaches
for hTERT inhibition have been investigated. The aim of this study was to
analyze the biological activity of ASODN to the hTERT mediated by retrovirus
vector, which was used as therapy for ovarian tumor. We constructed and
characterized a recombit retrovirus vector with full-length hTERT antisense
complementary DNA. The vector was introduced into ES-2 by lipofectamine-mediated
gene transfection. The cellular proliferation and telomerase activity of the
transformant cells were retarded. The hTERT gene expression and the telomerase
activity of the transformant cells were both decreased. The transformant cells
show partial reversion of the maligt phenotype. PT67 cells were also
transfected with the recombit vector and virus-producer cells were generated.
The retrovirus-containing supernatant effectively inhibited the growth of human
ovarian tumor xenografts in mouse models (subcutaneous tumor model), and
enhanced the mouse survival time. Telomerase is activated in human papillomavirus (HPV) positive cervical cancer
and targeting telomeres offers a novel anticancer therapeutic strategy. In this
study, the telomere targeting properties, the cytotoxic as well as the
pro-apoptotic effects of hexane (IV-HE) and dichloromethane (IV-DF) fractions
from Inula viscosa L. extracts were investigated on human cervical HeLa and SiHa
cancer cells. Our data demonstrate that IV-HE and IV-DF extracts were able to
inhibit cell growth in HeLa and SiHa cells in a dose-dependent manner and
studied resistant cell lines exhibited a resistance factor less than 2 when
treated with the extracts. IV-HE and IV-DF extracts were able to inhibit
telomerase activity and to induce telomere shortening as shown by telomeric
repeat amplification protocol and TTAGGG telomere length assay, respectively.
The sensitivity of fibroblasts to the extracts was increased when telomerase was
expressed. Finally, IV-HE and IV-DF were able to induce apoptosis as evidenced
by an increase in annexin-V labeling and caspase-3 activity. This study provides
the first evidence that the IV-HE and IV-DF extracts from Inula viscosa L.
target telomeres induce apoptosis and overcome drug resistance in tumor cells.
Future studies will focus on the identification of the molecules involved in the
anticancer activity. The expression of telomerase in approximately 85% of cancers and its absence in
the majority of normal cells makes it an attractive target for cancer therapy.
However the lag period between initiation of telomerase inhibition and growth
arrest makes direct inhibition alone an insufficient method of treatment.
However, telomerase inhibition has been shown to enhance cancer cell
radiosensitivity. To investigate the strategy of simultaneously inhibiting
telomerase while delivering targeted radionuclide therapy to cancer cells,
123I-radiolabeled inhibitors of telomerase were synthesized and their effects on
cancer cell survival studied. An 123I-labeled analogue of the telomerase
inhibitor MST-312 inhibited telomerase with an IC50 of 1.58 μM (MST-312 IC50:
0.23 μM). Clonogenic assays showed a dose dependant effect of 123I-MST-312 on
cell survival in a telomerase positive cell line, MDA-MB-435. |
What is DENdb? | DENdb is a centralized on-line repository of predicted enhancers derived from multiple human cell-lines. DENdb integrates enhancers predicted by five different methods generating an enriched catalogue of putative enhancers for each of the analysed cell-lines. DENdb provides information about the overlap of enhancers with DNase I hypersensitive regions, ChIP-seq regions of a number of transcription factors and transcription factor binding motifs, means to explore enhancer interactions with DNA using several chromatin interaction assays and enhancer neighbouring genes. DENdb is designed as a relational database that facilitates fast and efficient searching, browsing and visualization of information. | |
List scales that are used for scoring of patients with spinal metastasis? | Tokuhashi, Tomita, Bauer, and Oswestry scores are used for survival prediction of patients with spinal metastases. | Predicting prognosis is the key factor in selecting the proper treatment
modality for patients with spinal metastases. Therefore, various assessment
systems have been designed in order to provide a basis for deciding the course
of treatment. Such systems have been proposed by Tokuhashi, Sioutos, Tomita, Van
der Linden, and Bauer. The scores differ greatly in the kind of parameters
assessed. The aim of this study was to evaluate the prognostic value of each
score. Eight parameters were assessed for 69 patients (37 male, 32 female):
location, general condition, number of extraspinal bone metastases, number of
spinal metastases, visceral metastases, primary tumour, severity of spinal cord
palsy, and pathological fracture. Scores according to Tokuhashi (original and
revised), Sioutos, Tomita, Van der Linden, and Bauer were assessed as well as a
modified Bauer score without scoring for pathologic fracture. Nineteen patients
were still alive as of September 2006 with a minimum follow-up of 12 months. All
other patients died after a mean period of 17 months after operation. The mean
overall survival period was only 3 months for lung cancer, followed by prostate
(7 months), kidney (23 months), breast (35 months), and multiple myeloma (51
months). At univariate survival analysis, primary tumour and visceral metastases
were significant parameters, while Karnofsky score was only significant in the
group including myeloma patients. In multivariate analysis of all seven
parameters assessed, primary tumour and visceral metastases were the only
significant parameters. Of all seven scoring systems, the original Bauer score
and a Bauer score without scoring for pathologic fracture had the best
association with survival (P < 0.001). The data of the present study emphasize
that the original Bauer score and a modified Bauer score without scoring for
pathologic fracture seem to be practicable and highly predictive preoperative
scoring systems for patients with spinal metastases. However, decision for or
against surgery should never be based alone on a prognostic score but should
take symptoms like pain or neurological compromise into account. BACKGROUND CONTEXT: The incidence of metastatic spinal cord compression (MSCC)
is increasing, paralleling increasing life expectancy of patients. However,
management of MSCC and relevance of scoring systems remain controversial.
PURPOSE: The aims of our study were to analyze the feasibility and outcomes of
spinal surgery, to identify prognostic factors for survival, and to assess the
accuracy of scoring systems in patients with maligcies associated with MSCC.
STUDY DESIGN: Retrospective analysis of all patients with MSCC operated in our
institution.
METHODS: Outcomes of surgery, prognostic factors for survival, and relevance of
Tomita and Tokuhashi scores were investigated.
RESULTS: One hundred forty-eight patients were included: 66% were hyperalgic
(pain score >6) and Frankel score (FS) was decreased in 49%. Seventy-three
percent of patients had laminectomy with spinal fixation. After surgery, pain
decreased in 75% of cases, FS was improved in 31%, and 92% of patients were
ambulatory. Postoperative complication rate was 16%. Median overall survival
(OS) was 8.9 months (95% confidence interval, 4.4-13). Only Tokuhashi score was
relevant, but predictive accuracy of survival was just 51%. In univariate
analyses, hyperalgia (p=.001), primary tumor site, extrabone metastases
(p<.001), Karnofsky performance status (KPS) less than 70 (p<.001), poor
American Society of Anesthesiologist (ASA) score (p<.001) or FS (p=.01), and
absence of postoperative chemotherapy (p<.001) were associated with shorter OS.
In multivariate analysis, only extrabone metastases (p=.004), KPS (p=.001), and
ASA score (p=.007) remained significantly associated with OS.
CONCLUSIONS: Surgery for MSCC is associated with limited morbidity, improved
autonomy, and pain relief. Usual scores do not seem relevant, whereas ASA score,
KPS, and extrabone metastases are significantly associated with OS. BACKGROUND CONTEXT: The decision for operative treatment of patients with spinal
metastases is dependent on the patient's predicted survival. Tokuhashi, Tomita,
Bauer, and Oswestry scores have been devised for survival prediction; however,
none of these systems have been evaluated in nasopharyngeal carcinoma (NPC).
PURPOSE: To investigate the accuracy of these scoring systems in predicting
survival and to identify prognostic factors for survival of the patients with
spinal metastases from NPC.
STUDY DESIGN: Retrospective analysis of the patients with spinal metastases from
NPC who were treated in our institution.
PATIENT SAMPLE: The study included 87 patients with spinal metastases from NPC.
OUTCOME MEASURES: The primary outcome measure was the survival time of these
patients. The potential prognostic factors that are known to influence survival
such as general condition, extraspinal bone metastases, vertebral bone
metastases, visceral metastases, and neurologic assessment based on Frankel
score were also studied.
METHODS: The predicted survival according to the four scoring systems were
calculated and labeled as "A" scores. These patients were then rescored by
assigning NPC as a good prognostic tumor and labeled as "B" scores. The
predicted survival of scores A and B were compared with actual survival.
Potential prognostic factors of survival were investigated using univariate and
multivariate Cox regression analyses. For all scoring systems, Kaplan-Meier
survival estimates and log-rank tests were done; the predictive values were
calculated using postestimation after Cox regression analyses.
RESULTS: The median overall survival for the whole cohort was 13 (range 1-120)
months. In multivariate analysis, general condition (p<.01), visceral metastases
(p<.01), and vertebral metastases (p<.01) showed significant association with
survival. The absolute score of all scoring systems was significantly associated
with actual survival, which extended to the different prognostic subgroups of
each scoring systems. Log-rank test revealed significant differences in survival
between the different prognostic subgroups of all scoring systems (p<.01).
Predictive value of survival by modified Tokuhashi score was the highest among
all four scoring systems.
CONCLUSIONS: Patients with spinal metastases from NPC have relatively good
survival prognosis. All four scoring systems could be used to prognosticate
these patients. The modified Tokuhashi score is the best in doing so. BACKGROUND: We sought to identify preoperative factors significantly correlated
with survival. We also aimed to evaluate the validity of the prognostic scores
in the Tomita and Tokuhashi systems and discuss several aspects to improve the
predictive accuracy of these systems. Moreover, we suggest modified criteria for
selecting treatment strategies.
METHODS: In total, the outcomes of 112 patients with spinal metastasis who
underwent surgery between January 2006 and June 2011 were retrospectively
reviewed. The validity of the prognostic scores was assessed on the basis of
their correlation with survival. For various primary maligcies, new scoring
criteria were applied in each system according to the survival results obtained
in this study. Each revised scoring system was adjusted with a similar principle
of scoring as described previously. Patient survival according to each
preoperative factor was analyzed by the Kaplan-Meier method. The predictive
value of each scoring system was evaluated by the log-rank test and Cox
regression analysis.
RESULTS: The interval from the diagnosis of the primary maligcy to that of
spinal metastasis (p = 0.023) and the interval from the diagnosis of spinal
metastasis to surgery (p = 0.039) were significantly correlated with survival.
Regarding Tokuhashi scores, the correlation coefficient was 0.790 before
adjustment (p = 0.001) and 0.853 after adjustment (p < 0.001). For Tomita
scores, the correlation coefficient was -0.994 (p < 0.001) both before and after
adjustment.
CONCLUSIONS: Tomita scores more accurately predicted survival than Tokuhashi
scores. It is helpful to evaluate both scoring systems with adjustment for
primary maligcy depending on the clinical setting. Patients with Tomita
scores less than or equal to 8 and Tokuhashi scores greater than or equal to 6
are recommended to undergo surgical management. Author information:
(1)Department of Radiology, Valencian Oncology Institute Foundation, Valencia,
Spain; Research Institute in Health Services Foundation, Valencia, Spain;
Spanish Back Pain Research Network, Kovacs Foundation, Palma de Mallorca, Spain.
Electronic address: [email protected].
(2)Spanish Back Pain Research Network, Kovacs Foundation, Palma de Mallorca,
Spain; Scientific Department, Kovacs Foundation, Palma de Mallorca, Spain.
(3)Spanish Back Pain Research Network, Kovacs Foundation, Palma de Mallorca,
Spain; CIBER Epidemiology and Public Health (CIBERESP), Spain; Clinical
Biostatistics Unit, Hospital Ramón y Cajal, IRYCIS, Madrid, Spain.
(4)Spanish Back Pain Research Network, Kovacs Foundation, Palma de Mallorca,
Spain; Department of Radiology, Hospital Regional Universitario Carlos Haya,
Málaga, Spain.
(5)Spanish Back Pain Research Network, Kovacs Foundation, Palma de Mallorca,
Spain; Center for Biomaterials and Tissue Engineering, Universitat Politècnica
de València, Valencia, Spain.
(6)Spanish Back Pain Research Network, Kovacs Foundation, Palma de Mallorca,
Spain; CIBER Epidemiology and Public Health (CIBERESP), Spain; Clinical
Biostatistics Unit, Hospital Ramón y Cajal, IRYCIS, Madrid, Spain; Barts and the
London School of Medicine & Dentistry. Queen Mary University of London, London,
UK.
(7)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; CIBER Epidemiology and Public Health (CIBERESP),
Spain; Clinical Biostatistics Unit. Hospital Ramón y Cajal, IRYCIS. Ctra.
Colmenar Km. 9.1, 28034 Madrid, Spain.
(8)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario la Princesa, Madrid,
Spain.
(9)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Rey Juan Carlos,
Móstoles, Madrid, Spain.
(10)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Fundación Jiménez Díaz,
Madrid, Spain.
(11)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Central de Asturias,
Asturias, Spain.
(12)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Son Llàtzer, Palma de Mallorca, Spain.
(13)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Complejo Hospitalario Universitario de Albacete,
Albacete, Spain.
(14)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario y Politécnico La Fe,
Valencia, Spain.
(15)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Vall d'Hebron, Barcelona, Spain.
(16)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Nacional de Parapléjicos, Toledo,
Spain.
(17)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Fundación Instituto Valenciano de Oncología,
Valencia, Spain.
(18)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Instituto Oncológico Xanit, Benalmádena, Málaga,
Spain.
(19)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Reina Sofía, Córdoba,
Spain.
(20)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; OSATEK. Hospital de Galdakao, Vizcaya, Spain.
(21)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Consorcio hospitalario Provincial de Castellón,
Castellón, Spain.
(22)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Cruces, Barakaldo,
Vizcaya, Spain.
(23)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Puerta del Mar, Cádiz,
Spain.
(24)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Clínico Universitario de Valencia,
Valencia, Spain.
(25)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital General Universitario Gregorio Marañón,
Madrid, Spain.
(26)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Ramón y Cajal, Madrid,
Spain.
(27)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario la Paz, Madrid, Spain; HM
Universitario Sanchinarro, Madrid, Spain.
(28)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Intermutual de Levante, San Antonio de
Benagéber, Valencia, Spain.
(29)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Clinic de Barcelona, Barcelona, Spain.
(30)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital de Manacor, Manacor, Islas Baleares,
Spain.
(31)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Arnau de Vilanova, Valencia, Spain.
(32)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Puerta de Hierro
Majadahonda, Madrid, Spain.
(33)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; HM Universitario Sanchinarro, Madrid, Spain.
(34)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Marqués de Valdecilla,
Santander, Spain.
(35)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Fundación Alcorcón,
Alcorcón, Madrid, Spain.
(36)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Donostia, Donostia,
Spain.
(37)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Clínica Oncosur, Córdoba, Spain.
(38)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Clínica Oncosur, Granada, Spain.
(39)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital de la Santa Creu i Sant Pau, Barcelona,
Spain.
(40)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Doctor Peset, Valencia, Spain.
(41)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Duran i Reynals, l'Hospitalet de
Llobregat, Barcelona, Spain.
(42)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital POVISA, Vigo, Spain.
(43)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario de Salamanca, Salamanca,
Spain.
(44)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Virgen del Rocío,
Sevilla, Spain.
(45)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario de Canarias, Islas
Canarias, Spain.
(46)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Virgen de las Nieves,
Granada, Spain.
(47)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario la Paz, Madrid, Spain.
(48)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario 12 de Octubre, Madrid,
Spain.
(49)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital 9 de Octubre, Valencia, Spain.
(50)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Hospital Universitario Virgen de la Arrixaca,
Murcia, Spain.
(51)Spanish Back Pain Research Network. Kovacs Foundation, Paseo de Mallorca 36,
07012 Palma de Mallorca, Spain; Complejo Hospitalario de Navarra, Navarra,
Spain. BACKGROUND CONTEXT: Choosing appropriate surgical patients in the setting of
spinal metastases can be challenging. Existing scoring systems focus primarily
on patient selection or operative techniques. These scores are limited in their
capacity to predict postoperative survival.
PURPOSE: The aim was to model survival after spine surgery for metastastic
disease.
STUDY DESIGN: This was a retrospective multicenter study.
PATIENT SAMPLE: All patients who had undergone surgery for the treatment of
metastatic spinal disease at one of four tertiary care centers between 2007 and
2013 were included.
OUTCOME MEASURE: The outcome measure was 1-year survival after surgery.
METHODS: Demographic, medical, oncologic, surgical, and survival data were
abstracted from medical records. The effect of predictor variables on survival
was evaluated alone and in combination using stepwise logistic regression.
Multivariable logistic regression was subsequently used to adjust for
confounders. A predictive score was then developed and compared against that of
the modified Bauer score alone in terms of prognosticating 1-year survival after
surgery.
RESULTS: In the time period under investigation, 318 patients underwent surgical
intervention for metastastic disease involving the spine, with 307 having data
available for analysis. The survival rate at 1 year was 48% (n=142), with a
median survival of 10 months. In final adjusted analysis, preoperative modified
Bauer score (odds ratio [OR] 3.00; 95% confidence interval [CI] 1.80-5.01;
p<.001), ambulatory status (OR 2.47; 95% CI 1.48-4.14; p=.001), and serum
albumin (OR 2.80; 95% CI 1.66-4.72; p<.001) were all independent predictors of
1-year survival. The most parsimonious model weighted the modified Bauer score
with 2 points and intact ambulatory status and normal serum albumin level with 1
point each, with a ceiling score of 3. The final model using the predictive
score was able to explain 74% of the variation in 1-year survival. In contrast,
the modified Bauer score alone was only able to explain 64% of the variation in
1-year survival.
CONCLUSIONS: This study demonstrates the importance of including factors related
to the overall health of a patient, in addition to parameters surrounding their
cancer diagnosis, to better prognosticate survival. Our predictive score
performed better than the modified Bauer alone and may be used to predict
survival after surgical intervention for metastatic disease.
LEVEL OF EVIDENCE: III. STUDY DESIGN: A retrospective study of 180 patients with lung cancer spinal
metastases, wherein prognostic score-predicted survival was compared with actual
survival.
OBJECTIVE: To evaluate and compare the accuracy of prognostic scoring systems in
lung cancer spinal metastases.
SUMMARY OF BACKGROUND DATA: The modified Tokuhashi, Tomita, modified Bauer, and
Oswestry scores are currently used to guide decisions regarding operative
treatment of patients with spinal metastases. The best system for predicting
survival in patients with lung cancer spinal metastases remains undetermined.
The high incidence of spinal metastases from lung cancer and improved survival
of patients treated with systemic therapy warrants evaluation of these scoring
systems in this particular context.
METHODS: Patients with lung cancer spinal metastases treated at our institution
between May 2001 and August 2012 were studied. Fifty-one patients were treated
surgically. The primary outcome measure was survival from the time of diagnosis.
Scoring-predicted survival was compared with actual survival. Potential
prognostic factors were investigated using Cox regression analyses. Predictive
values of each scoring system for 3- and 6-month survival were measured via
receiver operating characteristic (ROC) curves.
RESULTS: Histological subtype (P = 0.015), sex (P = 0.001), Karnofsky
performance scale (P = 0.001), extent of neurological palsy (P = 0.002), and
visceral metastases (P = 0.037) are significant predictors of survival. Besides
the Oswestry spinal risk index, no significant differences were found between
different prognostic subgroups within the individual scoring systems. Although
the modified Bauer score was most accurate, all four scoring systems had areas
under the ROC curve 0.5 or less.
CONCLUSION: Although better prognostic scores correlated with longer survival,
all four scoring systems are inaccurate in prognosticating patients with lung
cancer spinal metastases. Specific lung cancer histology appears prognostic and
should be considered, especially given the increased survival of patients
receiving new targeted therapies appropriate to their disease.
LEVEL OF EVIDENCE: 3. BACKGROUND: Predicting survival after surgery for patients with metastatic spine
disease can be challenging, with multiple variables that can influence a
patient's overall survival. Predictive models have been developed to assist
clinicians in providing a prognosis for patients. Recently, Ghori et al.
reported a composite model taking into account a modified Bauer score,
preoperative albumin, and ambulatory status of patients with spinal metastasis.
Using an independent cohort, we sought to assess the reliability and validity of
this composite model to predict 1-year survival in patients diagnosed with
metastatic cancer to the spine.
PURPOSE: This study aimed to assess the reliability and validity of the Ghori
et al. composite model to predict 1-year survival in patients diagnosed with
metastatic cancer to the spine, using an independent cohort.
STUDY DESIGN/SETTING: A retrospective study was carried out.
PATIENT SAMPLE: The sample comprised 161 patients with spinal metastasis
undergoing surgery.
OUTCOME MEASURES: Patients' modified Bauer score, preoperative albumin, and
ambulatory status were assessed.
METHODS: This study used a retrospective analysis of 161 patients with spinal
metastasis who underwent surgical management from 2007 to 2013. The ability of
this composite model to predict 1-year survival was compared with actual patient
survival using multivariable logistic regression to control for confounders, as
well as post-regression diagnostics.
RESULTS: Our analysis revealed significantly lower 1-year mortality among
patients with higher composite scores as compared with those with lower scores.
Strong associations between scores and survival were appreciated in unadjusted
analysis. The final model was able to account for 80% of the variation in the
1-year survival, and there was no evidence of lack of fit.
CONCLUSION: This study demonstrates, in an independent cohort of spinal
metastases patients, that a composite model taking into account the ambulatory
status, serum albumin, and modified Bauer score is able to better predict
postoperative survival. These data serve to validate the use of this predictive
model in determining the prognosis of patients with spinal metastasis. STUDY DESIGN: Prospective propensity score-matched study.
OBJECTIVE: To compare the outcomes of minimal invasive surgery (MIS) and
conventional open surgery for spinal metastasis patients.
SUMMARY OF BACKGROUND DATA: There is lack of knowledge on whether MIS is
comparable to conventional open surgery in treating spinal metastasis.
METHODS: Patients with spinal metastasis requiring surgery from January 2008 to
December 2010 in two spine centers were recruited. The demographic,
preoperative, operative, perioperative and postoperative data were collected and
analyzed. Thirty MIS patients were matched with 30 open surgery patients using
propensity score matching technique with a match tolerance of 0.02 based on the
covariate age, tumor type, Tokuhashi score, and Tomita score.
RESULTS: Both groups had significant improvements in Eastern Cooperative
Oncology Group (ECOG), Karnofsky scores, visual analogue scale (VAS) for pain
and neurological status postoperatively. However, the difference comparing the
MIS and open surgery group was not statistically significant. MIS group had
significantly longer instrumented segments (5.5 ± 3.1) compared with open group
(3.8 ± 1.7). Open group had significantly longer decompressed segment
(1.8 ± 0.8) than MIS group (1.0 ± 1.0). Open group had significantly more blood
loss (2062.1 ± 1148.0 mL) compared with MIS group (1156.0 ± 572.3 mL). More
patients in the open group (76.7%) needed blood transfusions (with higher
average units of blood transfused) compared with MIS group (40.0%). Fluoroscopy
time was significantly longer in MIS group (116.1 ± 63.3 s) compared with open
group (69.9 ± 42.6 s). Open group required longer hospitalization (21.1 ± 10.8
days) compared with MIS group (11.0 ± 5.0 days).
CONCLUSION: This study demonstrated that MIS resulted in comparable outcome to
open surgery for patients with spinal metastasis but has the advantage of less
blood loss, blood transfusions, and shorter hospital stay.
LEVEL OF EVIDENCE: 3. |
Does NADPH oxidase 5 require any subunit for function? | No, NADPH oxidase 5 (NOX5) does not require any subunits for function. | The integral membrane protein p22phox is an indispensable component of the
superoxide-generating phagocyte NADPH oxidase, whose catalytic core is the
membrane-associated gp91phox (also known as Nox2). p22phox associates with
gp91phox and, through its proline-rich C terminus, provides a binding site for
the tandem Src homology 3 domains of the activating subunit p47phox. Whereas
p22phox is expressed ubiquitously, its participation in regulating the activity
of other Nox enzymes is less clear. This study investigates the requirement of
p22phox for Nox enzyme activity and explores the role of its proline-rich region
(PRR) for regulating activity. Coexpression of specific Nox catalytic subunits
(Nox1, Nox2, Nox3, Nox4, or Nox5) along with their corresponding regulatory
subunits (NOXO1/NOXA1 for Nox1; p47phox/p67phox/Rac for Nox2; NOXO1 for Nox3; no
subunits for Nox4 or Nox5) resulted in marked production of reactive oxygen.
Small interfering RNAs decreased endogenous p22phox expression and inhibited
reactive oxygen generation from Nox1, Nox2, Nox3, and Nox4 but not Nox5.
Truncated forms of p22phox that disrupted the PRR-inhibited reactive oxygen
generation from Nox1, Nox2, and Nox3 but not from Nox4 and Nox5. Similarly,
p22phox (P156Q), a mutation that disrupts Src homology 3 binding by the PRR,
potently inhibited reactive oxygen production from Nox1 and Nox2 but not from
Nox4 and Nox5. Expression of p22phox (P156Q) inhibited NOXO1-stimulated Nox3
activity, but co-expression of NOXA1 overcame the inhibitory effect. The P157Q
and P160Q mutations of p22phox showed selective inhibition of
Nox2/p47phox/p67phox, and selectivity was specific for the organizing subunit
(p47phox or NOXO1) rather than the Nox catalytic subunit. These studies stress
the importance of p22phox for the function of Nox1, Nox2, Nox3, and Nox4, and
emphasize the key role of the PRR for regulating Nox proteins whose activity is
dependent upon p47phox or NOXO1. Nox5 belongs to the calcium-regulated subfamily of NADPH oxidases (Nox). Like
other calcium-regulated Noxes, Nox5 has an EF-hand-containing calcium-binding
domain at its N-terminus, a transmembrane heme-containing region, and a
C-terminal dehydrogenase (DH) domain that binds FAD and NADPH. While Nox1-4
require regulatory subunits, including p22phox, Nox5 activity does not depend on
any subunits. We found that inactive point mutants and truncated forms of Nox5
(including the naturally expressed splice form, Nox5S) inhibit full-length Nox5,
consistent with formation of a domit negative complex. Oligomerization of
full-length Nox5 was demonstrated using co-immunoprecipitation of coexpressed,
differentially tagged forms of Nox5 and occurred in a manner independent of
calcium ion. Several approaches were used to show that the DH domain mediates
oligomerization: Nox5 could be isolated as a multimer when the calcium-binding
domain and/or the N-terminal polybasic region (PBR-N) was deleted, but deletion
of the DH domain eliminated oligomerization. Further, a chimera containing the
transmembrane domain of Ciona intestinalis voltage sensor-containing phosphatase
(CiVSP) fused to the Nox5 DH domain formed a co-immunoprecipitating complex
with, and functioned as a domit inhibitor of, full-length Nox5. Radiation
inactivation of Nox5 overexpressed in HEK293 cells and endogenously expressed in
human aortic smooth muscle cells indicated molecular masses of ∼350 and ∼300
kDa, respectively, consistent with a tetramer being the functionally active
unit. Thus, Nox5 forms a catalytically active oligomer in the membrane that is
mediated by its dehydrogenase domain. As a result of oligomerization, the short,
calcium-independent splice form, Nox5S, may function as an endogenous inhibitor
of calcium-stimulated ROS generation by full-length Nox5. |
What are the side effects during statins administration in patients with atherosclerosis? | The side effects during statins administration in patients with atherosclerosis are:
1) Myopathy
2) Transaminase elevations
3) Diabetes mellitus
4) Renal and neurologic adverse effects. | Statins are generally considered as safe drugs with a very favorable
cost-efficacy-ratio. Calculation of health expenses limits the prescription of
statins in primary prevention to persons on high risk (i.e. 20% risk of a
coronary infarction within the next 10 years). Prescription of statins in
secondary prevention of atherosclerosis is mandatory. Advanced age, impairment
of renal function or polypharmacotherapy increase the incidence of severe
side-effects, especially myopathy and rhabdomyolysis. These patients should be
given statins with lesser risk of these side-effects. Combination of statins
with fibrates has attracted public concern, but may be indicated in persons with
otherwise not treatable hyperlipidemia. These patients need intensive
monitoring, just as patients on other drugs that are metabolized via the enzyme
CYP3A4. Patients on statins should get dietary counseling, as an appropriate
diet increases the effect of statins. Recently, statin safety and potential drug interactions have received close
attention in the consumer and medical press. In particular, rosuvastatin, the
most recent statin introduced into the US market, has been the object of much
speculation. Many of these reports have lost sight of the proven efficacy of
statins in coronary disease prevention at a time when coronary heart disease is
the number one killer of adults, and have failed to frame the potential drug
toxicity in the context of this benefit. Summarized here are the conclusions of
the National Lipid Association's Statin Safety Assessment Task Force, which
reviewed extensive new drug application (NDA) and postmarketing data for all the
currently marketed statins regarding their effect on the liver, muscle, renal,
and neurologic systems. The task force found that, overall, hepatic, renal, or
neurologic function does not appear to be compromised by statin use. They do not
recommend routine monitoring of these systems but do recommend ongoing
surveillance of symptomatic patients. With respect to muscle toxicity, the task
force's Muscle Expert Panel concluded that the incidence of myopathy and
rhabdomyolysis is low and appears to be dose-related, rather than associated
with the degree of low-density lipoprotein cholesterol lowering and also appears
to be related to the individual statin used. For example, from the rosuvastatin
NDA database and additional postmarketing data, the overall incidence of
myopathy was found to be lower than that observed with other statins.
Administrative claims data of hospitalization rates for adverse events in statin
patients confirm the task force conclusion that, overall, statins are safe and
well tolerated when used as monotherapy. OBJECTIVES: Elderly patients are less likely to receive statin therapy because
of concerns about their side-effects. However, 80% of deaths related to coronary
heart disease occur in patients above the age of 65 years. This study evaluated
the potential benefit of early administration of statins in elderly patients
presenting with an acute coronary syndrome.
METHODS: This was a prospective cohort study of 774 elderly patients (>65 years)
with acute coronary syndrome. The patients were divided into two groups. The
first group, consisting of 611 patients, received statins within the first 24
hours of admission, while the second group, consisting of 163 patients, received
statins after the first 24 hours. The end points studied included death, heart
failure/pulmonary edema, stroke and recurrent myocardial infarction during
hospitalization.
RESULTS: Multivariable logistic regression analysis, adjusting for baseline
demographics, co-morbidities and chronic statin therapy, showed that the
occurrence of heart failure/pulmonary edema during hospitalization was
relatively lower among those who received statins within 24 hours of admission
(odds ratio: 0.5, 95% CI: 0.27-0.94, p=0.03). The C statistic for the model was
0.79.
CONCLUSION: Elderly patients presenting with acute coronary syndrome seem to
benefit from early statin therapy, and have significantly lower rates of heart
failure and pulmonary edema than those who are administered statins at a later
stage. The major public health concern worldwide is coronary heart disease, with
dyslipidemia as a major risk factor. Statin drugs are recommended by several
guidelines for both primary and secondary prevention. Rosuvastatin has been
widely accepted because of its efficacy, potency, and superior safety profile.
Inflammation is involved in all phases of atherosclerosis, with the process
beginning in early youth and advancing relentlessly for decades throughout life.
C-reactive protein (CRP) is a well-studied, nonspecific marker of inflammation
which may reflect general health risk. Considerable evidence suggests CRP is an
independent predictor of future cardiovascular events, but direct involvement in
atherosclerosis remains controversial. Rosuvastatin is a synthetic, hydrophilic
statin with unique stereochemistry. A large proportion of patients achieve
evidence-based lipid targets while using the drug, and it slows progression and
induces regression of atherosclerotic coronary lesions. Rosuvastatin lowers CRP
levels significantly. The Justification for Use of statins in Prevention: an
Intervention Trial Evaluating Rosuvastatin (JUPITER) trial was designed after
the observation that when both low density lipoprotein and CRP were reduced,
patients fared better than when only LDL was lowered. Advocates and critics
alike acknowledge that the benefits of rosuvastatin in JUPITER were real. After
a review, the US Food and Drug Administration extended the indications for
rosuvastatin to include asymptomatic JUPITER-eligible individuals with one
additional risk factor. The American Heart Association and Centers of Disease
Control and Prevention had previously recognized the use of CRP in persons with
"intermediate risk" as defined by global risk scores. The Canadian
Cardiovascular Society guidelines went further and recommended use of statins in
persons with low LDL and high CRP levels at intermediate risk. The JUPITER study
focused attention on ostensibly healthy individuals with "normal" lipid profiles
and high CRP values who benefited from statin therapy. The backdrop to JUPITER
during this period was an increasing awareness of a rising cardiovascular risk
burden and imperfect methods of risk evaluation, so that a significant number of
individuals were being denied beneficial therapies. Other concerns have been a
high level of residual risk in those who are treated, poor patient adherence, a
need to follow guidelines more closely, a dual global epidemic of obesity and
diabetes, and a progressively deteriorating level of physical activity in the
population. Calls for new and more effective means of reducing risk for coronary
heart disease are intensifying. In view of compelling evidence supporting
earlier and aggressive therapy in people with high risk burdens, JUPITER simply
offers another choice for stratification and earlier risk reduction in primary
prevention patients. When indicated, and in individuals unwilling or unable to
change their diet and lifestyles sufficiently, the benefits of statins greatly
exceed the risks. Two side effects of interest are myotoxicity and an increase
in the incidence of diabetes. We aim to study the prevalence of the side-effects of statins among Iranians
patients admitted to a cardiac-specialized hospital and had taken statins prior
to hospitalization. Data was collected between September 2007 and March 2008 and
200 patients were enrolled. A questionnaire was completed using the patients'
records and by interviewing the patients. The mean age of the participants was
61.5 (SD 12.3) years and 63% were males. The most commonly used statins was
atrovastatin (99% of the patients). In all, 63.5% of the participants reported
experiencing side-effects due to statins. The reported side-effects were
respiratory (18.5%), headache (16.5%), rash (0.5%) and allergic reactions (5%);
9.5% reported (4%) and gastrointestinal effect muscle-related side-effects such
as myalgia. Although, the clinical benefits outweigh the small risk of liver
failure and myopathy, clinicians should be aware of the side-effects of statins. OBJECTIVE: Statins, among the most commonly prescribed medications, are
associated with a wide range of musculoskeletal side effects. These include a
progressive autoimmune myopathy with anti-3-hydroxy-3-methylglutaryl-coenzyme A
reductase (anti-HMGCR) antibodies that requires immunosuppression. However, it
remains unknown whether these antibodies are found in statin users with and
without self-limited musculoskeletal side effects; this limits their diagnostic
utility. The current work assessed the prevalence of anti-HMGCR antibodies in
these groups of statin users.
METHODS: We determined the prevalence of anti-HMGCR antibodies in 1,966
participants (including 763 current statin users) in a substudy of the
community-based Atherosclerosis Risk in Communities (ARIC) Study and 98 French
Canadian subjects with familial hypercholesterolemia, including 51 with
documented statin intolerance.
RESULTS: No participant in the ARIC substudy, including those with past or
current statin exposure at the time of sample collection, had anti-HMGCR
antibodies. Similarly, none of 51 patients with self-limited statin intolerance
or 47 statin-tolerant patients receiving maximal statin therapy were anti-HMGCR
positive.
CONCLUSION: The majority of patients with and without statin exposure, including
those with self-limited statin intolerance, do not develop anti-HMGCR
antibodies. Therefore, anti-HMGCR antibodies are highly specific for those with
an autoimmune myopathy. BACKGROUND: The natural history of atherosclerosis might involve coronary plaque
rupture/erosion, thrombus formation and vessel lumen occlusion, clinically
recognized as acute coronary syndrome (ACS). International guidelines strongly
recommend early statin administration in patients admitted for ACS. In addition
to lowering circulating levels of low-density lipoprotein cholesterol (LDL-c),
statin treatment was shown to promote plaque stabilization or regression in
several ways, including reduction in necrotic lipid core, anti-inflammatory
effects and improvement in endothelial function. The aim of this review is to
summarize clinical evidence on the role of statins in secondary prevention of
ACS.
MATERIALS AND METHODS: This narrative review is based on the material found on
medline and pubmed up to August 2013. We looked for the terms 'statin, acute
coronary syndromes' in combination with 'atherosclerosis, acute myocardial
infarction, pathophysiology'.
RESULTS: This review article emphasizes the relevance of the timing of statin
administration to improve the outcomes after ACS. Early and continuous statin
administration has emerged as key features to prevent adverse events, especially
in patients admitted for ACS undergoing percutaneous coronary intervention.
Clinical trials matching the improved clinical outcome with the imaging of
atherosclerotic plaque stabilization/regression, further supporting the
effectiveness of statin therapy. However, the achievement of these goals
requires high dose of statins, thus increasing the risk of adverse events.
CONCLUSIONS: Although clinical trials and meta-analyses have provided
conflicting results, it is likely that in clinical practice, the rate of adverse
events is higher, so that many concerns still remain about a statin high-dose
approach in ACS patients. Rosuvastatin has been marketed for approximately a decade. In this review we
critically discuss available evidence on the benefits and risks from its use. In
clinical trials using rosuvastatin, 'lowest is best' was relevant for
on-treatment low-density lipoprotein cholesterol levels. Targeting levels
<50 mg/dl was associated with the greatest decrease in vascular
morbidity/mortality in the primary prevention setting. Also, such reduction can
induce atherosclerosis regression without increasing the risk of adverse
effects. Pooled data suggest that the safety profile of rosuvastatin is not
different from that of other statins. It was estimated that
rosuvastatin-associated absolute hazards of muscle-, liver- and renal-related
adverse effects are lower than the corresponding vascular benefits in moderate
vascular risk individuals. However, these data are subject to biases and need
confirmation on a prospective basis. Significant liver enzyme elevations are
rare. These often imply underlying non-alcoholic fatty liver disease (NAFLD),
which is associated with increased vascular risk. Rosuvastatin can improve
biochemical biomarkers and histological score of NAFLD. Whether this benefit is
associated with vascular risk reduction should be assessed by prospective
studies. Both chronic kidney disease and albuminuria independently predict
vascular morbidity and mortality. Rosuvastatin improved the estimated glomerular
filtration rate and decreased albuminuria in patients with moderately impaired
kidney function. Also, vascular morbidity and mortality might be reduced in
these patients. The same was not relevant in end-stage renal disease.
Rosuvastatin-induced proteinuria appears to be of tubular origin, not relating
to kidney injury. Rosuvastatin increases the risk of new-onset diabetes by
dose-dependently impairing insulin sensitivity. Obese individuals with
prediabetes appear to be predomitly affected. However, absolute vascular
benefits of rosuvastatin may counterbalance this risk. Rosuvastatin is effective
for the prevention and management of atherosclerotic vascular disease.
Individualization of its use can maximize benefits and reduce the risk of
adverse effects. Lowering serum lipid levels is part of the foundation of treating and preventing
clinically significant cardiovascular disease. Recently, the American Heart
Association/American College of Cardiology released cholesterol guidelines which
advocate for high efficacy statins rather than LDL-c goals for five patient
subgroups at high risk for cardiovascular disease. Therefore, it is critical
that clinicians have an approach for managing side-effects of statin therapy.
Statins are associated with myopathy, transaminase elevations, and an increased
risk of incident diabetes mellitus among some patients; connections between
statins and other processes, such as renal and neurologic function, have also
been studied with mixed results. Statin-related adverse effects might be
minimized by careful assessment of patient risk factors. Strategies to continue
statin therapy despite adverse effects include switching to another statin at a
lower dose and titrating up, giving intermittent doses of statins, and adding
non-statin agents. Non-statin lipid-lowering drugs have their own unique
limitations. Management strategies and algorithms for statin-associated
toxicities are available to help guide clinicians. Clinical practice should
emphasize tailoring therapy to address each individual's cholesterol goals and
risk of developing adverse effects on lipid-lowering drugs. BACKGROUND: Statins are currently the preferred pharmacological therapy in
individuals with familial hypercholesterolemia (FH) with the aim to prevent
premature atherosclerosis. In adults, these agents have been proven to be safe
and well tolerated; however, non-adherence is a significant clinical issue.
OBJECTIVES: In this study, we evaluated tolerability and adherence to statin
therapy in young adult FH patients 10 years after this was initiated in their
childhood.
METHODS: A questionnaire including items on medical history, adherence and
reasons for discontinuation was sent to 214 young adult FH patients that
initiated statin therapy at least 10 years ago. Tolerability was defined as 100%
minus the percentage of patients that discontinued statin therapy due to side
effects. Adherence was defined as the extent to which patients took their
medication as prescribed by their physician. We labelled patients adherent if
they took 80% or more of their pills in the month preceding our assessment.
RESULTS: Follow-up was successful in 205 (95.8%) subjects (age 18-30 years). A
history of side effects was reported by 40 (19.5%) of the patients, and mainly
consisted of muscle complaints and gastrointestinal symptoms. Three patients
(1.5%) discontinued statin therapy because of side effects. Rhadbomyolysis or
other serious adverse events were not reported. In fact, 169 (82.4%) of 205
patients remained on statin treatment and 78.7% (148 out of 188) were adherent.
None of the patient characteristics were significantly associated with
adherence.
CONCLUSIONS: Individuals with FH who started statin therapy in childhood
demonstrated good adherence during ten years of treatment. Furthermore, statin
therapy was well tolerated; only a small minority discontinued therapy because
of side effects and the side effects that were reported were mild in nature. Muscle problems and other adverse symptoms associated with statin use are
frequent reasons for non-adherence and discontinuation of statin therapy, which
results in inadequate control of hyperlipidemia and increased cardiovascular
risk. However, most patients who experience adverse symptoms during statin use
are able to tolerate at least some degree of statin therapy. Given the profound
cardiovascular benefits derived from statins, an adequate practical approach to
statin intolerance is, therefore, of great clinical importance. Statin
intolerance can be defined as the occurrence of myalgia or other adverse
symptoms that are attributed to statin therapy and that lead to its
discontinuation. In reality, these symptoms are actually unrelated to statin use
in many patients, especially in those with atypical presentations following long
periods of treatment. Thus, the first step in approaching patients with adverse
symptoms during the course of statin therapy is identification of those patients
for whom true statin intolerance is unlikely, since most of these patients would
probably be capable of tolerating adequate statin therapy. In patients with
statin intolerance, an altered dosing regimen of very low doses of statins
should be attempted and, if tolerated, should gradually be increased to achieve
the highest tolerable doses. In addition, other lipid-lowering drugs may be
needed, either in combination with statins, or alone, if statins are not
tolerated at all. Stringent control of other risk factors can aid in reducing
cardiovascular risk if attaining lipid treatment goals proves difficult. Recently, the European Society of Cardiology (ESC) and the European
Atherosclerosis Society (EAS) published a consensus paper giving guidance on the
definition and management of statin-associated muscle symptoms (SAMS), as well
as the use of proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors in
very high-risk patients. The occurrence of SAMS can have a major negative impact
on treatment adherence and, consequently, on the prognosis of cardiovascular
diseases. In addition, both the ESC guidelines on the prevention of
cardiovascular disease (CVD) in clinical practice with sections addressing
global strategies to minimise the burden of CVD at population and individual
levels, and the 2016 ESC/EAS guideline for the management of dyslipidaemias,
focus on evaluation and treatment of SAMS. The release of these guidelines was a
source of great interest to clinicians, as new emergent therapies, such as the
PCSK9 inhibitors, have been approved for the treatment of dyslipidaemias:
recently, both the US Food and Drugs Administration (FDA) and the European
Medicines Agency (EMA) approved the use of PCSK9 inhibitors as add-ons for the
treatment of hypercholesterolaemia in cases where low-density lipoprotein
cholesterol (LDL-C) target levels could not be reached with maximum tolerated
statin doses alone, or instead of statins in the event of SAMS. Because of the
relatively high cost of these new therapies, physicians need to justify the use
of PCSK9 inhibitors by demonstrating that their high-risk patients' LDL-C levels
have remained high (1) despite a well-conducted, but insufficiently effective
high-intensity statin therapy (e.g. rosuvastatin 10-20 mg or atorvastatin 40-80
mg), or (2) in the event of the patient developing side effects, in particular
severe SAMS, during treatment with at least three statins. In addition to SAMS,
the use of PCSK9 inhibitors may be considered in patients with documented
atherosclerotic cardiovascular disease or in patients with familial
hypercholesterolaemia and poorly controlled LDL-C under the combination of
maximum tolerated stain and ezetimibe. |
Entresto is composed of which two drugs? | Entresto is composed of sacubitril and valsartan. It is newly FDA-approved medication that dually inhibits angiotensin and neprilysin, in the treatment of heart failure. | Sacubitril/valsartan (Entresto) for chronic heart failure; brexpiprazole
(Rexulti) for major depressive disorder and schizophrenia; and
lumacaftor/ivacaftor (Orkambi) for cystic fibrosis involving specific CFTR
mutations. The PARADIGM-HF study, a large outcome trial in heart failure and reduced
ejection fraction (HFrEF), has recently shown improved cardiovascular outcomes
with sacubitril/valsartan (Entresto®, Novartis), still commonly referred to as
LCZ696, compared to ACE-inhibitor therapy, possibly leading us to a new era for
heart failure (HF) treatment. LCZ696 represents a first-in-class drug acting
through inhibition of angiotensin receptor and neprilysin, thus modulating the
renin angiotensin aldosterone system and vasoactive substances such as
natriuretic peptides. Based on the PARADIGM-HF results, the FDA recently
approved LCZ696 as an effective means to reduce the burden of HF with reduced
ejection fraction. Furthermore, these data also provided rationale to test
LCZ696 in an ongoing trial with HF and preserved ejection fraction. Despite the
enthusiasm regarding this novel compound, real world data on its efficacy and
safety are eagerly expected. This article summarizes all evidences regarding
LCZ696 and how to implement this new drug in the current HF armamentarium. Sacubitril/valsartan (Entresto™; LCZ696) is an orally administered
supramolecular sodium salt complex of the neprilysin inhibitor prodrug
sacubitril and the angiotensin receptor blocker (ARB) valsartan, which was
recently approved in the US and the EU for the treatment of chronic heart
failure (NYHA class II-IV) with reduced ejection fraction (HFrEF). In the large,
randomized, double-blind, PARADIGM-HF trial, sacubitril/valsartan reduced the
incidence of death from cardiovascular causes or first hospitalization for
worsening heart failure (composite primary endpoint) significantly more than the
angiotensin converting enzyme (ACE) inhibitor enalapril. Sacubitril/valsartan
was also superior to enalapril in reducing death from any cause and in limiting
the progression of heart failure. Sacubitril/valsartan was generally well
tolerated, with no increase in life-threatening adverse events. Symptomatic
hypotension was significantly more common with sacubitril/valsartan than with
enalapril; the incidence of angio-oedema was low. Therefore,
sacubitril/valsartan is a more effective replacement for an ACE inhibitor or an
ARB in the treatment of HFrEF, and is likely to influence the basic approach to
treatment. ▼ Sacubitril valsartan (Entresto-Novartis) is a new oral drug licensed for the
treatment of symptomatic chronic heart failure in adults with reduced ejection
fraction.(1) It is described as an angiotensin receptor neprilysin inhibitor and
contains the neprilysin inhibitor, sacubitril and the angiotensin II receptor
antagonist, valsartan.(1-3) Here, we review the evidence for sacubitril
valsartan and consider its place in the management of heart failure. In this article, we consider the new drugs approved for the European market in
2015. We present a summary of the new mechanisms of action introduced and
highlight three new mechanisms of action with a potentially high future impact:
PCSK9 inhibition (alirocumab (Praluent®) and evolocumab (Repatha®)) for
hypercholesterolaemia, neprilysin inhibition (sacubitril in combination with
valsartan (Entresto®)) for heart failure, and interleukin-5 inhibition
(mepolizumab (Nucala®)) for asthma. A 63-year-old woman previously stable on a regimen of atorvastatin 40 mg daily,
carvedilol 25 mg twice daily, digoxin 0.125 mg daily, furosemide 40 mg daily,
spironolactone 25 mg daily, rivaroxaban 15 mg daily, and enalapril 20 mg twice
daily for heart failure developed rhabdomyolysis 26 days after enalapril was
stopped and sacubitril/valsartan (Entresto™) started. The patient received
sacubitril/valsartan at 24/26 mg twice daily for heart failure; however, after
26 days she developed muscle and skin pain. Investigations revealed elevated
creatine kinase and liver function tests, and rhabdomyolysis with raised
transaminases was diagnosed. Sacubitril/valsartan and atorvastatin were
discontinued and the patient was hydrated. She returned to baseline in 23 days
and has not had any reoccurrence of rhabdomyolysis and elevated transaminases
for 46 days. A Naranjo assessment score of 5 was obtained, indicating a probable
relationship between the patient's rhabdomyolysis and her use of
sacubitril/valsartan. The Drug Interaction Probability Scale score was 3,
consistent with a possible interaction as a cause for the reaction, with
sacubitril/valsartan as the precipitant drug and atorvastatin as the object
drug. |
What is the doRiNA database? | doRina is a database of RNA interactions in post-transcriptional regulation. | In animals, RNA binding proteins (RBPs) and microRNAs (miRNAs)
post-transcriptionally regulate the expression of virtually all genes by binding
to RNA. Recent advances in experimental and computational methods facilitate
transcriptome-wide mapping of these interactions. It is thought that the
combinatorial action of RBPs and miRNAs on target mRNAs form a
post-transcriptional regulatory code. We provide a database that supports the
quest for deciphering this regulatory code. Within doRiNA, we are systematically
curating, storing and integrating binding site data for RBPs and miRNAs. Users
are free to take a target (mRNA) or regulator (RBP and/or miRNA) centric view on
the data. We have implemented a database framework with short query response
times for complex searches (e.g. asking for all targets of a particular
combination of regulators). All search results can be browsed, inspected and
analyzed in conjunction with a huge selection of other genome-wide data, because
our database is directly linked to a local copy of the UCSC genome browser. At
the time of writing, doRiNA encompasses RBP data for the human, mouse and worm
genomes. For computational miRNA target site predictions, we provide an update
of PicTar predictions. |
Is NSD-1015 an inhibitor of Aromatic L-Amino Acid Decarboxylase? | Yes, NSD-1015 is an ihnibitor of Aromatic L-Amino Decarboxylase. | The effect of haloperidol and apomorphine, and both drugs in combination, on the
first steps in the synthesis of catecholamines and 5-hydroxytryptamine (5-HT)
has been studied in three rat brain regions. The rate of formation of dopa and
5-hydroxytryptophan (5-HTP) was studied by measuring the accumulation of these
amino acids during 30 min after administration of the inhibitor of the aromatic
L-amino acid decarboxylase, NSD 1015 (3-hydroxybenzylhydrazine HCl). Haloperidol
caused an increase in dopa and no change in 5-HTP formation. The threshold dose
was severalfold higher in the noradrenaline-predominated hemisphere portion than
in the dopamine-rich striatal and limbic regions, suggesting a higher affinity
of haloperidol for dopamine than for noradrenaline receptors. Apomorphine caused
a decrease in dopa formation in all three brain regions studied, although the
effect was much more pronounced in the regions dominated by dopamine. The
threshold dose was about 30 microng/kg, i.e. an order of magnitude lower than
the threshold dose for apparent postsynaptic dopaminergic receptor activation.
This discrepancy is suggested to be due to preferential activation of inhibitory
dopaminergic autoreceptors by low apomorphine doses. This phenomenon may also
contribute to explain the complex dose-response curves of apomorphine. Low doses
of apomorphine caused a decrease and high doses an increase in 5-HTP formation.
These effects, like those on noradrenaline synthesis, are suggested to be
secondary to activation of dopaminergic pre- and post-synaptic receptors. The
interaction between apomorphine and haloperidol with respect to dopa formation
appears to be largely explicable on the assumption of a competition between an
agonist and an antagonist for dopaminergic receptors. However, very large doses
of apomorphine cause a haloperidol-resistant inhibition of tyrosine, and
probably also tryptophan, hydroxylation, which may be due to a direct inhibition
of the aromatic amino acid hydroxylase involved. DOPA was measured in the anterior pituitary and hypothalamic-hypophysial portal
blood after treatment with NSD-1015, a DOPA decarboxylase inhibitor. NSD-1015
caused DOPA to accumulate in the anterior pituitary of mice and rats, and
increased DOPA in the hypothalamic-hypophysial portal blood of rat. Serum
prolactin was also increased. Interruption of the anterior pituitary blood
supply from the hypothalamic-hypophysial system by cannulation of the entire
pituitary stalk eliminated the NSD-1015-induced DOPA accumulation in the rat
pituitary. We conclude that DOPA can be taken into the anterior pituitary from
the portal blood of NSD-1015-treated rodents and that the anterior pituitary
lacks tyrosine hydroxylase activity in both mice and rats. Addition of the aromatic amino acid decarboxylase inhibitor, NSD-1015 (10
microM), to Krebs'-Ringer phosphate (KRP) superfusion medium, significantly
increased the release of dopamine in vitro from superfused corpus striatum
tissue fragments of male rats. A dose-dependent increase in release of dopamine
was obtained in response to increasing concentrations of NSD-1015, with 1.0
microM being the minimally effective dose. In addition to the striatum, NSD-1015
also increased the release of dopamine from superfused hypothalamic tissue
fragments. This capacity of NSD-1015 to increase release of dopamine was
calcium-independent, appeared to be somewhat specific and could apparently
increase the release of dopamine in vivo, as indicated by increases in the
release of the metabolite of dopamine, DOPAC, under conditions of push-pull
perfusion. Although the putative role of NSD-1015 is as an aromatic amino acid
decarboxylase inhibitor, the present results demonstrate that, either as a
result of this function and/or in addition to this role, NSD-1015 is a potent
activator of the release of dopamine. In order to investigate the physiological importance of the membrane pump in
eliminating released dopamine (DA) we have studied the effects of the putative
selective dopamine re-uptake inhibitor, GBR 12909, on synthesis and metabolism
of monoamines in the rat striatum, limbic forebrain, cortical hemispheres and
substantia nigra (SN). The effects of the drug on the firing rate of
catecholamine containing neurons in the SN and locus coeruleus (LC) were also
investigated. For comparison we have investigated the effects of desipramine and
maprotiline. As a measure of the synthesis of noradrenaline (NA), DA and
5-hydroxytryptamine (5-HT) we determined the 3,4-dihydroxyphenylalanine (DOPA)
and 5-hydroxytryptophan (5-HTP) accumulation after inhibition of aromatic
L-amino acid decarboxylase by 3-hydroxy-benzylhydrazine (NSD 1015). As indirect
measurements of DA and NA release in vivo, we have assessed pargyline-induced
3-methoxytyramine (3-MT) and normetanephrine (NM) accumulation and disappearance
rates of DA and NA after inhibition of their synthesis by
alpha-methyl-p-tyrosine (alpha-MT). Administration of GBR 12909 (2.5, 5, 10, 20
or 40 mg/kg) decreased the NSD 1015-induced DOPA accumulation in the striatum
and in the limbic forebrain. In contrast, only minor effects of the drug were
seen on the DOPA accumulation in the cortical hemisphere and on the cerebral
5-HTP accumulation. GBR 12909 increased the 3-MT accumulation in the striatum,
limbic forebrain and the cortical hemispheres, an effect that was even more
pronounced in haloperidol-pretreated animals. However, GBR 12909 did not alter
the 3-MT accumulation in the SN either when given alone or when given to
haloperidol-pretreated rats. In haloperidol-pretreated rats GBR 12909 markedly
enhanced the DA disappearance in the striatum and in the limbic forebrain, but
not in the SN. Furthermore, GBR 12909 did not significantly affect the firing
rate of dopaminergic neurons in the SN or that of noradrenergic neurons in the
LC. Taken together, our results support the notion that GBR 12909 is a specific
DA uptake inhibitor without a transmitter releasing action. In addition, our
findings indicate that DA re-uptake is of physiological importance in the
elimination of DA from the synaptic cleft in the striatum, limbic forebrain and
cortical hemispheres, but not in the SN. Furthermore, a large part of the DA
taken up by the dopaminergic terminals in the striatum and in the limbic
forebrain seems to be re-incorporated into the storage vesicles. Cynomolgus and rhesus monkeys have been studied via PET with [18F]-L-6
fluorodopa tracer. Striatal fluorodopa uptake rate constants have been derived
by graphical analysis of transaxial slice images centered on the striata. The
differences between pairs of values of the rate constant, obtained from two
scans on the same monkey separated by two weeks or more, exhibited a relative
standard deviation of 34.4%. If the two scans were conducted one immediately
after the other, with the position of the monkey undisturbed, the standard
deviation was reduced to 14.0%. The utility of this technique was demonstrated
by comparing the effects on the scans of halothane and pentobarbital anesthesia
and by the administration of NSD 1015, a peripheral and central inhibitor of
L-aromatic amino-acid decarboxylase, between back-to-back scans. With NSD 1015,
the fluorodopa uptake constant was reduced by an average of 76.0%. Tryptophan hydroxylase (L-tryptophan, tetrahydropteridine:oxygen oxidoreductase
[5-hydroxylating]; EC 1.14.16.4; TPH), the initial and rate-limiting enzyme in
the biosynthesis of the neurotransmitter serotonin, was inhibited directly by
benserazide, an inhibitor of aromatic-L-amino-acid decarboxylase
(3,4-dihydroxy-L-phenylalanine carboxy-lyase; EC 4.1.1.28; AAAD). Benserazide
was a competitive inhibitor for the pterin cofactor tetrahydrobiopterin and an
uncompetitive inhibitor for the substrate tryptophan. NSD 1015, another
decarboxylase inhibitor, did not directly inhibit TPH. Other compounds with
catechol moieties in their structures such as 3,4-dihydroxyphenylalanine (DOPA),
dopamine, apomorphine, and SKF 38393 were also found to be potent inhibitors of
TPH. These results indicate that drugs or neurotransmitters with catechol
structures directly inhibit the activity of TPH and add to a growing body of
evidence indicating that endogenous dopamine can exert untoward effects on
serotonin neurons, including inhibition of TPH. Furthermore, the use of
decarboxylase inhibitors to cause the accumulation of 5-hydroxytryptophan as an
in vivo measure of TPH activity could be problematic, particularly when drugs
with catechol structures or dopamine-releasing compounds are also administered. Cathinone is an active ingredient in the leaves of the Khat shrub. Cathinone
affects behavior, neurochemistry and electrophysiology in a manner similar to
the stimulants amphetamine, cocaine and methylphenidate. The present study
extended these studies by evaluating the effects of (+/-)cathinone on dopamine
(DA) and 5-hydroxytryptamine (5-HT)-containing neurons in several regions of the
rat brain in vivo. An index of the rate of synthesis of DA and 5-HT in vivo was
determined in the nuclei caudatus putamen (CP), accumbens (NA), amygdaloideus
centralis (AC), septi lateralis (SL), preopticus pars suprachiasmatica (PSCN)
and dorsomedialis (hypothalami; DMN) of male rats (175-225 g) by measuring the
concentration of dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP)
after the administration of NSD 1015 (100 mg/kg, i.p.) an inhibitor of aromatic
L-amino acid decarboxylase. Concentrations of DA, 5-HT and their major
metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid
(5-HIAA), respectively, were analyzed by high pressure liquid chromatography
coupled to an electrochemical detector. Cathinone decreased levels of DOPAC in a
time- and dose-related manner in the caudatus putamen, accumbens, amygdaloideus
centralis and septi lateralis with the peak effect occurring 30-60 min after a
dose of 6 mg/kg (i.p.). Cathinone had no effect on DOPAC in the preopticus pars
suprachiasmatica or dorsomedialis (hypothalami). The drug also decreased the
accumulation of DOPA in the caudatus putamen, accumbens, amygdaloideus centralis
and septi lateralis, but in the preopticus pars suprachiasmatica and
dorsomedialis (hypothalami), there was no effect.(ABSTRACT TRUNCATED AT 250
WORDS) The activity of 5-hydroxytryptaminergic neurons has been estimated from
measurements of: concentrations of 5-hydroxyindoleacetic acid; the ratio of the
concentrations of 5-hydroxyindoleacetic acid to 5-hydroxytryptamine; the rate of
accumulation of 5-hydroxytryptophan following the administration of an aromatic
L-amino acid decarboxylase inhibitor (e.g., NSD 1015); the rate of accumulation
of 5-hydroxytryptamine, and the rate of decline of 5-hydroxyindoleacetic acid
following the administration of a monoamine oxidase inhibitor (e.g., pargyline).
The purpose of the present study was to compare these different methods under
conditions of changing neuronal impulse traffic produced by electrical
stimulation of 5-hydroxytryptaminergic neurons. Male rats anesthetized with
chloral hydrate were killed following 0, 15, or 30 min of electrical stimulation
of the dorsal raphe nucleus at a frequency of 0, 5, or 10 Hz. The concentrations
of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophan in
nucleus accumbens, amygdala, suprachiasmatic nucleus, and dorsomedial nucleus
were measured using HPLC coupled to an electrochemical detector. In each brain
region, stimulation elicited an increase in the concentration of
5-hydroxyindoleacetic acid and the 5-hydroxyindoleacetic
acid/5-hydroxytryptamine concentration ratio in saline-treated animals and an
increase in 5-hydroxytryptophan accumulation in NSD 1015-treated animals, but
did not alter the concentration of 5-hydroxytryptamine or 5-hydroxyindoleacetic
acid in pargyline-treated rats. The results o f this study indicate that
although the first three methods serve as valid indices of
5-hydroxytryptaminergic neuronal activity, the pargyline-dependent techniques
are not responsive to changes in the rate of 5-hydroxytryptamine nerve firing. Immunocytochemistry has revealed that nerve fibers within the neural and
intermediate lobes of the rat pituitary gland contain 5-hydroxytryptamine
(5-HT). Recent anatomical evidence suggests that the content of this amine in
the intermediate but not the neural lobe of the pituitary gland may represent
5-HT that has been taken up from the blood rather than synthesized
intraneuronally. The purpose of this study was to determine if 5-HT is
synthesized in neurons of the neurointermediate lobe of the pituitary gland.
5-HT synthesis was estimated by measuring the accumulation of the 5-HT
precursor, 5-hydroxytryptophan (5-HTP), in the neurointermediate lobe of male
Long-Evans rats following the administration of NSD 1015, an inhibitor of
aromatic L-amino acid decarboxylase. Thirty min following the injection of NSD
1015 (100 mg/kg, i.p.), 5-HTP accumulated in the neurointermediate lobe and the
rate of this accumulation was increased by the administration of the 5-HTP
precursor, tryptophan, and by electrical stimulation of the pituitary stalk. In
addition, repeated injections of the 5-HT uptake inhibitor, fluoxetine (10
mg/kg, i.p., every 12 h for a total of 7 injections), induced a marked depletion
of platelet 5-HT but did not alter the concentration of 5-HT in either the
neural or intermediate lobes of the pituitary gland. Taken together these
results indicate that much of the 5-HT in the neurointermediate lobe of the
pituitary gland does not represent 5-HT taken up from the blood, but rather the
amine is synthesized in neurons projecting to this region.(ABSTRACT TRUNCATED AT
250 WORDS) Dopa, catecholamines, and dopac were determined in superior cervical ganglia of
albino rats. The average amount of dopa in ganglia of control animals was
1.1--1.6 micrograms/g. The concentrations of catecholamines and dopac were
similar to values reported by others. The tyrosine hydroxylase inhibitor
alpha-methylparatyrosine methyl ester caused marked decrease in the dopa
concentration in the ganglia. The effect of reserpine was less pronounced. The
aromatic amino acid decarboxylase inhibitor NSD 1015 markedly increased the dopa
concentration. The ability of bicuculline, a GABA antagonist, to enhance dopamine (DA)
synthesis in retinas of rats 1, 4, 7, 15 and 60 days after eye opening was
assessed and compared to the time course of postnatal development of the
light-induced increase in DA synthesis. The accumulation of
dihydroxyphenylalanine (DOPA) following administration of the L-aromatic amino
acid decarboxylase inhibitor, NSD 1015, was used to estimate DA synthesis. In
dark-adapted rats, neither bicuculline nor light enhanced DOPA accumulation 1
day after eye opening, but on the remaining days either treatment significantly
augmented DA synthesis, and by day 15 the effects were as great as those
observed in adult retinas. At each time point, the magnitude of the drug effect
on DA synthesis in the dark was similar to that observed following light
exposure. These results suggest that an endogenous GABAergic input to the DA
neurons appears at the same time as the acquisition of the dopaminergic response
to light. The effect of bicuculline treatment on DA synthesis in light-exposed
animals was also assessed. At 4 and 7 days the drug significantly enhanced DOPA
accumulation over that produced by exposure to light alone, but on later days
bicuculline exerted no such additive effect. These data imply that early in the
maturation of the light response mechanisms other than removal of an inhibitory
GABAergic tone may be partially responsible for excitation of the DA neurons. A new method using high-performance liquid chromatography with electrochemical
detection (HPLC-ED) for the simultaneous determination of monoamines, their
precursor amino acids, and related major metabolites in small samples of brain
tissue weighing from 0.5 to 50 mg is described. The method is based on the
preliminary isolation of monoamines (dopamine, norepinephrine, epinephrine, and
serotonin), their precursor amino acids (tyrosine, 3,4-dihydroxyphenylalanine,
tryptophan and 5-hydroxytryptophan), and their major metabolites
(3-methoxytyramine, normetanephrine, 3,4-dihydroxyphenylacetic acid,
homovanillic acid, vanillylmandelic acid,
3-methoxy-4-hydroxyphenylethyleneglycol, and 5-hydroxyindoleacetic acid) by
chromatography on small columns of Amberlite CG-50 and Dowex 50W, and by ethyl
acetate extraction. All the compounds in the four isolated fractions were
measured by HPLC-ED on a reversed-phase column under four different conditions.
The sensitivity was from 0.1 to 40 pmol, depending on the substances analysed.
This newly established method was applied to the study of the effects of an
aromatic L-amino acid decarboxylase inhibitor (NSD-1015) and a monoamine oxidase
inhibitor (pargyline) on the levels of monoamines, their precursor amino acids
and their major metabolites in brain regions of mice. 6R-L-erythro-Tetrahydrobiopterin (6R-BH4) is a cofactor for aromatic L-amino
acid hydroxylases and nitric oxide synthase. Recently, we have reported that
independently of its cofactor activities, 6R-BH4 acts from the outside of
neurons in the brain to enhance the release of monoamine neurotransmitters such
as dopamine. To characterize the pharmacological properties of the action, we
examined the effects of 6S-BH4, a diastereoisomer of 6R-BH4, on dopamine release
in the rat striatum by using brain microdialysis and compared its effects with
those of 6R-BH4. Perfusion of 6S-BH4 or 6R-BH4 through the dialysis probe
increased extracellular dopamine levels (an index of in vivo dopamine release)
concentration dependently; the maximal increase by 6S-BH4, was one-sixth of that
by 6R-BH4. 6S-BH4 increased extracellular DOPA levels in the presence of NSD
1015, an inhibitor of aromatic L-amino acid decarboxylase (an index of in vivo
tyrosine hydroxylase activity), to an extent similar to the increase induced by
6R-BH4. The increase in the DOPA levels induced by either of the pteridines was
abolished after pretreatment of rats with alpha-methyl-p-tyrosine (an inhibitor
of tyrosine hydroxylase). Under the same conditions, the 6S-BH4-induced dopamine
release was abolished, but most of the 6R-BH4-induced increase persisted.
Coadministration of 6S-BH4 with 6R-BH4 inhibited the increase in dopamine
release induced by 6R-BH4 alone. These results show that 6R-BH4 stimulates
dopamine release by acting at the specific recognition site on the neuronal
membrane, and that 6S-BH4 acts as an antagonist of 6R-BH4 at this site, although
it has cofactor activities. Using adrenal dopamine as indicator we have previously obtained evidence that
quinpirole and several other agonists on dopamine D2-like receptors acutely
stimulate the synthesis of adrenal catecholamines. In the present study we
measured the effect of quinpirole and dopamine on the hydroxylation of tyrosine
in the adrenals, using the method of DOPA (3,4-dihydroxyphenylalanine)
accumulation following the administration of the inhibitor of aromatic L-amino
acid decarboxylase NSD 1015 (3-hydroxybenzylhydrazyne). In view of the large
amounts of catecholamines in the adrenal tissue samples, this necessitated a
modification of the method for analysing DOPA. Both quinpirole and dopamine
significantly enhanced the rate of DOPA accumulation in the adrenals, indicating
stimulation of adrenal tyrosine hydroxylase. The effect of dopamine was blocked
by domperidone, a dopamine D2 receptor antagonist that penetrates poorly into
the central nervous system. Thus the effect of dopamine, which itself penetrates
poorly into the central nervous system, was presumably mediated peripherally.
Similarly epinine, i.e. the N-methyl derivative of dopamine, appeared to enhance
adrenal catecholamine synthesis, as indicated by an elevated adrenal dopamine
level. The data support the view that stimulation of peripherally located
dopamine D2-like receptors can enhance the rate of adrenal catecholamine
synthesis by stimulating the activity of tyrosine hydroxylase. The in vivo rate of brain tryptophan hydroxylation was determined through
5-hydroxytryptophan accumulation (5-HTPacc) following the administration of NSD
1015, a L-aromatic amino-acid decarboxylase inhibitor. This measurement was
performed every 4 h throughout a 24 h hour period in 10 discrete brain areas of
rats maintained on a regular 12 h/12 h light-dark cycle. The concentrations of
5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were also
determined in untreated rats. Daily variations in 5-HTPacc were found in all the
areas studied, the 5-HTPacc being higher during the dark period in most
structures. These results strongly suggest that tryptophan hydroxylation is
involved in the control of the 5-HT biosynthesis circadian rhythm. However,
various patterns of 5-HT and 5-HIAA daily variations were observed, suggesting
that the circadian factors affecting serotonin metabolism can be different among
brain areas. Using a microdialysis technique, the rat striatum was perfused with NSD-1015, an
inhibitor of aromatic L-amino acid decarboxylase, and the amount of
L-3,4-dihydroxyphenylalanine (L-DOPA) and 5-hydroxytryptophan (5-HTP)
accumulating in dialysate was measured as an index of in vivo activities of
tyrosine hydroxylase and tryptophan hydroxylase. NSD-1015 increased the
concentration of L-DOPA much higher than that of 5-HTP in a dose-related manner
(1-100 mumol/L). In order to examine the relationship between dopaminergic and
serotonergic neurons in the striatum, either 5-HTP or L-DOPA was injected
intraperitoneally to rats pretreated with benserazide, an inhibitor of
peripheral decarboxylase. 5-HTP administration increased 5-HTP, but decreased
L-DOPA in a dose-dependent manner. Conversely, 5-HTP concentration decreased in
an association with the increased content of L-DOPA following L-DOPA
administration. The decrease of 5-HTP caused by L-DOPA administration was not as
remarkable as that of L-DOPA by 5-HTP injection. These results suggest that
L-DOPA and 5-HTP, the precursor amino acids for catecholamines and indoleamines,
could affect mutually each other neuronal activity through the inhibition of
their rate-limiting enzymes. 1. (-)-Deprenyl has been shown to potentiate rat striatal neurone responses to
dopamine agonists at doses not altering dopamine metabolism. Since there are a
number of effects of (-)-deprenyl which could result in this phenomenon, we have
investigated the effects of MDL 72,145 and Ro 19-6327, whose only common effect
with (-)-deprenyl is an inhibition of monoamine oxidase-B (MAO-B), on rat
striatal neurone responses to dopamine and on striatal dopamine metabolism. 2.
Using in vivo electrophysiology, i.p. injection of either MDL 72,145 or Ro
19-6327 was found to produce a dose-dependent potentiation of striatal neurone
responses to dopamine but not gamma-aminobutyric acid. 3. Neurochemical
investigations revealed that this occurred at doses (0.25-1 mg kg-1) which,
while not affecting levels of dopamine or its metabolites,
3,4-dihydroxyphenylacetic acid or homovanillic acid, did cause a significant,
dose-dependent, elevation in striatal levels of the putative neuromodulator,
2-phenylethylamine (PE). 4. Inhibition of PE synthesis by i.p. injection of the
aromatic L-amino acid decarboxylase inhibitor, NSD 1015, produced a reversal of
the effects of MDL 72,145 and Ro 19-6327. 5. Neurochemical analysis revealed
this to occur at a dose of NSD 1015 (10 mg kg-1) selective for reduction of
elevated PE levels. 6. These results suggest that PE can act as a neuromodulator
of dopaminergic responses and that MAO-B inhibitors may potentiate neuronal
responses to dopamine via the indirect mechanism of elevation of PE following
MAO-B inhibition. It has been suggested that ethanol may interact with the central nicotinic
acetylcholine receptor, thus providing a basis for the often observed high
consumption of both ethanol and nicotine. In the present in vivo microdialysis
study, ethanol (2.5 g/kg) moderately increased dopamine overflow in the rat
nucleus accumbens. The central nicotinic acetylcholine receptor antagonist
mecamylamine totally counteracted this effect in a dose (1.0 mg/kg) that did not
alter dopamine overflow per se. Ethanol also increased the overflow of
dihydroxyphenylacetic acid and homovanillic acid, but this effect was not
altered by mecamylamine (1.0 mg/kg). Furthermore, the ethanol-induced
enhancement of 3,4-dihydroxyphenylalanine accumulation in the mesolimbic
dopamine terminal area after NSD 1015 (an inhibitor of l-aromatic amino acid
decarboxylase) was completely antagonized by mecamylamine in doses (3.0 and 6.0
mg/kg) that exerted no effects per se. Neither ethanol nor mecamylamine changed
the catecholamine synthesis rate in the striatum or the cerebral cortex. These
results provide further evidence that ethanol-induced activation of the
mesolimbic dopamine system (increased dopamine synthesis and release) may be
mediated via stimulation of central nicotinic acetylcholine receptors. It is
suggested that antagonists of central nicotinic acetylcholine receptors may be
useful in the treatment of alcoholism. Rat pups suspended in air and administered L-DOPA engage in a locomotor behavior
termed air-stepping. The role of L-DOPA itself was investigated by administering
several doses of an aromatic L-amino acid decarboxylase inhibitor, NSD 1015,
prior to 100 mg/kg L-DOPA to 5-day-old rats. NSD 1015 dose-dependently increased
the latency to onset and decreased the duration of L-DOPA-induced air-stepping.
Thus L-DOPA induces air-stepping only after its conversion to dopamine and/or
noradrenaline. The comparative effects of L-3,4-dihydroxphenylalanine (L-DOPA) on dopamine
synthesis, release and behaviour were studied in the reserpine-treated rat.
Acute administration of L-DOPA (25-200 mg/kg) dose-dependently inhibited the
activity of aromatic L-amino acid decarboxylase (AADC) in the substantia nigra
and corpus striatum. The antiparkinsonian drugs budipine (10 mg/kg) and
amantadine (40 mg/kg) enhanced AADC activity in these regions, and prevented or
reversed AADC inhibition by L-DOPA. Dual probe dialysis revealed that low doses
of L-DOPA (25-50 mg/kg) dose-dependently stimulated the release of dopamine and
3,4-dihydroxyphenylacetic acid (DOPAC) in nigra and striatum, whilst high doses
of L-DOPA (100-200 mg/kg) completely suppressed the release of dopamine, but not
DOPAC. Sulpiride (50 microM) administered via the probes antagonized dopamine
release in response to 25 mg/kg L-DOPA, but greatly facilitated release by 200
mg/kg L-DOPA. Dopamine release was blocked by the centrally acting AADC
inhibitor NSD 1015, but facilitated by the central AADC activator budipine. In
behavioural tests L-DOPA (plus benserazide, 50 mg/kg) only reversed akinesia at
200 mg/kg, and not at 25-100 mg/kg. Pretreatment with either NSD 1015 (100
mg/kg) or budipine (10 mg/kg) markedly potentiated the motor stimulant action of
a threshold dose of L-DOPA (100 mg/kg). A combination of NSD 1015 (100 mg/kg)
and benserazide (50 mg/kg) potentiated L-DOPA behaviour more effectively than
either inhibitor alone. NSD 1015-facilitated L-DOPA behaviour was antagonized by
sulpiride (100 mg/kg) and not by SCH 23390 (1 mg/kg), whereas
budipine-facilitated L-DOPA behaviour was fully antagonized by SCH 23390 and
only partially by sulpiride. These results show that behaviourally active doses
of L-DOPA in the reserpinized rat are not accompanied by significant increases
in extracellular dopamine and are therefore probably not dopamine mediated. We
propose that L-DOPA is capable of directly stimulating dopamine D2 and possibly
non-dopamine receptors, thereby inhibiting dopamine efflux presynaptically and
promoting motor activation postsynaptically. A stimulant action of L-DOPA on
motor behaviour, preferentially mediated by D1 > D2 receptors, suggests that
L-DOPA may also be capable of yielding a dopamine-like response in the absence
of detectable dopamine release. These findings are incorporated into a new model
of L-DOPA's actions in the reserpinized rat, and their possible implications for
our understanding of L-DOPA in Parkinson's disease are discussed. Recently, we demonstrated that methylene blue partially inhibited
estradiol-benzoate-induced anterior pituitary hyperplasia in rats. Since central
dopaminergic systems participate in the regulation of estrogen-induced anterior
pituitary growth and tumor transformation, this study examined whether a 3-week
treatment with methylene blue could affect anterior pituitary levels of dopamine
(DA), dihydroxyphenylalanine (DOPA), and dihydroxyphenylacetic acid and dopamine
(D-2) receptors in male rats. Compared to controls, methylene blue significantly
decreased anterior pituitary weight, increased basal anterior pituitary DA
levels, and inhibited estradiol benzoate-induced decreases in anterior pituitary
DA concentrations. Furthermore, we found that methylene blue alone decreased
anterior pituitary D-2 receptor number. Methylene blue given in combination with
estradiol benzoate partially inhibited estradiol benzoate-induced anterior
pituitary growth and estradiol benzoate-induced increases in D-2 receptor
number. Estradiol benzoate-treated rats had significantly lower anterior
pituitary DOPA accumulation after intraperitoneal administration of
3,4-hydroxybenzyl-hydrazine dihydrochloride (NSD-1015), an irreversible
inhibitor of L-aromatic amino acid decarboxylase whereas methylene blue did not
affect anterior pituitary DOPA accumulation when compared to controls. Methylene
blue decreased anterior pituitary prolactin levels and inhibited increases in
anterior pituitary prolactin after estradiol benzoate administration. The
present results suggest that anterior pituitary DA may play an important role in
estrogen-induced anterior pituitary hyperplasia and tumor formation and that
antioxidant drugs such as methylene blue may attenuate estrogen-induced
pituitary growth. This may occur via increases in anterior pituitary DA levels
associated with down-regulation of anterior pituitary D-2 receptors. The effects of the peripheral aromatic amino acid decarboxylase (AADC)
inhibitors, carbidopa and benserazide, and the central AADC inhibitor,
3-hydroxybenzylhydrazine (NSD-1015) on peripheral and brain monoamine oxidase
(MAO) A and B activity were investigated in the rat. In vitro, carbidopa,
benserazide and NSD-1015 all potently inhibited hepatic MAO A and B activity
(IC(50) 10-50 micro M). In ex vivo studies following systemic drug
administration, NSD-1015 (100 mg/kg ip) produced 88% and 96% inhibition of
hepatic and striatal MAO A and B activity respectively. Carbidopa (12.5 mg/kg
i.p.) and benserazide (50 mg/kg i.p.) had no effect on striatal MAO A activity
or hepatic MAO B activity. However, they inhibited striatal MAO B activity by 45
+/- 10% and 36 +/- 10% respectively. In conclusion, carbidopa and benserazide
may not only protect L-DOPA from peripheral decarboxylation, but also increase
striatal dopamine content through MAO inhibition. NSD-1015 should not be used to
investigate the neuromodulatory role of L-DOPA as it potently inhibits rat
striatal MAO. The purpose of the present study was to determine whether the activation of NPY
receptors alters catecholamines (CA) synthesis in the central nervous system
and, if so, to identify the NPY receptor subtype(s) mediating this effect.
Tyrosine hydroxylation, the rate-limiting step in CA synthesis, was assessed by
measuring the accumulation of 3,4-dihydroxyphenyalanine (DOPA) by high pressure
liquid chromatography coupled to electrochemical detection (HPLC-EC) in rat
striatal dices following incubation of the tissue with the aromatic L-amino acid
decarboxylase inhibitor m-hydroxybenzyl hydrazine (NSD 1015). Treatment with NSD
1015 resulted in an increase in DOPA accumulation that was increased even
further following depolarization with a high potassium (KCl) buffer. PYY13-36
and NPY13-36 both produced a significant enhancement of the KCl-induced increase
in DOPA accumulation. The effect of PYY13-36 was completely attenuated by the
selective Y2 antagonist BIIE0246 suggesting that activation of Y2 receptors
enhanced the synthesis of dopamine. In contrast to the effects of NPY13-36 and
PYY13-36; NPY, PYY and PYY3-36 all produced a significant attenuation of the
KCl-induced increase in DOPA accumulation. The Y1 antagonist BIBO3304 and the
Y5-antagonist CGP71683A, both prevented the inhibitory effect of NPY converting
it to a stimulatory effect. The enhancement of the NPY induced increase in DOPA
accumulation observed by BIBO3304 was attenuated when examined in the presence
of the Y2 antagonist BIIE0246. These results suggest that activation of NPY
receptors can modulate the synthesis of CA in the rat striatum. The Y1 and Y5
receptor appear to be involved in attenuation, while Y2 receptors are involved
in the stimulation of synthesis. The aim of this study is to determine the effects of intrastriatal
administration of MnCl2, on the extracellular levels of dopamine (DA) and
metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in
basal conditions and stimulated by depolarization with KCl and pargyline
administration. Also, we studied the effect of MnCl2 on extracellular levels of
l-Dopa in the presence of aromatic amino acid decarboxylase (AADC) inhibitor
3-hydroxybencilhydracine-HCl (NSD 1015). This study concluded that MnCl2,
reduced the basal and K+-stimulated DA-release in striatum, without notably
affecting the DOPAC and HVA levels. Intraperitoneal injection of pargyline
increased striatal DA levels, decreasing DOPAC and HVA levels. The infusion of
MnCl2 removed the increase in DA levels, without affecting DOPAC and HVA levels.
Perfusion of NSD 1015 increased the extracellular levels of L-DOPA in striatum,
and MnCl2 increased the effect of NSD1015 on L-Dopa. The effects of daily late afternoon administration of the indoleamine,
melatonin, on the in situ activity of tyrosine hydroxylase (TH) and tryptophan
hydroxylase (TPH) were examined in the caudate nuclei of the striatum of male
Syrian hamsters. TH and TPH activities were determined in tissue extracts by
measuring the accumulation of L-Dopa and 5-HTP respectively, following the
administration of the aromatic L-amino acid decarboxylase inhibitor, NSD-1015.
Animals were sacrificed at 4 time points over the 24 light/dark cycle after 9.5
weeks of melatonin treatment. TH activity was significantly increased by
melatonin during the early part of the dark phase of the light/dark cycle. While
no significant effects of melatonin on TPH was observed, melatonin significantly
increased 5-HT concentrations, suggesting a melatonin-induced inhibition of 5-HT
release. The data suggest that the striatum may be a region in which
dopaminergic neurons are subject to significant regulation by melatonin, either
directly or through serotonergic neurons which synapse on dopaminergic neurons
in the striatum. To establish the neurotransmitter role(s) of L-3,4-dihydroxyphenylalanine (DOPA)
in its own right, we attempted to clarify whether i.p. injection of a DOPA
antagonist, DOPA cyclohexyl ester (CHE), would antagonize the behavioral
responses of conscious rats to DOPA in the presence of 3-hydroxybenzylhydrazine
(NSD-1015) (100 mg/kg i.p.), a central aromatic L-amino acid decarboxylase
(AADC) inhibitor. DOPA-CHE (40, 60 and 100 mg/kg) elicited a dose-dependent
partial antagonism against the increase in locomotor activity induced by DOPA
(100 mg/kg i.p.). A low dose of DOPA-CHE (10 mg/kg) elicited full antagonism
against the potentiating effect of a non-effective dose of DOPA (20 mg/kg) on
the increase in locomotor activity induced by a dopamine D(2) agonist quinpirole
(0.3 mg/kg s.c.). DOPA-CHE (100 mg/kg) elicited full antagonism against licking
behavior induced by DOPA (100 mg/kg). We confirmed that DOPA (100 mg/kg)
increased the striatal dopamine content but elicited no effect on locomotor
activity in the presence of benserazide (50 mg/kg i.p.), a peripheral AADC
inhibitor. DOPA also increased the dopamine content in the presence of NSD-1015
to a maximal degree similar to that in the presence of benserazide. Thus, we
conclude that DOPA-CHE is a suitable DOPA antagonist that would be available
under in vivo experimental conditions. DOPA plays a role in the neuromodulation
of behavior. Norepinephrine (NE) metabolism in brain regions was studied in the Purkinje cell
degeneration (pcd) mutant mouse. The Purkinje cells, which are target cells for
noradrenergic (NA) neurons, degenerate completely in the pcd mutant mouse
between 17 and 45 days of age, but the NA terminals remain intact. The purpose
of this study was to determine if cerebellar NE turnover is altered after
Purkinje cell loss. The concentrations of NE and of its major metabolite in
mouse brain, 3-methoxy-4-hydroxy-phenylglycol (MHPG) were measured by liquid
chromatography with electrochemical detection. The concentration of MHPG and the
concentration ratio MHPG/NE were taken as indices of NE turnover. Although the
cerebellar content of NE in the pcd mice was not different from control mice,
MHPG and the ratio MHPG/NE were decreased slightly in 3-month old, and more in 6
and 9-month old, mutants compared to controls. No decrease in MHPG content or in
the MHPG/NE ratio was detectable in younger mutants (22 or 45-day old). The
accumulation of dopa (3,4-dihydroxyphenylalanine) after administration of NSD
1015 to inhibit aromatic l-amino acid decarboxylase was determined as an index
of NE synthesis. Dopa accumulation was decreased in cerebellum to 69%, 53% and
37% of control values, respectively, at 3, 6 and 12 months in pcd mice, but was
not changed in brain stem and was only slightly decreased in hypothalamus. No
decrease in MHPG (content or concentration) or in the ratio MHPG/NE was found in
brain stem. In hypothalamus, NE concentration and content were slightly
increased in pcd mice at all ages, and the ratio MHPG/NE was decreased in 6 and
9-month old mice. We conclude that although NA axons in the cerebellum are
maintained in pcd mice after Purkinje cell degeneration, NE turnover is
decreased, suggesting that the synthesis and release of neurotransmitter does
not continue at normal rates when the target cells have degenerated. Tyrosine hydroxylation is considered to be the rate-limiting step in
catecholamine synthesis. It is also assumed that under usual conditions,
tyrosine 3-monooxygenase (EC 1.14.16.2) (tyrosine hydroxylase - TH) is close to
full saturation with its l-tyrosine substrate and hence that raising the
availability of l-tyrosine does not substantially increase
3,4-dihydroxyphenylalanine (DOPA) synthesis. We evaluated this in vivo by
reverse dialysis of the aromatic-l-amino-acid decarboxylase (EC 4.1.1.28)
inhibitor NSD-1015 (20μM) and selected concentrations of l- or d-tyrosine. In
striatum, extracellular DOPA levels increased linearly (maximum 250% control) as
l-tyrosine concentrations were raised from 0-1000μM. In medial prefrontal
cortex, DOPA levels reached a maximum (300% control) at l-tyrosine 62.5-125μM
but still remained significantly elevated (200% control) at higher l-tyrosine
concentrations (250-500μM). At the l-tyrosine concentrations tested, DOPA levels
were never below those of controls. d-tyrosine (62.5μM) did not affect DOPA
levels. The degree to which the elevation of DOPA levels represents a net
increase in tyrosine hydroxylation as opposed to heteroexchange of l-TYR for
intracellular DOPA remains to be determined. However, one interpretation of the
data is that under usual in vivo conditions brain TH may not be near full
saturation with l-tyrosine and that mechanisms other than tyrosine hydroxylation
may be more important in the acute regulation of brain catecholamine synthesis
than previously appreciated. This would have implications for the
pathophysiology and treatment of neuropsychiatric conditions in which
dysregulation of DA transmission and l-tyrosine transport have been implicated. Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric
neuro-metabolic disease in children. Due to the lack of an animal model, its
pathogenetic mechanism is poorly understood. To study the role of AADC in brain
development, a zebrafish model of AADC deficiency was generated. We identified
an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome.
Whole-mount in situ hybridization analysis showed that the ddc gene is expressed
in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and
raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of
Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO)
reduced brain volume and body length. We observed increased brain cell apoptosis
and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants
(ddc MO-injected embryos). Seizure-like activity was also detected in ddc
morphants in a dose-dependent manner. ddc morphants had less sensitive touch
response and impaired swimming activity that could be rescued by injection of
ddc plasmids. In addition, eye movement was also significantly impaired in ddc
morphants. Collectively, loss of Ddc appears to result in similar phenotypes as
that of ADCC deficiency, thus zebrafish could be a good model for investigating
pathogenetic mechanisms of AADC deficiency in children. |
Is pseudouridine a RNA modification? | Yes, pseudouridine (Ψ) is the most abundant of>150 nucleoside modifications in RNA. | The number and position of the pseudouridines of Haloarcula marismortui and
Deinococcus radiodurans large subunit RNA have been determined by a combination
of total nucleoside analysis by HPLC-mass spectrometry and pseudouridine
sequencing by the reverse transcriptase method and by LC/MS/MS. Three
pseudouridines were found in H. marismortui, located at positions 1956, 1958,
and 2621 corresponding to Escherichia coli positions 1915, 1917, and 2586,
respectively. The three pseudouridines are all in locations found in other
organisms. Previous reports of a larger number of pseudouridines in this
organism were incorrect. Three pseudouridines and one 3-methyl pseudouridine
(m3Psi) were found in D. radiodurans 23S RNA at positions 1894, 1898 (m3Psi),
1900, and 2584, the m3Psi site being determined by a novel application of mass
spectrometry. These positions correspond to E. coli positions 1911, 1915, 1917,
and 2605, which are also pseudouridines in E. coli (1915 is m3Psi). The
pseudouridines in the helix 69 loop, residues 1911, 1915, and 1917, are in
positions highly conserved among all phyla. Pseudouridine 2584 in D. radiodurans
is conserved in eubacteria and a chloroplast but is not found in archaea or
eukaryotes, whereas pseudouridine 2621 in H. marismortui is more conserved in
eukaryotes and is not found in eubacteria. All the pseudoridines are near, but
not exactly at, nucleotides directly involved in various aspects of ribosome
function. In addition, two D. radiodurans Psi synthases responsible for the four
Psi were identified. Pseudouridine is the most abundant of more than 100 chemically distinct natural
ribonucleotide modifications. Its synthesis consists of an isomerization
reaction of a uridine residue in the RNA chain and is catalyzed by pseudouridine
synthases. The unusual reaction mechanism has become the object of renewed
research effort, frequently involving replacement of the substrate uridines with
5-fluorouracil (f(5)U). f(5)U is known to be a potent inhibitor of pseudouridine
synthase activity, but the effect varies among the target pseudouridine
synthases. Derivatives of f(5)U have previously been detected, which are thought
to be either hydrolysis products of covalent enzyme-RNA adducts, or
isomerization intermediates. Here we describe the interaction of pseudouridine
synthase 1 (Pus1p) with f(5)U-containing tRNA. The interaction described is
specific to Pus1p and position 27 in the tRNA anticodon stem, but the enzyme
neither forms a covalent adduct nor stalls at a previously identified reaction
intermediate of f(5)U. The f(5)U27 residue, as analyzed by a DNAzyme-based assay
using TLC and mass spectrometry, displayed physicochemical properties unaltered
by the reversible interaction with Pus1p. Thus, Pus1p binds an f(5)U-containing
substrate, but, in contrast to other pseudouridine synthases, leaves the
chemical structure of f(5)U unchanged. The specific, but nonproductive,
interaction demonstrated here thus constitutes an intermediate of Pus turnover,
stalled by the presence of f(5)U in an early state of catalysis. Observation of
the interaction of Pus1p with fluorescence-labeled tRNA by a real-time readout
of fluorescence anisotropy and FRET revealed significant structural distortion
of f(5)U-tRNA structure in the stalled intermediate state of pseudouridine
catalysis. Pseudouridine (Ψ) is the most abundant of >150 nucleoside modifications in RNA.
Although Ψ was discovered as the first modified nucleoside more than half a
century ago, neither the enzymatic mechanism of its formation, nor the function
of this modification are fully elucidated. We present the consistent picture of
Ψ synthases, their substrates and their substrate positions in model organisms
of all domains of life as it has emerged to date and point out the challenges
that remain concerning higher eukaryotes and the elucidation of the enzymatic
mechanism. |
What does the human ABCC gene product do? | The important drug resistance-conferring members belong to three subfamilies of the human ABC family; these are ABCB1 (MDR1/P-glycoprotein of subfamily ABCB), subfamily ABCC (MRPs), and ABCG2 (BCRP of subfamily ABCG), which are expressed in various organs. The ATP-binding cassette (ABC) transporters constitute a large family of membrane proteins, which transport a variety of compounds through the membrane against a concentration gradient at the cost of ATP hydrolysis | Several years ago, we initiated a long-term project of cloning new human
ATP-binding cassette (ABC) transporters and linking them to various disease
phenotypes. As one of the results of this project, we present two new members of
the human ABCC subfamily, ABCC11 and ABCC12. These two new human ABC
transporters were fully characterized and mapped to the human chromosome 16q12.
With the addition of these two genes, the complete human ABCC subfamily has 12
identified members (ABCC1-12), nine from the multidrug resistance-like subgroup,
two from the sulfonylurea receptor subgroup, and the CFTR gene. Phylogenetic
analysis determined that ABCC11 and ABCC12 are derived by duplication, and are
most closely related to the ABCC5 gene. Genetic variation in some ABCC subfamily
members is associated with human inherited diseases, including cystic fibrosis
(CFTR/ABCC7), Dubin-Johnson syndrome (ABCC2), pseudoxanthoma elasticum (ABCC6)
and familial persistent hyperinsulinemic hypoglycemia of infancy (ABCC8). Since
ABCC11 and ABCC12 were mapped to a region harboring gene(s) for paroxysmal
kinesigenic choreoathetosis, the two genes represent positional candidates for
this disorder. Drug transporters are an important part of the defense of cells against
cytotoxic agents. One major group of transporters is known as multidrug
resistance associated proteins (MRP; ABCC gene family). The MRPs belong to the
ATP binding cassette transporter superfamily. One family member, ABCC4 (also
known as MRP4) functions as a cellular efflux pump for anti-HIV drugs, such as
9-(2-phoshoenylmethoxyethyl) adenine and azido-thymidine-monophosphate, an
antiviral nucleotide, ganciclovir-monophosphate, and anti-cancer agents such as
thiopurines. We isolated a ABCC4 cDNA encoding a non-functional protein, owing
to an insertion, and subsequently determined the ABCC4 gene structure. This
analysis revealed that the insertion was attributed to two additional exons that
would be predicted to produce premature termination codons (PTC) in ABCC4. The
highly similar mouse Abcc4 gene also contained these exons, which were
remarkable because their size and sequence identity were much higher than the
overall similarity between these genes. Further, a comparison of human, monkey
and rodent ABCC4 genes revealed that these same PTC-producing exons were also
highly conserved in evolution. As all the ABCC4 mRNA containing these PTC exons
might produce nonsense mRNA, we further tested the hypothesis that these mRNAs
were targets of nonsense-mediated mRNA decay (NMD). Protein synthesis inhibition
selectively stabilized PTC containing ABCC4 transcripts in human, monkey and
rodent cell lines. Moreover, the amount of PTC-containing ABCC4 transcripts was
critically dependent upon protein synthesis, as removal of the inhibitor
dramatically decreased expression, which correlated with the resumption of
protein synthesis. These are the first studies to indicate that the highly
conserved PTC exons of the ABCC4 gene may dictate its expression. The placenta functions both as site for nutrition and protection of the fetus.
Transport proteins, including members of the multidrug resistance protein
(MRP)/ABCC subfamily, have been recognized to contribute to the latter function.
MRP5 (ABCC5) was identified as transmembrane transport protein for cyclic
nucleotides, especially 3',5'-cyclic GMP (cGMP), indicating an additional role
in signal transduction and a potential role in placenta development. We
therefore studied expression, localization, and function of MRP5 in placenta of
different gestational ages. Quantitative real-time polymerase chain reaction
revealed expression of MRP5 in all 60 samples from pre-term and term placenta,
with a decreasing mean expression with gestational age (MRP5/18S-ratio x 1000; <
32 weeks: 2.91 +/- 0.73, n = 15; 32 to 37 weeks: 2.10 +/- 0.87, n = 15; > 37
weeks: 0.46 +/- 0.08, n = 30; P < 0.01). Immunofluorescence microscopy with an
anti-MRP5 antibody indicated localization of MRP5 preferentially in the basal
membrane of syncytiotrophoblasts and in and around fetal vessels. ATP-dependent
[(3)H]cGMP transport as evidence for MRP5 function could be demonstrated in
isolated basal membrane vesicles. Moreover, the influence of cellular
differentiation on MRP5 expression was studied in isolated trophoblasts,
revealing an increase of the MRP5 expression in parallel with the hCG production
(MRP5/18S-ratio x 1000 was 2.4 +/- 0.5 at day 5 of culture and 1.45 +/- 0.5 at
day 0 of culture, n = 3 preparations, significant difference with P < 0.05). In
conclusion, MRP5 expression depends on gestational age and varies throughout the
differentiation process. In view of the important role of cGMP for cellular
differentiation, MRP5 may play a role in placental development in context with a
specific need for cellular cGMP export. Genetic variations in drug metabolizing enzymes and targets are established
determits of adverse drug reactions and interactions, but less is known about
the role of genetic polymorphisms in membrane transport proteins. MRP1 (ABCC1)
is one of 13 polytopic membrane proteins that comprise the 'C' subfamily of the
ATP-binding cassette (ABC) superfamily of transport proteins. MRP1 and related
ABCC family members, including MRP2, 3, 4 and 5 (ABCC2, 3, 4 and 5), each have a
distinctive pattern of tissue expression and substrate specificity. Together,
these five transporters play important roles in the disposition and elimination
of drugs and other organic anions, and in maintece of blood-tissue barriers,
as confirmed by enhanced chemosensitivity of respective knockout mice. Moreover,
Mrp2 (Abcc2) deficient animals display mild conjugated hyperbilirubinemia,
corresponding to a human condition known as Dubin-Johnson syndrome (DJS).
Naturally occurring mutations in MRP/ABCC-related drug transporters have been
reported, some of which are non-synonymous single nucleotide polymorphisms. The
consequences of the resulting amino acid changes can sometimes be predicted from
in vitro site-directed mutagenesis studies or from knowledge of mutations of
analogous (conserved) residues in ABCC proteins that cause DJS, Pseudoxanthoma
elasticum (ABCC6), cystic fibrosis (CFTR/ABCC7) or persistent hyperinsulinemic
hypoglycemia of infancy (SUR1/ABCC8). Continual updating of databases of
sequence variants and haplotype analysis, together with in vitro biochemical
validation assays and pharmacological studies in knockout animals, should make
it possible to determine how genetic variation in the MRP-related transporters
contributes to the range of responses to drugs and chemicals observed in
different human populations. The ATP-binding cassette (ABC) transporters constitute a large family of
membrane proteins, which transport a variety of compounds through the membrane
against a concentration gradient at the cost of ATP hydrolysis. Substrates of
the ABC transporters include lipids, bile acids, xenobiotics, and peptides for
antigen presentation. As they transport exogenous and endogenous compounds, they
reduce the body load of potentially harmful substances. One by-product of such
protective function is that they also eliminate various useful drugs from the
body, causing drug resistance. This review is a brief summary of the structure,
function, and expression of the important drug resistance-conferring members
belonging to three subfamilies of the human ABC family; these are ABCB1
(MDR1/P-glycoprotein of subfamily ABCB), subfamily ABCC (MRPs), and ABCG2 (BCRP
of subfamily ABCG), which are expressed in various organs. In the text, the
transporter symbol that carries the subfamily name (such as ABCB1, ABCC1, etc.)
is used interchangeably with the corresponding original names, such as
MDR1P-glycoprotein, MRP1, etc., respectively. Both nomenclatures are maintained
in the text because both are still used in the transporter literature. This
helps readers relate various names that they encounter in the literature. It now
appears that P-glycoprotein, MRP1, MRP2, and BCRP can explain the phenomenon of
multidrug resistance in all cell lines analyzed thus far. Also discussed are the
gene structure, regulation of expression, and various polymorphisms in these
genes. Because genetic polymorphism is thought to underlie interindividual
differences, including their response to drugs and other xenobiotics, the
importance of polymorphism in these genes is also discussed. BACKGROUND: Biliary excretion is a major elimination route of many drugs and
their metabolites. Hepatobiliary elimination is a vectorial process involving
uptake transporters in the basolateral hepatocyte membrane, possibly Phase I and
Phase II metabolizing enzymes, and ATP-dependent efflux pumps in the apical
hepatocyte membrane.
OBJECTIVES: Because many drugs and their metabolites are anions, this review
focuses on transporters involved in their hepatocellular uptake (members of the
organic anion transporting polypeptide (OATP) family) and biliary elimination
(apical conjugate efflux pump ABCC2/MRP2).
METHODS: The molecular and functional characteristics of the human OATP and
ABCC/MRP transporters are presented, including a detailed overview of endogenous
and drug substrates. Examples illustrate the interplay of transporters with
Phase II conjugating enzymes. Model systems to study the vectorial transport of
organic anions are also discussed.
RESULTS/CONCLUSIONS: OATP uptake transporters, conjugating enzymes, and
ABCC2/MRP2 work in concert to enable the hepatobiliary elimination of anionic
drugs and their metabolites. It is increasingly important to understand how
genetic variants of these transporters and enzymes influence the interindividual
variability of drug elimination. The constituents of highly active anti-retroviral therapy (HAART) include HIV-1
protease inhibitors (HPIs) and nucleoside reverse transcriptase inhibitors
(NRTIs). Endothelial cell (EC) barriers, especially the blood-brain-barrier
(BBB) suppresses the entry of HAART drugs to subendothelial HIV-1 reservoirs.
The ATP binding cassette (ABC) transporter family members, multidrug resistant-1
(MDR-1) and multidrug resistance-associated proteins (MRPs) can efflux both HPIs
and NRTIs from intracellular compartments. Using brain derived ECs from
non-human sources, previous studies suggested a domit role for MDR-1 in HAART
efflux from the BBB. However, due to species variations in ABC-transporter
expression, drug-efflux functions using human brain ECs need to be investigated.
Furthermore, roles of ABC-transporters in drug-efflux from systemic EC barriers
need to be studied. We monitored the expression of ABC-transporters in primary
human ECs obtained from brain (HBMVECs), aorta (HAECs), pulmonary-artery
(HPAECs), dermal-microvessel (HDMVECs) and umbilical vein (HUVECs). Gene
expression for MDR-1 and MRPs (MRP-1 to MRP-5) were analyzed by reverse
transcriptase polymerase chain reaction (RT-PCR). Drug efflux functions were
determined by calcein retention assays. Intracellular accumulation of both
3H-saquinavir (an HPI) and 3H-zidovudine (an NRTI) were also monitored in HAECs
and HBMVECs. Both assays were carried out in presence of verapamil (20-60
microM) or MK-571 (12.5-50 microM) inhibitors of MDR-1 and MRPs, respectively in
presence of verapamil or MK-571. The HBMVECs expressed higher levels of MRPs
than MDR-1 and only MK-571 significantly (P<0.01) suppressed calcein efflux from
these cells. However, both HAECs and HPAECs showed MDR-1 and MRP expression and
calcein efflux was inhibited by both verapamil and MK-571. Both inhibitors
suppressed 3H-saqubinavir efflux from HAECs, but only MK-571 suppressed
saquinavir efflux from HBMVECs. In both ECs, 3H-zidovudine efflux was only
suppressed by MK-571. Thus, primary human ECs, especially brain derived ECs,
predomitly express MRPs and their specific inhibition may enhance HAART
efficacy in subendothelial HIV-1 reservoirs. 1. The adenosine triphosphate (ATP) binding cassette (ABC) transporters form one
of the largest protein families encoded in the human genome, and more than 48
genes encoding human ABC transporters have been identified and sequenced. It has
been reported that mutations of ABC protein genes are causative in several
genetic disorders in humans. 2. Many human ABC transporters are involved in
membrane transport of drugs, xenobiotics, endogenous substances or ions, thereby
exhibiting a wide spectrum of biological functions. According to the new
nomenclature of human ABC transporter genes, the 'ABCC' gene sub-family
comprises three classes involving multidrug resistance-associated proteins
(MRPs), sulfonylurea receptors (SURs), and a cystic fibrosis transmembrane
conductance regulator (CFTR). 3. Molecular cloning studies have identified a
total of ten members of the human MRP class including ABCC11, ABCC12, and ABCC13
(pseudo-gene) that have recently been characterized. 4. This review addresses
the historical background and discovery of the ATP-driven xenobiotic export
pumps (GS-X pumps) encoded by MRP genes, biological functions of ABC
transporters belonging to the MRP class, and regulation of gene expression of
MRPs by oxidative stress. Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug
resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4,
MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the
ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane
conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which
constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13
is clearly a pseudo-gene which encodes a truncated protein that is highly
expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but
without transporting activity. These transporters are localized to the apical
and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal
tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members
transport a structurally diverse array of important endogenous substances and
xenobiotics and their metabolites (in particular conjugates) with different
substrate specificity and transport kinetics. The human MRP/ABCC transporters
except MRP9/ABCC12 are all able to transport organic anions, such as drugs
conjugated to glutathione, sulphate or glucuronate. In addition, selected
MRP/ABCC members may transport a variety of endogenous compounds, such as
leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and
MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4,
MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids.
In vitro, the MRP/ABCC transporters can collectively confer resistance to
natural product anticancer drugs and their conjugated metabolites, platinum
compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical
and antimonial oxyanions, peptide-based agents, and in concert with alterations
in phase II conjugating or biosynthetic enzymes, classical alkylating agents,
alkylating agents. Several MRP/ABCC members (MRPs 1-3) are associated with tumor
resistance which is often caused by an increased efflux and decreased
intracellular accumulation of natural product anticancer drugs and other
anticancer agents. Drug targeting of these transporters to overcome
MRP/ABCC-mediated multidrug resistance may play a role in cancer chemotherapy.
Most MRP/ABCC transporters are subject to inhibition by a variety of compounds.
Based on currently available preclinical and limited clinical data, it can be
expected that modulation of MRP members may represent a useful approach in the
management of anticancer and antimicrobial drug resistance and possibly of
inflammatory diseases and other diseases. A better understanding of their
substrates and inhibitors has important implications in development of drugs for
treatment of cancer and inflammation. The identification of the transport proteins responsible for the uptake and the
efflux of nucleosides and their metabolites enables the characterization of
their vectorial transport and a better understanding of their absorption,
distribution, and elimination. Human concentrative nucleoside transporters
(hCNTs/SLC28A) are known to mediate the transport of natural nucleosides and
some nucleoside analogs into cells in a sodium-dependent and unidirectional
manner. On the other hand, several human multidrug resistance proteins [human
ATP-binding cassette transporter, subfamily C (ABCC)] cause resistance against
nucleoside analogs and mediate transport of phosphorylated nucleoside
derivatives out of the cells in an ATP-dependent manner. For the integrated
analysis of uptake and efflux of these compounds, we established a
double-transfected Madin-Darby canine kidney (MDCK) II cell line stably
expressing the human uptake transporter hCNT3 in the apical membrane and the
human efflux pump ABCC4 in the basolateral membrane. The direction of transport
was from the apical to the basolateral compartment, which is in line with the
unidirectional transport and the localization of both recombit proteins in
the MDCKII cells. Recombit hCNT3 mediated the transport of several known
nucleoside substrates, and we identified 5-azacytidine as a new substrate for
hCNT3. It is of interest that coexpression of both transporters was confirmed in
pancreatic adenocarcinomas, which represent an important clinical indication for
the therapeutic use of nucleoside analogs. Thus, our results establish a novel
cell system for studies on the vectorial transport of nucleosides and their
analogs from the apical to the basolateral compartment. The results contribute
to a better understanding of the cellular transport characteristics of
nucleoside drugs. The ABCC subfamily of the ATP binding cassette (ABC) transporters, which were
formerly known as multidrug resistance-related proteins (MRPs), consists of
closely related members found in all eukaryotic organisms. Although more than a
decade of intensive research has elapsed since the first MRP protein was
functionally characterised in Arabidopsis thaliana, knowledge of this particular
transporter family is still limited in plants. Although ABCC proteins were
originally defined as vacuolar pumps of glutathione-S (GS) conjugates, evidence,
as well as speculation, on their endogenous functions inside the cell ranges
from detoxification and heavy metal sequestration, to chlorophyll catabolite
transport and ion channel regulation. The characterisation of knockout mutants
in Arabidopsis has been pivotal for elucidation of different roles of ABCC
transporters. However, a functional annotation for the majority of these
transport proteins is still lacking, even in this model plant. On the one hand,
this problem seems to be caused by functional redundancy between family members,
which might lead to physiological complementation by a highly homologous gene in
the mutant lines. On the other hand, there is growing evidence that the
functional diversity of ABCC genes in Arabidopsis and other plants is far
greater than previously assumed. For example, analysis of microarray expression
data supports involvement of ABCC transporters in the response to biotic stress:
particular changes in ABCC transcript levels are found, which are
pathogen-specific and evoke distinct signalling cascades. Current knowledge
about plant ABCC transporters indicates that novel and unexpected functions and
substrates of these proteins are still waiting to be elucidated. BACKGROUND: Although the prognostic value of the ATP-binding cassette, subfamily
C (ABCC) transporters in childhood neuroblastoma is usually attributed to their
role in cytotoxic drug efflux, certain observations have suggested that these
multidrug transporters might contribute to the maligt phenotype independent
of cytotoxic drug efflux.
METHODS: A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived
(MYCN)-driven transgenic mouse neuroblastoma model was crossed with an
Abcc1-deficient mouse strain (658 hMYCN(1/-), 205 hMYCN(+/1) mice) or,
alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes
were suppressed using short interfering RNA or overexpressed by stable
transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were
then assessed for wound closure ability, clonogenic capacity, morphological
differentiation, and cell growth. Real-time quantitative polymerase chain
reaction was used to examine the clinical significance of ABCC family gene
expression in a large prospectively accrued cohort of patients (n = 209) with
primary neuroblastomas. Kaplan-Meier survival analysis and Cox regression were
used to test for associations with event-free and overall survival. Except where
noted, all statistical tests were two-sided.
RESULTS: Inhibition of ABCC1 statistically significantly inhibited neuroblastoma
development in hMYCN transgenic mice (mean age for palpable tumor: treated mice,
47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence
interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited
wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4
enhanced morphological differentiation (P < .001) and inhibited cell growth (P <
.001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast
with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with
reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to
4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing
recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover,
overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P <
.001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and
ABCC4 was associated with patients having an adverse event, such that of the 12
patients with the "poor prognosis" expression pattern, 10 experienced recurrence
or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001).
CONCLUSION: ABCC transporters can affect neuroblastoma biology independently of
their role in chemotherapeutic drug efflux, enhancing their potential as targets
for therapeutic intervention. |
What is the connection between furin and hepcidin? | The iron-regulatory peptide hepcidin is synthesized in the liver as an 84-aa pre-pro-hormone maturated by proteolysis through a consensus furin cleavage site to generate the bioactive 25-aa peptide secreted in the circulation. The hepatic prohormone convertase furin mediates the posttranslational processing of hepcidin. | Hepcidin is encoded as an 84 amino acid prepropeptide containing a typical
N-terminal 24 amino acid endoplasmic reticulum targeting signal sequence, and a
35 amino acid proregion (pro) with a consensus furin cleavage site immediately
followed by the C-terminal 25 amino acid bioactive iron-regulatory hormone
(mature peptide). We performed pulse-chase studies of posttranslational
processing of hepcidin in human hepatoma HepG2 cells and in primary human
hepatocytes induced with bone morphogenic protein (BMP-9). In some experiments,
the cells were treated with the furin protease inhibitor
decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) or furin siRNA. In the absence
of furin inhibitor, hepcidin was found to be processed in less than 1 h and
secreted as a 3 kDa form reactive with anti-mature but not anti-pro antibody. In
the presence of furin inhibitors or furin siRNA, a 6 kDa form reactive with both
anti-pro and anti-mature antibody was rapidly secreted into the medium.
Processing was not affected by inhibitors of the hypoxia inducible factor (HIF)
pathway, or by treatment with 30 microM holo- or apo-transferrin. In conclusion,
the hepatic prohormone convertase furin mediates the posttranslational
processing of hepcidin. The proteolytic cleavage of prohepcidin to hepcidin is
not regulated by iron-transferrin or the HIF pathway. BACKGROUND AND AIMS: Hepcidin is an iron homoeostasis regulator peptide.
Loss-of-function mutations cause juvenile haemochromatosis while its
over-expression results in anaemia. However, the mechanism and function of
preprohepcidin conversion to mature hepcidins (25, 22 and 20 amino acid
C-terminal peptides) are not well known. After removal of the signal peptide,
the first proteolytic cleavage occurs within the basic motif RRRRR(59)DT,
suggesting the involvement of proprotein convertase (PC) family members in this
process.
METHODS AND RESULTS: Using cell transfection experiments, the processing of
preprohepcidin in the human hepatocyte line Huh-7 was found to be inhibited by
the Furin inhibitors serpin alpha1-antitrypsin (alpha1-PDX) and prosegment
preproFurin (ppFurin). Site-directed mutagenesis analysis confirmed the
RRRRR(59)DT preprohepcidin cleavage site. In parallel, the lack of
preprohepcidin processing found in the PC activity-deficient cell line LoVo was
restored by the expression of Furin, paired basic amino acid cleaving enzyme 4
(PACE4), PC5 or PC7. This finding is consistent with the in vitro digestions of
a synthetic peptide mimicking the cleavage site of preprohepcidin. In addition,
during mouse embryonic development the major expression of hepcidin found in the
liver coincided with that of Furin. While hepcidin induces the degradation of
the iron transporter ferroportin, its RRRRR(59) to SSSSS(59) mutant is not
active.
CONCLUSIONS: These results demonstrate the key role of the convertases Furin,
PACE4, PC5 and/or PC7 in the generation and secretion of active hepcidin and
suggest that the control of hepcidin processing as a potential
therapeutic/diagnostic strategy in hepcidin-related disorders such as
haemochromatosis, inflammatory diseases, anaemia and cancer. Hepcidin is a small liver-derived peptide central in the regulation of systemic
iron homeostasis. Although the gene regulation has been extensively studied at
transcriptional level, the corresponding effects on the production of bioactive
peptide are largely unknown. We therefore applied a proteomics-based approach by
combining immunocapture with time-of-flight mass spectrometry to characterize
hepcidin-25 produced by hepatocyte-derived cell lines. Similar to its
transcriptional regulation, mature hepcidin-25 was strongly secreted upon
stimulation with BMPs and IL-6. The immunocaptured peptide down-modulated
iron-exporter ferroportin on the monocyte/macrophage surface. Further mass
spectrometry-based analyses indicated that hepcidin-25 in its bioactive
conformation was very stable in serum and urine and not converted into its
smaller isoforms. Hepcidin-25 was processed in the Golgi apparatus from its
precursor, while the unprocessed prohepcidin was secreted only when furin-like
protease activity was intracellularly inhibited. Furthermore, the amounts of
hepatocytic secretion of hepcidin-25 are highly correlated with the gene
transcript levels. An unexpected observation was the synergistic effect of BMPs
and IL-6 on hepcidin-25 secretion, which points towards cross-talk between iron
and inflammatory stimuli. The study underscores hepcidin-25 quantification as a
valuable tool to unravel regulatory pathways in iron metabolism. BACKGROUND/AIMS: The iron-regulatory peptide hepcidin is synthesized in the
liver as an 84-aa pre-pro-hormone maturated by proteolysis through a consensus
furin cleavage site to generate the bioactive 25-aa peptide secreted in the
circulation. This peptide regulates iron export from enterocytes and macrophages
by binding the membrane iron exporter, ferroportin, leading to its degradation.
Whether pro-hepcidin could be secreted and reflect hepcidin levels remains an
open question. However, the activity of the pro-peptide on ferroportin
degradation has never been addressed.
METHODS: To answer this question, we produced recombit pro-hepcidin, both the
wild-type form and a furin cleavage site mutant, and tested their activity on
ferroportin levels in macrophagic J774 cells. Furin activity was also modulated
using furin inhibitor or siRNA-mediated furin mRNA knockdown.
RESULTS: We found that pro-hepcidin could fully induce ferroportin degradation,
but only when processed by furin to generate the mature hepcidin-25 form.
Pro-hepcidin activity was abolished in the presence of furin inhibitor and
diminished after siRNA-mediated knockdown of furin mRNA. Furthermore, the
mutated version of pro-hepcidin was completely inefficient at degrading
ferroportin in macrophages.
CONCLUSIONS: Our results demonstrate that pro-hepcidin lacks biological
activity, unless fully maturated by a furin-dependent process to yield the
bioactive 25-aa peptide. Maintece of iron balance is essential for humans and requires the coordinate
regulation of iron transport into plasma from dietary sources in the duodenum,
from recycled senescent red cells in macrophages, and from storage in
hepatocytes. Hepcidin, a recently identified antimicrobial peptide produced in
the liver, has been shown to play a central role in the homeostatic regulation
of iron absorption and distribution [1]. It is a negative regulator of iron
absorption in the small intestine and of iron release from macrophages engaged
in the recycling of iron senescent erythrocytes [2]. The human hepcidin gene
contains three exons that encode a 72-aa precursor (pro-hepcidin) with a
characteristic furin cleavage site immediately N-terminal to the 25-aa major
hepcidin species found in plasma and urine [3]. Recently, hepcidin has been
shown to regulate iron homeostasis by interaction with ferroportin, an iron
cellular exporter highly expressed in absorptive enterocytes, macrophages,
hepatocytes, and placental cells [4]. BACKGROUND: Impaired regulation of hepcidin in response to iron is the cause of
genetic hemochromatosis associated with defects of HFE and transferrin receptor
2. However, the role of these proteins in the regulation of hepcidin expression
is unclear.
DESIGN AND METHODS: Hepcidin expression, SMAD and extracellular signal-regulated
kinase (Erk) phosphorylation and furin expression were analyzed in hepatic HepG2
cells in which HFE and transferrin receptor 2 were down-regulated or expressed,
or furin activity specifically inhibited. Furin expression was also analyzed in
the liver of transferrin receptor 2 null mice.
RESULTS: We showed that the silencing of HFE and transferrin receptor 2 reduced
both Erk phosphorylation and furin expression, that the exogenous expression of
the two enhanced the induction of phosphoErk1/2 and furin by holotransferrin,
but that this did not occur when the pathogenic HFE mutant C282Y was expressed.
Furin, phosphoErk1/2 and phosphoSMAD1/5/8 were down-regulated also in
transferrin receptor 2-null mice. Treatment of HepG2 cells with an inhibitor of
furin activity caused a strong suppression of hepcidin mRNA, probably due to the
inhibition of bone morphogenic protein maturation.
CONCLUSIONS: The data indicate that transferrin receptor 2 and HFE are involved
in holotransferrin-dependent signaling for the regulation of furin which
involved Erk phosphorylation. Furin in turn may control hepcidin expression. The first seven members of the proprotein convertase (PC) family activate
protein precursors by cleavage after basic residues. While PC7 has no known
specific substrates, it shows redundancy with other PCs. A genome-wide
association study suggested that circulating levels of shed human transferrin
receptor 1 (hTfR1) are regulated by PC7. We thus examined whether hTfR1
constitutes a specific substrate for PC7. Coexpression of hTfR1 with PCs in
several cell lines indicated that PC7 is the only convertase that sheds this
receptor into the medium. Site-directed mutagenesis showed that cleavage occurs
at the unusual site KTECER100 ↓LA, in which the P1 Arg100 and P6 Lys95 are
critical. Pharmacological treatments revealed that shedding of hTfR1 by PC7
requires endocytosis into acidic clathrin-coated vesicles. A PC7 chimera, in
which the transmembrane domain and the cytosolic tail of PC7 were replaced by
that of the convertase furin, lost its ability to cleave the receptor,
demonstrating the importance of these domains in the regulation of PC7 function.
Analysis of primary hepatocytes from mice lacking furin, PC5, PACE4, or PC7
revealed that hepcidin, which limits iron availability in the circulation, is
specifically generated by furin and not by PC7. Finally, depletion of iron in
the medium of hepatoma cell lines incubated with the iron chelator
desferrioxamine resulted in PC7 down-regulation.
CONCLUSION: Among the PC family members, only furin activates hepcidin in
hepatocytes, and uniquely the full-length membrane-bound PC7 can directly shed
hTfR1 by cleavage at Arg100 ↓. Our results support the notion that, when iron is
limiting, hTfR1 levels increase at least in part by way of the down-regulation
of PC7 expression. (HEPATOLOGY 2013;). The discovery of hepcidin clarified the basic mechanism of the control of
systemic iron homeostasis. Hepcidin is mainly produced by the liver as a
propeptide and processed by furin into the mature active peptide. Hepcidin binds
ferroportin, the only cellular iron exporter, causing the internalization and
degradation of both. Thus hepcidin blocks iron export from the key cells for
dietary iron absorption (enterocytes), recycling of hemoglobin iron (the
macrophages) and the release of storage iron from hepatocytes, resulting in the
reduction of systemic iron availability. The BMP/HJV/SMAD pathway is the major
regulator of hepcidin expression that responds to iron status. Also inflammation
stimulates hepcidin via the IL6/STAT3 pathway with a support of an active
BMP/HJV/SMAD pathway. In some pathological conditions hepcidin level is
inadequately elevated and reduces iron availability in the body, resulting in
anemia. These conditions occur in the genetic iron refractory iron deficiency
anemia and the common anemia of chronic disease (ACD) or anemia of inflammation.
Currently, there is no definite treatment for ACD. Erythropoiesis-stimulating
agents and intravenous iron have been proposed in some cases but they are
scarcely effective and may have adverse effects. Alternative approaches aimed to
a pharmacological control of hepcidin expression have been attempted, targeting
different regulatory steps. They include hepcidin sequestering agents
(antibodies, anticalins, and aptamers), inhibitors of BMP/SMAD or of IL6/STAT3
pathway or of hepcidin transduction (siRNA/shRNA) or ferroportin stabilizers. In
this review we summarized the biochemical interactions of the proteins involved
in the BMP/HJV/SMAD pathway and its natural inhibitors, the murine and rat
models with high hepcidin levels currently available and finally the progresses
in the development of hepcidin antagonists, with particular attention to the
role of heparins and heparin sulfate proteoglycans in hepcidin expression and
modulation of the BMP6/SMAD pathway. |
Which cells express CIDEC protein in humans? | The cell death-inducing DNA fragmentation factor alpha-like effector c (CIDEC) is a lipid droplet-associated protein that promotes intracellular triglyceride (TAG) storage. CIDEC is highly expressed in adipocytes, but undetectable in normal liver. However, its hepatic expression rises during fasting or under genetic or diet-induced hepatosteatosis in patients. | Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown
adipose tissues and increases in abundance by over 50-fold during adipogenesis.
However, its function in adipocytes has remained elusive since its discovery
over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid
droplets in cultured adipocytes and functions to promote lipid accumulation.
Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes
and colocalizes with the known lipid droplet protein perilipin. Immunostaining
of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid
droplets. FSP27-GFP expression also markedly increases lipid droplet size and
enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as
other cell types such as COS cells. Conversely, RNA interference-based
FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis
and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel
adipocyte lipid droplet protein that negatively regulates lipolysis and promotes
triglyceride accumulation. Storage of energy as triglyceride in large adipose-specific lipid droplets is a
fundamental need in all mammals. Efficient sequestration of fat in adipocytes
also prevents fatty acid overload in skeletal muscle and liver, which can impair
insulin signaling. Here we report that the Cide domain-containing protein Cidea,
previously thought to be a mitochondrial protein, colocalizes around lipid
droplets with perilipin, a regulator of lipolysis. Cidea-GFP greatly enhances
lipid droplet size when ectopically expressed in preadipocytes or COS cells.
These results explain previous findings showing that depletion of Cidea with
RNAi markedly elevates lipolysis in human adipocytes. Like perilipin, Cidea and
the related lipid droplet protein Cidec/FSP27 are controlled by peroxisome
proliferator-activated receptor gamma (PPARgamma). Treatment of lean or obese
mice with the PPARgamma agonist rosiglitazone markedly up-regulates Cidea
expression in white adipose tissue (WAT), increasing lipid deposition.
Strikingly, in both omental and s.c. WAT from BMI-matched obese humans,
expression of Cidea, Cidec/FSP27, and perilipin correlates positively with
insulin sensitivity (HOMA-IR index). Thus, Cidea and other lipid droplet
proteins define a novel, highly regulated pathway of triglyceride deposition in
human WAT. The data support a model whereby failure of this pathway results in
ectopic lipid accumulation, insulin resistance, and its associated comorbidities
in humans. Members of the cell death-inducing DFF45-like effector (CIDE) gene family have
been shown to regulate lipid metabolism. In this article, we report that the
third member of the human CIDE family, CIDEC, is down-regulated in response to a
reduced caloric intake. The down-regulation was demonstrated by microarray and
real-time polymerase chain reaction analysis of subcutaneous adipose tissue in 2
independent studies on obese patients undergoing treatment with a very low
calorie diet. By analysis of CIDEC expression in 65 human tissues, we conclude
that human CIDEC is predomitly expressed in subcutaneous adipocytes.
Together, these observations led us to investigate the effect of decreased CIDEC
expression in cultured 3T3-L1 adipocytes. Small interfering RNA-mediated
knockdown of CIDEC resulted in an increased basal release of nonesterified fatty
acids, decreased responsiveness to adrenergic stimulation of lipolysis, and
increased oxidation of endogenous fatty acids. Thus, we suggest that CIDEC is a
regulator of adipocyte lipid metabolism and may be important for the adipocyte
to adapt to changes in energy availability. The hepatic expression of the cell death-inducing DNA fragmentation factor
A-like effector family (CIDEA, CIDEB, and CIDEC) genes is markedly upregulated
in mouse models of obesity. We evaluated the expression of CIDE genes in liver
of obese human subjects undergoing gastric bypass surgery (GBS), at the time of
surgery and again 1 year later when subjects had lost 37.6 +/- 1.4% of their
initial body weight. At the time of GBS, the expression of CIDEA (r(2) = 0.20, P
= 0.04) and CIDEC (r(2) = 0.32, P = 0.01) was strongly correlated with BMI,
whereas CIDEB was not (r(2) = 0.01, P = 0.81). One year after surgery, CIDEC
expression had declined over 60% (P = 0.02), whereas CIDEA expression did not
change (P = 0.20). These data demonstrate that, consistent with previous studies
conducted in rodents, hepatic expression of CIDEA and CIDEC, but not CIDEB, is
increased in obese humans. Moreover, the hepatic expression of CIDEC is
downregulated by marked weight loss. Fat specific protein 27 (FSP27) was originally isolated by screen for genes
specifically expressed in fully differentiated mouse adipocytes. FSP27 and cell
death-inducing DFF45-like effector C (CIDEC), the human homologue of FSP27,
belong to the CIDE family. The FSP27 in adipocytes was recently reported to be a
lipid droplet (LD)-associated protein, that promotes the formation of unilocular
LDs. An FSP27 knockout mouse demonstrated lean phenotypes with atrophic adipose
tissue as a result of high-energy expenditure; this mouse line was also
resistant to diet-induced obesity and insulin resistance. Interestingly, FSP27
was also expressed in the steatoic liver of a type II diabetes model mouse. The
expression of FSP27 was markedly decreased in livers lacking the nuclear
receptor peroxisome proliferator-activated receptor gamma. Forced expression of
FSP27 in hepatocytes in vitro or in vivo led to an increase of LD through
increased triglyceride levels. The current status of the physiological roles of
FSP27/CIDEC in adipose tissue and liver are discussed along with its
significance as a factor involved in the development of metabolic disorders. A large number of macrophage-derived foam cells stores excessive neutral lipids
in intracellular droplets, and plays a major role during the development of
atherosclerosis. The formation and catabolism of intracellular lipid droplets
(LDs) are regulated by LD-associated proteins, a group of proteins which are
located on the surface of LDs and regulate the formation, morphology and
lipolysis of LDs. In order to illustrate the function of LD-associated proteins
during the process of atherosclerosis, the foam cell model is induced by
oxidized low-density lipoprotein (ox-LDL) in macrophages originated from the
THP-1 cell line, and cDNA microarrays are used to monitor the gene expression
profiles of LD-associated proteins. Gene expression data show that 2% of changed
genes are lipid binding genes during the transformation of foam cells. The major
candidate genes, the cell death-inducing DFF45-like effector (CIDE) family and
Perilipin, Adipophilin, and TIP47 (PAT) family, have different alterations
during the formation of foam cells. CIDEB, CIDEC, Adipophilin, S3-12 and LSDP5
were up-regulated, while TIP47 was down-regulated. There was no significant
change in CIDEA and Perilipin. These results were confirmed by real-time PCR and
immunoblotting. This study presents a comprehensive analysis of the gene
expression of LD-associated proteins during the differentiation of human foam
cells, which may play an important role in the process of atherosclerosis. Both insulin and the cell death-inducing DNA fragmentation factor-α-like
effector (CIDE) family play important roles in apoptosis and lipid droplet
formation. Previously, we reported that CIDEA and CIDEC are differentially
regulated by insulin and contribute separately to insulin-induced anti-apoptosis
and lipid droplet formation in human adipocytes. However, the upstream signals
of CIDE proteins remain unclear. Here, we investigated the signaling molecules
involved in insulin regulation of CIDEA and CIDEC expression. The
phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and PI-103 blocked
both insulin-induced downregulation of CIDEA and upregulation of CIDEC. The Akt
inhibitor API-2 and the c-Jun N-terminal kinase (JNK) inhibitor SP600125
selectively inhibited insulin regulation of CIDEA and CIDEC expression,
respectively, whereas the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor
SB203580 did not. Small interfering RNA-mediated depletion of Akt1/2 prevented
insulin-induced downregulation of CIDEA and inhibition of apoptosis. Depletion
of JNK2, but not JNK1, inhibited insulin-induced upregulation of CIDEC and lipid
droplet enlargement. Furthermore, insulin increased both Akt and JNK
phosphorylation, which was abrogated by the PI3K inhibitors. These results
suggest that insulin regulates CIDEA and CIDEC expression via PI3K, and it
regulates expression of each protein via Akt1/2- and JNK2-dependent pathways,
respectively, in human adipocytes. FSP27 [cell death-inducing DFFA-like effector c (CIDEC) in humans] is a protein
associated with lipid droplets that downregulates the fatty acid oxidation (FAO)
rate when it is overexpressed. However, little is known about its physiological
role in liver. Here, we show that fasting regulates liver expression of Fsp27 in
a time-dependent manner. Thus, during the initial stages of fasting, a maximal
induction of 800-fold was achieved, whereas during the later phase of fasting,
Fsp27 expression decreased. The early response to fasting can be explained by a
canonical PKA-CREB-CRTC2 signaling pathway because: i) CIDEC expression was
induced by forskolin, ii) Fsp27 promoter activity was increased by CREB, and
iii) Fsp27 expression was upregulated in the liver of Sirt1 knockout animals.
Interestingly, pharmacological (etomoxir) or genetic (Hmgcs2 interference)
inhibition of the FAO rate increases the in vivo expression of Fsp27 during
fasting. Similarly, CIDEC expression was upregulated in HepG2 cells by either
etomoxir or HMGCS2 interference. Our data indicate that there is a kinetic
mechanism of autoregulation between short- and long-term fasting, by which free
FAs delivered to the liver during early fasting are accumulated/exported by
FSP27/CIDEC, whereas over longer periods of fasting, they are degraded in the
mitochondria through the carnitine palmitoyl transferase system. Human adipocytes express high levels of two distinct lipid droplet proteins, fat
specific protein 27 (FSP27; also called CIDEC), a member of the CIDE family, and
perilipin1 (PLIN1), a member of the PAT family. Both proteins play a role in fat
metabolism in adipocytes, but how they interact is not known. Our present study
demonstrates that FSP27 and PLIN1 co-localize and interact in cultured human
primary adipocytes. We also found that the C-terminal domain of FSP27, aa
120-220, interacts with PLIN1. Individual expression of exogenous FSP27 or PLIN1
increased triglyceride content and decreased glycerol release (a measure of
lipolysis), but co-expression of both proteins did not further increase
triglyceride content or decrease lipolysis in human adipocytes. However, the
combination of PLIN1 and FSP27 increased the average size of lipid droplets or
caused the formation of unilocular adipocytes. Our data suggest that FSP27
interacts with PLIN1 to regulate lipid droplet size in human adipocytes in a
concerted manner. Cell death-inducing DFFA-like effector c (CIDEC) protein, also known as fat
specific protein 27 (Fsp27), is localized to lipid droplets. CIDEC protein is
required for unilocular lipid droplet formation and optimal energy storage in
addition to controlling lipid metabolism in adipocytes and hepatocytes. Research
found that Ad-36 could induce lipid droplets in the cultured skeletal muscle
cells and this process may be mediated by promoting CIDEC expression. The
content of intermuscular fat is an important index for evaluation of beef
quality, so the CIDEC gene appeared to be a candidate gene for regulation of
intermuscular fat, however similar research for the bovine CIDEC gene is
lacking. This paper examined the tissue expression profile of CIDEC gene in
cattle using real-time RT-PCR to suggest that bovine CIDEC is highly expressed
in adipose tissue. In addition, the Bovine CIDEC gene was cloned and inserted
into the eukaryotic expression vector pET-28a(+), whereupon recombit bovine
CIDEC protein was induced and identified by Western-blot. A phylogenetic
analysis showed that the animo acid sequence of bovine CIDEC was closer to
mammalian CIDEC than rasorial CIDEC. We found ten single nucleotide
polymorphisms sites (SNPs) in bovine CIDEC gene, of which SNP 2, 3, 4, 6 and 9,
and SNP 8 and 10 were in complete linkage disequilibrium, respectively. SNP 1, 2
and 10 were used in further haplotype studies. Eight different haplotypes were
identified in 973 cattle, of which haplotype 8 predominated with frequencies
ranging from 42.90 to 54.30 %. This research provides a basis for future
functional studies of CIDEC in cattle. OBJECTIVE: FSP27 KO mice showed enhanced expression of mitochondrial genes,
increased mitochondrial activity and smaller lipid droplets. Here, we aimed to
investigate lipid droplet protein (CIDEC/FSP27 and perilipinA (PLIN1)) gene
expression in human adipose tissue in association with obesity, insulin
resistance and mitochondrial gene expression.
DESIGN AND SUBJECTS: In cohort 1, CIDEC/FSP27, PLIN1, adipogenic (FASN, ACACA,
PPARG, GLUT4) and mitochondrial (PPARGC1A, PPARGC1B, TFAM, MT-CO3) gene
expression were analyzed in 171 adipose tissue samples (88 visceral adipose
tissue (VAT) and 83 subcutaneous adipose tissue (SAT) depots) and in a time
course experiment in human subcutaneous and visceral preadipocytes using
real-time PCR. In cohort 2, the effects of bariatric surgery-induced weight loss
were also evaluated in six caucasian morbidly obese women. Additionally, in
cohort 2 FSP27 and PLIN1 protein levels were measured using western blotting.
RESULTS: CIDEC/FSP27 (1.03±0.52 vs 0.49±0.23 relative gene expression unit
(R.U.), P<0.0001) and PLIN1 (1.32±0.82 vs 0.63±0.42 R.U., P<0.0001) gene were
significantly more expressed in SAT than in VAT. In VAT, CIDEC/FSP27 and PLIN1
gene expression decreased with body mass index, percent fat mass, fasting
glucose, fasting insulin, HOMA and were positively associated with adipogenic
(PPARG, GLUT4, FASN and ACACA) and mitochondrial biogenesis (PPARGC1A, PPARGC1B,
TFAM and MT-CO3)-related genes. Mitochondrial gene expression increased during
adipocyte differentiation in parallel to FSP27 and PLIN1 and other adipogenic
genes. After bariatric surgery-induced weight loss, PLIN1 and CIDEC/FSP27 gene
and protein expression in SAT increased significantly in parallel to adipogenic
and mitochondrial genes.
CONCLUSION: These findings suggest a positive functional interaction between
CIDEC/FSP27, PLIN1 and mitochondrial biogenesis-related genes in human adipose
tissue. OBJECTIVE: Obesity is a metabolic disorder that can lead to high blood pressure,
increased blood cholesterol and triglycerides, insulin resistance, and diabetes
mellitus. The aim was to study the effects of pioglitazone mediated
sensitization of peroxisome proliferator-activated receptor gamma (PPAR-γ) on
the relationship of Cell death-inducing DFFA-like effector C (CIDEC) with
obesity related changes in mice.
METHODS: Sixty C57B/L6 mice weighing 10-12g at 3 weeks of age were randomly
divided into 3 groups. Mice in Group 1 were fed on normal diet (ND) while Group
2 mice were given high fat diet (HFD), and Group 3 mice were given high fat diet
and treated with Pioglitazone (HFD+P). Body weight, length and level of blood
sugar were measured weekly. Quantitative real-time PCR, fluorescence microscopy,
and ELISA were performed to analyze the expression of CIDEC and PPAR-γ in
visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT).
RESULTS: Body weight and length of mice increased gradually with time in all
groups. Blood sugar in HFD mice started to increase significantly from the mid
of late phase of obesity while pioglitazone attenuated blood sugar level in
HFD+P mice. The mRNA expressions and protein levels of PPAR-γ and CIDEC genes
started to increase in HFD mice as compared to ND mice and decreased gradually
during the late phase of obesity in VAT. Pioglitazone enhanced the expression of
PPAR-γ and CIDEC genes in HFD+P mice even during the late phase of obesity.
CONCLUSION: It is insinuated that VAT is associated with late phase obesity
CIDEC decrease and insulin resistance, while pioglitazone enhances CIDEC through
activation of PPAR-γ, increases its expression, and decreases lipolysis, hence
preventing an increase of blood sugar in mice exposed to HFD. The CIDEC protein is located in lipid droplets (LDs) and the endoplasmic
reticulum (ER) and is induced in fat deposition. However, the binding domain,
the functional domain and the underlying mechanism of CIDEC in stimulating lipid
accumulation remain unclear. Here, we investigated the subcellular localization
and function of pig CIDEC and confirmed CIDEC promotes unilocular development of
LDs, reduces the specific surface area (SSA) of LDs and stimulates lipid
accumulation in HepG2 cells. By analyzing a series of CIDEC mutants, we showed
the N-domain (1-173 amino acid) is involved in LD localization and the C-domain
(174-238 amino acid) is necessary for LD fusion. Further analysis indicated that
the 106-173 amino acid region includes an ER-binding domain. Moreover, CIDEC
stayed in the ER under lipid-deficient conditions and translocated to LDs under
fatty acid stimulation suggesting that localization of CIDEC in the ER is before
the LD. Our data indicated additional fatty acids stimulated hepatic CIDEC
expression and an increasing level of CIDEC induced hepatic LD fusion and lipid
accumulation. Our work suggests that CIDEC protects LDs by decreasing the SSA of
LDs and is involved in the regulation of hepatic lipid deposition. White adipose tissue (WAT) functions as an energy reservoir where excess
circulating fatty acids are transported to WAT, converted to triglycerides, and
stored as unilocular lipid droplets. Fat-specific protein 27 (FSP27, CIDEC in
humans) is a lipid-coating protein highly expressed in mature white adipocytes
that contributes to unilocular lipid droplet formation. However, the influence
of FSP27 in adipose tissue on whole-body energy homeostasis remains unclear.
Mice with adipocyte-specific disruption of the Fsp27 gene (Fsp27(ΔAd)) were
generated using an aP2-Cre transgene with the Cre/LoxP system. Upon high-fat
diet feeding, Fsp27(ΔAd) mice were resistant to weight gain. In the small WAT of
these mice, small adipocytes containing multilocular lipid droplets were
dispersed. The expression levels of the genes associated with mitochondrial
abundance and brown adipocyte identity were increased, and basal lipolytic
activities were significantly augmented in adipocytes isolated from Fsp27(ΔAd)
mice compared with the Fsp27(F/F) counterparts. The impaired fat-storing
function in Fsp27(ΔAd) adipocytes and the resultant lipid overflow from WAT led
to marked hepatosteatosis, dyslipidemia, and systemic insulin resistance in
high-fat diet-treated Fsp27(ΔAd) mice. These results demonstrate a critical role
for FSP27 in the storage of excess fat in WAT with minimizing ectopic fat
accumulation that causes insulin-resistant diabetes and non-alcoholic fatty
liver disease. This mouse model may be useful for understanding the significance
of fat-storing properties of white adipocytes and the role of local FSP27 in
whole-body metabolism and estimating the pathogenesis of human partial
lipodystrophy caused by CIDEC mutations. BACKGROUND & AIMS: Alcoholic steatohepatitis (ASH) is the progressive form of
alcoholic liver disease and may lead to cirrhosis and hepatocellular carcinoma.
We studied mouse models and human tissues to identify molecules associated with
ASH progression and focused on the mouse fat-specific protein 27 (FSP-27)/human
cell death-inducing DFF45-like effector C (CIDEC) protein, which is expressed in
white adipose tissues and promotes formation of fat droplets.
METHODS: C57BL/6N mice or mice with hepatocyte-specific disruption of Fsp27
(Fsp27(Hep-/-) mice) were fed the Lieber-Decarli ethanol liquid diet (5%
ethanol) for 10 days to 12 weeks, followed by 1 or multiple binges of ethanol (5
or 6 g/kg) during the chronic feeding. Some mice were given an inhibitor
(GW9662) of peroxisome proliferator-activated receptor γ (PPARG). Adenoviral
vectors were used to express transgenes or small hairpin (sh) RNAs in cultured
hepatocytes and in mice. Liver tissue samples were collected from ethanol-fed
mice or from 31 patients with alcoholic hepatitis (AH) with biopsy-proved ASH
and analyzed histologically and immunohistochemically and by transcriptome,
immunoblotting, and real-time PCR analyses.
RESULTS: Chronic-plus-binge ethanol feeding of mice, which mimics the drinking
pattern of patients with AH, produced severe ASH and mild fibrosis. Microarray
analyses revealed similar alterations in expression of many hepatic genes in
ethanol-fed mice and humans with ASH, including up-regulation of mouse Fsp27
(also called Cidec) and human CIDEC. Fsp27(Hep-/-) mice and mice given
injections of adenovirus-Fsp27shRNA had markedly reduced ASH following
chronic-plus-binge ethanol feeding. Inhibition of PPARG and cyclic
AMP-responsive element binding protein H (CREBH) prevented the increases in
Fsp27α and FSP27β mRNAs, respectively, and reduced liver injury in this
chronic-plus-binge ethanol feeding model. Overexpression of FSP27 and ethanol
exposure had synergistic effects in inducing production of mitochondrial
reactive oxygen species and damage to hepatocytes in mice. Hepatic CIDEC mRNA
expression was increased in patients with AH and correlated with the degree of
hepatic steatosis and disease severity including mortality.
CONCLUSIONS: In mice, chronic-plus-binge ethanol feeding induces ASH that mimics
some histological and molecular features observed in patients with AH. Hepatic
expression of FSP27/CIDEC is highly up-regulated in mice following
chronic-plus-binge ethanol feeding and in patients with AH; this up-regulation
contributes to alcohol-induced liver damage. BACKGROUND: We previously showed that Cidec was localized on the surface of
lipid droplets and could promote the differentiation of human adipocytes, but
the molecular mechanism was still unknown.
METHODS & RESULTS: In this study, we first sought to identify proteins that
interact with Cidec using yeast two-hybrid system. The results revealed that
Cidec could directly interact with AMPKα1 subunit. We further showed that AMPKα
levels decreased while Cidec increased during the adipogenic differentiation of
human adipocytes. Meanwhile, we observed that the increased Cidec could reduce
AMPKα level in adipocytes, and the downregulation of AMPKα could help to promote
the differentiation of adipocytes. The results of co-immunoprecipitation and
immunofluorescent proved that Cidec biochemically interacted and co-localized
with AMPKα1, which meant Cidec was a regulator for AMPKα stability through an
ubiquitin-proteasome pathway.
CONCLUSION: Our data suggested that Cidec could interact with and down-regulate
AMPKα through an ubiquitin-proteasome degradation pathway, which provided a
possible mechanism of Cidec in promoting human adipocytes differentiation.
GENERAL SIGNIFICANCE: Our work proposed a new possible mechanism for human
adipogenesis, and also provided a potential role of AMPKα as a target in
treating obesity or obesity-related diseases. Peroxisome proliferator-activated receptor (PPAR) agonists are used for treating
hyperglycemia and type 2 diabetes. However, the mechanism of action of these
agonists is still under investigation. The lipid droplet-associated proteins
FSP27/CIDEC and LSDP5, regulated directly by PPARγ and PPARα, are associated
with hepatic steatosis and insulin sensitivity. Here, we evaluated the
expression levels of FSP27/CIDEC and LSDP5 and the regulation of these proteins
by consumption of a high-fat diet (HFD) or administration of PPAR agonists. Mice
with diet-induced obesity were treated with the PPARγ or PPARα agonist,
pioglitazone or fenofibrate, respectively. Liver tissues from db/db diabetic
mice and human were also collected. Interestingly, FSP27/CIEDC was expressed in
mouse and human livers and was upregulated in obese C57BL/6J mice. Fenofibrate
treatment decreased hepatic triglyceride (TG) content and FSP27/CIDEC protein
expression in mice fed an HFD diet. In mice, LSDP5 was not detected, even in the
context of insulin resistance or treatment with PPAR agonists. However, LSDP5
was highly expressed in humans, with elevated expression observed in the fatty
liver. We concluded that fenofibrate greatly decreased hepatic TG content and
FSP27/CIDEC protein expression in mice fed an HFD, suggesting a potential
regulatory role for fenofibrate in the amelioration of hepatic steatosis. OBJECTIVE: To investigate the impact of neuropeptide Y (NPY) at different
concentrations on the proliferation and differentiation of human adipose-derived
stem cells (hADSCs).
METHODS: The hADSCs were separated and incubated with NPY at a range of
concentrations 10(-6)-10(-15) mol/L. MTT assay was used to detect cell
proliferation. Following the treatment of complete medium, NPY and insulin
separately, cell differentiation was observed by Oil red O staining. In
addition, Western blotting was performed to exam the levels of peroxisome
proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer binding protein alpha
(C/EBPα), cell death-inducing dFF45-like effector c (Cidec) and receptor
interacting protein 140 (RIP140).
RESULTS: MTT assay revealed that 10(-11)-10(-15) mol/L NPY stimulated the
proliferation of hADSCs, while high-dose NPY (10(-6)-10(-10) mol/L) inhibited
the cell proliferation. NPY (10(-7), 10(-9), 10(-11) mol/L) induced the
differentiation of hADSCs. The expression levels of related adipocyte markers
(PPARγ, C/EBPα), mature white adipose tissue specific markers (Cidec, RIP140)
increased at the presence of NPY (10(-7), 10(-9), 10(-11) mol/L).
CONCLUSION: Low-dose NPY could stimulate the proliferation of hADSCs, and
high-dose NPY could inhibit the proliferation and promote the differentiation of
hADSCs. OBJECTIVE: Cell death-inducing DFF45-like effector C (CIDEC) is a lipid
droplet-coating protein that promotes triglyceride accumulation and inhibits
lipolysis. TNF-α downregulates CIDEC levels to enhance basal lipolysis, whereas
CIDEC overexpression could block this effect. This study aimed to investigate
the signaling pathway of TNF-α-mediated CIDEC downregulation in human
adipocytes.
METHODS: First CIDEC expression was detected in adipose tissue of lean and human
subjects with obesity. Next, the temporal- and dose-dependent effects of TNF-α
on CIDEC expression in human SW872 adipocytes were investigated. Selective
inhibitors or RNAi or constitutively active MEK1 mutant was used to suppress or
stimulate MEK/ERK cascade. Immunofluorescence and subcellular fractionation
technique were used to study PPARγ redistribution after TNF-α treatment.
Reporter assay was performed to confirm the direct effects of TNF-α on CIDEC
transcription.
RESULTS: CIDEC expression decreased in adipose tissue of subjects with obesity
and negatively correlated with adipose TNF-α levels and systemic lipolysis.
TNF-α reduced CIDEC expression in vitro, but suppression of MEK/ERK cascade
prevented TNF-α-mediated CIDEC downregulation. PPARγ, the transcription factor
of CIDEC, was phosphorylated and redistributed by TNF-α in a MEK/ERK-dependent
manner. Reporter assay confirmed that TNF-α reduced CIDEC transcription.
CONCLUSIONS: TNF-α downregulates CIDEC expression through phosphorylation and
nuclear export of PPARγ by MEK/ERK cascade. |
Which is the relation between coffee consumption and stroke risk? | The coffee paradox in stroke: Increased consumption linked with fewer strokes. | Current evidence from experimental studies in animals and humans along with
findings from prospective studies indicates beneficial effects of green and
black tea as well as chocolate on cardiovascular health, and that tea and
chocolate consumption may reduce the risk of stroke. The strongest evidence
exists for beneficial effects of tea and cocoa on endothelial function, total
and LDL cholesterol (tea only), and insulin sensitivity (cocoa only). The
majority of prospective studies have reported a weak inverse association between
moderate consumption of coffee and risk of stroke. However, there are yet no
clear biological mechanisms whereby coffee might provide cardiovascular health
benefits. Awaiting the results from further long-term RCTs and prospective
studies, moderate consumption of filtered coffee, tea, and dark chocolate seems
prudent. |
What is the purpose of the Orpington Prognostic Scale? | The Orpington Prognostic Scale (OPS) is used to predict futue functional status of stroke patients, to asses stroke severity, outcome and response to subacute rehabilitation. In patients with stroke, OPS and NIHSS had significant contribution to the estimation of the functional status and OPS was more effective than NIHSS. However, other reported that the OPS has limited predictive accuracy for discharge destination and is a poor predictor of follow-up services. | OBJECTIVE: To determine the validity of clinically derived prognostic scores in
targeting stroke rehabilitation in elderly patients.
DESIGN, SETTING AND PARTICIPANTS: One-year prospective cohort study in 96
hospitalized stroke patients over 75 years of age from a well defined
geographical area.
MEASUREMENTS: Edinburgh prognostic score (incorporating measures of motor
deficit, proprioception, and power), Orpington prognostic score (Edinburgh score
modified to include a measure of cognition), and Barthel ADL scores were
measured at 1, 2, and 4 weeks after stroke. These scores were correlated with
outcome and patients' Barthel ADL score at discharge or at 16 weeks if still in
hospital.
RESULTS: Edinburgh prognostic score measured at 2 weeks correlated significantly
with Barthel ADL score at discharge or at 16 weeks (r2 = 0.57, P < 0.001), and
Orpington prognostic scores showed greater correlation (r2 = 0.89 vs 0.57),
especially in patients with dementia (r2 = 0.81 vs 0.39). Barthel ADL scores at
2 weeks showed a weak correlation with Barthel ADL scores at discharge or 16
weeks (r2 = 0.58). Patients with Orpington Score < 3.2 were discharged within 3
weeks of stroke, whereas those scoring > 5.2 required long-term care. Most
patients (90%) with Orpington Score of 3-5 were eventually discharged home
although this was not always apparent on initial clinical assessment at the time
of admission.
CONCLUSIONS: The Orpington score when assessed at 2-weeks post-stroke is a
useful prognostic indicator with special suitability for the elderly and may
help to select patients most likely to benefit from stroke unit rehabilitation. BACKGROUND AND PURPOSE: This study compared the ability of 2 stroke impairment
scales, Orpington Prognostic Scale and National Institutes of Health (NIH)
Stroke Scale, to predict disability as measured by the Barthel activities of
daily living (ADL) Index and higher level of self-reported physical functioning
as measured by the SF-36 physical functioning index (PFI) at 1, 3, and 6 months
after stroke.
METHODS: The participants in this ongoing study are 184 individuals who
sustained an eligible stroke and were recruited for the Kansas City Stroke
Study. All patients were prospectively evaluated using standardized assessments
at enrollment (within 14 days of stroke onset) and followed at 1, 3, and 6
months after stroke. Coefficient of determination (R2) was used to assess the
ability of the 2 stroke scales to prognosticate outcomes.
RESULTS: Means and SDs of the Orpington Prognostic Scale and NIH Stroke Scale
measured at baseline were 3.6+/-1.31 and 5.5+/-4.58, respectively. The
Spearman's rank correlation between the 2 baseline measures was 0.83 (P=0.0001).
The Orpington Prognostic Scale and the NIH Stroke Scale explained well the
variance in Barthel ADL Index (P<0.001). However, the Orpington Prognostic Scale
explained more variance than did the NIH Stroke Scale. Similarly, the Orpington
Prognostic Score explained more variance in higher level of physical function
than did the NIH Stroke Scale. The amount of variance in Barthel ADL Index and
SF-36 PFI, which were explained by both stroke severity measures, decreased over
time.
CONCLUSIONS: Our results demonstrate that in a sample of mostly mild and
moderate strokes, the Orpington Prognostic Scale compared with the NIH Stroke
Scale is simpler to use and is a slightly better predictor of ADL and higher
levels of physical function. OBJECTIVE: To provide recovery rates after stroke for specific functions using
the Orpington Prognostic Scale (OPS).
DESIGN: Prospective cohort.
SETTING: Hospital and community.
PARTICIPANTS: 413 stroke survivors entered the study 3 to 14 days after
suffering a stroke.
MEASUREMENTS: A cohort of hospitalized stroke survivors were recruited 3 to 14
days after stroke and assessed at 1, 3, and 6 months poststroke for
neurological, functional, and health status. Baseline OPS score was used to
predict five functional outcomes at 3 and 6 months using development and
validation datasets and receiver operating characteristic (ROC) curves.
RESULTS: In 413 stroke survivors, functional recovery rates at 3 and 6 months
were similar. Baseline OPS predicted significant differences in recovery rates
for all five outcomes (P < .0001 for all five outcomes at 3 and 6 months).
Personal care dependence was present at 3 months in only 3% of persons with
baseline OPS scores of 3.2 or less compared with over 50% with OPS of 4.8 or
higher. Independent personal care, meal preparation, and self-administration of
medication were achieved by 80% who had baseline OPS scores of 2.4 or lower
compared with less than 20% when OPS scores were 4.4 or higher. Independent
community mobility was achieved in 50% of those who had OPS scores of 2.4 or
lower but only 3% of those with OPS scores of 4.4 or higher. The area under ROC
curves assessing OPS scores against each of the five outcomes ranged from 0.805
to 0.863 at 3 months and 0.74 to 0.806 at 6 months.
CONCLUSION: OPS scores can predict widely differing rates of functional recovery
in five important functional abilities. These estimates can be useful to
survivors, families, providers, and healthcare systems who need to plan for the
future. OBJECTIVE: To evaluate the immediate and long-term effects of 2 upper-extremity
rehabilitation approaches for stroke compared with standard care in participants
stratified by stroke severity.
DESIGN: Nonblinded, randomized controlled trial (baseline, postintervention,
9mo) design.
SETTING: Inpatient rehabilitation hospital and outpatient clinic.
PARTICIPANTS: Sixty-four patients with recent stroke admitted for inpatient
rehabilitation were randomized within severity strata (Orpington Prognostic
Scale) into 1 of 3 intervention groups. Forty-four patients completed the
9-month follow-up.
INTERVENTIONS: Standard care (SC), functional task practice (FT), and strength
training (ST). The FT and ST groups received 20 additional hours of
upper-extremity therapy beyond standard care distributed over a 4- to 6-week
period.
MAIN OUTCOME MEASURES: Performance measures of impairment (Fugl-Meyer
Assessment), strength (isometric torque), and function (Functional Test of the
Hemiparetic Upper Extremity [FTHUE]).
RESULTS: Compared with SC participants, those in the FT and ST groups had
significantly greater increases in Fugl-Meyer motor scores (P=.04) and isometric
torque (P=.02) posttreatment. Treatment benefit was primarily in the less severe
participants, where improvement in FT and ST group Fugl-Meyer motor scores more
than doubled that of the SC group. Similar results were found for the FTHEU and
isometric torque. During the long term, at 9 months, the less severe FT group
continued to make gains in isometric muscle torque, significantly exceeding
those of the ST group (P<.05).
CONCLUSIONS: Task specificity and stroke severity are important factors for
rehabilitation of arm use in acute stroke. Twenty hours of upper
extremity-specific therapy over 4 to 6 weeks significantly affected functional
outcomes. The immediate benefits of a functional task approach were similar to
those of a resistance-strength approach, however, the former was more beneficial
in the long-term. In the present study the potential for functional improvement in 103 stroke
patients attending a geriatric day hospital was evaluated using the Orpington
Prognostic Scale (OPS). This information may assist in coordinating the
patients' and caregivers' expectations, allocating resources efficiently, and
advance care planning. The Spearman correlation was used to calculate the
association between OPS score and Functional Independent Measure (FIM), the
Nottingham Extended ADL Index (NEAI), and the timed Get Up and Go test (TUG).
The studied population was divided into two groups: patients with mild
neurological impairment (OPS score<3.2) and those with moderate to severe
neurological impairment (OPS score> or =3.2) and their performance on the above
outcome measure were compared. The OPS score significantly correlated (p<0.001)
with discharge scores of FIM (r=-0.730), NEAI (r=-0.675), and TUG (r=0.448).
Patients with OPS score <3.2 achieved significantly higher discharge scores
(p<0.001) in all three measures (FIM, TUG, and NEAI) compared to patients with
OPS score > or =3.2. Nevertheless, patients with moderate to severe neurological
impairment achieved greater score changes in FIM and TUG than patients with mild
neurological impairment, although their discharge scores were less favorable.
These results indicate that stroke patients even the more impaired, may
continually recover and should be exposed to a prolonged rehabilitation program. PURPOSE: To determine the inter-rater and test-retest reliability of the
Orpington Prognostic Scale (OPS) in patients with stroke. Pilot data were
gathered to evaluate its predictive validity for discharge destination and
therapeutic services required on discharge.
METHOD: Ninety-four consecutive patients, admitted to hospital due to stroke
participated. Pairs of physiotherapists (PT) and occupational therapists (OT)
assessed patients using the OPS on days 7 and 14 post stroke. For inter-rater
reliability, one rater performed the OPS while the other observed, each scoring
the scale independently. For test-retest reliability, two different raters
tested the subjects separately within the same day. Data were gathered on the
discharge destination and the number of follow-up services prescribed.
RESULTS: The inter-rater reliability as measured by the intraclass correlation
coefficient (ICC) was 0.99 (95% CI 0.97 - 0.99). For test-retest reliability,
the ICC was 0.95 (95% CI 0.90 - 0.98). The accuracy for predicting discharge to
home using OPS 5.0 was 65% (95% CI 0.52 - 0.76). OPS scores were not related to
number of follow-up services prescribed.
CONCLUSIONS: Despite high inter-rater and test-retest reliability, the OPS has
limited predictive accuracy for discharge destination and is a poor predictor of
follow-up services. PURPOSE: The aim of our study is to compare the Orpington Prognostic Scale (OPS)
and the National Institutes of Health Stroke Scale (NIHSS) and to evaluate
whether they help us estimate the future functional status of patients with
stroke.
METHOD: Twenty-five patients with stroke were administered the OPS and NIHSS on
the 7th day of stroke in order to define the severity of the disease, and the
Barthel Index was performed in order to evaluate the functional status and the
activities of daily living (ADL) at the 1st, 3rd, and 6th months.
RESULTS: Both scales were statistically correlated (P = 0.0001). When the
predictability of these scales in terms of the ADL and functional status was
evaluated, the regression coefficient at the 1st month was -14.746, R(2) = 0.58,
P < 0.0001 and -4.885, R(2) = 0.50, P < 0.0001 for OPS and NIHSS, respectively,
the same coefficient at the 3rd month was -12.482, R(2) = 0.41, P = 0.001 for
OPS and -3.280, R(2) = 0.23, P = 0.016 for NIHSS, and at the 6th month it was
-11.662, R(2) = 0.38, P = 0.001 for OPS and -2.997, R(2) = 0.20, P = 0.02 for
NIHSS.
CONCLUSION: In patients with stroke, OPS and NIHSS had significant contribution
to the estimation of the functional status and OPS was more effective than
NIHSS. PURPOSE: Prediction of outcome and response to rehabilitation in patients with
stroke can be difficult, especially in the elderly. The purpose of this study
was to determine the ability of the Orpington Prognostic Scale (OPS) to predict
outcome and response to subacute rehabilitation in older patients with stroke.
METHODS: Twenty-two subjects in the subacute care setting diagnosed with acute
stroke were prospectively studied. The OPS was scored within 2 weeks of stroke,
and the Functional Independence Measure (FIM) motor subscale was scored at
admission and discharge.
RESULTS: Strong Spearman correlations with OPS scores were found for improvement
in FIM score [rs = -.74, 95% CI: (-.88, -.45), p = .0007] and discharge FIM
score [rs = -.81, 95% CI: (-.92, -.58), p = .0002].
CONCLUSIONS: The OPS scores were strong predictors of response to subacute
rehabilitation and discharge FIM motor subscale scores. The OPS may warrant a
broader application as a prognostic indicator for patients with stroke. This study investigates the prognostic ability of the Orpington Prognostic Scale
within 48 hours (OPS-1) after admission in predicting outcome at 6 months and 2
years in acute ischemic stroke and compares it with the 2 week OPS (OPS-2). All
consecutive ischemic stroke patients (n = 117) were scored on the OPS, Barthel
activities of daily living, Oxford handicap scale, European stroke scale, and
Rivermead motor assessment at 48 hours, 2 weeks, 6 months, and 2 years
post-stroke. Baseline OPS scores at 48 hours and 2 weeks were used to predict
outcomes at 6 months and 2 years. The OPS-1 was an excellent predictor of length
of hospital stay (P < .001), place of discharge (P < .01), and outcome at 6
months and 2 years (P < .0001, Fisher's exact). The OPS-2 was marginally better
than the OPS-1 though this benefit was outweighed by the earlier stratification
of the 48-hour measure. The sensitivity, specificity, and positive predictive
values (PPV) of the "good" OPS-1 versus the OPS-2 at predicting independence at
6 months were 85% vs 92%, 85% vs 63% and 87% vs 92%, respectively. The
sensitivity, specificity, and PPV of the "poor" OPS-1 versus OPS-2 were 48% v
35%, 97% v 100%, and 93% v 100% respectively. The OPS at 48 hours is a good
predictor of outcome at 6 months and 2 years after ischemic stroke and allows
early identification of 3 prognostic groups, which may help in identifying
patients most likely to benefit from intensive rehabilitation. |
Do IEG create a ripple effect of transcription? | Rapid induction of immediate-early genes (IEGs) in response to growth factor stimulations is accompanied by co-upregulation of their neighbouring genes. Profiling the primary transcripts in the nucleus with whole-genome tiling arrays delineated simultaneous activation of transcription centred on IEGs. | Transcriptional initiation of each gene is assumed to be independently
controlled in mammals. On the other hand, recent large-scale transcriptome
analyses have shown that the genome is pervasively transcribed, such that the
most of its DNA gives rise to RNAs. This raises the question of whether it is
possible to pinpoint and activate a particular locus without perturbing numerous
neighbouring transcripts. Here we show that intensive transcription at one locus
frequently spills over into its physical neighbouring loci. Rapid induction of
immediate-early genes (IEGs) in response to growth factor stimulations is
accompanied by co-upregulation of their neighbouring genes. Profiling the
primary transcripts in the nucleus with whole-genome tiling arrays delineated
simultaneous activation of transcription centred on IEGs. Even in surrounding
intergenic regions, transcriptional activation took place at the same time.
Acetylation levels of histone H3 and H4 are elevated along with the IEG
induction and neighbouring co-upregulation. Inhibition of the mitogen-activated
protein kinase (MAPK) pathway or the transcription factor SRF suppresses all
transcriptional upregulation. These results suggest that transcriptional
activation has a ripple effect, which may be advantageous for coordinated
expression. |
Which R package is used for the analysis of genome-wide DNA methylation profiles? | MethylKit is a comprehensive R package for the analysis of genome-wide DNA methylation profiles. MethylKit includes functions for clustering, sample quality visualization, differential methylation analysis and annotation features, thus automating and simplifying many of the steps for discerning statistically significant bases or regions of DNA methylation. | |
Do T-Cells regulate neuropathic pain? | Macrophage-T cell interactions can mediate neuropathic pain through the glucocorticoid-induced TNF | A catastrophic consequence of peripheral nerve injury is the development of
abnormal, chronic neuropathic pain. The inflammatory response at the injury site
is believed to contribute to the generation and maintece of such persistent
pain. However, the physiological significance and potential contribution of T
cells to neuropathic pain remains unclear. Here we show that T cells infiltrate
injured sciatic nerves following chronic constriction injury (CCI), but not
uninjured nerves. Congenitally athymic nude rats, which lack mature T cells,
developed a significantly reduced mechanical allodynia and thermal hyperalgesia
following CCI, compared with their heterozygous littermates. To understand
further the role played by different T-cell subsets, we generated polarized
populations of type 1 and type 2 T cells, with different cytokine secretion
profiles, from spleens of sciatic nerve-injured heterozygous rats. Passive
transfer of type 1 T cells, which produce proinflammatory cytokines, into nude
rats enhanced the recipients' pain hypersensitivity to a level similar to that
of heterozygous donor rats. In contrast, passive transfer of polarized type 2 T
cells, which produce anti-inflammatory cytokines, into heterozygous rats
modestly though significantly attenuated their pain hypersensitivity. Thus,
injection of type 1 and type 2 T-cell subsets produces opposing effects on
neuropathic pain. These findings suggest the modulation of the T-cell immune
response as a potential target for the treatment of neuropathic pain. Triptolide is a potent immunosuppressive drug capable of inhibiting T cell
activation and proliferation. Recent studies show that T cells play an important
role in neuropathic pain following nerve injury in rats. In this study, we
investigated the effect of triptolide on T cell activation and development of
neuropathic pain. Neuropathic pain by chronic constriction injury (CCI) was
induced by loose ligation of the sciatic nerve in Sprague-Dawley rats.
Triptolide (5 or 10 μg/kg) or vehicle (DMSO) was administered intrathecally
after surgery for 7 days (n=8 per group). The right hind paw withdrawal
threshold to von Frey filament stimuli and withdrawal latency to radiant heat
were determined before and after the surgery (days 0 to 7). NF-κB activation and
pro-inflammatory cytokine (TNF-α and IL-2) expression were determined by ELISA,
Western blot, and real time-PCR. CCI of the sciatic nerve induced mechanical
allodynia and thermal hyperalgesia in these rats. Intrathecal triptolide (5 and
10 μg/kg) suppressed the development of allodynia and thermal hyperalgesia. It
also inhibited CCI-induced inflammation and T cell activation, by decreasing
spinal cord TNF-α, IL-2 and NF-κB p65 levels. Motor dysfunction was not observed
after triptolide treatment. In the present study, we demonstrated the
suppressive effect of triptolide on the development of neuropathic pain.
Therefore, triptolide could be a promising immunosuppressive agent in the
treatment of neuropathic pain. Further studies are required to examine the
safety of intrathecal triptolide for clinical application. Microglia, which constitute the resident macrophages of the central nervous
system (CNS), are generally considered as the primary immune cells in the brain
and spinal cord. Microglial cells respond to various factors which are produced
following nerve injury of multiple aetiologies and contribute to the development
of neuronal disease. Chemokine (C-C motif) ligand 1 (CCL-1), a
well-characterized chemokine secreted by activated T cells, has been shown to
play an important role in neuropathic pain induced by nerve injury and is also
produced in various cell types in the CNS, especially in dorsal root ganglia
(DRG). However, the role of CCL-1 in the CNS and the effects on microglia
remains unclear. Here we showed the multiple effects of CCL-1 on microglia. We
first showed that CCR-8, a specific receptor for CCL-1, was expressed on primary
cultured microglia, as well as on astrocytes and neurons, and was upregulated in
the presence of CCL-1. CCL-1 at concentration of 1 ng/ml induced chemotaxis,
increased motility at a higher concentration (100 ng/ml), and increased
proliferation and phagocytosis of cultured microglia. CCL-1 also activated
microglia morphologically, promoted mRNA levels for brain-derived neurotrophic
factor (BDNF) and IL-6, and increased the release of nitrite from microglia.
These indicate that CCL-1 has a role as a mediator in neuron-glia interaction,
which may contribute to the development of neurological diseases, especially in
neuropathic pain. There is increasing evidence that CD4(+) T-cell-dependent responses are
associated with the maintece of neuropathic pain. However, little is known
about the precise mechanism(s) underlying the activation of CD4(+) T-cells. We
herein show that inhibition of cathepsin S (CatS) activity, either through
genetic deletion or via a pharmacological inhibitor, Z-Phe-Leu-COCHO (Z-FL),
significantly attenuated the maintece of tactile allodynia, splenic
hypertrophy, increased number of splenic CD4(+) T-cells and the final cleavage
step of the MHC class II-associated invariant chain following peripheral nerve
injury. It was also noted that splenectomy significantly attenuated the
peripheral nerve injury-induced tactile allodynia, whereas the adoptive transfer
of splenic CD4(+) T-cells from neuropathic wild-type mice significantly
increased the pain level of splenectomized wild-type or CatS(-/-) mice.
Furthermore, CatS deficiency or Z-FL treatment also significantly inhibited the
infiltration of CD4(+) T-cells that expressed interferon-γ (IFN-γ) in the dorsal
spinal cord. Signal transducer and activator of transcription 1, a molecule
downstream of IFN-γ receptor activation, was activated exclusively in microglia
7 d after peripheral nerve injury. Moreover, CatS deficiency, Z-FL treatment, or
splenectomy significantly attenuated the proliferation of microglia 14 d after
peripheral nerve injury. These results show a peripheral pivotal role of CatS in
the development of neuropathic pain through the antigen-specific activation of
CD4(+) T-cells. After activation, CD4(+) T-cells infiltrate into the dorsal
spinal cord and secrete IFN-γ to reactivate microglia, which contribute to the
transition of acute pain to a chronic pain state. BACKGROUND: The classification of pain into nociceptive and neuropathic pain is
based on characteristic symptoms and different pathophysiological mechanisms. In
a recent investigation, we found a disrupted TH17/Treg balance in patients
suffering from chronic unspecific low back pain (CLBP). These patients did not
show any signs of neuropathy. There is evidence for a considerable impact of the
immune system also in neuropathic pain. However, the role of the adaptive immune
system is still unclear. In the present study, we investigated systemic T-cell
subset responses and T-cell related cytokine profiles in patients with chronic
neuropathic pain.
METHODS: We analyzed T-cell subsets, mRNA expression and T-cell-related cytokine
profiles in 26 patients suffering from neuropathic pain in comparison to 26
healthy controls. Using multicolor flow cytometry (FACS), we quantified the
number of T helper cells 1 (TH1), TH2, TH17 and regulatory T-cells (Tregs).
Forkhead-Box-Protein 3 (FoxP3), Transforming growth factor-β (TGF-β) and
RAR-related orphan receptor-γT (ROR-γT) mRNA expression was determined by
quantitative real-time PCR (qPCR) and levels of pain-related cytokines were
measured by Human Cytokine Multiplex Immunoassay (Macrophage inflammatory
protein-1α (MIP-1α), Tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ),
Interleukin (IL) -4, IL-6, IL-10, IL-17, and IL-23).
RESULTS: We found a TH17/Treg imbalance with significantly increased
anti-inflammatory Tregs and decreased pro-inflammatory TH17 cells in patients
with neuropathic pain as compared to healthy controls. These results were
confirmed on mRNA level: Treg-related FoxP3 and TGF-β mRNA expression was
elevated, whereas expression of TH17-related RORγT was reduced. Cytokine
analyses revealed only marginal changes.
CONCLUSIONS: Our investigation revealed a clear shift of T-cell subsets towards
anti-inflammation in patients with neuropathic pain. Interestingly, this is
quite similar to our previous findings in CLBP patients, but even more
pronounced. Therefore, it remains to be elucidated in future investigations
whether the immune changes represent an underlying pathophysiological mechanism
or an epiphenomenon induced by ongoing pain and stress.
GERMAN CLINICAL TRIAL REGISTER (DRKS): Trial registration number: DRKS00005954. |
What is the incidence of new cases of X-linked adrenoleukodystrophy (ALD) in Australian and New Zealand in the late 1990's? | cases of ALD diagnosed in Australia and New Zealand between 1981 and 1996 and their families. We estimate that the combined incidence of ALD and its variants in Australasia is at least 1.6 per 100,000. | |
Can telomere length shortening be reversed by telomerase? | Yes, telomerase gene therapy rescues telomere length, bone marrow aplasia, and survival in mice with aplastic anemia. | Aplastic anemia is a fatal bone marrow disorder characterized by peripheral
pancytopenia and marrow hypoplasia. The disease can be hereditary or acquired
and develops at any stage of life. A subgroup of the inherited form is caused by
replicative impairment of hematopoietic stem and progenitor cells due to very
short telomeres as a result of mutations in telomerase and other telomere
components. Abnormal telomere shortening is also described in cases of acquired
aplastic anemia, most likely secondary to increased turnover of bone marrow stem
and progenitor cells. Here, we test the therapeutic efficacy of telomerase
activation by using adeno-associated virus (AAV)9 gene therapy vectors carrying
the telomerase Tert gene in 2 independent mouse models of aplastic anemia due to
short telomeres (Trf1- and Tert-deficient mice). We find that a high dose of
AAV9-Tert targets the bone marrow compartment, including hematopoietic stem
cells. AAV9-Tert treatment after telomere attrition in bone marrow cells rescues
aplastic anemia and mouse survival compared with mice treated with the empty
vector. Improved survival is associated with a significant increase in telomere
length in peripheral blood and bone marrow cells, as well as improved blood
counts. These findings indicate that telomerase gene therapy represents a novel
therapeutic strategy to treat aplastic anemia provoked or associated with short
telomeres. Telomeres progressively shorten throughout life. A hallmark of advanced
maligcies is the ability for continuous cell divisions that almost
universally correlates with the stabilization of telomere length by the
reactivation of telomerase. The repression of telomerase and shorter telomeres
in humans may have evolved, in part, as an anticancer protection mechanism.
Although there is still much we do not understand about the regulation of
telomerase, it remains a very attractive and novel target for cancer
therapeutics. This review focuses on the current state of advances in the
telomerase area, identifies outstanding questions, and addresses areas and
methods that need refinement.
SIGNIFICANCE: Despite many recent advances, telomerase remains a challenging
target for cancer therapy. There are few telomerase-directed therapies, and many
of the assays used to measure telomeres and telomerase have serious limitations.
This review provides an overview of the current state of the field and how
recent advances could affect future research and treatment approaches. Cancer
Discov; 6(6); 584-93. ©2016 AACR. Telomerase-mediated telomere elongation provides cell populations with the
ability to proliferate indefinitely. Telomerase is capable of recognizing and
extending the shortest telomeres in cells; nevertheless, how this mechanism is
executed remains unclear. Here, we show that, in the fission yeast
Schizosaccharomyces pombe, shortened telomeres are highly transcribed into the
evolutionarily conserved long noncoding RNA TERRA A fraction of TERRA produced
upon telomere shortening is polyadenylated and largely devoid of telomeric
repeats, and furthermore, telomerase physically interacts with this
polyadenylated TERRA in vivo We also show that experimentally enhanced
transcription of a manipulated telomere promotes its association with telomerase
and concomitant elongation. Our data represent the first direct evidence that
TERRA stimulates telomerase recruitment and activity at chromosome ends in an
organism with human-like telomeres. Telomere length is regulated around an equilibrium set point. Telomeres shorten
during replication and are lengthened by telomerase. Disruption of the length
equilibrium leads to disease; thus, it is important to understand the mechanisms
that regulate length at the molecular level. The prevailing protein-counting
model for regulating telomerase access to elongate the telomere does not explain
accumulating evidence of a role of DNA replication in telomere length
regulation. Here I present an alternative model: the replication fork model that
can explain how passage of a replication fork and regulation of origin firing
affect telomere length. High telomerase activity is detected in nearly all human cancers but most human
cells are devoid of telomerase activity. There is well-documented evidence that
reactivation of telomerase occurs during cellular transformation. In humans,
tumors can rely in reactivation of telomerase or originate in a telomerase
positive stem/progenitor cell, or rely in alternative lengthening of telomeres,
a telomerase-independent telomere-length maintece mechanism. In this review,
we will focus on the telomerase positive tumors. In this context, the recent
findings that telomerase reverse transcriptase (TERT) promoter mutations
represent the most common non-coding mutations in human cancer have flared up
the long-standing discussion whether cancer originates from telomerase positive
stem cells or telomerase reactivation is a final step in cellular
transformation. Here, we will discuss the pros and cons of both concepts in the
context of telomere length-dependent and telomere length-independent functions
of telomerase. Together, these observations may provoke a re-evaluation of
telomere and telomerase based therapies, both in telomerase inhibition for
cancer therapy and telomerase activation for tissue regeneration and anti-ageing
strategies. |
Is ABCE1 involved in ribosomal recycling? | Yes, recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea | To study the function of Rli1/ABCE1 in vivo, we used ribosome profiling and
biochemistry to characterize its contribution to ribosome recycling. When Rli1
levels were diminished, 80S ribosomes accumulated both at stop codons and in the
adjoining 3'UTRs of most mRNAs. Frequently, these ribosomes reinitiated
translation without the need for a canonical start codon, as small peptide
products predicted by 3'UTR ribosome occupancy in all three reading frames were
confirmed by western analysis and mass spectrometry. Eliminating the
ribosome-rescue factor Dom34 dramatically increased 3'UTR ribosome occupancy in
Rli1 depleted cells, indicating that Dom34 clears the bulk of unrecycled
ribosomes. Thus, Rli1 is crucial for ribosome recycling in vivo and controls
ribosome homeostasis. 3'UTR translation occurs in wild-type cells as well, and
observations of elevated 3'UTR ribosomes during stress suggest that modulating
recycling and reinitiation is involved in responding to environmental changes. Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1
can be considered as the final-or the first-step within the cyclic process of
protein synthesis, connecting translation termination and mRNA surveillance with
re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding
domains in ABCE1 transfers mechanical energy to the ribosome and tears the
ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA
translation. Here, we probed the so far unknown architecture of the 1-MDa PRC
(40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our
study reveals ABCE1 bound to the translational factor-binding (GTPase) site with
multiple cross-link contacts of the helix-loop-helix motif to the S24e ribosomal
protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12
substantiates an extreme lever-arm movement of the FeS cluster domain during
ribosome recycling. We were thus able to reconstitute and structurally analyse a
key complex in the translational cycle, resembling the link between translation
initiation and ribosome recycling. |
What are clinical features of the de Morsier syndrome? | Classic triad of the De Morsier syndrome (septooptic dysplasia) includes optic nerve hypoplasia, the absence of septum pellucidum, and pituitary hypoplasia. | A 12-year-old girl and a 30-year-old woman had bilateral optic disk hypoplasia
and bitemporal hemianopia. By using computed axial tomography on our patients,
we demonstrated the absence of the septum pellucidum, which confirmed the
diagnosis of septo-optic dysplasia, or the de Morsier syndrome. The de Morsier syndrome, or septo-optic dysplasia, is a developmental anomaly
characterized by involvement of the optic system, hypothalamic-pituitary axis
and septum pellucidum. Only a few anatomical observations are recorded. We
report three new cases and review the pertinent literature. The
neuropathological lesions varied as did the clinical features. The hypothalamic
nuclei were most commonly involved, followed by the optic system and the septum
pellucidum. Other lesions were found in the cerebral cortex, corpus callosum,
olfactory system and cerebellum. The hypopituitarism appeared to have been
secondary to hypothalamic damage rather than to intrinsic pituitary defect. A
virtually normal histology and the usual endocrine cell populations were
demonstrated by immunocytochemistry in the adenohypophysis. Damage to the
neurophysin-containing cells of the hypothalamus explains the various degrees of
clinically observed diabetes insipidus. The association of optic nerve hypoplasia and deficiency of the septum
pellucidum and hypopituitarism is known as the De Morsier syndrome or
septo-optic dysplasia. Four children with blindness and growth retardation due
to growth hormone deficiency are described. Frequent recurrent episodes of
severe hypoglycaemia, starting in the newborn period, were observed in three of
them. Although visual impairment was noted during the first year and growth
failure was detected between the first and sixth year of life in all four
patients, the diagnosis of septo-optic dysplasia was delayed for two to six
years. Early diagnosis and treatment with growth hormone should prevent attacks
of hypoglycaemia and their sequelae and bring about normal growth. Septo-optic dysplasia (or de Morsier syndrome) is a congenital disorder
characterised by anomalies in cerebral midline structures, optic nerve
hypoplasia, and hormonal deficiencies. Diagnosis should be made early, due to
the possibility of treating the hormonal disturbances. We describe here a case
with decreased visual acuity, one-sided hemianopia, nystagmus und agenesis of
the septum pellucidum and discuss the heterogeneous appearance of this syndrome.
There are two theories regarding its pathogenesis. The first postulates
simultaneous damage to both cerebral structures and optic nerve development
around the 6th week of gestation, while the other favours secondary degeneration
of optic nerve fibres due to a cerebral lesion. Septo-optic dysplasia (De Morsier syndrome) is a developmental anomaly of
mid-line brain structures and includes optic nerve hypoplasia, absence of the
septum pellucidum and hypothalamo-pituitary abnormalities. We describe seven
patients (four female, three male) who had at least two out of the three
features necessary for the diagnosis of septo-optic dysplasia. Four patients had
hypopituitarism and yet normal gonadotrophin secretion: one of these also had
anti-diuretic hormone insufficiency; three had isolated GH deficiency and yet
had premature puberty, with the onset of puberty at least a year earlier than
would have been expected for their bone age. In any progressive and evolving
anterior pituitary lesion it is extremely unusual to lose
corticotrophin-releasing hormone/ACTH and TRH/TSH secretion and yet to retain
gonadotrophin secretion. GnRH neurons develop in the nasal mucosa and migrate to
the hypothalamus in early fetal life. We hypothesise that the arrival of GnRH
neurons in the hypothalamus after the development of a midline hypothalamic
defect may explain these phenomena. Progress in spontaneous/premature puberty in
children with De Morsier syndrome may have important implications for
management. The combination of GH deficiency and premature puberty may allow an
apparently normal growth rate but with an inappropriately advanced bone age
resulting in impaired final stature. GnRH analogues may be a therapeutic option.
In conclusion, some patients with De Morsier syndrome appear to retain the
ability to secrete gonadotrophins in the face of loss of other hypothalamic
releasing factors. The migration of GnRH neurons after the development of the
midline defect may be an explanation. de Morsier syndrome, or septo-optic dysplasia, is a developmental malformation
complex characterized by optic nerve hypoplasia, dysgenesis of the septum
pellucidum, and hypothalamic-pituitary dysfunction. (1,2) In Duane retraction
syndrome, there is absence of the sixth nerve nucleus with congenital retraction
of the globe and narrowing of the lid fissure in adduction, frequent abduction
deficiency, and variable limitation to adduction of the affected eye. (3) The
purpose of this report is to present a patient with the uncommon and previously
unreported concurrence of both of these congenital malformation complexes,
presumably because of a common disturbance of neuronal development. INTRODUCTION: Septo optic syndrome, described by De Morsier in 1956, consists in
the hypoplasia of one or both optic nerves, mid line brain malformations and
hypothalamohypophysial dysfunction, which is inconstant. It is an infrequent,
but treatable, cause of hepatic and neurological damage, and it is important to
obtain an early diagnosis and to begin hormone replacement therapy.
CASE REPORT: We report the clinical case of a female baby who was diagnosed
early on as suffering from septo?optic dysplasia, after discovery of the
existence of cholestatic jaundice. In our case the three components of the
syndrome were present: hypothalamohypophysial dysfunction, bilateral hypoplasia
of the optic nerves and brain malformations with dysplasia of the transparent
septum. All this gives rise to complex clinical features and the predomice of
hypernatraemic dehydration secondary to insipid diabetes, nystagmus and serious
psychomotor retardation. Our patient died, as in other cases reported in the
literature, from an episode of sudden death.
DISCUSSION: Despite the importance of an early diagnosis of this disorder, it is
usually late. Most children who present hypopituitarism traits in the neonatal
period are not diagnosed at that time, with the subsequent risk of death or
brain damage. Some clinical findings, which appear early on and can provide
clues which aid us to reach a diagnosis, are the appearance of episodes of
hypoglycaemia in the neonatal period, the existence of micropenis and
cryptorchidism with hypoplasic testes, jaundice or the appearance of clinical
manifestations of insipid diabetes. Later on nystagmus and neurological symptoms
may appear. The final diagnosis is performed through the use of neuroimaging
techniques (CT or MRI) and hormonal studies. The term septooptic dysplasia was coined in 1956 by de Morsier, who pointed out
the association of optic nerve hypoplasia and absence of the septum pellucidum.
Patients with this condition may present with clinical features of
hypopituitarism, decreased visual acuity and neurodevelopmental disabilities
that lead to this diagnosis. The case that is presented here is unusual in that
this patient was initially diagnosed as having low tension glaucoma during a
routine screening examination and was treated for glaucoma for over a year
before he was discovered to have septooptic dysplasia, also known as de
Morsier's syndrome. INTRODUCTION: Septo-optic dysplasia (De Morsier syndrome) is defined as the
association between optic nerve hypoplasia, midline central nervous system
malformations and pituitary dysfunction.
CASE REPORT: Third child born to nonconsanguineous parents, female, adequate
pre-natal medical care, cesarean term delivery due to breech presentation, Apgar
score 3 at the first minute and 8 at 5 minutes, symptomatic hypoglycemia at 18
hours. Neurological follow-up identified a delay in acquisition of motor and
language developmental milestones. Epileptic generalized seizures began at 12
months and were controlled with phenobarbital. EEG was normal. MRI revealed
agenesis of the pituitary stalk, hypoplasia of the optic chiasm and
periventricular nodular heterotopia. Ophthalmologic evaluation showed bilateral
optic disk hypoplasia. Endocrine function laboratory tests revealed primary
hypothyroidism and hyperprolactinemia.
CONCLUSION: The relevance of this case report relies on its uniqueness, since
periventricular heterotopia had not been described in association with
septo-optic dysplasia until 2006. A 12-year-old boy presented with poor vision and nystagmus. Fundus examination
revealed bilateral optic nerve hypoplasia. An MRI of the brain demonstrated the
absence of the septum pellucidum, which confirmed a diagnosis of septo-optic
dysplasia or de Morsier syndrome. Septo-optic dysplasia, also known as de Morsier syndrome, is a rare congenital
entity almost always characterized by hypoplasia/dysplasia of the optical nerve,
chiasma or optic radiations and the complete or partial absence of the septum
pellucidum. It may also be accompanied by other malformations, including
multiple facial dysmorphism, midline defects, cleft lip and palate,
musculoskeletal and other non-neurological eye features. Various cases have been
reported which have presented various combinations of symptoms and stigmata of
the syndrome. We here present a unique case of septo-optic dysplasia with
familial repetition, a considerably early antenatal diagnosis and an
accompanying omphalocele, a feature never before connected with the syndrome. Septo-optic dysplasia (SOD), also referred to as de Morsier syndrome, is a rare
congenital condition, characterized by two of the classic triad features:
midline brain abnormalities, optic nerve hypoplasia (ONH) and pituitary
endocrine dysfunction. We report 5 children with SOD, originally referred to be
evaluated due to short stature, who also presented bilateral optic nerve
hypoplasia, nystagmus and development delay. In 4 of the patients, we identified
neuroimaging abnormalities of the hypothalamo-pituitary axis such as anterior
pituitary hypoplasia (3/5), ectopic posterior pituitary (4/5), thin or absent
stalk (3/5) and empty sella (1/5). We also encountered diverse pituitary
deficiencies: growth hormone (3/5), adrenocorticotropic hormone (3/5),
thyroid-stimulating hormone (2/5) and antidiuretic hormone (1/5). Only one child
presented intact pituitary function and anatomy. Although rare, SOD is an
important cause of congenital hypopituitarism and it should be considered in
children with optic nerve hypoplasia or midline brain abnormalities for early
diagnosis and treatment. BACKGROUND: Optic nerve hypoplasia (ONH) has been described as an increasingly
prevalent cause of congenital blindness. Its association with hypopituitarism
and absent septum pellucidum has been recognized for more than 40 years as
"septo-optic dysplasia" or "de Morsier syndrome." More recent studies have
suggested that these associations are independent of one another. This review
was designed to assess the historical and recent evidence for associations of
neuroradiologic, endocrinologic, and developmental problems in patients with
ONH.
EVIDENCE ACQUISITION: Historical and contemporary literature review.
RESULTS: The medical literature does not support the notion that Georges de
Morsier ever described a case of ONH or recognized its association with
hypopituitarism or missing septum pellucidum. Recognition of the critical
association of ONH with hypopituitarism should be attributed to William Hoyt.
Hypopituitarism and other more recently identified associations with ONH, such
as developmental delay, hypothalamic dysfunction, and autism, are independent of
septum pellucidum development. Other common neuroradiographic associations, such
as corpus callosum hypoplasia, gyrus dysplasia, and cortical heterotopia, may
have prognostic significance.
CONCLUSIONS: Children with ONH need to be monitored for many systemic,
developmental, and even life-threatening problems independent of the status of
the septum pellucidum. "Septo-optic dysplasia" and "de Morsier syndrome" are
historically inaccurate and clinically misleading terms that should be
abandoned. BACKGROUND: Optic nerve hypoplasia (ONH) has developed into a leading cause of
congenital blindness. The frequently associated features of hypopituitarism and
absent septum pellucidum were felt to have embryonic linkage as "septo-optic
dysplasia" or "de Morsier's syndrome." More recent studies have suggested these
associations are independent of one another. This review provides an assessment
of the historical and recent evidence linking neuroradiologic, endocrinologic
and developmental morbidity in patients with ONH. The prenatal risk factors,
heritability, and genetic mutations associated with ONH are described.
RESULTS: Recognition of the critical association of ONH with hypopituitarism
should be attributed to William Hoyt, not Georges de Morsier. De Morsier never
described a case of ONH or recognized its association with hypopituitarism or
missing septum pellucidum. Hypopituitarism is caused by hypothalamic
dysfunction. This, and other more recently identified associations with ONH,
such as developmental delay and autism, are independent of septum pellucidum
development. Other common neuroradiographic associations such as corpus callosum
hypoplasia, gyrus dysplasia, and cortical heterotopia may have prognostic
significance. The predomit prenatal risk factors for ONH are primiparity and
young maternal age. Presumed risk factors such as prenatal exposure to drugs and
alcohol are not supported by scrutiny of the literature. Heritability and
identified gene mutations in cases of ONH are rare.
CONCLUSION: Children with ONH require monitoring for many systemic,
developmental, and even life-threatening problems independent of the severity of
ONH and presence of brain malformations including abnormalities of the septum
pellucidum. "Septo-optic dysplasia" and "de Morsier's syndrome" are historically
inaccurate and clinically misleading terms. BACKGROUND: Septo-optic dysplasia (SOD), also known as de-Morsier's syndrome, is
a rare disorder characterized by any combination of optic nerve hypoplasia
(ONH), pituitary gland hypoplasia, and midline abnormalities of the brain
including absence of septum pellucidum and corpus callosum dysgenesis. It is
typically diagnosed in infancy and has a variable presentation that includes
visual, neurologic, and/or hypothalamic-pituitary endocrine deficits.
PURPOSE: To demonstrate the ophthalmic, endocrine, and neurologic spectrum of
SOD in five Omani children and address the crucial role of high-resolution
neuroimaging for its early and accurate diagnosis.
MATERIALS AND METHODS: A retrospective chart review was performed in 2010 of all
children in the pediatric ophthalmology database of Sultan Qaboos University
Hospital (SQUH) who were diagnosed to have ONH. All relevantdemographic,
ophthalmic, neurologic, endocrine, and neuro-radiological manifestations were
recorded in a data collection form. All previous neuroimaging results were
reviewed by a neuro-radiologist.
RESULTS: Five patients (four males, one female) with the diagnosis of ONH were
included in the study. They presented during the period 1998-2008. All patients
were born at term, with normal birth weights to healthy mothers with
insignificant antenatal history. Age at presentation ranged from three months to
one year. Manifestations at presentation included severe visual impairment
(5/5), neonatal hypoglycemia (3/5), seizure disorder (2/5), and failure to
thrive (4/5). ONH was bilateral in 3/5 patients and unilateral in (2/5). Brain
and orbit imaging revealed varying anomalies in all patients. These included
absent septum pellucidum (3/5), severe corpus callosum agenesis (1/5), ectopic
pituitary (5/5), falx cerebri deficiency (1/5), optic nerve hypoplasia (5/5),
optic chiasmal hypoplasia (5/5), and olfactory tract hypoplasia (1/5). Endocrine
deficits were detected in 4/5 patients (3 with panhypopituitarism, and 1 with
growth hormone deficiency) and necessitated replacement therapy.
CONCLUSION: SOD is a clinically heterogeneous disorder with a wide spectrum of
ophthalmic, endocrine, and neurologic manifestations. All features might not be
present in a single patient. A high consanguinity rate and lack of history of
alcohol and drug use were observed in our cohort. Most affected children present
first to the pediatrician with failure to thrive. Radiological confirmation of
ONH necessitates high-resolution imaging and interpretation by an experienced
neuro-radiologist. In our cohort, all patients with ONH had associated optic
chiasmal hypoplasia. Early detection and treatment reduces disease-related
morbidity, and can be life saving. BACKGROUND: Septo-optic dysplasia, also referred to as de Morsier syndrome, is a
congenital condition characterized by classic triad features: midline brain
abnormalities, optic nerve hypoplasia and pituitary endocrine dysfunction.
Sometimes, other various malformations appear within syndrome.
CASE PRESENTATION: An 11 and 1/2-year-old Caucasian Southeast European female
patient with earlier established diagnoses of growth hormone deficiency,
diabetes insipidus, seizures, mental retardation, optic nerve atrophy and right
ptosis, was directed to us for consultative examination.The girl of short
stature and low weight for her age had bilateral optic nerve hypoplasia, poor
vision, nystagmus and right eye oculomotor palsy. Electroencephalogram revealed
epileptic changes. Magnetic resoce imaging showed an empty sella syndrome,
partial hypoplasia of corpus callosum, cavum of pellucid septum and diffuse
polymicrogyria of the left temporal lobe. We found all elements of septo-optic
dysplasia plus syndrome with right oculomotor nerve involvement.
CONCLUSION: By earlier findings and evaluation, we established a diagnosis of
septo-optic dysplasia plus. The case confirms the existence of various
malformations within the syndrome and the need for the cooperation of several
specialists in the diagnosis and treatment of children with the syndrome. INTRODUCTION: Previous studies have described septooptic dysplasia (SOD) to
describe patients who have optic nerve hypoplasia, the absence of septum
pellucidum, and pituitary hypoplasia. Other rare ophthalmic associations have
been described, such as low-tension glaucoma. We report the ocular findings of a
patient with SOD who had high intraocular pressure (IOP) and glaucoma as a part
of the syndrome.
OBJECTIVES: To report the ocular findings in a Puerto Rican patient with SOD and
increased IOP.
PATIENTS AND METHODS: A patient with De Morsier syndrome underwent a
comprehensive eye examination, Humphrey visual fields, and Stratus optical
coherence tomography, and was referred for neuroradiologic examination. The
patient had increased IOP, visual field loss, and asymmetric optic nerve
hypoplasia. The IOP was lowered with topical hypotensive medications.
CONCLUSIONS: The patient with the De Morsier syndrome had poor visual acuity,
high IOP, visual field, and optical coherence tomography results that were all
compatible with glaucoma. Further studies comparing ocular findings in patients
with several mutations leading to De Morsier syndrome are warranted. To our
knowledge, this is the first report on a patient with glaucoma as a part of the
syndrome. |
Does oculocutaneous albinism show an autosomal recessive inheritance? | Yes, oculocutaneous albinism shows an autosomal recessive inheritance. | The segregation of brown (type IV) oculocutaneous albinism was analyzed in 18
Nigerian families. Analysis using the POINTER program showed that this type of
oculocutaneous albinism was inherited in an autosomal recessive pattern, with an
estimated gene frequency of 0.025 +/- 0.007 in this population. The enzyme
defect responsible for brown oculocutaneous albinism is unknown. We report on 2 related children, a boy and a girl, from a large Turkish clan.
Their parents are both first cousins and have several common ancestors. Both
children have tyrosinase-positive oculocutaneous albinism, recurrent bacterial
infections, granulocytopenia, intermittent thrombopenia, and microcephaly, a
protruding midface, rough and projecting hair, and mild mental retardation.
Chromosomes are normal. Metabolic disorders were excluded. None of 14 well-known
types of albinism, including Hermansky-Pudlak syndrome and Chediak-Higashi
syndrome, nor any other genetic syndrome, characterizes our patients
sufficiently. Thus, this combination of symptoms is considered a new autosomal
recessive syndrome. PURPOSE: The purpose of this study is to describe the heterogeneous phenotype of
individuals with an unusual type of albinism--minimal pigment oculocutaneous
albinism.
METHODS: Nine patients with minimal pigment oculocutaneous albinism were
identified and followed for up to 11 years. The criteria were the presence of
oculocutaneous albinism in association with low hairbulb tyrosinase activity in
the patient and disparate activity in the parents with one parent having normal
activity and the other having low tyrosinase activity. Changes in skin, hair,
and ocular pigment were followed as the patients matured. As a measure of ocular
pigment, iris transillumination and macular transparency were graded according
to a previously published scheme.
RESULTS: Patients were born with white scalp hair and skin, and nystagmus
developed. Visual acuity was reduced to 20/50 to 20/200 for the group, but in
one patient vision improved with maturity. Irides were blue. In seven patients,
iris pigment developed, which was detected by transillumination with slit-lamp
biomicroscopy, including the one patient with improved visual acuity. All
patients had foveal hypoplasia, and melanin pigment in the fundi could not be
detected by clinical examination. Visual acuity in the group did not correlate
directly with the presence or development of iris transillumination or macular
transparency. The pedigrees were consistent with an autosomal recessive
inheritance pattern.
CONCLUSION: This unique type of oculocutaneous albinism has heterogeneous
clinical features. Minimal pigment oculocutaneous albinism appears to represent
a new type of tyrosinase-related oculocutaneous albinism (OCA1MP). BACKGROUND: Type II (tyrosinase-positive) oculocutaneous albinism is an
autosomal recessive disorder that has recently been mapped to chromosome segment
15q11-q13. The frequency of this disorder is greatly increased in patients with
Prader-Willi or Angelman syndrome, both of which involve deletions of chromosome
15q. The P protein is a transmembrane polypeptide that may transport small
molecules such as tyrosine, the precursor of melanin. The P gene is located in
chromosome segment 15q11-q13.
METHODS: We studied the tyrosinase and P genes in three patients with type II
oculocutaneous albinism, one of whom also had Prader-Willi syndrome, and in one
patient with a milder syndrome known as autosomal recessive ocular albinism.
Individual exons of these genes were amplified from the DNA of each patient by
the polymerase chain reaction and screened for mutations by simultaneous
analyses of single-stranded conformation polymorphisms and heteroduplexes and
subsequent DNA sequencing.
RESULTS: Mutations of the P gene were identified in all four patients. These
included one frame shift, three missense mutations that result in amino acid
substitutions, and one mutation that affects RNA splicing. The patient with
Prader-Willi syndrome plus albinism had a typical deletion of the paternal
chromosome 15, rendering him hemizygous for a maternally inherited mutant allele
of the P gene. The child with ocular albinism was heterozygous for two different
mutations in the P gene.
CONCLUSIONS: Abnormalities of the P gene are associated with a wide range of
clinical phenotypes, including type II oculocutaneous albinism, albinism
associated with the Prader-Willi syndrome, and at least some cases of autosomal
recessive ocular albinism. Oculocutaneous albinism is an autosomal recessive genetic disorder. Several
types of oculocutaneous albinism are caused by mutation in related genes.
Oculocutaneous albinism 1 is associated with the tyrosinase gene. The human
tyrosinase gene (TYR) encodes tyrosinase, a key enzyme in melanin biosynthesis.
As exon 1 of the gene shows an MboI-RFLP within codon 192 in Caucasians, we
studied allele frequencies of MboI 192 polymorphism in 200 chromosomes from 100
unrelated normal Korean individuals. As a result, only one allele system, the
presence of the MboI 192 site, was detected in the Korean. A sister and brother, with oculocutaneous albinism and reduced bone density are
described. Autosomal recessive inheritance is possible. This association has not
been previously described. BACKGROUND: Oculocutaneous albinism type II (OCA2) is an autosomal recessively
inherited disorder, characterized by white hair and skin, and loss of pigment in
the eyes. Mutations in P gene have been shown to result in OCA2. So far, two
cases have been reported from Japan.
OBJECTIVE: We had an opportunity to examine a case of albinism, and screened the
mutations of tyrosinase and P gene.
METHODS: Genomic DNA was prepared from peripheral leukocytes. All of the exons
and flanking introns of tyrosinase and P gene were PCR-direct-sequenced.
RESULTS: Although no mutations were found in tyrosinase, we found two missense
substitutions, A481T and Q799H in P gene. The A481T has previously been shown to
result in partial function of the P protein.
CONCLUSION: The Q799H mutation is not a common polymorphism among normal
Japanese, seems most likely to be a pathological OCA2 mutation among Japanese
with this form of albinism. Chediak-Higashi syndrome (CHS) is a rare multiorgan disease entity with
autosomal recessive inheritance characterized by oculocutaneous albinism,
bleeding tendency, recurrent bacterial infections and various neurological
symptoms. Intracellular vesicle formation is deficient, resulting in giant
granules in many cells, e.g. giant melanosomes in the melanocytes. Diagnosis has
been based on morphological examination of peripheral blood and bone marrow,
with giant granules seen in cells of the myeloid lineage and in lymphocytes. The
ultimate diagnostic test is to look for a mutated LYST gene. Most patients
develop an accelerated phase of the disease with deposition of lymphohistiocytes
in the liver, spleen, lymph nodes and bone marrow, resulting in
hepatosplenomegaly, bone marrow infiltration and haemophagocytosis. Peripheral
blood neutropenia becomes more profound as anaemia and thrombocytopenia develop.
Most patients succumb before the age of 10 years. Four patients with CHS are
described, one of whom is a long-term survivor after successful allogeneic bone
marrow transplantation, two succumbed during the accelerated phase and one is
living with a chronic form of the disease.
CONCLUSION: Allogeneic bone marrow transplantation from an HLA-matched sibling
is the therapy of choice and should be performed early. If there is no matched
family donor, an unrelated donor or a placental blood graft is a good
alternative. The clinical picture of CHS is heterogeneous and therapeutic
decisions need to be made on an individual basis. Hearing impairment is frequently found associated with pigmentary disorders in
many syndromes. However, total oculocutaneous albinism (OCA) associated with
deafness has been described only once, by Ziprkowski and Adam (Arch Dermatol
89:151-155, 1964) in an inbred family. A syndrome associating deafness and OCA
was suggested by the authors, but two separate recessive genes segregating in
this inbred group were also proposed later by Fraser (OMIM # 220900). Combined
deafness and total OCA were also observed by us in a family originally reported
to be nonconsanguineous but in which haplotyping showed evidence of a common
ancestry: the proband was affected by both diseases, one of his sisters had only
OCA and another sister had only deafness. Both the proband and his deaf sister
were found to be homozygotes for the 35delG mutation (GJB2 gene), the most
frequent cause of hereditary deafness. Linkage analysis with markers close to
the four known OCA loci excluded linkage to OCA1, OCA2, and OCA3, and
homozygosity in markers near OCA4 locus was observed. Sequencing of the
corresponding gene (MATP) revealed a c.1121delT mutation, which leads to a stop
codon at position 397 (L374fsX397). Clearly, the combined occurrence of deafness
and albinism in this pedigree was due to mutations in two different genes,
showing autosomal recessive inheritance. We speculate that the putative syndrome
reported by Ziprkowski and Adam might have resulted from the co-occurrence of
autosomal recessive deafness and albinism in the same pedigree, as suggested by
Fraser. Tyrosinase (TYR) is a multifunctional copper-containing glycoenzyme
(approximately 80 kDa), which plays a key role in the rate-limiting steps of the
melanin biosynthetic pathway. This membrane-bound protein, possibly evolved by
the fusion of two different copper-binding proteins, is mainly expressed in
epidermal, ocular and follicular melanocytes. In the melanocytes, TYR functions
as an integrated unit with other TYR-related proteins (TYRP1, TYRP2),
lysosome-associated membrane protein 1 (LAMP1) and melanocyte-stimulating
hormone receptors; thus forming a melanogenic complex. Mutations in the TYR gene
(TYR, 11q14-21, MIM 606933) cause oculocutaneous albinism type 1 (OCA1, MIM
203100), a developmental disorder having an autosomal recessive mode of
inheritance. In addition, TYR can act as a modifier locus for primary congenital
glaucoma (PCG) and it also contributes significantly in the eye developmental
process. Expression of TYR during neuroblast division helps in later pathfinding
by retinal ganglion cells from retina to the dorsal lateral geniculate nucleus.
However, mutation screening of TYR is complicated by the presence of a
pseudogene-TYR like segment (TYRL, 11p11.2, MIM 191270), sharing approximately
98% sequence identity with the 3' region of TYR. Thus, in absence of a
full-proof strategy, any nucleotide variants identified in the 3' region of TYR
could actually be present in TYRL. Interestingly, despite extensive search, the
second TYR mutation in 15% of the OCA1 cases remains unidentified. Several
possible locations of these "uncharacterized mutations" (UCMs) have been
speculated so far. Based on the structure of TYR gene, its sequence context and
some experimental evidences, we propose two additional possibilities, which on
further investigations might shed light on the molecular basis of UCMs in TYR of
OCA1 patients; (i) partial deletion of the exons 4 and 5 region of TYR that is
homologous with TYRL and (ii) variations in the polymorphic GA complex repeat
located between distal and proximal elements of the human TYR promoter that can
modulate the expression of the gene leading to disease pathogenesis. PURPOSE: Oculocutaneous albinism (OCA) is an autosomal recessive disorder of
melanin biosynthesis that results in congenital hypopigmentation of ocular and
cutaneous tissues. It is also associated with common developmental abnormalities
of the eye. Mutations in the solute carrier family 45, member 2 gene (SLC45A2,
also called MATP) cause oculocutaneous albinism type 4 (OCA4), which is the
second most prevalent type of OCA in Japan. So far, 24 pathological mutations
have been reported in SLC45A2, but there is no report from India. Interestingly,
in almost 31% of the cases, the second mutation has never been found. The
purpose of this study was to investigate the molecular basis of OCA among
Indians using SLC45A2 as the candidate gene.
METHODS: From our patient pool, consisting of 50 unrelated OCA pedigrees
covering 17 ethnic groups of eastern and southern India, 20 patients (from 19
affected families) lacking any mutation in the tyrosinase gene (TYR) were
screened further for nucleotide variants in SLC45A2. All seven exons and
splice-site junctions of SLC45A2 were amplified and sequenced from the OCA
patients and from 50 ethnically matched healthy controls. Nucleotide changes
were detected by identifying 'double peaks' in the chromatogram due to
heterozygosity as well as by pairwise BLAST analysis of the sequence output data
with a normal copy of SLC45A2. Haplotype analysis was done among the affected
sibs using three newly identified microsatellite markers placed within and in
flanking regions of the SLC45A2 locus.
RESULTS: Four novel mutations (c.126G>A [Met42Ile], c.190G>A [Gly64Ser],
c.904A>T [Thr302Ser], and c.1042C>T [Arg348Cys]) and one reported mutation
(c.469G>A [Asp157Asn]) were identified in SLC45A2. All the novel changes
cosegregated with the disease and none were present in control samples.
Consistent with previous reports, we did not find the second mutant allele in
three unrelated patients. Haplotype analysis using microsatellite markers in the
family of one such proband suggested that the affected sibs inherited the mutant
allele (Arg348Cys) from their father but different SLC45A2 alleles from the
mother. In addition, five single nucleotide variants were identified which
included E272K and L374F polymorphisms that have been reported to be associated
with human ethnicities.
CONCLUSIONS: Our study reveals that 10% of the total OCA cases from eastern and
southern Indian ethnic groups carry mutations in SLC45A2. Among 10 variants
found in the gene, five are pathogenic changes. Our data, based on haplotype
analysis on a single family, suggest that the disease is caused in the affected
sibs either by a single mutation in SLC45A2 and a defect in another locus, or
SLC45A2 is not responsible for the disorder in the family, but the pathogenesis
is caused by a mutation in another gene not yet characterized in these patients. Mutations in the gene for tyrosinase, the key enzyme in melanin synthesis, are
responsible for oculocutaneous albinism type 1, and more than 100 mutations of
this gene have been identified. The c.1205G > A variant of the tyrosinase gene
(rs1126809) predicts p.R402Q and expression studies show thermolabile enzyme
activity for the variant protein. The Q402 allele has been associated with
autosomal recessive ocular albinism when it is in trans with a tyrosinase gene
mutation associated with oculocutaneous albinism type 1. We have identified 12
families with oculocutaneous albinism type 1 that exhibit segregation of the
c.1205G > A variant with a known pathologic mutation on the homologous
chromosome, and demonstrate no genetic association between autosomal recessive
oculocutaneous albinism and the Q402 variant. We conclude that the codon 402
variant of the tyrosinase gene is not associated with albinism. BACKGROUND: The mutation of the tyrosinase (TYR) gene results in oculocutaneous
albinism type 1 (OCA1), an autosomal recessive genetic disorder. OCA1 is the
most common type of OCA in the Chinese population. Hence, the TYR gene was
tested in this study. We also delineated the genetic analysis of OCA1 in a
Chinese family.
METHODS: Genomic DNA was isolated from the blood leukocytes of a proband and his
family. Mutational analysis at the TYR locus by DNA sequencing was used to
screen five exons, including the intron/exon junctions. A pedigree chart was
drawn and the fundus of the eyes of the proband was also examined.
RESULTS: A novel missense mutation p.I151S on exon 1, and homozygous TYR mutant
alleles were identified in the proband. None of the mutants was identified among
the 100 normal control subjects. Genetic analysis of the proband's wife showed
normal alleles in the TYR gene. Thus, the fetus was predicated a carrier of OCA1
with a normal appearance.
CONCLUSION: This study provided new information about a novel mutation, p.I151S,
in the TYR gene in a Chinese family with OCA1. Further investigation of the
proband would be helpful to determine the effects of this mutation on TYR
activity. BACKGROUND: Oculocutaneous albinism (OCA) is an autosomal recessive hereditary
pigmentation disorder affecting humans and several other animal species.
Oculocutaneous albinism was studied in a herd of Murrah buffalo to determine the
clinical presentation and genetic basis of albinism in this species.
RESULTS: Clinical examinations and pedigree analysis were performed in an
affected herd, and wild-type and OCA tyrosinase mRNA sequences were obtained.
The main clinical findings were photophobia and a lack of pigmentation of the
hair, skin, horns, hooves, mucosa, and iris. The results of segregation analysis
suggest that this disease is acquired through recessive inheritance. In the OCA
buffalo, a single-base substitution was detected at nucleotide 1,431 (G to A),
which leads to the conversion of tryptophan into a stop codon at residue 477.
CONCLUSION: This premature stop codon produces an inactive protein, which is
responsible for the OCA buffalo phenotype. These findings will be useful for
future studies of albinism in buffalo and as a possible model to study diseases
caused by a premature stop codon. BACKGROUND: Oculocutaneous albinism type1 (OCA1) is characterized by the absence
of melanin pigmentation. The mutation on TYR gene makes OCA1 as an autosomal
recessive genetic disorder. In this study, we delineated the genetic analysis of
an Iranian family with four members affected with OCA1.
METHODS: Clinical exams and paraclinical test were performed for all patients of
the case family, also proband, her husband, and her parents. Pedigree chart was
drawn too. We extracted the genomic DNA from the leukocytes of seven members of
the family. Haplotype analysis at the TYR locus was done and informative
microsatellite markers were employed. In order to amplify the entire coding
region of the TYR gene, for bidirectional direct sequencing mutation analysis,
eight sets of primers were used.
RESULTS: Our patients were diagnosed as affected with Oculocutaneous albinism
type1a. Analysis of pedigree pattern showed an autosomal recessive inheritance.
Analysis with different markers in chromosomes 5, 6, 9, 11 and 15 showed that
cause of albinism in our case family was on chromosome 11 (D11S1887 marker was
informative).
CONCLUSIONS: The results offered a more developed method of diagnosis for OCA1
carrier identification and genetic counseling for OCA1 affected families as
well; also submit a sample of mutation involved with oculocutaneous albinism in
Iran. Genetic analysis is necessary for determining the type of albinism in an
individual patient. Melanin biosynthesis is reduced in oculocutaneous albinism, an autosomal
recessive disorder. Hermansky-Pudlak syndrome is associated with oculocutaneous
albinism but also has systemic complications. The ocular and systemic phenotypes
vary, depending, in part, on the genetic mutations. This report presents a case
of a patient with Hermansky-Pudlak syndrome and the unique association of iris
heterochromia. BACKGROUND: Oculocutaneous albinism (OCA) is an autosomal recessive disorder of
abnormal melanin formation, which results in hypopigmentation of skin, hair and
eyes. OCA is classified into four types based on clinical and genetic findings.
OCA1 is the most severe form of albinism, and is caused by mutations in the
tyrosinase (TYR) gene, while OCA4 is caused due to mutations in SLC45A2.
METHODS: In total, 13 families with ≥ 3 members with OCA were enrolled. Family
history was ascertained and pedigrees were drawn up. Blood samples were
collected and processed for DNA extraction. Linkage analysis was performed by
typing three short tandem repeat markers in candidate regions of TYR and
SLC45A2. Sequence analysis was performed of all the coding exons and adjacent
intronic sequences of both genes.
RESULTS: Eight families showed linkage to OCA1 and one family showed linkage to
OCA4. Four missense substitutions (p.Arg239Trp, p.Ser192Tyr, p.Ser44Arg and
p.Arg77Gln) were identified in TYR in the families with OCA1 linkage, and
another missense substitution (p.Gln272Lys) was identified in the family with
OCA4 linkage. One of the identified missense substitution (p.Arg77Gln) in TYR
was found in five different families, which had a common haplotype.
CONCLUSIONS: We identified four missense substitutions in TYR and a single
missense substitution in SLC45A2. One missense substitution (p.Arg77Gln) in TYR
was found in five different families that originated from the same geographical
area and displayed a common haplotype, suggesting a single origin that then
spread to different geographical areas of Azad Kashmir, Pakistan. Oculocutaneous albinism (OCA) is an autosomal recessive disorder characterized
by hypopigmentation in eyes, hair and skin, accompanied with vision loss.
Currently, six genes have been identified as causative genes for non-syndromic
OCA (OCA-1∼4, 6, 7), and ten genes for syndromic OCA (HPS-1-9, CHS-1). Genetic
counseling of 51 Chinese OCA families (39 OCA-1 with mutations in the TYR gene,
6 OCA-2 with mutations in the OCA2 gene, 4 OCA-4 with mutations in the SLC45A2
gene, 1 HPS-1 (Hermansky-Pudlak syndrome-1) with mutation in the HPS1 gene, and
1 mixed OCA-1 and OCA-4) led us to perform the prenatal genetic testing of OCA
using amniotic fluid cells through the implementation of our optimized strategy.
In our cohort, eleven previously unidentified alleles (PUAs) (5 in TYR, 2 in
OCA2, and 4 in SLC45A2) were found. Three missense PUAs (p.C112R, p.H363R and
p.G379V of TYR) and one in-frame deletional PUA (p.S222del of SLC24A5) led to
fetuses with OCA when co-inherited with other disease causative alleles. Three
PUAs (p.P152H and p.W272X of TYR, p.A486T of SLC24A5) identified in the OCA
probands did not co-transmit with known pathological alleles and thus gave rise
to unaffected fetuses. Four PUAs (p.Q83X and p.A658T of TYR, p.G161R and p.G366R
of SLC24A5) did not transmit to the unaffected fetuses. In addition, the
in vitro transfection assays showed that the p.S192Y variant of TYR produced
less pigment compared to the wild-type allele. A fetus with a digenic carrier of
OCA-1 and OCA-4 was unaffected. In combination with functional assays, the
family inheritance pattern is useful for the evaluation of pathogenicity of PUAs
and genetic counseling of OCA. |
Which is the largest metabolic gene cluster in yeast? | The DAL cluster is the largest metabolic gene cluster in yeast and consists of six adjacent genes encoding proteins that enable Saccharomyces cerevisiae to use allantoin as a nitrogen source. | |
What is the applicability of the MCAST algorithm? | The MCAST algorithm uses a hidden Markov model with a P-value-based scoring scheme to identify candidate CRMs. | BACKGROUND: Cis-regulatory modules (CRMs) are short stretches of DNA that help
regulate gene expression in higher eukaryotes. They have been found up to 1
megabase away from the genes they regulate and can be located upstream,
downstream, and even within their target genes. Due to the difficulty of finding
CRMs using biological and computational techniques, even well-studied regulatory
systems may contain CRMs that have not yet been discovered.
RESULTS: We present a simple, efficient method (HexDiff) based only on hexamer
frequencies of known CRMs and non-CRM sequence to predict novel CRMs in
regulatory systems. On a data set of 16 gap and pair-rule genes containing 52
known CRMs, predictions made by HexDiff had a higher correlation with the known
CRMs than several existing CRM prediction algorithms: Ahab, Cluster Buster,
MSCAN, MCAST, and LWF. After combining the results of the different algorithms,
10 putative CRMs were identified and are strong candidates for future study. The
hexamers used by HexDiff to distinguish between CRMs and non-CRM sequence were
also analyzed and were shown to be enriched in regulatory elements.
CONCLUSION: HexDiff provides an efficient and effective means for finding new
CRMs based on known CRMs, rather than known binding sites. |
Which ApoE isoform is associated with atherosclerosis and Alzheimer's disease? | The ApoE4 isoform is associated with increased frequency of atherosclerosis and Alzheimer's disease (AD). | The apolipoprotein E type 4 allele is a susceptibility gene for late-onset
Alzheimer's disease. Apolipoprotein E is found in neurons, some of which contain
paired helical filaments made of the microtubule-associated protein tau.
Previous studies have demonstrated that the apoE3 isoform, but not the apoE4
isoform, binds tau with high avidity. Because the microtubule-associated protein
MAP2c also effects microtubule assembly and stability, we examined interactions
between apoE isoforms and MAP2c. Similar to the tau-binding results, apoE3, but
not apoE4, bound MAP2c. Binding was detectable down to 10(-9) M MAP2c and 10(-8)
M apoE3. Isoform-specific interactions of apoE with the microtubule-associated
proteins MAP2c and tau might affect intracellular maintece of microtubules
and could contribute to a time-dependent pathogenesis of Alzheimer's disease. BACKGROUND: The apolipoprotein E (apoE) type epsilon 4 isoform specifies
increased cerebral and cerebrovascular accumulation of amyloid-beta protein (A
beta) and contributes to the genetic susceptibility underlying a large
proportion (approximately 60%) of typical, sporadic Alzheimer disease.
Unfortunately, in vitro biochemical studies of direct apoE isoform-specific
interactions with A beta have been inconsistent, perhaps due to the use by
different research groups of apoE isoform preparations in different
conformational states (purified denatured versus native).
MATERIALS AND METHODS: In the current study, we have investigated the
possibility that synthetic A beta(1-40) preferentially associates with native
apoE of either the type epsilon 3 or the type epsilon 4 isoform.
RESULTS: Here, we demonstrate the preferential association of synthetic A
beta(1-40) with native apoE epsilon 3. The complex between apoE epsilon 3 and A
beta(1-40) could not be disrupted by sodium dodecyl sulfate. In a parallel
assay, no denaturant-resistant association of A beta(1-40) with apoE epsilon 4
was detectable.
CONCLUSIONS: These results support the notion that the apoE epsilon 4 isoform
may foster beta-amyloidogenesis because apoE epsilon 4 is inefficient in forming
complexes with A beta. The apolipoprotein E (APOE) E4 allele is associated with Alzheimer's disease,
cardiovascular disease, and decreased longevity. To probe the mechanism of these
associations, cell lines were created which secrete each apoE isoform. ApoE
conditioned media, purified apoE, and commercially obtained apoE protected B12
cells from hydrogen peroxide cytotoxicity with E2 > E3 > E4. Physiological
levels of apoE protected cells from beta-amyloid peptides, while higher doses of
apoE led to increased cytotoxicity. E2 > E3 > E4 possessed antioxidant activity,
and apoE bound certain metal ions. The decreased antioxidant activity of E4
could contribute to its association with Alzheimer's disease, cardiovascular
disease and decreased longevity. The inheritance of the apolipoprotein E (apoE) epsilon4 allele is a prevailing
risk factor for sporadic and familial Alzheimer's disease (AD). ApoE isoforms
bind directly to Alzheimer's amyloid beta (Abeta) peptides both in vitro and in
vivo. Recent studies suggest that association of apoE with lipids may modulate
its interaction with Abeta. We examined the binding of lipid-associated and
delipidated apoE3 and apoE4 isoforms to Abeta utilizing a solid-phase binding
assay and estimated the dissociation constants for the interaction of various
apoE and Abeta species. Using native apoE isoforms from stably transfected RAW
264 and human embryonic kidney 293 cells, apoE3 had greater affinity than apoE4
for both Abeta1-40 and Abeta1-42. Delipidation of apoE decreased its affinity
for Abeta peptides by 5-10-fold and abolished the isoform-specificity.
Conversely, incorporation of apoE isoforms produced by baculovirus-infected Sf9
cells into reconstituted human high-density-lipoprotein lipoparticles restored
the affinity values for Abeta peptides and resulted in preferential binding of
apoE3. The data demonstrate that native lipid-associated apoE3 binds to Abeta
peptides with 2-3-fold higher affinity than lipid-associated apoE4. Since the
isoforms' binding efficiency correlate inversely with the risk of developing
late-onset AD, the results suggest a possible involvement of apoE3 in the
clearance or routing out of Abeta from the central nervous system as one of the
mechanisms underlying the pathology of the disease. OBJECTIVES: There are three major isoforms of apolipoprotein E (apoE), namely
apoE2, apoE3, and apoE4, that are products of three alleles
(epsilon2,epsilon3,epsilon4) at a single gene locus on chromosome 19. It is well
known that the presence of apoE4 increases the risk for the development of
Alzheimer's disease and atherosclerosis. The aim of the study was to examine if
apoE polymorphism or apoE levels contribute to the severity of the disease in
patients with multiple sclerosis or the outcome of nerve damage in patients with
herpes zoster infection.
MATERIAL AND METHODS: We examined apoE phenotype of 105 MS patients and 41
patients with herpes zoster. We also measured serum and cerebrospinal fluid
(CSF) levels of apoE from 93 patients with definite MS using enzyme linked
immunosorbent assay.
RESULTS: There were no differences in apoE allele frequencies in MS or herpes
zoster patients compared to the allele frequencies of controls. The levels of
serum or CSF apoE did not differ from those of age-matched controls, nor did
they correlate with the disease activity.
CONCLUSION: We conclude that apoE does not contribute to the activity of MS or
the outcome of herpes zoster. First recognized as a major determit in lipoprotein metabolism and
cardiovascular disease, apolipoprotein (apo) E has emerged as an important
molecule in several biological processes not directly related to its lipid
transport function, including Alzheimer's disease and cognitive function,
immunoregulation, and possibly even infectious diseases. ApoE is a polymorphic
protein arising from three alleles at a single gene locus. The three major
isoforms, apoE4, apoE3, and apoE2, differ from one another only by single amino
acid substitutions, yet these changes have profound functional consequences at
both the cellular and molecular levels. ApoE3 seems to be the normal isoform in
all known functions, while apoE4 and apoE2 can each be dysfunctional. Isoform
(allele)-specific effects include the association of apoE2 with the genetic
disorder type III hyperlipoproteinemia and with both increased and decreased
risk for atherosclerosis and the association of apoE4 with increased risk for
both atherosclerosis and Alzheimer's disease, impaired cognitive function, and
reduced neurite outgrowth; isoform-specific differences in cellular signaling
events may also exist. Functional differences in the apoE isoforms that affect
(or did affect) survival before the reproductive years probably account, at
least in part, for the allele frequencies of the present day. Dendritic spines are postsynaptic sites of excitatory input in the mammalian
nervous system. Apolipoprotein (apo) E participates in the transport of plasma
lipids and in the redistribution of lipids among cells. A role for apoE is
implicated in regeneration of synaptic circuitry after neural injury. The apoE4
allele is a major risk factor for late-onset familial and sporadic Alzheimer's
disease (AD) and is associated with a poor outcome after brain injury. ApoE
isoforms are suggested to have differential effects on neuronal repair
mechanisms. In vitro studies have demonstrated the neurotrophic properties of
apoE3 on neurite outgrowth. We have investigated the influence of apoE genotype
on neuronal cell dendritic spine density in mice and in human postmortem tissue.
In order to compare the morphology of neurons developing under different apoE
conditions, gene gun labeling studies of dendritic spines of dentate gyrus (DG)
granule cells of the hippocampus were carried out in wild-type (WT), human
apoE3, human apoE4 expressing transgenic mice and apoE knockout (KO) mice; the
same dendritic spine parameters were also assessed in human postmortem DG from
individuals with and without the apoE4 gene. Quantitative analysis of dendritic
spine length, morphology, and number was carried out on these mice at 3 weeks, 1
and 2 years of age. Human apoE3 and WT mice had a higher density of dendritic
spines than human E4 and apoE KO mice in the 1 and 2 year age groups (P<0.0001),
while at 3 weeks there were no differences between the groups. These age
dependent differences in the effects of apoE isoforms on neuronal integrity may
relate to the increased risk of dementia in aged individuals with the apoE4
allele. Significantly in human brain, apoE4 dose correlated inversely with
dendritic spine density of DG neurons cell in the hippocampus of both AD
(P=0.0008) and aged normal controls (P=0.0015). Our findings provide one
potential explanation for the increased cognitive decline seen in aged and AD
patients expressing apoE4. Apolipoprotein E (apoE) is found in amyloid plaques and neurofibrillary tangles
(NFTs) in Alzheimer's disease (AD) brains, but its role in their pathogenesis is
unclear. Previously, we found C-terminal-truncated fragments of apoE in AD
brains and showed that such fragments can cause neurodegeneration and can induce
NFT-like inclusions in cultured neuronal cells and in transgenic mice. Here, we
analyzed apoE fragmentation in brain tissue homogenates from transgenic mice
expressing apoE3 or apoE4 in neurons [neuron-specific enolase (NSE)-apoE] or
astrocytes [glial fibrillary acidic protein (GFAP)-apoE] by Western blotting.
The C-terminal-truncated fragments of apoE accumulated, in an age-dependent
manner, in the brains of NSE-apoE4 and, to a significantly lesser extent,
NSE-apoE3 mice; however, no fragments were detected in GFAP-apoE3 or GFAP-apoE4
mice. In NSE-apoE mice, the pattern of apoE fragmentation resembled that seen in
AD brains, and the fragmentation was specific for certain brain regions,
occurring in the neocortex and hippocampus, which are vulnerable to AD-related
neurodegeneration, but not in the less vulnerable cerebellum. Excitotoxic
challenge with kainic acid significantly increased apoE fragmentation in
NSE-apoE4 but not NSE-apoE3 mice. Phosphorylated tau (p-tau) also accumulated in
an age-dependent manner in NSE-apoE4 mice and, to a much lesser extent, in
NSE-apoE3 mice but not in GFAP-apoE3 or GFAP-apoE4 mice. Intraneuronal p-tau
inclusions in the hippocampus were prominent in 21-month-old NSE-apoE4 mice but
barely detectable in NSE-apoE3 mice. Thus, the accumulation of potentially
pathogenic C-terminal-truncated fragments of apoE depends on both the isoform
and the cellular source of apoE. Neuron-specific proteolytic cleavage of apoE4
is associated with increased phosphorylation of tau and may play a key role in
the development of AD-related neuronal deficits. Human apolipoprotein E (apoE) is a 299-amino-acid protein with a molecular
weight of 34 kDa. The difference between the apoE3 and apoE4 isoforms is a
single residue substitution involving a Cys-Arg replacement at residue 112.
ApoE4 is positively associated with atherosclerosis and late-onset and sporadic
Alzheimer's disease (AD). ApoE4 and its C-terminal truncated fragments have been
found in the senile plaques and neurofibrillary tangles in the brain of AD
patients. However, detail structural information regarding isoform and domain
interaction remains poorly understood. We prepared full-length, N-, and
C-terminal truncated apoE3 and apoE4 proteins and studied their structural
variation. Sedimentation velocity and continuous size distribution analysis
using analytical ultracentrifugation revealed apoE3(72-299) as consisting of a
major species with a sedimentation coefficient of 5.9. ApoE4(72-299) showed a
wider and more complicated species distribution. Both apoE3 and E4 N-terminal
domain (1-191) existed with monomers as the major component together with some
tetramer. The oligomerization and aggregation of apoE protein increased when the
C-terminal domain (192-271) was incorporated. The structural influence of the
C-terminal domain on apoE is to assist self-association with no significant
isoform preference. Circular dichroism and fluorescence studies demonstrated
that apoE4(72-299) possessed a more alpha-helical structure with more
hydrophobic residue exposure. The structural variation of the N-terminal
truncated apoE3 and apoE4 protein provides useful information that helps to
explain the greater aggregation of the apoE4 isoform and thus has implication
for the involvement of apoE4 in AD. Amyloid-beta1-42 (Abeta1-42) is crucial to Alzheimer disease (AD) pathogenesis
but the conformation of the toxic Abeta species remains uncertain. AD risk is
increased by apolipoprotein E4 (apoE4) and decreased by apoE2 compared with the
apoE3 isoform, but whether inheritance of apoE4 represents a gain of negative or
a loss of protective function is also unresolved. Using hippocampal slices from
apoE knockout (apoE-KO) and human apoE2, E3, and E4 targeted replacement
(apoE-TR) mice, we found that oligomeric Abeta1-42 inhibited long-term
potentiation (LTP) with a hierarchy of susceptibility mirroring clinical AD risk
(apoE4-TR > apoE3-TR = apoE-KO > apoE2-TR), and that comparable doses of
unaggregated Abeta1-42 did not affect LTP. These data provide a novel link among
apoE isoform, Abeta1-42, and a functional cellular model of memory. In this
model, apoE4 confers a gain of negative function synergistic with Abeta1-42,
apoE2 is protective, and the apoE-Abeta interaction is specific to oligomeric
Abeta1-42. There are three major apolipoprotein E (apoE) isoforms. Although APOE-epsilon3
is considered a longevity gene, APOE-epsilon4 is a dual risk factor to
atherosclerosis and Alzheimer disease. We have expressed full-length and N- and
C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain
interactions to unveil structural and functional disturbances. The N-terminal
truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more
complicated or aggregated species than those of the corresponding apoE3
counterparts. This isoformic structural variation did not exist in the presence
of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and
apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the
dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein
(LDL) receptor binding ability, determined by a competition binding assay of
3H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with
N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal
truncation (apoE4-(1-191)) maintained greater receptor binding abilities than
their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299)
and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The
structural preference of apoE4 to remain functional in solution may explain the
enhanced opportunity of apoE4 isoform to display its pathophysiologic functions
in atherosclerosis and Alzheimer disease. After receptor-mediated endocytosis of apolipoprotein E (apoE)-containing
lipoproteins in hepatocytes, the isoform apoE3 is efficiently recycled in a
process which is associated with cholesterol efflux. Recycling and cholesterol
efflux are greatly reduced when apoE4 is the only isoform present. ApoE is the
main apolipoprotein in cerebrospinal fluid, and it plays a pivotal role in
maintaining cholesterol homeostasis in the brain. The isoform apoE4 is
associated with an increased risk of Alzheimer's disease and it has been
postulated that high intracellular cholesterol levels promote the amyloidogenic
processing of amyloid precursor protein. Therefore we investigated the cellular
processing of different apoE isoforms as well as the associated cholesterol
efflux in the murine neuronal cell line HT-22. Uptake of apoE3-containing
lipoproteins resulted in the expected recycling while, as seen in non-neuronal
cells, recycling of apoE4 was significantly reduced. However, despite these
differences in apoE recycling, there was no difference in rates of cholesterol
efflux. Therefore we conclude that in this neuronal cell model the reduced
recycling of apoE4 does not affect cellular cholesterol metabolism. BACKGROUND: Apolipoprotein E (ApoE) is a molecular scavenger in the blood and
brain. Aberrant function of the molecule causes formation of protein and lipid
deposits or "plaques" that characterize Alzheimer's disease (AD) and
atherosclerosis. There are three human isoforms of ApoE designated epsilon2,
epsilon3, and epsilon4. Each isoform differentially affects the structure and
function of the protein and thus the development of disease. Homozygosity for
ApoE epsilon4 is associated with atherosclerosis and Alzheimer's disease whereas
ApoE epsilon2 and epsilon3 tend to be protective. Furthermore, the epsilon2 form
may cause forms of hyperlipoproteinemia. Therefore, introduction of ApoE
epsilon3 may be beneficial to patients that are susceptible to or suffering from
these diseases. Mesenchymal stem cells or multipotent mesenchymal stromal cells
(MSCs) are adult progenitor cells found in numerous tissues. They are easily
expanded in culture and engraft into host tissues when administered
appropriately. Furthermore, MSCs are immunosuppressive and have been reported to
engraft as allogeneic transplants. In our previous study, mouse MSCs (mMSCs)
were implanted into the brains of ApoE null mice, resulting in production of
small amounts of ApoE in the brain and attenuation of cognitive deficits.
Therefore human MSCs (hMSCs) are a promising vector for the administration of
ApoE epsilon3 in humans.
RESULTS: Unlike mMSCs, hMSCs were found not to express ApoE in culture;
therefore a molecular screen was performed for compounds that induce expression.
PPARgamma agonists, neural stem cell conditioned medium, osteo-inductive media,
dexamethasone, and adipo-inductive media (AIM) were tested. Of the conditions
tested, only AIM or dexamethasone induced sustained secretion of ApoE in MSCs
and the duration of secretion was only limited by the length of time MSCs could
be sustained in culture. Upon withdrawal of the inductive stimuli, the ApoE
secretion persisted for a further 14 days.
CONCLUSION: The data demonstrated that pre-treatment and perhaps
co-administration of MSCs homozygous for ApoE epsilon3 and dexamethasone may
represent a novel therapy for severe instances of AD, atherosclerosis and other
ApoE-related diseases. Neurotoxic amyloid beta peptide (Abeta) accumulates in the brains of individuals
with Alzheimer disease (AD). The APOE4 allele is a major risk factor for
sporadic AD and has been associated with increased brain parenchymal and
vascular amyloid burden. How apoE isoforms influence Abeta accumulation in the
brain has, however, remained unclear. Here, we have shown that apoE disrupts
Abeta clearance across the mouse blood-brain barrier (BBB) in an
isoform-specific manner (specifically, apoE4 had a greater disruptive effect
than either apoE3 or apoE2). Abeta binding to apoE4 redirected the rapid
clearance of free Abeta40/42 from the LDL receptor-related protein 1 (LRP1) to
the VLDL receptor (VLDLR), which internalized apoE4 and Abeta-apoE4 complexes at
the BBB more slowly than LRP1. In contrast, apoE2 and apoE3 as well as
Abeta-apoE2 and Abeta-apoE3 complexes were cleared at the BBB via both VLDLR and
LRP1 at a substantially faster rate than Abeta-apoE4 complexes.
Astrocyte-secreted lipo-apoE2, lipo-apoE3, and lipo-apoE4 as well as their
complexes with Abeta were cleared at the BBB by mechanisms similar to those of
their respective lipid-poor isoforms but at 2- to 3-fold slower rates. Thus,
apoE isoforms differentially regulate Abeta clearance from the brain, and this
might contribute to the effects of APOE genotype on the disease process in both
individuals with AD and animal models of AD. Transforming growth factor-beta1 (TGF-beta1) has central functions in
development, tissue maintece, and repair and has been implicated in major
diseases. We discovered that TGF-beta1 contains several amphipathic helices and
hydrophobic domains similar to apolipoprotein E (apoE), a protein involved in
lipoprotein metabolism. Indeed, TGF-beta1 associates with lipoproteins isolated
from human plasma, cultured liver cells, or astrocytes, and its bioactivity was
highest in high-density lipoprotein preparations. Importantly, lipoproteins
containing the apoE3 isoform had higher TGF-beta levels and bioactivity than
those containing apoE4, a major genetic risk factor for atherosclerosis and
Alzheimer's disease. Because TGF-beta1 can be protective in these diseases an
association with apoE3 may be beneficial. Association of TGF-beta with different
types of lipoproteins may facilitate its diffusion, regulate signaling, and
offer additional specificity for this important growth factor. PURPOSE OF REVIEW: The purpose of this review is to provide insights into recent
advances in mechanisms linking apolipoprotein (apo) E isoforms to cardiovascular
and neurological diseases.
RECENT FINDINGS: Human apoE has three common isoforms (apoE2, apoE3, and apoE4)
with different structural and biophysical properties and different effects on
lipid and neuronal homeostasis. ApoE is a protein constituent of different
plasma lipoproteins and serves as a high-affinity ligand for several receptors.
By interacting with its receptors, apoE mediates the clearance of different
lipoproteins from the circulation. Absence or structural mutations of apoE cause
significant disorders in lipid metabolism and cardiovascular disease. ApoE also
has significant roles in neurobiology. ApoE4 is the major known genetic risk
factor for Alzheimer's disease. It increases the occurrence and lowers the age
of onset of Alzheimer's disease. ApoE4 carriers account for 65-80% of all
Alzheimer's disease cases, highlighting the importance of apoE4 in Alzheimer's
disease pathogenesis. ApoE4 has both amyloid beta-dependent and amyloid
beta-independent roles in Alzheimer's disease pathogenesis.
SUMMARY: Emerging data suggest that apoE isoforms, with their multiple cellular
origins and multiple structural and biophysical properties, contribute to
cardiovascular and neurological diseases by interacting with different factors
through various pathways. APOE alleles and apolipoprotein E isoforms control plasma cholesterol level on
population level. Among three ɛ2, ɛ3, ɛ4 alleles, ɛ4 allele is associated with
the increase in cholesterol level, risk of atherosclerosis and Alzheimer
disease, while ɛ2 allele is associated with the decrease in cholesterol level
and risk of atherosclerosis. The increase in plasma triglyceride is an
independent risk factor of atherosclerosis and triglyceride-high density
lipoprotein coupling determines the efficiency of reverse cholesterol transport.
The impairment of this coupling specifically at hypertriglyceridemia may be
followed by specific lipoprotein markers. The influence of major lipid-lowering
drugs on lipoprotein metabolism and association of apoE isoforms with the
efficiency of therapy by statins and fibrates are summarized both at isolated
and combined increase in plasma triglyceride and cholesterol. APOE polymorphism
seems to be a single genetic variant with a confirmed stratification both at
candidate gene and at wide genome analyses. The largest genetic risk for late-onset Alzheimer's disease (AD) resides at the
apolipoprotein E gene (APOE) locus, which has three common alleles (ɛ2, ɛ3, ɛ4)
that encode three isoforms (apoE2, apoE3, apoE4). The very strong association of
the APOE ɛ4 allele with AD risk and its role in the accumulation of amyloid β in
brains of people and animal models solidify the biological relevance of apoE
isoforms but do not provide mechanistic insight. The innate immune response is
consistently observed in AD and is a likely contributor to neuronal injury and
response to injury. Here we review emerging data showing that apoE isoform
regulation of multiple facets of the innate immune response in the brain may
alter AD not only through amyloid β-dependent mechanisms, but also through
other, amyloid β-independent mechanisms. OBJECTIVE: To examine the association between the circadian locomotor output
cycles kaput (CLOCK) gene rs1554483 G/C polymorphism and susceptibility to
Alzheimer's disease in Chinese people.
METHODS: This case-control study determined apolipoprotein E (APOE) and CLOCK
rs1554483 G/C genotypes using polymerase chain reaction restriction fragment
length polymorphism.
RESULTS: Unrelated patients with Alzheimer's disease (n = 130) and healthy
controls (n = 188) were analysed for an association between the CLOCK gene
rs1554483 G/C polymorphism and susceptibility to Alzheimer's disease. In the
whole sample and in APOE ε4 isoform noncarriers, the prevalence of CLOCK gene
rs1554483 G allele carriers was significantly higher in patients with
Alzheimer's disease than in controls. Among APOE ε4 carriers, the prevalence of
CLOCK rs1554483 G allele carriers was not significantly different between
patients with Alzheimer's disease and controls.
CONCLUSION: Among APOE ε4 noncarriers, but not APOE ε4 carriers, the CLOCK
rs1554483 G allele was associated with increased susceptibility to Alzheimer's
disease. BACKGROUND: Human apolipoprotein E (apoE) exists in three major isoforms: apoE2,
apoE3 and apoE4. In the brain, apoE is produced mostly by astrocytes and
transports cholesterol to neurons via apoE receptors. Among the gene alleles
encoding the three isoforms, the APOE4 allele is the strongest genetic risk
factor for late-onset Alzheimer's disease (AD), whereas APOE2 is protective.
ApoE4 confers a gain of toxic function, a loss of neuroprotective function or a
combination of both in AD pathogenesis. Given that therapeutic impacts of
modulating apoE expression may be isoform-dependent, we sought to investigate
the relationship between overexpressing apoE isoform and apoE-related functions
in apoE-targeted replacement (TR) mice. Specifically, apoE isoform expression
driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter
was built into an adeno-associated virus serotype 8 (AAV8) vector and injected
into the ventricles of postnatal day 2 (P2) apoE3-TR or apoE4-TR mice. Upon
confirmation of apoE isoform expression, effects on apoE lipidation and the
levels of amyloid-β (Aβ) in the brain were assessed.
RESULTS: AAV8-GFAP-apoE isoforms were specifically expressed in astrocytes
throughout all brain regions, which led to overall increased apoE levels in the
brain. Viral mediated overexpression of apoE4 in the apoE4-TR background
increased poorly-lipidated apoE lipoprotein particles and decreased
apoE-associated cholesterol in apoE4-TR mice. Conversely, apoE2 overexpression
in apoE4-TR mice enhanced apoE lipidation and associated cholesterol.
Furthermore, overexpression of apoE4 elevated the levels of endogenous Aβ,
whereas apoE2 overexpression trended to lower endogenous Aβ.
CONCLUSIONS: Overexpression of apoE isoforms induces differential effects in the
apoE4-TR background: apoE4 decreases apoE lipidation and enhances Aβ
accumulation, whereas apoE2 has the opposite effects. Our findings suggest that
increasing apoE2 in APOE4 carriers is a beneficial strategy to treat AD, whereas
increasing apoE4 in APOE4 carriers is likely harmful. We have also established
novel methods to express apoE isoforms in mouse brain to study apoE-related
pathways in AD and related dementia. The cholesterol transporting protein apolipoprotein E (ApoE) occurs in three
allelic variants in humans unlike in other species. The resulting protein
isoforms E2, E3 and E4 exhibit differences in lipid binding, integrating into
lipoprotein particles and affinity for lipoprotein receptors. ApoE isoforms
confer genetic risk for several diseases of aging including atherosclerosis,
Alzheimer's disease, and age-related macular degeneration (AMD). A single E4
allele increases the risk of developing Alzheimer's disease, whereas the E2
allele is protective. Intriguingly, the E4 allele is protective in AMD. Current
thinking about different functions of ApoE isoforms comes largely from studies
on Alzheimer's disease. These data cannot be directly extrapolated to AMD since
the primary cells affected in these diseases (neurons vs. retinal pigment
epithelium) are so different. Here, we propose that ApoE serves a fundamentally
different purpose in regulating cholesterol homeostasis in the retinal pigment
epithelium and this could explain why allelic risk factors are flipped for AMD
compared to Alzheimer's disease. Alzheimer's disease (AD) is the most common form of dementia in individuals over
the age of 65 years. The most prevalent genetic risk factor for AD is the ε4
allele of apolipoprotein E (ApoE4), and novel AD treatments that target ApoE are
being considered. One unresolved question in ApoE biology is whether ApoE is
necessary for healthy brain function. ApoE knock-out (KO) mice have synaptic
loss and cognitive dysfunction; however, these findings are complicated by the
fact that ApoE knock-out mice have highly elevated plasma lipid levels, which
may independently affect brain function. To bypass the effect of ApoE loss on
plasma lipids, we generated a novel mouse model that expresses ApoE normally in
peripheral tissues, but has severely reduced ApoE in the brain, allowing us to
study brain ApoE loss in the context of a normal plasma lipid profile. We found
that these brain ApoE knock-out (bEKO) mice had synaptic loss and dysfunction
similar to that of ApoE KO mice; however, the bEKO mice did not have the
learning and memory impairment observed in ApoE KO mice. Moreover, we found that
the memory deficit in the ApoE KO mice was specific to female mice and was fully
rescued in female bEKO mice. Furthermore, while the AMPA/NMDA ratio was reduced
in ApoE KO mice, it was unchanged in bEKO mice compared with controls. These
findings suggest that plasma lipid levels can influence cognition and synaptic
function independent of ApoE expression in the brain.
SIGNIFICANCE STATEMENT: One proposed treatment strategy for Alzheimer's disease
(AD) is the reduction of ApoE, whose ε4 isoform is the most common genetic risk
factor for the disease. A major concern of this strategy is that an animal model
of ApoE deficiency, the ApoE knock-out (KO) mouse, has reduced synapses and
cognitive impairment; however, these mice also develop dyslipidemia and severe
atherosclerosis. Here, we have shown that genetic restoration of plasma ApoE to
wild-type levels normalizes plasma lipids in ApoE KO mice. While this does not
rescue synaptic loss, it does completely restore learning and memory in the
mice, suggesting that both CNS and plasma ApoE are independent parameters that
affect brain health. |
Are Ultra-conserved elements (UCEs) enriched in segmental duplications? | ULEs are located in intergenic or intronic regions and are depleted from segmental duplications. In addition, here we show that these elements are preferentially found in pathogenic deletions (enrichment ratio 3.6 vs. 0.5 in duplications), and that this association is not related with a higher content of genes. | An earlier search in the human, mouse and rat genomes for sequences that are
100% conserved in orthologous segments and > or = 200 bp in length identified
481 distinct sequences. These human-mouse-rat sequences, which represent
ultraconserved elements (UCEs), are believed to be important for functions
involving DNA binding, RNA processing and the regulation of transcription and
development. In vivo and additional computational studies of UCEs and other
highly conserved sequences are consistent with these functional associations,
with some observations indicating enhancer-like activity for these elements.
Here, we show that UCEs are significantly depleted among segmental duplications
and copy number variants. Notably, of the UCEs that are found in segmental
duplications or copy number variants, the majority overlap exons, indicating,
along with other findings presented, that UCEs overlapping exons represent a
distinct subset. Ultraconserved elements (UCEs) are sequences that are identical between
reference genomes of distantly related species. As they are under negative
selection and enriched near or in specific classes of genes, one explanation for
their ultraconservation may be their involvement in important functions. Indeed,
many UCEs can drive tissue-specific gene expression. We have demonstrated that
nonexonic UCEs are depleted among segmental duplications (SDs) and copy number
variants (CNVs) and proposed that their ultraconservation may reflect a
mechanism of copy counting via comparison. Here, we report that nonexonic UCEs
are also depleted among 10 of 11 recent genomewide data sets of human CNVs,
including 3 obtained with strategies permitting greater precision in determining
the extents of CNVs. We further present observations suggesting that nonexonic
UCEs per se may contribute to this depletion and that their apparent dosage
sensitivity was in effect when they became fixed in the last common ancestor of
mammals, birds, and reptiles, consistent with dosage sensitivity contributing to
ultraconservation. Finally, in searching for the mechanism(s) underlying the
function of nonexonic UCEs, we have found that they are enriched in TAATTA,
which is also the recognition sequence for the homeodomain DNA-binding module,
and bounded by a change in A + T frequency. Ultraconserved elements (UCEs), stretches of DNA that are identical between
distantly related species, are enigmatic genomic features whose function is not
well understood. First identified and characterized in mammals, UCEs have been
proposed to play important roles in gene regulation, RNA processing, and
maintaining genome integrity. However, because all of these functions can
tolerate some sequence variation, their ultraconserved and ultraselected nature
is not explained. We investigated whether there are highly conserved DNA
elements without genic function in distantly related plant genomes. We compared
the genomes of Arabidopsis thaliana and Vitis vinifera; species that diverged
∼115 million years ago (Mya). We identified 36 highly conserved elements with at
least 85% similarity that are longer than 55 bp. Interestingly, these elements
exhibit properties similar to mammalian UCEs, such that we named them UCE-like
elements (ULEs). ULEs are located in intergenic or intronic regions and are
depleted from segmental duplications. Like UCEs, ULEs are under strong purifying
selection, suggesting a functional role for these elements. As their mammalian
counterparts, ULEs show a sharp drop of A+T content at their borders and are
enriched close to genes encoding transcription factors and genes involved in
development, the latter showing preferential expression in undifferentiated
tissues. By comparing the genomes of Brachypodium distachyon and Oryza sativa,
species that diverged ∼50 Mya, we identified a different set of ULEs with
similar properties in monocots. The identification of ULEs in plant genomes
offers new opportunities to study their possible roles in genome function,
integrity, and regulation. Ultraconserved elements (UCEs) are strongly depleted from segmental duplications
and copy number variations (CNVs) in the human genome, suggesting that deletion
or duplication of a UCE can be deleterious to the mammalian cell. Here we
address the process by which CNVs become depleted of UCEs. We begin by showing
that depletion for UCEs characterizes the most recent large-scale human CNV
datasets and then find that even newly formed de novo CNVs, which have passed
through meiosis at most once, are significantly depleted for UCEs. In striking
contrast, CNVs arising specifically in cancer cells are, as a rule, not depleted
for UCEs and can even become significantly enriched. This observation raises the
possibility that CNVs that arise somatically and are relatively newly formed are
less likely to have established a CNV profile that is depleted for UCEs.
Alternatively, lack of depletion for UCEs from cancer CNVs may reflect the
diseased state. In support of this latter explanation, somatic CNVs that are not
associated with disease are depleted for UCEs. Finally, we show that it is
possible to observe the CNVs of induced pluripotent stem (iPS) cells become
depleted of UCEs over time, suggesting that depletion may be established through
selection against UCE-disrupting CNVs without the requirement for meiotic
divisions. |
What organism causes woolsorter's disease | Woolsorter's disease is caused by the same organism as Anthrax, bacillus Anthrax. | Woolsorters' disease was a feared industrial disease associated primarily with
Yorkshire's textile industry of the nineteenth and early twentieth centuries.
Early occupational health methods were attempted locally before concerted
national efforts produced legislative measures. When its link with anthrax was
established, attention in prevention focused upon chemical disinfection methods.
Together, these factors were instrumental in decreasing the incidence of
woolsorters' disease. However, by the beginning of the Second World War, the
lack of treatment options for anthrax meant that the bacterium was experimented
upon as a potential war-winning weapon. Today, woolsorters' disease and other
industrial manifestations of anthrax are extremely rare, but the increasing
threat of bioterrorism means that the international dread and historical lessons
of this significant condition should never be forgotten. Consequently, this
paper reveals the history of woolsorters' disease in order to remind those
involved in occupational medicine today of the dread it caused both physicians
and workers in previous generations. Focusing on three Anglo-American outbreaks of industrial anthrax, this essay
engages the question of how local circumstances influenced the transmission of
scientific knowledge in the late nineteenth century. Walpole (Massachusetts),
Glasgow, and Bradford (Yorkshire) served as important nodes of transnational
investigation into anthrax. Knowledge about the morphology and behavior of
Bacillus anthracis changed little while in transit between these nodes, even
during complex debates about the nature of bacterial morphology, disease
causation, and spontaneous generation. Working independently of their more
famous counterparts (Robert Koch and Louis Pasteur), Anglo-American anthrax
investigators used visual representations of anthrax bacilli to persuade their
peers that a specific, identifiable cause produced all forms of
anthrax-maligt pustule (cutaneous anthrax), intestinal anthrax, and
woolsorter's disease (pneumonic anthrax). By the late 1870s, this point of view
also supported what we would today call an ecological notion of the disease's
origins in the interactions of people, animals, and microorganisms in the
context of global commerce. |
Which annotated database of A-to-I RNA editing is available? | RADAR is a rigorously annotated database of A-to-I RNA editing. RADAR includes a comprehensive collection of A-to-I RNA editing sites identified in humans (Homo sapiens), mice (Mus musculus) and flies (Drosophila melanogaster), together with extensive manually curated annotations for each editing site. RADAR also includes an expandable listing of tissue-specific editing levels for each editing site, which will facilitate the assignment of biological functions to specific editing sites. | We present RADAR--a rigorously annotated database of A-to-I RNA editing
(available at http://RNAedit.com). The identification of A-to-I RNA editing
sites has been dramatically accelerated in the past few years by high-throughput
RNA sequencing studies. RADAR includes a comprehensive collection of A-to-I RNA
editing sites identified in humans (Homo sapiens), mice (Mus musculus) and flies
(Drosophila melanogaster), together with extensive manually curated annotations
for each editing site. RADAR also includes an expandable listing of
tissue-specific editing levels for each editing site, which will facilitate the
assignment of biological functions to specific editing sites. |
Do normal cells express the protein TERT? | Νο, telomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells. Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. | Replication-selective tumor-specific viruses present a novel approach for
treatment of neoplastic disease. These vectors are designed to induce
virus-mediated lysis of tumor cells after selective viral propagation within the
tumor. For targeting cancer cells, there is a need for tissue- or cell-specific
promoters that can express in diverse tumor types and are silent in normal
cells. Recent advances in molecular biology have fostered remarkable insights
into the molecular basis of neoplasm. Telomerase activation is considered to be
a critical step in carcinogenesis and its activity correlates closely with human
telomerase reverse transcriptase (hTERT) expression. Since only tumor cells that
express telomerase activity would activate this promoter, the hTERT proximal
promoter allows for preferential expression of viral genes in tumor cells,
leading to selective viral replication. We constructed an attenuated adenovirus
5 vector (Telomelysin, OBP-301), in which the hTERT promoter element drives
expression of E1A and E1B genes linked with an internal ribosome entry site
(IRES). Telomelysin replicated efficiently and induced marked cell killing in a
panel of human cancer cell lines, whereas replication as well as cytotoxicity
was highly attenuated in normal human cells lacking telomerase activity. Thus,
the hTERT promoter confers competence for selective replication of Telomelysin
in human cancer cells, an outcome that has important implications for the
treatment of human cancers. This article reviews recent findings in this rapidly
evolving field: cancer therapeutic and cancer diagnostic approaches using the
hTERT promoter. Telomerase plays a pivotal role in cellular immortality and tumorigenesis. Its
activity is normally not detectable in most somatic cells while it is
reactivated in the vast majority of cancer cells. Therefore, inhibition of
telomerase has been viewed as a promising anticancer approach due to its
specificity for cancer cells. Studies so far have shown that telomerase
inhibition can inhibit the proliferation of cancer cells or cause apoptosis
while it has no effect on most normal cells. Strategies currently being applied
to induce telomerase inhibition target virtually all of the major components of
the ribonucleoprotein holoenzyme and related cell signal pathways that regulate
its activity. These strategies include inhibition of telomerase through
targeting at the telomerase reverse transcriptase (TERT) catalytic subunit, the
telomerase RNA (TR) component, and associated proteins. Other strategies have
been developed to target the proteins associated with telomerase at the
telomeric ends of chromosomes such as tankyrase. The specific mechanisms that
mediate those inhibition effects include small molecules, antisense RNA, and
ribozymes. Although the beneficial evidence of telomerase inhibition is obvious,
limitations of strategies remain to be resolved to increase the feasibility of
clinical application. This analysis will summarize recent developments of
strategies in telomerase inhibition. Since telomerase has been recognized as a relevant factor distinguishing cancer
cells from normal cells, it has become a very promising target for anti-cancer
therapy. A correlation between short telomere length and increased mortality was
revealed in many studies. The telomerase expression/activity appears to be one
of the most crucial factors to study to improve cancer therapy and prevention.
However, this multisubunit enzymatic complex can be regulated at various levels.
Thus, several strategies have been proposed to control telomerase in cancer
cells such as anti-sense technology against TR and TERT, ribozymes against TERT,
anti-estrogens, progesterone, vitamin D, retinoic acid, quadruplex stabilizers,
telomere and telomerase targeting agents, modulation of interaction with other
proteins involved in the regulation of telomerase and telomeres, etc. However,
the transcription control of key telomerase subunits seems to play the crucial
role in whole complexes activity and cancer cells immortality. Thus, the
research of telomerase regulation can bring significant insight into the
knowledge concerning stem cells metabolism but also ageing. This review
summarizes the current state of knowledge of numerous telomerase regulation
mechanisms at the transcription level in human that might become attractive
anti-cancer therapy targets. |
Which protein complexes recognize centromeric (CEN) DNA in yeast? | The Schizosaccharomyces pombe centromere-linked genes, LYS1 and CYH1 on chromosome I and TPS13 and RAN1 on chromosome II, have been isolated. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. | The Schizosaccharomyces pombe centromere-linked genes, LYS1 and CYH1 on
chromosome I and TPS13 and RAN1 on chromosome II, have been isolated. The
genetic order of these markers with respect to their centromeres was determined
to establish relative directionality on the genetic and physical maps.
Chromosome walking toward the centromeres reveals a group of repetitive
sequences that occur only in the centromere regions of chromosomes I and II and
at one other specific location in the S. pombe genome, presumably the centromere
of chromosome III. The major class of large repeated sequence elements is 6.4
kilobases (kb) long (repeat K), portions of which occur at least twice on
chromosome II and in several tandemly arranged intact copies at another
centromeric location. Repeat K in turn contains groups of smaller repeats.
Genetic recombination is strongly suppressed in the centromere II region, which
contains at least 30 kb of repeated sequences. Centromeric DNA organization is
much more complex in fission yeast than has been described in budding yeast
(Saccharomyces cerevisiae), possibly because of the larger more condensed nature
of the S. pombe chromosomes. During cell division, segregation of sister chromatids to daughter cells is
achieved by the poleward pulling force of microtubules, which attach to the
chromatids by means of a multiprotein complex, the kinetochore. Kinetochores
assemble at the centromeric DNA organized by specialized centromeric
nucleosomes. In contrast to other eukaryotes, which typically have large
repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp
sequence and assembles a single centromeric nucleosome. In budding yeast, as
well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as
CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome.
However, the exact composition of the CEN nucleosome remains a subject of
debate. We report the use of a novel ChIP approach to reveal the composition of
the centromeric nucleosome and its localization on CEN DNA in budding yeast.
Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A,
and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric
DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a
sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4
at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding
yeast centromere. Whether or not our model is correct, any future model will
have to account for the stable association of histone H3 with the centromeric
DNA. The structure of nucleosomes that contain the cenH3 histone variant has been
controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer
wraps the ∼80-bp Centromere DNA Element II (CDE II) sequence of each centromere
into a 'hemisome'. However, attempts to reconstitute cenH3 particles in vitro
have yielded exclusively 'octasomes', which are observed in vivo on chromosome
arms only when Cse4 (yeast cenH3) is overproduced. Here, we show that Cse4
octamers remain intact under conditions of low salt and urea that dissociate H3
octamers. However, particles consisting of two DNA duplexes wrapped around a
Cse4 octamer and separated by a gap efficiently split into hemisomes. Hemisome
dimensions were confirmed using a calibrated gel-shift assay and atomic force
microscopy, and their identity as tightly wrapped particles was demonstrated by
gelFRET. Surprisingly, Cse4 hemisomes were stable in 4 M urea. Stable Cse4
hemisomes could be reconstituted using either full-length or tailless histones
and with a 78-bp CDEII segment, which is predicted to be exceptionally stiff. We
propose that CDEII DNA stiffness evolved to favor Cse4 hemisome over octasome
formation. The precise correspondence between Cse4 hemisomes resident on CDEII
in vivo and reconstituted on CDEII in vitro without any other factors implies
that CDEII is sufficient for hemisome assembly. |
Is there any involvement of the long non-coding RNA Gomafu in schizophrenia? | Yes. The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing. | Author information:
(1)Institute for Molecular Bioscience, The University of Queensland, Brisbane,
QLD, Australia.
(2)Australian Institute for Bioengineering and Nanotechnology, The University of
Queensland, Brisbane, QLD, Australia.
(3)Department of Neuroscience, Neurology and Ophthalmology, Center for
High-Throughput Biology and Institute for Cell Engineering, Johns Hopkins
University School of Medicine, Baltimore, MD, USA.
(4)1] Schizophrenia Research Institute, Sydney, NSW, Australia [2] The
University of Newcastle, Callaghan, NSW, Australia.
(5)Queensland Brain Institute, The University of Queensland, Brisbane, QLD,
Australia.
(6)Queensland Centre for Medical Genomics, Institute for Molecular Bioscience,
The University of Queensland, St Lucia, QLD, Australia.
(7)Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, MD,
USA.
(8)RNA Biology Laboratory, RIKEN Advanced Research Institute, Wako, Saitama,
Japan.
(9)Isis Pharmaceuticals, Carlsbad, CA, USA.
(10)1] Institute for Molecular Bioscience, The University of Queensland,
Brisbane, QLD, Australia [2] Garvan Institute of Medical Research, Sydney, NSW,
Australia [3] St Vincent's Clinical School and School of Biotechnology and
Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia. |
Is butterfly rash a symptom of Systemic lupus erythematosus? | Yes, butterfly rash is symptom of Systemic lupus erythematosus. | The development of systemic lupus erythematosus (SLE) after 38 months of therapy
with recombit human interferon gamma (rIFN-gamma) was observed in a patient
with rheumatoid arthritis. In addition to glomerulonephritis and a butterfly
rash, previously negative tests for antinuclear, anti-dsDNA and anti-Sm
antibodies became positive. We assume that rIFN-gamma induced the de novo
development of SLE in our patient. According to the past reports, neuropsychiatric manifestations have been seen in
10-75% of patients with systemic lupus erythematosus and are second only to
renal involvement as a cause of death. The clinical feature is multiple. And
cerebrovascular diseases due to systemic lupus erythematosus are detected in
3-16% of the neuropsychiatric manifestations. Occlusion of the intracranial
major arteries is less frequently found in other cerebrovascular diseases. And
central nervous system involvement usually occurs at some intermediate or
terminal stage of systemic lupus erythematosus, so is rarely regarded as one of
the initial symptoms. We studied the case of a patient with systemic lupus
erythematosus with occlusion of the right middle cerebral artery indicated by
angitis and 'string of beads' appearance of the right internal carotid artery
indicated by fibromuscular dysplasia. The patient was a 38 year old female and
began to feel weakness in the left hand and developed mild-left hemiparesis due
to infarction of right temporo-parieto-occipital lesion which was revealed by CT
scan. Carotid angiograph showed irregularity at the right middle cerebral artery
and 'string of beads' appearance of the right internal carotid artery. Gradually
neurological manifestations improved, but a facial 'butterfly' rash, palmar
erythema and polyarthritis were detected.(ABSTRACT TRUNCATED AT 250 WORDS) BACKGROUND: Systemic lupus erythematosus (SLE) may involve any number of organ
systems and varies greatly in the severity and type of involvement. Cutaneous
manifestations of SLE are equally numerous and varied throughout the course of
the disease within an individual, as well as varying between patients. Cutaneous
manifestations of SLE are frequently the presenting symptoms, typically noted in
the classic malar "butterfly" rash; however, other cutaneous patterns are
frequently observed.
METHODS: We present here two patients who presented with what was thought to be
acne refractory to treatment.
RESULTS: These patients actually were found to have a facial eruption associated
with SLE as confirmed by skin biopsy.
CONCLUSIONS: The importance of investigating atypical or treatment-resistant
eruptions, especially in patients experiencing other symptoms, is emphasized. We reported a 54-year-old HTLV-I seropositive female patient with systemic lupus
erythematosus (SLE), who developed migrating radiculopathy but without chronic
progressive myelopathy. She occasionally noticed butterfly rash and
photosensitivity of the skin as well as painful episodes in different joints for
10 years. She developed pins and needles sensation on her trunk a few days after
she experienced lumbago with abrupt onset. Neurological examinations revealed
normal muscular strength, exaggerated deep tendon reflexes without Babinski
signs, and dysesthesia on her upper extremities and the trunk. The latter
symptom showed a segmental distribution of spinal nerve roots. And during the
course of the disease, it migrated in accord with a radicular pattern. This
sensory disturbance was fairly responsive to corticosteroid treatment. The
spinal tap yielded clear cerebrospinal fluid (CSF) which showed mononuclear
pleocytosis (16/mm3) with predomice of CD8+ cytotoxic cells and a positive
result for anti-HTLV-I antibody. A neurological status deteriorated in parallel
with non-neurological symptoms as SLE, when the patient had discontinued
corticosteroids in a tapering course by herself. We postulate that HTLV-I
infection in this patient modulated original autoimmune reactions as SLE, which
led to manifestation of migrating radiculopathy possibly due to autoimmunity
against ganglion cells. This is, to our knowledge, the first report of migrating
radiculopathy in an SLE patient associated with HTLV-I infection. An autopsied case of systemic lupus erythematosus with pulmonary hypertension is
reported. A 29-year-old woman with a seven-year history of polyarthralgia,
butterfly rash, nephrotic syndrome and Raynaud's phenomenon was admitted because
of progressive dyspnea on exertion. Tests for antinuclear antibody,
anti-cardiolipin antibody and lupus anticoagulant were positive.
Echocardiographic examination revealed right ventricular hypertrophy and a
moderate pericardial effusion. Estimated systolic pulmonary arterial pressure
was 53 mmHg. Despite treatment with corticosteroids including pulse
methylprednisolone therapy, lipo-PGE1 and warfarin, she died of progressive
congestive heart failure. Postmortem examination of the pulmonary vasculature
revealed findings consistent with plexogenic pulmonary arteriopathy, without
evidence of vasculitis, fibrinoid necrosis, or thromboemboli. OBJECTIVE: To identify individuals with antinuclear antibodies (ANA) not
fulfilling criteria for systemic lupus erythematosus (SLE) or other connective
tissue diseases (CTD); to describe their clinical and serological features, to
identify factors indicating evolution to SLE.
METHODS: A case-control study, based on retrospective evaluation of clinical
files. The cases were ANA positive individuals (n = 50) examined in a medical
outpatient setting, for symptoms compatible with SLE, but not fulfilling SLE
classification criteria. Two patients with SLE were matched to each case in
terms of age at initial symptom onset and sex. Thyroid autoimmunity was assessed
by detecting antithyroid antibodies. Fisher's exact test and conditional
logistic regression were used to evaluate the predictive ability of initial
findings for SLE development.
RESULTS: ANA positive individuals suspected of having a CTD present a wide
variety of symptoms and findings, usually at the 4th to 5th decade of life.
Antibodies to Sm and U1RNP were absent; anti-Ro(SSA) and anti-La(SSB) occurred
in 6%, while anti-dsDNA occurred in less than 10% of the cases. Arthritis,
butterfly and discoid rash, Raynaud's phenomenon, and anti-Ro/SSA antibodies are
the initial findings indicating evolution to SLE. Hematological abnormalities
such as leukopenia and thrombocytopenia as well as constitutional symptoms such
as easy fatigue and arthralgias are not associated with evolution to SLE.
Antithyroid antibodies were detected in 16% of the cases and 2.3% of controls.
CONCLUSION: ANA may connote a form of systemic autoimmunity that is expressed as
a wide variety of complaints, even in the absence of a definite diagnosis of
CTD. Arthritis, rash, Raynaud's phenomenon, and anti-Ro/SSA antibodies indicate
evolution to SLE. Autoimmune thyroid disease occurs in ANA positive individuals
not fulfilling SLE classification criteria rather than in patients with SLE. Kikuchi's disease (KD) is a benign and self-limiting lymphadenitis, particularly
affecting young women. KD is often associated with fever, arthralgia, and
leukopenia, features also found in systemic lupus erythematosus (SLE).
Lymphadenitis associated with SLE is indistinguishable from that in KD, and the
association of KD and SLE has been previously reported. We describe a case of KD
who developed a typical butterfly rash, reminiscent of SLE. However,
histological and laboratory findings excluded the diagnosis of SLE. This case
emphasizes that careful differential diagnosis between KD and SLE is required. Rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) are the most
common autoimmune disorders, although they each have very different
pathophysiology. In general, RA is considered to be a Th1-mediated disease,
while SLE is a Th2-mediated disease. Thus, their overlapping, in so called
"rhupus", is a rare condition. In Rembrandt van Rijn's (1606-1669) portrait of
the middle-aged Maria Bockenolle, we have what may be the earliest depiction of
a case of rhupus syndrome: the coexistence of a butterfly rash and digital
deformities. This suggests the possible historical importance of an RA epidemic
which took place in the early 17th century. We report a 12 years old female patient with an overlap syndrome involving
autoimmune hepatitis (AIH) and systemic lupus erythematosus (SLE). The patient
presented with jaundice, hepatosplenomegaly, malaise, polyarthralgia, arthritis
and butterfly rash on the face. Laboratory tests revealed severe liver
dysfunction, Coombs positive hemolytic anemia and a positive ANA/anti-dsDNA
test. Renal biopsy showed class IIA kidney disease, while liver biopsy showed
chronic hepatitis with severe inflammatory activity. The patient satisfied the
international criteria for both SLE and AIH. Clinical symptoms and laboratory
findings of SLE improved with high dose treatment with corticosteroids and
azathioprine, however, remission of the liver disease could not be achieved.
Repeat biopsy of the liver after three years of therapy revealed ongoing chronic
hepatitis with high level of inflammatory activity. The present case indicates
that children with liver dysfunction and SLE should be investigated for AIH.
There is much diagnostic and therapeutic dilemma in patients with AIH-SLE
overlap syndrome. This study demonstrates demographic, clinical and laboratory characteristics
with special reference to infections in Saudi patients with SLE. One-hundred and
ninety-nine patients with SLE treated at Riyadh Armed Forces Hospital, Saudi
Arabia over a period of 15 years (1990-2005) were retrospectively reviewed.
There were 162 females and 37 males (4.4 : 1) with an average age of 35 years at
onset of disease. Duration of diseases ranged from one to 23 years with a mean
of 7.23 years. Some of the clinical characteristics of SLE patients observed
were nephritis (53.7%), fever (53.26%), neuropsychological disorder (36.18%),
malar/butterfly rash (27.6%), pulmonary disorder (22.6%), photosensitivity
(21.6%), cardiac involvement (21.1%) and oral ulcers (19.09%). Infection was the
major complication with 58.79% of SLE patient having suffered from various
infections. A total of 22 species of pathogens including gram positive and gram
negative bacteria, viruses and fungi were isolated from 117 SLE patients. Single
to multiple episode of infection with various pathogens were recorded however,
majority of patients harboured one or two species of pathogens. Bacterial
infection was predomit (78.6%) followed by viral (28.2%) and fungal (28.2%)
infections. Forty-four percent of SLE patients were found to be infected with
organisms classified as opportunistic. The high incidence of infections in SLE
patients may be attributed to the multiple intrinsic and extrinsic risk factors
including deficiency of complement (C3 and C4), disease activity, renal
impairment, use of glucocorticoid and cytotoxic drugs. It is concluded that more
judicious use of corticosteroids and other immunosuppressive agents will be
critical to limit the infections in SLE and a high alert and close monitoring of
patients will ensure optimal patient outcome, both in terms of morbidity and
mortality. A 61-year-old male patient presented with petechiae on the arms and legs due to
a thrombocytopenia of 3 Gpt/l (3000/microl). A butterfly rash on the patient's
face suggested a diagnosis of systemic lupus erythematosus (SLE). The
thrombocytopenia was due to autoimmune thrombocytopenia. The diagnosis of SLE
could be excluded and the butterfly rash attributed to a laminar hemorrhage, an
ecchymosis due to the autoimmune thrombocytopenia. The prevalence of systemic lupus erythematosus (SLE) is 28 per 100,000. The
disease is most common in people of Caribbean or Asian descent. SLE mainly
affects adults and is common in women between the ages of 20 and 40 years, with
a female to male ratio of 9:1. The pathogenesis is multifactorial and
encompasses multiple immunological, vascular and inflammatory processes.
Diagnosing SLE can be challenging because of the myriad of clinical features and
substantial variability between patients. Cutaneous involvement is present in
about 60% of cases and typically manifests as a malar or butterfly rash. Joint
involvement is inflammatory in nature with arthralgia, arthritis and/or
tendinitis and occurs in about 90% of patients with SLE. Cardiorespiratory
symptoms are common with chest pain on inspiration due to lupus-induced pleurisy
or pericarditis, which may be associated with effusions. Lupus
glomerulonephritis is one of the most important systemic complications,
occurring in about 30% of patients with SLE in the UK. Careful screening tests
for renal disease need to be undertaken as it is asymptomatic. The diagnosis of
SLE is traditionally based on a combination of clinical features and laboratory
findings and any patient with suspected clinical features of lupus should be
investigated for the presence of autoantibodies. Treatment often includes
corticosteroids, by various routes, at different points in disease management.
In addition, some experts advocate the use of hydroxychloroquine, an
antimalarial, as a principal drug in all SLE patients. It is beneficial in the
management of mucocutaneous, musculoskeletal, serosal and constitutional
symptoms. Systemic lupus erythematosus (SLE) remains a challenging medical problem. The
integral approach to the analysis of underlying pathogenetic processes allows
identifying main symptom complexes of SLE and establishing relationship between
skin lesions and activity of the disease. We examined 84 patients with SLE (84%
women), their mean age was 42.3 +/- 2.3 yr duration of SLE 6.5 +/- 1.2 yr. The
subacute and chronic SLE variants were diagnosed in 30 (36%) and 54 (64%)
patients respectively. Grade 1 and 2-3 inflammatory process occurred in 53 (63%)
and 31 (37%) patients respectively. Symptom complexes "systemic inflammation",
"butterfly rash", "wrist petechiae", "ethema of the oral mucous membrane",
and other lesions were regarded as the markers of SLE activity. The relationship
of lupus-cheilitis and facial erythema with polyserositis and pericarditis
("visceral pathology-cardiovascular lesions") requires instrumental examination
of pericardium, pleural and abdominal cavities in the patients with the above
skin symptoms for diagnostics of polyserositis. At the same time, the presence
of teleangiectasia on the wrists (symptom complex "visceral
pathology-renoparenchymatous lesions") requires thorough examination of the
renal function. The presence of erythema at the major joints, mesh livedo, and
Raynaud's syndrome (symptom complex "musculoskeletal disorders") implies
specialized examination of the locomotor apparatus. This retrospective study aimed to collect data related to the clinical
manifestations and laboratory investigations of systemic lupus erythematosus
(SLE) patients in the eastern part of Saudi Arabia, in one of the tertiary-care
centers, King Fahd Hospital Al-Hasa, and to compare it with other regions of
Saudi Arabia. Forty-six patients fulfilling the American College of Rheumatology
1997 criteria (ACR) were collected over a period from January 2004 to December
2008. The results showed an average age of onset of 26.17 (±9.17). The most
common clinical features were nonspecific constitutional symptoms (fever,
fatigue and malaise) seen in 44 patients (95.7%). Musculoskeletal features seen
were mostly arthralgias (91.3%) and arthritis (76.1%). Nephritis was seen in
58.7% and hypertension in 52.2%. Mucocutaneous involvement included oral ulcers
(71.7%), hair loss (65.2%), butterfly rashes (67.4%), photosensitivity (47.8%)
and discoid lupus (13%). Neurologic manifestations showed psychosis in 17.4%,
depression in 15.2% and headache in 28.3%. The most common hematologic
presentation was leukopenia (58.7%) followed by hemolytic anemia and anemia of
chronic disease (47.8%). Antinuclear antibodies were positive in 44 (95.7%),
anti-dsDNA in 38 (42.6%), anti-Ro SSA and La SSB in 38 (82.6%). Anticardiolipin
antibodies and lupus anticoagulant were positive in eight (17.4%). Low
complement levels (C3 and C4) were seen in 41 (89.1%) of the patients with
active disease. The drugs used in treatment were NSAIDs (100%), antimalarials
(97.8%), steroids (100%), intermittent cyclophosphamide and other
immunosuppressive drugs (71.7%). We found that the age of onset and sex
distributions were different from other areas of Saudi Arabia, while clinical
manifestations were the same as in other areas. The prognosis of lupus was good
overall despite the multi-organ involvement. However, further studies based on
larger number of patients are needed. Neuromyelitis optica (NMO) is usually a relapsing demyelinating disease of the
central nervous system associated with optic neuritis, transverse myelitis
involving three or more contiguous spinal cord segments, and seropositivity for
NMO-IgG antibody. NMO is often mistaken for multiple sclerosis and there are
relatively sporadic publications about NMO and overlapping systemic or
organ-specific autoimmune diseases, such as systemic lupus erythematosus (SLE).
We described a unique case of a 25-year-old Arab young woman who was diagnosed
with SLE, depending on clinical, laboratory investigations and after she had
fulfilled the diagnostic criteria for SLE and had presented the following
findings: constitutional findings (fatigue, fever, and arthralgia); dermatologic
finding (photosensitivity and butterfly rash); chronic renal failure
(proteinuria up to 400 mg in 24 hours); hematologic and antinuclear antibodies
(positivity for antinuclear factor (ANF), anti-double-stranded DNA antibodies,
direct Coombs, ANA and anti-DNA, low C4 and C3, aCL by IgG and IgM). Recently,
she presented with several episodes of transverse myelitis and optic neuritis.
Clinical, radiological, and laboratory findings especially seropositivity for
NMO-IgG were compatible with NMO. Accurate diagnosis is critical to facilitate
initiation of immunosuppressive therapy for attack prevention. This case
illustrates that NMO may be associated with SLE. Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease with
highest prevalence among women of childbearing age. However, children younger
than 16 years also can develop SLE (childhood-onset lupus/juvenile-type SLE).
The aim of our study was to compare the clinical course of adult and
pediatric-onset SLE. Data from 342 adult patients followed at the University of
Debrecen, Hungary, and 79 children documented in the Hungarian National
Pediatric SLE registry were analyzed using hospital medical records. Organ
manifestations, laboratory parameters, and immunoserological characteristics
were reviewed and the results were evaluated using SPSS for Windows
software.Gender distribution was not significantly different between groups with
disease starting in childhood vs adulthood. The prevalence of the following
manifestations was significantly higher for pediatric than for adult-onset
disease including: lupus nephritis (43% pediatric vs 26.4% for adult-onset),
hematological disorders (57% vs 36.4%), photosensitivity (20% vs 9%), butterfly
rash (61% vs 35.5%) and mucosal ulceration (11.4% vs 4%). For adult-onset SLE,
neurological symptoms (30% vs 6%) and polyarthritis (86% vs 68%) occurred
significantly more frequently than in children. Anti-SSA, anti-SSB and
antiphospholipid antibodies were detected at significantly higher levels in
adult-onset patients compared to those in pediatrics. Children were more
commonly given high-dose intravenous immunoglobulin treatment (6.3% vs 0.6%) and
mycophenolate mofetil (15.2% vs 5.3%) than adults.These results suggest that
pediatric and adult-onset SLE differ in multiple aspects, and it is important to
recognize these differences for optimal treatment and prognosis of these
patients. BACKGROUND: Various different mucocutaneous symptoms may affect up to 80 % of
systemic lupus erythematosus (SLE) patients.
OBJECTIVES: To investigate, various unspecific, but otherwise typical clinical
symptoms of skin and mucous membranes that arise in SLE patients other than
those defined as SLE criteria such as butterfly rash, chronic cutaneous lupus
erythematosus, oral ulcers, and increased photosensitivity.
MATERIALS AND METHODS: Extensive search of peer-reviewed scientific articles was
performed, medical histories of several SLE patients seen in our department were
analyzed, and the rare disease courses in three SLE patients are presented.
RESULTS: Here we present a variety of unspecific but typical mucocutaneous
manifestations in SLE patients: periungual erythema, periungual telangiectasia
and periungual splinter hemorrhage, papules on the dorsum of the hands, scaling
erythema, sometimes associated with necrosis, especially of the ears, along with
complement deficiency, and the bizarre necroses of antiphospholipid syndrome.
Furthermore, we show the typical clinico-histological features of neutrophilic
urticarial dermatosis, as well as those of bullous SLE and finally a severe
course of bacterial sepsis with Neisseria flavescens/macacae.
CONCLUSIONS: Here we show several unspecific but rather typical mucocutaneous
symptoms in lupus patients that are indicative of SLE and thus may lead to an
early diagnosis. Also, life-threatening bacterial sepsis may occur with
microorganisms that are commonly considered "apathogenic", such as Neisseria
flavescens/macacae, which exclusively affect immunosuppressed patients. |
What is the function of yeast Clr4 on chromatin? | Clr4 is known to regulate silencing and switching at the mating-type loci and to affect chromatin structure at centromeres. The Clr4 methyltransferase determines the subnuclear localization of the mating-type region in fission yeast. Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. | Transcriptional silencing is known to occur at centromeres, telomeres and the
mating type region in the nucleus of fission yeast, Schizosaccharomyces pombe.
Mating-type silencing factors have previously been shown also to affect
transcriptional repression within centromeres and to some extent at telomeres.
Mutations in the clr4+, rik1+ and swi6+ genes dramatically reduce silencing at
certain centromeric regions and cause elevated chromosome loss rates. Recently,
Swi6p was found to co-localise with the three silent chromosomal regions. Here
the involvement of clr4+, rik1+ and swi6+ in centromere function is investigated
in further detail. Fluorescence in situ hybridisation (FISH) was used to show
that, as in swi6 mutant cells, centromeres lag on late anaphase spindles in clr4
and rik1 mutant cells. This phenotype is consistent with a role for these three
gene products in fission yeast centromere function. The Swi6 protein was found
to be delocalised from all three silent chromosomal regions, and dispersed
within the nucleus, in both clr4 and rik1 mutant cells. The phenotypic
similarity observed in all three mutants is consistent with the products of both
the clr4+ and rik1+ genes being required to recruit Swi6p to the centromere and
other silent regions. Mutations in clr4, rik1 and swi6 also result in elevated
sensitivity to reagents which destabilise microtubules and show a synergistic
interaction with a mutation in the beta-tubulin gene (nda3). These observations
suggest that clr4+ and rik1+ must play a role in the assembly of Swi6p into a
transcriptionally silent, inaccessible chromatin structure at fission yeast
centromeres which is required to facilitate interactions with spindle
microtubules and to ensure normal chromosome segregation. Heritable inactivation of specific regions of the genome is a widespread,
possibly universal phenomenon for gene regulation in eukaryotes.
Self-perpetuating, clonally inherited chromatin structure has been proposed as
the explanation for such phenomena as position-effect variegation (PEV) and
control of segment determination and differentiation in flies, X-chromosome
inactivation and parental imprinting in mammals, gene silencing by paramutation
in maize and silencing of the mating-type loci in yeasts. We have now found that
the clr4 gene, which is essential for silencing of centromeres and the
mating-type loci in Schizosaccharomyces pombe, encodes a protein with high
homology to the product of Su(var)3-9, a gene affecting PEV in Drosophila. Like
Su(var)3-9p, Clr4p contains SET and chromo domains, motifs found in proteins
that modulate chromatin structure. Site-directed mutations in the conserved
residues of the chromo domain confirm that it is required for proper silencing
and directional switching of the mating type, like SET domain. Surprisingly, RNA
differential display experiments demonstrated that clr4+ can mediate
transcriptional activation of certain other loci. These results show that clr4
plays a critical role in silencing at mating-type loci and centromeres through
the organization of repressive chromatin structure and demonstrate a new,
activator function for Clr4p. The encapsulation of otherwise transcribable loci within transcriptionally
inactive heterochromatin is rapidly gaining recognition as an important
mechanism of epigenetic gene regulation. In the fission yeast
Schizosaccharomyces pombe, heterochromatinization of the mat2/mat3 loci silences
the mating-type information encoded within these loci. Here, we present the
solution structure of the chromo domain from the cryptic loci regulator protein
Clr4. Clr4 is known to regulate silencing and switching at the mating-type loci
and to affect chromatin structure at centromeres. Clr4 and its human and
Drosophila homologs have been identified as histone H3-specific
methyltransferases, further implicating this family of proteins in chromatin
remodeling. Our structure highlights a conserved surface that may be involved in
chromo domain-ligand interactions. We have also analyzed two chromo domain
mutants (W31G and W41G) that previously were shown to affect silencing and
switching in full-length Clr4. Both mutants are significantly destabilized
relative to wild-type. In eukaryotes, heterochromatin mediates diverse processes including gene
silencing and regulation of long-range chromatin interactions. The formation of
heterochromatin involves a conserved array of histone modifications; in
particular, methylation of histone H3 at Lys 9 (H3K9me) is essential for
recruiting HP1/Swi6 proteins. In fission yeast, the Clr4 methyltransferase is
responsible for H3K9me across all heterochromatic domains. However, the
mechanism of Clr4 recruitment to these loci is poorly understood. We show that
Clr4 associates with Cul4, a cullin family protein that serves as a scaffold for
assembling ubiquitin ligases. Mutations in Cul4 result in defective localization
of Clr4 and loss of silencing at heterochromatic loci. This is accompanied by a
severe reduction in H3K9me and Swi6 levels, and accumulation of transcripts
corresponding to naturally silenced repeat elements within heterochromatic
domains. Moreover, heterochromatin defects in Cul4 mutants could not be rescued
by expression of Cul4 protein lacking Nedd8 modification, which is essential for
its ubiquitin ligase activity. Rik1, a protein related to DNA damage binding
protein DDB1 and required for H3K9me, also interacts with Cul4, the association
of which might serve to target Clr4 to heterochromatic loci. These analyses
uncover a role for Cul4-based protein ubiquitination in regulating H3K9me and
heterochromatin formation. The genome has a non-random spatial distribution in the cell nucleus. In
Schizosaccharomyces pombe, it has been shown that the centromeres, telomeres and
the mating-type region localize to the nuclear membrane (NM), the former by
attaching to the spindle pole body (SPB). In addition, reporter genes inserted
into these areas are transcriptionally repressed because of the formation of
specialized chromatin structures. Performing live cell analysis we found that in
a wild-type strain the mating-type region was positioned in the proximity of the
SPB, the location where the pericentromeric heterochromatin is also found. In a
strain lacking the histone methyltransferase Clr4, crucial for the formation of
heterochromatin, the mating-type region had a random localization in the
nucleus. Moreover, in a strain in which the two boundary elements IR-L and IR-R
had been deleted, the mating-type region was displaced from its position at the
proximity of the SPB, but remained in the vicinity of the NM. Moreover, in all
investigated strains with silencing deficiencies the distance between the
mating-type region and the SPB increased. This result indicates a correlation
between transcriptional derepression and displacement of the region. Two
different models of how the mating-type chromatin is organized in the nucleus
are discussed. Formation of centromeric heterochromatin in fission yeast requires the combined
action of chromatin modifying enzymes and small RNAs derived from centromeric
transcripts. Positive feedback mechanisms that link the RNAi pathway and the
Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in
requirements for H3K9 methylation for full siRNA production and for siRNA
production to achieve full histone methylation. Nonetheless, it has been
proposed that the Argonaute protein, Ago1, is the key initial trigger for
heterochromatin assembly via its association with Dicer-independent "priRNAs."
The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1.
Here we exploit an assay for heterochromatin assembly in which loss of silencing
by deletion of RNAi or Clr-C components can be reversed by re-introduction of
the deleted gene. We showed previously that a mutant version of the RITS complex
(Tas3(WG)) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits
maintece of heterochromatin, but prevents its formation when Clr4 is removed
and re-introduced. Here we show that the block occurs with mutants in Clr-C, but
not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors,
play a more critical role in assembly when the integrity of RITS is disrupted.
Consistent with previous reports, cells lacking Clr-C components completely lack
H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low
levels of H3K9me2. Further supporting the existence of RNAi-independent
mechanisms for establishment of centromeric heterochromatin, overexpression of
clr4(+) in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at
centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants
display indistinguishable low levels of H3K9me2 (in contrast to a previous
report) challenge the model that priRNAs trigger heterochromatin formation.
Instead, our results indicate that RNAi cooperates with RNAi-independent factors
in the assembly of heterochromatin. Heterochromatin assembly in fission yeast depends on the Clr4 histone
methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2
is required for Clr4 activity at telomeres, but acts redundantly with Clr3
histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is
critical for Clr4 function during de novo centromeric heterochromatin assembly.
We identified new targets of Sir2 and tested if their deacetylation is necessary
for Clr4-mediated heterochromatin establishment. Sir2 preferentially
deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic
acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2
also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations
exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why
H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that
recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest
that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we
demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in
RNAi-deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream
of Clr4 recruitment during heterochromatin assembly. |
What is the role of LIMT lncRNA? | LINC01089 (LncRNA Inhibiting Metastasis; LIMT) is a highly conserved lncRNA, which is depleted in basal-like and in HER2-positive tumors, and the low expression of which predicts poor patient prognosis. Interestingly, EGF rapidly downregulates LIMT expression by enhancing histone deacetylation at the respective promoter. LIMT inhibits extracellular matrix invasion of mammary cells in vitro and tumor metastasis in vivo. | Author information:
(1)Department of Biological Regulation, Weizmann Institute of Science, Rehovot,
Israel.
(2)Department of Cancer Genetics, Institute for Cancer Research, Oslo University
Hospital, The Norwegian Radium Hospital, Oslo, Norway K.G. Jebsen Centre for
Breast Cancer Research, Institute for Clinical Medicine, University of Oslo,
Oslo, Norway.
(3)Department of Computer Sciences, Technion-Israel Institute of Technology,
Haifa, Israel.
(4)Division of Molecular Genome Analysis, German Cancer Research Center,
Heidelberg, Germany.
(5)Division of RNA Biology & Cancer (B150), German Cancer Research Center
(DKFZ), Heidelberg, Germany Institute of Pathology, University Hospital
Heidelberg, Heidelberg, Germany.
(6)Department of Veterinary Resources, Weizmann Institute of Science, Rehovot,
Israel.
(7)Department of Oncology, Luxembourg Institute of Health, Luxembourg City,
Luxembourg.
(8)Division of RNA Biology & Cancer (B150), German Cancer Research Center
(DKFZ), Heidelberg, Germany Institute of Pathology, University Hospital
Heidelberg, Heidelberg, Germany German Cancer Consortium (DKTK), Freiburg,
Germany Division of Cancer Research, Department of Thoracic Surgery, Faculty of
Medicine, Medical Center - University of Freiburg, University of Freiburg,
Freiburg, Germany‡
(9)Department of Computer Sciences, Technion-Israel Institute of Technology,
Haifa, Israel Agilent Laboratories, Petach-Tikva, Israel.
(10)Department of Biological Regulation, Weizmann Institute of Science, Rehovot,
Israel [email protected]. |
Is infertility characteristic of individuals with Fanconi anemia? | Yes, infertility is characteristic of individuals with Fanconi anemia. | Fanconi anemia (FA) is a hereditary chromosomal instability syndrome with cancer
predisposition. Bone marrow failure resulting in pancytopenia is the main cause
of death of FA patients. Diagnosis of FA is based on their cellular
hypersensitivity to DNA crosslinking agents and chromosome breakages. Somatic
complementation experiments suggest the involvement of at least eight genes in
FA. The gene for complementation group A (FANCA) is defective in the majority of
FA patients. We show here that mice deficient of FANCA: are viable and have no
detectable developmental abnormalities. The hematological parameters showed a
slightly decreased platelet count and a slightly increased erythrocyte mean cell
volume in mice at young age, but this did not progress to anemia. Consistent
with the clinical phenotype of FA patients, both male and female mice showed
hypogonadism and impaired fertility. Furthermore, embryonic fibroblasts of the
knock-out mice exhibited spontaneous chromosomal instability and were
hyper-responsive to the clastogenic effect of the crosslinker mitomycin C. Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular
hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA
proteins share a common pathway with BRCA proteins. To study the in vivo role of
the FA group A gene (Fanca), gene-targeting techniques were used to generate
Fanca(tm1Hsc) mice in which Fanca exons 1-6 were replaced by a
beta-galactosidase reporter construct. Fanca(tm1.1Hsc) mice were generated by
Cre-mediated removal of the neomycin cassette in Fanca(tm1Hsc) mice.
Fanca(tm1.1Hsc) homozygotes display FA-like phenotypes including growth
retardation, microphthalmia and craniofacial malformations that are not found in
other Fanca mouse models, and the genetic background affects manifestation of
certain phenotypes. Both male and female mice homozygous for Fanca mutation
exhibit hypogonadism, and homozygous females demonstrate premature reproductive
senescence and an increased incidence of ovarian cysts. We showed that fertility
defects in Fanca(tm1.1Hsc) homozygotes might be related to a diminished
population of primordial germ cells (PGCs) during migration into the gonadal
ridges. We also found a high level of Fanca expression in pachytene
spermatocytes. Fanca(tm1Hsc) homozygous males exhibited an elevated frequency of
mispaired meiotic chromosomes and increased apoptosis in germ cells, implicating
a role for Fanca in meiotic recombination. However, the localization of Rad51,
Brca1, Fancd2 and Mlh1 appeared normal on Fanca(tm1Hsc) homozygous meiotic
chromosomes. Taken together, our results suggest that the FA pathway plays a
role in the maintece of reproductive germ cells and in meiotic recombination. Gonadal function is critically dependant on regulated secretion of the
gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin
biosynthesis and release is triggered by the binding of hypothalamic GnRH to
GnRH receptor expressed on the gonadotroph cell surface. The repertoire of
regulatory molecules involved in this process are still being defined. We used
the mouse L beta T2 gonadotroph cell line, which expresses both gonadotropin
hormones, as a model to investigate GnRH regulation of gene expression and
differential display reverse transcription-polymerase chain reaction (RT-PCR) to
identify and isolate hormonally induced changes. This approach identified
Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a
differentially expressed transcript. Mutations in Fanca account for the majority
of cases of Fanconi anemia (FA), a recessively inherited disease identified by
congenital defects, bone marrow failure, infertility, and cancer susceptibility.
We confirmed expression and hormonal regulation of Fanca mRNA by quantitative
RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca
mRNA. Fanca protein was also acutely upregulated after GnRH treatment of L beta
T2 cells. In addition, Fanca gene expression was confined to mature pituitary
gonadotrophs and adult mouse pituitary and was not expressed in the immature
alpha T3-1 gonadotroph cell line. Thus, this study extends the expression
profile of Fanca into a highly specialized endocrine cell and demonstrates
hormonal regulation of expression of the Fanca locus. We suggest that this
regulatory mechanism may have a crucial role in the GnRH-response mechanism of
mature gonadotrophs and perhaps the etiology of FA. BACKGROUND: To potentially reduce late effects of maligcy, chronic
graft-versus-host disease (GVHD), endocrinopathy, and infertility in patients
with Fanconi anemia (FA) undergoing HLA-matched related donor hematopoietic cell
transplantation (HCT), we developed a regimen using fludarabine (FLU),
cyclophosphamide (CY), and anti-thymocyte globulin (ATG) followed by infusion of
T-cell depleted (TCD) bone marrow (BM) or unmanipulated umbilical cord blood
(UCB). GVHD prophylaxis consisted of cyclosporine and short course
methylprednisolone.
PROCEDURE: Between April 2000 and June 2003, 11 patients (10 aplastic anemia
(AA), 1 myelodysplastic syndrome (MDS)) underwent HCT using this regimen. Stem
cell sources were BM and UCB in eight and three patients, respectively.
RESULTS: All patients demonstrated primary engraftment. Median days to
neutrophil and platelet engraftment were 11 days (range 9-21) and 38 days (range
19-381), respectively. No patient developed GVHD after primary HCT. The patient
with MDS relapsed with AML and a maternal donor recipient experienced secondary
graft failure. For the nine FA patients with AA who underwent HLA-identical
sibling donor HCT, the Kaplan-Meier estimates of overall survival and event-free
survival (EFS) at 2 years are 100% and 82%, respectively, at a median follow-up
of 2.9 years (range 1.9-4.8).
CONCLUSIONS: In summary, a FLU-based, non-irradiation approach is effective for
FA patients with AA undergoing HLA-identical sibling donor HCT. GnRH binds its cognate G protein-coupled GnRH receptor (GnRHR) located on
pituitary gonadotropes and drives expression of gonadotropin hormones. There are
two gonadotropin hormones, comprised of a common alpha- and hormone-specific
beta-subunit, which are required for gonadal function. Recently we identified
that Fanconi anemia a (Fanca), a DNA damage repair gene, is differentially
expressed within the LbetaT2 gonadotrope cell line in response to stimulation
with GnRH. FANCA is mutated in more than 60% of cases of Fanconi anemia (FA), a
rare genetically heterogeneous autosomal recessive disorder characterized by
bone marrow failure, endocrine tissue cancer susceptibility, and infertility.
Here we show that induction of FANCA protein is mediated by the GnRHR and that
the protein constitutively adopts a nucleocytoplasmic intracellular distribution
pattern. Using inhibitors to block nuclear import and export and a GnRHR
antagonist, we demonstrated that GnRH induces nuclear accumulation of FANCA and
green fluorescent protein (GFP)-FANCA before exporting back to the cytoplasm
using the nuclear export receptor CRM1. Using FANCA point mutations that locate
GFP-FANCA to the cytoplasm (H1110P) or functionally uncouple GFP-FANCA (Q1128E)
from the wild-type nucleocytoplasmic distribution pattern, we demonstrated that
wild-type FANCA was required for GnRH-induced activation of gonadotrope cell
markers. Cotransfection of H1110P and Q1128E blocked GnRH activation of the
alphaGsu and GnRHR but not the beta-subunit gene promoters. We conclude that
nucleocytoplasmic shuttling of FANCA is required for GnRH transduction of the
alphaGSU and GnRHR gene promoters and propose that FANCA functions as a
GnRH-induced signal transducer. Reduced fertility is one clinical manifestation among other well known Fanconi
anemia features. Most recipients of allogeneic hematopoietic stem cell
transplantation suffer from secondary infertility owing to gonadal damage from
myeloablative conditioning. In order to evaluate the rate of pregcy in
Fanconi anemia transplanted patients, we performed a retrospective analysis of
female patients transplanted in 15 centers from 1976 to 2008. Among 578
transplanted Fanconi anemia patients, we identified 285 transplanted females of
whom 101 patients were aged 16 years or over. Ten became pregt (4 twice).
Before hematopoietic stem cell transplantation all had confirmed Fanconi anemia
diagnosis. Median age at transplantation was 12 years (range 5-17 years).
Conditioning regimen consisted of cyclophosphamide with or without irradiation.
During follow up, 5 of 10 patients presented signs of ovarian failure. Among
those, 2 patients spontaneously recovered regular menses, and 3 received
hormonal replacement therapy. Pregcy occurred from four to 17 years after
hematopoietic stem cell transplantation. Three patients had preterm deliveries,
one patient had a hysterectomy for bleeding. All 14 newborns had normal growth
and development without congenital diseases. In conclusion, recovery of normal
ovarian function and a viable pregcy is a realistic but relatively rare
possibility even in Fanconi anemia patients following hematopoietic stem cell
transplantation. Mechanisms of fertility recovery are discussed. Fanconi anemia (FA) is a complex cancer susceptibility disorder associated with
DNA repair defects and infertility, yet the precise function of the FA proteins
in genome maintece remains unclear. Here we report that C. elegans FANCD2
(fcd-2) is dispensable for normal meiotic recombination but is required in
crossover defective mutants to prevent illegitimate repair of meiotic breaks by
nonhomologous end joining (NHEJ). In mitotic cells, we show that DNA repair
defects of C. elegans fcd-2 mutants and FA-deficient human cells are
significantly suppressed by eliminating NHEJ. Moreover, NHEJ factors are
inappropriately recruited to sites of replication stress in the absence of
FANCD2. Our findings are consistent with the interpretation that FA results from
the promiscuous action of NHEJ during DNA repair. We propose that a critical
function of the FA pathway is to channel lesions into accurate, as opposed to
error-prone, repair pathways. Fanconi anaemia (FA) is a rare recessive disorder marked by developmental
abnormalities, bone marrow failure, and a high risk for the development of
leukaemia and solid tumours. The inactivation of FA genes, in particular FANCF,
has also been documented in sporadic tumours in non-FA patients. To study
whether there is a causal relationship between FA pathway defects and tumour
development, we have generated a mouse model with a targeted disruption of the
FA core complex gene Fancf. Fancf-deficient mouse embryonic fibroblasts
displayed a phenotype typical for FA cells: they showed an aberrant response to
DNA cross-linking agents as manifested by G(2) arrest, chromosomal aberrations,
reduced survival, and an inability to monoubiquitinate FANCD2. Fancf homozygous
mice were viable, born following a normal Mendelian distribution, and showed no
growth retardation or developmental abnormalities. The gonads of Fancf mutant
mice functioned abnormally, showing compromised follicle development and
spermatogenesis as has been observed in other FA mouse models and in FA
patients. In a cohort of Fancf-deficient mice, we observed decreased overall
survival and increased tumour incidence. Notably, in seven female mice, six
ovarian tumours developed: five granulosa cell tumours and one luteoma. One
mouse had developed tumours in both ovaries. High-resolution array comparative
genomic hybridization (aCGH) on these tumours suggests that the increased
incidence of ovarian tumours correlates with the infertility in Fancf-deficient
mice and the genomic instability characteristic of FA pathway deficiency. Fanconi anemia (FA) is a human disease of bone marrow failure, leukemia,
squamous cell carcinoma, and developmental anomalies, including hypogonadism and
infertility. Bone marrow transplants improve hematopoietic phenotypes but do not
prevent other cancers. FA arises from mutation in any of the 15 FANC genes that
cooperate to repair double stranded DNA breaks by homologous recombination.
Zebrafish has a single ortholog of each human FANC gene and unexpectedly,
mutations in at least two of them (fancl and fancd1(brca2)) lead to
female-to-male sex reversal. Investigations show that, as in human, zebrafish
fanc genes are required for genome stability and for suppressing apoptosis in
tissue culture cells, in embryos treated with DNA damaging agents, and in
meiotic germ cells. The sex reversal phenotype requires the action of Tp53
(p53), an activator of apoptosis. These results suggest that in normal sex
determination, zebrafish oocytes passing through meiosis signal the gonadal soma
to maintain expression of aromatase, an enzyme that converts androgen to
estrogen, thereby feminizing the gonad and the individual. According to this
model, normal male and female zebrafish differ in genetic factors that control
the strength of the late meiotic oocyte-derived signal, probably by regulating
the number of meiotic oocytes, which environmental factors can also alter.
Transcripts from fancd1(brca2) localize at the animal pole of the zebrafish
oocyte cytoplasm and are required for normal oocyte nuclear architecture, for
normal embryonic development, and for preventing ovarian tumors. Embryonic DNA
repair and sex reversal phenotypes provide assays for the screening of small
molecule libraries for therapeutic substances for FA. CONTEXT: In females with Fanconi anemia (FA), infertility is often accompanied
by diminished ovarian reserve and hypergonadotropic amenorrhea before the age of
30 years, suggesting primary ovarian insufficiency (POI). POI is typically
diagnosed only after perimenopausal symptoms are observed.
OBJECTIVE: The objective of the study was to assess whether serum anti-Müllerian
hormone (AMH) levels can serve as a cycle-independent marker for the diagnosis
of POI in patients with FA.
DESIGN AND SETTING: This observational study used the National Cancer
Institute's inherited bone marrow failure syndrome cohort at the National
Institutes of Health Clinical Center.
PARTICIPANTS: The study included 22 females with FA, 20 unaffected female
relatives of patients with FA, and 21 unrelated healthy females under 41 years
of age.
MAIN OUTCOME MEASURE: Serum AMH, a marker of ovarian reserve, was measured in
all participants.
RESULTS: Females with FA had very low AMH levels (median 0.05 ng/mL; range
0-2.32 ng/mL; P < .001) when compared with unaffected relatives (median 2.10
ng/mL; range 0.04-4.73 ng/mL) and unrelated healthy females (median 1.92 ng/mL;
range 0.31-6.64 ng/mL). All patients with FA older than 25 years of age were
diagnosed with POI and had undetectable AMH levels.
CONCLUSIONS: AMH deficiency appears to be a shared trait across this
heterogeneous FA cohort. Substantially reduced AMH levels in females with FA
suggest a primary ovarian defect associated with reduced fertility. Measurement
of AMH at the time of FA diagnosis and subsequent monitoring of AMH levels at
regular intervals may be useful for the timely management of complications
related to POI such as subfertility/infertility, osteoporosis, and menopausal
symptoms. CONTEXT: Endocrine problems are common in patients with Fanconi anemia (FA).
About 80% of children and adults with FA have at least one endocrine
abnormality, including short stature, GH deficiency, abnormal glucose or insulin
metabolism, dyslipidemia, hypothyroidism, pubertal delay, hypogonadism, or
impaired fertility. The goal of this report is to provide an overview of
endocrine abnormalities and guidelines for routine screening and treatment to
allow early diagnosis and timely intervention.
EVIDENCE ACQUISITION: This work is based on a comprehensive literature review,
including relevant articles published between 1971 and 2014, and proceedings of
a Consensus Conference held by the Fanconi Anemia Research Fund in 2013.
EVIDENCE SYNTHESIS: The panel of experts collected published evidence and
discussed its relevance to reflect current information about the endocrine care
of children and adults with FA before the Consensus Conference and through
subsequent deliberations that led to the consensus.
CONCLUSIONS: Individuals with FA should be routinely screened for endocrine
abnormalities, including evaluation of growth; glucose, insulin, and lipid
metabolism; thyroid function; puberty; gonadal function; and bone mineral
metabolism. Inclusion of an endocrinologist as part of the multidisciplinary
patient care team is key to providing comprehensive care for patients with FA. |
What are Kupffer cells and what is their role? | Kupffer cells (KCs)are hepatic macrophages which can secrete matrix metalloproteinases (MMPs), and can contribute to decreased hepatic insulin sensitivity. KCs may play a role in the development of drug induced liver injury (DILI) | Kupffer cells (KC), by virtue of their ability to present antigen (AP) and
express major histocompatibility complex (MHC) class II antigen (Ia), play a
pivotal role in the host defence system against invading micro-organisms.
Although haemorrhagic shock depresses the above KC functions, it is not known
whether increased KC tumour necrosis factor (TNF) production and elevated TNF
plasma levels following haemorrhage are responsible for it. To study this,
C3H/HeN mice were pretreated intraperitoneally with either anti-murine TNF
antibody (anti-TNF Ab) or saline. Twenty hours later mice were bled and
maintained at a mean blood pressure of 35 mmHg for 60 min followed by adequate
fluid resuscitation. Two and 24 hr later, plasma was collected and KC were
isolated. AP was measured by co-culturing KC with the D10.G4.1 Th cell clone. Ia
expression was determined by direct immunofluorescence. Interleukin (IL)-1, IL-6
and TNF levels in KC supernatants and plasma were measured with bioassays or
ELISA. Haemorrhage increased circulating TNF levels by 215% at 2 hr and by 76%
at 24 hr (P less than 0.05), which was prevented by pretreatment with anti-TNF
Ab. Haemorrhage-induced increase of circulating IL-6 was abolished (P less than
0.05) at 2 hr but not at 24 hr in the anti-TNF Ab group. The suppression of KC
AP (P less than 0.05) and Ia expression (P less than 0.05) due to haemorrhage
was attenuated (P less than 0.05) in anti-TNF Ab-treated mice at 2 and 24 hr and
KC IL-1 and TNF synthesis was further (P less than 0.01) increased. These
results indicate that TNF plays a critical role in the initiation and regulation
of KC AP, Ia expression, and cytokine production following haemorrhage. Low-grade tissue inflammation induced by obesity can result in insulin
resistance, which in turn is a key cause of type 2 diabetes mellitus. Cells of
the innate immune system produce cytokines and other factors that impair insulin
signalling, which contributes to the connection between obesity and the onset of
type 2 diabetes mellitus. Here, we review the innate immune cells involved in
secreting inflammatory factors in the obese state. In the adipose tissue, these
cells include proinflammatory adipose tissue macrophages and natural killer
cells. We also discuss the role of innate immune cells, such as
anti-inflammatory adipose tissue macrophages, eosinophils, group 2 innate
lymphoid cells and invariant natural killer T cells, in maintaining an
anti-inflammatory and insulin-sensitive environment in the lean state. In the
liver, both Kupffer cells and recruited hepatic macrophages can contribute to
decreased hepatic insulin sensitivity. Proinflammatory macrophages might also
adversely affect insulin sensitivity in the skeletal muscle and pancreatic
β-cell function. Finally, this Review provides an overview of the mechanisms for
regulating proinflammatory immune responses that could lead to future
therapeutic opportunities to improve insulin sensitivity. Fibrosis is the result of the abnormal accumulation of the extracellular matrix
and ineffective clearance of fibroplasia. CD4(+)CD25(+)Foxp3(+) regulatory T
cells (Tregs) are immunosuppressive lymphocytes that are highly expressed in the
fibrotic tissues and peripheral blood of patients with cirrhosis or
hepatocellular carcinoma. The role of Tregs in the progression of liver fibrosis
is not well understood. Our experiments reveal that abundant of Tregs was
scattered around sites of fibroplasia. Conversely, the depletion of Tregs
promoted the resolution of liver fibrosis. As a consequence of Tregs depletion,
the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of
metalloproteinases (TIMPs) was altered; mmp9 and timp1 were reduced, whereas
mmp2 and mmp14 were enhanced. The mmp9/timp1, mmp13/timp1, and mmp14/timp2
ratios were significantly increased in association with fibrosis resolution.
Kupffer cells (KCs) are the main source of MMP. We observed that when KCs were
cocultured with Tregs, the Tregs were able to inhibit MMP expression of KCs even
at a low ratio; and anti-transforming growth factor-β (TGF-β) significantly
reversed the inhibition of Tregs on MMP. Meanwhile, we also found that after
Tregs depletion, TGF-β levels decreased in the mice liver, unlike in fibrosis.
Furthermore, double depletion of both KCs and Tregs did not cause fiber
resolution in mice. Thus, our results demonstrate that the persistence of liver
cirrhosis is maintained by increased Tregs in the sites of fibroplasia and the
subsequent regulation of the MMP/TIMP balance and that the suppression of
KC-mediated MMP expression contributed to the regulatory process. Macrophages represent a major cell type of innate immunity and have emerged as a
critical player and therapeutic target in many chronic inflammatory diseases.
Hepatic macrophages consist of Kupffer cells, which are originated from the
fetal yolk-sack, and infiltrated bone marrow-derived monocytes/macrophages.
Hepatic macrophages play a central role in maintaining homeostasis of the liver
and in the pathogenesis of liver injury, making them an attractive therapeutic
target for liver diseases. However, the various populations of hepatic
macrophages display different phenotypes and exert distinct functions. Thus,
more research is required to better understand these cells to guide the
development of macrophage-based therapeutic interventions. This review article
will summarize the current knowledge on the origins and composition of hepatic
macrophages, their functions in maintaining hepatic homeostasis, and their
involvement in both promoting and resolving liver inflammation, injury, and
fibrosis. Finally, the current strategies being developed to target hepatic
macrophages for the treatment of liver diseases will be reviewed. Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in
the development of drug induced liver injury (DILI). Furthermore, elevated
intracellular Ca2+ level of hepatocytes is considered as a common marker of
DILI. Here we applied an in vitro model based on hepatocyte mono- and
hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing
the GCaMP2 fluorescent Ca2+ sensor protein to investigate the effects of
polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5
Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following
dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity
assay and by Ca2+ imaging, while hepatocyte functions were studied by CYP2B1/2
inducibility, and bilirubin and taurocholate transport. G5 was significantly
more cytotoxic than G4.5 for hepatocytes and induced Ca2+ oscillation and
sustained Ca2+ signals at 1μM and10 μM, respectively both in hepatocytes and
KCs. Dendrimer-induced Ca2+ signals in hepatocytes were attenuated by
macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the
inducibility of CYP2B1/2, which was restored by depleting the KCs with
gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the
hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but
not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of
bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In
conclusion, H/KC provides a good model for the prediction of hepatotoxic
potential of drugs, especially of omaterials known to be trapped by
macrophages, activation of which presumably contributes to DILI. |
What is known about saponins in crops and human consumption? | Saponins are considered antinutrients for humans and have a bitter taste. They should be removed from the crops before consumption. | Seven seed samples of J. curcas, both in raw and roasted state, sold in some
villages in Quintana Roo state, Mexico for human consumption were analyzed for
physical characteristics, nutrients and antinutrients. The average seed weight
varied from 0.53 to 0.74 g and kernel weight as proportion of raw seed weight
was from 61 to 66%. The contents of crude protein, lipid and ash of kernels from
raw seeds were 27-30%, 55-62% and 3.7-5.2% respectively. The levels of
antinutrients in meal from the raw seeds were: trypsin inhibitor activity
(14.6-28.7 mg trypsin inhibited/g), lectin (25.6-52.2 unit; one unit is the
reverse of minimum amount of mg meal/ml assay which produced haemagglutination),
saponins (1.9-2.3% as diosgenin equivalent) and phytate (8.4-10%). Phorbol
esters in kernels from raw seeds were not detected in four samples and in other
three samples it ranged from 0.01 to 0.02 mg/g as phorbol-12-myristate
13-acetate equivalent. Roasting of seeds inactivated almost 100% of trypsin
inhibitor activity. Although lectin activity reduced on roasting, it was still
present in high amounts. Saponins, phytate and phorbol esters were not affected
by roasting. Quinoa cultivars currently grown in North America and Europe require removal of
bitter-tasting saponins from the grain prior to human consumption. This need for
postharvest processing is a barrier to expanding production of the crop outside
its Andean area of origin. Grain saponin content in quinoa shows continuous
variation and is considered to be a quantitative trait. However, segregation for
the presence or absence of grain saponin in F2 generations derived from crosses
between high- and low-saponin parents indicates a major gene effect, with plants
homozygous for a recessive allele spl having no detectable grain saponin.
Variation in saponin levels among F2 plants with detectable grain saponin was
consistent with polygenic inheritance. It appears that grain saponin level in
quinoa is both qualitatively and quantitatively controlled, with saponin
production requiring at least one domit allele at the Sp locus and the amount
of grain saponin being determined by an unknown number of additional
quantitative loci. Introgression of sp1 into day-neutral lines will facilitate
the development of short-season "sweet" quinoa cultivars which do not require
postharvest processing to remove grain saponin. BACKGROUND: Rotavirus is the leading cause of severe diarrhea disease in
newborns and young children worldwide, estimated to be responsible for over
300,000 childhood deaths every year, mostly in developing countries.
Rotavirus-related deaths represent approximately 5% of all deaths in children
younger than 5 years of age worldwide. Saponins are readily soluble in water and
are approved by the US FDA for inclusion in beverages intended for human
consumption. The addition of saponins to existing water supplies offers a new
form of intervention into the cycle of rotavirus infection. We believe that
saponins will 'coat' the epithelium of the host's small intestine and prevent
attachment of rotavirus.
DISCUSSION: This experiment provides in vitro data for the possibility of
including saponin in drinking water to prevent infections of rotavirus. We
demonstrate that microgram amounts of extract, while exhibiting no cell
cytotoxicity or direct virucidal activity, prevent rotavirus from infecting its
host cells. In addition, the presence of residual amounts of extract continue to
block viral infection and render cells resistant to infection for at least 16 h
after the removal of the extract from the cell culture media.
CONCLUSION: We demonstrate that two Quillaja extracts possess strong antiviral
activity at concentrations more than 1000-fold lower than concentrations
exhibiting cell cytotoxicity. Extract concentrations as high as 1000 μg/ml are
not cytotoxic, but concentrations as low as 1.0 μg/ml are able to block
rotavirus and reovirus attachment and infection. The fruit of the cherry tomato (Lycopersicon esculentum (Solanaceae)) was
analysed for mineral and antinutrient composition. Phosphorus (33.04 ± 0.21
mg/100g) was the most abundant mineral in the fruit, followed by calcium (32.04
± 0.06 mg/100 g), and potassium (11.9 ± 0.1 mg/100 g) and manganese (9.55 ± 0.28
mg/100 g) were also present in appreciable quantities. Antinutrients, including
phytate, glycoside, saponin and tannin, were screened and quantified. Phytate
(112.82 ± 0.1 mg/100 g), glycoside (2.33 ± 0.00 mg/100 g), saponin (1.31 ± 0.00
mg/100g) and tannin (0.21 ± 0.00 mg/100 g) were present in the fruit but
phlobatanin and glycosides with steroidal rings were not found. The calculated
calcium:phytate ratio of the fruits was below the critical value and the
calculated [calcium] [phytate]:[zinc] molar ratio was less than the critical
value. The calcium:phosphorus ratio (0.97 mg/100 g) shows the fruit to be a good
source of food nutrients, while the sodium:potassium value was less than 1. Ca/P
ratio below 0.5 indicates deficiency of these minerals while Na/K ratio above 1
is detrimental because of excessive sodium levels. The results of the study
generally revealed the fruit to be rich in minerals but containing insufficient
quantities of antinutrients to result in poor mineral bioavailability. |
Is golimumab effective for ulcerative colitis? | Yes. Golimumab is a TNF-blocking agent indicated as a second-line therapy in ulcerative colitis. | Centocor Inc and licensees Schering-Plough Corp, Mitsubishi Tanabe Pharma Corp
and Janssen Pharmaceutical KK are developing golimumab, a fully human mAb
antibody against TNFalpha, for the potential treatment of rheumatoid arthritis
(RA), psoriatic arthritis (PsA), ankylosing spondylitis (AS) and ulcerative
colitis. Golimumab is currently in phase III clinical trials for RA, PsA and AS
and preliminary data have shown an improvement in a number of physical
functions, disease activity, productivity and quality-of-life measurements. Golimumab is a human IgG monoclonal antibody specific for human tumor necrosis
factor alpha. Golimumab has been approved for use in rheumatological conditions;
however, its use in inflammatory bowel disease is still being evaluated in
clinical trials. We report a case of an exacerbation of ulcerative proctitis
after starting on golimumab for ankylosing spondylitis. BACKGROUND & AIMS: Little is known about the efficacy of golimumab, a fully
human monoclonal antibody to tumor necrosis factor (TNF) -α, for treatment of
ulcerative colitis (UC). We evaluated subcutaneous golimumab induction therapy
in TNF-α antagonist-naïve patients with moderate-to-severe UC despite
conventional treatment.
METHODS: We integrated double-blind phase 2 dose-finding and phase 3
dose-confirmation trials in a study of 1064 adults with UC (Mayo score: 6-12;
endoscopic subscore ≥ 2; 774 patients in phase 3). Patients were randomly
assigned to groups given golimumab doses of 100 mg and then 50 mg (phase 2
only), 200 mg and then 100 mg, or 400 mg and then 200 mg, 2 weeks apart. The
phase 3 primary end point was week-6 clinical response. Secondary end points
included week-6 clinical remission, mucosal healing, and Inflammatory Bowel
Disease Questionnaire (IBDQ) score change.
RESULTS: In phase 2, median changes from baseline in the Mayo score were -1.0,
-3.0, -2.0, and -3.0, in the groups given placebo, 100 mg/50 mg, 200/100 mg, and
400/200 mg golimumab, respectively. In phase 3, rates of clinical response at
week 6 were 51.0% and 54.9% among patients given 200 mg/100 mg and 400 mg/200 mg
golimumab, respectively, vs 30.3% among those given placebo (both, P ≤ .0001).
Rates of clinical remission and mucosal healing and mean changes in IBDQ scores
were significantly greater in both golimumab groups vs the placebo group (P ≤
.0014, all comparisons). Rates of serious adverse events were 6.1% and 3.0%, and
rates of serious infection were 1.8% and 0.5%, in the placebo and golimumab
groups, respectively. One patient in the 400 mg/200 mg group died as a result of
surgical complications of an ischiorectal abscess.
CONCLUSIONS: Treatment with subcutaneous golimumab induces clinical response,
remission, and mucosal healing, and increases quality of life in larger
percentages of patients with active UC than placebo. ClinicalTrials.gov Number:
NCT00487539. BACKGROUND & AIMS: Subcutaneous golimumab, a fully human monoclonal antibody to
tumor necrosis factor-α (TNFα), was evaluated as maintece therapy in TNFα
antagonist-naive adults with moderate-to-severe active ulcerative colitis,
despite conventional therapy, who responded to golimumab induction therapy.
METHODS: We performed a phase 3, double-blind trial of patients who completed
golimumab induction trials (Program of Ulcerative Colitis Research Studies
Utilizing an Investigational Treatment, eg, PURSUIT). Patients who responded to
induction therapy with golimumab (n = 464) were assigned randomly to groups
given placebo or injections of 50 or 100 mg golimumab every 4 weeks through week
52. Patients who responded to placebo in the induction study continued to
receive placebo. Nonresponders in the induction study received 100 mg golimumab.
The primary end point was clinical response maintained through week 54;
secondary end points included clinical remission and mucosal healing at both
weeks 30 and 54.
RESULTS: Clinical response was maintained through week 54 in 47.0% of patients
receiving 50 mg golimumab, 49.7% of patients receiving 100 mg golimumab, and
31.2% of patients receiving placebo (P = .010 and P < .001, respectively). At
weeks 30 and 54, a higher percentage of patients who received 100 mg golimumab
were in clinical remission and had mucosal healing (27.8% and 42.4%) than
patients given placebo (15.6% and 26.6%; P = .004 and P = .002, respectively) or
50 mg golimumab (23.2% and 41.7%, respectively). Percentages of serious adverse
events were 7.7%, 8.4%, and 14.3% among patients given placebo, 50 mg, or 100 mg
golimumab, respectively; percentages of serious infections were 1.9%, 3.2%, and
3.2%, respectively. Among all patients given golimumab in the study, 3 died
(from sepsis, tuberculosis, and cardiac failure, all in patients who received
100 mg golimumab) and 4 developed active tuberculosis.
CONCLUSIONS: Golimumab (50 mg or 100 mg) maintained clinical response through
week 54 in patients who responded to induction therapy with golimumab and had
moderate-to-severe active ulcerative colitis; patients who received 100 mg
golimumab had clinical remission and mucosal healing at weeks 30 and 54. Safety
was consistent with that reported for other TNFα antagonists and golimumab in
other approved indications. ClinicalTrials.gov number: NCT00488631. Golimumab, a human anti-TNF antibody, is effective in patients with ulcerative
colitis, according to new findings from an international phase III double-blind
trial. The addition of this drug makes a ménage à trois of available
drugs--comprising infliximab, adalimumab and golimumab--for the treatment of
ulcerative colitis. The presentations at Digestive Disease Week 2013 emphasized treatment safety.
Anti-tumor necrosis factor (TNF) agents and thiopurines are reasonably safe in
breastfeeding and pregcy. Several studies indicate that controlling the risk
of tuberculosis when anti-TNF agents are planned presents several problems, both
in the initial diagnosis of latent tuberculosis and in subsequent patient
follow-up, given that cases of tuberculosis continue to occur, despite
recommendations. Thiopurines increase the risk of lymphoma, but there is no
residual risk when these drugs are withdrawn. Despite increasing knowledge of
the risks and recommendations on how to avoid them, there remain considerable
shortfalls in the application of preventive measures and, more specifically, in
vaccinations. Infliximab and cyclosporin produce similar results when used to
treat severe outbreaks of ulcerative colitis. Thromboembolism prevention
continues to be deficient, and the barriers to effective prevention concern not
only physicians but can also involve nursing staff, for example. There is still
a wide margin for improvement in safety. New drugs under study (vedolizumab,
golimumab) have not shown any hitherto unknown signs of significant toxicity. PURPOSE OF REVIEW: Ulcerative colitis is a chronic inflammatory disease of the
colon of unknown cause that is characterized by alternating intervals of active
and inactive disease in 80-90% of patients. The primary goal of treatment is to
induce and maintain remission using therapy tailored to the individual patient.
The purpose of this review was to describe the management of ulcerative colitis
with emphasis on the use of anti-tumor necrosis factor (TNF) agents.
RECENT FINDINGS: Recent research has shown that new anti-TNF agents, adalimumab
(ADA) and golimumab, are effective in induction of remission and maintece of
remission in patients with extensive ulcerative colitis. In a recent study,
infliximab was found to have comparable efficacy to cyclosporine in treatment of
acute severe refractory to corticosteroids ulcerative colitis.
SUMMARY: Anti-TNF therapy should be initiated in patients with acute severe
refractory to corticosteroids ulcerative colitis and in patients with
moderate-to-severe ulcerative colitis who are not responsive to conventional
treatment with aminosalicylates, corticosteroids and immune modulators.
Alternatives to infliximab are ADA and golimumab. Future research is needed to
further assess the long-term efficacy and safety of ADA and golimumab in
ulcerative colitis. The complex pathogenesis of immune-mediated inflammatory diseases (IMIDs) has
been extensively investigated and dysregulation of cytokines, such as tumour
necrosis factor (TNF) has been shown to play a domit role in the pathogenesis
of various IMIDs, such as rheumatoid arthritis, ankylosing spondylitis, Crohn's
disease, ulcerative colitis, psoriasis and psoriatic arthritis. The subsequent
development of biological agents capable of blocking TNF has led to important
advances in the pharmacotherapy of such diseases and confirmed the concept of a
common pathophysiology among IMIDs with TNF having a predomit role. Five TNF
inhibitors have currently been approved for treatment of one or more IMIDs;
these include infliximab, etanercept, adalimumab, golimumab and certolizumab
pegol. Given the similarities in the pathogenic background of IMIDs, one could
expect that anti-TNF agents be similarly effective and with comparable
tolerability profiles; however, this may not be the case. Structural and
pharmacological differences among the anti-TNF drugs are likely to result in
differences in efficacy and tolerability among the agents in the different
IMIDs, together with differences in potency, therapeutic dose ranges, dosing
regimens, administration routes, and propensity for immunogenicity. Among the
five TNF inhibitors approved for treatment of IMIDs, adalimumab has the widest
range of indications. Data from controlled clinical trials of adalimumab,
showing its excellent efficacy and tolerability in a wide range of indications,
are supported by real-world long-term data from observational studies, which
confirm the value of adalimumab as a suitable choice in the management of IMIDs. BACKGROUND: Biological agents are emerging treatment options for the management
of ulcerative colitis (UC).
PURPOSE: To assess the comparative efficacy and harm of biological agents in
adult patients with moderately to severely active UC who are naive to biological
agents.
DATA SOURCES: MEDLINE, EMBASE, and Cochrane Library from inception through
December 2013, without language restrictions, and ClinicalTrials.gov, European
Medicines Agency, and U.S. Food and Drug Administration Web sites.
STUDY SELECTION: Randomized, placebo-controlled or head-to-head trials assessing
biological agents as induction or maintece therapy for moderately to severely
active UC.
DATA EXTRACTION: Two reviewers independently abstracted study data and outcomes
and rated each trial's risk of bias.
DATA SYNTHESIS: There were no head-to-head trials. There were 7 double-blind,
placebo-controlled trials that were rated as low risk of bias and showed that
all biological agents (adalimumab, golimumab, infliximab, and vedolizumab)
resulted in more clinical responses, clinical remissions, and mucosal healings
than placebo for induction therapy. The results of network meta-analysis
suggested that infliximab is more effective to induce clinical response (odds
ratio, 2.36 [95% credible interval, 1.22 to 4.63]) and mucosal healing (odds
ratio, 2.02 [95% credible interval, 1.13 to 3.59]) than adalimumab. No other
indirect comparison reached statistical significance. For maintece, 6
double-blind, placebo-controlled trials that were rated high risk of bias showed
that all biological agents have greater clinical efficacy than placebo. The
occurrence of adverse events was not different between biological agents and
placebo.
LIMITATION: Few trials, no head-to-head comparisons, and inadequate follow-up in
maintece trials.
CONCLUSION: Biological agents are effective treatments for UC, but head-to-head
trials are warranted to establish the best therapeutic option. BACKGROUND AND AIM: The aim of this systematic review was to evaluate the
efficacy and safety of biological agents (vedolizumab, abatacept, visilizumab,
golimumab) in patients with active moderate to severe ulcerative colitis.
METHODS: This paper was prepared according to the Preferred Reporting Items for
Systematic Reviews and Meta-Analysis guidelines. The systematic literature
search was performed in PubMed, Embase, Cochrane Library, and other databases
until December 27, 2013 to identify randomized controlled trials fulfilling the
established inclusion criteria for this review.
RESULTS: Eight randomized controlled trials were included in the systematic
review. Vedolizumab was significantly more effective compared with placebo (P <
0.05) increasing the percentage of patients with a clinical response, clinical
remission and mucosal healing in the induction phase, and patients with a
clinical remission and mucosal healing in the maintece phase. Similarly,
golimumab was significantly more effective than placebo (P < 0.05) regarding the
percentage of patients with a clinical response and mucosal healing in the
induction phase, and patients with a clinical response, clinical remission, and
mucosal healing in the maintece phase. The safety of these two biological
agents was comparable with placebo during the treatment (P > 0.05). However, the
efficacy of visilizumab or abatacept was related to the higher risk of treatment
failure and a worse safety profile than placebo.
CONCLUSIONS: The results of the systematic review demonstrated that the efficacy
and safety of particular biological agents are differentiated. Vedolizumab and
golimumab occurred more effective, and comparably as safe as placebo in patients
with active moderate to severe ulcerative colitis increasing the number of
available therapeutic options. Ulcerative colitis can cause debilitating symptoms and complications such as
colonic strictures, colonic dysplasia, colorectal cancer, and toxic megacolon or
perforation. Goals of treatment in ulcerative colitis include resolution of
gastrointestinal symptoms, healing of colonic mucosa, and prevention of disease
complications. Our treatment armamentarium has expanded dramatically over the
past 10 years, and we now have multiple biologic agents approved for the
treatment of moderate-severe disease, in addition to conventional therapies such
as 5-aminosalicylates, thiopurines, and corticosteroids. In this review, we will
provide a detailed discussion of the three tumor necrosis factor-alpha (TNF-α)
inhibitors currently approved for treatment of ulcerative colitis: infliximab,
adalimumab, and golimumab. All three agents are effective for inducing and
maintaining clinical response and remission in patients with ulcerative colitis,
and they have comparable safety profiles. There are no head-to-head trials
comparing their efficacy, and the choice of agent is most often based on
insurance coverage, route of administration, and patient preference. Combination
therapy with an immunomodulator is proven to be more effective than anti-TNF
monotherapy, and patients who lose response to an anti-TNF agent should undergo
dose intensification in order to regain clinical response. Despite therapeutic
optimization, a significant percentage of patients will not achieve clinical
remission with anti-TNF agents, and so newer therapies are on the horizon. Biological agents for inflammatory bowel diseases (IBD) targeting tumor necrosis
factor (TNF) have changed the way to treat IBD refractory to standard
medications and allowed us to reach new therapeutic goals such as mucosal
healing and deep remission. A better understanding of the components of the
pathological processes that are a hallmark of IBD has led to the development of
a new family of biological agents in Crohn's disease and ulcerative colitis.
Biosimilars, which are copy versions of currently licensed biological agents,
will be soon available. The biosimilar of infliximab is as effective and as safe
as its originator in rheumatologic conditions, while a new anti-TNF agent,
namely golimumab, has been recently approved for refractory ulcerative colitis.
Beyond TNF blockers, anti-adhesion molecules appear to be a potent drug class
for IBD. Vedolizumab was recently approved for both Crohn's disease and
ulcerative colitis. Numerous other compounds are in the pipeline. Ustekinumab
looks very promising for Crohn's disease. Smad7 antisense oligonucleotide might
enrich our armamentarium if preliminary data are confirmed in upcoming clinical
trials. Herein, we review the efficacy and safety of new and emerging biological
agents that are currently investigated in IBD clinical trials. Monoclonal antibodies directed against tumor necrosis factor alpha (anti-TNF-α
agents) have dramatically changed the therapeutical approach to inflammatory
bowel diseases, such as Crohn's disease and ulcerative colitis. A new anti-TNF
drug, golimumab, has recently been approved for patients with moderate to severe
ulcerative colitis. Its efficacy has been demonstrated by preclinical and
clinical studies and the drug showed an efficacy and safety profile in line with
the other anti-TNF agents, such as infliximab and adalimumab. This review gives
an overview on golimumab in the treatment of moderate to severe ulcerative
colitis. BACKGROUND AND AIMS: Golimumab has been approved recently to treat refractory
moderate-to-severe ulcerative colitis [UC]. To date it is not clear why a
considerable fraction of patients do not respond, or lose initial response, to
golimumab therapy. Our aim was to investigate whether a low golimumab serum
concentration and/or a positive anti-golimumab antibody status reduces the
efficacy of this drug in patients with UC.
METHODS: Serum samples of 21 patients with moderate-to-severe UC were collected
during the first 14 weeks of golimumab therapy. For measurement of golimumab
serum concentrations, both a tumour necrosis factor [TNF]-coated enzyme-linked
immunosorbent assay [ELISA] and a sandwich-type ELISA were developed.
Anti-golimumab antibodies were measured using a bridging ELISA and a
newly-developed drug-tolerant immunoassay. Clinical response and mucosal healing
were assessed 14 weeks after start of treatment.
RESULTS: Out of 21 patients, 10 [48%] reached partial clinical response at Week
14. Median [interquartile range] serum golimumab concentration was significantly
higher in partial clinical responders than in non-responders: 10.0 [7.8-10.5]
µg/ml versus 7.4 [4.8-8.3] µg/ml at Week 2 [p = 0.035] and 5.1 [4.0-7.9] µg/ml
versus 2.1 [1.8-4.2] µg/ml at week 6 [p = 0.037]. Four out of 21 UC patients
developed anti-golimumab antibodies, detectable only using a drug-tolerant
immunoassay, and three had a partial clinical response at that time. Clinical
non-responders had a significantly more severe colitis, indicated by a higher
endoscopic Mayo score at baseline compared with partial clinical responders [p =
0.048].
CONCLUSION: Adequate exposure to golimumab drives clinical response. A worse
disease at baseline influences clinical response rate negatively. BACKGROUND: Golimumab is a TNF-blocking agent indicated as a second-line therapy
in ulcerative colitis.
PURPOSE: To research the effectiveness and safety of golimumab in patients with
ulcerative colitis in clinical practice.
METHODS: Retrospective study of the effectiveness and safety of golimumab in
patients with ulcerative colitis. All patients received golimumab 200 mg
subcutaneously at week 0, and golimumab 100 mg subcutaneously at week 2. After
the induction treatment, each patient received 50 mg sc. every 4 weeks in
patients with body weight less than 80 kg, and 100 mg every 4 weeks in patients
with body weight greater than or equal to 80 kg.
RESULTS: Study of a group of 23 ulcerative colitis patients, 7 of whom were
naive to any anti-TNF therapy, and 16 patients who had previously been treated
with an anti-TNF agent other than golimumab (non-naive patients). The average
treatment time with golimumab was 14.3 weeks. Globally, withdrawal of
corticosteroids was observed in 74% of cases. Clinical response was observed in
85.5% of patients who had not received biological treatment previously, and in
patients who had previously received biological treatment the response rate was
75%.
CONCLUSIONS: In this short study, golimumab seems to be an alternative treatment
in naive and non-naive anti-TNF ulcerative colitis patients. It is also a safe
therapy, given that there were no adverse effects in the patients studied. Significant advances in the management of patients with ulcerative colitis (UC)
have been made since the introduction of anti-tumor necrosis factor (TNF)-alpha
agents, especially for those who fail or do not tolerate conventional therapies.
Two drugs, infliximab first, then adalimumab afterward, showed effectiveness in
inducing and maintaining long-term remission both in pivotal trials as well as
in clinical practice. However, approximately 25% of patients with UC, who fail
or do not tolerate all available therapies, require a colectomy for refractory
disease. The therapeutic scenario of UC has been recently upgraded by the
introduction of golimumab, the latest anti TNF-alpha agent to be approved.
Golimumab is a totally humanized monoclonal antibody, administered by a
subcutaneous injection every 4 weeks. Treatment with golimumab has shown to be
effective to induce sustained clinical benefit in tough-to-treat patients with
UC, including steroid and/or immunosuppressive refractory and steroid-dependent
patients. In this review, we summarize all available efficacy and safety data of
golimumab in UC, analyzing the potential therapeutic position for the treatment
of refractory patients with UC. Ulcerative colitis is a chronic inflammatory condition of the colon,
characterized by diffuse mucosal inflammation and blood-mixed diarrhea. The main
treatment has been 5-aminosalicylic acid, steroid, thiopurine, and anti-tumor
necrosis factor alpha (TNF-α) antibodies including infliximab, adalimumab, and
golimumab. Golimumab, a new anti-TNF-α agent has been recently approved for
patients with moderate to severe ulcerative colitis. Its efficacy and safety has
been demonstrated in line with infliximab and adalimumab in preclinical and
clinical studies. This review will focus on golimumab therapy in ulcerative
colitis. BACKGROUND AND AIMS: To assess golimumab pharmacokinetics [PK] and
exposure-response [ER] in adults with moderate-to-severe ulcerative colitis [UC]
from the Program of Ulcerative Colitis Research Studies Utilizing an
Investigational Treatment [PURSUIT] studies.
METHODS: We analysed golimumab PK and ER data of patients with
moderate-to-severe UC from the PURSUIT-subcutaneous induction [N = 1064] and
maintece [N = 464] studies. Induction analyses evaluated serum golimumab
concentration [SGC] and efficacy data through Week [wk] 6 following subcutaneous
doses at wk0 and wk2; maintece analyses assessed data through wk54 following
4-weekly dosing. ER relationships were assessed using trend, logistic
regression, and receiver-operating-characteristic curve analyses.
RESULTS: Median SGCs peaked at induction wk2 for golimumab 100/50mg, 200/100mg,
and 400/200mg. Wk6 median SGCs were 0.78, 1.78, and 4.01 μg/ml, respectively.
SGCs were sustained, reaching steady state approximately 8wks after golimumab
maintece commenced [wk14 of golimumab] regardless of induction dose. Median
trough SGCs from maintece wks8-44 ranged from 0.69 to 0.83 µg/ml [50 mg] and
1.33-1.58 µg/ml [100 mg]. SGCs were approximately dose proportional, and higher
SGCs were associated with higher efficacy response rates during induction and
maintece. Factors associated with golimumab exposure were body weight,
antibody-to-golimumab status, serum albumin, alkaline phosphatase, faecal
markers, C-reactive protein, and pancolitis. SGCs of 2.5 µg/ml [induction wk6]
and 1.4 µg/ml [maintece steady-state trough] are potential target
concentrations. Immunomodulators had no apparent impact on SGC with golimumab
100mg, whereas drug levels were slightly higher with golimumab 50mg with vs
without immunomodulators.
CONCLUSIONS: SGCs are approximately dose proportional, and a positive
SGC-efficacy relationship exists during induction/maintece golimumab
treatment of adult UC patients. Optimal SGC targets require validation in
prospective studies. INTRODUCTION: The aim of the study was to compare adalimumab or golimumab with
infliximab in patients with moderately-to-severely active ulcerative colitis
(UC).
MATERIAL AND METHODS: This paper was prepared according to the PRISMA
guidelines. The systematic literature search was performed in PubMed, Embase,
and Cochrane Library. No direct head-to-head comparisons for infliximab vs.
adalimumab or golimumab were available so an indirect comparison according to
the Bucher method was performed after a homogeneity evaluation of the included
studies.
RESULTS: Six RCTs were included in the systematic review. An indirect comparison
was performed, which revealed that infliximab was more effective in inducing
clinical response compared with both doses of adalimumab (160/80 mg or 80/40 mg;
p < 0.05), and, in clinical remission, infliximab was more effective than
adalimumab (only for a dosage regime of 80/40 mg; p < 0.05). No statistically
significant differences in clinical response and clinical remission were
observed between infliximab and golimumab in the induction phase. A significant
(p < 0.05) advantage only of infliximab compared with adalimumab at doses of
80/40 mg and 80/160 mg was seen in terms of clinical response in the maintece
phase (up to 52-54 weeks). The indirect comparison revealed that serious adverse
events were significantly more frequent among patients treated with a
maintece dose of 100 mg of golimumab compared with those treated with
infliximab (p < 0.05).
CONCLUSIONS: No significant differences in efficacy in the maintece phase
between infliximab and golimumab or adalimumab were revealed. Infliximab proved
to be more effective than adalimumab but of similar efficacy to that of
golimumab in the induction phase. Recently, several medical treatments for ulcerative colitis (UC) have been
developed, including 5-aminosalicylic acids (5-ASAs), corticosteroids,
thiopurine, calcineurin inhibitors, and anti-tumor necrosis factor (TNF) α
treatments. Treatment options including calcineurin inhibitors and anti-TNF
treatment for refractory UC are discussed in this article. Furthermore, upcoming
treatments are introduced, such as golimumab, vedolizumab, AJM300, tofacitinib.
Budesonide foamwill be used as one treatment option in patients with distal
colitis. Herbal medicine, such as Qing-Dai is also effective for active UC and
may be useful for patients who are refractory to anti-TNFα treatments. In the
near future, physicians will able to use many different treatments for UC
patients. However, we should not forget 5-ASA and corticosteroids as the
fundamental treatments for UC patients. Remarkable advances have been made in the treatment of inflammatory bowel
disease since the introduction of anti-tumor necrosis factor-α agents,
especially for patients who are refractory to or cannot tolerate conventional
therapies. Currently, infliximab, adalimumab, and golimumab are available in the
East Asian medical market, and these agents have been shown to be effective for
inducing and maintaining long-term remission of inflammatory bowel disease.
Despite their clinical benefits, anti-tumor necrosis factor therapy can also
lead to increased vulnerability to infections, development of autoimmune
diseases and maligcy, and decreased immunogenicity of vaccinations. Because
infectious diseases, such as tuberculosis, hepatitis, and influenza, remain
major health problems in East Asia, more cautious use of biologics is needed. To
further improve treatment efficacy and safety, close monitoring of inflammation,
regular surveillance for maligcy, and regularly scheduled vaccinations are
needed. Treatment strategies for biologics should be customized to meet the
needs of different patients. |
What is the role of histone variant H2A.W? | The histone variant H2A.W defines heterochromatin and promotes chromatin condensation in Arabidopsis. The histone variant H2A.W marks heterochromatin specifically and acts in synergy with heterochromatic marks H3K9me2 and DNA methylation to maintain transposon silencing. In vivo, H2A.W is required for heterochromatin condensation, demonstrating that H2A.W plays critical roles in heterochromatin organization. In vitro, H2A.W enhances chromatin condensation by promoting fiber-to-fiber interactions via its conserved C-terminal motif. In non-flowering land plants, we identify a new class of H2A variants and propose their possible role in the emergence of the H2A.W variant class in flowering plants. | Nucleosomal core histones (H2A, H2B, H3 and H4) must be assembled, replaced or
exchanged to preserve or modify chromatin organization and function according to
cellular needs. Histone chaperones escort histones, and play key functions
during nucleosome assembly/disassembly and in nucleosome structure
configuration. Because of their location at the periphery of nucleosome, histone
H2A-H2B dimers are remarkably dynamic. Here we focus on plant histone H2A/H2B
chaperones, particularly members of the NUCLEOSOME ASSEMBLY PROTEIN-1 (NAP1) and
FACILITATES CHROMATIN TRANSCRIPTION (FACT) families, discussing their molecular
features, properties, regulation and function. Covalent histone modifications
(e.g. ubiquitination, phosphorylation, methylation, acetylation) and H2A
variants (H2A.Z, H2A.X and H2A.W) are also discussed in view of their crucial
importance in modulating nucleosome organization and function. We further
discuss roles of NAP1 and FACT in chromatin-based processes, such as
transcription, DNA replication and repair. Specific functions of NAP1 and FACT
are evident when their roles are considered with respect to regulation of plant
growth and development and in plant responses to environmental stresses. Future
major challenges remain in order to define in more detail the overlapping and
specific roles of various members of the NAP1 family as well as differences and
similarities between NAP1 and FACT family members, and to identify and
characterize their partners as well as new families of chaperones to understand
histone variant incorporation and chromatin target specificity. Among eukaryotes, the four core histones show an extremely high conservation of
their structure and form nucleosomes that compact, protect, and regulate access
to genetic information. Nevertheless, in multicellular eukaryotes the two
families, histone H2A and histone H3, have diversified significantly in key
residues. We present a phylogenetic analysis across the green plant lineage that
reveals an early diversification of the H2A family in unicellular green algae
and remarkable expansions of H2A variants in flowering plants. We define motifs
and domains that differentiate plant H2A proteins into distinct variant classes.
In non-flowering land plants, we identify a new class of H2A variants and
propose their possible role in the emergence of the H2A.W variant class in
flowering plants. |
Which are the main brain dysfunctions caused by hyperbilirubinemia? | Bilirubin-induced neurologic dysfunction (BIND) and classical kernicterus are the main dysfunctions of hyperbilirubinemia, whenever bilirubin levels exceed the capacity of the brain defensive mechanisms in preventing its entrance and cytotoxicity. Bilirubin accumulation may lead to deficits in auditory, cognitive, and motor processing, due to neuronal cell death, reduced myelination and glial activation. | Astrocytic reaction to perinatal brain damage, which is caused by
hyperammonemia, liver disease, hyperbilirubinemia, and a few other conditions,
was studied using immunohistochemical methods for the demonstration of glial
fibrillary acidic protein (GFAP). We found no increase in GFAP expression in
those areas where Alzheimer II astrocytes usually proliferate. Diffuse
astrocytic proliferation in the white matter and focal reaction in gray matter,
which we ascribe to complicating factors, the foremost of which is anoxia, was
found in many of the cases. Two groups of neonates, 30 cases with hyperbilirubinemia (307.8 +/- 99.86
mumol/L) and 30 normal newborns (control group with bilirubin 80.37 +/- 35.74
mumol/L), were evaluated with Neonatal Behavioral Assessment Scale (NBAS) and
followed up at the age of 2 months and 5 months. All cases in the two groups
were evaluated with NBAS at ages of 2-3 days, 12-14 days and 26-28 days. The
results showed that there were statistically significant differences between the
two groups in the aspects of attention and social responsiveness, the ability to
modulate state of consciousness, the motor integrity and the reflexes. We also
found that while the level of bilirubin was below 205.2 mumol/L, there was no
correlation between the level of bilirubin and NBAS (r = -0.095, P > 0.02), but
when bilirubin was at or above the level of 205.2 mumol/L, there was significant
negative correlation (r = -0.53, P < 0.001) between the two. The follow-up
revealed that 9 of 23 jaundiced infants had abnormal auditory reactions or
abnormal muscle tonus at the age of 2 months; 8 of the 9 infants recovered, but
1 still had abnormal muscle tonus by the age of 5 months. The study suggests
that hyperbilirubinemia above the level of 205.2 mumol/L would affect neonatal
behavior and the greater the severity of jaundice, the more significant the
influence on neonatal behavior. The effects on some jaundiced infants may last 5
months or more. NBAS is a valuable scale for early detection of mild brain
dysfunctions which are less obvious on clinical examination. In 1994 the American Academy of Pediatrics recommended more liberal rules for
the treatment of hyperbilirubinemia in healthy term newborns. Yet, the safety of
moderate degrees of hyperbilirubinemia in healthy term newborns is debated. To
evaluate the safety of moderate degrees of hyperbilirubinemia, we assessed
neurologic condition of 20 healthy nonhemolytic term newborns with peak total
serum bilirubin levels of 233-444 micromol/L and 20 control infants matched for
sex and gestational age at birth. Neurologic condition was evaluated with
techniques focusing on the presence of minor neurologic dysfunction: in the
newborn period according to Prechtl, at 3 mo on the basis of the quality of
general movements, and at 12 mo according to Touwen. Moderate hyperbilirubinemia
turned out to be associated with a significant increase in minor neurologic
dysfunction throughout the first year of life. At 12 mo a strong dose-response
relationship between the degree of hyperbilirubinemia and the severity of minor
neurologic dysfunction was present. Our results indicate that total serum
bilirubin levels 335 micromol/L should be avoided. Bilirubin toxicity remains a significant problem despite recent advances in the
care of jaundiced (hyperbilirubinemic) neonates. A recent surge in reported
cases of classical kernicterus, due in part to earlier hospital discharge and
relaxation of treatment criteria for hyperbilirubinemia, and new reports of
hyperbilirubinemia-induced auditory dysfunction using evoked potential based
infant testing and hearing screening, underscore the need to better understand
how hyperbilirubinemia causes brain damage in some infants, especially because
the damage is preventable. Recent progress in understanding bilirubin binding
and neurotoxicity resulting from unbound or "free" unconjugated bilirubin, how
bilirubin affects the central nervous system in vivo and in vitro, and the use
of new clinical tools in neonates, for example magnetic resoce imaging
revealing bilateral lesions in globus pallidus and subthalamus, and abnormal
brainstem auditory evoked potentials with normal inner ear function, may lead to
improved detection and prevention of neurologic dysfunction and damage from
bilirubin. Finally, the concern is raised that partial or isolated neurologic
sequelae, for example auditory neuropathy and other central auditory processing
disorders, may result from excessive amount and duration of exposure to free,
unconjugated bilirubin at different stages of neurodevelopment. Nerve cell injury induced by unconjugated bilirubin (UCB) has been implicated in
brain damage during severe neonatal hyperbilirubinemia, although the molecular
mechanisms underlying UCB neurotoxicity are still not clarified. It has been
suggested recently that there is an association between hyperbilirubinemia and
long-term neurologic dysfunctions. We incubated immature neurons with UCB to
evaluate the short- and long-term effects of UCB on apoptotic death and on
neuritic outgrowth and ramification. We also evaluated whether mature neurons,
exposed previously to UCB in an early stage of differentiation, are more
sensitive to apoptosis or to neuritic breakdown when treated with inflammatory
agents, such as lipopolysaccharide and tumor necrosis factor-alpha. Results show
that exposure of immature neurons to UCB increased apoptosis and provoked a
reduction of both neurite extension and number of nodes. These injurious effects
observed in immature cells treated with UCB were increasingly perpetuated along
cell differentiation, as compared to neurons incubated in the absence of UCB. In
addition, neurons that were exposed to UCB when immature showed an increased
susceptibility to death by apoptosis, as well as an additional decrease in
neurite outgrowth when incubated with an inflammatory agent afterward. This work
shows, for the first time, that UCB induces neurite changes consistent with
neurodevelopment abnormalities. Furthermore, pre-exposure to UCB followed by an
inflammatory stimulus leads to an enhanced susceptibility to long-term
apoptosis, as well as a greater neuritic breakdown. These data support the
association between neonatal hyperbilirubinemia and the later development of
mental illness, such as schizophrenia. Hyperbilirubinemia may accompany harmful effects such as jaundice, brain
dysfunction, and pharmacokinetic alterations of drugs. Clinical drugs are the
important causes of hyperbilirubinemia, especially for patients with certain
pathologic conditions or with genetic variations. This article reviews
hyperbilirubinemic pathophysiology with respect to the effects of clinical
drugs. In addition, this review introduces a new formula that may be utilized to
estimate the annual occurrences of drug-induced hyperbilirubinemia in a
hospital. Variations in the genes of UDP-glucuronosyltransferases, organic
anion-transporting polypeptides and multidrug resistance proteins are the
predisposing factors for drug-induced hyperbilirubinemia; therefore, their
genetic and ethnic polymorphisms are discussed. INTRODUCTION: "Kernicterus" is a term currently used to describe bilirrubin
induced brain injury in the neuro-pathological studies. This is a confusing term
and nowadays we prefer bilirrubin encephalopathy or bilirrubin induced
neurological dysfunction. The clinical signs vary and it is clearly decreasing
in prevalence in developed countries.
MATERIAL AND METHODS: We review a series of 7 patients with bilirrubin
encephalopathy and variable neurological manifestations, who were seen in the
Neuropaediatric Department in the last 10 years. Only one patient died in the
neonatal period with hyperbilirubinaemia, sepsis and multi-organ failure.
RESULTS: Diverse aetiological factors were related to hyperbilirubinaemia. All
patients had clinical symptoms due to hyperbilirubinaemia. Neuroimaging during
the neonatal period showed involvement of the nucleus pallidus, with
hyperintensity in T1 in the brain MR scan as the most consistent finding. All
the patients who survived developed neurological signs and we try to correlate
them with biochemical, clinical, neuroimaging and neurophysiological parameters.
CONCLUSIONS: An increase in the number of patients with bilirrubin
encephalopathy has been observed over the last few years, and we attempt to find
out the causes. The increased survival of the low birth weight newborns, the
increase in the immigration population and the use of diagnostic neuroimaging
contribute to this increase. It is a great challenge for the neonatologist and
for neuropaediatricians to prevent its occurrence and to minimise the effects of
bilirrubin encephalopathy. OBJECTIVE: To explore the characteristics of auditory brainstem response (ABR)
in neonatal hyperbilirubinemia induced by different causes.
METHODS: A total of 88 neonates (176 ears) with hyperbilirubinemia were divided
into several groups according to the causes and followed up after 42 d, and 15
normal neonatus (30 ears) were measured ABR and analyzed the results.
RESULTS: The thresholds of ABR in glucose-6-phosphate dehydrogenase were the
highest in all the groups and had the longest incidence rate. The wave III, V
latencies and III-V, I-V interwave intervals of the ABR were significantly
difference and prolonged during test in comparison to the latencies in the
control group (P < 0.05). The neonatal infections group had the longest wave and
interwave intervals, followed by ABO incompatibility hemolytic diseases and the
unknown cause groups, while the breastfeeding jaundice were the shortest in the
groups of neonatal hyperbilirubinemia. The thresholds of ABR in the
hyperbilirubinemia caused by several etiologies were significant abnormality
when compared with the single etiology. However, they were similar in the wave
latencies and interwave intervals of ABR. During the follow up, the ABR wave
latencies and interwave intervals except for interwave latency I-III were
significantly shorter.
CONCLUSIONS: The toxicity of hyperbilirubinemia to the auditory nervous system
are related to the species and number of etiologies. The neonate
hyperbilirubinemia caused by glucose-6-phosphate dehydrogenase, infections, ABO
incompatibility hemolytic diseases and many etiologies are much more dangerous
to the auditory system than the breastfeeding jaundice. The damages of
hyperbilirubinemia to the auditory nervous system are reversible probably. BACKGROUND AND OBJECTIVES: Despite the implementation of screening guidelines to
identify infants at risk for hyperbilirubinemia, chronic bilirubin
encephalopathy (CBE) continues to be reported worldwide in otherwise healthy
infants. The incidence of CBE in Canada is unknown. The objectives of this study
were to establish the incidence of CBE in Canada and identify epidemiological
and medical risk factors associated with its occurrence.
METHODS: Data on infants were collected prospectively through the Canadian
Pediatric Surveillance Program. Infants born between January 1, 2007 and
December 31, 2008 were included if they either had symptoms of CBE and a history
of hyperbilirubinemia, or if they presented in the newborn period with severe
hyperbilirubinemia and an abnormal MRI finding as per the reporting physician.
RESULTS: During the study period, 20 cases were identified; follow-up data were
available for 14 of these. The causes for the hyperbilirubinemia included
glucose-6-phosphate dehydrogenase deficiency (n = 5), sepsis (n = 2), ABO
incompatibility and other red blood cell antibodies (n = 7). Fifteen infants had
abnormal brain MRI findings during the neonatal period. At follow-up, 5 infants
developed classic choreoathetoid cerebral palsy, 6 had spectrum of neurologic
dysfunction and developmental delay (as described by the reporting physician),
and 3 were healthy.
CONCLUSIONS: CBE continues to occur in Canada at an incidence that appears to be
higher than previously reported. High levels of serum unconjugated bilirubin (UCB) in newborns are associated
with axonal damage and glial reactivity that may contribute to subsequent
neurologic injury and encephalopathy (kernicterus). Impairments in myelination
and white matter damage were observed at autopsy in kernicteric infants. We have
recently reported that UCB reduces oligodendrocyte progenitor cell (OPC)
survival in a pure OPC in vitro proliferative culture. Here, we hypothesized
that neonatal hyperbilirubinemia may also impair oligodendrocyte (OL) maturation
and myelination. We used an experimental model of hyperbilirubinemia that has
been shown to mimic the pathophysiological conditions leading to brain
dysfunction by unbound (free) UCB. Using primary cultures of OL, we demonstrated
that UCB delays cell differentiation by increasing the OPC number and reducing
the number of mature OL. This finding was combined with a downregulation of
Olig1 mRNA levels and upregulation of Olig2 mRNA levels. Addition of UCB, prior
to or during differentiation, impaired OL morphological maturation, extension of
processes and cell diameter. Both conditions reduced active guanosine
triphosphate (GTP)-bound Rac1 fraction. In myelinating co-cultures of dorsal
root ganglia neurons and OL, UCB treatment prior to the onset of myelination
decreased oligodendroglial differentiation and the number of myelinating OL,
also observed when UCB was added after the onset of myelination. In both
circumstances, UCB decreased the number of myelin internodes per OL, as well as
the myelin internode length. Our studies demonstrate that increased
concentrations of UCB compromise myelinogenesis, thereby elucidating a potential
deleterious consequence of elevated UCB. Hyperbilirubinemia remains one of the most frequent clinical diagnoses in the
neonatal period. This condition may lead to the deposition of unconjugated
bilirubin (UCB) in the central nervous system, causing nerve cell damage by
molecular and cellular mechanisms that are still being clarified. To date, all
the studies regarding bilirubin-induced neurological dysfunction were performed
in monotypic nerve cell cultures. The use of co-cultures, where
astrocyte-containing culture inserts are placed on the top of neuron cultures,
provides the means to directly evaluate the cross-talk between these two
different cell types. Therefore, this study was designed to evaluate whether
protective or detrimental effects are produced by astrocytes over UCB-induced
neurodegeneration. Our experimental model used an indirect co-culture system
where neuron-to-astrocyte signaling was established concomitantly with the 24 h
exposure to UCB. In this model astrocytes abrogated the well-known UCB-induced
neurotoxic effects by preventing the loss of cell viability, dysfunction and
death by apoptosis, as well as the impairment of neuritic outgrowth. To this
protection it may have accounted the induced expression of the multidrug
resistance-associated protein 1 and the 3.5-fold increase in the values of
S100B, when communication between both cells was established independently of
UCB presence. In addition, the presence of astrocytes in the neuronal
environment preserved the UCB-induced increase in glutamate levels, but raised
the basal concentrations of nitric oxide and TNF-α although no UCB effects were
noticed. Our data suggest that bidirectional signalling during astrocyte-neuron
recognition exerts pro-survival effects, stimulates neuritogenesis and sustains
neuronal homeostasis, thus protecting cells from the immediate UCB injury. These
findings may help explain why irreversible brain damage usually develops only
after the first day of post-natal life. BACKGROUND: Hyperbilirubinemia in living liver donor is very common, but the
causes are still unclear.
AIMS: We sought to clarify the risk factors and predictors of nonobstructive
hyperbilirubinemia among living donors.
METHODS: We divided 210 consecutive right liver lobe donors into two groups
according to the peak total bilirubin postoperatively. We collected data on
preoperative, intraoperative, and postoperative biochemical measurements
retrospectively, performing multivariate logistic regression analysis adjusting
for potential confounders of the risk of hyperbilirubinemia.
RESULTS: There were significant differences between the two groups in donor age,
body mass index, operative time, blood loss, macrovescicular steatosis,
allogeneic blood transfusion rate, intensive care unit stay, hospital stay and
Clavien score after donation (P < .05). Age, graft/donor weight, operative time,
and blood loss were significantly associated with the risk of hyperbilirubinemia
upon logistic regression analysis.
CONCLUSION: Hyperbilirubinemia, one type of hepatic dysfunction after a living
donor procedure, was associated with multiple independent risk factors. BACKGROUND: Whether hyperbilirubinemia suppresses electrophysiological activity
of the neonatal auditory brainstem remains to be investigated.
AIM: To determine whether hyperbilirubinemia suppresses the brainstem auditory
electrophysiology in term neonates.
METHODS: Maximum length sequence brainstem auditory evoked response (MLS BAER)
was recorded shortly after confirming hyperbilirubinemia in 58 term neonates.
Wave amplitudes of the response were analyzed in detail.
RESULTS: Compared with age-matched term controls, the neonates with
hyperbilirubinemia showed a significant reduction in the amplitudes of MLS BAER
waves III and particularly V at all click rates 91-910/s. The reduction tended
to be more significant at higher than lower rates. Wave I amplitude was reduced
at 910/s. V/I amplitude ratio was decreased at all click rates. Therefore, the
amplitudes of MLS BAER, particularly later, waves were all reduced. The
amplitudes of all MLS BAER waves tended to be reduced with the increase in total
serum bilirubin level. All wave amplitudes were correlated with the level of
total serum bilirubin at some or most click rates.
CONCLUSIONS: Brainstem auditory electrophysiology is suppressed in neonates with
hyperbilirubinemia, which related to the severity of hyperbilirubinemia. Wave
amplitudes are valuable BAER variables to detect functional impairment of the
brainstem and auditory pathway in neonatal hyperbilirubinemia, and are
recommended to be used in assessing bilirubin neurotoxicity to the neonatal
brain. Bilirubin-induced neurologic dysfunction (BIND) and kernicterus has been used to
describe moderate to severe neurologic dysfunction observed in children exposed
to excessive levels of total serum bilirubin (TSB) during the neonatal period.
Here we use a new mouse model that targets deletion of the Ugt1 locus and the
Ugt1a1 gene in liver to promote hyperbilirubinemia-induced seizures and central
nervous system toxicity. The accumulation of TSB in these mice leads to diffuse
yellow coloration of brain tissue and a marked cerebellar hypoplasia that we
characterize as kernicterus. Histologic studies of brain tissue demonstrate that
the onset of severe neonatal hyperbilirubinemia, characterized by seizures,
leads to alterations in myelination and glia reactivity. Kernicterus presents as
axonopathy with myelination deficits at different brain regions, including pons,
medulla oblongata, and cerebellum. The excessive accumulation of TSB in the
early neonatal period (5 days after birth) promotes activation of the myelin
basic protein (Mbp) gene with an accelerated loss of MBP that correlates with a
lack of myelin sheath formation. These changes were accompanied by increased
astroglial and microglial reactivity, possibly as a response to myelination
injury. Interestingly, cerebellum was the area most affected, with greater
myelination impairment and glia burden, and showing a marked loss of Purkinje
cells and reduced arborization of the remaining ones. Thus, kernicterus in this
model displays not only axonal damage but also myelination deficits and glial
activation in different brain regions that are usually related to the neurologic
sequelae observed after severe hyperbilirubinemia. Author information:
(1)Cellular and Molecular Research Center, Iran University of Medical Sciences,
Tehran, Iran; Department of Neuroscience, School of Advanced Technologies in
Medicine, Tehran University of Medical Sciences, Tehran, Iran.
(2)Department of Neuroscience, School of Advanced Technologies in Medicine,
Tehran University of Medical Sciences, Tehran, Iran; Iranian National Center for
Addiction Studies, Tehran University of Medical Science, Tehran, Iran.
Electronic address: [email protected].
(3)Cellular and Molecular Research Center, Iran University of Medical Sciences,
Tehran, Iran; Department of Anatomy, Iran University of Medical Sciences,
Tehran, Iran.
(4)Cellular and Molecular Research Center, Iran University of Medical Sciences,
Tehran, Iran; Department of Tissue Engineering & Regenerative Medicine, Faculty
of Advanced Technologies in Medicine, Iran University of Medical Sciences,
Tehran, Iran.
(5)Audiology Department, Rehabilitation Faculty, Iran university of Medical
Sciences, Tehran, Iran.
(6)Neuroscience Department, Faculty of Advanced Technologies in Medicine, Iran
University of Medical Sciences, Tehran, Iran.
(7)Cellular and Molecular Research Center, Iran University of Medical Sciences,
Tehran, Iran; Department of Tissue Engineering & Regenerative Medicine, Faculty
of Advanced Technologies in Medicine, Iran University of Medical Sciences,
Tehran, Iran; Department of Tissue Engineering and Applied Cell Sciences, School
of Advanced Technologies in Medicine, Tehran University of Medical Sciences,
Tehran, Iran.
(8)Department of Neuroscience, School of Advanced Technologies in Medicine,
Tehran University of Medical Sciences, Tehran, Iran; Neuroscience Research
Center, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran.
(9)Cellular and Molecular Research Center, Iran University of Medical Sciences,
Tehran, Iran; Department of Tissue Engineering & Regenerative Medicine, Faculty
of Advanced Technologies in Medicine, Iran University of Medical Sciences,
Tehran, Iran; Neuroscience Department, Faculty of Advanced Technologies in
Medicine, Iran University of Medical Sciences, Tehran, Iran. Electronic address:
[email protected]. |
Does mTOR regulate the translation of MAPKAPK2? | Yes. mTOR regulates the translation of the MK2 (also known as MAPKAPK2) kinase through 4EBP1. In turn, MAPKAPK2 phosphorylates the RNA-binding protein ZFP36L1 during senescence, inhibiting its ability to degrade the transcripts of numerous senescence-associated secretory phenotype (SASP) components. Consequently, mTOR inhibition or constitutive activation of ZFP36L1 impairs the non-cell-autonomous effects of senescent cells in both tumour-suppressive and tumour-promoting contexts. | OBJECTIVES: The role of Ataxia-telangiectasia mutated (ATM) in response to DNA
damage has previously been studied, but its underlying mechanisms specific to
ionizing radiation (IR) have remained to be elucidated. In this study, function
of ATM on radiation-induced cell death in lung cancer H1299 cells was analysed.
MATERIALS AND METHODS: Human lung cancer cells, H1299, were used, and cell
models with ATM(-/-) and MAPK14(-/-) were established by genetic engineering.
Radiosensitivity was analysed using colony formation assays. Western blotting
and co-immunoprecipitation were implemented to detect protein expression and
interaction. MDC staining and GFP-LC3 relocalization were used to detect
autophagy.
RESULTS: Autophagy as well as phosphorylation of ATM was activated by ionizing
radiation. Both the inhibitor of ATM, KU55933 and ATM silencing reduced
phosphorylation of ATM and MAPKAPK2 expression. Both ATM(-/-) and MAPK14(-/-)
cells displayed hypersensitivity. IR increased autophagy level by more than 129%
in DMSO-treated cells, while only by 47% and 27% in KU55933-treated and ATM(-/-)
cells respectively. MAPK14 knock-down alone gave rise to the basal autophagy
level, but decreased notably after IR. KU55933 and ATM knock-down inhibited
IR-induced autophagy by activating mTOR pathways. Both Beclin1-PI3KIII and
Beclin1-MAPKAPK2 interactions as were remarkably affected by silencing either
ATM or MAPK14.
CONCLUSIONS: ATM promoted IR-induced autophagy via the MAPK14 pathway, mTOR
pathway and Beclin1/PI3KIII complexes. MAPK14 contributed to radiosensitization
of H1299 cells. Author information:
(1)Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College
London, Hammersmith Campus, London W12 0NN, UK.
(2)Epigenetics Section, MRC Clinical Sciences Centre, Imperial College London,
Hammersmith Campus, London W12 0NN, UK.
(3)Metabolic Signalling Group, MRC Clinical Sciences Centre, Imperial College
London, Hammersmith Campus, London W12 0NN, UK.
(4)Cancer Sciences Unit, Cancer Research UK Centre, Somers Building, University
of Southampton, Southampton SO16 6YD, UK.
(5)Division of Molecular Oncology of Solid Tumors, Department of Internal
Medicine I, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
(6)Proteomics Facility, MRC Clinical Sciences Centre, Imperial College London,
Hammersmith Campus, London W12 0NN, UK.
(7)Developmental Biology and Cancer Programme, Birth Defects Research Centre,
UCL Institute of Child Health, London WC1N 1EH, UK.
(8)Institute for Virology, Technische Universität München/Helmholtz Zentrum
München, 81675 Munich, Germany.
(9)Division of Chronic Inflammation and Cancer, German Cancer Research (DKFZ),
69121 Heidelberg, Germany.
(10)Department of Pathology and Geriatrics Center, University of Michigan, Ann
Arbor, Michigan 48109-2200, USA. |
What chromosome is affected in Turner's syndrome? | turner's syndrome (ts) is a chromosomal defect with partial or total absence of the x chromosome. | Seven women in three generations of a family have been affected by Turner
syndrome. Turner phenotype in this family is the result of deletion of the
entire short arm of one X chromosome. The short arm deletion is transmitted by
carriers of a balanced X-1 translocation. Autoradiographic findings showed that
the deleted X chromosome was late labeling in those persons with Turner
syndrome, whereas the normal X chromosome was late replicating in carriers of
the balanced translocation. The results of Xga typing of erythrocytes suggest
that the Xg locus is on the short arm of the X chromosome. Because of the
clinical implications, we believe that families of persons with structural
chromosomal abnormalities should be studied to exclude familial transmission. A 7-year-old girl was admitted to the hospital for anaemia, secondary to
intestinal blood los (melaena). She was found to have 45,X Turner's syndrome.
Her identical twin sister also had Turner's syndrome with a 45,X chromosome
complement. According to various criteria the probability of monozygosity was
0.9905. Although the incidence of twinning is greater than usual in families of
patients with Turner's syndrome, affected cases have only been observed in twin
sisters on six occasions. It seems therefore that the 45,X chromosome complement
itself is not a factor predisposing to twinning, but that in some families, a
factor is at play, which cuases either twinning or the 45,X aneuploidy, or both. The proposition that finger print variability between individuals might be
reduced by the absence of an X-chromosome in Turner's syndrome was rejected. In
the present study of 58 XO patients, aged 15-50 years, relatives of several
cases, unrelated female control samples and three unrelated male samples were
investigated. The higher mean value of the TRC among patients supported the
hypothesis forwarded by Penrose that an added X- or Y-chromosome reduces the TRC
and a missing one increasing it. The figures do not speak against the hypothesis
that genes affecting the TRC are located on the X-chromosome. A summary of the
major dermatoglyphic investigations in Turner's syndrome is presented. A case of mosaic Turner's syndrome with a 45,X/46,XX/47,XXX karyotype, who was
also a fragile X obligate carrier as the mother of an affected boy, was
identified by molecular diagnosis. Complete haplotyping and direct DNA analysis
showed that the X chromosome in all metaphases was the normal X. At the age of
57, she is mentally normal. Her external appearance was typical of Turner's
syndrome. This report shows that molecular studies in conjunction with
cytogenetic analysis can help in the clinical diagnosis of a rare case and can
show the uniqueness of a case such as the one here described. X-monosomy is a form of Turner syndrome (TS) in which an entire X chromosome is
missing. It is usually assumed that neuropsychological deficits in females with
TS result from insufficient dosage of gene products from alleles on the sex
chromosomes. If so, then parental origin of the single X chromosome should be
immaterial. However, if there are imprinted genes on the X chromosome affecting
brain development, neuropsychological development will depend on the parental
origin of the single X chromosome. We contrasted verbal and visuospatial memory
in females with a single paternal X chromosome (45,X(p)) and those with a single
maternal X (45,X(m)). Neither group showed any impairment on immediate story
recall; if anything, performance was above control levels. Groups did not differ
on a measure of delayed recall. However, when delayed recall was considered
after adjusting for level of immediate recall, 45,X(m) females showed enhanced
verbal forgetting relative to controls over a delay. On the Rey figure, both
groups were poor at copying the figure, but, after adjusting scores for initial
copy score and strategy, only the 45,X(p) females showed disproportionate
forgetting relative to controls. We propose there may be one or more imprinted
genes on the X chromosome that affect the development of lateralised brain
regions important for memory function. Turner syndrome is a chromosomal disorder in which all or part of one X
chromosome is missing. The meiotic or mitotic origin of most cases remains
unknown due to the difficulty in detecting hidden mosaicism and to the lack of
meiotic segregation studies. We analyzed 15 Turner patients, 10 with a 45,X
whereas the rest had a second cell line with abnormal X-chromosomes: a
pseudodicentric, an isochromosome, one large and one small ring, and the last
with a long arm deletion. Our aims were: to detect X cryptic mosaicism in
patients with a 45,X constitution; to determine the parental origin of the
abnormality; to infer the zygotic origin of the karyotype and to suggest the
timing and mechanism of the error(s) leading to the formation of abnormal X
chromosomes from maternal origin. Molecular investigation did not revealed
heterozygosity for any microsatellite, excluding X mosaicism in the 45,X cases.
Parental origin of the single X chromosome was maternal in 90% of these
patients. Three of the structurally abnormal Xs were maternally derived whereas
the other two were paternal. These results allowed us to corroborate breakpoints
in these abnormal X chromosomes and suggest that the pseudodicentric chromosome
originated from post-zygotic sister chromatid exchange, whereas the Xq deleted
chromosome probably arose after a recombination event during maternal meiosis. Women with Turner's syndrome (TS), who lack a complete X-chromosome, show an
impairment in remembering faces and in classifying "fear" in face images. Could
their difficulties extend to the processing of gaze? Three tasks, all of which
rely on the ability to make use of the eye-region of a pictured face, are
reported. Women with TS were impaired at judging mental state from images of the
upper face ("reading the mind in the eyes"). They were also specifically
impaired at interpreting "fear" from displays of the eye-region of the face.
However, they showed normal susceptibility to direction of gaze as an
attentional cue (social cueing), since they were as sensitive as controls to the
validity of the cue, under conditions where it should be ignored. In this task,
unlike those of reading the upper face for intention or expression, PIQ
accounted for a significant amount of individual variance in task performance.
The processing of displays of the eye region affording social and affective
information is specifically affected in TS. We speculate that amygdala
dysfunction is likely to be implicated in this anomalous behaviour. The presence
in the female karyotype of two complete X-chromosomes is protective for some
socio-cognitive abilities related to the modulation of behaviour by the
interpretation of gaze. CONTEXT: Turner syndrome (TS) is the most common genetic problem affecting women
and occurs when an X chromosome is completely deleted, portions of an X
chromosome are deleted, or chromosomal mosaicism occurs. Girls with TS may also
have occult Y chromosome sequences. Whereas some girls with TS are identified in
infancy or early childhood, many girls with TS are not detected until after 10
yr of age, resulting in delayed evaluation and treatment.
OBJECTIVE: To prevent the delayed recognition and treatment of TS, a
quantitative method of genotyping that can be performed as part of newborn
screening is needed.
DESIGN: To screen for sex chromosome abnormalities, we assembled a panel of
informative single nucleotide polymorphism (SNP) markers that span the X
chromosome from the dbSNP database. Pyrosequencing assays suitable for
quantitative assessment of signal strength from single nucleotides were designed
and used to genotype 46,XX; 46,XY; 45,X; and TS mosaics, examining zygosity and
signal strength for individual alleles. Pyrosequencing assays were also designed
for the detection of Y chromosome material.
RESULTS: With just four informative SNP markers for the X chromosome, all TS
girls with 45,X, partial X chromosome deletions, or mosaicism were identified
with 100% sensitivity. In mosaic individuals, Y chromosomal material was
detected with 100% sensitivity.
CONCLUSION: These results suggest that inexpensive high-throughput screening is
possible for TS and other sex chromosome disorders using quantitative genotyping
approaches. Turner's syndrome is defined as a congenital disease determining by quantitative
and/or structural aberrations of one from two X chromosomes with frequent
presence of mosaicism. Clinically it is characterized by growth and body
proportion abnormalities, gonadal dysgenesis resulting in sexual infantilism,
primary amenorrhoea, infertility, characteristic stigmata, anomalies of heart,
renal and bones and the presence of some diseases like Hashimoto thyroiditis
with hypothyroidism, diabetes mellitus type 2, osteoporosis, hypertension.
Turner's syndrome occurs in 1:2000 to 1:2500 female livebirth. The most frequent
X chromosome aberrations in patients with phenotype of Turner syndrome are as
follows: X monosomy - 45,X; mosaicism (50-75%), including 45,X/46,XX (10-15%),
45,X/46,XY (2-6%), 45,X/46,X,i(Xq), 45,X/46,X,del(Xp), 45,X/46,XX/47,XXX;
aberration of X structure: total or partial deletion of short arm of X
chromosome (46,X,del(Xp)) isochromosom of long arm of X chromosome
(46,X,(i(Xq)), ring chromosome (46, X,r(X)), marker chromosome (46,X+m).
Searching of X chromosome and mapping and sequencing of genes located at this
chromosome (such as SHOX, ODG2, VSPA, SOX 3) have made possible to look for
linkage between phenotypes and adequate genes or regions of X chromosome. In
this paper current data concerning correlation between phenotype and karyotype
in patients with TS have been presented. BACKGROUND: X monosomic mice (39,XO) have a remarkably mild phenotype when
compared to women with Turner syndrome (45,XO). The generally accepted
hypothesis to explain this discrepancy is that the number of genes on the mouse
X chromosome which escape X inactivation, and thus are expressed at higher
levels in females, is very small. However this hypothesis has never been tested
and only a small number of genes have been assayed for their X-inactivation
status in the mouse. We performed a global expression analysis in four somatic
tissues (brain, liver, kidney and muscle) of adult 40,XX and 39,XO mice using
the Illumina Mouse WG-6 v1_1 Expression BeadChip and an extensive validation by
quantitative real time PCR, in order to identify which genes are expressed from
both X chromosomes.
RESULTS: We identified several genes on the X chromosome which are overexpressed
in XX females, including those previously reported as escaping X inactivation,
as well as new candidates. However, the results obtained by microarray and qPCR
were not fully concordant, illustrating the difficulty in ascertaining modest
fold changes, such as those expected for genes escaping X inactivation.
Remarkably, considerable variation was observed between tissues, suggesting that
inactivation patterns may be tissue-dependent. Our analysis also exposed several
autosomal genes involved in mitochondrial metabolism and in protein translation
which are differentially expressed between XX and XO mice, revealing secondary
transcriptional changes to the alteration in X chromosome dosage.
CONCLUSIONS: Our results support the prediction that the mouse inactive X
chromosome is largely silent, while providing a list of the genes potentially
escaping X inactivation in rodents. Although the lower expression of X-linked
genes in XO mice may not be relevant in the particular tissues/systems which are
affected in human X chromosome monosomy, genes deregulated in XO mice are good
candidates for further study in an involvement in Turner Syndrome phenotype. Turner's syndrome, also known as 'monosomy X', is a genetic disorder that occurs
in 1/2,500 female births and is hypothesized to result from haploinsufficiency
of certain genes expressed from both sex chromosomes that escape X inactivation.
While the classic karyotype related to Turner's syndrome is 45,X, the majority
of those affected actually have a mosaic chromosomal complement, most often with
a second normal cell line (46,XX). The resulting phenotype is variable and
related to the underlying chromosomal pattern, but it is characterized by three
cardinal features: short stature (around 100%), ovarian failure (>90%) and
congenital lymphedema (>80%). In this paper we report a molecular and
cytogenetic investigation of a 26-year-old female with non-mosaic 45,X
karyotype, who has a stature of 170 cm without GH treatment, and whose only
apparent Turner feature is gonadal dysgenesis. The only possible explanation for
the absence of Turner phenotype is the hidden mosaicism combined with an
untreated gonadal dysgenesis. Our results support the theory that significant
ascertainment bias exists in our understanding of Turner's syndrome. Turner syndrome is a genetic disorder caused by the complete or partial absence
of an X chromosome in affected women. Individuals with TS show characteristic
difficulties with executive functions, visual-spatial and mathematical
cognition, with relatively intact verbal skills, and congruent abnormalities in
structural development of the posterior parietal cortex (PPC). The functionally
heterogeneous PPC has recently been investigated using connectivity-based
clustering methods, which sub-divide a given region into clusters of voxels
showing similar structural or functional connectivity to other brain regions. In
the present study, we extended this method to compare connectivity-based
clustering between groups and investigate whether functional networks
differentially recruit the PPC in TS. To this end, we parcellated the PPC into
sub-regions based on temporal correlations with other regions of the brain. fMRI
data were collected from 15 girls with TS and 14 typically developing (TD)
girls, aged 7-14, while they performed a visual-spatial task. Temporal
correlations between voxels in the PPC and a set of seed regions were
calculated, and the PPC divided into clusters of voxels showing similar
connectivity. It was found that in general the PPC parcellates similarly in TS
and TD girls, but that regions in bilateral inferior parietal lobules, and
posterior right superior parietal lobule, were reliably recruited by different
networks in TS relative to TD participants. These regions showed weaker
correlation in TS with a set of regions involved in visual processing. These
results suggest that abnormal development of visuospatial functional networks in
TS may relate to the well documented cognitive difficulties in this disorder. Children with chromosome 22q11.2 deletion syndrome (22q11.2DS), Fragile X
syndrome (FXS), or Turner syndrome (TS) are considered to belong to distinct
genetic groups, as each disorder is caused by separate genetic alterations. Even
so, they have similar cognitive and behavioral dysfunctions, particularly in
visuospatial and numerical abilities. To assess evidence for common underlying
neural microstructural alterations, we set out to determine whether these groups
have partially overlapping white matter abnormalities, relative to typically
developing controls. We scanned 101 female children between 7 and 14years old:
25 with 22q11.2DS, 18 with FXS, 17 with TS, and 41 aged-matched controls using
diffusion tensor imaging (DTI). Anisotropy and diffusivity measures were
calculated and all brain scans were nonlinearly aligned to population and
site-specific templates. We performed voxel-based statistical comparisons of the
DTI-derived metrics between each disease group and the controls, while adjusting
for age. Girls with 22q11.2DS showed lower fractional anisotropy (FA) than
controls in the association fibers of the superior and inferior longitudinal
fasciculi, the splenium of the corpus callosum, and the corticospinal tract. FA
was abnormally lower in girls with FXS in the posterior limbs of the internal
capsule, posterior thalami, and precentral gyrus. Girls with TS had lower FA in
the inferior longitudinal fasciculus, right internal capsule and left cerebellar
peduncle. Partially overlapping neurodevelopmental anomalies were detected in
all three neurogenetic disorders. Altered white matter integrity in the superior
and inferior longitudinal fasciculi and thalamic to frontal tracts may
contribute to the behavioral characteristics of all of these disorders. Turner's syndrome (TS) is a chromosomal defect with partial or total absence of
the X chromosome. Our objective is to report our experience in Greece with
patients suffering from TS and trying to conceive; therefore, we present four
patients with TS, who underwent In vitro fertilization (ICSI) with donor oocytes
in order to get pregt. Three out of four patients managed to conceive and
bring pregcy to completion. It was shown that patients diagnosed in childhood
or adolescence with TS have the possibility to undergo hormone replacement
therapy (HRT) and thus, secondary sexual characteristics as well as uterus of
almost normal size can develop. Assisted reproduction techniques (ART),
predomitly with donated oocytes, could give these patients the possibility to
have children. |
What is the aim of the TRAP assay? | Telomerase enzyme activity can be detected in whole cell lysates by a polymerase chain reaction (PCR)-based method referred to as the telomeric repeat amplification protocol (TRAP). | During erythropoiesis, some organelles such as mitochondria and nucleus are lost
by autophagy and enucleation processes in the presence of macrophages in vivo.
In vitro production of erythrocytes has raised many questions about the
mechanism of enucleation. The aim of this work was to study the DNA breakdown,
enucleation, hemoglobin synthesis and telomerase activity of K562 cells during
erythroid differentiation. For these purposes, K562 cells were induced to
differentiate by erythropoietin + rhGM-CSF, DMSO, and sodium butyrate separately
up to 14 d. In different time intervals, hemoglobin synthesis was evaluated by
benzidine staining and RT-PCR for γ-globin gene expression. DNA breakdown was
analyzed by 4',6-diamidino-2-phenylindole (DAPI) staining, DNA ladder
electrophoresis and comet assay. The telomerase activity was evaluated by TRAP
assay. Our result indicated that, sodium butyrate and DMSO inhibited K562 cell
growth about 50-60% in comparison to untreated control cells. The percentage of
benzidine-positive cells was about 45% in the presence of sodium butyrate after
10 d. Densitometric analysis of RT-PCR and calculated data indicated a 1.5-fold
increase in relative γ-globin gene expression at 96 h, in the presence of 1 mM
sodium butyrate in comparison with untreated cells. DAPI staining did not reveal
any evidence of internal lysis of the nucleus during erythroid differentiation
at first wk, but this was obvious in the second wk. DNA laddering pattern was
not observed in differentiated cells during 14 d. In comet assay, the percentage
of DNA in tail, tail length, and tail moment were significantly different
between untreated and treated cells (p < 0.05). Telomerase activity was
inhibited up to 90.3% during erythroid differentiation of these cells. Telomerase is the enzyme that extends the chromosome ends, thereby contributing
to eukaryotic cell genome stability. Telomerase is expressed in the majority of
cells that have an unlimited proliferation such as stem cells and cancer cells.
The increased interest in telomerase in cancer research, challenged by the low
cellular abundance of the enzyme, has led to the development of a reliable and
at the same time very sensitive approach to detect telomerase activity. The
telomeric repeat amplification protocol (TRAP) represents an easy and rapid
method for detection of telomerase activity in cells. A non-telomeric TS primer
is extended by telomerase in the first step followed by the PCR amplification of
the products. The PCR step renders this protocol very sensitive to detect
telomerase activity at the single cell level making it compatible with the
analysis of tumor samples. When run on a polyacrylamide gel, the PCR product is
a characteristic ladder of bands due to the repetitive nature of telomeric DNA
sequence. The densitometric analysis of the ladder allows the TRAP assay to be
used for comparative quantification of telomerase activity in different samples. BACKGROUND: Considering previous result in Non-Small Cell Lung Cancer (NSCLC),
we investigated in human cancer cells the role of PARP3 in the regulation of
telomerase activity.
METHODS: We selected A549 (lung adenocarcinoma cell line) and Saos-2
(osteosarcoma cell line), with high and low telomerase activity levels,
respectively. The first one was transfected using a plasmid construction
containing a PARP3 sequence, whereas the Saos-2 cells were submitted to shRNA
transfection to get PARP3 depletion. PARP3 expression on both cell systems was
evaluated by real-time quantitative PCR and PARP3 protein levels, by
Western-blot. Telomerase activity was determined by TRAP assay.
RESULTS: In A549 cells, after PARP3 transient transfection, data obtained
indicated that twenty-four hours after transfection, up to 100-fold increased
gene expression levels were found in the transfected cells with
pcDNA/GW-53/PARP3 in comparison to transfected cells with the empty vector.
Moreover, 48 hours post-transfection, telomerase activity decreased around 33%,
and around 27%, 96 hours post-transfection. Telomerase activity average ratio
was 0.67 ± 0.05, and 0.73 ± 0.06, respectively, with significant differences. In
Saos-2 cells, after shRNA-mediated PARP3 silencing, a 2.3-fold increase in
telomerase activity was detected in relation to the control.
CONCLUSION: Our data indicated that, at least in some cancer cells, repression
of PARP3 could be responsible for an increased telomerase activity, this fact
contributing to telomere maintece and, therefore, avoiding genome
instability. Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere
length and therefore promoting genomic integrity, proliferation, and lifespan.
In addition, telomerase protects the mitochondria from oxidative stress and
confers resistance to apoptosis, suggesting its possible importance for the
surviving of non-mitotic, highly active cells such as neurons. We previously
demonstrated the ability of novel telomerase activators to increase telomerase
activity and expression in the various mouse brain regions and to protect motor
neurons cells from oxidative stress. These results strengthen the notion that
telomerase is involved in the protection of neurons from various lesions. To
underline the role of telomerase in the brain, we here compare the activity of
telomerase in male and female mouse brain and its dependence on age. TRAP assay
is a standard method for detecting telomerase activity in various tissues or
cell lines. Here we demonstrate the analysis of telomerase activity in three
regions of the mouse brain by non-denaturing protein extraction using CHAPS
lysis buffer followed by modification of the standard TRAP assay. In this 2-step
assay, endogenous telomerase elongates a specific telomerase substrate (TS
primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase
reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp
increments. The analysis of the DNA ladder is made by 4.5% high resolution
agarose gel electrophoresis followed by staining with highly sensitive nucleic
acid stain. Compared to the traditional TRAP assay that utilize (32)P labeled
radioactive dCTP's for DNA detection and polyacrylamide gel electrophoresis for
resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP
assay for evaluating telomerase activity in the mouse brain, demonstrating the
ability to detect differences in telomerase activity in the various female and
male mouse brain region. Telomerase catalyzes telomeric DNA synthesis, an essential process to maintain
the length of telomere for continuous cell proliferation and genomic stability.
Telomerase is activated in gametes, stem cells, and most tumor cells, and its
activity is tightly controlled by a catalytic human telomerase reverse
transcriptase (hTERT) subunit and a collection of associated proteins. In the
present work, normal human testis tissue was used for the first time to identify
proteins involved in the telomerase regulation under normal physiological
conditions. Immunoprecipitation was performed using total protein lysates from
the normal testis tissue and the proteins of interest were identified by
microfluidic high-performance liquid chromatography and tandem mass spectrometry
(HPLC-Chip-MS/MS). The regulatory role of PCDH10 in telomerase activity was
confirmed by a telomeric repeat amplification protocol (TRAP) assay, and the
biological functions of it were characterized by in vitro proliferation,
migration, and invasion assays. A new in vivo hTERT interacting protein,
protocadherin 10 (PCDH10), was identified. Overexpression of PCDH10 in
pancreatic cancer cells impaired telomere elongation by inhibiting telomerase
activity while having no obvious effect on hTERT expression at mRNA and protein
levels. As a result of this critical function in telomerase regulation, PCDH10
was found to inhibit cell proliferation, migration, and invasion, suggesting a
tumor suppressive role of this protein. Our data suggested that PCDH10 played a
critical role in cancer cell growth, by negatively regulating telomerase
activity, implicating a potential value in future therapeutic development
against cancer. |
Describe clinical applications of the PIM2 scoring system. | The Pediatric Index of Mortality 2 (PIM2) is one of the most commonly used scoring systems to predict mortality in patients admitted to pediatric intensive care units. The PIM2 score adequately discriminates survivors from non-survivor | OBJECTIVE: Pediatric Index of Mortality 2 (PIM2) is an up-to-date mortality
prediction model in the public domain that has not yet been widely validated. We
aimed to evaluate this score in the population of patients admitted to our
pediatric intensive care unit.
DESIGN: Prospective cohort study.
SETTING: Multidisciplinary pediatric intensive care unit in a general university
hospital in Buenos Aires, Argentina.
PATIENTS: All consecutive patients admitted between January 1, 2004, and
December 31, 2005.
INTERVENTIONS: None.
MEASUREMENTS AND MAIN RESULTS: There were 1,574 patients included in the study.
We observed 41 (2.6%) deaths, and PIM2 estimated 48.1 (3.06) deaths.
Discrimination assessed by the area under the receiver operating characteristic
curve was 0.9 (95% confidence interval, 0.89-0.92). Calibration across five
conventional mortality risk intervals assessed by the Hosmer-Lemeshow
goodness-of-fit test showed chi5 = 12.2 (p = .0348). The standardized mortality
ratio for the whole population was 0.85 (95% confidence interval, 0.6-1.1).
CONCLUSIONS: PIM2 showed an adequate discrimination between death and survival
and a poor calibration assessed by the Hosmer-Lemeshow goodness-of-fit test. The
standardized mortality ratio and clinical analysis of the Hosmer-Lemeshow table
make us consider that PIM2 reasonably predicted the outcome of our patients. INTRODUCTION: Pediatric Index of Mortality 2 (PIM2) and Pediatric Risk of
Mortality (PRISM) are scoring systems to predict mortality likehood; thus, it is
necessary to validate such predictors in Pediatric Intensive Care Units'
population.
OBJECTIVE: To assess the validity of PRISM and PIM2 models of Mortality in
Pediatrics Intensive Care Units at Hospital Infantil de Córdoba (PICUHI).
POPULATION, MATERIAL AND METHODS: 435 critically ill admitted patients were
retrospectively analized in PICUHI from January 1st 2008 to January 31st 2008;
416 were included in the study, ruling out elective admitted patients with less
than 12 hour at PICU length stay. There were no deaths in this Group. Original
equations for each models, were used. Calibration was performed (p> 0.05) using
Hosmer- Lemeshow (HL) goodness-of-fit tests. Scores were assessed through
Standardized Mortality Ratio (SMR) and discrimination between patients alived
and dead, was estimated calculating the area under ROC curve.
RESULTS: 416 admitted patients were included, (55.04%) were male 55.04%, median
age was 3 years (1 month-17 years), with a median of 2 (1-76) admitted days in
PICU. Mortality was 6.66%. PIM2 had an area under ROC curve of 0.88 (CI 95%
0.82-0.95) and PRIMS: 0.85 (CI 95% 0.78-0.92), with p 0.3570 value. HL
calibration for PRISM was: x2 5.93 (p 0.54), and PIM2 was: x2 14.19 (p 0.07).
PRISM, Standardized Mortality Ratio (SMR) was: 1.00 (CI 95% 0.50-1.50) and PIM2
was 1.00 (CI 95% 0.55-1.55).
CONCLUSIONS: Both scores discriminated and calibrated well as the p-value of the
HL test, althougt the analysis of the HL table appears inadequate to PIM2
calibration, in terms of severity-adjusted mortality. INTRODUCTION: Blood lactate concentration predicts mortality in neonates,
infants, children and adults, with evidence that it has better predictive power
than other markers of acid-base status such as absolute base excess or pH.
OBJECTIVE: To investigate whether blood lactate concentration on admission
predicts mortality in paediatric intensive care and if its addition can improve
the performance of the Paediatric Index of Mortality 2 (PIM2) mortality
prediction score.
DESIGN AND SETTING: Retrospective cohort study in one 20-bed UK paediatric
intensive care unit (PICU) using data from the PICU clinical and blood gas
analyser databases between 2006 and 2010. Only cases with a blood lactate
concentration measured at the same time as the PIM2 variables were included.
Logistic regression was used to assess if blood lactate concentration predicted
mortality independently of PIM2, adjusting for potential confounders, and if it
could replace absolute base excess in the PIM2 model.
RESULTS: There were 155 deaths amongst 2,380 admissions (6.5 %). Admission
lactate in non-survivors was higher than in survivors (mean [standard deviation,
SD]) 6.6 [5.6] versus 3.0 [2.5] mmol/l, had a positive association with
mortality [adjusted odds ratio (OR) for death per unit (mmol/l)] increase 1.11
[95 % confidence interval (CI) 1.06-1.16; p < 0.001] and significantly improved
the model fit of PIM2 when it replaced absolute base excess (p < 0.001).
CONCLUSIONS: PICU admission blood lactate concentration predicts mortality
independently of PIM2. Given the limitations of this study, a prospective
multi-centre evaluation is required to establish whether it should be added to
the PIM2 model with or without replacement of base excess. The aim of the study was to explore the association between Glasgow Coma Scale
(GCS), Paediatric Index of Mortality (PIM2) and Injury Severity Score (ISS), and
the long-term outcome of children with injuries. The health related quality of
life (HRQL) was assessed by using the Royal Alexandra Hospital for children
Measure of Function (RAHC MOF), 12 months post discharge. Out of 118 children
with injuries (9% of all patients), 75 had injury of the head as the leading
injury. There were no significant differences at admission in the severity of
clinical condition, as expressed by PIM2 and ISS, between patients with head
injuries and patients with other injured leading body regions. Children with
head injuries had significantly worse HRQOL than children with other leading
injured body region (p < 0.045), and children from road traffic accidents had
significantly worse HRQL (p = 0.004), compared to other mechanisms of injury.
HRQL correlated significantly with GCS (p = 0.027), but not with ISS and PIM2.
As the conclusion, among all scoring systems applied, only GCS, which
demonstrates severity of head injury, showed significant impact on long-term
outcome of injured children. Although many inflammatory cytokines are prognostic in sepsis, the utility of
cytokines in evaluating disease severity in pediatric hematology/oncology
patients with septic shock was rarely studied. On the other hand, a single
particular cytokine is far from ideal in guiding therapeutic intervention, but
combination of multiple biomarkers improves the accuracy. In this prospective
observational study, 111 episodes of septic shock in pediatric
hematology/oncology patients were enrolled from 2006 through 2012. Blood samples
were taken for inflammatory cytokine measurement by cytometric bead array (CBA)
technology at the initial onset of septic shock. Interleukin (IL)-6 and IL-10
were significantly elevated in majority of patients, while tumor necrosis factor
(TNF)-α and interferon (IFN)-γ were markedly increased in patients with high
pediatric index of mortality 2 (PIM2) score and non-survivors. All the four
cytokines paralleled the PIM2 score and differentially correlated with
hemodynamic disorder and fatal outcomes. The pediatric multiplex cytokine score
(PMCS), which integrated the four cytokines into one score system, was related
to hemodynamic disorder and mortality as well, but showed more powerful
prediction ability than each of the four cytokines. PMCS was an independent
predictive factor for fatal outcome, presenting similar discriminative power
with PIM2, with accuracy of 0.83 (95% CI, 0.71-0.94). In conclusion, this study
develops a cytokine scoring system based on CBA technique, which performs well
in disease severity and fatality prediction in pediatric hematology/oncology
patients with septic shock. BACKGROUND AND AIMS: Pediatric index of mortality (PIM) 2 score is one of the
severity scoring systems being used for predicting outcome of patients admitted
to intensive care units (ICUs). The aim of the present study was to evaluate the
usefulness of PIM2 score in predicting mortality in a tertiary care pediatric
ICU (PICU) and to assess the associated factors in predicting mortality such as
presence of shock, need for assisted ventilation and Glasgow coma scale <8.
MATERIALS AND METHODS: This was a prospective observation study done at tertiary
care PICU from May 2011 to July 2011. Consecutive 119 patients admitted to PICU
(aged 1 month to 12 years) were enrolled in the study. PIM2 scoring was done for
all patients. The outcome was recorded as death or discharge. The associated
factors for mortality were analyzed with SPSS 17.
RESULTS: PIM2 score discriminated between death and survival at a 99.8 cut-off,
with area under receiver operating characteristic curve 0.843 with 95%
confidence interval (CI) (0.765, 0.903). Most patients were referred late to
this hospital, which explains higher death rate (46.2%), lesser length of
hospital stay (mean 2.98 days) in the mortality group, and increased rate of
mechanical ventilation (68.1%). Presence of shock was independently associated
with mortality, as evidenced by binary logistic regression.
CONCLUSION: PIM2 score discriminated well between survivors and death at PICU.
Presence of shock was significantly associated with mortality. INTRODUCTION: Mortality risk prediction scores are important for benchmarking
quality of care in paediatric intensive care units (PICUs). We aimed to
benchmark PICU outcomes at our hospital against the Pediatric Index of Mortality
2 (PIM2) mortality risk prediction score, and evaluate differences in diagnosis
on admission and outcomes between Malaysian and immigrant children.
METHODS: We prospectively collected demographic and clinical data on paediatric
medical patients admitted to the PICU of Sabah Women's and Children's Hospital
in Kota Kinabalu, Sabah, Malaysia. The PIM2 risk score for mortality was
tabulated.
RESULTS: Of the 131 patients who met the inclusion criteria, data was available
for 115 patients. The mean age of the patients was 2.6 ± 3.8 years, with 79% of
the cohort aged less than five years. Patients were mainly of Kadazan (38%) and
Bajau (30%) descent, and 26% of patients were non-citizens. Leading diagnoses on
admission were respiratory (37%), neurological (18%) and infectious (17%)
disorders. Out of the 29 patients who died, 23 (79%) were Malaysians and the
main mortality diagnostic categories were respiratory disorder (22%),
septicaemia (22%), haemato-oncological disease (17%) and neurological disorder
(13%). Calculated standardised mortality ratios (SMRs) were not significantly >
1 for any patient category for variables such as age and admission diagnosis.
However, infants less than two years old with comorbidities were significantly
worse (SMR 2.61, 95% confidence interval 1.02-6.66).
CONCLUSION: The patient profile at our centre was similar to that reported from
other PICUs in Asia. The PIM2 score is a useful mortality risk prediction model
for our population. INTRODUCTION: The Pediatric Index of Mortality 2 (PIM2) is one of the most
commonly used scoring systems to predict mortality in patients admitted to
pediatric intensive care units (PICU) in Argentina. The objective of this study
was to validate the PIM2 score in PICUs participating in the Quality of Care
Program promoted by the Argentine Society of Intensive Care.
POPULATION AND METHODS: Multicenter, prospective, observational, cross-sectional
study. All patients between 1 month and 16 years old admitted to participating
PICUs between January 1st, 2009 and December 31st, 2009 were included. The
discrimination and calibration of the PIM2 score were assessed in the entire
population and in different subgroups (risk of mortality, age, diagnoses on
admission).
RESULTS: Two thousand, eight hundred and thirty-two patients were included. PIM2
predicted 246 deaths; however, 297 patients died (p < 0.01). The standardized
mortality ratio was 1.20 (95% confidence interval [CI]: 1.01-1.43). The area
under the ROC curve was 0.84 (95% CI: 0.82-0.86). Statistically significant
differences were detected between the observed and the predicted mortality for
the entire population and for the different risk intervals (χ2: 71.02, df: 8, p
< 0.001). Statistically significant differences were also found between observed
and predicted mortality in adolescent patients (37/22, p = 0.03) and in those
hospitalized due to respiratory disease (105/81, p = 0.03).
CONCLUSIONS: The PIM2 score adequately discriminates survivors from
non-survivors. However, it underscores the overall risk of death, especially in
adolescent patients and those hospitalized due to respiratory disease. It is
critical to take such differences into account when interpreting results. BACKGROUND: Severity of illness is an important consideration in making the
decision to transfuse as it is the sicker patient that often needs a red cell
transfusion. Red blood cell (RBC) transfusions could potentially have direct
effects and interact with presenting illness by contributing to pathologies such
as multi-organ dysfunction and acute lung injury thus exerting a considerable
impact on overall morbidity and mortality. In this study, we examine if
transfusion is an independent predictor of mortality, or if outcomes are merely
a result of the initial severity as predicted by Pediatric Risk of Mortality
(PRISM) III, Pediatric Index of Mortality (PIM2), and day 1 Pediatric Logistic
Organ Dysfunction (PELOD) scores.
METHODS: A single center retrospective study was conducted using data from a
prospectively maintained transfusion database and center-specific data at our
pediatric ICU between January 2009 and December 2012. Multivariate regression
was used to control for the effects of clinical findings, therapy, and severity
scores, with mortality as the dependent variable. Likelihood ratios and area
under the curve were used to test the fidelity of severity scores by comparing
transfused vs. non-transfused patients.
RESULTS: There were 4975 admissions that met entry criteria. In multivariate
analysis, PRISM III scores and serum hemoglobin were significant predictors of
transfusion (p < 0.05). Transfused and non-transfused subjects were distinctly
disparate, so multivariate regression was used to control for differences.
Severity scores, age, volume transfused, and vasoactive agents were
significantly associated with mortality whereas hemoglobin was not. A
substantial number of transfusions (45 %) occurred in the first 24 h, and
patients transfused later (24-48 h) were more likely to die compared to this
earlier time point. Likelihood ratio testing revealed statistically significant
differences in severity scoring systems to predict mortality in transfused vs.
non-transfused patients.
CONCLUSIONS: This study suggests that RBC transfusion is an important risk
factor that is statistically independent of severity. The timing of transfusions
that related strongest to mortality remained outside the purview of severity
scoring, as these happened beyond the timing of data collection for most scoring
systems. |
Do histone variant mH2A (macro-H2A) levels decrease upon differentiation? | MacroH2A.1 was found to be present at low levels upon the establishment of pluripotency in the inner cell mass and epiblast, but it was highly enriched in the trophectoderm and differentiated somatic cells later in mouse development. Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the chromatin of regulatory regions of pluripotency genes in somatic cells such as mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic stem cells. In addition, overexpression of macroH2A isoforms prevented efficient reprogramming of epiblast stem cells to naïve pluripotency. | How various layers of epigenetic repression restrict somatic cell nuclear
reprogramming is poorly understood. The transfer of mammalian somatic cell
nuclei into Xenopus oocytes induces transcriptional reprogramming of previously
repressed genes. Here, we address the mechanisms that restrict reprogramming
following nuclear transfer by assessing the stability of the inactive X
chromosome (Xi) in different stages of inactivation. We find that the Xi of
mouse post-implantation-derived epiblast stem cells (EpiSCs) can be reversed by
nuclear transfer, while the Xi of differentiated or extraembryonic cells is
irreversible by nuclear transfer to oocytes. After nuclear transfer, Xist RNA is
lost from chromatin of the Xi. Most epigenetic marks such as DNA methylation and
Polycomb-deposited H3K27me3 do not explain the differences between reversible
and irreversible Xi. Resistance to reprogramming is associated with
incorporation of the histone variant macroH2A, which is retained on the Xi of
differentiated cells, but absent from the Xi of EpiSCs. Our results uncover the
decreased stability of the Xi in EpiSCs, and highlight the importance of
combinatorial epigenetic repression involving macroH2A in restricting
transcriptional reprogramming by oocytes. The importance of epigenetic mechanisms is most clearly illustrated during early
development when a totipotent cell goes through multiple cell fate transitions
to form the many different cell types and tissues that constitute the embryo and
the adult. The exchange of a canonical H2A histone for the 'repressive' macroH2A
variant is one of the most striking epigenetic chromatin alterations that can
occur at the level of the nucleosome. Here, we discuss recent data on macroH2A
in zebrafish and mouse embryos, in embryonic and adult stem cells and also in
nuclear reprogramming. We highlight the role of macroH2A in the establishment
and maintece of differentiated states and we discuss its still poorly
recognized function in transcriptional activation. How cell fate becomes restricted during somatic cell differentiation is a
long-lasting question in biology. Epigenetic mechanisms not present in
pluripotent cells and acquired during embryonic development are expected to
stabilize the differentiated state of somatic cells and thereby restrict their
ability to convert to another fate. The histone variant macroH2A acts as a
component of an epigenetic multilayer that heritably maintains the silent X
chromosome and has been shown to restrict tumor development. Here we show that
macroH2A marks the differentiated cell state during mouse embryogenesis.
MacroH2A.1 was found to be present at low levels upon the establishment of
pluripotency in the inner cell mass and epiblast, but it was highly enriched in
the trophectoderm and differentiated somatic cells later in mouse development.
Chromatin immunoprecipitation revealed that macroH2A.1 is incorporated in the
chromatin of regulatory regions of pluripotency genes in somatic cells such as
mouse embryonic fibroblasts and adult neural stem cells, but not in embryonic
stem cells. Removal of macroH2A.1, macroH2A.2 or both increased the efficiency
of induced pluripotency up to 25-fold. The obtained induced pluripotent stem
cells reactivated pluripotency genes, silenced retroviral transgenes and
contributed to chimeras. In addition, overexpression of macroH2A isoforms
prevented efficient reprogramming of epiblast stem cells to naïve pluripotency.
In summary, our study identifies for the first time a link between an epigenetic
mark and cell fate restriction during somatic cell differentiation, which helps
to maintain cell identity and antagonizes induction of a pluripotent stem cell
state. The chromatin template imposes an epigenetic barrier during the process of
somatic cell reprogramming. Using fibroblasts derived from macroH2A double
knockout (dKO) mice, here we show that these histone variants act cooperatively
as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms,
we further demonstrate that macroH2A2 is the predomit barrier to
reprogramming. Genomic analyses reveal that macroH2A1 and macroH2A2, together
with H3K27me3, co-occupy pluripotency genes in wild-type (wt) fibroblasts. In
particular, we find macroH2A isoforms to be highly enriched at target genes of
the K27me3 demethylase, Utx, which are reactivated early in iPS reprogramming.
Finally, while macroH2A dKO-induced pluripotent cells are able to differentiate
properly in vitro and in vivo, such differentiated cells retain the ability to
return to a stem-like state. Therefore, we propose that macroH2A isoforms
provide a redundant silencing layer or terminal differentiation 'lock' at
critical pluripotency genes that presents as an epigenetic barrier when
differentiated cells are challenged to reprogram. We have previously shown that macro histone variants (macroH2A) are expressed at
low levels in stem cells and are up-regulated during differentiation. Here we
show that the knockdown of macro histone variants impaired the in vitro and in
vivo differentiation of human pluripotent cells, likely through defects in the
silencing of pluripotency-related genes. ChIP experiments showed that during
differentiation macro histone variants are recruited to the regulatory regions
of pluripotency and developmental genes marked with H3K27me3 contributing to the
silencing of these genes. |
Which disease is associated with mutations in SLC40A1 gene? | Mutations in the SLC40A1 gene, which encodes the cellular iron exporter ferroportin, are associated with the autosomal dominant hemochromatosis type 4 or Ferroportin disease. The patients characteristically have hyperferritinemia and iron overload. | The product of the SLC40A1 gene, ferroportin 1, is a main iron export protein.
Pathogenic mutations in ferroportin 1 lead to an autosomal domit hereditary
iron overload syndrome characterized by high serum ferritin concentration,
normal transferrin saturation, iron accumulation predomitly in macrophages,
and marginal anemia. Iron overload occurs in both the African and the
African-American populations, but a possible genetic basis has not been
established. We analyzed the ferroportin 1 gene in 19 unrelated patients from
southern Africa (N = 15) and the United States (N = 4) presenting with primary
iron overload. We found a new c. 744 C-->T (Q248H) mutation in the SLC40A1 gene
in 4 of these patients (3 Africans and 1 African-American). Among 22 first
degree family members, 10 of whom were Q248H heterozygotes, the mutation was
associated with a trend to higher serum ferritin to amino aspartate transferase
ratios (means of 14.8 versus 4.3 microg/U; P = 0.1) and lower hemoglobin
concentrations (means of 11.8 versus 13.2 g/dL; P = 0.1). The ratio corrects
serum ferritin concentration for alcohol-induced hepatocellular damage. We also
found heterozygosity for the Q248H mutation in 7 of 51 (14%) southern African
community control participants selected because they had a serum ferritin
concentration below 400 microg/L and in 5 of 100 (5%) anonymous
African-Americans, but we did not find the change in 300 Caucasians with normal
iron status and 25 Caucasians with non-HFE iron overload. The hemoglobin
concentration was significantly lower in the African community controls with the
Q248H mutation than in those without it. We conclude that the Q248H mutation is
a common polymorphism in the ferroportin 1 gene in African populations that may
be associated with mild anemia and a tendency to iron loading. Ferroportin disease, autosomal-domit reticuloendothelial iron overload, may
be more prevalent than hemochromatosis in Japan. Hyperferritinemia of 822 ng/ml
with 24.8% transferrin saturation of iron was incidentally noted in a
43-year-old man. His iron overload was selective in Kupffer cells of the liver.
Subsequently, his father was found to have asymptomatic hyperferritinemia of
2,283 ng/ml with 62.1% saturation. These affected subjects were heterozygous for
1467A>C (R489S) in SLC40A1, and without other mutations of the hemochromatosis
genes. Here, we report a Japanese family with ferroportin disease, characterized
by hyperferritinemia with relatively low transferrin saturations of iron. Since the discovery of HFE gene in 1996, considerable progress has been made
concerning the iron-metabolism and its major abnormalities. Five types of
hereditary hemochromatosis are actually known: type 1 (HFE gene), type 2A (HJV
gene), type 2B (HAMP gene), type 3 (TfR2 gene), type 4 (SLC40A1 gene). The HFE
C282Y +/+ mutation is responsible for the most frequent type of hemochromatosis
in France. Various secondary causes can lead to iron-overload: associated
genetic diseases, exogenous iron intake, thalassaemia and refractory anaemia,
hepatic siderosis, alcoholic hepatitis, cutaneous porphyria and cirrhosis. The
deleterious consequences of iron-overload are due to the interactions of the
environmental factors. The role of HFE heterozygote mutations is still
discussed. In clinical practice, the interpretation of a serum ferritin increase
is a frequent problem that needs a careful evaluation based on the tranferrin
saturation measurement. Significant increase of both these factors is in favour
of an HFE C282Y +/+ hemochromatosis, after exclusion of a hepatocellular
insufficiency or a refractory anaemia. Nevertheless, high ferritin is not always
a marker of iron-overload. Thus, there are many disorders increasing the serum
ferritin levels without iron overload : cytolysis (hepatic...), inflammatory or
infectious syndromes, high alcohol intake, neoplasia... Looking for HFE
mutations help to separate type 1 hemochromatosis from other conditions mainly
hepatic siderosis (metabolic disorders). The identification of rare types of
hemochromatosis (types 2-4) is only required in particular cases. The evaluation
of the iron overload is now based on hepatic MRI determination rather than liver
biopsy. Repeated phlebotomies remain the essential way to decrease the iron
overload in HFE hemochromatosis and to prevent the occurrence of severe and
irreversible complications (cirrhosis, arthropathies, cardiac failure, and
diabetes). Because of the link established between the amount of iron-overload
and the occurrence of complications and the mortality over-risk in HFE C282Y +/+
hemochromatosis, venesections must be started when serum ferritin is higher than
300 microg/l in man and 200 microg/l in woman, whatever the clinical
manifestations are and obviously before the symptomatic phase of the disease. Mutations in the SLC40A1 gene result in a domit genetic disorder [ferroportin
disease; hereditary hemochromatosis type (HH) IV], characterized by iron
overload with two different clinical manifestations, normal transferrin
saturation with macrophage iron accumulation (the most prevalent type) or high
transferrin saturation with hepatocyte iron accumulation (classical
hemochromatosis phenotype). In previous studies, the mutational analysis of
SLC40A1 gene has been performed at the genomic DNA level by PCR amplification
and direct sequencing of all coding regions and flanking intron-exon boundaries
(usually in 9 PCR reactions). In this study, we analyzed the SLC40A1 gene at the
mRNA level, in two RT-PCR reactions, followed by direct sequencing and/or NIRCA
(non-isotopic RNase cleavage assay). This protocol turned out to be rapid,
sensitive and reliable, facilitating the detection of the SLC40A1 gene mutations
in two patients with hyperferritinemia, normal transferrin saturation and iron
accumulation predomitly in macrophages and Kupffer cells. The first one
displayed the well-described alteration V162 Delta and the second a novel
mutation (R178G) that was further detected in two relatives in a pedigree
analysis. The proposed procedure would facilitate the wide-range molecular
analysis of the SLC40A1 gene, contributing to better understanding the
pathogenesis of the ferroportin disease. Hereditary hemochromatosis is an iron overload disorder that can lead to the
impairment of multiple organs and is caused by mutations in one or more
different genes. Type 1 hemochromatosis is the most common form of the disease
and results from mutations in the HFE gene. Juvenile hemochromatosis (JH) is the
most severe form, usually caused by mutations in hemojuvelin (HJV) or hepcidin
(HAMP). The autosomal domit form of the disease, type 4, is due to mutations
in the SLC40A1 gene, which encodes for ferroportin (FPN). Hereditary
hemochromatosis is commonly found in populations of European origin. By
contrast, hemochromatosis in Asia is rare and less well understood and can be
masked by the presence of iron deficiency and secondary iron overload from
thalassemia. Here, we provide a comprehensive report of hemochromatosis in a
group of patients of Asian origin. We have identified novel mutations in HJV,
HAMP, and SLC40A1 in countries not normally associated with hereditary
hemochromatosis (Pakistan, Bangladesh, Sri Lanka, and Thailand). Our family
studies show a high degree of consanguinity, highlighting the increased risk of
iron overload in many countries of the developing world and in countries in
which there are large immigrant populations from these regions. Mutations in the SLC40A1 gene, which encodes ferroportin, are associated with
autosomal domit hemochromatosis. Ferroportin is inhibited directly by
hepcidin, a key iron-regulatory peptide, and functional consequences of SLC40A1
mutations account for observed phenotypic differences in patients with
ferroportin disease. We describe a large pedigree with a novel SLC40A1 mutation
and, through in vitro analysis, elucidate the associated molecular mechanism of
iron overload. The entire coding sequence of the SLC40A1 gene was sequenced in a
pedigree, presenting with autosomal domit hyperferritinemia. The functional
effects of a novel SLC40A1 mutation were studied by overexpression of wild-type
and mutant ferroportin fusion proteins in human embryonic kidney cells. Iron
export was studied in these cells using (59)Fe transport assays; subcellular
localization of ferroportin was examined by way of confocal microscopy. A novel
SLC40A1 mutation p.R489K segregated with iron overload in a family with clinical
and histopathological signs of macrophage-type ferroportin disease. Human
embryonic kidney cells overexpressing p.R489K ferroportin showed decreased iron
export capacity when compared with wild-type ferroportin overexpressing cells.
Subcellular localization studies demonstrated that p.R489K ferroportin was
retained abnormally within an intracellular compartment.
CONCLUSION: We report a novel pathological SLC40A1 variant associated with
abnormal cell surface expression of ferroportin due to intracellular retention
of the mutant protein. These findings predict macrophage-type ferroportin
disease, the phenotype observed in this kindred. Study of the molecular cell
biology of ferroportin and its mutants is key to understanding the pathogenesis
of this increasingly recognized form of hemochromatosis, which responds poorly
to conventional therapy. Aortic dilatation/dissection (AD) can occur spontaneously or in association with
genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS
type 2 and Loeys-Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and
Ehlers-Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations).
Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD
cases referred to us for molecular genetic testing, we have obtained negative
results for these genes in a large cohort of AD patients, suggesting the
involvement of additional genes or acquired factors. In this study we assessed
the effect of COL3A1 deletions/duplications in this cohort. Multiplex
ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients
identified one hemizygous deletion of the entire COL3A1 gene. Subsequent
microarray analyses and sequencing of breakpoints revealed the deletion size of
3,408,306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21
other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1,
ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1,
GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN)
have also been associated with an autosomal domit disorder (EDS classical
type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory
examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN,
but not that of SLC40A1, leads to a clinical phenotype. Our data not only
emphasize the impact/role of COL3A1 in AD patients but also extend the molecular
etiology of several disorders by providing hitherto unreported evidence for true
haploinsufficiency of the underlying gene. The most common form of hemochromatosis is caused by mutations in the HFE gene.
Rare forms of the disease are caused by mutations in other genes. We present a
patient with hyperferritinemia and iron overload, and facial flushing. Magnetic
resoce imaging was performed to measure hepatic iron overload, and a
molecular study of the genes involved in iron metabolism was undertaken. The
iron overload was similar to that observed in HFE hemochromatosis, and the
patient was double heterozygous for two novel mutations, c.-20G>A and c.718A>G
(p.K240E), in the HFE and ferroportin (FPN1 or SLC40A1) genes, respectively.
Hyperferritinemia and facial flushing improved after phlebotomy. Two of the
patient's children were also studied, and the daughter was heterozygous for the
mutation in the SLC40A1 gene, although she did not have hyperferritinemia. The
patient presented a mild iron overload phenotype probably because of the two
novel mutations in the HFE and SLC40A1 genes. BACKGROUND: p.C282Y mutation and rare variants in the HFE gene have been
associated with hereditary hemochromatosis (HH). HH is also caused by mutations
in other genes, such as the hemojuvelin (HJV), hepcidin (HAMP), transferrin
receptor 2 (TFR2) and ferroportin (SLC40A1). The low rate homozygous p.C282Y
mutation in Brazil is suggestive that mutations in non-HFE genes may be linked
to HH phenotype.
AIM: To screen exon-by-exon DNA sequences of HFE, HJV, HAMP, TFR2 and SLC40A1
genes to characterize the molecular basis of HH in a sample of the Brazilian
population.
MATERIALS AND METHODS: Fifty-one patients with primary iron overload
(transferrin saturation ≥50% in females and ≥60% in males) were selected.
Subsequent bidirectional DNA sequencing of HFE, HJV, HAMP, TFR2 and SLC40A1
exons was performed.
RESULTS: Thirty-seven (72.5%) out of the 51 patients presented at least one HFE
mutation. The most frequent genotype associated with HH was the homozygous
p.C282Y mutation (n=11, 21.6%). In addition, heterozygous HFE p.S65C mutation
was found in combination with p.H63D in two patients and homozygous HFE p.H63D
was found in two patients as well. Sequencing in the HJV and HAMP genes revealed
HJV p.E302K, HJV p.A310G, HJV p.G320V and HAMP p.R59G alterations. Molecular and
clinical diagnosis of juvenile hemochromatosis (homozygous form for the HJV
p.G320V) was described for the first time in Brazil. Three TFR2 polymorphisms
(p.A75V, p.A617A and p.R752H) and six SLC40A1 polymorphisms (rs13008848,
rs11568351, rs11568345, rs11568344, rs2304704, rs11568346) and the novel
mutation SLC40A1 p.G204S were also found.
CONCLUSIONS: The HFE p.C282Y in homozygosity or in heterozygosity with p.H63D
was the most frequent mutation associated with HH in this sample. The HJV
p.E302K and HAMP p.R59G variants, and the novel SLC40A1 p.G204S mutation may
also be linked to primary iron overload but their role in the pathophysiology of
HH remain to be elucidated. Hepcidin is an iron-regulatory hepatic peptide hormone encoded by the HAMP gene
that downregulates iron export from enterocytes and macrophages into the blood
plasma. In this study, we identified a novel mutation in the HAMP gene of a
58-year-old Japanese male patient with hemochromatosis. By direct sequencing of
the five hereditary hemochromatosis-related genes, HFE, HAMP, HJV, TFR2, and
SLC40A1, the previously unreported p.R75X mutation was identified, and the
patient was found to be homozygous for the mutation. No other potentially
pathogenic mutations were detected. In an LC-MS/MS analysis, hepcidin molecules
were not detected in the patient's serum or urine. These results indicate that
the p.R75X mutation causes iron overload by impairing the hepcidin system. We report on a 46-year-old black man who resided in Alabama with normal
transferrin saturation, mild hyperferritinemia, chronic hepatitis C, and 3+ iron
in hepatocytes and Kupffer cells. Exome sequencing revealed heterozygosity for
SLC40A1 D270V (exon 7, c.809A→T), a mutation previously reported only in 1 black
patient with iron overload who resided in the Republic of South Africa. The
present patient was also heterozygous for: heme transporter FLVCR1 novel allele
P542S (exon 10, 1624C→T); FLVCR1 T544M (rs3207090); hemopexin (HPX) R371W
(rs75307540); ferritin scavenger receptor (SCARA5) R471H (rs61737287); and
transferrin receptor (TFRC) G420S (rs41295879). He had no HFE, TFR2,HJV, or HAMP
mutations. D270V was not detected in 19 other African Americans with iron
overload who resided in Alabama. The allele frequency of SLC40A1 D270V in 258
African American adults who participated in a health appraisal clinic was 0.0019
(95% confidence interval 0-0.0057). D270V could explain 'classical' ferroportin
hemochromatosis phenotypes in some African Americans. Hereditary hemochromatosis causes iron overload and is associated with a variety
of genetic and phenotypic conditions. Early diagnosis is important so that
effective treatment can be administered and the risk of tissue damage avoided.
Most patients are homozygous for the c.845G>A (p.C282Y) mutation in the HFE
gene; however, rare forms of genetic iron overload must be diagnosed using a
specific genetic analysis. We studied the genotype of 5 patients who had
hyperferritinemia and an iron overload phenotype, but not classic mutations in
the HFE gene. Two patients were undergoing phlebotomy and had no iron overload,
1 with metabolic syndrome and no phlebotomy had mild iron overload, and 2
patients had severe iron overload despite phlebotomy. The patients' first-degree
relatives also underwent the analysis. We found 5 not previously published
mutations: c.-408_-406delCAA in HFE, c.1118G>A (p.G373D), c.1473G>A (p.E491E)
and c.2085G>C (p.S695S) in TFR2; and c.-428_-427GG>TT in SLC40A1. Moreover, we
found 3 previously published mutations: c.221C>T (p.R71X) in HFE; c.1127C>A
(p.A376D) in TFR2; and c.539T>C (p.I180T) in SLC40A1. Four patients were double
heterozygous or compound heterozygous for the mutations mentioned above, and the
patient with metabolic syndrome was heterozygous for a mutation in the TFR2
gene. Our findings show that hereditary hemochromatosis is clinically and
genetically heterogeneous and that acquired factors may modify or determine the
phenotype. Ferroportin (FPN) mediates iron export from cells and this function is modulated
by serum hepcidin. Mutations in the FPN gene (SLC40A1) lead to autosomal
domit iron overload diseases related either to loss or to gain of function,
and usually characterized by normal or low transferrin saturation versus
elevated transferrin saturation, respectively. However, for the same mutation,
the phenotypic expression may vary from one patient to another. Using in vitro
overexpression of wild-type or mutant FPN proteins, we characterized the
functional impact of five recently identified FPN gene mutations regarding FPN
localization, cell iron status, and hepcidin sensitivity. Our aim was to
integrate functional results and biological findings in probands and relatives.
We show that while the p.Arg371Gln (R371Q) mutation had no impact on studied
parameters, the p.Trp158Leu (W158L), p.Arg88Gly (R88G), and p.Asn185Asp (N185D)
mutations caused an iron export defect and were classified as loss-of-function
mutations. The p.Gly204Ser (G204S) mutation induced a gain of FPN function.
Functional studies are useful to determine whether or not a FPN gene mutation
found in an iron overloaded patient is deleterious and to characterize its
biological impact, especially when family studies are not fully informative
and/or additional confounding factors may affect bio-clinical expression. Ferroportin disease is an inherited disorder of iron metabolism and is caused by
mutations in the ferroportin gene (SLC40A1). We present a patient with
hyperferritinemia, iron overload in the liver with reticuloendothelial
distribution and also in the spleen, and under treatment with erythropheresis. A
molecular study of the genes involved in iron metabolism (HFE, HJV, HAMP, TFR2,
SLC40A1) was undertaken. In vitro functional studies of the novel mutation found
in the SLC40A1 gene was performed. The patient was heterozygous for a novel
mutation, c.386T>C (p.L129P) in the SLC40A1 gene; some of his relatives were
also heterozygous for this mutation. In vitro functional studies of the L129P
mutation on ferroportin showed it impairs its capacity to export iron from cells
but does not alter its sensitivity to hepcidin. These findings and the iron
overload phenotype of the patient suggest that the novel mutation c.386T>C
(p.L129P) in the SLC40A1 gene has incomplete penetrance and causes the classical
form of ferroportin disease. Hemochromatosis type 4 is a rare form of primary iron overload transmitted as an
autosomal domit trait caused by mutations in the gene encoding the iron
transport protein ferroportin 1 (SLC40A1). SLC40A1 mutations fall into two
functional categories (loss- versus gain-of-function) underlying two distinct
clinical entities (hemochromatosis type 4A versus type 4B). However, the vast
majority of SLC40A1 mutations are rare missense variations, with only a few
showing strong evidence of causality. The present study reports the results of
an integrated approach collecting genetic and phenotypic data from 44 suspected
hemochromatosis type 4 patients, with comprehensive structural and functional
annotations. Causality was demonstrated for 10 missense variants, showing a
clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups
of loss-of-function mutations were distinguished: one impairing cell-surface
expression and one altering only iron egress. Additionally, a new
gain-of-function mutation was identified, and the degradation of ferroportin on
hepcidin binding was shown to probably depend on the integrity of a large
extracellular loop outside of the hepcidin-binding domain. Eight further
missense variations, on the other hand, were shown to have no discernible
effects at either protein or RNA level; these were found in apparently isolated
patients and were associated with a less severe phenotype. The present findings
illustrate the importance of combining in silico and biochemical approaches to
fully distinguish pathogenic SLC40A1 mutations from benign variants. This has
profound implications for patient management. Ferroportin disease is a rare type of autosomal domitly inherited
hemochromatosis caused with mutations in the ferroportin gene (SLC40A1). The
patients characteristically have hyperferritinemia but normal transferin
saturations. Herein, we present a 15-year-old female whose chief complaint was
persistent nausea for the last one year. Extensive work-up including brain
imaging revealed nothing to explain the etiology of nausea. The serum ferritin
level of 1474ng/mL was suggestive for hemochromatosis syndromes and the
molecular testing revealed de-novo c.485_487delTTG (P.Val162del) ferroportin
gene mutation. Mild hepatic iron loading, in addition to the cumbersome nausea
were accepted as indications for chelation treatment in this particular patient
and deferasirox was initiated (10mg/kg/day) since family did not consent for
phlebotomy. Deferasirox was stopped by the 9th month of initiation, since nausea
subsided and hepatic iron content was normalized, in order to prevent over
chelation. There are no well-established guidelines for the chelation of
patients with hereditary hemochromatosis syndromes. However, lifelong
monitorization for iron loading and re-initiation of chelation when necessary
was planned in our patient. Hemochromatosis is a common cause of chronic liver disease and HFE genotyping
allows decisive and non-invasive diagnosis. Molecular and clinical genetic
studies have led to the identification of genes other than HFE in patients with
inherited diseases associated with increased hepatic iron storage that can cause
hemochromatosis, which adds complexity to a diagnostic approach to patients with
suspected hemochromatosis. Despite major advances in genetics, hepatic iron
quantification by non-invasive methods therefore remains the key to the
diagnosis of hemochromatosis. Although associated with homozygosity for the
C282Y polymorphism in the HFE gene in >80% of patients, hemochromatosis is a
complex genetic disease with strong environmental disease modifiers. Testing for
mutations in the non-HFE hemochromatosis genes transferrin receptor 2,
hemojuvelin, HAMP and SLC40A1 is complex, costly and time-consuming.
Demonstration of hepatic iron overload by liver biopsy or MRI is therefore
required before such complex tests are carried out. The pathogenesis of chronic
liver disease in hemochromatosis is mainly attributed to the redox potential of
tissue iron, and only the more recent studies have focused on the toxic
properties of circulating iron. Considering the fact that an increased
saturation of transferrin and high iron in plasma are the hallmark of all
hemochromatosis forms, an alternative view would be that toxic iron in the
circulation is involved in the pathogenesis of hemochromatosis. Recent studies
have shown an increased concentration of redox-active iron in plasma in patients
with increased transferrin saturation. This finding supports the hypothesis that
tissue iron may be the 'smoking gun' of iron-induced organ damage. Taken
together, caring for patients with suspected or established hemochromatosis
still remains a challenge, where understanding the genetics, biochemistry and
cell biology of hemochromatosis will aid better diagnosis and treatment of
affected individuals. |
Could divalent metal transporter 1 deficiency lead to anemia? | Yes, divalent metal transporter 1 (DMT1) deficiency could result in anemia, as DMT1 is a major iron transporter required for iron absorption and erythropoiesis. DMT1 deficiency impairs erythroid differentiation and induces apoptosis of erythroid cells. | NRAMP2 (natural resistance-associated macrophage protein 2)/DMT1 (divalent metal
transporter 1) is a divalent metal transporter conserved from prokaryotes to
higher eukaryotes that exhibits an unusually broad substrate range, including
Fe(2+), Zn(2+), Mn(2+), Cu(2+), Cd(2+), Co(2+), Ni(2+), and Pb(2+), and mediates
active proton-coupled transport. Recently, it has been shown that the microcytic
anemia (mk) mouse and the Belgrade (b) rat, which have inherited defects in iron
transport that result in iron deficiency anemia, have the same missense mutation
(G185R) in Nramp2. These findings strongly suggested that NRAMP2 is the apical
membrane iron transporter in intestinal epithelial cells and the endosomal iron
transporter in transferrin cycle endosomes of other cells. To investigate the
cellular functions of NRAMP2, we generated a polyclonal antibody against the
N-terminal cytoplasmic domain of human NRAMP2. The affinity-purified anti-NRAMP2
N-terminal antibody recognized a 90-116-kDa membrane-associated protein, and
this band was shifted to 50 kDa by deglycosylation with peptide N-glycosidase F.
Subcellular fractionation revealed that NRAMP2 co-sedimented with the late
endosomal and lysosomal membrane proteins and LAMP-1 (lysosome-associated
membrane protein 1), but not with the transferrin receptor in early endosomes.
The intracellular localization of endogenous NRAMP2 and recombit green
fluorescent protein (GFP)-NRAMP2 was examined by immunofluorescence staining and
by native fluorescence of GFP, respectively. Both endogenous and GFP-NRAMP2 were
detected in vesicular structures and were colocalized with LAMP-2, but not with
EEA1 (early endosome antigen 1) or the transferrin receptor. These results
indicated that NRAMP2 is localized to the late endosomes and lysosomes, where
NRAMP2 may function to transfer the endosomal free Fe(2+) into the cytoplasm in
the transferrin cycle. Microcytic anemia (mk) mice and Belgrade (b) rats are severely iron deficient
because of impaired intestinal iron absorption and defective iron metabolism in
peripheral tissues. Both animals carry a glycine to arginine substitution at
position 185 in the iron transporter known as Nramp2/DMT1 (divalent metal
transporter 1). DMT1 messenger RNA (mRNA) and protein expression has been
examined in the gastrointestinal tract of mk mice. Northern blot analysis
indicates that, by comparison to mk/+ heterozygotes, mk/mk homozygotes show a
dramatic increase in the level of DMT1 mRNA in the duodenum. This increase in
RNA expression is paralleled by a concomitant increase of the 100-kd DMT1
isoform I protein expression in the duodenum. Immunohistochemical analyses show
that, as for normal mice on a low-iron diet, DMT1 expression in enterocytes of
mk/mk mice is restricted to the duodenum. However, and in contrast to normal
enterocytes, little if any expression of DMT1 is seen at the apical membrane in
mk/mk mice. These results suggest that the G185R mutation, which was shown to
impair the transport properties of DMT1, also affects the membrane targeting of
the protein in mk/mk enterocytes. This loss of function of DMT1 is paralleled by
a dramatic increase in expression of the defective protein in mk/mk mice. This
is consistent with a feedback regulation of DMT1 expression by iron stores.
(Blood. 2000;96:3964-3970) Divalent metal transporter-1 (DMT1/DCT1/Nramp2) is the major Fe(2+) transporter
mediating cellular iron uptake in mammals. Phenotypic analyses of animals with
spontaneous mutations in DMT1 indicate that it functions at two distinct sites,
transporting dietary iron across the apical membrane of intestinal absorptive
cells, and transporting endosomal iron released from transferrin into the
cytoplasm of erythroid precursors. DMT1 also acts as a proton-dependent
transporter for other heavy metal ions including Mn(2+), Co(2+), and Cu(2), but
not for Mg(2+) or Ca(2+). A unique mutation in DMT1, G185R, has occurred
spontaneously on two occasions in microcytic (mk) mice and once in Belgrade (b)
rats. This mutation severely impairs the iron transport capability of DMT1,
leading to systemic iron deficiency and anemia. The repeated occurrence of the
G185R mutation cannot readily be explained by hypermutability of the gene. Here
we show that G185R mutant DMT1 exhibits a new, constitutive Ca(2+) permeability,
suggesting a gain of function that contributes to remutation and the mk and b
phenotypes. Belgrade rats exhibit microcytic, hypochromic anemia and systemic iron
deficiency due to a glycine-to-arginine mutation at residue 185 in a metal ion
transporter of a divalent metal transporter/divalent cation transporter/solute
carrier 11 group A member 2 or 3 (DMT1/DCT1/SLC11A2), a member of the
natural-resistance-associated macrophage protein (Nramp) family. By use of
rabbit duodenal tissue, a calcein fluorescence assay has previously been
developed to assess transport of divalent metal ions across the small-intestinal
brush border membrane (BBM). The assay was readily applied here to rat BBM to
learn if it detects DMT1 activity. The results demonstrate protein-mediated
transport across the BBM of all tested ions: Mn(2+), Fe(2+), and Ni(2+).
Transport into BBM vesicles (BBMV) from (b/b) Belgrade rats was below the
detection limit. BBMV of +/b origin had substantial activity. The kinetic rate
constant for Ni(2+) membrane transport for +/b BBMV was within the range for
normal rabbit tissue. Vesicles from +/b basolateral membranes (BLM) showed
similar activity to BBMV while b/b BLM vesicles (BLMV) lacked transport
activity. Immunoblots using isoform-specific antibodies demonstrated that
intestinal levels of b/b DMT1 were increased compared to +/b DMT1, reflecting
iron deficiency. Immunoblots on BBMV indicated that lack of activity in b/b
vesicles was not due to a failure of DMT1 to localize to the BBMV; an excess of
specific isoforms was present compared to +/b BBMV or duodenal extracts.
Immunoblots from BLMV also exhibited enrichment in DMT1 isoforms, despite their
distinct origin. Immunofluorescent staining of thin sections of b/b and +/b
proximal intestines confirmed that DMT1 localized similarly in mutant and
control enterocytes and showed that DMT1 isoforms have distinct distributions
within intestinal tissue. Exposure to bleomycin can result in an inflammatory lung injury. The biological
effect of this anti-neoplastic agent is dependent on its coordination of iron
with subsequent oxidant generation. In lung cells, divalent metal transporter 1
(DMT1) can participate in metal transport resulting in control of an oxidative
stress and tissue damage. We tested the postulate that metal import by DMT1
would participate in preventing lung injury after exposure to bleomycin.
Microcytic anemia (mk/mk) mice defective in DMT1 and wild-type mice were exposed
to either bleomycin or saline via intratracheal instillation and the resultant
lung injury was compared. Twenty-four h after instillation, the number of
neutrophils and protein concentrations after bleomycin exposure were
significantly elevated in the mk/mk mice relative to the wild-type mice.
Similarly, levels of a pro-inflammatory mediator were significantly increased in
the mk/mk mice relative to wild-type mice following bleomycin instillation.
Relative to wild-type mice, mk/mk mice demonstrated lower non-heme iron
concentrations in the lung, liver, spleen, and splenic, peritoneal, and liver
macrophages. In contrast, levels of this metal were elevated in alveolar
macrophages from mk/mk mice. We conclude that DMT1 participates in the
inflammatory lung injury after bleomycin with mk/mk mice having increased
inflammation and damage following exposure. This finding supports the hypothesis
that DMT1 takes part in iron detoxification and homeostasis in the lung. BACKGROUND: Hypochromic microcytic anemia associated with ineffective
erythropoiesis caused by recessive mutations in divalent metal transporter 1
(DMT1) can be improved with high-dose erythropoietin supplementation. The aim of
this study was to characterize and compare erythropoiesis in samples from a
DMT1-mutant patient before and after treatment with erythropoietin, as well as
in a mouse model with a DMT1 mutation, the mk/mk mice.
DESIGN AND METHODS: Colony assays were used to compare the in vitro growth of
pre-treatment and post-treatment erythroid progenitors in a DMT1-mutant patient.
To enable a comparison with human data, high doses of erythropoietin were
administered to mk/mk mice. The apoptotic status of erythroblasts, the
expression of anti-apoptotic proteins, and the key components of the bone
marrow-hepcidin axis were evaluated.
RESULTS: Erythropoietin therapy in vivo or the addition of a broad-spectrum
caspase inhibitor in vitro significantly improved the growth of human
DMT1-mutant erythroid progenitors. A decreased number of apoptotic erythroblasts
was detected in the patient's bone marrow after erythropoietin treatment. In
mk/mk mice, erythropoietin administration increased activation of signal
transducer and activator of transcription 5 (STAT5) and reduced apoptosis in
bone marrow and spleen erythroblasts. mk/mk mice propagated on the 129S6/SvEvTac
background resembled DMT1-mutant patients in having increased plasma iron but
differed by having functional iron deficiency after erythropoietin
administration. Co-regulation of hepcidin and growth differentiation factor 15
(GDF15) levels was observed in mk/mk mice but not in the patient.
CONCLUSIONS: Erythropoietin inhibits apoptosis of DMT1-mutant erythroid
progenitors and differentiating erythroblasts. Ineffective erythropoiesis
associated with defective erythroid iron utilization due to DMT1 mutations has
specific biological and clinical features. BACKGROUND/AIMS: Deficiency of the divalent metal transporter 1 (DMT1) leads to
hypochromic microcytic anemia. We have previously shown that DMT1 deficiency
impairs erythroid differentiation and induces apoptosis of erythroid cells. Here
we analyzed metabolic processes and survival of mature erythrocytes in order to
address potential involvement of erythrocyte defect in the pathophysiology of
the disease.
METHODS: FACS analysis was used to determine the half-life of erythrocytes (CFSE
fluorescence), phosphatidylserine exposure (Annexin V binding), cytosolic Ca(2+)
(Fluo3/AM fluorescence) and reactive oxygen species (ROS; DCF fluorescence).
Enzyme activities were determined by standard biochemical methods. The
concentration of ATP and ADP was measured on HPLC-MS/MS.
RESULTS: We observed an accelerated clearance of CFSE-labeled DMT1-mutant
erythrocytes from circulating blood when compared to wild-type erythrocytes. In
vitro, DMT1-mutant erythrocytes showed significantly increased Annexin V binding
after exposure to hyperosmotic shock and glucose depletion. Despite exaggerated
anti-oxidative defense, higher ROS levels were present in DMT1-mutant
erythrocytes. Accelerated anaerobic glycolysis and reduced ATP/ADP ratio
detected in DMT1-mutant erythrocytes indicate enhanced demand for ATP.
CONCLUSIONS: We propose that DMT1 deficiency negatively affects metabolism and
life span of mature erythrocytes; two other aspects of defective erythropoiesis
which contribute to the pathophysiology of the disease. Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring
membrane-transport protein that serves a critical role in erythroid iron
utilization. We have investigated its role in intestinal metal absorption by
studying a mouse model lacking intestinal DMT1 (i.e., DMT1(int/int)).
DMT1(int/int) mice exhibited a profound hypochromic-microcytic anemia,
splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was
demonstrated by the following observations in DMT1(int/int) mice: 1) blood iron
and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver
hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected
the anemia, and reversed the changes in blood iron, nonheme-iron stores, and
hepcidin expression levels. We observed decreased total iron content in multiple
tissues from DMT1(int/int) mice compared with DMT1(+/+) mice but no meaningful
change in copper, manganese, or zinc. DMT1(int/int) mice absorbed (64)Cu and
(54)Mn from an intragastric dose to the same extent as did DMT1(+/+) mice but
the absorption of (59)Fe was virtually abolished in DMT1(int/int) mice. This
study reveals a critical function for DMT1 in intestinal nonheme-iron absorption
for normal growth and development. Further, this work demonstrates that
intestinal DMT1 is not required for the intestinal transport of copper,
manganese, or zinc. The divalent metal transporter 1 (DMT1) is a major iron transporter required for
iron absorption and erythropoiesis. Loss of DMT1 function results in microcytic
anemia. While iron plays an important role in neural function, the behavioral
consequences of DMT1 deficiency are largely unexplored. The goal of this study
was to define the neurobehavioral and neurochemical phenotypes of homozygous
Belgrade (b/b) rats that carry DMT1 mutation and explore potential mechanisms of
these phenotypes. The b/b rats (11-12 weeks old) and their healthy littermate
heterozygous (+/b) Belgrade rats were subject to elevated plus maze tasks. The
b/b rats spent more time in open arms, entered open arms more frequently and
traveled more distance in the maze than +/b controls, suggesting increased
impulsivity. Impaired emotional behavior was associated with down-regulation of
GABA in the hippocampus in b/b rats. Also, b/b rats showed increased GABAA
receptor α1 and GABA transporter, indicating altered GABAergic function.
Furthermore, metal analysis revealed that b/b rats have decreased total iron,
but normal non-heme iron, in the brain. Interestingly, b/b rats exhibited
unusually high copper levels in most brain regions, including striatum and
hippocampus. Quantitative PCR analysis showed that both copper importer copper
transporter 1 and exporter copper-transporting ATPase 1 were up-regulated in the
hippocampus from b/b rats. Finally, b/b rats exhibited increased 8-isoprostane
levels and decreased glutathione/glutathione disulfide ratio in the hippocampus,
reflecting elevated oxidative stress. Combined, our results suggest that copper
loading in DMT1 deficiency could induce oxidative stress and impair GABA
metabolism, which promote impulsivity-like behavior. Iron-copper model:
Mutations in the divalent metal transporter 1 (DMT1) decrease body iron status
and up-regulate copper absorption, which leads to copper loading in the brain
and consequently increases metal-induced oxidative stress. This event disrupts
GABAergic neurotransmission and promotes impulsivity-like behavior. Our model
provides better understanding of physiological risks associated with imbalanced
metal metabolism in mental function and, more specifically, the interactions
with GABA and redox control in the treatment of emotional disorders. |
Does the hERG gene code for a protein which is part of a sodium channel? | The hERG AKA Human ether-a-go-go-related gene coded for a protein subunit of a potassium channel that conducts delayed rectifier K(+) current | BACKGROUND: The genes for the long QT syndrome (LQTS) linked to chromosomes 3
(LQT3) and 7 (LQT2) were identified as SCN5A, the cardiac Na+ channel gene, and
as HERG, a K+ channel gene. These findings opened the possibility of attempting
gene-specific control of ventricular repolarization. We tested the hypothesis
that the QT interval would shorten more in LQT3 than in LQT2 patients in
response to mexiletine and also in response to increases in heart rate.
METHODS AND RESULTS: Fifteen LQTS patients were studied. Six LQT3 and 7 LQT2
patients were treated with mexiletine, and its effects on QT and QTc were
measured. Mexiletine significantly shortened the QT interval among LQT3 patients
(QTc from 535 +/- 32 to 445 +/- 31 ms, P < .005) but not among LQT2 patients
(QTc from 530 +/- 79 to 503 +/- 60 ms, P = NS). LQT3 patients (n = 7) shortened
their QT interval in response to increases in heart rate much more than LQT2
patients (n = 4) and also more than 18 healthy control subjects (9.45 +/- 3.3
versus 3.95 +/- 1.97 and 2.83 +/- 1.33, P < .05; data expressed as percent
reduction in QT per 100-ms shortening in RR). Among these patients, there is
also a trend for LQT2 patients to have syncope or cardiac arrest under emotional
or physical stress and for LQT3 patients to have cardiac events either at rest
or during sleep.
CONCLUSIONS: This is the first study to demonstrate differential responses of
LQTS patients to interventions targeted to their specific genetic defect. These
findings also suggest that LQT3 patients may be more likely to benefit from Na+
channel blockers and from cardiac pacing because they would be at higher risk of
arrhythmia at slow heart rates. Conversely, LQT2 patients may be at higher risk
to develop syncope under stressful conditions because of the combined
arrhythmogenic effect of catecholamines with the insufficient adaptation of
their QT interval when heart rate increases. A human genetic defect associated with 'long Q-T syndrome', an abnormality of
cardiac rhythm involving the repolarization of the action potential, was
recently found to lie in the HERG gene, which codes for a potassium channel. The
HERG K+ channel is unusual in that it seems to have the architectural plan of
the depolarization-activated K+ channel family (six putative transmembrane
segments), yet it exhibits rectification like that of the inward-rectifying K+
channels, a family with different molecular structure (two transmembrane
segments). We have studied HERG channels expressed in mammalian cells and find
that this inward rectification arises from a rapid and voltage-dependent
inactivation process that reduces conductance at positive voltages. The
inactivation gating mechanism resembles that of C-type inactivation, often
considered to be the 'slow inactivation' mechanism of other K+ channels. The
characteristics of this gating suggest a specific role for this channel in the
normal suppression of arrhythmias. HERG (human eag-related gene) encodes an inward-rectifier potassium channel
formed by the assembly of four subunits. Since the truncated HERG protein in
patients with long QT syndrome induces a domit phenotype, that is, cardiac
sudden death, the assembly of nonfunctional complexes between wild-type and
mutated subunits was implicated in causing the disease. To understand
HERG-mediated cardiac sudden death at the molecular level, it is important to
determine which regions in the HERG protein participate in subunit interaction.
We therefore report the identification of a subunit interaction domain,
NAB(HERG), that is localized at the hydrophilic cytoplasmic N terminus and can
form a tetramer in the absence of the rest of the HERG protein. Truncated HERG
proteins containing NAB(HERG), including one that resulted from the delta1261
human mutation, inhibit the functional expression of the HERG channel in
transfected cells. Together, these results support the notion that the
expression of HERG in the human heart may be decreased in the presence of the
truncated subunit. Such a decrease of potassium channel expression can
contribute to the longer QT intervals observed in the patients with the HERG
mutation. BACKGROUND: Long-QT syndrome (LQTS) is an inherited cardiac arrhythmia that
causes sudden death in young, otherwise healthy people. Four genes for LQTS have
been mapped to chromosome 11p15.5 (LQT1), 7q35-36 (LQT2), 3p21-24 (LQT3), and
4q25-27 (LQT4). Genes responsible for LQT1, LQT2, and LQT3 have been identified
as cardiac potassium channel genes (KVLQT1, HERG) and the cardiac sodium channel
gene (SCN5A).
METHODS AND RESULTS: After studying 115 families with LQTS, we used
single-strand conformation polymorphism (SSCP) and DNA sequence analysis to
identify mutations in the cardiac potassium channel gene, KVLQT1. Affected
members of seven LQTS families were found to have new, previously unidentified
mutations, including two identical missense mutations, four identical splicing
mutations, and one 3-bp deletion. An identical splicing mutation was identified
in affected members of four unrelated families (one Italian, one Irish, and two
American), leading to an alternatively spliced form of KVLQT1. The 3-bp deletion
arose de novo and occurs at an exon-intron boundary. This results in a single
base deletion in the KVLQT1 cDNA sequence and alters splicing, leading to the
truncation of KVLQT1 protein.
CONCLUSIONS: We have identified LQTS-causing mutations of KVLQT1 in seven
families. Five KVLQT1 mutations cause the truncation of KVLQT1 protein. These
data further confirm that KVLQT1 mutations cause LQTS. The location and
character of these mutations expand the types of mutation, confirm a mutational
hot spot, and suggest that they act through a loss-of-function mechanism or a
domit-negative mechanism. The chromosome 7-linked form of congenital long QT syndrome (LQT2) is caused by
mutations in the human ether-a-go-go-related gene (HERG) that encodes the
rapidly activating delayed rectifier potassium channel. One mechanism for the
loss of normal channel function in LQT2 is defective protein trafficking, which
results in the failure of the channel protein to reach the plasma membrane. Here
we show that the N470D LQT2 mutant protein is trafficking-deficient when
expressed at 37 degrees C in HEK293 cells, whereas at 27 degrees C its
trafficking to the plasma membrane and channel function are markedly improved.
We further show that the antiarrhythmic drug E-4031, which selectively blocks
HERG channels, also corrects defective protein trafficking of the N470D mutant
and can restore the generation of HERG current. Similar findings were obtained
with the drugs astemizole and cisapride, as well as with high concentrations of
glycerol. The effect of E-4031 on HERG protein trafficking was
concentration-dependent and required low drug concentrations (saturation present
at 5 microM), developed rapidly with drug exposure, and occurred
post-translationally. These findings suggest that protein misfolding leading to
defective trafficking of some HERG LQT mutations may be corrected by specific
pharmacological strategies. The congenital long QT syndrome is characterised by the presence of syncopes due
to torsades de pointe which may degenerate to ventricular fibrillation and cause
sudden death. These syncopes occur in young subjects with electrocardiographic
abnormalities and prolongation of the QT interval. Patients with the autosomally
domit transmitted Romano-Ward syndrome with normal audition are classically
opposed to those with the Jervell and Lange-Nielsen autosomally recessive
syndrome who have bilateral total deafness. Our understanding of the congenital
long QT syndrome has improved in recent years with respect to the
physiopathology, diagnosis and treatment, due to research in the fields of
genetics, electrocardiography and electrophysiology. The diagnosis is based on
analysis of the phenotype and genotypes. A family enquiry is always necessary to
detect unrecognised forms. Five culprit genes have been identified for the
Romano-Ward syndrome. All code for subunits of sodium or potassium channels: two
a subunits of the potassium channels (QVLQT1 for LQT1, HERG for LQT2), the a
subunit of the sodium channel INa (SCN5A for LQT3), and two regulatory subunits
of potassium channels (KCNE1 for LQT5 regulating the KvLQT1 channel and MiRP1
regulating HERG). The concept of genetic heterogeneity of the congenital long QT
syndrome may thus be understood: different genes may be responsible for the same
phenotype. Except for specific cases, the usual treatment is life-long
betablocker therapy and the avoidance of a large number of drugs, the list of
which is continually updated. A multicentre trial is underway to validate
betablocker therapy for the prevention of cardiac events in a LQT1 genotype
population. Prospective studies will be necessary to assess gene-specific
treatments. The human ether-à-go-go-related gene (HERG) encodes the pore-forming subunit of
the rapidly activating delayed rectifier potassium channel in the heart. We
previously showed that HERG channel protein is modified by N-linked
glycosylation. HERG protein sequence contains two extracellular consensus sites
for N-linked glycosylation (N598, N629). In this study, we used the approaches
of site-directed mutagenesis and biochemical modification to inhibit N-linked
glycosylation and studied the role of glycosylation in the cell surface
expression and turnover of HERG channels. Our results show that N598 is the only
site for N-linked glycosylation and that glycosylation is not required for the
cell surface expression of functional HERG channels. In contrast, N629 is not
used for glycosylation, but mutation of this site (N629Q) causes a protein
trafficking defect, which results in its intracellular retention. Pulse-chase
experiments show that the turnover rate of nonglycosylated HERG channel is
faster than that of the glycosylated form, suggesting that N-linked
glycosylation plays an important role in HERG channel stability. OBJECTIVES: The aim of this study was to test whether a recently reported
polymorphism in the HERG gene coding for the rapidly activating delayed
rectifier K+ channel has influence on myocardial repolarization.
BACKGROUND: The length of myocardial repolarization, measured as the QT
interval, has a hereditary component, but no genes that would explain the
variability of repolarization have been identified in healthy subjects.
METHODS: QT intervals were measured from the 12-lead electrocardiogram in a
random middle-aged population (226 men/187 women). The longest QT interval at
any of the 12 leads (QTmax), QTV(2), and the Tpeak-Tend interval were used as
measures of repolarization. Deoxyribonucleic acid samples were genotyped for the
nucleotide 2690A>C variation of the HERG gene, corresponding to the HERG
K(lysine)897T(threonine) amino acid polymorphism.
RESULTS: The allele frequencies were 0.84 (A) and 0.16 (C). Females with the
genotype AC or CC had longer QTcmax (477 +/- 99 ms) and Tpeak-Tend intervals
(143 +/- 95 ms) than females with the genotype AA (441 +/- 69 ms and 116 +/- 65
ms, p = 0.005 and p = 0.025, respectively). In males, the QTcmax and the
Tpeak-Tend intervals did not differ between the genotypes. After adjustment for
echocardiographic and various laboratory variables, the HERG K897T polymorphism
remained as an independent predictor of QTcmax (p = 0.009) and the Tpeak-Tend
intervals (p = 0.026) in females. CONCLUSIONS; The common K897T polymorphism of
the HERG channel is associated with the maximal duration and transmural
dispersion of ventricular repolarization in middle-aged females. Long QT syndrome (LQTS) is a cardiac repolarization disorder that can lead to
arrhythmias and sudden death. Chromosome 7-linked inherited LQTS (LQT2) is
caused by mutations in human ether-a-go-go-related gene (HERG; KCNH2), whereas
drug-induced LQTS is caused primarily by HERG channel block. Many common
polymorphisms are functionally silent and have been traditionally regarded as
benign and without physiological consequence. However, the identification of
common nonsynonymous single nucleotide polymorphisms (nSNPs; i.e., amino-acid
coding variants) with functional phenotypes in the SCN5A Na(+) channel and MiRP1
K(+) channel beta-subunit have challenged this viewpoint. In this report, we
test the hypothesis that common missense HERG polymorphisms alter channel
physiology. Comprehensive mutational analysis of HERG was performed on genomic
DNA derived from a population-based cohort of sudden infant death syndrome and
two reference allele cohorts derived from 100 African American and 100 Caucasian
individuals. Amino acid-encoding variants were considered common polymorphisms
if they were present in at least two of the three study cohorts with an allelic
frequency >0.5%. Four nSNPs were identified: K897T, P967L, R1047L, and Q1068R.
Wild-type (WT) and polymorphic channels were heterologously expressed in human
embryonic kidney cells, and biochemical and voltage-clamp techniques were used
to characterize their functional properties. All channel types were processed
similarly, but several electrophysiological differences were identified: 1)
K897T current density was lower than the other polymorphic channels; 2) K897T
channels activated at more negative potentials than WT and R1047L; 3) K897T and
Q1068R channels inactivated and recovered from inactivation faster than WT,
P967L, and R1047L channels; and 4) K897T channels showed subtle differences
compared with WT channels when stimulated with an action potential waveform. In
contrast to K897T and Q1068R channels, P967L and R1047L channels were
electrophysiologically indistinguishable from WT channels. All HERG channels had
similar sensitivity to block by cisapride. Therefore, some HERG polymorphic
channels are electrophysiologically different from WT channels. BACKGROUND: Although KCNH2 (HERG) K897T polymorphism has been shown to be
associated with the QT interval measured from 12-lead electrocardiogram (ECG),
the functional significance of K897T polymorphism has been debated. The aim of
this study was to test whether the K897T polymorphism of the KCNH2 (HERG) gene
coding for the rapidly activating delayed rectifier K+ channel influences
cardiac repolarization assessed by principal component analysis (PCA) of T-wave
morphology.
METHODS: Twelve-lead ECGs were digitized and T-wave morphology was analyzed with
a PCA method in a population consisting of 228 healthy middle-aged subjects (121
women and 107 men). DNA samples were genotyped for the nucleotide 2690 A>C
variation of the KCNH2 gene, corresponding to the KCNH2 K(lysine)897T(threonine)
amino acid polymorphism.
RESULTS: The allele frequencies were 0.86 (K) and 0.14 (T). The KCNH2 K897T
polymorphism was associated with the total cosine R-to-T (TCRT), which reflects
the wave front direction between depolarization and repolarization. TCRT was
0.421 in the genotype KK and 0.300 in the genotypes KT and TT (P = 0.04). The
difference of TCRT was more marked between the KCNH2 K897T genotypes in women (P
= 0.03) than in men (P = 0.52).
CONCLUSIONS: The common K897T polymorphism of the cardiac potassium channel
KCNH2 has functional significance for cardiac electrical properties. Subjects
with a less common genotype, KT or TT, have smaller TCRT, which reflects
dyssynchrony between depolarization and repolarization and is associated with an
increased risk of cardiac mortality. BACKGROUND AND PURPOSE: Fluoxetine (Prozac) is a widely prescribed drug in
adults and children, and it has an active metabolite, norfluoxetine, with a
prolonged elimination time. Although uncommon, Prozac causes QT interval
prolongation and arrhythmias; a patient who took an overdose of Prozac exhibited
a prolonged QT interval (QTc 625 msec). We looked for possible mechanisms
underlying this clinical finding by analysing the effects of fluoxetine and
norfluoxetine on ion channels in vitro.
EXPERIMENTAL APPROACH: We studied the effects of fluoxetine and norfluoxetine on
the electrophysiology and cellular trafficking of hERG K+ and SCN5A Na+ channels
heterologously expressed in HEK293 cells.
KEY RESULTS: Voltage clamp analyses employing square pulse or ventricular action
potential waveform protocols showed that fluoxetine and norfluoxetine caused
direct, concentration-dependent, block of hERG current (IhERG). Biochemical
studies showed that both compounds also caused concentration-dependent
reductions in the trafficking of hERG channel protein into the cell surface
membrane. Fluoxetine had no effect on SCN5A channel or HEK293 cell endogenous
current. Mutations in the hERG channel drug binding domain reduced fluoxetine
block of IhERG but did not alter fluoxetine's effect on hERG channel protein
trafficking.
CONCLUSIONS AND IMPLICATIONS: Our findings show that both fluoxetine and
norfluoxetine at similar concentrations selectively reduce IhERG by two
mechanisms, (1) direct channel block, and (2) indirectly by disrupting channel
protein trafficking. These two effects are not mediated by a single drug binding
site. Our findings add complexity to understanding the mechanisms that cause
drug-induced long QT syndrome. The long QT syndrome gene human ether-a-go-go related gene (HERG) encodes a K(+)
channel critical to cardiac repolarization. It peculiarly overexpresses in
cancer cells of different histogenesis and promotes tumorigenesis. To decipher
the molecular mechanisms for HERG overexpression, we identified and
characterized the promoter region of the HERG gene, which contains cis-elements
for multiple oncoproteins and tumor suppressors. Oncoprotein Sp1 was found to be
essential to driving the HERG promoter thereby transcription. Another
oncoprotein NF-kappaB transactivated, while tumor suppressor Nkx3.1 repressed
HERG promoter activity and endogenous HERG transcription. Loss-of-function
mutations in the corresponding cis-elements rendered a loss of the ability of
the oncoproteins Sp1 and NF-kappaB to transactivate, and of the tumor repressor
Nkx3.1 to repress, HERG transcription. Either activation of Sp1 and NF-kappaB or
silencing of Nkx3.1 promoted tumor cell growth, and the effects were abrogated
by HERG inhibition or knockdown, but facilitated by overexpression of HERG,
indicating that HERG mediates the cell growth signals generated by activation of
oncoproteins or inactivation of tumor suppressors. Binding of Sp1, NF-kappaB,
and Nkx3.1 to their respective cis-elements in the HERG promoter in vitro and
their presence on the HERG promoter in vivo were confirmed. Therefore, the HERG
promoter region is characterized by multiple Sp1 binding sites that are
responsible for transcription initiation of the HERG gene and by binding sites
for multiple other oncogenes and tumor suppressor genes being important for
regulating HERG expression. The HERG K(+) channel is likely a mediator of
growth-promoting processes induced by oncoproteins and/or by silencing of tumor
suppressors. Rapidly activating component of delayed rectifier potassium current (I(Kr))
plays a key role in the repolarization phase of cardiac action potential. Human
ether-a-go-go-related gene (HERG) encodes the alpha subunit of this potassium
channel. Mutations of HERG gene induce genetic long QT syndrome (LQTS).
Furthermore, I(Kr)/HERG is the target of some drugs which may cause cardiac QT
interval prolongation. Some other drugs with different chemical structures also
may block the channel and prolong QT interval, which even developed into
acquired arrhythmias. This review summarized the recent progress of structure,
gating mechanisms and functions of I(Kr)/HERG channel, I(Kr)/HERG related
arrhythmias, interaction between K+ channel and drugs, and strategies of
grading-up the I(Kr)/HERG target. The problem of formation of cardiac arrhythmia such as "torsades de pointes"
because of prolongation of the QT interval is discussed in this article. It is
established 2 forms of the long QT syndrome, a congenital and an acquired one.
The congenital form has seven different known predisposing genes, six of which
are associated with the myocardial ion channels. The prevalence of the
congenital form is estimated at less than 1/10000. The molecular basis of
inherited disorders caused by a mutation in either the gene coding for a
particular potassium channel called HERG-or another gene, SCN5A, which codes for
the sodium channel and disruption of which results in a loss of inactivation of
the Na+ current. Among the congenital forms, particularly interest is focused on
the potassium channel coded by the HERG gene located on chromosome 7 and with a
key role in the normal electric cardiac activity. The potassium channel coded by
the HERG gene is partly responsible for the return of the electric cardiac
activity to the resting phase before the next myocardial electric activation
process. Disturbed function will prolong the return to resting phase, which is
thought to be an essential part of the development mechanism of myocardial
"torsades de pointes" tachycardia. There may also be correlation between the
strength of binding of the medicinal substance to the potassium channel coded by
the HERG gene and prolongation of the QT interval. The investigation revealed
that different groups of drugs may produce an effect on the QT interval, such as
some neuroleptics (haloperidol, droperidol, thioridazine) antidepressants
(paroxetine), narcotic analgesics (methadone), antiarrhythmics (class I),
antihistamines (H1 receptor blocking agents) and motility stimulants
(domperidone and cisapride). For the prevention of cardiac arrhythmia such as
"torsades de pointes". Human ether-à-go-go-related gene (hERG or Kv11.1) encodes the rapidly activated
delayed rectifier K(+) current (I(Kr)) in the human heart. Potential regulation
of hERG channel by protein tyrosine kinases (PTKs) is not understood. The
present study was designed to investigate whether this channel is modulated by
PTKs using whole-cell patch clamp technique, and immunoprecipitation and Western
blot analysis in HEK 293 cells stably expressing hERG gene. We found that the
broad-spectrum PTK inhibitor genistein (30 microM), the selective EGFR
(epidermal growth factor receptor) kinase inhibitor AG556 (10 microM) and the
Src-family kinase inhibitor PP2 (10 microM) remarkably inhibited hERG channel
current (I(hERG)), and the effects were significantly countered by the protein
tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation
and Western blot analysis demonstrated that membrane protein tyrosine
phosphorylation of hERG channels was reduced by genistein, AG556, and PP2. The
reduction of hERG channel phosphorylation level by genistein, AG556 or PP2 was
antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A
dramatically attenuated the inhibitory effect of I(hERG) by PP2 and/or AG556.
Our results demonstrate the novel information that I(hERG) is modulated not only
by Src-family kinases, but also by EGFR kinases. Y475 and/or Y611 are likely the
preferred phosphorylation sites. Regulation of hERG channels by PTKs modifies
the channel activity and thus likely alters electrophysiological properties
including action potential duration and cell excitability in human heart and
neurons. Several therapeutic compounds have been identified that prolong the QT interval
on the electrocardiogram and cause torsade de pointes arrhythmias not by direct
block of the cardiac potassium channel human ether-à-go-go-related gene (hERG)
but via disruption of hERG trafficking to the cell surface membrane. One example
of a clinically important compound class that potently inhibits hERG trafficking
are cardiac glycosides. We have shown previously that inhibition of hERG
trafficking by cardiac glycosides is initiated via direct block of Na(+)/K(+)
pumps and not via off-target interactions with hERG or any other protein.
However, it was not known how pump inhibition at the cell surface is coupled to
hERG processing in the endoplasmic reticulum. Here, we show that depletion of
intracellular K(+)-either indirectly after long-term exposure to cardiac
glycosides or directly after exposure to gramicidin in low sodium media-is
sufficient to disrupt hERG trafficking. In K(+)-depleted cells, hERG trafficking
can be restored by permeating K(+) or Rb(+) ions, incubation at low temperature,
exposure to the pharmacological chaperone astemizole, or specific mutations in
the selectivity filter of hERG. Our data suggest a novel mechanism for
drug-induced trafficking inhibition in which cardiac glycosides produce a
[K(+)](i)-mediated conformational defect directly in the hERG channel protein. The human ether-a-go-go-related gene (hERG) encodes the rapidly activating,
delayed rectifier potassium channel (IKr) important for cardiac repolarization.
Dysfunction of the hERG channel can cause Long QT Syndrome (LQTS). A wide
variety of structurally diverse therapeutic compounds reduce the hERG current by
acute direct inhibition of the hERG current or/and selective disruption of hERG
protein expression. Arsenic trioxide (As(2)O(3)), which is used to treat acute
promyelocytic leukemia, can cause LQTS type 2 (LQT2) by reducing the hERG
current through the diversion of hERG trafficking to the cytoplasmic membrane.
This cardiotoxicity limits its clinical applications. Our aim was to develop
cardioprotective agents to decrease As(2)O(3)-induced cardiotoxicity. We
reported that superfusion of hERG-expressing HEK293 (hERG-HEK) cells with
matrine (1, 10 μM) increased the hERG current by promoting hERG channel
activation. Long-term treatment with 1 μM matrine or oxymatrine increased
expression of the hERG protein and rescued the hERG surface expression disrupted
by As(2)O(3). In addition, Matrine and oxymatrine significantly shortened action
potential duration prolonged by As(2)O(3) in guinea pig ventricular myocytes.
These results were ascribed to the up-regulation of hERG at both mRNA and
protein levels via an increase in the expression of transcription factor Sp1, an
established transactivator of the hERG gene. Therefore, matrine and oxymatrine
may have the potential to cure LQT2 as a potassium channel activator by
promoting hERG channel activation and increasing hERG channel expression. The human ether-a-go-go-related gene (hERG) encodes the pore-forming α-subunit
of the rapidly activating delayed rectifier K(+) channel in the heart, which
plays a critical role in cardiac action potential repolarization. Dysfunction of
IKr causes long QT syndrome, a cardiac electrical disorder that predisposes
affected individuals to fatal arrhythmias and sudden death. The homeostasis of
hERG channels in the plasma membrane depends on a balance between protein
synthesis and degradation. Our recent data indicate that hERG channels undergo
enhanced endocytic degradation under low potassium (hypokalemia) conditions. The
GTPase Rab4 is known to mediate rapid recycling of various internalized proteins
to the plasma membrane. In the present study, we investigated the effect of Rab4
on the expression level of hERG channels. Our data revealed that overexpression
of Rab4 decreases the expression level of hERG in the plasma membrane. Rab4 does
not affect the expression level of the Kv1.5 or EAG K(+) channels.
Mechanistically, our data demonstrate that overexpression of Rab4 increases the
expression level of endogenous Nedd4-2, a ubiquitin ligase that targets hERG but
not Kv1.5 or EAG channels for ubiquitination and degradation. Nedd4-2 undergoes
self- ubiquitination and degradation. Rab4 interferes with Nedd4-2 degradation,
resulting in an increased expression level of Nedd4-2, which targets hERG. In
summary, the present study demonstrates a novel pathway for hERG regulation;
Rab4 decreases the hERG density at the plasma membrane by increasing the
endogenous Nedd4-2 expression. Escitalopram, a selective serotonin reuptake inhibitor, is the pharmacologically
active S-etiomer of the racemic mixture of RS-citalopram and is widely used
in the treatment of depression. The effects of escitalopram and citalopram on
the human ether-a-go-go-related gene (hERG) channels expressed in human
embryonic kidney cells were investigated using voltage-clamp and Western blot
analyses. Both drugs blocked hERG currents in a concentration-dependent manner
with an IC50 value of 2.6 μM for escitalopram and an IC50 value of 3.2 μM for
citalopram. The blocking of hERG by escitalopram was voltage-dependent, with a
steep increase across the voltage range of channel activation. However, voltage
independence was observed over the full range of activation. The blocking by
escitalopram was frequency dependent. A rapid application of escitalopram
induced a rapid and reversible blocking of the tail current of hERG. The extent
of the blocking by escitalopram during the depolarizing pulse was less than that
during the repolarizing pulse, suggesting that escitalopram has a high affinity
for the open state of the hERG channel, with a relatively lower affinity for the
inactivated state. Both escitalopram and citalopram produced a reduction of hERG
channel protein trafficking to the plasma membrane but did not affect the
short-term internalization of the hERG channel. These results suggest that
escitalopram blocked hERG currents at a supratherapeutic concentration and that
it did so by preferentially binding to both the open and the inactivated states
of the channels and by inhibiting the trafficking of hERG channel protein to the
plasma membrane. The human ERG protein (HERG or Kv 11.1) encoded by the human
ether-a-go-go-related gene (herg) is the pore-forming subunit of the cardiac
delayed rectifier potassium current (IKr) responsible for action potential (AP)
repolarization. Mutations in HERG lead to long-QT syndrome, a major cause of
arrhythmias. Protein-protein interactions are fundamental for ion channel
trafficking, membrane localization, and functional modulation. To identify
proteins involved in the regulation of the HERG channel, we conducted a yeast
two-hybrid screen of a human heart cDNA library using the C-terminus or
N-terminus of HERG as bait. Fifteen proteins were identified as HERG amino
terminal (HERG-NT)-interacting proteins, including Caveolin-1 (a membrane
scaffold protein with multiple interacting partners, including G-proteins,
kinases and NOS), the zinc finger protein, FHL2 and PTPN12 (a non-receptor
tyrosine phosphatase). Eight HERG carboxylic terminal (HERG-CT)-interacting
proteins were also identified, including the NF-κB-interacting protein
myotrophin, We have identified multiple potential interacting proteins that may
regulate cardiac IKr through cytoskeletal interactions, G-protein modulation,
phosphorylation and downstream second messenger and transcription cascades.
These findings provide further insight into dynamic modulation of HERG under
physiological conditions and arrhythmogenesis. Neurons regulate ionic fluxes across their plasma membrane to maintain their
excitable properties under varying environmental conditions. However, the
mechanisms that regulate ion channels abundance remain poorly understood. Here
we show that pickpocket 29 (ppk29), a gene that encodes a Drosophila
degenerin/epithelial sodium channel (DEG/ENaC), regulates neuronal excitability
via a protein-independent mechanism. We demonstrate that the mRNA 3'UTR of ppk29
affects neuronal firing rates and associated heat-induced seizures by acting as
a natural antisense transcript (NAT) that regulates the neuronal mRNA levels of
seizure (sei), the Drosophila homolog of the human Ether-à-go-go Related Gene
(hERG) potassium channel. We find that the regulatory impact of ppk29 mRNA on
sei is independent of the sodium channel it encodes. Thus, our studies reveal a
novel mRNA dependent mechanism for the regulation of neuronal excitability that
is independent of protein-coding capacity. DOI:
http://dx.doi.org/10.7554/eLife.01849.001. We investigated the effects of AT1 receptor stimulation by angiotensin II (Ang
II) on human ether-a-go-go-related gene (hERG) potassium channel protein in a
heterogeneous expression system with the human embryonic kidney (HEK) 293 cells
which stably expressed hERG channel protein and were transiently transfected
with the human AT1 receptors (HEK293/hERG). Western-blot analysis showed that
Ang II significantly decreased the expression of mature hERG channel protein
(155-kDa band) in a time- and dose-dependent manner without affecting the level
of immature hERG channel protein (135-kDa band). The relative intensity of
155-kDa band was 64.7±6.8% of control (P<0.01) after treatment of Ang II at
100nM for 24h. To investigate the effect of Ang II on the degradation of mature
hERG channel protein, we blocked forward trafficking from ER to Golgi with a
Golgi transit inhibitor brefeldin A (10μM). Ang II significantly enhanced the
time-dependent reduction of mature hERG channel protein. In addition, the
proteasomal inhibitor lactacystin (5μM) inhibited Ang II-mediated the reduction
of mature hERG channel protein, but the lysosomal inhibitor bafilomycin A1 (1μM)
had no effect on the protein. The protein kinase C (PKC) inhibitor
bisindolylmaleimide 1 (1μM) antagonized the reduction of mature hERG channel
protein induced by Ang II. The results indicate that sustained stimulation of
AT1 receptors by Ang II reduces the mature hERG channel protein via accelerating
channel proteasomal degradation involving the PKC pathway. Human ether-a-go-go related gene (herg) encoding HERG K(+) channel has been
demonstrated in many previous studies with its association to cell cycle
progression and growth in tumor cells. The upregulated expression of HERG K+
channels was determined in different tumor types. Furthermore, not only
full-length transcript herg1 but also a truncated isoform herg1b was shown to be
expressed in cancer cells. In this study, the expression levels of herg1 and
herg1b and the impact of K897T mutation on their expressions were investigated
in pediatric acute myeloid leukemia (pAML). Expression levels of herg1 and
herg1b isoforms were analyzed by quantitative real time polymerase chain
reaction (PCR) in pAML patients together with healthy donors, and their
expressions were confirmed by western blotting. The 2690 A>C nucleotide
variation in KCNH2 gene corresponding to K897T amino acid change was analyzed by
PCR followed by restriction enzyme digestion. herg1b overexpression was observed
in tumor cells compared to healthy controls (P = .0024). However, herg1
expression was higher in healthy control cells than tumor cells (P = .001). The
prevalence of polymorphic allele 897T was 26% in our patient group and 897T
carriers showed increased herg1b expression compared to wild-type allele
carriers. Our results demonstrate the presence of the increased levels of herg1b
expression in pAML. In addition, we report for the first time that, pAML
subgroup with HERG 897K/K genotype compared to 897K/T and T/T genotypes express
increased levels of herg1b. In conclusion, HERG 897K/K genotype with increased
level of herg1b expression might well be a prognostic marker for pAML. Donepezil is a potent, selective inhibitor of acetylcholinesterase, which is
used for the treatment of Alzheimer's disease. Whole-cell patch-clamp technique
and Western blot analyses were used to study the effects of donepezil on the
human ether-a-go-go-related gene (hERG) channel. Donepezil inhibited the tail
current of the hERG in a concentration-dependent manner with an IC50 of 1.3 μM.
The metabolites of donepezil, 6-ODD and 5-ODD, inhibited the hERG currents in a
similar concentration-dependent manner; the IC50 values were 1.0 and 1.5 μM,
respectively. A fast drug perfusion system demonstrated that donepezil
interacted with both the open and inactivated states of the hERG. A fast
application of donepezil during the tail currents inhibited the open state of
the hERG in a concentration-dependent manner with an IC50 of 2.7 μM. Kinetic
analysis of donepezil in an open state of the hERG yielded blocking and
unblocking rate constants of 0.54 µM(-1)s(-1) and 1.82 s(-1), respectively. The
block of the hERG by donepezil was voltage-dependent with a steep increase
across the voltage range of channel activation. Donepezil caused a reduction in
the hERG channel protein trafficking to the plasma membrane at low
concentration, but decreased the channel protein expression at higher
concentrations. These results suggest that donepezil inhibited the hERG at a
supratherapeutic concentration, and that it did so by preferentially binding to
the activated (open and/or inactivated) states of the channels and by inhibiting
the trafficking and expression of the hERG channel protein in the plasma
membrane. The cardiac electrical disorder long QT syndrome (LQTS) pre-disposes affected
individuals to ventricular arrhythmias and sudden death. Dysfunction of the
human ether-a-go-go-related gene (hERG)-encoded rapidly activating delayed
rectifier K(+) channel (IKr) is a major cause of LQTS. The expression of hERG
channels is controlled by anterograde trafficking of newly synthesized channels
to and retrograde degradation of existing channels from the plasma membrane. We
have previously shown that the E3 ubiquitin (Ub) ligase Nedd4-2 (neural
precursor cell expressed developmentally down-regulated protein 4-2) targets the
PY motif of hERG channels to initiate channel degradation. Although both
immature and mature hERG channels contain the PY motif, Nedd4-2 selectively
mediates the degradation of mature hERG channels. In the present study, we
demonstrate that Nedd4-2 is directed to specific cellular compartments by the
Nedd4 family interacting proteins, Nedd4 family-interacting protein 1 (Ndfip1)
and Ndfip2. Ndfip1 is primarily localized in the Golgi apparatus where it
recruits Nedd4-2 to mediate the degradation of mature hERG proteins during
channel trafficking to the plasma membrane. Although Ndfip2 directs Nedd4-2 to
the Golgi apparatus, it also recruits Nedd4-2 to the multivesicular bodies
(MVBs), which may impair MVB function and impede the degradation of mature hERG
proteins mediated by Nedd4-2. These findings extend our understanding of hERG
channel regulation and provide information which may be useful for the rescue of
impaired hERG function in LQTS. PURPOSE: The pathophysiology of Hirschsprung's disease (HSCR) is not entirely
understood. There is no clear explanation for the occurrence of the spastic or
tonically contracted aganglionic segment of bowel. Kv11.1 (hERG) channels play a
critical role in the regulation of the resting membrane potential as well as
affecting either the force or frequency of contraction of smooth muscles. We
designed this study to investigate the expression and distribution of hERG
channels in the normal colon and the colon of patients with HSCR.
METHODS: We investigated hERG protein expression in both the ganglionic and
aganglionic regions of HSCR patients (n = 10) versus normal control colon
(n = 10). Protein distribution was assessed using immunofluorescence and
confocal microscopy. Gene and protein expressions were quantified using
real-time polymerase chain reaction, western blot analysis and densitometry.
RESULTS: Confocal microscopy of the normal colon revealed strong hERG channel
expression in interstitial cells of Cajal, platelet-derived growth factor-alpha
receptor- (PDGFRα(+)) positive cells and enteric neurons. hERG expression was
markedly decreased in aganglionic bowel, whereas colonic hERG gene expression
levels were significantly decreased in aganglionic compared to ganglionic bowel
and controls (p < 0.05). Western blotting revealed decreased colonic hERG
protein expression in aganglionic HSCR specimens compared to controls.
CONCLUSIONS: We demonstrate, for the first time, the expression and distribution
of hERG channels in the human colon. The decreased expression of hERG in the
aganglionic colon may be responsible for the increased tone in the aganglionic
narrow spastic segment of bowel. Human ether-a-go-go-related gene (hERG) channels conduct delayed rectifier K(+)
current. However, little information is available on physiological situations
affecting hERG channel protein and function. In the present study we examined
the effects of intermittent hypoxia (IH), which is a hallmark manifestation of
sleep apnea, on hERG channel protein and function. Experiments were performed on
SH-SY5Y neuroblastoma cells, which express hERG protein. Cells were exposed to
IH consisting of alternating cycles of 30 s of hypoxia (1.5% O2) and 5 min of
20% O2. IH decreased hERG protein expression in a stimulus-dependent manner. A
similar reduction in hERG protein was also seen in adrenal medullary chromaffin
cells from IH-exposed neonatal rats. The decreased hERG protein was associated
with attenuated hERG K(+) current. IH-evoked hERG protein degradation was not
due to reduced transcription or increased proteosome/lysomal degradation. Rather
it was mediated by calcium-activated calpain proteases. Both COOH- and
NH2-terminal sequences of the hERG protein were the targets of calpain-dependent
degradation. IH increased reactive oxygen species (ROS) levels, intracellular
Ca(2+) concentration ([Ca(2+)]i), calpain enzyme activity, and hERG protein
degradation, and all these effects were prevented by
manganese-(111)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride, a
membrane-permeable ROS scavenger. These results demonstrate that activation of
calpains by ROS-dependent elevation of [Ca(2+)]i mediates hERG protein
degradation by IH. BACKGROUND: The human ether-à-go-go-related gene (hERG 1a) potassium channel is
critical for cardiac repolarization. hERG 1b, another variant subunit,
co-assembles with hERG 1a, modulates channel biophysical properties and plays an
important role in repolarization. Mutations of hERG 1a lead to type 2 long QT
syndrome (LQT2), and increased risk for fatal arrhythmias. The functional
consequences of these mutations in the presence of hERG 1b are not known.
OBJECTIVE: To investigate whether hERG 1a mutants exert domit negative gating
and trafficking defects when co-expressed with hERG 1b.
METHODS: Electrophysiology, co-immunoprecipitation, and fluorescence resoce
energy transfer (FRET) experiments in HEK293 cells and guinea pig cardiomyocytes
were used to assess the mutants on gating and trafficking. Mutations of 1a-G965X
and 1a-R1014X, relevant to gating and trafficking were introduced in the
C-terminus region.
RESULTS: The hERG 1a mutants when expressed alone did not result in decreased
current amplitude. Compared to wild-type hERG 1a currents, 1a-G965X currents
were significantly larger, whereas those produced by the 1a-R1014X mutant were
similar in magnitude. Only when co-expressed with wild-type hERG 1a and 1b did a
mutant phenotype emerge, with a marked reduction in surface expression, current
amplitude, and a corresponding positive shift in the V1/2 of the activation
curve. Co-immunoprecipitation and FRET assays confirmed association of mutant
and wild-type subunits.
CONCLUSION: Heterologously expressed hERG 1a C-terminus truncation mutants,
exert a domit negative gating and trafficking effect only when co-expressed
with hERG 1b. These findings may have potentially profound implications for LQT2
therapy. |
Is lenvatinib effective for renal cell carcinoma? | Yes, combination of lenvatinib and everolimus is approved to treat advanced or metastatic renal cell carcinoma. | PURPOSE: Lenvatinib is an oral multi-targeted tyrosine kinase inhibitor of
VEGFR1-3, FGFR1-4, PDGFRβ, RET, and KIT. Everolimus is an oral mammalian target
of rapamycin inhibitor approved for advanced renal cell carcinoma (RCC). This
phase 1b study assessed safety, maximum tolerated dose (MTD), and preliminary
antitumor activity of lenvatinib plus everolimus in metastatic RCC (mRCC)
patients.
METHODS: Patients with advanced unresectable or mRCC and Eastern Cooperative
Oncology Group performance status 0-1 were eligible (number of prior treatments
not restricted). Starting dose was lenvatinib 12 mg once daily with everolimus 5
mg once daily administered continuously in 28-day cycles using a conventional 3
+ 3 dose-escalation design. At the MTD, additional patients were enrolled in an
expansion cohort.
RESULTS: Twenty patients (mean 58.4 years) received lenvatinib [12 mg (n = 7);
18 mg (n = 11); 24 mg (n = 2)] plus everolimus 5 mg. MTD was established as once
daily lenvatinib 18 mg plus everolimus 5 mg. The most common treatment-related
treatment-emergent adverse events (all dosing cohorts) were fatigue 60 % (Grade
≥3: 10 %), mucosal inflammation 50 %, proteinuria (Grade ≥3: 15 %), diarrhea
(Grade ≥3: 10 %), vomiting (Grade ≥3: 5 %), hypertension, and nausea, each 40 %.
In MTD and lowest-dose cohorts (n = 18), best responses of partial response and
stable disease were achieved in 6 (33 %) and 9 (50 %) patients, respectively.
CONCLUSIONS: Lenvatinib 18 mg combined with everolimus 5 mg was associated with
manageable toxicity consistent with individual agents and no new safety signals.
Observed activity warrants further evaluation of the combination in advanced RCC
patients. INTRODUCTION: Metastatic clear-cell renal cell carcinoma (RCC) is a highly
vascularized tumor type that is often associated with inactivating mutations in
the von Hippel-Lindau gene that ultimately drives pro-angiogenic signaling
pathways, including the VEGF pathway. As such, new therapies indicated for RCC
have largely focused on blocking angiogenesis by inhibiting this pathway.
Despite the contribution of these agents to clinical outcomes in RCC, acquired
resistance that stimulates tumor regrowth and revascularization quickly emerges.
Resistance to VEGF inhibition appears to largely result from activation of
compensatory angiogenesis pathways (including the fibroblast growth factor [FGF]
pathway), providing a rationale to investigate their inhibition.
AREAS COVERED: This review explores the role of the FGF pathway in resistance to
VEGF-targeted therapy and rationale for targeting in RCC. PubMed, as well as
ASCO and ESMO congress abstracts, were searched for preclinical and clinical
data for FGF inhibitors in RCC.
EXPERT OPINION: The FGF pathway presents a logical target in RCC and trials of
the FGF receptor inhibitors regorafenib, dovitinib, nintedanib, lenvatinib and
cediranib demonstrated clinical activity. Clinical development should focus on
optimizing the use of this therapy by improving patient selection and evaluating
combined therapy. BACKGROUND: Currently, metastatic renal cell carcinoma is treated with
sequential single agents targeting VEGF or mTOR. Here, we aimed to assess
lenvatinib, everolimus, or their combination as second-line treatment in
patients with metastatic renal cell carcinoma.
METHODS: We did a randomised, phase 2, open-label, multicentre trial at 37
centres in five countries and enrolled patients with advanced or metastatic,
clear-cell, renal cell carcinoma. We included patients who had received
treatment with a VEGF-targeted therapy and progressed on or within 9 months of
stopping that agent. Patients were randomised via an interactive voice response
system in a 1:1:1 ratio to either lenvatinib (24 mg/day), everolimus (10
mg/day), or lenvatinib plus everolimus (18 mg/day and 5 mg/day, respectively)
administered orally in continuous 28-day cycles until disease progression or
unacceptable toxic effects. The randomisation procedure dynamically minimised
imbalances between treatment groups for the stratification factors haemoglobin
and corrected serum calcium. The primary objective was progression-free survival
in the intention-to-treat population. This study is closed to enrolment but
patients' treatment and follow-up is ongoing. This study is registered with
ClinicalTrials.gov, number NCT01136733.
FINDINGS: Between March 16, 2012, and June 19, 2013, 153 patients were randomly
allocated to receive either the combination of lenvatinib plus everolimus
(n=51), single-agent lenvatinib (n=52), or single-agent everolimus (n=50).
Lenvatinib plus everolimus significantly prolonged progression-free survival
compared with everolimus alone (median 14·6 months [95% CI 5·9-20·1] vs 5·5
months [3·5-7·1]; hazard ratio [HR] 0·40, 95% CI 0·24-0·68; p=0·0005), but not
compared with lenvatinib alone (7·4 months [95% CI 5·6-10·2]; HR 0·66, 95% CI
0·30-1·10; p=0·12). Single-agent lenvatinib significantly prolonged
progression-free survival compared with everolimus alone (HR 0·61, 95% CI
0·38-0·98; p=0·048). Grade 3 and 4 events occurred in fewer patients allocated
single-agent everolimus (25 [50%]) compared with those assigned lenvatinib alone
(41 [79%]) or lenvatinib plus everolimus (36 [71%]). The most common grade 3 or
4 treatment-emergent adverse event in patients allocated lenvatinib plus
everolimus was diarrhoea (ten [20%]), in those assigned single-agent lenvatinib
it was proteinuria (ten [19%]), and in those assigned single-agent everolimus it
was anaemia (six [12%]). Two deaths were deemed related to study drug, one
cerebral haemorrhage in the lenvatinib plus everolimus group and one myocardial
infarction with single-agent lenvatinib.
INTERPRETATION: Lenvatinib plus everolimus and lenvatinib alone resulted in a
progression-free survival benefit for patients with metastatic renal cell
carcinoma who have progressed after one previous VEGF-targeted therapy. Further
study of lenvatinib is warranted in patients with metastatic renal cell
carcinoma.
FUNDING: Eisai Inc. Current therapy for metastatic clear cell renal cell carcinoma (RCC) consists of
the serial administration of single agents. Combinations of VEGF and mTOR
inhibitors have been disappointing in previous randomized trials. However, the
combination of lenvatinib, a multitargeted agent that inhibits VEGF as well as
FGF receptors, and everolimus demonstrated promising results in a randomized
phase II trial. Moreover, the emergence of programmed cell death 1 (PD-1) and
programmed cell death ligand 1 (PD-L1) inhibitors has spawned the investigation
of combinations of these agents with VEGF inhibitors and cytotoxic T-lymphocyte
antigen 4 (CTLA-4) inhibitors. These ongoing phase III trials in conjunction
with the development of predictive biomarkers and agents inhibiting novel
therapeutic targets may provide much needed advances in this still largely
incurable disease. The FDA has approved the combination of lenvatinib and everolimus to treat
advanced or metastatic renal cell carcinoma. The approval marks the first time
that tyrosine kinase and mTOR inhibitors have been combined successfully as
second-line treatment for renal cell carcinoma following prior VEGF-targeted
therapy. Despite advances in metastatic renal cell carcinoma (mRCC) treatments, patients
eventually progress and develop resistance to therapies targeting a single
pathway. Lenvatinib inhibits VEGFR1-3, FGFR1-4, PDGFRβ, RET and KIT
proto-oncogenes. In a randomized, Phase II trial evaluating patients with mRCC
who had progressed after one prior VEGF-targeted therapy, progression-free
survival was significantly improved with lenvatinib alone or in combination with
everolimus versus everolimus alone. This review summarizes the clinical
development of lenvatinib in mRCC, and how simultaneous targeting of multiple
pathways involved in carcinogenesis and/or therapeutic resistance may improve
patient outcomes. Lenvatinib plus everolimus may be a promising second-line
treatment in patients with mRCC. PURPOSE OF REVIEW: Multiple agents, including vascular endothelial growth factor
(VEGF) inhibitors and mammalian target of rapamycin inhibitors have been
approved over the past decade for the treatment of metastatic renal cell
carcinoma (mRCC). Here, we focus on nivolumab, cabozantinib, and lenvatinib plus
everolimus, agents that have recently emerged with positive clinical data
leading to 'Food and Drug Administration approval or pending approval in mRCC.
We also review the development of novel agents of interest showing promise in
mRCC as part of combination therapy'.
RECENT FINDINGS: Nivolumab and cabozantinib both offer improved survival over
everolimus in the second-line treatment of mRCC. Lenvatinib plus everolimus has
similarly shown encouraging survival benefits in a phase II trial for the
second-line setting. Novel combinations in mRCC, including dual immune
checkpoint blockade, VEGF and programmed death 1 inhibition, VEGF and vaccine
therapy, dual angiogenic blockade, and VEGF-directed therapy with
oparticle-containing camptothecin have shown promising activity in
early-phase trials.
SUMMARY: Multiple promising agents are available in the treatment of mRCC. The
appropriate sequencing of agents in the treatment of mRCC may become further
elucidated by future studies that prospectively analyze potential biomarkers to
identify patients who will derive the greatest benefit from VEGF, mammalian
target of rapamycin, or checkpoint inhibitors. The landscape of systemic treatment for metastatic renal cell carcinoma (RCC)
has dramatically changed with the introduction of targeted agents including
vascular endothelial growth factor (VEGF) inhibitors. Recently, multiple new
agents including growth factor receptor antagonists and a checkpoint inhibitor
were approved for the treatment of refractory metastatic RCC based on
encouraging benefit shown in clinical trials. Areas covered: The background and
biological rationale of existing treatment options including a brief discussion
of clinical trials which led to their approval, is presented. This is followed
by reviewing the limitations of these therapeutic options, medical need to
develop new treatments and major goals of ongoing research. We then discuss two
recently approved growth factor receptor antagonists i.e. cabozantinib and
lenvatinib, and a recently approved checkpoint inhibitor, nivolumab, and issues
pertaining to drug development, and future directions in treatment of metastatic
RCC. Expert opinion: Recently approved growth factor receptor antagonists have
shown encouraging survival benefit but associated drug toxicity is a major
issue. Nivolumab, a programmed death 1 (PD-1) checkpoint inhibitor, has
similarly shown survival benefit and is well tolerated. With multiple options
now available in this patient population, the right sequence of these agents
remains to be determined. |
Which proteins form part of the NRD complex in S. cerevisiae? | The purification of an ATR complex allowed identification of chromodomain-helicase-DNA-binding protein 4 (CHD4) as an ATR-associated protein by tandem mass spectrometric sequencing. CHD4 (also called Mi-2beta) is a component of a histone-deacetylase-2 (HDAC2)-containing complex, the nucleosome remodeling and deacetylating (NRD) complex. Endogenous ATR, CHD4, and HDAC2 are shown to coimmunoprecipitate, and ATR and HDAC2 coelute through two biochemical purification steps. Other members of the NRD complex, HDAC1, MTA1, and MTA2, are also detectable in ATR immunoprecipitates. Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is also generally required for NRD-dependent transcription termination through the action of its C-terminal domain (CTD)-interacting domain (CID). | Ataxia telangiectasia mutated (ATM)- and Rad3-related protein (ATR) is a
phosphatidylinositol-kinase (PIK)-related kinase that has been implicated in the
response of human cells to multiple forms of DNA damage and may play a role in
the DNA replication checkpoint. The purification of an ATR complex allowed
identification of chromodomain-helicase-DNA-binding protein 4 (CHD4) as an
ATR-associated protein by tandem mass spectrometric sequencing. CHD4 (also
called Mi-2beta) is a component of a histone-deacetylase-2 (HDAC2)-containing
complex, the nucleosome remodeling and deacetylating (NRD) complex. Endogenous
ATR, CHD4, and HDAC2 are shown to coimmunoprecipitate, and ATR and HDAC2 coelute
through two biochemical purification steps. Other members of the NRD complex,
HDAC1, MTA1, and MTA2, are also detectable in ATR immunoprecipitates. ATR's
association with CHD4 and HDAC2 suggests that there may be a linkage between
ATR's role in mediating checkpoints induced by DNA damage and chromatin
modulation via remodeling and deacetylation. In Saccharomyces cerevisiae, short noncoding RNA (ncRNA) generated by RNA
polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1,
Nab3, and Sen1. We now show that Pcf11, a component of the cleavage and
polyadenylation complex (CPAC), is also generally required for NRD-dependent
transcription termination through the action of its C-terminal domain
(CTD)-interacting domain (CID). Pcf11 localizes downstream from Nrd1 on NRD
terminators, and its recruitment depends on Nrd1. Furthermore, mutation of the
Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA,
and restricted Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction.
Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar, as both
accumulate RNA:DNA hybrids and display Pol II pausing downstream from NRD
terminators. We predict a mechanism by which the exchange of Nrd1 and Pcf11 on
chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in
turn promotes Sen1 activity that is required for NRD-dependent transcription
termination in vivo. |
Which R / bioconductor package is used for performing SNP enrichment analysis? | traseR is an easy-to-use R Bioconductor package that performs enrichment analyses of trait-associated SNPs in arbitrary genomic intervals with flexible options, including testing method, type of background and inclusion of SNPs in LD. | Genome-wide association studies (GWASs) have successfully identified many
sequence variants that are significantly associated with common diseases and
traits. Tens of thousands of such trait-associated SNPs have already been
cataloged, which we believe form a great resource for genomic research. Recent
studies have demonstrated that the collection of trait-associated SNPs can be
exploited to indicate whether a given genomic interval or intervals are likely
to be functionally connected with certain phenotypes or diseases. Despite this
importance, currently, there is no ready-to-use computational tool able to
connect genomic intervals to phenotypes. Here, we present traseR, an easy-to-use
R Bioconductor package that performs enrichment analyses of trait-associated
SNPs in arbitrary genomic intervals with flexible options, including testing
method, type of background and inclusion of SNPs in LD.
AVAILABILITY AND IMPLEMENTATION: The traseR R package preloaded with up-to-date
collection of trait-associated SNPs are freely available in Bioconductor
CONTACT: [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. |
Does HuR bind to the untranslated regions (UTRs) of mRNAs? | Yes, the RNA-binding protein HuR binds at 3' untranslated regions (UTRs) of target transcripts, thereby protecting them against degradation. | Alterations in gene expression are central to the maligt phenotype. In this
issue, Al-Ahmadi et al elegantly demonstrate that one key mechanism that
determines invasiness in breast cancer is likely to be dysregulation of mRNA
stability. This is achieved by altered expression of the proteins TTP and HuR,
which bind 3' untranslated region (UTR) elements in cancer-related genes. The
authors link this to the expression of a miRNA, miR-29a, and show that
anti-miR-29a treatment can reverse the invasive phenotype in vitro. This further
highlights the important roles of mRNA UTRs in maligt transformation, and the
importance of investigating post-transcriptional disease mechanisms. Such
mechanisms have the potential to provide novel therapeutic targets, as well as
being vital to further our understanding of cancer biology. The RNA-binding protein HuR binds at 3' untranslated regions (UTRs) of target
transcripts, thereby protecting them against degradation. We show that HuR
directly interacts with cellular retinoic acid-binding protein 2 (CRABP2), a
protein known to transport RA from the cytosol to the nuclear retinoic acid
receptor (RAR). Association with CRABP2 dramatically increases the affinity of
HuR toward target mRNAs and enhances the stability of such transcripts,
including that of Apaf-1, the major protein in the apoptosome. We show further
that its cooperation with HuR contributes to the ability of CRABP2 to suppress
carcinoma cell proliferation. The data show that CRABP2 displays antioncogenic
activities both by cooperating with RAR and by stabilizing antiproliferative HuR
target transcripts. The observation that CRABP2 controls mRNA stabilization by
HuR reveals that in parallel to participating in transcriptional regulation, the
protein is closely involved in posttranscriptional regulation of gene
expression. Human antigen R (HuR) is a ubiquitous 32 kDa protein comprising three RNA
Recognition Motifs (RRMs), whose main function is to bind Adenylate and
uridylate Rich Elements (AREs) in 3' UnTranslated Regions (UTRs) of mRNAs. In
addition to binding RNA molecules, the third domain (RRM3) is involved in HuR
oligomerization and apoptotic signaling. The RRM3 monomer is able to dimerize,
with its self-binding affinity being dependent on ionic strength. Here we
provide a deeper structural insight into the nature of the encounter complexes
leading to the formation of RRM3 dimers by using Brownian Dynamics and Molecular
Dynamics. Our computational data show that the initial unspecific encounter
follows a downhill pathway until reaching an optimum conformation stabilized by
hydrophobic interactions. BACKGROUND/AIMS: The lncRNA Homeobox (HOX) transcript antisense RNA (HOTAIR) is
overexpressed in numerous cancers. HuR is also overexpressed during
tumourigenesis and is abnormally present within the cytoplasm, where it binds to
AU-rich elements in the 3'UTRs of target mRNA and post-transcriptionally
regulates the expression of its target genes. However, whether HOTAIR is
regulated and the mechanisms by which it affects head and neck squamous cell
carcinoma (HNSCC) are not well understood.
METHODS: MTT, cell cycle arrest and apoptotic assays were used to examine the
effects of HOTAIR and HuR on cell viability in SCC25 and FaDu cells. Wound
healing and transwell invasion analysis were performed to detect the effects of
HOTAIR and HuR on cell migration and invasion. The interaction between HuR and
HOTAIR was confirmed via qRT-PCR, western blots, luciferase reporter and RIP
assays. Finally, qRT-PCR analysis was used to detect the levels of HuR and
HOTAIR in HNSCC tumours and adjacent normal tissues.
RESULTS: Knockdown of HOTAIR and HuR decreased cell viability, cellular
migration and invasion. Moreover, HuR interacted and stabilized HOTAIR stability
and thus promoted HOTAIR expression. Notably, HOTAIR acted as a miRNA sponge for
HuR. HuR also reinforced HOTAIR sponge activity through miRNA recruitment, thus
enhancing HuR expression in turn. Finally, HuR and HOTAIR levels were positively
correlated and significantly up-regulated in tumours samples.
CONCLUSION: We demonstrated the existence of a regulatory loop in which the
expression of HOTAIR and HuR is reciprocally and temporally regulated during the
metastasis and progression of HNSCC. |
Describe Exploding head syndrome. | Exploding head syndrome is characterized by the perception of abrupt, loud noises when going to sleep or waking up. | Attention has recently been drawn to a condition termed the exploding head
syndrome, which is characterized by unpleasant, even terrifying sensations of
flashing lights and/or sounds during reported sleep. Nine patients complaining
of sensations of explosions in the head during sleep or drowsiness were
investigated with polysomnographic recordings. None of them had any neurological
disorder. Five patients reported explosions during the recording sessions.
According to the recordings, the attacks always took place when the patients
were awake and relaxed. In two cases abrupt electroencephalographic (EEG) and
electromyographic changes indicating increasing alertness were recorded at the
time of the reported attacks. In the remaining three cases no EEG changes were
seen. Thus, there were no indications of an epileptic etiology to the condition.
In all patients the symptoms ameliorated spontaneously with time. The severity
of the symptoms was reduced by reassurance of the harmlessness of the condition.
Clomipramine was prescribed to three patients who all reported immediate relief
of symptoms. It is concluded that symptoms of this type are probably not true
hypnagogic phenomena but may be an expression of emotional stress in the awake
state. The case is reported of a 47-year old female suffering from the exploding head
syndrome. This syndrome consists of a sudden awakening due to a loud noise
shortly after falling asleep, sometimes accompanied by a flash of light. The
patient is anxious and experiences palpitations and excessive sweating. Most
patients are more than fifty years of age. Further investigations do not reveal
any abnormality. The pathogenesis is unknown, and no therapy other than
reassurance is necessary. Fifty patients suffering from the "exploding head syndrome" are described. This
hitherto unreported syndrome is characterised by a sense of an explosive noise
in the head usually in the twilight stage of sleep. The associated symptoms are
varied, but the benign nature of the condition is emphasised and neither
extensive investigation nor treatment are indicated. This article reviews the features of an uncommon malady termed "the exploding
head syndrome." Sufferers describe terrorizing attacks of a painless explosion
within their head. Attacks tend to occur at the onset of sleep. The etiology of
attacks is unknown, although they are considered to be benign. Treatment with
clomipramine has been suggested, although most sufferers require only
reassurance that the spells are benign in nature. Exploding head syndrome (EHS) attacks are characterized by the sensation of
sudden loud banging noises, and are occasionally accompanied by the sensation of
a flash light. Although these attacks in themselves are usually not painful, it
is reported that EHS attacks may precede migraines and may be perceived as
auras. A 53-year-old woman, with a 40-year history of fulgurating migraines,
experienced 2 different types of EHS attacks. During most of the attacks, which
were not painful, she heard sounds like someone yelling or cars passing by. Only
1 episode was accompanied with the sensation of a flash light and of sounds
similar to those of an electrical short circuit. On the video-polysomnography,
video-polysomnography showed 11 EHS attacks occurred during stage N1 and stage
N2; these attacks were preceded by soft snoring. She also had moderate
obstructive sleep apnea syndrome (Apnea Hypopnea Index: 16.7) for which an oral
appliance was prescribed; the EHS attacks did not recur after this treatment.
The pathophysiology of EHS is still unclear. A detailed analysis of PSG data may
help in understanding the pathophysiology of this syndrome and also in the
selection of therapeutic strategies. Exploding head syndrome is a rare phenomenon but can be a significant disruption
to quality of life. We describe a 39-year-old female with symptoms of a loud
bang and buzz at sleep onset for 3 years. EEG monitoring confirmed these events
occurred in transition from stage 1 sleep. This patient reported improvement in
intensity of events with topiramate medication. Based on these results,
topiramate may be an alternative method to reduce the intensity of events in
exploding head syndrome. INTRODUCTION: Exploding head syndrome (EHS) is a rare parasomnia in which
affected individuals awaken from sleep with the sensation of a loud bang. The
etiology is unknown, but other conditions including primary and secondary
headache disorders and nocturnal seizures need to be excluded.
CASE PRESENTATION: A 57-year-old Indian male presented with four separate
episodes of awakening from sleep at night after hearing a flashing sound on the
right side of his head over the last 2 years. These events were described 'as if
there are explosions in my head'. A neurologic examination, imaging studies, and
a polysomnogram ensued, and the results led to the diagnosis of EHS.
CONCLUSION: EHS is a benign, uncommon, predominately nocturnal disorder that is
self-limited. No treatment is generally required. Reassurance to the patient is
often all that is needed. Exploding head syndrome is characterized by the perception of abrupt, loud
noises when going to sleep or waking up. They are usually painless, but
associated with fear and distress. In spite of the fact that its characteristic
symptomatology was first described approximately 150 y ago, exploding head
syndrome has received relatively little empirical and clinical attention.
Therefore, a comprehensive review of the scientific literature using Medline,
PsycINFO, Google Scholar, and PubMed was undertaken. After first discussing the
history, prevalence, and associated features, the available polysomnography data
and five main etiological theories for exploding head syndrome are summarized.
None of these theories has yet reached domice in the field. Next, the various
methods used to assess and treat exploding head syndrome are discussed, as well
as the limited outcome data. Finally, recommendations for future measure
construction, treatment options, and differential diagnosis are provided. BACKGROUND: Exploding head syndrome (EHS) is characterized by attacks of a
sudden noise or explosive feeling experienced in the head occurring during the
transition from wake to sleep or from sleep to wake.
METHODS: We present six new cases extending the clinical experience with the
syndrome. We also reviewed all available cases from the scientific literature
and evaluated the typical features of EHS.
RESULTS: The female to male ratio is 1.5 to 1. The median age at onset is 54. In
average, one attack per day to one attack per week occurs. Some patients suffer
from several attacks per night. In about half of all patients, a chronic time
course can be observed but episodic or sporadic occurrence is also common. The
most frequent accompanying symptoms beside the noise are fear and flashes of
light. Polysomnographic studies do not reveal any specific sleep pattern
associated with EHS. Tricyclic antidepressants are helpful in some patients.
However, most patients do not need treatment because of the benign nature of the
syndrome.
CONCLUSION: EHS is a well-defined disease entity with a benign nature. Exploding head syndrome is characterized by the perception of loud noises during
sleep-wake or wake-sleep transitions. Although episodes by themselves are
relatively harmless, it is a frightening phenomenon that may result in clinical
consequences. At present there are little systematic data on exploding head
syndrome, and prevalence rates are unknown. It has been hypothesized to be rare
and to occur primarily in older (i.e. 50+ years) individuals, females, and those
suffering from isolated sleep paralysis. In order to test these hypotheses, 211
undergraduate students were assessed for both exploding head syndrome and
isolated sleep paralysis using semi-structured diagnostic interviews: 18.00% of
the sample experienced lifetime exploding head syndrome, this reduced to 16.60%
for recurrent cases. Though not more common in females, it was found in 36.89%
of those diagnosed with isolated sleep paralysis. Exploding head syndrome
episodes were accompanied by clinically significant levels of fear, and a
minority (2.80%) experienced it to such a degree that it was associated with
clinically significant distress and/or impairment. Contrary to some earlier
theorizing, exploding head syndrome was found to be a relatively common
experience in younger individuals. Given the potential clinical impacts, it is
recommended that it be assessed more regularly in research and clinical
settings. |
Is H4K20 methylation associated with DNA replication? | We employed genetic, cytological, and genomic approaches to better understand the role of PR-Set7 and H4K20 methylation in regulating DNA replication and genome stability in Drosophila cells. Thus, coordinating the status of H4K20 methylation is pivotal for the proper selection of DNA replication origins in higher eukaryotes. The methylation state of lysine 20 on histone H4 (H4K20) has been linked to chromatin compaction, transcription, DNA repair and DNA replication. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). We review the signaling pathways and functions associated with a single residue, H4K20, as a model chromatin and clinically important mark that regulates biological processes ranging from the DNA damage response and DNA replication to gene expression and silencing. <CopyrightInformation>© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.</C In particular, the methylation states of H3K4, H3K36 and H4K20 have been associated with establishing active, repressed or poised origins depending on the timing and extent of methylation. 5BrC and 5ClC may cause aberrant methylation of cytosine during DNA replication and mimic the endogenous methylation signal associated with gene silencing. | The molecular biology of histone H4 lysine 20 (H4K20) methylation, like many
other post-translational modifications of histones, has been the subject of
intensive interest in recent years. While there is an emerging consensus linking
H4K20me1, H4K20me2, and H4K20me3 to transcription, repair, and constitutive
heterochromatin, respectively, the specific details of these associations and
the biological mechanisms by which the methylated histones are introduced and
function are now the subject of active investigation. Although a large number of
methylases capable of methylating H4K20 have been identified and characterized;
there is no known demethylase of H4K20, though the search is ongoing.
Additionally, many recent studies have been directed at understanding the role
of methylated H4K20 and other histone modifications associated with different
biological processes in the context of a combinatorial histone code. It seems
likely that continued study of the methylation of H4K20 will yield extremely
valuable insights concerning the regulation of histone modifications before and
during cell division and the impact of these modifications on subsequent gene
expression. PR-Set7 is the sole monomethyltransferase responsible for H4K20 monomethylation
(H4K20me1) that is the substrate for further methylation by Suv4-20h1/h2.
PR-Set7 is required for proper cell cycle progression and is subject to
degradation by the CRL4(Cdt2) ubiquitin ligase complex as a function of the cell
cycle and DNA damage. This report demonstrates that PR-Set7 is an important
downstream effector of CRL4(Cdt2) function during origin of DNA replication
licensing, dependent on Suv4-20h1/2 activity. Aberrant rereplication correlates
with decreased levels of H4K20me1 and increased levels of H4K20 trimethylation
(H4K20me3). Expression of a degradation-resistant PR-Set7 mutant in the mouse
embryo that is normally devoid of Suv4-20 does not compromise development or
cell cycle progression unless Suv4-20h is coexpressed. PR-Set7 targeting to an
artificial locus results in recruitment of the origin recognition complex (ORC)
in a manner dependent on Suv4-20h and H4K20me3. Consistent with this, H4K20
methylation status plays a direct role in recruiting ORC through the binding
properties of ORC1 and ORCA/LRWD1. Thus, coordinating the status of H4K20
methylation is pivotal for the proper selection of DNA replication origins in
higher eukaryotes. Maintece of genomic integrity is essential to ensure normal organismal
development and to prevent diseases such as cancer. Nuclear DNA is packaged into
chromatin, and thus genome maintece can be influenced by distinct chromatin
environments. In particular, post-translational modifications of histones have
emerged as key regulators of genomic integrity. Intense research during the past
few years has revealed histone H4 lysine 20 methylation (H4K20me) as critically
important for the biological processes that ensure genome integrity, such as DNA
damage repair, DNA replication and chromatin compaction. The distinct H4K20
methylation states are mediated by SET8/PR-Set7 that catalyses monomethylation
of H4K20, whereas SUV4-20H1 and SUV4-20H2 enzymes mediate further H4K20
methylation to H4K20me2 and H4K20me3. Disruption of these H4K20-specific histone
methyltransferases leads to genomic instability, demonstrating the important
functions of H4K20 methylation in genome maintece. In this review, we explain
molecular mechanisms underlying these defects and discuss novel ideas for
furthering our understanding of genome maintece in higher eukaryotes. Mammalian DNA replication starts at distinct chromosomal sites in a
tissue-specific pattern coordinated with transcription, but previous studies
have not yet identified a chromatin modification that correlates with the
initiation of DNA replication at particular genomic locations. Here we report
that a distinct fraction of replication initiation sites in the human genome are
associated with a high frequency of dimethylation of histone H3 lysine K79
(H3K79Me2). H3K79Me2-containing chromatin exhibited the highest genome-wide
enrichment for replication initiation events observed for any chromatin
modification examined thus far (23.39% of H3K79Me2 peaks were detected in
regions adjacent to replication initiation events). The association of H3K79Me2
with replication initiation sites was independent and not synergistic with other
chromatin modifications. H3K79 dimethylation exhibited wider distribution on
chromatin during S-phase, but only regions with H3K79 methylation in G1 and G2
were enriched in replication initiation events. H3K79 was dimethylated in a
region containing a functional replicator (a DNA sequence capable of initiating
DNA replication), but the methylation was not evident in a mutant replicator
that could not initiate replication. Depletion of DOT1L, the sole enzyme
responsible for H3K79 methylation, triggered limited genomic over-replication
although most cells could continue to proliferate and replicate DNA in the
absence of methylated H3K79. Thus, prevention of H3K79 methylation might affect
regulatory processes that modulate the order and timing of DNA replication.
These data are consistent with the hypothesis that dimethylated H3K79 associates
with some replication origins and marks replicated chromatin during S-phase to
prevent re-replication and preserve genomic stability. The transition between proliferation and quiescence is frequently associated
with changes in gene expression, extent of chromatin compaction, and histone
modifications, but whether changes in chromatin state actually regulate cell
cycle exit with quiescence is unclear. We find that primary human fibroblasts
induced into quiescence exhibit tighter chromatin compaction. Mass spectrometry
analysis of histone modifications reveals that H4K20me2 and H4K20me3 increase in
quiescence and other histone modifications are present at similar levels in
proliferating and quiescent cells. Analysis of cells in S, G2/M, and G1 phases
shows that H4K20me1 increases after S phase and is converted to H4K20me2 and
H4K20me3 in quiescence. Knockdown of the enzyme that creates H4K20me3 results in
an increased fraction of cells in S phase, a defect in exiting the cell cycle,
and decreased chromatin compaction. Overexpression of Suv4-20h1, the enzyme that
creates H4K20me2 from H4K20me1, results in G2 arrest, consistent with a role for
H4K20me1 in mitosis. The results suggest that the same lysine on H4K20 may, in
its different methylation states, facilitate mitotic functions in M phase and
promote chromatin compaction and cell cycle exit in quiescent cells. Faithful propagation of DNA methylation patterns during DNA replication is
critical for maintaining cellular phenotypes of individual differentiated cells.
Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring
finger domains 1; also known as Np95 and ICBP90) specifically binds to
hemi-methylated DNA through its SRA (SET and RING finger associated) domain and
has an essential role in maintece of DNA methylation by recruiting Dnmt1 to
hemi-methylated DNA sites, the mechanism by which Uhrf1 coordinates the
maintece of DNA methylation and DNA replication is largely unknown. Here we
show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in
the maintece DNA methylation. Using Xenopus egg extracts, we successfully
reproduce maintece DNA methylation in vitro. Dnmt1 depletion results in a
marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine
23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a
region previously identified as a replication foci targeting sequence. The RING
finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and
maintain DNA methylation in mammalian cultured cells. Our findings represent the
first evidence, to our knowledge, of the mechanistic link between DNA
methylation and DNA replication through histone H3 ubiquitylation. The delivery of site-specific post-translational modifications to histones
generates an epigenetic regulatory network that directs fundamental DNA-mediated
processes and governs key stages in development. Methylation of histone H4
lysine-20 has been implicated in DNA repair, transcriptional silencing, genomic
stability and regulation of replication. We present the structure of the histone
H4K20 methyltransferase Suv4-20h2 in complex with its histone H4 peptide
substrate and S-adenosyl methionine cofactor. Analysis of the structure reveals
that the Suv4-20h2 active site diverges from the canonical SET domain
configuration and generates a high degree of both substrate and product
specificity. Together with supporting biochemical data comparing Suv4-20h1 and
Suv4-20h2, we demonstrate that the Suv4-20 family enzymes take a previously
mono-methylated H4K20 substrate and generate an exclusively di-methylated
product. We therefore predict that other enzymes are responsible for the
tri-methylation of histone H4K20 that marks silenced heterochromatin. Nucleosome dynamics facilitated by histone turnover is required for
transcription as well as DNA replication and repair. Histone turnover is often
associated with various histone modifications such as H3K56 acetylation
(H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In
order to correlate histone modifications and transcription-dependent histone
turnover, we performed genome wide analyses for euchromatic regions in
G2/M-arrested fission yeast. The results show that transcription-dependent
histone turnover at 5' promoter and 3' termination regions is directly
correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1)
in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1
and antisense RNA production was observed in the absence of the histone H3K36
methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved
in the suppression of histone turnover within the coding regions. These results
together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for
transcription-dependent histone turnover in fission yeast. |
Describe crowned dens syndrome. | Crowned dens syndrome is a rare form of "crown-like" calcifications around the dens and often presents with recurrent neck pain, stiffness of neck, increased erythrocyte sedimentation rate, and episodes of fever. | The crowned dens syndrome has been termed as acute neck pain ascribed to CPPD
deposits associated with a tomographic appearance of calcification surrounding
the odontoid process. This rare entity resulting in cervical cord compression is
generally seen in older female patients. We present a 26-year-old woman with
cervical cord compression due to massive calcification in the periodontoid area
and discuss the X-ray and CT findings of the disease. INTRODUCTION: Crowned dens syndrome is due to a microcrystalline infringement
(hydroxyapatite or calcium pyrophosphate) of the retro-odontoidal ligament of
atlas, often leading to the erroneous diagnosis of meningitis or spondylitis. We
report on three new cases diagnosed from 1996 to 1999.
EXEGESIS: The patients complained of cervicalgies, headaches or fever. The
initially evoked diagnoses were meningitis, spondylodiscitis or endocarditis.
Clinical exam found meningism and an inflammatory syndrome in all patients.
Analysis of the cerebro-spinal fluid realised in two cases was normal. The
diagnosis of crowned dens syndrome was assessed in two cases by cervical CT scan
of C1/C2. In the third case, chondrocalcinosis of a wrist allowed this
diagnosis. We report a probably non fortuitous case of crowned dens syndrome
associated with genetic hemochromatosis. A non steroidal anti-inflammatory
treatment allowed a dramatic regression of clinical symptoms.
CONCLUSION: This entity should be better known; it can mimick numerous diagnosis
and be responsible for fever in the long course. We report a 70-year-old man who presented symptoms resembling those of
meningoencephalitis and who was subsequently diagnosed as having a crowned dens
syndrome. The patient exhibited severe neck pains, headache, high fever and a
pain in his knee joints together with symptoms of the central nervous system.
The patient's cerebrospinal fluid was almost clear and showed no sign of viral
infection. An analysis of the synovial fluid in the right knee joint revealed
typical calcium pyrophosphate dehydrate crystal deposition and a diagnosis of
pseudogout was therefore made. A tomographic examination of the neck showed
periodontoid calcification. The patient was first treated with non-steroidal
anti-inflammatory drug, but its effect was only minimal. On the other hand, the
administration of corticosteroid resulted in a dramatic improvement in his
condition. PURPOSE: The purpose of this study was to verify the value of computed
tomography (CT) in the diagnosis of the "crowned dens" syndrome, not only in
crystal deposition diseases, but also in other rheumatic or nonrheumatic
conditions.
MATERIALS AND METHODS: Thirty-eight patients (15 men and 23 women; mean age 55
years; age range 35-79) with neck pain were examined and divided into two
groups: (1) patients already identified as rheumatic and referred for further
investigation of the atlantoaxial region; (2) patients with symptoms confined to
the cervical spine, with inconclusive radiographic findings. Unenhanced CT of
the cervical spine (Tomoscan SR 7000 Philips, Eindhoven, Netherlands) was
performed in all patients. There were 11 cases of rheumatoid arthritis (ten
women and one man), two calcium pyrophosphate dihydrate crystal deposition
disease (both women), one of systemic sclerosis (a woman), one of osteoarthritis
(a man), one of seronegative arthritis (a man), four of neoplasm (one woman and
three men) with suspected cervical involvement, one (a man) of haematological
disease (lymphoma), one (a woman) of menopausal osteoporosis, ten (five men and
five women) of recent or previous trauma with suspected involvement of the skull
base and first cervical vertebrae and six of unknown painful cervical
dysfunction (three men and three women).
RESULTS: CT demonstrated calcific deposits around the dens in 12 patients (three
men and nine women), in the transverse and alar ligaments, and in the anterior
atlantooccipital membrane. CT revealed horseshoe- or crown-like calcification
surrounding the odontoid process. In our series, other rheumatic diseases,
especially rheumatoid arthritis, showed similar irregular calcifications of the
atlantoaxial joint. Discussion. In calcium pyrophosphate dihydrate (CPPD)
crystal deposition disease, the spine may be the only site of involvement,
generally asymptomatic. Crystals located in the transverse ligament of the atlas
give rise to the crowned dens syndrome, usually in patients affected by severe
degenerative lesions of the atlantoaxial joint and peripheral chondrocalcinosis.
Symptoms may be absent, or a neurological compressive syndrome may develop.
Symptoms tend to worsen with age. The diagnosis is not always easy, as the
symptoms are similar to those of other diseases, such as meningitis,
cervicobrachial pain, occipitotemporal headache, calcific tendinitis of the
longus colli muscle, spondylodiscitis and retropharyngeal abscess.
CONCLUSION: CT is the gold standard in identifying crowned dens syndrome, as it
is able to depict the shape and site of calcification and any bone erosions.
Radiography of other joints (wrist, knee, pubic symphysis) may help to ascertain
whether the disease is due to calcium pyrophosphate dihydrate or hydroxyapatite
crystals, and is therefore recommended for routine patient management. Magnetic
resoce imaging (MRI) is indicated for the study of neurological
complications. OBJECTIVE: To investigate the association between articular chondrocalcinosis
and calcification of the atlantoaxial region on a cervical computed tomography
(CT) scan and to explore the relation between such calcifications and neck pain.
MATERIALS AND METHODS: CT slices of the cervico-occipital junction were
performed routinely in 49 consecutive patients (male/female ratio 28/21; mean
age 70.4 yrs), diagnosed with calcium pyrophosphate dihydrate crystal deposition
disease (CPPD). Of these, 35 met criteria for definite CPPD and 14 met the
criteria for probable. The cervical CT scans were analyzed for the presence of
periodontoid calcifications by 2 independent musculoskeletal radiologists. Both
assessors were blinded to the disease status of the patients. Furthermore,
conventional radiographs of the upper cervical spine were performed. An ad hoc
designed protocol was used to register information at diagnosis, including age,
sex, location of pain and stiffness, fever, presence of synovitis and its
location.
RESULTS: CT scan of the cervico-occipital junction showed periodontoid calcified
deposits in 25 out of 49 patients (51%) with CPPD. In 10 of the 25 cases (40%)
with periodontoid calcified deposits, CT scanning showed osseous abnormalities
of the odontoid process, such as subchondral cysts or erosions. Conventional
radiographs showed calcification behind the odontoid process in 17 patients
(34.7%). Nine of CPPD cases (18.4%) presented with neck symptoms. In three
patients, articular chondrocalcinosis was revealed only by an acute attack of
neck pain with segmentary stiffness, fever, and an increased erythrocyte
sedimentation rate; in one of them initial clinical examination found cervical
stiffness with Kernig's and/or Brudzinski's sign. For the other two patients,
impairment of general condition, occipito-temporal and mandible pain and
weakness with inflammatory pain of the shoulder girdle was suggestive of giant
cell arteritis (GCA) and/or polymyalgia rheumatica (PMR). In the six additional
patients, questioning elicited a history of previous subacute or chronic neck
pain, from one week to one year before their admission to our ambulatory or
hospital.
CONCLUSIONS: These results suggest that CPPD deposition disease frequently
involves the cervical spine. Although such calcification often remains
asymptomatic, it may be associated with attacks of acute neck pain with
segmentary stiffness, fever, and an increased erythrocyte sedimentation rate,
sometimes mimicking PMR and/or GCA or neurological symptoms. Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is rare in
patients under the age of 40 in the absence of metabolic or familial
predisposition. A high incidence of involvement of the transverse ligament of
the atlas in CPPD deposition disease was reported. However, involvement of the
craniocervical junction is rarely symptomatic. We report a rare case in a young
male with severe idiopathic CPPD crystal deposition disease, including crowned
dens syndrome in the cervical spine. The crowned dens syndrome (CDS) is a clinicoradiological entity defined as acute
neck pain due to deposition of crystals surrounding the odontoid process.
Crystal deposition may occur without symptoms or exhibit myelopathy by
compressing the spinal cord. Neck pain on lateral rotation has been described in
the previous papers for CDS. We tested the validity of "painful neck on
rotation" as a potential diagnostic tool to differentiate CDS from meningeal
irritation. Patients 1 and 2 were relatively young females and showed acute
cervico-occipital pain, with marked limitation on neck rotation. Patient 2 was
suspected to have neck strain, since there was no fever and no elevated CRP. In
contrast, patient 3 was an older female and showed headache and limitation of
neck rotation. Cerebrospinal fluid was normal in spite of inflammatory reaction
on laboratory markers. All three patients showed absence of or only a mild
limitation on neck flexion. A computed tomography scan focused on the
craniocervical junction revealed mottled calcification around the odontoid
process. Painful neck on rotation may have diagnostic implications for CDS in
patients with cervico-occipital pain. Acute pseudogout of the neck, also known as the crowned dens syndrome, is a rare
cause of neck pain characterised by crystalline deposition in periodontoid
articular tissues. It is typified clinically by severe cervical pain and
stiffness, often in conjunction with pyrexia and raised inflammatory markers. As
such, it is often misdiagnosed. We report 2 cases of crowned dens syndrome
masquerading respectively as meningitis and polymyalgia rheumatica, and review
the literature with particular attention to the clinical and radiological
aspects of this under-recognised condition. BACKGROUND: Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is
one of the most common forms of crystal-associated arthropathy in the elderly.
However, CPPD deposition on the cervical spine is less well known, and only a
limited number of cases have been reported to date. Here, we report our recent
clinical experience with CPPD crystal deposition disease of the cervical spine
and describe the clinical features of this disease.
METHODS: Fourteen patients with clinically diagnosed CPPD crystal deposition
disease of the cervical spine at our department during the period from January
2005 to December 2008 were analyzed retrospectively.
RESULTS: Patients ranged in age from 54 to 92 (mean+/-SD, 77.5+/-8.5). Chief
symptoms of patients were acute posterior neck pain and fever. All patients had
markedly restricted neck rotation. Serum CRP level was highly elevated in all
patients (10.16+/-5.35 mg/dL). Computed tomography of the cervical spine
demonstrated linear calcific deposits in the transverse ligament of atlas
(crowned dens syndrome) in all patients. Calcific deposits were also found in
other periodontoid structures and the ligamenta flava in some patients.
Posterior neck pain, fever, and increased serum inflammatory indicators were
relieved within 1 to 3 weeks by nonsteroidal antiinflammatory drugs (NSAIDs) or
a combination of NSAIDs and prednisolone. Most of the patients were misdiagnosed
as having other diseases before consultation.
CONCLUSIONS: CPPD crystal deposition disease of the cervical spine is one of the
most common underrecognized causes of acute neck pain in the neurology
department, especially in elderly patients. We report a case of pseudogout manifested by severe posterior neck pain.
Pseudogout of the neck, also known as the crowned dens syndrome, causes acute
neck pain characterized by calcium pyrophosphate dehydrate deposition around the
odontoid process. Crowned dens syndrome is typified clinically by severe
cervical pain and stiffness, often in conjunction with raised inflammatory
markers. A 71-year-old man presented with severe neck pain. On admission,
elevation of serum CRP level was confirmed. Magnetic resoce images showed no
responsible abnormalities except for degenerating change of the spine. The
patient was diagnosed as having pseudogout caused by calcium pyrophosphate
dehydrate deposition based on cervical computed tomographic imaging, which
showed linear calcification in the transverse ligament of the axis. After
administration of non-steroidal anti-inflammatory drugs, the fever and neck pain
disappeared and the CRP level returned to within the normal range. Pseudogout of
the cervical spine should be considered as a differential diagnosis when we
examine patients with acute neck pain. Cervical spinal computed tomographic scan
is a more sensitive and useful examination method to diagnose this disease
rather than magnetic resoce images. Calcium pyrophosphate deposition (CPPD) disease is an arthropathy caused by
calcium pyrophosphate dihydrate (CPP) crystal deposits in articular tissues,
most commonly fibrocartilage and hyaline cartilage. According to EULAR, four
different clinical presentations can be observed: 1) asymptomatic CPPD; 2)
osteoarthritis (OA) with CPPD; 3) acute CPP crystal arthritis; 4) chronic CPP
inflammatory crystal arthritis. Acute CPP crystal arthritis is characterized by
sudden onset of pain, swelling and tenderness with overlying erythema, usually
in a large joint, most often the knee, wrist, shoulder, and hip. Occasionally,
ligaments, tendons, bursae, bone and the spine can be involved. CPPD of the
atlanto-occipital joint (crowned dens syndrome) can cause periodic acute
cervico-occipital pain with fever, neck stiffness and laboratory inflammatory
syndrome. Chronic inflammatory arthritis is characterized by joint swelling,
morning stiffness, pain, and high ESR and CRP. The relationship between OA and
CPPD is still unclear. The main problem is whether such crystals are directly
involved in the pathogenesis of OA or if they are the result of joint
degeneration. Diagnosis is based on evaluation of history and clinical features,
conventional radiology, and synovial fluid examination. Non-polarized light
microscopy should be used initially to screen for CPPD crystals based upon their
characteristic morphology, and compensated polarized light microscopy, showing
the crystals to be weakly positive birefringent, is recommended for definitive
identification, although this last pattern only occurs in about 20% of samples.
The main goals of CPPD therapy are control of the acute or chronic inflammatory
reaction and prevention of further episodes. We report a case of CPPD crowned dens syndrome in an 87 year white old male with
a known history of pseudogout, with clinical and radiological features
characteristic of this syndrome. Interestingly, there was significant mass
effect on the clivus, with clivus erosion and destruction, a finding that has
not previously been described with this syndrome. The clinical and radiological
characteristics of Crowned Dens syndrome, as well as CPPD are reviewed. We
suggest that CPPD crowned dens syndrome may be included in the differential
diagnosis when clivus destruction or erosion, in association with a soft tissue
mass with calcification, is seen. Crowned dens syndrome (CDS) is a rare but underrecognized cause of severe neck
pain in older adults. It is characterized by acute onset pain and stiffness of
the cervical spine. Accompanying fever and elevated inflammatory markers often
lead to misdiagnosis. It is frequently associated with calcium pyrophosphate
deposition disease, hydroxyapatite crystals, and sometimes other inflammatory
conditions. Periodontoid calcification is seen on cervical computed tomography
(CT) scan but is not typically visible on plain radiographs, making CT scanning
invaluable in diagnosis. We describe a case of CDS in a 59-year-old woman, who
presented with severe neck pain, elevated inflammatory markers, and progressive
evolution in the appearance of her CT scans. The pathophysiology, clinical and
radiographic findings, and dramatic response to corticosteroid therapy are
reviewed. BACKGROUND AND PURPOSE: Crowned dens syndrome is an ill-known etiology of acute
neck pain.
METHODS: We carried out a retrospective study of 18 cases of patients with
crowned dens syndrome, assessing clinical and radiological features.
RESULTS: The results of our study are comparable to data from the literature.
The clinical presentation of acute febrile neck pain, occipital headache and
multidirectional stiff neck especially affects women aged over 60. No
predisposing factor was recognized. However, a history of peripheral joint
chondrocalcinosis may reinforce the diagnosis. In more than 50% of cases,
laboratory tests showed a marked inflammatory syndrome. The diagnosis was
obtained with cervical CT-scan focusing on the C1/C2 joint. This gold standard
test was able to show a calcification of the cruciform ligament in connection
with deposits of calcium pyrophosphate crystals in almost 80% of cases. Other
imaging tests provided little information, including standard radiographs of the
cervical spine. MRI can eliminate some differential diagnoses such as infections
or neurological emergencies. Complications are infrequent. The standard
treatment is based on anti-inflammatory drugs (NSAID, colchicine) or
corticosteroids. These treatments are highly effective: a drammatic full
recovery of cervical mobility may be observed within 48 hours. In over half of
cases, a different diagnosis was initially made, responsible of unnecessary
additional tests and treatment.
CONCLUSION: A comprehensive consultation, a complete clinical examination and a
precise analysis of the imaging will avoid certain investigations and rule out
differential diagnoses. Spondylodiscitis are frequent and clinical challenge for practionners. Axial
calcium pyrophosphate dihydrate deposition disease (CPDD) is well known for
cervical spine involvement with the crowned dens syndrome but other
localisations are probably underdiagnosed in sterile spondylodiscitis. We report
a case of recurrent sterile spondylodiscitis with epidural abscess related to
CPDD proved by vertebral percutaneous needle biopsy with rapid favourable course
under colchicine therapy. Axial CPDD could mimic septic spondylodiscitis with
epidural abscess on MRI. Sterile spondylodiscitis are probably underdiagnosed
forms of microcrystalline disease. Investigations of the presence of
microcrystals should be systematically undertaken with tamponed formalin fixed
biopsies. If axial CPDD is suspected, colchicine therapy could be a good
therapeutic test and would avoid unnecessary antibiotic treatment. Basic calcium phosphate and pyrophosphate calcium crystals are the 2 main
calcium-containing crystals that can deposit in all skeletal tissues. These
calcium crystals give rise to numerous manifestations, including acute
inflammatory attacks that can mimic alarming and threatening differential
diagnoses, osteoarthritis-like lesions, destructive arthropathies, and calcific
tendinitis. Awareness of uncommon localizations and manifestations such as
intraspinal deposition (eg, crowned dens syndrome, tendinitis of longus colli
muscle, massive cervical myelopathy compression) prevents inappropriate
procedures and cares. Coupling plain radiography, ultrasonography, computed
tomography, and synovial fluid analysis allow accurate diagnosis by directly or
indirectly identifying the GRAAL of microcrystal-related symptoms. Study Design Case report and review of the literature. Objective A
retro-odontoid mass is a rare cause of cervical compression and myelopathy. The
differential diagnosis includes the following: metastatic disease, primary
tumor, collagen disorder, or inflammatory disease. Calcium pyrophosphate
dihydrate (CPPD) deposition has been referred to as "crowned dens syndrome" when
there are periodontoideal calcifications. There are only a few reported cases
where CPPD presents as a cystic retro-odontoid mass in the atlanto-dens
interval. In previous descriptions of surgical intervention, transoral resection
of the mass is associated with significant morbidity and usually requires
stabilization. The objective of this article is to report a case of an unusual
presentation of CPPD disease of C1/C2, where we used a novel, minimally invasive
surgical technique for decompression without fusion. Patients and Methods An
83-year-old female patient presented with progressive cervical myelopathy over a
3-month period. Computed tomography and magnetic resoce imaging demonstrated
a cystic odontoid mass with a separate retro-odontoid compressive mass. A novel,
minimally invasive transoral aspiration was performed. Histologic confirmation
of CPPD was obtained. Results Postop imaging showed satisfactory decompression,
which was maintained at the 6-month follow-up. This correlated with clinical
improvement postop and 6-month follow-up. Conclusion CPPD in the atlanto-dens
interval may present as a cystic retro-odontoideal mass and should be included
in the differential. We used a transoral minimally invasive approach to aspirate
the cyst. This novel technique avoided the need for a stabilization procedure or
morbid transoral resection and provided excellent results immediately and at 6
months. BACKGROUND: Crowned dens syndrome (CDS) is a clinical-radiological entity
characterized by acute attacks of neck pain with fever, rigidity, general signs
of inflammation, and calcification of the periodontoid articular structures.
METHODS: Case report with 42 months follow-up.
CASE DESCRIPTION: An 81-year-old man, who had never suffered from headache
before July 2010, developed strictly left-sided headaches. The pain was
restricted to the left side of the scalp and felt more intense in the frontal
area. The intensity was moderate to high with a throbbing quality. The pain had
an orthostatic component and was worsened by neck hyperextension and Valsalva
maneuvers. Neurological and general examinations were normal, except for a
reduced range of motion of the neck. He was prescribed indomethacin orally 25 mg
t.i.d. and had a partial response. After a week, he was given a dosage of 50 mg
t.i.d. with complete remission of the pain. Brain magnetic resoce imaging was
normal, while an magnetic resoce imaging of the cervical spine showed a
non-homogeneous mass behind the odontoid process of C2, narrowing the
subarachnoid space in C1, stretching the posterior longitudinal ligament, and
touching the left vertebral artery. A computed tomography scan showed
calcification of the soft tissue around the odontoid process and a thickening of
the left C2 root. After 4 months, the indomethacin dosage was reduced
step-by-step. Indomethacin was discontinued in March 2012. Since then, the
headache has not recurred.
DISCUSSION: We here present the case of a patient with headache and radiological
findings of crowned dens. However, the clinical presentation differed from
previous CDS cases in the literature in that the pain was unilateral with
frontal localization and throbbing quality, as well as an orthostatic component
and lack of either fever or inflammatory signs. The differential diagnosis also
includes a remitting form of hemicrania continua, presenting with an atypical
presentation, with neuroimaging incidental finding of CDS.
CONCLUSION: This case widens the spectrum of the clinical presentation of
crowned dens, a condition that should be kept in mind in cases of unilateral
headache in older patients. BACKGROUND: Pain is regarded as one of the most common nonmotor symptoms in
Parkinson's disease (PD). In particular, musculoskeletal pain has been reported
as the most common type of PD-associated pain. Crowned dens syndrome (CDS),
related to microcrystalline deposition in the periodontoid process, is the main
cause of acute or chronic cervical pain.
CASE PRESENTATION: This report describes the case of an 87-year-old woman who
had severe bradykinesia, muscle rigidity, gait disturbance and neck pain.
Laboratory examination revealed marked elevations of white blood cells
(10,100/µl) and C-reactive protein (CRP; 8.63 mg/dl). She was primarily
diagnosed with severe and untreated PD, corresponding to Hoehn and Yahr scale
score IV, with musculoskeletal pain and urinary tract infection. The patient was
treated with antiparkinsonism drugs, antibiotic agents and nonsteroidal
anti-inflammatory drugs, but they had only limited effects. Cervical plain
computed tomography (CT) scanning detected remarkable crown-like calcification
surrounding the odontoid process. Based on CT findings, the patient was
diagnosed as having CDS with PD, and was immediately treated with
corticosteroid. The severe neck rigidity with pain and the serum CRP level (0.83
mg/dl) of the patient were drastically improved within a week by the additional
corticosteroid therapy.
CONCLUSION: Severe neck rigidity and bradykinesia in this patient might have
strengthened the chondrocalcinosis around the odontoid process. Cervical plain
CT scan is necessary and useful for the definitive diagnosis of CDS. CDS should
be considered as a differential diagnosis of a possible etiology for
musculoskeletal pain related to rigidity and bradykinesia in PD. Crystal deposition in the cervical spine around the odontoid process may lead to
acute neck pain. This rare condition is called crowned dens syndrome and should
be considered in the differential diagnosis of a possible etiology for fever,
headache and cervical pain of unknown origin. The syndrome is often overlooked,
thus leading to misdiagnosis, invasive and useless investigations (lumbar
puncture, biopsy), inappropriate treatment (steroids, antibiotics, antiviral
drugs) and prolonged hospitalization. This can be prevented by imaging, based on
a cervical CT scan that allows an accurate diagnosis. The disease has a good
prognosis and symptoms usually subside within a few weeks. We describe a patient
with crowned dens syndrome which manifested with clinical (acute occipital
headache) and radiographic (calcium deposits in the alar ligament) features. Our
patient recovered in four days with symptomatic therapy. An 87-year-old woman with corticosteroid-resistant polymyalgia rheumatica
underwent ¹⁸F-FDG PET/CT for suspected giant cell arteritis or neoplastic
disease. FDG uptake in the immediate vicinity of the odontoid process, with a
crownlike calcification, was identified on the CT scan on the posterior side of
the dens, thus confirming the diagnosis of crowned dens syndrome. Because this
rare syndrome is frequently misdiagnosed, nuclear physicians should be aware of
the signs and symptoms of this condition, which may call for the use of PET/CT
imagery. BACKGROUND: Patients with crowned dens syndrome (CDS), which is pseudogout of
the atlantoaxial junction induced by "crown-like" calcifications around the
dens, present with symptoms of severe neck pain, rigidity, and high fever. CDS
patients are often misdiagnosed as having meningitis or polymyalgia rheumatica,
leading to potentially unnecessary invasive procedures for diagnosis and
treatment.
CASE REPORT: We report 3 patients with CDS who had characteristic findings on
computed tomography (CT), all of whom quickly recovered with nonsteroidal
antiinflammatory drug (NSAID) administration. In addition, we reviewed 72
published cases, including our patients. CDS typically occurs in elderly people
(mean age 71.4 years). Common symptoms include neck pain (100%), neck rigidity
(98%), and fever (80.4%), and most show elevated inflammatory markers (88.3%) on
serum laboratory tests. Neck pain on rotation is a characteristic and helpful
symptom in the diagnosis. The most useful modality is CT (97.1%), showing linear
calcium deposits around the dens, mostly in the transverse ligament of atlas
(TLA). CT number is especially helpful to distinguish a normal TLA (35-110 HU)
from a calcified one (202-258 HU) in our cases. The most effective treatment is
NSAID administration (85%), which usually leads to marked resolution of symptoms
within days or weeks. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Due
to acute and severe symptoms, CDS patients often present to an emergency
department. To avoid unnecessary invasive procedures for diagnosis and
treatment, CDS should be considered in the differential diagnosis of febrile
neck pain. BACKGROUND CONTEXT: Crowned dens syndrome (CDS) is a rare form of calcium
phosphate crystal depositions and often presents with recurrent neck pain,
stiffness of neck, increased erythrocyte sedimentation rate, and episodes of
fever.
PURPOSE: The goal of this report is to identify the early and late stages of CDS
and its consequences as the result of repeated attacks of CDS at cervical spine
in its late stage.
STUDY DESIGN: This is a case report.
METHODS: We reported one case of early-stage CDS and one late-stage CDS.
RESULTS: The two patients shared some common clinical features of acute attack
of CDS, such as increased erythrocyte sedimentation rate, C-reactive protein,
episode of fever, and increased white blood cells along with high blood glucose
levels. The first case showed early phase of CDS with computed tomography (CT)
scan that only showed mild calcification around the dens. The second case had
appearance of late stage of CDS with more severe chronic degenerative changes of
cervical spine.
CONCLUSIONS: Early stage of CDS can be difficult to identify because of mild
clinical symptoms, but CT scan is a preferable method to demonstrate densities
surrounding the top and sides of the odontoid process. In the late stage of CDS,
radiolographic features often include diffuse periodontoid calcifications,
diffuse destructive discopathies, and apophyseal joint destruction, and patient
might have severe neurological symptoms. An 88-year-old woman presented with fever and acute posterior neck pain. A CT
scan revealed calcification of the transverse ligament and crown-like
calcification around the odontoid process. According to the clinical and
radiological findings, she was diagnosed with crowned dens syndrome (CDS). Her
symptoms drastically improved following treatment with oral nonsteroidal
anti-inflammatory medication. An X-ray of her wrist, elbow, shoulder and knee
joints showed asymptomatic calcium deposits, suggesting underlying crystalline
deposition disease. CDS may occur as the initial presentation of crystalline
deposition disease. The measurement of procalcitonin and an X-ray survey of the
major joints may be helpful for the diagnosis of CDS. Crowned dens syndrome (CDS), a pseudogout attack involved with atlantoaxial
joint, mimics meningitis, because jolt accentuation of headache, a physical sign
for meningitis, is frequently considered mistakenly as 'positive' in CDS
patients. Our patient with CDS experienced multiple ambulance transports and
underwent lumbar puncture for suspected meningitis because of positive result of
jolt accentuation of headache. We found that the patient actually had jolt
accentuation of neck pain from CDS and treated her successfully. The
characteristic physical finding produced by axial neck rotation in CDS patients
is not headache, but a jolt accentuation of neck pain. We present a unique case of crowned dens syndrome (CDS) that developed after
endoscopic retrograde cholangiopancreatography (ERCP) in a patient who presented
with fever and neck pain. Administration of non-steroidal anti-inflammatory
drugs was extremely effective for relieving fever and neck pain, and in the
improvement of inflammatory markers. To the best of our knowledge, this is the
first case report of CDS caused by an ERCP procedure. In a patient with fever
and neck pain after an ERCP procedure, CDS should be considered in the
differential diagnosis. |
Does CRISPR inversion of CTCF sites alter genome topology? | Yes. CRISPR inversion of CTCF sites alters genome topology. | Author information:
(1)Center for Comparative Biomedicine, MOE Key Laboratory of Systems
Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center
of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240,
China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer
Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key
Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders
(MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai
200240, China.
(2)Department of Biochemistry and Molecular Biophysics, Columbia University
Medical Center, 701 West 168(th) Street, New York, NY 10032, USA.
(3)Ludwig Institute for Cancer Research and Department of Cellular and Molecular
Medicine, University of California, San Diego School of Medicine, 9500 Gilman
Drive, La Jolla, CA 92093, USA.
(4)Department of Molecular and Cell Biology, Center for Systems Biology,
University of Texas at Dallas, Richardson, TX 75080, USA; MOE Key Laboratory of
Bioinformatics and Bioinformatics Division, Center for Synthetic and System
Biology, TNLIST/Department of Automation, Tsinghua University, Beijing 100084,
China.
(5)Cold Spring Harbor Laboratory, 1 Bungtown Rd, NY 11724, USA.
(6)Department of Biochemistry and Molecular Biophysics, Columbia University
Medical Center, 701 West 168(th) Street, New York, NY 10032, USA. Electronic
address: [email protected].
(7)Center for Comparative Biomedicine, MOE Key Laboratory of Systems
Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center
of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240,
China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer
Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key
Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders
(MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai
200240, China. Electronic address: [email protected]. |
How are looped genes identified in yest? | Gene-loop formation is dependent on regulatory proteins localized at the 5' and 3' ends of genes, such as TFIIB | Gene looping juxtaposes the promoter and terminator regions of RNA polymerase
II-transcribed genes in yeast and mammalian cells. Here we report an
activator-dependent interaction of transcription initiation and termination
factors during gene looping in budding yeast. Chromatin analysis revealed that
MET16, INO1, and GAL1p-BUD3 are in a stable looped configuration during
activated transcription. Looping was nearly abolished in the absence of
transcription activators Met28, Ino2, and Gal4 of MET16, INO1, and GAL1p-BUD3
genes, respectively. The activator-independent increase in transcription was not
accompanied by loop formation, thereby suggesting an essential role for
activators in gene looping. The activators did not facilitate loop formation
directly because they did not exhibit an interaction with the 3' end of the
genes. Instead, activators physically interacted with the general transcription
factor TFIIB when the genes were activated and in a looped configuration. TFIIB
cross-linked to both the promoter and the terminator regions during the
transcriptionally activated state of a gene. The presence of TFIIB on the
terminator was dependent on the Rna15 component of CF1 3' end processing
complex. Coimmunoprecipitation revealed a physical interaction of Rna15 with
TFIIB. We propose that the activators facilitate gene looping through their
interaction with TFIIB during transcriptional activation of genes. Gene looping, defined as the physical interaction between the promoter and
terminator regions of a RNA polymerase II-transcribed gene, is widespread in
yeast and mammalian cells. Gene looping has been shown to play important roles
in transcription. Gene-loop formation is dependent on regulatory proteins
localized at the 5' and 3' ends of genes, such as TFIIB. However, whether other
factors contribute to gene looping remains to be elucidated. Here, we
investigated the contribution of intrinsic DNA and chromatin structures to gene
looping. We found that Saccharomyces cerevisiae looped genes show high DNA
bendability around middle and 3/4 regions in open reading frames (ORFs). This
bendability pattern is conserved between yeast species, whereas the position of
bendability peak varies substantially among species. Looped genes in human cells
also show high DNA bendability. Nucleosome positioning around looped ORF middle
regions is unstable. We also present evidence indicating that this unstable
nucleosome positioning is involved in gene looping. These results suggest a
mechanism by which DNA bendability and unstable nucleosome positioning could
assist in the formation of gene loops. |
Which are the causes of the Koebner phenomenon? | The causes of the Koebner phenomenon are:
1) Lichen sclerosus
2) Vitiligo
3) Psoriasis and
4) Physical stress. | In 1877, Dr Heinrich Koebner inflicted an experimental trauma on the uninvolved
skin of a psoriatic patient. This resulted in the appearance of a typical
psoriatic lesion at the site of trauma. This reaction, known as Koebner's
phenomenon (KP), has subsequently been associated with several skin diseases.
However, it has not been associated previously with necrobiosis lipoidica
diabeticorum (NBL), a rare skin manifestation of diabetes mellitus. This report
presents the unusual finding of NBL associated with KP in a patient with
diabetes mellitus. A 12-year-old boy was seen in our department with a three year history of
eruptions on his back consisting of normal colored or slightly reddish,
pinhead-sized papules, which had gradually spread to his trunk within the
previous year. Four days before the first visit, May 13, 1992, the eruption
involved his entire extremities. On the back side of his lumbar region,
Koebner's phenomenon was observed. He had received several medications for more
than two years to treat this eruption in several hospitals, but none were
effective. He was in good general health. Mantoux reaction was negative two
months after BCG injection. Histopathologic features of the papules included
focal epidermal hyperplasia and elongation of the rete ridges in a narrow area
and a well demarcated dense lymphohistiocytic infiltrate on the papillar layer.
Biscoclaurine alkaloids (20 mg/day) and Jumi-haidoku-to (TJ-6; 7.5 g/day) were
administered to the patient after the biopsy. No topical ointments were applied.
Two weeks after of these treatments, he reported moderate pruritus on the back.
The eruption diminished rapidly within 2 weeks after the therapy began. Almost
all the eruptions were cured within one year. Mantoux reaction developed 8 x 8
mm erythema two and half months after the treatment began, and it was
significantly positive (23 x 30 mm) 6 months later. In several cases of lupus erythematosus (SLE, SCLE, DLE), the Koebner phenomenon
could be seen in the apparently normal skin of patients following traumas,
scratching effects, operation scars, contact dermatitis, pressure from sock
tops, application of liquid nitrogen, exposure to strong sun light, etc. The
interval between exposure to such provocation factors and appearance of the
isomorphic reactions varied from case to case. In SLE patients receiving
systemic treatment with glucocorticosteroids, the Koebner phenomenon could not
be found, while it was detected in patients with generalized DLE who had never
received any systemic glucocorticoid hormones. It was not seen in patients with
localized DLE. There was no direct relationship between the appearance of
humoral autoantibodies and onset of the Koebner phenomenon. With regard to the
histopathogenesis of the Koebner phenomenon, it is thought that disturbances of
the local cellular repair processes and of the cytokine network, with expression
of adhesion molecules, might be involved. The Koebner phenomenon may be a
valuable model for the investigation of lupus erythematosus. INTRODUCTION: Former investigations of Koebner phenomenon had demonstrated its
higher incidence in patients with severe generalized and/or unstable forms of
psoriasis which expressed increased resistance to various treatment modalities.
The aim of this study was to establish the correlation between the presence of
Koebner phenomenon and the PUVA therapy effects, total number of PUVA
treatments, total dose of UVA radiation and the duration of remission after PUVA
therapy discontinuation.
MATERIAL AND METHODS: Sixty patients with severe clinical picture of psoriasis
vulgaris, treated with PUVA therapy, were included in this research. According
to the presence of Koebner phenomenon they were divided into two groups, 20
patients with positive and 40 patients with negative Koebner reaction, who were
the control group at the same time.
RESULTS AND DISCUSSION: 95% of patients treated with PUVA, were cleared of
psoriatic changes in the Koebner positive, as well as in the Koebner negative
group. There were also no differences between the Koebner positive and Koebner
negative group in the mean number of PUVA treatments, mean total dose and the
last dose of UVA radiation, which led up to the clinical remission of psoriasis.
Our results of investigation have demonstrated increased relapse of psoriasis,
during the first 6 months after cessation of PUVA therapy, in the Koebner
positive group, with a high statistical significance (p < 0.001), comparing with
Koebner negative group in the same period. Furthermore, the tendency of relapse
of Koebner positive and Koebner negative psoriatic patients was higher in
Koebner positive group even in the first 3 months after PUVA therapy.
CONCLUSIONS: PUVA therapy effects, total number of PUVA treatments, total dose
of UVA radiation didn't depend on presence of Koebner phenomenon. However,
Koebner phenomenon was a mark of high relapsing tendency of psoriasis in the
first 6 months after PUVA therapy cessation. Koebner phenomenon is the development of isomorphic pathological lesions in
distant wounds of patients with pre-existing cutaneous diseases. More frequent
in patients with psoriasis, it can occur in the presence of other cutaneous
pathologies. Surgeons should be aware of this entity and warn patients about its
possible occurrence. The Koebner phenomenon has been reported to develop in classic or acquired
immune deficiency syndrome-related Kaposi's sarcoma (KS). A 12-year-old kidney
transplant recipient who developed immunosuppression-related KS showed
reoccurrence of lesions in some previously intact incision sites following
removal of tumor, suggesting Koebner phenomenon. It is recommended that surgeons
be careful when planning surgical interventions in patients with certain skin
disorders in which Koebner phenomenon is known to develop. Pemphigus vulgaris is an autoimmune bullous disorder involving the skin and
sometimes the mucosa. Koebner's phenomenon is encountered when the typical
features of a dermatosis are observed on a part of the skin previously subject
to friction or trauma. A few cases of pemphigus vulgaris developing after damage
to the skin and especially scars have been described. To the best of our
knowledge, we report the first case where typical lesions of pemphigus vulgaris
appeared simultaneously on a recent and an old scar as the sign of the
reactivation of the disease. INTRODUCTION: Koebner phenomenon in psoriasis presents development of psoriatic
lesions, after injury of uninvolved skin, which are identical in morphology with
the previous trauma. The aim of this study was to establish the correlation of
Koebner phenomenon with sex and age distribution, clinical variants of psoriasis
vulgaris, age of onset and incidence in psoriasis among relatives of affected
patients.
MATERIAL AND METHODS: Sixty patients, with severe clinical picture, participated
in this study: 38 patients in acute flare of a chronic form; 10 with acute
exanthematic form; 8 with a chronic stable form; 3 with psoriatic changes on
palms and soles and one patient with psoriatic erythroderma. According to the
presence of Koebner phenomenon they were divided in two groups, one with
positive and the other with negative Koebner phenomenon which presented the
control group at the same time.
RESULTS AND DISCUSSION: The Koebner reaction is often thought to be more
frequent in actively spreading, severe psoriasis. Although this may be true, it
has to be established by prospective studies. According to our investigation,
Koebner phenomenon did not depend on clinical picture of psoriasis vulgaris.
This reaction also appears to be a marker for a subgroup of patients with a
tendency to early onset, but that was not confirmed by our study. In available
literature we did not find any data about relations of Koebner phenomenon to sex
and age or familiar incidence of psoriasis vulgaris. Our results demonstrated no
connection of Koebner phenomenon with sex and age structure. At the same time
its presence did not depend on familiar incidence of psoriasis vulgaris.
CONCLUSIONS: There is no relationship between Koebner phenomenon and sex and age
distribution. It does not depend on clinical picture and also does not predict
the age of onset and familiar incidence of psoriasis vulgaris. First described in 1877 as the appearance of psoriatic lesions in the uninvolved
skin of psoriatic patients as a consequence of trauma, the Koebner phenomenon
has since been described in numerous diseases. Other authors have tried to
implicate either infections or parasitic causes as the pathogenesis of this
phenomenon. Subsequent research by many authors have contributed to our poor
understanding of this reaction in the hope of understanding the pathogensis of
psoriasis. We present a review of the literature covering the following topics
as they relate to the Koebner phenomenon: diseases that koebnerize and their
possible causes, predisposing and provoking factors, type, site, depth and
degree of trauma, the all or none phenomenon, time lag, site preference,
medications, inhibition of koebnerization and reverse koebnerization. The isomorphic phenomenon belongs to the probably most well-known entities of
dermatology and is closely connected to the man who was the first to describe
it. Today the Koebner phenomenon is well documented in a number of skin diseases
and still of considerable interest. Heinrich Koebner first reported his
observation in 1872 and caused considerable diverse discussion about the origin
of psoriasis in the following years. Heinrich Koebner is not only well known as
"father" of the Koebner phenomenon, but also as a founder of the university
dermatology clinic and pioneer of dermatology in Breslau. We not only describe
the life of Heinrich Koebner, but also discuss the evolution of the term
"Koebner phenomenon" and its current status. Skin side effects following XRT take place more often in patients with skin
disorders. In this study six patients with psoriatic lesions were evaluated. The
total/daily XRT dose to the tumor site was 50-70/1.8-2.0 Gy. No debilitating
effect of XRT was observed in both the psoriatic lesions and in the surrounding
normal skin. The isomorphic response of Koebner can be observed not only in psoriasis, but
also in other diseases, such as lichen planus and some systemic diseases
including LE (lupus erythematosus) or sarcoidosis. Several clinical findings in
LE skin were presented and discussed in this review. The mutually-interactive-,
negative-, and internal-Koebner phenomena were introduced and discussed with
some speculative views. Many forms of environmental stress on the skin were
reported as provocating factors of the Koebner phenomenon, including trauma,
scratching, UV-exposure, and various types of dermatitis. Clinical observations
of the nature, localization, and movement of lesions should be carefully made.
The pathophysiology of the Koebner phenomenon may be classified into two steps.
A first non-specific inflammatory step and a second disease-specific step. The
inflammatory products released from the first step would be targeted in the
second step. In the first step, there could be many substances including
cytokines, stress proteins, adhesion molecules, or autoantigens translocated
from intra-cellular areas. In the second step after latent periods, there may be
disease-specific reactions, including ones by T-cells, B-cells, autoantibodies,
and immune deposits, under the restriction of genetic backgrounds. The Koebner
phenomenon may prove useful in understanding the pathophysiology of diseases of
unknown origin. The Koebner phenomenon originally described the appearance of psoriatic lesions
in the uninvolved skin of patients with psoriasis as a consequence of trauma. We
describe a case of concurrent lichen planus and sarcoidosis in the auditory
canal, which represents an unusual manifestation of the Koebner phenomenon. This
is the first case of concurrent lichen planus and sarcoidosis in the head and
neck region and highlights the need for biopsy to allow accurate
histopathological diagnosis and treatment. AIMS: To document the role of striae distensae and striae gravidarum in causing
Koebner phenomenon in cases of vitiligo, psoriasis and lichen planus.
RESULTS: Striae are documented to cause Koebner phenomenon in patients with
preexisting vitiligo, psoriasis and lichen planus, the three conditions where
'true kobenerisation' has been suggested according to Boyd and Nelder
classification.
CONCLUSIONS: Striae distensae and striae gravidarum are examples of blunt
trauma. Just as happens with penetrating trauma, striae too are shown to be
responsible for causing the Koebner phenomenon. The Koebner phenomenon is one of the most well-known entities in dermatology. It
was first described by Heinrich Koebner in 1876 as the formation of psoriatic
lesions in uninvolved skin of psoriatic patients after cutaneous trauma. This
isomorphic phenomenon is now known to involve numerous diseases, among them
vitiligo, lichen planus, and Darier disease. The pathogenesis of the Koebner
phenomenon is still obscure but may involve cytokines, stress proteins, adhesion
molecules, and autoantigens. This contribution reviews the clinical
manifestations of Koebner phenomenon, its provocative factors, suggested
pathogenesis mechanisms, and the various skin conditions that exhibit this
unique response. Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy associated with
psoriasis, and included among the seronegative spondyloarthropathies. The
presence of cutaneous psoriasis is very important for correct and early
diagnosis of PsA, because the cutaneous lesions precede the appearance of joint
manifestations. Thus, dermatologists are in a position to detect the condition
at its inception. PsA is clinically subdivided into asymmetric oligoarticular
arthritis, symmetric polyarthritis, distal interphalanges predomit, arthritis
mutilans, and spondylitis types. PsA has several unique characteristics, such as
enthesopathy, dactylitis and abnormal bone remodeling. Genetic, environmental,
and immunological factors are important in its development. The triggering role
of physical stress is seen in the "deep Koebner" phenomenon, which causes
inflammation in the synovial membrane and in enthesis, resulting in peripheral
arthritis. Cellular infiltrates such as activated T-cells and macrophages are
thought to play important roles in the induction of inflammatory and destructive
processes in joint tissues, as well as psoriatic skin. New ideas regarding the
involvement of the IL-23/Th17 axis have emerged, and the dramatic effects of
targeting therapies have highlighted the physiological role of key cytokines in
psoriasis. Current views on the pathogenesis of PsA are reviewed from a
dermatological perspective. The Koebner phenomenon (isomorphic response) is defined as the development of
typical lesions of a dermatosis that occur in areas of trauma in previously
uninvolved skin. Rare reports have suggested that striae are a form of injury to
the skin that can result in koebnerization of psoriasis, vitiligo, and lichen
planus. We herein report a 27-year-old female patient with plaque psoriasis who
developed psoriatic lesions along striae distensae. Lichen sclerosus is a chronic inflammatory disease, usually of the anogenital
area, that causes intractable itching and soreness. Less commonly, it may have
extragenital involvement in 15 to 20% of cases. Lichen sclerosus has been
reported at sites of injury as a Koebner phenomenon. We report a case of lichen
sclerosus at the site of a tattoo with simultaneous genital involvement. Both shingles and psoriasis are common cutaneous diseases. About 25% of the
psoriatic patients develop Koebner phenomenon (KP) after various injuries, and
in rare instance, KP may occur at the site of healed or healing shingles.We
report a 30-year-old man with 7-month history of scalp psoriasis who developed
KP at the areas of developing shingles. Cutaneous examination revealed scaly
erythematous papules and plaques located on the scalp and forehead, and groups
of clustered erythematous papules with silver scales in the dermatome
distributed on the right side of chest wall the prior herpes zoster lesions
involved. After removal of the scales on the papules, underlying bleeding points
were present.The lesions on chest had good response to anti-psoriatic therapies,
as the lesions on scalp did. After a year of follow-up, recurrent psoriasis
occurred, but the lesions were located only on the scalp, and the areas of prior
occurrence of shingles, because of which we considered diagnosis of recurrent
psoriasis rather than relapsing KP for the chest lesions.Not only the healing
and healed shingles can trigger KP in psoriasis, but also the developing
shingles can cause psoriatic KP at the site of herpes zoster lesions. Mycosis fungoides (MF) is the most frequent type of primary cutaneous
T-cell/NK-cell lymphoma. The Koebner phenomenon is defined as the appearance of
cutaneous lesions on previously noninvolved skin following trauma and is
observed in a series of cutaneous diseases including psoriasis, lichen planus,
viral warts, molluscum contagiosum, etc. In this case report, 3 patients with
longstanding MF are presented, the 1st with the appearance of a circumscribed
early-stage type MF lesion rapidly following a surgical excision of an
infundibular cyst, the 2nd with the appearance of a unique unilateral palmar
tumoral MF lesion at the pressure site of a crutch, and the 3rd presented
localized MF early stage lesions at the friction site of a belt. This report
suggests that some MF patients may experience Koebner phenomenon-induced MF
lesions and that MF should be added to the long list of skin diseases
potentially exhibiting the Koebner phenomenon. |
Is there a role of proton beam therapy in medulloblastoma treatment? | Yes, proton beam therapy is used for treatment of medulloblastoma. | The ability to vary the proton energy (depth of beam penetration) and modulate
the dose distribution at the end of range permits delivery of an increased dose
to the designated cancer-containing volume with a reduced dose to overlying
normal brain tissue. The evolution of childhood CNS maligcy following therapy
is reviewed to identify radiation response variables indicating where the proton
dose distribution will improve the therapeutic ratio. The review documents that
of the 1262 children expected to develop CNS maligcy in 1989, only 43% will
survive 5 years. About 75% of those with medulloblastoma and over 90% with
astrocytoma die from persistent (in-field) disease. When the patient has been
treated with radiation, it is accepted that disease persistence indicates the
cancer dose was insufficient. Potentially 536 children could show an improved
incidence of local control and improved survival from an increased cancer dose
available from proton irradiation. As the total dose and volume of brain
irradiated is increased about 1800 cGy, brain dysfunction increases, producing a
spectrum of functional and intellectual deficits which are age and volume
related. About 900 irradiated patients would have fewer in-field histologic and
functional changes if the dose to normal brain, or the volume of brain
irradiated, is reduced by an improved dose distribution. A proton beam treatment
plan, delivering a cancer dose of 7400 cGy, is simulated for a thalamic
astrocytoma. The dose distribution of this plan is compared with an x-ray plan
used to treat a patient, in which a dose of 5400 cGy was delivered to the
astrocytoma. Comparative isodose distributions and dose-volume histograms
indicate a decreased integral dose to normal brain and a decreased volume of
normal brain irradiated, even as the cancer dose is boosted 2000 cGy with
protons. PURPOSE: In this study factors are analyzed that may potentially influence the
site of failure in pediatric medulloblastoma. Patient-related, disease-related,
and treatment-related variables are analyzed with a special focus on
radiotherapy time-dose and technical factors.
METHODS AND MATERIALS: Eighty-six children and adolescents with a diagnosis of
medulloblastoma were treated in Switzerland during the period 1972-1991.
Postoperative megavoltage radiotherapy was delivered to all patients. Simulation
and portal films of the whole-brain irradiation (WBI) fields were
retrospectively reviewed in 77 patients. The distance from the field margin to
the cribiform plate and to the floor of the temporal fossa was carefully
assessed and correlated with supratentorial failure-free survival. In 19
children the spine was treated with high-energy electron beams, the remainder
with megavoltage photons. Simulation and port films of the posterior fossa
fields were also reviewed in 72 patients. The field size and the field limits
were evaluated and correlated with posterior fossa failure-free survival.
RESULTS: In 36 patients (47%) the WBI margins were judged to miss the inferior
portion of the frontal and temporal lobes. Twelve patients failed in the
supratentorial region and 9 of these patients belonged to the group of 36
children in whom the inferior portion of the brain had been underdosed. On
multivariate analysis only field correctness was retained as being significantly
correlated with supratentorial failure-free survival (p = 0.049). Neither the
total dose to the spinal theca nor the treatment technique (electron vs. photon
beams) were significantly correlated with outcome. Posterior fossa failure-free
survival was not influenced by total dose, overall treatment time, field size,
or field margin correctness. Overall survival was not influenced by any of the
radiotherapy-related technical factors.
CONCLUSION: A correlation between WBI field correctness and supratentorial
failure-free survival was observed. Treatment protocols should be considered
that limit supratentorial irradiation mainly to subsites at highest risk of
relapse. Optimized conformal therapy or proton beam therapy may help to reach
this goal. Treating the spine with electron beams was not deletereous. A
significant correlation between local control and other technical factors was
not observed, including those relating to posterior fossa treatment. The use of
small conformal tumor bed boost fields may be prefered to the larger posterior
fossa fields usually considered as the standard treatment approach. PURPOSE: One of the components of radiotherapy (RT) in medulloblastoma/primitive
neuroectodermal tumors is the prophylactic irradiation of the whole brain (WBI).
With the aim of reducing late neuropsychologic morbidity a CT-scan-based
dosimetric study was undertaken in which treatment was confined mainly or
exclusively to supratentorial sites considered at high risk for disease
recurrence.
METHODS AND MATERIALS: A comparative dosimetric study is presented in which a
three field (two laterals and one posterior) proton plan (spot scanning method)
is compared with a two-field conventional WBI 6 MV x-ray plan, to a 6-field
"hand-made" 6 MV x-ray plan, and to a computer-optimized 9-field "inverse" 15 MV
x-ray plan. For favorable patients, 30 Gy were delivered to the ventricles and
main cisterns, the subfrontal and subtemporal regions, and the posterior fossa.
For the unfavorable patients, 10 Gy WBI preceeded a boost to 30 Gy to the same
treatment volume chosen for favorable patients. The dose distribution was
evaluated with dose-volume histograms to examine the coverage of the targets as
well as the dose to the nontarget brain and optical structures. In addition, the
risks of radiation-related late neuropsychologic effects after WBI were
collected from the literature and used to predict normal tissue complication
probabilities (NTCPs) for an intelligence quotient deficit after treatment with
photon or proton beams.
RESULTS: Proton beams succeeded better in reducing the dose to the brain
hemispheres and eye than any of the photon plans. A 25.1% risk of an IQ score
<90 was predicted after 30 Gy WBI. Almost a 10% drop in the predicted risk was
observed when using proton beams in both favorable and unfavorable patients.
However, predicted NTCPs for both optimized photon plans ("hand made" and
"inverse") were only slightly higher (0.3-2.5%) than those of proton beams. An
age-modifying factor was introduced in the predictive NTCP model to assess for
IQ differences in relation with age at irradiation. Children with ages between
age 4 to 8 benefitted most from the dose reduction in this exercise (similar
NTCP predictions for both proton and "inverse" plans).
CONCLUSION: Modulated proton beams may help to significantly reduce the
irradiation of normal brain while optimally treating the supratentorial subsites
at higher risk for relapse. A decrease in morbidity can be expected from protons
and both optimized proton plans compared to WBI. PURPOSE: Conventional postoperative photon-beam radiotherapy to the spine in
children with medulloblastoma/PNET is associated with severe late effects. This
morbidity (growth and developmental) is related to the exit dose of the beams
and is particularly severe in young children. With the purpose of reducing this
toxicity, a dosimetric study was undertaken in which proton therapy was compared
to standard megavoltage photon treatment.
METHODS AND MATERIALS: The results of a comparative dosimetric study are
presented in such a way that the dose distribution achievable with a posterior
modulated 100 MeV proton beam (spot scanning method) is compared with that of a
standard set of posterior 6 MV x-ray fields. The potential improvements with
protons are evaluated, using dose-volume histograms to examine the coverage of
the target as well as the dose to the vertebral bodies (growth plates), lungs,
heart, and liver.
RESULTS: The target (i.e., the spinal dural sac) received the full prescribed
dose in both treatment plans. However, the proportions of the vertebral body
volume receiving > or = 50% of the prescribed dose were 100 and 20% for 6 MV
x-rays and protons, respectively. For 6 MV x-rays > 60% of the dose prescribed
to the target was delivered to 44% of the heart volume, while the proton beam
was able to completely avoid the heart, the liver, and in all likelihood the
thyroid and gonads as well.
CONCLUSION: The present study demonstrates a potential role of proton therapy in
decreasing the dose (and toxicity) to the critical structures in the irradiation
of the spinal neuraxis in medulloblastoma/PNET. The potential bone marrow and
growth arrest sparing effects make this approach specially attractive for
intensive chemotherapy protocols and for very young children. Sparing the
thyroid gland, the posterior heart wall, and the gonads may be additional
advantages in assuring a long-term posttreatment morbidity-free survival. PURPOSE: To assess the potential influence of improved dose distribution with
proton beams compared to conventional or intensity-modulated (IM) X-ray beams on
the incidence of treatment-induced secondary cancers in pediatric oncology.
METHODS AND MATERIALS: Two children, one with a parameningeal rhabdomyosarcoma
(RMS) and a second with a medulloblastoma, were used as models for the purpose
of this study. After defining the target and critical structures, treatment
plans were calculated and optimized, four for the RMS case (conventional X-ray,
IM X-rays, protons, and IM protons) and three for the irradiation of the spinal
axis in medulloblastoma (conventional X-ray, IM X-rays, protons). Secondary
cancer incidence was estimated using a model based on Publication No. 60 of the
International Commission on Radiologic Protection. This model allowed estimation
of absolute risks of secondary cancer for each treatment plan based on
dose-volume distributions for the nontarget organs.
RESULTS: Proton beams reduced the expected incidence of radiation-induced
secondary cancers for the RMS patient by a factor of >or=2 and for the
medulloblastoma case by a factor of 8 to 15 when compared with either IM or
conventional X-ray plans.
CONCLUSIONS: The potential for a significant reduction in secondary cancers with
pediatric cancers after using proton beams (forward planned or IM) in the
treatment of RMS and MBD in children and adolescents represents an additional
argument supporting the development of proton therapy for most radiotherapy
indications in pediatric oncology. BACKGROUND: Radiation therapy is an important component in the treatment of
medulloblastoma; however, in many patients, it is associated with risk of late
adverse events. Proton radiation therapy has potential to reduce the risk of
adverse events compared with conventional radiation, but it is associated with a
higher treatment cost. The objective of the current study was to assess the
cost-effectiveness of proton therapy compared with conventional radiation
therapy in the treatment of childhood medulloblastoma.
METHODS: The consequences of radiation therapy were evaluated using a Markov
simulation model. Children age 5 years with medulloblastoma were followed. The
patients were at risk of several types of adverse events, including hearing
loss, intelligence quotient (IQ) loss, hypothyroidism, growth hormone deficiency
(GHD), osteoporosis, cardiac disease, and secondary maligcies. The patients
also were at risk of death and were divided into risk groups for normal death,
death due to tumor recurrence, treatment-related cardiac death,
treatment-related subsequent tumor death, or treatment-related other death. A
review of the literature was conducted to estimate the parameters in the model.
RESULTS: The base-case results showed that proton therapy was associated with
23,600 in cost savings and 0.68 additional quality-adjusted life-years per
patient. The analyses showed that reductions in IQ loss and GHD contributed to
the greatest part of the cost savings and were the most important parameters for
cost-effectiveness.
CONCLUSIONS: The results of the current study indicated that proton radiation
therapy can be cost-effective and cost-saving compared with conventional
radiation therapy in the treatment of children with medulloblastoma if the
appropriate patients are selected for the therapy. However, there have been few
long-term follow-up studies, and more much information on the long-term
consequences of radiation therapy is needed. The aim of this treatment planning comparison study was to explore different
spinal irradiation techniques with respect to the risk of late side-effects,
particularly radiation-induced cancer. The radiotherapy techniques compared were
conventional photon therapy, intensity modulated x-ray therapy (IMXT),
conventional electron therapy, intensity/energy modulated electron therapy
(IMET) and proton therapy (IMPT).CT images for radiotherapy use from five
children, median age 8 and diagnosed with medulloblastoma, were selected for
this study. Target volumes and organs at risk were defined in 3-D. Treatment
plans using conventional photon therapy, IMXT, conventional electron therapy,
IMET and IMPT were set up. The probability of normal tissue complication (NTCP)
and the risk of cancer induction were calculated using models with
parameters-sets taken from published data for the general population; dose data
were taken from dose volume histograms (DVH). Similar dose distributions in the
targets were achieved with all techniques but the absorbed doses in the
organs-at-risk varied significantly between the different techniques. The NTCP
models based on available data predicted very low probabilities for side-effects
in all cases. However, the effective mean doses outside the target volumes, and
thus the predicted risk of cancer induction, varied significantly between the
techniques. The highest lifetime risk of secondary cancers was estimated for
IMXT (30%). The lowest risk was found with IMPT (4%). The risks associated with
conventional photon therapy, electron therapy and IMET were 20%, 21% and 15%,
respectively. This model study shows that spinal irradiation of young children
with photon and electron techniques results in a substantial risk of
radiation-induced secondary cancers. Multiple beam IMXT seems to be associated
with a particularly high risk of secondary cancer induction. To minimise this
risk, IMPT should be the treatment of choice. If proton therapy is not
available, advanced electron therapy may provide a better alternative. PURPOSE: To calculate treatment plans and compare the dose distributions and
dose-volume histograms (DVHs) for photon three-dimensional conformal radiation
therapy (3D-CRT), electron therapy, intensity-modulated radiation therapy
(IMRT), and standard (nonintensity modulated) proton therapy in three pediatric
disease sites.
METHODS AND MATERIALS: The tumor volumes from 8 patients (3 retinoblastomas, 2
medulloblastomas, and 3 pelvic sarcomas) were studied retrospectively to compare
DVHs from proton therapy with 3D-CRT, electron therapy, and IMRT. In
retinoblastoma, several planning techniques were analyzed: A single electron
appositional beam was compared with a single 3D-CRT lateral beam, a 3D-CRT
anterior beam paired with a lateral beam, IMRT, and protons. In medulloblastoma,
three posterior fossa irradiation techniques were analyzed: 3D-CRT, IMRT, and
protons. Craniospinal irradiation (which consisted of composite plans of both
the posterior fossa and craniospinal components) was also evaluated, primarily
comparing spinal irradiation using 3D-CRT electrons, 3D-CRT photons, and
protons. Lastly, in pelvic sarcoma, 3D-CRT, IMRT, and proton plans were
assessed.
RESULTS: In retinoblastoma, protons resulted in the best target coverage
combined with the most orbital bone sparing (10% was the mean orbital bone
volume irradiated at > or =5 Gy for protons vs. 25% for 3D-CRT electrons, 69%
for IMRT, 41% for a single 3D lateral beam, 51% for a 3D anterolateral beam with
a lens block, and 65% for a 3D anterolateral beam without a lens block). A
single appositional electron field was the next best technique followed by other
planning approaches. In medulloblastoma, for posterior fossa and craniospinal
irradiation, protons resulted in the least dose to the cochlea (for only
posterior fossa irradiation at > or =20 Gy, 34% was the mean cochlear volume
irradiated for protons, 87% for IMRT, 89% for 3D-CRT) and hypothalamus-pituitary
axis (for only posterior fossa irradiation at > or =10 Gy, 21% was the mean
hypothalamus-pituitary volume irradiated for protons, 81% for IMRT, 91% for
3D-CRT); additional dose reductions to the optic chiasm, eyes, vertebrae,
mandible, thyroid, lung, kidneys, heart, and liver were seen.
Intensity-modulated radiotherapy appeared to be the second best technique for
posterior fossa irradiation. For spinal irradiation 3D-CRT electrons were better
than 3D-CRT photons in sparing dose to the thyroid, heart, lung, kidney, and
liver. With pelvic sarcoma, protons were superior in eliminating any dose to the
ovaries (0% of mean ovarian volume was irradiated at > or =2 Gy with protons)
and to some extent, the pelvic bones and vertebrae. Intensity-modulated
radiotherapy did show more bladder dose reduction than the other techniques in
pelvic sarcoma irradiation.
CONCLUSIONS: In the diseases studied, using various techniques of 3D-CRT,
electrons, IMRT, and protons, protons are most optimal in treating
retinoblastomas, medulloblastomas (posterior fossa and craniospinal), and pelvic
sarcomas. Protons delivered superior target dose coverage and sparing of normal
structures. As dose-volume parameters are expected to correlate with acute and
late toxicity, proton therapy should receive serious consideration as the
preferred technique for the treatment of pediatric tumors. A group of Swedish oncologists and hospital physicists have estimated the number
of patients in Sweden suitable for proton beam therapy. The estimations have
been based on current statistics of tumour incidence, number of patients
potentially eligible for radiation treatment, scientific support from clinical
trials and model dose planning studies and knowledge of the dose-response
relations of different tumours and normal tissues. It is estimated that in
paediatric cancers, proton beams are of potential importance in 80-100 children
annually in Sweden. About 20 of the patients have medulloblastoma. The main
purpose is to reduce late sequelae, but these are also increased chances to
avoid myelosupression during e.g. concomitant chemo-radiation and to further
intensify the chemotherapy. OBJECTIVE: To improve medulloblastoma proton therapy. Although considered ideal
for proton therapy, there are potential disadvantages. Expected benefits include
reduced radiation-induced cancer and circulatory complications, while avoiding
small brain volumes of dose in-homogeneity when compared with conventional
X-rays. Several aspects of proton therapy might contribute to reduced tumour
control due to (a) the use of more homogenous dose levels which can result in
under-dosage, (b) differences in relative biological effectiveness (RBE) between
that prescription RBE of 1.1 and the RBE of brain and spinal cord (likely to
exceed 1.1) and in medulloblastoma cells (where RBE is likely to be below 1.1).
Such changes, although speculative for RBE, might result in potential
underdosage of tumour cells and a higher bio-effect in brain tissue.
METHODS: Dose distributions for X-ray and proton treatment are compared, with
allocation of likely RBE values for fast growing medullolastoma cells and stable
central nervous system tissue.
RESULTS: These physical and radiobiological factors are shown to combine to give
a higher risk of tumour recurrence with further risks on tumour control when
dose reduction schedules used for X-ray therapy are replicated for proton
therapy for "low-risk" patients.
CONCLUSION: The dose distributions and prescribed doses of proton therapy,
taking into account RBE, in children and adults with medulloblastoma, need to be
reconsidered. BACKGROUND: The aim of this study is to evaluate the cost-effectiveness of
proton beam therapy with cochlear dose reduction compared with conventional
X-ray radiotherapy for medulloblastoma in childhood.
METHODS: We developed a Markov model to describe health states of 6-year-old
children with medulloblastoma after treatment with proton or X-ray radiotherapy.
The risks of hearing loss were calculated on cochlear dose for each treatment.
Three types of health-related quality of life (HRQOL) of EQ-5D, HUI3 and SF-6D
were used for estimation of quality-adjusted life years (QALYs). The incremental
cost-effectiveness ratio (ICER) for proton beam therapy compared with X-ray
radiotherapy was calculated for each HRQOL. Sensitivity analyses were performed
to model uncertainty in these parameters.
RESULTS: The ICER for EQ-5D, HUI3 and SF-6D were $21 716/QALY, $11 773/QALY, and
$20 150/QALY, respectively. One-way sensitivity analyses found that the results
were sensitive to discount rate, the risk of hearing loss after proton therapy,
and costs of proton irradiation. Cost-effectiveness acceptability curve analysis
revealed a 99% probability of proton therapy being cost effective at a societal
willingness-to-pay value.
CONCLUSIONS: Proton beam therapy with cochlear dose reduction improves health
outcomes at a cost that is within the acceptable cost-effectiveness range from
the payer's standpoint. BACKGROUND AND PURPOSE: Brain tumours are the most frequent solid tumours in
children and the most frequent radiotherapy indications in paediatrics, with
frequent late effects: cognitive, osseous, visual, auditory and hormonal. A
better protection of healthy tissues by improved beam ballistics, with particle
therapy, is expected to decrease significantly late effects without decreasing
local control and survival. This article reviews the scientific literature to
advocate indications of protontherapy and carbon ion therapy for childhood
central nervous system cancer, and estimate the expected therapeutic benefits.
MATERIALS AND METHODS: A systematic review was performed on paediatric brain
tumour treatments using Medline (from 1966 to March of 2014). To be included,
clinical trials had to meet the following criteria: age of patients 18 years or
younger, treated with radiation, and report of survival. Studies were also
selected according to the evidence level. A secondary search of cited references
found other studies about cognitive functions, quality of life, the comparison
of photon and proton dosimetry showing potential dose escalation and/or sparing
of organs at risk with protontherapy; and studies on dosimetric and technical
issues related to protontherapy.
RESULTS: A total of 7051 primary references published were retrieved, among
which 40 clinical studies and 60 papers about quality of life, dose distribution
and dosimetry were analysed, as well as the ongoing clinical trials. These
papers have been summarized and reported in a specific document made available
to the participants of a final 1-day workshop. Tumours of the meningeal envelop
and bony cranial structures were excluded from the analysis. Protontherapy
allows outstanding ballistics to target the tumour area, while substantially
decreasing radiation dose to the normal tissues. There are many indications of
protontherapy for paediatric brain tumours in curative intent, either for
localized treatment of ependymomas, germ-cell tumours, craniopharyngiomas,
low-grade gliomas; or panventricular irradiation of pure non-secreting
germinoma; or craniospinal irradiation of medulloblastomas and metastatic pure
germinomas. Carbon ion therapy is just emerging and may be studied for highly
aggressive and radioresistant tumours, as an initial treatment for diffuse
brainstem gliomas, and for relapse of high-grade gliomas.
CONCLUSION: Both protontherapy and carbon ion therapy are promising for
paediatric brain tumours. The benefit of decreasing late effects without
altering survival has been described for most paediatric brain tumours with
protontherapy and is currently assessed in ongoing clinical trials with
up-to-date proton devices. Unfortunately, in 2015, only a minority of paediatric
patients in France can receive protontherapy due to the lack of equipment. |
What is the function of the DGAT1 gene product? | Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in the acyl-CoA-dependent triacylglycerol biosynthesis | Acyl CoA:diacylgycerol acyltransferase (EC; DGAT) catalyzes the final step in
the production of triacylglycerol. Two polypeptides, which co-purified with DGAT
activity, were isolated from the lipid bodies of the oleaginous fungus
Mortierella ramanniana with a procedure consisting of dye affinity,
hydroxyapatite affinity, and heparin chromatography. The two enzymes had
molecular masses of 36 and 36.5 kDa, as estimated by gel electrophoresis, and
showed a broad activity maximum between pH 6 and 8. Based on partial peptide
sequence information, polymerase chain reaction techniques were used to obtain
full-length cDNA sequences encoding the purified proteins. Expression of the
cDNAs in insect cells conferred high levels of DGAT activity on the membranes
isolated from these cells. The two proteins share 54% homology with each other
but are unrelated to the previously identified DGAT gene family (designated
DGAT1), which is related to the acyl CoA:cholesterol acyltransferase gene
family, or to any other gene family with ascribed function. This report
identifies a new gene family, including members in fungi, plants and animals,
which encode enzymes with DGAT function. To distinguish the two unrelated
families we designate this new class DGAT2 and refer to the M. ramanniana genes
as MrDGAT2A and MrDGAT2B. Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) catalyzes the final step of
triglyceride synthesis in mammalian cells. Data obtained from DGAT1-knockout
mice have indicated that this enzyme plays an important role in energy
homeostasis. We investigated the regulation of the expression and function of
DGAT1 in mouse 3T3-L1 cell as a model for mammalian adipocytes. We demonstrated
that the DGAT1 protein level increased by approximately 90-fold following
differentiation of 3T3-L1 into mature adipocytes, a change that was accompanied
by approximately 7-fold increase in DGAT1 mRNA. On the other hand, forced
overexpression of DGAT1 mRNA by >20-fold via a recombit adenovirus only
resulted in approximately 2-fold increase in DGAT1 protein in mature adipocytes
and little increase in preadipocytes. These results indicated that gene
expression of DGAT1 in adipocytes is subjected to rigorous posttranscriptional
regulation, which is modulated significantly by the differentiation status of
3T3-L1 cells. Protein stability is not a significant factor in the control of
DGAT1 expression. The steady-state levels of DGAT1 were unaffected by blockage
of proteolytic pathways by ALLN. However, translational control was suggested by
sequence analysis of the 5'-untranslated region of human DGAT1 (hDGAT1) mRNA. We
found that the level of DGAT1 activity was predomitly a function of the
steady-state level of DGAT1 protein. No significant functional changes were
observed when the conserved tyrosine phosphorylation site in hDGAT1 was mutated
by a single base pair substitution. Despite only a approximately 2-fold increase
in DGAT1 protein caused by recombit viral transduction, a proportionate
increase in cellular triglyceride synthesis resulted without affecting the
triglyceride lipolysis rate, leading to >2-fold increase in intracellular
triglyceride accumulation. No change in adipocyte morphology or in the
expression levels of lipoprotein lipase, proxisomal proliferation-activating
receptor-gamma, and aP2 was evident secondary to DGAT1 overexpression at
different stages in 3T3-L1 differentiation. These data suggest that
dysregulation of DGAT1 may play a role in the development of obesity, and
manipulation of the steady-state level of DGAT1 protein may offer a potential
means to treat or prevent obesity. Acyl-CoA:diacylglycerol acyltransferases (DGATs) catalyze the last step in
triglyceride (TG) synthesis. The genes for two DGAT enzymes, DGAT1 and DGAT2,
have been identified. To examine the roles of liver DGAT1 and DGAT2 in TG
synthesis and very low density lipoprotein (VLDL) secretion, liver DGAT1- and
DGAT2-overexpressing mice were created by adenovirus-mediated gene transfection.
DGAT1-overexpressing mice had markedly increased DGAT activity in the presence
of the permeabilizing agent alamethicin. This suggests that DGAT1 possesses
latent DGAT activity on the lumen of the endoplasmic reticulum.
DGAT1-overexpressing mice showed increased VLDL secretion, resulting in
increased gonadal (epididymal or parametrial) fat mass but not subcutaneous fat
mass. The VLDL-mediated increase in gonadal fat mass might be due to the 4-fold
greater expression of the VLDL receptor protein in gonadal fat than in
subcutaneous fat. DGAT2-overexpressing mice had increased liver TG content, but
VLDL secretion was not affected. These results indicate that DGAT1 but not DGAT2
has a role in VLDL synthesis and that increased plasma VLDL concentrations may
promote obesity, whereas increased DGAT2 activity has a role in steatosis. DGAT1 is a microsomal enzyme that catalyses the final step in triglycerides
synthesis. DGAT1-deficient mice are viable, lean, fertile and resistant to diet
induced obesity. We have previously identified a quantitative trait loci (QTL)
on chromosome 4 that affects fatty acid composition in an F2 cross between
Iberian x Landrace. The human DGAT1 gene is located on chromosome 8q24.3, this
region aligns to porcine chromosome 4, making the pig DGAT1 gene a suggestive
positional candidate gene for the QTL. In this study, we sequenced 1679 bp of
the mRNA from animals of five pig breeds (Iberian, Landrace, Large White,
Piétrian and Meishan) to identify genetic variants. One of the polymorphisms
found creates a polymorphic HinfI restriction site and it was genotyped by
PCR-RFLP in these five pig breeds. Allele A was not found in the analysed
Iberian and Landrace populations, whereas Meishan population presents the
highest frequency (35%). The DGAT1 gene was located by radiation hybrid mapping
to the porcine chromosome 4, outside the confidence interval for the fatty acid
composition QTL and excludes it as a positional candidate gene. A quantitative trait locus (QTL) affecting milk fat percentage has been mapped
to the centromeric end of the bovine chromosome 14 (BTA14). This genomic area
includes the DGAT1 gene, which encodes acyl-CoA:diacylglycerol acyltransferase
1, the key enzyme of triglyceride biosynthesis. Genetic and biochemical studies
led to the identification of the nonconservative DGAT1-K232A polymorphism as a
causal mutation for the QTL. In addition to this, another polymorphism in the
5'-regulatory region of this gene, the DGAT1 variable number of tandem repeat
(VNTR), also showed a strong association with milk fat percentage. This promoter
VNTR polymorphism affects the number of potential Sp1 binding sites and
therefore might have an impact on DGAT1 expression and also milk fat content.
Hence, the DGAT1 VNTR polymorphism might be another causal mutation for the
BTA14 QTL. However, evidence for Sp1 binding to this polymorphic site and for
the capability of DGAT1 VNTR alleles to stimulate gene expression was lacking.
In the current work Sp1-VNTR interactions were analyzed by EMSA. In addition,
effects of DGAT1 VNTR alleles on gene expression were measured with reporter
gene analyses. Conclusions from the results are that 1) the DGAT1 VNTR sequence
is indeed a target for Sp1 binding; 2) DGAT1 VNTR alleles can stimulate gene
expression in vitro and probably in vivo as well; and 3) although the
stimulating effects of the different DGAT1 VNTR alleles did not show significant
differences in vitro, their effects on transcription might be different in the
chromatin context existing in vivo. We report molecular cloning and single nucleotide polymorphism detection of the
buffalo DGAT1 gene. Diacylglycerol acyltransferase (DGAT1) is considered the key
enzyme in controlling the rate of synthesis of triglycerides. The DGAT1 gene was
recently identified as a strong functional candidate gene affecting milk yield
and composition in cattle. A full-length buffalo DGAT1 genomic DNA was amplified
by iterative PCR based on homolog cloning. The buffalo DGAT1 gene comprises 17
exons and spans approximately 8.3 kb. The genomic structures of DGAT1 are highly
conserved among mammal species. The deduced protein of buffalo DGAT1 contains
489 amino acids, showing high-sequence similarity with mammal homologs. Through
PCR-SSCP analysis and sequencing, seven polymorphic positions were detected in
the complete genomic region of buffalo DGAT1, and their frequencies were
observed from a collection of 117 buffalo. The SNP (C/T) detected at position
11785 in exon 17 creates a substitution change for the amino acid sequence,
resulting in an Ala residue (GCG) transition to a Val residue (GTG) in position
484 of buffalo DGAT1 protein. Information provided in this study will be useful
in further studies to determine the role DGAT1 plays in the regulation of milk
fat synthesis and quality improvement for milk in buffalo. Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein
of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols.
Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in
lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of
synthesizing diacylglycerol, retinyl, and wax esters in addition to
triacylglycerol. Here, we report the membrane topology for murine DGAT1 using
protease protections assays and indirect immunofluorescence in conjunction with
selective permeabilization of cellular membranes. Topology models based on
prediction algorithms suggested that DGAT1 had eight transmembrane domains. In
contrast, our data indicate that DGAT1 has three transmembrane domains with the
N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which
accounts for ∼50% of the protein, is present in the endoplasmic reticulum lumen
and contains a highly conserved histidine residue (His-426) that may be part of
the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1
to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro
acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is
not required for the catalytic activity of DGAT1 but, instead, may be involved
in regulating enzyme activity and dimer/tetramer formation. BACKGROUND: The involvement of muscle triacylglycerol (TAG) storage in the onset
of insulin resistance is questioned and the attention has shifted towards
inhibition of insulin signalling by the lipid intermediate diacylglycerol (DAG).
The enzyme 1,2-acylCoA:diacylglyceroltransferase-1 (DGAT1) esterifies a fatty
acyl-CoA on DAG to form TAG. Therefore, the aim of the present study was to
investigate if unilateral overexpression of DGAT1 in adult rat Tibialis anterior
(TA) muscle will increase conversion of the lipid intermediate DAG into TAG,
thereby improving muscle insulin sensitivity.
METHODOLOGY/PRINCIPAL FINDINGS: The DGAT1 gene construct was injected in the
left TA muscle of male rats on chow or high-fat (45% kcal) diet for three weeks,
followed by application of one 800 V/cm and four 80 V/cm pulses, using the
contralateral leg as sham-electroporated control. Seven days after
electroporation, muscle specific insulin sensitivity was assessed with a
hyperinsulinemic euglycemic clamp using 2-deoxy-[3H]glucose. Here, we provide
evidence that unilateral overexpression of DGAT1 in TA muscle of male rats is
associated with an increased rather than decreased DAG content. Strikingly, this
increase in DAG content was accompanied by improved muscle insulin sensitivity.
Interestingly, markers of muscle lipolysis and mitochondrial function were also
increased in DGAT1 overexpressing muscle.
CONCLUSIONS/SIGNIFICANCE: We conclude that unilateral DGAT1 overexpression can
rescue insulin sensitivity, possibly by increasing DAG and TAG turnover in
skeletal muscle. In case of a proper balance between the supply and oxidation of
fatty acids in skeletal muscle, the lipid intermediate DAG may not exert harmful
effects on insulin signalling. Diacylglyceroltransferase-1 (DGAT1) expresses in nearly all tissues, including
the mammary gland. Mice lacking DGAT1 exhibit decreased triglyceride content in
mammary tissue, and are resistant to diet-induced obesity and diabetes mellitus.
Thus, DGAT1 has received considerable attention. In the present study, the
function of DGAT1 was examined by liposome mediated RNA interference (RNAi) to
knockdown the expression of endogenous DGAT1 expression in bovine mammary
epithelial cells (BMEC) and the changes of the biological functions of cells
were analyzed. The mRNA and protein levels, intracellular triglyceride (TG)
content, and total protein of BMECs were analyzed by real-time PCR, Western
blot, TG kit, and ultraviolet spectrophotometer, respectively, before and after
RNAi treatment. The results indicated that knockdown of DGAT1 expression
significantly reduced TG content in BMECs. This study further confirmed the
importance of DGAT1 in triglyceride synthesis in bovine mammary tissue. Diacylglycerol-O-acyltransferase (DGAT1) gene encodes the rate-limiting enzyme
of triglyceride synthesis. A polymorphism in this gene, DGAT1 K232A, has been
associated with milk production and composition in taurine breeds. However, this
polymorphism is not a good tool for ascertaining the effects of this QTL in Bos
indicus (Zebu), since the frequency of the DGAT1 232A allele is too low in these
breeds. We sequenced the 3'-untranslated region of DGAT1 gene in a sample of
bulls of the breeds Guzerá (Bos indicus) and Holstein (Bos taurus) and, using in
silico analysis, we searched for genetic variation, evolutionary conservation,
regulatory elements, and possible substitution effects. Six single nucleotide
(SNPs) and one insertion-deletion (INDEL) polymorphisms were found in the Guzerá
bulls. Additionally, we developed a preliminary association study, using this
INDEL polymorphism as a genetic marker. A significant association was detected
(P ≤ 0.05) between the INDEL (DGAT1 3'UTR INDEL) and the breeding values (BV)
for protein, fat, and milk yields over a 305-day lactation period. The DGAT1 3'
UTR INDEL genotype I/I (I, for insertion) was associated with lower BVs (-38.77
kg for milk, -1.86 kg for fat, and -1.48 kg for protein yields), when compared
to the genotype I/D (D, for deletion). I/D genotype was lower D/D genotype
(-34.98 kg milk, -1.73 kg fat, and -1.09 kg protein yields). This study reports
the first polymorphism of DGAT1 3'UTR in the Guzerá breed, as well as its
association with BV for milk protein, fat, and milk yields. |
Which histone mutation is associated with gliomas? | Pediatric central nervous system tumors are the most common solid tumor of childhood. Over 70% of diffuse intrinsic pediatric gliomas, an aggressive brainstem tumor, harbor heterozygous mutations that create a K27M amino acid substitution (methionine replaces lysine 27) in the tail of histone H3.3. | Sequencing of pediatric gliomas has identified missense mutations Lys27Met
(K27M) and Gly34Arg/Val (G34R/V) in genes encoding histone H3.3 (H3F3A) and H3.1
(HIST3H1B). We report that human diffuse intrinsic pontine gliomas (DIPGs)
containing the K27M mutation display significantly lower overall amounts of H3
with trimethylated lysine 27 (H3K27me3) and that histone H3K27M transgenes are
sufficient to reduce the amounts of H3K27me3 in vitro and in vivo. We find that
H3K27M inhibits the enzymatic activity of the Polycomb repressive complex 2
through interaction with the EZH2 subunit. In addition, transgenes containing
lysine-to-methionine substitutions at other known methylated lysines (H3K9 and
H3K36) are sufficient to cause specific reduction in methylation through
inhibition of SET-domain enzymes. We propose that K-to-M substitutions may
represent a mechanism to alter epigenetic states in a variety of pathologies. Recent studies have identified a Lys 27-to-methionine (K27M) mutation at one
allele of H3F3A, one of the two genes encoding histone H3 variant H3.3, in 60%
of high-grade pediatric glioma cases. The median survival of this group of
patients after diagnosis is ∼1 yr. Here we show that the levels of H3K27 di- and
trimethylation (H3K27me2 and H3K27me3) are reduced globally in H3.3K27M patient
samples due to the expression of the H3.3K27M mutant allele. Remarkably, we also
observed that H3K27me3 and Ezh2 (the catalytic subunit of H3K27
methyltransferase) at chromatin are dramatically increased locally at hundreds
of gene loci in H3.3K27M patient cells. Moreover, the gain of H3K27me3 and Ezh2
at gene promoters alters the expression of genes that are associated with
various cancer pathways. These results indicate that H3.3K27M mutation
reprograms epigenetic landscape and gene expression, which may drive
tumorigenesis. INTRODUCTION: Mutations in H3F3A, which encodes histone H3.3, commonly occur in
pediatric glioblastoma. Additionally, H3F3A K27M substitutions occur in gliomas
that arise at midline locations (eg, pons, thalamus, spine); moreover, this
substitution occurs mainly in tumors in children and adolescents. Here, we
sought to determine the association between H3F3A mutations and adult thalamic
glioma.
METHODS: Genomic H3F3A was sequenced from 20 separate thalamic gliomas.
Additionally, for 14 of the 20 gliomas, 639 genes--including cancer-related
genes and chromatin-modifier genes--were sequenced, and the Infinium
HumanMethylation450K BeadChip was used to examine DNA methylation across the
genome.
RESULTS: Of the 20 tumors, 18 were high-grade thalamic gliomas, and of these 18,
11 were from patients under 50 years of age (median age, 38 y; range, 17-46),
and 7 were from patients over 50 years of age. The H3F3A K27M mutation was
present in 10 of the 11 (91%) younger patients and absent from all 7 older
patients. Additionally, H3F3A K27M was not detected in the 2 diffuse
astrocytomas. Further sequencing revealed recurrent mutations in TP53, ATRX,
NF1, and EGFR. Gliomas with H3F3A K27M from pediatric or young adult patients
had similar, characteristic DNA methylation profiles. In contrast, thalamic
gliomas with wild-type H3F3A had DNA methylation profiles similar to those of
hemispheric glioblastomas.
CONCLUSION: We found that high-grade thalamic gliomas from young adults, like
those from children and adolescents, frequently had H3F3A K27M. Histone H3 lysine(27)-to-methionine (H3K27M) gain-of-function mutations occur in
highly aggressive pediatric gliomas. We established a Drosophila animal model
for the pathogenic histone H3K27M mutation and show that its overexpression
resembles polycomb repressive complex 2 (PRC2) loss-of-function phenotypes,
causing derepression of PRC2 target genes and developmental perturbations.
Similarly, an H3K9M mutant depletes H3K9 methylation levels and suppresses
position-effect variegation in various Drosophila tissues. The histone H3K9
demethylase KDM3B/JHDM2 associates with H3K9M-containing nucleosomes, and its
misregulation in Drosophila results in changes of H3K9 methylation levels and
heterochromatic silencing defects. We have established histone
lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation
pathways that also function as biochemical reagents for capturing site-specific
histone-modifying enzymes, thus providing molecular insight into chromatin
signaling pathways. Brain tumors are the most common solid tumors in children. Pediatric high-grade
glioma (HGG) accounts for ∼8-12 % of these brain tumors and is a devastating
disease as 70-90 % of patients die within 2 years of diagnosis. The failure to
advance therapy for these children over the last 30 years is largely due to
limited knowledge of the molecular basis for these tumors and a lack of disease
models. Recently, sequencing of tumor cells revealed that histone H3 is
frequently mutated in pediatric HGG, with up to 78 % of diffuse intrinsic
pontine gliomas (DIPGs) carrying K27M and 36 % of non-brainstem gliomas carrying
either K27M or G34R/V mutations. Although mutations in many chromatin modifiers
have been identified in cancer, this was the first demonstration that histone
mutations may be drivers of disease. Subsequent studies have identified
high-frequency mutation of histone H3 to K36M in chondroblastomas and to G34W/L
in giant cell tumors of bone, which are diseases of adolescents and young
adults. Interestingly, the G34 mutations, the K36M mutations, and the majority
of K27M mutations occur in genes encoding the replacement histone H3.3. Here, we
review the peculiar characteristics of histone H3.3 and use this information as
a backdrop to highlight current thinking about how the identified mutations may
contribute to disease development. Brainstem and thalamic gliomas are rare, and they are poorly understood in
adults. Genetic aberrations that occur in these tumors are still unknown. In
this study, we investigated whether thalamic gliomas have different genetic
aberrations and clinical outcomes compared with brainstem gliomas in adults.
Forty-three glioma samples were selected, including 28 brainstem and 15 thalamic
gliomas. The frequency of the K27M mutation in adult midline gliomas was 58.1%.
High-grade gliomas in the thalamus were statistically significantly more
numerous than brainstem gliomas. Patients with K27M mutant brainstem gliomas had
a significantly shorter overall survival than patients with wild-type tumors (P
= .020) by Cox regression after adjustment for other independent risk factors.
However, there was no statistical tendency toward a poorer overall survival in
thalamic gliomas containing the K27M mutation compared with wild-type tumors.
The presence of the K27M mutation significantly corresponded with mutations in
TP53 in thalamic gliomas. Interestingly, the K27M mutation was mutually
exclusive with mutations in IDH1, which was detected only in brainstem gliomas.
The microarray data identified 86 differentially expressed genes between
brainstem and thalamic gliomas with the K27M mutation. The cyclin-dependent
kinase 6 (CDK6) gene, which plays an important role in cancer pathways, was
found to be differentially expressed between brainstem and thalamic gliomas with
K27M mutations. Although the K27M mutation was frequently observed in adult
brainstem and thalamic gliomas, this mutation tended to be associated with a
poorer prognosis in brainstem gliomas but not in thalamic gliomas. Brainstem
gliomas may present different genetic aberrations from thalamic gliomas. These
differences may provide guidance for therapeutic decisions for the treatment of
adult brainstem and thalamic gliomas, which may have different molecular
targets. Somatic mutations of the H3F3A and HIST1H3B genes encoding the histone H3
variants, H3.3 and H3.1, were recently identified in high-grade gliomas arising
in the thalamus, pons and spinal cord of children and young adults. However, the
complete range of patients and locations in which these tumors arise, as well as
the morphologic spectrum and associated genetic alterations remain undefined.
Here, we describe a series of 47 diffuse midline gliomas with histone H3-K27M
mutation. The 25 male and 22 female patients ranged in age from 2 to 65 years
(median = 14). Tumors were centered not only in the pons, thalamus, and spinal
cord, but also in the third ventricle, hypothalamus, pineal region and
cerebellum. Patients with pontine tumors were younger (median = 7 years) than
those with thalamic (median = 24 years) or spinal (median = 25 years) tumors. A
wide morphologic spectrum was encountered including gliomas with giant cells,
epithelioid and rhabdoid cells, primitive neuroectodermal tumor (PNET)-like
foci, neuropil-like islands, pilomyxoid features, ependymal-like areas,
sarcomatous transformation, ganglionic differentiation and pleomorphic
xanthoastrocytoma (PXA)-like areas. In this series, histone H3-K27M mutation was
mutually exclusive with IDH1 mutation and EGFR amplification, rarely co-occurred
with BRAF-V600E mutation, and was commonly associated with p53 overexpression,
ATRX loss (except in pontine gliomas), and monosomy 10. It has become increasingly evident that morphologically similar gliomas may have
distinct clinical phenotypes arising from diverse genetic signatures. To date,
glial tumours from the Tunisian population have not been investigated. To
address this, we correlated the clinico-pathology with molecular data of 110
gliomas by a combination of HM450K array, MLPA and TMA-IHC. PTEN loss and EGFR
amplification were distributed in different glioma histological groups. However,
1p19q co-deletion and KIAA1549:BRAF fusion were, respectively, restricted to
Oligodendroglioma and Pilocytic Astrocytoma. CDKN2A loss and EGFR overexpression
were more common within high-grade gliomas. Furthermore, survival statistical
correlations led us to identify Glioblastoma (GB) prognosis subtypes. In fact,
significant lower overall survival (OS) was detected within GB that
overexpressed EGFR and Cox2. In addition, IDH1R132H mutation seemed to provide a
markedly survival advantage. Interestingly, the association of IDHR132H mutation
and EGFR normal status, as well as the association of differentiation markers,
defined GB subtypes with good prognosis. By contrast, poor survival GB subtypes
were defined by the combination of PTEN loss with PDGFRa expression and/or EGFR
amplification. Additionally, GB presenting p53-negative staining associated with
CDKN2A loss or p21 positivity represented a subtype with short survival. Thus,
distinct molecular subtypes with individualised prognosis were identified.
Interestingly, we found a unique histone mutation in a poor survival young adult
GB case. This tumour exceptionally associated the H3F3A G34R mutation and MYCN
amplification as well as 1p36 loss and 10q loss. Furthermore, by exhibiting a
remarkable methylation profile, it emphasised the oncogenic power of G34R
mutation connecting gliomagenesis and chromatin regulation. Pediatric central nervous system tumors are the most common solid tumor of
childhood. Of these, approximately one-third are gliomas that exhibit diverse
biological behaviors in the unique context of the developing nervous system.
Although low-grade gliomas predominate and have favorable outcomes, up to 20% of
pediatric gliomas are high-grade. These tumors are a major contributor to
cancer-related morbidity and mortality in infants, children, and adolescents,
with long-term survival rates of only 10 to 15%. The recent discovery of somatic
oncogenic mutations affecting chromatin regulation in pediatric high-grade
glioma has markedly improved our understanding of disease pathogenesis, and
these findings have stimulated the development of novel therapeutic approaches
targeting epigenetic regulators for disease treatment. We review the current
perspective on pediatric high-grade glioma genetics and epigenetics, and discuss
the emerging and experimental therapeutics targeting the unique molecular
abnormalities present in these deadly childhood brain tumors. Advances in our understanding of the biology of paediatric central nervous
system (CNS) tumours have encouraged pathologists to use molecular markers
alongside histopathological analysis for disease classification or
prognostication and treatment stratification. In this article, we review
molecular genetic alterations in paediatric CNS tumours, including those in
low-grade and high-grade gliomas, ependymomas, and embryonal tumours. Some of
these molecular changes with clinicopathological utility have been used for the
first time in the most recent edition of the World Health Organization (WHO)
classification of CNS tumours to define entities like ependymoma, RELA
fusion-positive or diffuse midline glioma, H3 K27M-mutant. The classification of
paediatric CNS tumours is entering a new era when histopathologists must work
with molecular genetic data and their molecular pathology colleagues to provide
an optimal diagnostic evaluation for their patients and clinical colleagues.
Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by
John Wiley & Sons, Ltd. |
Are there canonical marks of active chromatin in developmentally regulated genes? | No. The transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association also observed in mammals. Consequently, chromatin marking is associated with the stable production of RNA, whereas unmarked chromatin would permit rapid gene activation and deactivation during development. In the latter case, regulation by transcription factors would have a comparatively more important regulatory role than chromatin marks. | The interplay of active and repressive histone modifications is assumed to have
a key role in the regulation of gene expression. In contrast to this generally
accepted view, we show that the transcription of genes temporally regulated
during fly and worm development occurs in the absence of canonically active
histone modifications. Conversely, strong chromatin marking is related to
transcriptional and post-transcriptional stability, an association that we also
observe in mammals. Our results support a model in which chromatin marking is
associated with the stable production of RNA, whereas unmarked chromatin would
permit rapid gene activation and deactivation during development. In the latter
case, regulation by transcription factors would have a comparatively more
important regulatory role than chromatin marks. |
What is the purpose of HaploReg v4? | HaploReg v4 enables the systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and disease. | More than 90% of common variants associated with complex traits do not affect
proteins directly, but instead the circuits that control gene expression. This
has increased the urgency of understanding the regulatory genome as a key
component for translating genetic results into mechanistic insights and
ultimately therapeutics. To address this challenge, we developed HaploReg
(http://compbio.mit.edu/HaploReg) to aid the functional dissection of
genome-wide association study (GWAS) results, the prediction of putative causal
variants in haplotype blocks, the prediction of likely cell types of action, and
the prediction of candidate target genes by systematic mining of comparative,
epigenomic and regulatory annotations. Since first launching the website in
2011, we have greatly expanded HaploReg, increasing the number of chromatin
state maps to 127 reference epigenomes from ENCODE 2012 and Roadmap Epigenomics,
incorporating regulator binding data, expanding regulatory motif disruption
annotations, and integrating expression quantitative trait locus (eQTL) variants
and their tissue-specific target genes from GTEx, Geuvadis, and other recent
studies. We present these updates as HaploReg v4, and illustrate a use case of
HaploReg for attention deficit hyperactivity disorder (ADHD)-associated SNPs
with putative brain regulatory mechanisms. OBJECTIVE: To explore the association between DNA damage-related genetic
variants and lung cancer susceptibility in a Han Chinese population.
METHODS: This case-control study enrolled patients from the Cancer Hospital of
Jiangsu Province and Jiangsu Province Hospital from 2003 to 2009. Controls were
randomly selected from individuals who visited the same hospital or a
community-based health examination program during the same time period. A 5 ml
venous blood sample was obtained from each participant and epidemiological
information was collected on a standard questionnaire. Illumina Infinium(®)
BeadChip was used for genotyping of 35 DNA damage-related single nucleotide
variations (SNVs), which were identified in our previous study. Multivariate and
binary logistic regressions were used to calculate the OR and 95%CI for lung
cancer risk. HaploReg V4.1 and Regulome DB were used to understand functional
annotation on important SNV.
RESULTS: The distributions of age (61.06±10.15) vs. (61.32±11.07) years;
t=-0.72, P=0.473) and sex (χ(2)=1.81, P=0.179) were similar between cases and
controls. However, the case group had a higher frequency of smokers (61.08% vs.
48.54%; χ(2)=50.04, P<0.001) and heavy smokers (42.28% vs. 24.07%; χ(2)=122.32,
P<0.001). Among the 34 SNVs that passed quality control, two SNVs were
significantly associated with lung cancer risk after adjustments for age, sex
and cumulative smoking dose: rs9267576 C>A (CA genotype/CC genotype, OR=1.56,
95% CI: 1.01-2.40) and rs3130683 A>G (AG genotype/AA genotype, OR=1.87, 95%CI:
1.13-3.09). After step-wise logistic regression analysis, only the rs3130683 SNV
was retained in the model, indicating that the association between rs9267576 and
lung cancer may be due to the effect of rs3130683. Functional annotation
indicated that rs3130683 was located in the promoter and enhancer regions, and
was an expression quantitative trait loci of HLA. The Cancer Genome Atlas
indicated that expression of HLA-C, DQB1, DRB1 and DRB5 in lung cancer tissue
was significantly lower than in paired normal tumor-adjacent tissue, with
down-regulation of the four respective genes in 81.3%, 88.8%, 90.7% and 90.7% of
lung cancer tissues (P-values were 6.68×10(-15), 2.21×10(-13), 2.20×10(-16),
2.58×10(-13), respectively).
CONCLUSIONS: The SNV rs3130683 (A>G) was associated with the risk of lung cancer
in a Han Chinese population. This SNV may affect the risk of lung cancer by
regulating HLA expression. |
Which are the typical symptoms of Ménière's disease? | The typical symptoms of Ménière's disease are:
1) sensorineural hearing loss,
2) vertigo and
3) tinnitus. | Among 93 patients presenting the typical symptoms of a Ménière's disease
associating an unilateral fluctuating hearing loss of sensorineural type,
tinnitus and vertiginous attacks lasting minutes to hours, 40 patients (43%)
presented in their personal history a particular otologic insult in the ear
which later on developed into the full Ménière's symptomatology, or a particular
systemic disease with otologic manifestations. The Ménière's triad appeared in
these patients six months to twenty nine years after the initial otologic or
systemic lesion. Among these initial lesions were 16 cases of sudden partial or
complete deafness related to viral or bacterial infection, 3 cases of sudden
cochleo-vestibular deficit and 1 case of vestibular neuritis, 5 cases of
temporal bone fractures and 4 cases of significant acoustic trauma, 2 cases of
otosclerosis, 1 case of chronicotitis media and 1 case of severe hearing loss
after otologic surgery, 5 cases of meningo-encephalitis and 2 cases of acquired
syphilis. These particular lesion could be, in our opinion, the releasing factor
of the inner ear dysfonction leading eventually to a secondary Ménière's
syndrome. Vertigo is the most important symptom of Ménière's disease both from the
standpoint of follow-up and indication for surgery. But although vertigo is an
alerting symptom for both the patient and the physician, we believe that the
hearing level is the most reliable and even the single sign in determining the
recent status of the disease. Between 1983-1989, 42 patients with various types
of Ménière's disease (MD) (34 typical MD, 3 cochlear MD and 3 vestibular MD)
underwent endolymphatic sac surgery at ENT department of Gazi University School
of Medicine. In the typical MD group, patients with a duration of symptoms of
less than one year prior to surgery revealed better postoperative results; 91%
fell into class A and B, whereas this rate was found to be lower (40%) in
patients with symptomatology lasting for more than one year. In conclusion,
especially in bilateral cases, given the importance of the hearing, early sac
surgery is thoroughly recommended for the conservation of hearing. 140 patients with various forms of sensorineural hearing loss were examined via
the glycerol test (Klockhoff). In 66% of the cases with Menière's disease, a
typical temporary threshold shift could be seen. 17 patients with fluctuating
hearing loss of unknown etiology without vestibular symptoms showed a similar
effect; they were classified as belonging to the group of the cochlear type of
Menière's disease. In 34% of cases with clinically assured diagnosis of
Ménière's disease, glycerol did not induce improvement in hearing. Repeated
glycerol tests revealed varying results; shortly after a Menière attack, the
glycerol test was usually negative, whereas shortly before the next attack the
glycerol test changed to positive. These data support the specificity of the
glycerol test: The results depend on the presence of endolymphatic hydrops at
the time of examination. Positive results of the glycerol test were also seen in
the cases of reduced sensorineural hearing associated with syphilis, as well as
in cases of cerebrospinal pressure changing synchronously with fluctuating
hearing loss (Lindsay and Zajtschuk). In all other cases of sensorineural
hearing losses, no positive effect of the glycerol test has been found. BACKGROUND: Auditory and vestibular symptoms and signs are common in patients
with migraine, yet little is known about the pathogenesis of these symptoms and
signs.
OBJECTIVE: To perform clinicopathological correlation in a patient with
migraine, sudden deafness, and delayed endolymphatic hydrops.
METHODS: A patient with long-standing migraine with aura developed sudden
hearing loss in the left ear at the age of 50 years and Ménière disease on the
right side at age 73. At age 76, he had a flurry of sudden drop attacks typical
of otolithic crisis. He died of unrelated causes at age 81. The brain and
temporal bones were removed approximately 24 hours after death. The cochlea and
vestibular end organs were dissected after the surrounding bone was carefully
removed.
RESULTS: The brain and cerebrovasculature were normal. The left cochlea showed
prominent fibrosis consistent with an old infarction. The right inner ear showed
hydrops, with relatively good preservation of the hair cells in the cochlea,
saccular macule, and cristae of the semicircular canals. However, the utricular
macule was denuded of hair cells.
CONCLUSIONS: The sudden left-sided deafness likely resulted from ischemia,
possibly due to migraine-associated vasospasm. Presumably, the right ear
suffered only minimal damage when the patient was 50 years old, but this damage
later led to the development of delayed endolymphatic hydrops on the right.
Otolithic crises are thought to result from pressure changes across the
utricular macule. We speculate that loss of hair cells in the utricular macule
resulted from a collapse of the utricular membrane onto the macule. The effects of middle ear pressure pulses were studied in 37 patients with a
diagnosis of definite Meniere's disease and active vestibular symptoms, 31 of
whom had failed to respond to medical treatment including diuretics prior to
pressure treatment. The number of vertigo spells during the 6 months prior to
treatment and for 6, 18 and 24 months after the start of treatment were
evaluated. The functionality level scores were three or worse according to the
American Academy of Otolaryngology--Head and Neck Surgery (AAO-HNS) criteria.
The results of the study were as follows: 19 patients experienced freedom from
vertigo spells; 15 patients reported a significant decrease in the frequency of
vertigo spells; and 3 patients did not respond to pressure treatment and were
subjected to gentamicin injections, I of whom consequently became deaf in the
affected ear. No patient became worse in connection with pressure treatment.
With the exception of the three patients who did not respond to pressure
treatment, all patients reported an improvement in functionality of at least two
levels according to the AAO-HNS functionality scale. No side effects or adverse
events were observed during the 2 years of pressure treatment. Overall, the
results indicate that pressure treatment provides efficient control of symptoms
in patients with intractable forms of Ménière's disease without producing any
adverse effects on the inner ear. From an otological standpoint, Lyme disease can manifest itself as Ménière's
disease both clinically and electrophysiologically. The aim of this study was to
describe the findings of routine clinical, auditory and vestibular tests in
patients with Ménière's and Lyme disease and to use electrocochleography (ECoG)
to assess the presence of endolymphatic hydrops (EH) in both diseases.
Transtympanic ECoG was performed in 91 patients with Ménière's disease and in 11
patients with confirmed Lyme disease. In both diseases the majority of patients
had more than one complaint. There was one case with isolated hearing loss in
the Lyme disease group. Typical clinical manifestations of Ménière's disease
(vertigo, sensorineural hearing loss and tinnitus) were found in 6/11 patients
(54.5%) in the Lyme disease group. The ECoG results indicated that there were
65/91 patients (71.4%) with Ménière's disease and 5 patients (45.5%) with Lyme
disease who presented with EH. No statistically significant difference was found
between the incidence of different symptoms of Ménière's and Lyme disease. On
the basis of these results, patients with Lyme disease should undergo careful
examination and investigation, especially in endemic regions. The presence of EH
does not exclude the presence of infection with borreliosis as a cause of
Ménière's disease-like symptoms. OBJECTIVE: To evaluate the association between migraine, episodic vertigo, and
Ménière's disease in families.
STUDY DESIGN: Clinical report.
SETTING: University Neurotology Clinic.
PATIENTS: Index patients identified with Ménière's disease and migraine and
their family members.
INTERVENTION: Structured interview to assess a diagnosis of migraine, episodic
vertigo, and Ménière's disease in 6 families. Genotyping was performed on 3 sets
of twins to analyze monozygosity or dizygosity.
MAIN OUTCOME MEASURES: Clinical history of migraine, episodic vertigo, and
Ménière's disease.
RESULTS: Six index patients and 57 family members were interviewed either by a
senior neurologist in person or over the phone by a trained study coordinator.
An additional 6 family members completed questionnaires by mail. All 6 index
patients had Ménière's disease and migraine. Twenty-six (41%) of the 63
relatives met International Classification of Headache Disorders II criteria for
migraine headaches. Thirteen (50%) of these 26 experienced migraine with aura.
Three others experienced typical aura without headache. Seventeen (27%) of 63
family members experienced recurrent spells of spontaneous episodic vertigo.
There was one twin pair in each of 3 families; 2 pairs were monozygotic and one
was dizygotic. In each twin pair, one twin had migraine and Ménière's disease,
whereas the other experienced migraine and episodic vertigo without auditory
symptoms.
CONCLUSION: The frequent association of episodic vertigo, migraine, and
Ménière's disease in closely related individuals, including identical twins
supports the heritability of a migraine-Ménière's syndrome, with variable
expression of the individual features of hearing loss, episodic vertigo, and
migraine headaches. Ménière's diseasenot only includes the symptom complex consisting of attacks of
vertigo, low-frequency hearing loss, and tinnitus but comprises symptoms related
to the eustachian tube, the upper cervical spine, the temporomandibular joints,
and the autonomic nervous system. Quantifiable experience shows that the
insertion of a middle-ear ventilation tube can alleviate Ménière's disease
symptoms, suggesting that eustachian tube dysfunction is a contributing feature.
Clinical practice also shows that treating disorders of the upper cervical spine
and temporomandibular joints can lessen Ménière's disease symptoms, suggesting a
relationship. Similarly, stellate ganglion blocks can be beneficial in
controlling Ménière's disease symptoms, highlighting the influence of the
autonomic nervous system. Thus, contrasting symptoms associated with the
eustachian tube, the upper cervical spine, the temporomandibular joints, and the
autonomic nervous system relate to Ménière's disease, but the possible reflex
pathway by which a link is established is unclear. We made an attempt in this
study to describe a hypothetical reflex pathway that links joint injury and the
autonomic nervous system, where eustachian tube function is under their
influence and is the critical link. In this hypothetical reflex pathway,
irritation of facet joints can first lead to an activated anterior cervical
sympathetic system via an independent pathway in the mediolateral cell column;
it can simultaneously lead to an axon reflex involving nociceptive neurons,
resulting in neurogenic inflammation and the prospect of a eustachian tube
dysfunction. The eustachian tube dysfunction is responsible for a disturbed
middle ear-inner ear pressure relationship, circumstances that have the
potential to develop into secondary Ménière's disease. This reflex pathway is
supported by recent animal experiments. HYPOTHESIS: The goal of this study was to assess the impact of dizziness
handicap, illness intrusiveness (in relation to vertigo, tinnitus, and hearing
problems), and illness uncertainty on depression in people with the symptoms of
Ménière's disease.
BACKGROUND: Ménière's disease is a progressive disease of the inner ear, the
symptoms of which are vertigo, tinnitus, hearing loss, and aural fullness.
Although pharmacologic treatments may reduce acute vertigo spells and dizziness,
they rarely disappear entirely. Previous research shows that Ménière's disease
is unpredictable and has a negative impact on patients' quality of life.
METHODS: Questionnaires measuring Dizziness Handicap, Illness Intrusiveness,
Illness Uncertainty, and Depression were completed by 74 people with
self-reported symptoms of Ménière's disease. Bivariate correlations,
repeated-measures analysis of variance, and multiple regression analyses were
used to assess the contribution of dizziness handicap, illness intrusiveness,
and illness uncertainty to depression.
CONCLUSION: Vertigo was more intrusive than tinnitus, hearing problems, and most
other comparator illnesses. The intrusiveness of the symptoms of Ménière's
disease accounted for 32% of the variance in depression scores, which were high;
illness uncertainty did not account for additional variance. Dizziness handicap
accounted for 31% of the variation in depression. Although the symptoms of
Ménière's disease may not be alleviated by psychological methods, programs that
target cognitions in relation to the embarrassment in front of others, and the
feeling of being handicapped, may lessen the psychosocial impact of the symptoms
of Ménière's disease, which may reduce some of the depression felt in this
group. OBJECTIVES: To evaluate the onset of vertigo, hearing loss and tinnitus in
Ménière's disease and the associated endolymphatic hydrops (EH) of the inner
ear.
DESIGN: Multicentre evaluation of three patient groups.
SETTINGS: Disease-specific symptoms were reviewed among referred patients in a
tertiary referral hospital in Finland and in members of a Finnish Ménière
Association in Finland. The MRI of a separate group of patients was undertaken
in a tertiary referral centre in Japan.
PARTICIPANTS: 340 patients were reviewed in the referral hospital along with 740
members of the Ménière Association. MRI was undertaken in 224 patients in Japan.
PRIMARY AND SECONDARY OUTCOME MEASURES: Latency and symptom development in
Ménière's disease, and the appearance of EH of the inner ear in monosymptomatic
patients and in Ménière's disease.
RESULTS: The mean age of the first symptom was 43.8 years, with 10% of the
patients being older than 65 years. The time delay between hearing loss and
vertigo was more than 5 years in 20% of the members and of the patients.
Gadolinium-contrasted MRI demonstrated EH in 90% of the patients with Ménière's
disease, in which 75% was bilateral among patients with unilateral symptoms. In
monosymptomatic patients with vertigo, tinnitus or hearing loss; EH was
demonstrated in 55-90% of the patients either in the cochlea and/or the
vestibulum of the symptomatic ear.
CONCLUSIONS: Ménière's disease often shows bilateral EH and comprises a
continuum from a monosymptomatic disease to the typical symptom complex of the
disease. We suggest that a 3T MRI measurement should be carried out in patients
with sensory-neural hearing loss, vertigo and tinnitus, 4 h after the
intravenous injection of a gadolinium-contrast agent to verify the inner ear
pathology. This may lead to a better management of the condition. |
What tissue is most affected in Ehlers-Danlos syndromes? | the ehlers-danlos syndromes (eds) are a group of connective tissue disorders characterized by triad of joint hypermobility, skin extensibility, and tissue fragility. | The Ehlers-Danlos syndromes (EDS) are a group of heritable connective tissue
disorders that share the common features of skin hyperextensibility, articular
hypermobility, and tissue fragility. Considerable clinical and genetic
heterogeneity exists, and more than nine separate forms have been recognized.
Recent advances in the molecular analysis of EDS have identify defects
responsible for EDS VI (homozygous and compound heterozygous mutations in the
lysyl-hydroxylase gene), EDS VIIA and EDS VIIB (mutations in the type I
collagene genes), EDS VIIC (deficiency of procollagen N-proteinase), EDS IX
(mutations in the MNK gene), and EDS IV (mutations in the type III collagen
gene). Of the various types of Ehlers-Danlos syndrome the most severe is type IV
(EDS IV). Early studies showed that fibroblasts from EDS IV patients secreted
lower than normal amounts of type III procollagen (Pope et al., 1975). Later,
the disease was linked to COL3A1, the gene encoding this protein. More recently,
with the publication of full length cDNA and partial characterisation of the
gene structure, detailed analysis of mutations in EDS IV patients has become
possible. Nineteen different mutations in the type III procollagen gene have
been reported in different families with EDS IV. Recent results support the
hypothesis that in EDS IV, domit inheritance should be assumed, in sporadic
cases also, unless proven otherwise. Very little is known about the genetics or
biochemicals defects responsible for the others EDS subtypes, but with the
applications of the tools of molecular biology, analysis of these defects if now
within reach. Ehlers-Danlos syndrome denotes a group of inherited connective tissue diseases
comprising nine types. Type IV Ehlers-Danlos syndrome is the most
life-threatening form. It is characterized by a type III collagen deficiency
resulting in arterial fragility and death from vascular rupture or bowel
perforation. This disease involves a col 3A1 gene mutation. We report the case
of a 44 year-old woman with type IV Ehlers-Danlos syndrome. The medical history
of our patient included bowel necrosis and two vascular ruptures. We indicate
data required to establish Ehlers-Danlos syndrome diagnosis and guidelines for
patient management. BACKGROUND: The Ehlers-Danlos syndromes (EDS) comprise a heterogenous group of
heritable disorders of connective tissue, characterized by joint hypermobility,
skin hyperextensibility and tissue fragility. Most EDS types are caused by
mutations in genes encoding different types of collagen or enzymes, essential
for normal processing of collagen.
METHODS: Oral health was assessed in 31 subjects with EDS (16 with hypermobility
EDS, nine with classical EDS and six with vascular EDS), including signs and
symptoms of temporomandibular disorders (TMD), alterations of dental hard
tissues, oral mucosa and periodontium, and was compared with matched controls.
RESULTS: All EDS subjects were symptomatic for TMD and reported recurrent
temporomandibular joint (TMJ) dislocations. Abnormal pulp shape (13%) and pulp
calcification (78%) were observed in subjects affected with classical EDS.
Caries experience was higher in EDS compared with controls and was related to
poor oral hygiene, influenced by increased mucosal fragility and restraint of
(wrist) joint mobility. The overall periodontal status in EDS was poor, with 62%
of EDS subjects presenting high periodontal treatment needs (community
periodontal index for treatment need, CPITN = II).
CONCLUSION: Oral health may be severely compromised in EDS as a result of
specific alterations of collagen in orofacial structures. When considering
dental treatment in EDS, a number of tissue responses (mucosa, periodontium,
pulp) and precautions (TMJ dislocation) should be anticipated. BACKGROUND: The Ehlers-Danlos syndrome (EDS) comprises a group of hereditary
connective tissue disorders. Periventricular nodular heterotopia (PNH) is a
human neuronal migration disorder characterised by seizures and conglomerates of
neural cells around the lateral ventricles of the brain, caused by FLNA
mutations. FLNA encodes filamin A, an actin binding protein involved in
cytoskeletal organisation. The amino-terminal actin binding domain (ABD) of
filamins contains two tandem calponin homology domains, CHD1 and CHD2.
OBJECTIVE: To report clinical and genetic analyses in a Spanish family affected
by a connective tissue disorder suggestive of EDS type III and PNH.
METHODS: A clinical and molecular study was undertaken in the three affected
women. Clinical histories, physical and neurological examinations, brain
magnetic resoce imaging studies, and skin biopsies were done. Genetic
analysis of the FLNA gene was undertaken by direct sequencing and restriction
fragment length polymorphism analysis.
RESULTS: Mutation analysis of the FLNA gene resulted in the identification of a
novel mutation in exon 3 (c.383C-->T) segregating with the combination of both
syndromes. This mutation results in a substitution of an alanine residue (A128V)
in CHD1.
CONCLUSIONS: The findings suggest that the Ala128Val mutation causes the dual
EDS-PNH phenotype. This association constitutes a new variant within the EDS
spectrum. This is the first description of a familial EDS-PNH association with a
mutation in FLNA. Ehlers-Danlos syndrome (EDS), a heterogeneous group of inheritable connective
tissue disorders, is attributed to mutations in connective tissue genes. These
mutations cause defects in collagen. Collagen, a connective tissue protein that
acts like glue, gives strength to the body and provides support and elasticity
for movement. Thus, the altered gene affects the mechanical properties of skin,
joints, ligaments, and blood vessels. Ehlers-Danlos syndrome is transmitted
through autosomal domit, autosomal recessive, or x-linked patterns of
inheritance. The life expectancy of an affected infant varies with the type of
EDS. This article provides an overview of the 6 major classifications of EDS,
their unique clinical presentations, a focused physical assessment guide,
considerations for nursing care, and resources for parents. Ehlers-Danlos
syndrome can be a potentially debilitating syndrome. It requires preventative
and protective measures starting at birth to preserve joint function to improve
infant outcomes. Caring for patients with EDS requires an understanding of the
potential associated complications to help minimize the physical and emotional
impact of the syndrome and improve the quality of life for affected individuals. Ehlers-Danlos syndrome is a complex hereditary connective tissue disorder that
is characterized by abnormalities of the skin and joints and visceral and
neurological manifestations. At present, at least 11 forms are recognized on the
basis of their clinical characteristics, methods of transmission, and
biochemical defect. The neurologic manifestations include cerebrovascular
disease, peripheral neuropathy, plexopathy, periventricular subependymal
heterotopias, and epilepsy. Previously, 2 females were reported to be affected
with subependimal periventricular heterotopias and Ehlers-Danlos syndrome type
1. The authors report a new case of a 12-year-old girl with similar clinical and
neuroradiological features. Classic Ehlers-Danlos syndrome is a heritable connective tissue disorder
characterized by skin hyperextensibility, fragile and soft skin, delayed wound
healing with formation of atrophic scars, easy bruising, and generalized joint
hypermobility. It comprises Ehlers-Danlos syndrome type I and Ehlers-Danlos
syndrome type II, but it is now apparent that these form a continuum of clinical
findings and differ only in phenotypic severity. It is currently estimated that
approximately 50% of patients with a clinical diagnosis of classic Ehlers-Danlos
syndrome harbor mutations in the COL5A1 and the COL5A2 gene, encoding the α1 and
the α2-chain of type V collagen, respectively. However, because no prospective
molecular studies of COL5A1 and COL5A2 have been performed in a clinically
well-defined patient group, this number may underestimate the real proportion of
patients with classic Ehlers-Danlos syndrome harboring a mutation in one of
these genes. In the majority of patients with molecularly characterized classic
Ehlers-Danlos syndrome, the disease is caused by a mutation leading to a
nonfunctional COL5A1 allele and resulting in haploinsufficiency of type V
collagen. A smaller proportion of patients harbor a structural mutation in
COL5A1 or COL5A2, causing the production of a functionally defective type V
collagen protein. Most mutations identified so far result in a reduced amount of
type V collagen in the connective tissues available for collagen
fibrillogenesis. Inter- and intrafamilial phenotypic variability is observed,
but no genotype-phenotype correlations have been observed. No treatment for the
underlying defect is presently available for Ehlers-Danlos syndrome. However, a
series of preventive guidelines are applicable. The Ehlers-Danlos syndromes comprise a clinically and genetically heterogeneous
group of heritable connective tissue disorders characterized by articular
hypermobility, skin extensibility, and tissue fragility. Surgical treatment of
scoliosis associated with Ehlers-Danlos syndrome poses a challenge to spine
surgeons because of the high risk of major complications. There is a paucity of
evidence in the literature on surgical treatment for scoliosis in the
Ehlers-Danlos syndrome patient.This article describes 3 adolescent patients
diagnosed with Ehlers-Danlos syndrome, kyphoscoliosis type, which was treated by
posterior spinal fusion only. After unsuccessful conservative treatment for at
least 1 year, the patients underwent posterior spinal surgery for the correction
of spinal deformity. A satisfactory correction in the spinal curve was achieved,
with no obvious loss of correction during follow-up. No intra- or postoperative
major complications were observed.Our experience supports that a satisfactory
correction of scoliosis can be achieved by posterior spinal fusion only in
patients with Ehlers-Danlos syndrome, kyphoscoliosis type. Ehlers-Danlos syndrome type 4, the vascular type, is a rare, life-threatening
inherited disorder of the connective tissue. Affected patients are at risk of
arterial, bowel and uterine rupture during pregcy. Generally, this syndrome
remains undiagnosed until a sudden, acute presentation with organ rupture, and
results in premature death, even if the patients survive the first and second
major complications. An early diagnosis with genetic assays can help to plan the
best treatment, which is often challenging due to the frailty of the arterial
tissue. We report on a 28-year-old lady who presented with spontaneous rupture
of a pseudoaneurysm of the posterior tibial artery. The Ehlers-Danlos Syndrome (EDS) is a rare connective tissue disorder
characterised by fragility of the soft connective tissues and widespread
manifestations in skin, ligaments, joints, blood vessels and internal organs. We
report a case of a 12-year-old boy, previously diagnosed with
kyphoscoliosis-type EDS (type VI), presenting with a left brachial artery
pseudo-aneursym with history of multiple spontaneous and post-traumatic arterial
ruptures. Surgical management of this patient was performed successfully by
primary repair of brachial artery lesion. The Ehlers-Danlos syndrome (EDS), inherited disorder of connective tissue,
frequently leads to impairment of various functional areas, including
employment. In 35 subjects with classic type EDS, 14 hypermobile, 3 vascular was
administered 7 visual analogical scales (pain, stiffness, activities of daily
living, instrumental activities of daily living, work, social relations). An
impairment of particular significance in total score and in individual areas
emerges is in the hypermobile group, followed by classic, less for the
vasculature. Overall there is a significant alteration of the quality of life
that deserves proper evaluation to facilitate the definition of fitness and the
improvement of job insertion in patients with EDS. BACKGROUND AND OBJECTIVES: Ehlers-Danlos syndromes (EDS) are a heterogeneous
group of heritable connective tissue disorders. Gastrointestinal manifestations
in EDS have been described but their frequency, nature and impact are poorly
known. We aimed to assess digestive features in a national cohort of EDS
patients.
METHODS: A questionnaire has been sent to 212 EDS patients through the French
patient support group, all of which had been formally diagnosed according to the
Villefranche criteria. The questionnaire included questions about digestive
functional symptoms, the GIQLI (Gastrointestinal Quality of Life Index), KESS
scoring system and the Rome III criteria.
RESULTS: Overall, 135 patients (64% response rate) completed the questionnaire
and 134 were analyzable (123 women; 91%). Mean age and Body Mass Index were
respectively 35±14.7 years and 24.3±6.1 kg/m(2). The most common EDS subtype was
hypermobility form (n=108; 80.6%). GIQLI and KESS median values were
respectively 63.5 (27-117) and 19 [13.5-22]. Eighty four percent of patients had
functional bowel disorders (FBD) according to the Rome III criteria. An
irritable bowel syndrome according to the same criteria was observed in 64
patients (48%) and 48 patients (36%) reported functional constipation. A
gastro-esophageal reflux disease (GERD) was reported in 90 patients (68.7%),
significantly associated with a poorer GIQLI (60.5±16.8 versus 75.9±20.3;
p<0.0001). GIQLI was also negatively impacted by the presence of an irritable
bowel syndrome or functional constipation (p=0.007). There was a significant
correlation between FBD and GERD.
CONCLUSIONS: Natural frequency of gastrointestinal manifestations in EDS seems
higher than previously assessed. FBD and GERD are very common in our study
population, the largest ever published until now. Their impact is herein shown
to be important. A systematic clinical assessment of digestive features should
be recommended in EDS. The classic form of Ehlers-Danlos syndrome (cEDS) is an inherited connective
tissue disorder, where mutations in type V collagen-encoding genes result in
abnormal collagen fibrils. Thus the cEDS patients have pathological connective
tissue morphology and low stiffness, but the rate of connective tissue protein
turnover is unknown. We investigated whether cEDS affected the protein synthesis
rate in skin and tendon, and whether this could be stimulated in tendon tissue
with insulin-like growth factor-I (IGF-I). Five patients with cEDS and 10
healthy, matched controls (CTRL) were included. One patellar tendon of each
participant was injected with 0.1 ml IGF-I (Increlex, Ipsen, 10 mg/ml) and the
contralateral tendon with 0.1 ml isotonic saline as control. The injections were
performed at both 24 and 6 h prior to tissue sampling. The fractional synthesis
rate (FSR) of proteins in skin and tendon was measured with the stable isotope
technique using a flood-primed continuous infusion over 6 h. After the infusion
one skin biopsy and two tendon biopsies (one from each patellar tendon) were
obtained. We found similar baseline FSR values in skin and tendon in the cEDS
patients and controls [skin: 0.005 ± 0.002 (cEDS) and 0.007 ± 0.002 (CTRL);
tendon: 0.008 ± 0.001 (cEDS) and 0.009 ± 0.002 (CTRL) %/h, mean ± SE]. IGF-I
injections significantly increased FSR values in cEDS patients but not in
controls (delta values: cEDS 0.007 ± 0.002, CTRL 0.001 ± 0.001%/h). In
conclusion, baseline protein synthesis rates in connective tissue appeared
normal in cEDS patients, and the patients responded with an increased tendon
protein synthesis rate to IGF-I injections. Vascular Ehlers-Danlos Syndrome (VEDS), previously called Ehlers-Danlos syndrome
type-IV, is a heterogeneous group of heritable connective tissue disorders
characterized by thin, translucent skin, easy bruising, arterial, intestinal,
and/or uterine fragility. There is large vessel involvement that leads to
arterial rupture often preceded by aneurysm, arteriovenous fistulae, or
dissection. Noninvasive imaging studies such as CT angiography and MR
angiography are preferred as diagnostic studies for this condition. We are
reporting a 4 years old girl who was presented with right sided unilateral
convulsions and hypertension. CT angiogram showed stenosis with post-stenotic
dilatation of coeliac and superior mesenteric arteries. There were extensive
calcified plaques with atherosclerotic changes in the segment of right common
iliac artery with aneurysmal dilatation of celiac, superior mesenteric and
common iliac artery. Radiological findings were consistent with vascular
Ehlers-Danlos syndrome. She was successfully managed with anti-hypertensive and
anticonvulsants. Ehlers-Danlos syndromes (EDS) are a heterogeneous group of hereditary connective
tissue disorders characterized by joint hypermobility, widespread
musculoskeletal pain and tissue fragility. Psychiatric disorders and
psychosocial impairment are common, yet poorly characterized, findings in EDS
patients. We investigated the frequency and types of psychiatric disorders and
their relationship to systemic manifestations in a cohort of 106 classic and
hypermobility type EDS patients. In this retrospective study, extensive medical
chart review was performed for patients referred at two genetics clinics who
were diagnosed with EDS. Statistical analysis was undertaken to determine the
frequency of psychiatric disorders and association with systemic findings.
Psychiatric disorders were found in 42.5% of the EDS cohort, with 22.7% of
patients affected with 2 or more psychiatric diagnoses. Anxiety and depression
were most commonly reported, with frequencies of 23.6 and 25.5%, respectively. A
variety of other psychiatric diagnoses were also identified. Abdominal pain
[odds ratio (OR) 7.38], neuropathic pain (OR 4.07), migraines (OR 5.21), joint
pain (OR 2.85) and fatigue (OR 5.55) were significantly associated with the
presence of a psychiatric disorder. The presence of any pain symptom was
significantly associated with having a psychiatric disorder (OR 9.68). Muscle
pain (OR 2.79), abdominal pain (OR 5.78), neuropathic pain (OR 3.91), migraines
(OR 2.63) and fatigue (OR 3.78) were significantly associated with having an
anxiety or mood disorder. Joint hypermobility and the classic dermatological
features of EDS showed no significant association with having a psychiatric
disorder. Our findings demonstrate a high frequency of psychiatric disorders and
an association with pain symptoms in EDS. The Ehlers-Danlos syndromes (EDS) are a group of connective tissue disorders
characterized by triad of joint hypermobility, skin extensibility, and tissue
fragility. Ehlers-Danlos syndrome type IV places patients at risk for
life-threatening, spontaneous, vascular or visceral rupture due to reduced or
abnormal secretion of type III collagen. We present an adolescent male who was
found to have a perisigmoid abscess with a fistula connecting to adjacent
sigmoid colon secondary to undiagnosed EDS type IV. Conservative management with
antibiotics and bowel rest was pursued to allow for elective resection for his
acute complicated diverticulitis at a safer time. The Ehlers-Danlos syndrome is characterized by abnormal connective tissue but
bone involvement is debated. We found a reduced BMD and bone quality and
increased prevalence of asymptomatic vertebral fractures in eugonadal patients
with Ehlers-Danlos syndrome. These findings suggest the need of a bone health
evaluation in these patients.
INTRODUCTION: The Ehlers-Danlos (EDS) syndrome is characterized by abnormalities
of the connective tissue leading to ligamentous laxity and skin and tissue
fragility. We evaluated the bone metabolism, bone mineral density (BMD) and bone
quality (measured by trabecular bone score, TBS), and the prevalence of
vertebral fractures (VFx) in a group of eugonadal adult EDS patients.
METHODS: Fifty consecutive Caucasian patients, aged 30-50 years (36 females, 14
males) with classical or hypermobility EDS and 50 age-, gender-, and body mass
index (BMI)-matched control subjects were enrolled. In all subjects'
calcium-phosphorous metabolism, bone turnover, BMD at the lumbar spine (LS) and
femur (femoral neck, FN and total femur, FT) and TBS by dual-energy X-ray
absorptiometry, and the VFx presence by spine radiograph were assessed.
RESULTS: Patients showed reduced BMD (Z-scores LS -0.45 ± 1.00, FN -0.56 ± 1.01,
FT -0.58 ± 0.92) and TBS (1.299 ± 0.111) and increased prevalence of
morphometric VFx (32 %) than controls (Z-scores LS 0.09 ± 1.22, FN 0.01 ± 0.97,
FT 0.08 ± 0.89; TBS 1.382 ± 0.176; VFx 8 %, p <0.05 for all comparisons), while
vitamin D levels, calcium-phosphorous metabolism, and bone turnover were
comparable. Fractured EDS patients showed lower TBS values than non-fractured
ones (1.245 ± 0.138 vs 1.325 ± 0.086, p < 0.05), despite comparable BMD. In EDS
patients, the VFx presence was significantly associated with TBS even after
adjusting for sex, age, BMD, EDS type, and falls frequency.
CONCLUSIONS: EDS patients have reduced BMD and bone quality (as measured by TBS)
and increased prevalence of VFx. BACKGROUND/AIMS: Ehlers-Danlos syndromes (EDSs) constitute a rare group of
inherited connective tissue diseases, characterized by multisystemic
manifestations and general tissue fragility. Most severe complications include
vascular and gastrointestinal (GI) emergencies requiring acute surgery. The
purpose of this systematic review was to assess the causes of GI-related surgery
and related mortality and morbidity in patients with EDSs.
METHODS: A systematic search was conducted in PubMed, Embase, and Scopus to
identify relevant studies. Preferred Reporting Items for Systematic Reviews and
Meta-Analysis guidelines for systematic reviews were followed. According to
eligibility criteria, data were extracted and systematically screened by 2
authors.
RESULTS: Screening process identified 11 studies with a total of 1,567 patients.
Findings indicated that patients with EDSs had a higher occurrence of surgery
demanding GI manifestations, including perforation, hemorrhage, rupture of
intra-abdominal organs, and rectal prolapse. Most affected was the vascular
subtype, of which up to 33% underwent GI surgery and suffered from a lowered
average life expectancy of 48 years (range 6-78). Secondary complications of
surgery were common in all patients with EDSs.
CONCLUSION: Studies suggested that patients with EDSs present an increased need
for GI surgery, but also an increased risk of surgery-related complications,
most predomitly seen in the vascular subtype. |
What is Mondor's disease? | Mondor's disease is a rare benign and self-limiting condition characterized by thrombophlebitis of the superficial veins of the anterolateral thoracoabdominal wall and genital area. | Mondor's disease is a superficial thrombophlebitis of the thoracic wall
frequently affecting the female breast. In most cases the etiology is unknown,
although operation, direct and indirect trauma, are known as causative factors.
This material comprises five women, all with Mondor's disease of the breast. One
patient did not return for follow-up, in one patient biopsy was performed after
2 weeks. In the remaining three patients the lesion had disappeared after 9 and
10 weeks. Mondor's disease has no relationship to cancer or systemic disease,
and no treatment apart from observation is required. Mondor disease, or superficial thrombophlebitis of the breast, is an uncommon
disorder that occurs only rarely in pregt women. One such case is presented
here followed by a review of the clinicopathologic features of the disease.
Accurate diagnosis of Mondor disease is based almost entirely on careful
physical examination of the breast, and no specific treatment is required. Its
major clinical significance lies in the need to distinguish it from maligcy
of the breast. Mondor's disease, superficial thrombophebitis of the breast, is an uncommon
self-limiting condition. Surgical procedures and trauma were the common known
causes. The objective of this study was to evaluate the incidence of Mondor's
disease in different breast operations in lower risk of breast cancer area over
a 6-year period and to identify its causes, clinical features, related surgical
factors and associated breast cancer. Eighty-four cases of Mondor's disease were
obtained from 9657 new patients in the breast clinic of Kaohsiung Medical
University Hospital between January 1991 and December 1996. The incidence per
year was close (0.84%-0.96%) although the number has been increasing each year.
In 23 cases, no definite cause was diagnosed, whereas in 61 cases, the disorder
was secondary because the pathogenesis could be discerned. The identified causes
included forty-three cases caused by breast surgery, two cases associated with
breast cancer and sixteen cases with other benign causes. Although the incidence
did not differ significantly between breast surgery (0.95%) and non-surgical
causes (0.79%), the highest incidence, 1.52%, occurred when excision through
circumareolar incision and tunnel procedure for cosmesis (25 cases in 1634
excisions) were used, and the lowest 0.69% when excisions through direct
incision (14 cases in 2004 excisions) were performed. (P < 0.05) The other
incidence rates were 1.56% in breast conserving surgery which is higher than
0.37% following mastectomy. The incidence of the disease was higher (4.28%) when
the distance of the breast lesion was more than 3 cm from the areolar edge,
compared to 1.20% for the 2 cm group and 0.32% for the 1 cm group (P < 0.05) in
tunnel procedures. The incidence of Mondor's disease during breast surgery was
not significantly different in different breast quardrants. Although Mondor's
disease is a benign, self-limiting condition, a high incidence developed in the
excision biopsy through circumareolar incision with tunnel procedure when the
distance from the breast lesion to the areolar edge was more than 3 cm. To
prevent this complication, the tunnel procedure in breast biopsy should be
avoided. The incidence of Mondor's disease associated with breast cancer was low
(2.4%) in the lower-incidence breast cancer area from this series, but awareness
of the condition is recommended. OBJECTIVE: Mondor reported the first superficial venous thrombosis on the chest
wall in 1939. This condition is usually a benign and self-limited process,
requiring only symptomatic treatment. Mondor's disease of the penis is an
uncommon condition, which usually involves the superficial dorsal veins, it was
first described by Braun-Falco in 1955. Isolated superficial dorsal
vein-thrombosis was reported in 1958 by Helm et al. Since then around fifty
cases have been reported. Patients experience a cord or string-like induration
along the penile superficial dorsal vein, which is often painful and accompanied
by localized inflammatory changes. This condition is benign and self-limited in
most patients with complete resolution after 6 to 8 weeks of conservative
management although sometimes surgery is indicated when it is associated with
chronic or severe local pain.
MATERIAL AND METHODS: We report on a 23-year sold man with Mondor's disease of
the penis following a normal sexual intercourse, who recently underwent
microsurgical left varicocelectomy.
RESULTS: Treatment consisted in NSAID Aulin (100 mg orally twice a day for 3
weeks) Ciproxin (500 mg orally twice a day for 10 days), Reparil 1 x 3 orally
for 25 days and Lansox 30 mg orally 1 per day for 21 days. The patient was
advised to abstain from sexual intercourse or masturbation until the thrombosis
had completely resolved. In the follow-up visit there was the complete
resolution of the disease with no evidence of superficial dorsal vein thrombosis
or palpable penile plaque 30 days later. The patient was also able to have
sexual intercourse without problems.
CONCLUSION: Although penile Mondors' disease is rare, proper clinical diagnosis
and consequent reassurance can help the patient to dissipate the anxiety and the
following erectile dysfunction. Ultrasound and Doppler Ultrasonography
examination was not useful for diagnosis but helped the clinician to show the
patient that the disease is a benign condition. This report describes the color and pulsed Doppler US findings of penile
Mondor's disease. The pulsed Doppler US findings of penile Mondor's disease have
not been previously published, so we report here for the first time on the
cavernosal arterial flow signal pattern of penile Mondor's disease. Penile
Mondor's disease is rare disease that's characterized by thrombosis in the
dorsal vein of the penis. The previous reports on penile Mondor's disease are
concerned with the color Doppler US finding without the flow signals in this
area, but these findings are insufficient to understand the hemodynamics in
penile Mondor's disease. We report for the first time on a cavernosal artery
flow signal pattern of low peak systolic velocity and high-resistance. Thrombophlebitis of the thoracoepigastric system of veins is a benign disease
and, despite its localized involvement and presentation, the condition is known
as Mondor disease (MD). A transverse incision made on the thoracoabdominal wall
divides the axially arranged superficial veins at a right angle and the presence
of unidirectional valves prevents retrograde blood flow, leading to stasis and
thrombus formation. The incidence of MD in oncologic breast cases and aesthetic
mammaplasties is reported to be 0.95% and 1.07%, respectively. Siliconeadenitis
of axillary nodes, on the other hand, is uncommon and has only been reported
occasionally. Extensive MD of the left axilla and inner arm is presented
following excision of axillary nodes secondary to siliconeadenitis after
cohesive gel silicone breast implant rupture. Mondor's disease is a rare cause of superficial thrombophlebitis, which is very
exceptionally observed in the penis. Usually a benign condition, careful
etiological search is needed to avoid missing exceptional causes. Mondor's
disease is generally treated with non-steroidal anti-inflammatory drugs or
low-molecular-weight heparin and resolves without sequelae. Mondor's disease and
superficial venous thrombosis of the penis may or may not be a unique clinical
entity. A favorable outcome with no precise etiology would favor penile Mondor's
disease. INTRODUCTION: Mondor's disease is a peculiar form of thrombophlebitis, involving
a superficial vein in the subcutaneous fat of the breast or anterior chest wall.
CASE PRESENTATION: The author presents a case of a 35-year-old male Japanese
patient with cord-like induration in the right lateral thoracic wall. This
lesion was diagnosed as traumatic funicular phlebitis, resembling Mondor's
disease.
CONCLUSION: Traumatic funicular phlebitis, resembling Mondor's disease, is a
clinical entity which may give suggestive insight to the etiology of Mondor's
disease itself. OBJECTIVE: To describe clinical features and ultrasound findings of three cases
of a little-known and relatively infrequent entity in daily clinical activity,
which is often unnoticed and under-reported: penile Mondor's disease or
superficial penile veins thrombophlebitis.
METHODS: We are reporting the cases of three patients aged 33, 25 and 39 years
who were referred to our department, the first case with suspicion of inguinal
hernia, the second one to rule out testicular pathology because of pubic and
perineal discomfort, and the third one for painful induration of the dorsal
region of the penis. The three patients underwent Doppler-ultrasound examination
(Toshiba®, using a 13-18MHz linear transducer) to establish definitive
diagnosis, and had a favorable evolution with conservative management.
RESULTS: Ultrasound examination revealed: Case 1. Penile superficial dorsal vein
and lateral superficial veins thrombosis. Case 2. Thrombosis of the right branch
of the superficial dorsal vein and its perineal distal connections. Case 3.
Penile superficial dorsal vein thrombosis. Definitive diagnosis of the three
cases was Mondor's disease.
CONCLUSIONS: Mondor's disease is an often under-reported entity in daily
clinical activity. Doppler-ultrasound findings (echogenic material within veins,
lack of any response after compression by the transducer and absence of color
flow) confirm de diagnosis. This disease has a favorable evolution and
functional prognosis. Knowledge of Mondor's disease by echographists is basic to
avoid false-negative results in radiologic examination. PATIENT: A 37-year-old woman presented with Mondor's thrombophlebitis 13 years
after augmentation mammaplasty with subpectoral saline implants. She presented
complaining of 1 week of "band-like" cords and pain involving the
thoracoepigastric and lateral thoracic vessels. She was evaluated and ruled out
for other etiologies of her breast symptoms.
BACKGROUND: Mondor's disease is a benign and self-limiting disease characterized
by thrombophlebitis of the subcutaneous veins of the chest and abdominal wall.
The inflammation causes painful superficial cords manifesting as skin
retraction. Mondor's disease has been described in aesthetic breast surgery
literature, but most cases occur within the first few postoperative weeks.
CONCLUSIONS: Mondor's disease may be idiopathic, iatrogenic, or a manifestation
of underlying pathology such as breast cancer. The diagnosis of iatrogenic
Mondor's disease can be made with high clinical certainty when following
aesthetic breast surgery in the early postoperative period. However, in late
presentations that are not immediately related to surgery, Mondor's disease
remains a diagnosis of exclusion, and other underlying pathologic etiologies
must be ruled out. Penile and scrotal emergencies are uncommon, but when they do occur, urgent or
emergent diagnosis and treatment are necessary. Emergent conditions of the male
genitalia are primarily infectious, traumatic, or vascular. Infectious
conditions, such as epididymitis and epididymo-orchitis, are well evaluated at
ultrasonography (US), and their key findings include heterogeneity and
hyperemia. Pyocele and abscess may also be seen at US. Fournier gangrene is best
evaluated at computed tomography, which depicts subcutaneous gas. Vascular
conditions, such as testicular torsion, infarction, penile Mondor disease, and
priapism, are well evaluated at duplex Doppler US. The key imaging finding of
testicular torsion and infarction is a lack of blood flow in the testicle or a
portion of the testicle. Penile Mondor disease is characterized by a lack of
flow to and noncompressibility of the superficial dorsal vein of the penis.
Clinical examination and history are usually adequate for diagnosis of priapism,
but Doppler US may help confirm the diagnosis. Traumatic injuries of the penis
and scrotum are initially imaged with US, which depicts whether the penile
corpora and testicular seminiferous tubules are contained by the tunicae
albuginea; herniation of contents and discontinuity of the tunica albuginea
indicate rupture. In some cases, magnetic resoce imaging may be performed
because of its ability to directly depict discontinuity of the tunica albuginea.
Radiologists must closely collaborate with emergency physicians, surgeons, and
urologists to quickly and efficiently diagnose or rule out emergent conditions
of the male genitalia to facilitate prompt and appropriate treatment. Mondor's disease is a rare benign and self-limiting condition characterized by
thrombophlebitis of the superficial veins of the anterolateral thoracoabdominal
wall. We describe a case of Mondor's disease of the breast as a complication of
an ultrasound-guided core needle biospy. The patient presented with a palpable
cord-like structure in her left breast, associated with severe pain and
tenderness. She was treated concervatively and complete resolution was observed
after four weeks. We conclude that emergency clinicians and interventional
breast radiologists should be aware of Mondor's disease as a potential
complication of ultrasound-guided core needle biopsy of the breast. Mondor's disease is a rare, benign condition characterised by thrombophlebitis
affecting subcutaneous veins of the chest and/or abdomen without an accompanying
inflammatory response. The disease has a multifactorial etiology and its course
is benign. It is usually self-limiting or it is eliminated by local treatment.
Mondor's disease in the thoracoepigastric region may be a rare complication of
mammotome biopsy. The case presentation describes a 32-year-old patient with
Mondor's disease in the thoracoepigastric region after an ultrasound-guided
mammotome biopsy of a breast. In the histopathological examination the lesion
was diagnosed as fibroadenoma. Regardless of the disease's etiology, it is
recommended to carry out diagnostic examinations to exclude co-occurring breast
cancer. INTRODUCTION: Mondor disease (MD), a superficial thrombophlebitis of the
thoraco-epigastric veins and their confluents is rarely reported in the
literature. The superior epigastric vein is the most affected vessel but
involvement of the inferior epigastric vessels or their branches have also been
described. There is no universal consensus on treatment in the literature but
most authors suggest symptomatic treatment with non-steroid anti-inflammatory
drugs (NSAIDs).
CASE REPORT: We report the case of a marathon runner who presented with right
iliac fossa pain mimicking the clinical symptomatology of an acute appendicitis.
The history and the calculated Alvarado score were not in favor of an acute
appendicitis. This situation motivated multiple investigations and we finally
arrived at the diagnosis of MD.
DISCUSSION: Acute appendicitis (AA) is the most common cause of surgical
emergencies and one of the most frequent indications for an urgent abdominal
surgical procedure around the world. In some cases, right lower quadrant pain
remains unclear in spite of US, CT scan, and exclusion of urological and
gynecological causes, thus we need to think of some rare pathologies like MD.
CONCLUSION: MD is often mentioned in the differential diagnosis of breast
pathologies but rarely in abdominal pain assessment. It should be mentioned in
the differential diagnosis of the right lower quadrant pain when the clinical
presentation is unclear and when acute appendicitis has been excluded. Awareness
of MD can avoid misdiagnosis and decrease extra costs by sparing unnecessary
imaging. |
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