question
stringlengths 13
215
| ground_truth
stringlengths 2
3.15k
| context
stringlengths 0
157k
|
|---|---|---|
List metalloenzyme inhibitors. | Foscarnet
VT-1129
VT-1161
BB-3497
hydroxamate molecules
siderophores | By screening a library of metalloenzyme inhibitors, the N-formyl-hydroxylamine
derivative BB-3497 was identified as a potent inhibitor of Escherichia coli
peptide deformylase with antibacterial activity both in vitro and in vivo. The
homochiral synthesis of BB-3497, involving a novel asymmetric Michael addition
reaction is described. Foscarnet (phosphonoformate trisodium salt), an antiviral used for the treatment
of HIV and herpes virus infections, also acts as an activator or inhibitor of
the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Interaction of the drug
with 11 CA isozymes has been investigated kinetically, and the X-ray structure
of its adduct with isoform I (hCA I-foscarnet complex) has been resolved. The
first CA inhibitor possessing a phosphonate zinc-binding group is thus
evidenced, together with the factors governing recognition of such small
molecules by a metalloenzyme active site. Foscarnet is also a clear-cut example
of modulator of an enzyme activity which can act either as an activator or
inhibitor of a CA isozyme. The desire to inhibit zinc-dependent matrix metalloproteinases (MMPs) has, over
the course of the last 30 years, led to the development of a plethora of MMP
inhibitors that bind directly to the active-site metal. With one exception, all
of these drugs have failed in clinical trials, due to many factors, including an
apparent lack of specificity for MMPs. To address the question of whether these
inhibitors are selective for MMPs in a biological setting, a cell-based
screening method is presented to compare the relative activities of zinc, heme
iron, and non-heme iron enzymes in the presence of these compounds using the
RAW264.7 macrophage cell line. We screened nine different zinc-binding groups
(ZBGs), four established MMP inhibitors (MMPis), and two novel MMP inhibitors
developed in our laboratory to determine their selectivities against five
different metalloenzymes. Using this model, we identified two nitrogen donor
compounds--2,2'-dipyridylamine (DPA) and triazacyclonoe (TACN)--as the most
selective ZBGs for zinc metalloenzyme inhibitor development. We also
demonstrated that the model could predict known nonspecific interactions of some
of the most commonly used MMPis, and could also give cross-reactivity
information for newly developed MMPis. This work demonstrates the utility of
cell-based assays in both the design and the screening of novel metalloenzyme
inhibitors. BACKGROUND AND PURPOSE: Based on their ability to chelate metals, hydroxamate
molecules and siderophores have been successfully used as metalloenzyme
inhibitors. As the anthrax toxin lethal factor (LF) is a zinc
(Zn)-metallopeptidase, an investigation of the ability of some small
non-siderophore hydroxamate compounds, 5 hydroxamate-containing siderophores,
and 1 catecholate siderophore was undertaken to determine whether these
compounds would inhibit LF. In addition, salmon sperm protamine and
ethylenediaminetetraacetic acid were investigated.
METHODS: A spectrophotometric assay of LF activity, based on its reaction with
the substrate
(Ac-gly-tyr-betaala-arg-arg-arg-arg-arg-arg-arg-arg-val-leu-arg-p-nitroanilide),
was used to assess the degree of inhibition of LF by the putative inhibitors.
Procedures were implemented to avoid iron contamination of the test solutions
and non-ferrated siderophores and hydroxamates were used as potential
inhibitors.
RESULTS: The hydroxamate-containing siderophores displayed limited capacities to
inhibit LF, as did the low molecular weight hydroxamate compounds. In contrast,
the catecholate siderophore enterobactin and the cationic polyamine salmon sperm
protamine demonstrated notable inhibition of LF at concentrations ranging from
approximately 10 to 200 microM.
CONCLUSIONS: The polyamine salmon sperm protamine which mimics the target site
of proteins cleaved by LF, was the most effective inhibitor of the molecules
examined, while the small molecule hydroxamates and the hydroxamate siderophores
were among the poorest. If chelation of the Zn of LF results in LF inhibition by
the molecules examined, it is most likely secondary to binding of the putative
inhibitors to the active site of LF. The 50th annual Interscience Conference on Antimicrobial Agents and Chemotherapy
(ICAAC), held in Boston, included topics covering new therapeutic developments
in the field of infectious disease. This conference report highlights selected
presentations on research with novel antimicrobial agents. Investigational drugs
discussed include the chitin synthase inhibitor nikkomycin Z (Valley Fever
Solutions/University of Arizona), the glycosylphosphatidylinositol biosynthesis
inhibitor E-1210 (Eisai), the β-1,3-d-glucan synthesis inhibitor MK-3118 (Merck
& Co/SCYNEXIS), the metalloenzyme inhibitors VT-1129 and VT-1161 (both Viamet
Pharmaceuticals), and the anti-inflammatory oemulsion NB-003 (NanoBio). Fragment-based lead design (FBLD) has been used to identify new metal-binding
groups for metalloenzyme inhibitors. When screened at 1 mM, a chelator fragment
library (CFL-1.1) of 96 compounds produced hit rates ranging from 29% to 43% for
five matrix metalloproteases (MMPs), 24% for anthrax lethal factor (LF), 49% for
5-lipoxygenase (5-LO), and 60% for tyrosinase (TY). The ligand efficiencies (LE)
of the fragment hits are excellent, in the range of 0.4-0.8 kcal/mol. The MMP
enzymes all generally elicit the same chelators as hits from CFL-1.1; however,
the chelator fragments that inhibit structurally unrelated metalloenzymes (LF,
5-LO, TY) vary considerably. To develop more advanced hits, one hit from
CFL-1.1, 8-hydroxyquinoline, was elaborated at four different positions around
the ring system to generate new fragments. 8-Hydroxyquinoline fragments
substituted at either the 5- or 7-positions gave potent hits against MMP-2, with
IC(50) values in the low micromolar range. The 8-hydroxyquinoline represents a
promising new chelator scaffold for the development of MMP inhibitors that was
discovered by use of a metalloprotein-focused chelator fragment library. A series of HIV integrase (HIV-1 IN) inhibitors were synthesized to evaluate the
role of the metal-binding group (MBG) in this class of metalloenzyme inhibitors.
A total of 21 different raltegravir-chelator derivative (RCD) compounds were
prepared that differed only in the nature of the MBG. These IN strand-transfer
inhibitors (INSTIs) were evaluated in vitro in cell-free enzyme activity assays,
and the in vitro results were further validated in cell culture experiments. All
of the active compounds showed selective inhibition of the strand-transfer
reaction over 3'-processing, suggesting a common mode of action with
raltegravir. The results of the in vitro activity suggest that the nature of the
MBG donor atoms, the overall MBG structure, and the specific arrangement of the
MBG donor atom triad are essential for obtaining maximal HIV-1 IN inhibition. At
least two compounds (RCD-4, RCD-5) containing a hydroxypyrone MBG were found to
display superior strand-transfer inhibition when compared to an abbreviated
analogue of raltegravir (RCD-1). By isolating and examining the role of the MBG
in a series of INSTIs, we have identified a scaffold (hydroxypyrones) that may
provide access to a unique class of HIV-1 IN inhibitors, and may help overcome
rising raltegravir resistance. The inhibitory activity of a broad group of known metalloenzyme inhibitors
against a panel of metalloenzymes was evaluated. Clinically approved inhibitors
were selected as well as several other reported metalloprotein inhibitors in
order to represent a broad range of metal binding groups (MBGs), including
hydroxamic acid, carboxylate, hydroxypyridinonate, thiol, and N-hydroxyurea
functional groups. A panel of metalloenzymes, including carbonic anhydrase
(hCAII), several matrix metalloproteinases (MMPs), angiotensin converting enzyme
(ACE), histone deacetylase (HDAC-2), and tyrosinase (TY), was selected based on
their clinical importance for a range of pathologies. In addition, each
inhibitor was evaluated for its ability to remove Fe(3+) from holo-transferrin
to gauge the ability of the inhibitors to access Fe(3+) from a primary transport
protein. The results show that the metalloenzyme inhibitors are quite selective
for their intended targets, suggesting that despite their ability to bind metal
ions, metalloprotein inhibitors are not prone to widespread off-target enzyme
inhibition activity. |
How are immediate early genes (IEG) defined? | this class of genes is experimentally defined by their transcription following primary infection or reactivation in the presence of inhibitors of protein synthesis. Immediate-early (IE) genes are the first class of viral genes expressed after primary infection or reactivation. | In situ hybridization was used to measure the expression of members of the
Fos/Jun family of immediate-early genes in hypothalamic neurons in vivo
following defined stimuli that utilize different afferent pathways. Only c-jun
messenger RNA was expressed in the hypothalamic supraoptic and paraventricular
nuclei of control animals. Intravenous infusions of sodium chloride solutions of
different tonicity produced a range of plasma osmolalities within physiological
limits. While the induction of c-fos and jun B messenger RNAs followed the
stimulus intensity, the expression of c-jun was repressed at low levels of
stimulation. A higher level of osmotic stimulation was able to co-induce c-jun
with the c-fos, jun B and fos B genes, suggesting that other signalling pathways
may then be activated. Parturition or systemic administration of
cholecystokinin, that activate supraoptic and paraventricular neurons via
ascending afferent pathways from the brainstem, both induced c-fos, but not the
other genes, in the magnocellular nuclei. Use of double in situ hybridization
confirmed that, unlike with osmotic stimulation, induction of c-fos only
occurred in oxytocin neurons. These two stimuli did not cause a concomitant
repression of c-jun messenger RNA expression in magnocellular oxytocin neurons.
These patterns of induction provide evidence for the differential regulation of
members of this family of genes in a physiological context. The lungs are a major organ site of cytomegalovirus (CMV) pathogenesis, latency,
and recurrence. Previous work on murine CMV latency has documented a high load
and an even distribution of viral genomes in the lungs after the resolution of
productive infection. Initiation of the productive cycle requires expression of
the ie1/3 transcription unit, which is driven by the immediate-early (IE)
promoter P(1/3) and generates IE1 and IE3 transcripts by differential splicing.
Latency is molecularly defined by the absence of IE3 transcripts specifying the
essential transactivator protein IE3. In contrast, IE1 transcripts were found to
be generated focally and randomly, reflecting sporadic P(1/3) activity.
Selective generation of IE1 transcripts implies molecular control of latency
operating after ie1/3 transcription initiation. P(1/3) is regulated by an
upstream enhancer. It is widely assumed that the viral transcriptional program
is started by activation of the enhancer through the binding of transcription
factors. Accordingly, stochastic transcription during latency might reflect
episodes of enhancer activation by the "noise" activity of intrinsic
transcription factors. In addition to ie1/3, the enhancer controls gene ie2,
which has its own promoter, P(2), and is transcribed in opposite direction. We
show here that ie2 is also randomly transcribed during latency. Notably,
however, ie1 and ie2 were found to be expressed independently. We infer from
this finding that expression of the major IE genes is regulated asymmetrically
and asynchronously via the combined control unit P(1/3) -E-P(2). Our data are
consistent with a stochastic nature of enhancer action as it is proposed by the
"binary" or probability model. Immediate-early (IE) genes are the first class of viral genes expressed after
primary infection or reactivation. As transcription of IE genes does not require
prior viral protein synthesis, this class of genes is experimentally defined by
their transcription following primary infection or reactivation in the presence
of inhibitors of protein synthesis. This chapter describes an approach to
identify IE genes in a novel herpesvirus genome. Transcription of IE genes is
selectively induced with sodium butyrate in the presence of the protein
synthesis inhibitor cycloheximide. The transcripts of the induced genes are
identified by using a cDNA subtraction-based method of gene expression
screening. Visual inputs from the 2 eyes in most primates activate alternating bands of
cortex in layer 4C of primary visual cortex, thereby forming the well-studied
ocular domice columns (ODCs). In addition, the enzymatic reactivity of
cytochrome oxidase (CO) reveals "blob" structures within the supragranular
layers of ODCs. Here, we present evidence for compartments within ODCs that have
not been clearly defined previously. These compartments are revealed by the
activity-dependent mRNA expression of immediate-early genes (IEGs), zif268 and
c-fos, after brief periods of monocular inactivation (MI). After a 1-3-h period
of MI produced by an injection of tetrodotoxin, IEGs were expressed in a patchy
pattern that included infragranular layers, as well as supragranular layers,
where they corresponded to the CO blobs. In addition, the expressions of IEGs in
layer 4C were especially high in narrow zones along boundaries of ODCs, referred
to here as the "border strips" of the ODCs. After longer periods of MI (>5 h),
the border strips were no longer apparent. When either eyelid was sutured,
changes in IEG expression were not evident in layer 4C; however, the patchy
pattern of the expression in the infragranular and supragranular layers
remained. These changes of IEG expression after MI indicate that cortical
circuits involving the CO blobs of the supragranular layers include aligned
groups of neurons in the infragranular layers and that the border strip neurons
of layer 4C are highly active for a 3-h period after MI. |
Which proteins remove H2A.Z in the yeast Saccharomyces cerevisiae? | Removal of H2A.Z by INO80 promotes homologous recombination Budding yeast INO80 can remove H2A.Z/H2B dimers from chromatin and replace them with H2A/H2B dimers. Here, we show that H2A.Z in human cells is indeed rapidly removed from chromatin flanking DNA damage by INO80. | The mammalian INO80 remodelling complex facilitates homologous recombination
(HR), but the mechanism by which it does this is unclear. Budding yeast INO80
can remove H2A.Z/H2B dimers from chromatin and replace them with H2A/H2B dimers.
H2A.Z is actively incorporated at sites of damage in mammalian cells, raising
the possibility that H2A.Z may need to be subsequently removed for resolution of
repair. Here, we show that H2A.Z in human cells is indeed rapidly removed from
chromatin flanking DNA damage by INO80. We also report that the histone
chaperone ANP32E, which is implicated in removing H2AZ from chromatin, similarly
promotes HR and appears to work on the same pathway as INO80 in these assays.
Importantly, we demonstrate that the HR defect in cells depleted of INO80 or
ANP32E can be rescued by H2A.Z co-depletion, suggesting that H2A.Z removal from
chromatin is the primary function of INO80 and ANP32E in promoting homologous
recombination. |
Which library is used for fixed-length approximate string matching? | libFLASM is a free open-source C++ software library for solving fixed-length approximate string matching under both the edit and the Hamming distance models. | BACKGROUND: Approximate string matching is the problem of finding all factors of
a given text that are at a distance at most k from a given pattern. Fixed-length
approximate string matching is the problem of finding all factors of a text of
length n that are at a distance at most k from any factor of length ℓ of a
pattern of length m. There exist bit-vector techniques to solve the fixed-length
approximate string matching problem in time [Formula: see text] and space
[Formula: see text] under the edit and Hamming distance models, where w is the
size of the computer word; as such these techniques are independent of the
distance threshold k or the alphabet size. Fixed-length approximate string
matching is a generalisation of approximate string matching and, hence, has
numerous direct applications in computational molecular biology and elsewhere.
RESULTS: We present and make available libFLASM, a free open-source C++ software
library for solving fixed-length approximate string matching under both the edit
and the Hamming distance models. Moreover we describe how fixed-length
approximate string matching is applied to solve real problems by incorporating
libFLASM into established applications for multiple circular sequence alignment
as well as single and structured motif extraction. Specifically, we describe how
it can be used to improve the accuracy of multiple circular sequence alignment
in terms of the inferred likelihood-based phylogenies; and we also describe how
it is used to efficiently find motifs in molecular sequences representing
regulatory or functional regions. The comparison of the performance of the
library to other algorithms show how it is competitive, especially with
increasing distance thresholds.
CONCLUSIONS: Fixed-length approximate string matching is a generalisation of the
classic approximate string matching problem. We present libFLASM, a free
open-source C++ software library for solving fixed-length approximate string
matching. The extensive experimental results presented here suggest that other
applications could benefit from using libFLASM, and thus further maintece and
development of libFLASM is desirable. |
Are there methods for generating highly multiplexed ChIP-seq libraries? | Yes. There are methods for generating highly multiplexed ChIP-seq libraries. | |
Is Doxorubicin cardiotoxic? | Doxorubicin (DOXO) is widely used to treat solid tumors. However, its clinical use is limited by side effects including serious cardiotoxicity due to cardiomyocyte damage | Doxorubicin is an effective antineoplastic agent, but it frequently causes
dose-related cardiotoxic effects. Because the atrial natriuretic peptide (ANP)
level is elevated in children with heart defects, the authors measured the ANP
levels in children to determine whether ANP might serve as a simple diagnostic
indicator of cardiotoxic effects. Sixteen patients, 5 to 19 years of age, who
were being treated with doxorubicin (45 mg/m2 body surface area) for various
maligcies had ANP levels measured in plasma. There was a group of six
children, with a significant peak of plasma ANP (pANP) levels 3 weeks after the
administration of the drug. Of these six patients, five had received high
cumulative doses of doxorubicin (160 to 370 mg/m2), and two of them went into
congestive heart failure without a previous decline in left ventricular ejection
fraction, a standard technique for monitoring cardiac function during treatment
with doxorubicin. The other ten patients had normal ANP levels throughout the
study, and signs of cardiac dysfunction did not develop. None of the patients in
the control group who had cancer and were not treated with doxorubicin and none
of the healthy volunteers had elevated ANP levels. These preliminary results
suggest that pANP may be useful as an early and sensitive indicator for
doxorubicin-related myocardial damage. Human neutrophils exposed to 10(-4) M doxorubicin and the derivatives epirubicin
and thepirubicin revealed a different intracellular penetration and distribution
pattern as demonstrated by fluorescence microscopy and fluorimetric
determination of drug intracellular concentration. While doxorubicin was found
to be a potent inducer of superoxide generation from resting cells, epirubicin
exhibited less superoxide-inducing power. Thepirubicin on the contrary did not
show any superoxide-inducing effect. Moreover the anthracyclines tested all
inhibited the phorbol ester-stimulated chemiluminescent response to the same
extent, which suggested a common target for the drug action.
Anthracycline-stimulated superoxide production seems to correlate with the
cardiotoxic effects. The most cardiotoxic drug, doxorubicin, is the most potent
inducer of superoxide generation, while epirubicin, which is less cardiotoxic,
has a relatively limited effect on superoxide production. Thepirubicin which has
been shown not to induce delayed cardiomyopathy has no effect on superoxide
release from the cells. Doxorubicin (Adriamycin) is an unquestionably effective anticancer agent for
many types of tumors, including advanced breast cancer. However, cardiotoxic
effects of the drug have limited its use. Methods of reducing or preventing
doxorubicin-induced cardiotoxicity have been suggested, including an
investigational doxorubicin analog, mitoxantrone ( Novantrone ). Mitoxantrone
was developed to reduce the cardiotoxicity associated with doxorubicin while
maintaining effective antitumoral effects. Initial reports suggested that
mitoxantrone might lack cardiotoxic potential; however, recent studies indicate
that the drug can produce adverse cardiac effects (congestive heart failure),
perhaps with similar frequency to that observed with doxorubicin. It is doubtful
that mitoxantrone will be a significant advance over doxorubicin if direct
comparative studies reveal a similar incidence of cardiomyopathy. Verapamil reverses multidrug resistance acquired by cancer cells during
treatment with chemotherapeutic agents such as doxorubicin by inhibiting the
function of P-glycoprotein. Verapamil has also been suggested to potentiate the
cardiotoxicity of doxorubicin. We have recently demonstrated that selective
inhibition of cardiac muscle gene expression is among the earliest events in
doxorubicin cardiotoxicity. To explore the influence of verapamil on doxorubicin
cardiotoxicity, we evaluated [14C]-doxorubicin accumulation, cardiac muscle gene
expression by Northern blot analysis, and ultrastructural changes in cultured
cardiomyocytes in the presence and absence of verapamil. Treatment with a
combination of doxorubicin and verapamil for 24 h did not augment doxorubicin
accumulation in cardiomyocytes, although substantial augmentation of doxorubicin
accumulation by verapamil in cardiac fibroblasts was observed. Further,
treatment with verapamil for 24 h did not augment the decrease in expression of
muscle genes induced by doxorubicin (myosin light chain 2 slow, troponin I, M
isoform creatine kinase). However, we found that verapamil reduced alpha-actin
gene expression in a direct, doxorubicin-independent manner. Furthermore, the
effect of doxorubicin plus verapamil on alpha-actin gene expression was additive
over a wide range of doxorubicin and verapamil concentrations, resulting in a
selective augmentation of doxorubicin-induced inhibition of gene expression for
this single muscle protein gene. This was reflected in a substantial increase in
cardiac myocyte damage when treatment with verapamil and doxorubicin was
compared to treatment with doxorubicin alone by thin section electron
microscopy. This suggests a possible mechanism by which verapamil may potentiate
doxorubicin cardiotoxicity. Attempts to reduce the incidence of congestive heart failure following
anthracycline therapy include the replacement of the parent compounds
(especially doxorubicin) by less cardiotoxic analogs. Among these analogs,
idarubicin (4-demethoxy-daunorubicin) was shown to be less cardiotoxic than
doxorubicin in phase II clinical trials, but its actual cardiotoxicity has never
been evaluated in large series and has never been compared to that of
doxorubicin in relevant experimental models. Using the isolated perfused rat
heart model, we compared the cardiac effects (developed pressure, contractility
and relaxation of the left ventricle) induced by idarubicin to those induced by
doxorubicin. Drugs were administered i.v. every other day for 11 days at doses
of 1, 2, 2.5 and 3 mg/kg per injection for doxorubicin and 0.5, 0.75 and 1 mg/kg
per injection for idarubicin. We confirmed that similar general toxicity
symptoms were obtained for a dose ratio of 1:4 (idarubicin:doxorubicin).
However, at the maximum tolerated doses of both drugs (3 mg/kg per injection for
doxorubicin and 0.75 mg/kg per injection for idarubicin), the cardiac toxicity
of idarubicin remained significantly lower than that of doxorubicin.
Anthracycline cardiac accumulation was evaluated in parallel and revealed a
lower cardiac accumulation of idarubicin, which could explain the reduced
cardiac toxicity of this analog. Direct perfusion of the drugs in the isolated
hearts of untreated animals revealed that idarubicin was taken up more readily
than doxorubicin in the cardiac tissue, despite the fact that it had less
deleterious effects on cardiac function. This indicates that idarubicin also had
less intrinsic cardiotoxicity than doxorubicin in this model. Besides its cardiotoxic effect, doxorubicin also elicits inflammatory effects in
vivo. 7-Monohydroxyethylrutoside (monoHER) has recently been used as a protector
against doxorubicin-induced cardiotoxicity in vivo. It is not known yet whether
monoHER can also protect against doxorubicin-induced inflammatory effects. The
aim of the present study was (1) to illustrate the inflammatory effects of
doxorubicin in vitro and (2) to evaluate a possibly protective effect of
monoHER. In order to demonstrate the inflammatory effects of doxorubicin and the
possible protection of monoHER, proliferating human umbilical cord vascular
endothelial cells (HUVECs) were incubated with different concentrations of
doxorubicin ranging from 12.5 to 600 nM with(out) 200 micro M monoHER. Resting
(confluent) HUVECs were incubated with (0.5-25 micro M) doxorubicin with(out)
monoHER (0.2-1.2 mM) and the viability of endothelial cells and their propensity
to adhere to neutrophils were measured 24 h after treatment. The localisation of
adhered neutrophils was determined with immunofluorescence microscopy. To
further characterise the mechanism of doxorubicin-induced neutrophil adhesion,
the expression of the HUVECs surface adhesion molecules was determined after
doxorubicin treatment. Doxorubicin decreased the viability and proliferation
capacity of HUVECs in a concentration-dependent manner. The proliferating HUVECs
were much more sensitive to doxorubicin (IC(50)=60.0+/-20.8 nM) than resting
cells (LC(50)=4.0+/-0.3 micro M). Doxorubicin also increased the adhesion of
neutrophils reaching a plateau value at a doxorubicin concentration of > or =0.4
micro M (P=0.0113). The induced neutrophil adhesion was accompanied by
overexpression of VCAM and E-selectin but not ICAM. Although monoHER did not
reverse the effect of doxorubicin on the proliferation of endothelial cells, it
significantly protected resting HUVECs against the cytotoxic effect of
doxorubicin (< or =25 micro M, P<0.0015). In addition, monoHER completely
protected against the stimulatory effect of doxorubicin on neutrophil adhesion,
and inhibited the doxorubin-induced expression of VCAM and E-selectin on the
surface of treated HUVECs. This study illustrates that monoHER, which protects
against doxorubicin's cardiotoxic effect, can also protect against
doxorubicin-induced inflammatory effects. These data prompt further
investigation about the possible link between doxorubicin-induced inflammatory
effects and its cardiotoxicity in vivo. Doxorubicin is a chemotherapeutic agent successfully used in the treatment of a
wide range of cancers. However, with cumulative doses, doxorubicin also is known
to have cardiotoxic effects, including cardiomyopathy and heart failure.
Research is targeted at maximizing the antitumor effects of doxorubicin while
attenuating the potential cardiotoxicity. Concurrent therapies under study are
combinations of doxorubicin with drugs such as probucol, carvedilol (Coreg,
GlaxoSmithKline, Research Triangle Park, NC), dexrazoxane (Zinecard, Pfizer, New
York, NY), and antioxidant nutrients. As patient advocates, nurses must be aware
of current research, treatment options, and evidence-based patient resources and
be diligent in assessing and educating patients before, during, and after
treatment with doxorubicin. Doxorubicin is among the most effective and widely used anticancer drugs in the
clinic. However, cardiotoxicity is one of the life-threatening side effects of
doxorubicin-based therapy. Dexrazoxane (Zinecard, also known as ICRF-187) has
been used in the clinic as a cardioprotectant against doxorubicin
cardiotoxicity. The molecular basis for doxorubicin cardiotoxicity and the
cardioprotective effect of dexrazoxane, however, is not fully understood. In the
present study, we showed that dexrazoxane specifically abolished the DNA damage
signal gamma-H2AX induced by doxorubicin, but not camptothecin or hydrogen
peroxide, in H9C2 cardiomyocytes. Doxorubicin-induced DNA damage was also
specifically abolished by the proteasome inhibitors bortezomib and MG132 and
much reduced in top2beta(-/-) mouse embryonic fibroblasts (MEF) compared with
TOP2beta(+/+) MEFs, suggesting the involvement of proteasome and DNA
topoisomerase IIbeta (Top2beta). Furthermore, in addition to antagonizing Top2
cleavage complex formation, dexrazoxane also induced rapid degradation of
Top2beta, which paralleled the reduction of doxorubicin-induced DNA damage.
Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced
DNA damage through its interference with Top2beta, which could implicate
Top2beta in doxorubicin cardiotoxicity. The specific involvement of proteasome
and Top2beta in doxorubicin-induced DNA damage is consistent with a model in
which proteasomal processing of doxorubicin-induced Top2beta-DNA covalent
complexes exposes the Top2beta-concealed DNA double-strand breaks. BACKGROUND AND AIM: The clinical use of doxorubicin (DOX) and other
anthracyclines is limited by a dosage-dependent cardiotoxicity, which can lead
to cardiomyopathy. The role of the individual genetic makeup in this disorder is
poorly understood. Alterations in genes encoding cardiac cytoskeleton or
sarcolemma proteins may increase the susceptibility to doxorubicin-related
cardiotoxicity.
METHODS: Female dystrophin-deficient mice (MDX) and age-matched wild-type mice
underwent chronic treatment with doxorubicin. Cardiac function and tissue damage
were assessed by echocardiography and histopathology, respectively. Gene
expression changes were investigated using microarrays.
RESULTS: DOX treatment resulted in mortality, cardiac insufficiency, and cardiac
interstitial fibrosis. These alterations were more pronounced in DOX-treated MDX
mice than in DOX-treated wild-type mice. Changes in gene expression were more
numerous in MDX mice, including genes involved in cell adhesion, oxidative
stress, cytoskeleton organization, inflammatory and immune response and cell
death.
CONCLUSIONS: Dystrophin deficiency facilitates the development and progression
of doxorubicin-induced cardiac injury. The underlying mechanisms may involve
changes in cell adhesion, in cytoskeleton, as well as in inflammatory and immune
responses. Genetic variants of cytoskeletal proteins in humans may affect the
individual susceptibility to doxorubicin. Cardiotoxic drugs may accelerate the
manifestation of pre-clinical cardiomyopathies caused by deficiencies in
cytoskeletal or sarcolemma proteins. Clinical uses of doxorubicin (DOX), a highly active anticancer agent, are
limited by its severe cardiotoxic side effects associated with increased
oxidative stress and apoptosis. In this study we investigated whether aged
garlic has protective effects against doxorubicin-induced free radical
production and cardiotoxicity in male rats. A single dose of doxorubicin
(25mg/kg) caused increased both serum cardiac enzymes LDH and CPK activities and
a significant increase malonyldialdehyde (MDA) in plasma. However, pretreatment
of rats with aged garlic extract (250 mg/kg) for 27 days before doxorubicin
therapy, reduced the activity of both enzymes, and significantly decreased of
MDA production in plasma. Total antioxidant activity was increased after aged
garlic extract administration. Histopathological examination of heart tissue
showed that DOX treatment resulted in alteration of cardiac tissue structure in
the form of peri arterial fibrosis and apoptotic changes in cardiomyocytes.
Pretreatment with aged garlic extract for 27 days ameliorated the effect of DOX
administration on cardiac tissue; cardiomyocytes looked more or less similar to
those of control. However, still vascular dilatation, mild congestion and
interstitial edemas were observed. Our results suggest that aged garlic extract
is potentially protective against doxorubicin-induced cardiotoxicity. BACKGROUND: Doxorubicin (DOX) is a potent chemotherapeutic agent used in the
treatment of several tumors but its cardiac toxicity prevents its use at a
maximum dose, representing an important problem. Increased reactive oxygen
species (ROS) and imbalance in nitric oxide (NO) production have been implicated
in the cardiotoxicity of doxorubicin. Hesperidin is a citrus bioflavonoid that
possesses a potent antioxidant and NO modulating activities.
OBJECTIVES: Therefore, the aim of this study was to investigate the possible
protective role of hesperidin against doxorubicin-induced cardiac toxicity.
METHODS: Four groups of animals were used in this study. First group served as a
control and injected with the vehicle. Second group was given 200 mg/kg of
hesperidin orally for seven consecutive days. The third group was injected with
a single dose (20 mg/kg) of doxorubicin intraperitoneally and was sacrificed
after 48 h. The fourth group was treated with hesperidin for seven days but on
day five, 1-hour after hesperidin treatment, rats were injected with the single
dose of doxorubicin. On day seven, the rats were scarified by decapitation.
Blood was collected and processed for determination of serum lactate
dehydrogenase (LDH), creatine kinase (CK) and NO. The hearts were removed and
processed for both histopathological examination and determination of oxidative
stress parameters like reduced glutathione (GSH), lipid peroxide (TBARS) levels
and superoxide dismutase (SOD) activity.
RESULTS: Our results showed that doxorubicin produced severe cardiotoxicity as
indicated from increase in serum LDH, CK activities and NO level.
Histopathological examination of DOX-treated rats revealed degenerative changes
in heart tissues. The significant decrease in GSH levels, SOD activity and
increase in TBARS levels, indicated that DOX-induced cardiotoxicity was mediated
through ROS generation. On the other hand, pretreatment of rats with hesperidin
protected cardiac tissues against the cardiotoxic effects of doxorubicin as
evidenced from amelioration of histopathological changes and normalization of
cardiac biochemical parameters.
CONCLUSION: Hesperidin may have a protective effect against DOX-induced
cardiotoxicity. The anthracycline chemotherapeutic agent doxorubicin is converted by the enzyme
carbonyl reductase 1 (CBR1) into its cardiotoxic metabolite doxorubicinol.
Cbr1+/- mice have been shown to be protected from doxorubicin-induced
cardiotoxicity, and the inhibition of CBR1 activity may be a useful means of
ameliorating the side effects of doxorubicin in patients undergoing
chemotherapy. Because reduced conversion to doxorubicinol increases circulating
levels of the more effective parent drug doxorubicin, it was hypothesized that
therapeutic efficacy against tumors might also be enhanced. Cbr1+/- mice were
bred to mice transgenic for the polyomavirus middle T antigen (PyVT) to create
offspring with palpable mammary tumors. Latency to initial tumor formation was
similar in Cbr1+/- and Cbr1+/+ animals. Tumor regression was improved in Cbr1+/-
animals, but only in male mice. Western blotting showed a marked sex difference
in protein levels, with a much higher expression of Cbr1 in the female kidney
and liver. Thus, the combined effects of a naturally low expression and the
heterozygous Cbr1 null allele seem to have enhanced tumor regression in Cbr1+/-
males. Future efforts to design a clinical CBR1 inhibitor to protect patients
from the cardiac side effects of doxorubicin treatment should evaluate the
effect of sex on anticancer efficacy. Doxorubicin is an effective anticancer drug with known cardiotoxic side effects.
It has been hypothesized that doxorubicin-dependent cardiotoxicity occurs
through ROS production and possibly cellular iron accumulation. Here, we found
that cardiotoxicity develops through the preferential accumulation of iron
inside the mitochondria following doxorubicin treatment. In isolated
cardiomyocytes, doxorubicin became concentrated in the mitochondria and
increased both mitochondrial iron and cellular ROS levels. Overexpression of
ABCB8, a mitochondrial protein that facilitates iron export, in vitro and in the
hearts of transgenic mice decreased mitochondrial iron and cellular ROS and
protected against doxorubicin-induced cardiomyopathy. Dexrazoxane, a drug that
attenuates doxorubicin-induced cardiotoxicity, decreased mitochondrial iron
levels and reversed doxorubicin-induced cardiac damage. Finally, hearts from
patients with doxorubicin-induced cardiomyopathy had markedly higher
mitochondrial iron levels than hearts from patients with other types of
cardiomyopathies or normal cardiac function. These results suggest that the
cardiotoxic effects of doxorubicin develop from mitochondrial iron accumulation
and that reducing mitochondrial iron levels protects against doxorubicin-induced
cardiomyopathy. PURPOSE: Doxorubicin is one of the most effective chemotherapeutic agents.
However, up to 30% of the patients treated with doxorubicin suffer from
congestive heart failure. The mechanism of doxorubicin cardiotoxicity is likely
multifactorial and most importantly, the genetic factors predisposing to
doxorubicin cardiotoxicity are unknown. On the basis of the fact that mtDNA
lesions and mitochondrial dysfunctions have been found in human hearts exposed
to doxorubicin and that mitochondrial topoisomerase 1 (Top1mt) specifically
controls mtDNA homeostasis, we hypothesized that Top1mt knockout (KO) mice might
exhibit hypersensitivity to doxorubicin.
EXPERIMENTAL DESIGN: Wild-type (WT) and KO Top1mt mice were treated once a week
with 4 mg/kg doxorubicin for 8 weeks. Heart tissues were analyzed one week after
the last treatment.
RESULTS: Genetic inactivation of Top1mt in mice accentuates mtDNA copy number
loss and mtDNA damage in heart tissue following doxorubicin treatment. Top1mt KO
mice also fail to maintain respiratory chain protein production and
mitochondrial cristae ultrastructure organization. These mitochondrial defects
result in decreased O2 consumption, increased reactive oxygen species
production, and enhanced heart muscle damage in animals treated with
doxorubicin. Accordingly, Top1mt KO mice die within 45 days after the last
doxorubicin injection, whereas the WT mice survive.
CONCLUSIONS: Our results provide evidence that Top1mt, which is conserved across
vertebrates, is critical for cardiac tolerance to doxorubicin and adaptive
response to doxorubicin cardiotoxicity. They also suggest the potential of
Top1mt single-nucleotide polymorphisms testing to investigate patient
susceptibility to doxorubicin-induced cardiotoxicity. Anthracyclines are active clinical agents that have multiple mechanisms of
cytotoxicity. Cardiotoxicity by anthracyclines limits the therapeutic potential
of these agents, but mechanisms leading to cardiotoxicity remain controversial.
Transgenic mice that lack mitochondrial topoisomerase I are hypersensitive to
doxorubicin cardiotoxicity, providing support for cardiotoxicity arising from
damage of mitochondrial DNA. Doxorubicin (DOX), a highly active chemotherapeutic drug, faces limitations in
clinical application due to severe cardiotoxic effects (mainly through increased
oxidative stress). Therefore, its effect is exacerbated in subjects with
ischemic heart disease. We have recently reported that saffron extract (SAF), a
natural compound mainly consisting of safranal and corcins, exerts a protective
effect against DOX oxidative cytotoxicity in isolated rabbit hearts. Here, we
aimed to investigate whether SAF exerts cardioprotection against combined
ischemia-reperfusion (I/R) and DOX toxicity in H9c2 cardiomyocytes. H9c2 were
subjected to simulated I/R, with or without DOX treatment at reperfusion, in the
presence or absence of SAF prior to ischemia or at reperfusion. We evaluated the
effects of these treatments by MTT, LDH and western blot analysis. Apoptosis was
assessed by Hoechst 33258 staining, tetramethyl rhodamine methyl ester
fluorescence and caspase activity. The results showed that I/R and DOX
significantly decreased cardiomyocytes viability, inhibited reperfusion injury
salvage kinase cardioprotective pathway, reduced contractile proteins
(α-Actinine, Troponine C and MLC), increased caspase-3 expression and induced
loss of mitochondrial membrane potential. These effects were remarkably
inhibited by treatment with SAF (10 μg/mL) at reperfusion. SAF activated
AKT/P70S6K and ERK1/2, restored contractile proteins expression, inhibited
mitochondrial permeability transition pore and decreased caspase-3 activity. In
conclusion, our findings indicate that SAF treatment exerted cardioprotection
against I/R and DOX toxicity by reducing oxidative stress (LDH assay). Thereby,
SAF offers a potential novel antioxidant therapeutic strategy to counteract I/R
and DOX cardiotoxicity, paving the way for future clinical trials. BACKGROUND: Before this study started, the standard postoperative chemotherapy
regimen for stage II-III Wilms' tumour pretreated with chemotherapy was to
include doxorubicin. However, avoidance of doxorubicin-related cardiotoxicity
effects is important to improve long-term outcomes for childhood cancers that
have excellent prognosis. We aimed to assess whether doxorubicin can be omitted
safely from chemotherapy for stage II-III, histological intermediate-risk Wilms'
tumour when a newly defined high-risk blastemal subtype was excluded from
randomisation.
METHODS: For this international, multicentre, open-label, non-inferiority, phase
3, randomised SIOP WT 2001 trial, we recruited children aged 6 months to 18
years at the time of diagnosis of a primary renal tumour from 251 hospitals in
26 countries who had received 4 weeks of preoperative chemotherapy with
vincristine and actinomycin D. Children with stage II-III intermediate-risk
Wilms' tumours assessed after delayed nephrectomy were randomly assigned (1:1)
by a minimisation technique to receive vincristine 1·5 mg/m(2) at weeks 1-8, 11,
12, 14, 15, 17, 18, 20, 21, 23, 24, 26, and 27, plus actinomycin D 45 μg/kg
every 3 weeks from week 2, either with five doses of doxorubicin 50 mg/m(2)
given every 6 weeks from week 2 (standard treatment) or without doxorubicin
(experimental treatment). The primary endpoint was non-inferiority of event-free
survival at 2 years, analysed by intention to treat and a margin of 10%.
Assessment of safety and adverse events included systematic monitoring of
hepatic toxicity and cardiotoxicity. This trial is registered with EudraCT,
number 2007-004591-39, and is closed to new participants.
FINDINGS: Between Nov 1, 2001, and Dec 16, 2009, we recruited 583 patients, 341
with stage II and 242 with stage III tumours, and randomly assigned 291 children
to treatment including doxorubicin, and 292 children to treatment excluding
doxorubicin. Median follow-up was 60·8 months (IQR 40·8-79·8). 2 year event-free
survival was 92·6% (95% CI 89·6-95·7) for treatment including doxorubicin and
88·2% (84·5-92·1) for treatment excluding doxorubicin, a difference of 4·4% (95%
CI 0·4-9·3) that did not exceed the predefined 10% margin. 5 year overall
survival was 96·5% (94·3-98·8) for treatment including doxorubicin and 95·8%
(93·3-98·4) for treatment excluding doxorubicin. Four children died from a
treatment-related toxic effect; one (<1%) of 291 receiving treatment including
doxorubicin died of sepsis, three (1%) of 292 receiving treatment excluding
doxorubicin died of varicella, metabolic seizure, and sepsis during treatment
for relapse. 17 patients (3%) had hepatic veno-occlusive disease. Cardiotoxic
effects were reported in 15 (5%) of 291 children receiving treatment including
doxorubicin. 12 children receiving treatment including doxorubicin, and ten
children receiving treatment excluding doxorubicin, died, with the remaining
deaths from tumour recurrence.
INTERPRETATION: Doxorubicin does not need to be included in treatment of stage
II-III intermediate risk Wilms' tumour when the histological response to
preoperative chemotherapy is incorporated into the risk stratification.
FUNDING: See Acknowledgments for funders. BACKGROUND AND PURPOSE: Doxorubicin is a powerful antineoplastic agent for
treating a wide range of cancers. However, doxorubicin cardiotoxicity of the
heart has largely limited its clinical use. All-trans retinoic acid (ATRA) plays
an important role in many cardiac biological processes, but its protective
effects on doxorubicin-induced cardiotoxicity remain unknown. Here, we studied
the effect of ATRA on doxorubicin cardiotoxicity and the underlying mechanisms.
EXPERIMENTAL APPROACHES: Cellular viability assays, Western blotting and
mitochondrial respiration analyses were employed to evaluate the cellular
response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR and
gene knockdown were performed to investigate the underlying molecular mechanisms
of ATRA's effects on doxorubicin cardiotoxicity.
KEY RESULTS: ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2
cells and primary cardiomyocytes. ATRA was more effective against doxorubicin
cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive
oxygen species generation and restored expression levels of mRNA and proteins in
the phase II detoxifying enzyme system: nuclear factor-E2-related factor 2,
manganese superoxide dismutase, haem oxygenase-1, and mitochondrial function
(mitochondrial membrane integrity, mitochondrial DNA copy numbers and
mitochondrial respiration capacity, biogenesis and dynamics). Both a ERK1/2
inhibitor (U0126) and ERK2 siRNA, but not ERK1 siRNA, abolished the protective
effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably,
ATRA did not compromise the anticancer efficacy of doxorubicin in gastric
carcinoma cells.
CONCLUSIONS AND IMPLICATIONS: ATRA protected cardiomyocytes against
doxorubicin-induced toxicity, by activating the ERK2 pathway, without
compromising its anticancer efficacy. Therefore, ATRA is a promising candidate
as a cardioprotective agent against doxorubicin cardiotoxicity. Author information:
(1)Laboratory of Regenerative Medicine and Stem Cell Biology, Department of
Physiology, Medical Research Institute, School of Medicine, Pusan National
University, Yangsan 50612, Korea. [email protected].
(2)Cellular Therapeutics Team, Bio Reseach and Development Center, Daewoong
Pham. Co., Ltd., Seoul 06170, Korea. [email protected].
(3)Department of Thoracic and Cardiovascular Surgery; Pusan National University
Yangsan Hospital, Yangsan 50612, Korea. [email protected].
(4)Laboratory of Regenerative Medicine and Stem Cell Biology, Department of
Physiology, Medical Research Institute, School of Medicine, Pusan National
University, Yangsan 50612, Korea. [email protected].
(5)Laboratory of Regenerative Medicine and Stem Cell Biology, Department of
Physiology, Medical Research Institute, School of Medicine, Pusan National
University, Yangsan 50612, Korea. [email protected].
(6)Research Institute of Convergence Biomedical Science and Technology, Pusan
National University School of Medicine, Yangsan 50612, Korea.
[email protected].
(7)Division of Cardiology, Seoul St. Mary's Hospital, School of Medicine, The
Catholic University of Korea, Seoul 06591, Korea. [email protected].
(8)Laboratory of Regenerative Medicine and Stem Cell Biology, Department of
Physiology, Medical Research Institute, School of Medicine, Pusan National
University, Yangsan 50612, Korea. [email protected].
(9)Research Institute of Convergence Biomedical Science and Technology, Pusan
National University School of Medicine, Yangsan 50612, Korea.
[email protected]. |
Which diseases are associated with Primary intestinal lymphangiectasia (PIL)? | Primary intestinal lymphangiectasia (PIL) is associated with:
1) Waldmann's disease and
2) Hennekam syndrome (HS). | Primary intestinal lymphangiectasia (Waldmann's disease) is characterized by
protein-losing enteropathy occurring more frequently in childhood. Chronic
diarrhea and diffuse edema are the main clinical manifestations. Peripheral
lymphedema may also be associated. Lymphedema is usually present at the time of
diagnosis or appears later in the course of the disease. We report the
observation of a 31-year-old man suffering from an upper, lower limb and genital
lymphedema many years before diagnosis of primary intestinal lymphangiectasia
was established. Lower limb lymphoscintigraphy confirmed lymphedema and duodenal
biopsies lymphangiectasia. Hypoproteinemia, lymphopenia and
hypogammaglobulinemia were also noted. Treatment of lymphedema included low
stretch bandaging and elastic stocking. No dietary management with a low-fat
diet was added. Search for primary intestinal lymphangiectasia with biological
parameters would be useful when primary lymphedema is present. Especially since
primary intestinal lymphangiectasia may be complicated by occurrence of B cell
lymphoma. Primary intestinal lymphangiectasia (PIL), so-called Waldmann's disease, is an
uncommon condition, characterized by dilated intestinal submucosal and
subserosal lymphatics of the gastrointestinal tract. Protein-losing enteropathy
is the most common manifestation of this supposed congenital disease. Since the
initial description in 1961, 11 cases of lymphoma have been reported suggesting
that PIL predisposes to lymphoma. Here, we report the first case of primary
nodal location lymphoma during PIL with recovery of the protein-losing
enteropathy after its treatment by radiochemotherapy. Primary intestinal lymphangiectasis (PIL), also known as Waldmann's disease, is
a rare protein-losing enteropathy characterized by abnormal enlargement of the
lymphatic ducts in the bowel wall. The symptoms usually start in early infancy.
We report a case of osteomalacia in a 63-year-old patient with delayed-onset of
PIL, for which she was on dietary treatment. She presented with a 3-year history
of mechanical pain in the back and pelvis. Mild ascites and edema with
functional impairment of the lower limbs were noted. The neurological evaluation
was normal. Blood tests showed hypocalcemia, hypophosphatemia, alkaline
phosphatase elevation, and evidence of intestinal malabsorption. Radiographs of
the pelvis disclosed a fracture, Looser's zones in the iliopubic rami and left
femoral neck, and a washed-out appearance of the vertebras. Dual-energy X-ray
absorptiometry showed bone loss with T-score values of -1.2SD at the lumbar
spine and -2.5SD at the femoral necks. A diagnosis of osteomalacia related to
vitamin D deficiency was given. Serum 25-OH-vitamin D was 18.2ng/ml (normal,
20-40ng/ml) and serum parathyroid hormone was 620pg/ml (normal, 15-65pg/ml),
suggesting secondary hyperparathyroidism. Intramuscular vitamin D was given,
together with oral calcium and an adequate diet. At follow-up 8 months later,
small improvements were noted in the symptoms and absorptiometry findings. Primary intestinal lymphangiectasia (PIL) is a rare disorder characterized by
dilated intestinal lacteals resulting in lymph leakage into the small bowel
lumen and responsible for protein-losing enteropathy leading to lymphopenia,
hypoalbuminemia and hypogammaglobulinemia. PIL is generally diagnosed before 3
years of age but may be diagnosed in older patients. Prevalence is unknown. The
main symptom is predomitly bilateral lower limb edema. Edema may be moderate
to severe with anasarca and includes pleural effusion, pericarditis or chylous
ascites. Fatigue, abdominal pain, weight loss, inability to gain weight,
moderate diarrhea or fat-soluble vitamin deficiencies due to malabsorption may
also be present. In some patients, limb lymphedema is associated with PIL and is
difficult to distinguish lymphedema from edema. Exsudative enteropathy is
confirmed by the elevated 24-h stool alpha1-antitrypsin clearance. Etiology
remains unknown. Very rare familial cases of PIL have been reported. Diagnosis
is confirmed by endoscopic observation of intestinal lymphangiectasia with the
corresponding histology of intestinal biopsy specimens. Videocapsule endoscopy
may be useful when endoscopic findings are not contributive. Differential
diagnosis includes constrictive pericarditis, intestinal lymphoma, Whipple's
disease, Crohn's disease, intestinal tuberculosis, sarcoidosis or systemic
sclerosis. Several B-cell lymphomas confined to the gastrointestinal tract
(stomach, jejunum, midgut, ileum) or with extra-intestinal localizations were
reported in PIL patients. A low-fat diet associated with medium-chain
triglyceride supplementation is the cornerstone of PIL medical management. The
absence of fat in the diet prevents chyle engorgement of the intestinal
lymphatic vessels thereby preventing their rupture with its ensuing lymph loss.
Medium-chain triglycerides are absorbed directly into the portal venous
circulation and avoid lacteal overloading. Other inconsistently effective
treatments have been proposed for PIL patients, such as antiplasmin, octreotide
or corticosteroids. Surgical small-bowel resection is useful in the rare cases
with segmental and localized intestinal lymphangiectasia. The need for dietary
control appears to be permanent, because clinical and biochemical findings
reappear after low-fat diet withdrawal. PIL outcome may be severe even
life-threatening when maligt complications or serous effusion(s) occur. Primary intestinal lymphangiectasia (PIL) is a rare disorder characterized by
dilated intestinal lymphatics and the development of protein-losing enteropathy.
Patients with PIL develop hypoalbuminemia, hypocalcemia, lymphopenia and
hypogammaglobulinemia, and present with bilateral lower limb edema, fatigue,
abdominal pain and diarrhea. Endoscopy reveals diffusely elongated,
circumferential and polypoid mucosae covered with whitish enlarged villi, all of
which indicate intestinal lymphangiectasia. Diagnosis is confirmed by
characteristic tissue pathology, which includes dilated intestinal lymphatics
with diffusely swollen mucosa and enlarged villi. The prevalence of PIL has
increased since the introduction of capsule endoscopy. The etiology and
prevalence of PIL remain unknown. Some studies have reported that several genes
and regulatory molecules for lymphangiogenesis are related to PIL. We report the
case of a patient with PIL involving the entire small bowel that was confirmed
by capsule endoscopy and double-balloon enteroscopy-guided tissue pathology who
carried a deletion on chromosome 4q25. The relationship between this deletion on
chromosome 4 and PIL remains to be investigated. Primary intestinal lymphangiectasia (PIL) or Waldmann's disease is a rare
protein-losing gastroenteropathy of unknown etiology. Less than 200 cases have
been reported globally. Patients may be asymptomatic or present edema,
lymphedema, diarrhea, ascites and other manifestations. We report two pediatric
cases with PIL with extremely different outcome in a 3-year follow-up period.
The first patient presented with persistent diarrhea, hypoalbuminemia and
failure to thrive, while the second patient presented with an abrupt eyelid
edema. Hypoproteinemia was the common laboratory finding for the two patients
and upper gastrointestinal endoscopy established the diagnosis. The first
patient relapsed five times during the follow-up period after the diagnosis had
been made and required intravenous albumin administration and micronutrient
supplementation. The second patient revealed normal gastrointestinal endoscopy 4
months after the diagnosis had been established; he followed an unrestricted
diet and remained asymptomatic throughout the follow-up period. PIL can be
either severe, affecting the entire small bowel, leading to lifetime disease, or
sometimes affects part of the small bowel, leading to transient disorder. The rare combination of intestinal lymphangiectasia with malrotation of the
duodenum in a child of three months of life is described. Basing on the
literature review only 3 similar cases were described in the world practice. The
boy with protein-losing enteropathy was examined at Moscow Scientific Centre of
Children's Health. The child had vomiting, diarrhea, loss in body weight,
hypoproteinemia, lymphopenia. The infectious nature of the disease was excluded.
It had been suggested the Waldman desease (primary intestinal lymphangiectasia).
The prognosis for such disease is unfavorable. An examination of the child was
continued against the backdrop of ongoing symptomatic therapy. Complete physical
examination included monitoring laboratory blood tests, X-ray examination with
contrast, CT-scan, gastroduodenoscopy with biopsy of the mucosa of the small
intestine. Malrotation duodenum with the recurrent mid-gut volvulus with the
development of secondary intestinal lymphangiectasia was diagnosed. Modern
methods of examination and multidisciplinary approach made it possible to
diagnose the case. Operation to eliminate fixation duodenum resulted in the
recovery of the patient. At the present time the child grows and develops
according to age and does not require treatment. The prognosis for this disease
is regarded as favorable. BACKGROUND: Primary intestinal lymphangiectasia (Waldmann's disease) is a rare
disease characterized by dilated lymphatics in the small bowel leading to an
exudative enteropathy with lymphopenia, hypoalbuminemia and
hypogammaglobulinemia.
CASE PRESENTATION: We report the case of a 23 year-old male who presented with
chronic anemia and in whom primary intestinal lymphangiectasia was diagnosed. A
low-fat diet along with nutritional therapy with medium-chain triglyceride
supplementation improved the protein-losing enteropathy, but did not solve the
anemia. Octreotide was also unsuccessful, and after attempting angiographic
embolization therapy, limited small bowel resection together with antiplasmin
therapy managed to correct the anemia and control the exudative enteropathy.
CONCLUSIONS: Although primary intestinal lymphangiectasia is usually adequately
managed by nutritional therapy, complications such as anemia can occur and can
prove to be a therapeutic challenge. Primary intestinal lymphangiectasia (PIL), also known as Waldmann's disease, is
an exudative enteropathy resulting from morphologic abnormalities in the
intestinal lymphatics. In this article, we describe a 12-year-old boy with PIL
that led to protein-losing enteropathy characterized by diarrhea,
hypoalbuminemia associated with edema (serum albumin level: 1.0 g/dL), and
hypogammaglobulinemia (serum IgG level: 144 mg/dL). Severe hypoalbuminemia,
electrolyte abnormalities, and tetany persisted despite a low-fat diet and
propranolol. Everolimus (1.6 mg/m(2)/day) was added to his treatment as an
antiangiogenic agent. With everolimus treatment, the patient's diarrhea resolved
and replacement therapy for hypoproteinemia was less frequent. Hematologic and
scintigraphy findings also improved (serum albumin level: 2.5 g/dL). There were
no adverse reactions during the 12-month follow-up. To the best of our
knowledge, this is the first report of everolimus use in a patient with PIL. Primary intestinal lymphangiectasia (PIL) is rare disorder characterized by
congenital malformation or obstruction of intestinal lymphatic drainage; it is
responsible for protein losing enteropathy leading to lymphopenia,
hypoalbuminemia and hypogammaglobulinemia. A low-fat diet associated with
medium-chain triglyceride supplementation is the cornerstone of PIL management.
The administration of intravenous immunoglobulins does not always lead to
satisfactory plasma levels and therefore the replacement therapy with
immunoglobulins is controversial. We describe here the case of a patient with
PIL and severe hypogammaglobulinemia treated with immunoglobulins. The striking
aspect of this case is the clinical and serological benefit obtained with the
subcutaneous compared to the intravenous immunoglobulins administration. |
Is the mouse Sry gene locus free of repetitive sequences? | We demonstrate that the presence of long inverted repeats (IR) flanking the mouse Sry gene leads to the formation of the Sry circular transcript in cultured cells. We have found that in an in vitro assay, the SRY protein binds to several sites of the Sry gene and especially to a (CA)25 sequence and to a (CAG)30 repeat. | The testis-determining gene Sry is located on the short arm of the mouse Y
chromosome in a region known to have undergone duplications and rearrangements
in comparison with the equivalent portion of the human Y chromosome. Detailed
analysis of the Sry genomic locus reveals a further difference in that the mouse
Sry open reading frame lies within 2.8 kilobases of unique sequence at the
center of a large inverted repeat. This repeat, which is found in both Mus
musculus musculus and Mus musculus domesticus Y chromosomes, is not present at
the human SRY locus. Recombination involving the repeat region may have led to
an 11-kilobase deletion, precisely excising Sry in a line of XY female mice. Circular non-polyadenylated RNA molecules have been identified as stable
transcription products of the human ETS-1 and mouse Sry genes. RNA
circularization has been proposed to require two steps. The first step utilizes
intramolecular base pairing to produce a transient stem-loop structure. The
second step involves splicing a downstream donor splice site (DSS) to a now
closely appositioned upstream acceptor splice site (ASS) within the loop. We
demonstrate that the presence of long inverted repeats (IR) flanking the mouse
Sry gene leads to the formation of the Sry circular transcript in cultured
cells. Circularization requires the presence of both IR. As few as 400
complementary nt are necessary for this process. The presence of the IR does not
significantly stimulate intermolecular annealing and trans-splicing in vivo. The mouse testis determining gene Sry is expressed in somatic cells of the
differentiating male gonad as a linear transcript, encoding a transcription
factor containing an HMG box. In the adult mouse testis, Sry expression occurs
in meiotic and postmeiotic germ cells. The mouse genomic Sry locus is
characterized by two arms of a large inverted repeat, flanking a unique region
that, between an acceptor and a donor splice site, contains a single exon
encoding the Sry protein. In germ cells from the adult mouse testis, Sry RNA is
a circular molecule, which is generated by an inverted splicing event that
utilizes the above-mentioned splice sites. Thus, a circular exon is spliced out
starting from a large linear RNA precursor containing both arms of the inverted
repeat, which pair and generate a large stem-loop structure. Using reverse
transcription-polymerase chain reaction and an RNase protection assay, we have
now mapped the 5' end of this precursor RNA in the 5' arm of the inverted
repeat. Gel mobility shift assay and in vitro transcription with nuclear
extracts from adult germ cells further confirm that a region immediately 5'
upstream of two transcriptional initiation sites of the precursor RNA contains a
promoter sequence in which two consensus Sry binding sequences are specifically
recognized by nuclear factors present in adult germ cells but not in Sertoli
cells. We also show that the linear precursor of the Sry circular transcript and
its splicing product are specifically expressed not only in adult germ cells but
also in male embryonal gonads between 11.5 and 13.5 days postcoitum, immediately
after the expression of the linear transcript starting from the unique region. The Q-rich domain of the mouse sex determining gene, Sry, is encoded by an
in-frame insertion of a repetitive sequence composed of mostly CAG repeats. The
exact function of this Q-rich domain is unknown. Studies on the polymorphisms
within this Q-rich domain among different domesticus and musculus mouse strains
suggest a possible role for this domain in sex determination. Using the
farwestern protein-blotting technique and recombit fusion proteins containing
the Sry Q-rich domain as probes, three Sry interactive proteins of 94, 32 and 28
kDa apparent molecular weight (Sip-1, -2 and -3 respectively) were consistently
detected in adult testis. Sip expression was detected in somatic cells and was
associated with the spermatogenic activity of the testis. During embryogenesis,
Sips were readily detected in total tissue extracts of embryos as early as E8.5
day. In fetal gonads of both sexes, their expression peaked around E11.5-13.5
day, at the time of sex determination and differentiation, and decreased
drastically towards late stages of gestation. These observations support the
hypothesis that the Q-rich domain may contribute to the biological function(s)
of mouse Sry through a protein-protein interactive role(s). One of the first questions asked about excavated human skeletal remains is the
sex. As the morphological sex determination is complicated in cases involving
fragmentary bones and in skeletons from infants and children, the development of
DNA-based techniques has led to improvements in sex determination. This study is
focused on sex determination from ancient DNA obtained from 25 skeletons found
in Middle Aged burials in western Slovakia. We performed separate amplifications
of DXZ4 repetitive satellite sequences on the X chromosome, and SRY gene -
testis determined factor on the Y chromosome, using nested PCR. Our results
showed that DXZ4 was amplified in the case of 23 individuals. With newly
designed internal and external primer sets for SRY detection with internal PCR
products in lengths of 102 bp and 85 bp we succeeded in detecting the SRY locus
in 9 samples. Finally, the gender was determined in 23 individuals (14 females
and 9 males). In 20 samples, the gender was determined by morphological and
molecular methods. Sex determination of 17 samples using nested PCR matched the
morphological one, providing evidence of the authenticity and ancient origin of
the PCR amplifications. The DXZ4/SRY nested PCR method represents a useful
technique in sex determination of medieval human remains and it is a critical
addition to anthropological studies. |
Is Meis1 implicated in microphthalmia? | Yes. Meis1 coordinates a network of genes implicated in eye development and microphthalmia. Haploinsufficiency of Meis1, which encodes a transcription factor with evolutionarily conserved expression in the embryonic trunk, brain and sensory organs, including the eye, causes microphthalmic traits and visual impairment in adult mice. | Microphthalmos is a rare congenital anomaly characterized by reduced eye size
and visual deficits of variable degree. Sporadic and hereditary microphthalmos
have been associated with heterozygous mutations in genes fundamental for eye
development. Yet, many cases are idiopathic or await the identification of
molecular causes. Here we show that haploinsufficiency of Meis1, which encodes a
transcription factor with evolutionarily conserved expression in the embryonic
trunk, brain and sensory organs, including the eye, causes microphthalmic traits
and visual impairment in adult mice. By combining analysis of Meis1
loss-of-function and conditional Meis1 functional rescue with ChIP-seq and
RNA-seq approaches we show that, in contrast to its preferential association
with Hox-Pbx BSs in the trunk, Meis1 binds to Hox/Pbx-independent sites during
optic cup development. In the eye primordium, Meis1 coordinates, in a
dose-dependent manner, retinal proliferation and differentiation by regulating
genes responsible for human microphthalmia and components of the Notch signaling
pathway. In addition, Meis1 is required for eye patterning by controlling a set
of eye territory-specific transcription factors, so that in Meis1(-/-) embryos
boundaries among the different eye territories are shifted or blurred. We
propose that Meis1 is at the core of a genetic network implicated in eye
patterning/microphthalmia, and represents an additional candidate for syndromic
cases of these ocular malformations. |
Can Pentraxin 3 predict outcomes of sepsis? | Yes, Pentraxin 3 s an objective biochemical marker in diagnosis of sepsis; it is helpful in assessment of severity and prognosis of sepsis; it also has a certain clinical value in the assessment of sepsis cardiovascular function damage. | OBJECTIVE: To evaluate the recently discovered long pentraxin PTX3 in plasma of
critically ill patients and to compare it with the classic short pentraxin
C-reactive protein and with other indicators of inflammation.
DESIGN: A cohort study on plasma samples.
SETTING: Medical intensive care unit (ICU) of the University Hospital of Basel.
PATIENTS: A total of 101 consecutive critically ill patients admitted to the
medical ICU.
INTERVENTIONS: Venous blood samples were routinely obtained at entry, on day 2,
and at discharge or before death.
MEASUREMENTS AND MAIN RESULTS: Plasma samples were obtained from 101 consecutive
critically ill patients admitted to the ICU with systemic inflammatory response
syndrome, sepsis, or septic shock. PTX3 plasma levels were measured by
enzyme-linked immunosorbent assay. PTX3 was elevated in critically ill patients,
with a gradient from systemic inflammatory response syndrome to septic shock.
PTX3 levels correlated with clinical scores reflecting severity of disease
(e.g., Acute Physiology and Chronic Health Evaluation II: p =.00097). In
addition, high levels of PTX3 were associated with unfavorable outcome.
CONCLUSIONS: The long pentraxin PTX3 is elevated in critically ill patients and
correlates with severity of disease and infection. Compared with the short
pentraxin C-reactive protein, PTX3 may be a more direct indicator of tissue
involvement by inflammatory and infectious processes. PURPOSE: Pentraxin 3 (PTX3) is an inflammatory mediator produced by neutrophils,
macrophages, myeloid dendritic and endothelial cells. During sepsis a massive
inflammatory activation and coagulation/fibrinolysis dysfunction occur. PTX3, as
a mediator of inflammation, may represent an early marker of severity and
outcome in sepsis.
METHODS: This study is based on a prospective trial regarding the impact of
glycemic control on coagulation in sepsis. Ninety patients admitted to three
general intensive care units were enrolled when severe sepsis or septic shock
was diagnosed. At enrollment, we recorded sepsis signs, disease severity,
coagulation activation [prothrombin fragments 1 + 2 (F(1+2))] and fibrinolysis
inhibition [plasminogen activator inhibitor-1 (PAI-1)]. We measured plasma PTX3
levels at enrollment, everyday until day 7, then at days 9, 11, 13, 18, 23 and
28. Mortality was recorded at day 90.
RESULTS: Although not different on day 1, PTX3 remained significantly higher in
non-survivors than in survivors over the first 5 days (p = 0.002 by general
linear model). On day 1, PTX3 levels were higher in septic shock than in
severely septic patients (p = 0.029). Day 1 PTX3 was significantly correlated
with platelet count (p < 0.001), SAPS II score (p = 0.006) and SOFA score (p <
0.001). Day 1 PTX3 was correlated with F(1+2) concentration and with PAI-1
activity and concentration (p < 0.05 for all).
CONCLUSIONS: Persisting high levels of circulating PTX3 over the first days from
sepsis onset may be associated with mortality. PTX3 correlates with severity of
sepsis and with sepsis-associated coagulation/fibrinolysis dysfunction. INTRODUCTION: Long pentraxin 3 (PTX3) is an acute-phase protein secreted by
various cells, including leukocytes and endothelial cells. Like C-reactive
protein (CRP), it belongs to the pentraxin superfamily. Recent studies indicate
that high levels of PTX3 may be associated with mortality in sepsis. The
prognostic value of plasma PTX3 in bacteremic patients is unknown.
METHODS: Plasma PTX3 levels were measured in 132 patients with bacteremia caused
by Staphylococcus aureus, Streptococcus pneumoniae, β-hemolytic streptococcae
and Escherichia coli, using a commercial solid-phase enzyme-linked immunosorbent
assay (ELISA). Values were measured on days 1-4 after positive blood culture, on
day 13-18 and on recovery.
RESULTS: The maximum PTX3 values on days 1-4 were markedly higher in
nonsurvivors compared to survivors (44.8 vs 6.4 ng/ml, p<0.001) and the AUC(ROC)
in the prediction of case fatality was 0.82 (95% CI 0.73-0.91). PTX3 at a
cut-off level of 15 ng/ml showed 72% sensitivity and 81% specificity for fatal
disease. High PTX3 (>15 ng/ml) was associated with hypotension (MAP <70 mmHg)(OR
7.9;95% CI 3.3-19.0) and high SOFA score (≥4)(OR 13.2; 95% CI 4.9-35.4). The CRP
level (maximum value on days 1 to 4) did not predict case fatality at any
cut-off level in the ROC curve (p = 0.132). High PTX3 (>15 ng/ml) remained an
independent risk factor for case fatality in a logistic regression model
adjusted for potential confounders.
CONCLUSIONS: PTX3 proved to be a specific independent prognostic biomarker in
bacteremia. PTX3 during the first days after diagnosis showed better prognostic
value as compared to CRP, a widely used biomarker in clinical settings. PTX3
measurement offers a novel opportunity for the prognostic stratification of
bacteremia patients. BACKGROUND: Early diagnostic and prognostic stratification of patients with
suspected infection is a difficult clinical challenge. We studied plasma
pentraxin 3 (PTX3) upon admission to the emergency department in patients with
suspected infection.
METHODS: The study comprised 537 emergency room patients with suspected
infection: 59 with no systemic inflammatory response syndrome (SIRS) and without
bacterial infection (group 1), 67 with bacterial infection without SIRS (group
2), 54 with SIRS without bacterial infection (group 3), 308 with sepsis (SIRS
and bacterial infection) without organ failure (group 4) and 49 with severe
sepsis (group 5). Plasma PTX3 was measured on admission using a commercial
solid-phase enzyme-linked immunosorbent assay (ELISA).
RESULTS: The median PTX3 levels in groups 1-5 were 2.6 ng/ml, 4.4 ng/ml, 5.0
ng/ml, 6.1 ng/ml and 16.7 ng/ml, respectively (p<0.001). The median PTX3
concentration was higher in severe sepsis patients compared to others (16.7 vs.
4.9 ng/ml, p<0.001) and in non-survivors (day 28 case fatality) compared to
survivors (14.1 vs. 5.1 ng/ml, p<0.001). A high PTX3 level predicted the need
for ICU stay (p<0.001) and hypotension (p<0.001). AUC(ROC) in the prediction of
severe sepsis was 0.73 (95% CI 0.66-0.81, p<0.001) and 0.69 in case fatality
(95% CI 0.58-0.79, p<0.001). PTX3 at a cut-off level for 14.1 ng/ml (optimal
cut-off value for severe sepsis) showed 63% sensitivity and 80% specificity. At
a cut-off level 7.7 ng/ml (optimal cut-off value for case fatality) showed 70%
sensitivity and 63% specificity in predicting case fatality on day 28.In
multivariate models, high PTX3 remained an independent predictor of severe
sepsis and case fatality after adjusting for potential confounders.
CONCLUSIONS: A high PTX3 level on hospital admission predicts severe sepsis and
case fatality in patients with suspected infection. The long pentraxin-3 (PTX3) is a key component of the humoral arm of the innate
immune system. PTX3 is produced locally in response to pro-inflammatory stimuli.
To investigate PTX3 levels and its use as a biomarker in patients with systemic
inflammation, we developed a solid-phase enzyme-linked immunosorbent assay based
on novel anti-PTX3 monoclonal antibodies detecting PTX3 with high sensitivity.
The assay was applied on 261 consecutive patients admitted to an intensive care
unit prospectively monitored with the systemic inflammatory response syndrome
(SIRS). 100 blood donors were included as controls. PTX3 levels were elevated in
patients (median = 71.3 ng/ml) compared with the controls (median = 0 ng/ml)
(Mann-Whitney, p<0.0001). ROC analysis showed that PTX3 levels were
significantly specific (85.0%) and sensitive (89.1%) to discriminate between
healthy controls and patients (area under the curve (AUC) 0.922 (95% CI 0.892 to
0.946, p<0.0001)). Higher levels of PTX3 were associated with the development of
sepsis, severe sepsis and septic shock (p = 0.0001). The serum levels of PTX3
correlated significantly with SAPS2 score (Spearman's rho 0.28, p<0.0001).
Patients with high levels of PTX3 at admission did have a higher 90 day
mortality rate than patients with the 25% lowest levels (Cox regression
analysis, hazard ratio 3.0, p = 0.0009). In conclusion, we have established a
highly sensitive and robust assay for measurement of PTX3 and found that its
serum concentrations correlated with disease severity and mortality in patients
with SIRS and sepsis. In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for
biomarker discovery identified long pentraxin 3 (PTX3) as the lead candidate for
inflamed myocardium. When the redox-sensitive oligomerization state of PTX3 was
further investigated, PTX3 accumulated as an octamer as a result of
disulfide-bond formation in heart, kidney, and lung-common organ dysfunctions
seen in patients with sepsis. Oligomeric moieties of PTX3 were also detectable
in circulation. The oligomerization state of PTX3 was quantified over the first
11 days in critically ill adult patients with sepsis. On admission day, there
was no difference in the oligomerization state of PTX3 between survivors and
non-survivors. From day 2 onward, the conversion of octameric to monomeric PTX3
was consistently associated with a greater survival after 28 days of follow-up.
For example, by day 2 post-admission, octameric PTX3 was barely detectable in
survivors, but it still constituted more than half of the total PTX3 in
non-survivors (p < 0.001). Monomeric PTX3 was inversely associated with cardiac
damage markers NT-proBNP and high-sensitivity troponin I and T. Relative to the
conventional measurements of total PTX3 or NT-proBNP, the oligomerization of
PTX3 was a superior predictor of disease outcome. BACKGROUND: Pentraxin 3 (PTX3) is an acute phase reactant which has been used to
detect intra-amniotic infections (IAI) in pregcy, but the prognostic value of
PTX3 concentrations on neonates has not been studied. We aimed to investigate
the relationship between maternal PTX3-neonatal PTX3 concentrations and early
neonatal outcome.
METHODS: The mothers diagnosed with preterm prelabor rupture of membranes
(PPROM) (n = 28) and their preterm infants (n = 28) were included in the study.
PTX3 concentrations were studied in plasma in the maternal peripheral blood and
umbilical/peripheral vein in the neonates. The relationship between the
mPTX3-nPTX3 concentrations and neonatal outcome were investigated using
non-parametric tests and binary logistic regression analysis.
RESULTS: The mean mPTX3 concentration was 10.35 ± 7.82 μg/L. Ten (35.7%) of all
mothers were within the normal range and 18 (64.3%) in high percentile (≥ 97.5
percentile). There was no relation between mPTX3 concentrations and clinical or
histologic chorioamnionitis, latency of PPROM, and early neonatal outcome. Mean
nPTX3 concentrations was 9.18 ± 7.83 μg/L and high nPTX3 concentrations were
detected in five (17.8%) neonates. nPTX3 concentrations were inversely
correlated with gestational age and correlated with rate of intraventricular
hemorrhage (IVH) and mortality. Neonates with high nPTX3 concentrations also
have lowered APGAR scores, increased rate of respiratory distress syndrome,
clinical sepsis, IVH, necrotizing enterocolitis and prolonged NICU stay.
CONCLUSION: High PTX3 concentrations of the newborns are associated with some
worsened early neonatal outcome including lower gestational age at delivery,
increased rate of IVH and mortality. Maternal PTX3 concentrations are not an
adequate marker in defining clinical or histologic chorioamnionitis and early
neonatal outcome. Author information:
(1)Department of Quantitative Biology and Medicine, Research Center for Advanced
Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.
(2)Laboratory of Medical Proteomics, Institute of Medical Science, The
University of Tokyo, Tokyo 108-8639, Japan.
(3)Division of Cellular and Molecular Pathology, Department of Cellular
Function, Niigata University Graduate School of Medical and Dental Sciences,
Niigata 951-8510, Japan. Department of Pathology, Niigata University Medical and
Dental Hospital, Niigata 951-8520, Japan.
(4)Division of Cellular and Molecular Pathology, Department of Cellular
Function, Niigata University Graduate School of Medical and Dental Sciences,
Niigata 951-8510, Japan.
(5)Laboratory for Vascular Biology, Research Center for Advanced Science and
Technology, The University of Tokyo, Tokyo 153-8904, Japan.
(6)Department of Emergency and Critical Care Medicine, Juntendo University
Nerima Hospital, Tokyo 177-8521, Japan.
(7)Department of Cardiology, Juntendo University Nerima Hospital, Tokyo
177-8521, Japan.
(8)Division of Cellular and Molecular Pathology, Department of Cellular
Function, Niigata University Graduate School of Medical and Dental Sciences,
Niigata 951-8510, Japan. Niigata College of Medical Technology, Niigata
950-2076, Japan. Perseus Proteomics Inc., Tokyo 153-0041, Japan.
(9)Department of Quantitative Biology and Medicine, Research Center for Advanced
Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.
[email protected]. OBJECTIVE: To evaluate prognostic value of pentraxin3 ( PTX3 ) content combining
with extravascular lung water index ( EVLWI ) in patients with sepsis.
METHODS: A retrospective analysis of complete clinical data of septic patients
admitted to Department of Critical Care Medicine of the First Affiliated
Hospital of Zhengzhou University from February 2013 to February 2014 was
conducted. These patients were divided into two groups, survival group and death
group, according to the outcome on the 28th day. Pulse index continuous cardiac
output ( PiCCO ) was used to record the levels of EVLWI on the 1st, 2nd and 3rd
day of intensive care unit ( ICU ) admission. The plasma level of PTX3 was
measured simultaneously by enzyme-linked immunosorbent assay ( ELISA ). At the
same time, acute physiology and chronic health evaluation II ( APACHEII) score
and sequential organ failure assessment ( SOFA ) were calculated. Correlation
analysis between plasma PTX3 and EVLWI values was performed, receiver operating
characteristic curve ( ROC ) was drawn, and the prognostic value of each
parameter was assessed finally.
RESULTS: A total of 74 septic patients were enrolled, with 41 cases in the
survival group and 33 cases in the non-survival group. Blood lactate, APACHEII,
SOFA scores in the non-survival group were significantly higher than those of
the survival group at ICU admission, and the length of ICU stay was
significantly shorter than that of the survival group, while differences of the
other clinical characteristics between two groups were not statistically
significant. The plasma PTX3 level gradually declined with time in both groups,
and plasma PTX3 at 1, 2, 3 days after ICU admission in non-survival group were
significantly higher than those in survival group [ PTX3 ( μg/L ) at 1 day:
46.3±10.5 vs. 19.4±6.5, t = -13.486, P = 0.000; 2 days: 34.8±10.7 vs. 17.7±8.4,
t = -8.284, P = 0.000; 3 days: 23.9±11.2 vs. 15.6±7.9, t = -5.036, P = 0.000 ].
EVLWI gradually declined in survival group, but increased in death group. EVLWI
at 1, 2, 3 days after ICU admission in non-survival group were significantly
higher than those in survival group [ EVLWI ( mL/kg ) at 1 day: 12.12±4.31 vs.
10.02±2.87, t = -2.502, P = 0.023; 2 days: 13.67±4.95 vs. 9.08±2.89, t = -5.188,
P = 0.000; 3 days: 14.51±5.06 vs. 8.09±2.50, t = -7.126, P = 0.000 ]. PTX3 at 1,
2, 3 days after ICU admission showed a significant positive correlation with
EVLWI ( r1 = 0.747, r2 = 0.719, r3 = 0.705, all P = 0.000 ). ROC curve analysis
showed that the area under the ROC ( AUC ) of PTX3 at 1 day was 0.845±0.045, at
the cut-off point of 23.0 μg/L, PTX3 showed a sensitivity of 84.8%, a
specificity of 74.1%, a negative predictive value of 85.81%, and a positive
predictive value of 72.42%. AUC of EVLWI at 3 days was 0.838±0.048, at the
cut-off point of 10.5 mL/kg, EVLWI showed a sensitivity of 83.9%, a specificity
of 82.9%, a negative predictive value of 86.45%, and a positive predictive value
of 79.79%. Their sensitivities and specificities were found to be better than
APACHEII, SOFA score. AUC of PTX3 combined with EVLWI at 1 day was 0.886±0.038.
On the 1st day after ICU admission, with combination of the two indicators,
cut-off point was found to be 0.312, a sensitivity of 86.8%, a specificity of
85.4%, a negative predictive value of 88.93%, and a positive predictive value of
82.72%. On the 3rd day after ICU admission, AUC of PTX3 combined with EVLWI was
0.856±0.046, and showed a cut-off of 0.471 for the prognosis of sepsis, a
sensitivity of 85.8%, a specificity of 85.4%, a negative predictive value of
87.97%, and a positive predictive value of 82.50%. Compared with other single
index, a combination of above mentioned two indexes showed a better sensitivity
and specificity.
CONCLUSIONS: PTX3 can serve as a novel prognostic indicator at early stage in
septic patients. Combined with EVLWI, it shows important value in predicting
prognosis of septic patients, and it also provides guidance in treatment of
high-risk patients. BACKGROUND: Pentraxin-3 (PTX3) and soluble tumor necrosis factor (TNF)-like weak
inducer of apoptosis (sTWEAK) are new candidate prognostic markers for
comorbidities and mortality in various inflammatory diseases. Acute
decompensation of cirrhosis is characterized by acute exacerbation of chronic
systemic inflammation. Recently, increased circulating PTX3 levels have been
reported in nonalcoholic steatohepatitis patients and positively correlated with
disease severity. This study aims to explore serum PTX3/sTWEAK levels and their
relationship with clinical outcomes in cirrhotic patients with acute
decompensation.
METHODS: We analyzed serum PTX3/sTWEAK levels in relation to inhospital and
3-month new clinical events and survivals in cirrhotic patients with acute
decompensation.
RESULTS: During admission, serum PTX3/sTWEAK levels were significantly higher in
acute decompensated cirrhotic patients than controls and positively correlated
with protein-energy wasting (PEW), new infections, long hospital stays, high
medical costs, and high mortality. During a 3-month follow-up, acute
decompensated cirrhotic patients with high serum PTX3/sTWEAK levels had more
episodes of unplanned readmission and high 3-month mortality. On multivariate
analysis, high PTX3/sTWEAK levels and PEW were independent risk factors for high
mortality.
CONCLUSION: High serum PTX3/sTWEAK levels and PEW are common in cirrhotic
patients with acute decompensation. As compared with low serum PTX3 and sTWEAK
cases, cirrhotic patients with high serum PTX3/sTWEAK levels a have higher
probability of new severe infections, severe sepsis, septic shock, type 1
hepatorenal syndrome, in-hospital, and 3-month follow-up mortalities. Therefore,
high serum PTX3/sTWEAK levels on hospital admission predict disease severity and
case fatality in cirrhotic patients with acute decompensation. BACKGROUND: The long pentraxin PTX3 is a key component of the humoral arm of
innate immunity related to sepsis severity and mortality. We evaluated the
clinical and prognostic significance of circulating PTX3 in the largest cohort
ever reported of patients with severe sepsis or septic shock.
MATERIALS AND METHODS: Plasma PTX3 was measured on days 1, 2 and 7 after
randomization of 958 patients to albumin or crystalloids for fluid resuscitation
in the multicentre Albumin Italian Outcome Sepsis (ALBIOS) trial. We tested the
association of PTX3 and its changes over time with clinical severity, prevalent
and incident organ dysfunctions, 90-day mortality and treatment.
RESULTS: PTX3 was high at baseline (72 [33-186] ng/mL) and rose with the
severity and number of organ dysfunctions (P < 0·001) and the incidence of
subsequent new failures. The PTX3 concentration dropped from day 1 to 7, but
this decrease was less pronounced in patients with septic shock (P = 0·0004).
Higher concentrations of PTX3 on day 1 predicted incident organ dysfunctions.
Albumin supplementation was associated with lower levels of PTX3 in patients
with septic shock (P = 0·005) but not in those without shock. In a fully
adjusted multivariable model, PTX3 on day 7 predicted 90-day mortality. Smaller
drops in PTX3 predicted higher 90-day mortality.
CONCLUSIONS: In severe sepsis and septic shock, early high PTX3 predict
subsequent new organ failures, while a smaller drop in circulating PTX3 over
time predicts an increased risk of death. Patients with septic shock show lower
levels of PTX3 when assigned to albumin than to crystalloids. |
Which polymerase transcribes pri-miRNAs? | Recent evidence indicates that miRNA genes are transcribed by RNA polymerase II (Pol II) we have established a robust in vivo system in which pri-miRNA is transcribed by RNAP II and processed to pre-miRNA in HeLa cell nuclear extracts. | MicroRNAs (miRNAs) constitute a large family of noncoding RNAs that function as
guide molecules in diverse gene silencing pathways. Current efforts are focused
on the regulatory function of miRNAs, while little is known about how these
unusual genes themselves are regulated. Here we present the first direct
evidence that miRNA genes are transcribed by RNA polymerase II (pol II). The
primary miRNA transcripts (pri-miRNAs) contain cap structures as well as poly(A)
tails, which are the unique properties of class II gene transcripts. The
treatment of human cells with alpha-amanitin decreased the level of pri-miRNAs
at a concentration that selectively inhibits pol II activity. Furthermore,
chromatin immunoprecipitation analyses show that pol II is physically associated
with a miRNA promoter. We also describe, for the first time, the detailed
structure of a miRNA gene by determining the promoter and the terminator of
mir-23a approximately 27a approximately 24-2. These data indicate that pol II is
the main, if not the only, RNA polymerase for miRNA gene transcription. Our
study offers a basis for understanding the structure and regulation of miRNA
genes. The factors regulating the expression of microRNAs (miRNAs), a ubiquitous family
of approximately 22-nt noncoding regulatory RNAs, remain undefined. However, it
is known that miRNAs are first transcribed as a largely unstructured precursor,
termed a primary miRNA (pri-miRNA), which is sequentially processed in the
nucleus, to give the approximately 65-nt pre-miRNA hairpin intermediate, and
then in the cytoplasm, to give the mature miRNA. Here we have sought to identify
the RNA polymerase responsible for miRNA transcription and to define the
structure of a full-length human miRNA. We show that the pri-miRNA precursors
for nine human miRNAs are both capped and polyadenylated and report the sequence
of the full-length, approximately 3433-nt pri-miR-21 RNA. This pri-miR-21 gene
sequence is flanked 5' by a promoter element able to transcribe heterologous
mRNAs and 3' by a consensus polyadenylation sequence. Nuclear processing of
pri-miRNAs was found to be efficient, thus largely preventing the nuclear export
of full-length pri-miRNAs. Nevertheless, an intact miRNA stem-loop precursor
located in the 3' UTR of a protein coding gene only moderately inhibited
expression of the linked open reading frame, probably because the 3' truncated
mRNA could still be exported and expressed. Together, these data show that human
pri-miRNAs are not only structurally similar to mRNAs but can, in fact, function
both as pri-miRNAs and mRNAs. MicroRNAs (miRNAs) are a recently discovered group of small RNA molecules
involved in the regulation of gene expression. Analogously to mRNAs, the
non-protein-encoding pri-miRNAs are synthesized by RNA polymerase II and
post-transcriptionally modified by addition of a 5'-cap and a 3'-poly (A) tail.
Subsequently, the pri-miRNA undergoes a number of processing steps in the
nucleus and cytoplasm, and ends up as a mature approximately 22 nt miRNA, which
can exert its function by binding to the 3'-untranslated region of a subset of
mRNAs. Binding of the miRNA to the mRNA results in a reduced translation rate
and/or increased degradation of the mRNA. In this way a large number of cellular
pathways, such as cellular proliferation, differentiation, and apoptosis, are
regulated by mi-RNAs. As corruption of these pathways is the hallmark of many
cancers, dysregulation of miRNA biogenesis or expression levels may lead to
tumorigenesis. The mechanisms that alter the expression of miRNAs are similar to
those that change the expression levels of mRNAs of tumor suppressor- and
oncogenes, i.e. gross genomic aberrations, epigenetic changes, and minor
mutations affecting the expression level, processing, or target-interaction
potential of the miRNA. Furthermore, expression profiling of miRNAs has been
found to be useful for classification of different tumor types. Taken together,
miRNAs can be classified as onco-miRs or tumor suppressor-miRs, and may turn out
to be potential targets for cancer therapy. MicroRNAs (miRNAs) are 21 nt RNAs that regulate many biological processes in
plants by mediating translational inhibition or cleavage of target transcripts.
Arabidopsis mutants defective in miRNA biogenesis have overlapping and highly
pleiotropic phenotypes including serrated leaves and ABA hypersensitivity.
Recent evidence indicates that miRNA genes are transcribed by RNA polymerase II
(Pol II). Since Pol II transcripts are capped, we hypothesized that CBP
(cap-binding protein) 20 and 80 may bind to capped primary miRNA (pri-miRNA)
transcripts and play a role in their processing. Here, we show that cbp20 and
cbp80 mutants have reduced miRNA levels and increased pri-miRNA levels.
Co-immunoprecipitation experiments revealed that pri-miRNAs 159, 166, 168 and
172 could be associated with CBP20 and CBP80. We found that CBP20 and CBP80 are
stabilized by ABA by a post-translational mechanism, and these proteins are
needed for ABA induction of miR159 during seed germination. The lack of miR159
accumulation in ABA-treated seeds of cbp20/80 mutants leads to increased MYB33
and MYB101 transcript levels, and presumably higher levels of these positive
regulators result in ABA hypersensitivity. Genetic and molecular analyses show
that CBP20 and 80 have overlapping function in the same developmental pathway as
SE and HYL1. Our results identify new components in miRNA biogenesis. Canonical primary microRNA (pri-miRNA) precursors are transcribed by RNA
polymerase II and then processed by the Drosha endonuclease to generate
approximately 60 nt pre-miRNA hairpins. Pre-miRNAs in turn are cleaved by Dicer
to generate mature miRNAs. Previously, some short introns, called miRtrons, were
reported to fold into pre-miRNA hairpins after splicing and debranching, and
miRNAs can also be excised by Dicer cleavage of rare endogenous short hairpin
RNAs. Here we report that the miRNAs encoded by murine gamma-herpesvirus 68
(MHV68) are also generated via atypical mechanisms. Specifically, MHV68 miRNAs
are transcribed from RNA polymerase III promoters located within adjacent viral
tRNA-like sequences. The resultant pri-miRNAs, which bear a 5' tRNA moiety, are
not processed by Drosha but instead by cellular tRNase Z, which cleaves 3' to
the tRNA to liberate pre-miRNA hairpins that are then processed by Dicer to
yield the mature viral miRNAs. The majority of human miRNA genes is transcribed by polymerase II and can be
classified as class II genes similar to protein-coding genes. Whereas current
research on miRNAs has focused on the physiological and pathological functions,
the molecular mechanisms underlying their transcriptional regulation are largely
unknown. We recently reported that lipopolysaccharide (LPS) alters mature miRNA
expression profile in human biliary epithelial cells. In this study, we tested
the role of transcription factor NF-kappaB in LPS-induced transcription of
select miRNA genes. Of the majority of LPS-up-regulated mature miRNAs in
cultured human biliary epithelial cells, potential NF-kappaB binding sites were
identified in the putative promoter elements of their corresponding genes.
Inhibition of NF-kappaB activation by SC-514, an IKK2 inhibitor, blocked
LPS-induced up-regulation of a subset of pri-miRNAs, including pri-miR-17-92,
pri-miR-125b-1, pri-miR-21, pri-miR-23b-27b-24-1, pri-miR-30b, pri-miR-130a and
pri-miR-29a. Moreover, direct binding of NF-kappaB p65 subunit to the promoter
elements of mir-17-92, mir-125b-1, mir-21, mir-23b-27b-24-1, mir-30b and
mir-130a genes was identified by chromatin immunoprecipitation analysis and
confirmed by the luciferase reporter assay. Thus, a subset of miRNA genes is
regulated in human biliary epithelial cells through NF-kappaB activation induced
by LPS, suggesting a role of the NF-kappaB pathway in the transcriptional
regulation of miRNA genes. MicroRNAs (miRNA) participate in regulating diverse biological pathways by
translational repression in animals. They have attracted increasing attention
recently. However, little work has been done on the miRNA genes in
agriculturally important pests. Because the transcripts of most miRNA genes are
the products of type-II RNA polymerase, pri-miRNA has a poly(A) tail and appears
in expressed sequence tags (EST). We developed a computational pipeline to
identify miRNA genes from insect ESTs. First, 980,697 ESTs from 63 insects were
collected and used to search the nr database. The ESTs which did not share
significant similarities with any known protein-coding genes were treated as
non-coding ESTs. Next, known mature miRNAs were used to align with non-coding
ESTs. The ESTs which contain the sequence of mature miRNA were treated as
candidate ESTs. Finally, putative precursors were extracted flanking the mature
miRNA region in candidate ESTs and evaluated by the Triplet-SVM algorithm. As a
result, 86 miRNAs from 30 insect species were found based on a strict criterion
while 330 miRNAs from 51 species were found based on a loose criterion.
Evolution analysis indicated that mir-467, mir-297 and mir-466 were the highest
conserved miRNA families in insects. To confirm the reliability of putative
insect miRNAs, the expression profile of nine predicted miRNAs in Locusta
migratoria was investigated. Eight miRNAs were successfully detected by RT-PCR.
Most miRNAs were expressed ubiquitously at all examined tissues and
developmental stages whereas Lmi-mir-509 was specifically expressed in the
thorax of the 2nd, 4th and 5th instars and adult locust. In all, our work
reported an efficient computational strategy for predicting miRNA genes from
insect ESTs and presented tens of miRNAs in diverse insect species which are
expected to participate in many important physiological processes. Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool
in profiling gene expression levels. One of its many advantages is a lower
detection limit compared to other methods of gene expression profiling while
using smaller amounts of input for each assay. Automated qPCR setup has improved
this field by allowing for greater reproducibility. Its convenient and rapid
setup allows for high-throughput experiments, enabling the profiling of many
different genes simultaneously in each experiment. This method along with
internal plate controls also reduces experimental variables common to other
techniques. We recently developed a qPCR assay for profiling of pre-microRNAs
(pre-miRNAs) using a set of 186 primer pairs. MicroRNAs have emerged as a novel
class of small, non-coding RNAs with the ability to regulate many mRNA targets
at the post-transcriptional level. These small RNAs are first transcribed by RNA
polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved
into the precursor miRNA (pre-miRNA). Pre-miRNAs are exported to the cytoplasm
where Dicer cleaves the hairpin loop to yield mature miRNAs. Increases in miRNA
levels can be observed at both the precursor and mature miRNA levels and
profiling of both of these forms can be useful. There are several commercially
available assays for mature miRNAs; however, their high cost may deter
researchers from this profiling technique. Here, we discuss a cost-effective,
reliable, SYBR-based qPCR method of profiling pre-miRNAs. Changes in pre-miRNA
levels often reflect mature miRNA changes and can be a useful indicator of
mature miRNA expression. However, simultaneous profiling of both pre-miRNAs and
mature miRNAs may be optimal as they can contribute nonredundant information and
provide insight into microRNA processing. Furthermore, the technique described
here can be expanded to encompass the profiling of other library sets for
specific pathways or pathogens. In plant, primary transcripts (pri-miRNAs) transcribed from miRNA genes by RNA
polymerase II are first processed into stem-loop pre-miRNAs and further chopped
into ∼21 nt long miRNAs by RNase III-like enzyme DCL1. SERRATE (SE) protein is
an essential component for miRNA processing by assisting DCL1 for accurate
cleavage. Here we report the crystal structure of Arabidopsis SE core (residues
194-543) at 2.7 Å. SE core adopts the 'walking man-like' topology with
N-terminal α helices, C-terminal non-canonical zinc-finger domain and novel
Middle domain resembling the leading leg, the lagging leg and the body,
respectively. Pull-down assay shows that SE core provides the platform for HYL1
and DCL1 binding, whereas in vitro RNA binding and in vivo mutant rescue
experiments suggest that the non-canonical zinc-finger domain coupled with
C-terminal tail binds miRNA precursors. SE presumably works as a scaffold-like
protein capable of binding both protein and RNA to guide the positioning of
miRNA precursor toward DCL1 catalytic site within miRNA processing machinery in
plant. BACKGROUND: MicroRNAs (miRNAs) are non-coding RNAs with important roles in
regulating gene expression. Recent studies indicate that transcription and
cleavage of miRNA are coupled, and that chromatin structure may influence miRNA
transcription. However, little is known about the relationship between the
chromatin structure and cleavage of pre-miRNA from pri-miRNA.
RESULTS: By analysis of genome-wide nucleosome positioning data sets from human
and Caenorhabditis elegans (C. elegans), we found an enrichment of positioned
nucleosome on pre-miRNA genomic sequences, which is highly correlated with GC
content within pre-miRNA. In addition, obvious enrichments of three histone
modifications (H2BK5me1, H3K36me3 and H4K20me1) as well as RNA Polymerase II
(RNAPII) were observed on pre-miRNA genomic sequences corresponding to the
active-promoter miRNAs and expressed miRNAs.
CONCLUSION: Our results revealed the chromatin structure characteristics of
pre-miRNA genomic sequences, and implied potential mechanisms that can recognize
these characteristics, thus improving pre-miRNA cleavage. MicroRNAs (miRNAs) are non-coding, short (21-23nt) regulators of protein-coding
genes that are generally transcribed first into primary miRNA (pri-miR),
followed by the generation of precursor miRNA (pre-miR). This finally leads to
the production of the mature miRNA. A large amount of information is available
on the pre- and mature miRNAs. However, very little is known about the pri-miRs,
due to a lack of knowledge about their transcription start sites (TSSs). Based
on the genomic loci, miRNAs can be categorized into two types --intragenic
(intra-miR) and intergenic (inter-miR). While it is already an established fact
that intra-miRs are commonly transcribed in conjunction with their host genes,
the transcription machinery of inter-miRs is poorly understood. Although it is
assumed that miRNA promoters are similar in structure to gene promoters, since
both are transcribed by RNA polymerase II (Pol II), computational validations
exhibit poor performance of gene promoter prediction methods on miRNAs. In this
paper, we concentrate on the problem of TSS prediction for miRNAs. The present
study begins with the identification of positive and negative promoter samples
from recently published data stemming from RNA-sequencing studies. From these
samples of experimentally validated miRNA TSSs, a number of standard sequence
features are extracted. Furthermore, to account for potential footprints related
to promoter regulation by CpG dinucleotide targeted DNA methylation, a number of
novel features are defined. We develop a support vector machine (SVM) with RBF
kernel for the prediction of miRNA TSSs trained on human miRNA promoters. A
novel feature reduction technique based on archived multi-objective simulated
annealing (AMOSA) identifies the final set of features. The resulting model
trained on miRNA promoters shows improved performance over the one trained on
protein-coding gene promoters in terms of classification accuracy, sensitivity
and specificity. Results are also reported for a completely independent
biologically validated test set. In a part of the investigation, the proposed
approach is used to predict protein-coding gene TSSs. It shows a significantly
improved performance when compared to previously published gene TSS prediction
methods. MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of
gene expression. Mammalian miRNA biogenesis begins with cotranscriptional
cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor
complex. Although most miRNAs are located within introns of protein-coding
transcripts, a substantial minority of miRNAs originate from long noncoding
(lnc) RNAs, for which transcript processing is largely uncharacterized. We show,
by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide
analysis in human cell lines, that most lncRNA transcripts containing miRNAs
(lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway
but instead use Microprocessor cleavage to terminate transcription.
Microprocessor inactivation leads to extensive transcriptional readthrough of
lnc-pri-miRNA and transcriptional interference with downstream genes.
Consequently we define a new RNase III-mediated, polyadenylation-independent
mechanism of Pol II transcription termination in mammalian cells. MicroRNAs (miRNAs) regulate many biological processes such as development,
metabolism, and others. They are processed from their primary transcripts called
primary miRNA transcripts (pri-miRNAs) by the processor complex containing the
RNAse III enzyme, DICER-LIKE1 (DCL1), in plants. Consequently, miRNA biogenesis
is controlled through altering pri-miRNA accumulation and processing, which is
crucial for plant development and adaptation to environmental changes. Plant
pri-miRNAs are transcribed by DNA-dependent RNA polymerase II (Pol II) and their
levels are determined through transcription and degradation, whereas pri-miRNA
processing is affected by its structure, splicing, alternative splicing, loading
to the processor and the processor activity, which involve in many accessory
proteins. Here, we summarize recent progresses related to pri-miRNA
transcription, stability, and processing in plants. Previous studies in vivo reported that processing of primary microRNA
(pri-miRNA) is coupled to transcription by RNA polymerase II (RNAP II) and can
occur co-transcriptionally. Here we have established a robust in vivo system in
which pri-miRNA is transcribed by RNAP II and processed to pre-miRNA in HeLa
cell nuclear extracts. We show that both the kinetics and efficiency of
pri-miRNA processing are dramatically enhanced in this system compared to that
of the corresponding naked pri-miRNA. Moreover, this enhancement is general as
it occurs with multiple pri-miRNAs. We also show that nascent pri-miRNA is
efficiently processed before it is released from the DNA template. Together, our
work directly demonstrates that transcription and pri-miRNA processing are
functionally coupled and establishes the first in vivo model systems for this
functional coupling and for co-transcriptional processing. |
Which mutated enzyme is responsible for oculocutaneous 1 (OCA1)-type albinism? | Mutations in the gene for tyrosinase (TYR), the key enzyme in melanin synthesis, are responsible for oculocutaneous 1 (OCA1)-type albinism. | Mutations in the gene for the pigment-producing enzyme tyrosinase are
responsible for type IA (tyrosinase-negative) oculocutaneous albinism (OCA).
Most reported mutations have been single base substitutions. We now report three
different frameshift mutations in three unrelated individuals with type IA OCA.
The first individual has a single base deletion within a series of five
guanidines, resulting in a premature stop codon in exon I on one allele and a
missense mutation at codon 382 in exon III on the homologous allele. The second
individual is a genetic compound of two separate frameshift mutations, including
both the same exon I single base deletion found in the first individual and a
deletion of a thymidine-guanidine pair, within the sequence GTGTG, forming a
termination codon (TAG) in exon I on the homologous allele. The third individual
has a single base insertion in exon I on one allele and a missense mutation at
codon 373 in exon III on the homologous allele. The two missense mutations occur
within the copper Bbinding region and may interfere with either copper binding
to the enzyme or oxygen binding to the copper. These five different mutations
disrupt tyrosinase function and are associated with a total lack of melanin
biosynthesis. Mutations of the tyrosinase gene associated with a partial or complete loss of
enzymatic activity are responsible for tyrosinase related oculocutaneous
albinism (OCA1). A large number of mutations have been identified and their
analysis has provided insight into the biology of tyrosinase and the
pathogenesis of these different mutations. Missense mutations produce their
effect on the activity of an enzyme by altering an amino acid at a specific
site. The location of these mutations in the peptide can be used to indicate
potential domains important for enzymatic activity. Missense mutations of the
tyrosinase polypeptide cluster in four regions, suggesting that these are
important functional domains. Two of the potential domains involve the copper
binding sites while the others are likely involved in substrate binding. More
critical analysis of the copper binding domain of tyrosinase can be gained by
analyzing the structure of hemocyanin, a copper-binding protein with a high
degree of homology to tyrosinase in the copper binding region. This analysis
indicates a single catalytic site in tyrosinase for all enzymatic activities. Most types of human oculocutaneous albinism (OCA) result from mutations in the
gene for tyrosinase (OCA1) or the P protein (OCA2), although other types of OCA
have been described but have not been mapped to specific loci. Melanocytes were
cultured from an African-American with OCA, who exhibited the phenotype of Brown
OCA, and his normal fraternal twin. Melanocytes cultured from the patient with
OCA and the normal twin appeared brown versus black, respectively. Melanocytes
from both the patient with OCA and the normal twin demonstrated equal amounts of
NP-40-soluble melanin; however, melanocytes from the patient with OCA contained
only 7% of the amount of insoluble melanin found from the normal twin.
Tyrosinase- related protein-1 (TRP-1) was not detected in the OCA melanocytes by
use of various anti-TRP-1 probes. Furthermore, transcripts for TRP-1 were absent
in cultured OCA melanocytes. The affected twin was homozygous for a single-bp
deletion in exon 6, removing an A in codon 368 and leading to a premature stop
at codon 384. Tyrosine hydroxylase activity of the OCA melanocytes was
comparable to controls when assayed in cell lysates but was only 30% of controls
when assayed in intact cells. We conclude that this mutation of the human TRP-1
gene affects its interaction with tyrosinase, resulting in dysregulation of
tyrosinase activity, promotes the synthesis of brown versus black melanin, and
is responsible for a third genetic type of OCA in humans, which we classify as
"OCA3." Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme that catalyzes several
reactions in the biosynthesis of melanin pigments and is deficient in patients
with type I oculocutaneous albinism (OCA1). Tyrosinase is thought to bind two
copper ions, one at each of two conserved sequence motifs, termed CuA and CuB,
but to date this has been directly proved only for the Neurospora and mushroom
enzyme. Here, we demonstrate that mammalian tyrosinase directly binds copper,
and that the CuA and CuB sites are both required for copper binding and for
catalytic activity. We show that in human tyrosinase, copper binding by the CuB
site is most likely coordinated by residues His363, His367, and His389, and that
copper binding may be cooperative, with copper binding at one site facilitating
copper binding by the other site. Furthermore, correct folding of the tyrosinase
polypeptide appears to be necessary for copper binding, and a number of human
OCA1 mutations disrupt copper binding and thus catalytic function of tyrosinase. Albinism, caused by a deficiency of melanin pigment in the skin, hair, and eye
(oculocutaneous albinism [OCA]), or primarily in the eye (ocular albinism [OA]),
results from mutations in genes involved in the biosynthesis of melanin pigment.
The lack of melanin pigment in the developing eye leads to fovea hypoplasia and
abnormal routing of the optic nerves. These changes are responsible for the
nystagmus, strabismus, and reduced visual acuity common to all types of
albinism. Mutations in six genes have been reported to be responsible for
different types of oculocutaneous and ocular albinism, including the tyrosinase
gene (TYR) and OCA1 (MIM# 203100), the OCA2 gene and OCA2 (MIM# 203200), the
tyrosinase-related protein-1 gene (TYRP1) and OCA3 (MIM# 203290), the HPS gene
and Hermansky-Pudlak syndrome (MIM# 203300), the CHS gene (CHS1), and
Chediak-Higashi syndrome (MIM# 214500), and the X-linked ocular albinism gene
and OA1 (MIM#300500). The function of only two of the gene products is known
tyrosinase and tyrosinase-related protein-1 both of which are enzymes in the
melanin biosynthetic pathway. Continued mutational analysis coupled with
function/structure studies should aid our understanding of the function of the
remaining genes and their role in albinism. Mutation and polymorphism data on
these genes are available from the International Albinism Center Albinism
Database web site (http://www.cbc.umn.edu/tad). Type I oculocutaneous albinism (OCA1) is an autosomal recessive disorder, which
is caused by the reduction or the absence of tyrosinase activity in melanocytes
of the skin, hair and eyes. Although tyrosinase mutations of OCA1 have been
extensively analyzed in most populations worldwide, there is no systemic study
of OCA1 mutation in Chinese patients. By use of single strand conformation
polymorphism and direct sequencing, we had detected 21 mutant alleles out of 24
OCA1 chromosomes screened (87.5%). Detected mutant alleles include one splicing
site, three insertion/deletion and five missense mutations, of which the
splicing site nucleotide alteration (IVS 1-3C>G) and two each of the
insertion/deletion (232-233 ins GGG and 861-862 del TT) and missense mutations
(Cys 289 Gly and Trp 400 Leu) are novel. The ins/del mutations accounts for
about 37.5% in Chinese OCA1 alleles. The 232-233 ins GGG, one of the novel
mutations, was found to be most frequent (25%) among the OCA1 alleles in
Chinese. Through this study, we found that while some of the OCA mutant alleles
were identified in other populations, ethnic difference still exists. Hum Mutat
14:542, 1999. Mutations in the human tyrosinase gene produce tyrosinase-related oculocutaneous
albinism (OCA1, MIM #203100). Tyrosinase is a copper containing enzyme and is
responsible for catalyzing the rate limiting step in melanin biosynthesis, the
hydroxylation of tyrosine to dopaquinone. We report 13 new mutations in the
tyrosinase gene associated with OCA1A (without pigment) and OCA1B (with pigment)
including 9 missense mutations (H19Q, R521, R77C, G97R, C289R, L312V, P313R,
F340L and H404P), two nonsense mutations (W80X and R116X) and two frameshift
mutations (53delG and 223 delG). Our previous work has defined clusters of
missense mutations that appear to represent functional domains of the enzyme,
and three of the missense mutations fall into these clusters including two
(F340L and H404P) that flank the copper B bindng site and the missense mutation
R52I that is located in the amino terminal end cluster of the protein. The G97R
missense mutation is the first identified within the epidermal growth factor
(EGF)-like sequence and the H19Q missense mutation alters the cleavage site of
the signal peptide sequence. Mutational analysis can provide a definitive
diagnosis of the type of OCA as well as help structure/function analysis. Mutations in the human P gene lead to oculocutaneous albinism type 2 (OCA2, MIM
#203200), the most common type of albinism in humans. The P gene encodes a 110
kDa protein that is associated with melonosomal membranes and contains 12
potential membrane spanning domains. The specific function of the P protein is
currently unknown. We report 7 new mutations in the P gene associated with OCA2.
This includes 6 missense mutations (S86R, C112F, A368V, T592I, A724P and A787V)
and one frameshift mutation (1047del7). We also report 8 polymorphisms including
one amino acid substitution, D/A257. We and others have found many polymorphisms
of the P gene in the coding region, several of which result in amino acid
substitutions, making molecular diagnosis problematic. In contrast to this is
the tyrosinase gene associated with OCA1, with a limited number of polymorphic
variations in the coding region. There is also no apparent clustering of P gene
missense mutations in contrast to the clustering observed by the tyrosinase gene
missense mutations that define functional domains of the protein. Further
mutational analysis is needed to help define the critical functional domains of
the P protein and to allow a definitive diagnosis of OCA2. Through the last century there has been a steady progression in our
understanding of the biology of melanin biosynthesis. Much of this work includes
the analysis of coat color mutations of the mouse and albinism in man. Our
understanding has been greatly enhanced in the last 10 years, as the molecular
pathogenesis of albinism has been better understood. Different mutations of the
tyrosinase gene (TYR) , and their association with oculocutaneous albinism type
1 (OCA1) has provided insight into the biology of tyrosinase, including protein
trafficking and structure/function analysis. Several questions still remain,
including cryptic mutations that affect tyrosinase activity and the minimum
amount of pigment required for normal optic development. The next 10 years
should prove just as exciting as the last. The sequence of the tyrosinase (Tyr) gene coding tracts has been obtained for
the gorilla (Gorilla gorilla gorilla). The five exons of the gene were sequenced
in three gorillas and in a normally pigmented human. The tyrosinase gene has
been found to be a very conserved locus with a very low substitution rate. Some
nucleotide and amino acid differences were found between the gorilla and human
tyrosinase coding sequences. One of the gorillas included in the study is the
only known case of albinism in a gorilla ('Snowflake'). Mutations of the TYR
gene lead to Oculocutaneous Albinism type 1 (OCA1), the most common type of
albinism in humans (OMIM accession number 203100). The TYR gene encodes the
tyrosinase enzyme (E.C. 1.14.18.1), whose activity was found to be completely
lacking in 'Snowflake', indicating that a mutation in the Tyr gene is the likely
cause of his albinism. Nonetheless, no nucleotide changes were detected that
could account for the lack of Tyr product or tyrosinase activity in Snowflake,
and explanations of these findings are discussed. Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disease
resulting from mutations of the tyrosinase gene (TYR). To elucidate the
molecular basis of OCA1 phenotypes, we analysed the early processing and
maturation of several different types of mutant tyrosinase with various degrees
of structural abnormalities (i.e. two large deletion mutants, two missense
mutants that completely destroy catalytic function and three missense mutants
that have a temperature-sensitive phenotype). When expressed in COS7 cells, all
mutant tyrosinases were sensitive to endoglycosidase H digestion, and
immunostaining showed their localization in the endoplasmic reticulum (ER) and
their failure to be sorted further to their target organelles. Pulse-chase
experiments showed that all mutant tyrosinases were retained by calnexin in the
ER and that they were degraded at similarly rapid rates, which coincided with
their dissociation from calnexin. Temperature-sensitive mutant enzymes were
sorted more efficiently at 31 degrees C than at 37 degrees C, and their
degradation was accelerated at 37 degrees C compared with 31 degrees C. Thus in
contrast to the current concept that mutant tyrosinases are transported to
melanosomes but are functionally inactive there, our results suggest that mutant
tyrosinases may not be transported to melanosomes in the first place. We
conclude that a significant component of mutant tyrosinase malfunction in OCA1
results from their retention and degradation in the ER compartment. This
quality-control process is highly sensitive to minimal changes in protein
folding, and so even relatively minor mutations in peripheral sequences of the
enzyme not involved with catalytic activity may result in a significant
reduction of functional enzyme in melanosomes. Oculocutaneous albinism (OCA) affects approximately 1/20,000 people worldwide.
All forms of OCA exhibit generalized hypopigmentation. Reduced pigmentation
during eye development results in misrouting of the optic nerves, nystagmus,
alternating strabismus, and reduced visual acuity. Loss of pigmentation in the
skin leads to an increased risk for skin cancer. Two common forms and one
infrequent form of OCA have been described. OCA1 (MIM 203100) is associated with
mutations of the TYR gene encoding tyrosinase (the rate-limiting enzyme in the
production of melanin pigment) and accounts for approximately 40% of OCA
worldwide. OCA2 (MIM 203200), the most common form of OCA, is associated with
mutations of the P gene and accounts for approximately 50% of OCA worldwide.
OCA3 (MIM 203290), a rare form of OCA and also known as "rufous/red albinism,"
is associated with mutations in TYRP1 (encoding tyrosinase-related protein 1).
Analysis of the TYR and P genes in patients with OCA suggests that other genes
may be associated with OCA. We have identified the mouse underwhite gene (uw)
and its human orthologue, which underlies a new form of human OCA, termed
"OCA4." The encoded protein, MATP (for "membrane-associated transporter
protein") is predicted to span the membrane 12 times and likely functions as a
transporter. Research on human albinism has been central to many of the major discoveries in
human genetics. These include the first evidence that Mendel's rules of genetic
segregation apply to humans, first published in 1903. Contrary to initial
thought that albinism is caused by mutations in a single gene, we now know that
the genetics of albinism are complex. The complexity of albinism was hinted at,
in early publications, but has only recently been fully appreciated with the
advent of molecular techniques. Currently, 12 different genes have been
identified, that when mutated, result in a different type of albinism.
Oculocutaneous albinism type 1 (OCA1), resulting from mutations of the
tyrosinase gene, is genetically and biochemically the best understood type of
albinism. Though much of the research in albinism has involved OCA1, there are
many uswered questions about OCA1 and albinism, in general. The next 100 yr
should still provide many surprises as did the first 100 yr. Oculocutaneous albinism (OCA) in man may be caused by mutations within the
tyrosinase gene (TYR) resulting in OCA1. Analysing patients with recessively
inherited albinism we found DNA variations in 82 unrelated individuals. 53 out
of 78 mutations and polymorphisms revealed by this study are not published
previously. The changes include 68 nucleotide substitutions resulting in amino
acid changes, stop mutations and polymorphisms as well as four nucleotide
insertions and six deletions. Furthermore, we found an accumulation of three to
five mutations in 17 patients with OCA1. BACKGROUND: Oculocutaneous albinism (OCA) is a heterogeneous congenital
disorder. Tyrosinase is a key enzyme in melanin biosynthesis, and tyrosinase
gene mutations cause the OCA1 subtype.
OBJECTIVE: This study was intended evaluate the frequency and details of
tyrosinase gene mutations in Japanese OCA patients.
PATIENTS AND METHODS: We examined nine non-consanguineous OCA families,
sequenced the tyrosinase gene of the patients and also confirmed a splicing site
mutation using exon trapping system.
RESULTS: Tyrosinase gene mutations were identified in five out of nine OCA
families (55%). IVS2-10deltt-7t-a was present in 3 out of 18 alleles in three
families (16%), P310insC was present in three alleles in three families (16%)
and R278X was found in three alleles (16%), including those in one heterozygous
and one compound homozygous patient. G97V (290 G-T) was found in 1 out of 18
alleles, and we could not find G97V in the mutation database. We have added this
mutation as 9th mutation of Japanese OCA1 patients. In 8 of 18 alleles, four
families, no tyrosinase mutations were identified. They were presumed not to be
OCA1, but other subtypes of OCA. Exon trapping system demonstrated
IVS2-10deltt-7t-a mutation generated the abnormal splicing site, and inserted
the codon 4 bases in mRNA level resulting in premature termination codon
downstream.
CONCLUSION: This study provided new information about OCA1 mutations, and
highlights the requirement of broader detailed search to make precise diagnosis
of OCA. PURPOSE: Oculocutaneous albinism type 1 (OCA1) patients demonstrate a partial or
total lack of melanin in the skin, hair and eye. OCA1 is an autosomal recessive
genetic disorder caused by mutations in the TYR gene located at chromosome band
11q14-q25. The purpose of this study was to carry out genetic analysis of OCA1
in Indian families.
METHODS: Genomic DNA was isolated from blood leukocytes of all the individuals
in this study. Haplotype analysis was performed at the TYR locus using
informative microsatellite markers. Eight sets of primers were used to amplify
the entire coding region of the TYR gene for bidirectional direct sequencing
mutation analysis.
RESULTS: Two novel deletions (c.937del8, c.1379del2) and a previously known
nonsense mutation (R278X) in the TYR gene were identified from a total of 8
oculocutaneous albinism patients in India.
CONCLUSIONS: Our study reports the distribution of two novel frameshift and a
previously reported nonsense mutations in four OCA1 families from the Indian
population. These findings will contribute to the development of a diagnostic
method for OCA1 carrier status and genetic counseling for OCA1 affected
families. Tyrosinase serves as a key enzyme in the synthesis of melanin. In humans
mutations in the TYR gene are associated with type 1 oculocutaneous albinism
(OCA1) that leads to reduced or absent pigmentation of skin, hair and eye.
Various mutations causing OCA in man, mouse, rabbit and cattle have been
identified throughout the Tyrosinase gene including nonsense, missense,
frameshift and splice site alterations. Here we report a missense substitution
at codon R299H in exon 2 of the Tyr gene in the albino Wistar rat. As this very
exchange has already been described in OCA patients, our findings reinforce the
significance of this region for normal catalytic activity of tyrosinase protein. Oculocutaneous albinism type 1 (OCA1) results from mutations in the tyrosinase
gene, which lead to partial or complete loss of activity of the corresponding
enzyme. A large number of mutations have been identified worldwide, providing
insight into the pathogenesis of the disorder. We performed ophthalmic and
dermatological exams on 30 Lebanese subjects with oculocutaneous albinism, then
screened for mutations in the tyrosinase gene in an effort to establish the
molecular basis of the disorder in our population and correlate it with
phenotypic findings. The five exons of the gene together with the exon-intron
boundaries and part of the promoter region were sequenced. Mutations were found
in a total of 14 patients (47%) while no mutation was identified in the
sequenced regions in 53% of patients. Fourteen different mutations were
identified of which eight were novel while six had been previously reported.
Mutations were mainly seen in patients with clinical findings, suggestive of
OCA1A (64% of patients with OCA1A versus 25% of patients with OCA1B); therefore,
the absence of mutations in some of the other patients may indicate the
involvement of other genes. 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. 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. Mutation of the tyrosinase gene (TYR) causes oculocutaneous albinism, type 1
(OCA1), a condition characterized by reduced skin and eye melanin pigmentation
and by vision loss. The retinal pigment epithelium influences postnatal visual
development. Therefore, increasing ocular pigmentation in patients with OCA1
might enhance visual function. There are 2 forms of OCA1, OCA-1A and OCA-1B.
Individuals with the former lack functional tyrosinase and therefore lack
melanin, while individuals with the latter produce some melanin. We hypothesized
that increasing plasma tyrosine concentrations using nitisinone, an FDA-approved
inhibitor of tyrosine degradation, could stabilize tyrosinase and improve
pigmentation in individuals with OCA1. Here, we tested this hypothesis in mice
homozygous for either the Tyrc-2J null allele or the Tyrc-h allele, which model
OCA-1A and OCA-1B, respectively. Only nitisinone-treated Tyrc-h/c-h mice
manifested increased pigmentation in their fur and irides and had more pigmented
melanosomes. High levels of tyrosine improved the stability and enzymatic
function of the Tyrc-h protein and also increased overall melanin levels in
melanocytes from a human with OCA-1B. These results suggest that the use of
nitisinone in OCA-1B patients could improve their pigmentation and potentially
ameliorate vision loss. 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. OBJECTIVE: To explore the patients' genotypes and the mutation spectrum of
Tyrosinase (TYR) gene and the effects on protein structure and function in
oculocutaneous albinism type 1 (OCA1).
METHODS: The polymerase chain reaction (PCR) and sequencing techniques were
applied to amplify and analyze the regions of exon, exonintron and promoter of
TYR gene of 15 OCA1 probands and some of their parents. The protein structure
and function were forecasted and analyzed by bioinformatics software.
RESULTS: Sequencing result showed 11 kinds of mutations, including 5 missense
mutations (W400L, R299H, E294K, R77Q and K142M), 3 nonsense mutations (R116X,
R278X and G295X), 2 insertion mutation (929insC and 232insGGG) and 1 splice site
mutation (IVS1-3C > G). The nosogenesis was related to the change of protein
structure and function in four pathological mutations.
CONCLUSION: It seemes that W400L is the frequent mutations, which accounted for
about 30.0% in Chinese mainland OCA1 alleles. It is doable to make some
reasonable interpretation about TYR gene nosogenesis by bioinformatics method. OBJECTIVE: To evaluate the feasibility of genetic analysis of tyrosinase gene
(TYR) in oculocutaneous albinism type I (OCA1). Mutation analysis and prenatal
genetic diagnosis of TYR gene for seven pedigrees with OCA1 were performed.
METHODS: PCR was used to amplify the exons, exon-intron boundaries and promoter
of the TYR gene in the probands and/or their parents. The products were further
analyzed by direct sequencing. Prenatal genetic diagnoses were performed by
chorionic villus sampling after the genotypes of the probands or their parents
were determined.
RESULTS: Compound heterozygous mutations were detected in all pedigrees, which
included 9 mutations, namely R76Q, c.232insGGG, R116X, R278X, R299H,
c.929-930insC, IVS2-11delTT, Q399X and W400L. Among these, R76Q and Q399X were
identified for the first time. Seven families have requested prenatal diagnoses.
One fetus was detected with double mutations of TYR gene, and the parents have
decided to have therapeutic abortion. Two fetuses did not carry the mutations
identified in the probands, whilst other four fetuses were carriers of
heterozygous mutations. Six families decided to carry on with the pregcies.
And the neonates did not show any symptoms of OCA after birth.
CONCLUSION: Direct sequencing of the TYR gene is helpful for genetic counseling,
prenatal diagnosis and carriers screening of OCA1. 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. INTRODUCTION: Oculocutaneus albinism is a pigment-related inherited disorder
characterized by hypopigmentation of the skin, hair and eyes, foveal hypoplasia
and low vision. To date, 230 mutations in the TYR gene have been reported as
responsible for oculocutaneus albinism type 1 worldwide. TYR gene encodes the
enzyme tyrosinase involved in the metabolic pathway of melanin synthesis.
OBJECTIVES: Mutations were identified in the TYR gene as responsible for
oculocutaneous albinism type 1 in five Colombian individuals, and a new
ophthalmic system was tested that corrected visual defects and symptoms in a
patient with oculocutaneous albinism.
MATERIALS AND METHODS: Samples were taken from 5 individuals, four of whom
belong to a single family, along with a fifth individual not related to the
family. Five exons in the TYR gene were sequenced to search for the gene
carriers in the family and in the non-related individual. In addition, clinical
ophthalmological evaluation and implementation of an new oculo-visual system was
undertaken.
RESULTS: A G47D and 1379delTT mutation was identified in the family. The
unrelated individual carried a compound heterozygote for the G47D and D42N
mutations. The oculo-visual corrective system was able to increase visual acuity
and to diminish the nystagmus and photophobia.
CONCLUSIONS: This is the first study in Colombia where albinism mutations are
reported. The methods developed will enable future molecular screening studies
in Colombian populations. BACKGROUND: Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin
production and its gene (TYR) is mutated in many cases of oculocutaneous
albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients
with reduced TYR activity are classified as OCA1B; some OCA1B mutations are
temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part,
by the absence of purified, active, recombit wild-type and mutant human
enzymes.
METHODOLOGY/PRINCIPAL FINDINGS: The intra-melanosomal domain of human tyrosinase
(residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and
R422W were expressed in insect cells and produced in T. ni larvae. The short
trans-membrane fragment was deleted to avoid potential protein insolubility,
while preserving all other functional features of the enzymes. Purified
tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The
protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and
neutral pH. The two purified mutants when compared to the wild-type protein were
less active and temperature sensitive. These differences are associated with
conformational perturbations in secondary structure.
CONCLUSIONS/SIGNIFICANCE: The intramelanosomal domains of recombit wild-type
and mutant human tyrosinases are soluble monomeric glycoproteins with activities
which mirror their in vivo function. This advance allows for the structure -
function analyses of different mutant TYR proteins and correlation with their
corresponding human phenotypes; it also provides an important tool to discover
drugs that may improve tyrosinase activity and treat OCA1. BACKGROUND: Oculocutaneous albinism (OCA) is a congenital genetic disorder
characterized by defects in melanin production. OCA type 1 (OCA1) is the most
serious and common type of OCA. This study characterized mutations associated
with OCA1 in a series of Chinese patients.
METHODS: We recruited 41 unrelated patients with OCA and 100 healthy subjects
from the Chinese Han population. Genomic DNA was extracted from their blood
samples. Mutational analysis of tyrosinase (TYR) genes was conducted using
polymerase chain reaction (PCR) and direct sequencing, specifically to test the
100 control subjects and exclude the possibility of polymorphism. Mutational
analysis and bioinformatics study were performed in TYR mutations.
RESULTS: Among the 24 (58.5%) patients with OCA1, 21 different TYR mutations
were identified, including three previously unidentified alleles (PUAs): one
frameshift mutation (c.216delA) and two missense mutations (A241T and N364K).
The proband mutation A241T carries three possible mutations in complex OCA.
CONCLUSION: The findings of this study expand current knowledge and data of
mutations associated with OCA1 in China and allow us to estimate or explore the
mutation spectrum and relative frequencies of the TYR gene in the Chinese
population. The TYR gene (MIM #6069333) is located at position 11q14.3 on the human
chromosome, and encodes tyrosinase, which is expressed in melanocytes and
controls the biosynthesis of melanin. Most TYR mutations eliminate the activity
of tyrosinase, preventing melanocytes from producing any melanin throughout
life. People with this form of albinism have white hair, light-coloured eyes and
very pale skin. Some mutations in TYR reduce but do not completely eliminate
tyrosinase activity, and allow some melanin to be produced. We report a
Pakistani family with four members affected by oculocutaneous albinism (OCA).
Blood samples were collected from all affected individuals, normal siblings and
their parents. Genomic DNA was extracted, and sequence analysis of all the
coding exons and adjacent intronic sequences of TYR was performed, which
identified a novel missense substitution (p.Ile198Thr). Sequencing of TYR in 90
unrelated healthy individuals showed no sequence variant at this location. Our
study expands the mutational spectrum of OCA1. Oculocutaneous albinism (OCA) is an autosomal recessive disorder caused by
either complete lack of or a reduction of melanin biosynthesis in the
melanocytes. The OCA1A is the most severe type with a complete lack of melanin
production throughout life, while the milder forms OCA1B, OCA2, OCA3, and OCA4
show some pigment accumulation over time. Mutations in TYR, OCA2, TYRP1, and
SLC45A2 are mainly responsible for causing oculocutaneous albinism. Recently,
two new genes SLC24A5 and C10orf11 are identified that are responsible to cause
OCA6 and OCA7, respectively. Also a locus has been mapped to the human
chromosome 4q24 region which is responsible for genetic cause of OCA5. In this
paper, we summarized the clinical and molecular features of OCA genes. Further,
we reviewed the screening of pathological mutations of OCA genes and its
molecular mechanism of the protein upon mutation by in silico approach. We also
reviewed TYR (T373K, N371Y, M370T, and P313R), OCA2 (R305W), TYRP1 (R326H and
R356Q) mutations and their structural consequences at molecular level. It is
observed that the pathological genetic mutations and their structural and
functional significance of OCA genes will aid in development of personalized
medicine for albinism patients. Oculocutaneous albinism (OCA) is a heterogeneous group of autosomal recessive
disorders resulting from mutations of the tyrosinase (TYR) gene and presents
with either complete or partial absence of pigment in the skin, hair and eyes
due to a defect in an enzyme involved in the production of melanin. In this
study, mutations in the TYR gene of 30 unrelated Iranian OCA1 patients and 100
healthy individuals were examined using PCR-sequencing. Additionally, in order
to predict the possible effects of new mutations on the structure and function
of tyrosinase, these mutations were analyzed by SIFT, PolyPhen and I-Mutant 2
software. Here, two new pathogenic p.C89S and p.H180R mutations were detected in
two OCA1 patients. Moreover, the R402Q and S192Y variants, which are common
non-pathogenic polymorphisms, were detected in 17.5% and 35% of the patients,
respectively. The outcome of this study has extended the genotypic spectrum of
OCA1 patients, which paves the way for more efficient carrier detection and
genetic counseling. Oculocutaneous albinism (OCA) is a heterogeneous autosomal recessive genetic
disorder that affects melanin synthesis. OCA results in reduced or absent
pigmentation in the hair, skin and eyes. Type 1 OCA (OCA1) is the result of
tyrosinase (TYR) gene mutations and is a severe disease type. This study
investigated TYR mutations in a Chinese cohort with OCA1. This study included
two parts: patient genetic study and prenatal genetic diagnosis. A total of 30
OCA1 patients were subjected to TYR gene mutation analysis. Ten pedigrees were
included for prenatal genetic diagnosis. A total of 100 unrelated healthy
Chinese individuals were genotyped for controls. The coding sequence and the
intron/exon junctions of TYR were analysed by bidirectional DNA sequencing. In
this study, 20 mutations were identified, four of which were novel. Of these 30
OCA1 patients, 25 patients were TYR compound heterozygous; two patients carried
homozygous TYR mutations; and three were heterozygous. Among the ten prenatally
genotyped fetuses, three fetuses carried compound heterozygous mutations and
seven carried no mutation or only one mutant allele of TYR and appeared normal
at birth. In conclusion, we identified four novel TYR mutations and showed that
molecular-based prenatal screening to detect TYR mutations in a fetus at risk
for OCA1 provided essential information for genetic counselling of couples at
risk. A feral donkey population (Equus asinus), living in the Asinara National Park
(an island north-west of Sardinia, Italy), includes a unique white albino donkey
subpopulation or colour morph that is a major attraction of this park.
Disrupting mutations in the tyrosinase (TYR) gene are known to cause recessive
albinisms in humans (oculocutaneous albinism Type 1; OCA1) and other species. In
this study, we analysed the donkey TYR gene as a strong candidate to identify
the causative mutation of the albinism of these donkeys. The TYR gene was
sequenced from 13 donkeys (seven Asinara white albino and six coloured animals).
Seven single nucleotide polymorphisms were identified. A missense mutation
(c.604C>G; p.His202Asp) in a highly conserved amino acid position (even across
kingdoms), which disrupts the first copper-binding site (CuA) of functional
protein, was identified in the homozygous condition (G/G or D/D) in all Asinara
white albino donkeys and in the albino son of a trio (the grey parents had
genotype C/G or H/D), supporting the recessive mode of inheritance of this
mutation. Genotyping 82 donkeys confirmed that Asinara albino donkeys had
genotype G/G whereas all other coloured donkeys had genotype C/C or C/G.
Across-population association between the c.604C>G genotypes and the albino coat
colour was highly significant (P = 6.17E-18). The identification of the
causative mutation of the albinism in the Asinara white donkeys might open new
perspectives to study the dynamics of this putative deleterious allele in a
feral population and to manage this interesting animal genetic resource. BACKGROUND: Oculocutaneous albinism type 1 (OCA1), caused by pathogenic
variations in the tyrosinase gene (TYR), is the most frequent and severe form of
hypopigmentary disorder worldwide. While OCA1A manifests as a complete loss of
melanin pigment, patients with OCA1B show residual pigmentation of the skin,
hair and eyes. Limited experimental evidence suggests retention of TYR in the
endoplasmic reticulum (ER) causes OCA1 pathogenesis. However, a comprehensive
functional analysis of TYR missense variations and correlation with genotype is
lacking.
OBJECTIVES: Functional characterization of nonsynonymous tyrosinase variants in
patients with OCA1 reported in the Albinism Database, dbSNP and the published
literature, and an attempt to correlate them with reported and predicted
phenotypes.
METHODS: Thirty-four reported missense variants of TYR were subcloned by
site-directed mutagenesis, and the dual-enzyme activities of the variant
proteins were compared with the wild-type. The degree of ER retention was also
checked for each of the variants through endoglycosidase H (Endo H) digestion
followed by immunoprecipitation and densitometric analysis.
RESULTS: Functional studies revealed one reported OCA1A variation with nearly
100% enzyme activity, 10 OCA1B variants lacking any enzyme activity, eight
nonsynonymous single-nucleotide polymorphisms (SNPs) with ~30-70% of enzyme
activity, and three SNPs that completely lacked activity altogether. The Endo H
assay corroborated these results.
CONCLUSIONS: Loss of enzyme activity of TYR variants was completely in agreement
with ER retention across all variants examined. The results of the assay clearly
established that determination of the biological activity of identified variants
in patients with OCA is essential to correlate the identified suspect genotype
with the obvious phenotype of the disease. Oculocutaneous albinism (OCA) is an autosomal recessive disorder characterized
by either complete lack of or a reduction in melanin biosynthesis in the skin,
hair, and eyes. OCA1, the most common and severe type, is caused by mutations in
the tyrosinase (TYR) gene. In this study, we report a Chinese family with two
members affected by OCA. Blood samples were collected from all family members.
Genomic DNA was isolated from blood leukocytes, and all coding exons and
adjacent intronic sequences of the TYR gene were examined for mutation analysis
using polymerase chain reaction (PCR)-based sequencing. A pedigree chart was
drawn, and clinical examinations and paraclinical tests were performed. Compound
heterozygous mutations in TYR (c.832C>T and c.929_930insC, which resulted in
p.Arg278* and p.Arg311Lysfs*7, respectively) were identified in the two patients
with milky skin, white hair, photophobia, and reduced visual acuity, while other
family members only carried one of two heterozygous mutations. In addition, a
homozygous missense mutation c.814G>A (p.Glu272Lys) in the solute carrier family
45 member 2 (SLC45A2) gene was found in both patients and unaffected family
members, suggesting that this may not be a causative mutation. The findings of
this study expand the mutational spectrum of OCA. Compound heterozygous
mutations (c.832C>T and c.929_930insC) in the TYR gene may be responsible for
partial clinical manifestations of OCA, while the homozygous missense mutation
c.814G>A (p.Glu272Lys) in the SLC45A2 gene may not be associated with OCA. |
Please list 2 treatments for a torn rotator cuff | to compare clinical outcomes between conventional en masse repair and separate double-layer double-row repair for the treatment of delaminated rotator cuff tears. | BACKGROUND: Many approaches exist for managing rotator cuff tears.
PURPOSE: To compare the benefits and harms of nonoperative and operative
interventions on clinically important outcomes in adults with rotator cuff
tears.
DATA SOURCES: 12 electronic databases (1990 to September 2009), gray literature,
trial registries, and reference lists were searched.
STUDY SELECTION: Controlled and uncontrolled studies that assessed nonoperative
or operative treatments or postoperative rehabilitation for adults with
confirmed rotator cuff tears were included. Operative studies in
English-language publications and nonoperative and postoperative rehabilitation
studies in English, French, or German were considered. Studies were assessed in
duplicate.
DATA EXTRACTION: 2 reviewers assessed risk for bias by using the Cochrane Risk
of Bias tool and the Newcastle-Ottawa Scale. One reviewer rated the evidence by
using a modified GRADE (Grading of Recommendations Assessment, Development, and
Evaluation) approach. Data were extracted by one reviewer and verified by
another.
DATA SYNTHESIS: 137 studies met eligibility criteria. All trials had high risk
for bias. Cohort and uncontrolled studies were of moderate quality. Reported
functional outcomes did not differ between open versus mini-open repair,
mini-open versus arthroscopic repair, arthroscopic repair with versus without
acromioplasty, or single-row versus double-row fixation. Earlier return to work
was reported for mini-open repair versus open repair and for continuous passive
motion with physical therapy versus physical therapy alone. Open repairs showed
greater improvement in function than did arthroscopic debridement. Complication
rates were low across all interventions.
LIMITATIONS: Limited evidence, which was often of low quality, precluded
conclusions for most comparisons. Language restrictions may have excluded some
relevant studies, and selective outcome reporting may have introduced bias.
CONCLUSION: Evidence on the comparative effectiveness and harms of various
operative and nonoperative treatments for rotator cuff tears is limited and
inconclusive.
PRIMARY FUNDING SOURCE: Agency for Healthcare Research and Quality. PURPOSE: To evaluate the effectiveness of arthroscopic debridement (DB), partial
(PR), and complete repair (CR) for massive rotator cuff tears (mRCT) in terms of
functional and subjective parameters, and repair integrity.
METHODS: For this single-centre study, 68 consecutive shoulders with mRCT
involving at least three tendons and treated with arthroscopic DB (n = 23), PR
(n = 22), and CR (n = 23) were included. All patients (52-81 years) were
prospectively assessed before and at a mean of 45 months after surgery using
functional and subjective parameters. Preoperative tendon rupture pattern and
post-operative repair integrity were assessed by MRI. A coding system describing
accurately rotator cuff rupture, treatment, and integrity was established.
RESULTS: All treatment groups improved significantly from pre- to post-operative
(P < 0.01), while preoperative parameters, except fatty degeneration, were not
significantly different. However, post-operative comparisons revealed similar
scores with DB (constant score, CS 65.8 ± 14.7, qDASH 24.1 ± 20.6) and PR (CS
67.5 ± 9.9, P = n.s.; qDASH 20.5 ± 14.4, P = n.s.), while CR were significantly
better (CS 80.3 ± 8.9; qDASH 7.0 ± 8.7; P ≤ 0.001). Force couple restoration of
PR did not significantly influence outcome. Re-tear rates with CR (29 %) were
lower compared to PR (53 %). Intact CR compared to intact PR showed better CS
(83.4 ± 7.3 vs. 68.5 ± 10.6, P = 0.009) and qDASH (5.4 ± 8.3 vs. 21.2 ± 9.5,
P = 0.006). The vast majority of patients were satisfied with their arthroscopic
procedure (DB 87 %; PR 86 %; CR 91 %).
CONCLUSION: Arthroscopic DB, PR, and CR were effective in treating mRCT
involving at least three tendons. Reparability of mRCT was influenced by fatty
degeneration of the muscles. However, CR showed the most favourable short-term
improvements.
LEVEL OF EVIDENCE: IV. PURPOSE: The purpose was to investigate whether surgical repair earlier or later
than 3 months after injury may result in similar outcomes and patient
satisfaction.
METHODS: Seventy-three patients (75 shoulders, 58 males, mean age 59) who had
undergone surgical intervention for traumatic rotator cuff tears from 1999 to
2011 were assessed by MRI, clinical examination and Western Ontario Rotator Cuff
Index (WORC) as a primary outcome measure and Oxford Shoulder score (OSS),
Constant-Murley score (CS) and EQ-5D as secondary. The patients treated less
than 3 months after injury (n = 39) were compared with patients treated more
than 3 months after injury (n = 36). The average follow-up time was 56 months
(range 14-149), and the average time from injury to repair for all patients was
16 weeks (range 3-104). A single senior radiologist performed a blinded
evaluation of all the MRIs. Rotator cuff integrity, presence of arthritis, fatty
degeneration and muscle atrophy were evaluated.
RESULTS: No differences were found for any of the assessed outcomes (WORC, OSS,
CS and EQ-5D) between the two groups. The mean WORC % was 77 % for both groups.
Re-tear frequency was 24 %, nine in both groups. Patients with re-tear reported
less satisfaction with their outcome.
CONCLUSIONS: The surgical treatment of symptomatic traumatic rotator cuff tears
repairable later than 3 months after injury yields a good functional outcome, a
high level of subjective patient satisfaction, and at the same level for
patients receiving earlier treatment. Based on our findings, surgical repair
could be encouraged whenever technically possible.
LEVEL OF EVIDENCE: Retrospective Comparative Study, Level III. BACKGROUND: The rotator cuff tendon is known to exert a shear force between the
superficial and deep layers. Owing to this characteristic, separate repair of
delaminated rotator cuff tears has been introduced for the restoration of the
physiological biomechanics of the rotator cuff. However, whether conventional en
masse repair or separate repair is superior is controversial in terms of
outcomes.
PURPOSE: To compare clinical outcomes between conventional en masse repair and
separate double-layer double-row repair for the treatment of delaminated rotator
cuff tears.
STUDY DESIGN: Randomized controlled study; Level of evidence, 2.
METHODS: Between August 2007 and March 2014, a total of 82 patients who
underwent arthroscopic rotator cuff repair of a delaminated tear were enrolled
and randomized into 2 groups. In group 1 (n = 48), arthroscopic conventional en
masse repair was performed. In group 2 (n = 34), separate double-layer
double-row repair was performed. The American Shoulder and Elbow Surgeons score,
Constant score, Simple Shoulder Test score, and visual analog scale (VAS) score
for pain and range of motion (ROM) were assessed before surgery; at 3, 6, and 12
months after surgery; and at the last follow-up visit. Magnetic resoce
imaging (MRI) was performed at 12 months postoperatively to examine the retear
rate and pattern.
RESULTS: There was no significant difference between groups in the preoperative
demographic data, including patient age, sex, symptom duration, tear size, and
functional scores (P > .05). The mean follow-up period was 25.9 ± 1.2 months.
Significant improvements in functional and pain scores were observed in both
groups at the last follow-up visit. However, no significant differences in
functional scores and ROM were found between the 2 groups at each time point,
except that group 2 had significantly lower VAS pain scores (P < .05) at 3, 6,
and 12 months postoperatively. Eight (17%) of 48 patients in group 1 and 6 (18%)
of 34 patients in group 2 showed retears on MRI at 12-month follow-up (P > .05).
CONCLUSION: Both conventional en masse repair and separate double-layer
double-row repair were effective in improving clinical outcomes in the treatment
of delaminated rotator cuff tears. Lower pain scores were seen in patients who
underwent separate double-layer double-row repair. PURPOSE: To investigate the alteration of passive stiffness in the supraspinatus
muscle after double-row (DR) and knotless transosseous-equivalent (KL-TOE)
repair techniques, using shear wave elastography (SWE) in cadavers with rotator
cuff tears. We also aimed to compare altered muscular stiffness after these
repairs to that obtained from shoulders with intact rotator cuff tendon.
METHODS: Twelve fresh-frozen cadaveric shoulders with rotator cuff tear (tear
size: small [6], medium-large [6]) were used. Passive stiffness of 4 anatomic
regions in the supraspinatus muscle was measured based on an established SWE
method. Each specimen underwent DR and KL-TOE footprint repairs at 30°
glenohumeral abduction. SWE values, obtained at 0°, 10°, 20°, 30°, 60°, and 90°
abduction, were assessed in 3 different conditions: preoperative (torn) and
postoperative conditions with the 2 techniques. The increased ratio of SWE
values after repair was compared among the 4 regions to assess stiffness
distribution. In addition, SWE values were obtained on 12 shoulders with intact
rotator cuff tendons as control.
RESULTS: In shoulders with medium-large-sized tears, supraspinatus muscles
showed an increased passive stiffness after rotator cuff repairs, and this was
significantly observed at adducted positions. KL-TOE repair showed uniform
stiffness changes among the 4 regions of the supraspinatus muscle (mean, 189% to
218% increase after repair), whereas DR repair caused a significantly
heterogeneous stiffness distribution within the muscle (mean, 187% to 319% after
repair, P = .002). Although a repair-induced increase in muscle stiffness was
observed also in small-sized tears, there were no significant differences in
repaired stiffness changes between DR and KL-TOE (mean, 127% to 138% and 127% to
130% after repairs, respectively). Shoulders with intact rotator cuff tendon
showed uniform SWE values among the 4 regions of the supraspinatus muscle (mean,
38.2 to 43.0 kPa).
CONCLUSIONS: Passive stiffness of the supraspinatus muscle increases after
rotator cuff repairs for medium-large-sized tears. KL-TOE technique for the
medium-large-sized tear provided a more uniform stiffness distribution across
the repaired supraspinatus muscles compared with the DR technique.
CLINICAL RELEVANCE: Based on this insight, investigating rotator cuff muscle
stiffness changes, further studies using SWE may determine the optimal repair
technique for various sizes of rotator cuff tears. |
What is the role of Laser Interstitial Thermal Therapy in glioma treatment? | Laser Interstitial Thermal Therapy (LITT) is used in treatment of gliomas. LITT can be used effectively for treatment of difficult-to-access high-grade gliomas. More complete coverage of tumor improves progression free survival which can be translated as the extent of resection concept for surgery. | Supratentorial primitive neuroectodermal tumors (PNETs) are rare tumors that
carry a poorer prognosis than those arising from the infratentorial compartment
(such as medulloblastoma). The overall prognosis for these patients depends on
several factors including the extent of resection, age at diagnosis, CSF
dissemination, and site in the supratentorial space. The authors present the
first case of a patient with a newly diagnosed supratentorial PNET in which
cytoreduction was achieved with MR-guided laser-induced thermal therapy. A
10-year-old girl presented with left-sided facial weakness and a large right
thalamic mass extending into the right midbrain. The diagnosis of supratentorial
PNET was made after stereotactic biopsy. Therapeutic options for this lesion
were limited because of the risks of postoperative neurological deficits with
resection. The patient underwent MR-guided laser-induced thermal ablation of her
tumor. Under real-time MR thermometry, thermal energy was delivered to the tumor
at a core temperature of 90°C for a total of 960 seconds. The patient underwent
follow-up MR imaging at regular intervals to evaluate the tumor response to the
thermal ablation procedure. Initial postoperative scans showed an increase in
the size of the lesion as well as the amount of the associated edema. Both the
size of the lesion and the edema stabilized by 1 week and then decreased below
preablation levels at the 3-month postsurgical follow-up. There was a slight
increase in the size of the lesion and associated edema at the 6-month follow-up
scan, presumably due to concomitant radiation she received as part of her
postoperative care. The patient tolerated the procedure well and has had
resolution of her symptoms since surgery. Further study is needed to assess the
role of laser-induced thermal therapy for the treatment of intracranial tumors.
As such, it is a promising tool in the neurosurgical armamentarium.
Postoperative imaging has shown no evidence of definitive recurrence at the
6-month follow-up period, but longer-term follow-up is required to assess for
late recurrence. BACKGROUND: Surgical treatments for deep-seated intracranial lesions have been
limited by morbidities associated with resection. Real-time magnetic resoce
imaging-guided focused laser interstitial thermal therapy (LITT) offers a
minimally invasive surgical treatment option for such lesions.
OBJECTIVE: To review treatments and results of patients treated with LITT for
intracranial lesions at Washington University School of Medicine.
METHODS: In a review of 17 prospectively recruited LITT patients (34-78 years of
age; mean, 59 years), we report demographics, treatment details, postoperative
imaging characteristics, and peri- and postoperative clinical courses.
RESULTS: Targets included 11 gliomas, 5 brain metastases, and 1 epilepsy focus.
Lesions were lobar (n = 8), thalamic/basal ganglia (n = 5), insular (n = 3), and
corpus callosum (n = 1). Mean target volume was 11.6 cm, and LITT produced 93%
target ablation. Patients with superficial lesions had shorter intensive care
unit stays. Ten patients experienced no perioperative morbidities. Morbidities
included transient aphasia, hemiparesis, hyponatremia, deep venous thrombosis,
and fatal meningitis. Postoperative magnetic resoce imaging showed blood
products within the lesion surrounded by new thin uniform rim of contrast
enhancement and diffusion restriction. In conjunction with other therapies, LITT
targets often showed stable or reduced local disease. Epilepsy focus LITT
produced seizure freedom at 8 months. Preliminary overall median
progression-free survival and survival from LITT in tumor patients were 7.6 and
10.9 months, respectively. However, this small cohort has not been followed for
a sufficient length of time, necessitating future outcomes studies.
CONCLUSION: Early peri- and postoperative clinical data demonstrate that LITT is
a safe and viable ablative treatment option for intracranial lesions, and may be
considered for select patients. Surgical extent-of-resection has been shown to have an impact on high-grade
glioma (HGG) outcomes; however, complete resection is rarely achievable in
difficult-to-access (DTA) tumors. Controlled thermal damage to the tumor may
have the same impact in DTA-HGGs. We report our multicenter results of laser
interstitial thermal therapy (LITT) in DTA-HGGs. We retrospectively reviewed 34
consecutive DTA-HGG patients (24 glioblastoma, 10 anaplastic) who underwent LITT
at Cleveland Clinic, Washington University, and Wake Forest University (May
2011-December 2012) using the NeuroBlate(®) System. The extent of thermal damage
was determined using thermal damage threshold (TDT) lines: yellow TDT line (43
°C for 2 min) and blue TDT line (43°C for 10 min). Volumetric analysis was
performed to determine the extent-of-coverage of tumor volume by TDT lines.
Patient outcomes were evaluated statistically. LITT was delivered as upfront in
19 and delivered as salvage in 16 cases. After 7.2 months of follow-up, 71% of
cases demonstrated progression and 34% died. The median overall survival (OS)
for the cohort was not reached; however, the 1-year estimate of OS was 68 ± 9%.
Median progression-free survival (PFS) was 5.1 months. Thirteen cases who met
the following two criteria-(1) <0.05 cm(3) tumor volume not covered by the
yellow TDT line and (2) <1.5 cm(3) additional tumor volume not covered by the
blue TDT line-had better PFS than the other 21 cases (9.7 vs. 4.6 months; P =
0.02). LITT can be used effectively for treatment of DTA-HGGs. More complete
coverage of tumor by TDT lines improves PFS which can be translated as the
extent of resection concept for surgery. Magnetic resoce imaging-guided laser interstitial thermal therapy (LITT) is a
minimally invasive treatment modality with recent increasing use to ablate brain
tumors. When originally introduced in the late 1980s, the inability to precisely
monitor and control the thermal ablation limited the adoption of LITT in
neuro-oncology. Popularized as a means of destroying maligt hepatic and renal
metastatic lesions percutaneously, its selective thermal tumor destruction and
preservation of adjacent normal tissues have since been optimized for use in
neuro-oncology. The progress made in real-time thermal imaging with MRI, laser
probe design, and computer algorithms predictive of tissue kill has led to the
resurgence of interest in LITT as a means to ablate brain tumors. Current LITT
systems offer a surgical option for some inoperable brain tumors. We discuss the
origins, principles, current indications, and future directions of MRI-guided
LITT in neuro-oncology. Evolving research has demonstrated that surgical cytoreduction of a high-grade
glial neoplasm is an important factor in improving the prognosis of these
difficult tumors. Recent advances in intraoperative imaging have spurred the use
of stereotactic laser ablation (laser interstitial thermal therapy [LITT]) for
intracranial lesions. Among other targets, laser ablation has been used in the
focal treatment of high-grade gliomas (HGGs). The revived application of laser
ablation for gliomas parallels major advancements in intraoperative adjuvants
and groundbreaking molecular advances in neuro-oncology. The authors review the
research on stereotactic LITT for the treatment of HGGs and provide a potential
management algorithm for HGGs that incorporates LITT in clinical practice. Laser interstitial thermal therapy (LITT) is a minimally invasive technique for
treating intracranial tumors, originally introduced in 1983. Its use in
neurosurgical procedures was historically limited by early technical
difficulties related to the monitoring and control of the extent of thermal
damage. The development of magnetic resoce thermography and its application
to LITT have allowed for real-time thermal imaging and feedback control during
laser energy delivery, allowing for precise and accurate provision of tissue
hyperthermia. Improvements in laser probe design, surgical stereotactic
targeting hardware, and computer monitoring software have accelerated acceptance
and clinical utilization of LITT as a neurosurgical treatment alternative.
Current commercially available LITT systems have been used for the treatment of
neurosurgical soft-tissue lesions, including difficult to access brain tumors,
maligt gliomas, and radiosurgery-resistant metastases, as well as for the
ablation of such lesions as epileptogenic foci and radiation necrosis. In this
review, the authors aim to critically analyze the literature to describe the
advent of LITT as a neurosurgical, laser excision tool, including its
development, use, indications, and efficacy as it relates to neurosurgical
applications. Minimally invasive stereotactic tumor ablation is a viable option for the
treatment of benign and maligt intracranial lesions. Although surgical
excision constitutes first-line therapy for various brain pathologies, it can
cause irreversible neurologic deficits. Additionally, many patients who may
benefit from surgery do not qualify as surgical candidates due to multiple
comorbidities. Recent advancements in laser interstitial thermal therapy, namely
the ability to monitor ablation in real-time under MR imaging, have improved the
safety and efficacy of the procedure. MRI-guided laser interstitial thermal
therapy is currently used as a minimally invasive treatment for brain
metastases, radiation necrosis, glioma, and epilepsy. This article will discuss
the principles, suggested indications, complications, and imaging
characteristics of MRI-guided laser interstitial thermal therapy as they pertain
to the treatment of brain pathology. Author information:
(1)Department of Nuclear Medicine, Institut Bergonié, 229 cours de l'Argonne,
Bordeaux 33000, France. Electronic address: [email protected].
(2)Department of Nuclear Medicine, Institut Bergonié, 229 cours de l'Argonne,
Bordeaux 33000, France.
(3)Non-Vascular IR Department, University Hospital of Strasbourg, Nouvel hopital
civil, 1 Place de l'hopital, BP 426, Strasbourg 67091, France.
(4)Department of Interventional Radiology, Institut Bergonié, 229 cours de
l'Argonne, Bordeaux 33000, France. BACKGROUND: Pediatric low-grade gliomas (LGGs) account for approximately half of
all pediatric central nervous system tumors. The low-grade gliomas' first line
of treatment is gross total resection. However, when gross total resection is
not possible, options for adjuvant therapy are limited. MRI-guided laser
ablation (magnetic resoce-guided laser interstitial thermal therapy
(MRgLITT)) offers a new option for treatment in selected cases. We present a
description of the current MRgLITT technology and an exemplary case-series
review of our experience in its use in LGGs.
CASE DESCRIPTION: A 19-month-old male was referred to the pediatric neurosurgery
clinic with an incidental left temporal lesion discovered on a prenatal
ultrasound. An MRI of the brain revealed a diffuse mesial temporal lesion.
Electroencephalogram (EEG) showed generalized activity arising from the lesion.
The patient underwent a navigation-guided biopsy then, two bolts were secured to
the skull, and laser ablation was performed with intraoperative MR guidance.
Pathology was consistent with ganglioglioma. Follow-up images 13 months after
ablation showed a significant volumetric reduction in size of the tumor.
DISCUSSION: It is important to achieve maximal resection of low-grade gliomas in
children, lessening the need for adjuvant chemotherapy and radiotherapy, while
minimizing the length of hospital stay and disruption to the child's life. Of
our nine LGGs patients treated with this technology, six had undergone previous
surgery and MRgLITT proved itself to be a safe surgical treatment option to
achieve further cytoreduction. While most of the cases are pilocytic
astrocytomas, the location of the tumors was surgically challenging. Eight of
the nine cases required a single trajectory-laser-while our case example
requires two lasers. Only a case of a midbrain-thalamic tumor presented a
post-ablation significant brain edema as perioperative complication [1]. Eight
of the nine tumors did not require any coadjuvant therapy or further surgical
treatment to date.
CONCLUSION: MRIgLITT is a successful option for treatment for selected de novo
or recurrent low-grade gliomas in children. It can be combined with other
therapies offering the advantages of a minimally invasive procedure. LITT may be
added to the current pediatric neuro-oncology protocols, but larger prospective
series are needed to show the effectiveness of LITT and to standardize
indications and protocols. OBJECTIVE Glioblastoma (GBM) is the most common and deadly maligt primary
brain tumor. Better surgical therapies are needed for newly diagnosed GBMs that
are difficult to resect and for GBMs that recur despite standard therapies. The
authors reviewed their institutional experience of using laser interstitial
thermal therapy (LITT) for the treatment of newly diagnosed or recurrent GBMs.
METHODS This study reports on the pre-LITT characteristics and post-LITT
outcomes of 8 patients with newly diagnosed GBMs and 13 patients with recurrent
GBM who underwent LITT. RESULTS Compared with the group with recurrent GBMs, the
patients with newly diagnosed GBMs who underwent LITT tended to be older (60.8
vs 48.9 years), harbored larger tumors (22.4 vs 14.6 cm3), and a greater
proportion had IDH wild-type GBMs. In the newly diagnosed GBM group, the median
progression-free survival and the median survival after the procedure were 2
months and 8 months, respectively, and no patient demonstrated radiographic
shrinkage of the tumor on follow-up imaging. In the 13 patients with recurrent
GBM, 5 demonstrated a response to LITT, with radiographic shrinkage of the tumor
following ablation. The median progression-free survival was 5 months, and the
median survival was greater than 7 months. CONCLUSIONS In carefully selected
patients with recurrent GBM, LITT may be an effective alternative to surgery as
a salvage treatment. Its role in the treatment of newly diagnosed unresectable
GBMs is not established yet and requires further study. OBJECTIVE Laser interstitial thermal therapy (LITT), sometimes referred to as
"stereotactic laser ablation," has demonstrated utility in a subset of high-risk
surgical patients with difficult to access (DTA) intracranial neoplasms.
However, the treatment of tumors larger than 10 cm3 is associated with
suboptimal outcomes and morbidity. This may limit the utility of LITT in dealing
with precisely those large or deep tumors that are most difficult to treat with
conventional approaches. Recently, several groups have reported on minimally
invasive transsulcal approaches utilizing tubular retracting systems. However,
these approaches have been primarily used for intraventricular or
paraventricular lesions, and subtotal resections have been reported for
intraparenchymal lesions. Here, the authors describe a combined approach of LITT
followed by minimally invasive transsulcal resection for large and DTA tumors.
METHODS The authors retrospectively reviewed the results of LITT immediately
followed by minimally invasive, transsulcal, transportal resection in 10
consecutive patients with unilateral, DTA maligt tumors > 10 cm3. The
patients, 5 males and 5 females, had a median age of 65 years. Eight patients
had glioblastoma multiforme (GBM), 1 had a previously treated GBM with radiation
necrosis, and 1 had a melanoma brain metastasis. The median tumor volume treated
was 38.0 cm3. RESULTS The median tumor volume treated to the yellow thermal dose
threshold (TDT) line was 83% (range 76%-92%), the median tumor volume treated to
the blue TDT line was 73% (range 60%-87%), and the median extent of resection
was 93% (range 84%-100%). Two patients suffered mild postoperative neurological
deficits, one transiently. Four patients have died since this analysis and 6
remain alive. Median progression-free survival was 280 days, and median overall
survival was 482 days. CONCLUSIONS Laser interstitial thermal therapy followed
by minimally invasive transsulcal resection, reported here for the first time,
is a novel option for patients with large, DTA, maligt brain neoplasms. There
were no unexpected neurological complications in this series, and operative
characteristics improved as surgeon experience increased. Further studies are
needed to elucidate any differences in survival or quality of life metrics. Magnetic resoce-guided laser interstitial thermal therapy (MRgLITT) is a
novel minimally invasive modality that uses heat from laser probes to destroy
tissue. Advances in probe design, cooling mechanisms, and real-time MR
thermography have increased laser utilization in neurosurgery. The authors
perform a systematic analysis of two commercially available MRgLITT systems used
in neurosurgery: the Visualase® thermal therapy and NeuroBlate® Systems. Data
extraction was performed in a blinded fashion. Twenty-two articles were included
in the quantitative synthesis. A total of 223 patients were identified with the
majority having undergone treatment with Visualase (n=154, 69%). Epilepsy was
the most common indication for Visualase therapy (n=8 studies, 47%). Brain mass
was the most common indication for NeuroBlate therapy (n=3 studies, 60%). There
were no significant differences, except in age, wherein the NeuroBlate group was
nearly twice as old as the Visualase group (p<0.001). Frame, total
complications, and length-of-stay (LOS) were non-significant when adjusted for
age and number of patients. Laser neurosurgery has evolved over recent decades.
Clinical indications are currently being defined and will continue to emerge as
laser technologies become more sophisticated. Head-to-head comparison of these
systems was difficult given the variance in indications (and therefore patient
population) and disparate literature. BACKGROUND: Magnetic resoce-guided laser-interstitial thermotherapy (MR-LITT)
is a minimally invasive technique that shows promise in neuro-oncology because
of its superiority in delivering precise minimally invasive thermal energy with
minimal collateral damage.
OBJECTIVE: In this analysis, we investigate initial data on the use of MR-LITT
in the treatment of newly diagnosed high-grade gliomas.
METHODS: With the use of the PubMed, OVID, and Google-scholar database systems,
a comprehensive search of the English literature was performed. Eighty-five
articles were identified plus 1 that is pending publication. Four articles were
accounted for in this review, including 25 patients with newly diagnosed
high-grade gliomas who underwent MR-LITT treatment. We evaluated safety,
progression-free survival, and overall survival.
RESULTS: Twenty-five patients with a mean age of 53.8 years underwent LITT
treatments. On average, 82.9% of the pretreatment lesion volume was ablated. The
average tumor volume treated was 16.5 cm. The mean follow-up time was 7.6
months. Median overall survival was found to be 14.2 months (range 0.1-23
months). The median progression-free survival was 5.1 months (range 2.4-23
months); however, these data are limited by the relatively short follow-up of
the patients reviewed and small sample size of only 25 patients. There was 1
(3.4%) major perioperative complication, which was a central nervous system
infection.
CONCLUSION: MR-LITT is a promising technology for the treatment of small, yet
difficult-to-treat newly diagnosed high-grade gliomas. This study demonstrates
that MR-LITT is safe, and future randomized studies are needed to evaluate its
role as a treatment adjunct for newly diagnosed high-grade gliomas.
ABBREVIATIONS: BBB, blood-brain barrierHGG, high-grade gliomaLITT,
laser-interstitial thermal therapyWHO, World Health Organization. BACKGROUND: The value of maximal safe cytoreductive surgery in recurrent
high-grade gliomas (HGGs) is gaining wider acceptance. However, patients may
harbor recurrent tumors that may be difficult to access with open surgery. Laser
interstitial thermal therapy (LITT) is emerging as a technique for treating a
variety of brain pathologies, including primary and metastatic tumors, radiation
necrosis, and epilepsy.
OBJECTIVE: To review the role of LITT in the treatment of recurrent HGGs, for
which current treatments have limited efficacy, and to discuss the possible role
of LITT in the disruption of the blood-brain barrier to increase delivery of
chemotherapy locoregionally.
METHODS: A MEDLINE search was performed to identify 17 articles potentially
appropriate for review. Of these 17, 6 reported currently commercially available
systems and as well as magnetic resoce thermometry to monitor the ablation
and, thus, were thought to be most appropriate for this review. These studies
were then reviewed for complications associated with LITT. Ablation volume,
tumor coverage, and treatment times were also reviewed.
RESULTS: Sixty-four lesions in 63 patients with recurrent HGGs were treated with
LITT. Frontal (n = 34), temporal (n = 14), and parietal (n = 16) were the most
common locations. Permanent neurological deficits were seen in 7 patients (12%),
vascular injuries occurred in 2 patients (3%), and wound infection was observed
in 1 patient (2%). Ablation coverage of the lesions ranged from 78% to 100%.
CONCLUSION: Although experience using LITT for recurrent HGGs is growing,
current evidence is insufficient to offer a recommendation about its role in the
treatment paradigm for recurrent HGGs.
ABBREVIATIONS: BBB, blood-brain barrierFDA, US Food and Drug AdministrationGBM,
glioblastoma multiformeHGG, high-grade gliomaLITT, laser interstitial thermal
therapy. |
Which protein mediates the replacement of H2A by H2A.Z in the yeast Saccharomyces cerevisiae? | Saccharomyces cerevisiae Swr1, a Swi2/Snf2-related ATPase, is the catalytic core of a multisubunit chromatin remodeling enzyme, called the SWR1 complex, that efficiently replaces conventional histone H2A in nucleosomes with histone H2A.Z. We identified a complex containing 13 different polypeptides associated with a soluble pool of H2A.Z in Saccharomyces cerevisiae. | The histone variant H2AZ is incorporated preferentially at specific locations in
chromatin to modulate chromosome functions. In Saccharomyces cerevisiae,
deposition of histone H2AZ is mediated by the multiprotein SWR1 complex, which
catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Here, we
define interactions between SWR1 components and H2AZ, revealing a link between
the ATPase domain of Swr1 and three subunits required for the binding of H2AZ.
We discovered that Swc2 binds directly to and is essential for transfer of H2AZ.
Swc6 and Arp6 are necessary for the association of Swc2 and for nucleosome
binding, whereas other subunits, Swc5 and Yaf9, are required for H2AZ transfer
but neither H2AZ nor nucleosome binding. Finally, the C-terminal alpha-helix of
H2AZ is crucial for its recognition by SWR1. These findings provide insight on
the initial events of histone exchange. The SWR1 complex (SWR1C) in yeast catalyzes the replacement of nucleosomal H2A
with the H2AZ variant, which ensures full activation of underlying genes. We
compared the phenotype of mutants in the homologs of SWR1C components in
Arabidopsis thaliana. Mutations in Arabidopsis SWC6 (AtSWC6), SUPPRESSOR OF
FRIGIDA 3 (SUF3) and PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1), homologs
of SWC6, ARP6 and SWR1, respectively, caused similar developmental defects,
including leaf serration, weak apical domice, flowers with extra petals and
early flowering by reduction in expression of FLOWERING LOCUS C (FLC), a strong
floral repressor. Chromatin immunoprecipitation assays showed that AtSWC6 and
SUF3 bind to the proximal region of the FLC promoter, and protoplast
transfection assays showed that AtSWC6 colocalizes with SUF3. Protein
interaction analyses suggested the formation of a complex between PIE1, SUF3,
AtSWC6 and AtSWC2. In addition, H2AZ, a substrate of SWR1C, interacts with both
PIE1 and AtSWC2. Finally, knockdown of the H2AZ genes by RNA interference or
artificial microRNA caused a phenotype similar to that of atswc6 or suf3. Our
results strongly support the presence of an SWR1C-like complex in Arabidopsis
that ensures proper development, including floral repression through full
activation of FLC. Variant histone H2AZ-containing nucleosomes are involved in the regulation of
gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone
H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes
ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work
defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5,
Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement
activity. Here we examined the function of three additional SWR1 subunits,
bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing
affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4
(arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In
contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex
integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in
vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1
subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary
to the basic histone replacement activity. The N-terminal region of the Swr1
ATPase subunit is necessary and sufficient to direct association of Bdf1 and
Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an
additional H2AZ-H2B specific binding site, distinct from the previously
identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be
capable of binding two H2AZ-H2B dimers, and provide further insight on the
hierarchy and interdependency of molecular interactions within the SWR1 complex. Chromatin can be modified by posttranslational modifications of histones,
ATP-dependent remodeling, and incorporation of histone variants. The
Saccharomyces cerevisiae protein Yaf9 is a subunit of both the essential histone
acetyltransferase complex NuA4 and the ATP-dependent chromatin remodeling
complex SWR1-C, which deposits histone variant H2A.Z into euchromatin. Yaf9
contains a YEATS domain, found in proteins associated with multiple
chromatin-modifying enzymes and transcription complexes across eukaryotes. Here,
we established the conservation of YEATS domain function from yeast to human,
and determined the structure of this region from Yaf9 by x-ray crystallography
to 2.3 A resolution. The Yaf9 YEATS domain consisted of a beta-sandwich
characteristic of the Ig fold and contained three distinct conserved structural
features. The structure of the Yaf9 YEATS domain was highly similar to that of
the histone chaperone Asf1, a similarity that extended to an ability of Yaf9 to
bind histones H3 and H4 in vitro. Using structure-function analysis, we found
that the YEATS domain was required for Yaf9 function, histone variant H2A.Z
chromatin deposition at specific promoters, and H2A.Z acetylation. The SWR1 complex replaces the canonical histone H2A with the variant H2A.Z (Htz1
in yeast) at specific chromatin regions. This dynamic alteration in nucleosome
structure provides a molecular mechanism to regulate transcription, gene
silencing, chromosome segregation and DNA repair. Here we show that genetic
instability, sensitivity to drugs impairing different cellular processes and
genome-wide transcriptional misregulation in htz1Delta can be partially or
totally suppressed if SWR1 is not formed (swr1Delta), if it forms but cannot
bind to chromatin (swc2Delta) or if it binds to chromatin but lacks histone
replacement activity (swc5Delta and the ATPase-dead swr1-K727G). These results
suggest that in htz1Delta the nucleosome remodelling activity of SWR1 affects
chromatin integrity because of an attempt to replace H2A with Htz1 in the
absence of the latter. This would impair transcription and, either directly or
indirectly, other cellular processes. Specifically, we show that in htz1Delta,
the SWR1 complex causes an accumulation of recombinogenic DNA damage by a
mechanism dependent on phosphorylation of H2A at Ser129, a modification that
occurs in response to DNA damage, suggesting that the SWR1 complex impairs the
repair of spontaneous DNA damage in htz1Delta. In addition, SWR1 causes DSBs
sensitivity in htz1Delta; consistently, in the absence of Htz1 the SWR1 complex
bound near an endonuclease HO-induced DSB at the mating-type (MAT) locus impairs
DSB-induced checkpoint activation. Our results support a stepwise mechanism for
the replacement of H2A with Htz1 and demonstrate that a tight control of this
mechanism is essential to regulate chromatin dynamics but also to prevent the
deleterious consequences of an incomplete nucleosome remodelling. Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic
promoters. This incorporation is mediated by the conserved SWR1 complex, which
replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent
manner. Here, we show that promoter-proximal nucleosomes are highly
heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial
representation of nucleosomes containing one, two, or zero H2A.Z molecules.
SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and
unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic
nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products.
The ATPase activity of SWR1 is specifically stimulated by H2A-containing
nucleosomes without ensuing histone H2A eviction. Remarkably, further addition
of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction
of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that
the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as
both effector and substrate for SWR1 governs the specificity and outcome of the
replacement reaction. The histone variant H2A.Z is a universal mark of gene promoters, enhancers, and
regulatory elements in eukaryotic chromatin. The chromatin remodeler SWR1
mediates site-specific incorporation of H2A.Z by a multi-step histone
replacement reaction, evicting histone H2A-H2B from the canonical nucleosome and
depositing the H2A.Z-H2B dimer. Binding of both substrates, the canonical
nucleosome and the H2A.Z-H2B dimer, is essential for activation of SWR1. We
found that SWR1 primarily recognizes key residues within the α2 helix in the
histone-fold of nucleosomal histone H2A, a region not previously known to
influence remodeler activity. Moreover, SWR1 interacts preferentially with
nucleosomal DNA at superhelix location 2 on the nucleosome face distal to its
linker-binding site. Our findings provide new molecular insights on recognition
of the canonical nucleosome by a chromatin remodeler and have implications for
ATP-driven mechanisms of histone eviction and deposition. Author information:
(1)Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research,
National Cancer Institute, Building 37, Room 6114, Bethesda, MD 20892, USA.
(2)Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive,
Ashburn, VA 20147, USA.
(3)Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research,
National Cancer Institute, Building 37, Room 6114, Bethesda, MD 20892, USA.
Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive,
Ashburn, VA 20147, USA. [email protected]. |
Which are the key players on radial glial specification to ependymal cells? | Mcidas and GemC1/Lynkeas specify embryonic radial glial cells. Both proteins were initially described as cell cycle regulators revealing sequence similarity to Geminin. They are expressed in radial glial cells committed to the ependymal cell lineage during embryogenesis, while overexpression and knock down experiments showed that are sufficient and necessary for ependymal cell generation. | Multiciliated cells are abundant in the epithelial surface of different tissues,
including cells lining the walls of the lateral ventricles in the brain and the
airway epithelium. Their main role is to control fluid flow and defects in their
differentiation are implicated in many human disorders, such as hydrocephalus,
accompanied by defects in adult neurogenesis and mucociliary disorder in the
airway system. Here we show that Mcidas, which is mutated in human mucociliary
clearance disorder, and GemC1 (Gmnc or Lynkeas), previously implicated in cell
cycle progression, are key regulators of multiciliated ependymal cell generation
in the mouse brain. Overexpression and knockdown experiments show that Mcidas
and GemC1 are sufficient and necessary for cell fate commitment and
differentiation of radial glial cells to multiciliated ependymal cells.
Furthermore, we show that GemC1 and Mcidas operate in hierarchical order,
upstream of Foxj1 and c-Myb transcription factors, which are known regulators of
ependymal cell generation, and that Notch signaling inhibits GemC1 and Mcidas
function. Our results suggest that Mcidas and GemC1 are key players in the
generation of multiciliated ependymal cells of the adult neurogenic niche. Ependymal cells are multiciliated cells located in the wall of the lateral
ventricles of the adult mammalian brain and are key components of the
subependymal zone niche, where adult neural stem cells reside. Through the
movement of their motile cilia, ependymal cells control the cerebrospinal fluid
flow within the ventricular system from which they receive secreted molecules
and morphogens controlling self-renewal and differentiation decisions of adult
neural stem cells. Multiciliated ependymal cells become fully differentiated at
postnatal stages however they are specified during mid to late embryogenesis
from a population of radial glial cells. Here we discuss recent findings
suggesting that 2 novel molecules, Mcidas and GemC1/Lynkeas are key players on
radial glial specification to ependymal cells. Both proteins were initially
described as cell cycle regulators revealing sequence similarity to Geminin.
They are expressed in radial glial cells committed to the ependymal cell lineage
during embryogenesis, while overexpression and knock down experiments showed
that are sufficient and necessary for ependymal cell generation. We propose that
Mcidas and GemC1/Lynkeas are key components of the molecular cascade that
promotes radial glial cells fate commitment toward multiciliated ependymal cell
lineage operating upstream of c-Myb and FoxJ1. |
Is ocular melanosis a risk factor for uveal melanoma? | Yes, ocular melanosis (melanosis oculi) is a risk factor for uveal melanoma. | The patient aged 75 years presents since her youth, in the left eye, ocular
melanosis with abnormal pigmentation of the sclerotic, hyperpigmentation of the
iris and abnormal pigmentation of the fundus oculi. Six months ago, she noticed
that she is not able to see with this eye, and the fundus oculi examination
emphasizes a maligt choroidal melanoma, with a fixed detachment of the
retina. The authors show that the denomination Bourquin's benign ocular
melanosis, under which the affection is known, is improper, as 10% of the cases
of ocular melanosis degenerate into melanosarcomas. The term melanosis oculi is
the most adequate one. Maligt melanomas may arise in the uveal tract, the conjunctiva, the skin of
the eyelid, or the orbit. Risk factors so far identified include pre-existing
choroidal naevi for uveal melanomas, primary acquired melanosis (PAM) for
conjunctival tumours, and ocular and oculodermal melanocytosis for uveal and
orbital lesions. The atypical mole syndrome (AMS) is associated with uveal and
conjunctival melanomas, especially when the ocular lesions are multiple or
familial. AMS patients should be screened for ocular melanomas. Conjunctival
melanomas are managed by excision with or without adjunctive beta-irradiation.
Circumscribed tumours have a better prognosis than diffuse and multifocal
lesions arising in acquired melanosis and attempts should be made to limit the
progress of the latter variant of the disease by treating PAM with cryotherapy.
The most significant prognostic factor in uveal melanoma is the size of the
tumour at presentation. Early dissemination is the rule and every effort should
be made to distinguish a melanoma from a naevus as soon as possible. Small and
medium-sized melanomas respond well to focal treatments chosen according to the
size and location of the tumour. The techniques employed include
photocoagulation, radioactive plaque therapy, proton beam radiotherapy and
surgical resection. OBJECTIVE: In the white population, an association between oculo(dermal)
melanocytosis (ODM) and uveal melanoma is well recognized. However, the lifetime
prevalence of uveal melanoma in the ODM population is not known. This study was
designed to determine the lifetime prevalence of uveal melanoma among patients
with ocular melanocytosis.
DESIGN: Fifty-six white patients manifesting ODM with uveal melanoma formed the
basis of the study.
MAIN OUTCOME MEASURES: Published prevalence rates of ODM and uveal melanoma were
used for calculations using Bayes' theorem.
RESULTS: The lifetime prevalence of uveal melanoma in white patients with ODM is
estimated to be 2.6 x 10(-3). The median age at diagnosis of uveal melanoma in
the ODM population was similar to a randomly selected population (60.5 years and
62.5 years, respectively). In the vast majority of patients (90%) with
ODM-associated uveal melanoma, the uveal melanoma was diagnosed between the ages
of 31 years and 80 years.
CONCLUSIONS: One of about 400 patients with ODM followed for life is estimated
to develop uveal melanoma. Excessive melanocytes in the uveal tract in ODM may
provide the biologic basis for susceptibility to the development of uveal
melanoma. Patients with ODM should be monitored ophthalmoscopically, especially
during the susceptible period, for the development of uveal melanoma. The
authors suggest that a national registry of ODM patients be created and
prospective data collected to better assess the risk of developing uveal
melanoma. CASES: We present four cases of ocular melanosis. Choroidal melanoma was
detected in all of them. Three eyes had decreased visual acuity and were
enucleated because of their large, active tumours. In the fourth case the
melanoma was detected in a routine examination and we were able to apply a
preserving treatment with I125 brachytherapy.
DISCUSSION: Melanosis oculi is often underestimated as a risk factor for uveal
melanoma and glaucoma. Ophthalmic surveillance, every 6 or 12 months is
important, in patients with ocular melanocytosis for early detection of high
risk diseases. OBJECTIVE: To describe the features of phacomatosis pigmentovascularis
(cesioflammea type).
DESIGN: Noninterventional retrospective case series composed of 7 patients.
RESULTS: Nevus flammeus combined with ipsilateral ocular melanocytosis or
melanosis was seen in all 7 patients. Additional contralateral nevus flammeus
was observed in 3 patients. Nevus flammeus (unilateral in 4 patients and
bilateral in 3 patients) was distributed in trigeminal nerves V1 (n = 3), V2 (n
= 7), and V3 (n = 5). Related findings included diffuse choroidal hemangioma (n
= 1) and glaucoma (n = 1), with no patients having brain hemangioma or seizures.
Ocular pigmentary abnormalities (unilateral in all 7 patients) included
congenital ocular melanocytosis (n = 6) and conjunctival acquired melanosis (n =
1). Pigmentation was sectorial (partial) in 5 patients and complete in 2
patients. Melanocytosis involved the periocular skin in 1 patient, sclera in 2
patients, iris in 2 patients, and choroid in 4 patients. In 3 of 6 patients,
melanocytosis was visible in the choroid only on dilated fundus evaluation.
Related tumors included choroidal melanoma (n = 3), optic disc melanocytoma (n =
1), and conjunctival melanoma in situ (primary acquired melanosis) (n = 1).
Melanoma metastasis developed in 1 patient.
CONCLUSIONS: Phacomatosis pigmentovascularis shows features of nevus flammeus
and more serious ocular pigmentary abnormalities (uveoscleral melanocytosis and
conjunctival melanosis). Melanocytosis may be detected only by dilated ocular
fundus examination, as found in 3 of 6 patients. Furthermore, choroidal melanoma
can develop from melanocytosis, as noted in 3 of our 6 patients (50%). All
patients with nevus flammeus should be examined for phacomatosis
pigmentovascularis by an ophthalmologist because ocular melanocytosis and uveal
melanoma may remain hidden within the eye. IMPORTANCE: Ocular/oculodermal (oculo[dermal]) melanocytosis is a congenital
periocular pigmentary condition that can lead to the development of uveal
melanoma, estimated at 1 in 400 affected patients. In this study, patients with
melanocytosis who developed uveal melanoma were found to have double the risk
for metastasis compared with those without melanocytosis.
OBJECTIVE: To determine the relationship of oculo(dermal) melanocytosis to the
prognosis of patients with uveal melanoma.
DESIGN, SETTING, AND PATIENTS: Retrospective chart review of 7872 patients with
uveal melanoma treated at the Ocular Oncology Service, Wills Eye Institute, from
August 25, 1970, through August 27, 2008.
EXPOSURES: Enucleation, plaque radiotherapy, local resection, or thermotherapy.
MAIN OUTCOMES AND MEASURES: Metastasis and death.
RESULTS: Of 7872 patients with uveal melanoma, oculo(dermal) melanocytosis was
present in 230 (3%). The melanocytosis involved the sclera (92%), iris (17%),
choroid (12%), eyelid (8%), and temporal fossa (1%). Eyes with melanoma and
oculo(dermal) melanocytosis had a relative risk for metastasis 1.6 times greater
compared with those with no melanocytosis (P < .001). Metastasis of uveal
melanoma was 2.8 times higher in patients with iris melanocytosis (P < .001),
2.6 times higher with choroidal melanocytosis (P = .02), and 1.9 times higher
with scleral melanocytosis (P < .001). By Kaplan-Meier estimates, metastasis in
patients with oculo(dermal) melanocytosis vs no melanocytosis was 2% vs 1.8% at
1 year, 27% vs 15% at 5 years, and 48% vs 24% at 10 years (P < .001). By
multivariable analysis, the factors predictive of metastasis in patients
harboring uveal melanoma associated with oculo(dermal) melanocytosis were
increased tumor thickness (P = .001) and the presence of subretinal fluid (P =
.05), and the only factor predictive of death was increased tumor thickness (P =
.009). CONCLUSIONS AND RELEVANCE Patients with uveal melanoma associated with
oculo(dermal) melanocytosis have double the risk for metastasis compared with
those with no melanocytosis. All patients with oculo(dermal) melanocytosis
should undergo ophthalmic examination and imaging on a twice-yearly basis
because this could help with the early detection of melanoma. |
What is known about telomere length shortening and stress? | A significant relationship between more perceived stress and shorter telomere length is consistent with theoretical frameworks positing that stress induces physiological changes that result in shortened telomeres. | Telomere length, a reliable predictor of disease pathogenesis, can be affected
by genetics, chronic stress and health behaviors. Cross-sectionally, highly
stressed postmenopausal women have shorter telomeres, but only if they are
inactive. However, no studies have prospectively examined telomere length change
over a short period, and if rate of attrition is affected by naturalistic
factors such as stress and engagement in healthy behaviors, including diet,
exercise, and sleep. Here we followed healthy women over 1 year to test if major
stressors that occurred over the year predicted telomere shortening, and whether
engaging in healthy behaviors during this period mitigates this effect. In 239
postmenopausal, non-smoking, disease-free women, accumulation of major life
stressors across a 1-year period predicted telomere attrition over the same
period-for every major life stressor that occurred during the year, there was a
significantly greater decline in telomere length over the year of 35 bp
(P<0.05). Yet, these effects were moderated by health behaviors (interaction
B=0.19, P=0.04). Women who maintained relatively higher levels of health
behaviors (1 s.d. above the mean) appeared to be protected when exposed to
stress. This finding has implications for understanding malleability of telomere
length, as well as expectations for possible intervention effects. This is the
first study to identify predictors of telomere length change over the short
period of a year. BACKGROUND: Accelerated telomere shortening is associated with stress-related
cell damage and aging. Patients with depression have been shown to have
shortened life expectancy and to be associated with multiple age-related
systemic diseases. Previous studies have examined leukocyte telomere length
(LTL) in patients with depression, but have shown inconsistent results.
METHODS: We conducted meta-analyses by pooling relevant results strictly from
all eligible case-control studies for cross-sectional comparison of LTL between
depressive patients and control subjects (16 studies involving 7207 subjects).
The effect sizes (shown as Hedges' g) of each individual study were synthesized
by using a random effects model.
RESULTS: Our analysis revealed telomere length is significantly shorter in
subjects with depression in comparison to healthy controls (Hedges' g = -0.42,
p = 1 × 10(-5), corresponding to r = -0.21). Significant heterogeneity among
studies examining LTL in subjects with depression was found (Q = 116.07,
df = 16, I(2) = 86.21%, p < 1 × 10(-8)), which can possibly be explained by
methods used in measuring telomere length (Q = 18.42, df = 2, p = 1 × 10(-4)).
There was no significant publication bias, nor moderating effect of age, female
percentage, or illness duration of depression on synthesized results.
CONCLUSIONS: Our results support the hypothesis that depression is associated
with accelerated cell aging. Future studies are required to clarify whether the
association is mediated through environmental stress, and whether effective
treatment can halt cell aging. |
What fruit causes Jamaican vomiting sickness? | Jamaican Vomiting Sickness is caused by ingestion of the unripe arils of the Ackee fruit, its seeds and husks. | Thirty experimental and fifteen control Wistar rats were studied to determine
whether hypoglycin A influences insulin levels in the body to contribute to the
state of hypoglycemia usually observed in Jamaican vomiting sickness, a
condition arising after ingestion of unripe ackees. This fruit also grows in
other Caribbean islands, as well as North and Central America. Hypoglycin A is
one of the toxic compounds found in unripe ackees and is capable of inducing
hypoglycemia. A fall in blood glucose occurred after administration of
hypoglycin A. The lowest level of 42.60 +/- 4.84 mg/dl was attained 3 hr after
administration of the drug. This alteration of blood glucose from the fasting
level of 80.31 +/- 5.20 mg/dl was significant (P less than 0.01). The blood
glucose level in the control rats showed no significant change from the fasting
level. The insulin level in portal and peripheral blood showed no significant
change. Results showed that, although hypoglycin A induced severe hypoglycemia
after intravenous application, there was no significant change in insulin
levels. This observation suggests that hypoglycin A has a mechanism of action
other than an alteration in insulin levels to induce hypoglycemia. An acute illness (Jamaican vomiting sickness) which affected two adults after
eating unripe ackee fruit was investigated. Analyses of serum and urine samples
were performed to compare the patterns of organic acidaemia and aciduria with
those reported from childhood cases. The main conclusion from the comparison is
that the toxic ackee constitutent, hypoglycin, produces essentially the same
metabolic effects in adults as in children. The endemic illness of Jamaica known as ackee poisoning is reported for the
first time in the United States. The toxic exposure resulted from the
consumption of canned ackee. The epidemiology, diagnosis, theoretical mechanism,
and possible therapy of this disease are discussed. We report a case of fulmit liver failure resulting in emergent liver
transplantation following 3 weeks of nausea, vomiting, and malaise from Jamaican
Vomiting Sickness. Jamaican Vomiting Sickness is caused by ingestion of the
unripe arils of the Ackee fruit, its seeds and husks. It is characterized by
acute gastrointestinal illness and hypoglycemia. In severe cases, central
nervous system depression can occur. In previous studies, histologic sections
taken from patients with Jamaican Vomiting Sickness have shown hepatotoxicity
similar to that seen in Reye syndrome and/or acetaminophen toxicity. We
highlight macroscopic and microscopic changes in the liver secondary to
hepatoxicity of Ackee fruit versus those caused by a previously unknown sickle
cell trait. We discuss the clinical variables and the synergistic hepatotoxic
effect of Ackee fruit and ischemic injury from sickled red blood cells, causing
massive hepatic necrosis in this patient. |
Which gene is mutated in the Karak syndrome? | PLA2G6 gene is mutated in the Karak syndrome. | Neurodegenerative disorders with high brain iron include Parkinson disease,
Alzheimer disease and several childhood genetic disorders categorized as
neuroaxonal dystrophies. We mapped a locus for infantile neuroaxonal dystrophy
(INAD) and neurodegeneration with brain iron accumulation (NBIA) to chromosome
22q12-q13 and identified mutations in PLA2G6, encoding a calcium-independent
group VI phospholipase A2, in NBIA, INAD and the related Karak syndrome. This
discovery implicates phospholipases in the pathogenesis of neurodegenerative
disorders with iron dyshomeostasis. Mutations in PLA2G6 gene have variable phenotypic outcome including infantile
neuroaxonal dystrophy, atypical neuroaxonal dystrophy, idiopathic
neurodegeneration with brain iron accumulation and Karak syndrome. The cause of
this phenotypic variation is so far unknown which impairs both genetic diagnosis
and appropriate family counseling. We report detailed clinical,
electrophysiological, neuroimaging, histologic, biochemical and genetic
characterization of 11 patients, from 6 consanguineous families, who were
followed for a period of up to 17 years. Cerebellar atrophy was constant and the
earliest feature of the disease preceding brain iron accumulation, leading to
the provisional diagnosis of a recessive progressive ataxia in these patients.
Ultrastructural characterization of patients' muscle biopsies revealed focal
accumulation of granular and membranous material possibly resulting from
defective membrane homeostasis caused by disrupted PLA2G6 function. Enzyme
studies in one of these muscle biopsies provided evidence for a relatively low
mitochondrial content, which is compatible with the structural mitochondrial
alterations seen by electron microscopy. Genetic characterization of 11 patients
led to the identification of six underlying PLA2G6 gene mutations, five of which
are novel. Importantly, by combining clinical and genetic data we have observed
that while the phenotype of neurodegeneration associated with PLA2G6 mutations
is variable in this cohort of patients belonging to the same ethnic background,
it is partially influenced by the genotype, considering the age at onset and the
functional disability criteria. Molecular testing for PLA2G6 mutations is,
therefore, indicated in childhood-onset ataxia syndromes, if neuroimaging shows
cerebellar atrophy with or without evidence of iron accumulation. The PLA2G6 gene encodes a group VIA calcium-independent phospholipase A2 beta
enzyme that selectively hydrolyses glycerophospholipids to release free fatty
acids. Mutations in PLA2G6 have been associated with disorders such as infantile
neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type II
and Karak syndrome. More recently, PLA2G6 was identified as the causative gene
in a subgroup of patients with autosomal recessive early-onset
dystonia-parkinsonism. Neuropathological examination revealed widespread Lewy
body pathology and the accumulation of hyperphosphorylated tau, supporting a
link between PLA2G6 mutations and parkinsonian disorders. Here we show that
knockout of the Drosophila homologue of the PLA2G6 gene, iPLA2-VIA, results in
reduced survival, locomotor deficits and organismal hypersensitivity to
oxidative stress. Furthermore, we demonstrate that loss of iPLA2-VIA function
leads to a number of mitochondrial abnormalities, including mitochondrial
respiratory chain dysfunction, reduced ATP synthesis and abnormal mitochondrial
morphology. Moreover, we show that loss of iPLA2-VIA is strongly associated with
increased lipid peroxidation levels. We confirmed our findings using cultured
fibroblasts taken from two patients with mutations in the PLA2G6 gene. Similar
abnormalities were seen including elevated mitochondrial lipid peroxidation and
mitochondrial membrane defects, as well as raised levels of cytoplasmic and
mitochondrial reactive oxygen species. Finally, we demonstrated that deuterated
polyunsaturated fatty acids, which inhibit lipid peroxidation, were able to
partially rescue the locomotor abnormalities seen in aged flies lacking
iPLA2-VIA gene function, and restore mitochondrial membrane potential in
fibroblasts from patients with PLA2G6 mutations. Taken together, our findings
demonstrate that loss of normal PLA2G6 gene activity leads to lipid
peroxidation, mitochondrial dysfunction and subsequent mitochondrial membrane
abnormalities. Furthermore we show that the iPLA2-VIA knockout fly model
provides a useful platform for the further study of PLA2G6-associated
neurodegeneration. |
What is LedPred? | LedPred is an R/bioconductor package to predict regulatory sequences using support vector machines. LedPred is provided as an R/Bioconductor package connected to an online server to avoid installation of non-R software. Due to the heterogeneous CRM feature integration, LedPred excels at the prediction of regulatory sequences in Drosophila and mouse datasets compared with similar SVM-based software. | Supervised classification based on support vector machines (SVMs) has
successfully been used for the prediction of cis-regulatory modules (CRMs).
However, no integrated tool using such heterogeneous data as position-specific
scoring matrices, ChIP-seq data or conservation scores is currently available.
Here, we present LedPred, a flexible SVM workflow that predicts new regulatory
sequences based on the annotation of known CRMs, which are associated to a large
variety of feature types. LedPred is provided as an R/Bioconductor package
connected to an online server to avoid installation of non-R software. Due to
the heterogeneous CRM feature integration, LedPred excels at the prediction of
regulatory sequences in Drosophila and mouse datasets compared with similar
SVM-based software.
AVAILABILITY AND IMPLEMENTATION: LedPred is available on GitHub:
https://github.com/aitgon/LedPred and Bioconductor:
http://bioconductor.org/packages/release/bioc/html/LedPred.html under the MIT
license.
CONTACT: [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. |
Is siltuximab effective for Castleman disease? | Yes, siltuximab , a chimeric human-mouse monoclonal antibody to IL6, is approved for the treatment of patients with multicentric Castleman disease who are human immunodeficiency virus negative and human herpesvirus-8 negative. | PURPOSE: Interleukin-6 (IL-6) has emerged as a key factor in the pathogenesis of
the atypical lymphoproliferative disorder Castleman's disease (CD). Siltuximab
is a new anti-IL-6, chimeric monoclonal antibody with potential therapeutic
benefit in patients with CD.
METHODS: We report interim results from an open-label, dose-finding,
seven-cohort, phase I study in which patients with symptomatic, multicentric or
unresectable, unicentric CD received siltuximab at 1-, 2-, or 3-week intervals.
The main efficacy end point of clinical benefit response (CBR) was defined as a
composite of clinical and laboratory measures relevant to the management of CD.
In addition, radiologic response was independently assessed by using modified
Cheson criteria.
RESULTS: Eighteen (78%) of 23 patients (95% CI, 56% to 93%) achieved CBR, and 12
patients (52%) demonstrated objective tumor response. All 11 patients (95% CI,
72% to 100%) treated with the highest dose of 12 mg/kg achieved CBR, and eight
patients (73%) achieved objective tumor response. Overall objective-response
duration ranged from 44 to > or = 889 days, and one patient had complete
response for > or = 318 days. Hemoglobin increased markedly in 19 patients
(median increase, 2.1 g/dL; range, 0.2 to 4.7 g/dL) in the absence of
transfusion or erythropoiesis-stimulating agents. No dose-limiting toxicity was
reported, and only three patients had grade 3 or higher adverse events after a
median exposure of 331 days (range, 1 to 1,148 days).
CONCLUSION: These interim results strongly suggest that siltuximab is an
effective treatment with favorable safety for the management of CD. An
additional study is planned to fully evaluate safety and efficacy at the
recommended dose of 12 mg/kg every 3 weeks. PURPOSE: To evaluate the safety and pharmacokinetics of siltuximab, an
anti-interleukin-6 chimeric monoclonal antibody (mAb) in patients with B-cell
non-Hodgkin lymphoma (NHL), multiple myeloma, or Castleman disease.
EXPERIMENTAL DESIGN: In an open-label, dose-finding, 7 cohort, phase I study,
patients with NHL, multiple myeloma, or symptomatic Castleman disease received
siltuximab 3, 6, 9, or 12 mg/kg weekly, every 2 weeks, or every 3 weeks.
Response was assessed in all disease types. Clinical benefit response (CBR;
composite of hemoglobin, fatigue, anorexia, fever/night sweats, weight, largest
lymph node size) was also evaluated in Castleman disease.
RESULTS: Sixty-seven patients received a median of 16 siltuximab doses for a
median of 8.5 (maximum 60.5) months; 29 were treated 1 year or longer. There was
no dose-limiting toxicity, antibodies to siltuximab, or apparent dose-toxicity
relationship. The most frequently reported possible drug-related adverse events
were thrombocytopenia (25%), hypertriglyceridemia (19%), neutropenia (19%),
leukopenia (18%), hypercholesterolemia (15%), and anemia (10%). None of these
events led to dose delay/discontinuation except for neutropenia and
thrombocytopenia (n = 1 each). No treatment-related deaths occurred. C-reactive
protein (CRP) suppression was most pronounced at 12 mg/kg every 3 weeks. Mean
terminal-phase half-life of siltuximab ranged 17.73 to 20.64 days. Thirty-two of
37 (86%) patients with Castleman disease improved in 1 or more CBR component; 12
of 36 evaluable Castleman disease patients had radiologic response [complete
response (CR), n = 1; partial response (PR), n = 11], including 8 of 19 treated
with 12 mg/kg; 2 of 14 (14%) evaluable NHL patients had PR; 2 of 13 (15%)
patients with multiple myeloma had CR.
CONCLUSION: No dose-related or cumulative toxicity was apparent across all
disease indications. A dose of 12 mg/kg every 3 weeks was recommended on the
basis of the high response rates in Castleman disease and the sustained CRP
suppression. Randomized studies are ongoing in Castleman disease and multiple
myeloma. BACKGROUND: Multicentric Castleman's disease is a rare lymphoproliferative
disorder driven by dysregulated production of interleukin 6. No randomised
trials have been done to establish the best treatment for the disease. We
assessed the safety and efficacy of siltuximab-a chimeric monoclonal antibody
against interleukin 6-in HIV-negative patients with multicentric Castleman's
disease.
METHODS: We did this randomised, double-blind, placebo-controlled study at 38
hospitals in 19 countries worldwide. We enrolled HIV-negative and human
herpesvirus-8-seronegative patients with symptomatic multicentric Castleman's
disease. Treatment allocation was randomised with a computer-generated list,
with block size six, and stratification by baseline corticosteroid use. Patients
and investigators were masked to treatment allocation. Patients were randomly
assigned (2:1) to siltuximab (11 mg/kg intravenous infusion every 3 weeks) or
placebo; all patients also received best supportive care. Patients continued
treatment until treatment failure. The primary endpoint was durable tumour and
symptomatic response for at least 18 weeks for the intention-to-treat
population. Enrolment has been completed. The study is registered with
ClinicalTrials.gov, number NCT01024036.
FINDINGS: We screened 140 patients, 79 of whom were randomly assigned to
siltuximab (n=53) or placebo (n=26). Durable tumour and symptomatic responses
occurred in 18 (34%) of 53 patients in the siltuximab group and none of 26 in
the placebo group (difference 34·0%, 95% CI 11·1-54·8, p=0·0012). The incidence
of grade 3 or more adverse events (25 [47%] vs 14 [54%]) and serious adverse
events (12 [23%] vs five [19%]) was similar in each group despite longer median
treatment duration with siltuximab than with placebo (375 days [range 1-1031] vs
152 days [23-666]). The most common grade 3 or higher were fatigue (five vs
one), night sweats (four vs one), and anaemia (one vs three). Three (6%) of 53
patients had serious adverse events judged reasonably related to siltuximab
(lower respiratory tract infection, anaphylactic reaction, sepsis).
INTERPRETATION: Siltuximab plus best supportive care was superior to best
supportive care alone for patients with symptomatic multicentric Castleman's
disease and well tolerated with prolonged exposure. Siltuximab is an important
new treatment option for this disease.
FUNDING: Janssen Research & Development. Dysregulated secretion of IL-6 plays a pivotal role in the pathogenesis of
Castleman disease (CD), a rare lymphoproliferative disorder. In contrast to
unicentric CD for which surgery is considered the treatment of choice, there is
no standard therapeutic approach for multicentric CD (MCD). Siltuximab (trade
name: Sylvant, formerly known as CNTO 328) is a chimeric monoclonal antibody
with high binding affinity for human IL-6. In a recent randomized
placebo-controlled Phase II trial, subjects with HIV-negative, HHV8-negative MCD
who received siltuximab demonstrated a significantly higher rate of durable
tumor and symptomatic response with a tolerable safety profile, leading to its
approval for the treatment of HIV-negative HHV8-negative MCD by the US FDA and
the European Commission in April and May 2014, respectively. This article will
cover the current treatment options of MCD, the drug profile of siltuximab and
future directions in the management of MCD. BACKGROUND: Castleman disease is an uncommon lymphoproliferative disorder
characterized as either unicentric or multicentric. Unicentric Castleman disease
(UCD) is localized and carries an excellent prognosis, whereas multicentric
Castleman disease (MCD) is a systemic disease occurring most commonly in the
setting of HIV infection and is associated with human herpesvirus 8. MCD has
been associated with considerable morbidity and mortality, and the therapeutic
landscape for its management continues to evolve.
METHODS: The available medical literature on UCD and MCD was reviewed. The
clinical presentation and pathological diagnosis of Castleman disease was
reviewed, along with associated disorders such as certain maligcies and
autoimmune complications.
RESULTS: Surgical resection remains the standard therapy for UCD, while systemic
therapies are required for the management of MCD. Rituximab monotherapy is the
mainstay of therapy; however, novel therapies targeting interleukin 6 may
represent a treatment option in the near future. Antiviral strategies as well as
single-agent and combination chemotherapy with glucocorticoids are established
systemic therapies. The management of Castleman disease also requires careful
attention to potential concomitant infections, maligcies, and associated
syndromes.
CONCLUSIONS: UCD and MCD constitute uncommon but well-defined clinicopathologic
entities. Although UCD is typically well controlled with local therapy, MCD
continues to pose formidable challenges in management. We address historical
chemotherapy-based approaches to this disease as well as recently developed
targeted therapies, including rituximab and siltuximab, that have improved the
outcome for newly diagnosed patients. Ongoing research into the management of
MCD is needed. On April 22, 2014, the FDA granted full approval to siltuximab (SYLVANT for
injection; Janssen Biotech, Inc.), a chimeric human-mouse monoclonal antibody to
IL6, for the treatment of patients with multicentric Castleman disease (MCD) who
are human immunodeficiency virus (HIV) negative and human herpesvirus-8 (HHV-8)
negative. The approval was primarily based on the results of a randomized,
double-blind trial in which 79 symptomatic patients with MCD were allocated
(2:1) to siltuximab plus best supportive care (BSC) or to placebo plus BSC. The
primary efficacy endpoint was the proportion of patients in each arm achieving a
durable tumor and symptomatic response that persisted for a minimum of 18 weeks
without treatment failure. Tumor response was based on independent review of CT
scans using the revised Response Criteria for Maligt Lymphoma, and
symptomatic response was defined as complete resolution or stabilization of 34
MCD-related signs and symptoms as reported by the investigator. Thirty-four
percent of patients in the siltuximab arm and no patients in the placebo arm met
the primary endpoint (P = 0.0012). The most common adverse reactions (>10%
compared with placebo) during treatment with siltuximab were pruritus, increased
weight, rash, hyperuricemia, and upper respiratory tract infection. BACKGROUND: Multicentric Castleman's disease (MCD) is a rare lymphoproliferative
disorder driven by dysregulated interleukin-6 production. MCD has a poor
prognosis, and treatment is generally noncurative and aimed at symptom relief.
Siltuximab is a novel, monoclonal interleukin-6 antibody recently shown to be
effective in a registration clinical trial. MCD symptoms, such as fatigue, pain,
and weakness, are most appropriately quantified using patient-reported outcome
(PRO) measures. We assessed the effect of siltuximab on patient perception of
symptoms, functional status, and wellbeing using PRO instruments.
METHODS: We analyzed results of a randomized, double-blind trial comparing
siltuximab 11 mg/kg every 3 weeks with placebo to treat MCD. Subjects (N = 79)
completed the recently developed MCD-Symptom Scale (MCD-SS), the Functional
Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) scale, and the
Short Form (SF)-36 at predetermined time points throughout the treatment period.
Scores were compared at baseline and over time between the treatment arms and
PRO instruments.
RESULTS: At baseline, the mean number of symptoms reported was 9.2 (standard
deviation 3.76) out of 16 total, as measured by the MCD-SS. Fatigue was a key
symptom across all PRO instruments. Siltuximab-treated subjects reported early
improvements in symptoms compared with subjects in the placebo arm on both the
MCD-SS and FACIT-Fatigue scale. Statistically significant improvements in five
SF-36 domains were observed in siltuximab-treated patients, namely role
physical, role emotional, vitality, bodily pain, and mental health.
CONCLUSIONS: Patients with MCD commonly report impairments in functioning,
wellbeing, and fatigue at baseline. Siltuximab-treated patients reported
significant improvements in these outcomes after treatment. We describe a case of multicentric Castleman disease with generalized
lymphadenopathy and splenomegaly, accompanied by typical B symptoms - loss of 15
kg, fever of non-infectious origin, night sweats, symptoms of anemia.
Histological examination of the nodes with the highest accumulation of
fluorodeoxyglucose, taken from mediastinum by thoracoscopy, revealed
plasmocellular type of Castleman disease. Tests for HIV and human herpesvirus 8
(HHV-8) were negative. Three recurrences of herpes zoster indicating an
alteration of immunity preceded the dia-gnosis of disease. Treatment was
initiated with combination of thalidomide, dexamethasone, and cyclophosphamide.
The response after 2 months therapy was not clear and patient doesn't tolerated
the therapy well. Therefore, this treatment was terminated and R-CHOP (Mabthera
- rituximab, cyclophosphamide, adriamycin, vincristine, and prednisone) was
selected as a second-line therapy. Lymphadenopathy and splenomegaly were reduced
during the 2 cycles of treatment, however, serious infectious complications
accompanied the therapy. Therefore, only use of Mabthera monotherapy 375 mg /m2
was administered in 28-day intervals. This treatment has shown efficacy and
tolerability. PET-CT scan has demonstrated disappearance of lymphadenopathy and
splenomegaly, in addition, normalized accumulation of fluorodeoxyglucose.
Monotherapy with Mabthera has proved to be effective and well tolerated drug in
this case. Currently, there are more effective therapeutic alternatives in
multicentric Castleman disease: treatment with monotherapy of rituximab or in
combination therapy with immunomodulatory drugs (thalidomide or lenalidomide,
treatment with anti-IL-6 (siltuximab) or against its receptor (tocilizumab). In
the case of ineffectiveness of one treatment option must be tested other
alternative. In this case the therapy based on thalidomide wasn't successful,
whereas the treatment with Mabthera has achieved disappearance of disease
symptoms. INTRODUCTION: Multicentric Castleman's disease is a rare lymphoproliferative
disorder whose hallmark is atypical lymph node hyperplasia. Symptoms can include
fever, splenomegaly, and abnormal blood cell counts. High levels of interleukin
6 (IL-6) are observed frequently in this disorder and are believed to drive the
disease. Recently, therapies that target interleukin-6 or its receptor have been
shown to be effective in Castleman's disease.
CASE PRESENTATION: We report the case of a 76-year-old Caucasian man with
aggressive biopsy-proven Castleman's disease who experienced pulmonary and lymph
node involvement, as well as fever and weight loss. He was treated with
siltuximab, a chimeric anti-interleukin-6 antibody. After 5 months,
fluorodeoxyglucose positron emission tomography computed tomography scans showed
marked improvement in his lungs, but worsening mediastinal disease, consistent
with a mixed response. Biopsy of the mediastinal disease revealed
lymphoplasmacytic infiltrate with non-caseating, ill-defined granulomas and
scarring consistent with sarcoidosis. Prednisone 50mg by mouth daily was
started, which was tapered to 2.0-5.0mg daily. Siltuximab was continued. A
subsequent fluorodeoxyglucose positron emission tomography computed tomography
scan showed near-complete resolution of lung and mediastinal disease, now
ongoing for 3.5+ years without serious adverse events.
CONCLUSIONS: Lymphomas have previously been reported to coexist with
sarcoidosis, albeit rarely, but there has been only a single previous case of
this type with Castleman's disease. Of importance, early recognition of the
presence of sarcoidosis in our patient prevented discontinuation of siltuximab
therapy due to "progression". Our experience may also have broader implications
in that it suggests that etiology of "mixed responses" should be confirmed by
performing biopsies on the progressive tumor. PURPOSE: Siltuximab (IL6 antibody) is approved for the treatment of multicentric
Castleman disease (MCD). Effects of IL6 inhibition on the inflammatory milieu
accompanying MCD have not been characterized.
EXPERIMENTAL DESIGN: Trends in inflammatory- and anemia-associated markers,
measured over the course of a placebo-controlled study of siltuximab (11 mg/kg
q3w) in patients with MCD (n = 79), were characterized.
RESULTS: Baseline IL6 and C-reactive protein (CRP) levels were significantly
correlated (r = 0.708; P < 0.0001). CRP levels decreased (median, 92%) by cycle
1 day 8 (C1D8), remaining suppressed during siltuximab treatment while remaining
stable in the placebo group. There were no associations between baseline CRP or
IL6 and MCD symptom burden, histologic subtype, ethnicity, maximum CRP decrease,
and response parameters. A hemoglobin response (change ≥ 15 g/L at week 13) was
observed with siltuximab (61%; P = 0.0002). Median hepcidin decrease from
baseline at C1D8 with siltuximab was 47% versus median 11% increase with
placebo. Maximum post-baseline changes in hepcidin levels among siltuximab
recipients were correlated with maximum changes for hemoglobin (r = -0.395; P =
0.00607), total iron-binding capacity (TIBC; r = -0.354; P = 0.01694), and
ferritin (r = 0.599; P = 0.0001). Greater median changes from baseline in
ferritin, hemoglobin, and TIBC were observed in anemic siltuximab-treated
patients.
CONCLUSIONS: IL6 neutralization with siltuximab resulted in sustained CRP
suppression and improvement of anemia, in part, by hepcidin pathway inhibition. Siltuximab (Sylvant™), an interleukin (IL)-6 chimeric immunoglobulin Gк
monoclonal antibody, is a currently the only agent approved to treat human
idiopathic (herpesvirus-8 negative) multicentric Castleman disease (iMCD), which
is a rare lymphoproliferative disorder. iMCD is caused by dysregulated
production of IL-6 in the lymph nodes, and is associated with high morbidity,
and potentially fatal consequences. Siltuximab binds to human IL-6 with high
affinity and specificity, thereby preventing it from binding to IL-6 receptors,
and neutralizing IL-6 bioactivity. In clinical trials in patients with iMCD,
siltuximab reduced levels of C-reactive protein (a biomarker for IL-6), and
provided clinical responses. Relative to placebo, the addition of siltuximab to
best supportive care improved tumor- and symptom-related outcomes, with patients
also reporting improvements in MCD symptoms, functional status, and well-being.
Siltuximab has an acceptable tolerability profile, with the majority of
treatment-emergent adverse events being manageable and/or of mild severity. In
the absence of a cure, siltuximab represents a significant achievement in the
management of this difficult-to-treat orphan disease. Human herpes virus-8 (HHV-8)-negative or idiopathic multicentric Castleman
disease (iMCD) is a rare and deadly disorder that sits at the nexus of
hematology/oncology, virology and immunology. Management of iMCD has been
challenging due to limited understanding of etiology and pathogenesis and few
treatment options. The recent approvals in North America, Europe and Brazil of
siltuximab, a monoclonal antibody against IL-6, for iMCD now provide a safe and
effective therapy that targets a key aspect of pathogenesis. In the first ever
randomized, placebo-controlled trial in iMCD, siltuximab significantly reduced
disease burden and symptoms in a large portion (34%) of patients. The optimal
dose is 11 mg/kg intravenously every 3 weeks. At this time, duration of
treatment is often life-long or until treatment failure. Additional research is
needed to identify biomarkers that may assist with predicting treatment
effectiveness in iMCD and to investigate the role of siltuximab in
HHV-8-positive MCD and pediatric iMCD patients. Castleman's disease is not just a single disease but rather an uncommon,
heterogeneous group of nonclonal lymphoproliferative disorders, which have a
broad spectrum of clinical expression. Three histological types have been
reported, along with several clinical forms according to clinical presentation,
histological substrate and associated diseases. Interleukin-6, its receptor
polymorphisms, the human immunodeficiency virus and the human herpes virus 8 are
involved in the etiopathogenesis of Castleman's disease. The study of this
disease has shed light on a syndrome whose incidence is unknown. Despite recent
significant advances in our understanding of this disease and the increasing
therapeutic experience with rituximab, tocilizumab and siltuximab, there are
still difficult questions concerning its aetiology, prognosis and optimal
treatment. Castleman disease (CD) is a rare, heterogeneous lymphoproliferative disorder for
which no standard of care currently exists. Evidence that the pathophysiology of
CD is fueled by excessive interleukin-6 (IL-6) has led to considerable interest
in therapeutic targeting of this cytokine. Siltuximab, a chimeric monoclonal
antibody to IL-6, has thus emerged as a promising treatment option in a disease
lacking efficacious therapy. Here, we review the findings of recent studies
evaluating single-agent siltuximab treatment in CD, including the first-ever
randomized clinical trial in this disease. Although much more work is needed to
establish a standardized treatment approach, siltuximab appears to be a safe and
effective treatment for patients with newly diagnosed and previously treated CD. Multicentric Castleman's disease is a rare lymphoproliferative disorder
characterised by disseminated lymphadenopathy. Symptoms and outcomes differ
widely from one patient to another. The median survival time is about 2.5 years.
There is no consensus on treatment. Siltuximab, a monoclonal antibody that
antagonises interleukin-6, has been authorised in the European Union for
patients with multicentric Castleman's disease who are not infected with HIV or
HHV-8. In a randomised, double-blind trial in 79 patients, most of whom had mild
or moderate symptoms, the estimated one-year survival rate was 100% in the
siltuximab group versus 92% in the placebo group after a median follow-up of 60
weeks. However, half of the patients in the placebo group received siltuximab
after disease progression. Symptoms disappeared for at least 18 weeks in
one-quarter of patients in the siltuximab group versus none of those in the
placebo group; the median symptom-free period in the siltuximab group was about
16 months. The known adverse effects of siltuximab are related to its
immunosuppressive effect. They include frequent infections, neutropenia and
thrombocytopenia. Reactions can occur during the infusion, and mouth sores have
been reported. Other adverse events that appear to be more frequent with
siltuximab include cutaneous disorders, oedema, renal and cardiac disorders, and
peripheral neuropathy. Cases of gastrointestinal perforation have been reported
in trials in other clinical settings. Siltuximab can mask the signs and symptoms
of acute inflammation, in particular by suppressing fever and acute-phase
markers such as C-reactive protein. Interleukin-6 is a cytochrome P450
inhibitor. Siltuximab activates its isoenzymes and can thus reduce the
effectiveness of the numerous drugs that are substrates of this enzyme system.
This risk of interactions is likely to persist up to several weeks after
siltuximab withdrawal, because of its long plasma elimination half-life (about
16 days). In practice, the only available trial suggests that siltuximab has a
marked impact on the symptoms of mild or moderate multicentric Castleman's
disease in patients who are not infected with HIV or HHV-8. Siltuximab seems to
be a reasonable option in this setting, despite its numerous adverse effects,
provided patients are fully informed of the many unknowns and are closely
monitored. |
What are Degrons? | Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. | The N-degrons, a set of degradation signals recognized by the N-end rule
pathway, comprise a protein's destabilizing N-terminal residue and an internal
lysine residue. We show that the strength of an N-degron can be markedly
increased, without loss of specificity, through the addition of lysine residues.
A nearly exhaustive screen was carried out for N-degrons in the lysine
(K)-asparagine (N) sequence space of the 14-residue peptides containing either K
or N (16 384 different sequences). Of these sequences, 68 were found to function
as N-degrons, and three of them were at least as active and specific as any of
the previously known N-degrons. All 68 K/N-based N-degrons lacked the lysine at
position 2, and all three of the strongest N-degrons contained lysines at
positions 3 and 15. The results support a model of the targeting mechanism in
which the binding of the E3-E2 complex to the substrate's destabilizing
N-terminal residue is followed by a stochastic search for a sterically suitable
lysine residue. Our strategy of screening a small library that encompasses the
entire sequence space of two amino acids should be of use in many settings,
including studies of protein targeting and folding. The ubiquitin-proteasome system degrades an enormous variety of proteins that
contain specific degradation signals, or 'degrons'. Besides the degradation of
regulatory proteins, almost every protein suffers from sporadic biosynthetic
errors or misfolding. Such aberrant proteins can be recognized and rapidly
degraded by cells. Structural and functional data on a handful of degrons allow
several generalizations regarding their mechanism of action. We focus on
different strategies of degron recognition by the ubiquitin system, and contrast
regulatory degrons that are subject to signalling-dependent modification with
those that are controlled by protein folding or assembly, as frequently occurs
during protein quality control. The presence of two basic amino acids strategically located within a single
spanning transmembrane region has previously been shown to act as a signal for
the endoplasmic reticulum associated degradation (ERAD) of several polypeptides.
In contrast, the functionality of this degron motif within the context of a
polytopic membrane protein has not been established. Using opsin as a model
system, we have investigated the consequences of inserting the degron motif in
the first of its seven transmembrane (TM) spans. Whilst these basic residue
reduce the binding of the targeting factor, signal recognition particle, to the
first TM span, this has no effect on membrane integration in vitro or in vivo.
This most likely reflects the presence of multiple TM spans that can act as
targeting signals within in the nascent opsin chain. We find that the degron
motif leads to the efficient retention of mutant opsin chains at the endoplasmic
reticulum. The mutant opsin polypeptides are degraded via a proteasomal pathway
that involves the actions of the E3 ubiquitin ligase HRD1. In contrast,
wild-type opsin remains stable for a prolonged period even when artificially
accumulated at the endoplasmic reticulum. We conclude that a single dibasic
degron motif is sufficient to initiate both the ER retention and subsequent
degradation of ospin via an ERAD pathway. The N-end rule pathway is a proteolytic system in which recognition components
(N-recognins) recognize a set of N-terminal residues as part of degradation
signals (N-degrons). Two studies in this issue report the structures of Ubr
boxes, a substrate recognition domain of eukaryotic N-recognins. We discuss how
eukaryotic and prokaryotic N-recognins use a similar molecular principle to
recognize a different set of N-degrons. BACKGROUND: Tools for in vivo manipulation of protein abundance or activity are
highly beneficial for life science research. Protein stability can be
efficiently controlled by conditional degrons, which induce target protein
degradation at restrictive conditions.
RESULTS: We used the yeast Saccharomyces cerevisiae for development of a
conditional, bidirectional degron to control protein stability, which can be
fused to the target protein N-terminally, C-terminally or placed internally.
Activation of the degron is achieved by cleavage with the tobacco etch virus
(TEV) protease, resulting in quick proteolysis of the target protein. We found
similar degradation rates of soluble substrates using destabilization by the N-
or C-degron. C-terminal tagging of essential yeast proteins with the
bidirectional degron resulted in deletion-like phenotypes at non-permissive
conditions. Developmental process-specific mutants were created by N- or
C-terminal tagging of essential proteins with the bidirectional degron in
combination with sporulation-specific production of the TEV protease.
CONCLUSIONS: We developed a system to influence protein abundance and activity
genetically, which can be used to create conditional mutants, to regulate the
fate of single protein domains or to design artificial regulatory circuits.
Thus, this method enhances the toolbox to manipulate proteins in systems biology
approaches considerably. The N-end rule relates the regulation of the in vivo half-life of a protein to
the identity of its N-terminal residue. Degradation signals (degrons) that are
targeted by the N-end rule pathway include a set called N-degrons. The main
determit of an N-degron is a destabilizing N-terminal residue of a protein.
In eukaryotes, the N-end rule pathway is a part of the ubiquitin system and
consists of two branches, the Ac/N-end rule and the Arg/N-end rule pathways. The
Ac/N-end rule pathway targets proteins containing N(α) -terminally acetylated
(Nt-acetylated) residues. The Arg/N-end rule pathway recognizes unacetylated
N-terminal residues and involves N-terminal arginylation. Together, these
branches target for degradation a majority of cellular proteins. For example,
more than 80% of human proteins are cotranslationally Nt-acetylated. Thus most
proteins harbor a specific degradation signal, termed (Ac)N-degron, from the
moment of their birth. Specific N-end rule pathways are also present in
prokaryotes and in mitochondria. Enzymes that produce N-degrons include
methionine-aminopeptidases, caspases, calpains, Nt-acetylases, Nt-amidases,
arginyl-transferases and leucyl-transferases. Regulated degradation of specific
proteins by the N-end rule pathway mediates a legion of physiological functions,
including the sensing of heme, oxygen, and nitric oxide; selective elimination
of misfolded proteins; the regulation of DNA repair, segregation and
condensation; the signaling by G proteins; the regulation of peptide import, fat
metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis,
neurogenesis, and cardiovascular development; and the functioning of adult
organs, including the pancreas and the brain. Discovered 25 years ago, this
pathway continues to be a fount of biological insights. The N-end rule pathway is a proteolytic system in which destabilizing N-terminal
amino acids of short lived proteins are recognized by recognition components
(N-recognins) as an essential element of degrons, called N-degrons. In
eukaryotes, the major way to generate N-degrons is through arginylation by ATE1
arginyl-tRNA-protein transferases, which transfer Arg from aminoacyl-tRNA to
N-terminal Asp and Glu (and Cys as well in mammals). We have shown previously
that ATE1-deficient mice die during embryogenesis with defects in cardiac and
vascular development. Here, we characterized the arginylation-dependent N-end
rule pathway in cardiomyocytes. Our results suggest that the cardiac and
vascular defects in ATE1-deficient embryos are independent from each other and
cell-autonomous. ATE1-deficient myocardium and cardiomyocytes therein, but not
non-cardiomyocytes, showed reduced DNA synthesis and mitotic activity ~24 h
before the onset of cardiac and vascular defects at embryonic day 12.5
associated with the impairment in the phospholipase C/PKC-MEK1-ERK axis of
Gα(q)-mediated cardiac signaling pathways. Cardiac overexpression of Gα(q)
rescued ATE1-deficient embryos from thin myocardium and ventricular septal
defect but not from vascular defects, genetically dissecting vascular defects
from cardiac defects. The misregulation in cardiovascular signaling can be
attributed in part to the failure in hypoxia-sensitive degradation of RGS4, a
GTPase-activating protein for Gα(q). This study is the first to characterize the
N-end rule pathway in cardiomyocytes and reveals the role of its arginylation
branch in Gα(q)-mediated signaling of cardiomyocytes in part through
N-degron-based, oxygen-sensitive proteolysis of G-protein regulators. It is useful to artificially control the expression levels of a protein of
interest (POI), not only for its characterization in vivo, but also for the
modulation of biological pathways. Overexpression of a POI is relatively easy
because it is possible to drive its expression from a transgene encoding the POI
under the control of a strong promoter. However, it is more challenging to
reduce or deplete the expression of a POI. A protein domain called "degron",
which induces rapid proteolysis by the proteasome, can be used for this purpose.
Degron-based technologies for the conditional depletion of POI--degron fusion
proteins have been developed by exploiting various pathways leading to
proteasomal degradation. Compared with other depletion technologies that control
the expression levels of POIs at the DNA or mRNA levels, these protein-depletion
approaches are advantageous in terms of specificity, reversibility, and the time
required for depletion. Current conditional degron-based technologies are
described and discussed. Protein degradation in normal living cells is precisely regulated to match the
cells' physiological requirements. The selectivity of protein degradation is
determined by an elaborate degron-tagging system. Degron refers to an amino acid
sequence that encodes a protein degradation signal, which is oftentimes a
poly-ubiquitin chain that can be transferred to other proteins. Current
understanding of ubiquitination dependent and independent protein degradation
processes has expanded the application of degrons for targeted protein
degradation and novel cell engineering strategies. Recent findings suggest that
small molecules inducing protein association can be exploited to create degrons
that target proteins for degradation. Here, recent applications of degron-based
targeted protein degradation in eukaryotic organisms are reviewed. The degron
mediated protein degradation represents a rapidly tunable methodology to control
protein abundance, which has broad application in therapeutics and cellular
function control and monitoring. 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. |
Which disease is associated with mutated Sox2? | SOX2 anophthalmia syndrome is an uncommon autosomal dominant syndrome caused by mutations in the SOX2 gene and clinically characterized by severe eye malformations (anophthalmia/microphthalmia) and extraocular anomalies mainly involving brain, esophagus, and genitalia. | Anophthalmia and microphthalmia (A/M) are developmental ocular malformations
defined as the complete absence or reduction in size of the eye. A/M is a highly
heterogeneous disorder with SOX2 and FOXE3 playing major roles in domit and
recessive pedigrees, respectively; however, the majority of cases lack a genetic
etiology. We analyzed 28 probands affected with A/M spectrum (without mutations
in SOX2/FOXE3) by whole-exome sequencing. Analysis of 83 known A/M factors
identified pathogenic/likely pathogenic variants in PAX6, OTX2 and NDP in three
patients. A novel heterozygous likely pathogenic variant in PAX6, c.767T>C,
p.(Val256Ala), was identified in two brothers with bilateral microphthalmia,
coloboma, primary aphakia, iris hypoplasia, sclerocornea and congenital
glaucoma; the unaffected mother appears to be a mosaic carrier. While A/M has
been reported as a rare feature, this is the first report of congenital primary
aphakia in association with PAX6 and the identified allele represents the first
variant in the PAX6 homeodomain to be associated with A/M. A novel pathogenic
variant in OTX2, c.651delC, p.(Thr218Hisfs*76), in a patient with syndromic
bilateral anophthalmia and a hemizygous pathogenic variant in NDP, c.293 C>T,
p.(Pro98Leu), in two brothers with isolated bilateral microphthalmia and
sclerocornea were also identified. Pathogenic/likely pathogenic variants were
not discovered in the 25 remaining A/M cases. This study underscores the utility
of whole-exome sequencing for identification of causative mutations in highly
variable ocular phenotypes as well as the extreme genetic heterogeneity of A/M
conditions. SOX2 anophthalmia syndrome is an uncommon autosomal domit syndrome caused by
mutations in the SOX2 gene and clinically characterized by severe eye
malformations (anophthalmia/microphthalmia) and extraocular anomalies mainly
involving brain, esophagus, and genitalia. In this work, a patient with the SOX2
anophthalmia syndrome and exhibiting a novel dental anomaly is described. SOX2
genotyping in this patient revealed an apparently de novo c.70del20 deletion, a
commonly reported SOX2 mutation. A review of the phenotypic variation observed
in patients carrying the recurrent SOX2 c.70del20 mutation is presented.
Although dental anomalies are uncommonly reported in the SOX2 anophthalmia
syndrome, we suggest that a dental examination should be performed in patients
with SOX2 mutations. Author information:
(1)CHU Toulouse, Service de Génétique Médicale, Hôpital Purpan, 31059 Toulouse,
France; Université Paul-Sabatier Toulouse III, EA-4555, 31000 Toulouse, France;
Inserm U1056, 31000 Toulouse, France;
(2)Center for Human Disease Modeling, Duke University Medical Center, Durham,
North Carolina 27701, USA; Department of Pediatrics and Department of Cell
Biology, Duke University Medical Center, Durham, North Carolina 27701, USA;
(3)Center for Human Disease Modeling, Duke University Medical Center, Durham,
North Carolina 27701, USA;
(4)Université Paul-Sabatier Toulouse III, EA-4555, 31000 Toulouse, France; CHU
Toulouse, Service d'Ophtalmologie, Hôpital Purpan, 31059 Toulouse, France;
(5)Institut de Génétique et Développement, CNRS UMR6290, Université de Rennes 1,
IFR140 GFAS, Faculté de Médecine, 35043 Rennes, France; Laboratoire de Génétique
Moléculaire, CHU Pontchaillou, 35043 Rennes Cedex, France;
(6)Université Paul-Sabatier Toulouse III, EA-4555, 31000 Toulouse, France;
(7)Université de Toulouse; INSA, UPS, INP, LISBP, F-31077 Toulouse, France;
INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400
Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France; Plateforme Biopuces
de la Génopole de Toulouse Midi Pyrénées, INSA/DGBA 135, 31077 Toulouse, France;
(8)Service de Génétique Médicale, Hôpital Jeanne de Flandre, 59037 Lille,
France;
(9)Service de Génétique Clinique, Hôpital Sud, 35200 Rennes, France;
(10)Service de Génétique Médicale, Hôpital Arnaud de Villeneuve, 34295
Montpellier, France;
(11)Service de Génétique Médicale, Hôpital Pellegrin, 33076 Bordeaux Cedex,
France; Université Bordeaux Segalen, Laboratoire MRGM, 33076 Bordeaux, France;
(12)Service de Génétique, Hospices Civils de Lyon, Groupement Hospitalier Est,
69677 Bron, France; INSERM U1028 UMR CNRS 5292, UCBL, CRNL TIGER Team, 69677
Bron Cedex, France;
(13)Service d'Ophtalmologie, Hôpital Necker Enfants Malades, 75015 Paris,
France;
(14)Service de Génétique Médicale, Hôpitaux Universitaires de Strasbourg, 67091
Strasbourg, France;
(15)INSERM U781 & Department of Genetics, Paris Descartes University, 75015
Paris, France;
(16)Université Paul-Sabatier Toulouse III, EA-4555, 31000 Toulouse, France;
INSERM, UMR_S910, Aix-Marseille University, Faculté de Médecine, 13385
Marseille, France;
(17)INSERM unit 1048, I2MC, Team 12, 31432 Toulouse, France. |
Is the enzyme EPRS phosphorylated? | Yes, EPRS is dually phosphorylated by cyclin-dependent kinase 5 (Cdk5) at Ser(886) and then by a Cdk5-dependent-AGC kinase at Ser(999) | The gamma interferon (IFN-γ)-activated inhibitor of translation (GAIT) complex
in human myeloid cells is heterotetrameric, consisting of glutamyl-prolyl-tRNA
synthetase (EPRS), NS1-associated protein 1 (NSAP1), ribosomal protein L13a, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The complex binds a structural
GAIT element in the 3' untranslated region of VEGF-A and other
inflammation-related transcripts and inhibits their translation. EPRS is dually
phosphorylated by cyclin-dependent kinase 5 (Cdk5) at Ser(886) and then by a
Cdk5-dependent-AGC kinase at Ser(999); L13a is phosphorylated at Ser(77) by
death-associated protein kinases DAPK and ZIPK. Because profound differences in
inflammatory responses between mice and humans are known, we investigated the
GAIT system in mouse macrophages. The murine GAIT complex is heterotrimeric,
lacking NSAP1. As in humans, IFN-γ activates the mouse macrophage GAIT system
via induced phosphorylation of EPRS and L13a. Murine L13a is phosphorylated at
Ser(77) by the DAPK-ZIPK cascade, but EPRS is phosphorylated only at Ser(999).
Loss of EPRS Ser(886) phosphorylation prevents NSAP1 incorporation into the GAIT
complex. However, the triad of Ser(999)-phosphorylated EPRS,
Ser(77)-phosphorylated L13a, and GAPDH forms a functional GAIT complex that
inhibits translation of GAIT target mRNAs. Thus, translational control by the
heterotrimeric GAIT complex in mice exemplifies the distinctive species-specific
responses of myeloid cells to inflammatory stimuli. Phosphorylation of many aminoacyl tRNA synthetases (AARSs) has been recognized
for decades, but the contribution of post-translational modification to their
primary role in tRNA charging and decryption of genetic code remains unclear. In
contrast, phosphorylation is essential for performance of diverse noncanonical
functions of AARSs unrelated to protein synthesis. Phosphorylation of
glutamyl-prolyl tRNA synthetase (EPRS) has been investigated extensively in our
laboratory for more than a decade, and has served as an archetype for studies of
other AARSs. EPRS is a constituent of the IFN-γ-activated inhibitor of
translation (GAIT) complex that directs transcript-selective translational
control in myeloid cells. Stimulus-dependent phosphorylation of EPRS is
essential for its release from the parental multi-aminoacyl tRNA synthetase
complex (MSC), for binding to other GAIT complex proteins, and for regulating
the binding to target mRNAs. Importantly, phosphorylation is the common driving
force for the context- and stimulus-dependent release, and non-canonical
activity, of other AARSs residing in the MSC, for example, lysyl tRNA synthetase
(KARS). Here, we describe the concepts and experimental methodologies we have
used to investigate the influence of phosphorylation on the structure and
function of EPRS. We suggest that application of these approaches will help to
identify new functional phosphorylation event(s) in other AARSs and elucidate
their possible roles in noncanonical activities. |
Which disease the skin condition Necrobiosis lipoidica diabeticorum is associated to? | Necrobiosis lipoidica diabeticorum (NLD) is a rare, granulomatous inflammatory skin disease of unknown origin, sometimes associated with diabetes mellitus. | Necrobiosis lipoidica is a skin disorders with an interesting predisposition for
areas of trauma such as the anterior shins. In this report a patient with
diabetes mellitus and generalized necrobiosis lipoidica diabeticorum with
localization in surgical scars is described. A brief review of other skin
disorders occurring in scars is also included. 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. Necrobiosis lipoidica diabeticorum is an unusual dermatologic condition with a
characteristic clinical appearance and a clear association with diabetes
mellitus. There is currently no treatment that reverses the atrophic changes
associated with this lesion. We have carried out a clinicopathologic study on 15
subjects and, in addition, have reviewed 10 further biopsy specimens of
necrobiosis lipoidica diabeticorum. We found a frequent association of
necrobiosis lipoidica diabeticorum with other chronic complications of diabetes
mellitus, including limited joint mobility. It is possible that nonenzymatic
glucosylation or other changes in collagen may be important in the etiology of
necrobiosis lipoidica diabeticorum and the limited joint mobility. We confirmed
that cutaneous anesthesia is usually present in the necrobiosis lipoidica
diabeticorum lesions. With the use of an antibody to S100 protein and an
immunohistochemical method, there was an apparent decreased number of nerves in
the skin lesions. We suggest that sensory loss results from local destruction of
cutaneous nerves by the inflammatory process. Finally, in six elliptical
biopsies extending into clinically normal skin, we demonstrated that the
inflammatory infiltrate of necrobiosis lipoidica diabeticorum extended from the
lesion into apparently normal skin surrounding clinically active lesions. Thus,
intradermal steroids might be administered to perilesional areas surrounding
active lesions in the hope of halting progression. The prevalence of persistent microalbuminuria, retinopathy, and peripheral and
autonomic neuropathy was assessed in 18 children and adolescents with type 1
(insulin-dependent) diabetes mellitus (IDDM) who suffered from necrobiosis
lipoidica diabeticorum (NLD) and in 40 diabetics without NLD, matched for sex,
age, duration of disease, and metabolic control. The mean +/- SD age of the
patients was 15.1 +/- 8.6 years (range 7.9-23.9 yrs) and their duration of IDDM
was 10.9 +/- 8.1 years (range 7.1-21.0 yrs). Their mean glycosylated hemoglobin
level was 9.9 +/- 5.0% (7.3-16.6%) and their fructosamine level was 274 +/- 180
mumol/L (199-466 mumol/L). Patients with NLD had a higher frequency of
persistent microalbuminuria (p < 0.001) and retinopathy (p < 0.001) than those
without NLD. Our study suggests that children as well as adult diabetics with
NLD can be at high risk for nephropathy and retinopathy; NLD can be a clue for
diabetic nephropathy and retinopathy. In order to evaluate patients with necrobiosis lipoidica diabeticorum and to
compare them with age, sex, and duration of diabetes matched controls, 15
patients with necrobiosis were each matched with 5 control subjects with
diabetes mellitus. Complications of diabetes, glycaemic control, and proteinuria
were measured. Patients with necrobiosis (mean age 40, range 18-74 years) had a
mean duration of diabetes of 14 (range 3-36) years; 8 patients were male, and 7
were female. For necrobiosis versus controls, background retinopathy (67% vs
27%, p = 0.009), proteinuria (53% vs 17%, p = 0.006), and smoking (60% vs 20%, p
= 0.003) were all more common with necrobiosis. There were no significant
differences between patients with necrobiosis and control patients in the
prevalence of vascular disease and neuropathy. Glycosylated haemoglobin
concentrations were higher in patients with necrobiosis (p = 0.02). Blood
pressure measurements were similar in both groups. We conclude that smoking,
proteinuria, and retinopathy were more prevalent in diabetic patients with
necrobiosis; the skin lesion may therefore share common aetiological factors
which affect the microvascular circulation, leading to damage to basement
membranes and vascular endothelial cells. Necrobiosis lipoidica diabeticorum is a rare skin disorder, usually considered a
marker for diabetes mellitus. More than half of the patients with necrobiosis
lipoidica diabeticorum have diabetes mellitus, but less than one per cent of
diabetes mellitus patients have necrobiosis lipoidica diabeticorum. In the
diabetes and dermatology literature, we find the position that there is no
effect of glucose control on either the appearance of necrobiosis lipoidica
diabeticorum or the clinical course of the lesion. We base our challenge to this
position on a critical review of the original data. And conclude on the
contrary, that necrobiosis lipoidica diabeticorum is usually associated with
poor glucose control and that tighter glucose control, as currently practised,
might improve or prevent the disorder. The etiologies of a variety of skin conditions associated with diabetes have not
been fully explained. One possible etiological factor is diabetic
microangiopathy, which is known to affect the eyes and kidneys in patients with
diabetes. There are many mechanisms by which diabetes may cause microangiopathy.
These include excess sorbitol formation, increased glycation end products,
oxidative damage, and protein kinase C overactivity. All of these processes
occur in the skin, and the existence of a cutaneous diabetic microangiopathy has
been well demonstrated. These microangiopathic changes are associated with
abnormalities of skin perfusion. Because the skin plays a thermoregulatory role,
there is significant capillary redundancy in normal skin. In diabetic patients,
loss of capillaries is associated with a decrease in perfusion reserve. This
lost reserve is demonstrable under stressed conditions, such as thermal
stimulation. The associated failure of microvascular perfusion to meet the
requirements of skin metabolism may result in diverse skin lesions in patients
with diabetes. Many skin conditions peculiar to diabetes are fairly rare.
Necrobiosis lipoidica diabeticorum (NLD) and diabetic bullae occur very
infrequently as compared with diabetic retinopathy and nephropathy. Conversely,
there is a correlation between diabetic microvascular disease and NLD. This
correlation also exists with more common skin conditions, such as diabetic
dermopathy. This relationship suggests that diabetic microangiopathy may
contribute to these conditions even if it is not primarily causal. Clinically,
the major significance of diabetic cutaneous microangiopathy is seen in skin
ulceration which is very common and has a major impact on diabetic patients.
Many factors contribute to the development of diabetic foot ulcers. Neuropathy,
decreased large vessel perfusion, increased susceptibility to infection, and
altered biomechanics all play a role, but there is no doubt that inadequate
small blood vessel perfusion is a major cause of the inability to heal small
wounds that eventually results in ulcer formation. The accessibility of skin
capillaries makes cutaneous diabetic microangiopathy an attractive model for
research on the evolution of microvascular disease in diabetic patients. Necrobiosis lipoidica dibeticum (NLD) is a granulomatous skin disease mostly
associated with diabetes mellitus. NLD has been reported in patients with other
systemic disease. Also, the lesions of NLD may be clinically, and sometimes even
histologically indistinguishable from other inflammatory skin lesions. We
described three patients with established diagnosis of sarcoidosis that
developed skin lesions consistent with NLD. The association of NLD-like skin
lesion in sarcoidosis is not widely appreciated. The subject of NLD and
sarcoidosis is reviewed. Many of the complications of the diabetes are well studied but robust research
documenting the cutaneous effects of the disease remains sparse. Various studies
have suggested that the majority of patients with diabetes will suffer a skin
disorder during the course of their disease and for some, the skin changes may
even precede the diagnosis of diabetes. Cutaneous pathology of the diabetic foot
and lower leg can arise as a result of the direct or indirect effects of
diabetic complications. The most common manifestations include fungal and
bacterial skin infection, nail disease and diabetic dermopathy. Other less
commonly observed conditions include diabetic bullae, necrobiosis lipoidica
diabeticorum (NLD), granuloma annulare and reddening of the soles. For many of
the less common disorders, there is little in the way of effective treatment.
However, much can be done in the clinical setting in the management of the more
common manifestations such as bacterial and fungal infection. Fungal infection,
in particular, although relatively inconspicuous, is a very common foot problem
and if left untreated can threaten tissue viability in the diabetic foot leading
to secondary bacterial infection and cellulitis. Management of fungal disease is
often considered difficult due to high relapse and re-infection rates, although
by introducing a combination of therapies including mechanical and
pharmacological the success in treating this stubborn condition can be greatly
improved. BACKGROUND: Necrobiosis lipoidica diabeticorum (NLD) is a granulomatous skin
reaction found in < 1% of diabetic patients. Our purpose was to determine if NLD
represented areas of cutaneous ischemia.
METHODS: Using laser Doppler flowmetry, we measured cutaneous blood flow in nine
diabetic patients at NLD lesions and at contiguous uninvolved sites. Flow values
were also determined at several reference sites noncontiguous with the NLD
lesions and compared to age- and sex-matched controls: 24 diabetic subjects
without skin abnormalities, 18 diabetic patients with dermopathy, and 40
nondiabetic subjects.
RESULTS: NLD lesions exhibited significantly higher blood flow (4.8 +/- 0.7
ml/min/100 g) than areas of unaffected skin close to the lesions (1.2 +/- 0.1
ml/min/100 g) (P < 0.01 for both comparisons). There were no significant
differences in flow between normal skin sites in NLD patients and normal sites
in diabetic patients without skin lesions.
CONCLUSIONS: Our findings refute the hypothesis that NLD is a manifestation of
microvascular ischemic disease of the skin. The increased blood flow seen in NLD
lesions suggests an ongoing inflammatory process. INTRODUCTION: Necrobiosis lipoidica diabeticorum is a rare disease of unclear
etiology, that occurs in about 1% of diabetic patients.
CASE REPORT: We present case of granulomatosis disciformis chronica et
progressiva Miescher with good response to systemic corticosteroids
therapy.Patient 45 years old woman, with primary yellow-brown areas skin
lesions, with foci well separated from surroundings on both lower legs, that
occurred 5 years ago. In laboratory tests there was no abnormalities. Because of
advance suggestion (after last admit in dermatological ward) of observation
according to xantogranuloma necrobioticum tests for paraproteinemia were made.
Immunoelectrophoresis, IgG, IgM, IgA levels, kappa light chain, lambda heavy
chain; were correct, Bence-Johns protein-negative. During hospitalization in
Clinic methylprednisolone in dose of 32 mg od, vascular drugs and local
steroidotherapy was applied with good therapeutic response.
CONCLUSION: We described case of typical clinical and histological characters of
necrobiosis lipoidica. without diabetes-granulomatosis disciformis chronica et
progressiva Miescher that despite of suspicion of proper diagnosis for a long
time was not treat effective. BACKGROUND: Diabetes mellitus (DM) is a clinical syndrome characterized by
hyperglycaemia due to absolute or relative insulin deficiency. The aim of this
study was to evaluate the frequency of skin manifestations in patients with
diabetes mellitus of this area. This descriptive study was conducted in medical
out patient door of District Headquarter Hospital Battgram from January 2008 to
July 2008.
METHODS: A total of 350 diabetic (types 1 and 2) patients over 15 years of age
attending the medical OPD of DHQ Hospital were examined in detail for skin
manifestations of the disease.
RESULTS: Three hundred and fifty diabetic (type-1 and type-2) patients (193
females and 157 males) enrolled in this study. Mean age of the patients was 54
+/- 8.53 years. Duration of diabetes was between 1-12 years; 320 patients had
type-2 and 30 patients had type-1 diabetes mellitus. Patients with uncontrolled
disease were 327 and 23 patients showed adequate glycaemic control. Seventy-six
percent of patients had cutaneous manifestations. The skin manifestations
observed were: skin infections 30.9%, foot gangrene and ulcers 12.9%, pruritus
7.1%, vitiligo 5.7%, yellow skin 4.2%, diabetic dermopathy 4.2%, skin tags 3.7%,
acanthosis nigricans 2.9%, eruptive xanthomas 2.6%, necrobiosis lipoidica
diabeticorum 1.4%, diabetic bullae 0.6%, and pigmented purpuras in 0.3%
patients.
CONCLUSIONS: Cutaneous manifestations were quite common in the diabetics of this
area. Diabetes mellitus is associated with a range of dermatologic presentations,
including granuloma annulare and necrobiosis lipoidica diabeticorum. Granuloma
annulare occurs earlier than necrobiosis lipoidica diabeticorum and the
association with diabetes mellitus is much weaker. We describe two children with
diabetes who both developed granuloma annulare and later, necrobiosis lipoidica
diabeticorum. We postulate that the early onset and transient nature of
granuloma annulare, compared with the later onset and persistence of necrobiosis
lipoidica diabeticorum, might account for the different apparent rates of
association with diabetes mellitus. Necrobiosis lipoidica is a rare inflammatory granulomatous skin disease of
unknown etiology which is associated with diabetes mellitus in about 60 % of the
patients. In up to 30 % of these patients extremely painful and often
hard-to-heal ulcerations occur in the course of the disease. We present a new
therapeutic option using adalimumab to treat refractory ulcerated necrobiosis
lipoidica non diabeticorum. The clinical efficacy of adalimumab probably
reflects an immunomodulatory effect through the specific TNF-α inhibition which
is one central aspect of the underlying inflammation. Thus, adalimumab could
represent promising new treatment option, especially for patients with otherwise
therapy-refractory ulcerated necrobiosis lipoidica. A 32-year-old woman with type 2 diabetes mellitus suffering from morbid obesity
with BMI 45,14 kg/m(2) was operated on. Not only the type 2DM but also one of
its complication known as necrobiosis lipoidica diabeticorum remitted
postoperatively. Obesity should no longer be regarded simply as a cosmetic
problem affecting certain individuals but an epidemic that threatens global
well-being. It causes or exacerbates many health problems, and in particular, it
is associated with the type 2 diabetes. Necrobiosis lipoidica is a granulomatous
skin disease of unknown etiology, associated mainly with diabetes mellitus. We
presented in this paper a morbid obese case of necrobiosis lipoidica
diabeticorum with dramatic good response to bariatric surgery. BACKGROUND: Necrobiosis lipoidica is a rare complication of diabetes mellitus.It
is said to occur more often in people with diabetes,a family history of
diabetes,tendency to develop diabetes mellitus and those with insulin dependent
diabetes.
METHOD: We report an evaluated case of necrobiosis lipoidica diabeticorum
residing in the northern part of Nigeria.
RESULT: The patient was treated for 3 weeks in the hospital on admission and was
followed up in the general-out-patient department (GOPD) and has been in good
health.
AIM AND OBJECTIVE: To bring to the fore of clinicians this dermal complication
of diabetes mellitus, the different medical treatments available and the medical
treatment employed in our index patient.
CONCLUSION: That necrobiosis lipoidica diabeticorum does exist in our
environment and requires a high index of suspicion and scrupplelousness in
making the diagnosis and treating the patient. INTRODUCTION: Necrobiosis lipoidica (NL) is a rare chronic granulomatous
dermatitis that usually appears in the lower extremities. It affects about
0.3-1.2% of diabetic patients, the majority of whom have type 1 diabetes. The
etiology and pathogenesis of this disorder are still unclear. NL is
characterized by skin rash that usually affects the shins. The average onset is
30 years, with females being affected more commonly. There are very few reported
cases of necrobiosis lipoidica in children.
CASE REPORT: We report a case of a 16 year old girl affected by type 1 diabetes
mellitus (15 years disease duration) who developed an erythematous nodular rash
on the lower extremities and interscapular area. In the suspect of necrobiosis
lipoidica, a skin biopsy was performed (lower extremities and interscapular
area). The microscopic evaluation of the pretibial lesions was suggestive of
necrobiosis lipoidica. The smaller lesions in the interscapular area showed
signs of perivascular dermatitis which could be consistent with early stages of
necrobiosis lipoidica. Local treatment with tacrolimus determined a progressive
improvement of the lesions.
CONCLUSION: In patients with T1DM, diagnosis of NL of the lower legs is usually
unequivocal. However, diagnosis may be more challenging in the presence of
lesions with recent onset and/or atypical clinical presentation and unusual
site. In these cases, NL must always be taken in consideration in order to avoid
misdiagnosis, wrong/late treatment decisions and progression to ulceration. AIMS: Diabetic Foot Syndrome (DFS) is the most costly and devastating
complication of diabetes mellitus (DM), which early effective assessment can
reduce the severity of complications including ulceration and amputations. This
study aimed to review dermatological and musculoskeletal assessment of diabetic
foot.
MATERIALS AND METHODS: In this review article, we searched for articles
published between March 1, 1980 and July 28, 2015 in PubMed, Science Direct,
Embase, Web of Science, and Scopus, for both English and non-English language
articles with the following keywords: "Diabetic foot syndrome", "Ulceration",
"Amputation", "Foot assessment", "Skin disorders" and "Musculoskeletal
deformities".
RESULTS: In dermatological dimension, most studies focused on elucidated changes
in skin temperature, color, hardiness and turgor as well as common skin
disorders such as Diabetic Dermopathy (DD), Necrobiosis Lipoidica Diabeticorum
(NLD) and Diabetic Bullae (DB), which are common in diabetic patients and have
high potential for leading to limb-threatening problems such as ulceration and
infection. In musculoskeletal dimension, most studies focused on range of motion
and muscle strength, gait patterns and as well as foot deformities especially
Charcot osteoarthropathy (COA), which is the most destructive musculoskeletal
complication of diabetes.
CONCLUSION: DFS as a common condition in DM patients lead to ulceration and
lower limb amputation frequently unless a prompt and comprehensive assessment
was taken. So that dermatological and musculoskeletal assessments are usually
neglected in primary health care, these assessments should be done frequently to
reduce the high risk of serious complications. |
What is the role of Lysine 2-hydroxyisobutyrylation? | Lysine 2-hydroxyisobutyrylation (Khib) is a widely distributed active histone mark. This histone mark was initially identified by MS and then validated by chemical and biochemical methods. Histone Khib shows distinct genomic distributions from histone Kac or histone Kcr during male germ cell differentiation. In male germ cells, H4K8hib is associated with active gene transcription in meiotic and post-meiotic cells. In addition, H4K8ac-associated genes are included in and constitute only a subfraction of H4K8hib-labeled genes. The histone Khib mark is conserved and widely distributed, has high stoichiometry and induces a large structural change. | We report the identification of a new type of histone mark, lysine
2-hydroxyisobutyrylation (Khib), and identify the mark at 63 human and mouse
histone Khib sites, including 27 unique lysine sites that are not known to be
modified by lysine acetylation (Kac) and lysine crotonylation (Kcr). This
histone mark was initially identified by MS and then validated by chemical and
biochemical methods. Histone Khib shows distinct genomic distributions from
histone Kac or histone Kcr during male germ cell differentiation. Using
chromatin immunoprecipitation sequencing, gene expression analysis and
immunodetection, we show that in male germ cells, H4K8hib is associated with
active gene transcription in meiotic and post-meiotic cells. In addition,
H4K8ac-associated genes are included in and constitute only a subfraction of
H4K8hib-labeled genes. The histone Khib mark is conserved and widely
distributed, has high stoichiometry and induces a large structural change. These
findings suggest its critical role on the regulation of chromatin functions. |
List diseases associated with the Dopamine Receptor D4 (DRD4). | The 5-repeat allele of a common length polymorphism in the gene that encodes the dopamine D4 receptor (DRD4) is robustly associated with the risk of attention deficit hyperactivity disorder (ADHD). | The dopaminergic system plays a pivotal role in the central nervous system via
its five diverse receptors (D1-D5). Dysfunction of dopaminergic system is
implicated in many neuropsychological diseases, including attention deficit
hyperactivity disorder (ADHD), a common mental disorder that prevalent in
childhood. Understanding the relationship of five different dopamine (DA)
receptors with ADHD will help us to elucidate different roles of these receptors
and to develop therapeutic approaches of ADHD. This review summarized the
ongoing research of DA receptor genes in ADHD pathogenesis and gathered the past
published data with meta-analysis and revealed the high risk of DRD5, DRD2, and
DRD4 polymorphisms in ADHD. INTRODUCTION: The variable number of tandem repeats (VNTR) of the dopamine
receptor D4 (DRD4) gene among humans may elucidate individual differences in
susceptibility to neuropsychiatric diseases. Dopamine dysfunction may be
involved with Attention Deficit Hyperactivity Disorder (ADHD) symptoms. In this
study, we report the association between the phenotype of ADHD, a condition
characterized by inattentiveness, hyperactivity, and impulsiveness, and a
48-base pair VNTR in exon 3 of the DRD4 polymorphism.
SUBJECTS AND METHODS: We used a case control approach conducted on 29 ADHD and
31 ethnically matched control Egyptian children (ages 6-12 years). Cases were
assessed by a psychiatric semi-structured interview and the Conners' Parent
Rating Scale. VNTR polymorphisms of the DRD4 gene were done by touchdown PCR
program using exon 3-specific primers followed by agarose gel electrophoresis.
RESULTS: We observed a significant association between the existence of D4.4
allele of DRD4 and ADHD (P, 0.002); 6.9% of cases showed a single D4.4 and 10.3%
showed a double D4.4 as compared to controls in whom D4.4 has never been
detected.
CONCLUSION: Children with smaller number of repeat alleles (two to four repeats)
of the DRD4 gene have higher possibility to develop ADHD in Egyptian children. |
Which bacteria cause diphtheria? | Diphtheria is caused by the bacteria:
1) Corynebacterium ulcerans and
2) Corynebacterium diphtheriae. | It was shown in the passive hemagglutination test (PHAT) with a type-specific
somatic antigen on 147 carriers of toxigenic diphtheria bacilli that the PHAT
titres of 1/80 and over were determined in 64% of bacteria. In the process of
carrier state of toxigenic bacteria antimicrobial antibodies were detected in
79% of the children; after the release from the carrier state, the percentage
was from 57 to 26, depending on the time lapse after it. Among the carriers of
nontoxigenic diphtheria bacilli the PHAT titre of 1/80 and over was established
in up to 20% of children, and only in those which were in the focus of toxigenic
bacilli carriers. The applied test could be used for epidemiological purpose to
determine the spread of the carrier state of toxigenic bacteria in a collective
body. The public immunization program against diphtheria, established in 1941, has
almost eradicated the disease in Denmark, and 1956 became the first year without
any notified cases. Since then, toxigenic strains have only been isolated five
times--three cases of clinical diphtheria due to Corynebacterium diphtheriae
biovar. mitis and two cases of tonsillitis/pharyngitis due to Corynebacterium
ulcerans. The source of the infection was not identified in any of the cases.
The first case of diphtheria in 1968 was imported from abroad. The following two
cases in 1983 and 1985 were due to strains of the same phage type and peptide
profile as the strains isolated during the epidemic in Sweden in 1984-1986. This
indicates that the Danish cases and the Swedish epidemic derived from the same
source. The diphtheria immunity of the Danish population is decreasing, and the
level of protection is approaching the Swedish level. The impact is that a
situation like that in Sweden may be anticipated with diphtheria epidemic in the
lowest socio-economical groups--the skid row dwellers, alcoholics and drug
abusers--if the immunization program against diphtheria is not intensified. A high-density growth approach was utilized to produce mutated diphtheria toxin
from two strains of Corynebacterium diphtheria: C7 (beta)(tox-201,tox-9) and C7
(beta)(tox-107). The cross-reacting mutants (CRM) of the diphtheria toxin are
CRM9 and CRM107; both of them carry the mutation in their binding site and, as a
result, have 1/300 of the systemic toxicity of the wild-type diphtheria toxin.
Since iron inhibits diphtheria toxin production, the traditional approach has
been to grow the bacteria in a very low iron concentration. The procedure
described here involved the use of a modified, non-deferrated, growth medium
that provided fast and high-density growth of the bacteria, and which, when
associated with simultaneous depletion of glucose and iron, enhanced the toxin
production. Oxygen-enriched air was supplied to enable the bacteria to grow to a
cell density giving an absorbance of 70 at 600 nm (15-20 g/l dry weight). The
maximum toxin concentration in the culture supernatant was 150 mg/l. The CRM
products, which remained stable following microfiltration and ultrafiltration,
could be easily purified using a two-step chromatography procedure. Diphtheria toxin (tox) and its regulatory element (dtxR) from 72 Corynebacterium
diphtheriae strains isolated in Russia and Ukraine before and during the current
diphtheria epidemic were studied by PCR-single-strand conformation polymorphism
analysis (PCR-SSCP). Twelve sets of primers were constructed (eight for tox and
four for dtxR), and three regions within tox and all four regions of dtxR showed
significant variations in the number and/or sizes of the amplicons. Two to four
different SSCP patterns were identified in each of the variable regions;
subsequently, tox and dtxR could be classified into 6 and 12 different types,
respectively. The great majority of epidemic strains from both Russia and
Ukraine had tox types 3 and 4, and only in a single preepidemic strain isolated
in Russia were all eight tox regions identical to those of C. diphtheriae
Park-Williams No. 8 (tox type 1). Epidemic strains from Ukraine can easily be
identified by dtxR type 5, while the majority of the Russian epidemic strains
have dtxR of types 2 and 8. No differences in the tox regions between mitis and
gravis biotype strains were observed. However, dtxR types 2, 5, and 8 were
identified only in the gravis biotype, and dtxR type 1 was characteristic for
the mitis biotype strains. PCR-SSCP is a simple and rapid method for the
identification of variable tox and dtxR regions that allows for the clear
association of tox and dtxR types with strains of distinct temporal and/or
geographic origins. Respiratory diphtheria was one of the most common causes of death among children
in the pre-vaccine era. Since the introduction of diphtheria toxoid vaccine in
1920s, and its widespread use by the late 1940s, diphtheria became increasingly
rare in the United States. However, through the 1970s diphtheria remained
endemic in some states, with reported incidence rates > 1.0 per million
population in six states (Alaska, Arizona, Montana, New Mexico, South Dakota,
and Washington). Starting in 1980, less than five cases have been reported each
year in the United States. The majority of culture-confirmed cases have been
associated with importation from other countries. Toxigenic Corynebacterium
diphtheriae, the organism causing diphtheria, was thought to have become rare or
even have disappeared from previously endemic areas such as South Dakota.
However, during four months in 1996, 11 persons (one index case, six patients
and four household contacts) in an American Indian community in South Dakota
were found to be infected by C. diphtheriae; six of these isolates were
toxigenic. The findings in this report indicate that despite 20 years without
reported respiratory diphtheria cases, toxigenic C. diphtheriae is still present
in South Dakota. The continuous circulation of toxigenic strains of C.
diphtheriae emphasizes the need for health care providers throughout South
Dakota to promote timely vaccination against diphtheria among persons of all
ages and ethnic groups, to be aware of the clinical signs and symptoms of
diphtheria so that cases can be promptly diagnosed and treated, and further
public health measures can be taken to contain this serious disease. Extrapharyngeal infections caused by Corynebacterium ulcerans have rarely been
reported previously, and diphtheria toxin production has usually not been
addressed. This case demonstrates that strains of C. ulcerans that produce
diphtheria toxin can cause infections of the skin that completely mimic typical
cutaneous diphtheria, thereby potentially providing a source of bacteria capable
of causing life-threatening diseases in the patient's environment. Therefore, it
is recommended to screen wound swabs for coryneform bacteria, identify all
isolates, carefully assess possible toxin production, and send questionable
strains to a specialist or a reference laboratory. A 58-year-old woman was admitted due to a pseudomembranous pharyngitis. The
patient had not been vaccinated against diphtheria. Corynebacterium ulcerans was
cultured from a throat swab. The production of diphtheria toxin by these
bacteria was demonstrated with PCR and an immunoprecipitation test. The patient
was cared for in respiratory isolation and was treated with benzylpenicillin.
She quickly recovered and was discharged four days after admission. A contact
investigation did not reveal any dissemination of the toxin-producing C.
ulcerans and a source was not found. In spite of the large-scale vaccination
against diphtheria which has taken place in the Netherlands since 1953, a
physician has to consider diphtheria in the differential diagnosis of patients
who present with a clinical syndrome compatible with this disease. Either
Corynebacterium diphtheriae or C. ulcerans could be the pathogen responsible. Diphtheria toxin and exotoxin A are well-characterized members of the
ADP-ribosyltransferase toxin family that function as virulence factors in the
pathogenic bacteria Corynebacterium diphtheriae and Pseudomonas aeruginosa.
Recent high-resolution structural data of the Michaelis (enzyme-substrate)
complex of the P. aeruginosa toxin with an NAD(+) analog and eukaryotic
elongation factor 2 (eEF2) have provided insights into the mechanism of
inactivation of protein synthesis caused by these protein factors. In addition,
rigorous steady-state and stopped-flow kinetic analyses of the toxin-catalyzed
reaction, in combination with inhibitor studies, have resulted in a quantum leap
in our understanding of the mechanistic details of this deadly enzyme mechanism.
It is now apparent that these toxins use stealth and molecular mimicry in
unleashing their toxic strategy in the infected host eukaryotic cell. Infections caused by Corynebacterium diphtheriae frequently induce situations in
which very small doses of antigens injected intradermally can cause strong
inflammatory reactions. This bacterium secretes the diphtheria toxin (DT), a
virulence factor that can be lethal to the human organism at doses below 0.1
μg/kg of body weight. The present work proposes alternative methods of DT
purification using affinity chromatography and of DT detoxification through
conjugating with the polymer methoxypolyethylene glycol activated (mPEG). Tests
were performed to evaluate: the formation of edemas and the presence of
dermonecrotic activity, in vitro cytotoxicity to Vero cells, the neutralizing
activity of serum from guinea pigs immunized with the diphtheria toxoid
inactivated with mPEG, and the immunogenic activity of the purified and modified
toxin. The results indicated that purification with Blue Sepharose was an
efficient method, yielding antigen purity equivalent to 2600 Lf/mg of protein
nitrogen. The modification of the Purified Toxin with mPEG did not result in the
formation of edema or necrosis although it was immunogenic and stimulated the
formation of antibodies that could neutralize the Purified Toxin. The toxoid
obtained from the purified toxin maintained its immunogenic characteristics,
inducing antibodies with neutralizing activity; edema and necrosis were still
observed, however. Diphtheria is a serious upper respiratory tract disease caused by the diphtheria
toxin (DT) secreted from the bacteria Corynebacterium diphtheriae. Vaccination
is the best way to protect against this infectious disease. Diphtheria vaccines
are prepared by isolating, purifying and chemically deactivating DT. Although
toxoids have been used for decades in immunization, there is still little
understanding at the molecular level of the process of toxoid preparation, and
how chemical treatment enhances their immunogenicity. We have undertaken an NMR
study of the catalytic domain as a first step in understanding the molecular
details involved in vaccine antigen preparation. Here we report a near complete
assignment for the backbone and side chain resoces of the diphtheria toxin
catalytic domain. Diphtheria, caused by toxigenic strains of Corynebacterium diphtheriae, is an
ancient disease with high incidence and mortality that has always been
characterized by epidemic waves of occurrence. Whilst towards the beginning of
the 1980s, many European countries were progressing towards the elimination of
diphtheria, an epidemic re-emergence of diphtheria in the Russian Federation and
the Newly Independent States of the former Soviet Union demonstrated a
continuous threat of the disease into the 1990s. At present, the epidemic is
under control and only sporadic cases are observed in Europe. However, the
circulation of toxigenic strains is still observed in all parts of the world,
posing a constant threat to the population with low levels of seroprotection.
More recently, Corynebacterium ulcerans has been increasingly isolated as
emerging zoonotic agent of diphtheria from companion animals such as cats or
dogs, indicating the enduring threat of this thought-to-be controlled disease. BACKGROUND: Corynebacterium ulcerans can cause a diphtheria-like illness,
especially when the bacterium is lysogenized with a tox gene-carrying
bacteriophage that produces diphtheria toxin. Acquisition of toxigenicity upon
phage lysogenization is a common feature of C. ulcerans and C. diphtheriae.
However, because of a lack of C. ulcerans genome information, a detailed
comparison of prophages has not been possible between these two clinically
important and closely related bacterial species.
RESULTS: We determined the whole genome sequence of the toxigenic C. ulcerans
0102 isolated in Japan. The genomic sequence showed a striking similarity with
that of Corynebacterium pseudotuberculosis and, to a lesser extent, with that of
C. diphtheriae. The 0102 genome contained three distinct prophages. One of
these, ΦCULC0102-I, was a tox-positive prophage containing genes in the same
structural order as for tox-positive C. diphtheriae prophages. However, the
primary structures of the individual genes involved in the phage machinery
showed little homology between the two counterparts.
CONCLUSION: Taken together, these results suggest that the tox-positive prophage
in this strain of C. ulcerans has a distinct origin from that of C. diphtheriae
NCTC 13129. Diphtheria is caused by diphtheria toxin-producing Corynebacterium species.
While classical respiratory diphtheria is transmitted by droplets, cutaneous
diphtheria often results from minor trauma. This report concerns the first case
of sexually transmitted diphtheria in a patient with non-gonococcal urethritis
after orogenital contact. Diphtheria, an acute infectious condition caused by Corynebacterium diphtheriae,
was once a major killer of children. Although the mortality rates dropped
dramatically in the mid-twentieth century, due to a combination of improved
standards of living and immunization programs, outbreaks are still occurring.
Two children, aged four and five years respectively, are reported to demonstrate
characteristic features of lethal cases. Death in case 1 was due to an extensive
upper airway pseudomembrane causing acute respiratory failure. The diagnosis of
diphtheria was only made at postmortem. Death in case 2 was due to acute cardiac
failure with heart block complicating diphtheria. Other mechanisms in fatal
cases involve disseminated intravascular coagulation, renal and endocrine
failure. Declining levels of immunity among adults has resulted in a change in
the epidemiological pattern of the disease with an older age of victims in
recent outbreaks. As a result of population shifts and failure to immunize
children it is likely that forensic pathologists may see more cases of
diphtheria in the future. Due to the rarity of cases in Western communities and
atypical presentations, the diagnosis may only be established at autopsy. Corynebacterium diphtheriae is the etiological agent of diphtheria, a potential
fatal disease caused by a corynephage toxin. The expression of this diphtheria
toxin is controlled via an iron-dependent repressor with various functions
(DtxR). Some mutations in the dtxR gene are associated with diminished activity
or even with total loss of DtxR function. We conducted a molecular study to
characterize the dtxR alleles harbored by 34 isolates of C. diphtheriae
recovered from Romanian patients between 1961 and 2007. Three of the seven
alleles identified in this study have not previously been described. Two new
DtxR types were identified, one of which has an unusual polypeptide length. All
the new DtxR types were found in toxigenic isolates, suggesting that they
effectively regulate the expression of diphtheria toxin. Furthermore, one of the
new DtxR identified was also found in a non-toxigenic isolate, making it a
potential source of toxigenic isolates after lysogenic conversion. OBJECTIVES: Clinical diphtheria is on the increase worldwide, mainly affecting
developing countries. We sought to understand its presentation among patients at
Sir Ronald Ross Institute of Tropical and Communicable Diseases in Hyderabad,
Andhra Pradesh, India.
METHODS: Diphtheria patients presented with fever, pharyngitis, and a patch in
the throat. Data collected for each patient included age, clinical presentation,
morbidity, mortality, bacteria isolated from culture, and immunization status.
RESULTS: Of 61 950 admissions from January 2008 to December 2012, 2925 (4.7%)
had clinical diphtheria; 1194 had been immunized and 1731 were non-immunized.
Immunized patients had a milder disease. Culture-positive immunized patients
were positive for Corynebacterium other than diphtheriae (COD; n=104) or
Corynebacterium diphtheriae (CD; n=23); these patients suffered mild disease and
recovered completely. In contrast, culture-positive non-immunized patients were
positive for COD (n=11) or CD (n=412). Eighty-one patients (3%) died, 77 of whom
were non-immunized; death was usually as a result of myocarditis. Seventy-three
percent of deaths were in patients aged <5 years.
CONCLUSIONS: The clinical presentation of diphtheria and its severity and
morbidity differ considerably in immunized and non-immunized patients. Disease
caused by CD can be deadly, while disease due to COD is mild and responds to
treatment. The bacterial genus Corynebacteria contains several pathogenic species that
cause diseases such as diphtheria in humans and "cheesy gland" in goats and
sheep. Thus, identifying new therapeutic targets to treat Corynebacteria
infections is both medically and economically important. CG2496, a functionally
uncharacterized protein from Corynebacterium glutamicum, was evaluated using an
NMR ligand-affinity screen. A total of 11 compounds from a library of 460
biologically active compounds were shown to selectively bind CG2496 in a highly
conserved region of the protein. The best binder was identified to be
methiothepin (KD =54 ± 19 µM), an FDA-approved serotonin receptor antagonist.
Methiothepin was also shown to inhibit the growth of C. glutamicum, but not
bacteria that lack CG2496 homologs. Our results suggest that CG2496 is a novel
therapeutic target and methiothepin is a potential lead compound or structural
scaffold for developing new antibiotics specifically targeting Corynebacteria. Corynebacterium ulcerans may cause diphtheria in humans and caseous
lymphadenitis in animals. We isolated nontoxigenic tox-bearing C. ulcerans from
13 game animals in Germany. Our results indicate a role for game animals as
reservoirs for zoonotic C. ulcerans. Corynebacterium pseudodiphtheriticum is a common commensal flora of the upper
respiratory tract in humans. Though the pathogenicity of C. pseudodiphtheriticum
is not rare, its role as an opportunistic pathogen is mainly limited to the
lower respiratory tract, particularly in patients with underlying systemic
conditions or immune-compromisation. We hereby present the first case of C.
pseudodiphtheriticum causing diphtheria-like illness affecting the upper
respiratory tract of a 6-year-old fully immunised otherwise healthy child. In
countries with very low incidence of diphtheria, C. pseudodiphtheriticum should
be included in differential diagnosis for a child presenting with
diphtheria-like illness. Simple, rapid screening tests should be used to
differentiate it from C. diphtheriae and hence, to prevent unnecessary concern
in community. Human-to-human-transmitted Corynebacterium diphtheriae was historically the main
pathogen causing diphtheria and has therefore been studied extensively in the
past. More recently, diphtheria caused by toxigenic Corynebacterium ulcerans is
an emerging disease in several industrial countries, including the United
Kingdom, the United States, France, and Germany. However, toxigenic C. ulcerans
has so far been almost neglected in the development of epidemiologic tools. One
of the most important tools in modern epidemiology to understand transmission
pathways is sequence typing of pathogens. Here, we provide a protocol for
multilocus sequence typing (MLST) to type C. ulcerans strains rapidly and
relatively cost-effectively. Applying MLST to C. ulcerans for the first time, we
show that related sequence types (STs) might be associated with the presence of
the diphtheria toxin gene, which encodes diphtheria toxin (DT), the most
important diphtheria-causing virulence factor. Interestingly, we found only two
very closely related STs in the isolates derived from six dogs. Additionally,
our data show that all STs derived from animals which were at least twice
present in our analysis were found in humans as well. This finding is congruent
with zoonotic transmission of C. ulcerans. Corynebacterium ulcerans (toxigenic C. ulcerans) produces the diphtheria toxin,
which causes pharyngeal and cutaneous diphtheria-like disease in people, and
this bacterium is commonly detected in dogs and cats that are reared at home. It
is considered dangerous when a carrier animal becomes the source of infection in
people. To investigate the carrier situation of toxigenic C. ulcerans of cats
bred in Japan, bacteria were isolated from 37 cats with a primary complaint of
rhinitis in 16 veterinary hospitals in Osaka. Toxigenic C. ulcerans was detected
in two of the cats. By drug sensitivity testing, the detected bacterium was
sensitive to all investigated drugs, except clindamycin. It appears necessary to
create awareness regarding toxigenic C. ulcerans infection in pet owners because
this bacterium is believed to be the causative organism for rhinitis in cats. BACKGROUND: Toxigenic Corynebacterium ulcerans can cause a diphtheria-like
illness in humans and have been found in domestic animals, which were suspected
to serve as reservoirs for a zoonotic transmission. Additionally, toxigenic C.
ulcerans were reported to take over the leading role in causing diphtheria in
the last years in many industrialized countries.
METHODS: To gain deeper insights into the tox gene locus and to understand the
transmission pathway in detail, we analyzed nine isolates derived from human
patients and their domestic animals applying next generation sequencing and
comparative genomics.
RESULTS: We provide molecular evidence for zoonotic transmission of C. ulcerans
in four cases and demonstrate the superior resolution of next generation
sequencing compared to multi-locus sequence typing for epidemiologic research.
Additionally, we provide evidence that the virulence of C. ulcerans can change
rapidly by acquisition of novel virulence genes. This mechanism is exemplified
by an isolate which acquired a prophage not present in the corresponding isolate
from the domestic animal. This prophage contains a putative novel virulence
factor, which shares high identity with the RhuM virulence factor from
Salmonella enterica but which is unknown in Corynebacteria so far. Furthermore,
we identified a putative pathogenicity island for C. ulcerans bearing a
diphtheria toxin gene.
CONCLUSION: The novel putative diphtheria toxin pathogenicity island could
provide a new and alternative pathway for Corynebacteria to acquire a functional
diphtheria toxin-encoding gene by horizontal gene transfer, distinct from the
previously well characterized phage infection model. The novel transmission
pathway might explain the unexpectedly high number of toxigenic C. ulcerans. Author information:
(1)Landesbetrieb Hessisches Landeslabor, Schubertstr. 60, 35392, Gießen,
Germany. [email protected].
(2)Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/2, 70736,
Fellbach, Germany. [email protected].
(3)Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/2, 70736,
Fellbach, Germany. [email protected].
(4)Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/2, 70736,
Fellbach, Germany. [email protected].
(5)Institute of Hygiene and Infectious Diseases of Animals, Justus Liebig
University Giessen, Frankfurter Strasse 85-89, 35392, Giessen, Germany.
[email protected].
(6)Landesbetrieb Hessisches Landeslabor, Schubertstr. 60, 35392, Gießen,
Germany. [email protected].
(7)Landesbetrieb Hessisches Landeslabor, Schubertstr. 60, 35392, Gießen,
Germany. [email protected].
(8)Zoo Frankfurt, Bernhard-Grzimek-Allee 1, 60316, Frankfurt, Germany.
[email protected].
(9)Zoo Frankfurt, Bernhard-Grzimek-Allee 1, 60316, Frankfurt, Germany.
[email protected].
(10)National Consiliary Laboratory on Diphtheria, Bayerisches Landesamt für
Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764,
Oberschleißheim, Germany. [email protected].
(11)National Consiliary Laboratory on Diphtheria, Bayerisches Landesamt für
Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764,
Oberschleißheim, Germany. [email protected].
(12)National Consiliary Laboratory on Diphtheria, Bayerisches Landesamt für
Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764,
Oberschleißheim, Germany. [email protected]. |
What is the function of calcium-sensing receptor (CaSR)? | The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that plays an essential role in maintaining calcium homeostasis.
The CaSR is a key regulator for such diverse processes as hormone secretion, gene expression, inflammation, proliferation, differentiation, and apoptosis. Due to this pleiotropy, the CaSR is able to regulate cell fate and is implicated in the development of many types of benign or malignant tumours of the breast, prostate, parathyroid, and colon. | OBJECTIVE: Genetic disorders of calcium metabolism arise in a familial or
sporadic setting. The calcium-sensing receptor (CASR) plays a key role in
maintaining calcium homeostasis and study of the CASR gene can be clinically
useful in determining etiology and appropriate therapeutic approaches. We report
two cases of novel CASR gene mutations that illustrate the varying clinical
presentations and discuss these in terms of the current understanding of CASR
function.
PATIENTS AND METHODS: A 16-year-old patient had mild hypercalcemia associated
with low-normal urinary calcium excretion and normal-to-high parathyroid hormone
(PTH) levels. Because of negative family history, familial hypocalciuric
hypercalcemia was originally excluded. The second patient was a 54-year-old man
with symptomatic hypocalcemia, hyperphosphatemia, low PTH, and mild
hypercalciuria. Familial investigation revealed the same phenotype in the
patient's sister. The coding region of the CASR gene was sequenced in both
probands and their available first-degree relatives.
RESULTS: The first patient had a novel heterozygous inactivating CASR mutation
in exon 4, which predicted a p.A423K change; genetic analysis was negative in
the parents. The second patient had a novel heterozygous activating CASR
mutation in exon 6, which predicted a p.E556K change; the affected sister of the
proband was also positive.
CONCLUSIONS: We reported two novel heterozygous mutations of the CASR gene, an
inactivating mutation in exon 4 and the first activating mutation reported to
date in exon 6. These cases illustrate the importance of genetic testing of CASR
gene to aid correct diagnosis and to assist in clinical management. Expression and function of the CaSR have been shown in some mammalian taste buds
and basal cells of the esophagus. Signaling cascades responsible for
CaSR-mediated stimulation of H(+)-K(+)-ATPase on human parietal cells have been
defined. Transgenic mice and reductionistic cell culture models have shown that
the CaSR promotes gastrin secretion from G cells, cholecystokinin (CCK)
secretion from duodenal I cells and BMP-2 secretion from sub-epithelial
myofibroblasts. In addition, the CaSR mediates a novel paracrine relationship
between myofibroblasts and overlying epithelial cells in the colon. Thus, CaSR
activators stimulate secretion of Wnt5a from myofibroblasts and expression of
the Wnt5a receptor Ror2 in epithelial cells. CaSR-mediated Wnt5a/Ror2 engagement
stimulates epithelial differentiation and reduces expression of the receptor for
tumor necrosis factor (TNFR1). CaSR activators also modulate intestinal
motility, inhibit Cl(-) secretion and stimulate Na(+) absorption in both the
small intestine and colon. Colonic epithelia from conditional and global CaSR
knockout mice exhibit increased proliferation with increased Wnt/β-catenin
signaling, demonstrating that the CaSR negatively modulates colonic epithelial
growth. G protein-coupled receptors (GPCRs) are cell surface receptors that detect a
wide range of extracellular messengers and convey this information to the inside
of cells. Extracellular calcium-sensing receptor (CaSR) and ovarian cancer gene
receptor 1 (OGR1) are two GPCRs that sense extracellular Ca(2+) and H(+),
respectively. These two ions are key components of the interstitial fluid, and
their concentrations change in an activity-dependent manner. Importantly, the
interstitial fluid forms part of the microenvironment that influences cell
function in health and disease; however, the exact mechanisms through which
changes in the microenvironment influence cell function remain largely unknown.
We show that CaSR and OGR1 reciprocally inhibit signaling through each other in
central neurons, and that this is lost in their transformed counterparts.
Furthermore, strong intracellular acidification impairs CaSR function, but
potentiates OGR1 function. Thus, CaSR and OGR1 activities can be regulated in a
seesaw manner, whereby conditions promoting signaling through one receptor
simultaneously inhibit signaling through the other receptor, potentiating the
difference in their relative signaling activity. Our results provide insight
into how small but consistent changes in the ionic microenvironment of cells can
significantly alter the balance between two signaling pathways, which may
contribute to disease progression. The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that plays
an essential role in maintaining calcium homeostasis. In the present study, we
analyzed the CaSR gene in a Korean family with familial hypocalciuric
hypercalcemia (FHH). Genetic studies were performed by direct sequence analysis
of the CaSR gene in genomic DNA obtained from peripheral leukocytes. A novel
heterozygous G to T substitution at nucleotide position 1711 in exon 6,
resulting in the G571W mutation, was identified in the CaSR gene in a
26-year-old female with asymptomatic hypercalcemia, a low calcium/creatinine
clearance ratio, and normal intact parathyroid hormone. To study CaSR
expression, the mutation was introduced by site-directed mutagenesis into a
wild-type (WT) CaSR-expressing pCR3.1 vector, and COS-7 cells were transfected
with either the WT or mutant CaSR-containing vector. Transfected cells loaded
with Fura-2/AM, a fluorescent indicator of Ca2+, were assessed for CaSR function
by the change in intracellular calcium [as measured by the 340 nm/380 nm
fluorescence intensity ratio (F340/F380)] made in response to challenge with
extracellular Ca2+. Both WT and G571W cells had equivalent amounts of CaSR
protein in the cell membrane. However, after challenge with extracellular Ca2+,
cells transfected with G571W CaSR responded with a lower F340/F380 ratio than
those transfected with WT CaSR and showed decreased sensitivity to extracellular
Ca2+ concentrations. The G571W mutation had therefore impaired the CaSR
function. In conclusion, we identified a novel loss-of-function mutation, G571W,
in the CaSR gene in a Korean family with FHH. The calcium-sensing receptor (CaSR) plays a pivotal role in systemic calcium
metabolism by regulating parathyroid hormone secretion and urinary calcium
excretion. The CaSR is ubiquitously expressed, implying a wide range of
functions regulated by this receptor. Abnormal CaSR function affects the
development of both calciotropic disorders such as hyperparathyroidism, and
non-calciotropic disorders such as cardiovascular disease and cancer, which are
the leading causes of mortality worldwide. The CaSR is able to bind a plethora
of ligands; it interacts with multiple G protein subtypes, and regulates highly
divergent downstream signalling pathways, depending on the cellular context. The
CaSR is a key regulator for such diverse processes as hormone secretion, gene
expression, inflammation, proliferation, differentiation, and apoptosis. Due to
this pleiotropy, the CaSR is able to regulate cell fate and is implicated in the
development of many types of benign or maligt tumours of the breast,
prostate, parathyroid, and colon. In cancer, the CaSR appears to have
paradoxical roles, and depending on the tissue involved, it is able to prevent
or promote tumour growth. In tissues like the parathyroid or colon, the CaSR
inhibits proliferation and induces terminal differentiation of the cells.
Therefore, loss of the receptor, as seen in colorectal or parathyroid tumours,
confers maligt potential, suggestive of a tumour suppressor role. In
contrast, in prostate and breast tumours the expression of the CaSR is increased
and it seems that it favours metastasis to the bone, acting as an oncogene.
Deciphering the molecular mechanism driving the CaSR in the different tissues
could lead to development of new allosteric drug compounds that selectively
target the CaSR and have therapeutic potential for cancer. This article is part
of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques
Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. |
List drug that were evaluated in the CHAMP trial for migraine. | Childhood and Adolescent Migraine Prevention (CHAMP) trial evaluated effectiveness of amitriptyline and topiramate in the prevention of migraine in children and adolescents. | BACKGROUND: Migraine is one of the most common health problems for children and
adolescents. If not successfully treated, it can impact patients and families
with significant disability due to loss of school, work, and social function.
When headaches become frequent, it is essential to try to prevent the headaches.
For children and adolescents, this is guided by extrapolation from adult
studies, a limited number of small studies in children and adolescents and
practitioner preference. The aim of the Childhood and Adolescent Migraine
Prevention (CHAMP) study is to determine the most effective preventive agent to
use in children and adolescents.
METHODS: CHAMP is a double-blinded, placebo-controlled, multicenter, comparative
effectiveness study of amitriptyline and topiramate for the prevention of
episodic and chronic migraine, designed to mirror real-world practice, sponsored
by the US National Institute of Neurological Disorders and Stroke/National
Institutes of Health (U01NS076788). The study will recruit 675 subjects between
the ages of 8 and 17 years old, inclusive, who have migraine with or without
aura or chronic migraine as defined by the International Classification of
Headache Disorders, 2nd Edition, with at least 4 headaches in the 28 days prior
to randomization. The subjects will be randomized in a 2:2:1 (amitriptyline:
topiramate: placebo) ratio. Doses are weight based and will be slowly titrated
over an 8-week period to a target dose of 1 mg/kg of amitriptyline and 2 mg/kg
of topiramate. The primary outcome will be a 50% reduction in headache frequency
between the 28-day baseline and the final 28 days of treatment (weeks 20-24).
CONCLUSIONS: The goal of the CHAMP study is to obtain level 1 evidence for the
effectiveness of amitriptyline and topiramate in the prevention of migraine in
children and adolescents. If this study proves to be positive, it will provide
information to the practicing physician as how to best prevent migraine in
children and adolescents and subsequently improve the disability and outcomes. OBJECTIVE: To describe baseline headache characteristics of children and
adolescents participating in a multicenter, randomized, double-blinded,
placebo-controlled, comparative effectiveness study of amitriptyline,
topiramate, and placebo for the prevention of migraine (CHAMP Study).
METHODS: Children and adolescents (age 8-17 years old, inclusive) diagnosed with
migraine with or without aura, having headaches at least four times per month
were enrolled from 2012 through 2014. The trial involved a baseline period
(minimum of 28 days) during which prospective diaries were completed and
demographics and headache features obtained.
RESULTS: A total of 488 children and adolescents (mean age 14.0 ± 2.4 years)
agreed to participate in the trial, with 361 randomized and 127 not randomized.
Randomized subjects had a 5.5 ± 3.1 year history of headaches, with 15.1 ± 7.1
headache days per month (based upon retrospective report at screening visit).
Prospective diaries reported 11.5 ± 6.1 headache days per 28 day baseline.
Across this 28 day period, reported headache days per week were stable (about 3
headache days per week). Recording of individual headache features by diary
(n = 4136 headache days) showed characteristics consistent with migraine (mean
duration 10.5 ± 8.1 hours, mean severity 6.0 ± 2.1, 60% throbbing, 55% with
activity worsening headaches, 55% with photophobia, and 47% with phonophobia).
CONCLUSIONS: Baseline data from the CHAMP Study suggested that the randomized
sample was representative of the real world population of children and
adolescents that present for treatment of migraine. Headaches in children and
adolescents recorded during a 28 day prospective baseline period in this
multi-site comparative effectiveness study did not change over the course of the
baseline period, even though a clear diagnosis, recommendation for effective
acute treatment, and standardized education about healthy habits occurred prior
to the diary collection period. BACKGROUND: Which medication, if any, to use to prevent the headache of
pediatric migraine has not been established.
METHODS: We conducted a randomized, double-blind, placebo-controlled trial of
amitriptyline (1 mg per kilogram of body weight per day), topiramate (2 mg per
kilogram per day), and placebo in children and adolescents 8 to 17 years of age
with migraine. Patients were randomly assigned in a 2:2:1 ratio to receive one
of the medications or placebo. The primary outcome was a relative reduction of
50% or more in the number of headache days in the comparison of the 28-day
baseline period with the last 28 days of a 24-week trial. Secondary outcomes
were headache-related disability, headache days, number of trial completers, and
serious adverse events that emerged during treatment.
RESULTS: A total of 361 patients underwent randomization, and 328 were included
in the primary efficacy analysis (132 in the amitriptyline group, 130 in the
topiramate group, and 66 in the placebo group). The trial was concluded early
for futility after a planned interim analysis. There were no significant
between-group differences in the primary outcome, which occurred in 52% of the
patients in the amitriptyline group, 55% of those in the topiramate group, and
61% of those in the placebo group (amitriptyline vs. placebo, P=0.26; topiramate
vs. placebo, P=0.48; amitriptyline vs. topiramate, P=0.49). There were also no
significant between-group differences in headache-related disability, headache
days, or the percentage of patients who completed the 24-week treatment period.
Patients who received amitriptyline or topiramate had higher rates of several
adverse events than those receiving placebo, including fatigue (30% vs. 14%) and
dry mouth (25% vs. 12%) in the amitriptyline group and paresthesia (31% vs. 8%)
and weight loss (8% vs. 0%) in the topiramate group. Three patients in the
amitriptyline group had serious adverse events of altered mood, and one patient
in the topiramate group had a suicide attempt.
CONCLUSIONS: There were no significant differences in reduction in headache
frequency or headache-related disability in childhood and adolescent migraine
with amitriptyline, topiramate, or placebo over a period of 24 weeks. The active
drugs were associated with higher rates of adverse events. (Funded by the
National Institutes of Health; CHAMP ClinicalTrials.gov number, NCT01581281 ). |
What is the role of IL-18BP? | IL-18 binding protein (IL-18BP) is a natural inhibitor of IL-18. The balance between IL-18 and IL-18BP has an important role in the inflammatory setting. | AIM: In patients, an association exists between pulmonary diseases and diastolic
dysfunction of the left ventricle (LV). We have previously shown that alveolar
hypoxia in mice induces LV diastolic dysfunction and that mice exposed to
hypoxia have increased levels of circulating interleukin-18 (IL-18), suggesting
involvement of IL-18 in development of diastolic dysfunction. IL-18 binding
protein (IL-18BP) is a natural inhibitor of IL-18. In this study, we
hypothesized that neutralization of IL-18 during alveolar hypoxia would improve
LV diastolic function.
METHODS: Mice were exposed to 10% oxygen for 2 weeks while treated with IL-18BP
or vehicle. Cardiac function and morphology were measured using
echocardiography, intraventricular pressure measurements and magnetic resoce
imaging (MRI). For characterization of molecular changes in the heart, both
real-time PCR and Western blotting were performed. ELISA technique was used to
measure levels of circulating cytokines.
RESULTS: As expected, exposure to hypoxia-induced LV diastolic dysfunction, as
shown by prolonged time constant of isovolumic relaxation (τ). Improved
relaxation with IL-18BP treatment was demonstrated by a significant reduction
towards control τ values. Decreased levels of phosphorylated phospholamban
(P-PLB) in hypoxia, but normalization by IL-18BP treatment suggest a role for
IL-18 in regulation of calcium-handling proteins in hypoxia-induced diastolic
dysfunction. In addition, MRI showed less increase in right ventricular (RV)
wall thickness in IL-18BP-treated animals exposed to hypoxia, indicating an
effect on RV hypertrophy.
CONCLUSION: Neutralization of IL-18 during alveolar hypoxia improves LV
diastolic function and partly prevents RV hypertrophy. PURPOSE: Severe local and systemic tissue damage called ischemia/reperfusion
(IR) injury occurs during the period of reperfusion. Free oxygen radicals and
proinflammatory cytokines are responsible for reperfusion injury. IL-18 binding
protein (IL-18BP) is a natural inhibitor of IL-18. The balance between IL-18 and
IL-18BP has an important role in the inflammatory setting. The present study
aimed to investigate whether IL-18BP had a protective role in remote organ
hepatic IR injury.
METHODS: Wistar-Albino rats were divided into three groups that contained seven
rats. Group I (sham): Laparotomy and infrarenal abdominal aorta (AA) dissection
were done but no clamping was done. Group II (I/R): The infrarenal AA was
clamped by atraumatic microvascular clamp for 30 minutes and then was exposed to
90 minutes of reperfusion. Group III (IR + IL-18BP): 75 µg/kg of IL-18BP in 0.9%
saline (1 mL) was administered 30 minutes before infrarenal AA dissection and
clamping; 30 minutes of ischemia was applied and then was exposed to 90 minutes
of reperfusion.
RESULTS: Serum AST, ALT, and LDH levels were remarkably higher in IR group and
returned to normal levels in treatment group. The proinflammatory cytokine
levels had decreased in treatment group, and was statistically significant
compared with the IR group. Serum levels of total oxidant status and oxidative
stress index decreased and levels of total antioxidant status increased by
IL-18BP.
CONCLUSION: This study suggested that IL-18BP has antioxidant, anti-inflammatory
and hepatoprotective effects in cases of IR with infrarenal AA induced liver
oxidative damage. Lung cancer is the leading cause of cancer death, and it is widely accepted that
chronic inflammation is an important risk for the development of lung cancer.
Now, it is recognized that the nucleotide-binding and oligomerization domain
(NOD) like receptors (NLRs)-containing inflammasomes are involved in
cancer-related inflammation. This study was designed to investigate the effects
of NLR family pyrin domain containing protein 3 (NLRP3) inflammasome on the
proliferation and migration of lung adenocarcinoma cell line A549. Using
5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, scratch assay, and
Transwell migration assay, we showed that activation of the NLRP3 inflammasome
by LPS+ATP enhanced the proliferation and migration of A549 cells. Western blot
analysis showed that activation of phosphorylation of Akt, ERK1/2, CREB and the
expression of Snail increased, while the expression of E-cadherin decreased
after the activation of NLRP3 inflammasome. Moreover, these effects were
inhibited by the following treatments: i) downregulating the expression of NLRP3
by short hairpin RNA (shRNA) interference, ii) inhibiting the activation of
NLRP3 inflammasome with a caspase-1 inhibitor, iii) blocking the interleukin-1β
(IL-1β) and IL-18 signal transduction with IL-1 receptor antagonist (IL-1Ra) and
IL-18 binding protein (IL-18BP). Collectively, these results indicate that NLRP3
inflammasome plays a vital role in regulating the proliferation and migration of
A549 cells and it might be a potential target for the treatment of lung cancer. IL-18 is a pleiotropic and multifunctional cytokine that belongs to the IL-1
family. It is produced as a biologically inactive precursor, which is cleaved
into its active mature form mainly by caspase-1. The caspase becomes active from
its inactive precursor (procaspase-1) upon assembly of an inflammasome. Because
of IL-18's potential pro-inflammatory and tissue destructive effects, its
biological activities are tightly controlled in the body by its naturally
occurring antagonist called IL-18BP. The antagonist is produced in the body both
constitutively and in response to an increased production of IL-18 as a negative
feedback mechanism. Under physiological conditions, most of IL-18 in the
circulation is bound with IL-18BP and is inactive. However, an imbalance in the
production of IL-18 and its antagonist (an increase in the production of IL-18
with a decrease, no increase or an insufficient increase in the production of
IL-18BP) has been described in many chronic inflammatory diseases in humans. The
imbalance results in an increase in the concentrations of free IL-18 (unbound
with its antagonist) resulting in increased biological activities of the
cytokine that contribute towards pathogenesis of the disease. In this article,
we provide an overview of the current biology of IL-18 and its antagonist,
discuss how the imbalance occurs in HIV infections and how it contributes
towards development of AIDS and other non-AIDS-associated clinical conditions
occurring in HIV-infected individuals undergoing combination anti-retroviral
therapy (cART). Finally, we discuss challenges facing immunotherapeutic
strategies aimed at restoring balance between IL-18 and its antagonist in these
patients. |
Which protein mediates gene loop formation in the yeast S. cerevisiae? | Moreover, looping is dependent upon the general transcription factor TFIIB: the E62K (glutamic acid 62 --> lysine) form of TFIIB adversely affects looping at every gene tested, including BLM10, SAC3, GAL10, SEN1, and HEM3. TFIIB crosslinks to both the promoter and terminator regions of the PMA1 and BLM10 genes, and its association with the terminator, but not the promoter, is adversely affected by E62K and by depletion of the Ssu72 component of the CPF 3' end processing complex, and is independent of TBP. | Most nucleosomes are well-organized at the 5' ends of S. cerevisiae genes where
"-1" and "+1" nucleosomes bracket a nucleosome-free promoter region (NFR). How
nucleosomal organization is specified by the genome is less clear. Here we
establish and inter-relate rules governing genomic nucleosome organization by
sequencing DNA from more than one million immunopurified S. cerevisiae
nucleosomes (displayed at http://atlas.bx.psu.edu/). Evidence is presented that
the organization of nucleosomes throughout genes is largely a consequence of
statistical packing principles. The genomic sequence specifies the location of
the -1 and +1 nucleosomes. The +1 nucleosome forms a barrier against which
nucleosomes are packed, resulting in uniform positioning, which decays at
farther distances from the barrier. We present evidence for a novel 3' NFR that
is present at >95% of all genes. 3' NFRs may be important for transcription
termination and anti-sense initiation. We present a high-resolution genome-wide
map of TFIIB locations that implicates 3' NFRs in gene looping. 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 interaction of the promoter and the terminator
regions of a gene during transcription, requires transcription factor IIB
(TFIIB). We have earlier demonstrated association of TFIIB with the distal ends
of a gene in an activator-dependent manner (El Kaderi, B., Medler, S.,
Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The
presence of TFIIB at the 3' end of a gene required its interaction with cleavage
factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity
chromatography and glycerol gradient centrifugation, we show that TFIIB
associates with poly(A) polymerase and the entire CF1 complex in yeast cells.
The factors required for general transcription such as TATA-binding protein, RNA
polymerase II, and TFIIH are not a component of the TFIIB complex. This
holo-TFIIB complex was resistant to MNase digestion. The complex was observed
only in the looping-competent strains, but not in the looping-defective sua7-1
strain. The requirement of Rna15 in gene looping has been demonstrated earlier.
Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits
Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes.
Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the
mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where
holo-TFIIB complex is not formed, the kinetics of activated transcription is
altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1
exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and
Pap1 is crucial for gene looping and transcriptional regulation. TFIIB is essential for transcription initiation by RNA polymerase II. TFIIB also
cross-links to terminator regions and is required for gene loops that juxtapose
promoter-terminator elements in a transcription-dependent manner. The
Saccharomyces cerevisiae sua7-1 mutation encodes an altered form of TFIIB (E62K)
that is defective for both start site selection and gene looping. Here we report
the isolation of an ssl2 mutant, encoding an altered form of TFIIH, as a
suppressor of the cold-sensitive growth defect of the sua7-1 mutation. Ssl2
(Rad25) is orthologous to human XPB and is a member of the SF2 family of
ATP-dependent DNA helicases. The ssl2 suppressor allele encodes an arginine
replacement of the conserved histidine residue (H508R) located within the
DEVH-containing helicase domain. In addition to suppressing the TFIIB E62K
growth defect, Ssl2 H508R partially restores both normal start site selection
and gene looping. Moreover, Ssl2, like TFIIB, associates with promoter and
terminator regions, and the diminished association of TFIIB E62K with the PMA1
terminator is restored by the Ssl2 H508R suppressor. These results define a
novel, functional interaction between TFIIB and Ssl2 that affects start site
selection and gene looping. 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 two drugs were compared in the ARISTOTLE Trial? | Apixaban for Reduction In Stroke and Other Thromboembolic Events in Atrial Fibrillation (ARISTOTLE) trial compared apixaban and warfarin. | The objective of this review is to summarize data from the Apixaban for
Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation
(ARISTOTLE) and Apixaban Versus Acetylsalicylic Acid to Prevent Stroke in Atrial
Fibrillation Patients Who Have Failed or Are Unsuitable for Vitamin K Antagonist
Treatment (AVERROES) trials of apixaban for stroke prevention in patients with
atrial fibrillation (AF). The ARISTOTLE trial compared apixaban with warfarin in
18 201 patients with AF and ≥ 1 additional risk factor for stroke. The AVERROES
trial compared apixaban with aspirin in 5599 patients with AF who were at
increased risk of stroke and for whom vitamin K antagonists were unsuitable. In
ARISTOTLE, apixaban reduced the risk of stroke or systemic embolism by 21%
compared with warfarin (1.27% vs 1.60% per year; hazard ratio, 0.79; 95%
confidence interval, 0.66-0.95). The reduction was significant and demonstrated
the superiority of apixaban over warfarin for the primary outcome of preventing
stroke or systemic embolism (P = 0.01 for superiority). Apixaban also reduced
all-cause mortality by 11% (P = 0.047) and major bleeding by 31% (P < 0.001)
compared with warfarin. The benefits of apixaban observed in ARISTOTLE are
further supported by the results from AVERROES, which demonstrated a 55%
reduction in the risk of stroke or systemic embolism compared with aspirin. Risk
of major bleeding was not significantly different between apixaban and aspirin.
Subgroup analyses in both trials demonstrated that the effects of apixaban are
highly consistent across various patient subpopulations. Discontinuation of
study medication was significantly lower with apixaban than with either warfarin
in ARISTOTLE or aspirin in AVERROES. Apixaban is the first new oral
anticoagulant that has been shown to be superior to warfarin in reducing stroke
or systemic embolism, all-cause mortality, and major bleeding in patients with
AF. Moreover, in patients with AF who are considered unsuitable for warfarin
therapy, apixaban was more effective than aspirin for stroke prevention and had
a similar rate of major bleeding. Warfarin has long been considered the gold standard for stroke prevention in
patients with atrial fibrillation (AF). Recently, three major trials comparing
the efficacy and safety of new drugs: a thrombin inhibitor dabigatran and two
inhibitors of factor Xa - rivaroxaban and apixaban, with that of warfarin, have
been published. The aim of this paper is to present the main results of the
RE-LY, ROCKET AF and ARISTOTLE trials, compare study populations and outcomes,
and discuss clinical implications of their results for the long-term
anticoagulation in patients with nonvalvular AF. OBJECTIVE: The randomized clinical trials, RE-LY, ROCKET-AF, and ARISTOTLE,
demonstrate that the novel oral anticoagulants (NOACs) are effective options for
stroke prevention among non-valvular atrial fibrillation (AF) patients. This
study aimed to evaluate the medical cost reductions associated with the use of
individual NOACs instead of warfarin from the US payer perspective.
METHODS: Rates for efficacy and safety clinical events for warfarin were
estimated as the weighted averages from the RE-LY, ROCKET-AF and ARISTOTLE
trials, and event rates for NOACs were determined by applying trial hazard
ratios or relative risk ratios to such weighted averages. Incremental medical
costs to a US health payer of an AF patient experiencing a clinical event during
1 year following the event were obtained from published literature and inflation
adjusted to 2010 cost levels. Medical costs, excluding drug costs, were
evaluated and compared for each NOAC vs warfarin. Sensitivity analyses were
conducted to determine the influence of variations in clinical event rates and
incremental costs on the medical cost reduction.
RESULTS: In a patient year, the medical cost reduction associated with NOAC
usage instead of warfarin was estimated to be -$179, -$89, and -$485 for
dabigatran, rivaroxaban, and apixaban, respectively. When clinical event rates
and costs were allowed to vary simultaneously, through a Monte Carlo simulation,
the 95% confidence interval of annual medical costs differences ranged between
-$424 and +$71 for dabigatran, -$301 and +$135 for rivaroxaban, and -$741 and
-$252 for apixaban, with a negative number indicating a cost reduction. Of the
10,000 Monte-Carlo iterations 92.6%, 79.8%, and 100.0% were associated with a
medical cost reduction >$0 for dabigatran, rivaroxaban, and apixaban,
respectively.
CONCLUSIONS: Usage of the NOACs, dabigatran, rivaroxaban, and apixaban may be
associated with lower medical (excluding drug costs) costs relative to warfarin,
with apixaban having the most substantial medical cost reduction. Collaborators: Granger CB, Wallentin L, Alexander JH, Ansell J, Diaz R, Easton
JD, Gersh BJ, Hanna M, Horowitz J, Hylek EM, McMurray JJ, Mohan P, Verheugt FW,
Diaz R, Bahit MC, Aylward P, Amerena J, Huber K, Bartunek J, Avezum A, Ezekowitz
JA, Dorian P, Lanas F, Lisheng L, Zhu J, Isaza D, Jansky P, Husted S, Harjola
VP, Steg PG, Hohnloser SH, Keltai M, Pais P, Xavier D, Lewis BS, De Caterina R,
Goto S, Hermosillo AG, Alings AM, Atar D, Segura L, Ruzyllo W, Vinereanu D,
Varshavsky S, Golitsyn S, Oh BH, Commerford P, Lopez-Sendon JL, Rosenquist M,
Erol C, McMurray JJ, Parkhomenko A, Flaker G, Garcia D, Pfeffer MA, Diener HC,
Maggione A, Pocock S, Rouleau JL, Wyse G, Alexander JH, Al-Khatib S, Lopes RD,
Held C, Hylek EM, Bushnell C, Terent A, Leonardi S, Subherwal S, Eapen Z,
Vavalle J, Zomorodi A, Kolls B, Berger J, Vergara J, Parikh D, Zia S, Stashenko
G, Lombardi C, Matthews R, Hagstrom E, Akerblom A, Varenhorst C, Berntsson SG,
Stenborg A, Lundstrom E, Guimaraes H, Flato U, Nacif S, Barros P, Echenique L,
Rodrigues P, Armaganijan L, Lopes AC, Albrecht A, Vico M, Mackinnon I, Vogel D,
Vico M, Gabito A, Cassettari A, Zaidman C, Montaña O, Hrabar A, Jure H, Lastiri
H, Poy C, Caccavo A, Cuneo C, Colombo H, Rolandi F, Hershson A, Garrido M,
Sanchez A, Bruno ML, Piskorz D, Cuadrado J, Hasbani E, Serra J, Cartasegna L,
Schygiel P, Muratore C, Marino J, Sosa Liprandi MI, Guerrero RA, Ramos H,
Mercado D, Guzman L, Beneitez C, Estepo J, Torrijos R, Retyk E, Vita N, Luciardi
H, Casey M, Orlando S, Labarta MB, Santos D, Amerena J, Purnell P, Horowitz J,
Salem H, Liu A, Zimmet L, Roger S, de Looze F, de Looze F, Lehman R, de Looze F,
Jackson B, Ashby D, Heddle W, Rogers J, Brieger D, Martin P, Cross D, Walters D,
Waites J, Counsell J, Lowy A, de Looze F, Huber K, Stockenhuber F, Pieske B,
Striekwold H, Wollaert B, Nachtergaele H, Vijgen J, El Allaf D, Mairesse G,
Boxho G, De Deyn PP, Vanderheyden M, Semeraro O, Desfontaines P, Leroy J,
Provenier F, Bruneel B, Vrolix M, Peeters A, Deceuninck O, Saraiva JF, Reis G,
Rossi P, Zimmermann S, Jaber J, Botelho R, Manenti E, Jorge J, Maia L, Leães P,
Villaça Guimaraes Filho F, Fichino M, De Paola A, Indio do Brasil CK,
Albuquerque D, Bodanese L, Matsubara L, Mourilhe Rocha R, Genta P, Meneghelo Z,
Oliveira L, Lorga Filho A, Garbelini B Jr, Oliveira G, Teixeira M, Precoma D,
Pelloso E, Muniz A, Valéria Braile MC, Ueda R, Rabelo Alves A Jr, Pimentel Filho
P, Zimerman L, Coutinho M, Silveira J, Reis H, Moreira D, Paiva M, Aziz JL, Gois
J, Dutra O, Yao L, Syan G, Coutu B, Chehayeb R, Sabe-Affaki G, Fortin C, Borts
D, Bhargava R, Fell D, Cha J, Pandey A, Boucher P, Sabbah E, Ma P, Talbot P,
Spence D, Wade A, Green M, Berlingieri J, Vizel S, Chan YK, Blostein M, Talajic
M, Sterns L, Grondin F, Hruczkowski T, Labonte R, O'Mahony M, Rupka D, Mangat I,
Dowell A, Kelly A, St Maurice F, Henein S, Saunders K, Lasko B, Sami M,
MacKinnon R, Rizvi Q, O'Keefe D, Ricci J, Gervais B, Hart R, Bose S, Nawaz S,
Connors S, Winkler L, Boileau M, Healey J, Collette R, Rebane T, Ramjattan B,
Senior R, Therrien R, Wells P, Raffo C, Cobos J, Potthoff S, Stockins B,
Pincetti C, Corbalan R, Vejar M, Potthoff S, Li W, Zhao S, Chen X, Wu S, Tan H,
Wu S, Qu P, Jiang X, Wei M, Yang X, Li J, Ma S, Gu S, Dai QY, Li L, Yu B, Yin Y,
Wang N, Gao L, Zhou SX, Wang JA, Li ZQ, Bai F, Zhang F, Lu G, Chen Y, Zhang Y,
Jiang D, Zonggui W, Li H, Cao K, Lu Q, Li L, Hu T, Li H, Wang X, Botero R, Reyes
A, Jaramillo N, Urina M, Velez S, Gomez E, Pava L, Isaza D, Dunaj M, Hudcovic M,
Jerabek O, Brat R, Spinar J, Dedek V, Zidkova E, Podpera I, Cech M, Gorican K,
Micko M, Stribrna M, Frost L, Torp-Pedersen C, Toftager Larsen C, Tuxen C, May
O, Pedersen KE, Jensen G, Nielsen T, Nyvad O, Husted S, Gilså Hansen M, Egstrup
K, Lomholdt J, Skagen K, Joen Jakobsen T, Olsen M, Grande P, Melin J, Tynni M,
Harjola VP, Strand J, Parikka H, Jääskeläinen T, Corbelli JL, Gosse P, Mirode A,
Mansourati J, Lavabre G, Defaye P, Steg PG, Kahrmann G, Horacek T, Bauer A,
Spitzer S, Schlegl M, Winkelmann B, Vöhringer HF, Stenzel G, Haverkamp W,
Harenberg J, Schumacher M, Wunderlich J, Natour M, Rieker W, Gass S, Brachmann
J, Jung J, Poppert H, Häge R, Lickfett L, Schoeller R, Goedel-Meinen L, Utech A,
Buerke M, Maschke M, Haberl R, von Hodenberg E, Griewing B, Schlachetzki F,
Hamer H, Hoch T, Zabel M, Jordan R, Stögbauer F, Hetzel A, Weimar C, Schauerte
P, Fischer D, Mügge A, Bittersohl A, Mügge A, Lee K, Yu C, Lakatos F, Vértes A,
Kovács A, Takács J, Pálinkás A, Bakai J, Papp A, Illés Á, Tomcsányi J, Szakál I,
László Z, Katona A, Jobbágy L, Keltai K, Dézsi C, Lupkovics G, Cziraki A,
Mohácsi A, Simon K, Gupta S, Agarwal D, Fulwani M, Naik A, Nambiar A,
Chidambaram N, Srinivasasastry B, Joseph J, Padinhare M, Khanna P, Grant P,
Arneja J, Gadkari M, Garg N, Pothineni RB, Sathe S, Ramesh S, Malipeddi B,
Bharani A, Gowdappa HB, Prakash VS, Dharmadhikari A, Bhandari S, Desai N,
Banerjee S, Ghaisas N, Ramagiri B, Sinha S, Gojanur G, Duggal J, Jain V, Dani S,
Singh P, Srikanthan V, Puri VK, Gopal R, Viskin S, Shochat M, Hayek T, Reisin L,
Rosenheck S, Lewis B, Zimlichman R, Weiss A, Marmor A, Turgeman Y, Klainman E,
Francis A, Lahav M, Omary M, Morris M, Olivieri C, Santonastaso M, Fenici R, Mos
L, Ghirarduzzi A, De Caterina R, Novo S, Richiardi E, Testa S, Pini M, D'Angelo
A, Barsotti A, Donati M, Chiariello M, Galli M, Casolo G, Moretti L, Atsushi Y,
Yamamoto K, Goto S, Kihara H, Akihiko T, Saito T, Yoshii H, Sasaki T, Suwa M,
Adachi S, Usada K, Nakamura Y, Hayashida K, Yamada T, Iwasawa T, Kawase Y, Sugi
K, Murakami T, Satake K, Iwao T, Maemura K, Koretsune Y, Tsubokou Y, Yamashita
M, Sato Y, Kouichi F, Yura S, Matsushima A, Iwade K, Kamakura S, Tanaka S,
Murata H, Yamamoto S, Kobayashi Y, Higashi Y, Shinozaki T, Ikeda H, Hisaoka T,
Node K, Takagi H, Ong TK, Abidin IZ, Yusof Z, Yusoff K, Maskon O, De los Rios
Ibarra M, Alcocer Gamba M, Lopez Rosas E, Calvo Vargas C, Fajardo Campos P,
Cordero-Cabra JA, JerJes-Sanchez C, Herdez Santamaria I, Molina L, Olvera
Ruiz R, Arean Martinez C, Gonzalez Guerra J, Morales Gonzalez I,
Cervantes-Escarcega J, Chuquiure-Valenzuela EJ, Riojas C, Gonzalez Hermosillo J,
Galicia A, Nierop P, Verheugt F, Daniels M, Lok D, Alings A, Scholten M, Plomp
J, Derksen R, Bredero A, Zwart P, Schaap A, Michels H, Van der Zwaan C, Hysing
J, Elle S, Omland T, Rønnevik P, Øie B, Otterstad JE, Bogale N, Kjærnli T,
Halvorsen S, Bryhni B, Vikenes K, Cabrera W, Rodriguez A, Chavez C, Gamboa R,
Segura L, Araoz O, Azanero R, Toce L, Medina F, Bustamante G, Rios Vasquez C,
Zubiate M, Collado F, Morales-Palomares E, Sy R, Morales D, Rogelio G, Abola MT,
Ramos E, Yamamoto M, Collado F, De Leon F, Abelardo N, Kania G, Szpajer M,
Rajzer M, Janion M, Bronisz M, Ruzyllo W, Piepiorka M, Wendland M, Czerski T,
Korzeniak R, Budaj A, Wawrzynska L, Ogorek M, Miekus P, Tracz W,
Ocicka-Kozakiewicz A, Stepinska J, Kiedrowicz Z, Pasierski T, Kasprzak J,
Galewicz M, Podogrodzka B, Krauze-Wielicka M, Chmielinski A,
Boruczkowska-Kaszkowiak A, Piotrowski W, Vinereanu D, Stamate S, Cinteza M,
Tanaseanu CM, Dan GA, Benedek I, Ionescu RM, Lighezan D, Salajan A, Carasca E,
Capalneanu R, Pop C, Fierbinteanu-Braticevici CG, Zdrenghea DT, Gaita D, Manitiu
I, Nanea T, Arsenescu Georgescu C, Chizhov P, Kastanayan A, Oleynikov V,
Treshkur T, Govorin A, Zhelninova T, Barbarash O, Novikova T, Reshetko O,
Sivkova E, Obrezan A, Panchenko E, Popov S, Ivleva A, Zotov D, Gordienko A,
Kamalov G, Chernichka I, Levin A, Zrazhevsky K, Golitsyn S, Shilkina N, Golovach
A, Filonenko G, Bondarev S, Orlov V, Bragina A, Koziolova N, Svistov A, Shustov
S, Libov I, Kisliak O, Privalova E, Aleksandrov O, Mazur E, Karpov Y, Belenky D,
Kostenko V, Shubik Y, Ruda M, Arutyunov G, Eltishcheva V, Sidorenko B, Oseshnyuk
R, Boyarkin M, Pozdnyakov Y, Staxhinskiy N, Sinopalnikov A, Yakushin S,
Koniakhin A, Yarohno N, Sizova Z, Zadionchenko V, Klimov I, Duplyakov D,
Kanorsky S, Mareev V, Terekhov V, Arkhipov V, Sotnikova T, Yakusevich V, Lesnov
V, Gordeev I, Gratsiansky N, Shalaev S, Sulimov V, Talibov O, Zemtsovsky E,
Azarin O, Tan RS, Thorne J, De Jong D, Basson M, van Zyl L, Commerford P, Weich
H, van Nieuwenhuizen E, Roos J, Kelbe D, Viljoen J, Hong TJ, Oh BH, Park KS,
Jeong MH, Rhim CY, Choi DJ, Sung JH, Kim YN, Bae JH, Tahk SJ, Ryu KH, Kwon SU,
Rho TH, Cha TJ, Shin DG, Álvarez García P, Vida M, Galve E, Bruguera J, Roquer
J, Olías de la Cruz F, Paz Bermejo MA, Segura T, Gómez Gómez JH, Ugarriza A,
Plaza Pérez I, Villacastin J, Bertomeu V, Vargas R, Serena Leal J, Egido Herrero
J, Bethencourt González A, Albert X, Gonzalez Juanatey C, Medrano V, Vivancos J,
Andersson T, Lindholm CJ, Al-Khalili F, Höglund C, Rosenqvist M, Bergström O,
Herlitz J, Rasmanis G, Johansson L, Christersson C, Fredholm O, Al-Khalili F,
Callander M, Po H, Pan JP, Tseng CD, Shyu KG, Chu SH, Kultursay H, Gorenek B,
Erol C, Lip G, Albazzaz M, Kadr H, Cohen A, Cooke J, Agarwal A, Brack M, Pye M,
Anderson M, Dunn F, Wong YK, Glen S, Ford G, Alwail A, Yousef Z, Blagden M,
Murdoch D, McInnes G, Camm J, Ridsdill Smith W, McMurray JJ, Senior R, Webster
J, Dutka D, McClements B, Yousef Z, Levy T, Yogasundram S, Moriarty A, McCullagh
M, Flather J, Gumbley M, Findlay I, Davies J, Cooke A, Ahsan A, Cannon J, Bakhai
A, Jacob A, Trouton T, Ajala A, Bartkowiak A, Humiston D, Kufs W, Klancke K,
Jetty P, Gupta D, Nadar V, Yousuf K, Sosa-Suarez G, Gill S, Wukelic M, Mayer N,
Colan D, Haskel E, Grena P, Haught W, Walsh R, Carr K, Tahirkheli N, Jardula M,
Cebe J, Bloom S, Bilazarian S, Gilmore R, Jaffrani N, Goldscher D, Lebowitz A,
Friedlander I, Stein M, Promisloff S, Mago A, Dean J, Gerber J, Perloff D,
Cohen Y, Meholick A, Tobin T, Acheatel R, Levanovich P, Ip J, Porterfield J,
Seshadri N, McKenzie M, Alfieri A, Gazmuri R, Beanblossom B, Van Hamersveld D,
McCriskin J, Gelernt M, Bowden W, Sotolongo C, Lang J, Kaatz S, Agnone F,
Hassman D, Flores E, Albrecht F, Hassel C, Quartner J, LaFata J, Bedwell N,
Herzog W, Amin J, Usedom J, Brockmyre A, Abadier R, Corder C, Marple R, Lovell
C, Henry W 3rd, Chhabra A, Baker S, Bedoya R, Sandoval R, Nathan M, Garcia D,
Vora K, Sloan S, Kosinski E, Foster R, Salacata A, Weachter R, Bredlau C, Mehta
P, Russo C, Swint R Sr, Rosenthal S, Bybee K, Peart B, Chaudhuri P, Iteld B,
Staab M, Rhodes D, Kappler J, Mandviwala M, Niedermaier O, Sofley C, Heiman M,
Gips S, Harris R, East C, Nguyen V, Huling R, Thadani U, Travis D, Maislos F,
West M, Castello R, Cohen K, Karlsberg R, Schmedtje J Jr, Gottlieb D, MacKinnon
A, Noble G, O'Neill P, Roth J, Kobayashi J, Ho M, Hanovich G, Ehrlich S,
Wilson W, Warner A, Rubin A, Rivera E, Leu S, Smith L, Agaiby J, Jan M, Gould R,
Weiner S, Fleming J, Hemphill J, Parr K, Stoddard M, Curran P, Kurrelmeyer K,
Desai V, Kereiakes D, Schwarz E, Lillestol M, Vazquez-Tanus J, Vicari R, White
J, Shroff R, Fierer R, Vijay N, Hamad A, Kozinn M, Young D, Fenton S, Ouyang P,
Wellford A, Zwerner P, Meyer P, Foley J, Boyle A, Dotani M, Skolnick A,
Rodriguez-Fierro C, Hattler B, Rubin M, Hartley P, Tami L, Ball E, McPherson C,
Eisenberg S, Niazi I, Orlov M, Bachhuber B, Massey C, Phillips W, Mouhayar E,
Griffin J, Blumberg E, Slabic S, Danisa K, Samal A, Rohrbeck S, McCullough P,
Dohan D, Aycock G, Jones A, Anderson J, Silverman R, Smith W, Mishkel D,
Patterson N, Moore H, Kabour A, Muttreja M, Jaffrani W, Vidic T, Kent S, Platt
B, Goldberg R, Kulback S, Patel R, Bazzi A, Goldman S, Roman A, Levin C,
Lachterman B, Chandrashekhar Y, Aisiku I, Powers C, Engeron E, Bernstein R, Shah
S, Strobel J, Punatar H, Garcia R, Linden D, Al-Mudamgha A, Quick A, Kramer J,
Feldman G, Stapleton D, Davis M, Graff J, Galizia J, Wassmer P, Chiu D, Ison R,
Curry K, Finneran M, Solomon A, Schifferdecker B, Seger J, Alexander A, Yates S,
Ashley R Jr, Cordero-Sepulveda J, Cowen P, Ayres T, Kowalski B, Anton S,
Phillips J, Donovan D, Patel A, Smith R, Updegrove J, Buxton A, Clark D,
Margolis J, Brooks G, Pernenkil R, Walters J, Richards M, Maccaro P, Pieniek M,
Garcia Pulido J, Lee CW, Ilvento J, Riff D, Blue B, Welker J, Karunaratne H,
Santucci P, Vatutin M, Kraiz I, Mostovoy Y, Karpenko O, Tseluyko V, Kononenko L,
Volkov V, Parkhomenko A, Popik G, Chopey I, Burmak Y, Pavlyk S, Vizir V, Barna
O, Sychov O, Vykhovanyuk I, Tashchuk V, Khomazjuk I, Andrievskaya S,
Telyatnikova Z, Bondarchuk O, Netiazhenko V, Seredyuk N, Koval O, Kovalsky I,
Bazylevych A, Lysenko G, Borschivsky M, Yagensky A, Dzyak G, Bereznyakov I,
Rudenko L, Ignatenko G, Amosova K, Voloshyna O, Ivanova L, Tykhonova S, Suprun
E. Stroke prevention in atrial fibrillation (AF) has been challenging over decades,
mostly due to a number of difficulties associated with oral vitamin K
antagonists (VKAs), which have been the most effective stroke prevention
treatment for a long time. The oral direct thrombin inhibitors (e.g.,
dabigatran) and oral direct inhibitors of factor Xa (e.g., rivaroxaban,
apixaban) have emerged recently as an alternative to VKAs for stroke prevention
in AF. These drugs act rapidly, and have a predictable and stable dose-related
anticoagulant effect with a few clinically relevant drug-drug interactions. The
novel oral anticoagulants are used in fixed doses with no need for regular
laboratory monitoring of anticoagulation intensity. However, each of these drugs
has distinct pharmacological properties that could influence optimal use in
clinical practice. The following phase 3 randomized trials with novel oral
anticoagulants versus warfarin for stroke prevention in AF have been completed:
the Randomized Evaluation of Long-term Anticoagulant therapy (RE-LY) trial with
dabigatran, the Rivaroxaban Once daily oral direct Factor Xa inhibition Compared
with vitamin K antagonism for prevention of stroke and Embolism Trial in Atrial
Fibrillation (ROCKET-AF) trial with rivaroxaban, and the Apixaban for Reduction
of Stroke and Other Thromboembolism Events in Atrial Fibrillation (ARISTOTLE)
trial with apixaban. Moreover, the Apixaban Versus Acetylsalicylic Acid to
prevent Strokes (AVERROES) trial included patients with AF who have failed or
were unsuitable for warfarin, and compared apixaban versus aspirin for stroke
prevention in AF. Overall, apixaban has two large trials for stroke prevention
in AF showing benefits not only over warfarin, but also over aspirin among those
patients who have failed or refused warfarin. In the ARISTOTLE trial, apixaban
was superior to warfarin in the reduction of stroke or systemic embolism, major
bleeding, intracranial hemorrhage, and all-cause mortality, with a similar
reduction in the rate of ischemic stroke and better tolerability. When compared
with aspirin in the AVERROES trial, apixaban was associated with more effective
reduction of stroke, a similar risk of major bleeding, and better tolerability.
In this review article, the authors summarize the current knowledge on novel
oral anticoagulants and discuss the clinical aspects of their use for stroke
prevention in AF, with particular emphasis on apixaban. The incidence and prevalence of atrial fibrillation are quickly increasing,
mainly due to the ageing of the population. Atrial fibrillation is, to date, a
problem of public health. Atrial fibrillation is associated to a five-fold risk
of stroke, which may be identified by score risks, such as CHADS(2) score. The
classical antithrombotic treatment of atrial fibrillation is based on vitamin K
antagonists. Trials made in the 90's have clearly shown that vitamin K
antagonists were able to decrease stroke risk by about 60%. New oral
anticoagulants are now available on the market to treat patients with atrial
fibrillation. These drugs are dabigatran which has demonstrated an interest in
the RE-LY trial. Two doses may be prescribed, 110 mg bid and 150 mg bid. Anti Xa
have also demonstrated an interest : rivaroxaban in the ROCKET AF trial and
apixaban in the AVERROES (versus aspirin) and ARISTOTLE trials. In the future
these drugs will have a major place in the armamentarium used to treat patients
with atrial fibrillation. In all these trials a decrease in intra cranial
haemorrhages has been demonstrated. In the everyday practice it will be
necessary to be very cautious in patients with impaired renal function, as all
these drugs are eliminated by kidneys. Alternative anticoagulants to warfarin (dabigatran, rivaroxaban, and apixaban)
are becoming available for the prevention of thromboembolic stroke in atrial
fibrillation (AF), but there is a lack of information on their comparative
effectiveness. Using a discrete event simulation method adopting a lifetime
horizon of analysis, we made an indirect comparison of the RE-LY, ROCKET-AF, and
ARISTOTLE trial results for AF patients in the US population. Over a lifetime,
apixaban, dabigatran, and rivaroxaban accrued 0.130 (95% central range (CR)
-0.030 to 0.264), 0.106 (95% CR -0.048 to 0.248), and 0.095 (95% CR -0.052 to
0.242) more quality-adjusted life-years (QALYs), respectively, than warfarin,
with apixaban having a 55% probability of accruing the highest total QALYs. In
the absence of a definitive trial, and acknowledging the limitations of an
indirect comparison, the available evidence suggests apixaban to be the most
effective anticoagulant. BACKGROUND: Clinical event rates may differ among patients treated in the real
world (RW) compared to randomized controlled trials (RCTs). When translating the
efficacy of new treatments to RW, the relative risk reductions (RRRs) from RCTs
may produce different absolute risk reductions in RW.
OBJECTIVE: To estimate the absolute effect of apixaban on stroke and major
bleeding (MB) rates in a RW non-valvular atrial fibrillation (NVAF) population.
METHODS: NVAF patients were selected during 2007-2010 from a population of U.S.
commercial and Medicare health plans using the Medco claims database. Pharmacy
claims were used to define warfarin exposure periods. Stroke and MB were
identified using diagnosis codes. RW event rates were calculated during periods
of warfarin exposure. The numbers of stroke and MB events estimated to be
avoided in RW with apixaban versus warfarin were calculated by applying RRRs
from the ARISTOTLE trial to RW rates from the Medco database. The Medco data did
not contain information for patients receiving apixaban as it was not on the
market at the time of analysis.
RESULTS: Stroke and MB rates among RW NVAF patients during warfarin exposure
were higher compared with event rates in patients treated with warfarin in
ARISTOTLE (stroke: 5.29 vs. 1.51 per 100 person years (PYs); MB: 10.78 vs. 3.09
per 100 PYs). If RRRs from trials persist in RW, apixaban vs. warfarin would
result in greater absolute risk reductions (ARRs) and a lower number needed to
treat (NNT) in RW vs. ARISTOTLE (stroke: 91 vs. 313; MB: 30 vs. 105).
CONCLUSION: The impact of apixaban, as an alternative to warfarin in RW may be
greater than in RCTs. The NNT with apixaban versus warfarin in RW may be lower
versus ARISTOTLE if RRRs from the trial persists in RW and if baseline stroke
and MB rates among RW patients are higher compared to trial participants. Given the increasing prevalence of atrial fibrillation, the need for safe and
effective stroke prophylaxis will continue to rise. Warfarin has been around for
many years and has proven efficacy in preventing stroke, but it has major
limitations due to its variable dosing, food and drug interactions, and
requirement for regular monitoring. Newer agents which include dabigatran,
rivaroxaban, and apixaban have recently or will soon be available and may
provide an improved efficacy in stroke prevention, an improved safety profile,
and improved user-friendliness. Dabigatran was the first of the agents to be
widely available, and in the RE-LY study, dabigatran (150 mg dose) showed
superiority to warfarin in preventing ischemic stroke and a significant
reduction in intracranial bleeding. Rivaroxaban was studied in the ROCKETAF
trial, and with once daily dosing, it showed noninferiority to warfarin in
preventing stroke with a significant reduction in intracranial bleeding. The
ARISTOTLE trial showed apixaban was superior to warfarin for stroke prevention,
significantly reduced all major bleeding, and resulted in a significant
reduction in all-cause mortality. While all three trials have important
limitations, they were very large randomized trials with more than 14,000
patients each and show a clear overall net clinical benefit when compared with
warfarin. Key features of the drugs as well as an individual's preferences and
stability on warfarin will help guide the ultimate drug choice for any given
patient, but these newer anticoagulant agents are likely to usher in a new era
in stroke prevention in atrial fibrillation. OBJECTIVES: Based on clinical trials the oral anticoagulants (OACs) apixaban,
dabigatran, and rivaroxaban are efficacious for reducing stroke risk for
non-valvular atrial fibrillation (NVAF) patients. Based on the clinical trials,
this study evaluated the medical costs for clinical events among NVAF patients
≥75 and <75 years of age treated with individual OACs vs warfarin.
METHODS: Rates for primary and secondary efficacy and safety outcomes (i.e.,
clinical events) among NVAF patients receiving warfarin or each of the OACs were
determined for NVAF populations aged ≥75 years and <75 years of age from the OAC
vs warfarin trials. One-year incremental costs among patients with clinical
events were obtained from published literature and inflation adjusted to 2010
costs. Medical costs, excluding medication costs, for clinical events associated
with each OAC and warfarin were then estimated and compared.
RESULTS: Among NVAF patients aged ≥75, compared to warfarin, use of either
apixaban or rivaroxaban was associated with a reduction in medical costs per
patient year (apixaban = -$825, rivaroxaban =-$23), while dabigatran use was
associated with increased medical costs of $180 per patient year. Among NVAF
patients <75 years of age medical costs per patient year were estimated to be
reduced -$254, -$367, and -$88, for apixaban, dabigatran, and rivaroxaban,
respectively, in comparison to warfarin.
LIMITATIONS: This economic analysis was based on clinical trial data and,
therefore, the direct application of the results to routine clinical practice
will require further assessment.
CONCLUSIONS: Difference in medical costs between OAC and warfarin treated NVAF
patients vary by age group and individual OACs. Although reductions in medical
costs for NVAF patients aged ≥75 and <75 were observed for those using either
apixaban or rivaroxaban vs warfarin, the reductions were greater per patient
year for both the older and younger NVAF populations using apixaban. OBJECTIVE: The Apixaban for Reduction in Stroke and Other Thromboembolic Events
in Atrial Fibrillation (ARISTOTLE) trial demonstrated that apixaban was
effective in reducing the risk of stroke and major bleeding in non-valvular
atrial fibrillation (NVAF) patients. Medical cost avoidance studies for oral
anticoagulants have used warfarin event rates from clinical trials, which may
not reflect the real-world (RW) setting. This study aimed to estimate the
difference in medical costs associated with apixaban instead of warfarin in RW
NVAF patients.
METHODS: This study selected patients with NVAF diagnosis during 2007-2010 from
a Medco population of US commercial and Medicare health plans. Stroke and major
bleeding excluding intracranial hemorrhage (MBEIH) were identified using
diagnosis codes. Pharmacy claims were used to define warfarin exposure periods.
Rates of stroke and MBEIH were calculated during warfarin exposure. To estimate
the absolute risk reduction (ARR) between warfarin and apixaban in RW, the
relative risk reductions (RRR) from ARISTOTLE were multiplied by the event rates
observed in RW during warfarin exposure. Medical cost reductions associated with
apixaban were calculated by applying the ARR to the 1-year incremental cost for
each event. Stroke and MBEIH costs were obtained from the literature and
adjusted to 2011 levels.
RESULTS: During a patient year, the use of apixaban instead of warfarin resulted
in medical cost reductions of $493 for stroke and $752 for MBEIH and $1245 for
the combined outcome of both events. The medical costs avoided were greater as
baseline stroke risk increased.
CONCLUSION: If RRRs demonstrated in ARISTOTLE persist in RW, the use of apixaban
will be associated with lower medical costs vs warfarin. Main limitations of
this study were: identification of clinical events using administrative codes
rather than confirmatory clinical data, inability to evaluate the level of
international normalized ratio (INR) control, and not including INR monitoring
and drug costs. OBJECTIVE: To determine the cost-effectiveness of apixaban versus warfarin in
patients with atrial fibrillation (AF) with a moderate to severe risk of stroke,
from an Australian government-perspective.
METHODS: A decision-analytic Markov model was constructed to assess the
cost-effectiveness of apixaban versus warfarin, based on data from the Apixaban
for Reduction in Stroke and Other Thromboembolic Events in AF (ARISTOTLE) trial.
The model comprised five health states: 'Alive, no major bleeding or stroke',
'Alive, no major bleeding, post stroke/systemic embolism', 'Alive, post major
bleeding, no stroke', 'Alive, post-major bleeding and stroke' and 'Dead'.
Disease cost data was derived from the North-East Melbourne Stroke Incidence
Study and the Australian Refined Diagnose Related Groups. Costs of medications
were based on data from the Pharmaceutical Benefit Scheme. Utility data was
derived from published sources, and an annual discount rate of 5% was applied to
costs and benefits. The main outcome of interest was incremental
cost-effectiveness ratios per life year gained (LYG) and quality adjusted life
years (QALYs) gained.
RESULTS: Over 20 years, in the sample of 1000 subjects the model predicted that
compared to warfarin, apixaban led to a (discounted) of 0.33 LYG and 0.31 QALYs
gained, at a net cost of $4,308 per-person. These equated to ICERs of $AUD12,
914 per LYG and $AUD13, 679 per QALY gained. Probabilistic sensitivity analysis
demonstrated that apixaban was cost-effective at 99.0% probability using
willingness to pay thresholds of $AUD45 000 per LYG and QALY.
CONCLUSION: Compared to warfarin, apixaban is likely to represent a
cost-effective means of preventing stroke-related morbidity and mortality in
patients with AF. AIMS: The risk of stroke in patients with atrial fibrillation (AF) increases
with age. In the ARISTOTLE trial, apixaban when compared with warfarin reduced
the rate of stroke, death, and bleeding. We evaluated these outcomes in relation
to patient age.
METHODS AND RESULTS: A total of 18 201 patients with AF and a raised risk of
stroke were randomized to warfarin or apixaban 5 mg b.d. with dose reduction to
2.5 mg b.d. or placebo in 831 patients with ≥2 of the following criteria: age
≥80 years, body weight ≤60 kg, or creatinine ≥133 μmol/L. We used Cox models to
compare outcomes in relation to patient age during 1.8 years median follow-up.
Of the trial population, 30% were <65 years, 39% were 65 to <75, and 31% were
≥75 years. The rates of stroke, all-cause death, and major bleeding were higher
in the older age groups (P < 0.001 for all). Apixaban was more effective than
warfarin in preventing stroke and reducing mortality across all age groups, and
associated with less major bleeding, less total bleeding, and less intracranial
haemorrhage regardless of age (P interaction >0.11 for all). Results were also
consistent for the 13% of patients ≥80 years. No significant interaction with
apixaban dose was found with respect to treatment effect on major outcomes.
CONCLUSION: The benefits of apixaban vs. warfarin were consistent in patients
with AF regardless of age. Owing to the higher risk at older age, the absolute
benefits of apixaban were greater in the elderly. OBJECTIVES: This study sought to characterize major bleeding on the basis of the
components of the major bleeding definition, to explore major bleeding by
location, to define 30-day mortality after a major bleeding event, and to
identify factors associated with major bleeding.
BACKGROUND: Apixaban was shown to reduce the risk of major hemorrhage among
patients with atrial fibrillation in the ARISTOTLE (Apixaban for Reduction in
Stroke and Other Thromboembolic Events in Atrial Fibrillation) trial.
METHODS: All patients who received at least 1 dose of a study drug were
included. Major bleeding was defined according to the criteria of the
International Society on Thrombosis and Haemostasis. Factors associated with
major hemorrhage were identified using a multivariable Cox model.
RESULTS: The on-treatment safety population included 18,140 patients. The rate
of major hemorrhage among patients in the apixaban group was 2.13% per year
compared with 3.09% per year in the warfarin group (hazard ratio [HR] 0.69, 95%
confidence interval [CI]: 0.60 to 0.80; p < 0.001). Compared with warfarin,
major extracranial hemorrhage associated with apixaban led to reduced
hospitalization, medical or surgical intervention, transfusion, or change in
antithrombotic therapy. Major hemorrhage followed by mortality within 30 days
occurred half as often in apixaban-treated patients than in those receiving
warfarin (HR 0.50, 95% CI: 0.33 to 0.74; p < 0.001). Older age, prior
hemorrhage, prior stroke or transient ischemic attack, diabetes, lower
creatinine clearance, decreased hematocrit, aspirin therapy, and nonsteroidal
anti-inflammatory drugs were independently associated with an increased risk.
CONCLUSIONS: Apixaban, compared with warfarin, was associated with fewer
intracranial hemorrhages, less adverse consequences following extracranial
hemorrhage, and a 50% reduction in fatal consequences at 30 days in cases of
major hemorrhage. BACKGROUND: The perceived risk of serious bleeding is an obstacle to the use of
oral anticoagulation in East Asia. The efficacy and safety of apixaban in East
Asian patients with atrial fibrillation are unknown.
METHODS: ARISTOTLE included 18,201 patients with nonvalvular atrial fibrillation
randomized to apixaban 5mg twice daily or warfarin. The efficacy and safety of
apixaban and warfarin among patients recruited from East Asia (n = 1,993) were
compared with those recruited from outside East Asia (n = 16,208).
RESULTS: Compared with warfarin, apixaban resulted in a consistent reduction in
stroke or systemic embolism in East Asian (hazard ratio [HR] 0.74, 95% CI
0.50-1.10) and non-East Asian (HR 0.81, 95% CI 0.66-0.99) patients (interaction
P = .70). Consistent benefits of apixaban over warfarin were also seen for major
bleeding in East Asian (HR 0.53, 95% CI 0.35-0.80) and non-East Asian (HR 0.72,
95% CI 0.62-0.83) patients (interaction P = .17). There was a greater reduction
in major or clinically relevant nonmajor bleeding with apixaban compared with
warfarin in East Asian (HR 0.49, 95% CI 0.35-0.67) than in non-East Asian (HR
0.71, 95% CI 0.63-0.79) patients (interaction P = .03). Numerically higher rates
of intracranial bleeding were seen in East Asian patients with warfarin but not
with apixaban.
CONCLUSIONS: Apixaban resulted in similar reductions in stroke or systemic
embolism and major bleeding and greater reductions in major or clinically
relevant nonmajor bleeding in patients from East Asia. Warfarin is associated
with more intracranial bleeding, particularly in patients from East Asia. OBJECTIVES: A comparative analysis of three major clinical trials with factor Xa
inhibitor oral anticoagulant (XOAC) drugs versus warfarin in atrial
fibrillation-Rocket-AF (rivaroxaban), Aristotle (apixaban) and Engage AF Timi 48
(edoxaban; two different doses and sets of data)-was carried out.
METHODS: Data were extracted from the original reports (study level) and a
meta-analysis was carried out.
RESULTS: When compared with warfarin, XOAC therapy was associated with a
decrease in haemorrhagic stroke, with a similar pattern for all regimens and
meta-analysis showing a risk ratio of 0.488 (95% CI 0.396 to 0.601). Regarding
total mortality, a favourable pattern was seen for all four regimens and
meta-analysis showed a risk ratio of 0.892 (95% CI 0.840 to 0.947). Major
bleeding and gastrointestinal bleeding provided two examples regarding which
heterogeneity would seem to exist, when XOAC drugs are compared with warfarin.
In what concerns the incidence of myocardial infarction, the primary end point
(stroke plus systemic embolism) and ischaemic stroke, the situation is less
clear. These results are inconsistent with a putative 'group effect' for all the
seven parameters under study, and for some of them it would probably be best to
look at each of the individual trial data rather than at the meta-analysis data
(which seem to lack a clear biological meaning).
CONCLUSIONS: Apixaban, rivaroxaban and edoxaban have shown interesting effects,
when compared with warfarin in clinical trials, in patients with atrial
fibrillation, particularly with regard to haemorrhagic stroke and to the
mortality rate. No other consistent conclusions concerning a putative 'group
effect' can be reached at the present stage. Concerns regarding adherence to
therapy, possible drug interactions, cost and current absence of antidotes may
be taken into consideration when choosing an anticoagulant drug. BACKGROUND: We sought to assess the occurrence of events after blinded study
drug discontinuation and transition to open-label vitamin K antagonist (VKA) in
ARISTOTLE.
METHODS: At the end of ARISTOTLE, blinded study drug was stopped, and open-label
VKA was recommended. For patients completing the trial on blinded study drug, a
2-day bridging period with apixaban or apixaban placebo was recommended (while
beginning open-label VKA). Outcomes were assessed during the 30 days after
stopping blinded study drug.
RESULTS: Of the 6,809 patients in the apixaban group and 6,588 in the warfarin
group who completed the trial on study drug, there were 21 strokes or systemic
emboli (4.02%/year) and 26 major bleeding (4.97%/year) events in the apixaban
group (transitioning to VKA) and 5 strokes or systemic emboli (0.99%/year) and
10 major bleeding (1.97%/year) events in the warfarin group (continuing on VKA),
with most of the imbalance between groups being after the first week. Similar
results were seen in the first 30 days of the trial where warfarin-naive
patients starting warfarin had a higher rate of stroke or systemic emboli
(5.41%/year) than warfarin-experienced patients (1.42%/year), a pattern not seen
when starting apixaban. No similar increase in events with apixaban versus
warfarin was seen during temporary or permanent study drug discontinuation
during the trial.
CONCLUSIONS: The excess in thrombotic and bleeding events in the apixaban group
after study drug discontinuation appears to be related to an increased risk
associated with the initiation of a VKA rather than a direct effect of apixaban.
Whether ≥2 days of apixaban bridging improves outcomes during VKA transition is
unknown and deserves further evaluation. INTRODUCTION AND OBJECTIVES: Cost-effectiveness analysis of apixaban (5 mg twice
daily) vs acenocoumarol (5mg/day) in the prevention of stroke in patients with
nonvalvular atrial fibrillation in Spain.
METHODS: Markov model covering the patient's entire lifespan with 10 health
states. Data on the efficacy and safety of the drugs were provided by the
ARISTOTLE trial. Warfarin and acenocoumarol were assumed to have therapeutic
equivalence.
PERSPECTIVES: The Spanish National Health System and society. Information on the
cost of the drugs, complications, and the management of the disease was obtained
from Spanish sources.
RESULTS: In a cohort of 1000 patients with nonvalvular atrial fibrillation,
administration of apixaban rather than acenocoumarol would avoid 18 strokes, 71
hemorrhages (28 intracranial or major), 2 myocardial infarctions, 1 systemic
embolism, and 23 related deaths. Apixaban would prolong life (by 0.187 years)
and result in more quality-adjusted life years (by 0.194 years) per patient.
With apixaban, the incremental costs for the Spanish National Health System and
for society would be € 2,488 and € 1,826 per patient, respectively.
Consequently, the costs per life year gained would be € 13,305 and € 9,765 and
the costs per quality-adjusted life year gained would be € 12,825 and € 9,412
for the Spanish National Health System and for society, respectively. The
stability of the baseline case was confirmed by sensitivity analyses.
CONCLUSIONS: According to this analysis, apixaban may be cost-effective in the
prevention of stroke in patients with nonvalvular atrial fibrillation compared
with acenocoumarol. BACKGROUND: Atrial fibrillation (AF), the most common cardiac arrhythmia, is a
major risk factor for stroke. Rivaroxaban, an oral factor Xa inhibitor, is
approved for the prevention of stroke in patients with non-valvular AF. In the
pivotal phase III trial ROCKET AF, rivaroxaban demonstrated non-inferiority
compared with warfarin for reducing the risk of stroke or systemic embolism (SE)
in patients with AF (intention-to-treat analysis), without an increased risk of
major bleeding. Superior efficacy vs. warfarin was achieved while patients were
on study medication. Other direct oral factor Xa inhibitors have completed phase
III clinical trials in this indication. Compared with warfarin, apixaban (in the
ARISTOTLE trial) and edoxaban (in the ENGAGE-AF trial) were shown to be superior
or non-inferior, respectively, for reduction in stroke or SE risk in patients
with AF. Baseline stroke risk, as indicated by CHADS2 scores, was lower in
patients in the ARISTOTLE and ENGAGE-AF trials than in ROCKET AF.
OBJECTIVES: This review discusses the main findings from ROCKET AF, specifically
examining recent subgroup analyses investigating rivaroxaban use across various
patient types at high risk for adverse outcomes, including those with prior
stroke or transient ischaemic attack, reduced renal function, prior myocardial
infarction, peripheral artery disease, heart failure or patients aged ≥ 75 years
and those resident in East Asia.
CONCLUSIONS: These subgroup analyses demonstrate that the treatment effect for
rivaroxaban vs. warfarin is broadly consistent across a wide range of patient
groups, with respect to both efficacy and safety. Apixaban is a new oral anticoagulant (NOACs: Novel Oral Anticoagulant), like
dabigatran, rivaroxaban, and edoxaban. All of them are prescribed to patients
with non valvular atrial fibrillation or venous thromboembolism, to replace
warfarin, because of the lower probability of bleeding, however they can cause
bleeding by themselves. Bleeding is an adverse event in patients taking
anticoagulants. It is associated with a significant increase of morbidity and
risk of death. However, these drugs should be used only for the time when
anticoagulation is strictly required, especially when used for preventing deep
vein thrombosis. Prolonged use increases the risk of bleeding. In the ARISTOTLE
Trial Apixaban, compared with warfarin, was associated with a lower rate of
intracranial hemorrhages and less adverse consequences following extracranial
hemorrhage. Many physicians still have limited experience with new oral
anticoagulants and about bleeding risk managment. We reviewed the available
literature on extracranial and intracranial bleeding concerning apixaban. OBJECTIVE: To determine whether the treatment effect of apixaban versus
warfarin differs with increasing numbers of concomitant drugs used by patients
with atrial fibrillation.
DESIGN: Post hoc analysis performed in 2015 of results from ARISTOTLE (apixaban
for reduction in stroke and other thromboembolic events in atrial
fibrillation)-a multicentre, double blind, double dummy trial that started in
2006 and ended in 2011.
PARTICIPANTS: 18 201 ARISTOTLE trial participants.
INTERVENTIONS: In the ARISTOTLE trial, patients were randomised to either 5 mg
apixaban twice daily (n=9120) or warfarin (target international normalised ratio
range 2.0-3.0; n=9081). In the post hoc analysis, patients were divided into
groups according to the number of concomitant drug treatments used at baseline
(0-5, 6-8, ≥9 drugs) with a median follow-up of 1.8 years.
MAIN OUTCOME MEASURES: Clinical outcomes and treatment effects of apixaban
versus warfarin (adjusted for age, sex, and country).
RESULTS: Each patient used a median of six drugs (interquartile range 5-9);
polypharmacy (≥5 drugs) was seen in 13 932 (76.5%) patients. Greater numbers of
concomitant drugs were used in older patients, women, and patients in the United
States. The number of comorbidities increased across groups of increasing
numbers of drugs (0-5, 6-8, ≥9 drugs), as did the proportions of patients
treated with drugs that interact with warfarin or apixaban. Mortality also rose
significantly with the number of drug treatments (P<0.001), as did rates of
stroke or systemic embolism (1.29, 1.48, and 1.57 per 100 patient years, for
0-5, 6-8, and ≥9 drugs, respectively) and major bleeding (1.91, 2.46, and 3.88
per 100 patient years, respectively). Relative risk reductions in stroke or
systemic embolism for apixaban versus warfarin were consistent, regardless of
the number of concomitant drugs (Pinteraction=0.82). A smaller reduction in
major bleeding was seen with apixaban versus warfarin with increasing numbers of
concomitant drugs (Pinteraction=0.017). Patients with interacting (potentiating)
drugs for warfarin or apixaban had similar outcomes and consistent treatment
effects of apixaban versus warfarin.
CONCLUSIONS: In the ARISTOTLE trial, three quarters of patients had
polypharmacy; this subgroup had an increased comorbidity, more interacting
drugs, increased mortality, and higher rates of thromboembolic and bleeding
complications. In terms of a potential differential response to anticoagulation
therapy in patients with atrial fibrillation and polypharmacy, apixaban was more
effective than warfarin, and is at least just as safe.Trial
registration ARISTOTLE trial, ClinicalTrials.gov NCT00412984. IMPORTANCE: In the Apixaban for Reduction of Stroke and Other Thromboembolic
Complications in Atrial Fibrillation (ARISTOTLE) trial, the standard dose of
apixaban was 5 mg twice daily; patients with at least 2 dose-reduction
criteria-80 years or older, weight 60 kg or less, and creatinine level 1.5 mg/dL
or higher-received a reduced dose of apixaban of 2.5 mg twice daily. Little is
known about patients with 1 dose-reduction criterion who received the 5 mg twice
daily dose of apixaban.
OBJECTIVE: To determine the frequency of 1 dose-reduction criterion and whether
the effects of the 5 mg twice daily dose of apixaban on stroke or systemic
embolism and bleeding varied among patients with 1 or no dose-reduction
criteria.
DESIGN, SETTING, AND PARTICIPANTS: Among 18 201 patients in the ARISTOTLE trial,
17 322 were included in this analysis. Annualized event rates of stroke or
systemic embolism and major bleeding and hazard ratios (HRs) and 95% CIs were
evaluated. Interactions between the effects of apixaban vs warfarin and the
presence of 1 or no dose-reduction criteria were assessed. The first patient was
enrolled in the ARISTOTLE trial on December 19, 2006, and follow-up was
completed on January 30, 2011. Data were analyzed from January 2015 to May 30,
2016.
MAIN OUTCOMES AND MEASURES: Analysis of major bleeding included events during
study drug treatment. Analysis of stroke or systemic embolism was based on
intention to treat.
RESULTS: Of the patients with 1 or no dose-reduction criteria assigned to
receive the 5 mg twice daily dose of apixaban or warfarin, 3966 had 1
dose-reduction criterion; these patients had higher rates of stroke or systemic
embolism (HR, 1.47; 95% CI, 1.20-1.81) and major bleeding (HR, 1.89; 95% CI,
1.62-2.20) compared with those with no dose-reduction criteria (n = 13 356). The
benefit of the 5 mg twice daily dose of apixaban (n = 8665) compared with
warfarin (n = 8657) on stroke or systemic embolism in patients with 1
dose-reduction criterion (HR, 0.94; 95% CI, 0.66-1.32) and no dose-reduction
criterion (HR, 0.77; 95% CI, 0.62-0.97) were similar (P for interaction = .36).
Similarly, the benefit of 5 mg twice daily dose of apixaban compared with
warfarin on major bleeding in patients with 1 dose-reduction criterion (HR,
0.68; 95% CI, 0.53-0.87) and no dose-reduction criterion (HR, 0.72; 95% CI,
0.60-0.86) were similar (P for interaction = .71). Similar patterns were seen
for each dose-reduction criterion and across the spectrum of age, body weight,
creatinine level, and creatinine clearance.
CONCLUSIONS AND RELEVANCE: Patients with atrial fibrillation and isolated
advanced age, low body weight, or renal dysfunction have a higher risk of stroke
or systemic embolism and major bleeding but show consistent benefits with the 5
mg twice daily dose of apixaban vs warfarin compared with patients without these
characteristics. The 5 mg twice daily dose of apixaban is safe, efficacious, and
appropriate for patients with only 1 dose-reduction criterion.
TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00412984. |
What is REVIGO? | REVIGO summarizes and visualizes long lists of gene ontology terms. | Outcomes of high-throughput biological experiments are typically interpreted by
statistical testing for enriched gene functional categories defined by the Gene
Ontology (GO). The resulting lists of GO terms may be large and highly
redundant, and thus difficult to interpret.REVIGO is a Web server that
summarizes long, unintelligible lists of GO terms by finding a representative
subset of the terms using a simple clustering algorithm that relies on semantic
similarity measures. Furthermore, REVIGO visualizes this non-redundant GO term
set in multiple ways to assist in interpretation: multidimensional scaling and
graph-based visualizations accurately render the subdivisions and the semantic
relationships in the data, while treemaps and tag clouds are also offered as
alternative views. REVIGO is freely available at http://revigo.irb.hr/. |
What is the mechanism of action of onartuzumab? | Onartuzumab is monoclonal antibody targeting MET. It works by inhibiting MET. Onartuzumab was tested for treatment of non-small cell lung carcinoma, adenocarcinoma of the stomach and gastroesophageal Junction, and recurrent glioblastoma. | The objective of this study was to evaluate circulating hepatocyte growth factor
(cHGF) as a pharmacodynamic biomarker of Met inhibition for onartuzumab (MetMAb,
OA5D5v2) in a phase I trial in patients with advanced cancers and a phase II
trial in non-small cell lung cancer (NSCLC). The phase I study was a dose
escalation trial with onartuzumab administered i.v. once every three weeks. The
phase II study was a randomized two-arm trial in which onartuzumab or placebo
was administered in combination with erlotinib in 137 patients with second and
third line (2/3L) NSCLC. cHGF levels were evaluated by ELISA at multiple time
points over the treatment period. Onartuzumab administration resulted in an
acute and sustained rise in cHGF in both the phase I and phase II studies.
Elevation in cHGF was independent of dose or drug exposure and was restricted to
onartuzumab treatment. Neither higher baseline nor elevated change in cHGF
levels upon treatment could simply be attributed to tumor burden or number of
liver metastasis. We have shown that elevated cHGF can consistently and
reproducibly be measured as a pharmacodynamic biomarker of onartuzumab activity.
The elevation in cHGF is independent of tumor type, dose administered, or dose
duration. Although these studies were not powered to directly address the
contribution of cHGF as a predictive, on-treatment, circulating biomarker, these
data suggest that measurement of cHGF in future expanded studies is warranted. BACKGROUND: Dysregulation of the hepatocyte growth factor (HGF)/MET pathway is
associated with poor prognosis, more aggressive biological characteristics of
the tumor, and shortened survival in patients with metastatic colorectal cancer
(mCRC). Onartuzumab (MetMAb) is a recombit humanized monovalent monoclonal
antibody directed against MET. We present the treatment rationale and protocol
for an ongoing randomized multicenter placebo-controlled phase II study designed
to evaluate the efficacy and safety of MetMAb combined with bevacizumab and
mFOLFOX-6 (5-fluoruracil, leucovorin, and oxaliplatin).
PATIENTS AND METHODS: Eligible patients with previously untreated mCRC are
randomized 1:1 to either mFOLFOX-6 combined with bevacizumab and placebo
followed by 5-fluorouracil/leucovorin plus bevacizumab and placebo or mFOLFOX6,
bevacizumab plus MetMAb followed by 5 FU/LV, bevacizumab, and MetMAb. The
primary end point of this study is progression-free survival (PFS) in the
intent-to-treat (ITT) population. Secondary end points include overall survival
(OS), objective response rate, and safety. Subanalyses will be performed to
evaluate the effect of MET receptor expression on study primary and secondary
end points. Correlative studies will be performed on tissue- and blood-derived
biomarkers related to both HGF/MET signaling and other associated pathway
markers. Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is
implicated in the maligt process of multiple cancers, making disruption of
this interaction a promising therapeutic strategy. However, targeting MET with
bivalent antibodies can mimic HGF agonism via receptor dimerization. To address
this limitation, we have developed onartuzumab, an Escherichia coli-derived,
humanized, and affinity-matured monovalent monoclonal antibody against MET,
generated using the knob-into-hole technology that enables the antibody to
engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF
binding and receptor phosphorylation and signaling and has antibody-like
pharmacokinetics and antitumor activity. Biochemical data and a crystal
structure of a ternary complex of onartuzumab antigen-binding fragment bound to
a MET extracellular domain fragment, consisting of the MET Sema domain fused to
the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF
β-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain
(but not β-chain) binding to MET. These data suggest a likely binding site of
the HGF α-chain on MET, which when dimerized leads to MET signaling.
Onartuzumab, therefore, represents the founding member of a class of therapeutic
monovalent antibodies that overcomes limitations of antibody bivalency for
targets impacted by antibody crosslinking. PURPOSE: We characterized the pharmacokinetics of onartuzumab (MetMAb) in
animals and determined a concentration-effect relationship in tumor-bearing mice
to enable estimation of clinical pharmacokinetics and target doses.
EXPERIMENTAL DESIGN: A tumor growth inhibition model was used to estimate
tumoristatic concentrations (TSC) in mice. Human pharmacokinetic parameters were
projected from pharmacokinetics in cynomolgus monkeys by the species-invariant
time method. Monte Carlo simulations predicted the percentage of patients
achieving steady-state trough serum concentrations (Ctrough ss) ≥TSC for every
3-week (Q3W) dosing.
RESULTS: Onartuzumab clearance (CL) in the linear dose range was 21.1 and 12.2
mL/d/kg in mice and cynomolgus monkeys with elimination half-life at 6.10 and
3.37 days, respectively. The estimated TSC in KP4 pancreatic xenograft
tumor-bearing mice was 15 μg/mL. Projected CL for humans in the linear dose
range was 5.74 to 9.36 mL/d/kg with scaling exponents of CL at 0.75 to 0.9.
Monte Carlo simulations projected a Q3W dose of 10 to 30 mg/kg to achieve
Ctrough ss of 15 μg/mL in 95% or more of patients.
CONCLUSIONS: Onartuzumab pharmacokinetics differed from typical bivalent
glycosylated monoclonal antibodies with approximately 2-times faster CL in the
linear dose range. Despite this higher CL, xenograft efficacy data supported
dose flexibility with Q1W to Q3W dose regimens in the clinical setting with a
TSC of 15 μg/mL as the Ctrough ss target. The projected human efficacious dose
of 10 to 30 mg/kg Q3W should achieve the target TSC of 15 μg/mL. These data show
effective pharmacokinetic/pharmacodynamic modeling to project doses to be tested
in the clinic. Onartuzumab is a unique, humanized, monovalent (one-armed) monoclonal antibody
(mAb) against the MET receptor. The intravenous (IV) pharmacokinetics (PK) of
onartuzumab were investigated in a phase I study and a phase II study in
recurrent non-small cell lung cancer (NSCLC) patients. The potential for
drug-drug interaction (DDI) was assessed during co-administration of IV
onartuzumab with oral erlotinib, by measuring the PK of both drugs. The
concentration-time profiles of onartuzumab were adequately described using a
two-compartment model with linear clearance (CL) at doses between 4 and
30 mg/kg. The estimates for CL, central compartment volume (V1 ), and median
terminal half-life were 0.439 L/day, 2.77 L, and 13.4 days, respectively.
Statistically significant covariates included creatinine clearance (CrCL) on
clearance, weight and gender on V1 , and weight on peripheral compartment volume
(V2 ), but the clinical relevance of these covariates needs to be further
evaluated. The current analysis did not indicate obvious DDI between onartuzumab
and erlotinib. MET diagnostic status did not impact the exposure of either
agent. Despite the slightly faster clearance compared with typical bivalent
mAbs, the PK of onartuzumab support dosing regimens of 15 mg/kg every 3 weeks or
doses equivalent to achieve the target minimum tumoristatic concentration in
patients. Onartuzumab, a humanized, monovalent monoclonal anti-MET antibody, antagonizes
MET signaling by inhibiting binding of its ligand, hepatocyte growth factor
(HGF). We investigated the effects of onartuzumab on cell-associated and
circulating (shed) MET (sMET) and circulating HGF in vitro and nonclinically to
determine their utility as pharmacodynamic biomarkers for onartuzumab. Effects
of onartuzumab on cell-associated MET were assessed by flow cytometry and
immunofluorescence. sMET and HGF were measured in cell supernatants and in serum
or plasma from multiple species (mouse, cynomolgus monkey, and human) using
plate-based immunoassays. Unlike bivalent anti-MET antibodies, onartuzumab
stably associates with MET on the surface of cells without inducing MET
internalization or shedding. Onartuzumab delayed the clearance of human
xenograft tumor-produced sMET from the circulation of mice, and endogenous sMET
in cynomolgus monkeys. In mice harboring MET-expressing xenograft tumors, in the
absence of onartuzumab, levels of human sMET correlated with tumor size, and may
be predictive of MET-expressing tumor burden. Because binding of sMET to
onartuzumab in circulation resulted in increasing sMET serum concentrations due
to reduced clearance, this likely renders sMET unsuitable as a pharmacodynamic
biomarker for onartuzumab. There was no observed effect of onartuzumab on
circulating HGF levels in xenograft tumor-bearing mice or endogenous HGF in
cynomolgus monkeys. Although sMET and HGF may serve as predictive biomarkers for
MET therapeutics, these data do not support their use as pharmacodynamic
biomarkers for onartuzumab. PURPOSE: In a recent phase II study of onartuzumab (MetMAb), patients whose
non-small cell lung cancer (NSCLC) tissue scored as positive for MET protein by
immunohistochemistry (IHC) experienced a significant benefit with onartuzumab
plus erlotinib (O+E) versus erlotinib. We describe development and validation of
a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers
as predictors of patient benefit.
EXPERIMENTAL DESIGN: Biomarkers related to MET and/or EGF receptor (EGFR)
signaling were measured by IHC, FISH, quantitative reverse transcription PCR,
mutation detection techniques, and ELISA.
RESULTS: A positive correlation between IHC, Western blotting, and MET mRNA
expression was observed in NSCLC cell lines/tissues. An IHC scoring system of
MET expression taking proportional and intensity-based thresholds into
consideration was applied in an analysis of the phase II study and resulted in
the best differentiation of outcomes. Further analyses revealed a nonsignificant
overall survival (OS) improvement with O+E in patients with high MET copy number
(mean≥5 copies/cell by FISH); however, benefit was maintained in "MET
IHC-positive"/MET FISH-negative patients (HR, 0.37; P=0.01). MET, EGFR,
amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant
benefit with onartuzumab; a nonsignificant OS improvement was observed in
patients with high tumor MET mRNA levels (HR, 0.59; P=0.23). Patients with low
baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519
(P=0.09) in favor of onartuzumab treatment.
CONCLUSIONS: MET IHC remains the most robust predictor of OS and
progression-free survival benefit from O+E relative to all examined exploratory
markers. The MET/hepatocyte growth-factor (HGF) signaling pathway plays a key role in the
processes of embryogenesis, wound healing, and organ regeneration. Aberrant
activation of MET/HGF occurs through multiple mechanisms including gene
amplification, mutation, protein overexpression, and abnormal gene splicing
interrupting autocrine and paracrine regulatory feedback mechanisms. In many
cancers including non-small-cell lung cancer, colorectal, gastric, renal, and
hepatocellular cancer, dysregulation of MET may lead to a more aggressive cancer
phenotype and may be a negative prognostic indicator. Successful therapeutic
targeting of the MET/HGF pathway has been achieved using monoclonal antibodies
against the MET receptor and its ligand HGF in addition to MET-specific and
multitargeted small-molecule tyrosine-kinase inhibitors with several drugs in
late-phase clinical trials including onartuzumab, rilotumumab, tivantinib, and
cabozantinib. MET frequently interacts with other key oncogenic tyrosine kinases
including epidermal growth-factor receptor (EGFR) and HER-3 and these
interactions may be responsible for resistance to anti-EGFR therapies.
Similarly, resistance to MET inhibition may be mediated through EGFR activation,
or alternatively by increasing levels of MET amplification or acquisition of
novel "gatekeeper" mutations. In order to optimize development of effective
inhibitors of the MET/HGF pathway clinical trials must be enriched for patients
with demonstrable MET-pathway dysregulation for which robustly standardized and
validated assays are required. Pancreatic adenocarcinoma (PDAC) is a major unmet medical need and a deeper
understanding of molecular drivers is needed to advance therapeutic options for
patients. We report here that p21-activated kinase 1 (PAK1) is a central node in
PDAC cells downstream of multiple growth factor signalling pathways, including
hepatocyte growth factor (HGF) and MET receptor tyrosine kinase. PAK1 inhibition
blocks signalling to cytoskeletal effectors and tumour cell motility driven by
HGF/MET. MET antagonists, such as onartuzumab and crizotinib, are currently in
clinical development. Given that even highly effective therapies have resistance
mechanisms, we show that combination with PAK1 inhibition overcomes potential
resistance mechanisms mediated either by activation of parallel growth factor
pathways or by direct amplification of PAK1. Inhibition of PAK1 attenuated in
vivo tumour growth and metastasis in a model of pancreatic adenocarcinoma. In
human tissues, PAK1 is highly expressed in a proportion of PDACs (33% IHC score
2 or 3; n = 304) and its expression is significantly associated with MET
positivity (p < 0.0001) and linked to a widespread metastatic pattern in
patients (p = 0.067). Taken together, our results provide evidence for a
functional role of MET/PAK1 signalling in pancreatic adenocarcinoma and support
further characterization of therapeutic inhibitors in this indication. Onartuzumab is a monovalent, humanized, monoclonal antibody that showed
significant survival benefits in combination with erlotinib in MET-positive
non-small-cell lung cancer (NSCLC) in pre-specified subgroup analyses of a
randomized phase II study. We conducted a two-stage, open-label, multicenter,
phase I study of onartuzumab in Japanese patients. Stage 1 investigated the
safety, tolerability, pharmacokinetics (PK), and recommended dose of onartuzumab
in patients with solid tumors, and Stage 2 determined the safety, tolerability,
and PK of onartuzumab plus erlotinib in patients with MET-positive NSCLC. Nine
patients received onartuzumab monotherapy (4, 15, or 30 mg/kg on Day 1 of each
21-day cycle) in Stage 1, and six patients received onartuzumab (15 mg/kg) plus
erlotinib (150 mg/day) in Stage 2. There were no dose-limiting toxicities in
either stage. Serious adverse events (AEs) occurred in one patient in Stage 1
(convulsion), and two patients in Stage 2 (once case each of diarrhea, vomiting,
and pulmonary embolism), but there were no grade 4 AEs or AEs leading to death.
Onartuzumab PKs were linear in the dose range of 4 to 30 mg/kg, and were not
affected by co-administration with erlotinib. PK parameters of onartuzumab were
similar to those reported in non-Japanese patients. A partial response was
observed in a patient with MET immunohistochemistry 3+ NSCLC without MET gene
amplification. Based on these results, the recommended dose of onartuzumab in
Japanese patients with solid tumors is 15 mg/kg every 21 days. The combination
of onartuzumab with erlotinib is feasible in Japanese patients with MET-positive
lung cancer. Companion diagnostics assay interpretation can select patients with the greatest
targeted therapy benefits. We present the results from a prospective study
demonstrating that pathologists can effectively learn immunohistochemical
assay-interpretation skills from digital image-based electronic training
(e-training). In this study, e-training was used to train board-certified
pathologists to evaluate non-small cell lung carcinoma for eligibility for
treatment with onartuzumab, a MET-inhibiting agent. The training program
mimicked the live training that was previously validated in clinical trials for
onartuzumab. A digital interface was developed for pathologists to review
high-resolution, static images of stained slides. Sixty-four pathologists
practicing in the United States enrolled while blinded to the type of training.
After training, both groups completed a mandatory final test using glass slides.
The results indicated both training modalities to be effective. Overall, 80.6%
of e-trainees and 72.7% of live trainees achieved passing scores (at least 85%)
on the final test. All study participants reported that their training
experience was "good" and that they had received sufficient information to
determine the adequacy of case slide staining to score each case. This study
established that an e-training program conducted under highly controlled
conditions can provide pathologists with the skills necessary to interpret a
complex assay and that these skills can be equivalent to those achieved with
face-to-face training using conventional microscopy. Programs of this type are
scalable for global distribution and offer pathologists the potential for
readily accessible and robust training in new companion diagnostic assays linked
to novel, targeted, adjuvant therapies for cancer patients. BACKGROUND: The phase II YO28252 study (NCT01590719) examined first-line
onartuzumab plus mFOLFOX6 in patients with metastatic, human epidermal growth
factor receptor 2-negative adenocarcinoma of the stomach or gastroesophageal
junction. MET immunohistochemistry expression as a biomarker of onartuzumab
activity was also examined.
PATIENTS AND METHODS: Patients were randomized 1:1 to receive standard mFOLFOX6
plus onartuzumab (10 mg/kg) or placebo in 2-week cycles for 12 cycles, followed
by onartuzumab or placebo until disease progression. Coprimary endpoints were
progression-free survival (PFS) in intent-to-treat (ITT) and MET-positive
populations. The target hazard ratio (HR) was 0.70 for patients in the ITT group
and 0.60 in the MET-positive population. Secondary endpoints were overall
survival (OS), overall response rate (ORR), and safety.
RESULTS: Overall, 123 patients were enrolled (n = 62 onartuzumab, n = 61
placebo). Median PFS was 6.77 versus 6.97 months for onartuzumab versus placebo,
respectively (HR, 1.08; 95% confidence interval [CI], 0.71-1.63; p = .71). In
the MET-positive population, median PFS was 5.95 versus 6.80 months, onartuzumab
versus placebo (HR, 1.38; 95% CI, 0.60-3.20; p = .45). Median OS was 10.61
months for onartuzumab versus 11.27 months for placebo) (HR, 1.06, 0.64-1.75; p
= .83). In the MET-positive population, median OS was 8.51 versus 8.48 months
for onartuzumab versus placebo, respectively (HR, 1.12, 95% CI, 0.45-2.78; p =
.80). ORR was 60.5% for the onartuzumab group and 57.1% for placebo. Grade 3-5
adverse events (AEs) were seen in 88.3% of patients receiving onartuzumab and in
78.3% of patients receiving placebo, with serious AEs in 55% and 40%,
respectively.
CONCLUSION: The addition of onartuzumab to mFOLFOX6 in gastric cancer did not
improve efficacy in an unselected population or in a MET
immunohistochemistry-positive population.
IMPLICATIONS FOR PRACTICE: The YO28252 study demonstrated that the addition of
the anti-MET agent onartuzumab to mFOLFOX6 for treatment of gastric cancer did
not improve efficacy in an overall study population or those selected for
positive MET status by immunohistochemistry. This highlights the importance of
correctly selecting biomarkers for targeted therapies. A multivariate analysis
suggested that MET positivity may still be prognostic for worse median overall
survival in gastric cancer; therefore, it is important to continue investigation
into the optimal approach to inhibit MET signaling in gastric cancer. BACKGROUND: Onartuzumab is a monovalent monoclonal antibody that binds with the
extracellular domain of the MET receptor. Given the role of MET in
non-small-cell lung cancer (NSCLC), we investigated whether onartuzumab added to
first-line chemotherapy efficacy in non-squamous NSCLC.
METHODS: Patients with untreated stage IIIB/IV non-squamous NSCLC, stratified by
MET diagnostic status, were randomized to receive onartuzumab (15 mg/kg
intravenously every 3 weeks) or placebo in combination with either
paclitaxel/platinum/bevacizumab (bevacizumab cohort), or in combination with
platinum/pemetrexed (pemetrexed cohort) with maintece bevacizumab or
pemetrexed and onartuzumab/placebo as appropriate. Co-primary endpoints of this
phase II study were progression-free survival (PFS) in all patients and in MET+
patients (2+/3+), defined by the Ventana immunohistochemistry assay; secondary
endpoints included overall survival (OS), objective response rate (ORR), safety,
and pharmacokinetics.
RESULTS: Efficacy data were available for 139 and 120 patients in the
bevacizumab and pemetrexed cohorts, respectively. No benefit was seen in the PFS
endpoint in the intent-to treat population of either cohort, but was numerically
worse in the onartuzumab arm of the MET+ subgroup of the bevacizumab cohort. The
onartuzumab and placebo arms had similar ORR and OS results in both cohorts. A
higher incidence of some adverse events was observed with onartuzumab versus
placebo, including peripheral edema (30% vs. 3%, bevacizumab cohort; 48% vs.
14%, pemetrexed cohort) and venous thromboembolic events (bevacizumab cohort
only, 15% vs. 6%).
CONCLUSION: Onartuzumab does not appear to provide any additional clinical
benefit when given in combination with current first-line standard-of-care
chemotherapy for non-squamous NSCLC. Purpose Bevacizumab regimens are approved for the treatment of recurrent
glioblastoma in many countries. Aberrant mesenchymal-epithelial transition
factor (MET) expression has been reported in glioblastoma and may contribute to
bevacizumab resistance. The phase II study GO27819 investigated the monovalent
MET inhibitor onartuzumab plus bevacizumab (Ona + Bev) versus placebo plus
bevacizumab (Pla + Bev) in recurrent glioblastoma. Methods At first recurrence
after chemoradiation, bevacizumab-naïve patients with glioblastoma were randomly
assigned 1:1 to receive Ona (15 mg/kg, once every 3 weeks) + Bev (15 mg/kg, once
every 3 weeks) or Pla + Bev until disease progression. The primary end point was
progression-free survival by response assessment in neuro-oncology criteria.
Secondary end points were overall survival, objective response rate, duration of
response, and safety. Exploratory biomarker analyses correlated efficacy with
expression levels of MET ligand hepatocyte growth factor, O6-methylguanine-DNA
methyltransferase promoter methylation, and glioblastoma subtype. Results Among
129 patients enrolled (Ona + Bev, n = 64; Pla + Bev, n = 65), baseline
characteristics were balanced. The median progression-free survival was 3.9
months for Ona + Bev versus 2.9 months for Pla + Bev (hazard ratio, 1.06; 95%
CI, 0.72 to 1.56; P = .7444). The median overall survival was 8.8 months for Ona
+ Bev and 12.6 months for Pla + Bev (hazard ratio, 1.45; 95% CI, 0.88 to 2.37; P
= .1389). Grade ≥ 3 adverse events were reported in 38.5% of patients who
received Ona + Bev and 35.9% of patients who received Pla + Bev. Exploratory
biomarker analyses suggested that patients with high expression of hepatocyte
growth factor or unmethylated O6-methylguanine-DNA methyltransferase may benefit
from Ona + Bev. Conclusion There was no evidence of further clinical benefit
with the addition of onartuzumab to bevacizumab compared with bevacizumab plus
placebo in unselected patients with recurrent glioblastoma in this phase II
study; however, further investigation into biomarker subgroups is warranted. IMPORTANCE: Dysregulation of the mesenchymal-epithelial transition (MET)
signaling pathway is associated with poor prognosis in gastroesophageal
adenocarcinoma (GEC). We report results of METGastric, a phase 3 trial of the
MET inhibitor onartuzumab plus standard first-line chemotherapy for human
epidermal growth factor receptor 2 (HER2)-negative, MET-positive, advanced GEC.
OBJECTIVE: To determine whether the addition of onartuzumab to first-line
fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) improves efficacy compared
with mFOLFOX6 plus placebo in HER2-negative, MET-positive GEC.
DESIGN, SETTING, AND PARTICIPANTS: Randomized, double-blind, multicenter trial
conducted from November 2012 to March 2014. Patients were 18 years or older with
an adenocarcinoma of the stomach or gastroesophageal junction with metastatic
disease not amenable for curative therapy. Tumor samples were centrally tested
for MET expression using Ventana anti-Total c-MET (SP44) rabbit monoclonal
antibody, HER2 status, and Lauren histologic subtype. MET-positive tumors were
defined as at least 50% of tumor cells showing weak, moderate, and/or strong
staining intensity (MET 1+/2+/3+, respectively) by immunohistochemistry.
INTERVENTIONS: Patients with HER2-negative, MET-positive GEC were enrolled and
randomized 1:1 to receive mFOLFOX6 with or without onartuzumab (10 mg/kg).
MAIN OUTCOMES AND MEASURES: Co-primary end points: overall survival in the
intent-to-treat (ITT) population and in patients with MET 2+/3+ GEC. Secondary
end points: progression-free survival (PFS), overall response rate (ORR), and
safety.
RESULTS: Enrollment was stopped early due to sponsor decision, which was agreed
with an independent data monitoring committee. At the data cutoff (April 25,
2014) there were 562 patients in the ITT population (n = 283 placebo plus
mFOLFOX6 [median age, 58 y; 65% male]; n = 279 onartuzumab plus mFOLFOX6 [median
age, 60 y; 67% male]); 109 (38.5%) and 105 (37.6%) of the ITT population were
MET 2+/3+, respectively. Addition of onartuzumab to mFOLFOX6 did not
significantly improve OS, PFS, or ORR vs placebo plus mFOLFOX6 in the ITT (OS
hazard ratio [HR], 0.82; 95% CI, 0.59-1.15; P = .24; PFS HR, 0.90; 95% CI,
0.71-1.16; P = .43; ORR, 46.1% vs 40.6%) or MET 2+/3+ populations (OS HR, 0.64;
95% CI, 0.40-1.03; P = .06; PFS HR, 0.79; 95% CI, 0.54-1.15; P = .22; ORR, 53.8%
vs 44.6%). Safety was as expected for onartuzumab.
CONCLUSIONS AND RELEVANCE: Addition of onartuzumab to first-line mFOLFOX6 did
not significantly improve clinical benefits in the ITT or MET 2+/3+ populations.
TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01662869. Purpose The phase III OAM4971g study (METLung) examined the efficacy and safety
of onartuzumab plus erlotinib in patients with locally advanced or metastatic
non-small-cell lung cancer selected by MET immunohistochemistry whose disease
had progressed after treatment with a platinum-based chemotherapy regimen.
Patients and Methods Patients were randomly assigned at a one-to-one ratio to
receive onartuzumab (15 mg/kg intravenously on day 1 of each 21-day cycle) plus
daily oral erlotinib 150 mg or intravenous placebo plus daily oral erlotinib 150
mg. The primary end point was overall survival (OS) in the intent-to-treat
population. Secondary end points included median progression-free survival,
overall response rate, biomarker analysis, and safety. Results A total of 499
patients were enrolled (onartuzumab, n = 250; placebo, n = 249). Median OS was
6.8 versus 9.1 months for onartuzumab versus placebo (stratified hazard ratio
[HR], 1.27; 95% CI, 0.98 to 1.65; P = .067), with a greater number of deaths in
the onartuzumab arm (130 [52%] v 114 [46%]). Median progression-free survival
was 2.7 versus 2.6 months (stratified HR, 0.99; 95% CI, 0.81 to 1.20; P = .92),
and overall response rate was 8.4% and 9.6% for onartuzumab versus placebo,
respectively. Exploratory analyses using MET fluorescence in situ hybridization
status and gene expression showed no benefit for onartuzumab; patients with EGFR
mutations showed a trend toward shorter OS with onartuzumab treatment (HR, 4.68;
95% CI, 0.97 to 22.63). Grade 3 to 5 adverse events were reported by 56.0% and
51.2% of patients, with serious AEs in 33.9% and 30.7%, for experimental versus
control arms, respectively. Conclusion Onartuzumab plus erlotinib did not
improve clinical outcomes, with shorter OS in the onartuzumab arm, compared with
erlotinib in patients with MET-positive non-small-cell lung cancer. |
Is there a relationship between B cells and Multiple Sclerosis? | MS patients with high neurodegeneration have changes in B cells characterized by down-regulation of B-cell-specific genes and increased activation status | Two-color flow-cytometric analysis on peripheral blood lymphocytes of 46
untreated multiple sclerosis patients (MS), 36 other medical disease patients
(OMD) and 19 healthy control subjects (HC) was performed to know the
relationships between T and B cell subpopulations. In MS patients we observed an
increase of total lymphocyte count and an increase of CD4+CD29+ cells, which are
adjuvant to B cell in antibody production. We hypothesized this change is
related to the reduction of CD21+ cells, expressing B2 antigen which disappears
after B cell activation. The unperfect balance of immune system in MS was also
demonstrated by the increased level of CD25+ cells in relapsing-remitting
patients and by the decreased level of CD4+ CD45RA+ (suppressor inducer) cells
in progressive patients. Multiple sclerosis is a chronic inflammatory and demyelinating disorder of the
CNS with an unknown aetiology. Although intrathecal immunoglobulin G (IgG)
synthesis is a key feature of the disease, little is still known about the B
cell response in the CNS of multiple sclerosis patients. We analysed the
phenotype and kinetics of different B cell subsets in patients with multiple
sclerosis, infectious disease (IND) and non-inflammatory neurological disease
(NIND). B cells were detected in the CSF of multiple sclerosis and IND patients,
but were largely absent in NIND patients. In the CSF, the majority of B cells
had a phenotype of memory B cells and short-lived plasma blasts (PB); plasma
cells were absent from the compartment. The proportion of PB was highest in
multiple sclerosis patients and patients with acute CNS infection. While PB
disappeared rapidly from the CSF after resolution of infection in IND patients,
these cells were present at high numbers throughout the disease course in
multiple sclerosis patients. CSF PB numbers in multiple sclerosis patients
strongly correlated with intrathecal IgG synthesis and inflammatory parenchymal
disease activity as disclosed by MRI. This study identifies short-lived plasma
blasts as the main effector B cell population involved in ongoing active
inflammation in multiple sclerosis patients. Strategies for treating autoimmune disorders are increasingly employing targeted
therapies rather than non-specific, multitargeted treatments. Accumulating
evidence on the involvement of B lymphocytes in the pathophysiology of
autoimmune demyelinating disease has led to a renewed interest in B cells as
potential therapeutic targets. In particular, antigen presentation between B
cells and T cells, increased trafficking of B cells across the blood-brain
barrier, and autoantibodies produced by plasma cells may contribute to the
pathophysiology of autoimmune disorders such as multiple sclerosis. Several
B-cell-targeted, depletion therapies are currently in development, including
rituximab, epratuzumab, diphtheria toxin-single chain Fv (DC2219), belimumab,
atacicept, abatacept, and abetimus sodium. Of these agents, only rituximab and
abatacept have been evaluated in multiple sclerosis patients. Preliminary
results of a phase II trial of rituximab in multiple sclerosis suggest that
rituximab is well tolerated and significantly reduces the number of gadolinium
enhancing lesions over 24 weeks of treatment. Results of an exploratory analysis
suggest the potential promise of abatacept 10 mg/kg for multiple sclerosis. It
is expected that future clinical trials will establish a role for
B-cell-targeted therapies in the treatment of multiple sclerosis and other
autoimmune neurological diseases. This article describes the mechanism of action
behind B-cell-targeted depletion therapies in development and reviews available
clinical data. Multiple sclerosis is a common neurological disorder that represents a
significant source of disability. B cells have recently emerged as a novel
therapeutic target for multiple sclerosis. The natural development of B cells is
characterized by an antigen-independent phase that occurs in the bone marrow and
an antigen-dependent phase that takes place in the peripheral lymphoid tissue.
The stage of B-cell development can be identified by the presence of specific
cell surface markers. Checkpoints are in place to prevent self-reactive B cells
from further development and activation. Some self-reactive B cells are able to
escape these checkpoints, resulting in a loss of tolerance. B cells may
contribute to systemic autoimmunity and the development of autoimmune disease
via cytokine production, antigen presentation, and complement activation. In
addition, B cells may trigger autoimmune disease via molecular mimicry, which
occurs when a single B-cell receptor recognizes both a non-self antigen molecule
and a self-molecule. Accumulating data suggest that ectopic proliferation of B
cells in the central nervous system may also play a role. Further research is
needed to elucidate the pathology of B cells and their role in central nervous
system autoimmune diseases, including multiple sclerosis. Multiple sclerosis is an inflammatory demyelinating disease of the central
nervous system with no clear etiology. Until recently, most studies have
emphasized the role of T cells in the pathogenesis of multiple sclerosis. Data
suggesting that B cells play a role in the pathogenesis of multiple sclerosis
have been accumulating for the past five decades, demonstrating that the
cerebrospinal fluid and central nervous system tissues of multiple sclerosis
patients contain B cells, plasma cells, antibodies, and immunoglobulins. Data
suggest that B cells are involved in antigen capture and presentation to T
cells, cytokine production, antibody secretion, demyelination, tissue damage,
and remyelination in multiple sclerosis. These advances in the understanding of
B-cell and antibody roles in the pathophysiology of multiple sclerosis provide a
strong rationale for B-cell-targeted therapies. BACKGROUND: Accumulating evidence supports a major role of B cells in multiple
sclerosis (MS) pathogenesis. How B cells are recruited to the CNS is
incompletely understood. Our objective was to study B-cell chemokine
concentrations in MS, their relationship with disease activity, and how
treatment with methylprednisolone and natalizumab affected the concentration in
CSF.
METHODS: Using a cross-sectional design, CSF and blood samples were obtained
from cohorts of patients with clinically isolated syndromes (CIS),
relapsing-remitting MS (RRMS), primary progressive MS (PPMS), or secondary
progressive MS (SPMS), and noninflammatory neurologic disease control subjects.
Some patients with RRMS were studied before and after treatment with
methylprednisolone or natalizumab.
RESULTS: In CSF, concentrations of CXCL13, but not CXCL12, were higher in
patients with CIS, RRMS, SPMS, and PPMS than in controls. CSF concentrations of
CXCL13 correlated with the CSF B-cell count, with markers of immune activation,
and with disease activity in patients with CIS and RRMS. CSF concentrations of
CXCL13 decreased after treatment with high-dose methylprednisolone and
natalizumab. High CSF concentrations of CXCL13 correlated with low expression of
messenger RNA encoding the immunoregulatory cytokines interleukin 10 and
transforming growth factor beta1, but not with the expression of T-helper type 1
(Th1) and Th17 factors.
CONCLUSION: The chemokine CXCL13 may play a major role in recruitment of B cells
and T-cell subsets expressing the chemokine receptor CXCR5 to the CNS in
multiple sclerosis (MS), and may be a useful biomarker for treatment effects in
MS. Furthermore, CXCL13 or its receptor CXCR5 should be considered as
therapeutic targets in MS. A cardinal feature of multiple sclerosis (MS) is the persistent intrathecal
synthesis of antibodies. Our previous finding that a large fraction of B cells
infiltrating the MS brain are infected with Epstein-Barr virus (EBV) raises the
possibility that this virus, because of its ability to establish a latent
infection in B cells and interfere with their differentiation, contributes to
B-cell dysregulation in MS. The aim of this study was to gain further insight
into EBV latency programs and their relationship to B-cell activation in the MS
brain. Immunohistochemical analysis of postmortem MS brain samples harboring
large EBV deposits revealed that most B cells in white matter lesions, meninges,
and ectopic B-cell follicles are CD27+ antigen-experienced cells and coexpress
latent membrane protein 1 and latent membrane protein 2A, 2 EBV-encoded proteins
that provide survival and maturation signals to B cells. By combining
laser-capture microdissection with preamplification reverse
transcription-polymerase chain reaction techniques, EBV latency transcripts
(latent membrane protein 2A, EBV nuclear antigen 1) were detected in all MS
brain samples analyzed. We also found that B cell-activating factor of the tumor
necrosis factor family is expressed in EBV-infected B cells in acute MS lesions
and ectopic B-cell follicles. These findings support a role for EBV infection in
B-cell activation in the MS brain and suggest that B cell-activating factor of
the tumor necrosis factor family produced by EBV-infected B cells may contribute
to this process resulting in viral persistence and, possibly, disruption of
B-cell tolerance. Recent years have substantially broadened our view on the pathogenesis of
multiple sclerosis (MS). While earlier concepts focused predomitly on T
lymphocytes as the key cell type to mediate inflammatory damage within central
nervous system (CNS) lesions, emerging evidence suggests that B lymphocytes may
play a comparably important role both as precursors of antibody-secreting plasma
cells and as antigen-presenting cells (APCs) for the activation of T cells. With
greater appreciation of this pathogenic B-cell function in MS, B-cell-directed
therapies, and in particular B-cell-depleting monoclonal antibodies targeting
the CD20 molecule, have gained enormous interest over recent years. Clinical
trials demonstrated that anti-CD20 treatment, which depletes immature and mature
B cells but spares CD20 negative plasma cells, rapidly reduces formation of new
inflammatory CNS lesions. While these findings clearly corroborate a pathogenic
contribution of B cells, recent experimental but also clinical findings indicate
that not all B cells contribute in an equally pathogenic manner and that certain
subsets may in contrast mediate anti-inflammatory effects. In this review, we
summarize current findings in support of pathogenic B-cell function in MS,
including the encouraging clinical data which derived from anti-CD20 MS trials.
Further, we review novel findings suggestive of regulatory properties of B-cell
subsets which may be collaterally abolished by pan-CD20 depletion. In
conclusion, we aim to provide an outlook on how this currently differentiating
concept of pro- and anti-inflammatory B-cell function could be harnessed to
further improve safety and effectiveness of B-cell-directed therapeutic
approaches in MS. BACKGROUND: Fingolimod is an oral drug approved for multiple sclerosis (MS) with
an ability to trap central memory T cells in secondary lymphoid tissues;
however, its variable effectiveness in individual patients indicates the need to
evaluate its effects on other lymphoid cells.
OBJECTIVE: To clarify the effects of fingolimod on B-cell populations in
patients with MS.
METHODS: We analysed blood samples from 9 fingolimod-treated and 19 control
patients with MS by flow cytometry, to determine the frequencies and activation
states of naive B cells, memory B cells, and plasmablasts.
RESULTS: The frequencies of each B-cell population in peripheral blood
mononuclear cells (PBMC) were greatly reduced 2 weeks after starting fingolimod
treatment. Detailed analysis revealed a significant reduction in activated
memory B cells (CD38(int-high)), particularly those expressing Ki-67, a marker
of cell proliferation. Also, we noted an increased proportion of activated
plasmablasts (CD138(+)) among whole plasmablasts, in the patients treated with
fingolimod.
CONCLUSIONS: The marked reduction of Ki-67(+) memory B cells may be directly
linked with the effectiveness of fingolimod in treating MS. In contrast, the
relative resistance of CD138(+) plasmablasts to fingolimod may be of relevance
for understanding the differential effectiveness of fingolimod in individual
patients. BACKGROUND: Five different G protein-coupled sphingosine-1-phosphate (S1P)
receptors (S1P1-S1P5) regulate a variety of physiologic and pathophysiologic
processes, including lymphocyte circulation, multiple sclerosis (MS), and
cancer. Although B-lymphocyte circulation plays an important role in these
processes and is essential for normal immune responses, little is known about
S1P receptors in human B cells.
OBJECTIVE: To explore their function and signaling, we studied B-cell lines and
primary B cells from control subjects, patients with leukemia, patients with S1P
receptor inhibitor-treated MS, and patients with primary immunodeficiencies.
METHODS: S1P receptor expression was analyzed by using multicolor
immunofluorescence microscopy and quantitative PCR. Transwell assays were used
to study cell migration. S1P receptor internalization was visualized by means of
time-lapse imaging with fluorescent S1P receptor fusion proteins expressed by
using lentiviral gene transfer. B-lymphocyte subsets were characterized by means
of flow cytometry and immunofluorescence microscopy.
RESULTS: Showing that different B-cell populations express different
combinations of S1P receptors, we found that S1P1 promotes migration, whereas
S1P4 modulates and S1P2 inhibits S1P1 signals. Expression of CD69 in activated B
lymphocytes and B cells from patients with chronic lymphocytic leukemia
inhibited S1P-induced migration. Studying B-cell lines, normal B lymphocytes,
and B cells from patients with primary immunodeficiencies, we identified Bruton
tyrosine kinase, β-arrestin 2, LPS-responsive beige-like anchor protein,
dedicator of cytokinesis 8, and Wiskott-Aldrich syndrome protein as critical
signaling components downstream of S1P1.
CONCLUSION: Thus S1P receptor signaling regulates human B-cell circulation and
might be a factor contributing to the pathology of MS, chronic lymphocytic
leukemia, and primary immunodeficiencies. Multiple sclerosis (MS) is a unique central nervous system (CNS) inflammatory
disease with a broad spectrum of clinical presentations, which are time- and
disease progression-related. It usually affects young adults, with a female
predomice of 3:1. Men are more likely to develop symptoms at a slightly older
age with a more progressive disease course. Diagnosis relies on a combination of
clinical, radiological, and laboratory investigations, with a central role of
magnetic resoce imaging (MRI). Although the exact etiology is still obscure,
the leading hypothesis behind MS relapses is acute inflammatory attacks on CNS
myelin and axons. This complex process involves B and T cells together with
macrophages and microglia. Genetic and environmental factors are thought to be
major contributors to the disease's evolution. MS therapies consist of long-term
(immunomodulatory) management, focusing on disease modification, and short-term
symptomatic control. Symptomatic treatment includes pharmacological and
non-pharmacological methods to protect function and restore quality of life
(QoL). The introduction and development of disease-modifying medications provide
opportunities to change the face of this disease, enhancing QoL over the
long-term. Interferon (INF) and Glatiramer acetate (GLAT) represent first line
medications with limited effect and relatively fair safety profile. Newer
medications with improved efficacy along with a more hazardous side effect
profile are now considered second line therapy.
CONCLUSIONS: The present review summarizes current knowledge of this frequent
disease. Urologists must acquire a deeper understanding for better integration
of practice recommendations. OBJECTIVE: To evaluate the influence of Fingolimod treatment on B-cell subset
composition and function in multiple sclerosis patients and its potential
clinical relevance.
METHODS: Subset composition and cytokine production of B cells derived from
peripheral blood mononuclear cells from multiple sclerosis patients under
Fingolimod treatment, untreated multiple sclerosis patients and healthy controls
were analyzed by flow cytometry and ELISA. Migration of lymphocyte subsets
across primary human brain microvascular endothelial cells was assessed in an in
vitro transmigration assay. Cell numbers and composition of B-cell subsets in
cerebrospinal fluid and peripheral blood were determined by flow cytometry.
Regulatory B-cell frequencies were correlated with parameters of disease
stability.
RESULTS: Within the peripheral B-cell compartment of Fingolimod-treated
patients, the proportion of regulatory B cells (CD38(+)CD27(-)CD24(+)CD5(+)) was
significantly increased as compared to treatment-naïve multiple sclerosis
patients and to healthy controls, and significantly more regulatory B cells
produced Interleukin-10. Fingolimod treatment enhanced the capacity of
regulatory B cells to transmigrate across brain endothelial cells in an in vitro
model of the blood-brain-barrier. In line with these findings, the cerebrospinal
fluid/blood ratio of total B cells and regulatory B cells was strongly increased
by Fingolimod treatment, and patients exhibited increased regulatory B-cell
frequencies in the cerebrospinal fluid. Finally, elevated regulatory B-cell
percentages in the periphery significantly correlated with clinical and
paraclinical disease stability.
INTERPRETATION: These data suggest a novel and as yet unrecognized role of
Fingolimod in correction of the imbalance between regulatory and effector B-cell
functions in multiple sclerosis both by direct effects and indirect partitioning
effects on B-cell subpopulations. B cells are increasingly regarded as integral to the pathogenesis of multiple
sclerosis, in part as a result of the success of B cell-depletion therapy.
Multiple B cell-dependent mechanisms contributing to inflammatory demyelination
of the CNS have been explored using experimental autoimmune encephalomyelitis
(EAE), a CD4 T cell-dependent animal model for multiple sclerosis. Although B
cell Ag presentation was suggested to regulate CNS inflammation during EAE,
direct evidence that B cells can independently support Ag-specific autoimmune
responses by CD4 T cells in EAE is lacking. Using a newly developed murine model
of in vivo conditional expression of MHC class II, we reported previously that
encephalitogenic CD4 T cells are incapable of inducing EAE when B cells are the
sole APC. In this study, we find that B cells cooperate with dendritic cells to
enhance EAE severity resulting from myelin oligodendrocyte glycoprotein (MOG)
immunization. Further, increasing the precursor frequency of MOG-specific B
cells, but not the addition of soluble MOG-specific Ab, is sufficient to drive
EAE in mice expressing MHCII by B cells alone. These data support a model in
which expansion of Ag-specific B cells during CNS autoimmunity amplifies cognate
interactions between B and CD4 T cells and have the capacity to independently
drive neuroinflammation at later stages of disease. Multiple sclerosis is caused by a complex interaction between genetic
predisposition and environmental factors. Epstein-Barr virus (EBV) is an
environmental risk factor that is strongly related to multiple sclerosis (MS),
since EBV seropositivity is linked to a significant risk of developing MS. EBV
may be involved in the pathogenesis of the disease and it is possibly a
prerequisite for the development of MS. EBV infection persists in B-cells during
the lifetime of the host and can modulate their function. In addition, MS
patients might have a deficient capacity to eliminate latent EBV infection in
the central nervous system and this would promote the accumulation of infected B
cells. Several mechanisms of pathogenesis, including a direct and indirect
function of infected B cells, have been postulated in inflammation and
neurodegeneration. A relationship between EBV and human endogenous retroviruses
in the pathogenesis of MS has also been reported. If EBV is important in the
pathogenesis of MS, different therapeutic strategies seem possible for MS
treatment. |
Which virus type causes Molluscum contagiosum? | Molluscum contagiosum virus (MCV) is a human poxvirus that causes tumor-like skin lesions. | We report here the clinical and immunological findings in two patients with
molluscum contagiosum poxvirus infection and the acquired immunodeficiency
syndrome (AIDS). These cases support earlier evidence that the molluscum
contagiosum virus may act as cases support earlier evidence that the molluscum
contagiosum virus may act as an opportunistic pathogen. There is now evidence
that members of all five families of double stranded DNA-containing human
viruses have been associated with unusual clinical manifestations in AIDS
patients, and the significance of DNA virus infections in patients with AIDS is
discussed. BACKGROUND: Molluscum contagiosum virus (MCV) causes molluscum contagiosum (MC)
in both children and adults. Recent studies have revealed that the DNA of MCV
can be classified into two major types by restriction enzyme cleavage patterns;
however, the relationship between MCV types and the clinical features has not
been fully understood. Our study was conducted to examine whether there are
geographic differences in the incidence of MCV types and whether a correlation
exists between MCV types and the age, sex, and clinical status of the patients.
METHODS: Specimens were obtained from 171 Japanese patients. The total DNA was
extracted and digested with the restriction enzymes, BamH I, Hind III, and Cla
I, respectively. Specimens were then electrophoresed in agarose gels. The gels
were stained with ethidium bromide and photographs were taken under
transillumination.
RESULTS: Six different cleavage patterns were observed; they were classified
into two major types, MCV 1 and MCV 2, consisting of two MCV 1-variants, and MCV
2 prototype, and three MCV 2-variants. The ratio of MCV 1 to MCV 2 was 13:1. MCV
1 was commonly detected in children (98%) and adult women (92%). MCV 2 was more
frequently isolated from adult men (44%) and from patients with human
immunodeficiency virus (HIV) infection (75%).
CONCLUSION: MCV types found in Japanese children and adult women were
predomitly MCV 1 and less frequently MCV 2. This pattern is similar to that
observed in European countries and Australia, suggesting a high frequency and
world-wide distribution of MCV 1. The higher incidence of MCV 2 among adult men
and HIV-positive patients may indicate that transmission routes of MCV 1 and MCV
2 is somewhat different, of which the latter may be in part by sexual contact. Molluscum contagiosum virus (MCV) is a member of the family Poxviridae and
pathogenic to humans. MCV causes benign epidermal tumors mainly in children and
young adults and is a common pathogen in immunecompromised individuals. The
viral DNA polymerase is the essential enzyme involved in the replication of the
genome of DNA viruses. The identification and characterization of the gene
encoding the DNA polymerase of molluscum contagiosum virus type 1 (MCV-1) was
carried out by PCR technology and nucleotide sequence analysis. Computer-aided
analysis of known amino acid sequences of DNA polymerases from two members of
the poxvirus family revealed a high amino acid sequence homology of about 49.7%
as detected between the DNA polymerases of vaccinia virus (genus Orthopoxvirus)
and fowlpoxvirus (genus Avipoxvirus). Specific oligonucleotide primers were
designed and synthesized according to the distinct conserved regions of amino
acid sequences of the DNA polymerases in which the codon usage of the MCV-1
genome was considered. Using this technology a 228 bp DNA fragment was amplified
and used as hybridization probe for identifying the corresponding gene of the
MCV-1 genome. It was found that the PCR product was able to hybridize to the
BamHI MCV-1 DNA fragment G (9.2 kbp, 0.284 to 0.332 map units). The nucleotide
sequence of this particular region of the MCV-1 genome (7267 bp) between map
coordinates 0.284 and 0.315 was determined. The analysis of the DNA sequences
revealed the presence of 22 open reading frames (ORFs-1 to -22). ORF-13 (3012
bp; nucleotide positions 6624 to 3612) codes for a putative protein of a
predicted size of 115 kDa (1004 aa) which shows 40.1% identity and 35%
similarity to the amino acid sequences of the DNA polymerases of vaccinia,
variola, and fowlpoxvirus. In addition significant homologies (30% to 55%) were
found between the amino acid sequences of the ORFs 3, -5, -9, and -14 and the
amino acid sequences of the E6R, E8R, E10R, and a 7.3 kDa protein of vaccinia
and variola virus, respectively. Comparative analysis of the genomic positions
of the loci of the detected viral genes including the DNA polymerases of MCV-1,
vaccinia, and variola virus revealed a similar gene organization and
arrangement. Molluscum contagiosum virus (MCV), the only member of the Molluscipoxvirus
genus, causes benign papules in healthy people but disfiguring lesions in
immunocompromised patients. The sequence of MCV has been completed, revealing
that MCV encodes a probable type I topoisomerase enzyme. All poxviruses
sequenced to date also encode type I topoisomerases, and in the case of vaccinia
virus the topoisomerase has been shown to be essential for replication. Thus,
inhibitors of the MCV topoisomerase might be useful as antiviral agents. We have
cloned the gene for MCV topoisomerase, overexpressed and purified the protein,
and begun to characterize its activities in vitro. Like other eukaryotic type I
topoisomerases, MCV topoisomerase can relax both positive and negative
supercoils. An analysis of the cleavage of plasmid and oligonucleotide
substrates indicates that cleavage by MCV topoisomerase is favored just 3' of
the sequence 5' (T/C)CCTT 3', resulting in formation of a covalent bond to the
3' T residue, as with other poxvirus topoisomerases. We identified solution
conditions favorable for activity and measured the rate of formation and decay
of the covalent intermediate. MCV topoisomerase is sensitive to inhibition by
coumermycin A1 (50% inhibitory concentration, 32 microM) but insensitive to five
other previously reported topoisomerase inhibitors. This work provides the point
of departure for studies of the mechanism of function of MCV topoisomerase and
the development of medically useful inhibitors. Molluscum contagiosum virus (MCV) is a common, human poxvirus that causes small
papular skin lesions that persist for long periods without signs of
inflammation. Previous studies revealed that MCV encodes a family of proteins
with homology to mammalian IL-18 binding proteins. IL-18 is a proinflammatory
cytokine that induces synthesis of interferon gamma, activates NK cells, and is
required for a T-lymphocyte helper type 1 response. We expressed and purified
the proteins encoded by the MC53L and MC54L genes of MCV, as well as their human
and murine homologs. All four recombit proteins were able to bind with high
affinity to human and murine IL-18 molecules and inhibited IL-18 mediated
interferon gamma production in a dose-dependent manner. The pirating of IL-18
binding proteins by poxviruses and their use as decoy receptors is consistent
with the critical role of IL-18 in defense against virus infections and provides
a mechanism for evasion of the immune system by MCV. All poxviruses studied encode a type 1B topoisomerase that introduces transient
nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of
the protein domains of the topoisomerase of the poxvirus molluscum contagiosum
(MCV), which allows us to specify DNA contacts made by different domains.
Partial proteolysis of the enzyme revealed two stable domains separated by a
protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal
domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV
topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro,
albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was
sensitive to sequences in the favored 5'-(T/C)CCTT-3' recognition site and also
flanking DNA, indicating that some of the sequence-specific contacts are made by
residues 82-323. Assays of initial binding and covalent catalysis by
MCV-TOP(82-323) identified the contacts flanking the 5'-CCCTT-3' sequence at
+10, +9, -2, and -3 to be important. Tests with substrates containing a
5-bridging phosphorothiolate that trap the cleaved complex revealed that correct
contacts to the flanking sequences were important in the initial cleavage step.
MCV-TOP(82-323) differed from the full-length protein in showing reduced
sensitivity to mutations at a position within the 5'-(T/C)CCTT-3' recognition
site, consistent with a model in which the amino-terminal domain contacts this
region. These findings provide insight into the division of labor within the
MCV-TOP enzyme. Molluscum contagiosum is a benign contagious disease caused by a poxvirus. The
virus proliferates within keratinocytes and forms intracytoplasmic Molluscum
bodies. Though it is a common clinical condition, histologically is not yet
reported from this region of Mymensingh. We received a skin biopsy specimen in a
pathology laboratory for histological examination. The Haematoxylin and Eosin
stained sections revealed typical intracytoplasmic Molluscum bodies in
keratinocytes. The lesions were in the trunk, which is a common site for
Molluscum Contagiosum (MC). As the diagnosis of Molluscum contagiosum is easy by
histological examination, every patient suspected to be this disease is
recommended to be examined histologically to exclude other similar types of
lesions. Infection with molluscum contagiosum virus, a poxvirus, normally has a typical
clinical presentation; therefore, laboratory confirmation is infrequently sought
and the virus is rarely isolated in culture. As reported herein, viral culture
of specimens from atypical lesions may produce an abortive infection in limited
cell lines and a cytopathic effect suggestive of herpes simplex virus. Molluscum contagiosum is a virus that causes characteristic pearly lesions on
the surface of the skin. Small clusters of mollusca are a nuisance rather than a
serious health problem. However, the mollusca can be more widespread and
disfiguring in people with impaired cell-mediated immunity. Molluscum
contagiosum virus is common in children. In adults it can also be contracted
during sexual activity and might indicate a need for diagnostic testing for
other, more serious sexually transmitted infections in young, sexually active
adults. Molluscum contagiosum is a viral infection of the skin and mucous membranes that
is caused by infection with the molluscum contagiosum virus. Molluscum
contagiosum can be acquired from skin to skin contact which may be during play,
in a swimming pool, or through sexual contact. Sexually acquired molluscum is
rare in younger children, but becomes quite common during adolescence and young
adulthood, after the sexual debut. It has been long known that the human
papillomavirus, which causes genital warts, i.e., condyloma accuminatum, can be
vertically transmitted through an infected genital tract. Children may not
manifest condyloma lesions for a few years. The entity of congenital molluscum
has been debated in the literature and only three cases of suspected congenital
molluscum have been reported. We report on four more infants with congenital
molluscum, two children with congenital lesions, and two children with onset of
lesions at 6 weeks of age. Two children had single cutaneous lesions on the
extremities and two had lesions of the scalp consistent with the site of
cervical pressure. Congenital molluscum appears to be a more common entity than
previously reported. Vertical transmission of molluscum should be considered for
all infantile cases of molluscum. Molluscum contagiosum poxvirus (MCV) type 1 and type 2 encode two chemokine-like
proteins MC148R1 and MC148R2. It is believed that MC148R proteins function by
blocking the inflammatory response. However, the mechanism of the proposed
biological activities of MC148R proteins and the role of the additional
C-terminal cysteines that do not exist in other chemokines are not understood.
Here, we demonstrated in two different assay systems that His-tagged MC148R1
displaces the interaction between CXCL12α and CXCR4. The N-terminal cysteines
but not the additional C-terminal cysteines modulate this displacement.
His-tagged MC148R1 blocked both CXCL12α-mediated and MIP-1α-mediated chemotaxis.
In contrast, MC148R2 blocked MIP-1α-mediated but not CXCL12α-mediated
chemotaxis. Immunoprecipitation by antibodies to MC148R1 or CXCL12α followed by
immunoblotting and detection by antibodies to the other protein demonstrated
physical interaction of His-tagged CXCL12α and His-tagged MC148R1. Interaction
with chemokines might mask the receptor interaction site resulting in decreased
binding and impairment of the biological activities. Molluscum contagiosum virus (MCV) causes persistent neoplasms in healthy and
immunocompromised people. Its ability to persist likely is due to its arsenal of
viral immunoevasion proteins. For example, the MCV MC159 protein inhibits
TNF-R1-induced NF-κB activation and apoptosis. The MC159 protein is a viral FLIP
and, as such, possesses two tandem death effector domains (DEDs). We show in
this article that, in human embryonic kidney 293 T cells, the expression of
wild-type MC159 or a mutant MC159 protein containing the first DED (MC159 A)
inhibited TNF-induced NF-κB, or NF-κB activated by PMA or MyD88 overexpression,
whereas a mutant protein lacking the first DED (MC159 B) did not. We
hypothesized that the MC159 protein targeted the IκB kinase (IKK) complex to
inhibit these diverse signaling events. Indeed, the MC159 protein, but not MC159
B, coimmunoprecipitated with IKKγ. MC159 coimmunoprecipitated with IKKγ when
using mouse embryonic fibroblasts that lack either IKKα or IKKβ, suggesting that
the MC159 protein interacted directly with IKKγ. MC159-IKKγ
coimmunoprecipitations were detected during infection of cells with either MCV
isolated from human lesions or with a recombit MC159-expressing vaccinia
virus. MC159 also interacts with TRAF2, a signaling molecule involved in NF-κB
activation. However, mutational analysis of MC159 failed to reveal a correlation
between MC159-TRAF2 interactions and MC159's inhibitory function. We propose
that MC159-IKK interactions, but not MC159-TRAF2 interactions, are responsible
for inhibiting NF-κB activation. Molluscum contagiosum virus (MCV), a poxvirus pathogenic for humans, replicates
well in human skin in vivo, but not in vitro in standard monolayer cell
cultures. In order to determine the nature of the replication deficiency in
vitro, the MCV infection process in standard culture has to be studied step by
step. The method described in this chapter uses luciferase and GFP reporter
constructs to measure poxviral mRNA transcription activity in cells in standard
culture infected with known quantities of MCV or vaccinia virus. Briefly, MCV
isolated from human tissue specimen is quantitated by PCR and used to infect
human HEK293 cells, selected for ease of transfection. The cells are
subsequently transfected with a reporter plasmid encoding firefly luciferase
gene under the control of a synthetic early/late poxviral promoter and a control
plasmid encoding a renilla luciferase reporter under the control of a eukaryotic
promoter. After 16 h, cells are harvested and tested for expression of
luciferase. MCV genome units are quantitated by PCR targeting a genome area
conserved between MCV and vaccinia virus. Using a GFP reporter plasmid, this
method can be further used to infect a series of epithelial and fibroblast-type
cell lines of human and animal origin to microscopically visualize MCV-infected
cells, to assess late promoter activation, and, using these parameters, to
optimize MCV infectivity and gene expression in more complex eukaryotic cell
culture models. In recent years, our understanding of the role of natural killer (NK) cells in
the response to viral infection has grown rapidly. Not only do we realize
viruses have many immune-evasion strategies to escape NK cell responses, but
that stimulation of NK cell subsets during an antiviral response occurs through
receptors seemingly geared directly at viral products and that NK cells can
provide a memory response to viral pathogens. Tremendous knowledge has been
gained in this area through the study of herpes viruses, but appreciation for
the significance of NK cells in the response to other types of viral infections
is growing. The function of NK cells in defense against poxviruses has emerged
over several decades beginning with the early seminal studies showing the role
of NK cells and the NK gene complex in susceptibility of mouse strains to
ectromelia, a poxvirus pathogen of mice. More recently, greater understanding
has emerged of the molecular details of the response. Given that human diseases
caused by poxviruses can be as lethal as smallpox or as benign as Molluscum
contagiosum, and that vaccinia virus, the prototypic member of the pox family,
persists as a mainstay of vaccine design and has potential as an oncolytic virus
for tumor therapy, further research in this area remains important. This review
focuses on recent advances in understanding the role of NK cells in the immune
response to poxviruses, the receptors involved in activation of NK cells during
poxvirus infection, and the viral evasion strategies poxviruses employ to avoid
the NK response. Molluscum contagiosum is a common skin and mucosal disease of viral origin,
caused by molluscum contagiosum virus (MCV) virus of poxvirus family. With the
eradication of smallpox, MCV is now the only member of the poxvirus family that
causes substantial disease in humans. Though frequently reported, its unusual
clinical presentation makes its diagnosis a challenging task. We discuss a case
of molluscum contagiosum in a 30-year-old woman along with a review of
aetiology, histopathology and different possible treatment modalities. BACKGROUND AND AIMS: Molluscum contagiosum is caused by the molluscum
contagiosum virus (MCV) and is a very common skin disorder mainly involving
young children Cryotherapy, curettage or some topical therapies have been
applied for MC, but all of these treatments need several sessions, can be
somewhat ineffective, and very painful. The present study assessed the impact of
a single session of pulsed dye laser treatment of MC lesions which had proved
resistant to other approaches Subjects and methods: Fifteen children comprised
the study subjects, 11 boys and 4 girls, 3-5 years of age (mean 4.2 yr) with
recalcitrant MC. Lesions were counted at baseline, and a single shot from a 585
nm pulsed dye laser was applied to each lesion (3 mm, 300 ms, 8.0 J/cm(2)).
Lesions were counted again at 1 week post-treatment and followed for up to 3
months thereafter.
RESULTS: All patients completed the study and no patient dropped out through
pain or discomfort. Purpura was seen at each treated lesion immediately after
irradiation, but at 1 week after treatment, lesion clearance was virtually
complete which was maintained for 1 month, and no recurrence was seen at 3
months in 8 of the 15 patients who remained available for followup.
CONCLUSIONS: A single treatment of MC lesions with the pulsed dye laser
successfully cured even recalcitrant lesions with no recurrence on follow up,
and was well tolerated by the young subjects. INTRODUCTION: Molluscum contagiosum is a common superficial skin infection
caused by the poxvirus, Molluscum Contagiosum virus. The study objective is to
obtain a better understanding of physician practices and experiences with
molluscum contagiosum in order to focus informational and guidance material.
METHODS: A cross-sectional survey to assess medical practitioners' knowledge and
practices with molluscum contagiosum was conducted using the 2009 DocStyles
survey. Questions regarding category and number of molluscum contagiosum
patients seen, treatments used and advice given to patients were included in the
survey.
RESULTS: Dermatologists saw the most cases, with the majority seeing 51-100
molluscum contagiosum cases/year. The most common cases seen were children with
multiple lesions and adults with genital lesions. Respondents were most likely
to recommend treatment to immunocompromised individuals, HIV patients, adults
with genital lesions and children with multiple lesions. Cryotherapy was the top
choice for all specialties with the exception of OB/GYNs, whose top choice was
curettage. "Avoid intimate contact until lesions resolve", "Avoid touching
lesions to reduce further spread", and "Don't be concerned, this will go away"
were the top advice choices.
DISCUSSION: Most survey respondents have dealt with molluscum contagiosum in
their practice during the previous year. Overall, respondents picked appropriate
choices for treatment and advice given; however some ineffective or unnecessary
treatments were chosen and recommendations to prevent spread were chosen
infrequently. Knowledge gaps for appropriate transmission precaution advice
might cause unnecessary spread or autoinoculation. This survey has demonstrated
that molluscum contagiosum is a common infection seen by many types of
practitioners and therefore guidance on treatment considerations and infection
control is valuable. BACKGROUND: Molluscum contagiosum virus (MCV) causes an innocuous yet persistent
skin infection in immunocompetent individuals and is spread by contact with
lesions. Studies point to atopic dermatitis (AD) as a risk factor for MCV
infection; however, there are no longitudinal studies that have evaluated this
hypothesis.
METHODS: Outpatient visit data from fiscal years 2001-2009 for American Indian
and Alaska Native (AI/AN) children were examined to describe the incidence of
molluscum contagiosum (MC). We conducted a case-control study of patients <5
years old at an Indian Health Service (IHS) clinic to evaluate dermatological
risk factors for infection.
RESULTS: The incidence rate for MC in children <5 years old was highest in the
West and East regions. MC cases were more likely to have a prior or co-occurring
diagnosis of eczema, eczema or dermatitis, impetigo, and scabies (p<0.05)
compared to controls; 51.4% of MC cases had a prior or co-occurring diagnosis of
eczema or dermatitis.
CONCLUSIONS: The present study is the first demonstration of an association
between AD and MC using a case-control study design. It is unknown if the
concurrent high incidence of eczema and MC is related, and this association
deserves further investigation. BACKGROUND: Molluscum contagiosum (MC) is caused by a DNA virus of the poxvirus
group. It is common in children, and is also found in sexually active adults and
HIV-seropositive patients. Cellular immunity is essential to controlling MC
virus infection. We report the first observation of a patient with stage IV
Sezary syndrome, who presented multiple molluscum contagiosum, spread and
surrounded by a pale halo.
CASE REPORT: A woman aged 70 presented with aggravation of Sezary syndrome
diagnosed in 2009 and treated with topical corticosteroids. The examination
showed a generalized pruritic exanthem and multiple flesh-coloured papules from
1 to 3 mm, spread over the entire skin surface and surrounded by a white halo.
Histological examination of a lesion showed the presence of infected cells with
intracytoplasmic inclusions infected in an acanthotic epidermis, surrounded by a
melaninopenic hypomelanosis with a normal melanocyte density. There was no
inflammatory character. The diagnosis of multiple molluscum contagiosum was
given, the application of clobetasol propionate was suspended and treatment with
chlorambucil 4 mg/day and prednisone 0.5 mg/kg/day was started. The evolution of
the rash and pruritus was rapidly favourable. After 3 months, the rash and
pruritus had regressed. There was no molluscum contagiosum or clear halo.
CONCLUSION: We report the original observation of a patient with stage IV Sezary
syndrome, who presented multiple molluscum contagiosum, spread and surrounded by
a pale halo, without inflammation, eczema or disappearance of melanocytes. This
halo could be due to the secretion of a protein by molluscum contagiosum
inhibiting inflammation around this MC. To our knowledge, this phenomenon
reported in a patient with severe atopic dermatitis associated with Sezary
syndrome has not previously been described. Molluscum contagiosum (MC) is a very common benign self-limiting cutaneous viral
infection caused by molluscum contagiosum virus. Disease is self-limiting in
immunocompetent individuals, while it is severe and prolonged when associated
with Human Immunodeficiency Virus (HIV) infection. The widespread and refractory
mollusca of HIV disease occur especially on the face. In advanced stages of
immunosuppression, giant or verrucous forms of MC may occur. Molluscum
contagiosum tends to take a chronic course and is usually not responsive to
various treatments in immunocompromised patients. Here, we present a HIV
positive male patient with extensive papulonodular lesions over face, neck,
bilateral upper limbs since 2 months, diagnosed as giant molluscum contagiosum,
treated with cryotherapy with little improvement for few weeks after which
patient did not turn up. |
Which disease(s) are caused by HEX A deficiency? | Mutations in the HEX A gene, encoding the alpha-subunit of beta-hexosaminidase A (Hex A), are the cause of Tay-Sachs disease as well as of juvenile, chronic, and adult GM2 gangliosidoses. | Tay-Sachs disease is a genetically determined neurodegenerative disorder,
resulting from mutations of the hexosaminidase (Hex) A gene coding for the
alpha-subunit of beta-D-N-acetyl-hexosaminidase. Clinically, there is severe
encephalomyelopathy leading to death within the first few years of life. Hex A
activity is usually absent in tissue and body fluids of these patients. Juvenile
and adult Hex A deficiencies are less severe but rare variants with some
residual Hex A activity. All these variants are most prevalent among Ashkenazi
Jews. We describe a non-Jewish family in which four adult brothers and sisters
had markedly reduced Hex A activities and onset of symptoms in the second decade
of life. The phenotypical expression was remarkably homogeneous, consisting in a
combination of slowly progressive motor neuron disease, ataxia and ocular motor
disturbances. None of the patients were demented at this stage of their illness.
Magnetic resoce studies showed severe cerebellar atrophy, but were otherwise
normal. Hex A deficiency was established by biochemical measurements in the
serum and skin fibroblasts using the fluorogenic substrates 4-MUG and 4-MUGS as
well as by gel electrophoresis. Molecular genetic studies revealed that the
patients are compound heterozygotes for the 'adult' mutation Gly269 --> Ser and
the 'infantile' 4-base insertion in exon 11 of the Hex A gene. We have characterized the molecular basis of beta-hexosaminidase A (HEX A)
deficiency in a patient ascertained through an ophthalmologic examination that
revealed cherry red spots on his retina. The absence of neurological deficit in
this child until 3 3/4 years of age indicated residual HEX A must be present.
Three HEXA mutations, 10T > C (S4P) and 972T > A (V324V) on the maternal allele,
and 1A > T (M1L) on the paternal allele were identified. The effects of the
amino acid substitutions on HEX A expressed in COS-7 cells were analyzed; as
expected, no HEX A activity was associated with the M1L mutation but
surprisingly, the S4P mutation resulted in 59% of the HEX A activity expressed
by the wild type cDNA. The effect of the S4P change was much less than that of
another HEXA mutation, G269S, associated with an adult onset form of G(M2)
gangliosidosis. This indicated that the S4P change was not the cause of disease
and suggested that one of the mutations on the maternal allele, 10T > C or 972T
> A, had its effect at the mRNA level. This was confirmed by Northern blot
analysis that showed only 7% of the normal level of HEXA mRNA in proband
fibroblasts. Analysis of the residual mRNA by RT/PCR and sequencing revealed
normal transcripts from both the maternal and paternal allele, as well as a low
abundance aberrant transcript from the maternal allele. Sequencing of this
aberrant transcript revealed a new exon 8 donor site created by the 972T > A
mutation that resulted in a 17 bp deletion and destabilization of the resulting
abnormal transcript. The remaining normal mRNA produced from the 972T > A allele
must account for the delayed onset of clinical symptoms in this child. Tay-Sachs disease (TSD) is a recessively inherited neurodegenerative disorder
due to mutations in the HEXA gene resulting in a beta-hexosaminidase A (Hex A)
deficiency. The purpose of this study was to characterize the molecular
abnormalities in patients with infantile or later-onset forms of the disease.
The complete sequencing of the 14 exons and flanking regions of the HEXA gene
was performed with a unique technical condition in 10 unrelated TSD patients.
Eleven mutations were identified, including five splice mutations, one
insertion, two deletions and three single-base substitutions. Four mutations
were novel: two splice mutations (IVS8+5G>A, IVS2+4delAGTA), one missense
mutation in exon 6 (c.621T>G (p.D207E)) and one small deletion
(c.1211-1212delTG) in exon 11 resulting in a premature stop codon at residue
429. The c.621T>G missense mutation was found in a patient presenting an
infantile form. Its putative role in the pathogenesis of TSD is suspected as
residue 207 is highly conserved in human, mouse and rat. Moreover, structural
modelling predicted changes likely to affect substrate binding and catalytic
activity of the enzyme. The time-saving procedure reported here could be useful
for the characterization of Tay-Sachs-causing mutations, in particular in
non-Ashkenazi patients mainly exhibiting rare mutations. Tay-Sachs disease (TSD) is a recessively inherited neurodegenerative disorder
caused by mutations in the HEXA gene resulting in β-hexosaminidase A (HEX A)
deficiency and neuronal accumulation of GM2 ganglioside. We describe the first
patient with Tay-Sachs disease in the Cypriot population, a juvenile case which
presented with developmental regression at the age of five. The diagnosis was
confirmed by measurement of HEXA activity in plasma, peripheral leucocytes and
fibroblasts. Sequencing the HEXA gene resulted in the identification of two
previously described mutations: the nonsense mutation c.78G>A (p.Trp26X) and the
silent mutation c.1305C>T (p.=). The silent mutation was reported once before in
a juvenile TSD patient of West Indian origin with an unusually mild phenotype.
The presence of this mutation in another juvenile TSD patient provides further
evidence that it is a disease-causing mutation. Successful preimplantation
genetic diagnosis (PGD) and prenatal follow-up were provided to the couple. |
What is Behçet's disease | Behet's disease (BD) is a complex chronic relapsing inflammatory disorder of unknown etiology. | PURPOSE: Behçet disease is a systemic disease of young adults characterized by
venous occlusion in both the deep venous and retinal circulations. In severe
ocular disease, blindness may occur despite immunosuppressive treatment. The
most common inherited risk factor for the development of idiopathic venous
thrombosis is the presence of the Factor V (FV Leiden) mutation, which confers
resistance to activated protein C. The association of FV Leiden with Behçet
disease has been reported, but its influence on ocular disease is not known. We
therefore investigated the prevalence of this mutation in patients with Behçet
disease to determine its contribution to the presence and severity of ocular
disease.
METHODS: One hundred and six Middle Eastern patients satisfying international
criteria, and 120 healthy control subjects without a history of venous
thrombosis were included in the study, and patients underwent standard
examination by two ophthalmologists with an interest in Behçet disease. Genomic
DNA was extracted from peripheral blood leukocytes and screened for the FV
Leiden mutation with the polymerase chain reaction method with sequence-specific
primers (PCR-SSP).
RESULTS: FV Leiden was detected in 19% (23/120) of the control population
compared with 27% (29/106) of all patients with Behçet disease (P = .13).
However, among patients with Behçet disease who had ocular disease (75/106), the
prevalence of FV Leiden was significantly higher (32%) than it was in control
subjects (P = .04). Furthermore, ocular patients with Behçet disease in whom
retinal occlusive disease was observed (25/75) had the highest expression of FV
Leiden (44%).
CONCLUSIONS: These data suggest that FV Leiden may be an additional risk factor
for the development of ocular disease and, in particular, retinal
vaso-occlusion, and it may contribute to the poor visual outcome in these
patients. BACKGROUND: Behçet's disease is a multisystem disease featuring mucocutaneous,
ocular, articular, vascular, intestinal, urogenital, and neurologic involvement
and occurs with a high prevalence in the Mediterranean including Turkey. Higher
incidence of severe clinical course and systemic involvement is observed in male
patients.
OBJECTIVE: To determine the influence of sex on the clinical course of Behçet' s
disease.
METHODS: We retrospectively evaluated the clinical findings of 2313 Behçet
patients followed up at the multidisciplinary Behçet's Disease Center at Ankara
University.
RESULTS: The male/female patient ratio was 1.03. Oral aphthae was seen in all
patients. In male Behçet patients, the prevalence of mucocutaneous lesions and
systemic manifestations was as follows: 85.6% genital aphthae, 45.5% erythema
nodosum, 59.5% papulopustular lesions, 17.5% thrombophlebitis, 38.1% ocular
involvement, 11.3% articular involvement, 11.7% vascular involvement, 3.3%
neurologic involvement, 1.4% gastrointestinal involvement, and 1.8% pulmonary
involvement. In female Behçet patients, the prevalence of manifestations were as
follows: 91% genital aphthae, 49.8% erythema nodosum, 48.3% papulopustular
lesions, 3.5% thrombophlebitis, 19.8% ocular involvement, 11.8% articular
involvement, 2.1% vascular involvement, 1.3% neurologic involvement, 1.4%
gastrointestinal involvement, and 0.03% pulmonary involvement.
CONCLUSIONS: Only genital aphthae and erythema nodosum were more frequent in
females. On the other hand papulopustular eruptions, thrombophlebitis, ocular,
neurologic, pulmonary and vascular involvement were more frequent in males.
While female patients had the best prognosis, male patients had a worse overall
prognosis than females. BACKGROUND: Behçet's disease is a systemic immunoinflammatory disease of young
adults characterized by systemic vasculitis of arteries and veins. Although many
studies have been published since its discovery in 1937, the etiopathogenesis of
this unique disorder is still unclear.
OBJECTIVE: To assess the relationship between stress factors, psychological and
somatic symptoms, and coping mechanisms in patients with Behcet's disease.
METHOD: Thirty-four patients with Behcet's disease and 43 control subjects were
compared by using sociodemographic data collection forms, a psychosocial and
environmental problems list, the Beck Anxiety Inventory (BAI), Hamilton
Depression Rating Scale (HAM-D) and Toronto Alexithymia Scale (TAS).
RESULTS: Twenty-four patients (70.6%) defined stress factors in the first stage
of the disease. Twenty-seven (79.4%) out of 34 patients stated that the
recurrence period of the disease was related to the stress factors. Fear was
expressed by 10 (29.4%) patients, sadness by 11 (32.3%), and fear plus sadness
by 13 (38.2%) when they first learnt the diagnosis. While coping with these
emotions 14 (41.2%) revealed active-reliance strategy. A statistically
significant difference was present between the Behcet's patients and control
subjects regarding TAS (P < 0.05), HAM-D (P < 0.001) and BAI (P < 0.001) scores.
CONCLUSION: It seems that stressful life events have important implications in
both relapsing and remission periods of Behçet's disease via secondary problems. Behçet's disease is a systemic inflammatory disease presented with recurrent
oral aphtha, cutaneous manifestations, uveitis, and genital ulcer. The etiology
of Behçet's disease is still unknown, but both genetic background and
environmental factors are thought to be important in the pathogenesis of
Behçet's disease. Behçet's disease has long been regarded as a Th1 type
autoimmune disease, because of the association with HLA-B51 and hyperreactivity
against streptococcal antigen. However, it was recently found that Behçet's
disease and autoinflammatory diseases share several clinical features.
Furthermore, increased activity of neutrophils and elevated levels of
interleukin-1β are observed in both Behçet's disease and autoinflammatory
diseases. The relationship between Behçet's disease and autoinflammatory
diseases, especially Familial Mediterranean fever, is speculated, because both
diseases are prevalent in the Mediterranean basin and treated with colchicine.
Genetic researches on Behçet's disease and FMF suggest that the MEFV gene
mutated in Familial Mediterranean fever is a probable susceptibility gene for
Behçet's disease. Although many observations suggest that Behçet's disease might
be autoinflammatory, there is evidence implying autoimmune pathogenesis of
Behçet's disease. For example, some symptoms of Behçet's disease is treated with
T cell suppressing agents. Recent data suggest that a novel subset of T cells,
Th17, plays a crucial role in pathogenesis of Behçet's disease, and genome-wide
association researches verified it. IL-17, which is the secreted from of Th17
activates neutrophils. Hence, IL-17 might cause the symptoms resembling
autoinflammatory diseases. Recently, Anti-IL-1 treatment proved to be effective
and other susceptibility genes are being investigated. These new findings will
shed light on the long-sought pathogenesis of Behçet's disease. Behçet disease is a chronic relapsing inflammatory disease affecting many
different organs. Ocular involvement is quite common in the course of Behçet
disease and is frequently manifested by bilateral panuveitis and retinal
vasculitis. Medications such as corticosteroids and immunosuppressive agents are
used to reduce inflammation in patients with posterior or panuveitis. Chronic
immunosuppression is a risk factor for systemic infections. We report a case of
choroidal tuberculoma associated with tuberculosis in a patient with ocular
Behçet disease. A 25-year-old female with known ocular Behçet disease contracted
tuberculosis 3 months earlier. She had been receiving methotrexate and oral
steroids. Funduscopy of the left eye revealed a choroidal tuberculoma located
superonasally to the optic disc. Fluorescein angiography showed a central area
of hypofluorescence surrounded by a hyperfluorescent zone. Since she was already
receiving antituberculosis treatment combined with oral steroids, the same
treatment was continued. Diagnosis of the other diseases that may cause uveitis
in patients with Behçet disease should not be missed. This is especially
important since immunosuppressive drugs, that cause an increased incidence of
systemic infections, are the common treatment of choice for patients with Behçet
disease. Behçet syndrome is a chronic disease hallmarked by inflammation of the blood
vessels that is related to an autoimmune reaction caused by inherited
susceptibility due to specific genes and environmental factors, probably
components of infectious microorganisms, which turn on or get going the disease
in genetically susceptible subjects. The more common clinical expression of the
disease is represented by a triple-symptom complex of recurrent oral aphthous
ulcers, genital ulcers, and uveitis, sometimes associated with inflammatory
arthritis, phlebitis, iritis, as well as inflammation of the digestive tract,
brain, and spinal cord. The treatment strategies used to manage the
manifestations of Behçet syndrome have gradually progressed, and a number of new
therapeutic resources have been implemented in recent years, allowing better
control of pathogenic mechanisms, reducing symptoms and suffering, and
ameliorating patient's outcome. OBJECTIVE: Behcet's disease is a multisystem inflammatory disorder, and its
etiology has not been defined clearly yet. In this study, we aimed to
investigate the antistreptolysin O (ASO) levels of patients with Behcet's
disease.
MATERIALS AND METHODS: Thirty patients with Behcet's disease and 30 healthy
controls were enrolled in this study. We measured erythrocyte sedimentation rate
(ESR), serum C-reactive protein (CRP), and ASO levels in both groups.
RESULTS: There was no statistically significant difference between the two
groups with respect to demographic data (p>0.05). The ASO levels of the patients
and the controls were 288.4±145.7 and 170.6±142.4 ng/ml, respectively. In the
patients with Behcet's disease, ASO (p<0.01) and ESR (p<0.05) values were
significantly higher than in the healthy controls. There was no other
significant difference in serum CRP levels between the patients and the
controls. We could not find any correlation among ASO, CRP, and ESR values.
CONCLUSION: Our results suggest that serum ASO levels may increase in patients
with Behcet's disease. Further studies are needed in order to define the
relationship between ASO levels and inflammation status in Behcet's disease. Behçet disease (BD) is a rare relapsing, multisystem vasculitis characterised by
recurrent oral and genital ulcers, and uveitis. As an autoimmune small vessel
vasculitis, BD can involve other organs including the skin, joints, nervous
system, kidney and the gastrointestinal tract. This report describes a
40-year-old woman who presented with an uncommon feature of BD, namely myositis,
and who went on to develop myocarditis and polymicrobial necrotising fasciitis.
To the best of our knowledge, this is the first reported case of an
immunocompromised-associated infection occurring in BD without concurrent
immunosuppressive therapy. |
Does Yersinia pestis causes a respiratory infection? | Inhalation of Yersinia pestis results in primary pneumonic plague. | Plague is an infectious disease caused by the Yersinia pestis microorganism,
which is transmitted to the human host from a natural reservoir (different
rodent species) by a flea bite. Plague is still encountered in humans in the
areas of its enzootic prevalence in local rodent populations. Infection by flea
bite results in a bubonic or septicemic plague, possibly complicated by
secondary pneumonia. The person with pneumonic symptoms may be a source of a
droplet-borne inhalatory infection for other people who consequently develop
primary pneumonic plague. Despite a clinical form, plague is a severe infection
characterized by a short incubation period, rapid onset and quick progress with
mortality exceeding 50% if not treated properly. The pneumonic plague is
associated with a particularly rapid progress and the mortality rate of almost
100% if not treated properly. As Yersinia pestis can be easily obtained and
cultured and is highly pathogenic for humans, it poses a serious threat of being
used for bioterrorism purposes. Artificially created aerosol containing plague
bacilli can cause numerous and almost simultaneous cases of primary pulmonic
plague in an exposed population. Persons exposed would most likely develop
severe pneumonia with rapidly progressing respiratory and circulatory failure.
The use of the Yersinia pestis strains resistant to antibiotics typically
applied cannot be excluded. Pulmonary infection by Yersinia pestis causes pneumonic plague, a rapidly
progressing and often fatal disease. To aid the development of safe and
effective pneumonic plague vaccines, we are deciphering mechanisms used by the
immune system to protect against lethal pulmonary Y. pestis infection. In murine
pneumonic plague models, passive transfer of convalescent-phase sera confers
protection, as does active vaccination with live Y. pestis. Here, we demonstrate
that protection by either protocol relies upon both gamma interferon (IFN-gamma)
and tumor necrosis factor alpha (TNF-alpha) cytokines classically associated
with type 1 cellular immunity. In both protocols, abrogating IFN-gamma or
TNF-alpha activity significantly decreases survival and increases the bacterial
burden in pulmonary, splenic, and hepatic tissues. Neutralization of either
cytokine also counteracts challenge-induced, vaccination-dependent upregulation
of nitric oxide synthase 2 (NOS2). Moreover, genetic depletion of NOS2
suppresses protection conferred by serotherapy. We conclude that IFN-gamma,
TNF-alpha, and NOS2, key elements of cellular immunity, perform critical
protective functions during humoral defense against lethal pulmonary Y. pestis
challenge. These observations strongly suggest that plague vaccines should
strive to maximally prime both cellular and humoral immunity. Plague is an acute bacterial infection caused by Gram negative organism Yersinia
pestis. This bacteria is subdivided into three classical biotypes: Orientalis,
Medievalis and Antiqua. Plague is transmitted via flea vectors from rodents to
humans and by respiratory droplets from animals to humans or humans to humans.
This agent is on the top of the CDC list of "Critical Biological
Agents"--category A. It appears to be a good candidate agent for a bioterrorist
attack. Type III secretion (TTS) is a mechanism by which Y. pestis communicates
with eukaryotic cells by injecting bacterial proteins across cellular membranes
into the cytosol of these cells. These bacterial proteins take control of the
host cells by hijacking their intracellular machinery. A laboratory diagnostics
of plague is based on: staining techniques, culture on media,
immunochromatography, hemagglutination, immunofluorescence, ELISA, phage tests
and genetical techniques including: PCR, multiplex and nested PCR, real time
PCR, VNTR, PFGE, ISBF and Microarray. The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, a
rapidly fatal disease. At present, no plague vaccines are available for use in
the United States. One candidate for the development of a subunit vaccine is the
Y. pestis virulence (V) antigen, a protein that mediates the function of the
Yersinia outer protein virulence factors and suppresses inflammatory responses
in the host. On the basis of the knowledge that adenovirus (Ad) gene-transfer
vectors act as adjuvants in eliciting host immunity against the transgene they
carry, we tested the hypothesis that a single administration of a
replication-defective Ad gene-transfer vector encoding the Y. pestis V antigen
(AdsecV) could stimulate strong protective immune responses without a
requirement for repeat administration. AdsecV elicited specific T cell responses
and high IgG titers in serum within 2 weeks after a single intramuscular
immunization. Importantly, the mice were protected from a lethal intranasal
challenge of Y. pestis CO92 from 4 weeks up to 6 months after immunization with
a single intramuscular dose of AdsecV. These observations suggest that an Ad
gene-transfer vector expressing V antigen is a candidate for development of an
effective anti-plague vaccine. Pneumonic plague is a deadly respiratory disease caused by Yersinia pestis. The
bacterial protease Pla contributes to disease progression and manipulation of
host immunity, but the mechanisms by which this occurs are largely unknown. Here
we show that Pla degrades the apoptotic signaling molecule Fas ligand (FasL) to
prevent host cell apoptosis and inflammation. Wild-type Y. pestis, but not a Pla
mutant (Δpla), degrades FasL, which results in decreased downstream caspase-3/7
activation and reduced apoptosis. Similarly, lungs of mice challenged with
wild-type Y. pestis show reduced levels of FasL and activated caspase-3/7
compared to Δpla infection. Consistent with a role for FasL in regulating immune
responses, Δpla infection results in aberrant proinflammatory cytokine levels.
The loss of FasL or inhibition of caspase activity alters host inflammatory
responses and enables enhanced Y. pestis outgrowth in the lungs. Thus, by
degrading FasL, Y. pestis manipulates host cell death pathways to facilitate
infection. Inhalation of Yersinia pestis results in primary pneumonic plague, a highly
lethal and rapidly progressing necrotizing pneumonia. The disease begins with a
period of extensive bacterial replication in the absence of disease symptoms,
followed by the sudden onset of inflammatory responses that ultimately prove
fatal. Very little is known about the bacterial and host factors that contribute
to the rapid biphasic progression of pneumonic plague. In this work, we analyzed
the in vivo transcription kinetics of 288 bacterial open reading frames
previously shown by microarray analysis to be dynamically regulated in the lung.
Using this approach combined with bacterial genetics, we were able to identify
five Y. pestis genes that contribute to the development of pneumonic plague.
Deletion of one of these genes, ybtX, did not alter bacterial survival but
attenuated host inflammatory responses during late-stage disease. Deletion of
ybtX in another lethal respiratory pathogen, Klebsiella pneumoniae, also
resulted in diminished host inflammation during infection. Thus, our in vivo
transcriptional screen has identified an important inflammatory mediator that is
common to two Gram-negative bacterial pathogens that cause severe pneumonia.
IMPORTANCE: Yersinia pestis is responsible for at least three major pandemics,
most notably the Black Death of the Middle Ages. Due to its pandemic potential,
ease of dissemination by aerosolization, and a history of its weaponization,
Y. pestis is categorized by the Centers for Disease Control and Prevention as a
tier 1 select agent most likely to be used as a biological weapon. To date,
there is no licensed vaccine against Y. pestis. Importantly, an early "silent"
phase followed by the rapid onset of nondescript influenza-like symptoms makes
timely treatment of pneumonic plague difficult. A more detailed understanding of
the bacterial and host factors that contribute to pathogenesis is essential to
understanding the progression of pneumonic plague and developing or enhancing
treatment options. Pneumonic plague is a fatal disease caused by Yersinia pestis that is associated
with a delayed immune response in the lungs. Because neutrophils are the first
immune cells recruited to sites of infection, we investigated the mechanisms
responsible for their delayed homing to the lung. During the first 24 hr after
pulmonary infection with a fully virulent Y. pestis strain, no significant
changes were observed in the lungs in the levels of neutrophils infiltrate,
expression of adhesion molecules, or the expression of the major neutrophil
chemoattractants keratinocyte cell-derived chemokine (KC), macrophage
inflammatory protein 2 (MIP-2) and granulocyte colony stimulating factor
(G-CSF). In contrast, early induction of chemokines, rapid neutrophil
infiltration and a reduced bacterial burden were observed in the lungs of mice
infected with an avirulent Y. pestis strain. In vitro infection of lung-derived
cell-lines with a YopJ mutant revealed the involvement of YopJ in the inhibition
of chemoattractants expression. However, the recruitment of neutrophils to the
lungs of mice infected with the mutant was still delayed and associated with
rapid bacterial propagation and mortality. Interestingly, whereas KC, MIP-2 and
G-CSF mRNA levels in the lungs were up-regulated early after infection with the
mutant, their protein levels remained constant, suggesting that Y. pestis may
employ additional mechanisms to suppress early chemoattractants induction in the
lung. It therefore seems that prevention of the early influx of neutrophils to
the lungs is of major importance for Y. pestis virulence. Indeed, pulmonary
instillation of KC and MIP-2 to G-CSF-treated mice infected with Y. pestis led
to rapid homing of neutrophils to the lung followed by a reduction in bacterial
counts at 24 hr post-infection and improved survival rates. These observations
shed new light on the virulence mechanisms of Y. pestis during pneumonic plague,
and have implications for the development of novel therapies against this
pathogen. Yersinia pestis causes the fatal respiratory disease pneumonic plague. Y. pestis
recently evolved from the gastrointestinal pathogen Y. pseudotuberculosis;
however, it is not known at what point Y. pestis gained the ability to induce a
fulmit pneumonia. Here we show that the acquisition of a single gene encoding
the protease Pla was sufficient for the most ancestral, deeply rooted strains of
Y. pestis to cause pneumonic plague, indicating that Y. pestis was primed to
infect the lungs at a very early stage in its evolution. As Y. pestis further
evolved, modern strains acquired a single amino-acid modification within Pla
that optimizes protease activity. While this modification is unnecessary to
cause pneumonic plague, the substitution is instead needed to efficiently induce
the invasive infection associated with bubonic plague. These findings indicate
that Y. pestis was capable of causing pneumonic plague before it evolved to
optimally cause invasive infections in mammals. |
List 3 indications for Bupropion | Bupropion is used to treat Obesity, for smoking cessation and for depression | Sustained release bupropion (amfebutamone) is a non-nicotine agent that is
indicated as an aid to smoking cessation. In 2 large well designed clinical
trials, sustained release bupropion 300 mg/day (the recommended dose) for 7 or 9
weeks was associated with considerably and significantly higher smoking
abstinence rates (continuous abstinence and 7-day point prevalence rates) than
placebo during treatment and at follow-up at 6 and 12 months. Point prevalence
rates at 12 months in 2 studies were 23.1 and 30.3% with bupropion, whereas
values for placebo were 12.4 and 15.6%. Continuous abstinence rates at 12
months, available from 1 trial, were 18.4% with bupropion and 5.6% with placebo.
Furthermore, bupropion was associated with significantly higher quitting rates
than nicotine patch in a comparative study. Combination therapy with bupropion
and nicotine patch provided slightly higher abstinence rates than bupropion
alone, although differences were not statistically significant. The combination
was superior to nicotine patch alone. Data from a preliminary report of long
term bupropion treatment (52 weeks) showed that the drug was associated with
significantly higher continuous abstinence rates than placebo only to 6 months.
However, point prevalence abstinence rates were significantly higher with
bupropion than placebo to 18 months. Bupropion 300 mg/day recipients reported
nicotine withdrawal symptoms during treatment; however, the symptoms were
significantly less severe with bupropion than placebo. Patients receiving
bupropion 300 mg/day or bupropion in combination with nicotine patch for smoking
cessation generally gained less bodyweight than placebo recipients. The benefits
of bupropion for preventing weight gain persisted after the completion of long
term, but not short term therapy. Bupropion was well tolerated in clinical
trials, and the only adverse events that were significantly more common with
bupropion than placebo were insomnia and dry mouth. Data published so far
suggest that sustained release bupropion has a low potential for inducing
seizures (seizure rate approximately 0.1% in patients with depression).
CONCLUSIONS: Bupropion is an effective and well tolerated smoking cessation
intervention. Further studies with long term follow-up will be useful in
determining whether abstinence rates are maintained with bupropion. In addition,
clarification of its efficacy in comparison with other therapies used for
smoking cessation would help to establish its clinical value. The reduced
potential for weight gain with bupropion and the ability to use bupropion in
combination with nicotine replacement therapy make the drug a useful treatment
option for smoking cessation. The discovery that bupropion is an effective treatment for tobacco dependence
has triggered a rapid increase in development of potential new non-nicotine
pharmacotherapies, including bromocriptine, glucose, GTS-21, reboxetine,
rimonabant, selegeline and varenicline. Successful new products will need to
have excellent side-effect profiles in addition to proven efficacy. New faster
delivery nicotine replacement products have the promise of addressing a broader
list of indications, including treatment of nicotine withdrawal during temporary
abstinence and long-term nicotine maintece. Nicotine vaccines will need to
demonstrate efficacy and also improve certain consumer acceptability
characteristics (e.g., frequency of injections required) before they can become
widely used and successful therapies. The best hope of improved treatment comes
from combining existing and new pharmacotherapies with effective behavioural
therapy. Smoking is a risk factor for complications during and after surgery, but most
smokers are unable to quit before elective surgery. We tested the efficacy of
bupropion in improving smoking cessation rates in this setting by enrolling 47
patients from the elective surgery waiting list in a double-blind randomised
controlled trial. Patients receiving bupropion had a lower daily cigarette
consumption at the time of hospital admission, median (IQR) cigarettes per day:
6 (2-7) vs. 15 (9-20), p = 0.046. They also had a reduction in end-expired
carbon monoxide (p = 0.004), a known contamit of cigarette smoke, and
increased arterial oxygen saturation on pulse oximetry (p = 0.011). They were
more likely to have stopped smoking at the 3-week visit (p = 0.036), but not at
the 6-week visit (p = 0.25) or at the time of hospital admission for surgery (p
> 0.99). This study found that smokers waiting for elective surgery are more
likely to reduce or stop smoking when treated with bupropion. BACKGROUND: Bupropion has been available in the United States since 1989.
Initially a thrice-daily immediate-release formulation, a twice-daily
sustained-release formulation followed in 1996, and, in August 2003, a
once-daily extended-release formulation was introduced. On the 15th anniversary
of its introduction, we undertook a review of the background/history, mechanism
of action, formulations, and clinical profile of bupropion.
DATA SOURCES: Major efficacy trials and other reports were obtained and reviewed
from MEDLINE searches, review of abstracts from professional meetings, and the
bupropion SR manufacturer's databases. Searches of English-language articles
were conducted from June 2003 through August 2004. No time limit was specified
in the searches, which were conducted using the search terms bupropion,
bupropion SR, and bupropion XL.
DATA SYNTHESIS: Bupropion inhibits the re-uptake of norepinephrine and dopamine
neurotransmission without any significant direct effects on serotonin
neurotransmission. Bupropion is an effective antidepressant with efficacy
comparable to selective serotonin reuptake inhibitors and other antidepressants.
It is well tolerated in short-and longer-term treatment. Headache, dry mouth,
nausea, insomnia, constipation, and dizziness are the most common adverse
events. Seizure and allergic reactions are medically important adverse events
associated with bupropion and are reported rarely. Among all the newer
antidepressants in the United States, bupropion appears to have among the lowest
incidence of sexual dysfunction, weight gain, and somnolence. Although not U.S.
Food and Drug Administration approved for these indications, bupropion has also
been used as an adjunctive treatment to reverse antidepressant-induced sexual
dysfunction and to augment anti-depressant efficacy in partial responders and
non-responders to other agents.
CONCLUSION: Bupropion has played and will continue to play an important role as
a treatment for major depressive disorder in adults, as well as for other
related disorders. According to the US Public Health Service, all patients attempting to quit
smoking should be encouraged to use one or more effective pharmacotherapy agents
for cessation except in the presence of special circumstances. This article
provides an overview of the pharmacologic agents for acute and critical care
nurses to consider when intervening with tobacco-dependent patients. Medications
addressed in this article include (1) first-line agents (nicotine replacement
therapy, sustained-release bupropion) that have proven efficacy and are approved
by the Food and Drug Administration (FDA) for smoking cessation, (2) second-line
agents (nortriptyline, clonidine) that have proven efficacy but no FDA
indication for smoking cessation, (3) approaches that use of combination or
high-dose therapy, (4) herbal therapies, and (5) emerging therapies that are
currently under investigation. Over the past decade, bupropion has become a major pharmacotherapy for smoking
cessation in the Western world. Unlike other smoking cessation
pharmacotherapies, bupropion is a non-nicotine treatment. Compared with a
placebo control, bupropion approximately doubles smoking quit rates. Most
smoking cessation pharmacotherapies are thought to work, in part, by reducing
nicotine withdrawal and craving. This article reviews preclinical, human
laboratory and clinical trial studies of the effect of bupropion on nicotine
withdrawal and craving. Preclinical studies demonstrate that in rats undergoing
nicotine withdrawal, bupropion can dose-dependently lower changes in
brain-reward threshold and somatic signs of nicotine withdrawal. Human
laboratory studies have demonstrated that bupropion can alleviate some nicotine
withdrawal symptoms, including depressed mood, irritability, difficulty
concentrating and increased appetite. Moreover, bupropion has shown some
efficacy in alleviating craving to smoke. Clinical trials of bupropion have
offered mixed support of its ability to reduce nicotine withdrawal, weight gain
during treatment and craving. Strong mediational evidence of bupropion's action
through relief of withdrawal and craving in smoking cessation is growing.
Greater understanding of the psychological mechanisms of bupropion action will
likely be obtained through advances in the conceptualization and measurement of
withdrawal and craving. Improvements in the efficacy of bupropion may be
achieved through pharmacogenetic studies, with particular emphasis on its
metabolites. Ultimately, the efficacy of bupropion may be augmented by
combination with other agents that target withdrawal and craving through
complementary neurobiological processes. Bupropion hydrochloride ((+/-)-2-tert-butylamino)-3'-chloropropiophenone x HCl)
is a nonselective inhibitor of the dopamine transporter (DAT) and the
norepinephrine transporter (NET) and is also an antagonist at neuronal nicotinic
acetylcholine receptors (nAChRs). In animal models used commonly to screen for
antidepressant activity, bupropion shows a positive response. Also using animal
models, bupropion has been shown to attenuate nicotine-induced unconditioned
behaviors, to share or enhance discriminative stimulus properties of nicotine
and to have a complex effect on nicotine self-administration, i.e., low doses
augmenting nicotine self-administration and high doses attenuating
self-administration. Current studies show that bupropion facilitates the
acquisition of nicotine conditioned place preference in rats, further suggesting
that bupropion enhances the rewarding properties of nicotine. Bupropion has been
shown to attenuate the expression of nicotine withdrawal symptoms in both animal
models and human subjects. With respect to relapse, current studies show that
bupropion attenuates nicotine-induced reinstatement in rats, but large
individual differences are apparent. Clinically, bupropion is used as a
treatment for two indications, as an antidepressant, the indication for which it
was developed, and as a tobacco use cessation agent. In clinical trials,
bupropion is being tested as a candidate treatment for psychostimulant drug
abuse, attention-deficit hyperactivity disorder (ADHD) and obesity. Bupropion is
available in three bioequivalent oral formulations, immediate release (IR),
sustained release (SR), and extended release (XL). Extensive hepatic metabolism
of bupropion produces three pharmacologically active metabolites, which may
contribute to its clinical profile. PURPOSE: Hydroxylation of the antidepressant and smoking deterrent drug
bupropion is a clinically important bioactivation and elimination pathway.
Bupropion hydroxylation is catalyzed selectively by cytochrome P4502B6 (CYP2B6).
CYP2B6-catalyzed bupropion hydroxylation has been used as an in vitro and in
vivo phenotypic probe for CYP2B6 activity and CYP2B6 drug interactions.
Bupropion is chiral, used clinically as a racemate, and disposition is
stereoselective. Nevertheless, it is unknown whether CYP2B6-catalyzed bupropion
hydroxylation is stereoselective.
METHODS: Hydroxylation of racemic bupropion by recombit CYP2B6 and human
liver microsomes was evaluated using a stereoselective assay.
RESULTS: At therapeutic concentrations, hydroxylation of (S)-bupropion was
threefold and 1.5-greater than (R)-bupropion, respectively, by recombit
CYP2B6 and human liver microsomes. In vitro intrinsic clearances were likewise
different for bupropion etiomers.
CONCLUSIONS: Stereoselective bupropion hydroxylation may have implications for
the therapeutic efficacy of bupropion as an antidepressant or smoking cessation
therapy, and for the use of bupropion as an in vivo phenotypic probe for CYP2B6
activity. OBJECTIVE: To compare the usage of bupropion hydrochloride and nicotine
replacement in Australia between 2001 and 2005.
DESIGN AND SETTING: We analysed aggregate data on the utilisation of: (1)
bupropion under the Pharmaceutical Benefit Scheme (PBS) between 2001 and 2005;
(2) bupropion and nicotine replacement therapy (NRT) on the Repatriation
Pharmaceutical Benefit Scheme (RPBS) between 1995 and 2005; and (3) NRT
aggregate sales data from GlaxoSmithKline for 2001 - 05. The National Drug
Strategy Household Survey (NDSHS) 2004 was used to estimate the proportion of
smokers who received a bupropion prescription in each year.
MAIN OUTCOME MEASURES: Numbers of annual prescriptions for bupropion on the PBS
and buproprion and NRT on the RPBS; annual sales figures on NRT patches (2001 -
05); and the estimated proportion of Australian smokers who used bupropion in
2003.
RESULTS: The number of bupropion prescriptions on the PBS peaked at 351 772 in
2001 (costing the PBS $83 million). This declined by 72% to 97 173 in 2005 (a
cost of $12 million). The estimated percentage of smokers in Australia who used
bupropion in a year fell from 11% in 2001 to 3.6% in 2005. The annual number of
bupropion prescriptions on the RPBS fell from 3786 in 2001 to 1173 in 2005,
while there was no change in the number of NRT prescriptions (3793 in 2001 and
3886 in 2005). Sales data from the leading market supplier of NRT also indicated
that NRT use continued to grow in Australia while bupropion use declined.
Conclusions. Bupropion usage has fallen by 72% since a peak in the year of first
listing on the PBS, while the utilisation of NRTs appears to have increased,
despite the price differential in favour of bupropion.
IMPLICATIONS: Given the greater interest among smokers in NRT than bupropion
(and evidence of the effectiveness and cost-effectiveness of NRT), the
Australian government should reconsider its decision not to list NRT on the PBS. There are 3 first-line medications for smoking cessation i.e. nicotine
replacement therapy (NRT), varenicline (a partial nicotine receptor agonist) and
slow-release (SR) bupropion. All 3 agents approximately double 1-year quit rates
when used for 3 months, although varenicline seems to be a little more
efficacious than bupropion SR. An un-blinded study comparing varenicline with
nicotine patches are analysed in details and it is concluded that the validity
of that study is low regarding the relative efficacy of varenicline versus NRT.
Depression and suicidal attempts have been reported with varenicline use but it
is probably not induced by varenicline but by the quitting process per se. It is
recommended that the first agent to be used in smoking cessation should be NRT
as it is the best documented product with mild side effects. It might be optimal
to combine the patch with either gum, inhaler, sublingual tablets or nasal
spray. In subjects that have failed with NRT, varinicline should be the choice.
Bupropion SR is preferred to subjects with depression or smokers who have failed
with the previous two agents, due to the many contra-indications and side
effects of bupropion SR. With one of the 3 agents combined with follow-up visits
with counselling, one can expect a 1-year quit rate around 20-25%. Tobacco use is the leading cause of preventable death and disability in the
world. Although gradually declining in most developed countries, the prevalence
of tobacco use has increased among developing countries. Treatment for tobacco
use and dependence is effective, although long-term abstinence rates remain
disappointingly low. In response, new treatments continue to be developed. In
addition, many of the pharmacotherapies that have been available for years have
found new applications with the use of medication combinations, higher doses and
a longer duration of therapy for approved medications. There are now seven
medications (nicotine patch, nicotine gum, nicotine lozenge, nicotine inhaler,
nicotine nasal spray, bupropion sustained release and varenicline) approved for
tobacco dependence treatment in most countries, and many national and
professional society practice guidelines recommend their use. Although each of
the medications used for tobacco dependence treatment has been rigorously tested
for efficacy and safety, broader experience in clinical trials and in
observational population-based studies suggests that adverse events associated
with these medications are relatively common. Since 2008, two of the medications
(varenicline and bupropion) have come under increasing scrutiny because of
reports of unexplained serious adverse events (SAEs), including behaviour
change, depression, self-injurious thoughts and suicidal behaviour. To date,
this association has not been shown to be caused by these medications, but
concerns about medication safety continue. Prescribers require a working
knowledge of the common adverse effects for all of these medications as well as
a more detailed knowledge of the SAE potential. Nicotine replacement therapy
(NRT) has been rigorously tested in clinical trials for over 30 years. A number
of adverse effects are commonly associated with NRT use, although SAEs are rare.
The adverse effects associated with NRT are due to the pharmacological action of
nicotine as well as the mode and site of the NRT application. Bupropion has been
tested in over 40 controlled clinical trials and has been associated with higher
rates of treatment discontinuation due to adverse events than NRTs. A number of
SAEs are associated with bupropion and new warnings were recently added to
bupropion prescribing information because of observed neuropsychiatric symptoms
including suicidal thoughts and behaviours. Varenicline is the most recently
approved medication for tobacco dependence treatment and, although proven safe
in clinical testing, new safety concerns have arisen based on post-marketing
reports. Warnings have been added to the prescribing information for varenicline
because of neuropsychiatric symptoms also including suicidal thoughts and
behaviours. Informed decision making regarding the use of pharmacotherapy for
the treatment of tobacco dependence requires knowledge about the risks of drug
treatment that is weighed against the risks of continued tobacco use and the
benefits of treatment. Over half of all long-term smokers will die of a
tobacco-related disease and the risk of a serious or life-threatening adverse
event with tobacco cessation pharmacotherapy is vanishingly small.
Pharmacotherapy for tobacco dependence is also among the most cost-effective
preventive health interventions. Given these factors, the benefit : risk ratio
is strongly in favour of pharmacotherapy for tobacco dependence treatment in
virtually all smokers who are motivated to quit. OBJECTIVE: To assess a possible relationship between treatment with bupropion
(vs placebo) and expressed suicidal ideation and behavior.
DATA SOURCES: This analysis, based on the US Food and Drug Administration's
(FDA's) analysis of antidepressant suicidality data, included 8,953 adult
subjects receiving bupropion and 6,520 adult subjects receiving placebo from
randomized, placebo-controlled trials with bupropion conducted between 1976 and
2006 across multiple indications, including major depressive disorder (MDD). A
text string search of the adverse event database and case report form comments
was performed to identify potential suicidal events. FDA search criteria
included the following text strings: accident-, injur-, suic-, or overdos-,
including all events coded as accidental overdose, attempt, cut, gas, hang,
hung, jump, mutilat-, self damag-, self harm, self inflict-, shoot, slash,
poison, asphyxiation, suffocation, firearm, burn, drown, gun, immolat-, and
monoxide, and the following terms were added by GlaxoSmithKline to the search
criteria: accident, lacerat-, MVA, and hospital. The database search included
data beginning from the first dose of study medication through 1 day following
the last dose.
DATA EXTRACTION: Suicidal event narratives were classified using the Columbia
Classification Algorithm for Suicide Assessment. Additionally, changes on rating
scale items for depressed mood and suicidality were analyzed.
DATA SYNTHESIS: In the MDD population, the incidence of suicidal behavior or
ideation was 17/3,179 (0.53%) versus 11/2,310 (0.48%) for the bupropion and
placebo groups, respectively (OR = 1.28; 95% CI, 0.59-2.86). For suicidal
behavior, the incidence was 8/3,179 (0.25%) versus 2/2,310 (0.09%), respectively
(OR = 3.52; 95% CI, 0.81-24.48). No suicidal behavior event was noted in the
other indications, and no completed suicides were reported during treatment. No
significant worsening was observed for bupropion relative to placebo on the
rating scale items. No differential treatment effects were observed by gender or
age; regardless of treatment, however, the 18- to 24-year-old group had the
greatest odds of having a suicide event.
CONCLUSIONS: Although no statistically significant differences were observed
between bupropion and placebo in expressed suicidal ideation or behavior, we
believe that all patients treated with antidepressants should receive careful
monitoring for clinical worsening, suicidality, or unusual changes in behavior. BACKGROUND AND AIMS: Bupropion was introduced for smoking cessation following a
pivotal trial showing that it gave improved efficacy over the nicotine patch and
also suggesting combination treatment was beneficial. We tested in clinical
practice for an effectiveness difference between bupropion and nicotine
replacement therapy (NRT), whether the combination improves effectiveness and
whether either treatment might be more beneficial for certain subgroups of
smokers.
DESIGN: Open-label randomized controlled trial with 6-month follow-up.
SETTING: Four UK National Health Service (NHS) smoking cessation clinics.
PARTICIPANTS: Smokers (n = 1071) received seven weekly behavioural support
sessions and were randomized to an NRT product of their choice (n = 418),
bupropion (n = 409) or NRT plus bupropion (n = 244).
MEASURES: The primary outcome was self-reported cessation over 6 months, with
biochemical verification at 1 and 6 months. Also measured were baseline
demographics, health history, smoking characteristics and unwanted events during
treatment.
FINDINGS: Abstinence rates for bupropion (27.9%) and NRT (24.2%) were not
significantly different (odds ratio = 1.21, 95% confidence interval =
0.883-1.67), and the combination rate (24.2%) was similar to that for either
treatment alone. There was some evidence that the relative effectiveness of
bupropion and NRT differed according to depression (χ(2) = 2.86, P = 0.091),
with bupropion appearing more beneficial than NRT in those with a history of
depression (29.8 versus 18.5%). Several unwanted symptoms were more common with
bupropion.
CONCLUSION: There is no difference in smoking cessation effectiveness among
bupropion, nicotine replacement therapy and their combination when used with
behavioural support in clinical practice. There is some evidence that bupropion
is more beneficial than nicotine replacement therapy for smokers with a history
of depression. Obesity is a major correlate of cardiovascular disease. Weight loss improves
cardiovascular risk factors and has the potential to improve outcomes. Two
drugs, phentermine plus topiramate and lorcaserin, have recently been approved
by the US Food and Drug Administration for the indication of obesity; a third,
bupropion plus naltrexone, is under consideration for approval. In clinical
trials, these drugs cause weight loss and improve glucose tolerance, lipid
profile, and, with the exception of bupropion plus naltrexone, blood pressure.
However, their effect on cardiovascular outcomes is unknown. In defining
appropriate roles for these drugs in preventive cardiology, it is important to
remember the checkered history of drugs for obesity. New weight-loss drugs share
the serotonergic and sympathomimetic mechanisms that proved harmful in the cases
of Fen-Phen and sibutramine, respectively, albeit with significant differences.
Given these risks, randomized cardiovascular outcomes trials are needed to
establish the safety, and potential benefit, of these drugs. This review will
discuss the history of pharmacotherapy for obesity, existing efficacy and safety
data for the novel weight-loss drugs, and issues in the design of postapproval
clinical trials. For the past 30 years, research examining predictors of successful smoking
cessation treatment response has focused primarily on clinical variables, such
as levels of tobacco dependence, craving, and self-efficacy. However, recent
research has begun to determine biomarkers (such as genotype, nicotine and
metabolite levels, and brain imaging findings) that may have utility in
predicting smoking cessation. For genotype, genes associated with nicotinic
acetylcholine receptors (nAChRs) and related proteins have been found to predict
response to first-line medications (e.g. nicotine replacement therapy [NRT],
bupropion, or varenicline) or quitting over time without a controlled treatment
trial. For nicotine and metabolite levels, function of the cytochrome P450 2A6
liver enzyme, which can be assessed with the nicotine metabolite ratio or via
genotype, has been found to predict response, with slow nicotine metabolizers
having less severe nicotine dependence and a greater likelihood of quitting with
NRT than normal metabolizers. For brain imaging, decreased activation of brain
regions associated with emotion regulation and increased connectivity in emotion
regulation networks, increased responsiveness to pleasant cues, and altered
activation with the Stroop effect have been found in smokers who quit with the
first-line medications listed above or counseling. In addition, our group
recently demonstrated that lower pre-treatment brain nAChR density is associated
with a greater chance of quitting smoking with NRT or placebo. Several of these
studies found that specific biomarkers may provide additional information for
predicting response beyond subjective symptom or rating scale measures, thereby
giving an initial indication that biomarkers may, in the future, be useful for
guiding smoking cessation treatment intensity, duration, and type. Obesity is considered the most concerning and blatantly visible--yet most
neglected--public health problem by the WHO. The steadily increasing number of
overweight and obese people has reached 2.3 billion and 700 million worldwide,
respectively. Obesity is a complex condition, one that presents serious health
risks with respect to type 2 diabetes, ischemic heart disease, and hypertension,
therefore controlling the global obesity epidemic decreases not only health
problems, but also expenditure. The underlying cause of obesity is a metabolic
disorder of genetic, central nervous system or endocrine etiology that manifests
in increased nutritional intake and/or decreased physical activity ultimately
leading to excessive lipogenesis. The natural treatment of obesity, that is
often advised, is comprised of healthy lifestyle choices, namely low-calorie
diet and exercise. However, the pharmaceutic treatment of obesity is just as
important; having a better compliance rate, anti-obesity drugs also improve
quality of life and patient-care outcome concerning accompanying diseases. In
most countries only one drug is currently available against obesity: orlistat,
which is a specific and irreversible lipase inhibitor. One of the reasons for
the scarce number of anti-obesity drugs is the complex pathomechanism involved
in obesity. Interference with the intricate biochemical processes that govern
alimentation may lead to widespread adverse effects. The advances of the field
however, have prompted novel drug leads. In the past few years FDA has approved
new drugs for the treatment of obesity, recently liraglutide in 2014. The
approval of drug combinations, such as phentermine/topiramate and
bupropion/naltrexone are also noteworthy, the components of which have been
previously approved, but not necessarily for obesity as main indication.
Furthermore, there are many anti-obesity drug candidates currently in clinical
phase trials, with promisingly modest adverse effect profiles; hence the
expansion of the anti-obesity agents in the near future can be foreseen. The
present work summarizes the central and peripheral regulatory pathways of energy
consumption, nutrition, and appetite. The possible drug targets, the currently
available and novel anti-obesity agents, and the new trends in obesity research
are also discussed. Remission rates for Major Depressive Disorder (MDD) are low and unpredictable
for any given antidepressant. No biological or clinical marker has demonstrated
sufficient ability to match individuals to efficacious treatment. Biosignatures
developed from the systematic exploration of multiple biological markers, which
optimize treatment selection for individuals (moderators) and provide early
indication of ultimate treatment response (mediators) are needed. The rationale
and design of a multi-site, placebo-controlled randomized clinical trial of
sertraline examining moderators and mediators of treatment response is
described. The target sample is 300 participants with early onset (≤30 years)
recurrent MDD. Non-responders to an 8-week trial are switched double blind to
either bupropion (for sertraline non-responders) or sertraline (for placebo
non-responders) for an additional 8 weeks. Clinical moderators include anxious
depression, early trauma, gender, melancholic and atypical depression, anger
attacks, Axis II disorder, hypersomnia/fatigue, and chronicity of depression.
Biological moderator and mediators include cerebral cortical thickness,
task-based fMRI (reward and emotion conflict), resting connectivity, diffusion
tensor imaging (DTI), arterial spin labeling (ASL), electroencephalograpy (EEG),
cortical evoked potentials, and behavioral/cognitive tasks evaluated at baseline
and week 1, except DTI, assessed only at baseline. The study is designed to
standardize assessment of biomarkers across multiple sites as well as institute
replicable quality control methods, and to use advanced data analytic methods to
integrate these markers. A Differential Depression Treatment Response Index
(DTRI) will be developed. The data, including biological samples (DNA, RNA, and
plasma collected before and during treatment), will become available in a public
scientific repository.
CLINICAL TRIAL REGISTRATION: Establishing Moderators and Biosignatures of
Antidepressant Response for Clinical Care for Depression (EMBARC). Identifier:
NCT01407094. URL: http://clinicaltrials.gov/show/NCT01407094. |
What is the function of BAX | BAX is a central death regulator that controls apoptosis in normal and cancer cells | The E1B 19K protein is a potent apoptosis inhibitor and the putative adenovirus
Bcl-2 homolog. To investigate the mechanism of apoptosis regulation,
19K-interacting cellular proteins were identified using the yeast two-hybrid
system, and Bax was one of seven 19-K interacting clones. Residues 50-78 of Bax
containing a conserved region designated Bcl-2 homology region 3 (BH3) were
sufficient for specific binding to both the E1B 19K and Bcl-2 proteins. The
Bax-E1B 19K interaction was detectable in vitro and in lysates from mammalian
cells, and Bax expression antagonized E1B 19K protein function. bax mRNA and
protein levels were p53-inducible with kinetics identical to that of
p21/Waf-1/Cip-1, and E1B 19K and Bcl-2 expression did not affect Bax or
p21/Waf-1/Cip-1 accumulation. In cells where p53 was mutant, Bax expression
induced apoptosis, suggesting that Bax was sufficient for apoptosis, and acted
downstream of p53. p53 may simultaneously activate the transcription of genes
required for both growth arrest (p21/Waf-1/Cip-1) and death (bax), and E1B 19K
and Bcl-2 may act distally and function through interaction with and antagonism
of Bax to prevent apoptosis. With the death pathway disabled, induction of
growth arrest by p53 can then be manifested. Adenovirus E1B 19-kDa protein (19K) is a member of the Bcl-2 family of
suppressors of apoptosis. The suppressors function through heterodimerization
with the death promoters, Bax and related proteins, thus establishing a set
point within the cell that determines whether or not apoptosis is executed in
response to a death signal. Sequence similarities between 19K and Bcl-2 are
largely restricted to short Bcl-2 homology (BH) domains that mediate interaction
with Bax. The BH1 sequence in 19K is degenerate but nevertheless contains a
conserved glycine residue found in all family members that when mutated to
alanine in Bcl-2 results in loss of Bcl-2 function and ability to dimerize with
Bax (Yin, X.-M., Oltvai, Z. N., and Korsmeyer, S. J. (1994) Nature 369,
321-323). Here, we show that the analogous mutation in BH1 of 19K also abrogates
the anti-apoptotic properties of 19K and its ability to interact with Bax, thus
establishing the critical importance of this residue within BH1 and the likely
similarity of Bcl-2 and 19K function. In distinct contrast to Bcl-2, however,
19K interaction was not detected with Bad, a Bcl-2/Bcl-XL dimerizing protein
that can potentially regulate a Bax middle dotBcl-2/Bcl-XL survival set point
and reinstate susceptibility to a death signal. Furthermore, the anti-apoptotic
function of 19K was not overcome by enforced expression of Bad in transfected
cells. This feature of 19K may provide adenovirus with a selective advantage in
evading premature induction of apoptosis by the host cell. Bax is a proapoptotic member of the Bcl-2 family of proteins which localizes to
and uses mitochondria as its major site of action. Bax normally resides in the
cytoplasm and translocates to mitochondria in response to apoptotic stimuli, and
it promotes apoptosis in two ways: (i) by disrupting mitochondrial membrane
barrier function by formation of ion-permeable pores in mitochondrial membranes
and (ii) by binding to antiapoptotic Bcl-2 family proteins via its BH3 domain
and inhibiting their functions. A hairpin pair of amphipathic alpha-helices
(alpha5-alpha6) in Bax has been predicted to participate in membrane insertion
and pore formation by Bax. We mutagenized several charged residues in the
alpha5-alpha6 domain of Bax, changing them to alanine. These substitution
mutants of Bax constitutively localized to mitochondria and displayed a
gain-of-function phenotype when expressed in mammalian cells. Furthermore,
substitution of 8 out of 10 charged residues in the alpha5-alpha6 domain of Bax
resulted in a loss of cytotoxicity in yeast but a gain-of-function phenotype in
mammalian cells. The enhanced function of this Bax mutant was correlated with
increased binding to Bcl-X(L), through a BH3-independent mechanism. These
observations reveal new functions for the alpha5-alpha6 hairpin loop of Bax: (i)
regulation of mitochondrial targeting and (ii) modulation of binding to
antiapoptotic Bcl-2 proteins. A popular model of BCL-2 and BAX involvement in apoptosis suggests that upon
apoptosis induction cytosolic BAX translocates to the mitochondria, where it
displays the pro-apoptotic function, which involves its homodimerization. BCL-2
exerts anti-apoptotic function by forming heterodimers with BAX, thus
neutralizing the pro-apoptotic activity of the latter. We have shown that
irradiation of the human myeloma cell line RPMI-8226 induced apoptosis as
determined by DNA degradation, cytochrome c release into cytoplasm and BCL-2
caspase-mediated cleavage. BCL-2 protein was present only in the membrane
fraction, whereas BAX was found both in cytosol and membranes isolated from
non-irradiated cells. Radiation induced moderate redistribution of BAX from
cytosol to membranes with a concomitant increase in BCL-2/BAX heterodimer
formation. Rapid and transient BCL-2 phosphorylation in membrane fractions of
irradiated cells did not affect BCL-2/BAX heterodimerization. We failed to
detect any BAX/BAX homodimers in apoptotic cells. Our findings show that in
irradiated RPMI-8226 cells the formation of BCL-2/BAX heterodimers correlates
with apoptosis. We conclude that BCL-2/BAX heterodimers are negative regulators
of death protection, and our data agree with those who propose that BCL-2 does
not require BAX to exert its survival function. Adenovirus infection and expression of E1A induces both proliferation and
apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B
19K. The mechanism of apoptosis induction and the role that it plays in
productive infection are not known. Unlike apoptosis mediated by death
receptors, infection with proapoptotic E1B 19K mutant viruses did not induce
cleavage of Bid but nonetheless induced changes in Bak and Bax conformation,
Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In
wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B
19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax
conformation. Infection with E1B 19K mutant viruses induced apoptosis in
wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those
deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically
increased E1A expression and virus replication. Thus, Bax- and Bak-mediated
apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak
function as an antiviral response at the cellular level. The murine proapoptotic protein Bax was expressed in Kluyveromyces lactis to
investigate its effect on cell survival and production of reactive oxygen
species (ROS). Bax expression decreased the number of cells capable of growing
and forming colonies, and it increased the number of cells producing ROS, as
detected by both dihydrorhodamine 123 fluorescence and the intracellular content
of SH groups. Mutation in the beta-subunit of F(1)-ATPase, or mitochondrial
deficiency resulting from deletion of mtDNA (rho(0) mutant), increased the
sensitivity to Bax, indicating that Bax cytotoxicity does not require
mitochondrial respiratory-chain functions. The antiapoptotic protein Bcl-x(L),
when co-expressed with Bax, localized to the mitochondria and prevented Bax
cytotoxicity. However, this co-expression did not prevent the production of ROS.
These data suggest that in K. lactis cells expressing Bax, ROS are not the sine
qua non of cell death and that the antiapoptotic function of Bcl-x(L) is not
limited to its antioxidant property. Bax (Bcl2-associated X protein) is an apoptosis-inducing protein that
participates in cell death during normal development and in various diseases.
Bax resides in an inactive state in the cytosol of many cells. In response to
death stimuli, Bax protein undergoes conformational changes that expose
membrane-targeting domains, resulting in its translocation to mitochondrial
membranes, where Bax inserts and causes release of cytochrome c and other
apoptogenic proteins. It is unknown what controls conversion of Bax from the
inactive to active conformation. Here we show that Bax interacts with humanin
(HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes.
HN prevents the translocation of Bax from cytosol to mitochondria. Conversely,
reducing HN expression by small interfering RNAs sensitizes cells to Bax and
increases Bax translocation to membranes. HN peptides also block Bax association
with isolated mitochondria, and suppress cytochrome c release in vitro. Notably,
the mitochondrial genome contains an identical open reading frame, and the
mitochondrial version of HN can also bind and suppress Bax. We speculate
therefore that HN arose from mitochondria and transferred to the nuclear genome,
providing a mechanism for protecting these organelles from Bax. Nicotine-induced cell survival is associated with chemoresistance of human lung
cancer cells, but our understanding of the intracellular mechanism(s) is
fragmentary. Bax is a major proapoptotic member of the Bcl2 family and a
molecule required for apoptotic cell death. Growth factor (i.e.
granulocyte-macrophage colony-stimulating factor)-induced phosphorylation of Bax
has been reported to negatively regulate its proapoptotic function. Because Bax
is ubiquitously expressed in both small cell lung cancer and non-small cell lung
cancer cells, nicotine may mimic growth factor(s) to regulate the activity of
Bax. We found that nicotine potently induces Bax phosphorylation at Ser-184,
which results in abrogation of the proapoptotic activity of Bax and increased
cell survival. AKT, a known physiological Bax kinase, is activated by nicotine,
co-localizes with Bax in the cytoplasm, and can directly phosphorylate Bax in
vitro. Treatment of cells with the phosphatidylinositol 3-kinase (PI3K)
inhibitor LY294002 or specific depletion of AKT expression by RNA interference
can block both nicotine-induced Bax phosphorylation and cell survival.
Importantly, nicotine-induced Bax phosphorylation potently blocks stress-induced
translocation of Bax from cytosol to mitochondria, impairs Bax insertion into
mitochondrial membranes, and reduces the half-life of Bax protein (i.e. from
9-12 h to <6 h). Because knockdown of Bax expression by gene silencing results
in prolonged cell survival following treatment with cisplatin in the absence or
presence of nicotine, Bax may be an essential component in the nicotine survival
signaling pathway. Thus, nicotine-induced survival and chemoresistance of human
lung cancer cells may occur in a novel mechanism involving activation of
PI3K/AKT that directly phosphorylates and inactivates the proapoptotic function
of Bax. Bax is a major proapoptotic member of the Bcl2 family that is required for
apoptotic cell death. We have recently discovered that Bax phosphorylation at
serine 184 induced by nicotine through activation of protein kinase AKT
abolishes its proapoptotic function in human lung cancer cells. Here we found
that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor
okadaic acid or specific disruption of PP2A activity by expression of SV40 small
tumor antigen enhanced Bax phosphorylation, whereas C(2)-ceramide, a potent PP2A
activator, reduced nicotine-induced Bax phosphorylation, suggesting that PP2A
may function as a physiological Bax phosphatase. PP2A co-localized and
interacted with Bax. Purified, active PP2A directly dephosphorylated Bax in
vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppressed
nicotine-stimulated Bax phosphorylation in association with increased apoptotic
cell death. By contrast, depletion of PP2A/C by RNA interference enhanced Bax
phosphorylation and prolonged cell survival. Mechanistically
C(2)-ceramide-induced Bax dephosphorylation caused a conformational change by
exposure of the 6A7 epitope (amino acids 13-19) that is normally hidden at its N
terminus that promoted the insertion of Bax into mitochondrial membranes and
formation of Bax oligomers leading to cytochrome c release and apoptosis. In
addition, PP2A directly disrupted the Bcl2/Bax association to liberate Bax from
the heterodimer complex. Thus, PP2A may function as a physiological Bax
regulatory phosphatase that not only dephosphorylates Bax but also activates its
proapoptotic function. Vaccinia virus, the prototypic member of the orthopoxvirus genus, encodes the
mitochondrial-localized protein F1L that functions to protect cells from
apoptotic death and inhibits cytochrome c release. We previously showed that F1L
interacts with the pro-apoptotic Bcl-2 family member Bak and inhibits activation
of Bak following an apoptotic stimulus (Wasilenko, S. T., Banadyga, L., Bond,
D., and Barry, M. (2005) J. Virol. 79, 14031-14043). In addition to Bak, the
pro-apoptotic protein Bax is also capable of initiating cytochrome c release
suggesting that vaccinia virus infection could also inhibit Bax activity. Here
we show that F1L inhibits the activity of the pro-apoptotic protein Bax by
inhibiting oligomerization and N-terminal activation of Bax. F1L expression also
inhibited the subcellular redistribution of Bax to the mitochondria and the
insertion of Bax into the outer mitochondrial membrane. The ability of F1L to
inhibit Bax activation does not require Bak, because F1L expression inhibited
cytochrome c release and Bax activation in Bak-deficient cells. No interaction
between Bax and F1L was detected during infection, suggesting that F1L functions
upstream of Bax activation. Notably, F1L was capable of interacting with the
BH3-only protein BimL as shown by co-immunoprecipitation, and F1L expression
inhibited apoptosis induced by BimL. These studies suggest that, in addition to
interacting with the pro-apoptotic protein Bak, F1L also functions to indirectly
inhibit the activation of Bax, likely by interfering with the pro-apoptotic
activity of BH3-only proteins such as BimL. Prion protein (PrP) inhibits the activation of proapoptotic Bax in primary human
neurons and MCF-7 cells. Because neuronal apoptosis occurs in human prion
diseases, here we examine the anti-Bax function of familial PrP mutants. All
Creutzfeldt-Jakob disease and fatal familial insomnia-associated prion protein
mutations partially or completely lose the anti-Bax function in human neurons
and, except for A117V and V203I, in MCF-7 cells. The ability of the mutants to
protect against Bax-mediated cell death is divided into three groups: (1) group
I, retention of anti-Bax function in both the Val129 and Met129 mutants; (2)
group II, retention of anti-Bax function only in Val129 mutants; and (3) group
III, reduction or no anti-Bax function in Val129 and Met129 mutants. The loss of
anti-Bax function in these PrP mutants correlates completely with a significant
decrease in the production of cytosolic PrP, a form of PrP shown previously to
have anti-Bax function in human neurons. Cotransfection of the full-length PrP
mutants with wild-type or mutant cytosolic PrP, but not with wild type
full-length PrP, rescues the anti-Bax function of PrP. The results show that the
failure of PrP mutants to produce cytosolic PrP is responsible for the loss of
anti-Bax function and that the effect of the PrP mutants is domit over
wild-type PrP. Furthermore, these results imply that misfolded PrP that escapes
retrotranslocation could accumulate at the cell surface and cause neuronal
dysfunction. BACKGROUND: Bcl-2 family proteins are key regulators of mitochondrial integrity
and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member
localizes as monomers in the cytosol of healthy cells and accumulates as
oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain
regulates interactions within the family, but regions other than BH3 are also
critical for Bax function. Thus, the N-terminus has been variously implicated in
targeting to mitochondria, interactions with BH3-only proteins as well as
conformational changes linked to Bax activation. The transmembrane (TM) domains
(alpha5-alpha6 helices in the core and alpha9 helix in the C-terminus) in Bax
are implicated in localization to mitochondria and triggering cytotoxicity. Here
we have investigated N-terminus modulation of TM function in the context of
regulation by the anti-apoptotic protein Bcl-xL.
RESULTS: Deletion of 29 amino acids in the Bax N-terminus (Bax 30-192) caused
constitutive accumulation at mitochondria and triggered high levels of
cytotoxicity, not inhibited by Bcl-xL. Removal of the TM domains (Bax 30-105)
abrogated mitochondrial localization but resulted in Bcl-xL regulated activation
of endogenous Bax and Bax-Bak dependent apoptosis. Inclusion of the
alpha5-alpha6 helices/TMI domain (Bax 30-146) phenocopied Bax 30-192 as it
restored mitochondrial localization, Bcl-xL independent cytotoxicity and was not
dependent on endogenous Bax-Bak. Inhibition of function and localization by
Bcl-xL was restored in Bax 1-146, which included the TM1 domain. Regardless of
regulation by Bcl-xL, all N-terminal deleted constructs immunoprecipitated
Bcl-xLand converged on caspase-9 dependent apoptosis consistent with
mitochondrial involvement in the apoptotic cascade. Sub-optimal sequence
alignments of Bax and Bcl-xL indicated a sequence similarity between the
alpha5-alpha6 helices of Bax and Bcl-xL. Alanine substitutions of three residues
(T14A-S15A-S16A) in the N-terminus (Bax-Ala3) attenuated regulation by the
serine-threonine kinase Akt/PKB but not by Bcl-xL indicative of distinct
regulatory mechanisms.
CONCLUSION: Collectively, the analysis of Bax deletion constructs indicates that
the N-terminus drives conformational changes facilitating inhibition of
cytotoxicity by Bcl-xL. We speculate that the TM1 helices may serve as
'structural antagonists' for BH3-Bcl-xL interactions, with this function being
regulated by the N-terminus in the intact protein. Although Bcl-XL and Bax are structurally similar, activated Bax forms large
oligomers that permeabilize the outer mitochondrial membrane, thereby committing
cells to apoptosis, whereas Bcl-XL inhibits this process. Two different models
of Bcl-XL function have been proposed. In one, Bcl-XL binds to an activator,
thereby preventing Bax activation. In the other, Bcl-XL binds directly to
activated Bax. It has been difficult to sort out which interaction is important
in cells, as all three proteins are present simultaneously. We examined the
mechanism of Bax activation by tBid and its inhibition by Bcl-XL using
full-length recombit proteins and measuring permeabilization of liposomes and
mitochondria in vitro. Our results demonstrate that Bcl-XL and Bax are
functionally similar. Neither protein bound to membranes alone. However, the
addition of tBid recruited molar excesses of either protein to membranes,
indicating that tBid activates both pro- and antiapoptotic members of the Bcl-2
family. Bcl-XL competes with Bax for the activation of soluble, monomeric Bax
through interaction with membranes, tBid, or t-Bid-activated Bax, thereby
inhibiting Bax binding to membranes, oligomerization, and membrane
permeabilization. Experiments in which individual interactions were abolished by
mutagenesis indicate that both Bcl-XL-tBid and Bcl-XL-Bax binding contribute to
the antiapoptotic function of Bcl-XL. By out-competing Bax for the interactions
leading to membrane permeabilization, Bcl-XL ties up both tBid and Bax in
nonproductive interactions and inhibits Bax binding to membranes. We propose
that because Bcl-XL does not oligomerize it functions like a domit-negative
Bax in the membrane permeabilization process. Bax is a pro-apoptotic member of the Bcl-2 family proteins involved in the
release of apoptogenic factors from mitochondria to the cytosol. Recently, it
has been shown both in mammals and yeast that Bax insertion in the mitochondrial
outer membrane involves at least two distinct mechanisms, one of which uses the
TOM complex. Here, we show that in Drosophila, heterozygous loss of function
mutations of Tom22 or Tom70, two receptors of the TOM complex, attenuates
bax-induced phenotypes in vivo. These results argue that the TOM complex may be
used as a mitochondrial Bax receptor in Drosophila. Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob
disease (CJD)-associated prion protein (PrP) mutants that are unable to generate
cytosolic PrP (CyPrP). To determine if the anti-Bax function of PrP modulates
the manifestation of prion diseases, we further investigated the anti-Bax
function of eight familial Gerstmann-Sträussler-Scheinker Syndrome
(GSS)-associated PrP mutants. These PrP mutants contained their respective
methionine ((M)) or valine ((V)) at codon 129. All of the mutants lost their
ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in
primary human neurons. In the breast carcinoma MCF-7 cells, the F198S(V),
D202N(V), P102L(V) and Q217R(V) retained, whereas the P102L(M), P105L(V),
Y145stop(M) and Q212P(M) PrP mutants lost their ability to inhibit Bax-mediated
condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended
on the ability of the mutants to generate cytosolic PrP. However, except for the
P102L(V), none of the mutants significantly inhibited Bax-mediated caspase
activation. These results show that the cytosolic PrP generated from the GSS
mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell
death. Furthermore, these results indicate that the anti-Bax function is also
disrupted in GSS-associated PrP mutants and is not associated with the
difference between CJD and GSS. OBJECTIVE: The Bcl-2 associated X protein (Bax), belonging to the Bcl-2 family,
plays a pivotal role in mitochondria-dependent apoptosis. The aims of this study
are to revalidate the functional significance of Bax G(-248)A polymorphism, and
investigate its association with lung cancer risk in Chinese population.
METHODS: The biological function of Bax G(-248)A was tested by luciferase
assays, and its effects on lung cancer risk was determined by case-control
analysis of 989 patients with lung cancer and 990 controls.
RESULTS: Bax -248A allele exhibited significantly higher transcriptional
activity compared with G allele. The Bax-248A allele carriers yielded a
significantly decreased risk of lung cancer, compared with -248G allele carriers
(OR = 0.68, 95% CI = 0.48 approximately 0.90, P = 0.01). Furthermore, a
significant gene-smoking interaction between Bax G(-248)A polymorphism and
smoking existed among the light smokers.
CONCLUSION: Bax G(-248)A polymorphism is associated with lung cancer
susceptibility in Chinese population. Members of the Bcl-2 family play key roles as proapoptotic (e.g., Bax) and
antiapoptotic (e.g., Bcl-x(L)) regulators of programmed cell death. We
previously identified the mitochondrial potassium channel Kv1.3 as a novel
target of Bax. Incubating Kv1.3-positive isolated mitochondria with Bax
triggered apoptotic events, whereas Kv1.3-deficient mitochondria were resistant
to this stimulus. Mutation of Bax at lysine 128 (BaxK128E) abrogated its effects
on Kv1.3 and the induction of apoptotic changes in mitochondria. These data
indicate a toxin-like action of Bax on Kv1.3 to trigger at least some of the
mitochondrial changes typical for apoptosis. To gain insight into the mechanism
of Bax-Kv1.3 interaction, we mutated Glu158 of Bcl-x(L) (corresponding to K128
in Bax) to lysine. This substitution turned Bcl-x(L) proapoptotic. Transfection
of double knockout (Bax(-/-)/Bak(-/-)) mouse embryonic fibroblasts (DKO MEFs)
with either wild-type Bax, BaxK128E, or Bcl-x(L)E158K showed that apoptosis
induced by various stimuli was defective in DKO MEFs and BaxK128E-transfected
cells, but was recovered upon transfection with Bcl-xLE158K or wild-type Bax.
Both wild-type Bax and BaxK128E can form similar ion-conducting pores upon
incorporation into planar lipid bilayers. Our results point to a physiologically
relevant interaction of Bax with Kv1.3 and further indicate a crucial role of a
distinct lysine in determining the proapoptotic character of Bcl2-family
proteins. BACKGROUND: One of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to
permeabilize the mitochondrial outer membrane during apoptosis. While Bax is
mostly cytosolic and translocates to mitochondria following an apoptotic
stimulus, Bak is constitutively integrated within the outer membrane. Membrane
anchorage occurs via a C-terminal transmembrane domain that has been studied in
Bax but not in Bak, therefore what governs their distinct subcellular
distribution is uncertain. In addition, whether the distinct subcellular
distributions of Bak and Bax contributes to their differential regulation during
apoptosis remains unclear.
METHODOLOGY/PRINCIPAL FINDINGS: To gain insight into Bak and Bax targeting to
mitochondria, elements of the Bak C-terminus were mutated, or swapped with those
of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of
three basic residues within the C-segment destabilized Bak. Replacing the Bak
C-segment with that from Bax rescued stability and function, but unexpectedly
resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol,
both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in
their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS
translocated to the mitochondrial outer membrane, inserted its transmembrane
domain, oligomerized, and released cytochrome c. Despite this Bax-like
subcellular distribution, Bak/BaxCS retained Bak-like regulation following
targeting of Mcl-1.
CONCLUSIONS/SIGNIFICANCE: Residues in the C-segment of Bak and of Bax contribute
to their distinct subcellular localizations. That a semi-cytosolic form of Bak,
Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates
that Bak and Bax share a conserved mode of activation. In addition, the
differential regulation of Bak and Bax by Mcl-1 is predomitly independent of
the initial subcellular localizations of Bak and Bax. Bax and Bak (Bax/Bak) are essential pro-apoptotic proteins of the Bcl-2 family
that trigger mitochondrial outer membrane permeabilization (MOMP) in a
Bcl-2/Bcl-xL-inhibitable manner. We recently discovered a new stress-related
function for Bax/Bak-regulation of nuclear protein redistribution (NPR) from the
nucleus to cytoplasm. This effect was independent of Bax/Bak N-terminus exposure
and not inhibited by Bcl-xL over-expression. Here, we studied the molecular
mechanism governing this novel non-canonical response. Wild-type (WT) and mutant
versions of Bax were re-expressed in Bax/Bak double-knockout mouse embryonic
fibroblasts and their ability to promote NPR, apoptotic events, and changes in
lamin A mobility was examined. Our results show that, in this system, Bax
expression was sufficient to restore NPR such as in WT cells undergoing
apoptosis. This activity of Bax was uncoupled from cytochrome c release from the
mitochondria (indicative of MOMP) and required its membrane localization, α
helices 5/6, and the Bcl-2 homology 3 (BH3) domain. Moreover, enrichment of Bax
in the nuclear envelope by the so-called Klarsicht/ANC-1/Syne-1 homology domain
effectively triggered NPR as in WT Bax, but without inducing MOMP or cell death.
Bax-induced NPR was associated with impairment in lamin A mobility, implying a
connection between these two nuclear envelope-associated events. Overall, the
results indicate a new MOMP-independent, stress-induced Bax function on the
nuclear envelope. The multi-BCL-2 homology domain pro-apoptotic BCL-2 family members BAK and BAX
have critical roles in apoptosis. They are essential for mitochondrial
outer-membrane permeabilization, leading to the release of apoptogenic factors
such as cytochrome-c, which promote activation of the caspase cascade and
cellular demolition. The BOK protein has extensive amino-acid sequence
similarity to BAK and BAX and is expressed in diverse cell types, particularly
those of the female reproductive tissues. The BOK-deficient mice have no readily
discernible abnormalities, and its function therefore remains unresolved. We
hypothesized that BOK may exert functions that overlap with those of BAK and/or
BAX and examined this by generating Bok(-/-)Bak(-/-) and Bok(-/-)Bax(-/-) mice.
Combined loss of BOK and BAK did not elicit any noticeable defects, although it
remains possible that BOK and BAK have critical roles in developmental cell
death that overlap with those of BAX. In most tissues examined, loss of BOK did
not exacerbate the abnormalities caused by loss of BAX, such as defects in
spermatogenesis or the increase in neuronal populations in the brain and retina.
Notably, however, old Bok(-/-)Bax(-/-) females had abnormally increased numbers
of oocytes from different stages of development, indicating that BOK may have a
pro-apoptotic function overlapping with that of BAX in age-related follicular
atresia. The function Bax and/or Bak in constituting a gateway for mitochondrial
apoptosis in response to apoptotic stimuli has been unequivocally demonstrated.
However, recent work has suggested that Bax/Bak may have unrecognized
nonapoptotic functions related to mitochondrial function in nonstressful
environments. Wild-type (WT) and Bax/Bak double knockout (DKO) mice were used to
determine alternative roles for Bax and Bak in mitochondrial morphology and
protein import in skeletal muscle. The absence of Bax and/or Bak altered
mitochondrial dynamics by regulating protein components of the organelle fission
and fusion machinery. Moreover, DKO mice exhibited defective mitochondrial
protein import, both into the matrix and outer membrane compartments, which was
consistent with our observations of impaired membrane potential and attenuated
expression of protein import machinery (PIM) components in intermyofibrillar
mitochondria. Furthermore, the cytosolic chaperones heat-shock protein 90
(Hsp90) and binding immunoglobulin protein (BiP) were markedly increased with
the deletion of Bax/Bak, indicating that the cytosolic environment related to
protein folding may be changed in DKO mice. Interestingly, endurance training
fully restored the deficiency of protein import in DKO mice, likely via the
upregulation of PIM components and through improved cytosolic chaperone protein
expression. Thus our results emphasize novel roles for Bax and/or Bak in
mitochondrial function and provide evidence, for the first time, of a curative
function of exercise training in ameliorating a condition of defective
mitochondrial protein import. Author information:
(1)Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai,
One Gustave L. Levy Place, Box 1130, New York, NY 10029, USA; Department of
Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place,
Box 1130, New York, NY 10029, USA; The Tisch Cancer Institute, Icahn School of
Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1130, New York, NY
10029, USA; The Metabolism Institute, Icahn School of Medicine at Mount Sinai,
One Gustave L. Levy Place, Box 1130, New York, NY 10029, USA.
(2)Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai,
One Gustave L. Levy Place, Box 1130, New York, NY 10029, USA; Department of
Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place,
Box 1130, New York, NY 10029, USA; The Tisch Cancer Institute, Icahn School of
Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1130, New York, NY
10029, USA; The Metabolism Institute, Icahn School of Medicine at Mount Sinai,
One Gustave L. Levy Place, Box 1130, New York, NY 10029, USA; The Graduate
School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, One
Gustave L. Levy Place, Box 1130, New York, NY 10029, USA. Electronic address:
[email protected]. AIMS: Disturbance of the balance between mitochondrial fission and fusion has
been implicated in cerebral ischemia and several neurodegenerative diseases,
whereas the underlying mechanisms remain poorly understood. In the present
study, we attempted to investigate the role of dynamin-related protein 1 (Drp1),
a key mitochondrial fission protein, in the pathogenesis of cerebral ischemia.
METHODS: Using Drp1 siRNA or Mdivi-1, a small molecule inhibitor of Drp1, we
examined the effect of Drp1 knockdown or inhibition on oxygen-glucose
deprivation (OGD)-induced mitochondrial dysfunction and death of SH-SY-5Y cells.
Cell death and viability were evaluated with LDH and MTT assays, respectively,
and mitochondrial morphology, mitochondrial membrane potential (Δψm), and ATP
production were assessed using epifluorescence microscopy, flow cytometry, and
HPLC, respectively. Moreover, to examine the effect of Drp1 inhibition on
ischemic brain injury, middle cerebral artery occlusion (MCAO) mice were
injected (i.p.) with Mdivi1, and blood-brain barrier permeability, brain water
content, and cell apoptosis were assessed.
RESULTS: Knockdown or inhibition of Drp1 by Mdivi-1 significantly attenuated
OGD-induced cell death in SH-SY-5Y cells, associated with reduced morphological
change of mitochondria and attenuated Bax insertion,oligomerization. Moreover,
treatment of the MCAO mice with Mdivi-1 remarkably reduced the infarct volume
and neurological deficits in a dose-dependent manner, associated with marked
reduction of mitochondrial fragmentation and BAX expression.
CONCLUSIONS: Down-regulation or inhibition of Drp1 may reduce cerebral ischemic
damage through maintaining normal mitochondrial morphology and function, and
decreasing Bax insertion and oligomerization in mitochondria. BAX protein plays a key role in the mitochondria-mediated apoptosis. However, it
remains unclear by what mechanism BAX is triggered to initiate apoptosis. Here,
we reveal the mechanism using electron spin resoce (ESR) techniques. An
inactive BAX monomer was found to exhibit conformational heterogeneity and exist
at equilibrium in two conformations, one of which has never been reported. We
show that upon apoptotic stimulus by BH3-only peptides, BAX can be induced to
convert into either a ligand-bound monomer or an oligomer through a
conformational selection mechanism. The kinetics of reaction is studied by means
of time-resolved ESR, allowing a direct in situ observation for the
transformation of BAX from the native to the bound states. In vitro
mitochondrial assays provide further discrimination between the proposed BAX
states, thereby revealing a population-shift allosteric mechanism in the
process. BAX's apoptotic function is shown to critically depend on excursions
between different structural conformations. The aim of this study was to determine the anti-tumour activity of tanshinone
IIA in SKOV3 cells. Results suggested that tanshinone IIA could significantly
inhibit (IC50 value = 19.6 μM) the proliferation and induce apoptosis of SKOV3
cells as demonstrated by flow cytometry analysis. In addition, tanshinone IIA
treatment induced G2/M phase cell cycle arrest in SKOV3 cells. The results of
Western blotting indicated that tanshinone IIA can suppress the expression of
anti-apoptotic protein Bcl-2, increase (0.28 vs. 0.62) the expression of
pro-apoptotic protein Bax (0.83 vs. 0.24) in SKOV3 cells. It can be concluded
that the tanshinone IIA may be a possible therapeutic candidate having cytotoxic
and anti-tumour potential. Bax, a central cell death regulator, is an indispensable gateway to
mitochondrial dysfunction and a major proapoptotic member of the B-cell lymphoma
2 (Bcl-2) family proteins that control apoptosis in normal and cancer cells.
Dysfunction of apoptosis renders the cancer cell resistant to treatment as well
as promotes tumorigenesis. Bax activation induces mitochondrial membrane
permeabilization, thereby leading to the release of apoptotic factor cytochrome
c and consequently cancer cell death. A number of drugs in clinical use are
known to indirectly activate Bax. Intriguingly, recent efforts demonstrate that
Bax can serve as a promising direct target for small-molecule drug discovery.
Several direct Bax activators have been identified to hold promise for cancer
therapy with the advantages of specificity and the potential of overcoming
chemo- and radioresistance. Further investigation of this new class of drug
candidates will be needed to advance them into the clinic as a novel means to
treat cancer. Publisher: ANTECEDENTES/OBJETIVO: El objetivo de este estudio fue determinar si
se producía un incremento de la expresión de Bax (proapoptótico) y una
disminución de la expresión de Blc-2A1 (antiapoptótico) en el intestino de los
recién nacidos con enterocolitis necrosante.
MATERIALES Y MÉTODOS: Comparamos a ocho pacientes recién nacidos de manera
consecutiva sometidos a resección intestinal debido a enterocolitis necrosante
con ocho recién nacidos sometidos a resección intestinal debido a atresia ileal.
La evaluación histopatológica de la lesión tisular y la apoptosis se realizó
mediante microscopía óptica y el método TUNEL. El nivel de ARNm en los genes
apoptóticos (CASP3, CASP6, CASP7, Bax, BIRC2) y antiapoptóticos se evaluó con el
método de matriz de RCP (PCR array). La expresión de proteínas se evaluó
mediante inmunohistoquímica.
RESULTADOS: Los puntajes de las lesiones tisulares y los puntajes medios de
apoptosis fueron significativamente más altos en el grupo con enterocolitis
necrosante en comparación con el grupo de referencia (p < 0,01). La expresión de
los genes proapoptóticos aumentó significativamente en el grupo con
enterocolitis necrosante frente al grupo de referencia (p < 0,01). La expresión
del gen Bcl-2A1 (antiapoptótico) disminuyó significativamente en el grupo con
enterocolitis necrosante (p < 0,01). La expresión de las proteínas Bax y CASP3
aumentó significativamente en el grupo con enterocolitis necrosante (p < 0,01).
CONCLUSIONES: Según nuestros datos, la alteración del equilibrio entre la
expresión de Bax (proapoptótico) y la expresión de Bcl-2A1 (antiapoptótico) en
el lugar de la lesión es un posible mecanismo de la patogenia en recién nacidos
que presentan enterocolitis necrosante. |
What is the link between Ctf4 and Chl1 in cohesion establishment? | Ctf4 links DNA replication with sister chromatid cohesion establishment by recruiting the Chl1 helicase to the replisome. The Eco1 acetyltransferase, helped by factors including Ctf4 and Chl1, concomitantly acetylates the chromosomal cohesin complex to stabilize its cohesive links. | Cohesion between sister chromatids mediated by a multisubunit complex called
cohesin is established during DNA replication and is essential for the orderly
segregation of chromatids during anaphase. In budding yeast, a specialized
replication factor C called RF-C(Ctf18/Dcc1/Ctf8) and the
DNA-polymerase-alpha-associated protein Ctf4 are required to maintain
sister-chromatid cohesion in cells arrested for long periods in mitosis. We show
here that CTF8, CTF4 and a helicase encoded by CHL1 are required for efficient
sister chromatid cohesion in unperturbed mitotic cells, and provide evidence
that Chl1 functions during S-phase. We also show that, in contrast to mitosis,
RF-C(Ctf18/Dcc1/Cft8), Ctf4 and Chl1 are essential for chromosome segregation
during meiosis and for the viability of meiotic products. Our finding that cells
deleted for CTF8, CTF4 or CHL1 undergo massive meiosis II non-disjunction
suggests that the second meiotic division is particularly sensitive to cohesion
defects. Using a functional as well as a cytological assay, we demonstrate that
CTF8, CHL1 and CTF4 are essential for cohesion between sister centromeres during
meiosis but dispensable for cohesin's association with centromeric DNA. Our
finding that mutants in fission yeast ctf18 and dcc1 have similar defects
suggests that the involvement of the alternative RF-C(Ctf18/Dcc1/Ctf8) complex
in sister chromatid cohesion might be highly conserved. Deletion mutants of CHL1 or CTF4, which are required for sister chromatid
cohesion, showed higher sensitivity to the DNA damaging agents methyl
methanesulfonate (MMS), hydroxyurea (HU), phleomycin, and camptothecin, similar
to the phenotype of mutants of RAD52, which is essential for recombination
repair. The levels of Chl1 and Ctf4 associated with chromatin increased
considerably after exposure of the cells to MMS and phleomycin. Although the
activation of DNA damage checkpoint did not affected in chl1 and ctf4 mutants,
the repair of damaged chromosome was inefficient, suggesting that Chl1 and Ctf4
act in DNA repair. In addition, MMS-induced sister chromatid recombination in
haploid cells, and, more importantly, MMS-induced recombination between
homologous chromosomes in diploid cells were impaired in these mutants. Our
results suggest that Chl1 and Ctf4 are directly involved in homologous
recombination repair rather than acting indirectly via the establishment of
sister chromatid cohesion. Cohesion between sister chromatids, mediated by the chromosomal cohesin complex,
is a prerequisite for their alignment on the spindle apparatus and segregation
in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then,
during DNA replication in S-phase, the replication fork-associated
acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin's DNA
binding resistant to destabilization by the Wapl protein. Whether stabilization
of cohesin molecules that happen to link sister chromatids is sufficient to
build sister chromatid cohesion, or whether additional reactions are required to
establish these links, is not known. In addition to Eco1, several other factors
contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1
and Mrc1, but little is known about their roles. Here, we show that each of
these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and
Chl1, but not of the other factors, causes a synthetic growth defect in cells
lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in
ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously
thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment
processing, or of Okazaki fragment processing in sister chromatid cohesion.
Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that
might hold important clues as to the mechanism of sister chromatid cohesion
establishment. DNA replication during S phase is accompanied by establishment of sister
chromatid cohesion to ensure faithful chromosome segregation. The Eco1
acetyltransferase, helped by factors including Ctf4 and Chl1, concomitantly
acetylates the chromosomal cohesin complex to stabilize its cohesive links. Here
we show that Ctf4 recruits the Chl1 helicase to the replisome via a conserved
interaction motif that Chl1 shares with GINS and polymerase α. We visualize
recruitment by EM analysis of a reconstituted Chl1-Ctf4-GINS assembly. The Chl1
helicase facilitates replication fork progression under conditions of nucleotide
depletion, partly independently of Ctf4 interaction. Conversely, Ctf4
interaction, but not helicase activity, is required for Chl1's role in sister
chromatid cohesion. A physical interaction between Chl1 and the cohesin complex
during S phase suggests that Chl1 contacts cohesin to facilitate its
acetylation. Our results reveal how Ctf4 forms a replisomal interaction hub that
coordinates replication fork progression and sister chromatid cohesion
establishment. |
How is primary intestinal lymphangiectasia (PIL) caused? | Primary intestinal lymphangiectasia (PIL) is a rare disorder characterized by diffuse or localized dilation and eventual rupture of the enteric lymphatic vessels in mucosa, submucosa, and/or subserosa. Lymph, rich in all kinds of proteins and lymphocytes, leaks into the gastrointestinal tract via the affected lymphatic vessels causing hypoproteinemia and lymphopenia. | Primary intestinal lymphangiectasia (PIL), first described in 1961, is a rare
disease of childhood. Oedema, hypoproteinaemia and diarrhoea are characteristic
symptoms. Bioptic demonstration of dilated lymphatic capillary vessels in
intestinal villi and increased intestinal protein loss are diagnostic. Two
patients successfully treated with a low fat diet, containing medium chain
triglycerides (MCT) are reported. Primary intestinal lymphangiectasia (PIL), so-called Waldmann's disease, is an
uncommon condition, characterized by dilated intestinal submucosal and
subserosal lymphatics of the gastrointestinal tract. Protein-losing enteropathy
is the most common manifestation of this supposed congenital disease. Since the
initial description in 1961, 11 cases of lymphoma have been reported suggesting
that PIL predisposes to lymphoma. Here, we report the first case of primary
nodal location lymphoma during PIL with recovery of the protein-losing
enteropathy after its treatment by radiochemotherapy. Primary intestinal lymphangiectasia (PIL) is a rare disorder characterized by
dilated intestinal lacteals resulting in lymph leakage into the small bowel
lumen and responsible for protein-losing enteropathy leading to lymphopenia,
hypoalbuminemia and hypogammaglobulinemia. PIL is generally diagnosed before 3
years of age but may be diagnosed in older patients. Prevalence is unknown. The
main symptom is predomitly bilateral lower limb edema. Edema may be moderate
to severe with anasarca and includes pleural effusion, pericarditis or chylous
ascites. Fatigue, abdominal pain, weight loss, inability to gain weight,
moderate diarrhea or fat-soluble vitamin deficiencies due to malabsorption may
also be present. In some patients, limb lymphedema is associated with PIL and is
difficult to distinguish lymphedema from edema. Exsudative enteropathy is
confirmed by the elevated 24-h stool alpha1-antitrypsin clearance. Etiology
remains unknown. Very rare familial cases of PIL have been reported. Diagnosis
is confirmed by endoscopic observation of intestinal lymphangiectasia with the
corresponding histology of intestinal biopsy specimens. Videocapsule endoscopy
may be useful when endoscopic findings are not contributive. Differential
diagnosis includes constrictive pericarditis, intestinal lymphoma, Whipple's
disease, Crohn's disease, intestinal tuberculosis, sarcoidosis or systemic
sclerosis. Several B-cell lymphomas confined to the gastrointestinal tract
(stomach, jejunum, midgut, ileum) or with extra-intestinal localizations were
reported in PIL patients. A low-fat diet associated with medium-chain
triglyceride supplementation is the cornerstone of PIL medical management. The
absence of fat in the diet prevents chyle engorgement of the intestinal
lymphatic vessels thereby preventing their rupture with its ensuing lymph loss.
Medium-chain triglycerides are absorbed directly into the portal venous
circulation and avoid lacteal overloading. Other inconsistently effective
treatments have been proposed for PIL patients, such as antiplasmin, octreotide
or corticosteroids. Surgical small-bowel resection is useful in the rare cases
with segmental and localized intestinal lymphangiectasia. The need for dietary
control appears to be permanent, because clinical and biochemical findings
reappear after low-fat diet withdrawal. PIL outcome may be severe even
life-threatening when maligt complications or serous effusion(s) occur. Primary intestinal lymphangiectasia is a rare cause of protein-losing
enteropathy and usually presents with intermittent diarrhea or malnutrition.
Diagnosis depends largely on its pathologic condition demonstrating greatly
dilated lymphatics mainly in the lamina propria of the mucosa. We report a case
of primary intestinal lymphangiectasia, of the diffuse type, presenting with
abdominal pain and voluminous diarrhea in a previously healthy 8-year-old boy.
He had periumbilical pain for 3 months before presentation. He was managed by
segmental bowel resections and end-to-end anastomoses. The histopathologic
condition of the resected small intestine showed lymphatic dilation limited
mainly to the subserosa and mesentery but was not prominent in the mucosa.
Abdominal pain and diarrhea subsided postoperatively. The present case is the
fourth report describing a response to operative resection. Primary intestinal lymphangiectasia (PIL) is a rare disorder characterized by
dilated intestinal lymphatics and the development of protein-losing enteropathy.
Patients with PIL develop hypoalbuminemia, hypocalcemia, lymphopenia and
hypogammaglobulinemia, and present with bilateral lower limb edema, fatigue,
abdominal pain and diarrhea. Endoscopy reveals diffusely elongated,
circumferential and polypoid mucosae covered with whitish enlarged villi, all of
which indicate intestinal lymphangiectasia. Diagnosis is confirmed by
characteristic tissue pathology, which includes dilated intestinal lymphatics
with diffusely swollen mucosa and enlarged villi. The prevalence of PIL has
increased since the introduction of capsule endoscopy. The etiology and
prevalence of PIL remain unknown. Some studies have reported that several genes
and regulatory molecules for lymphangiogenesis are related to PIL. We report the
case of a patient with PIL involving the entire small bowel that was confirmed
by capsule endoscopy and double-balloon enteroscopy-guided tissue pathology who
carried a deletion on chromosome 4q25. The relationship between this deletion on
chromosome 4 and PIL remains to be investigated. Primary intestinal lymphangiectasia (PIL) is a protein-losing, exsudative
gastroenteropathy causing lymphatic obstruction. Diagnosis depends on clinical
examination and histological findings. Conservative treatment modalities include
a low-fat diet and enteral nutritional therapy in order to reduce enteric
protein loss and to improve fat metabolism. Other treatment options consist of
administration of antiplasmin or octreotide to lower lymph flow and secretion.
We report on a 58-year-old patient who underwent exploratory laparotomy due to a
worsening physical status, recurrent chylaskos and leg oedema under conservative
dietary therapy. Intraoperative findings showed a typical PIL of the jejunum
about 20 cm distal to the Treitz's ligament. Histological examinations confirmed
this diagnosis. One year after segmental small bowel resection (105 cm) with
end-to-end anastomosis the patient is healthy, free of symptoms, has gained
weight and his serum protein level has increased. Intraabdominal ascites and leg
oedema have not reoccurred since. Primary intestinal lymphangiectasia (PIL) or Waldmann's disease is a rare
protein-losing gastroenteropathy of unknown etiology. Less than 200 cases have
been reported globally. Patients may be asymptomatic or present edema,
lymphedema, diarrhea, ascites and other manifestations. We report two pediatric
cases with PIL with extremely different outcome in a 3-year follow-up period.
The first patient presented with persistent diarrhea, hypoalbuminemia and
failure to thrive, while the second patient presented with an abrupt eyelid
edema. Hypoproteinemia was the common laboratory finding for the two patients
and upper gastrointestinal endoscopy established the diagnosis. The first
patient relapsed five times during the follow-up period after the diagnosis had
been made and required intravenous albumin administration and micronutrient
supplementation. The second patient revealed normal gastrointestinal endoscopy 4
months after the diagnosis had been established; he followed an unrestricted
diet and remained asymptomatic throughout the follow-up period. PIL can be
either severe, affecting the entire small bowel, leading to lifetime disease, or
sometimes affects part of the small bowel, leading to transient disorder. Primary intestinal lymphangiectasia (PIL) is a protein-losing enteropathy
characterized by tortuous and dilated lymph channels of the small bowel. The
main symptoms are bilateral lower limb edema, serosal effusions, and vitamin D
malabsorption resulting in osteoporosis. We report here a case of long-lasting
misdiagnosed PIL with a peculiar liver picture, characterized by a very high
stiffness value at transient elastography, which decreased with clinical
improvement. The complex interplay between lymphatic and hepatic circulatory
system is discussed. Primary intestinal lymphangiectasia (PIL) is a rare disorder, especially in
adults. It causes a local disruption of chylus transport and is part of the
exudative gastroenteropathies. Conservative therapy includes dietary measures or
somatostatin medication. Taking the differential diagnosis of PIL into
consideration is a major challenge, since patients suffering from PIL may
present with diarrhoea and lymphedema or chylous ascites. This can be explained
by the chronic lymphedema of the bowel leading to dilation of the vessels
(intraluminal loss) and sometimes even to a rupture (peritoneal loss). Push-pull
enteroscopy and capsule endoscopy are the proper interventional diagnostic tools
to discover PIL. Exploratory laparoscopy may be useful in unclear cases.
Surgical resection of the altered intestine has been described with positive
results. Exploratory laparoscopy may even be a diagnostic tool in unclear cases.
Resection of the altered intestine is a treatment option in symptomatic and
treatment-refractory cases. Primary intestinal lymphangiectasia (PIL) is a rare protein-losing enteropathy
with lymphatic leakage into the small intestine. Dilated lymphatics in the small
intestinal wall and mesentery are observed in this disease. Laboratory tests of
PIL patients revealed hypoalbuminemia, lymphocytopenia, hypogammaglobulinemia
and increased stool α-1 antitrypsin clearance. Cell-mediated immunodeficiency is
also present in PIL patients because of loss of lymphocytes. As a result, the
patients are vulnerable to chronic viral infection and lymphoma. However, cases
of PIL with chronic viral infection, such as human papilloma virus-induced
warts, are rarely reported. We report a rare case of PIL with generalized warts
in a 36-year-old male patient. PIL was diagnosed by capsule endoscopy and
colonoscopic biopsy with histological tissue confirmation. Generalized warts
were observed on the head, chest, abdomen, back, anus, and upper and lower
extremities, including the hands and feet of the patient. BACKGROUND: Primary intestinal lymphangiectasia (Waldmann's disease) is a rare
disease characterized by dilated lymphatics in the small bowel leading to an
exudative enteropathy with lymphopenia, hypoalbuminemia and
hypogammaglobulinemia.
CASE PRESENTATION: We report the case of a 23 year-old male who presented with
chronic anemia and in whom primary intestinal lymphangiectasia was diagnosed. A
low-fat diet along with nutritional therapy with medium-chain triglyceride
supplementation improved the protein-losing enteropathy, but did not solve the
anemia. Octreotide was also unsuccessful, and after attempting angiographic
embolization therapy, limited small bowel resection together with antiplasmin
therapy managed to correct the anemia and control the exudative enteropathy.
CONCLUSIONS: Although primary intestinal lymphangiectasia is usually adequately
managed by nutritional therapy, complications such as anemia can occur and can
prove to be a therapeutic challenge. Primary intestinal lymphangiectasia (PIL), also known as Waldmann's disease, is
an exudative enteropathy resulting from morphologic abnormalities in the
intestinal lymphatics. In this article, we describe a 12-year-old boy with PIL
that led to protein-losing enteropathy characterized by diarrhea,
hypoalbuminemia associated with edema (serum albumin level: 1.0 g/dL), and
hypogammaglobulinemia (serum IgG level: 144 mg/dL). Severe hypoalbuminemia,
electrolyte abnormalities, and tetany persisted despite a low-fat diet and
propranolol. Everolimus (1.6 mg/m(2)/day) was added to his treatment as an
antiangiogenic agent. With everolimus treatment, the patient's diarrhea resolved
and replacement therapy for hypoproteinemia was less frequent. Hematologic and
scintigraphy findings also improved (serum albumin level: 2.5 g/dL). There were
no adverse reactions during the 12-month follow-up. To the best of our
knowledge, this is the first report of everolimus use in a patient with PIL. |
What are congenital disorders of glycosylation? | Congenital disorders of glycosylation (CDG) are a growing group of inherited metabolic disorders where enzymatic defects in the formation or processing of glycolipids and/or glycoproteins lead to variety of different diseases.
More than 100 rare human genetic disorders that result from deficiencies in the different glycosylation pathways are known today.
The patients have hundreds of misglycosylated products, which afflict a myriad of processes, including cell signaling, cell-cell interaction, and cell migration. | Congenital disorders of glycosylation (CDG) are a growing group of inherited
metabolic disorders where enzymatic defects in the formation or processing of
glycolipids and/or glycoproteins lead to variety of different diseases. The
deficiency of GDP-Man:GlcNAc2-PP-dolichol mannosyltransferase, encoded by the
human ortholog of ALG1 from yeast, is known as ALG1-CDG (CDG-Ik). The
phenotypical, molecular and biochemical analysis of a severely affected ALG1-CDG
patient is the focus of this paper. The patient's main symptoms were feeding
problems and diarrhea, profound hypoproteinemia with massive ascites, muscular
hypertonia, seizures refractory to treatment, recurrent episodes of apnoea,
cardiac and hepatic involvement and coagulation anomalies. Compound
heterozygosity for the mutations c.1145T>C (M382T) and c.1312C>T (R438W) was
detected in the patient's ALG1-coding sequence. In contrast to a previously
reported speculation on R438W we confirmed both mutations as disease-causing in
ALG1-CDG. This review presents principles of glycosylation, describes the relevant
glycosylation pathways and their related disorders, and highlights some of the
neurological aspects and issues that continue to challenge researchers. More
than 100 rare human genetic disorders that result from deficiencies in the
different glycosylation pathways are known today. Most of these disorders impact
the central and/or peripheral nervous systems. Patients typically have
developmental delays/intellectual disabilities, hypotonia, seizures, neuropathy,
and metabolic abnormalities in multiple organ systems. Among these disorders
there is great clinical diversity because all cell types differentially
glycosylate proteins and lipids. The patients have hundreds of misglycosylated
products, which afflict a myriad of processes, including cell signaling,
cell-cell interaction, and cell migration. This vast complexity in glycan
composition and function, along with the limited availability of analytic tools,
has impeded the identification of key glycosylated molecules that cause
pathologies. To date, few critical target proteins have been pinpointed. Congenital disorders of glycosylation form a rapidly growing group of inherited
metabolic diseases. As glycosylation affects proteins all over the organism, a
mutation in a single gene leads to a multisystemic disorder. We describe a
patient with TMEM165-CDG with facial dysmorphism, nephrotic syndrome, cardiac
defects, enlarged cerebral ventricles, feeding problems, and neurological
involvement. Having confirmed the diagnosis via prenatal diagnostics, we were
able to observe the glycosylation right from birth, finding a pathological
pattern already on the first day of life. Within the next few weeks,
hypoglycosylation progressed to less sialylated and then also to
hypogalactosylated isoforms. On the whole, there has not been much published
evidence concerning postnatal glycosylation and its adaptational process. This
is the first paper reporting changes in glycosylation patterns over the first
postnatal weeks in TMEM165-CDG. Congenital disorder of glycosylation (CDG), formerly representing a group of
diseases due to defects in the biosynthetic pathway of protein N-glycosylation,
currently covers a wide range of disorders affecting glycoconjugates. Since its
first application to serum transferrin from a CDG patient with
phosphomannomutase-2 deficiency in 1992, mass spectrometry (MS) has been playing
a key role in identification and characterization of glycosylation defects
affecting glycoproteins. MS of native transferrin detects a lack of glycans
characteristic to the classical CDG-I type of molecular abnormality.
Electrospray ionization MS of native transferrin, especially, allows glycoforms
to be analyzed precisely but requires basic knowledge regarding deconvolution of
multiply-charged ions which may generate ghost signals upon transformation into
a singly-charged form. MS of glycopeptides from tryptic digestion of transferrin
delineates site-specific glycoforms and reveals a delicate balance of
donor/acceptor substrates or the conformational effect of nascent proteins in
cells. Matrix-assisted laser desorption ionization MS of apolipoprotein C-III is
a simple method of elucidating the profiles of mucin-type core 1 O-glycans
including site occupancy and glycoforms. In this technological review, the
principle and pitfalls of MS for CDG are discussed and mass spectra of various
types of CDG are presented. Glycosylation is an integral part in health and disease, as emphasized by the
growing number of identified glycosylation defects. In humans, proteins are
modified with a diverse range of glycoforms synthesized in complex biosynthetic
pathways. Glycosylation disorders have been described in congenital disorders of
glycosylation (CDG) as well as in acquired disease conditions such and
non-alcoholic fatty liver disease (NAFLD). A hallmark in a subset of CDG cases
is the reduced glycosylation site occupancy of asparagine-linked glycans. Using
an optimized method protocol, we determined the glycosylation site occupancy
from four proteins of hepatic and lymphatic origin from CDG and NAFLD patients.
We found variable degrees of site occupancy, depending on the tissue of origin
and the disease condition. In CDG glycosylation sites of IgG2 and IgA1 were
occupied to normal levels. In NAFLD haptoglobin and transferrin glycosylation
sites were hyper-glycosylated, a property qualifying for its use as a potential
biomarker. Furthermore, we observed, that glycosylation sites of
liver-originating transferrin and haptoglobin are differentially occupied under
physiological conditions, a further instance not noticed in serum proteins to
date. Our findings suggest the use of serum protein hyperglycosylation as a
biomarker for early stages of NAFLD. |
Which are the additions of the JASPAR 2016 open-access database of transcription factor binding profiles? | Compared to the JASPAR CORE collection, JASPAR 2016 has been expanded with 494 new TF binding profiles (315 in vertebrates, 11 in nematodes, 3 in insects, 1 in fungi and 164 in plants) and 59 profiles (58 in vertebrates and 1 in fungi) have been updated. The introduced profiles represent an 83% expansion and 10% update when compared to the previous release. The structural annotation of the TF DNA binding domains (DBDs) has been updated following a published hierarchical structural classification. In addition, 130 transcription factor flexible models trained on ChIP-seq data for vertebrates, which capture dinucleotide dependencies within TF binding sites were introduced . The new JASPAR release is accompanied by a new web tool to infer JASPAR TF binding profiles recognized by a given TF protein sequence. Moreover, users are provided with a Ruby module complementing the JASPAR API to ease programmatic access and use of the JASPAR collection of profiles. JASPAR2016 R/Bioconductor data package is also provided with the data of this release. | JASPAR (http://jaspar.genereg.net) is an open-access database storing curated,
non-redundant transcription factor (TF) binding profiles representing
transcription factor binding preferences as position frequency matrices for
multiple species in six taxonomic groups. For this 2016 release, we expanded the
JASPAR CORE collection with 494 new TF binding profiles (315 in vertebrates, 11
in nematodes, 3 in insects, 1 in fungi and 164 in plants) and updated 59
profiles (58 in vertebrates and 1 in fungi). The introduced profiles represent
an 83% expansion and 10% update when compared to the previous release. We
updated the structural annotation of the TF DNA binding domains (DBDs) following
a published hierarchical structural classification. In addition, we introduced
130 transcription factor flexible models trained on ChIP-seq data for
vertebrates, which capture dinucleotide dependencies within TF binding sites.
This new JASPAR release is accompanied by a new web tool to infer JASPAR TF
binding profiles recognized by a given TF protein sequence. Moreover, we provide
the users with a Ruby module complementing the JASPAR API to ease programmatic
access and use of the JASPAR collection of profiles. Finally, we provide the
JASPAR2016 R/Bioconductor data package with the data of this release. |
What is the function of the protein tafazzin? | Tafazzin is a phospholipid transacylase that transfers acyl chains with unsaturated fatty acids from phospholipids to monolysocardiolipin to generate cardiolipin with unsaturated fatty acids on mitochondrial membrane. | Barth syndrome is an X-linked disorder characterized by cardiomyopathy, skeletal
myopathy, neutropenia, organic aciduria, and growth retardation caused by
mutations in tafazzin. The sequence similarity of tafazzin to acyltransferases
suggests a role in mitochondrial phospholipid metabolism. To study the role of
tafazzin in heart function and development, we created a knockdown zebrafish
model. Zebrafish tafazzin mRNA is first evident at 7 hours post-fertilization
(hpf). At 10 and 24 hpf, tafazzin mRNA is ubiquitous, with highest levels in the
head. By 51 hpf, expression becomes cardiac restricted. The tafazzin knockdown
created by antisense morpholino yolk injection resulted in dose-dependent
lethality, severe developmental and growth retardation, marked bradycardia and
pericardial effusions, and generalized edema, signs that resemble human Barth
syndrome heart failure. This knockdown phenotype was rescued by concomitant
injection of normal tafazzin mRNA. Abnormal cardiac development, with a linear,
nonlooped heart, and hypomorphic tail and eye development proves that tafazzin
is essential for overall zebrafish development, especially of the heart. The
tafazzin knockdown zebrafish provides an animal model similar to Barth syndrome
to analyze the severity of human mutants and to test potential treatments. Tafazzin is a putative enzyme that is involved in cardiolipin metabolism, it may
carry mutations responsible for Barth syndrome. To identify the biochemical
reaction catalyzed by tafazzin, we expressed the full-length isoform of
Drosophila melanogaster tafazzin in a baculovirus-Sf9 insect cell system.
Tafazzin expression induced a new enzymatic function in Sf9 cell mitochondria,
namely 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine:monolysocardiolipin
linoleoyltransferase. We also found evidence for the reverse reaction, because
tafazzin expression caused transfer of acyl groups from phospholipids to
1-[14C]palmitoyl-2-lyso-phosphatidylcholine. An affinity-purified tafazzin
construct, tagged with the maltose-binding protein, catalyzed both forward and
reverse transacylations between cardiolipin and phosphatidylcholine, but was
unable to utilize CoA or acyl-CoA as substrates. Whereas tafazzin supported
transacylations between various phospholipid-lysophospholipid pairs, it showed
the highest rate for the phosphatidylcholine-cardiolipin transacylation.
Transacylation activities were about 10-fold higher for linoleoyl groups than
for oleoyl groups, and they were negligible for arachidonoyl groups. The data
show that Drosophila tafazzin is a CoA-independent, acyl-specific phospholipid
transacylase with substrate preference for cardiolipin and phosphatidylcholine. The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved
in cardiolipin metabolism, but it is not known why it forms multiple transcripts
as a result of alternative splicing. Here we studied the intracellular
localization, enzymatic activity, and metabolic function of four isoforms of
human tafazzin and three isoforms of Drosophila tafazzin upon expression in
different mammalian and insect systems. When expressed in HeLa cells, all
isoforms were localized in mitochondria except for the B-form of Drosophila
tafazzin, which was associated with multiple intracellular membranes. Among the
human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5
(Delta5) had transacylase activity, and only these two isoforms were able to
restore a normal cardiolipin pattern, normal respiratory activity of
mitochondria, and male fertility in tafazzin-deficient flies. Both FL and Delta5
were associated with large protein complexes in 293T cell mitochondria, but
treatment with alkali and proteinase K suggested that the Delta5 isoform was
more integrated into the hydrophobic core of the membrane than the FL isoform.
Although all Drosophila isoforms showed transacylase activity in vitro, only the
A-form supported cardiolipin remodeling in flies. The data suggest that humans
express two mitochondrial isoenzymes of tafazzin that have similar transacylase
activities but different membrane topologies. Furthermore, the data show that
the expression of human tafazzin in flies creates cardiolipin with a Drosophila
pattern, suggesting that the characteristic fatty acid profile of cardiolipin is
not determined by the substrate specificity of tafazzin. Cardiolipin (CL) that is synthesized de novo is deacylated to
monolysocardiolipin (MLCL), which is reacylated by tafazzin. Remodeled CL
contains mostly unsaturated fatty acids. In eukaryotes, loss of tafazzin leads
to growth and respiration defects, and in humans, this results in the
life-threatening disorder Barth syndrome. Tafazzin deficiency causes a decrease
in the CL/MLCL ratio and decreased unsaturated CL species. Which of these
biochemical outcomes contributes to the physiological defects is not known.
Yeast cells have a single CL-specific phospholipase, Cld1, that can be exploited
to distinguish between these outcomes. The cld1Δ mutant has decreased
unsaturated CL, but the CL/MLCL ratio is similar to that of wild type cells. We
show that cld1Δ rescues growth, life span, and respiratory defects of the taz1Δ
mutant. This suggests that defective growth and respiration in
tafazzin-deficient cells are caused by the decreased CL/MLCL ratio and not by a
deficiency in unsaturated CL. CLD1 expression is increased during respiratory
growth and regulated by the heme activator protein transcriptional activation
complex. Overexpression of CLD1 leads to decreased mitochondrial respiration and
growth and instability of mitochondrial DNA. However, ATP concentrations are
maintained by increasing glycolysis. We conclude that transcriptional regulation
of Cld1-mediated deacylation of CL influences energy metabolism by modulating
the relative contribution of glycolysis and respiration. Structure and function of mitochondria are intimately linked. In a search for
components that participate in building the elaborate architecture of this
complex organelle we have identified Aim24, an inner membrane protein. Aim24
interacts with the MICOS complex that is required for the formation of crista
junctions and contact sites between inner and outer membranes. Aim24 is
necessary for the integrity of the MICOS complex, for normal respiratory growth
and mitochondrial ultrastructure. Modification of MICOS subunits Mic12 or Mic26
by His-tags in the absence of Aim24 leads to complete loss of cristae and
respiratory complexes. In addition, the level of tafazzin, a cardiolipin
transacylase, is drastically reduced and the composition of cardiolipin is
modified like in mutants lacking tafazzin. In conclusion, Aim24 by interacting
with the MICOS complex plays a key role in mitochondrial architecture,
composition and function. DOI: http://dx.doi.org/10.7554/eLife.01684.001. BACKGROUND: Tafazzin (TAZ), a transmembrane protein contributes in mitochondrial
structural and functional modifications through cardiolipin remodeling. TAZ
mutations are associated with several diseases, but studies on the role of TAZ
protein in carcinogenesis and radiotherapy (RT) response is lacking. Therefore
we investigated the TAZ expression in rectal cancer, and its correlation with
RT, clinicopathological and biological variables in the patients participating
in a clinical trial of preoperative RT.
METHODS: 140 rectal cancer patients were included in this study, of which 65
received RT before surgery and the rest underwent surgery alone. TAZ expression
was determined by immunohistochemistry in primary cancer, distant, adjacent
normal mucosa and lymph node metastasis. In-silico protein-protein interaction
analysis was performed to study the predictive functional interaction of TAZ
with other oncoproteins.
RESULTS: TAZ showed stronger expression in primary cancer and lymph node
metastasis compared to distant or adjacent normal mucosa in both non-RT and RT
patients. Strong TAZ expression was significantly higher in stages I-III and
non-mucinious cancer of non-RT patients. In RT patients, strong TAZ expression
in biopsy was related to distant recurrence, independent of gender, age, stages
and grade (p = 0.043, HR, 6.160, 95% CI, 1.063-35.704). In silico
protein-protein interaction study demonstrated that TAZ was positively related
to oncoproteins, Livin, MAC30 and FXYD-3.
CONCLUSIONS: Strong expression of TAZ protein seems to be related to rectal
cancer development and RT response, it can be a predictive biomarker of distant
recurrence in patients with preoperative RT. Tafazzin (EC 2.3.1.23) is a Phospholipid Transacylase involved in Cardiolipin
remodeling on mitochondrial membrane and coded by TAZ gene (Cytogenetic
Location: Xq28) in human. Its mutations cause Barth syndrome (MIM ID:
#302060)/3-Methyl Glutaconyl Aciduria Type II, an inborn error of metabolism
often leading to foetal or infantile fatality. Nevertheless, some mis-sense
mutations result in mild clinical symptoms. To evaluate the rationale of mild
symptoms and for an insight of Tafazzin active site, sequence based and
structure based ramifications of wild and mutant Tafazzins were compared
in-silico. Sequence based domain predictions, surface accessibilities on
substitution & conserved catalytic sites with statistical drifts, as well as
thermal stability changes for the mutations and the interaction analysis of
Tafazzin were performed. Crystal structure of Tafazzin is not yet resolved
experimentally, therefore 3D coordinates of Tafazzin and its mutants were
spawned through homology modeling. Energetically minimized and structurally
validated models were used for comparative docking simulations. We analyzed
active site geometry of the models in addition to calculating overall substrate
binding efficiencies for each of the enzyme-ligand complex deduced from binding
energies instead of comparing only the docking scores. Also, individual binding
energies of catalytic residues on conserved HX4D motif of Acyltransferase
superfamily present in Tafazzins were estimated. This work elucidates the basis
of mild symptoms in patients with mis-sense mutations, identifies the most
pathogenic mutant among others in the study and also divulges the critical role
of HX4D domain towards successful transacylation by Taffazin. The in-silico
observations are in complete agreement with clinical findings reported for the
patients with mutations. Tafazzin, a mitochondrial acyltransferase, plays an important role in
cardiolipin side chain remodeling. Previous studies have shown that dysfunction
of tafazzin reduces cardiolipin content, impairs mitochondrial function, and
causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS)
have been implicated in the development of cardiomyopathy and are also the
obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown
increases ROS production from mitochondria, and a mitochondria-targeted
antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac
dysfunction. We employed cardiac myocytes transduced with an adenovirus
containing tafazzin shRNA as a model to investigate the effects of the
mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady
state levels of cardiolipin and increased mitochondrial ROS. Treatment of
cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced
mitochondrial ROS production and cellular ATP decline. Mito-Tempo also
significantly abrogated tafazzin knockdown induced cardiac hypertrophy,
contractile dysfunction, and cell death. We conclude that mitochondria-targeted
antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in
cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth
syndrome and other dilated cardiomyopathies resulting from mitochondrial
oxidative stress. Tafazzin is a transacylase that affects cardiolipin fatty acid composition and
mitochondrial function. Mutations in human tafazzin cause Barth syndrome yet the
enzyme has mostly been characterized in yeast. To study tafazzin in higher
organisms, we isolated mitochondria from Drosophila and mammalian cell cultures.
Our data indicate that tafazzin binds to multiple protein complexes in these
organisms, and that the interactions of tafazzin lack strong specificity. Very
large tafazzin complexes could only be detected in the presence of cardiolipin,
but smaller complexes remained intact even upon treatment with phospholipase A2.
In mammalian cells, tafazzin had a half-life of only 3-6h, which was much
shorter than the half-life of other mitochondrial proteins. The data suggest
that tafazzin is a transient resident of multiple protein complexes. Cardiolipin (CL) is a phospholipid with many unique characteristics. CL is
synthesized in the mitochondria and resides almost exclusively within the
mitochondrial inner membrane. Unlike most phospholipids that have two fatty acyl
chains, CL possesses four fatty acyl chains resulting in unique biophysical
characteristics that impact several biological processes including membrane
fission and fusion. In addition, several proteins directly bind CL including
proteins within the electron transport chain, the ADP/ATP carrier, and proteins
that mediate mitophagy. Tafazzin is an enzyme that remodels saturated fatty acyl
chains within CL to unsaturated fatty acyl chains, loss of function mutations in
the TAZ gene encoding tafazzin are causal for the inherited cardiomyopathy Barth
syndrome. Cells from Barth syndrome patients as well as several models of Barth
have reduced mitochondrial functions including impaired electron transport chain
function and increased reactive oxygen species (ROS) production. Mitochondria in
cells from Barth syndrome patients, as well as several model organism mimics of
Barth syndrome, are large and lack cristae consistent with the recently
described role of CL participating in the generation of mitochondrial membrane
contact sites. Cells with an inactive TAZ gene have also been shown to have a
decreased capacity to undergo mitophagy when faced with stresses such as
increased ROS or decreased mitochondrial quality control. This review describes
CL metabolism and how defects in CL metabolism cause Barth syndrome, the
etiology of Barth syndrome, and known modifiers of Barth syndrome phenotypes
some of which could be explored for their amelioration of Barth syndrome in
higher organisms. |
What are assassin bugs? | The family Reduviidae (Hemiptera: Heteroptera), or assassin bugs, is among the most diverse families of the true bugs, with more than 6,000 species. | The complete mitochondrial genome of Peirates arcuatus (Stål) is a typical
double-stranded circular molecule 16,176 bp long with 37 genes usually present
in animal mitochondrial genomes and a control region. Gene order is identical to
that of the putative ancestral arrangement of insects and other assassin bugs.
Thirteen protein-coding genes initiate with ATN codons and mostly terminate with
TAN codons except for COII and COIII use a single T residue as the termination
codon. All tRNA sequences can be folded into classic clover-leaf secondary
structure except that the dihydrouridine (DHU) arm of tRNA(Ser(AGN)) forms a
simple loop, and their length range from 63 to 69 bp. The control region is 1552
bp long and includes two complete 118-bp tandem repeats and one partial copy of
anterior repeat unit. The complete mitochondrial genome (mitogenome) of Opistoplatys sp. was
determined, which was the first representation from the assassin bug subfamily
Tribelocephalinae. The mitogenome is a typical circular DNA molecule of
15,615 bp and contains 37 genes and a putative control region. All the 13
protein-coding genes initiate with ATN codons and mostly terminate with TAA or
TAG codons except for COII, COIII, and ND5 genes use a single T residue as the
termination codon. All the 22 tRNAs have the clover-leaf structure except for
the tRNASer(AGN) with the length ranging from 60 to 72 bp. The control region is
1068 bp in length and includes 456 bp tandem repeat sequence with four 94-bp
tandem repeat units and a partial fifth (80 bp). In the sampled subfamilies of
Reduviidae, Peiratinae + the remaining subfamilies, Triatominae + Stenopodainae,
Tribelocephalinae + Ectrichodiinae, are recovered in phylogenetic analyses with
high supports. The rate of discovery of new species of Reduviidae (Insecta: Heteroptera) from
North America has slowed in the 21st century. This is not surprising, given the
conspicuousness and large distribution ranges of many Nearctic assassin bug
species that are often collected using general insect collecting techniques.
Nevertheless, biodiversity discovery in Nearctic Reduviidae is ongoing. We here
describe a new species, Reduvius frommeri, n. sp., from Southern California that
is so far only known from a small endemic range in the Sonoran Desert. With
about 197 species, the genus Reduvius Fabricius is one of the most speciose
genera of Reduviidae. The majority of species occur in arid- and semi-arid areas
in the Afrotropical, Oriental, and Palearctic regions and only three species are
New World endemics. A fourth species that occurs in the United States, Reduvius
personatus Fabricius, is cosmopolitan and has been introduced to the Western
Nearctic. The new species of Reduvius stands out amongst the four other Nearctic
Reduvius species by the small size and pale body coloration with a contrasting
dark head. Image plates documenting habitus and selected morphological details
and maps are provided for the five species in the Nearctic. We conclude that
efforts to document species diversity and distribution ranges even for
conspicuous insects such as assassin bugs in fairly well studied biogeographic
regions need to continue. BACKGROUND: The family Reduviidae (Hemiptera: Heteroptera), or assassin bugs, is
among the most diverse families of the true bugs, with more than 6,000 species.
The subfamily Triatominae (kissing bugs) is noteworthy not simply because it is
the only subfamily of the Reduviidae whose members feed on vertebrate blood but
particularly because all 147 known members of the subfamily are potential Chagas
disease vectors. Due to the epidemiological relevance of these species and the
lack of an efficient treatment and vaccine for Chagas disease, it is more common
to find evolutionary studies focusing on the most relevant vectors than it is to
find studies aiming to understand the evolution of the group as a whole. We
present the first comprehensive phylogenetic study aiming to understand the
events that led to the diversification of the Triatominae.
METHODOLOGY/PRINCIPAL FINDINGS: We gathered the most diverse samples of
Reduviidae and Triatominae (a total of 229 Reduviidae samples, including 70
Triatominae species) and reconstructed a robust dated phylogeny with several
fossil (Reduviidae and Triatominae) calibrations. Based on this information, the
possible role of geological events in several of the major cladogenetic events
within Triatominae was tested for the first time. We were able to not only
correlate the geological changes in the Neotropics with Triatominae evolution
but also add to an old discussion: Triatominae monophyly vs. paraphyly.
CONCLUSIONS/SIGNIFICANCE: We found that most of the diversification events
observed within the Rhodniini and Triatomini tribes are closely linked to the
climatic and geological changes caused by the Andean uplift in South America and
that variations in sea levels in North America also played a role in the
diversification of the species of Triatoma in that region. |
Are the genes for marneral biosynthesis scattered in the genome of A. thaliana? | These clusters are unlikely to have arisen by horizontal gene transfer, and the mechanisms behind their formation are poorly understood. Here we characterize a second operon-like triterpene cluster (the marneral cluster) from A. thaliana, compare the features of these two clusters, and investigate the evolutionary events that have led to cluster formation. | Operon-like arrangements of genes occur in eukaryotes ranging from yeasts and
filamentous fungi to nematodes, plants, and mammals. In plants, several examples
of operon-like gene clusters involved in metabolic pathways have recently been
characterized, e.g. the cyclic hydroxamic acid pathways in maize, the avenacin
biosynthesis gene clusters in oat, the thalianol pathway in Arabidopsis
thaliana, and the diterpenoid momilactone cluster in rice. Such operon-like gene
clusters are defined by their co-regulation or neighboring positions within
immediate vicinity of chromosomal regions. A comprehensive analysis of the
expression of neighboring genes therefore accounts a crucial step to reveal the
complete set of operon-like gene clusters within a genome. Genome-wide
prediction of operon-like gene clusters should contribute to functional
annotation efforts and provide novel insight into evolutionary aspects acquiring
certain biological functions as well. We predicted co-expressed gene clusters by
comparing the Pearson correlation coefficient of neighboring genes and randomly
selected gene pairs, based on a statistical method that takes false discovery
rate (FDR) into consideration for 1469 microarray gene expression datasets of A.
thaliana. We estimated that A. thaliana contains 100 operon-like gene clusters
in total. We predicted 34 statistically significant gene clusters consisting of
3 to 22 genes each, based on a stringent FDR threshold of 0.1. Functional
relationships among genes in individual clusters were estimated by sequence
similarity and functional annotation of genes. Duplicated gene pairs (determined
based on BLAST with a cutoff of E<10(-5)) are included in 27 clusters. Five
clusters are associated with metabolism, containing P450 genes restricted to the
Brassica family and predicted to be involved in secondary metabolism.
Operon-like clusters tend to include genes encoding bio-machinery associated
with ribosomes, the ubiquitin/proteasome system, secondary metabolic pathways,
lipid and fatty-acid metabolism, and the lipid transfer system. |
Is Dupilumab used for treatment of atopic dermatitis? | Yes, patients treated with dupilumab had marked and rapid improvement in all the evaluated measures of atopic dermatitis disease activity. | INTRODUCTION: Current treatment options for moderate-to-severe atopic dermatitis
(AD) are limited and have potentially dangerous side effects. Dupilumab is a
novel monoclonal antibody that was recently studied in adult patients with
moderate-to-severe AD. Dupilumab inhibits interleukin-4 (IL-4) and
interleukin-13 (IL-13) signaling and was previously found to be effective in
asthma. Considering that both AD and asthma are Th2 cell-mediated inflammatory
processes, it is reasonable to suspect that dupilumab would be beneficial in
AD.`
AREAS COVERED: This article is a review of the one major clinical trial that
assessed the efficacy of dupilumab in patients with AD. Its goal is to provide a
comparison to the current modalities for the treatment of AD and expert insight
regarding future studies.
EXPERT OPINION: The results of this study are a significant therapeutic
advancement. Dupilumab was shown to provide a mean percent change in Eczema Area
and Severity Index score of -74% ± 3.6, in addition to, statistically and
clinically significant reductions the severity, symptomatology, and morbidity
associated with AD. However, the small sample size makes it difficult to assess
the magnitude of this effect. As a result, dupilumab will likely be reserved for
cases of severe AD unresponsive to traditional modalities. Asthma is a heterogeneous inflammatory disease. Most patients respond to current
standard of care, i.e., bronchodilators, inhaled glucocorticosteroids and other
anti-inflammatory drugs, but in some adequate asthma control cannot be achieved
with standard treatments. These difficult-to-treat patients would be the target
population for new biological therapies. At present, omalizumab is the only
biological agent approved for the treatment of early-onset, severe IgE-dependent
asthma. It is safe, effective, and well tolerated. Also, discovery of asthma
subtypes suggests new treatments. Half of patients with severe asthma have
T-helper type 2 (Th-2) inflammation and they are expected to benefit from
monoclonal antibody-based treatments. The efficacy of the investigational
monoclonal antibody mepolizumab which targets IL-5 has been well documented in
late onset non-atopic asthma with persistent eosinophilic airway inflammation.
Anti-IL-4 and IL-13 agents (dupilumab, lebrikizumab, and tralokinumab) which
block different Th-2 inflammatory pathways and agents targeting the Th-17
inflammatory pathway in severe refractory asthma are under development. In
clinical trials, these drugs reduce disease activity and improve lung function,
asthma symptoms, and quality of life. However, studies on larger groups of
patients are needed to confirm their safety and efficacy. Collaborators: Nikolova-Pavlova E, Stoyanova B, Vlaeva T, Alavi A, Gauvreau G,
Henein S, Poulos E, Yang W, Lepage F, Wiseman M, Bissonnette R, Agner T,
Deleuran M, Jemec G, Skov L, Kingo K, Konno P, Pender K, Põder A, Vahlberg A,
Oksman R, Pasternack R, Remitz A, Bieber T, Dominicus R, Gerlach B, Kardorff B,
Toader AL, Kleinheinz A, Gellrich S, Kreutzer K, Leitz N, Offers M, Pauser S,
Radtke M, Roloff E, Rosenbach T, Schwarz B, Sell S, Simon JC, Staubach P, Weigel
US, Werfel T, Wohlrab J, Wollenberg A, Rothenberger C, Walter A, Yazdi A, Aihara
M, Hide M, Kataoka Y, Katoh N, Kawashima M, Kobayashi S, Mitsui H, Nakahara T,
Saeki H, Sueki H, Arai S, Ikeda M, Kabashima K, Kawachi Y, Kume A, Moriwaki S,
Natsuaki Y, Ogata F, Omi T, Seishima M, Sugaya M, Tsukamoto K, Tsuruta D, Urano
S, Watanabe D, Yoshioka A, Furukawa F, Katoh A, Ang CC, Aw DC, Tang M, Lee HY,
Orpinell FB, Hernández GC, De La Cueva P, Foraster CF, Iranzo P, Serra AJ, Luna
PL, Moya SM, Ramírez DM, Muñoz JP, Carazo JS, Soong W, Hull C, Johnson S, Bhatia
N, Limova M, Raikhel M, Sher L, Sofen H, Spector S, Tan R, Yamauchi P, Weber R,
Kimura S, Nelson C, Randhawa S, Rendon M, Trevino M, Ling M, Rice Z, Silverberg
J, Siri D, Fretzin S, Fowler JF, Boh E, Merola J, Murakawa G, Korenblat P,
Campbell J, Bagel J, Beck L, Hazan C, Kalb R, Smith C, Bardelas J, Gawchik S,
Schenkel E, Krause R, Allison D, Browning J, Davis S, Lee M, Duffin K, Fisher
CT, Pariser D, Gower RG, Adams S, Sapijaszko MJ, Wasel N, Albrecht L, Hong CH,
Gulliver W, Landells I, Adam D, Gooderham M, Lomaga M, Lynde C, Rosoph L, Raman
M, Robern M, Sapra S, Toth D, Poulin Y, Bagot M, Barbarot S, Grob JJ, Guillet G,
Lacour JP, Khemis A, Misery L, Staumont-Sallé D, Brüning H, Darsow U,
Ekanayake-Bohlig S, Herbst R, Hoffmann M, Homey B, Niesmann J, Pinter A, Radny
P, Reich K, Sattler G, Sebastian M, Thaçi D, Weidinger S, Wildfeuer T, Worm M,
Chan H, Chan J, Amerio P, Carlesimo M, Di Lernia V, Emilia R, Didona B, Fargnoli
M, Ferrucci SM, Naldi L, Papini M, Parodi A, Pellacani G, Peris K, Pimpinelli N,
Romanelli M, Talamonti M, Bylaite-Bucinskiene M, Cesiene J, Narbutas R,
Sidlauskiene RB, Kucinskiene V, Adamski Z, Bystrzanowska D, Dyczek A, Hofman T,
Leszniewska L, Nowicki R, Owczarek W, Slowinska M, Sobieszek-Kundro A, Weglowska
J, Zakrzewski M, Ahn HH, Ahn KJ, Chang SE, Choi GS, Kim MB, Kim KH, Lee KH, Park
YM, Park CW, Park GH, Nahm DH, Park YL, Roh J, Seo SJ, Ameen M, Ardern-Jones M,
Bewley A, Cooper H, Cork MJ, Guha-Niyogi B, Khan M, Marshall M, Foerster J,
Smith C, Appell M, Elewski B, Haynes S, Jazayeri SS, Crowley J, Dhawan S, Ellis
M, Kim S, Meltzer S, Mitchell J, Pearlman D, Moss J, Ehrlich A, Forman S,
Kuttner B, Penate F, Vaca C, Hamilton T, Paull W, Weisman J, Glazer S, Mehlis S,
Guenthner S, Lockshin B, Kimball A, Rosmarin D, Pickett-Baisden T, Halverson P,
Kaiser H, Martin A, Stone M, Davis K, Mirkil V, Nossa R, Bretton E, Alexis A,
Guttman-Yassky E, Peredo M, Weinberg J, Fleischer A, George R, Lugo-Somolinos A,
Nasir A, Hussain I, Blauvelt A, Simpson E, Kalafer M, Hampton M, Humeniuk JM,
Rupp N, Carrasco D, MacGillivray B, Moore A, Teller C, Tyring S, Harris D,
Jenkin P. |
Where is base J found in the genome of Leishmania tarentolae? | Base J (β-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes in Trypanosoma brucei. | DNA from Kinetoplastida contains the unusual modified base
beta-D-glucosyl(hydroxymethyl)uracil, called J. Base J is found predomitly in
repetitive DNA and correlates with epigenetic silencing of telomeric variant
surface glycoprotein genes in Trypanosoma brucei. We have now identified a
protein in nuclear extracts of bloodstream stage T.brucei that binds
specifically to J-containing duplex DNA. J-specific DNA binding was also
observed with extracts from the kinetoplastids Crithidia fasciculata and
Leishmania tarentolae. We purified the 90 kDa C.fasciculata J-binding protein 50
000-fold and cloned the corresponding gene from C.fasciculata, T.brucei and
L.tarentolae. Recombit proteins expressed in Escherichia coli demonstrated
J-specific DNA binding. The J-binding proteins show 43-63% identity and are
unlike any known protein. The discovery of a J-binding protein suggests that J,
like methylated cytosine in higher eukaryotes, functions via a protein
intermediate. Base J or beta-d-glucosylhydroxymethyluracil is a DNA modification replacing a
fraction of thymine in the nuclear DNA of kinetoplastid parasites and of
Euglena. J is located in the telomeric sequences of Trypanosoma brucei and in
other simple repeat DNA sequences. In addition, J was found in the inactive
variant surface glycoprotein (VSG) expression sites, but not in the active
expression site of T. brucei, suggesting that J could play a role in
transcription silencing in T. brucei. We have now looked at the distribution of
J in the genomes of other kinetoplastid parasites. First, we analyzed the DNA
sequences immunoprecipitated with a J-antiserum in Leishmania major Friedlin.
Second, we investigated the co-migration of J- and telomeric repeat-containing
DNA sequences of various kinetoplastids using J-immunoblots and Southern blots
of fragmented DNA. We find only approximately 1% of J outside the telomeric
repeat sequences of Leishmania sp. and Crithidia fasciculata, in contrast to the
substantial fraction of non-telomeric J found in T. brucei, Trypanosoma
equiperdum and Trypanoplasma borreli. Our results suggest that J is a telomeric
base modification, recruited for other (unknown) functions in some
kinetoplastids and Euglena. Base J is a hypermodified DNA base localized primarily to telomeric regions of
the genome of Trypanosoma brucei. We have previously characterized two
thymidine-hydroxylases (TH), JBP1 and JBP2, which regulate J-biosynthesis. JBP2
is a chromatin re-modeling protein that induces de novo J-synthesis, allowing
JBP1, a J-DNA binding protein, to stimulate additional J-synthesis. Here, we
show that both JBP2 and JBP1 are capable of stimulating de novo J-synthesis. We
localized the JBP1- and JBP2-stimulated J by anti-J immunoprecipitation and
high-throughput sequencing. This genome-wide analysis revealed an enrichment of
base J at regions flanking polymerase II polycistronic transcription units (Pol
II PTUs) throughout the T. brucei genome. Chromosome-internal J deposition is
primarily mediated by JBP1, whereas JBP2-stimulated J deposition at the
telomeric regions. However, the maintece of J at JBP1-specific regions is
dependent on JBP2 SWI/SNF and TH activity. That similar regions of Leishmania
major also contain base J highlights the functional importance of the modified
base at Pol II PTUs within members of the kinetoplastid family. The regulation
of J synthesis/localization by two THs and potential biological function of J in
regulating kinetoplastid gene expression is discussed. Base J, β-d-glucosyl-hydroxymethyluracil, is an epigenetic modification of
thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida.
J is enriched at sites involved in RNA polymerase (RNAP) II initiation and
termination. Reduction of J in Leishmania tarentolae via growth in BrdU resulted
in cell death and indicated a role of J in the regulation of RNAP II
termination. To further explore J function in RNAP II termination among
kinetoplastids and avoid indirect effects associated with BrdU toxicity and
genetic deletions, we inhibited J synthesis in Leishmania major and Trypanosoma
brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in
transcription termination at the end of polycistronic gene clusters and the
generation of antisense RNAs, without cell death. In contrast, loss of J in T.
brucei did not lead to genome-wide termination defects; however, the loss of J
at specific sites within polycistronic gene clusters led to altered
transcription termination and increased expression of downstream genes. Thus, J
regulation of RNAP II transcription termination genome-wide is restricted to
Leishmania spp., while in T. brucei it regulates termination and gene expression
at specific sites within polycistronic gene clusters. |
Which tool is available for predicting regulatory interactions from ChIP-seq data? | CisMapper predicts the regulatory targets of a TF using the correlation between a histone mark at the TF's bound sites and the expression of each gene across a panel of tissues. CisMapper is more accurate at predicting the target genes of a TF than the distance-based approaches currently used, and is particularly advantageous for predicting the long-range regulatory interactions typical of tissue-specific gene expression. CisMapper also predicts which TF binding sites regulate a given gene more accurately than using genomic distance. Unlike distance-based methods, CisMapper can predict which transcription start site of a gene is regulated by a particular binding site of the TF. | 1. |
Where are the unipolar brush cells localized? | Unipolar brush cells (UBCs) are glutamatergic interneurons localized in granule cell regions of the cochlear nucleus and the vestibulocerebellum of cerebellum. | 1. The synaptic activation by mossy fibers (MFs) of unipolar brush cells (UBCs)
in the vestibular cerebellum (nodulus and uvula) was examined using patch-clamp
recording methods in thin, rat cerebellar slices with Lucifer yellow-filled
pipettes for subsequent fluorescence microscopic verification of the cell
morphology. 2. UBCs were distinguished from adjacent granule cells in thin
cerebellar slices in the uvula and nodulus regions by their larger soma
diameters and short dendritic brush, greater whole-cell capacitance, and a
prolonged, biphasic excitatory postsynaptic current (EPSC) to stimulation of
MFs. 3. Thin-section transmission electron micrographs of the MF-UBC synapse
displayed an unusually extensive area of synaptic apposition estimated to
measure 12-40 microns2. The majority of UBCs was innervated by a single MF. At
high magnification, individual clusters of presynaptic vesicles could be
discerned, separated by regions of presynaptic membrane lacking vesicles, but
apposed to continuous regions of postsynaptic density. Thus, after release,
transmitter diffusion from the synaptic cleft must traverse considerable
stretches of postsynaptic membrane before escape into extracellular space. In
contrast, MF-granule cell synapses in these cerebellar regions resembled
glutamate synapses in other brain regions in that the total synaptic area
measured < or = 4 microns2. These synaptic junctions were flanked by short
stretches of unspecialized plasma membrane, providing a short (0.5 micron)
diffusional path from the site of neurotransmitter release to a branch point of
the extracellular space. 4. The MF-evoked EPSC in UBCs was composed of a fast
(10-90% rise time: 0.70 ms) and slow (10-90% rise time: 395 ms; 10-90% decay
time: 3.1 s) component. The fast component was blocked by the
alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/KA)
antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and displayed linear
current-voltage (I-V) relations in the presence or absence of external
magnesium. 5. The slow EPSC was also mediated by glutamate receptors, but in
most neurons both AMPA/KA and N-methyl-D-aspartate (NMDA) receptors contributed
to the slow EPSC, with the contribution of NMDA receptors predominating in the
majority of cells. Consequently, although all cells displayed linear I-V
relations in Mg(2+)-free saline, cells in which the slow EPSC was predominently
mediated by NMDA receptors exhibited voltage-dependent rectification in the
presence of external Mg2+ (1 mM). 6. With increasing postnatal age (10-30 d),
the contribution made to the slow EPSC by NMDA receptors declined, with a
reciprocal increase in the contribution being made by AMPA/KA
receptors.(ABSTRACT TRUNCATED AT 400 WORDS) The present study provides a survey of the immunolocalization of ionotropic
glutamate receptor subunits throughout the rat and cat cerebellar cortex, with
emphasis on the unipolar brush cell (UBC), a hitherto neglected cerebellar cell
that is densely concentrated in the granular layer of the vestibulocerebellum
and that forms giant synapses with mossy fibers. An array of nine previously
characterized antibodies has been used, each of which stained a characteristic
profile of cerebellar cells. The UBCs of both rat and cat were strongly
immunostained by an antibody against the
alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptor subunits,
GluR2 and GluR3; were moderately immunostained by a monoclonal antibody to
kainate receptor subunits, GluR5/6/7; were weakly immunostained by antibodies to
NR1 subunits; and were not stained by antibodies to GluR1, GluR4, GluR6/7, KA-2,
and NR2A/B. Postsynaptic densities of the giant mossy fiber-UBC synapses were
GluR2/3, GluR5/6/7, and NR1 immunoreactive. The other cerebellar neurons were
all immunolabeled to some extent with the GluR2/3 and NR1 antibodies. In
addition, Purkinje cells were immunopositive for GluR1 and GluR5/6/7; granule
cells were immunopositive for GluR5/6/7, GluR6/7, KA-2, and NR2A/B. The
Golgi-Bergmann glia was densely stained by GluR1 and GluR4 antibodies, whereas
astrocytes of the granular layer were stained by the GluR4 antiserum. Data
provided herein may guide further electrophysiological and pharmacological
studies of cerebellar cells in general and the UBCs in particular. A neglected type of neuron, termed the unipolar brush cell, was recently
characterized in the granular layer of the mammalian cerebellar cortex with
several procedures, including light and electron microscopic immunocytochemistry
utilizing antibodies to calretinin and neurofilament proteins. Although certain
features of the unipolar brush cells were highlighted in these studies, the
internal fine structure was partially obfuscated by immunoreaction product. In
this study, rat cerebella were prepared for electron microscopy after perfusion
fixation and Araldite embedding, and folia of the vestibulo-cerebellum, where
unipolar brush cells are known to be enriched, were studied by light microscopy
in semithin (0.5-1 micron) sections and by electron microscopy in ultrathin
sections. Unipolar brush cells were easily identified in semithin sections
immunostained with antibodies to GABA and/or glycine, and counterstained with
toluidine blue. The unipolar brush cells have a pale cytoplasm and are GABA and
glycine negative, while Golgi cells are darker and appear positive for GABA and,
for the most part, also for glycine. Sets of identification criteria to
differentiate unipolar brush cells from granule and Golgi cells in standard
electron micrographs are presented. The unipolar brush cells possess many
distinctive features that make them easily distinguishable from other cerebellar
neurons and form unusually conspicuous and elaborate synapses with mossy
rosettes. The unipolar brush cell has a deeply indented nucleus containing
little condensed chromatin. The Golgi apparatus is large and the cytoplasm is
rich in neurofilaments, microtubules, mitochondria, and large dense core
vesicles, but contains few cisterns of granular endoplasmic reticulum. In
addition, unipolar brush cells contain an unusual inclusion, which invariably
lacks a limiting membrane and is made up of peculiar ringlet subunits. The cell
body usually emits a thin axon and is provided with a single, large dendritic
trunk that terminates with a paintbrush-like bunch of branchlets. Numerous
nonsynaptic appendages emanate from the cell body, the dendritic stem, and the
branchlets. The appendages contain rare organelles and lack neurofilaments. The
branchlets contain numerous mitochondria, neurofilaments, large dense core
vesicles, and clusters of clear, small, and round synaptic vesicles. They form
extensive asymmetric synaptic junctions with one or two mossy fibers, which
indicates minimal convergence of excitatory inputs. Under the postsynaptic
densities, the branchlet cytoplasm displays a microfilamentous web. Besides
their contact with mossy rosettes, the branchlets form symmetric and asymmetric
synaptic junctions with presumed Golgi cell boutons that contain pleomorphic
synaptic vesicles, indicating that the unipolar brush cells receive an
inhibitory modulation. Some of these junctions are unusually extensive.(ABSTRACT
TRUNCATED AT 400 WORDS) The unipolar brush cell (UBC) is a medium-sized neuron located in the granular
layer of the cerebellar cortex and morphologically characterized by a single
dendritic shaft terminated by a brush-like tuft [Mugnaini and Floris, J. Comp.
Neurol., in press]. This neuronal class is mostly encountered in the vestibular
part of the cerebellum, although also present in the rest of the vermal folia.
The UBC axon, as seen in Golgi impregnated material, has a tortuous course
throughout the granular layer and gives off number of collaterals of variable
diameter. Some of the axon collaterals enter the white matter only for a short
distance, after which they ascend into the granular layer. Astonishingly, some
of the thick granular collaterals of these axons display characteristic
expansions resembling the rosette excrescences seen on mossy fibers. In our
series these rosettes are mostly of the simple type [Palay, S. and Chan-Palay,
V., Cerebellar Cortex. Cytology and Organization, Springer, Berlin, 1974], i.e.
short swellings with simple globular contours. The exact projection targets and
functional effect of the UBC axon and granular rosettes have still to be
demonstrated. We describe with a variant of the Golgi method a new type of neuron that is
prominently represented in the granular layer of the mammalian
vestibulocerebellum but is presently neglected in all major accounts on the
cerebellum. These neurons, here termed unipolar brush cells, are intermediate in
size between granule cells and Golgi cells. They typically have a thin and
presumably myelinated axon, and a single and stubby dendrite whose tip forms a
tightly packed group of branchlets resembling a paintbrush. The branchlets often
intertwine with the digitiform claws of granule cell dendrites and are
occasionally approached by Golgi cell dendrites, indicating that the unipolar
brush cells may share the input of the other granular layer neurons. Branchlets
of neighboring unipolar brush cells converging into the same neuropil island
also occur. The brush-like tip of the unipolar cell engulfs one or two mossy
fiber rosettes to form an extensive synapse that appears to close recurrent
loops involving the vestibular nuclei. Positive feedback in these loops could
help to explain several motor responses and drive mechanisms of extended
duration that are controlled by the ventral cerebellum. We demonstrate that the metabotropic glutamate receptor mGluR1 alpha is enriched
in two interneuron cell populations in the dorsal division of the cochlear
nucleus. Electron microscopic analysis confirms that mGluR1 alpha
immunoreactivity is concentrated in the dendritic spines of cartwheel cells and
in dendrites of the recently described unipolar brush cells. The cartwheel
cells, which have many similarities to the Purkinje cells of the cerebellum,
participate in a local neuronal circuit that modulates the output of the dorsal
cochlear nucleus. Immunostained unipolar brush cells were observed in granule
cell regions of the cochlear nucleus and the vestibulocerebellum. The presence
of analogous cell types with similar patterns of immunolabeling in the
cerebellum and in the dorsal cochlear nucleus suggests that a shared but as yet
unknown mode of processing may occur in both structures. The unipolar brush cell (UBC) is a novel type of small neuron that is
characterized by sets of morphological and chemical phenotypes. UBCs occur in
the granular layer of the mammalian cerebellar cortex, particularly in folia of
the vestibulocerebellum, and in the granule cell domains of the dorsal cochlear
nucleus. The UBC is characterized by a single dendrite that terminates with a
brush-like tip of dendrioles. The soma, the dendritic stem, and especially the
dendrioles emit short, non-synaptic appendages. The dendrioles represent the
main synaptic apparatus of the UBC and articulate tightly with a single mossy
fiber rosette forming a glomerular array characterized by an extraordinarily
extensive synaptic contact. Electron microscopic and electrophysiological
observations indicate that the unusual synaptic ultrastructure may produce
entrapment of neurotransmitter in the synaptic cleft. While ionotropic glutamate
receptors are enriched in correspondence of the postsynaptic density,
metabotropic glutamate receptors are situated extrasynaptically and are
particularly enriched at the appendages, which usually do not bear synaptic
junctions. Some of the UBCs receive their input from choline
acetyltransferase-positive mossy rosettes originating from the vestibular
nuclei, suggesting that ACh and glutamate are co-released at these synapses. The
UBC brush occupies a glomerulus where granule cell dendrites are intermixed with
the UBC dendrioles, both of which receive synapses from the same mossy fiber
rosette and portions of the Golgi axonal plexus. In addition, the dendrioles are
presynaptic to granule cell dendrites, forming dendrodendritic contacts that
display features of excitatory synapses. Branches of the UBC axon in the
granular layer bear large endings resembling mossy fibers. The UBCs may
represent an extraordinary device for feedforward, excitatory links along the
mossy fiber pathways of cerebellum and dorsal cochlear nucleus. Unipolar brush cells are a class of interneurons in the granular layer of the
mammalian cerebellum that receives excitatory mossy fiber synaptic input in the
form of a giant glutamatergic synapse. Previously, it was shown that the
unipolar brush cell axon branches within the granular layer, giving rise to
large terminals. Single mossy fiber stimuli evoke a prolonged burst of firing in
unipolar brush cells, which would be distributed to postsynaptic targets within
the granular layer. Knowledge of the ultrastructure of the unipolar brush cell
terminals and of the cellular identity of its postsynaptic targets is required
to understand how unipolar brush cells contribute to information processing in
the cerebellar circuit. To investigate the unipolar brush cell axon and its
targets, unipolar brush cells were patch-clamped in fresh parasagittal slices
from rat cerebellar vermis with electrodes filled with Lucifer Yellow and
Biocytin, and examined by confocal fluorescence and electron microscopy.
Biocytin was localized with diaminobenzidine chromogen or gold-conjugated,
silver-intensified avidin. Light microscopic examination revealed a single thin
axon emanating from the unipolar brush cell soma that gave rise to 2-3 axon
collaterals terminating in mossy fiber-like rosettes in the granular layer,
typically within a few hundred microm of the soma. In some cases, axon
collaterals crossed the white matter within the same folium before terminating
in the adjacent granular layer. Electron microscopic examination of serial
ultrathin sections revealed that proximal unipolar brush cell axons and axon
collaterals were unmyelinated and devoid of synaptic contacts. However, the
rosette-shaped enlargements of each collateral formed the central component of
glomeruli where they were surrounded by dendrites of granule cells and/or other
unipolar brush cells, with which they formed asymmetric synaptic contacts. A
long-latency repetitive burst of polysynaptic activity was observed in granule
cells in this cerebellar region following white matter stimulation. The unipolar
brush cell axons, therefore, form a system of cortex-intrinsic mossy fibers. The
results indicate that synaptic excitation of unipolar brush cells by mossy
fibers will drive a large population of granule cells, and thus will contribute
a powerful form of distributed excitation within the basic circuit of the
cerebellar cortex. The unipolar brush cell (UBC), a small interneuron occurring at high density in
the granular layer of the mammalian vestibulocerebellum, receives a giant
glutamatergic synapse from a single mossy fiber (MF) rosette, usually on a brush
of dendritic branchlets. MF stimulation produces a current in the UBC several
orders of magnitude greater in duration than at other glutamatergic synapses. We
assumed that the cytoskeleton would have a special role in plasticity of the
MF-UBC synapse. Neurofilaments and microtubules are enriched in the UBC
somatodendritic compartment but are conspicuously absent in close proximity to
the giant synapse, where standard electron microscopy reveals a
granulo-flocculent material. Because osmium tetroxide fixation during sample
preparation for standard electron microscopy destabilizes actin filaments, we
hypothesized that this subsynaptic granulo-flocculent material is actin-based.
After actin stabilization, we observed prominent, but loosely organized, bundles
of microfilaments at the subsynaptic region of the MF-UBC synapse that linked
the postsynaptic density with the cytoskeletal core of the dendritic branchlets.
Confocal fluorescence microscopy and pre- and postembedding immunogold labeling
with phalloidin and actin antibodies showed that these microfilaments consist of
f-actin and contain little beta-actin. This extraordinary postsynaptic actin
apparatus is ideally situated to form a dynamic framework for glutamate
receptors and other postsynaptic molecules, and to mediate activity-dependent
plastic rearrangements of the giant synapse. The unipolar brush cells are excitatory, cerebellar granular layer interneurons
that receive mossy fiber input on their dendritic brushes in the form of a giant
glutamatergic synapse. We investigated the postnatal development of the brush of
the unipolar brush cell in lobules IX and X by light microscopy and defined the
maturation of mossy fiber-unipolar brush cell synapses and mossy fiber-granule
cell synapses by electron microscopy using calretinin immunocytochemistry to
identify unipolar brush cells. During the first postnatal week, unipolar brush
cells possessed one or two short, branched dendrites. The brush differentiated
primarily during the successive 21 postnatal (P) days, during which it underwent
progressive maturation. This developmental process was subdivided into stages
1-4, which were descriptively termed protodendritic unipolar brush cell (P2-12),
filopodial brush (P12-16), intermediate brush (P16-21), and dendriolar brush
(P21-28) stages. Electron microscopic measurements of individual mossy
fiber-unipolar brush cell and mossy fiber-granule cell synaptic junctions were
made at P12, 16, 21, and 28. While the average length of mossy fiber-unipolar
brush cell synapses increased during development, that of mossy fiber-granule
cell synapses decreased. Comparisons of the lengths of mossy fiber-unipolar
brush cell and mossy fiber-granule cell synapses demonstrated that mossy
fiber-unipolar brush cell synapses were longer on average than mossy
fiber-granule cell synapses for all ages. Frequency distribution histograms also
showed that the percentage of mossy fiber-unipolar brush cell synapses longer
than 0.5 microm was lower in the pooled P12-P16 groups than in the pooled
P21-P28 groups (8 versus 20%). In contrast, mossy fiber-granule cell synapses
longer than 0.5 microm were a small minority at P12, 16, and 21, and occurred
rarely at P28. The present study indicates that mossy fiber-unipolar brush cell
synapses increase in length with the differentiation of the brush dendrioles,
while that of mossy fiber-granule cell synapses decrease with differentiation of
the granule cell dendritic claws. The finding that mossy fiber-unipolar brush
cell synapses were generally longer than mossy fiber-granule cell synapses may
indicate that the properties of the postsynaptic targets play a major role in
shaping synaptic appositions within cerebellar glomeruli. Neurogranin (NG) is a brain-specific protein kinase C substrate involved in the
regulation of calcium signaling and neuronal plasticity. A rostrocaudal
expression profile, with large amounts in telencephalic brain regions and low
expression levels in phylogenetically older brain structures, was reported
previously. In the cerebellum, expression of NG has not been described. By using
immunocytochemistry and in situ hybridization, we found that NG is expressed in
the mouse (C57Bl/6), rat (Wistar), and monkey (Cercopithecus aetiops) cerebella.
In the mouse cerebellum, Golgi cells were strongly immunoreactive for NG,
whereas other cerebellar neurons were devoid of this protein. Cell counts showed
1.6-fold more immunopositive Golgi cells in the hemispheres (61.1 +/- 8.0
cells/mm(2)) than in the vermis (37.5 +/- 3.3 cells/mm(2)). Developmental
studies showed detectable NG in the mouse cerebellum as early as on postnatal
day 10 (P10). In contrast to the mouse, in the rat cerebellum we found only a
few Golgi cells containing NG (hemispheres, 2.4 +/- 0.5 cells/mm(2); vermis, 1.5
+/- 0.3 cells/mm(2)). In the monkey cerebellum, unipolar brush cells, localized
in the granular layer, were heavily labeled, whereas Golgi cells were devoid of
NG. This study demonstrated that NG is strongly expressed in specific
gamma-aminobutyric acidergic neurons in the rodent cerebellum. In addition, NG
expression in the primate cerebellum by brush cells, which are excitatory,
showed remarkable cell type-specific and species-specific expression patterns of
a postsynaptic protein mediating calcium signaling mechanisms. Different isoforms of a vesicular glutamate transporter (VGLUT) mediate
glutamate uptake into synaptic vesicles of excitatory neurons. There is
agreement that the VGLUTs are differentially expressed in brain, and that two
isoforms, VGLUT1 and VGLUT2, are localized to excitatory axon terminals in the
cerebellar cortex. While granule cells express solely VGLUT1, there is no report
about the VGLUT(s) of the unipolar brush cell (UBC), the second type of
glutamatergic interneuron residing in the cerebellar granular layer. In the
mouse, UBCs are particularly numerous in the uvula (lobule IX) and nodulus
(lobule X). These folia contain two distinct subsets of UBCs: one kind expresses
the calcium-binding protein calretinin (CR), and the other kind expresses the
metabotropic glutamate receptor (mGluR) 1alpha. UBCs give rise to an extensive
system of intrinsic mossy fibers (MF), whose terminals innervate granule cells
and other UBCs, altogether similar to those formed by the extrinsic MFs. The
presence of both extrinsic and intrinsic MFs in the vestibulocerebellum makes it
difficult to determine which type of VGLUT is contained in MFs formed by the UBC
axons. Hence, the nodulus was isolated from sagittal cerebellar slices from
postnatal day 10 mice, and cultured for 15-20 days in vitro. Double
immunofluorescence and confocal microscopy showed that mossy terminals of
CR-positive (CR(+)) UBCs were immunoreactive for VGLUT1 and VGLUT2, while mossy
terminals of mGluR1alpha-positive (mGluR1alpha(+)) UBCs were provided with
VGLUT1 only. Moreover, CR(+) dendritic brushes were contacted by mossy terminals
provided with both transporters, while mGluR1alpha(+) dendritic brushes were
contacted by mossy terminals immunopositive for VGLUT1 and immunonegative for
VGLUT2. These data indicate that the two UBC subsets use different modalities of
vesicular glutamate storage and form separate networks. We consider it possible
that expressions of CR with VGLUT1/VGLUT2 and mGluR1alpha(+) with VGLUT1 in the
two subsets of vestibulocerebellar UBCs are determined by specific vestibular
inputs, carried by groups of primary and/or secondary vestibular afferents. Immediate early gene expression in the cerebellar vermis of cats and squirrel
monkeys was stimulated by prolonged whole body rotations. Continuous,
earth-horizontal axis rotations that excited only otoliths or high velocity
vertical axis rotations that excited only semicircular canals resulted in c-fos
immunoreactive nuclei concentrated in the granular layer of lobules X and
ventral IX (the nodulus and ventral uvula), which represent the medial parts of
the vestibulo-cerebellum. Large clusters of labeled nuclei consisting mainly of
granule cells and calretinin-positive unipolar brush cells were present in the
granular layer, whereas Purkinje cell nuclei were unlabeled, and labeled basket
and stellate cell nuclei were scattered in the molecular layer. In other vermal
lobules there was a significant but less dense label than in the nodulus and
ventral uvula. Generally, the extent of c-fos labeling of molecular layer
interneurons was in relation to nuclear labeling of granular layer neurons:
labeling of both basket and stellate cells accompanied nuclear labeling of
neurons throughout the depth of the granular layer, whereas only stellate cells
were labeled when nuclear labeling was restricted to the superficial granular
layer. Yaw horizontal or roll vertical rotations each stimulated c-fos
expression in the cat medial vestibulo-cerebellum to approximately the same
extent. Low-velocity rotations resulted in much less c-fos expression. Similar,
albeit less intense, patterns of c-fos activation were observed in monkeys.
Concentrated c-fos expression in the medial vestibulo-cerebellum after exposure
to a strong head velocity signal that could originate from either otolith or
canal excitation suggests that granule and unipolar brush cells participate in a
neuronal network for estimating head velocity, irrespective of the signal
source. We have studied the temporal and spatial characteristics of the development of
unipolar brush cells (UBCs) in the human cerebellar vermis. Consistently with
previous studies in rodents and cat, we have found that unipolar brush cells
appear at a relatively late phase of cerebellar development and their
development continues up to and beyond the first postnatal year. A series of 23
normal human brains, including 5 adult and 18 fetal or infant brains (between
the 24th gestational week and the 11th postnatal month) were used. In order to
visualize unipolar brush cells, calretinin-immunocytochemistry was performed on
formaldehyde-fixed, paraffin-embedded blocks of the cerebellar vermis. Our
results show that calretinin-immunoreactive unipolar brush cells are not yet
present in the cerebellar vermis at the 28th gestational week. At birth, they
are present in a relatively small number, mostly in the vestibular lobules. At
the 3rd, 5th, 8.5th and 11th postnatal months the number of
calretinin-immunoreactive unipolar brush cells gradually increase, first
appearing in the vestibular lobules, followed by the invasion of the later
developing vermal lobules, spreading in a rostro-caudal and proximo-distal
direction. Although at the 11th postnatal month unipolar brush cells exhibited
adult-like morphological and distributional features, their number appeared to
be lower than in the adult cerebellum. The late maturation of unipolar brush
cells implies that the cytoarchitectonical development of the human cerebellum
is not completed by the end of the first postnatal year. Cerebellar morphogenesis occurs through a complex interplay of cell
proliferation and migration that in mouse and rat begins about midgestation and
ends in the third postnatal week. Cerebellar cells derive from germinative
matrices in the ventricular zone and the external granular layer. Like granule
cells, unipolar brush cells (UBCs) are excitatory interneurons situated in the
granular layer of the cortex and innervated by mossy fibers. While granule cells
are produced from the external granular layer, the generation of UBCs is still
controversial. We utilized the reeler mutant mouse, which has widespread
misplacement of neurons due to lack of Reelin protein, to ascertain the origin
of UBCs. In the reeler cerebellum, which is small and lacks foliation, Purkinje
cells are greatly reduced in number and in large part are located ectopically in
deep cerebellar masses. Granule cells are also reduced in number and form an
irregular granule cell layer. In this study we demonstrate that the reeler
mutation influences the positioning of UBCs and also significantly reduces their
number. Both subsets of UBCs identified in normal mouse, the calretinin-positive
and the metabotropic glutamate receptor 1alpha-positive subsets, are affected in
the reeler. About 40% of the calretinin-positive UBCs are ectopically situated
in the deep cerebellar regions and the immediate vicinity of the ependyma of the
fourth ventricle. Ectopic UBCs have discrete, although somewhat looser brushes
than granular layer UBCs, but form synaptic junctions with complex axon
terminals, possibly belonging to mossy fibers and UBC axons, like their normally
situated counterpart. The observed displacement of UBCs in the reeler suggests
that they originate from the ventricular zone. Epidermal growth factor receptor pathway substrate 8 (Eps8) is a widely
expressed multidomain signaling protein that coordinates two disparate
GTPase-dependent mechanisms: actin reorganization via Ras/Rac pathways and
receptor trafficking via Rab5. Expression of Eps8, the gene encoding the
founding member of the Eps8 family of proteins, was found in cerebellum by
virtual Northern analysis and in situ hybridization. Because the cerebellum has
a well-known cellular architecture and is a favored model to study synaptic
plasticity and actin dynamics, we sought to analyze Eps8 localization in rat
cerebellar neurons and synapses by light and electron microscopy. Specificity of
Eps8-antibody was demonstrated by immunoblots and in brain sections. In
cerebellum, unipolar brush cells (UBCs) were densely Eps8 immunopositive and
granule cells were moderately immunostained. In both types of neuron
immunoreaction product was localized to the somatodendritic and axonal
compartments. Postsynaptic immunostained foci were demonstrated in the glomeruli
in correspondence of the synapses formed by mossy fiber terminals with granule
cell and UBC dendrites. These foci appeared especially evident in the UBC brush,
which contains an extraordinary postsynaptic apparatus of actin microfilaments
facing synaptic junctions of the long and segmented varieties. Eps8
immunoreactivity was conspicuously absent in Purkinje cells and their actin-rich
dendritic spines, in all types of inhibitory interneurons of the cerebellum,
cerebellar nuclei neurons, and astrocytes. In conclusion, Eps8 protein in
cerebellum is expressed exclusively by excitatory cortical interneurons and is
intracellularly compartmentalized in a cell-class specific manner. This is the
first demonstration of the presence of a member of the Eps8 protein family in
UBCs and its enrichment at postsynaptic sites. Unipolar brush cells (UBCs) are a class of excitatory interneuron found in the
granule cell layer of the vestibulocerebellum. Mossy fibers form excitatory
inputs on to the paint brush shaped dendrioles in the form of giant,
glutamatergic synapses, activation of which results in prolonged bursts of
action potentials in the postsynaptic UBC. The axons of UBCs themselves form
mossy fiber contacts with other UBCs and granule cells, forming an excitatory,
intrinsic cerebellar network that has the capacity to synchronize and amplify
mossy fiber inputs to potentially large populations of granule cells. In this
paper, we demonstrate that UBCs in rat cerebellar slices express low voltage
activated (LVA) fast-inactivating and high voltage activated (HVA) slowly
inactivating calcium channels. LVA calcium currents are mediated by T-type
calcium channels and they are associated with calcium increases in the dendrites
and to a lesser extent the cell soma. HVA currents, mediated by L-type calcium
channels, are slowly inactivating and they produce larger overall increases in
intracellular calcium but with a similar distribution pattern. We review these
observations alongside several recent papers that examine how intrinsic membrane
properties influence UBCs firing patterns and we discuss how UBC signaling may
affect downstream cerebellar processing. Unipolar brush cells (UBCs) are excitatory interneurons with their somata
located in the granular layer. Recently, T-brain factor 2 (Tbr2) was shown to be
expressed in a subset of UBCs in mouse cerebellum. Scrambler mice exhibit severe
cerebellum abnormalities, including the failure of embryonic Purkinje cell
dispersal and a complete absence of foliation due to a mutation in the
disabled-1 adaptor protein. Since most UBC markers are expressed postnatally, it
has proven difficult to identify the relationship between developing Purkinje
cell clusters and migrating UBCs. Because scrambler mice closely mimic normal
embryonic day 18 cerebellum, we examined whether Tbr2-positive UBCs are
associated with Purkinje cell cluster markers such as zebrin II, which is the
most studied compartmentation marker in the cerebellum. We investigated the
distribution of Tbr2-positive UBCs in this mutant by using anti-Tbr2
immunocytochemistry. The data revealed that Tbr2 immunoreactivity was
exclusively present in the nucleus of UBCs in scrambler cerebellum. Based on
expression data, a Tbr2-positive UBC map was constructed. In addition,
Tbr2-positive UBCs are found associated with ectopic zebrin II-immunoreactive
Purkinje cell clusters in scrambler cerebellum. These data suggest that UBCs use
Purkinje cell compartmentation to migrate into their final position through
interactions with the embryonic array of specific Purkinje cell subtypes. |
Which driver mutations have been identified for Diffuse Intrinsic Pontine Glioma (DIPG)? | We found conservation of heterozygous K27M mutations in H3F3A (n = 4) or HIST1H3B (n = 3) across all primary, contiguous, and metastatic tumor sites in all DIPGs | Diffuse intrinsic pontine glioma (DIPG) is a highly morbid form of pediatric
brainstem glioma. Here, we present the first comprehensive protein, mRNA, and
methylation profiles of fresh-frozen DIPG specimens (n = 14), normal brain
tissue (n = 10), and other pediatric brain tumors (n = 17). Protein profiling
identified 2,305 unique proteins indicating distinct DIPG protein expression
patterns compared to other pediatric brain tumors. Western blot and
immunohistochemistry validated upregulation of Clusterin (CLU), Elongation
Factor 2 (EF2), and Talin-1 (TLN1) in DIPGs studied. Comparisons to mRNA
expression profiles generated from tumor and adjacent normal brain tissue
indicated two DIPG subgroups, characterized by upregulation of Myc (N-Myc) or
Hedgehog (Hh) signaling. We validated upregulation of PTCH, a membrane receptor
in the Hh signaling pathway, in a subgroup of DIPG specimens. DNA methylation
analysis indicated global hypomethylation of DIPG compared to adjacent normal
tissue specimens, with differential methylation of 24 genes involved in Hh and
Myc pathways, correlating with protein and mRNA expression patterns. Sequencing
analysis showed c.83A>T mutations in the H3F3A or HIST1H3B gene in 77 % of our
DIPG cohort. Supervised analysis revealed a unique methylation pattern in
mutated specimens compared to the wild-type DIPG samples. This study presents
the first comprehensive multidimensional protein, mRNA, and methylation
profiling of pediatric brain tumor specimens, detecting the presence of two
subgroups within our DIPG cohort. This multidimensional analysis of DIPG
provides increased analytical power to more fully explore molecular signatures
of DIPGs, with implications for evaluating potential molecular subtypes and
biomarker discovery for assessing response to therapy. Here, we review the recent literature on molecular discoveries in ependymomas
and pediatric diffuse gliomas. Ependymomas can now be categorized into three
location-related subgroups according to their biological profile: posterior
fossa ependymomas, group A (PFA) and B (PFB), and supratentorial ependymomas.
Although no recurrently mutated genes were found throughout these groups of
ependymomas, PFA exhibited a CpG island methylator phenotype, PFB was associated
with extensive chromosomal aberrations, and the C11orf95-RELA fusion gene was
frequently observed in supratentorial ependymomas. Meanwhile, it has now become
apparent that pediatric diffuse gliomas have a distinct genetic status from
their adult counterparts, even though they share an indistinguishable histology.
In pediatric low-grade diffuse gliomas, an intragenic duplication of the portion
of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of
MYB/MYBL1 were found recurrently and mutually exclusively. As for non-brainstem
high-grade tumors, in addition to H3F3A, TP53, and ATRX mutations, which were
frequently observed in older children, recurrent fusions involving NTRK1, NTRK2,
and NTRK3 were reported in infants younger than 3 years of age. Moreover, in
diffuse intrinsic pontine gliomas (DIPG), recurrent somatic mutations of ACVR1
were found in association with HIST1H3B mutations. 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. Author information:
(1)Department of Human Genetics, McGill University, Montreal, Québec, Canada H3A
1B1.
(2)McGill University and Génome Québec Innovation Centre, Montreal, Québec,
Canada H3A 0G1.
(3)Research Center for Genetic Medicine, Children's National Health System,
Washington, District Of Columbia 20010, USA.
(4)Institute for Biomedical Sciences, George Washington University School of
Medicine and Health Sciences, Washington, District Of Columbia 20052, USA.
(5)Department of Pediatrics, McGill University and McGill University Heath
Centre Research Institute, Montreal, Québec, Canada H4A 3J1.
(6)Division of Pathology, Children's National Health System, Washington,
District Of Columbia 20010, USA.
(7)Center for Cancer and Blood Disorders, Children's National Health System,
Washington, District Of Columbia 20010, USA.
(8)The Department of Neurological Surgery, George Washington University School
of Medicine and Health Sciences, Washington, District Of Columbia 20052, USA.
(9)Center for Molecular Oncologic Pathology, Department of Medical Oncology,
Dana-Farber Cancer Institute, Boston, Massachusett 02115, USA.
(10)Department of Pathology, CHU Ste-Justine, Université de Montréal, Montreal,
Québec, Canada H3T 1C5.
(11)UQ Child Health Research Centre, The University of Queensland, Brisbane,
Queensland 4101, Australia.
(12)University of Queensland Diamantina Institute, The University of Queensland,
Brisbane, Queensland 4102, Australia.
(13)Oncology Service, Children's Health Queensland Hospital and Health Service,
Brisbane, Queensland 4101, Australia.
(14)National Cancer Institute, National Institute of Health, Bethesda, Maryland
20892, USA.
(15)Department of Pathology, Montreal Neurological Hospital, McGill University,
Montreal, Québec, Canada H3A 2B4.
(16)Brain Tumour Institute, Center for Neuroscience and Behavioral Medicine,
Children's National Health System, Washington, District Of Columbia, 20010, USA.
(17)Department of Integrative Systems Biology, George Washington University
School of Medicine and Health Sciences, Washington, District Of Columbia 20052,
USA. |
Does Jarid2 play a role in early embryo development? | Yes. Jarid2 coordinates Nanog expression and PCP/Wnt signaling required for efficient ESC differentiation and early embryo development. | Jumonij (JMJ)/Jarid2 plays important roles in embryonic development and
functions as a transcriptional repressor. Using yeast two-hybrid screening, we
have identified a cofactor of JMJ, the zinc finger protein 496 (Zfp496) that
contains a SCAN, KRAB and zinc finger domain. Our molecular analyses indicate
that Zfp496 functions as a transcriptional activator. Further, Zfp496 inhibits
the transcriptional repression of JMJ and JMJ represses the transcriptional
activation of Zfp496. This study demonstrates that JMJ physically and
functionally interacts with Zfp496, which will provide important insights into
endogenous target gene regulation by both factors. The Polycomb group (PcG) proteins have an important role in controlling the
expression of genes essential for development, differentiation and maintece
of cell fates. The Polycomb repressive complex 2 (PRC2) is believed to regulate
transcriptional repression by catalysing the di- and tri-methylation of lysine
27 on histone H3 (H3K27me2/3). At present, it is unknown how the PcG proteins
are recruited to their target promoters in mammalian cells. Here we show that
PRC2 forms a stable complex with the Jumonji- and ARID-domain-containing
protein, JARID2 (ref. 4). Using genome-wide location analysis, we show that
JARID2 binds to more than 90% of previously mapped PcG target genes. Notably, we
show that JARID2 is sufficient to recruit PcG proteins to a heterologous
promoter, and that inhibition of JARID2 expression leads to a major loss of PcG
binding and to a reduction of H3K27me3 levels on target genes. Consistent with
an essential role for PcG proteins in early development, we demonstrate that
JARID2 is required for the differentiation of mouse embryonic stem cells. Thus,
these results demonstrate that JARID2 is essential for the binding of PcG
proteins to target genes and, consistent with this, for the proper
differentiation of embryonic stem cells and normal development. |
Is PUVA therapy indicated for eczema treatment? | Yes, PUVA (psoralen plus UVA) therapy is effective for eczema treatment and has relatively few side effects. | PUVA therapy successfully cleared various dermatoses mainly confined to the
palms and soles in 18 of 20 patients treated. The conditions treated were:
plaque-type psoriasis, pustular psoriasis, endogenous eczema and persistent
palmoplantar pustulosis. Seventeen patients were treated in a controlled study
of PUVA therapy versus no treatment at all and in 16 of these patients the
disease was cleared in the PUVA-treated areas while the untreated areas remained
unchanged or deteriorated. Twelve of the 18 patients were maintained in a clear
state by continued maintece PUVA treatment over 6--31 months while 3 patients
had a spontaneous remission and are free of disease off all treatment. Topical photochemotherapy with psoralen and its derivatives
4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP), with UVA
irradiation, was evaluated with regard to minimum phototoxic dose,
concentration, timing of UVA irradiation and systemic and local side-effects, in
healthy volunteers. Psoralen (0.005%) in aqueous gel was found to be superior to
TMP and 8-MOP in aqueous gel. No hyperpigmentation was seen after topical PUVA
treatment with psoralen in aqueous gel. Patients with plaque-type psoriasis (n =
7), palmoplantar psoriasis (n = 7) and hyperkeratotic eczema (n = 2) were
treated. Topical PUVA therapy was effective in most psoriasis patients, without
the occurrence of local or systemic side-effects. Moreover, hyperkeratotic
eczema patients who did not respond to conventional therapy showed partial
remission. These results indicate that topical PUVA therapy with psoralen in
aqueous gel is a useful therapeutic modality for treatment of psoriasis
patients, and patients with recalcitrant dermatoses such as palmoplantar
psoriasis and hyperkeratotic eczema. BACKGROUND: Topical PUVA therapy has become a useful alternative for patients
who cannot tolerate the systemic side effects of nausea and headache or are
concerned about the ophthalmologic risk associated with oral PUVA therapy.
However, there is no study to date on the systemic absorption of psoralen after
the localized application of topical paint PUVA.
OBJECTIVE: This study was designed to assess the plasma level of
8-methoxypsoralen (8-MOP) after paint PUVA therapy for patients with
palmoplantar psoriasis or eczema.
METHODS: Reverse-phase high-pressure liquid chromatography was used to determine
8-MOP plasma levels in eight patients with palmoplantar psoriasis and two with
eczema. Three patients receiving oral PUVA therapy served as the control group.
RESULTS: Plasma levels of 8-MOP taken 1, 6, and 24 hours after topical PUVA
treatments of patients with palmoplantar psoriasis were undetectable. One
patient with hand eczema consistently had detectable 8-MOP levels 1 hour after
topical PUVA treatments.
CONCLUSION: This report indicates that there is minimal, if any, systemic
absorption of 8-MOP after topical PUVA treatment of patients with palmoplantar
psoriasis. Seventeen patients with persistent chronic hand eczema were treated with topical
0.1% 8-methoxypsoralen and UVA (PUVA) for 8 weeks. Significant improvement was
achieved in 5 cases (29%), moderate improvement in 9 (53%), and little
improvement in 3 (18%). The mean number of PUVA treatments was 22.2, and the
mean total UVA dose was 63.5 J/cm2. There was no association between clinical
response and duration of hand eczema, positive patch test reaction, or atopic
status. Since topical PUVA has no risk of systemic side effects, it should be
considered as an alternative treatment for patients with chronic hand eczema who
are resistant to other topical medications. BACKGROUND: Vesicular dyshidrotic palmoplantar eczema is a common disorder but
treatment is difficult. Localized photochemotherapy (cream psoralen-UVA [PUVA])
has widely been used for therapy. Although the efficacy of cream PUVA therapy is
well known, potential side effects may occur. Therefore, a more standardized
safe and effective UV therapy should be carried out.
OBJECTIVE: This study compared the effects of localized high-dose UVA1
irradiation versus topical cream PUVA for treatment of chronic vesicular
dyshidrotic eczema.
METHODS: On the basis of the assessment of the Dyshidrotic Area and Severity
Index, the decrease of score points on the UVA1-treated side was compared with
the decrease on the cream PUVA-treated side in 27 patients. In addition,
analysis of serum markers was performed.
RESULTS: Of 27 patients, 24 showed a good response to localized UVA1 irradiation
or cream PUVA. Dyshidrotic Area and Severity Index scores significantly
decreased on both sides and were reduced to half of the pretreatment values. No
statistically significant differences between localized UVA1 irradiation or
cream PUVA could be detected.
CONCLUSION: This study demonstrates that localized UVA1 phototherapy is easy to
perform and appears to be an effective and safe treatment for vesicular
dyshidrotic eczema. OBJECTIVE: To study whether oral psoralen-UV-A (PUVA) with a portable tanning
unit at home is as effective as hospital-administered bath PUVA in patients with
chronic hand eczema.
DESIGN: Open-label randomized controlled trial, with a 10-week treatment period
and an 8-week follow-up period.
SETTING: Two university hospital dermatology departments in the Netherlands,
specializing in hand eczema.
PATIENTS: One hundred fifty-eight patients with moderate to severe chronic hand
eczema (more than 1 year in duration).
INTERVENTIONS: Oral PUVA using methoxsalen capsules and a simple portable
commercial facial tanning unit, or hospital-administered bath PUVA with
trioxsalen.
MAIN OUTCOME MEASURES: The primary outcome was clinical assessment by a hand
eczema score (evaluation of desquamation, erythema, vesiculation, infiltration,
fissures, itch, and pain, each on a 4-point scale) after 10 weeks of treatment.
The secondary outcome was hand eczema score at 8 weeks of follow-up, after
completion of treatment. The tertiary outcome was travel cost and time off work.
RESULTS: Both groups showed a comparable and substantial decrease in hand eczema
score (meaningful clinical improvement). This decrease was maintained during the
follow-up period. Patients treated with oral PUVA at home had lower travel costs
and less time off work.
CONCLUSIONS: Oral PUVA at home has a clinically relevant efficacy, similar to
that of hospital-administered bath PUVA. This effect was maintained during an
8-week follow-up period. It resulted in lower travel costs and less time off
work. BACKGROUND: Hand eczema is a chronic skin disorder characterized by a poor
response to conventional therapies. Although local PUVA has been proven to be
effective in the treatment of chronic hand eczema, little is known about the
efficacy and safety of local narrowband UVB (TL-01) for this condition. The aim
of our study was to compare the efficacy and safety of local narrowband UVB
phototherapy with paint-PUVA in patients with chronic hand eczema of dry and
dyshidrotic types unresponsive to conventional therapies.
PATIENTS/METHODS: Fifteen patients (nine men and six women) with chronic hand
eczema of dry and dyshidrotic types was included in this prospective,
comparative study based on a left to right comparison pattern. The treatments
were administered with local narrowband UVB irradiation on one hand and local
paint-PUVA using 0.1% 8-methoxypsoralen gel on the other hand three times a week
over a 9-week period. The NB-UVB irradiation was administered using a local
NB-UVB system equipped with TL-01 lamps. The initial dose was 150 mJ/cm(2) for
each patient. An increasing percentile dose schedule based on an increase of 20%
was used in every session, until a final dose of 2000 mJ/cm(2) was reached.
Evaluation of clinical scores was carried out every 3 weeks during the treatment
period.
RESULTS: Twelve of the 15 recruited patients completed the study. There was a
statistically significant decrease in the mean clinical score at the third,
sixth and nineth week in both groups. The difference in clinical response
between the two treatment modalities was not statistically significant at the
end of the 9-week treatment period. In the narrowband UVB-treated side, the
tolerance of all the patients to the treatment was good all patients
well-tolerated the treatment with the exception of mild xerosis that responded
to topical emollients.
CONCLUSION: Local narrowband UVB phototherapy regimen is as effective as
paint-PUVA therapy in patients with chronic hand eczema of dry and dyshidrotic
types. BACKGROUND: Both oral and bath PUVA with 8-methoxypsoralen (8-MOP) have been
shown to be effective in the treatment of chronic palmoplantar eczema. However,
most studies were retrospective and did not include longer follow-up periods.
AIM: To compare the therapeutic efficacy, tolerability and duration of remission
after oral vs. bath PUVA using 8-MOP in patients with chronic palmoplantar
eczema.
METHODS: Twenty-nine patients were randomly allocated to treatment with oral or
bath PUVA. Treatment was given thrice weekly for a maximum of 20 weeks. The
primary outcome measure was the improvement in eczema score at the end of
treatment. After clearing patients were followed up until relapse or up to 40
months.
RESULTS: Overall, both PUVA modalities appeared comparably effective. However,
after stratifying according to eczema type, significant differences in
therapeutic outcome in general as well as in response to the two regimes were
found. Dyshidrotic eczema responded better to both treatments (P=0.048) and
remained longer in remission than hyperkeratotic eczema. Hyperkeratotic eczema
cleared significantly better with oral than with bath PUVA (P=0.03).
CONCLUSION: Oral PUVA is preferable for patients with hyperkeratotic eczema and
bath PUVA for patients with dyshidrotic eczema. BACKGROUND: Numerous studies have confirmed the short-term effectiveness of
8-methoxypsoralen bath PUVA therapy in patients with chronic palmoplantar
dermatoses; however, little is known about long-term results.
PATIENTS AND METHODS: In this retrospective study we examined the long-term
results in 79 patients (mean age: 48 years) with chronic palmoplantar dermatoses
who were treated with bath PUVA three times a week over an 8-year period. A good
clinical response (a reduction of more than 50% of the skin lesions) occurred
after a mean of 23 treatments and a mean cumulative UVA dose of 39 J/cm(2) in 51
patients (65%). In 2007 a questionnaire was sent to these 51 patients to assess
the long-term outcome.
RESULTS: With bath PUVA treatment, the best results were found in patients with
hyperkeratotic eczema (17/22; 77% good clinical response) followed by patients
with palmoplantar psoriasis (26/41; 63%) and patients with dyshidrotic eczema
(8/16; 50%). Thirty-four patients (67%) answered the questionnaire after a mean
follow-up interval of 4.3 years (10-87 months). Among these patients, 36%
reported an improved course of disease after PUVA therapy with reduced frequency
and/or intensity of the skin rash, and 29% of patients reported continued
complete clearance. 79% of our patients reported a long-term reduction in the
use of topical corticosteroids during the follow-up period (mean: 4.3 years). In
addition, 67% of patients reported a lasting improvement in quality of life.
CONCLUSIONS: These data show that bath PUVA may have a long-term, beneficial
influence on the course of disease in a majority of patients with recalcitrant
chronic palmoplantar dermatoses. The aim of this study was to investigate the effect of iontophoresis combined
with local psoralen plus ultraviolet A (PUVA) therapy in chronic foot eczema. A
randomized, observer-blinded, multi-centre study was conducted in 48 patients
with chronic moderate-to-severe foot eczema randomized to one of 3 groups: In
the iontophoresis group local bath-PUVA was preceded by iontophoresis. In the
PUVA group only local PUVA was given. The corticosteroid group was treated with
fluticasone. All treatments were given for 8 weeks, with an 8-week follow-up
period. The primary efficacy parameter was eczema score described by Rosén et
al. Secondary efficacy parameters were a global impression by the patient, and
the Dermatology Life Quality Index (DLQI). The eczema score and the DLQI
decreased significantly over time. There were no significant differences in the
decrease in eczema score (p=0.053) and DLQI values (p=0.563) between the 3
treatments. The DLQI values in our chronic foot eczema patients were high. There
was no obvious advantage of local bath-PUVA with or with-out iontophoresis over
local steroid therapy. |
What is DECKO? | DECKO (Double Excision CRISPR Knockout) is a dual CRISPR tool, which is cloned using a single starting oligonucleotide, thereby affording simplicity and scalability to CRISPR knockout studies of non-coding genomic elements, including long non-coding RNAs. | Author information:
(1)Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and
Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. [email protected].
(2)Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain.
[email protected].
(3)Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88,
08003, Barcelona, Spain. [email protected].
(4)Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and
Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. [email protected].
(5)Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain.
[email protected].
(6)Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88,
08003, Barcelona, Spain. [email protected].
(7)Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and
Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. [email protected].
(8)Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain.
[email protected].
(9)Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88,
08003, Barcelona, Spain. [email protected].
(10)Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and
Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. [email protected].
(11)Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain.
[email protected].
(12)Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88,
08003, Barcelona, Spain. [email protected].
(13)Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and
Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. [email protected].
(14)Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain.
[email protected].
(15)Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88,
08003, Barcelona, Spain. [email protected].
(16)Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and
Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. [email protected].
(17)Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain.
[email protected].
(18)Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88,
08003, Barcelona, Spain. [email protected]. |
List genes associated with hypolipidemia. | PCSK9
APOB
ANGPTL3
ANGPTL4
MTP | BACKGROUND: Angiopoietin-like protein 3 (ANGPTL3) affects lipid metabolism by
inhibiting the activity of lipoprotein and endothelial lipases. Angptl3 knockout
mice have marked hypolipidemia, and heterozygous carriers of ANGPLT3,
loss-of-function mutations were found among individuals in the lowest quartile
of plasma triglycerides in population studies. Recently, 4 related individuals
with primary hypolipidemia were found to be compound heterozygotes for ANGPTL3
loss-of-function mutations.
METHODS AND RESULTS: We resequenced ANGPTL3 in 4 members of 3 kindreds
originally identified for very low levels of low-density lipoprotein cholesterol
and high-density lipoprotein cholesterol (0.97±0.16 and 0.56±0.20 mmol/L,
respectively) in whom no mutations of known candidate genes for monogenic
hypobetalipoproteinemia and hypoalphalipoproteinemia had been detected. These
subjects were found to be homozygous or compound heterozygous for ANGPTL3
loss-of-function mutations (p.G400VfsX5, p.I19LfsX22/p.N147X) associated with
the absence of ANGPTL3 in plasma. They had reduced plasma levels of
triglyceride-containing lipoproteins and of HDL particles that contained only
apolipoprotein A-I and pre-β-high-density lipoprotein. In addition, their
apolipoprotein B-depleted sera had a reduced capacity to promote cell
cholesterol efflux through the various pathways (ABCA1-, SR-BI-, and
ABCG1-mediated efflux); however, these subjects had no clinical evidence of
accelerated atherosclerosis. Heterozygous carriers of the ANGPTL3 mutations had
low plasma ANGPTL3 and moderately reduced low-density lipoprotein cholesterol
(2.52±0.38 mmol/L) but normal plasma high-density lipoprotein cholesterol.
CONCLUSIONS: Complete ANGPTL3 deficiency caused by loss-of-function mutations of
ANGPTL3 is associated with a recessive hypolipidemia characterized by a
reduction of apolipoprotein B and apolipoprotein A-I-containing lipoproteins,
changes in subclasses of high-density lipoprotein, and reduced cholesterol
efflux potential of serum. Partial ANGPTL3 deficiency is associated only with a
moderate reduction of low-density lipoprotein. Dyslipidemia is a commonly encountered clinical condition and is an important
determit of cardiovascular disease. Although secondary factors play a role in
clinical expression, dyslipidemias have a strong genetic component. Familial
hypercholesterolemia is usually due to loss-of-function mutations in LDLR, the
gene coding for low density lipoprotein receptor and genes encoding for proteins
that interact with the receptor: APOB, PCSK9 and LDLRAP1. Monogenic
hypertriglyceridemia is the result of mutations in genes that regulate the
metabolism of triglyceride rich lipoproteins (eg LPL, APOC2, APOA5, LMF1,
GPIHBP1). Conversely familial hypobetalipoproteinemia is caused by inactivation
of the PCSK9 gene which increases the number of LDL receptors and decreases
plasma cholesterol. Mutations in the genes APOB, and ANGPTL3 and ANGPTL4 (that
encode angiopoietin-like proteins which inhibit lipoprotein lipase activity) can
further cause low levels of apoB containing lipoproteins. Abetalipoproteinemia
and chylomicron retention disease are due to mutations in the microsomal
transfer protein and Sar1b-GTPase genes, which affect the secretion of apoB
containing lipoproteins. Dysbetalipoproteinemia stems from dysfunctional apoE
and is characterized by the accumulation of remts of chylomicrons and very
low density lipoproteins. ApoE deficiency can cause a similar phenotype or
rarely mutations in apoE can be associated with lipoprotein glomerulopathy. Low
HDL can result from mutations in a number of genes regulating HDL production or
catabolism; apoAI, lecithin: cholesterol acyltransferase and the ATP-binding
cassette transporter ABCA1. Patients with cholesteryl ester transfer protein
deficiency have markedly increased HDL cholesterol. Both common and rare genetic
variants contribute to susceptibility to dyslipidemias. In contrast to rare
familial syndromes, in most patients, dyslipidemias have a complex genetic
etiology consisting of multiple genetic variants as established by genome wide
association studies. Secondary factors, obesity, metabolic syndrome, diabetes,
renal disease, estrogen and antipsychotics can increase the likelihood of
clinical presentation of an individual with predisposed genetic susceptibility
to hyperlipoproteinemia. The genetic profiles studied are far from complete and
there is room for further characterization of genes influencing lipid levels.
Genetic assessment can help identify patients at risk for developing
dyslipidemias and for treatment decisions based on 'risk allele' profiles. This
review will present the current information on the genetics and pathophysiology
of disorders that cause dyslipidemias. |
What is the enzymatic activity of PARL? | the mitochondrial protease presenilin-associated rhomboid-like (PARL). Rhomboids are a recently discovered family of widely distributed intramembrane serine proteases. | Multiple factors promote insulin resistance. In this study, we evaluated the
mRNA levels of presenilins-associated rhomboid-like protein (PARL) and
mitochondrial content and enzyme activity from skeletal muscle isolated from
insulin-resistant rats. Rats fed a high-fat diet for 35 days developed moderate
insulin resistance, which was determined by an increase in plasma glucose and
insulin concentrations following an oral glucose tolerance test. The PARL mRNA
level was lower in the insulin-resistant rats than in control animals, and is
associated with low mitochondrial content and reduced mitochondrial enzyme
activity in the skeletal muscle from the insulin-resistant rats. The results
suggest that high-fat-diet-induced insulin resistance is associated with
mitochondrial dysfunction in skeletal muscle, and may be the result of the
decreased expression of the PARL gene, which encodes the protein with functional
significance in mitochondria. The mitochondrial rhomboid protease Parl governs apoptosis, morphology,
metabolism and might be implicated in Parkinson's disease, but the structural
basis of its activity and complex regulation remain unknown. We report the
discovery of γ-cleavage, a proteolytic event on the loop connecting the first
transmembrane helix (TMH) of Parl to the 6-TMH catalytic rhomboid domain of the
protease. This cleavage disrupts the '1+6' structure that defines every
mitochondrial rhomboid and generates a new form of Parl, PROD
(Parl-rhomboid-domain). Structure-function analysis of Parl suggests that
γ-cleavage could be implicated in eliminating Parl proteolytic activity, and
structural modeling of PROD reveals structural conservation with the bacterial
rhomboid GlpG. However, unlike bacterial rhomboids, which employ a diad-based
mechanism of catalysis, Parl appears to use a conserved mitochondrial
rhomboid-specific Asp residue on TMH-5 in a triad-based mechanism of catalysis.
This work provides unexpected insights into the structural determits
regulating Parl stability and activity in vivo, and reveals a complex cascade of
proteolytic events controlling the function of the protease in the
mitochondrion. |
Do brown fat cells produce heat? | Yes, brown fat cells produce heat. | Heat production of isolated brown-fat cells by addition of noradrenaline and
glucagon was measured in warm-acclimated control, cold-acclimated and
heat-acclimated rats by use of a twin-type conduction microcalorimeter.
Noradrenaline and glucagon induced maximum heat production per 10(6) cells in
dose of 1 microgram/ml. Heat produced by maximum thermogenic response to
glucagon was twice as much as that to noradrenaline. Thermogenic response to
noradrenaline was markedly increased in cold-acclimated brown adipocytes, while
it was reduced in heat-acclimated ones. Thermogenic response to glucagon was
significantly reduced in heat-acclimated brown adipocytes, while it was not
affected in cold-acclimated brown adipocytes. In 134 out of 180 perirenal fat samples (74%) derived from Japanese necropsy
cases aged from 1 month to 86 years, the brown fat tissue persisted in variable
amounts. Brown fat cells were classified into 6 types: Type 1 cells are
fat-depleted cells filled with granular cytoplasm and are believed to be
produced after oxidation of fat for heat production. Type 2 cells are
small-locular cells suitable for rapid oxidation of fat droplets. Type 3
(middle-locular) and 4 (large-locular) represent fat-storage cells containing
large amounts of fat. Type 5 cells are thought to be transitional forms between
multilocular brown fat cells and monolocular white fat cells. Type 6
(cytoplasm-rich multilocular) cells, usually found together with Type 1 cells,
are thought to be fat-depleting or -consuming cells, since in them fat droplets
are reduced in number and size probably in consequence of oxidation of fat, but
by contrast granular cytoplasm is increased in amount separating the individual
fat droplets by thick cytoplasmic septa. The occurrence of Types 1 and/or 6
cells that has been revealed in 65 out of the total 180 samples (36%), suggests
that the oxidation of fat for the thermogenesis proceeds in the brown fat tissue
and that brown fat cells partially undergo fat depletion. In the present study,
the thermogenesis of human brown fat tissue was suggested chiefly with regard to
the occurrence of Types 1 and/or 6 cells. In the majority of perirenal fat
samples from infants (1-11 months) relatively numerous Types 1 and 6 cells were
frequently revealed together with Type 2 cells, suggesting rapid and active heat
production in support of the view that in human infants the brown fat tissue may
be thermogenetically active to maintain body temperature. In the same manner,
marked ability to produce a considerable amount of heat was evidenced in brown
fat tissue of children and teenagers. In younger and elderly adults the
frequency of occurrence and the amount of the perirenal brown fat tissue were
decreased but Types 1 and/or 6 cells could be found in 17-40% of them,
infrequently together, with Type 2 cells, suggesting persistence of the
thermogenic activity with occasional large heat production especially in younger
adults (20-39 years). Thus, the results obtained in this study have clarified
that the human brown fat tissue can respond to stimuli given to the body by
oxidation of stored fat even in the latest decades of life. In cases of death
from burning, drowning, bleeding, drug poisoning etc., numerous Types 1 and/or 6
cells were found, suggesting that an active fat oxidation would take place in
brown fat tissue assumedly as the result of the raised noradrenalin level in
this tissue. The so-called small cytoplasmic cells found in perirenal fats from
cases of death from liver cirrhosis and other causes were assumed to be atrophic
fat-depleted brown fat cells. This review deals with the comparative observations of brown fat tissue in the
bat, a hibernator, and in man, a nonhibernator. In both mammals, the brown fat
is distributed in restricted portions of the body and brought into a
thermogenetic activity by an acute drop in ambient temperature. Light
microscopic examination was performed on the interscapular brown fat of bats
captured monthly throughout one year; electron microscopic observations were
made using a bat captured in April and another in September. Human perirenal
brown fat was investigated light-microscopically on tissues derived from 215
fresh necropsy cases (Japanese) of both sexes aged from one month to 93 years.
Brown adipose tissue was recognized only in 162 (75%) of the 215 samples,
because brown fat cells were reduced by transformation into white fat cells.
Human brown fat cells were classified into six types according to the
morphological features of their lipid droplets. These reflect different
functional states of intracellular heat production. The Type 1 (D) cell is a
fat-depleted brown fat cell with a darkly stained cytoplasm; it is specific to
humans. Human perirenal brown fat cells begin to show a transformation into
white fat cells already at the infantile stage. This change occurs from the
peripheral toward the central portion of the lobule, so that various functioning
cell types remain only in the central area of the lobules. In contrast to
humans, bat interscapular brown fat cells exhibit regular seasonal changes in
size and lipid droplet content, so that the cells could not be classified as in
humans into definite types according to the features of their lipid droplets.
The most conspicuous difference between brown fat tissue in the nonhibernator
and hibernator is that in the latter, white fat cells never occur within the
brown fat tissue. In the hibernator, thermogenesis in the brown fat is necessary
for both the arousal from hibernation and the maintece of hibernation as well
as rutting. In human perirenal brown fat tissue, darkly stained fat-depleted
cells (D) occupy, with other cell types (CR, CR'), an important part in the
reversible heat production cycle of the brown fat tissue. The "hypothermic" or
"cold syndrome (cold injury)" is a disorder affecting inadequately protected
infants in severely cold seasons, accompanied by lethargy, hypothermia and
lactation refusal and revealing hemorrhagic pneumonia in necropsy. The brown fat
tissue in such infants is composed exclusively of fat-depleted brown fat
cells.(ABSTRACT TRUNCATED AT 400 WORDS) The maximum thermogenic capacity of brown fat cells from control and cold
acclimated rats was measured using a continuous-flow microcalorimetric system.
The content of the 32.000 D, brown fat specific protein, thermogenin, was
measured in the cells used for heat production measurements by competitive
ELISA. The ratio between the maximal thermogenic capacity and the amount of
thermogenin for control and cold acclimated rats was compared. It was found that
the ratio between the two parameters decreased during cold acclimation due to a
decrease in maximal thermogenic capacity and an increase in the amount of
thermogenin, indicating regulation of heat production either at thermogenin or
receptor level. Intracellular potentials of brown fat cells in lightly anesthetized
cold-acclimated rats were measured in vivo. The effects of adrenergic agonists
and antagonists on these potentials were examined in an attempt to relate the
electrical activity of the cells to the adrenergic-induced stimulation of brown
fat thermogenesis.Norepinephrine, the physiological mediator of brown fat heat
production, significantly depolarized the membrane of these cells in vivo. This
was effected either upon norepinephrine administration (3-100 mug/kg body wt) or
excitation of the transsected nerve trunk to the interscapular fat pad and
appreciably inhibited (55%) by doses of propranolol (1 mg/kg) sufficient to
abolish the temperature increase of the tissue. Since theophylline (325 mum/kg)
did not depolarize the cells, although it stimulated thermogenesis in the
tissue, the depolarizing effect of norepinephrine is interpreted as being at
least partially associated with biochemical events terminating in the activation
of adenylate cyclase. However, the norepinephrine-induced electrical changes and
the ensuing increase in brown fat thermogenesis appear to be causally
independent and experimentally separable. On the other hand, our data do not
preclude the speculation that the membrane phenomenon, if accompanied by
increased Na(+), may serve partially to regulate the metabolic rate of brown fat
during long-term physiological stimulation (e.g., cold stress) by increasing the
rate of ATP utilization via the Na(+)/K(+) pump. The ability of cells from the stromal-vascular fraction of rat brown adipose
tissue to develop into adipocytes in primary cell cultures was investigated.
Comparison was made with precursor cells isolated by the same procedure from the
white adipose tissue of the same animals and cultured in parallel under
identical conditions. The culture procedure used allowed the cells isolated from
both tissues to rapidly proliferate and differentiate. During the first week in
culture the brown fat cells grew to confluence and accumulated fat in a
multilocular way. During the second week, further fat was accumulated, but the
cells remained multilocular. Analysis of the parallel white fat cell cultures
revealed clear differences between the two adipocyte types, although the rates
of cell growth were identical. Measurement of the size of the cellular lipid
inclusions as a function of the time in culture indicated a much higher number
of fat droplets larger than 30 micron in the white adipocytes. Moreover, after
isolation of pelleting fractions of both cultured cell types, comparative
functional analysis of their mitochondria by oxygen consumption measurement, as
well as direct cytochrome-c-oxidase determinations, showed a significantly
higher amount of mitochondria in the brown fat cell fractions than in the white
fat cell fractions. It was concluded that mature brown fat contains precursor
cells which can proliferate and develop into adipocytes in monolayer cell
culture and which have inherent characteristics distinct from those of white fat
precursor cells. Thermogenin is the purine-nucleotide binding polypeptide in brown adipose tissue
mitochondria (Mr 32 000) which confers upon these mitochondria the ability to
produce heat. An enzyme-linked immunosorbent assay (ELISA) has been developed to
demonstrate and quantitate the occurrence of thermogenin antigen in small
amounts of tissue, and thus to characterize different depots of fat tissue as
white or brown. The extreme sensitivity of the method allows determination of
thermogenin in samples equivalent to less than 1 mg tissue. The results indicate
that thermogenin seems to be exclusively localised in brown fat mitochondria (as
compared to white fat, liver or heart muscle mitochondria), and thermogenin
antigen could only be found in brown adipocytes (as compared to white
adipocytes). Thus, brown and white adipose tissue are probably ontogenetically
different. BACKGROUND: In infants, nonshivering thermogenesis from brown adipose tissue
provides an important source of heat for thermoregulation. Infants are known to
have a high susceptibility to hypothermia during anesthesia. To investigate
whether this could be due to an inhibition of nonshivering thermogenesis by
anesthetics, the effect of preincubation with volatile anesthetics on the
norepinephrine-induced heat production of brown adipocytes was investigated.
METHODS: Brown adipocytes from hamsters were isolated with a collagenase
digestion method and preincubated with volatile anesthetics. The cells were
stimulated with norepinephrine, and heat production, measured as oxygen
consumption, was monitored polarographically.
RESULTS: Norepinephrine addition led to a 20-fold increase in the rate of oxygen
consumption (thermogenesis). However, preincubation of cells with 3% halothane
reduced the response to norepinephrine by more than 70%. The potency of
norepinephrine (the median effective concentration) was not affected by
halothane. Full effect of halothane was reached quickly, and after halothane
withdrawal, the thermogenic response recovered, although rather slowly.
Halothane, isoflurane, and enflurane were approximately equipotent inhibitors of
thermogenesis, with concentrations of approximately 0.7% resulting in 50%
inhibition. The inhibitory effect of 1% halothane was unaffected by the presence
of 74% nitrous oxide, but nitrous oxide alone also reduced thermogenesis.
CONCLUSIONS: Volatile anesthetics severely attenuated the thermogenic response
to norepinephrine of isolated brown-fat cells. It is inferred that
brown-adipose-tissue heat production is reduced during (and probably also some
time after) anesthesia. Because infants are dependent on brown-fat-derived
nonshivering thermogenesis for thermal balance, the inhibition by volatile
anesthetic agents of brown-adipocyte heat production may at least partly explain
the susceptibility of infants to hypothermia during and after anesthesia. CONTEXT: CD31 (platelet-endothelial cell adhesion molecule-1; PECAM-1), an
adhesion molecule involved in the process of angiogenesis, is used as a marker
of normal and neoplastic vascularization. During the assessment of angiogenesis
and vascular invasion in a thymic carcinoid tumor, we observed unexpected
immunostaining for CD31 in perithymic brown fat nests.
OBJECTIVE: To determine whether CD31 is expressed by normal and neoplastic cells
of brown fat, a tissue whose thermogenetic activity depends heavily on high
perfusion.
DESIGN: Formalin-fixed, paraffin-embedded archival tissues were immunostained by
the labeled avidin-biotin method using antibodies against CD31 (clones JC70A and
1A10) after retrieval of heat-induced epitopes. Archival tissues included
perithymic, periadrenal, axillary, and neck adipose tissue in which were
embedded nests of brown fat (n = 15), hibernoma (n = 3), lipoma (n = 6),
well-differentiated liposarcoma (n = 4), and myxoid liposarcoma (n = 4).
RESULTS: Invariably, multivacuolated and univacuolated adipocytes of normal
brown fat and hibernomas were intensely positive for the CD31 antigen. The
immunostaining "decorated" cell membranes and the membranes of intracytoplasmic
vacuoles. No expression of CD31 was found in normal adipocytes of white fat, in
neoplastic cells of lipomas, or in multivacuolated lipoblasts of
well-differentiated and myxoid liposarcomas.
CONCLUSIONS: The spectrum of cell types that express CD31 is expanded to include
normal and neoplastic brown fat cells. We speculate that the expression of CD31
may play a role in the development and maintece of the vascular network
characterizing this specialized adipose tissue. Moreover, CD31 may inhibit the
Bax-mediated apoptosis of brown fat cells. For practical purposes, CD31 may be
used as an immunohistochemical marker for distinguishing between white and brown
fat and for diagnosing hibernoma in paraffin sections. The white adipose organ is composed of both subcutaneous and several
intra-abdominal depots. Excess abdominal adiposity is a major risk factor for
metabolic disease in rodents and humans, while expansion of subcutaneous fat
does not carry the same risks. Brown adipose produces heat as a defense against
hypothermia and obesity, and the appearance of brown-like adipocytes within
white adipose tissue depots is associated with improved metabolic phenotypes.
Thus, understanding the differences in cell biology and function of these
different adipose cell types and depots may be critical to the development of
new therapies for metabolic disease. Here, we found that Prdm16, a brown adipose
determination factor, is selectively expressed in subcutaneous white adipocytes
relative to other white fat depots in mice. Transgenic expression of Prdm16 in
fat tissue robustly induced the development of brown-like adipocytes in
subcutaneous, but not epididymal, adipose depots. Prdm16 transgenic mice
displayed increased energy expenditure, limited weight gain, and improved
glucose tolerance in response to a high-fat diet. shRNA-mediated depletion of
Prdm16 in isolated subcutaneous adipocytes caused a sharp decrease in the
expression of thermogenic genes and a reduction in uncoupled cellular
respiration. Finally, Prdm16 haploinsufficiency reduced the brown fat phenotype
in white adipose tissue stimulated by β-adrenergic agonists. These results
demonstrate that Prdm16 is a cell-autonomous determit of a brown fat-like
gene program and thermogenesis in subcutaneous adipose tissues. Thyroid hormone is a major regulator of thermogenesis, acting both in peripheral
organs and on central autonomic pathways. Mice heterozygous for a point mutation
in thyroid hormone receptor α1 display increased thermogenesis as a consequence
of high sympathetic brown fat stimulation. Surprisingly, despite the
hypermetabolism, their body temperature is not elevated. Here we show, using
isolated tail arteries, that defective thyroid hormone receptor α1 signaling
impairs acetylcholine-mediated vascular relaxation as well as
phenylephrine-induced vasoconstriction. Using infrared thermography on conscious
animals, we demonstrate that these defects severely interfere with appropriate
peripheral heat conservation and dissipation, which in turn leads to
compensatory alterations in brown fat activity. Consequently, when the
vasoconstrictive defect in mice heterozygous for a point mutation in thyroid
hormone receptor α1 was reversed with the selective α1-adrenergic agonist
midodrine, the inappropriate heat loss over their tail surface was reduced,
normalizing brown fat activity and energy expenditure. Our analyses demonstrate
that thyroid hormone plays a key role in vascular heat conservation and
dissipation processes, adding a unique aspect to its well-documented functions
in thermoregulation. The data thus facilitate understanding of temperature
hypersensitivity in patients with thyroid disorders. Moreover, the previously
unrecognized connection between cardiovascular regulation and metabolic activity
revealed in this study challenges the interpretation of several experimental
paradigms and questions some of the currently derived hypotheses on the role of
thyroid hormone in thermogenesis. Brown adipocytes oxidize fatty acids to produce heat in response to cold or
caloric overfeeding. The motivation and function of the development of brown fat
may thus counteract obesity, though this remains uncertain. We investigated the
brown adipocyte proteome by two-dimensional gel electrophoresis followed by mass
spectrometry. Comparative analyses of proteins focused on total protein spots to
filter differentially expressed proteins during the differentiation of mouse
primary brown preadipocytes. A Western blot analysis was performed to verify the
target proteins. The results indicated that 10 protein spots were differentially
expressed with significant changes, including the three up-regulated proteins of
prohibitin, hypoxanthine-guanine phosphoribosyltransferase, and enoyl-CoA
hydratase protein; the 5 down-regulated proteins of triosephosphate isomerase,
elongation factor 2, α-tropomyosin slow, endophilin-B1, and cofilin-1 (CFL1);
and the two unequivocally expressed proteins of peroxiredoxin-1 and collagen
α-1(i) chain precursor. We found that during brown adipogenesis, CFL1 has an
inhibitory effect on brown adipocyte differentiation. The overexpression of CFL1
inhibited the brown fat deposition and repressed the brown marker genes UCP1,
PRDM16, PGC-1α and PPARγ via actin dynamics and polymerization. These
observations may be novel findings that bring new insight into the detailed
mechanisms of brown adipogenesis and identify possible therapeutic targets for
anti-obesity.
BIOLOGICAL SIGNIFICANCE: We use 2-DE to compare the proteomes of adipocytes
during the brown adipogenesis of primary mouse preadipocytes. We identified 10
proteins that are differentially expressed. Among these, we found that the actin
binding protein CFL1 inhibits the differentiation of brown preadipocytes. CFL1
overexpressing cells showed lower deposition of brown fat droplets, and the
brown marker genes of UCP1, PRDM16, PGC-1α and PPARγ were decreased through
actin dynamics and polymerization. Adipose tissue is a major metabolic organ, and it has been traditionally
classified as either white adipose tissue (WAT) or brown adipose tissue (BAT).
WAT and BAT are characterized by different anatomical locations, morphological
structures, functions, and regulations. WAT and BAT are both involved in energy
balance. WAT is mainly involved in the storage and mobilization of energy in the
form of triglycerides, whereas BAT specializes in dissipating energy as heat
during cold- or diet-induced thermogenesis. Recently, brown-like adipocytes were
discovered in WAT. These brown-like adipocytes that appear in WAT are called
beige or brite adipocytes. Interestingly, these beige/brite cells resemble white
fat cells in the basal state, but they respond to thermogenic stimuli with
increased levels of thermogenic genes and increased respiration rates. In
addition, beige/brite cells have a gene expression pattern distinct from that of
either white or brown fat cells. The current epidemic of obesity has increased
the interest in studying adipocyte formation (adipogenesis), especially in
beige/brite cells. This review summarizes the developmental process of adipose
tissues that originate from the mesenchymal stem cells and the features of these
three different types of adipocytes. Current efforts to treat obesity and associated disorders focus on the
stimulation of energy expenditure by increasing thermogenesis, for instance
through activating brown adipose tissue or more recently "beige" or "brite" fat,
a relatively novel type of adipose tissue with putative thermogenic potential.
In this commentary, we aim to provide an alternative perspective on the current
trend of analyzing and manipulating thermogenesis, brought about by our recent
publication, in which we investigated the unexpected hypermetabolic phenotype of
an animal model with defective thyroid hormone receptor α1 signaling. These mice
display elevated brown adipose tissue thermogenesis; surprisingly, however,
their body temperature is lower, pointing to a defect in heat conservation.
Using infrared thermography and wire myograph experiments, we revealed that the
tail arteries of the mutant mice are less sensitive to contractile stimuli,
which leads to insufficient peripheral vasoconstriction and heat loss over the
tail surface. This heat loss in turn lowers body temperature and triggers the
additional thermogenesis. Our findings add a new aspect to the role of thyroid
hormone in thermoregulation, and encourage a more holistic view in future
studies in the field of thermogenesis, including the often-overlooked heat
dissipation and recordings of body temperature. Obesity develops when energy intake chronically exceeds energy expenditure.
Because brown adipose tissue (BAT) dissipates energy in the form of heat,
increasing energy expenditure by augmenting BAT-mediated thermogenesis may
represent an approach to counter obesity and its complications. The ability of
BAT to dissipate energy is dependent on expression of mitochondrial uncoupling
protein 1 (UCP1). To facilitate the identification of pharmacological modulators
of BAT UCP1 levels, which may have potential as antiobesity medications, we
developed a transgenic model in which luciferase activity faithfully mimics
endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic
screening of a library using cells derived from this model yielded a small
molecule that increases UCP1 expression in brown fat cells and mice. Upon
adrenergic stimulation, compound-treated mice showed increased energy
expenditure. These tools offer an opportunity to identify pharmacologic
modulators of UCP1 expression and uncover regulatory pathways that impact
BAT-mediated thermogenesis. Brown adipose tissue (BAT), a specialized fat that dissipates energy to produce
heat, plays an important role in the regulation of energy balance. Two types of
thermogenic adipocytes with distinct developmental and anatomical features exist
in rodents and humans: classical brown adipocytes and beige (also referred to as
brite) adipocytes. While classical brown adipocytes are located mainly in
dedicated BAT depots of rodents and infants, beige adipocytes sporadically
reside with white adipocytes and emerge in response to certain environmental
cues, such as chronic cold exposure, a process often referred to as "browning"
of white adipose tissue. Recent studies indicate the existence of beige
adipocytes in adult humans, making this cell type an attractive therapeutic
target for obesity and obesity-related diseases, including type 2 diabetes. This
Review aims to cover recent progress in our understanding of the anatomical,
developmental, and functional characteristics of brown and beige adipocytes and
discuss emerging questions, with a special emphasis on adult human BAT. The ability of brown adipocytes (fat cells) to dissipate energy as heat shows
great promise for the treatment of obesity and other metabolic disorders.
Employing pluripotent stem cells, with an emphasis on directed differentiation,
may overcome many issues currently associated with primary fat cell cultures. In
addition, three-dimensional (3D) cell culture systems are needed to better
understand the role of brown adipocytes in energy balance and treating obesity.
To address this need, we created 3D "Brown-Fat-in-Microstrands" by microfluidic
synthesis of alginate hydrogel microstrands that encapsulated cells and directly
induced cell differentiation into brown adipocytes, using mouse embryonic stem
cells (ESCs) as a model of pluripotent stem cells, and brown preadipocytes as a
positive control. Brown adipocyte differentiation within microstrands was
confirmed by immunocytochemistry and qPCR analysis of the expression of the
brown adipocyte-defining marker uncoupling protein 1 (UCP1), as well as other
general adipocyte markers. Cells within microstrands were responsive to a
β-adrenergic agonist with an increase in gene expression of thermogenic UCP1,
indicating that these "Brown-Fat-in-Microstrands" are functional. The ability to
create "Brown-Fat-in-Microstrands" from pluripotent stem cells opens up a new
arena to understanding brown adipogenesis and its implications in obesity and
metabolic disorders. Adult humans have heat-producing and energy-consuming brown adipose tissue in
the clavicular region of the neck. There are two types of brown adipose cells,
the so-called classic and beige adipose cells. Brown adipose cells produce heat
by means of uncoupler protein 1 (UCP1) from fatty acids and sugar. By applying
positron emission tomography (PET) measuring the utilization of sugar, the
metabolism of brown fat has been shown to multiply in the cold, presumably
influencing energy consumption. Active brown fat is most likely present in young
adults, persons of normal weight and women, least likely in obese persons. Brown fat is a thermogenic tissue that generates heat to maintain body
temperature in cold environments and dissipate excess energy in response to
overfeeding. We have addressed the role of the IGFIR in the brown fat
development and function. Mice lacking IGFIR exhibited normal brown adipose
tissue/body weight in knockout (KO) vs control mice. However, lack of IGFIR
decreased uncoupling protein 1 expression in interscapular brown fat and beige
cells in inguinal fat. More importantly, the lack of IGFIR resulted in an
impaired cold acclimation. No differences in the total fat volume were found in
the KO vs control mice. Epididymal fat showed larger adipocytes but with a lower
number of adipocytes in KO vs control mice at age 12 months. In addition, KO
mice showed a sustained moderate hyperinsulinemia and hypertriglyceridemia upon
time and hepatic insulin insensitivity associated with lipid accumulation, with
the outcome of a global insulin resistance. In addition, we found that the
expression of uncoupling protein 3 in the skeletal muscle was decreased and its
expression was increased in the heart in parallel with the expression of beta-2
adrenergic receptors. Upon nonobesogenic high-fat diet, we found a severe
insulin resistance in the liver and in the skeletal muscle, but unchanged
insulin sensitivity in the heart. In conclusion, our data suggest that IGFIR it
is not an essential growth factor in the brown fat development in the presence
of the IR and very high plasma levels of IGF-I, but it is indispensable for full
brown fat functionality. Brown fat is a specialized fat depot that can increase energy expenditure and
produce heat. After the recent discovery of the presence of active brown fat in
human adults and novel transcription factors controlling brown adipocyte
differentiation, the field of the study of brown fat has gained great interest
and is rapidly growing. Brown fat expansion and/or activation results in
increased energy expenditure and a negative energy balance in mice and limits
weight gain. Brown fat is also able to utilize blood glucose and lipid and
results in improved glucose metabolism and blood lipid independent of weight
loss. Prolonged cold exposure and beta adrenergic agonists can induce browning
of white adipose tissue. The inducible brown adipocyte, beige adipocyte evolving
by thermogenic activation of white adipose tissue have different origin and
molecular signature from classical brown adipocytes but share the
characteristics of high mitochondria content, UCP1 expression and thermogenic
capacity when activated. Increasing browning may also be an efficient way to
increase whole brown fat activity. Recent human studies have shown possibilities
that findings in mice can be reproduced in human, making brown fat a good
candidate organ to treat obesity and its related disorders. The worldwide epidemic of obesity and type 2 diabetes has greatly increased
interest in the biology and physiology of adipose tissues. Adipose (fat) cells
are specialized for the storage of energy in the form of triglycerides, but
research in the last few decades has shown that fat cells also play a critical
role in sensing and responding to changes in systemic energy balance. White fat
cells secrete important hormone-like molecules such as leptin, adiponectin, and
adipsin to influence processes such as food intake, insulin sensitivity, and
insulin secretion. Brown fat, on the other hand, dissipates chemical energy in
the form of heat, thereby defending against hypothermia, obesity, and diabetes.
It is now appreciated that there are two distinct types of thermogenic fat
cells, termed brown and beige adipocytes. In addition to these distinct
properties of fat cells, adipocytes exist within adipose tissue, where they are
in dynamic communication with immune cells and closely influenced by innervation
and blood supply. This review is intended to serve as an introduction to adipose
cell biology and to familiarize the reader with how these cell types play a role
in metabolic disease and, perhaps, as targets for therapeutic development. All mammals own two main forms of fat. The classical white adipose tissue builds
up energy in the form of triglycerides and is useful for preventing fatigue
during periods of low caloric intake and the brown adipose tissue instead of
inducing fat accumulation can produce energy as heat. Since adult humans possess
significant amounts of active brown fat depots and their mass inversely
correlates with adiposity, brown fat might play an important role in human
obesity and energy homeostasis. New evidence suggests two types of thermogenic
adipocytes with distinct developmental and anatomical features: classical brown
adipocytes and beige adipocytes. Beige adipocyte has recently attracted special
interest because of its ability to dissipate energy and the possible ability to
differentiate itself from white adipocytes. Importantly, adult human brown
adipocyte appears to be mainly composed of beige-like adipocytes, making this
cell type an attractive therapeutic target for obesity and obesity-related
diseases. Because many epigenetic changes can affect beige adipocyte
differentiation, the knowledge of the circumstances that affect the development
of beige adipocyte cells may be important for therapeutic strategies. In this
review we discuss some recent observations arising from the great physiological
capacity of these cells and their possible role as ways to treat obesity and
diabetes mellitus type 2. Brown and beige adipocytes expend chemical energy to produce heat and are
therefore important in regulating body temperature and body weight. Brown
adipocytes develop in discrete and relatively homogenous depots of brown adipose
tissue, whereas beige adipocytes are induced to develop in white adipose tissue
in response to certain stimuli - notably, exposure to cold. Fate-mapping
analyses have identified progenitor populations that give rise to brown and
beige fat cells, and have revealed uticipated cell-lineage relationships
between vascular smooth muscle cells and beige adipocytes, and between skeletal
muscle cells and brown fat. In addition, non-adipocyte cells in adipose tissue,
including neurons, blood vessel-associated cells and immune cells, have crucial
roles in regulating the differentiation and function of brown and beige fat. |
What is a mimotope vaccine? | A mimotope vaccine contains peptide mimics of specific antigen epitopes, which alter the antigen presentation and/or T cell activation to increase the expansion of tumor-specific T cells and are able to induce polyclonal antibodies response. | We have previously reported the identification, using human immune sera, of
mimotopes of human hepatitis B virus surface Ag (HBsAg) displayed on filamentous
phage. To test if these mimotopes could be useful in developing a vaccine
against the human hepatitis B virus (HBV), we have compared the humoral immune
response of animals immunized either with a recombit HBsAg vaccine, or with
mimotopes. Immunogens were prepared by fusing the mimotopes on different carrier
molecules (phage coat protein pIII and pVIII, recombit human H ferritin, HBV
core peptide) and by synthesizing multiple antigenic peptides carrying the
mimotopes' amino acid sequences. These immunogens were injected into mice and
rabbits and sera were collected and tested for the presence of HBsAg-specific
Abs. Our data confirm that mimotopes can induce a humoral immune response
resembling that induced by the original Ag, and HBsAg mimotopes displayed on
phage prove to be the best immunogens, inducing the most reproducible and potent
immunization. Mimotopes that react as HBV subtype-specific Ags do not show this
specificity as immunogen and induce a nonsubtype-restricted response.
Furthermore, mimotopes displayed on phage elicit a strong response to HBsAg in a
strain of mouse reported to show a low response to it. These results indicate
that mimotopes identified from random peptide libraries through utilizing human
immune sera could be important leads for the derivation of new vaccines. Peptide mimics of a conformational epitope that is recognized by a mAb with
antitumor activity are promising candidates for formulations of anticancer
vaccines. These mimotope vaccines are able to induce a polyclonal Ab response
focused to the determit of the mAb. Such attempts at cancer immunotherapy are
of special interest for maligt melanoma that is highly resistant to
chemotherapy and radiotherapy. In this study, we describe for the first time the
design and immunogenicity of a vaccine containing a mimotope of the human high
m.w. melanoma-associated Ag (HMW-MAA) and the biological potential of the
induced Abs. Mimotopes were selected from a pVIII-9mer phage display peptide
library with the anti-HMW-MAA mAb 225.28S. The mimotope vaccine was then
generated by coupling the most suitable candidate mimotope to tetanus toxoid as
an immunogenic carrier. Immunization of rabbits with this vaccine induced a
specific humoral immune response directed toward the epitope recognized by the
mAb 225.28S on the native HMW-MAA. The induced Abs inhibited the in vitro growth
of the melanoma cell line 518A2 up to 62%. In addition, the Abs mediated 26%
lysis of 518A2 cells in Ab-dependent cellular cytotoxicity. Our results indicate
a possible application of this mimotope vaccine as a novel immunotherapeutic
agent for the treatment of maligt melanoma. Carbohydrate mimetic peptides of tumor associated carbohydrate antigens (TACA)
are T-cell-dependent antigens and, therefore, immunization with these surrogates
is predicted to overcome the low immunogenicity of carbohydrate antigens.
Consistent with this hypothesis, we show that among the potential immune cells
involved, peptide immunization led to an increase in T-cell populations. While
peptide mimetics may also function as TLR binding ligands, we did not observe
evidence of involvement of NK cells. Examining tumor challenged animals, we
observed that peptide immunization and not tumor cells rendered IL-12
responsiveness to T-cells, as T-cells from peptide-immunized mice produced
IFN-gamma upon stimulation with IL-12. Cyclophosphamide administration enhanced
the anti-tumor efficacy of the vaccine, which was achieved by enhancing T-cell
responses with no effect on NK cell population. Prophylactic immunization of
mice with a DNA construct encoding carbohydrate mimetic peptides indicated a
specific role for the mimotope vaccine in anti-tumor immune responses. These
data suggest a role for both CD4(+) and CD8(+) T-cells induced by mimotopes of
TACA in protective immunity against tumor cells. CD20 is expressed strictly by B-cells and is ubiquitously expressed at high
surface densities of maligt human B-cells. This suggests that CD20 may be a
tumor target for immunotherapy of B-cell lymphomas. Rituximab, a chimeric
monoclonal antibody directed against CD20, has been demonstrated to be an
effective treatment for non-Hodgkin's lymphoma (NHL) and some autoimmune
diseases. In the current study, we used the phage display technique to generate
mimotopes that complemented the screening Ab Rituximab. A total of seven
candidate mimotopes were isolated from a 12-mer peptide library from which one
mimotope was conjugated to keyhole limpet hemocyanin (KLH) or tetanus toxoid
(TT). The immunogenicity of the two vaccines generated was examined in BALB/c
mice. Sera from the vaccinated mice demonstrated high-titer specific antibodies
to the mimotope conjugates. Antibody binding to native CD20 and Ab-mediated
cytotoxicity (CDC, complement-dependent cytotoxicity) were also analyzed. Our
data suggest that a Rituximab mimotope may be a useful tool for the construction
of a functional vaccine to treat B-cell maligcy as well as some CD20 related
autoimmune disorders. To explore the mimotope vaccine approach against infectious bursal disease virus
(IBDV), five IBDV-specific monoclonal antibodies (mAbs) were prepared and their
binding peptides were screened against a phage-displayed 12-mer peptide library.
After three rounds of biopanning, 12 phages were selected for each mAbs and
their specificity to IBDV was verified by sandwich and competitive inhibition
ELISAs. Seven phages per mAb were sequenced and their amino acid sequences were
deduced. The five representative sequences of mimotopes corresponding mAbs were
determined. An artificial gene, designated 5epis (5 epitopes) and consisting of
the five mimotopes arranged in tandem (F1-F7-B34-2B1-2G8) with four GGGS
spacers, was chemically synthesized and cloned into a prokaryotic expression
plasmid pET28b. The protein, designated r5EPIS, was efficiently expressed in
Escherichia coli and showed a size of 10kDa in SDS-PAGE. The r5EPIS protein
reacted with anti-IBDV mAbs and polyclonal antibodies in Western blot
immunoassays. Immunization of SPF chickens with r5EPIS protein (with Freund
adjuvant, 50mug per injection on day 0 and 14) evoked high levels of antibody
(12,800 by ELISA/1600 by virus neutralizing assay at day 21) and protected 100%
of the chickens against a challenge of 200 ELD(50) of IBDV GX8/99 strain, which
sharply contrasted with the, respectively, 13.3% and 6.6% survival rate in the
adjuvant group and the untreated group. The multi-mimotope protein r5EPIS
promises to be a novel subunit vaccine candidate for IBDV. The GD2 ganglioside expressed on neuroectodermal tumor cells is weakly
immunogenic in tumor-bearing patients and induces predomitly IgM antibody
responses in the immunized host. Using a syngeneic mouse challenge model with
GD2-expressing NXS2 neuroblastoma, we investigated novel strategies for
augmenting the effector function of GD2-specific antibody responses induced by a
mimotope vaccine. We demonstrated that immunization of A/J mice with DNA vaccine
expressing the 47-LDA mimotope of GD2 in combination with IL-15 and IL-21 genes
enhanced the induction of GD2 cross-reactive IgG2 antibody responses that
exhibited cytolytic activity against NXS2 cells. The combined immunization
regimen delivered 1 day after tumor challenge inhibited subcutaneous (s.c.)
growth of NXS2 neuroblastoma in A/J mice. The vaccine efficacy was reduced after
depletion of NK cells as well as CD4(+) and CD8(+) T lymphocytes suggesting
involvement of innate and adaptive immune responses in mediating the antitumor
activity in vivo. CD8(+) T cells isolated from the immunized and cured mice were
cytotoxic against syngeneic neuroblastoma cells but not against allogeneic EL4
lymphoma, and exhibited antitumor activity after adoptive transfer in
NXS2-challenged mice. We also demonstrated that coimmunization of
NXS2-challenged mice with the IL-15 and IL-21 gene combination resulted in
enhanced CD8(+) T cell function that was partially independent of CD4(+) T cell
help in inhibiting tumor growth. This study is the first demonstration that the
mimotope vaccine of a weakly immunogenic carbohydrate antigen in combination
with plasmid-derived IL-15 and IL-21 cytokines induces both innate and adaptive
arms of the immune system leading to the generation of effective protection
against neuroblastoma challenge. PURPOSE: The high molecular weight melanoma-associated antigen (HMW-MAA) is an
attractive target for immunotherapy of maligt melanoma. We have recently
generated a vaccine based on the HMW-MAA mimotope 225D9.2+ that was able to
induce anti-HMW-MAA antibodies with antitumor activity in vitro. Here, we
investigated the antitumor activity of these antibodies in a human melanoma
xenotransplant severe combined immunodeficient (SCID) mouse model.
EXPERIMENTAL DESIGN: Tumors were established by injecting the human melanoma
518A2 cells into C.B.17 SCID/SCID mice. In tumor prevention experiments, 200
microg purified total IgG antibodies were injected intravenously the same day or
on day 5 in therapeutic experiments. Antibody administration was repeated every
fourth day and tumor volumes were measured. Antibody specificity and tumor
infiltration by macrophages were investigated by immunohistochemistry.
RESULTS: Within 35 days after cell inoculation, antibody treatment reduced tumor
growth up to 40% in the therapeutic and up to 62% in the tumor prevention
experiments compared with the control mice. In tumors of all groups, a similar
distribution of the HMW-MAA and no differences in infiltration of macrophages
were detected by immunohistochemistry.
CONCLUSIONS: Here, we showed that antibodies induced by the 225D9.2+ mimotope
effectively inhibited melanoma tumor growth. Additional mechanisms besides
antibody-dependent cell cytotoxicity like disruption of interactions of melanoma
cells mediated by extracellular matrix components seem to be involved in tumor
growth inhibition. Based on our findings, we suggest that active immunization
with this mimotope might be a promising strategy for treatment of melanoma. BACKGROUND: Mimotopes are peptides mimicking protein, carbohydrates or lipid
epitopes and can be generated by phage display technology. When selected by
antibodies, they represent exclusively B-cell epitopes and are devoid of
antigen/allergen-specific T-cell epitopes. Coupled to carriers or presented in a
multiple antigenic peptide form mimotopes achieve immunogenicity and induce
epitope-specific antibody responses upon vaccination.
OBJECTIVE/METHODS: In allergy IgG antibodies may block IgE binding to allergens,
whereas other IgG antibody specificities enhance this and support the
anaphylactic reaction. In cancer, inhibitory antibody specificities prevent
growth signals derived from overexpressed oncogenes, whereas growth-promoting
specificities enhance signalling and proliferation. Therefore, the mimotope
concept is applicable to both fields for epitope-specific vaccination and
analysis of conformational B-cell epitopes for the allergen/antigen.
RESULTS/CONCLUSIONS: Mimotope technology is a relatively young theme in
allergology and oncology. Still, proof of concept studies testing allergen and
tumour mimotope vaccines suggest that mimotopes are ready for clinical trials. The likelihood of identifying peptides of sufficient quality for the development
of effective cancer vaccines by screening of phage display libraries is low.
Here, we introduce the sequential application of systematic amino acid
substitution by SPOT synthesis. After the substitution of two amino acids within
the sequence of a phage display-derived mimotope of disialoganglioside GD2
(mimotope MA), the novel mimotope C3 showed improved GD2 mimicry in vitro.
Peptide vaccination with the C3 mimotope induced an 18-fold increased anti-GD2
serum response associated with reduction of primary tumour growth and
spontaneous metastasis in contrast to MA mimotope controls in a syngeneic
neuroblastoma model. In summary, SPOT provides an ideal optimisation tool for
the development of phage display-derived cancer vaccines. A major challenge for inducing antitumor immune responses with native or
modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient
uptake and processing by dendritic cells (DCs) to activate immune effector cells
and limit the generation of regulatory T cell activity. We analyzed the ability
of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2
ganglioside, 47-LDA, to selectively expand adoptively transferred,
tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice.
Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice
were lymphodepleted by nonmyeloablative total body irradiation or a
myeloablative regimen that required bone marrow transplantation. The 47-LDA
mimotope was presented to DCs either as a linear polypeptide in conjunction with
universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment
(47-LDA-Fcgamma2a) to deliver the antigenic cassette to the activating Fcgamma
receptors. We demonstrate that immunization of adoptively transferred T cells in
tumor-bearing mice with the 47-LDA mimotope expressed in the context of the
activating Fc fusion protein induced higher levels of antitumor immune responses
and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of
the therapeutic 47-LDA-Fcgamma2a-DC vaccine was comparable to that achieved by a
virotherapy-associated cancer vaccine using a recombit oncolytic vaccinia
virus expressing the 47-LDA-Fcgamma2a fusion protein. The latter treatment,
however, did not require total body irradiation or adoptive cell transfer and
resulted in induction of antitumor immune responses in the setting of
established tolerance, paving the way for testing novel anticancer treatment
strategies. Vaccination has become an important therapeutic approach to the treatment of
Alzheimer's disease (AD), however, immunization with Abeta amyloid can have
unwanted, potentially lethal, side effects. Here we demonstrate an alternative
peptide-mimotope vaccine strategy using the SDPM1 peptide. SDPM1 is a 20 amino
acid peptide bounded by cysteines that binds tetramer forms of Abeta(1-40)- and
Abeta(1-42)-amyloids and blocks subsequent Abeta amyloid aggregation.
Immunization of mice with SDPM1 induced peptide-mimotope antibodies with the
same biological activity as the SDPM1 peptide. When done prior to the onset of
amyloid plaque formation, SDPM1 vaccination of APPswePSEN1(A246E) transgenic
mice reduced amyloid plaque burden and Abeta(1-40) and Abeta(1-42) levels in the
brain, improved cognitive performance in Morris water maze tests, and resulted
in no increased T cell responses to immunogenic or Abeta peptides or brain
inflammation. When done after plaque burden was already significant, SDPM1
immunization still significantly reduced amyloid plaque burden and
Abeta(1-40/1-42) peptide levels in APPswePSEN1(A246E) brain without inducing
encephalitogenic T cell responses or brain inflammation, but treatment at this
stage did not improve cognitive function. These experiments demonstrate the
efficacy of a novel vaccine approach for Alzheimer's disease where immunization
with an Abeta(1-40/1-42) amyloid-specific binding and blocking peptide is used
to inhibit the development of neuropathology and cognitive dysfunction. To date, passive immunotherapy with monoclonal antibodies is a well-established
option in clinical oncology. By contrast, anticancer vaccines are less advanced,
with the exception of successfully applied prophylactic vaccines against
oncogenic virus infections. The creation of therapeutic vaccines is still a
great challenge mostly due to the self-nature of tumor antigens. Therapeutic
vaccines may be based on patient-specific material including pulsed effector
cells, or tumor-associated antigens and derivatives thereof, such as peptides,
mimotopes and nucleic acids. The latter represents a more universal approach,
which would set an ideal economic framework resulting in broad patient access.
In this article we focus on cancer vaccines for antibody production, in
particular mimotope vaccines. The collected evidence suggests that they will
open up new treatment options in minimal residual disease and early stage
disease. A major hurdle in vaccine development is the difficulty in identifying relevant
target epitopes and then presenting them to the immune system in a context that
mimics their native conformation. We have engineered novel virus-like-particle
(VLP) technology that is able to display complex libraries of random peptide
sequences on a surface-exposed loop in the coat protein without disruption of
protein folding or VLP assembly. This technology allows us to use the same VLP
particle for both affinity selection and immunization, integrating the power of
epitope discovery and epitope mimicry of traditional phage display with the high
immunogenicity of VLPs. Previously, we showed that using affinity selection with
our VLP platform identifies linear epitopes of monoclonal antibodies and
subsequent immunization generates the proper antibody response. To test if our
technology could identify immunologic mimotopes, we used affinity selection on a
monoclonal antibody (AP4-24H11) that recognizes the Staphylococcus aureus
autoinducing peptide 4 (AIP4). AIP4 is a secreted eight amino acid, cyclized
peptide produced from the S. aureus accessory gene regulator (agrIV)
quorum-sensing operon. The agr system coordinates density dependent changes in
gene expression, leading to the upregulation of a host of virulence factors, and
passive transfer of AP4-24H11 protects against S. aureus agrIV-dependent
pathogenicity. In this report, we identified a set of peptides displayed on VLPs
that bound with high specificity to AP4-24H11. Importantly, similar to passive
transfer with AP4-24H11, immunization with a subset of these VLPs protected
against pathogenicity in a mouse model of S. aureus dermonecrosis. These data
are proof of principle that by performing affinity selection on neutralizing
antibodies, our VLP technology can identify peptide mimics of non-linear
epitopes and that these mimotope based VLP vaccines provide protection against
pathogens in relevant animal models. To date, several small molecule inhibitors and monoclonal-antibodies (like
ICR-62) have been used to treat tumors over-expressing epidermal growth factor
receptor (EGFR). However, the limitations associated with these conventional
applications accentuate the necessity of alternative approaches. Mimotopes as
compelling molecular tools could rationally be employed to circumvent these
drawbacks. In the present study, an M13 phage displaying ICR-62 binding peptide
mimotope is exploited as a vaccine candidate. It exhibited high affinity towards
ICR62 and polyclonal anti-P-BSA antibodies. Following the mice immunization,
phage-based mimotope vaccine induced humoral immunity. Elicited anti-EGFR
mimotope antibodies were detected using ELISA method. Moreover, the phage
vaccine was tested on the Lewis lung carcinoma mice model to investigate the
prophylactic and therapeutic effects. The tumor volume was measured and recorded
in different animal groups to evaluate the anti-tumor effects of the vaccine.
Our data indicate that the reported phage-based mimotope could potentially
elicit specific antibodies resulting in low titers of EGFR-specific antibodies
and reduced tumor growth. However, in vivo experiments of prophylactic or
therapeutic vaccination showed no specific advantage. Furthermore,
phage-mimotope vaccine might be a promising approach in the field of cancer
immunotherapy. |
Describe clinical manifestation of the Mal de debarquement syndrome. | Mal de debarquement syndrome (MdDS) is a disorder of chronic self-motion perception that occurs though entrainment to rhythmic background motion, such as from sea voyage, and involves the perception of low-frequency rocking that can last for months or years. | Mal de debarquement syndrome is defined by a persistent sensation of rocking and
swaying commonly felt with sea travel that is first noted on return to land. Mal
de debarquement syndrome is not to be confused with seasickness, which causes
nausea, vomiting, diaphoresis, and headache. Four female patients with mal de
debarquement syndrome are reviewed. Mal de debarquement is normally a
short-lived phenomenon. The need for extensive evaluations may be avoided by an
awareness of this entity. Mal de debarquement (MDD) is a common, benign, and self-limited syndrome
suffered by many people after disembarkation from an oceangoing vessel. It is
characterized by a continuing sensation of being on an unsteady pitching and
rolling deck, even after a return to solid ground. Symptoms typically dissipate
over several hours or days, but can linger for weeks. There is no effective
treatment for MDD, no work-up is required, and patients can be reassured that
the symptoms are transient. We present a case of MDD in a previously healthy
22-year-old male, and discuss the approach to MDD in the emergency department
setting. Mal de debarquement syndrome (MdDS) is a disorder of phantom perception of
self-motion of unknown cause. The purpose of this work was to describe the
quality of life (QOL) of patients with MdDS and to estimate the economic costs
associated with this disorder. A modified version of a QOL survey used for
another neurological disease (multiple sclerosis; MSQOL-54) was used to assess
the impact of MdDS on QOL in 101 patients. The estimated economic costs were
based on self-reported direct and indirect costs of individuals living in the
United States using Medicare reimbursement payment rates for 2011 in 79
patients. Patients with MdDS reported a poor overall QOL as indicated by a mean
composite QOL score of 59.26 ± 1.89 (out of 100). The subcategories having the
lowest QOL rating were role limitations due to physical problems (18.32 ± 3.20),
energy (34.24 ± 1.47), and emotional problems (36.30 ± 4.00). The overall
physical health composite score including balance was 49.40 ± 1.69, and the
overall mental health composite score was 52.40 ± 1.83. The cost to obtain a
diagnosis was $2,997 ± 337, which included requiring an average of 19 physician
visits per patient. The direct cost of MdDS medical care was $826 ± 140 per
patient per year, which mainly included diagnostic imaging and physician visits.
The indirect costs (i.e., lost wages) were $9,781 ± 2,347 per patient per year.
Among 65 patients who were gainfully employed when they acquired MdDS, the
indirect costs were $11,888 ± 2,786 per patient per year. Thus, the total annual
cost of the disorder ranged from $11,493 ± 2,341 to $13,561 ± 2,778 per patient
per year depending on employment status prior to developing MdDS. MdDS
negatively and dramatically impacts QOL, and also imposes a substantial economic
burden on MdDS patients. These findings underscore the need for further basic
and clinical research on MdDS. BACKGROUND: Mal de debarquement (MdD) literally means "sickness of
disembarkation", and refers to the illusion of movement perceived as an
after-effect of traveling on a boat, train, or airplane. The pathophysiology of
MdD is currently unknown.
CASE REPORT: A 20-year-old man presented with dizziness and swaying sensation
for 3 days after a boat trip. Compared with the follow-up EEG without symptoms,
the EEG recorded while having MdD symptoms disclosed a significantly decreased
alpha-band current source density at the precentral gyrus of the left frontal
lobe and increased beta-2 activity at the parahippocampal gyrus of right mesial
temporal region.
CONCLUSIONS: Our results provide evidence of deranged cortical activity in MdD.
To the best of our knowledge this is the first study to document cortical
correlates of MdD using an EEG source-localization method. OBJECTIVE: Mal de debarquement syndrome (MdDS) is a chronic disorder of
imbalance characterized by a feeling of rocking and swaying. The disorder starts
after prolonged exposure to passive motion such as from a boat or plane. All
medical treatment is palliative and symptoms that persist beyond 6 months show
low likelihood of remission. This pilot study explored the feasibility and
tolerability of repetitive transcranial magnetic stimulation (rTMS) as potential
treatment for MdDS.
PATIENTS/INTERVENTION: Ten subjects (8 women) with persistent MdDS lasting from
10 to 91 months were given 1 session each of 4 counterbalanced protocols: left
10 Hz (high frequency), left 1 Hz (low frequency), right 10 Hz, and right 1 Hz
rTMS over the dorsolateral prefrontal cortex (DLPFC).
MAIN OUTCOME MEASURE: Reduction of rocking sensation reported on a visual
analogue scale.
RESULTS: 1) Right-handers improved most with 10-Hz stimulation over the left
DLPFC while left-handers improved most with 10 Hz stimulation over the right
DLPFC; 2) low-frequency DLPFC stimulation was associated with symptom worsening
in some subjects; 3) duration of symptoms was negatively correlated with
treatment response; 4) rTMS was well tolerated in MdDS subjects, showing similar
rates of headache (10 of 40 sessions) as for other studies; and 5) fatigue
occurred after 6 sessions usually with low-frequency stimulation.
CONCLUSION: rTMS was well tolerated in subjects with MdDS with promising
short-term symptom improvement. Future studies of rTMS in MdDS may consider
sequential days of stimulation, longer post-rTMS observation periods, formal
measurement of post-TMS fatigue, and randomization with a sham condition. BACKGROUND: Individuals with mal de debarquement syndrome (MdDS) experience a
chronic illusion of self-motion triggered by prolonged exposure to passive
motion, such as from sea or air travel. The experience is one of rocking
dizziness similar to when the individual was originally on the motion trigger
such as a boat or airplane. MdDS represents a prolonged version of a normal
phenomenon familiar to most individuals but which persists for months or years
in others. It represents a natural example of the neuroplasticity of motion
adaptation. However, the localization of where that motion adaptation occurs is
unknown. Our goal was to localize metabolic and functional connectivity changes
associated with persistent MdDS.
METHODS: Twenty subjects with MdDS lasting a median duration of 17.5 months were
compared to 20 normal controls with (18)F FDG PET and resting state fMRI.
Resting state metabolism and functional connectivity were calculated using age,
grey matter volume, and mood and anxiety scores as nuisance covariates.
RESULTS: MdDS subjects showed increased metabolism in the left entorhinal cortex
and amygdala (z>3.3). Areas of relative hypometabolism included the left
superior medial gyrus, left middle frontal gyrus, right amygdala, right insula,
and clusters in the left superior, middle, and inferior temporal gyri. MdDS
subjects showed increased connectivity between the entorhinal cortex/amygdala
cluster and posterior visual and vestibular processing areas including middle
temporal gyrus, motion sensitive area MT/V5, superior parietal lobule, and
primary visual cortex, while showing decreased connectivity to multiple
prefrontal areas.
CONCLUSION: These data show an association between resting state metabolic
activity and functional connectivity between the entorhinal cortex and amygdala
in a human disorder of abnormal motion perception. We propose a model for how
these biological substrates can allow a limited period of motion exposure to
lead to chronic perceptions of self-motion. Mal de debarquement syndrome (MdDS) is a poorly characterized and understood
disorder of perceived motion. We sought to characterize postural control and the
psychological impact of MdDS. Additionally, we explored whether patients with
MdDS exhibit altered corticospinal and intracortical excitability. In a
case-control study we compared patients with MdDS to age- and sex-matched
controls (n=8/group). Postural stability (σr) was quantified from plane phase
plots based on center or pressure, and psychological indices of depression,
fatigue and kinesiophobia were obtained. Transcranial magnetic stimulation (TMS)
was used to assess corticospinal excitability by quantifying the motor evoked
potential (MEP) amplitude of the flexor carpi radialis, and intracortical
excitability was assessed by quantifying indices of intracortical facilitation
(ICF), and short-interval and long-interval intracortical inhibition using a
paired-pulse TMS paradigm. The patients with MdDS exhibited greater mean
(±standard error of the mean) σr during semi-tandem stance (10.9 ± 1.5 compared
to 7.1 ± 0.7, p=0.04), higher levels of kinesiophobia (41.6 ± 2.8 compared to
27.3 ± 2.2), and higher levels of fatigue (27.0 ± 4.1 compared to 48.4 ± 1.0).
Patients with MdDS exhibited a higher mean motor threshold (MT) (58.1 ± 2.5
compared to 47.4 ± 2.7% of stimulator output), and larger MEP (13.1 ± 3.1
compared to 5.1 ± 1.2% of maximal compound muscle action potential) but there
was no difference in measures of intracortical excitability. These findings
suggest that patients with MdDS exhibit impaired postural stability, and high
levels of kinesiophobia and fatigue. Additionally, we observed that patients
with MdDS exhibit higher MT and large MEP amplitudes, but do not exhibit
differences in measures of intracortical excitability, compared to controls.
These findings help characterize MdDS, and provide insight into the physiology
of MdDS. BACKGROUND: Exposure to unfamiliar motion patterns commonly results in motion
sickness and a false perception of motion, termed mal de debarquement, on the
return to stable conditions.
OBJECTIVE: To investigate whether motion sickness severity is correlated with
the duration and severity of mal de debarquement; to study the possible
preventive effect of projecting earth-referenced scenes (an artificial horizon)
during exposure to motion on the development of mal de debarquement.
METHODS: Thirty subjects were exposed to the recorded motion profile of a boat
in a 3-degrees-of-freedom ship motion simulator. During the simulated voyage,
the study participants were repeatedly put through a performance test battery
and completed a motion sickness susceptibility questionnaire, while
self-referenced and earth-referenced scenes were projected inside the simulator
cabin. Six hours post disembarkation, subjects completed a questionnaire on mal
de debarquement duration and severity.
RESULTS: Mal de debarquement, mostly of mild severity, was reported following
59% of the exposures to the provocative motion profile, and in 79% of cases
lasted less than 6 hours. The incidence of mal de debarquement, its duration,
and the severity of symptoms did not differ between the various artificial
horizon projection modes. Significant correlations were found between motion
sickness severity and the duration and severity of the mal de debarquement that
followed.
CONCLUSIONS: The significant correlations found between motion sickness severity
and mal de debarquement duration and severity imply that both syndromes might
stem from a failure to adapt to new motion conditions. There is a disparity
between the previously reported reduction in motion sickness symptoms by an
artificial horizon, and its failure to influence the duration and symptoms of
mal de debarquement. This might be explained by the different response in the
two syndromes, physical versus cognitive. The mal de debarquement syndrome (MdDS), a continuous feeling of swaying,
rocking, and/or bobbing, generally follows travel on the sea. The associated
symptoms cause considerable distress. The underlying neural mechanisms are
unknown, and to date there have been no effective treatments for this condition.
Results in monkeys and humans suggested that MdDS was caused by maladaptation of
the vestibulo-ocular reflex (VOR) to roll of the head during rotation. We
studied 24 subjects with persistent MdDS (3 males, 21 females;
19.1 ± 33 months). Physical findings included body oscillation at 0.2 Hz,
oscillating vertical nystagmus when the head was rolled from side-to-side in
darkness, and unilateral rotation during the Fukuda stepping test. We posited
that the maladapted rocking and the physical symptoms could be diminished or
extinguished by readapting the VOR. Subjects were treated by rolling the head
from side-to-side while watching a rotating full-field visual stimulus.
Seventeen of the 24 subjects had a complete or substantial recovery on average
for approximately 1 year. Six were initially better, but the symptoms recurred.
One subject did not respond to treatment. Thus, readaptation of the VOR has led
to a cure or substantial improvement in 70% of the subjects with MdDS. We
conclude that the adaptive processes associated with roll-while-rotating are
responsible for producing MdDS, and that the symptoms can be reduced or resolved
by readapting the VOR. Mal de Debarquement Syndrome is a neurological disorder of motion perception,
triggered by external motion. This study aimed to determine the importance of
psychosocial factors in relation to depression and quality of life in Mal de
Debarquement Syndrome. A total of 66 participants with self-reported Mal de
Debarquement Syndrome completed quality-of-life, symptom severity, stigma,
depression, and illness intrusiveness measurements in this naturalistic
correlational study. Mal de Debarquement Syndrome was associated with high
levels of depression and illness intrusiveness. Illness intrusiveness mediated
between stigma and quality of life; also the level of stigma moderated the
effect of illness intrusiveness on quality of life. Targeted interventions aimed
at alleviating psychological distress may improve quality of life in Mal de
Debarquement Syndrome. Mal de debarquement syndrome (MdDS) is a chronic disorder of imbalance
characterized by a feeling of rocking and swaying. The medical treatment for
MdDS is still limited. Motivated by our previous pilot study that demonstrates
the promising clinical efficacy of repetitive transcranial stimulation (rTMS) in
MdDS patients, a novel rTMS paradigm, i.e., 1 Hz stimulation over ipsilateral
dorsal lateral prefrontal cortex (DLPFC) with respect to the domit hand
followed by 10 Hz stimulation over contralateral DLPFC, was proposed and
conducted in MdDS in the present study. To evaluate the potential efficacy, we
examined the changes before and after rTMS in both subjective reported symptom
using visual analogue scale (VAS) and direct brain activity in resting state
electroencephalography (rsEEG). To disentangle activity from distinct brain
substrates and/or local networks in rsEEG signals, a group-wise independent
component analysis was employed and the corresponding spectral power changes
were examined in the identified components. In general, reduction in rocking
sensation was reported in five of ten subjects (with dramatic reductions
(changes > 30) in three subjects) after rTMS using the present paradigm, while
no changes and slight increases in rocking sensation were reported in the
remaining subjects. In rsEEG, significant elevated spectral powers in low
frequency bands (i.e., theta and alpha) over broad areas of occipital, parietal,
motor, and prefrontal cortices were induced by rTMS, reflecting the enhancement
of cortical inhibition over these areas. Meanwhile, the significant correlations
between changes in rsEEG and VAS scores were detected in the high frequency
bands (i.e., high alpha and beta) over posterior parietal and left visual areas,
reflecting the suppression of spatial information processing. Therefore, the
present findings demonstrate the promising clinical efficacy of a new rTMS
paradigm for MdDS, and suggest its merit for further studies in more patients. Mal de Debarquement Syndrome (MdDS) is an enigmatic neurotological disorder with
high morbidity, psychosocial burden, and few treatment options. Fortunately,
there has been recent growth in scientific interest in understanding the
biological basis of and in treating MdDS. Recent studies using functional
neuroimaging have shown increased glucose metabolism in the left entorhinal
cortex (EC) and amygdala in the setting of decreased prefrontal and temporal
cortex metabolism in subjects with persistent MdDS. The EC is a key player in
processing and gating spatial information to be stored in the hippocampus and is
a major driver of brain oscillations. A limbic focus may also be key to
spontaneous MdDS-like symptoms occurring in individuals with a history of
anxiety or chronic stress. Treatment with repetitive transcranial magnetic
stimulation over the dorsolateral prefrontal cortex can decrease the rocking
dizziness of MdDS, with successful responses associated with decreases in the
coherence between brain networks with nodes in the parietal and occipital lobes.
A new theory of MdDS is proposed as pathology secondary to entrainment of
intrinsic brain networks driven by oscillatory motion exposure coupled with an
inability to subsequently desynchronize the activity of these nodes. Future
treatment strategies may be directed toward unyoking these networks. BACKGROUND: Mal de debarquement syndrome (MdDS) is a disorder of chronic
self-motion perception that occurs though entrainment to rhythmic background
motion, such as from sea voyage, and involves the perception of low-frequency
rocking that can last for months or years. The neural basis of this persistent
sensory perception abnormality is not well understood.
METHODS: We investigated grey matter volume differences underlying persistent
MdDS by performing voxel-based morphometry on whole brain and pre-specified ROIs
in 28 individuals with MdDS and comparing them to 18 age, sex, and handedness
matched controls.
RESULTS: MdDS participants exhibited greater grey matter volume in the left
inferior parietal lobule, right inferior occipital gyrus (area V3v), right
temporal pole, bilateral cerebellar hemispheric lobules VIII/IX and left lobule
VIIa/VIIb. Grey matter volumes were lower in bilateral inferior frontal,
orbitofrontal, pregenual anterior cingulate cortex (pgACC) and left superior
medial gyri (t = 3.0, p<0.005uncorr). In ROI analyses, there were no volume
differences in the middle occipital gyrus (region of V5/MT) or parietal
operculum 2 (region of the parietoinsular vestibular cortex). Illness duration
was positively related to grey matter volume in bilateral inferior frontal
gyrus/anterior insula (IFG/AI), right posterior insula, superior parietal
lobule, left middle occipital gyrus (V5/MT), bilateral postcentral gyrus,
anterior cerebellum, and left cerebellar hemisphere and vermian lobule IX. In
contrast, illness duration was negatively related to volume in pgACC, posterior
middle cingulate gyrus (MCC), left middle frontal gyrus (dorsolateral prefrontal
cortex-DLPFC), and right cerebellar hemispheric lobule VIIIb (t = 3.0,
p<0.005uncorr). The most significant differences were decreased volume in the
pgACC and increased volume in the left IFG/AI with longer illness duration
(qFDRcorr <0.05). Concurrent medication use did not correlate with these
findings or have a relationship with duration of illness. MdDS participants
showed positive correlations between grey matter volume in pgACC and bilateral
cerebellar lobules VIII/IX, which was not seen in controls.
CONCLUSIONS: Individuals with MdDS show brain volume differences from healthy
controls as well as duration of illness dependent volume changes in (a)
visual-vestibular processing areas (IPL, SPL, V3, V5/MT), (b) default mode
network structures (cerebellar IX, IPL, ACC), (c) salience network structures
(ACC and IFG/AI) (d) somatosensory network structures (postcentral gyrus, MCC,
anterior cerebellum, cerebellar lobule VIII), and (e) a structure within the
central executive network (DLPFC). The identification of these associations may
enhance future investigations into how exposure to oscillating environments can
modulate brain function and affect motion perception as well cognitive and
affective control. Mal de Debarquement Syndrome (MDS) is a rare, understudied, underdiagnosed, and
self-limiting condition. Etiology and incidence are unknown. It is characterized
by abnormal sensation of motion/balance reported after travel by air, land, and
sea; being reexposed to motion/activity relieves it. Symptoms may last from
minutes to years. Workup though required reveals no findings; it is a diagnosis
of exclusion. While no efficacious treatment exists, amitriptyline and
benzodiazepines as well as supportive therapy have proved to be useful. We have
described a 40-year-old Caucasian female who presented for the evaluation of
persistent rocking and swaying sensation after a ship cruise which lasted for
one week. Patient was treated with benzodiazepines after extensive workup and is
now stable. A high index of suspicion is required to make a diagnosis. Mal de debarquement (MdD) is a subjective perception of self-motion after
exposure to passive motion, in most cases sea travel, hence the name. Mal de
debarquement occurs quite frequently in otherwise healthy individuals for a
short period of time (several hours). However, in some people symptoms remain
for a longer period of time or even persist and this is then called mal de
debarquement syndrome (MdDS). The underlying pathogenesis is poorly understood
and therefore, treatment options are limited. In general, limited studies have
focused on the topic, but the past few years more and more interest has been
attributed to MdDS and its facets, which is reflected by an increasing number of
papers. Till date, some interesting reviews on the topic have been published,
but a systematic review of the literature is lacking and could help to address
the shortcomings and flaws of the current literature. We here present a
systematic review of MdD(S) based on a systematic search of medical databases
employing predefined criteria, using the terms "mal de debarquement" and "sea
legs". Based on this, we suggest a list of criteria that could aid healthcare
professionals in the diagnosis of MdDS. Further research needs to address the
blank gaps by addressing how prevalent MdD(S) really is, by digging deeper into
the underlying pathophysiology and setting up prospective, randomized
placebo-controlled studies to evaluate the effectiveness of possible treatment
strategies. OBJECTIVE: Mal de debarquement syndrome (MdDS) is a balance disorder that
typically starts after an extended exposure to passive motion, such as a boat or
plane ride. Management is typically supportive (e.g. physical therapy), and
symptoms that persist beyond 6 months have been described as unlikely to remit.
This study was conducted to evaluate the response of patients with MdDS to
management with migraine prophylaxis, including lifestyle changes and medical
therapy.
STUDY DESIGN: Prospective review.
SETTING: Ambulatory setting at a tertiary care medical center.
METHODS: Clinical history, detailed questionnaires, and audiograms were used to
diagnose patients with MdDS. Those patients with the diagnosis of the MdDS were
placed on our institutional vestibular migraine management protocol. Treatment
response was assessed with a quality-of-life (QOL) survey and visual analog
scale.
RESULTS: Fifteen patients were diagnosed with MdDS, with a predomice of
females (73%) and a mean age of 50 ± 13 years. Eleven patients (73%) responded
well to management with a vestibular migraine protocol, which included lifestyle
changes, as well as pharmacotherapy with verapamil, nortriptyline, topiramate,
or a combination thereof. In comparison, a retrospective control group of 17
patients demonstrated a lower rate of improvement when treated with vestibular
rehabilitation and physical therapy.
CONCLUSION: Management of MdDS as vestibular migraine can improve patients'
symptoms and increase the QOL. Nearly all the patients suffering from MdDS had a
personal or family history of migraine headaches or had signs or symptoms
suggestive of atypical migraine.
LEVEL OF EVIDENCE: 4 Laryngoscope, 127:1670-1675, 2017. |
Is Melioidosis caused by the bacterium Burkholderia pseudomallei? | Burkholderia pseudomallei is the causative agent of melioidosis | The mechanisms involved in the pathogenesis of melioidosis, caused by the
intracellular bacterium Burkholderia pseudomallei, are unclear. C57BL/6 mice are
resistant to infection, while BALB/c mice are highly susceptible. Previous
studies have demonstrated that peritoneal exudate cell preparations enriched for
macrophages are capable of effectively eliminating intracellular pathogens. In
this study we present evidence showing that interaction of macrophages with
lymphocytes is necessary for efficient anti-B. pseudomallei activity. Melioidosis is a rare tropical disease caused by infection with the bacterium
Burkholderia pseudomallei. It occurs predomitly in south-east Asia, northern
and central Australia and central and south America. Patients often present to
the internal medicine physicians with a severe, potentially fatal sepsis. We
report three patients who presented to our dermatology department with cutaneous
melioidosis. Melioidosis is an infectious disease caused by a saprophytic bacterium,
Burkholderia pseudomallei. It is endemic to Southeast Asia and Northern
Australia. The spectrum of melioidosis in humans varies from sub-clinical to
overwhelming protean manifestations resembling other acute and chronic bacterial
infections. Disseminated septicaemia melioidosis presenting as a masticator
space infection is reported here. This is germane to those treating diabetic
patients with deep neck infections living in, or having visited, areas endemic
for B. pseudomallei. Melioidosis is a tropical infectious disease caused by the gram-negative
bacterium Burkholderia pseudomallei. It is endemic in many parts of the world,
including Southeast Asia, and has a mortality rate of about 45%. We report a
case of localized nonfatal cutaneous melioidosis presenting as a persistent
ulcer in an otherwise healthy young woman. Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia
pseudomallei. Individuals with subclinical melioidosis have no apparent clinical
signs or symptoms, and are identified only by positive serology. The present
study is the first to investigate cell-mediated immune (CMI) responses following
in vitro stimulation with B. pseudomallei antigens in peripheral blood
mononuclear cells (PBMC), collected under field conditions in Papua New Guinea
(PNG) from individuals with exposure to B. pseudomallei (n = 13). While five had
a clinical history of melioidosis (C(+)), the remaining individuals (n = 8) were
seropositive, yet healthy with no clinical history of melioidosis (S(+)/C(-)).
Proliferation and IFN-gamma production were significantly greater in lymphocyte
cultures from S(+)/C(-) individuals compared to C(+) individuals (P < 0.001 and
P < 0.05, respectively). These findings demonstrate that compared to C(+)
patients, individuals with subclinical melioidosis have a stronger CMI response
to B. pseudomallei antigens in vitro. Such a response may be essential for
protection against disease progression. Melioidosis is an infection caused by the gram-negative bacterium Burkholderia
pseudomallei. This disease is endemic in Southeast Asia and North Australia with
sporadic occurrence in temperate countries, mostly imported from travellers. Any
organ can be involved in melioidosis whereby Burkholderia pseudomallei causes an
acute inflammatory reaction with rapid development of small abcesses which tend
to coalesce to form larger abscesses. Cutaneous manifestations vary greatly. We
report a man with systemic melioidosis who presented with cutaneous lesions. Melioidosis is a potentially fatal disease caused by the bacterium, Burkholderia
pseudomallei. The current study was carried out to determine the mechanisms
involved in the development of protective immunity in a murine model of
melioidosis. Following intravenous infection with B. pseudomallei, both C57BL/6
and BALB/c mice demonstrated delayed-type hypersensitivity responses and
lymphocyte proliferation towards B. pseudomallei antigens, indicating the
generation of B. pseudomallei-specific lymphocytes. Adoptive transfer of these
lymphocytes to naïve C57BL/6 mice was demonstrated by a delayed-type
hypersensitivity response. Mice were not protected from a subsequent lethal
challenge with a highly virulent strain of B. pseudomallei, suggesting that a
single intravenous dose of the bacterium is insufficient to induce a protective
adaptive immune response. Attempts to induce resistance in susceptible BALB/c
mice used repetitive low-dose exposure to live B. pseudomallei. Immune responses
and resistance following subcutaneous immunization with live B. pseudomallei
were compared with exposure to heat-killed, culture filtrate and sonicated B.
pseudomallei antigens. Compared to heat-killed B. pseudomallei, significant
protection was generated in BALB/c mice following immunization with live
bacteria. Our studies also demonstrate that the type of immune response
generated in vivo is influenced by the antigenic preparation of B. pseudomallei
used for immunization. Melioidosis is a suppurative chronic infection caused by a gramnegative
bacterium, Burkholderia pseudomallei. We report two patients who presented with
isolated liver abscesses caused by this pathogen. Both patients presented with
high-grade fever and abdominal pain. On examination they were toxic and had
tender hepatomegaly. Investigations showed leucocytosis and a shift to the left.
Early diagnosis of melioidosis was made by culture and growth of Burkholderia
pseudomallei from aspirated pus from the abscesses and the patients were treated
with ceftazidime and co-trimoxazole. Despite institution of antibiotics both the
patients succumbed to their illness. Melioidosis is an emerging infection in the
Indian subcontinent and can cause isolated liver abscesses. BACKGROUND: Melioidosis, a lethal tropical infection that is endemic in
southeast Asia and northern Australia, is caused by the saprophytic
Gram-negative bacterium Burkholderia pseudomallei. Overall mortality approaches
40% yet little is known about mechanisms of host defense. Toll-like receptors
(TLRs) are host transmembrane receptors that recognize conserved pathogen
molecular patterns and induce an inflammatory response. The lipopolysaccharide
(LPS) of Gram-negative bacteria is a potent inducer of the host innate immune
system. TLR4, in association with MD-2, is the archetype receptor for LPS
although B. pseudomallei LPS has been previously identified as a TLR2 agonist.
We examined TLR signaling induced by B. pseudomallei, B. pseudomallei LPS, and
B. pseudomallei lipid A using gain-of-function transfection assays of NF-kappaB
activation and studies of TLR-deficient macrophages.
RESULTS: In HEK293 cells transfected with murine or human TLRs, CD14, and MD-2,
heat-killed B. pseudomallei activated TLR2 (in combination with TLR1 or TLR6)
and TLR4. B. pseudomallei LPS and lipid A activated TLR4 and this TLR4-mediated
signaling required MD-2. In TLR2-/- macrophages, stimulation with heat-killed B.
pseudomallei augmented TNF-alpha and MIP-2 production whereas in TLR4-/- cells,
TNF-alpha, MIP-2, and IL-10 production was reduced. Cytokine production by
macrophages stimulated with B. pseudomallei LPS or lipid A was entirely
dependent on TLR4 but was increased in the absence of TLR2. TLR adaptor molecule
MyD88 strongly regulated TNF-alpha production in response to heat-killed B.
pseudomallei.
CONCLUSION: B. pseudomallei activates TLR2 and TLR4. In the presence of MD-2, B.
pseudomallei LPS and lipid A are TLR4 ligands. Although the macrophage cytokine
response to B. pseudomallei LPS or lipid A is completely dependent on TLR4, in
TLR2-/- macrophages stimulated with B. pseudomallei, B. pseudomallei LPS or
lipid A, cytokine production is augmented. Other MyD88-dependent signaling
pathways may also be important in the host response to B. pseudomallei
infection. These findings provide new insights into critical mechanisms of host
defense in melioidosis. BACKGROUND: The soil-dwelling saprophyte bacterium Burkholderia pseudomallei is
the cause of melioidosis, a severe disease of humans and animals in southeast
Asia and northern Australia. Despite the detection of B. pseudomallei in various
soil and water samples from endemic areas, the environmental habitat of B.
pseudomallei remains unclear.
METHODOLOGY/PRINCIPAL FINDINGS: We performed a large survey in the Darwin area
in tropical Australia and screened 809 soil samples for the presence of these
bacteria. B. pseudomallei were detected by using a recently developed and
validated protocol involving soil DNA extraction and real-time PCR targeting the
B. pseudomallei-specific Type III Secretion System TTS1 gene cluster.
Statistical analyses such as multivariable cluster logistic regression and
principal component analysis were performed to assess the association of B.
pseudomallei with environmental factors. The combination of factors describing
the habitat of B. pseudomallei differed between undisturbed sites and
environmentally manipulated areas. At undisturbed sites, the occurrence of B.
pseudomallei was found to be significantly associated with areas rich in
grasses, whereas at environmentally disturbed sites, B. pseudomallei was
associated with the presence of livestock animals, lower soil pH and different
combinations of soil texture and colour.
CONCLUSIONS/SIGNIFICANCE: This study contributes to the elucidation of
environmental factors influencing the occurrence of B. pseudomallei and raises
concerns that B. pseudomallei may spread due to changes in land use. PURPOSE OF REVIEW: Largely due to its recognition as a biological threat agent,
current knowledge on melioidosis, caused by the Gram-negative bacterium
Burkholderia pseudomallei, has increased tremendously over the last years. This
review summarizes current understanding on the molecular characterization of B.
pseudomallei and the immunology of melioidosis.
RECENT FINDINGS: The genome of B. pseudomallei is composed of two chromosomes of
which the largest part represents the B. pseudomallei core genome, whereas the
remaining accessory genome has been associated with bacterial virulence.
Virulence factors, most notably quorum sensing, type III secretion system,
lipopolysaccharide and other surface polysaccharides, flagella and various
factors essential for the intracellular life cycle of B. pseudomallei, have been
further characterized. The neutrophils play a critical in host defense, which is
initiated by the Toll-like receptors. The proinflammatory immune
response--including the activation of coagulation-- and its regulation have been
further dissected.
SUMMARY: Severe melioidosis can probably be seen as the clinical manifestation
of a pathogen recognition receptor mediated dysregulation of the immune response
to invading B. pseudomallei. B. pseudomallei employs numerous tactics to evade
the immune response. Studies on host-pathogen interactions in melioidosis have
identified a whole range of potential new treatment targets. Melioidosis, an infectious disease caused by the Gram-negative bacterium
Burkholderia pseudomallei, is now recognized as an important public health
problem in Southeast Asia and tropical northern Australia. Although B.
pseudomallei has been detected in various water and soil samples in southeast
China, the enviromental distribution of B. pseudomallei in China is unclear. In
the winter months of 2007, 154 and 130 soil and water samples, respectively,
were collected from several locations in Guangxi, China. The samples were
screened for B. pseudomallei by bacterial culture and identification and
confirmed by PCR for species-specific 16S rDNA and flagellin genes. B.
pseudomallei was detected in 8.4% of the soil samples but in none of the water
samples. All positive samples were confined to a single low-lying region from
rice paddy fields. Counts of B. pseudomallei ranged from 23 to 521 c.f.u./g
soil. This is the first geographical distribution survey of B. pseudomallei in
soil in Guangxi, China, and the data are of importance for further evaluating
the impact of this pathogen on melioidosis in this region. Melioidosis is an infectious disease caused by the Gram-negative bacterium
Burkholderia pseudomallei. Interest in the molecular identification of B.
pseudomallei has increased after its classification as a category B agent by the
US Centers for Disease Control and Prevention. The present article reports a
diagnosis of B. pseudomallei directly in a bronchoalveolar lavage by polymerase
chain reaction amplification. The results obtained show that direct detection of
the 16-23s spacer sequence in bronchoalveolar lavage is a quick and specific
test to diagnose melioidosis. Urokinase receptor (urokinase-type plasminogen activator receptor [uPAR], CD87),
a GPI-anchored protein, is considered to play an important role in inflammation
and fibrinolysis. The Gram-negative bacterium Burkholderia pseudomallei is able
to survive and replicate within leukocytes and causes melioidosis, an important
cause of pneumonia-derived community-acquired sepsis in Southeast Asia. In this
study, we investigated the expression and function of uPAR both in patients with
septic melioidosis and in a murine model of experimental melioidosis. uPAR mRNA
and surface expression was increased in patients with septic melioidosis in/on
both peripheral blood monocytes and granulocytes as well as in the pulmonary
compartment during experimental pneumonia-derived melioidosis in mice.
uPAR-deficient mice intranasally infected with B. pseudomallei showed an
enhanced growth and dissemination of B. pseudomallei when compared with
wild-type mice, corresponding with increased pulmonary and hepatic inflammation.
uPAR knockout mice demonstrated significantly reduced neutrophil migration
toward the pulmonary compartment after inoculation with B. pseudomallei. Further
in vitro experiments showed that uPAR-deficient macrophages and granulocytes
display a markedly impaired phagocytosis of B. pseudomallei. Additional studies
showed that uPAR deficiency did not influence hemostatic and fibrinolytic
responses during severe melioidosis. These data suggest that uPAR is crucially
involved in the host defense against sepsis caused by B. pseudomallei by
facilitating the migration of neutrophils toward the primary site of infection
and subsequently facilitating the phagocytosis of B. pseudomallei. BACKGROUND: Melioidosis is a frequently fatal infectious disease caused by the
soil dwelling Gram-negative bacterium Burkholderia pseudomallei. Environmental
sampling is important to identify geographical distribution of the organism and
related risk of infection to humans and livestock. The aim of this study was to
evaluate spatial distribution of B. pseudomallei in soil and consider the
implications of this for soil sampling strategies.
METHODS AND FINDINGS: A fixed-interval sampling strategy was used as the basis
for detection and quantitation by culture of B. pseudomallei in soil in two
environmental sites (disused land covered with low-lying scrub and rice field)
in northeast Thailand. Semivariogram and indicator semivariogram were used to
evaluate the distribution of B. pseudomallei and its relationship with range
between sampling points. B. pseudomallei was present on culture of 80/100
sampling points taken from the disused land and 28/100 sampling points from the
rice field. The median B. pseudomallei cfu/gram from positive sampling points
was 378 and 700 for the disused land and the rice field, respectively (p =
0.17). Spatial autocorrelation of B. pseudomallei was present, in that samples
taken from areas adjacent to sampling points that were culture positive
(negative) for B. pseudomallei were also likely to be culture positive
(negative), and samples taken from areas adjacent to sampling points with a high
(low) B. pseudomallei count were also likely to yield a high (low) count. Ranges
of spatial autocorrelation in quantitative B. pseudomallei count were 11.4
meters in the disused land and 7.6 meters in the rice field.
CONCLUSIONS: We discuss the implications of the uneven distribution of B.
pseudomallei in soil for future environmental studies, and describe a range of
established geostatistical sampling approaches that would be suitable for the
study of B. pseudomallei that take account of our findings. Melioidosis is an emerging infectious disease of humans and animals in the
tropics caused by the soil bacterium Burkholderia pseudomallei. Despite high
fatality rates, the ecology of B.pseudomallei remains unclear. We used a
combination of field and laboratory studies to investigate B.pseudomallei
colonization of native and exotic grasses in northern Australia. Multivariable
and spatial analyses were performed to determine significant predictors for
B.pseudomallei occurrence in plants and soil collected longitudinally from field
sites. In plant inoculation experiments, the impact of B.pseudomallei upon these
grasses was studied and the bacterial load semi-quantified. Fluorescence in situ
hybridization and confocal laser scanning microscopy were performed to localize
the bacteria in plants. Burkholderia pseudomallei was found to inhabit not only
the rhizosphere and roots but also aerial parts of specific grasses. This raises
questions about the potential spread of B.pseudomallei by grazing animals whose
droppings were found to be positive for these bacteria. In particular,
B.pseudomallei readily colonized exotic grasses introduced to Australia for
pasture. The ongoing spread of these introduced grasses creates new habitats
suitable for B.pseudomallei survival and may be an important factor in the
evolving epidemiology of melioidosis seen both in northern Australia and
elsewhere globally. Melioidosis is an emerging infectious disease caused by the soil bacterium
Burkholderia pseudomallei. In diagnostic and forensic settings, molecular
detection assays need not only high sensitivity with low limits of detection but
also high specificity. In a direct comparison of published and newly developed
TaqMan PCR assays, we found the TTS1-orf2 assay to be superior in detecting B.
pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay
(targeting the Yersinia-like fimbrial/Burkholderia thailandensis-like flagellum
and chemotaxis region) also showed high diagnostic sensitivity and provides
additional information on possible geographic origin. Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia
pseudomallei. Type 2 diabetes (T2D) is the most common comorbidity associated
with melioidosis. B. pseudomallei isolates from melioidosis patients with T2D
are less virulent in animal models than those from patients with melioidosis and
no identifiable risk factors. We developed an ex vivo whole-blood assay as a
tool for comparison of early inflammatory profiles generated by T2D and
nondiabetic (ND) individuals in response to a B. pseudomallei strain of low
virulence. Peripheral blood from individuals with T2D, with either poorly
controlled glycemia (PC-T2D [n = 6]) or well-controlled glycemia (WC-T2D [n =
8]), and healthy ND (n = 13) individuals was stimulated with B. pseudomallei.
Oxidative burst, myeloperoxidase (MPO) release, expression of pathogen
recognition receptors (TLR2, TLR4, and CD14), and activation markers (CD11b and
HLA-DR) were measured on polymorphonuclear (PMN) leukocytes and monocytes.
Concentrations of plasma inflammatory cytokine (interleukin-6 [IL-6], IL-12p70,
tumor necrosis factor alpha [TNF-α], monocyte chemoattractant protein 1 [MCP-1],
IL-8, IL-1β, and IL-10) were also determined. Following stimulation, oxidative
burst and MPO levels were significantly elevated in blood from PC-T2D subjects
compared to controls. Differences were also observed in expression of Toll-like
receptor 2 (TLR2), CD14, and CD11b on phagocytes from T2D and ND individuals.
Levels of IL-12p70, MCP-1, and IL-8 were significantly elevated in blood from
PC-T2D subjects compared to ND individuals. Notably, differential inflammatory
responses of PC-T2D, WC-T2D, and ND individuals to B. pseudomallei occur
independently of bacterial load and confirm the efficacy of this model of
T2D-melioidosis comorbidity as a tool for investigation of dysregulated PMN and
monocyte responses to B. pseudomallei underlying susceptibility of T2D
individuals to melioidosis. OBJECTIVE: Melioidosis is a frequent cause of severe sepsis in Southeast Asia
caused by the gram-negative bacterium Burkholderia pseudomallei. Patients with
melioidosis have elevated circulating levels of tissue-type plasminogen
activator, an important regulator of fibrinolysis. In this study, we aimed to
investigate the role of tissue-type plasminogen activator during melioidosis.
DESIGN: Animal study.
SETTING: University research laboratory.
SUBJECTS: Wild-type and tissue-type plasminogen activator-deficient C57BL/6
mice.
INTERVENTIONS: Mice were intranasally infected with viable Burkholderia
pseudomallei and killed after 24, 48, or 72 hrs for harvesting of lungs, liver,
and blood. Additionally, survival studies were performed.
MEASUREMENTS AND MAIN RESULTS: Experimentally induced melioidosis was associated
with elevated levels of tissue-type plasminogen activator in lungs of infected
wild-type mice. During infection with Burkholderia pseudomallei, tissue-type
plasminogen activator-deficient mice were protected when compared to wild-type
mice as demonstrated by a strongly decreased mortality (62% vs. 100% amongst
wild-type mice, p < .0001), together with decreased pulmonary bacterial loads,
less severe histopathological scores, and decreased fibrinolysis. These results
were accompanied with an early increase in cytokine levels in tissue-type
plasminogen activator-deficient mice.
CONCLUSIONS: During severe gram-negative sepsis caused by Burkholderia
pseudomallei, endogenous tissue-type plasminogen activator has harmful effects
with respect to survival and pulmonary bacterial growth. These effects are
related to tissue-type plasminogen activator-associated plasmin-induced
fibrinolysis and/or a tissue-type plasminogen activator-associated decrease in
proinflammatory cytokine production. Melioidosis is a disease of humans caused by opportunistic infection with the
soil and water bacterium Burkholderia pseudomallei. Melioidosis can manifest as
an acute, overwhelming infection or as a chronic, recurrent infection. At
present, it is not clear where B. pseudomallei resides in the mammalian host
during the chronic, recurrent phase of infection. To address this question, we
developed a mouse low-dose mucosal challenge model of chronic B. pseudomallei
infection and investigated sites of bacterial persistence over 60 days.
Sensitive culture techniques and selective media were used to quantitate
bacterial burden in major organs, including the gastrointestinal (GI) tract. We
found that the GI tract was the primary site of bacterial persistence during the
chronic infection phase, and was the only site from which the organism could be
consistently cultured during a 60-day infection period. The organism could be
repeatedly recovered from all levels of the GI tract, and chronic infection was
accompanied by sustained low-level fecal shedding. The stomach was identified as
the primary site of GI colonization as determined by fluorescent in situ
hybridization. Organisms in the stomach were associated with the gastric mucosal
surface, and the propensity to colonize the gastric mucosa was observed with 4
different B. pseudomallei isolates. In contrast, B. pseudomallei organisms were
present at low numbers within luminal contents in the small and large intestine
and cecum relative to the stomach. Notably, inflammatory lesions were not
detected in any GI tissue examined in chronically-infected mice. Only low-dose
oral or intranasal inoculation led to GI colonization and development of chronic
infection of the spleen and liver. Thus, we concluded that in a mouse model of
melioidosis B. pseudomallei preferentially colonizes the stomach following oral
inoculation, and that the chronically colonized GI tract likely serves as a
reservoir for dissemination of infection to extra-intestinal sites. The Gram-negative bacterium Burkholderia pseudomallei is the causative agent of
melioidosis, a serious infectious disease of humans and animals. Once considered
an esoteric tropical disease confined to Southeast Asia and northern Australia,
research on B. pseudomallei has recently gained global prominence due to its
classification as a potential bioterrorism agent by countries such as the United
States and also by increasing numbers of case reports from regions where it is
not endemic. An environmental bacterium typically found in soil and water,
assessing the true global prevalence of melioidosis is challenged by the fact
that clinical symptoms associated with B. pseudomallei infection are extremely
varied and may be confused with diverse conditions such as lung cancer,
tuberculosis, or Staphyloccocus aureus infection. These diagnostic challenges,
coupled with lack of awareness among clinicians, have likely contributed to
underdiagnosis and the high mortality rate of melioidosis, as initial treatment
is often either inappropriate or delayed. Even after antibiotic treatment,
relapses are frequent, and after resolution of acute symptoms, chronic
melioidosis can also occur, and the symptoms can persist for months to years. In
a recent article, Price et al. [mBio 4(4):e00388-13, 2013,
doi:10.1128/mBio.00388-13] demonstrate how comparative genomic sequencing can
reveal the repertoire of genetic changes incurred by B. pseudomallei during
chronic human infection. Their results have significant clinical ramifications
and highlight B. pseudomallei's ability to survive in a wide range of potential
niches within hosts, through the acquisition of genetic adaptations that
optimize fitness and resource utilization. Melioidosis is a pyogenic infection with high mortality caused by the bacterium
Burkholderia pseudomallei. As the clinical presentation is not distinctive, a
high index of clinical suspicion is required for diagnosis. We present a case of
a 50-year-old farmer who was diabetic and a chronic alcoholic, who presented to
us with pneumonia, followed by septic arthritis. He was ultimately diagnosed as
having melioidosis. Melioidosis is a potentially fatal disease caused by the saprophytic bacterium
Burkholderia pseudomallei. Resistance to gentamicin is generally a hallmark of
B. pseudomallei, and gentamicin is a selective agent in media used for diagnosis
of melioidosis. In this study, we determined the prevalence and mechanism of
gentamicin susceptibility found in B. pseudomallei isolates from Sarawak,
Malaysian Borneo. We performed multilocus sequence typing and antibiotic
susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis
patients in Sarawak district hospitals. Whole-genome sequencing was used to
identify the mechanism of gentamicin susceptibility. A novel allelic-specific
PCR was designed to differentiate gentamicin-sensitive isolates from wild-type
B. pseudomallei. A reversion assay was performed to confirm the involvement of
this mechanism in gentamicin susceptibility. A substantial proportion (86%) of
B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be
susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions
where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to
genetically related strains belonging to sequence type 881 or its single-locus
variant, sequence type 997. Whole-genome sequencing identified a novel
nonsynonymous mutation within amrB, encoding an essential component of the
AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in
conferring aminoglycoside and macrolide sensitivity by reversion of this
mutation to the wild-type sequence. Our study demonstrates that alternative B.
pseudomallei selective media without gentamicin are needed for accurate
melioidosis laboratory diagnosis in Sarawak. This finding may also have
implications for environmental sampling of other locations to test for B.
pseudomallei endemicity. Melioidosis, infection caused by the Gram-negative bacterium Burkholderia
pseudomallei, is a common cause of sepsis in northeast Thailand. In white North
Americans, common functional genetic variation in TLR1 is associated with organ
failure and death from sepsis. We hypothesized that TLR1 variants would be
associated with outcomes in Thais with melioidosis. We collated the global
frequencies of three TLR1 variants that are common in white North American
populations: rs5743551 (-7202A/G), rs4833095 (742A/G), and rs5743618 (1804G/T).
We noted a reversal of the minor allele from white North American subjects to
Asian populations that was particularly pronounced for rs5743618. In the Utah
residents of European ancestry, the frequency of the rs5743618 T allele was 17%
whereas in Vietnamese subjects the frequency was >99%. We conducted a genetic
association study in 427 patients with melioidosis to determine the association
of TLR1 variation with organ failure or death. We genotyped rs5743551 and
rs4833095. The variants were in high linkage disequilibrium but neither variant
was associated with organ failure or in-hospital death. In 300 healthy Thai
individuals we further tested the association of TLR1 variation with ex vivo
blood responses to Pam3CSK4, a TLR1 agonist. Neither variant was robustly
associated with blood cytokine responses induced by Pam3CSK4. We identified
additional common variation in TLR1 by searching public databases and the
published literature and screened three additional TLR1 variants for
associations with Pam3CSK4-induced responses but found none. We conclude that
the genetic architecture of TLR1 variation differs substantially in southeast
Asians compared to other populations and common variation in TLR1 in Thais is
not associated with outcome from melioidosis or with altered blood responses to
Pam3CSK4. Our findings highlight the need for additional studies of TLR1 and
other innate immune genetic modulators of the inflammatory host response and
determits of sepsis in southeast Asian populations. Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is
a dreadful disease common in South-East Asia and Northern Australia and is
characterized by chronic suppurative lesions and pneumonia. Melioidosis may
evolve into severe sepsis with multi-organ failure with high mortalities,
despite proper antibiotic therapy. Besides activation of a strong
pro-inflammatory host response, the coagulation system plays an important role
during melioidosis, which is thought to be host-protective. In particular, a
procoagulant state together with downregulation of anticoagulant pathways and
activation of fibrinolysis are present, all closely interrelated with parameters
of inflammation. This review presents an overview of recent studies in which the
role of coagulation, anti-coagulation and fibrinolysis during melioidosis was
investigated both in patients and in experimental settings. Melioidosis is caused by the environmental bacterium Burkholderia pseudomallei
and can present with severe sepsis. Predisposing risk factors are present in 80%
of cases. Monoclonal antibodies are increasingly prescribed for varied medical
conditions. This report describes the first known case of melioidosis in a
patient whose only risk factor for disease is treatment with a monoclonal
antibody. Prescribers of monoclonal antibodies and other immunosuppressants
should ensure that their patients are aware of the potential risk of melioidosis
prior to travel and the precautions that should be taken. BACKGROUND: Melioidosis is an increasingly recognised cause of sepsis and death
across South East Asia and Northern Australia, caused by the bacterium
Burkholderia pseudomallei. Risk factors include diabetes, alcoholism and renal
disease, and a vaccine targeting at-risk populations is urgently required. A
better understanding of the protective immune response in naturally infected
patients is essential for vaccine design.
METHODS: We conducted a longitudinal clinical and immunological study of 200
patients with melioidosis on admission, 12 weeks (n = 113) and 52 weeks (n = 65)
later. Responses to whole killed B. pseudomallei were measured in peripheral
blood mononuclear cells (PBMC) by interferon-gamma (IFN-γ) ELIspot assay and
flow cytometry and compared to those of control subjects in the region with
diabetes (n = 45) and without diabetes (n = 43).
RESULTS: We demonstrated strong CD4+ and CD8+ responses to B. pseudomallei
during acute disease, 12 weeks and 52 weeks later. 28-day mortality was 26% for
melioidosis patients, and B. pseudomallei-specific cellular responses in fatal
cases (mean 98 IFN-γ cells per million PBMC) were significantly lower than those
in the survivors (mean 142 IFN-γ cells per million PBMC) in a multivariable
logistic regression model (P = 0.01). A J-shaped curve association between
circulating neutrophil count and mortality was seen with an optimal count of
4000 to 8000 neutrophils/μl. Melioidosis patients with known diabetes had poor
diabetic control (median glycated haemoglobin HbA1c 10.2%, interquartile range
9.2-13.1) and showed a stunted B. pseudomallei-specific cellular response during
acute illness compared to those without diabetes.
CONCLUSIONS: The results demonstrate the role of both CD4+ and CD8+ T-cells in
protection against melioidosis, and an interaction between diabetes and cellular
responses. This supports development of vaccine strategies that induce strong
T-cell responses for the control of intracellular pathogens such as B.
pseudomallei. Melioidosis is a tropical disease of high mortality caused by the environmental
bacterium, Burkholderia pseudomallei. We have collected clinical isolates from
the highly endemic Northern Territory of Australia routinely since 1989, and
animal and environmental B. pseudomallei isolates since 1991. Here we provide a
complete record of all B. pseudomallei multilocus sequence types (STs) found in
the Northern Territory to date, and distribution maps of the eight most common
environmental STs. We observed surprisingly restricted geographic distributions
of STs, which is contrary to previous reports suggesting widespread
environmental dissemination of this bacterium. Our data suggest that B.
pseudomallei from soil and water does not frequently disperse long distances
following severe weather events or by migration of infected animals. Melioidosis is an infectious disease caused by Burkholderia pseudomallei, a
bacterium endemic in Southeast Asia and northern Australia. In New Caledonia,
sporadic cases were first described in 2005; since then, more cases have been
identified. To improve our understanding of melioidosis epidemiology in New
Caledonia, we compared the local cases and B. pseudomallei isolates with those
from endemic areas. Nineteen melioidosis cases have been diagnosed in New
Caledonia since 1999, mostly severe and with frequent bacteraemia, leading to
three (16%) fatalities. All but one occurred in the North Province. Besides
sporadic cases caused by non-clonal strains, we also identified a hotspot of
transmission related to a clonal group of B. pseudomallei that is
phylogenetically related to Australian strains. The Darwin region in northern Australia has experienced rapid population growth
in recent years, and with it, an increased incidence of melioidosis. Previous
studies in Darwin have associated the environmental presence of Burkholderia
pseudomallei, the causative agent of melioidosis, with anthropogenic land usage
and proximity to animals. In our study, we estimated the occurrence of B.
pseudomallei and Burkholderia spp. relatives in faecal matter of wildlife,
livestock and domestic animals in the Darwin region. A total of 357 faecal
samples were collected and bacteria isolated through culture and direct DNA
extraction after enrichment in selective media. Identification of B.
pseudomallei, B. ubonensis, and other Burkholderia spp. was carried out using
TTS1, Bu550, and recA BUR3-BUR4 quantitative PCR assays, respectively. B.
pseudomallei was detected in seven faecal samples from wallabies and a chicken.
B. cepacia complex spp. and Pandoraea spp. were cultured from wallaby faecal
samples, and B. cenocepacia and B. cepacia were also isolated from livestock
animals. Various bacteria isolated in this study represent opportunistic human
pathogens, raising the possibility that faecal shedding contributes to the
expanding geographical distribution of not just B. pseudomallei but other
Burkholderiaceae that can cause human disease. BACKGROUND: Melioidosis, caused by the gram-negative bacterium Burkholderia
pseudomallei, is a common cause of community-acquired sepsis in Southeast Asia
and Northern Australia. The NLRP3 inflammasome and its downstream product
interleukin-1 beta (IL-1β) have been proposed to play crucial roles in
melioidosis. In this study, we characterized the role of IL-1β more closely and
we assessed its therapeutic potential.
METHODS: mRNA expression of inflammasome components was determined in isolated
leukocytes of 32 healthy controls and 34 patients with sepsis caused by B
pseudomallei.Wild-type (WT), NLRP3-deficient (Nlrp3), and Asc mice were infected
with B pseudomallei. In additional experiments, infected WT mice were treated
with an anti-IL-1β antibody. After 24, 48, and 72 hours (h) mice were sacrificed
and organs were harvested. Furthermore, survival studies were performed.
RESULTS: Patients with melioidosis exhibited lower mRNA levels of caspase-1,
NLRP3, and ASC. Bacterial dissemination and organ damage were increased in B
pseudomallei-infected Nlrp3 and Asc mice, together with a reduced pulmonary cell
influx. Anti-IL-1β treatment of B pseudomallei challenged mice resulted in
strongly reduced bacterial counts, organ damage, and pulmonary granulocyte
influx together with reduced mortality. Postponement of anti-IL-1β treatment for
24 h postinfection still protected mice during melioidosis.
CONCLUSION: Expression of caspase-1, NLRP3, and ASC is altered in melioidosis
patients. In mice, both NLRP3 and ASC contribute to the host defense against
melioidosis. Anti-IL-1β treatment protects mice against B pseudomallei infection
and might be a novel treatment strategy in melioidosis. Burkholderia pseudomallei, an environmental bacterium that causes the deadly
disease melioidosis, is endemic in northern Australia and Southeast Asia. An
increasing number of melioidosis cases are being reported in other tropical
regions, including Africa and the Indian Ocean islands. B. pseudomallei first
emerged in Australia, with subsequent rare dissemination event(s) to Southeast
Asia; however, its dispersal to other regions is not yet well understood. We
used large-scale comparative genomics to investigate the origins of three
B. pseudomallei isolates from Madagascar and two from Burkina Faso. Phylogenomic
reconstruction demonstrates that these African B. pseudomallei isolates group
into a single novel clade that resides within the more ancestral Asian clade.
Intriguingly, South American strains reside within the African clade, suggesting
more recent dissemination from West Africa to the Americas. Anthropogenic
factors likely assisted in B. pseudomallei dissemination to Africa, possibly
during migration of the Austronesian peoples from Indonesian Borneo to
Madagascar ~2,000 years ago, with subsequent genetic diversity driven by
mutation and recombination. Our study provides new insights into global patterns
of B. pseudomallei dissemination and adds to the growing body of evidence of
melioidosis endemicity in Africa. Our findings have important implications for
melioidosis diagnosis and management in Africa. IMPORTANCE Sporadic melioidosis
cases have been reported in the African mainland and Indian Ocean islands, but
until recently, these regions were not considered areas where B. pseudomallei is
endemic. Given the high mortality rate of melioidosis, it is crucial that this
disease be recognized and suspected in all regions of endemicity. Previous work
has shown that B. pseudomallei originated in Australia, with subsequent
introduction into Asia; however, the precise origin of B. pseudomallei in other
tropical regions remains poorly understood. Using whole-genome sequencing, we
characterized B. pseudomallei isolates from Madagascar and Burkina Faso. Next,
we compared these strains to a global collection of B. pseudomallei isolates to
identify their evolutionary origins. We found that African B. pseudomallei
strains likely originated from Asia and were closely related to South American
strains, reflecting a relatively recent shared evolutionary history. We also
identified substantial genetic diversity among African strains, suggesting
long-term B. pseudomallei endemicity in this region. BACKGROUND: The environmental bacterium Burkholderia pseudomallei causes the
infectious disease melioidosis with a high case-fatality rate in tropical and
subtropical regions. Direct pathogen detection can be difficult, and therefore
an indirect serological test which might aid early diagnosis is desirable.
However, current tests for antibodies against B. pseudomallei, including the
reference indirect haemagglutination assay (IHA), lack sensitivity, specificity
and standardization. Consequently, serological tests currently do not play a
role in the diagnosis of melioidosis in endemic areas. Recently, a number of
promising diagnostic antigens have been identified, but a standardized,
easy-to-perform clinical laboratory test for sensitive multiplex detection of
antibodies against B. pseudomallei is still lacking.
METHODS AND PRINCIPAL FINDINGS: In this study, we developed and validated a
protein microarray which can be used in a standard 96-well format. Our array
contains 20 recombit and purified B. pseudomallei proteins, previously
identified as serodiagnostic candidates in melioidosis. In total, we analyzed
196 sera and plasmas from melioidosis patients from northeast Thailand and 210
negative controls from melioidosis-endemic and non-endemic regions. Our protein
array clearly discriminated between sera from melioidosis patients and controls
with a specificity of 97%. Importantly, the array showed a higher sensitivity
than did the IHA in melioidosis patients upon admission (cut-off IHA titer
≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from
single patients at 0, 12 and 52 weeks post-admission revealed that protein
antigens induce either a short- or long-term antibody response.
CONCLUSIONS: Our protein array provides a standardized, rapid, easy-to-perform
test for the detection of B. pseudomallei-specific antibody patterns. Thus, this
system has the potential to improve the serodiagnosis of melioidosis in clinical
settings. Moreover, our high-throughput assay might be useful for the detection
of anti-B. pseudomallei antibodies in epidemiological studies. Further studies
are needed to elucidate the clinical and diagnostic significance of the
different antibody kinetics observed during melioidosis. BACKGROUND: Melioidosis, an often fatal infectious disease in Northeast
Thailand, is caused by skin inoculation, inhalation or ingestion of the
environmental bacterium, Burkholderia pseudomallei. The major underlying risk
factor for melioidosis is diabetes mellitus. Recommendations for melioidosis
prevention include using protective gear such as rubber boots and gloves when in
direct contact with soil and environmental water, and consuming bottled or
boiled water. Only a small proportion of people follow such recommendations.
METHODS: Nine focus group discussions were conducted to evaluate barriers to
adopting recommended preventive behaviours. A total of 76 diabetic patients from
northeast Thailand participated in focus group sessions. Barriers to adopting
the recommended preventive behaviours and future intervention strategies were
identified using two frameworks: the Theoretical Domains Framework and the
Behaviour Change Wheel.
RESULTS: Barriers were identified in the following five domains: (i) knowledge,
(ii) beliefs about consequences, (iii) intention and goals, (iv) environmental
context and resources, and (v) social influence. Of 76 participants, 72 (95%)
had never heard of melioidosis. Most participants saw no harm in not adopting
recommended preventive behaviours, and perceived rubber boots and gloves to be
hot and uncomfortable while working in muddy rice fields. Participants reported
that they normally followed the behaviour of friends, family and their
community, the majority of whom did not wear boots while working in rice fields
and did not boil water before drinking. Eight intervention functions were
identified as relevant for the intervention: (i) education, (ii) persuasion,
(iii) incentivisation, (iv) coercion, (v) modeling, (vi) environmental
restructuring, (vii) training, and (viii) enablement. Participants noted that
input from role models in the form of physicians, diabetic clinics, friends and
families, and from the government via mass media would be required for them to
change their behaviours.
CONCLUSION: There are numerous barriers to the adoption of behaviours
recommended for melioidosis prevention. We recommend that a multifaceted
intervention at community and government level is required to achieve the
desired behaviour changes. Burkholderia pseudomallei is a soil-dwelling bacterium and the cause of
melioidosis, which kills an estimated 89,000 people per year worldwide.
Agricultural workers are at high risk of infection due to repeated exposure to
the bacterium. Little is known about the soil physicochemical properties
associated with the presence or absence of the organism. Here, we evaluated the
soil physicochemical properties and presence of B. pseudomallei in 6,100 soil
samples collected from 61 rice fields in Thailand. The presence of B.
pseudomallei was negatively associated with the proportion of clay, proportion
of moisture, level of salinity, percentage of organic matter, presence of
cadmium, and nutrient levels (phosphorus, potassium, calcium, magnesium, and
iron). The presence of B. pseudomallei was not associated with the level of soil
acidity (P = 0.54). In a multivariable logistic regression model, the presence
of B. pseudomallei was negatively associated with the percentage of organic
matter (odds ratio [OR], 0.06; 95% confidence interval [CI], 0.01 to 0.47; P =
0.007), level of salinity (OR, 0.06; 95% CI, 0.01 to 0.74; P = 0.03), and
percentage of soil moisture (OR, 0.81; 95% CI, 0.66 to 1.00; P = 0.05). Our
study suggests that B. pseudomallei thrives in rice fields that are nutrient
depleted. Some agricultural practices result in a decline in soil nutrients,
which may impact the presence and amount of B. pseudomallei bacteria in affected
areas.
IMPORTANCE: Burkholderia pseudomallei is an environmental Gram-negative bacillus
and the cause of melioidosis. Humans acquire the disease following skin
inoculation, inhalation, or ingestion of the bacterium in the environment. The
presence of B. pseudomallei in soil defines geographic regions where humans and
livestock are at risk of melioidosis, yet little is known about the soil
properties associated with the presence of the organism. We evaluated the soil
properties and presence of B. pseudomallei in 61 rice fields in East, Central,
and Northeast Thailand. We demonstrated that the organism was more commonly
found in soils with lower levels of organic matter and nutrients, including
phosphorus, potassium, calcium, magnesium, and iron. We also demonstrated that
crop residue burning after harvest, which can reduce soil nutrients, was not
uncommon. Some agricultural practices result in a decline in soil nutrients,
which may impact the presence and amount of B. pseudomallei bacteria in affected
areas. |
What is the mechanism of action of verubecestat? | Verubecestat (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a potent, selective, structurally unique BACE1 inhibitor that reduced plasma, cerebrospinal fluid (CSF), and brain concentrations of Aβ40, Aβ42, and sAPPβ (a direct product of BACE1 enzymatic activity). | Alzheimer's disease (AD) is the primary cause of dementia in the elderly. It
remains incurable and poses a huge socio-economic challenge for developed
countries with an aging population. AD manifests by progressive decline in
cognitive functions and alterations in behaviour, which are the result of the
extensive degeneration of brain neurons. The AD pathogenic mechanism involves
the accumulation of amyloid beta peptide (Aβ), an aggregating protein fragment
that self-associates to form neurotoxic fibrils that trigger a cascade of
cellular events leading to neuronal injury and death. Researchers from academia
and the pharmaceutical industry have pursued a rational approach to AD drug
discovery and targeted the amyloid cascade. Schemes have been devised to prevent
the overproduction and accumulation of Aβ in the brain. The extensive efforts of
the past 20 years have been translated into bringing new drugs to advanced
clinical trials. The most progressed mechanism-based therapies to date consist
of immunological interventions to clear Aβ oligomers, and pharmacological drugs
to inhibit the secretase enzymes that produce Aβ, namely β-site amyloid
precursor-cleaving enzyme (BACE) and γ-secretase. After giving an update on the
development and current status of new AD therapeutics, this review will focus on
BACE inhibitors and, in particular, will discuss the prospects of verubecestat
(MK-8931), which has reached phase III clinical trials. Currently available drugs against Alzheimer's disease (AD) target cholinergic
and glutamatergic neurotransmissions without affecting the underlying disease
process. Putative disease-modifying drugs are in development and target
β-amyloid (Aβ) peptide and tau protein, the principal neurophatological
hallmarks of the disease. Areas covered: Phase III clinical studies of emerging
anti-Aβ drugs for the treatment of AD were searched in US and EU clinical trial
registries and in the medical literature until May 2016. Expert opinion: Drugs
in Phase III clinical development for AD include one inhibitor of the
β-secretase cleaving enzyme (BACE) (verubecestat), three anti-Aβ monoclonal
antibodies (solanezumab, gantenerumab, and aducanumab), an inhibitor of receptor
for advanced glycation end products (RAGE) (azeliragon) and the combination of
cromolyn sodium and ibuprofen (ALZT-OP1). These drugs are mainly being tested in
subjects during early phases of AD or in subjects at preclinical stage of
familial AD or even in asymptomatic subjects at high risk of developing AD. The
hope is to intervene in the disease process when it is not too late. However,
previous clinical failures with anti-Aβ drugs and the lack of fully
understanding of the pathophysiological role of Aβ in the development of AD, put
the new drugs at substantial risk of failure. Author information:
(1)Department of Neuroscience, Merck Research Laboratories, Kenilworth, NJ
07033, USA. [email protected] [email protected]
[email protected] [email protected].
(2)Department of Global Chemistry, Merck Research Laboratories, Kenilworth, NJ
07033, USA. [email protected] [email protected]
[email protected] [email protected].
(3)Department of Neuroscience, Merck Research Laboratories, Kenilworth, NJ
07033, USA.
(4)Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck
Research Laboratories, Kenilworth, NJ 07033, USA.
(5)Department of Global Chemistry, Merck Research Laboratories, Kenilworth, NJ
07033, USA.
(6)Department of Clinical Research, Merck Research Laboratories, Kenilworth, NJ
07033, USA.
(7)PAREXEL, Glendale, CA 91206, USA.
(8)Department of Safety Assessment, Merck Research Laboratories, West Point, PA
19446, USA.
(9)Department of Biostatistics, Merck Research Laboratories, Kenilworth, NJ
07033, USA.
(10)Translational Biomarkers Department, Merck Research Laboratories,
Kenilworth, NJ 07033, USA.
(11)Department of Translational Medicine, Merck Research Laboratories,
Kenilworth, NJ 07033, USA.
(12)Department of Translational Medicine, Merck Research Laboratories,
Kenilworth, NJ 07033, USA. [email protected] [email protected]
[email protected] [email protected]. Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazie
1,1-dioxide derivative, is a high-affinity β-site amyloid precursor protein
cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical
evaluation for the treatment of mild to moderate and prodromal Alzheimer's
disease. Although not selective over the closely related aspartyl protease
BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl
proteases, notably cathepsin D, and profoundly lowers CSF and brain Aβ levels in
rats and nonhuman primates and CSF Aβ levels in humans. In this annotation, we
describe the discovery of 3, including design, validation, and selected SAR
around the novel iminothiadiazie dioxide core as well as aspects of its
preclinical and Phase 1 clinical characterization. Verubecestat is an inhibitor of β-secretase being evaluated for the treatment of
Alzheimer's disease. The first-generation route relies on an amide coupling with
a functionalized aniline, the preparation of which introduces synthetic
inefficiencies. The second-generation route replaces this with a
copper-catalyzed C-N coupling, allowing for more direct access to the target.
Other features of the new route include a diastereoselective Mannich-type
addition into an Ellman sulfinyl ketimine and a late-stage guanidinylation. |
How are triple negative gliomas characterized? | of these markers - 1p/19q deletions , mgmt methylation status , and mutations in the idh1 gene - are so potent that a new brain tumor subtype , the "triple negative" glioma (1p/19q intact , mgmt unmethylated , idh1 non-mutated) has entered common parlance . | Adult gliomas are most often infiltrative. The World Health Organization (WHO)
has classed them into three major groups according to the presomptive cell of
origin: astrocytoma, oligodendroglioma and mixed oligoastrocytoma. Depending on
the presence or absence of a small number of signs of anaplasia (mitosis,
nuclear atypia, cell density, microvascular proliferation and necrosis) the WHO
distinguishes grade II (LGG), III (anaplastic), and IV (glioblastomas, GBM).
Mutation in the isocitrate deshydrogenase I and II (IDH1 and 2) genes
distinguishes grade II, III and secondary GBM from primary GBM. Moreover two
additional genetic alterations are recorded in grade II and III gliomas: TP53
mutations that characterize astrocytomas and 1p19q codeletion (as the result of
t(1;19)(q10;p10) translocation) recorded in oligodendrogliomas. Mixed gliomas,
the most non-reproducible category, share with astrocytomas and
oligodendrogliomas the same genetic alterations. Interestingly TP53 mutation
(p53+) and 1p19q codeletion (1p19q+) are mutually exclusive and involve IDH
mutated (IDH+) glial precursor cells. According to IDH, TP53, and 1p19q status,
four major subtypes of LGG are recorded: IDH+/p53-/1p19q-, IDH+/p53+/1p19q-,
IDH+/p53-/1p19q+ and triple negative, this last subgroup having the worst
prognosis. Interestingly, p53 expression and internexin alpha (INA) expression
can replace to some extent TP53 mutation and 1p19 codeletion, respectively.
Moreover the antibody directed against the IDH1R132H isoform is highly specific.
Because this mutation is the most frequent it is sufficient to assess IDH status
in more than 80% of grade II and III gliomas. Taken together these three
immunohistochemical markers are contribute greatly to the classification of
gliomas and should be tested routinely as diagnostic markers. Finally, although
GBM are genetically heterogeneous, the vast majority display EGFR amplification,
often associated with EGFR expression, which can be helpful for diagnosis in
certain cases. Adult grade II low-grade gliomas (LGG) are classified according to the WHO as
astrocytomas, oligodendrogliomas or mixed gliomas. TP53 mutations and 1p19q
codeletion are the main molecular abnormalities recorded, respectively, in
astrocytomas and oligodendrogliomas and in mixed gliomas. Although IDH mutations
(IDH1 or IDH2) are recorded in up to 85 % of low-grade gliomas, IDH negative
gliomas do occur. We have searched for p53 expression, 1p19q codeletion and IDH
status (immunohistochemical detection of the common R132H IDH1 mutation and IDH
direct sequencing). Internexin alpha (INA) expression previously recorded to be
associated with 1p19q codeletion (1p19q+) gliomas was also analysed. Low-grade
gliomas were accurately classified into four groups: group 1, IDH+/p53-/1p19q-;
group 2, IDH+/p53-/1p19q+; group 3, IDH+/p53+/1p19q-; and group 4, triple
negative gliomas. In contrast to the WHO classification, this molecular
classification predicts overall survival on uni- and multivariate analysis (P =
0.001 and P = 0.007, respectively). Group 4 carries the worst prognosis and
group 2 the best. Interestingly, p53 +/INA- expression predicts lack of 1p19q
codeletion (specificity 100 %, VPP 100 %). The combined use of these three
molecular markers allow for an accurate prediction of survival in LGG. These
findings could significantly modify LGG classification and may represent a new
tool to guide patient-tailored therapy. Moreover, immunohistochemical detection
of p53, INA and mR132H IDH1 expression could represent an interesting
prescreening test to be performed before 1p19q codeletion, IDH1 minor mutation
and IDH2 mutation detection. Primary brain tumors constitute a substantial public health problem with 66,290
cases diagnosed in the US in 2012, and 13,700 deaths recorded. With discovery of
genetic factors associated with specific brain tumor subtypes, the goal of
therapy is changing from treating a class of tumors to developing individualized
therapies catering to the molecular composition of the actual tumor. For
oligodendrogliomas, the loss of 1p/19q due to an unbalanced translocation
improves both survival and the response to therapy, and is thus both a
prognostic and a predictive marker. Several additional genetic alterations such
as EGFR amplification, MGMT methylation, PDGFR activation, and 9p and 10q loss,
have improved our understanding of the characteristics of these tumors and may
help guide therapy in the future. For astrocytic tumors, MGMT is associated with
a better prognosis and an improved response to temozolomide, and for all glial
tumors, mutations in the IDH1 gene are possibly the most potent of good
prognostic markers. Three of these markers - 1p/19q deletions, MGMT methylation
status, and mutations in the IDH1 gene - are so potent that a new brain tumor
subtype, the "triple negative" glioma (1p/19q intact, MGMT unmethylated, IDH1
non-mutated) has entered common parlance. Newer markers, such as CD 133, require
additional investigation to determine their prognostic and predictive utility.
In medulloblastomas, markers of WNT activation, MYCC/MCYN amplification, and
TrkC expression levels are reliable prognostic indicators, but do not yet drive
specific treatment selection. Many other proposed markers, such as 17q gain,
TP53 mutations, and hMOF protein expression show promise, but are not yet ready
for prime time. In this chapter, we focus on the markers that have shown
convincing prognostic, predictive, and diagnostic value, and discuss potential
markers that are being currently being intensively investigated. We also discuss
serum profiling of tumors in an effort to discover additional potential markers. |
What is ectopia lentis? | Ectopia Lentis is dislocation of the optic lens in the eye. | Ectopia lentis is a genetically heterogeneous condition that is characterized by
the subluxation of the lens resulting from the disruption of the zonular fibers.
Patients with ectopia lentis commonly present with a marked loss in visual
acuity in addition to a number of possibly accompanying ocular complications
including cataract, myopia, and retinal detachment. We here describe an isolated
form of ectopia lentis in a large inbred family that shows autosomal-recessive
inheritance. We map the ectopia lentis locus in this family to the
pericentromeric region on chromosome 1 (1p13.2-q21.1). The linkage region
contains well more than 60 genes. Mutation screening of four candidate genes
revealed a homozygous nonsense mutation in exon 11 of ADAMTSL4 (p.Y595X;
c.1785T-->G) in all affected individuals that is absent in 380 control
chromosomes. The mutation would result in a truncated protein of half the
original length, if the mRNA escapes nonsense-mediated decay. We conclude that
mutations in ADAMTSL4 are responsible for autosomal-recessive simple ectopia
lentis and that ADAMTS-like4 plays a role in the development and/or integrity of
the zonular fibers. |
Can glyburide reduce cerebral edema? | Yes. Glyburide, a selective inhibitor of sulfonylurea receptor 1-transient receptor potential melastatin 4, is effective in preventing and attenuating cerebral edema. | Inhibition of sulfonylurea receptor 1 (SUR1) by glyburide has been shown to
decrease edema after subarachnoid hemorrhage. We investigated if inhibiting SUR1
reduces cerebral edema due to metastases, the most common brain tumor, and
explored the putative association of SUR1 and the endothelial tight junction
protein, zona occludens-1 (ZO-1). Nude rats were intracerebrally implanted with
small cell lung carcinoma (SCLC) LX1 or A2058 melanoma cells (n = 36). Rats were
administered vehicle, glyburide (4.8 µg twice, orally), or dexamethasone (0.35
mg, intravenous). Blood-tumor barrier (BTB) permeability (K (trans)) was
evaluated before and after treatment using dynamic contrast-enhanced magnetic
resoce imaging. SUR1 and ZO-1 expression was evaluated using
immunofluorescence and Western blots. In both models, SUR1 expression was
significantly increased (P < .05) in tumors. In animals with SCLC, control mean
K (trans) (percent change ± standard error) was 101.8 ± 36.6%, and both
glyburide (-21.4 ± 14.2%, P < .01) and dexamethasone (-14.2 ± 13.1%, P < .01)
decreased BTB permeability. In animals with melanoma, compared to controls
(117.1 ± 43.4%), glyburide lowered BTB permeability increase (3.2 ± 15.4%, P <
.05), while dexamethasone modestly lowered BTB permeability increase (63.1 ±
22.1%, P > .05). Both glyburide (P < .001) and dexamethasone (P < .01) decreased
ZO-1 gap formation. By decreasing ZO-1 gaps, glyburide was at least as effective
as dexamethasone at halting increased BTB permeability caused by SCLC and
melanoma. Glyburide is a safe, inexpensive, and efficacious alternative to
dexamethasone for the treatment of cerebral metastasis-related vasogenic edema. BACKGROUND: Brain edema is a serious complication of ischemic stroke that can
lead to secondary neurological deterioration and death. Glyburide is reported to
prevent brain swelling in preclinical rodent models of ischemic stroke through
inhibition of a non-selective channel composed of sulfonylurea receptor 1 and
transient receptor potential cation channel subfamily M member 4. However, the
relevance of this pathway to the development of cerebral edema in stroke
patients is not known.
METHODS: Using a case-control design, we retrospectively assessed neuroimaging
and blood markers of cytotoxic and vasogenic edema in subjects who were enrolled
in the glyburide advantage in maligt edema and stroke-pilot (GAMES-Pilot)
trial. We compared serial brain magnetic resoce images (MRIs) to a cohort
with similar large volume infarctions. We also compared matrix
metalloproteinase-9 (MMP-9) plasma level in large hemispheric stroke.
RESULTS: We report that IV glyburide was associated with T2 fluid-attenuated
inversion recovery signal intensity ratio on brain MRI, diminished the lesional
water diffusivity between days 1 and 2 (pseudo-normalization), and reduced blood
MMP-9 level.
CONCLUSIONS: Several surrogate markers of vasogenic edema appear to be reduced
in the setting of IV glyburide treatment in human stroke. Verification of these
potential imaging and blood biomarkers is warranted in the context of a
randomized, placebo-controlled trial. BACKGROUND AND PURPOSE: Preclinical and retrospective clinical data indicate
that glyburide, a selective inhibitor of sulfonylurea receptor 1-transient
receptor potential melastatin 4, is effective in preventing edema and improving
outcome after focal ischemia. We assessed the feasibility of recruiting and
treating patients with severe stroke while obtaining preliminary information on
the safety and tolerability of RP-1127 (glyburide for injection).
METHODS: We studied 10 patients with acute ischemic stroke, with baseline
diffusion-weighted imaging lesion volumes of 82 to 210 cm3, whether treated with
intravenous recombit tissue-type plasminogen activator, age 18 to 80 years,
and time to RP-1127≤10 hours.
RESULTS: Recruitment was completed within 10 months. The mean age was 50.5
years, and baseline diffusion-weighted image lesion volume was 102±23 cm3. There
were no serious adverse events related to drug and no symptomatic hypoglycemia.
The increase in ipsilateral hemisphere volume was 50±33 cm3. The proportion of
90-day modified Rankin Scale≤4 was 90% (40% modified Rankin Scale, ≤3).
CONCLUSIONS: RP-1127 at a dose of 3 mg/d was well tolerated and did not require
any dose reductions. A clinical trial of RP-1127 is feasible.
CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique
identifier: NCT01268683. Metastatic disease to the brain results in significant morbidity because of
edema in the central nervous system. Current anti-edema therapies are either
expensive or result in unwanted long-term side effects. Sulfonylurea receptor 1
(Sur1) is a transmembrane protein that, when activated in the central nervous
system, allows for unregulated sodium influx into cells, a process that has been
linked to cytotoxic edema formation in ischemic stroke, subarachnoid hemorrhage,
spinal cord injury, traumatic brain injury, and, most recently, brain
metastases. In this focused review, we explore preclinical data linking Sur1
channel formation to development of edema and reference evidence suggesting that
the antidiabetic sulfonylurea drug glyburide (a Sur1 inhibitor) is an
inexpensive and well-tolerated agent that can be clinically tested to reduce or
prevent maligcy and/or treatment-associated edema. BACKGROUND: Maligt infarction is characterized by the formation of cerebral
edema, and medical treatment is limited. Preclinical data suggest that
glyburide, an inhibitor of SUR1-TRPM4, is effective in preventing edema. We
previously reported feasibility of the GAMES-Pilot study, a two-center
prospective, open label, phase IIa trial of 10 subjects at high risk for
maligt infarction based on diffusion weighted imaging (DWI) threshold of 82
cm(3) treated with RP-1127 (glyburide for injection). In this secondary
analysis, we tested the hypothesis that RP-1127 may be efficacious in preventing
poor outcome when compared to controls.
METHODS: Controls suffering large hemispheric infarction were obtained from the
EPITHET and MMI-MRI studies. We first screened subjects for controls with the
same DWI threshold used for enrollment into GAMES-Pilot, 82 cm(3). Next, to
address imbalances, we applied a weighted Euclidean matching. Ninety day mRS
0-4, rate of decompressive craniectomy, and mortality were the primary clinical
outcomes of interest.
RESULTS: The mean age of the GAMES cohort was 51 years and initial DWI volume
was 102 ± 23 cm(3). After Euclidean matching, GAMES subjects showed similar
NIHSS, higher DWI volume, younger age and had mRS 0-4-90% versus 50% in controls
p = 0.049; with a similar trend in mRS 0-3 (40 vs. 25%; p = 0.43) and trend
toward lower mortality (10 vs. 35%; p = 0.21).
CONCLUSIONS: In this pilot study, RP-1127-treated subjects showed better
clinical outcomes when compared to historical controls. An adequately powered
and randomized phase II trial of patients at risk for maligt infarction is
needed to evaluate the potential efficacy of RP-1127. BACKGROUND: Patients with large territory infarction are at high risk of
cerebral edema and neurological deterioration, including death. Preclinical
studies have shown that a continuous infusion of glyburide blocks edema
formation and improves outcome. We hypothesize that treatment with RP-1127
(Glyburide for Injection) reduces formation of brain edema in patients after
large anterior circulation infarction.
METHODS: GAMES-RP is a prospective, randomized, double-blind, multicenter trial
designed to evaluate RP-1127 in patients at high risk for the development of
maligt cerebral edema. The study population consisted of subjects with a
clinical diagnosis of acute severe anterior circulation ischemic stroke with a
baseline diffusion-weighted image lesion between 82 and 300 cm(3) who are
18-80 years of age. The target time from symptom onset to start of study
infusion was ≤10 h. Subjects were randomized to RP-1127 (glyburide for
injection) or placebo and treated with a continuous infusion for 72 h.
RESULTS: The primary efficacy outcome was a composite of the modified Rankin
Scale and the incidence of decompressive craniectomy, assessed at 90 days.
Safety outcomes were the frequency and severity of adverse events, with a focus
on cardiac- and glucose-related serious adverse events.
CONCLUSIONS: GAMES-RP was designed to provide critical information regarding
glyburide for injection in patients with large hemispheric stroke and will
inform the design of future studies. |
What is the function of gasdermin D? | The gasdermin-N domains of the gasdermin proteins can bind membrane lipids, phosphoinositides and cardiolipin to produce membrane-disrupting cytotoxicity. | Inflammatory caspases drive a lytic form of cell death called pyroptosis in
response to microbial infection and endogenous damage-associated signals. Two
studies now demonstrate that cleavage of the substrate gasdermin D by
inflammatory caspases necessitates eventual pyroptotic demise of a cell. Inflammasome is an intracellular signaling complex of the innate immune system.
Activation of inflammasomes promotes the secretion of interleukin 1β (IL-1β) and
IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the
canonical and non-canonical inflammasome pathways, respectively, are crucial for
inflammasome-mediated inflammatory responses. Here we report that gasdermin D
(GSDMD) is another crucial component of inflammasomes. We discovered the
presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a
quantitative mass spectrometry-based analysis. Gene deletion of GSDMD
demonstrated that GSDMD is required for pyroptosis and for the secretion but not
proteolytic maturation of IL-1β in both canonical and non-canonical inflammasome
responses. It was known that GSDMD is a substrate of caspase-1 and we showed its
cleavage at the predicted site during inflammasome activation and that this
cleavage was required for pyroptosis and IL-1β secretion. Expression of the
N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal
modification such as tagging with Flag sequence disrupted the function of GSDMD.
We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not
essential for GSDMD to function. Further analyses of LPS plus nigericin- or
Salmonella typhimurium-treated macrophage cell lines and primary cells showed
that apoptosis became apparent in Gsdmd(-/-) cells, indicating a suppression of
apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other
inflammasome receptors and ASC, and caspase-1 may partially contribute to the
activation of apoptotic caspases in Gsdmd(-/-) cells. These data provide new
insights into the molecular mechanisms of pyroptosis and reveal an unexpected
interplay between apoptosis and pyroptosis. Inflammatory caspases cleave the gasdermin D (GSDMD) protein to trigger
pyroptosis, a lytic form of cell death that is crucial for immune defences and
diseases. GSDMD contains a functionally important gasdermin-N domain that is
shared in the gasdermin family. The functional mechanism of action of gasdermin
proteins is unknown. Here we show that the gasdermin-N domains of the gasdermin
proteins GSDMD, GSDMA3 and GSDMA can bind membrane lipids, phosphoinositides and
cardiolipin, and exhibit membrane-disrupting cytotoxicity in mammalian cells and
artificially transformed bacteria. Gasdermin-N moved to the plasma membrane
during pyroptosis. Purified gasdermin-N efficiently lysed
phosphoinositide/cardiolipin-containing liposomes and formed pores on membranes
made of artificial or natural phospholipid mixtures. Most gasdermin pores had an
inner diameter of 10–14 nm and contained 16 symmetric protomers. The crystal
structure of GSDMA3 showed an autoinhibited two-domain architecture that is
conserved in the gasdermin family. Structure-guided mutagenesis demonstrated
that the liposome-leakage and pore-forming activities of the gasdermin-N domain
are required for pyroptosis. These findings reveal the mechanism for pyroptosis
and provide insights into the roles of the gasdermin family in necrosis,
immunity and diseases. Pyroptosis was long regarded as caspase-1-mediated monocyte death in response to
certain bacterial insults. Caspase-1 is activated upon various infectious and
immunological challenges through different inflammasomes. The discovery of
caspase-11/4/5 function in sensing intracellular lipopolysaccharide expands the
spectrum of pyroptosis mediators and also reveals that pyroptosis is not cell
type specific. Recent studies identified the pyroptosis executioner, gasdermin D
(GSDMD), a substrate of both caspase-1 and caspase-11/4/5. GSDMD represents a
large gasdermin family bearing a novel membrane pore-forming activity. Thus,
pyroptosis is redefined as gasdermin-mediated programmed necrosis. Gasdermins
are associated with various genetic diseases, but their cellular function and
mechanism of activation (except for GSDMD) are unknown. The gasdermin family
suggests a new area of research on pyroptosis function in immunity, disease, and
beyond. |
What is TOPAZ1? | TOPAZ1 is a novel germ cell-specific expressed gene conserved during evolution across vertebrates. Its PAZ-domain protein is abundantly expressed in the gonads during germ cell meiosis. The expression pattern of TOPAZ1, and its high degree of conservation, suggests that it may play an important role in germ cell development. Further characterization of TOPAZ1 may elucidate the mechanisms involved in gametogenesis, and particularly in the RNA silencing process in the germ line. | BACKGROUND: We had previously reported that the Suppression Subtractive
Hybridization (SSH) approach was relevant for the isolation of new mammalian
genes involved in oogenesis and early follicle development. Some of these
transcripts might be potential new oocyte and granulosa cell markers. We have
now characterized one of them, named TOPAZ1 for the Testis and Ovary-specific
PAZ domain gene.
PRINCIPAL FINDINGS: Sheep and mouse TOPAZ1 mRNA have 4,803 bp and 4,962 bp open
reading frames (20 exons), respectively, and encode putative TOPAZ1 proteins
containing 1,600 and 1653 amino acids. They possess PAZ and CCCH domains. In
sheep, TOPAZ1 mRNA is preferentially expressed in females during fetal life with
a peak during prophase I of meiosis, and in males during adulthood. In the
mouse, Topaz1 is a germ cell-specific gene. TOPAZ1 protein is highly conserved
in vertebrates and specifically expressed in mouse and sheep gonads. It is
localized in the cytoplasm of germ cells from the sheep fetal ovary and mouse
adult testis.
CONCLUSIONS: We have identified a novel PAZ-domain protein that is abundantly
expressed in the gonads during germ cell meiosis. The expression pattern of
TOPAZ1, and its high degree of conservation, suggests that it may play an
important role in germ cell development. Further characterization of TOPAZ1 may
elucidate the mechanisms involved in gametogenesis, and particularly in the RNA
silencing process in the germ line. Topaz1 (Testis and Ovary-specific PAZ domain gene 1) is a germ cell specific
gene highly conserved in vertebrates. The putative protein TOPAZ1 contains a PAZ
domain, specifically found in PIWI, Argonaute and Zwille proteins. Consequently,
Topaz1 is supposed to have a role during gametogenesis and may be involved in
the piRNA pathway and contribute to silencing of transposable elements and
maintece of genome integrity. Here we report Topaz1 inactivation in mouse.
Female fertility was not affected, but male sterility appeared exclusively in
homozygous mutants in accordance with the high expression of Topaz1 in male germ
cells. Pachytene Topaz1--deficient spermatocytes progress through meiosis
without either derepression of retrotransposons or MSCI dysfunction, but become
arrested before the post-meiotic round spermatid stage with extensive apoptosis.
Consequently, an absence of spermatids and spermatozoa was observed in
Topaz1(-/-) testis. Histological analysis also revealed that disturbances of
spermatogenesis take place between post natal days 15 and 20, during the first
wave of male meiosis and before the generation of haploid germ cells.
Transcriptomic analysis at these two stages showed that TOPAZ1 influences the
expression of one hundred transcripts, most of which are up-regulated in mutant
testis at post natal day 20. Our results also showed that 10% of these
transcripts are long non-coding RNA. This suggests that a highly regulated
balance of lncRNAs seems to be essential during spermatogenesis for induction of
appropriate male gamete production. |
Which are the symptoms of glucose-6-phosphate dehydrogenase (G6PD) deficiency? | Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) is the most common red blood cell (RBC) enzyme disorder. The decrease as well as the absence of the enzyme increase RBC vulnerability to oxidative stress caused by exposure to certain medications or intake of fava beans. Among the most common symptoms of this condition are:
1) acute hemolysis,
2) chronic hemolysis,
3) neonatal hyperbilirubinemia. | Kinetic and electrophoretic properties of 230--300 fold purified preparations of
glucose-6-phosphate dehydrogenase (G6PD) from red cells of donors and patients
with acute drug hemolytic anemia due to G6PD deficiency were studied. A new
abnormal variant of G6PD isolated from red cell of a patient with acute drug
hemolytic anemia, which was not described in literature, has been discovered.
The abnormal enzyme differs from the normal by decreased Michaelis constant for
glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP), by
increased utilization of analogues of substrates--2-deoxy-glucose-6-phosphate
and particularly deamino-NADP, by low thermal stability, by the character of
pH-dependence, by the appearance of a single band of G6PD activity in
polyacrylamide gel electrophoresis. The purpose of this study was to evaluate the antipyretic action of tolfenamic
acid, as well as its possible adverse reactions, especially in children with
severe or partial form of glucose-6-phosphate dehydrogenase (G6PD) deficiency.
In the study 55 febrile children were included, whose mean age was +/- SD 3.5
+/- 3.3 years, range 0.5-15. Ten of them had severe or partial form of G6PD
deficiency. Fifty-three of the patients responded with a decrease of temperature
which lasted at least 6 hours, though in 2 of them the temperature decrease
lasted less than 6 hours. The tolerance of the drug was good and no side-effects
were noted. None of the patients with or without G6PD deficiency showed
symptoms, signs, or laboratory findings indicating hemolysis before
administration of the drug and 4 days thereafter. In conclusion, tolfenamic acid
is a strong antipyretic agent with excellent tolerance and high safety in
children. Glucose 6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a
housekeeping X-linked gene whose main function is to produce NADPH, a key
electron donor in the defense against oxidizing agents and in reductive
biosynthetic reactions. Inherited G6PD deficiency is associated with either
episodic hemolytic anemia (triggered by fava beans or other agents) or life-long
hemolytic anemia. We show here that an evolutionary analysis is a key to
understanding the biology of a housekeeping gene. From the alignment of the
amino acid (aa) sequence of 52 glucose 6-phosphate dehydrogenase (G6PD) species
from 42 different organisms, we found a striking correlation between the aa
replacements that cause G6PD deficiency in humans and the sequence conservation
of G6PD: two-thirds of such replacements are in highly and moderately conserved
(50-99%) aa; relatively few are in fully conserved aa (where they might be
lethal) or in poorly conserved aa, where presumably they simply would not cause
G6PD deficiency. This is consistent with the notion that all human mutants have
residual enzyme activity and that null mutations are lethal at some stage of
development. Comparing the distribution of mutations in a human housekeeping
gene with evolutionary conservation is a useful tool for pinpointing amino acid
residues important for the stability or the function of the corresponding
protein. In view of the current explosive increase in full genome sequencing
projects, this tool will become rapidly available for numerous other genes. Rasburicase is a new treatment for hyperuricemia, a metabolic manifestation of
tumor lysis syndrome (TLS). Rasburicase has a unique mechanism of action that
allows uric acid byproducts to be easily excreted in the urine. Clinical trials
have shown that rasburicase has a rapid onset of action that allows chemotherapy
to be delivered on time and prevents hyperuricemia-related complications,
including renal compromise. The drug has been used successfully in adults and
children. The main side effect of rasburicase is the potential for a
hypersensitivity reaction. The drug is contraindicated in patients with glucose
6-phosphate dehydrogenase (G6PD) deficiency because this can precipitate
hemolytic anemia. The drug has not been studied in patients with a history of
allergies or asthma. Oncology nurses play a major role in the assessment and
management of TLS-related complications. They must assess patients for G6PD
deficiency and signs and symptoms of hypersensitivity reaction before and during
chemotherapy or other therapeutic interventions. Nurses play a direct role in
preventing complications related to TLS and contributing to the quality of life
in this patient population. Glucosephosphate isomerase (GPI) deficiency in humans is an autosomal recessive
disorder, which results in nonspherocytic hemolytic anemia of variable clinical
expression. A 4-year-old female with severe congenital hemolytic anemia had low
red cell GPI activity of 15.5 IU/g Hb (50% of normal mean) indicating GPI
deficiency. Subsequent DNA sequence analysis revealed a novel homozygous 921C to
G mutation in the GPI gene sequence, predicting a Phe307 to Leu replacement.
Strikingly, the red cell GPI activity in this patient was higher than that found
in a second patient expressing the same GPI variant, with a more severe clinical
phenotype. We propose that the hemolysis in the first patient may be modified by
an accompanying deficiency of glucose-6-phosphate dehydrogenase (G6PD). The
proband's red cell G6PD activity was reduced at 4.5 IU/g Hb (50% of normal mean)
and molecular studies revealed heterozygosity for the G6PD Viangchan mutation
and a skewed pattern of X-chromosome inactivation, producing almost exclusive
expression of the mutated allele. The G6PD Viangchan variant is characterised by
severe enzyme deficiency, but not chronic hemolysis. This study suggests that
the metabolic consequences of a combined deficiency of GPI and G6PD might be
responsible for a different clinical outcome than predicted for either defect in
isolation. Possible causes for a normocytic hyperregeneratory anemia are beside an
incomplete treatment of iron deficiency, vitamin B12 deficiency or folic acid
deficiency notably a hemolysis. After exclusion of other causes of hemolysis
like immune hemolytic anemias, microangiopathic hemolytic anemias and
hemoglobinopathies, an enzyme deficiency of erythrocytes should be considered.
By far the most common form worldwide is the Glucose-6-phosphate deficiency. In
the most frequent variants of this disease hemolysis occurs only during stress,
imposed for example by infection, "oxidative" drugs or after ingestion of fava
beans. The most serious clinical complication of the Glucose-6-phosphate
deficiency is the rarely observed neonatal icterus. Some enzyme variants can
cause chronic hemolysis which is described as chronic nonsperocytic hemolytic
anemia. This form of chronic anemia can also be caused by other enzyme
deficiencies, most frequently by the Pyruvate kinase deficiency. All other
deficiencies of glycolytic enzymes are even rarer. It should be noted that in
some of these very rare forms neurological rather than hematological symptoms
predominate the clinical syndrome. If there is suspicion, on the basis of
clinical symptoms and/or familial history, diagnosis of an enzyme deficiency can
be achieved relatively easy by measurement of the enzyme activity. Accurate
diagnosis might be helpful in therapeutic decisions (e.g. splenectomy in certain
forms) and it is essential for genetic counseling, since certain deficiencies
are transmitted as autosomal recessive disorders (e.g. pyruvate kinase
deficiency), while the most common form, the glucose-6-phosphate dehydrogenase
deficiency is linked to the X-chromosome. Although glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common
enzyme disorder worldwide, it has rarely been reported among Korean. The patient
was previously healthy 39 yr old male who showed severe hemolytic anemia and
acute renal failure accompanied by hyperbilirubinemia after hepatitis A
infection. The additional studies for differential diagnosis of hemolytic anemia
showed a moderate deficiency of G6PD enzyme. Because hepatitis A infection in
patient with G6PD deficiency present much more severe clinical symptoms, G6PD
enzyme should be examined in patients with triggering factors of hemolysis such
as hepatitis A infection. Dermatitis artefacta is a disorder in which the skin is the target of
self-inflicted injury. We report a case of dermatitis artefacta, in which the
patient developed skin lesions, after taking each and every medication.
Additionally he also had red coloured urine after taking certain group of
medications, which, on further investigations, was found to be associated with
glucose 6-phosphate dehydrogenase (G6PD) deficiency. This case illustrates the
presence of factitious dermatitis and physical co-morbidity simultaneously,
which was missed before psychiatric referral. Every symptom in a patient with a
factitious disorder should not be labelled as feigned without a proper workup. PURPOSE: The case of a patient who developed aseptic meningitis, hemolytic
anemia, hepatitis, and orthostatic hypotension simultaneously during treatment
with trimethoprim-sulfamethoxazole is described.
BACKGROUND: A healthy 37-year-old African- American man was receiving treatment
with trimethoprim-sulfamethoxazole double strength. This was the patient's first
experience with trimethoprim- sulfamethoxazole, and he was not taking any other
medications during the treatment period. He had been taking
trimethoprim-sulfamethoxazole for approximately eight days when he revisited his
family physician, complaining of headaches, dizziness, difficulty with speech,
weakness, and itching on the trunk of his body and legs, where a maculopapular
rash was noted. Orthostatic hypotension was also noted at that visit, with a
standing blood pressure measurement of 95/80 mm Hg. Based on these findings and
since the patient had no signs of infection, his physician instructed him to
discontinue the drug. The patient was admitted to the emergency department of a
local hospital within two days due to ongoing headache, elevated temperature,
and nuchal rigidity, symptoms suggestive of meningitis. Because of the presence
of hemolysis, the patient underwent testing for glucose-6-phosphate
dehydrogenase (G6PD) deficiency, for which he tested positive. The patient was
discharged five days after admission and referred to a hematology clinic for
follow-up. The patient has since returned to his routines of daily living and
has reported no fatigue or other lingering adverse symptoms.
CONCLUSION: A 37-year-old African- American man with G6PD deficiency developed
hemolytic anemia, hepatitis, orthostatic hypotension, and aseptic meningitis
simultaneously after using trimethoprim-sulfamethoxazole. The current interest in malaria elimination has led to a renewed interest in
drugs that can be used for mass administration to minimize malaria transmission.
Primaquine (PQ) is the only generally available drug with a strong activity
against mature Plasmodium falciparum gametocytes, the parasite stage responsible
for transmission. Despite concerns about PQ-induced hemolysis in
glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals, a single dose of
PQ may be safe and efficacious in clearing gametocytes that persist after
conventional treatment. As part of a mass drug intervention, we determined the
hemolytic effect of sulfadoxine-pyrimethamine (SP) plus artesunate (AS) plus a
single dose of primaquine (PQ; 0.75 mg/kg of body weight) in children aged 1 to
12 years. Children were randomized to receive SP+AS+PQ or placebo; those with a
hemoglobin (Hb) level below 8 g/dl were excluded from receiving PQ and received
SP+AS. The Hb concentration was significantly reduced 7 days after SP+AS+PQ
treatment but not after placebo or SP+AS treatment. This reduction in Hb was
most pronounced in G6PD-deficient (G6PD A-) individuals (-2.5 g/dl; 95%
confidence interval [95% CI], -1.2 to -3.8 g/dl) but was also observed in
heterozygotes (G6PD A) (-1.6 g/dl; 95% CI, -0.9 to -2.2 g/dl) and individuals
with the wild-type genotype (G6PD B) (-0.5 g/dl; 95% CI, -0.4 to -0.6 g/dl).
Moderate anemia (Hb level of <8 g/dl) was observed in 40% (6/15 individuals) of
the G6PD A-, 11.1% (3/27 individuals) of the G6PD A, and 4.5% (18/399
individuals) of the G6PD B individuals; one case of severe anemia (Hb level of
<5 g/dl) was observed. PQ may cause moderate anemia when coadministered with
artemisinins, and excluding individuals based on G6PD status alone may not be
sufficient to prevent PQ-induced hemolysis. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common
hereditary enzymopathies worldwide. Mostly G6PD deficient cases are asymptomatic
though they may have the risk of neonatal jaundice (NNJ) and acute intravascular
hemolysis during oxidative stress. Chronic nonspherocytic hemolytic anemia
(CNSHA) due to G6PD deficiency is rare. In Thailand, one case was reported
40 years ago and by biochemical study this G6PD was reported to be a new variant
G6PD Bangkok. We, herein, report two families with CNSHA due to G6PD deficiency.
In the first family, we have been following up the clinical course of the
patient with G6PD Bangkok. In addition to chronic hemolysis, he had three acute
hemolytic episodes requiring blood transfusions during childhood period.
Multiple gallstones were detected at the age of 27. His two daughters who
inherited G6PD Bangkok from him and G6PD Vanua Lava from his wife are
asymptomatic. Both of them had NNJ and persistent evidences of compensated
hemolysis. Molecular analysis revealed a novel missense mutation 825 G→C
predicting 275 Lys→Asn causing G6PD Bangkok. In the second family, two male
siblings are affected. They had NNJ and several hemolytic episodes which
required blood transfusions. On follow-up they have been diagnosed with chronic
hemolysis as evidenced by reticulocytosis and indirect hyperbilirubinemia.
Molecular analysis revealed combined missense mutations in exons 12 and 13. The
first mutation was 1376 G→T predicting 459 Arg→Leu (known as G6PD Canton) and
the second one was 1502 T→G predicting 501 Phe→Cys. We designated the resulting
novel G6PD variant, G6PD Bangkok Noi. OBJECTIVE: To evaluate the risk of hemolysis in children with
glucose-6-phosphate dehydrogenase (G6PD) deficiency after short-term
administration of analgesics, such as paracetamol, ibuprofen, tramadol,
sufentanil, and parecoxib.
STUDY DESIGN: This was a prospective study of children with G6PD deficiency who
were treated with analgesics for 3 days after undergoing surgery. Hemoglobin
(Hb) concentration, reticulocyte count, unconjugated bilirubin level, lactate
dehydrogenase level, and the presence of Heinz bodies on blood smear microscopy
were assessed at baseline and after analgesic treatment. Telephone interviews
and clinical reviews were provided during a 7-day study period. The primary
outcome was evidence of hemolysis. Statistical analyses were done using the
paired Student t test or Wilcoxon signed-rank test as appropriate.
RESULTS: Ten male infants (mean age, 4.3 ± 1.3 years) completed the study. The
mean decrease in (Hb) concentration was -0.2 g/dL (P, not significant). The mean
reticulocyte count increased by 0.1% (95% CI, 0.08%-0.2%; P = .001). However,
the change in reticulocyte count was not correlated with the changes in Hb
concentration or other laboratory results and was not accompanied by the
clinical signs and symptoms of hemolysis.
CONCLUSION: Short-term administration of paracetamol, ibuprofen, tramadol,
sufentanil, and parecoxib in therapeutic dosages did not increase the risk of
hemolysis in children with G6PD deficiency. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human
enzymopathy that affects cellular redox status and may lower flux into
nonoxidative pathways of glucose metabolism. Oxidative stress may worsen
systemic glucose tolerance and cardiometabolic syndrome. We hypothesized that
G6PD deficiency exacerbates diet-induced systemic metabolic dysfunction by
increasing oxidative stress but in myocardium prevents diet-induced oxidative
stress and pathology. WT and G6PD-deficient (G6PDX) mice received a standard
high-starch diet, a high-fat/high-sucrose diet to induce obesity (DIO), or a
high-fructose diet. After 31 wk, DIO increased adipose and body mass compared
with the high-starch diet but to a greater extent in G6PDX than WT mice (24 and
20% lower, respectively). Serum free fatty acids were increased by 77% and
triglycerides by 90% in G6PDX mice, but not in WT mice, by DIO and high-fructose
intake. G6PD deficiency did not affect glucose tolerance or the increased
insulin levels seen in WT mice. There was no diet-induced hypertension or
cardiac dysfunction in either mouse strain. However, G6PD deficiency increased
aconitase activity by 42% and blunted markers of nonoxidative glucose pathway
activation in myocardium, including the hexosamine biosynthetic pathway
activation and advanced glycation end product formation. These results reveal a
complex interplay between diet-induced metabolic effects and G6PD deficiency,
where G6PD deficiency decreases weight gain and hyperinsulinemia with DIO, but
elevates serum free fatty acids, without affecting glucose tolerance. On the
other hand, it modestly suppressed indexes of glucose flux into nonoxidative
pathways in myocardium, suggesting potential protective effects. Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is
classically manifested by hemolytic anemia in human. More recently, it has been
shown that mild G6PD deficiency moderately affects cardiac function, whereas
severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency
affects organisms has not been fully elucidated due to the lack of a suitable
animal model. In this study, G6PD-deficient Caenorhabditis elegans was
established by RNA interference (RNAi) knockdown to delineate the role of G6PD
in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly
hampered in C. elegans in parallel with increased oxidative stress and DNA
oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis
(2-fold increase), reduced egg production (65% of mock), and hatching (10% of
mock). To determine whether oxidative stress is associated with G6PD
knockdown-induced reproduction defects, C. elegans was challenged with a
short-term hydrogen peroxide (H2O2). The early phase egg production of both mock
and G6PD-knockdown C. elegans were significantly affected by H2O2. However,
H2O2-induced germ cell apoptosis was more dramatic in mock than that in
G6PD-deficient C. elegans. To investigate the signaling pathways involved in
defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53
and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the
upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and
hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation
significantly affected egg production, whereas sek-1 mutation further decreased
egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of
sek-1 or mek-1 dramatically rescued defective hatching (8.3- and 9.6-fold
increase, respectively) induced by G6PD knockdown. Taken together, these
findings show that G6PD knockdown reduces egg production and hatching in C.
elegans, which are possibly associated with enhanced oxidative stress and
altered MAPK pathways, respectively. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human
enzymopathy and in Sub-Saharan Africa, is a significant cause of infection- and
drug-induced hemolysis and neonatal jaundice. Our goals were to determine the
prevalence of G6PD deficiency among Nigerian children of different ethnic
backgrounds and to identify predictors of G6PD deficiency by analyzing vital
signs and hematocrit and by asking screening questions about symptoms of
hemolysis. We studied 1,122 children (561 males and 561 females) aged 1 month to
15 years. The mean age was 7.4 ± 3.2 years. Children of Yoruba ethnicity made up
the largest group (77.5%) followed by those Igbo descent (10.6%) and those of
Igede (10.2%) and Tiv (1.8%) ethnicity. G6PD status was determined using the
fluorescent spot method. We found that the overall prevalence of G6PD deficiency
was 15.3% (24.1% in males, 6.6% in females). Yoruba children had a higher
prevalence (16.9%) than Igede (10.5%), Igbo (10.1%) and Tiv (5.0%) children. The
odds of G6PD deficiency were 0.38 times as high in Igbo children compared to
Yoruba children (p=0.0500). The odds for Igede and Tiv children were not
significantly different from Yoruba children (p=0.7528 and 0.9789 respectively).
Mean oxygen saturation, heart rate and hematocrit were not significantly
different in G6PD deficient and G6PD sufficient children. The odds of being G6PD
deficient were 2.1 times higher in children with scleral icterus than those
without (p=0.0351). In conclusion, we determined the prevalence of G6PD
deficiency in Nigerian sub-populations. The odds of G6PD deficiency were
decreased in Igbo children compared to Yoruba children. There was no association
between vital parameters or hematocrit and G6PD deficiency. We found that a
history of scleral icterus may increase the odds of G6PD deficiency, but we did
not exclude other common causes of icterus such as sickle cell disease or
malarial infection. Red blood cells carry oxygen in the body and Glucose-6-Phosphate Dehydrogenase
protects these cells from oxidative chemicals. If there is a lack of
Glucose-6-Phosphate Dehydrogenase, red blood cells can go acute hemolysis.
Convulsion is a rare presentation for acute hemolysis due to Glucose-6-Phosphate
Dehydrogenase deficiency. Herein, we report a case report of a
Glucose-6-Phosphate Dehydrogenase deficiency diagnosed patient after
presentation with convulsion. A 70 year-old woman patient had been hospitalized
because of convulsion and fatigue. She has not had similar symptoms before. She
had ingested fava beans in the last two days. Her hypophyseal and brain magnetic
resoce imaging were normal. Blood transfusion was performed and the patient
recovered. INTRODUCTION: Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) is
the most common red blood cell (RBC) enzyme disorder. The decrease as well as
the absence of the enzyme increase RBC vulnerability to oxidative stress caused
by exposure to certain medications or intake of fava beans. Among the most
common clinical manifestations of this condition, acute hemolysis, chronic
hemolysis, neonatal hyperbilirubinemia, and an asymptomatic form are observed.
OBJECTIVE: To analyze the case of a child who presented hemolytic crisis due to
favism.
CASE REPORT: A 2 year and 7 month old boy with a history of hyperbilirubinemia
during the newborn period with no apparent cause, no family history of hemolytic
anemia or parental consanguinity. He presented a prolonged neonatal jaundice and
severe anemia requiring RBC transfusion. An intake of fava beans 48 h prior to
onset of symptoms was reported. G6PD qualitative determination was compatible
with this enzyme deficiency.
CONCLUSION: G6PD deficiency can be highly variable in its clinical presentation,
so it is necessary to keep it in mind during the diagnosis of hemolytic anemia
at any age. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive
genetic defect that can cause hemolytic crisis. However, this disease affects
both males and females. In Turkey, the frequency of this enzyme deficiency was
reported to vary, from 0.25 to 18%, by the geographical area. Its prevalence in
the northern Black Sea region of Turkey is unknown. The aims of this study were
to assess the prevalence of G6PD deficiency in the northern region Turkey in
children and adults with hyperbilirubinemia and hemolytic anemia. This report
included a total of 976 G6PD enzyme results that were analyzed between May 2005
and January 2014. G6PD deficiency was detected in 5.0% of all patients. G6PD
deficiency was significantly less frequent in females (1.9%, 6/323) than in
males (6.6%, 43/653). G6PD deficiency was detected in 3.7% of infants with
hyperbilirubinemia, 9.2% of children, and 4.5% of adults with hemolytic anemia.
In both the newborn group and the group of children, G6PD deficiency was
significantly more frequent in males. In the combined group of children (groups
I and II), the proportion of males was 74% and 67% in all groups (P = .0008). In
conclusion, in northern region of Turkey, G6PD deficiency is an important cause
of neonatal hyperbilirubinemia and hemolytic crisis in children and adults. This
study suggests that most pediatricians thought that G6PD deficiency is
exclusively a male disease. For this reason, some female patients may have been
undiagnosed. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive
hemolytic anemia caused by a mutation in the G6PD gene on Xq28. Herein, we
describe a Korean boy with G6PD deficiency resulting from a novel mutation in
G6PD. A 20-month-old boy with hemolytic anemia was referred for molecular
diagnosis. He had no relevant family history. The G6PD activity was severely
decreased at 0.2 U/g Hb (severe deficiency). Direct sequencing analyses on the
G6PD gene revealed that he was hemizygous for a novel missense variant,
c.1187C>G (p.Pro396Arg), in exon 10 of G6PD. Family study involving his parents
revealed the de novo occurrence of the mutation. This is the first report of
genetically confirmed G6PD deficiency in Korea. |
Has the gorilla genome been determined? | Yes, the gorilla genome has been sequenced. | DNA sequencing reveals that the genomes of the human, gorilla and chimpanzee
share more than 98% homology. Comparative chromosome painting and gene mapping
have demonstrated that only a few rearrangements of a putative ancestral
mammalian genome occurred during great ape and human evolution. However,
interspecies representational difference analysis (RDA) of the gorilla between
human and gorilla revealed gorilla-specific DNA sequences. Cloning and
sequencing of gorilla-specific DNA sequences indicate that there are repetitive
elements. Gorilla-specific DNA sequences were mapped by fluorescence in-situ
hybridization (FISH) to the subcentromeric/centromeric regions of three pairs of
gorilla submetacentric chromosomes. These sequences could represent either
ancient sequences that got lost in other species, such as human and orang-utan,
or, more likely, recent sequences which evolved or originated specifically in
the gorilla genome. Accurate sequence and assembly of genomes is a critical first step for studies
of genetic variation. We generated a high-quality assembly of the gorilla genome
using single-molecule, real-time sequence technology and a string graph de novo
assembly algorithm. The new assembly improves contiguity by two to three orders
of magnitude with respect to previously released assemblies, recovering 87% of
missing reference exons and incomplete gene models. Although regions of large,
high-identity segmental duplications remain largely unresolved, this
comprehensive assembly provides new biological insight into genetic diversity,
structural variation, gene loss, and representation of repeat structures within
the gorilla genome. The approach provides a path forward for the routine
assembly of mammalian genomes at a level approaching that of the current quality
of the human genome. The increasing availability of complete genome data is facilitating the
acquisition of phylogenomic data sets, but the process of obtaining orthologous
sequences from other genomes and assembling multiple sequence alignments remains
piecemeal and arduous. We designed software that performs these tasks and
outputs anonymous loci (AL) or anchored enrichment/ultraconserved element loci
(AE/UCE) data sets in ready-to-analyze formats. We demonstrate our program by
applying it to the hominoids. Starting with human, chimpanzee, gorilla, and
orangutan genomes, our software generated an exhaustive data set of 292 ALs (∼1
kb each) in ∼3 h. Not only did analyses of our AL data set validate the program
by yielding a portrait of hominoid evolution in agreement with previous studies,
but the accuracy and precision of our estimated ancestral effective population
sizes and speciation times represent improvements. We also used our program with
a published set of 512 vertebrate-wide AE "probe" sequences to generate data
sets consisting of 171 and 242 independent loci (∼1 kb each) in 11 and 13 min,
respectively. The former data set consisted of flanking sequences 500 bp from
adjacent AEs, while the latter contained sequences bordering AEs. Although our
AE data sets produced the expected hominoid species tree, coalescent-based
estimates of ancestral population sizes and speciation times based on these data
were considerably lower than estimates from our AL data set and previous
studies. Accordingly, we suggest that loci subjected to direct or indirect
selection may not be appropriate for coalescent-based methods. Complete in
silico approaches, combined with the burgeoning genome databases, will
accelerate the pace of phylogenomics. |
Is vemurafenib used for thyroid cancer? | Yes. Vemurafenib, a selective BRAF inhibitor, appears to have promising clinical activity in patients with papillary thyroid cancer (PTC) harboring the BRAF(V600E) mutation. | BACKGROUND: Clinical benefit from cytotoxic chemotherapy for metastatic
papillary thyroid carcinoma (PTC) is disappointing, and effective therapeutic
approaches for these patients are urgently needed. Because kinase-activating
mutations in the BRAF proto-oncogene commonly occur in advanced PTC, and
inhibition of BRAF(V600E) has shown promising clinical activity in melanoma,
BRAF inhibitor therapy may be an effective strategy to treat metastatic PTC.
METHODS: The dose escalation portion of a first-in-human, phase I study of
vemurafenib, a selective RAF inhibitor, included three patients with metastatic
PTC harboring the BRAF(V600E) mutation. Vemurafenib was initially dosed at
240-360 mg twice a day, later escalated to 720 mg twice a day. Response
evaluation was performed every 8 weeks per Response Evaluation Criteria in Solid
Tumors (RECIST).
RESULTS: Among the three patients, one had a confirmed partial response with
reduction of pulmonary target lesions by 31%, and the duration of response was
7.6 months before the disease progressed in the lungs and the bones. The time to
progression was 11.7 months. The other two patients had stable disease, and the
time to progression was 13.2 and 11.4 months, respectively.
CONCLUSIONS: Vemurafenib appears to have a promising clinical activity in
patients with metastatic PTC, and our data suggest that the BRAF(V600E) mutant
kinase is a relevant target for therapy in this patient population. Further
investigation of inhibitors of mutated BRAF kinase in patients with PTC in a
phase II study is warranted. BACKGROUND: Mouse models of metastatic human cancers are important tools in
preclinical studies for testing new systematic therapies and studying effectors
of cancer metastasis. The major drawbacks of current mouse models for metastatic
thyroid cancer are that they have low metastasis rates and do not allow in vivo
tumor detection. Here, we report and characterize an in vivo detectable
metastasis mouse model of human thyroid cancer using multiple thyroid cancer
cell lines.
METHODS: Human anaplastic thyroid cancer cell lines 8505C, C-643, SW-1736, and
THJ-16T; follicular thyroid cancer cell lines FTC-133, FTC-236, and FTC-238; and
Hürthle cell carcinoma cell line XTC-1 were transfected with a linearized
pGL4.51[luc2/CMV/Neo] vector or transduced with lentivirus containing Luc2-eGFP
reporter genes. The stably transfected cells were injected intravenously into
NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. Tumors were detected with an in vivo
imaging system-Xenogen IVIS. Vemurafenib, a BRAF inhibitor, was used to treat
lung metastases generated from 8505C-Luc2 cells with a BRAF(V600E) mutation to
test the accuracy of the model to evaluate response to therapy.
RESULTS: Intravenous injection of as few as 30,000 8505C-Luc2 cells produced
lung metastases in 100% of the injected mice, and many of these mice also
developed bone metastases at a later stage of the disease. Similarly, metastatic
tumors also developed in all mice injected with C-643-Luc2, THJ-16T-Luc2,
FTC-133-Luc2, FTC-236-Luc2, FTC-238-Luc2, and XTC-1-Luc2 cells. The metastases
were easily detectable in vivo, and tumor progression could be dynamically and
accurately followed and correlated with the actual tumor burden. Furthermore,
disease progression could be easily controlled by adjusting the number of
injected cells. The in vivo treatment of 8505C xenograft lung metastases with
vemurafenib dramatically reduced the growth and signal intensity with good
correlation with actual tumor burden.
CONCLUSIONS: Herein we report an in vivo detectable mouse model of metastatic
human thyroid cancer that is reliable and reproducible. It will serve as a
useful tool in the preclinical testing of alternative systematic therapies for
metastatic thyroid cancer, and for functional studies of thyroid cancer tumor
biology in vivo. CONTEXT: For patients with metastatic papillary thyroid carcinoma (PTC)
refractory to radioactive iodine (RAI) treatment, systemic chemotherapy has
limited efficacy. Such tumors frequently harbor BRAF V600E, and this alteration
may predict responsiveness to vemurafenib treatment.
OBJECTIVE: We report a metastatic PTC patient refractory to RAI treatment that
underwent genomic profiling by next-generation sequencing. The sole genomic
alteration identified was BRAF V600E on a near diploid genome with trisomy 1q.
With vemurafenib treatment, the patient experienced a dramatic radiographic and
clinical improvement, with the duration of an ongoing antitumor response
exceeding 23 months.
DESIGN: Hybridization capture of 3,769 exons of 236 cancer-related genes and the
introns of 19 genes frequently rearranged in cancer was applied to >50 ng of DNA
extracted from a formalin-fixed, paraffin-embedded biopsy of a lymph node
containing metastatic PTC and was sequenced to a high, uniform coverage of ×616.
RESULTS: A BRAF V600E alteration was identified with no other somatic genomic
alterations present within a near diploid tumor genome. The patient initially
received vemurafenib at 960 mg twice daily that was reduced to 480 mg twice
daily due to rash and diarrhea and has experienced an ongoing antitumor response
exceeding 23 months by both PET-CT and dedicated CT imaging.
CONCLUSIONS: Genomic profiling in metastatic, RAI-refractory PTC can reveal a
targetable BRAF V600E alteration without compounding somatic alterations, and
such patients may derive a more prolonged benefit from vemurafenib treatment.
Prospective clinical trials are ongoing to confirm our preliminary observation. CONTEXT: Vemurafenib, a selective BRAF inhibitor, appears to have promising
clinical activity in patients with papillary thyroid cancer (PTC) harboring the
BRAF(V600E) mutation.
OBJECTIVE: To determine the efficacy and safety of vemurafenib when used outside
of a clinical trial.
DESIGN: A retrospective review at MD Anderson Cancer Center.
METHODS: The best responses were evaluated using RECIST v1.1. A single
radiologist reviewed all images. Adverse events (AEs) were evaluated using CTCAE
v.4.0.
RESULTS: We identified 17 patients with advanced PTC harboring the BRAF(V600E)
mutation who were treated with vemurafenib outside of a clinical trial. Median
age at diagnosis was 63 years, and 53% were male. At vemurafenib start, 3 (18%)
patients had disease confined to the neck, and 14 (72%) had distant metastases.
Tyrosine kinase inhibitors had been previously administered to 4 (24%) patients.
Two (12%) patients discontinued vemurafenib because of AEs before restaging.
Best response: partial response (PR) in 7/15 (47%) and stable disease (SD) in
8/15(53%) patients. The rate of durable response (PR plus SD ≥ 6 months) was
67%. Median time to treatment failure was 13 months. There was no association
between change in thyroglobulin and tumor size. Drug discontinuation, drug
interruptions, and dose reductions were needed in 5 (29%), 13 (76%), and 10
(59%) patients, respectively. Most common AEs were fatigue (71%), weight loss
(71%), anorexia (65%), arthralgias (59%), hair loss (59%), rash (59%), hand-foot
syndrome (53%), calluses (47%), diarrhea (47%), fever (41%), dry mouth (35%),
nausea (35%), and verrucous keratosis (35%). Grade ≥ 3 AEs were present in 8
(47%) patients.
CONCLUSIONS: Vemurafenib is a potentially effective and well-tolerated treatment
strategy in patients with advanced PTC harboring the BRAF(V600E) mutation. Our
results are similar to those reported in a phase II clinical trial and support
the potential role of vemurafenib in this patient population. The US Food and Drug Administration-approved BRAF inhibitors, vemurafenib and
dabrafenib, have demonstrated superior efficacy in patients with BRAF-mutant
melanomas but have limited efficacy in BRAF-mutant colorectal cancer. Little is
known at this time regarding BRAF inhibitors in thyroid cancer. Initial reports
in patients with progressive, radioactive iodine-refractory BRAF-mutant
papillary thyroid cancer suggest response rates of approximately 30-40%. In this
review, we discuss BRAF inhibitors in the context of thyroid cancer, the
toxicities associated with BRAF inhibitors, and the suggested management of
those toxicities. The management of vemurafenib and dabrafenib toxicities is
applicable across all tumor types and may serve as a practical guide to their
use. Treatment options for advanced metastatic thyroid cancer patients are limited.
Vemurafenib, a BRAFV600E inhibitor, has shown promise in clinical trials
although cellular resistance occurs. Combination therapy that includes BRAFV600E
inhibition and avoids resistance is a clinical need. We used an in vitro model
to examine combination treatment with vemurafenib and mammalian target of
rapamycin (mTOR) inhibitors, metformin and rapamycin. Cellular viability and
apoptosis were analyzed in thyroid cell lines by trypan blue exclusion and TUNEL
assays. Combination of vemurafenib and metformin decreased cell viability and
increased apoptosis in both BCPAP papillary thyroid cancer cells and 8505c
anaplastic thyroid cancer cells. This combination was also found to be active in
vemurafenib-resistant BCPAP cells. Changes in expression of signaling molecules
such as decreased mTOR expression in BCPAP and enhanced inhibition of
phospho-MAPK in resistant BCPAP and 8505c were observed. The second combination
of vemurafenib and rapamycin amplified cell death in BCPAP cells. We conclude
that combination of BRAFV600E and mTOR inhibition forms the basis of a treatment
regimen that should be further investigated in in vivo model systems. Metformin
or rapamycin adjuvant treatment may provide clinical benefits with minimal side
effects to BRAFV600E-positive advanced thyroid cancer patients treated with
vemurafenib. BRAF(V600E) mutation exerts an essential oncogenic function in many tumors,
including papillary thyroid carcinoma (PTC). Although BRAF(V600E) inhibitors are
available, lack of response has been frequently observed. To study the mechanism
underlying intrinsic resistance to the mutant BRAF(V600E) selective inhibitor
vemurafenib, we established short-term primary cell cultures of human
metastatic/recurrent BRAF(V600E)-PTC, intrathyroidal BRAF(V600E)-PTC, and normal
thyroid (NT). We also generated an early intervention model of human
BRAF(V600E)-PTC orthotopic mouse. We find that metastatic BRAF(V600E)-PTC cells
elicit paracrine-signaling which trigger migration of pericytes, blood
endothelial cells and lymphatic endothelial cells as compared to BRAF(WT)-PTC
cells, and show a higher rate of invasion. We further show that vemurafenib
therapy significantly suppresses these aberrant functions in non-metastatic
BRAF(V600E)-PTC cells but lesser in metastatic BRAF(V600E)-PTC cells as compared
to vehicle treatment. These results concur with similar folds of down-regulation
of tumor microenvironment-associated pro-metastatic molecules, with no effects
in BRAF(WT)-PTC and NT cells. Our early intervention preclinical trial shows
that vemurafenib delays tumor growth in the orthotopic BRAF(WT/V600E)-PTC mice.
Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss
of CDKN2A (P16, chromosome 9p) in metastatic BRAF(V600E)-PTC cells which are
associated with resistance to vemurafenib treatment. Critically, we demonstrate
that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic
BRAF(V600E)-PTC cell death and ameliorates response to vemurafenib treatment as
compared to single agent treatment. In conclusion, short-term PTC and NT
cultures offer a predictive model for evaluating therapeutic response in
patients with PTC. Our PTC pre-clinical model suggests that combined targeted
therapy might be an important therapeutic strategy for metastatic and refractory
BRAF(V600E)-positive PTC. CONTEXT: Use of BRAF V600E inhibitors to restore thyroid iodide-handling gene
expression and radioactive iodine (RAI) avidity is an attractive therapeutic
strategy for RAI-refractory thyroid cancer, but recent initial clinical
responses were modest. Given histone deacetylation at the sodium/iodide
symporter promoter by histone deacetylase (HDAC) as a mechanism, simultaneously
targeting BRAF V600E and HDAC could be a more effective strategy.
OBJECTIVES: The objective of the study was to test whether suppressing both BRAF
V600E and HDAC could more effectively induce thyroid gene expression and RAI
uptake in thyroid cancer cells.
RESEARCH DESIGN: We tested the BRAF V600E inhibitor PLX4032 (vemurafenib) and
the HDAC inhibitor SAHA (vorinostat), two major anticancer drugs currently
approved for clinical use, in inducing thyroid gene expression and RAI uptake in
thyroid cancer cells.
RESULTS: PLX4032 alone induced a modest expression of thyroid genes and RAI
uptake preferentially in thyroid cancer cells harboring BRAF V600E. SAHA showed
an effect in a genetic-independent manner in all the cells. A robust synergistic
effect on thyroid gene expression and RAI uptake was observed in BRAF
V600E-positive thyroid cancer cells when the two inhibitors were simultaneously
used. This was dramatically enhanced further by TSH; triple combination of
PLX4032, SAHA, and TSH showed the most robust effect on thyroid gene expression
and RAI uptake in cells harboring BRAF V600E. Abundant sodium/iodide symporter
protein expression in thyroid cancer cells under these conditions was confirmed
by immunofluorescent microscopy.
CONCLUSIONS: Simultaneously suppressing BRAF V600E and HDAC, particularly when
cotreated with TSH, induced a far more robust expression of thyroid genes and
RAI uptake in thyroid cancer cells than suppressing BRAF V600E alone. Triple
combination of PLX4032, SAHA, and TSH is a specific robust regimen to restore
RAI avidity in RAI-refractory BRAF V600E-positive thyroid cancer, which warrants
clinical trials to confirm. Treatment options for advanced metastatic or progressive thyroid cancers are
limited. Although targeted therapy specifically inhibiting intracellular kinase
signaling pathways has markedly changed the therapeutic landscape, side-effects
and resistance of single agent targeted therapy often leads to termination of
the treatment. The objective of the present study was to identify the antitumor
property of the non-selective β-adrenergic receptor antagonist propranolol for
thyroid cancers. Human thyroid cancer cell lines 8505C, K1, BCPAP and BHP27 were
used in the present study. Broad β-blocker propranolol and β2-specific
antagonist ICI118551, but not β1-specific antagonist atenolol, inhibited the
growth of 8505C and K1 cells. Propranolol treatment inhibited growth and induced
apoptosis of 8505C cells in vitro and in vivo, which are closely associated with
decreased expressions of cyclin D1 and anti-apoptotic Bcl-2. Expression of
hexokinase 2 (HK2) and glucose transporter 1 (GLUT1) also decreased following
propranolol intervention. 18F-FDG PET/CT imaging of the 8505C xenografts
validated shrinkage of the tumors in the propranolol-treated group when compared
to the phosphate‑buffered saline treated group. Finally, we found that
propranolol can amplify the cytotoxicity of vemurafenib and sensitize thyroid
cancer cells to cytotoxic effect of vemurafenib. Our present results suggest
that propranolol has potential activity against thyroid cancers and
investigation of the combination with targeted molecular therapy for progressive
thyroid cancers could be beneficial. BACKGROUND: About half of patients with papillary thyroid cancer have tumours
with activating BRAF(V600E) mutations. Vemurafenib, an oncogenic BRAF kinase
inhibitor approved for BRAF-positive melanoma, showed clinical benefit in three
patients with BRAF(V600E)-positive papillary thyroid cancer in a phase 1 trial.
We aimed to establish the activity of vemurafenib in patients with
BRAF(V600E)-positive papillary thyroid cancer.
METHODS: We did an open-label, non-randomised, phase 2 trial at ten academic
centres and hospitals worldwide in patients aged 18 years or older with
histologically confirmed recurrent or metastatic papillary thyroid cancer
refractory to radioactive iodine and positive for the BRAF(V600E) mutation.
Participants either had never received a multikinase inhibitor targeting VEGFR
(cohort 1) or had been treated previously with a VEGFR multikinase inhibitor
(cohort 2). Patients received vemurafenib 960 mg orally twice daily. The primary
endpoint was investigator-assessed best overall response in cohort 1 (confirmed
on two assessments 4 weeks or longer apart). Analyses were planned to have a
minimum median follow-up of 15 months (data cutoff April 18, 2014) and were done
in safety, intention-to-treat, and per-protocol populations. This trial is
closed and is registered at ClinicalTrials.gov, number NCT01286753.
FINDINGS: Between June 23, 2011, and Jan 15, 2013, 51 patients were enrolled to
the study, 26 in cohort 1 and 25 in cohort 2. Median duration of follow-up was
18·8 months (IQR 14·2-26·0) in cohort 1 and 12·0 months (6·7-20·3) in cohort 2.
Partial responses were recorded in ten of 26 patients in cohort 1 (best overall
response 38·5%, 95% CI 20·2-59·4). Grade 3 or 4 adverse events were recorded in
17 (65%) of 26 patients in cohort 1 and 17 (68%) of 25 patients in cohort 2; the
most common grade 3 and 4 adverse events were squamous cell carcinoma of the
skin (seven [27%] in cohort 1, five [20%] in cohort 2), lymphopenia (two [8%] in
each cohort), and increased γ-glutamyltransferase (one [4%] in cohort 1, three
[12%] in cohort 2). Two individuals in cohort 2 died due to adverse events, one
from dyspnoea and one from multiorgan failure, but neither was treatment
related. Serious adverse events were reported for 16 (62%) of 26 patients in
cohort 1 and 17 (68%) of 25 patients in cohort 2.
INTERPRETATION: Vemurafenib showed antitumour activity in patients with
progressive, BRAF(V600E)-positive papillary thyroid cancer refractory to
radioactive iodine who had never been treated with a multikinase inhibitor. As
such, this agent represents a potential new treatment option for these patients.
FUNDING: F Hoffmann-La Roche. A new study shows that the BRAF inhibitor vemurafenib may induce partial
responses and stable disease in patients with papillary thyroid cancer that has
become refractory to radioactive iodine. However, the drug led to a variety of
serious adverse events in roughly 65% of patients, including the development of
some squamous cell carcinomas. |
Are mutations in the C9orf72 gene associated with macular degeneration? | Amyotrophic lateral sclerosis (ALS) is characterized by motor neurone loss resulting in muscle weakness, spasticity and ultimately death. 5-10% are caused by inherited mutations, most commonly C9ORF72, SOD1, TARDBP and FUS. | Two studies recently identified a GGGGCC hexanucleotide repeat expansion in a
non-coding region of the chromosome 9 open-reading frame 72 gene (C9ORF72) as
the cause of chromosome 9p-linked amyotrophic lateral sclerosis (ALS) and
frontotemporal dementia (FTD). In a cohort of 231 probands with ALS, we
identified the C9ORF72 mutation in 17 familial (27.4%) and six sporadic (3.6%)
cases. Patients with the mutation presented with typical motor features of ALS,
although subjects with the C9ORF72 mutation had more frequent bulbar onset,
compared to those without this mutation. Dementia was significantly more common
in ALS patients and families with the C9ORF72 mutation and was usually
early-onset FTD. There was striking clinical heterogeneity among the members of
individual families with the mutation. The associated neuropathology was a
combination of ALS with TDP-ir inclusions and FTLD-TDP. In addition to
TDP-43-immunoreactive pathology, a consistent and specific feature of cases with
the C9ORF72 mutation was the presence of ubiquitin-positive, TDP-43-negative
inclusions in a variety of neuroanatomical regions, such as the cerebellar
cortex. These findings support the C9ORF72 mutation as an important newly
recognized cause of ALS, provide a more detailed characterization of the
associated clinical and pathological features and further demonstrate the
clinical and molecular overlap between ALS and FTD. The identification of a hexanucleotide repeat expansion in the C9ORF72 gene as
the cause of chromosome 9-linked frontotemporal dementia and motor neuron
disease offers the opportunity for greater understanding of the relationship
between these disorders and other clinical forms of frontotemporal lobar
degeneration. In this study, we screened a cohort of 398 patients with
frontotemporal dementia, progressive non-fluent aphasia, semantic dementia or
mixture of these syndromes for mutations in the C9ORF72 gene. Motor neuron
disease was present in 55 patients (14%). We identified 32 patients with C9ORF72
mutations, representing 8% of the cohort. The patients' clinical phenotype at
presentation varied: nine patients had frontotemporal dementia with motor neuron
disease, 19 had frontotemporal dementia alone, one had mixed semantic dementia
with frontal features and three had progressive non-fluent aphasia. There was,
as expected, a significant association between C9ORF72 mutations and presence of
motor neuron disease. Nevertheless, 46 patients, including 22 familial, had
motor neuron disease but no mutation in C9ORF72. Thirty-eight per cent of the
patients with C9ORF72 mutations presented with psychosis, with a further 28%
exhibiting paranoid, deluded or irrational thinking, whereas <4% of non-mutation
bearers presented similarly. The presence of psychosis dramatically increased
the odds that patients carried the mutation. Mutation bearers showed a low
incidence of motor stereotypies, and relatively high incidence of complex
repetitive behaviours, largely linked to patients' delusions. They also showed a
lower incidence of acquired sweet food preference than patients without C9ORF72
mutations. Post-mortem pathology in five patients revealed transactive response
DNA-binding protein 43 pathology, type A in one patient and type B in three.
However, one patient had corticobasal degeneration pathology. The findings
indicate that C9ORF72 mutations cause some but not all cases of frontotemporal
dementia with motor neuron disease. Other mutations remain to be discovered.
C9ORF72 mutations are associated with variable clinical presentations and
pathology. Nevertheless, the findings highlight a powerful association between
C9ORF72 mutations and psychosis and suggest that the behavioural characteristics
of patients with C9ORF72 mutations are qualitatively distinct. Mutations in the
C9ORF72 gene may be a major cause not only of frontotemporal dementia with motor
neuron disease but also of late onset psychosis. An expanded hexanucleotide repeat in the C9ORF72 gene has recently been
identified as a major cause of familial frontotemporal lobar degeneration and
motor neuron disease, including cases previously identified as linked to
chromosome 9. Here we present a detailed retrospective clinical, neuroimaging
and histopathological analysis of a C9ORF72 mutation case series in relation to
other forms of genetically determined frontotemporal lobar degeneration
ascertained at a specialist centre. Eighteen probands (19 cases in total) were
identified, representing 35% of frontotemporal lobar degeneration cases with
identified mutations, 36% of cases with clinical evidence of motor neuron
disease and 7% of the entire cohort. Thirty-three per cent of these C9ORF72
cases had no identified relevant family history. Families showed wide variation
in clinical onset (43-68 years) and duration (1.7-22 years). The most common
presenting syndrome (comprising a half of cases) was behavioural variant
frontotemporal dementia, however, there was substantial clinical heterogeneity
across the C9ORF72 mutation cohort. Sixty per cent of cases developed clinical
features consistent with motor neuron disease during the period of follow-up.
Anxiety and agitation and memory impairment were prominent features (between a
half to two-thirds of cases), and domit parietal dysfunction was also
frequent. Affected individuals showed variable magnetic resoce imaging
findings; however, relative to healthy controls, the group as a whole showed
extensive thinning of frontal, temporal and parietal cortices, subcortical grey
matter atrophy including thalamus and cerebellum and involvement of long
intrahemispheric, commissural and corticospinal tracts. The neuroimaging profile
of the C9ORF72 expansion was significantly more symmetrical than progranulin
mutations with significantly less temporal lobe involvement than
microtubule-associated protein tau mutations. Neuropathological examination in
six cases with C9ORF72 mutation from the frontotemporal lobar degeneration
series identified histomorphological features consistent with either type A or B
TAR DNA-binding protein-43 deposition; however, p62-positive (in excess of TAR
DNA-binding protein-43 positive) neuronal cytoplasmic inclusions in hippocampus
and cerebellum were a consistent feature of these cases, in contrast to the
similar frequency of p62 and TAR DNA-binding protein-43 deposition in 53 control
cases with frontotemporal lobar degeneration-TAR DNA-binding protein. These
findings corroborate the clinical importance of the C9ORF72 mutation in
frontotemporal lobar degeneration, delineate phenotypic and neuropathological
features that could help to guide genetic testing, and suggest hypotheses for
elucidating the neurobiology of a culprit subcortical network. Frontotemporal lobar degeneration (FTLD) is a genetically heterogenous syndrome
and has been associated most recently with a hexanucleotide repeat expansion
within the C9ORF72 gene. Pathogenic TDP-43 gene (TARDBP) mutations have been
identified in amyotrophic lateral sclerosis, but the role of TARDBP mutations in
FTLD is more contradictory. To investigate the role of TARDBP mutations in a
clinical series of Finnish FTLD patients, we sequenced TARDBP exons 1 to 6 in 77
FTLD patients. No evident pathogenic mutations were found. We identified a novel
heterozygous c.876_878delCAG sequence variant in 2 related patients with
behavioral variant frontotemporal dementia without amyotrophic lateral
sclerosis. The variant is predicted to cause an amino acid deletion of serine at
position 292 (p.Ser292del). However, p.Ser292del was also found in 1 healthy
middle-aged control. Interestingly, both patients carried the C9ORF72 expansion.
Therefore, the TARDBP variant p.Ser292del might be considered a rare
polymorphism and the C9ORF72 repeat expansion the actual disease-causing
mutation in the family. Our results suggest that TARDBP mutations are a rare
cause of FTLD. However, the interaction of several genetic factors needs to be
taken into account when investigating neurodegenerative diseases. Recent works have demonstrated an expansion of the GGGGCC hexanucleotide repeat
in the first intron of chromosome 9 open reading frame 72 (C9ORF72), encoding an
unknown C9ORF72 protein, which was responsible for an unprecedented large
proportion of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia
(FTD) cases of European ancestry. C9ORF72 is expressed in most tissues including
the brain. Emerging evidence has demonstrated that C9ORF72 mutations could
reduce the level of C9ORF72 variant 1, which may influence protein expression
and the formation of nuclear RNA foci. The spectrum of mutations is broad and
provides new insight into neurological diseases. Clinical manifestations of
diseases related with C9ORF72 mutations can vary from FTD, ALS, primary lateral
sclerosis (PLS), progressive muscular atrophy (PMA), Huntington disease-like
syndrome (HDL syndrome), to Alzheimer's disease. In this article, we will review
the brief characterizations of the C9ORF72 gene, the expansion mutations, the
related disorders, and their features, followed by a discussion of the
deficiency knowledge of C9ORF72 mutations. Based on the possible pathological
mechanisms of C9ORF72 mutations in ALS and FTD, we can find new targets for the
treatment of C9ORF72 mutation-related diseases. Future studies into the
mechanisms, taking into consideration the discovery of those disorders, will
significantly accelerate new discoveries in this field, including targeting
identification of new therapy. Expansion of a hexanucleotide repeat in the C9ORF72 gene has been identified as
the most common pathogenic mutation in families with autosomal domit
frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis.
Herein we investigated frequency and penetrance of the C9ORF72 hexanucleotide
repeat pathological expansion in a large cohort of familial and sporadic FTLD
and related disorders (FTLD and related disorders, n = 388; Controls, n = 201).
Moreover, we weighed the impact of C9ORF72 genotype on clinical phenotype taking
into account the hexanucleotide repeat units number as a possible disease
modifier. In our cohort, the C9ORF72 pathological expansion: (i) showed a
prevalence of 7.5%; (ii) showed a full penetrance by the age of 80; (iii) was
rarely found in sporadic patients; (iv) was solely associated with FTLD; (v) was
mainly associated with bvFTD clinical subtype; and (vi) was associated with
earlier age of onset in the youngest generation compared with the previous
generation within a pedigree. Interestingly, intermediate C9ORF72 expansion had
a risk effect in familial/sporadic FTLD. Eventually, the C9ORF72 repeat units
number influenced the disease phenotype in terms of age of onset and associated
clinical subtype. Genome-wide studies in well characterized clinical cohorts
will be essential in order to decipher pathways of disease expression in
C9ORF72-associated neurodegeneration. Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72)
have recently been linked to frontotemporal lobar degeneration (FTLD) and
amyotrophic lateral sclerosis, and may be the most common genetic cause of both
neurodegenerative diseases. Genetic variants at TMEM106B influence risk for the
most common neuropathological subtype of FTLD, characterized by inclusions of
TAR DNA-binding protein of 43 kDa (FTLD-TDP). Previous reports have shown that
TMEM106B is a genetic modifier of FTLD-TDP caused by progranulin (GRN)
mutations, with the major (risk) allele of rs1990622 associating with earlier
age at onset of disease. Here, we report that rs1990622 genotype affects age at
death in a single-site discovery cohort of FTLD patients with C9orf72 expansions
(n = 14), with the major allele correlated with later age at death (p = 0.024).
We replicate this modifier effect in a 30-site international neuropathological
cohort of FTLD-TDP patients with C9orf72 expansions (n = 75), again finding that
the major allele associates with later age at death (p = 0.016), as well as
later age at onset (p = 0.019). In contrast, TMEM106B genotype does not affect
age at onset or death in 241 FTLD-TDP cases negative for GRN mutations or
C9orf72 expansions. Thus, TMEM106B is a genetic modifier of FTLD with C9orf72
expansions. Intriguingly, the genotype that confers increased risk for
developing FTLD-TDP (major, or T, allele of rs1990622) is associated with later
age at onset and death in C9orf72 expansion carriers, providing an example of
sign epistasis in human neurodegenerative disease. BACKGROUND: Mutations in C9ORF72 are an important cause of frontotemporal
dementia (FTD) and motor neuron disease. Accumulating evidence suggests that FTD
associated with C9ORF72 mutations (C9ORF72-FTD) is distinguished clinically by
early prominent neuropsychiatric features that might collectively reflect
deranged body schema processing. However, the pathophysiology of C9ORF72-FTD has
not been elucidated.
METHODS: We undertook a detailed neurophysiological investigation of five
patients with C9ORF72-FTD, in relation to patients with FTD occurring
sporadically and on the basis of mutations in the microtubule-associated protein
tau gene and healthy older individuals. We designed or adapted behavioural tasks
systematically to assess aspects of somatosensory body schema processing
(tactile discrimination, proprioceptive and body part illusions and
self/non-self differentiation).
RESULTS: Patients with C9ORF72-FTD selectively exhibited deficits at these
levels of body schema processing in relation to healthy individuals and other
patients with FTD.
CONCLUSIONS: Altered body schema processing is a novel, generic
pathophysiological mechanism that may link the distributed cortico-subcortical
network previously implicated in C9ORF72-FTD with a wide range of
neuropsychiatric and behavioural symptoms, and constitute a physiological marker
of this neurodegenerative proteinopathy. Amyotrophic lateral sclerosis (ALS) is characterized by motor neurone loss
resulting in muscle weakness, spasticity and ultimately death. 5-10% are caused
by inherited mutations, most commonly C9ORF72, SOD1, TARDBP and FUS. Rarer
genetic causes of ALS include mutation of optineurin (mt OPTN). Furthermore,
optineurin protein has been localized to the ubiquitylated aggregates in several
neurodegenerative diseases, including ALS. This study: (i) investigated the
frequency of mt OPTN in ALS patients in England; (ii) characterized the clinical
and neuropathological features of ALS associated with a mt OPTN; and (iii)
investigated optineurin neuropathology in C9ORF72-related ALS (C9ORF72-ALS). We
identified a heterozygous p.E322K missense mutation in exon 10 of OPTN in one
familial ALS patient who additionally had a C9ORF72 mutation. This patient had
bulbar, limb and respiratory disease without cognitive problems. Neuropathology
revealed motor neurone loss, trans-activation response DNA protein 43
(TDP-43)-positive neuronal and glial cytoplasmic inclusions together with
TDP-43-negative neuronal cytoplasmic inclusions in extra motor regions that are
characteristic of C9ORF72-ALS. We have demonstrated that both TDP-43-positive
and negative inclusion types had positive staining for optineurin by
immunohistochemistry. We went on to show that optineurin was present in
TDP-43-negative cytoplasmic extra motor inclusions in C9ORF72-ALS cases that do
not carry mt OPTN. We conclude that: (i) OPTN mutations are associated with ALS;
(ii) optineurin protein is present in a subset of the extramotor inclusions of
C9ORF72-ALS; (iii) It is not uncommon for multiple ALS-causing mutations to
occur in the same patient; and (iv) studies of optineurin are likely to provide
useful dataregarding the pathophysiology of ALS and neurodegeneration. |
What is the Genome 10K Project? | The Genome 10K Project was established in 2009 by a consortium of biologists and genome scientists determined to facilitate the sequencing and analysis of the complete genomes of 10,000 vertebrate species. | The Genome 10K project aims to sequence the genomes of 10,000 vertebrates,
representing approximately one genome for each vertebrate genus. Since fishes
(cartilaginous fishes, ray-finned fishes and lobe-finned fishes) represent more
than 50% of extant vertebrates, it is planned to target 4,000 fish genomes. At
present, nearly 60 fish genomes are being sequenced at various public funded
labs, and under a Genome 10K and BGI pilot project. An additional 100 fishes
have been identified for sequencing in the next phase of Genome 10K project. |
Which gene controls the expression of GATA-1 isoforms? | In this study, we report a transcriptional network in which PU.1 positively regulates GATA-1 expression in mast cell development. This isoform contains an alternatively spliced first exon (IB) that is distinct from the first exon (IE) incorporated in the major erythroid mRNA transcript. | GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one
another in the regulation of erythroid versus myeloid gene transcription and
development. The CCAAT enhancer binding protein epsilon (C/EBPepsilon) is
expressed as multiple isoforms and has been shown to be essential to myeloid
(granulocyte) terminal differentiation. We have defined a novel synergistic, as
opposed to antagonistic, combinatorial interaction between GATA-1 and PU.1, and
a unique repressor role for certain C/EBPepsilon isoforms in the transcriptional
regulation of a model eosinophil granulocyte gene, the major basic protein
(MBP). The eosinophil-specific P2 promoter of the MBP gene contains GATA-1,
C/EBP, and PU.1 consensus sites that bind these factors in nuclear extracts of
the eosinophil myelocyte cell line, AML14.3D10. The promoter is transactivated
by GATA-1 alone but is synergistically transactivated by low levels of PU.1 in
the context of optimal levels of GATA-1. The C/EBPepsilon(27) isoform strongly
represses GATA-1 activity and completely blocks GATA-1/PU.1 synergy. In vitro
mutational analyses of the MBP-P2 promoter showed that both the GATA-1/PU.1
synergy, and repressor activity of C/EBPepsilon(27) are mediated via
protein-protein interactions through the C/EBP and/or GATA-binding sites but not
the PU.1 sites. Co-immunoprecipitations using lysates of AML14.3D10 eosinophils
show that both C/EBPepsilon(32/30) and epsilon(27) physically interact in vivo
with PU.1 and GATA-1, demonstrating functional interactions among these factors
in eosinophil progenitors. Our findings identify novel combinatorial
protein-protein interactions for GATA-1, PU.1, and C/EBPepsilon isoforms in
eosinophil gene transcription that include GATA-1/PU.1 synergy and repressor
activity for C/EBPepsilon(27). Mutations in the hematopoietic transcription factor GATA-1 alter the
proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2
interfere with the synthesis of the full-length isoform of GATA-1 and lead to
the production of a shortened isoform, GATA-1s. These mutations have been found
in patients with Diamond-Blackfan anemia (DBA), a congenital erythroid aplasia
typically caused by mutations in genes encoding ribosomal proteins. We sequenced
GATA-1 in 23 patients that were negative for mutations in the most frequently
mutated DBA genes. One patient showed a c.2T > C mutation in the initiation
codon leading to the loss of the full-length GATA-1 isoform. |
What is MIRA-seq? | MIRA-seq is a reliable, genome-scale DNA methylation analysis platform for scoring DNA methylation differences at CpG-rich genomic regions. The method is not limited by primer or probe design and is cost effective. | BACKGROUND: By comparing fibroblasts collected from animals at 5-months or
16-months of age we have previously found that the cultures from older animals
produce much more IL-8 in response to lipopolysaccharide (LPS) stimulation. We
now expand this finding by examining whole transcriptome differences in the LPS
response between cultures from the same animals at different ages, and also
investigate the contribution of DNA methylation to the epigenetic basis for the
age-dependent increases in responsiveness.
RESULTS: Age-dependent differences in IL-8 production by fibroblasts in response
to LPS exposure for 24 h were abolished by pretreatment of cultures with a DNA
demethylation agent, 5-aza-2'deoxycytidine (AZA). RNA-Seq analysis of
fibroblasts collected from the same individuals at either 5 or 16 months of age
and exposed in parallel to LPS for 0, 2, and 8 h revealed a robust response to
LPS that was much greater in the cultures from older animals. Pro-inflammatory
genes including IL-8, IL-6, TNF-α, and CCL20 (among many other immune associated
genes), were more highly expressed (FDR < 0.05) in the 16-month old cultures
following LPS exposure. Methylated CpG island recovery assay sequencing
(MIRA-Seq) revealed numerous methylation peaks spread across the genome,
combined with an overall hypomethylation of gene promoter regions, and a
remarkable similarity, except for 20 regions along the genome, between the
fibroblasts collected at the two ages from the same animals.
CONCLUSIONS: The fibroblast pro-inflammatory response to LPS increases
dramatically from 5 to 16 months of age within individual animals. A better
understanding of the mechanisms underlying this process could illuminate the
physiological processes by which the innate immune response develops and
possibly individual variation in innate immune response arises. In addition,
although relatively unchanged by age, our data presents a general overview of
the bovine fibroblast methylome as a guide for future studies in cattle
epigenetics utilizing this cell type. AIM: To develop a reliable method for whole genome analysis of DNA methylation.
MATERIALS & METHODS: Genome-scale analysis of DNA methylation includes
affinity-based approaches such as enrichment using methyl-CpG-binding proteins.
One of these methods, the methylated-CpG island recovery assay (MIRA), is based
on the high affinity of the MBD2b-MBD3L1 complex for CpG-methylated DNA. Here we
provide a detailed description of MIRA and combine it with next generation
sequencing platforms (MIRA-seq).
RESULTS: We assessed the performance of MIRA-seq and compared the data with
whole genome bisulfite sequencing.
CONCLUSION: MIRA-seq is a reliable, genome-scale DNA methylation analysis
platform for scoring DNA methylation differences at CpG-rich genomic regions.
The method is not limited by primer or probe design and is cost effective. Using MIRA-seq, we have characterized the DNA methylome of metastatic melanoma
and normal melanocytes. Individual tumors contained several thousand
hypermethylated regions. We discovered 179 tumor-specific methylation peaks
present in all (27/27) melanomas that may be effective disease biomarkers, and
3113 methylation peaks were seen in >40% of the tumors. We found that 150 of the
approximately 1200 tumor-associated methylation peaks near transcription start
sites (TSSs) were marked by H3K27me3 in melanocytes. DNA methylation in melanoma
was specific for distinct H3K27me3 peaks rather than for broadly covered
regions. However, numerous H3K27me3 peak-associated TSS regions remained devoid
of DNA methylation in tumors. There was no relationship between BRAF mutations
and the number of methylation peaks. Gene expression analysis showed upregulated
immune response genes in melanomas presumably as a result of lymphocyte
infiltration. Down-regulated genes were enriched for melanocyte differentiation
factors; e.g., KIT, PAX3 and SOX10 became methylated and downregulated in
melanoma. BACKGROUND: We have previously found substantial animal-to-animal and
age-dependent variation in the response of Holstein fibroblast cultures
challenged with LPS. To expand on this finding, fibroblast cultures were
established from dairy (Holstein) and beef (Angus) cattle and challenged with
LPS to examine breed-dependent differences in the innate immune response. Global
gene expression was measured by RNA-Seq, while an epigenetic basis for
expression differences was examined by methylated CpG island recovery assay
sequencing (MIRA-Seq) analysis.
RESULTS: The Holstein breed displayed a more robust response to LPS than the
Angus breed based on RNA-Seq analysis of cultures challenged with LPS for 0, 2,
and 8 h. Several immune-associated genes were expressed at greater levels (FDR <
0.05) in Holstein cultures including TLR4 at all time points and a number of
pro-inflammatory genes such as IL8, CCL20, CCL5, and TNF following LPS exposure.
Despite extensive breed differences in the transcriptome, MIRA-Seq unveiled
relatively similar patterns of genome-wide DNA methylation between breeds, with
an overall hypomethylation of gene promoters. However, by examining the genome
in 3Kb windows, 49 regions of differential methylation were discovered between
Holstein and Angus fibroblasts, and two of these regions fell within the
promoter region (-2500 to +500 bp of the transcription start site) of the genes
NTRK2 and ADAMTS5.
CONCLUSIONS: Fibroblasts isolated from Holstein cattle display a more robust
response to LPS in comparison to cultures from Angus cattle. Different selection
strategies and management practices exist between these two breeds that likely
give rise to genetic and epigenetic factors contributing to the different immune
response phenotypes. |
How does Ssu72 mediate gene looping? | Investigation of chromosome folding in mutants confirms roles for RSC, "gene looping" factor Ssu72, Mediator, H3K56 acetyltransferase Rtt109, and the N-terminal tail of H4 in folding of the yeast genome. The essential N terminus of the Pta1 scaffold protein is required for snoRNA transcription termination and Ssu72 function but is dispensable for pre-mRNA 3'-end processing. TFIIB crosslinks to both the promoter and terminator regions of the PMA1 and BLM10 genes, and its association with the terminator, but not the promoter, is adversely affected by E62K and by depletion of the Ssu72 component of the CPF 3' end processing complex, and is independent of TBP. We propose a model for RNAP II transcription in which promoter and terminator regions are juxtaposed, and that the resulting gene loops facilitate transcription reinitiation by the same molecule of RNAP II in a manner dependent upon Ssu72-mediated CTD dephosphorylation. The first 300 amino acids of Pta1 are sufficient for interactions with Ssu72, which is needed for pre-mRNA cleavage. | The prevailing view of the RNA polymerase II (RNAP II) transcription cycle is
that RNAP II is recruited to the promoter, transcribes a linear DNA template,
then terminates transcription and dissociates from the template. Subsequent
rounds of transcription are thought to require de novo recruitment of RNAP II to
the promoter. Several recent findings, including physical interaction of 3'-end
processing factors with both promoter and terminator regions, challenge this
concept. Here we report a physical association of promoter and terminator
regions of the yeast BUD3 and SEN1 genes. These interactions are
transcription-dependent, require the Ssu72 and Pta1 components of the CPF 3'-end
processing complex, and require the phosphatase activity of Ssu72. We propose a
model for RNAP II transcription in which promoter and terminator regions are
juxtaposed, and that the resulting gene loops facilitate transcription
reinitiation by the same molecule of RNAP II in a manner dependent upon
Ssu72-mediated CTD dephosphorylation. Saccharomyces cerevisiae Pta1 is a component of the cleavage/polyadenylation
factor (CPF) 3'-end processing complex and functions in pre-mRNA cleavage,
poly(A) addition, and transcription termination. In this study, we investigated
the role of the N-terminal region of Pta1 in transcription and processing. We
report that a deletion of the first 75 amino acids (pta1-Delta75) causes
thermosensitive growth, while the deletion of an additional 25 amino acids is
lethal. The pta1-Delta75 mutant is defective for snoRNA termination, RNA
polymerase II C-terminal domain Ser5-P dephosphorylation, and gene looping but
is fully functional for mRNA 3'-end processing. Furthermore, different regions
of Pta1 interact with the CPF subunits Ssu72, Pti1, and Ysh1, supporting the
idea that Pta1 acts as a scaffold to organize CPF. The first 300 amino acids of
Pta1 are sufficient for interactions with Ssu72, which is needed for pre-mRNA
cleavage. By the degron-mediated depletion of Pta1, we show that the removal of
this essential region leads to a loss of Ssu72, yet surprisingly, in vitro
cleavage and polyadenylation remain efficient. In addition, a fragment
containing amino acids 1 to 300 suppresses 3'-end processing in wild-type
extracts. These findings suggest that the amino terminus of Pta1 has an
inhibitory effect and that this effect can be neutralized through the
interaction with Ssu72. Functional links connecting gene transcription and condensin-mediated chromosome
condensation have been established in species ranging from prokaryotes to
vertebrates. However, the exact nature of these links remains misunderstood.
Here we show in fission yeast that the 3' end RNA processing factor Swd2.2, a
component of the Cleavage and Polyadenylation Factor (CPF), is a negative
regulator of condensin-mediated chromosome condensation. Lack of Swd2.2 does not
affect the assembly of the CPF but reduces its association with chromatin. This
causes only limited, context-dependent effects on gene expression and
transcription termination. However, CPF-associated Swd2.2 is required for the
association of Protein Phosphatase 1 PP1(Dis2) with chromatin, through an
interaction with Ppn1, a protein that we identify as the fission yeast homologue
of vertebrate PNUTS. We demonstrate that Swd2.2, Ppn1 and PP1Dis2 form an
independent module within the CPF, which provides an essential function in the
absence of the CPF-associated Ssu72 phosphatase. We show that Ppn1 and Ssu72,
like Swd2.2, are also negative regulators of condensin-mediated chromosome
condensation. We conclude that Swd2.2 opposes condensin-mediated chromosome
condensation by facilitating the function of the two CPF-associated phosphatases
PP1 and Ssu72. We describe a Hi-C-based method, Micro-C, in which micrococcal nuclease is used
instead of restriction enzymes to fragment chromatin, enabling nucleosome
resolution chromosome folding maps. Analysis of Micro-C maps for budding yeast
reveals abundant self-associating domains similar to those reported in other
species, but not previously observed in yeast. These structures, far shorter
than topologically associating domains in mammals, typically encompass one to
five genes in yeast. Strong boundaries between self-associating domains occur at
promoters of highly transcribed genes and regions of rapid histone turnover that
are typically bound by the RSC chromatin-remodeling complex. Investigation of
chromosome folding in mutants confirms roles for RSC, "gene looping" factor
Ssu72, Mediator, H3K56 acetyltransferase Rtt109, and the N-terminal tail of H4
in folding of the yeast genome. This approach provides detailed structural maps
of a eukaryotic genome, and our findings provide insights into the machinery
underlying chromosome compaction. |
Is the number of described human nuclear mutations less than 50000? | No, The number of known mutations in human nuclear genes, underlying or associated with human inherited disease, has now exceeded 100,000 in more than 3700 different genes (Human Gene Mutation Database). | The Human Gene Mutation Database (HGMD) constitutes a comprehensive core
collection of data on germ-line mutations in nuclear genes underlying or
associated with human inherited disease (http://www.hgmd.org). Data cataloged
include single-base-pair substitutions in coding, regulatory, and
splicing-relevant regions, micro-deletions and micro-insertions, indels, and
triplet repeat expansions, as well as gross gene deletions, insertions,
duplications, and complex rearrangements. Each mutation is entered into HGMD
only once, in order to avoid confusion between recurrent and
identical-by-descent lesions. By March 2012, the database contained in excess of
123,600 different lesions (HGMD Professional release 2012.1) detected in 4,514
different nuclear genes, with new entries currently accumulating at a rate in
excess of 10,000 per annum. ∼6,000 of these entries constitute
disease-associated and functional polymorphisms. HGMD also includes cDNA
reference sequences for more than 98% of the listed genes. |
What is the role of peptide aptamers? | Peptide aptamers are artificial short peptides which are able to specifically bind to defined functional domains, track, and inhibit a given target molecule with high affinity, even molecules with poor immunogenicity or high toxicity. They represent a remarkable alternative to antibodies in many different applications. | RNA aptamers were selected against an affinity column containing a farnesylated
peptide modeled after the carboxyl terminus of K ras, the major oncogenic form
of this small G protein family. After 10-rounds of selection, 25% of the RNA
applied to the column could be specifically eluted. Sequence analysis of the
binding RNA aptamers revealed two consensus sequences--GGGUGGG and GGGAGG.
Quantitative fluorescence binding studies on two of the high-affinity aptamers,
showed a binding affinities of 139 nM and 0.93 microM, respectively for the
farnesylated peptide. Binding to the nonfarnesylated peptide was at least
10-fold weaker, showing that the aptamers can recognize the hydrophobic farnesyl
moiety. High affinity aptamers could be useful in specifically interfering with
oncogenic ras function in particular, and G proteins in general. The p16-cyclin D-pRB-E2F pathway is frequently deregulated in human tumors. This
critical regulatory pathway controls the G1/S transition of the mammalian cell
cycle by positive and negative regulation of E2F-responsive genes required for
DNA replication. To assess the value of the transcription factors E2Fs as
targets for antiproliferative strategies, we have initiated a program aiming to
develop inhibitors targeting specifically these proteins in vitro and in vivo.
The cellular activity of E2F is the result of the heterodimeric association of
two families of proteins, E2Fs and DPs, which then bind DNA. Here, we use a two
hybrid approach to isolate from combinatorial libraries peptide aptamers that
specifically interact with E2Fs DNA binding and dimerization domains. One of
these is a potent inhibitor of E2F binding activity in vitro and in mammalian
fibroblasts, blocks cells in G1, and the free variable region from this aptamer
has the same effect. Our experiments argue that the variable region of this
aptamer is structured, and that it functions by binding E2F with a motif that
resembles a DP heterodimerization region, and blocking E2F's association with
DP. These results show that cell proliferation can be inhibited using
genetically-selected synthetic peptides that specifically target protein-protein
interaction motifs within cell cycle regulators. These results also emphasize
the critical role of the E2F pathway for cell proliferation and might allow the
design of novel antiproliferative agents targeting the cyclin/CDK-pRB-E2F
pathway. The nuclear division cycles of early Drosophila embryogenesis have a number of
unique features that distinguish them from later cell cycles. These features
include the lack of some checkpoints that operate in later cell cycles, the
absence of gap phases, and very rapid DNA synthesis phases. The molecular
mechanisms that control these rapid nuclear division cycles are poorly
understood. Here we describe analysis of cyclin J, a previously uncharacterized
cyclin which has an RNA expression pattern that suggests a possible role in
early embryogenesis. We show that the cyclin J protein is present in early
embryos where it forms active kinase complexes with cyclin-dependent kinase
(Cdk) 2. To determine whether cyclin J plays a role in controlling the early
nuclear cycles we isolated peptide aptamers that specifically bind to cyclin J
and inhibit its ability to activate Cdks. We injected the inhibitory aptamers
into syncytial Drosophila embryos and demonstrated that they caused defects in
chromosome segregation and progression through mitosis. We obtained similar
results by injecting cyclin J antibodies into embryos. Our results suggest that
a cyclin J-associated kinase activity is required for the early embryonic
division cycles. Peptide aptamers have emerged as powerful new tools for molecular medicine. They
can specifically bind to and functionally inactivate a given target molecule
under intracellular conditions. Typically, peptide aptamers are generated by
screening a randomized peptide expression library, displayed from the
Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide
moieties from defined TrxA-based peptide aptamers to alternative scaffold
proteins, such as the green fluorescent protein and staphylococcal nuclease.
Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show
that the TrxA scaffold can be a major determit for the binding of peptide
aptamers. In addition, we demonstrate that TrxA can correctly display peptide
sequences that correspond to the binding domains of natural interaction
partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated
by two-hybrid screening from randomized expression libraries, should also be
useful to find cellular binding partners for a given target protein, by
homology. The insulin-like growth factor 1 receptor (IGF-1R) is an important signaling
molecule in cancer cells and plays an essential role in the establishment and
maintece of the transformed phenotype. Inhibition of IGF-1R signaling thus
appears to be a promising strategy to interfere with the growth and survival of
cancer cells. Different classes of molecules, e.g., antisense RNA, monoclonal
antibodies and domit negative IGF-1R gene variants, have been employed
towards this aim. These agents have been able to reverse the transformed
phenotype in several rodent and human cancer cell lines. The application of
peptide aptamers specifically binding to the IGF-1R represents a novel approach
to target IGF-1R signaling. The integration of peptide aptamers into targeted
protein degradation vehicles and their transduction into cells allows the
temporary elimination of the receptor protein. This review summarizes recently
published data about inhibition of IGF-1R signaling and provides a perspective
on upcoming possibilities. FLASH protein is a component of death-inducing signaling complex and might be
involved in death receptor-mediated extrinsic apoptosis. Here we developed the
peptide aptamer against death effecter domain recruiting domain (DRD) of FLASH
protein and showed that the peptide bound to FLASH protein in vitro.
Intracellular expression of the DRD-binding peptide aptamer specifically
suppressed receptor-mediated extrinsic apoptosis but not intrinsic pathway,
which was recapitulated by the antisense oligonucleotides for FLASH. These data
suggest that DRD-binding peptide is not only a novel inhibitor modulating
receptor-mediated apoptosis but also a tool for elucidating the roles of FLASH
in apoptosis. The AL1 protein of tomato golden mosaic virus (TGMV), a member of the
geminivirus family, is essential for viral replication in plants. Its N terminus
contains three conserved motifs that mediate origin recognition and DNA cleavage
during the initiation of rolling-circle replication. We used the N-terminal
domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide
aptamer library constrained in the active site of the thioredoxin A (TrxA) gene.
The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1
protein. Plant expression cassettes corresponding to the TrxA peptides and a
TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and
viral DNA replication was monitored by semiquantitative PCR. In these assays, 31
TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA
levels to 13 to 64% of those of the wild type. All of the interfering aptamers
also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the
20-mer peptides revealed that their sequences are not random. The alignments
detected seven potential binding motifs, five of which are more highly
represented among the interfering peptides. One motif was present in 18
peptides, suggesting that these peptides interact with a hot spot in the AL1 N
terminus. The peptide aptamers characterized in these studies represent new
tools for studying AL1 function and can serve as the basis for the development
of crops with broad-based resistance to single-stranded DNA viruses. The ErbB2 receptor tyrosine kinase is overexpressed in approximately 30% of
breast tumor cases and its overexpression correlates with an unfavorable
prognosis. A major contributor for this course of the disease is the
insensitivity of these tumors toward chemotherapy. Monoclonal antibodies,
inhibiting the ligand-induced activation of the receptor and tyrosine kinase
inhibitors acting on the intrinsic enzymatic activity of the intracellular
domain, have been developed as targeted drugs. Both have been shown to be
beneficial for breast cancer patients. We targeted a third aspect of receptor
function: its association with intracellular signaling components. For this
purpose, we selected peptide aptamers, which specifically interact with defined
domains of the intracellular part of the receptor. The peptide aptamers were
selected from a random peptide library using a yeast two-hybrid system with the
intracellular tyrosine kinase domain of ErbB2 as a bait construct. The peptide
aptamer AII-7 interacts with high specificity with the ErbB2 receptor in vitro
and in vivo. The aptamers colocalized with the intracellular domain of ErbB2
within cells. We investigated the functional consequences of the aptamer
interaction with the ErbB2 receptor within tumor cells. The aptamer sequences
were either expressed intracellularly or introduced into the cells as
recombit aptamer proteins. The phosphorylation of p42/44 mitogen-activated
protein kinase was nearly unaffected and the activation of signal transducers
and activators of transcription-3 was only modestly reduced. In contrast, they
strongly inhibited the induction of AKT kinase in MCF7 breast cancer cells
treated with heregulin, whereas AKT activation downstream of insulin-like growth
factor I or epidermal growth factor receptor was not or only slightly affected.
High AKT activity is responsible for the enhanced resistance of
ErbB2-overexpressing cancer cells toward chemotherapeutic agents. Peptide
aptamer interference with AKT activation resulted in the restoration of regular
sensitivity of breast cancer cells toward Taxol. Prion diseases are rare and obligatory fatal neurodegenerative disorders caused
by the accumulation of a misfolded isoform (PrPSc) of the host-encoded prion
protein (PrPc). Prophylactic and therapeutic regimens against prion diseases are
very limited. To extend such strategies we selected peptide aptamers binding to
PrP from a combinatorial peptide library presented on the Escherichia coli
thioredoxin A (trxA) protein as a scaffold. In a yeast two-hybrid screen
employing full-length murine PrP (aa 23-231) as a bait we identified three
peptide aptamers that reproducibly bind to PrP. Treatment of prion-infected
cells with recombitly expressed aptamers added to the culture medium
abolished PrPSc conversion with an IC50 between 350 and 700 nM. For expression
in eukaryotic cells, peptide aptamers were fused to an N-terminal signal peptide
for entry of the secretory pathway. The C terminus was modified by a
glycosyl-phosphatidyl-inositol-(GPI) anchoring signal, a KDEL retention motif
and the transmembrane and cytosolic domain of LAMP-I, respectively. These
peptide aptamers retained their binding properties to PrPc and, depending on
peptide sequence and C-terminal modification, interfered with endogenous PrPSc
conversion upon expression in prion-infected cells. Notably, infection of cell
cultures could be prevented by expression of KDEL peptide aptamers. For the
first time, we show that trxA-based peptide aptamers can be targeted to the
secretory pathway, thereby not losing the affinity for their target protein.
Beside their inhibitory effect on prion conversion, these molecules could be
used as fundament for rational drug design. BACKGROUND/AIMS: Hepatitis C virus infection is a major worldwide health
problem, causing chronic hepatitis, cirrhosis and primary liver cancer. In
addition to its role in the viral polyprotein-processing, the viral NS3 serine
protease has been implicated in interactions with various cell constituents
resulting in phenotypic changes including maligt transformation. NS3 is
currently regarded a prime target for anti-viral drugs thus specific inhibitors
of its activities should be important. With the aim of inhibiting NS3 protease
activity as a means to inhibit HCV replication we used a novel bacterial genetic
screen to isolate NS3-inhibiting peptide aptamers.
METHODS: We have isolated and characterized seven NS3-inhibiting peptide
aptamers. We investigated the phenotypic changes that SEAP-secreting subgenomic
RNA replicons undergo upon intracellular expression of these peptide aptamers,
assayed by real-time RT-PCR and inhibition of SEAP secretion by transfected
replicon cells.
RESULTS AND CONCLUSIONS: The peptide aptamers inhibited NS3 protease activity in
vitro with an IC50 in the low micromolar range. Upon transfection, aptamers
inhibited the replication of SEAP-secreting genotype 1b subgenomic RNA
replicons. Aptamer-based intracellular immunization may emerge as a promising
antiviral approach to interfere with the life cycle and pathogenicity of HCV. Peptide aptamers are the newest in the class of "genetic" agents that aid in the
analysis of cellular processes. Peptide aptamers interact with and can
inactivate gene products, but don't mutate the DNA that encodes them. Reverse
analysis with peptide aptamers involves isolating aptamers that interact with a
specific protein and monitoring the resulting aptamer-induced phenotype.
Conversely, forward analysis with peptide aptamers involves expressing
combinatorial libraries of aptamers within an organism and screening for
aptamer-induced variations in their phenotypes. The two-hybrid system is used in
both processes to help identify the protein interactions, and variations from
the basic procedure as described in UNIT are presented here. Peptide aptamers are specific binders that are artificially created using in
vitro evolution systems. The peptide aptamers that can detect maligt cells in
vivo, or can bind surfaces of various materials will enable us to develop novel
types of cancer diagnosis and cancer therapy agents by combining with various
o-carriers. Here, the development of peptide aptamer research is described in
terms of artificial protein research. Also, two examples of o-carriers are
introduced, i. e., a ferritin, a natural caged protein, and carbon ohorns,
members of the carbon omaterial family, by focusing their potential roles in
cancer diagnosis and cancer therapy. The early detection and diagnosis of cancer lies central to successful treatment
and improved patient outcome. Current techniques are limited by the nature of
the biological receptor and the assays available. This paper reports the use of
novel biological probes, peptide aptamers, in detecting cyclin-dependent protein
kinases (CDKs) whose activity is important in proliferating and cancerous cells.
We describe, specifically, the optimization of an orientated peptide aptamer
surface and its utilization in establishing a highly specific, low-omolar
sensitive, detection protocol for the active form of CDK2. In comparing target
binding affinity of two different aptamers (pep6 and pep9), both constructed
through the insertion of peptide sequences into the surface of a scaffold
protein, one was observed to be consistently more effective. Significantly, the
pep9 aptamers were able to detect subtle changes in the conformation of CDK2
associated with activation of its catalytic activity that may be caused by the
phosphorylation of a single amino acid (threonine 160). A typical response
toward the inactive form of CDK2 was in the range of 0.5-2% of the binding of
the active form of CDK2 in the concentration range from 2 to 20 nM. Although
antibodies are occasionally able to recognize conformations in their targets,
this is the first time that a notibody protein probe has been used to detect
an active protein isoform. Because peptide aptamers are usually raised against
full-length proteins, this raises the possibility that peptide aptamers will be
able to extend the repertoire of probes that recognize protein conformations,
post-translational modifications (PTMs), or conformations stabilized by PTMs. BACKGROUND: Peptide aptamers are combinatorial protein reagents that bind to
targets with a high specificity and a strong affinity thus providing a molecular
tool kit for modulating the function of their targets in vivo.
RESULTS: Here we report the isolation of a peptide aptamer named swiggle that
interacts with the very short (21 amino acid long) intracellular domain of
membrane type 1-metalloproteinase (MT1-MMP), a key cell surface protease
involved in numerous and crucial physiological and pathological cellular events.
Expression of swiggle in mammalian cells was found to increase the cell surface
expression of MT1-MMP by impairing its internalisation. Swiggle interacts with
the LLY573 internalisation motif of MT1-MMP intracellular domain, thus
disrupting the interaction with the mu2 subunit of the AP-2 internalisation
complex required for endocytosis of the protease. Interestingly,
swiggle-mediated inhibition of MT1-MMP clathrin-mediated internalisation was
also found to promote MT1-MMP-mediated cell migration.
CONCLUSIONS: Taken together, our results provide further evidence that peptide
aptamers can be used to dissect molecular events mediated by individual protein
domains, in contrast to the pleiotropic effects of RNA interference techniques. Peptide aptamers are combinatorial protein molecules with specific bind affinity
to given target proteins under intracellular conditions. The typical structure
of peptide aptamers is a short peptide region inserted within a scaffold
protein. The short peptide region is responsible for binding with its target
protein and the scaffold protein helps to enhance the binding affinity and
specificity through restriction on the conformation of the binding peptide. This
unique structural feature allows peptide aptamers to bind with their target
proteins with strong affinity and high specificity. Applications of peptide
aptamers thus vary from in vitro detection of various proteins in a complex
mixture to in vivo modulation on proteins and cell functions. Peptide aptamers
have also been considered as therapeutic molecules because of their anticancer
and antivirus activity. Due to the importance of peptide aptamers, a general
review on the structure, selection and applications of peptide aptamers in
biological study as well as in therapeutics will be presented in this paper. Several engineered protein scaffolds have been developed recently to circumvent
particular disadvantages of antibodies such as their large size and complex
composition, low stability, and high production costs. We previously identified
peptide aptamers containing one or two disulfide-bonds as an alternative ligand
to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in
length) were screened from a random peptide library by in vitro peptide
selection using the evolutionary molecular engineering method "cDNA display". In
this report, the antagonistic activity of the peptide aptamers were examined by
an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an
IL-6-dependent cell proliferation assay. The results revealed that a
disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with
similar efficacy to an anti-IL-6R monoclonal antibody. Aptamers are a group of molecules, which can specifically bind, track, and
inhibit target molecules, comprising DNA aptamers, RNA aptamers, and peptide
aptamers. So far, there are much progress about developing novel aptamers and
their expansile applications. This prospect systematically introduces the
composition and technological evolution of aptamers, and then focuses on the
application of aptamers in cancer diagnosis, imaging, and therapy. Following
this, we discuss the potential to harness aptamers in discovering the biomarker
of stem cells, which is favorable for us to study the normal developmental or
abnormal pathological process of tissue and to deliver drugs into target cells
or tissues in the future. Peptide aptamers are small proteins containing a randomized peptide sequence
embedded into a stable protein scaffold, such as Thioredoxin. We developed a
robust method for building a Combinatorial Library of Improved Peptide aptamers
(CLIPs) of high complexity, containing ≥3×10(10) independent clones, to be used
as a molecular tool in the study of biological pathways. The Thioredoxin
scaffold was modified to increase solubility and eliminate aggregation of the
peptide aptamers. The CLIPs was used in a yeast two-hybrid screen to identify
peptide aptamers that bind to various domains of the Receptor for Advanced
Glycation End products (RAGE). NMR spectroscopy was used to identify interaction
surfaces between the peptide aptamers and RAGE domains. Cellular functional
assays revealed that in addition to directly interfering with known binding
sites, peptide aptamer binding distal to ligand sites also inhibits RAGE
ligand-induced signal transduction. This finding underscores the potential of
using CLIPs to select allosteric inhibitors of biological targets. Peptide aptamers are artificial short peptides that potentially interfere with
the biological roles of their target proteins; however, this technology has not
yet been applied to plant functional genomics. MAGO and Y14, the two core
subunits of the exon junction complex (EJC), form obligate heterodimers in
eukaryotes. In Oryza sativa L. (rice), each of the two genes has two homologs,
designated OsMAGO1 and OsMAGO2, and OsY14a and OsY14b, respectively. Here, we
characterized a 16-amino acida peptide aptamer (PAP) for the rice MAGO proteins.
PAP and rice Y14 bound competitively to rice MAGO proteins. Specifically
targeting the MAGO proteins by expressing the aptamer in transgenic rice plants
did not affect the endogenous synthesis and accumulation of MAGO proteins;
however, the phenotypic variations observed in multiple organs phenocopied those
of transgenic rice plants harboring RNA interference (RNAi) constructs in which
the accumulation of MAGO and/or OsY14a transcripts and MAGO proteins was
downregulated severely. Morphologically, the aptamer transgenic plants were
short with abnormally developed flowers, and the stamens exhibited reduced
degradation and absorption of both the endothecium and tapetum, thus confirming
that EJC core heterodimers play essential roles in rice development, growth and
reproduction. This study reveals that as a complementary approach of RNAi,
peptide aptamers are powerful tools for interfering with the function of
proteins in higher plants. The FUR protein (ferric uptake regulator) is an iron-dependent global
transcriptional regulator. Specific to bacteria, FUR is an attractive
antibacterial target since virulence is correlated to iron bioavailability.
Recently, four anti-FUR peptide aptamers, composed of 13 amino acid variable
loops inserted into a thioredoxinA scaffold, were identified, which were able to
interact with Escherichia coli FUR (EcFUR), inhibit its binding to DNA and to
decrease the virulence of pathogenic E. coli in a fly infection model. The first
characterization of anti-FUR linear peptides (pF1 6 to 13 amino acids) derived
from the variable part of the F1 anti-FUR peptide aptamer is described herein.
Theoretical and experimental approaches, in original combination, were used to
study interactions of these peptides with FUR in order to understand their
mechanism of inhibition. After modeling EcFUR by homology, docking with Autodock
was combined with molecular dynamics simulations in implicit solvent to take
into account the flexibility of the partners. All calculations were
cross-checked either with other programs or with experimental data. As a result,
reliable structures of EcFUR and its complex with pF1 are given and an
inhibition pocket formed by the groove between the two FUR subunits is proposed.
The location of the pocket was validated through experimental mutation of key
EcFUR residues at the site of proposed peptide interaction. Cyclisation of pF1,
mimicking the peptide constraint in F1, improved inhibition. The details of the
interactions between peptide and protein were analyzed and a mechanism of
inhibition of these anti-FUR molecules is proposed. Aptamers, including DNA, RNA and peptide aptamers, are a group of promising
recognition units that can specifically bind to target molecules and cells. Due
to their excellent specificity and high affinity to targets, aptamers have
attracted great attention in various fields in which selective recognition units
are required. They have been used in biosensing, drug delivery, disease
diagnosis and therapy (especially for cancer treatment). In this review, we
summarized recent applications of DNA and RNA aptamers in cancer theranostics.
The specific binding ability of aptamers to cancer-related markers and cancer
cells ensured their high performance for early diagnosis of cancer. Meanwhile,
the efficient targeting ability of aptamers to cancer cells and tissues provided
a promising way to deliver imaging agents and drugs for cancer imaging and
therapy. Furthermore, with the development of oscience and otechnology,
the conjugation of aptamers with functional omaterials paved an exciting way
for the fabrication of theranostic agents for different types of cancers, which
might be a powerful tool for cancer treatment. Aptasensors utilize aptamers as bioreceptors. Aptamers are highly efficient,
have a high specificity and are reusable. Within the biosensor the aptamers are
immobilized to maximize their access to target molecules. Knowledge of the
orientation and location of the aptamer and peptide during binding could be
gained through computational modeling. Experimentally, the aptamer (anti-MUC1
S2.2) has been identified as a bioreceptor for breast cancer biomarker mucin 1
(MUC1) protein. However, within this protein lie several peptide variants with
the common sequence APDTRPAP that are targeted by the aptamer. Understanding
orientation and location of the binding region for a peptide-aptamer complex is
critical in their biosensor applicability. In this study, we investigate through
computational modeling how this peptide sequence and its minor variants affect
the peptide-aptamer complex binding. We use molecular dynamics simulations to
study multiple peptide-aptamer systems consisting of MUC1 (APDTRPAP) and MUC1-G
(APDTRPAPG) peptides with the anti-MUC1 aptamer under similar physiological
conditions reported experimentally. Multiple simulations of the MUC1 peptide and
aptamer reveal that the peptide interacts between 3' and 5' ends of the aptamer
but does not fully bind. Multiple simulations of the MUC1-G peptide indicate
consistent binding with the thymine loop of the aptamer, initiated by the
arginine residue of the peptide. We find that the binding event induces
structural changes in the aptamer by altering the number of hydrogen bonds
within the aptamer and establishes a stable peptide-aptamer complex. In all
MUC1-G cases the occurrence of binding was confirmed by systematically studying
the distance distributions between peptide and aptamers. These results are found
to corroborate well with experimental study reported in the literature that
indicated a strong binding in the case of MUC1-G peptide and anti-MUC1 aptamer.
Present MD simulations highlight the role of the arginine residue of MUC1-G
peptide in initiating the binding. The addition of the glycine residue to the
peptide, as in the case of MUC1-G, is shown to yield a stable binding. Our study
clearly demonstrates the ability of MD simulations to obtain molecular insights
for peptide-aptamer binding, and to provide details on the orientation and
location of binding between the peptide-aptamer that can be instrumental in
biosensor development. In recent years, peptide aptamers have emerged as novel molecular tools that
have attracted the attention of researchers in various fields of basic and
applied science, ranging from medicine to analytical chemistry. These artificial
short peptides are able to specifically bind, track, and inhibit a given target
molecule with high affinity, even molecules with poor immunogenicity or high
toxicity, and represent a remarkable alternative to antibodies in many different
applications. Their use is on the rise, driven mainly by the medical and
pharmaceutical sector. Here we discuss the enormous potential of peptide
aptamers in both basic and applied aspects of plant biotechnology and food
safety. The different peptide aptamer selection methods available both in vivo
and in vitro are introduced, and the most important possible applications in
plant biotechnology are illustrated. In particular, we discuss the generation of
broad-based virus resistance in crops, "reverse genetics" and aptasensors in
bioassays for detecting contaminations in food and feed. Furthermore, we suggest
an alternative to the transfer of peptide aptamers into plant cells via genetic
transformation, based on the use of cell-penetrating peptides that overcome the
limits imposed by both crop transformation and Genetically Modified Organism
commercialization. |
Which are the clinical symptoms of left ventricular noncompaction? | The clinical symptoms of left ventricular noncompaction are:
1) heart failure,
2) systemic thromboembolic events,
3) ventricular arrhythmias and
4) sudden cardiac death. | Isolated noncompaction of the left ventricular myocardium is a rare cardiac
disorder due to an arrest in myocardial morphogenesis. It is characterized by
prominent and excessive trabeculation in a ventricular wall segment, with deep
intertrabecular spaces perfused from the ventricular cavity. Echocardiographic
findings are important clues for the diagnosis. Clinical symptoms include signs
of left ventricular systolic dysfunction even to the point of heart failure,
ventricular arrhythmias, and embolic events. We describe an adult case in whom
the only clinical symptoms were life-threatening ventricular arrhythmias.
Transthoracic echocardiography did not contribute to the diagnosis, which was
made thanks to left ventricular contrast angiography. Electrophysiological
testing induced a fast monomorphic sustained ventricular tachycardia, with
hemodynamic impairment, that was refractory to pharmacological treatment, and
for this reason a permanent cardioverter-defibrillator was implanted. A
subsequently performed transesophageal echocardiographic examination showed a
localized, regional increase in left ventricular wall thickness and degree of
trabeculation. The causes and electrophysiological mechanisms of arrhythmias in
noncompaction are still unknown: grossly irregular branching and connecting of
myocardial fascicles in the noncompacted segments, isometric contraction with
increased wall stress, and localized coronary perfusion impairment can all
induce disorganized or delayed activation and increase the potential for
arrhythmias. This is the first reported case of noncompaction in which an
implantable defibrillator was used to control life-threatening arrhythmias. Noncompaction of the ventricular myocardium is a rare congenital disorder
characterized by the presence of numerous prominent trabeculations and deep
intertrabecular recessess which communicate with the left ventricular cavity.
The disease uniformly affects the left ventricle, and sometimes also affects the
right ventricle. Echocardiographic findings are important clues for the
diagnosis. Clinical symptoms include signs of left ventricular systolic
dysfunction even to the point of heart failure, ventricular arrhythmias, and
embolic events. We describe an illustrative case of isolated noncompaction of
the left ventricular myocardium in a two-year-old child with the typical
clinical and echocardiographic features of the disease. The literature on the
topic is reviewed. Myocardial noncompaction is a rare type of cardiomyopathy which can be an
isolated entity or in association with other congenital heart diseases. We
present three children with myocardial noncompaction: one male with isolated
left ventricular noncompaction, another with right ventricular noncompaction and
dysplastic tricuspid valve, and the last with left ventricular noncompaction,
ventricular septal defect and coarctation of aorta, to stress especially the
different clinical forms of the disorder and the importance of early diagnosis,
as it may result in a fatal outcome. Noncompaction of the ventricular myocardium is a recently recognized genetic
cardiomyopathy. The left ventricle is the most affected site, but right
ventricular involvement has been reported in some cases. Diagnosis is made with
2-dimensional echocardiography or cardiac magnetic resoce imaging. The major
clinical manifestations are heart failure, arrhythmias and embolic events. A
20-year old man had left and right ventricular noncompaction complicated by
severe pulmonary hypertension, which is one of the first cases of biventricular
noncompaction associated with severe pulmonary hypertension. Pulmonary
hypertension may be a consequence of increased pulmonary venous pressures caused
by systolic and diastolic heart dysfunction secondary to noncompaction. INTRODUCTION: Isolated left ventricular noncompaction is a rare form of
cardiomyopathy. Heart failure with deteriorated systolic function is the
hallmark of this cardiomyopathy. Albeit it may cause ventricular tachycardia
(VT) and systemic embolism, it is a rarity to see these complications in a
patient with noncompaction and normal systolic functions.
CASE REPORT: A 78-year old female patient with a history of cerebrovascular
accident admitted to our hospital with palpitation and subsequently developed
cardiopulmonary arrest. Her ECG showed ventricular tachycardia degenerated into
fibrillation. Echocardiography and cardiac magnetic resoce (CMR) revealed a
small noncompacted segment in left ventricular apex. Ventricular tachycardia was
induced in electrophysiologic study and an implantable
cardioverter-defibrillator was implanted.
DISCUSSION: Patients with isolated left ventricular noncompaction usually
present with heart failure symptoms and subsequently diagnosed with
echocardiography. Rarely, it may cause ventricular tachycardia and systemic
embolism in a patient with normal systolic functions and a small noncompacted
segment. Noncompaction should be carefully sought in unexplained ventricular
tachycardia and cerebrovascular accidents, even if heart failure is not present. Isolated left ventricular non-compaction (LVNC) is a rare genetic form of
cardiomyopathy (CM) characterized by prominent left ventricular wall
trabeculation and intertrabecular recesses communicating with the ventricular
cavity. Clinical signs are variable, ranging from lack of symptoms to severe
manifestations including heart failure, sustained ventricular arrhythmias,
cardioembolism and sudden death. The diagnosis of LVNC is frequently missed, due
to limited awareness in the medical community. Contemporary diagnostic
sensitivity has been enhanced by the introduction of specific morphologic
criteria by high resolution echocardiography and cardiac magnetic resoce. As
a consequence, LVNC has been diagnosed more frequently in association with other
disorders such as congenital heart disease or genetic CM. The clinical relevance
of regional non-compaction in the context of other cardiac diseases is still
uncertain. Recent evidence points to an overlapping genetic background
encompassing LVNC, hypertrophic and dilated CM, suggesting a continuum of
disease associated with sarcomere protein gene mutations. This concept may prove
relevant to the understanding of common pathogenetic mechanisms of CM and offer
novel research opportunities. BACKGROUND: Isolated left ventricular noncompaction (ILVNC) is a cardiomyopathy
caused by intrauterine failure of the myocardium to compact. Common clinical
complications are heart failure, arrhythmias, and cardioembolism. A paucity of
data exists relating to clinical and echocardiographic features of ILVNC in
Africans.
METHODS AND RESULTS: This study is a single-center, prospective case-control
study, whereby subjects attending a dedicated cardiomyopathy clinic were
screened for and diagnosed with ILVNC, provided they had no other associated
structural heart disease and fulfilled all the accompanying echocardiographic
criteria: (1) end-systolic ratio of noncompacted layer to compacted layer >2,
(2) presence of >3 prominent apical trabeculations, and (3) deep intertrabecular
recesses that fill with blood from the ventricular cavity visualized using color
Doppler ultrasound. Fifty-four subjects were identified, age 45.4±13.1 years
(mean±SD), 95% confidence interval 3.6 to 10.2, 55.6% male, and 63.0% New York
Health Association Class II, and prevalence of LVNC in our clinic was 6.9%, 95%
confidence interval 3.6 to 10.2. Heart failure because of systolic dysfunction
was the most common clinical presentation (53 subjects, 98.1%). Left ventricular
end-diastolic diameter was 61.4±7.2 mm (mean±SD) and ejection fraction
26.7±11.9% (mean±SD). Common sites of noncompaction were the apical (100%),
midinferior (74.1%), and midlateral (64.8%) walls. Right ventricular
noncompaction occurred in 12 subjects (22.2%). Pulmonary hypertension was
documented in 45 cases (83.3%). Right ventricular dilation was noted in 40
subjects (74.1%), while right ventricular function was depressed in 32 (59.3%).
Tricuspid S' was 9.6±2.8 cm/s (mean±SD). No echocardiographic features
suggestive of ILVNC were noted in a healthy control group of African descent.
CONCLUSIONS: ILVNC in patients of African descent can be characterized by
biventricular abnormality and pulmonary hypertension, in addition to isolated
left-sided abnormality. The major clinical features of myocardial noncompaction are heart failure,
arrhythmias, and thromboembolic events. Prominent myocardial trabeculae and deep
recesses characteristic of myocardial noncompaction can cause stagt blood
flow and the formation of left ventricular clots. We describe the case of a
62-year-old woman who presented with symptoms of heart failure secondary to left
ventricular noncompaction. Transthoracic and transesophageal echocardiography
revealed multiple left ventricular thrombi, which had formed despite the
patient's long-term therapy with aspirin. Anticoagulative therapy should be
considered for patients with myocardial noncompaction who also have risk factors
for thromboembolism, such as atrial fibrillation, a history of systemic
embolism, or severe left ventricular systolic dysfunction. However, chronic
antiplatelet therapy may not sufficiently prevent clot formation in patients who
have myocardial noncompaction and severe left ventricular systolic dysfunction. Left ventricular noncompaction (LVNC) represents arrest of the normal myocardial
compaction process and results in the persistence of multiple prominent
ventricular trabeculations and deep intertrabecular recesses. LVNC can be
classified into 2 forms: isolated LVNC in the absence of other cardiac anomalies
and non-isolated LVNC associated with congenital heart disease. The clinical
presentation and the natural history of LVNC are highly variable, ranging from
no symptoms to congestive heart failure, arrhythmias, and systemic
thromboemboli. LVNC is genetically heterogeneous and can be inherited as an
autosomal domit or X-linked recessive disorder. It is also linked to
mutations in several genes, encoding the sarcomeric proteins, such as myosin
heavy chain 7 (MYH7). MYH7 encodes the β-myosin heavy chain, expressed in the
cardiac muscle. The operative indication for patients with non-isolated LVNC is
unclear. Here, we report the first successful case of surgical repair of a
ventricular septal defect (VSD) in an infant with non-isolated LVNC associated
with a novel MYH7 mutation. This mutation leads to the substitution of 7 amino
acid residues (671-677) in the actin-binding region of the protein. After the
VSD operation, the patient's congestive heart failure and pulmonary hypertension
improved. His condition has remained stable for 18 months with pharmacotherapy
comprising diuretics, an angiotensin converting enzyme inhibitor, and a
β-blocker. Although the postsurgical observational period was short, the
findings indicate that LVNC mutation analyses may facilitate surgical decisions
and help predict clinical courses. Isolated left ventricular noncompaction in adults is uncommon. The most frequent
clinical manifestations are heart failure due to left ventricular systolic
dysfunction and supraventricular and ventricular arrhythmias, which may be
sustained and associated with sudden death. Thromboembolic complications are
also possible. We report the case of an adult patient with isolated left
ventricular noncompaction who came to our observation because of acute cerebral
ischemia, an initial presentation of the disease only rarely described. BACKGROUND: Left ventricular noncompaction is a cardiomyopathy characterized by
excessive trabeculation of the left ventricle, progressive myocardial
dysfunction, and early mortality. Left ventricular noncompaction has a
heterogeneous clinical presentation that includes arrhythmia and sudden cardiac
death.
METHODS AND RESULTS: We retrospectively reviewed all children diagnosed with
left ventricular noncompaction at Texas Children's Hospital from January 1990 to
January 2009. Patients with congenital cardiac lesions were excluded. Two
hundred forty-two children were diagnosed with isolated left ventricular
noncompaction over the study period. Thirty-one (12.8%) died, and 13 (5.4%) were
received a transplant. One hundred fifty (62%) presented with or developed
cardiac dysfunction. The presence of cardiac dysfunction was strongly associated
with mortality (hazard ratio, 11; P<0.001). ECG abnormalities were present in
87%, with ventricular hypertrophy and repolarization abnormalities occurring
most commonly. Repolarization abnormalities were associated with increased
mortality (hazard ratio, 2.1; P=0.02). Eighty children (33.1%) had an
arrhythmia, and those with arrhythmias had increased mortality (hazard ratio,
2.8; P=0.002). Forty-two (17.4%) had ventricular tachycardia, with 5 presenting
with resuscitated sudden cardiac death. In total, there were 15 cases of sudden
cardiac death in the cohort (6.2%). Nearly all patients with sudden death (14 of
15) had abnormal cardiac dimensions or cardiac dysfunction. No patient with
normal cardiac dimensions and function without preceding arrhythmias died.
CONCLUSIONS: Left ventricular noncompaction has a high mortality rate and is
strongly associated with arrhythmias in children. Preceding cardiac dysfunction
or ventricular arrhythmias are associated with increased mortality. Children
with normal cardiac dimensions and normal function are at low risk for sudden
death. Isolated left ventricular noncompaction is a rare form of cardiomyopathy
characterized by prominent left ventricular trabeculations and intertrabecular
recesses. The typical clinical manifestations are severe systolic and diastolic
dysfunction, conduction abnormalities, and cardiac embolic events theorized to
result from thrombus formation within the intertrabecular recesses.
Evidence-based recommendations for preventing thromboembolic events in isolated
left ventricular noncompaction have not been established. We report the case of
a woman who, at 10 years of age, had been diagnosed with hypertrophic
cardiomyopathy without systolic dysfunction. At age 30, she presented with left
hemiparesis consequent to a large right-hemispheric ischemic stroke, and she was
diagnosed with isolated left ventricular noncompaction. In addition to
discussing the patient's case, we review the medical literature that pertains to
isolated left ventricular noncompaction. The first case of noncompaction was described in 1932 after an autopsy performed
on a newborn infant with aortic atresia/coronary-ventricular fistula. Isolated
noncompaction cardiomyopathy was first described in 1984. A review on
selected/relevant medical literature was conducted using Pubmed from 1984 to
2013 and the pathogenesis, clinical features, and management are discussed. Left
ventricular noncompaction (LVNC) is a relatively rare congenital condition that
results from arrest of the normal compaction process of the myocardium during
fetal development. LVNC shows variability in its genetic pattern,
pathophysiologic findings, and clinical presentations. The genetic
heterogeneity, phenotypical overlap, and variety in clinical presentation raised
the suspicion that LVNC might just be a morphological variant of other
cardiomyopathies, but the American Heart Association classifies LVNC as a
primary genetic cardiomyopathy. The familiar type is common and follows a
X-linked, autosomal-domit, or mitochondrial-inheritance pattern (in
children). LVNC can occur in isolation or coexist with other cardiac and/or
systemic anomalies. The clinical presentations are variable ranging from
asymptomatic patients to patients who develop ventricular arrhythmias,
thromboembolism, heart failure, and sudden cardiac death. Increased awareness
over the last 25 years and improvements in technology have increased the
identification of this illness and improved the clinical outcome and prognosis.
LVNC is commonly diagnosed by echocardiography. Other useful diagnostic
techniques for LVNC include cardiac magnetic resoce imaging, computerized
tomography, and left ventriculography. Management is symptom based and patients
with symptoms have a poorer prognosis. LVNC is a genetically heterogeneous
disorder which can be associated with other anomalies. Making the correct
diagnosis is important because of the possible associations and the need for
long-term management and screening of living relatives. Ventricular noncompaction (VNC) of the myocardium is a rare genetic
cardiomyopathy caused by a disorder during endocardial morphogenesis and could
be accompanied by life-threatening complications. The major clinical
manifestations of VNC are heart failure, arrhythmias, and embolic events. The
left ventricle is the most commonly reported affected site, but a few cases of
right ventricular involvement have also been reported. We report a case of
biventricular noncompaction cardiomyopathy in a 31-year-old woman presenting
with a new onset seizure. On the second day of her telemetry-monitored
hospitalization, she suffered a witnessed ventricular fibrillation arrest
requiring emergency direct-current cardioversion and induced hypothermia.
Transthoracic echocardiography (TTE) showed isolated left ventricular (LV)
noncompaction and depressed LV systolic function. Subsequent cardiac magnetic
resoce imaging (MRI) revealed both left and right ventricular noncompaction.
This unusual presentation highlights the importance of a complete and thorough
evaluation of patients even when presenting with apparently noncardiac
symptom(s). Isolated left ventricular noncompaction (LVNC) is a rare primary genetic
cardiomyopathy characterized by prominent trabeculation of the left ventricular
wall and intertrabecular recesses. Perioperative management of the patient with
LVNC might be challenging due to the clinical symptoms of heart failure,
systemic thromboembolic events, and fatal left ventricular arrhythmias. We
conducted real time intraoperative transesophageal echocardiography in a patient
with LVNC undergoing general anesthesia for ovarian cystectomy. |
What is the Glasgow Coma score? | Glasgow coma sore is used to determine injury severity on admission to a hospital emergency department or by the duration of unconsciousness. | An instrument in neurologic rehabilitation for the assessment of a person's
clarity of consciousness must fulfill the need to provide the rehabilitation
team with some information regarding the structuring of the rehabilitation
process. The Vienna Vigilance Score is oriented toward cooperation within the
rehabilitation team with the specific advantage of focus on the subject status
of the patient on the basis of the dialogue. It does not use painful stimuli and
is oriented toward the next developmental step of the rehabilitation process.
The first extensive application of the Vienna Vigilance Score was carried out in
a 3-year study of 38 children and adolescents. We report the comparison with
other coma scales (Glasgow Coma Scale, Children's Coma Score, and Modified
Glasgow Coma Scale) on 24 children and adolescents (1.4-16.8 years of age). The
results showed satisfying statistical parameters: measuring range comparable
with other scales; a Kendall tau correlation of .59 on both the Modified Glasgow
Coma Scale and the Children's Coma Scale and of .66 on the Glasgow Coma Scale; a
Spearman's rank order correlation of .70 to both the Modified Glasgow Coma Scale
and the Children's Coma Scale and of .78 on the Glasgow Coma Scale. By accepting
the Glasgow Coma Scale as a gold standard for classification of the level of
coma, we can confirm satisfactory measuring qualities for the Vienna Vigilance
Score. INTRODUCTION: Our aim in this study was to assess whether the new Glasgow Coma
Scale, Age, and Systolic Blood Pressure (GAP) scoring system, which is a
modification of the Mechanism, Glasgow Coma Scale, Age, and Arterial Pressure
(MGAP) scoring system, better predicts in-hospital mortality and can be applied
more easily than previous trauma scores among trauma patients in the emergency
department (ED).
METHODS: This multicenter, prospective, observational study was conducted to
analyze readily available variables in the ED, which are associated with
mortality rates among trauma patients. The data used in this study were derived
from the Japan Trauma Data Bank (JTDB), which consists of 114 major emergency
hospitals in Japan. A total of 35,732 trauma patients in the JTDB from 2004 to
2009 who were 15 years of age or older were eligible for inclusion in the study.
Of these patients, 27,154 (76%) with complete sets of important data (patient
age, Glasgow Coma Scale (GCS) score, systolic blood pressure (SBP), respiratory
rate and Injury Severity Score (ISS)) were included in our analysis. We
calculated weight for the predictors of the GAP scores on the basis of the
records of 13,463 trauma patients in a derivation data set determined by using
logistic regression. Scores derived from four existing scoring systems (Revised
Trauma Score, Triage Revised Trauma Score, Trauma and Injury Severity Score and
MGAP score) were calibrated using logistic regression models that fit in the
derivation set. The GAP scoring system was compared to the calibrated scoring
systems with data from a total of 13,691 patients in a validation data set using
c-statistics and reclassification tables with three defined risk groups based on
a previous publication: low risk (mortality < 5%), intermediate risk, and high
risk (mortality > 50%).
RESULTS: Calculated GAP scores involved GCS score (from three to fifteen
points), patient age < 60 years (three points) and SBP (> 120 mmHg, six points;
60 to 120 mmHg, four points). The c-statistics for the GAP scores (0.933 for
long-term mortality and 0.965 for short-term mortality) were better than or
comparable to the trauma scores calculated using other scales. Compared with
existing instruments, our reclassification tables show that the GAP scoring
system reclassified all patients except one in the correct direction. In most
cases, the observed incidence of death in patients who were reclassified matched
what would have been predicted by the GAP scoring system.
CONCLUSIONS: The GAP scoring system can predict in-hospital mortality more
accurately than the previously developed trauma scoring systems. OBJECTIVE: To determine the strength of the correlation between the Hunt and
Hess scale, Fisher score, Brussels coma score, World Federation of Neurosurgeons
score, and Glasgow coma score and health-related quality of life.
METHODS: Evaluable questionnaires from 236 patients (5.6 years [± standard
deviation, 2.854 years] on average after hemorrhage) were included in the
analysis. Quality of life was documented using the MOS-36 item short form health
survey. Because of the ordinal nature of the variables, Kendall tau was used for
calculation. Significance was established as P ≤ 0.05.
RESULTS: Weak and very weak correlations were found in general (r ≤ 0.28). The
strongest correlations were found between the Glasgow coma score and quality of
life (r = 0.236, P = 0.0001). In particular, the "best verbal response" achieved
the strongest correlations in the comparison, at r = 0.28/P = 0.0001. The Fisher
score showed very weak correlations (r = -0.148/P = 0.012). The Brussels coma
score (r = -0.216/P = 0.0001), Hunt and Hess scale (r = -0.197/P = 0.0001), and
the World Federation of Neurosurgeons score (r = -0.185/P = 0.0001) revealed
stronger correlations, especially in terms of the physical aspects of quality of
life.
CONCLUSIONS: The Glasgow coma scale revealed the strongest, and the Fisher score
showed the weakest correlations. Thus the Fisher score, as an indicator of the
severity of a hemorrhage, has little significance in terms of health-related
quality of life. OBJECTIVE: There are limited data regarding concussion among youth skiers and
snowboarders. The objective of this study was to examine the frequency of
concussion among helmeted and unhelmeted youth skiers and snowboarders
presenting to trauma centers.
METHODS: Subjects 18 years or younger with a ski- or snowboard-related injury
were studied using data from the National Trauma Data Bank from 2009 to 2010. We
further selected those with head/neck injuries and stratified based on helmet
status. Concussive injuries were identified from International Classification of
Diseases, 9th Revision codes. Severity analysis was based on the Glasgow Coma
Scale and Injury Severity Score.
RESULTS: A total of 1001 subjects met inclusion criteria with 678 subjects
having documented helmet status. Subjects 12 years or younger were more likely
to use helmets compared to 13-18 year-olds (odds ratio, 2.21; 95% confidence
interval [95% CI], 1.52-3.21). Skiers were more likely to use helmets compared
to snowboarders (odds ratios, 1.60; 95% CI, 1.16-2.19). Snowboarders had a
greater likelihood of concussion (estimated-β, 2.1; 95% CI, 1.48-2.85) after
adjusting for helmet status and age. There was no significant difference in the
frequency of concussion among helmeted compared to unhelmeted subjects. Imputing
missing values for helmets status had no effect on outcome for concussion. We
found no difference in injury severity among helmeted compared to unhelmeted
subjects.
CONCLUSIONS: Among youth skiers and snowboarders who present to trauma centers
with a head injury, the likelihood of that injury involving a concussion was not
associated with helmet use. In this study, we investigated the influence of children's level of executive
functioning on two types of metamemory knowledge following a traumatic brain
injury (TBI). For this purpose, 22 children (aged 7 to 14 years) who had
sustained a moderate to severe TBI and 44 typically developing children were
recruited. The children with TBI were divided into two groups according to the
severity of their executive impairment. Injury severity was determined by the
Glasgow Coma Scale (GCS) score on admission or by the duration of
unconsciousness. All children were then tested on both their knowledge of
general memory functioning and their level of memory self-awareness,
respectively assessed using the total number of correct responses on an adapted
version of a metamemory interview and a self-other discrepancy score on a
questionnaire evaluating everyday memory abilities. Data analyses revealed that
participants with TBI who suffered impaired executive functions demonstrated
less general metamemory knowledge, and underestimated the frequency of their
memory problems, compared with children with TBI who had preserved executive
functions and with control participants. Considering the well-established effect
of metamemory knowledge on people's spontaneous implementation of strategies,
the interest and the importance of these findings on both theoretical and
clinical grounds are discussed. |
Andexanet Alfa is an antidote of which clotting factor inhibitors? | Andexanet alfa is a specific reversal agent for Factor Xa inhibitors. | The new oral anticoagulants have many advantages over vitamin K antagonists, but
they are still associated with a troublesome incidence of major bleeding.
Additionally, the absence of a reversal agent for the new oral anticoagulants is
a barrier to their more widespread use. Currently, there are 3 potential
reversal agents in development: idarucizumab is a humanized murine monoclonal
antibody fragment directed specifically at dabigatran; andexanet alfa is a
recombit modified decoy factor Xa that binds to factor Xa inhibitors; and
PER977 is a small molecule that binds to factor Xa and IIa inhibitors and to
heparin-based anticoagulants through charge interaction. These agents have
undergone phase I clinical testing, appear to be well tolerated in healthy
volunteers, and are effective in neutralizing their respective targets. All 3
are currently undergoing or entering into a phase II or III clinical study. This
article reviews the available data for idarucizumab, andexanet alfa, and PER977. Thrombin inhibitor dabigatran and factor Xa inhibitors rivaroxaban, apixaban and
edoxaban form a new class of non-vitamin K antagonist oral anticoagulants and
have been extensively studied in patients with venous thromboembolism and atrial
fibrillation. They offer anticoagulation that is as effective and at least as
safe compared to warfarin without the need for routine laboratory monitoring;
however, no reversal strategies are currently validated in case of a non-vitamin
K antagonist oral anticoagulant-associated bleed. In emergency situations,
laboratory drug measurement and well-defined management for non-vitamin K
antagonist oral anticoagulant-induced hemorrhage may improve clinical outcome.
In this review, the merits and limitations of the routine coagulation tests and
some of the more specific laboratory assays are compared. Furthermore,
prohemostatic measures are reviewed and the recommended strategies in case of
bleeding are summarized. Specific reversal agents are currently under
development (idarucizumab for dabigatran, andexanet alfa for Xa inhibitors, and
PER977 for both Xa- and thrombin inhibitors), which will facilitate clinical
management of severe bleeding and emergency surgery. In the last decade, several direct oral anticoagulants (DOAC; dabigatran,
rivaroxaban, apixaban, edoxaban) have been marketed for prophylaxis and/or
treatment of thromboembolism without having specific antidotes available for
their reversal. Current management of bleeding associated to DOAC includes the
removal of all antithrombotic medications and supportive care. Non-specific
procoagulant agents (prothrombin complex concentrates and activated factor VIIa)
have been used in case of serious bleeding. Currently, some specific antidotes
for the DOAC are under development. Idarucizumab (BI 655075; Boehringer
Ingelheim) is a fragment of an antibody (Fab), which is a specific antidote to
the oral direct thrombin inhibitor dabigatran. Andexanet alfa (r-Antidote,
PRT064445; Portola Pharmaceuticals) is a truncated form of enzymatically
inactive factor Xa, which binds and reverses the anticoagulant action of the
factor Xa inhibitors (e.g.: rivaroxaban, apixaban and edoxaban). Aripazine
(PER-977, ciraparantag; Perosphere Inc.) is a synthetic small molecule (~500 Da)
that reverses oral dabigatran, apixaban, rivaroxaban, as well as subcutaneous
fondaparinux and LMWH in vivo. These antidotes could provide an alternative for
management of life-threatening bleeding events occurring with the
above-mentioned anticoagulants. In addition, the specific antidote anivamersen
(RB007; Regado Biosciences Inc.) is an RNA aptamer in clinical development to
reverse the anticoagulant effect of the parenteral factor IXa inhibitor
pegnivacogin, which is also in development. This anticoagulant-antidote pair may
provide an alternative in situations in which a fast onset and offset of
anticoagulation is needed, like in patients undergoing cardiac surgery with
extracorporeal circulation, as an alternative to the heparin/protamine pair.
This patent review includes a description of the pharmacological characteristics
of the novel specific antidotes, the available results from completed
non-clinical and clinical studies and the description of ongoing clinical trials
with the new compounds. Three target-specific oral anticoagulants (TSOACs)-dabigatran, rivaroxaban, and
apixaban-have been approved by the FDA to reduce the risk of stroke and systemic
embolism in patients with nonvalvular atrial fibrillation; however, no agents
are currently approved to reverse the anticoagulant effects of these TSOACs in
cases of active bleeding. This review discusses the benefits and risks of these
TSOACs from a clinician's perspective, with a focus on the interruption of
treatment for either elective or emergent surgery, monitoring, and reversal of
anticoagulation. Available coagulation assays are not ideal for monitoring the
effects of TSOACs and do not provide reliable quantitative measurement of their
anticoagulant effects. When necessary, activated partial thromboplastin time
(aPTT) may provide qualitative information on dabigatran, and prothrombin time
(PT) may provide qualitative assessment of the presence of the factor Xa
inhibitors, rivaroxaban and apixaban. Current recommendations for reversal of
TSOACs are based largely on limited and sometimes conflicting data from in vitro
or in vivo animal models, and clinical experience with these recommendations is
also limited. Methods that have been investigated for effectiveness for reversal
of the pharmacodynamic effects of the TSOACs include dialysis, activated
charcoal, prothrombin complex concentrate (PCC), and recombit activated
factor VII. It is important to note that even within a class of anticoagulant
drugs, compounds respond differently to reversal agents; therefore,
recommendations for one agent should not be extrapolated to another, even if
they are from the same therapeutic class. New antidotes are being explored,
including a mouse monoclonal antibody to dabigatran; andexanet alfa, a potential
universal factor Xa inhibitor reversal agent; and a synthetic small molecule
(PER977) that may be effective for the reversal of factor Xa inhibitors and
direct thrombin inhibitors. Given the short half-lives of TSOACs, watchful
waiting, rather than reversal, may be the best approach in some circumstances. The non-vitamin K antagonist oral anticoagulants (NOACs) are used for
thromboembolic prophylaxis of patients with atrial fibrillation and in the
treatment as well as secondary prophylaxis of patients with venous
thromboembolism. Even though NOACs have a better safety profile than vitamin K
antagonists (VKAs), there will still be bleeding complications on NOAC
treatment. In some cases, stopping the NOAC and non-drug-related management such
as manual compression and interventional endoscopy will be sufficient to stop
the bleeding. In more serious bleeding events and before acute surgery,
coagulation factor concentrates or NOAC-specific antidotes could be used.
Coagulation factor concentrates can be used in patients with haemophilia and to
reverse the effect of VKAs but, in NOAC-treated patients, results are
inconsistent and these agents could potentially have pro-thrombotic effects.
Specific antidotes for NOACs are expected to be on the market soon. Phase III
clinical trials with a humanized antibody fragment directed against dabigatran
(idarucizumab) and recombit, modified factor Xa (andexanet alfa) are ongoing.
A molecule (aripazine) with broad activity against various anticoagulants
including NOACs is currently undergoing phase II trials. For use of these
specific antidotes, it is desirable that measurements for coagulation activity
with a short response delay are widely available for the different NOACs and
further research in this field is needed. Furthermore, guidelines for antidote
use, including general measures for the treatment of NOAC-related bleeding,
should be available. BACKGROUND: Bleeding is a complication of treatment with factor Xa inhibitors,
but there are no specific agents for the reversal of the effects of these drugs.
Andexanet is designed to reverse the anticoagulant effects of factor Xa
inhibitors.
METHODS: Healthy older volunteers were given 5 mg of apixaban twice daily or 20
mg of rivaroxaban daily. For each factor Xa inhibitor, a two-part randomized
placebo-controlled study was conducted to evaluate andexanet administered as a
bolus or as a bolus plus a 2-hour infusion. The primary outcome was the mean
percent change in anti-factor Xa activity, which is a measure of factor Xa
inhibition by the anticoagulant.
RESULTS: Among the apixaban-treated participants, anti-factor Xa activity was
reduced by 94% among those who received an andexanet bolus (24 participants), as
compared with 21% among those who received placebo (9 participants) (P<0.001),
and unbound apixaban concentration was reduced by 9.3 ng per milliliter versus
1.9 ng per milliliter (P<0.001); thrombin generation was fully restored in 100%
versus 11% of the participants (P<0.001) within 2 to 5 minutes. Among the
rivaroxaban-treated participants, anti-factor Xa activity was reduced by 92%
among those who received an andexanet bolus (27 participants), as compared with
18% among those who received placebo (14 participants) (P<0.001), and unbound
rivaroxaban concentration was reduced by 23.4 ng per milliliter versus 4.2 ng
per milliliter (P<0.001); thrombin generation was fully restored in 96% versus
7% of the participants (P<0.001). These effects were sustained when andexanet
was administered as a bolus plus an infusion. In a subgroup of participants,
transient increases in levels of d-dimer and prothrombin fragments 1 and 2 were
observed, which resolved within 24 to 72 hours. No serious adverse or thrombotic
events were reported.
CONCLUSIONS: Andexanet reversed the anticoagulant activity of apixaban and
rivaroxaban in older healthy participants within minutes after administration
and for the duration of infusion, without evidence of clinical toxic effects.
(Funded by Portola Pharmaceuticals and others; ANNEXA-A and ANNEXA-R
ClinicalTrials.gov numbers, NCT02207725 and NCT02220725.). Non-vitamin K oral anticoagulants (NOACs) have been a major addition to our
therapeutic armamentarium. They are at least as effective as warfarin in the
thromboprophylaxis of non-valvular atrial fibrillation and management of
thromboembolic disease, with a more favorable safety profile. Their predictable
pharmacokinetics and pharmacodynamics allow for a fixed oral dosing without the
need for anticoagulation monitoring. A major concern regarding NOACs is the lack
of a readily available antidote to reverse their anticoagulation effect in case
of life-threatening bleeding or need for emergent surgery. In this review, we
summarize preclinical and clinical data on (a) hemostatic agents used to reverse
NOACs, and (b) novel, target-specific NOACs reversal agents under development.
The prothrombin complex concentrates, activated prothrombin complex concentrates
and recombit activated factor VII are hemostatic agents that have been
assessed in reversing NOACs. Preclinical studies with hemostatic agents report
variable results and there is only limited clinical data available to date.
Idarucizumab and andexanet alfa are NOAC-specific reversal agents designed to
reverse dabigatran and factor Xa inhibitors accordingly. Aripazine is a
universal anticoagulation reversal agent. Preclinical studies show promising
results and these agents are already in different stages of clinical
development. Phase I and II clinical trials demonstrate efficacy in reversing
NOACs without major side effects. Until these agents become commercially
available, management of patients receiving NOACs who present with major
bleeding or require emergent surgery should focus on (a) immediate
discontinuation of NOACs, (b) supportive measures or postponing surgery for
12-24 h after the last NOAC dose, and/or Andexanet alfa is a specific reversal agent for Factor Xa inhibitors. The
molecule is a recombit protein analog of factor Xa that binds to Factor Xa
inhibitors and antithrombin:LMWH complex but does not trigger prothrombotic
activity. In ex vivo, animal, and volunteer human studies, andexanet alfa (AnXa)
was able to dose-dependently reverse Factor Xa inhibition and restore thrombin
generation for the duration of drug administration. Further trials are underway
to examine its safety and efficacy in the population of patients experiencing
FXa inhibitor-related bleeding. The vitamin K antagonists (VKAs) have been the standard (and only) oral
anticoagulants used for the long-term treatment or prevention of venous
thromboembolism or stroke in patients with atrial fibrillation. The coagulopathy
induced by VKAs can be reversed with vitamin K, and in urgent situations, the
vitamin K-dependent coagulation factors can be replaced by transfusion. In the
last decade, a new class of oral anticoagulants has been developed, direct oral
anticoagulants that bind to a specific coagulation factor and neutralize it.
These compounds were shown to be effective and safe compared with the VKAs and
were licensed for specific indications, but without a specific reversal agent.
The absence of a reversal agent is a barrier to more widespread use of these
agents. Currently, for the management of major life-threatening bleeding with
the direct oral anticoagulants, most authorities recommend the use of four
factor prothrombin complex concentrates. There are now three reversal agents in
development and poised to enter the market. Idarucizumab is a specific antidote
targeted to reverse the direct thrombin inhibitor, dabigatran, which was
recently approved for use in the USA. Andexanet alfa is an antidote targeted to
reverse the oral direct factor Xa inhibitors as well as the indirect inhibitor
enoxaparin. Ciraparantag is an antidote targeted to reverse the direct thrombin
and factor Xa inhibitors as well as the indirect inhibitor enoxaparin. The Vitamin K antagonist warfarin was the only oral anticoagulant available for
decades for the treatment of thrombosis and prevention of thromboembolism until
Direct Oral Anticoagulants (DOACs); a group of new oral anticoagulants got
approved in the last few years. Direct thrombin inhibitor: dabigatran and factor
Xa inhibitors: apixaban, rivaroxaban, and edoxaban directly inhibit the
coagulation cascade. DOACs have many advantages over warfarin. However, the
biggest drawback of DOACs has been the lack of specific antidotes to reverse the
anticoagulant effect in emergency situations. Activated charcoal, hemodialysis,
and activated Prothrombin Complex Concentrate (PCC) were amongst the nonspecific
agents used in a DOAC associated bleeding but with limited success.
Idarucizumab, the first novel antidote against direct thrombin inhibitor
dabigatran was approved by US FDA in October 2015. It comprehensively reversed
dabigatran-induced anticoagulation in a phase I study. A phase III trial on
Idarucizumab also complete reversal of anticoagulant effect of dabigatran.
Andexanet alfa (PRT064445), a specific reversal agent against factor Xa
inhibitors, showed a complete reversal of anticoagulant activity of apixaban and
rivaroxaban within minutes after administration without adverse effects in two
recently completed parallel phase III trials ANNEXA-A and ANNEXA-R respectively.
It is currently being studied in ANNEXA-4, a phase IV study. Aripazine
(PER-977), the third reversal agent, has shown promising activity against
dabigatran, apixaban, rivaroxaban, as well as subcutaneous fondaparinux and
LMWH. This review article summarizes pharmacological characteristics of these
novel antidotes, coagulation's tests affected, available clinical and
preclinical data, and the need for phase III and IV studies. PURPOSE: The properties of three oral anticoagulant-specific reversal agents are
reviewed, and guidance is presented to assist pharmacists in planning for the
agents' introduction to the market.
SUMMARY: Idarucizumab, which received Food and Drug Administration approval in
October 2015, is a humanized monoclonal antibody fragment that immediately
neutralizes the anticoagulant effect of dabigatran, as evidenced by reduced
unbound dabigatran concentrations and normalized coagulation tests. Preliminary
Phase III trial results demonstrated a median maximum reversal of 100%, a median
time to bleeding cessation of 11.4 hours, and normal intraoperative hemostasis
in 92% of patients requiring anticoagulation reversal before an urgent
procedure. Andexanet alfa is a factor Xa (FXa) decoy that binds to direct and
indirect FXa inhibitors. In Phase III trials in healthy volunteers, andexanet
alfa reduced anti-FXa activity by more than 90%, reduced the concentration of
unbound direct FXa inhibitor, and inhibited thrombin generation. Ciraparantag is
a reversal agent under development for reversal of anticoagulation with direct
and indirect FXa inhibitors and certain factor IIa inhibitors; it exerts its
effect through hydrogen bonding. Concerns for thromboembolic events directly
related to administration of idarucizumab, andexanet alfa, or ciraparantag have
not arisen. Pharmacists need to begin preparing for the introduction of these
specific reversal agents through protocol development and provider education; in
addition, pharmacy departments need to plan for procurement and storage. The
specific reversal agents should be incorporated into antithrombotic stewardship
or other clinical pharmacy programs for surveillance.
CONCLUSION: As agents that provide rapid reversal of direct oral anticoagulant
activity become available, advance planning will help hospitals to optimize
their use. 1. Oral Factor Xa (FXa) inhibitors, a growing class of direct-acting
anticoagulants, are frequently used to prevent stroke and systemic embolism in
patients with atrial fibrillation and to prevent and treat venous
thromboembolism. These drugs reduce the risk of clotting at the expense of
increasing the risk of bleeding, and currently they have no specific reversal
agent. However, andexanet alfa, a recombit modified FXa decoy molecule, is in
a late-phase clinical trial in bleeding patients, and ciraparantag, a small
molecule that appears to reverse many anticoagulants including the FXa
inhibitors, is in development. This review summarizes the published data to date
on both drugs, which have the potential to change the management approach to
patients with FXa inhibitor-associated major hemorrhage. BACKGROUND: Four nonvitamin K antagonist oral anticoagulants (NOACs) are
approved for the prevention of stroke in patients with nonvalvular atrial
fibrillation and for the treatment of venous thromboembolism. These include the
direct thrombin inhibitor dabigatran and the direct factor Xa inhibitors
rivaroxaban, apixaban, and edoxaban. Bleeding is a complication for all
anticoagulants and concerns regarding bleeding risk and the suitability of
effective reversal strategies may be a barrier to their prescription. Despite
the reduced risk of bleeding compared with vitamin K antagonists, questions
persist regarding the management of bleeding related to NOAC use.
MAIN TEXT: To date, although a number of assays are responsive to NOACs, no
single routine laboratory test has been identified to accurately measure the
clinical anticoagulation state of patients on NOACs or established as a reliable
predictor of bleeding risk. In addition, the establishment of a reliable human
bleeding model to test novel inhibitors of the coagulation cascade has proved
challenging. Although routine monitoring of anticoagulant levels is not
necessary in patients taking NOACs, anticoagulant reversal and a means of
measuring reversal may be required for patients who present with bleeding or
require urgent surgery. Prothrombin complex concentrates are pooled plasma
products containing varying amounts of inactive vitamin K-dependent clotting
factors in addition to vitamin K-dependent proteins and can replenish factors in
the intrinsic and extrinsic coagulation cascade, reversing an anticoagulant
effect. Only one agent, idarucizumab, has been approved for rapid reversal of
dabigatran-induced anticoagulation and one more agent, andexanet alfa, has been
submitted for approval to reverse the anticoagulatory effects of direct and
indirect factor Xa inhibitors.
CONCLUSIONS: This review discusses the laboratory tests available for assessing
anticoagulation, human models of bleeding, and the use of current
strategies-including prothrombin complex concentrates for reversal of
anticoagulation by NOACs-to manage bleeding in patients. Oral Factor Xa (FXa) inhibitors, a growing class of direct-acting
anticoagulants, are frequently used to prevent stroke and systemic embolism in
patients with atrial fibrillation and to prevent and treat venous
thromboembolism. These drugs reduce the risk of clotting at the expense of
increasing the risk of bleeding, and currently they have no specific reversal
agent. However, andexanet alfa, a recombit modified FXa decoy molecule, is in
a late-phase clinical trial in bleeding patients, and ciraparantag, a small
molecule that appears to reverse many anticoagulants including the FXa
inhibitors, is in development. This review summarizes the published data to date
on both drugs, which have the potential to change the management approach to
patients with FXa inhibitoreassociated major hemorrhage. Bleeding is the most common complication of all anticoagulants. Any bleeding
patient on an anticoagulant should be risk-stratified based on hemodynamic
instability, source of bleeding, and degree of blood loss. Although minor bleed
may be managed with discontinuation of anticoagulant, major bleed may require
transfusion of blood products and use of specific antidote. The residual effects
of each anticoagulant may be monitored with distinct coagulation assay.
Intravenous or oral vitamin K can reverse the effect of warfarin within 24 to 48
hours and is indicated for any bleeding, international normalized ratio of >10
or 4.5 to 10 in patients with other risk factors for bleeding. Fresh frozen
plasma or prothrombin complex concentrate (PCC) may be necessary in major
bleeding related to warfarin. Protamine sulfate reverses the effect of
unfractionated heparin completely and of low-molecular-weight heparin (LMWH)
partially. Idarucizumab has recently been approved in United States for
dabigatran reversal, whereas andexanet alfa is expected to get approved in the
near future for reversal of oral factor Xa inhibitors. The PCC may reverse the
effect of rivaroxaban to some extent, but no data are available regarding
reversal of apixaban and edoxaban. Aripazine has shown promising results to
reverse the effects of LMWH, fondaparinux, and direct oral anticoagulants but is
still in the developmental phase. |
Which is the main cause of the Patau syndrome? | Patau syndrome is caused by trisomy 13. | The authors examined the soluble proteins of the brain frontal lobes in the
newborn with trisomias of the 13th, 18th, and 21st chromosomes (Down's, Patau's,
and Edwards' syndromes). The examinations were carried out on autopsy material
(the post-mortem period not exceeding 24 hours) by the method of disc
electrophoresis in polyacrylamide gel. The brain tissue was taken from 17
newborn infants with Down's syndrome; 9 infants with Patau's syndrome; and 7
infants with Edwards' syndrome. For the control the brain of 21 newborn infants
without defects of the CNS development (the death cause being analogous) was
taken. In all the syndromes studied diversely directed but relatively specific
shifts were revealed on the proteinograms. It was the albumin section which
appeared to be the most sensitive to the chromosomal pathology: in cases of
Down's and Patau's syndromes the protein content in it was reduced, whereas in
cases of Edwards' syndrome it was increased. In the latter syndrome the relative
amount of neuronines S-5 and S-6, and in Patau's syndrome the amount of
neuronine S-6 were lowered, this lowering being statistically significantly. In
all the trisomias a tendency to a diminution of the zone of the acidic
neurospecific cerebral proteins was noted. This is, possibly, due to the lower
level of the CNS functional activity in chromosomal pathologies. Autosomal trisomies are associated with major congenital malformations that may
result in prolonged hospitalization of the newborn. Knowledge about these
chromosomal abnormalities is important for nurses in neonatal practice. This
article identifies the causes and manifestations of most of these trisomies:
trisomy 13 (Patau syndrome), trisomy 18 (Edwards syndrome), and trisomy 21 (Down
syndrome). More detailed description of the manifestations, associated
abnormalities, and outcomes of the most common of these, trisomy 21, is
provided. Wolf-Hirschhorn syndrome (WHS) and Patau syndrome are two of the most severe
conditions resulting from chromosome abnormalities. WHS is caused by a deletion
of 4p16, while Patau syndrome is caused by trisomy for some or all regions of
chromosome 13. Though the etiologies of these syndromes differ, they share
several features including pre- and postnatal growth retardation, microcephaly,
cleft lip and palate, and cardiac anomalies. We present here a female fetus with
deletion of 4p16 --> pter and duplication of 13q32 --> qter due to unbalanced
segregation of t(4;13)(p16;q32) in the father. She displayed overlapping
features of both of these syndromes on ultrasound. To the best of our knowledge,
this is the first report of a fetus with both partial trisomy 13 and deletion of
4p16, the critical region for WHS. Known as D trisomy, Patau syndrome is the third chromosomopathy according to
frequency. One of the 5000 newborn carries the trisomy 13. In over 80% cases
there is fresh mutation with non separation in myeosis of older mother. The
mosaic or translocation forms are not rare. The mail newborn with Patau syndrome
is shown in this article. We notice: microcephalia, dolihocephalia,
microphthalmia, cheilognatopalatoshisis, polydactilia, and found ultrasound
changes at the brain, hearth and genitourinary system. Cytogenetic finding show:
mail cariotype with aberrations 47, XY + 13, Sy Patau. We describe the management of the eyelid anomaly associated with Patau syndrome.
Trisomy 13 is the genotype of the syndrome's phenotype. The eyelid anomaly was a
tarsal kink, a congenital malformation of the tarsus that causes entropion. A
2-month-old white girl presented with unilateral upper eyelid entropion and
central corneal ulceration. To correct this condition, two 6-0 polyglactin
sutures were passed through the gray line of the upper and lower eyelids and
tied. Correction of the entropion and improvement in the corneal condition were
achieved after surgery. No recurrence of the entropion or corneal ulceration was
noted after 2 months of follow-up. This simple technique, which corrected the
eyelid malposition, providing an excellent cosmetic result without incision of
the tarsus, has been previously reported in other cases of tarsal kink but not
in a patient with Patau syndrome. Patau syndrome (trisomy 13) is very rare in live-born babies. Individuals with
this chromosomal syndrome have a short lifespan and are rarely seen beyond
infancy. This study is aimed at the clinical spectrum, natural history, and
survival of patients with trisomy 13. We reviewed the detailed data of 13 Patau
syndrome live-born babies. Among them two individuals were delivered from
continuation of pregcy even after prenatal diagnosis. The remaining 11
patients were born to younger mothers who did not undergo amniocentesis because
no major anomalies except for cleft lip/palate were found on prenatal sonograms.
The common features of Patau syndrome including the clinical triad
(microphthalmia, cleft lip/palate, and polydactyly) and non-cyanotic heart
defects were always found in our series. However, certain serious central
defects (holoprosencephaly, omphalocele, and single umbilical artery), which are
easily recognized from prenatal sonogram, occurred less frequently than those
stated in the literature. The median survival time was 95 days and was longer
than that previously reported. There were two infants with trisomic mosaicism
with different outcomes in both clinical spectrum and survival. Otherwise, we
also found the increased recurrence risks of aneuploidy in two individuals, and
the longest survivor (84 months) of non-mosaic trisomy 13 in Taiwan. We thus
suggest that long-term survival in our series is strongly correlated with
different expressivity after prenatal selection, in addition to cytogenetic
mosaicism. Less associated anomalies such as polyhydramnios, oligohydramnios,
intrauterine growth retardation, single umbilical artery, eye defects,
holoprosencephaly, omphalocele, and polycystic kidney may contribute to their
clinical courses. Among full autosomal trisomies, only trisomies of chromosome 21 (Down syndrome),
18 (Edwards syndrome) and 13 (Patau syndrome) are compatible with postnatal
survival. But the mechanisms, how a supernumerary chromosome disrupts the normal
development and causes specific phenotypes, are still not fully explained. As an
alternative to gene dosage effect due to the trisomic chromosome a genome-wide
transcriptional dysregulation has been postulated. The aim of this study was to
define the transcriptional changes in trisomy 13, 18, and 21 during early fetal
development in order to obtain more insights into the molecular etiopathology of
aneuploidy. Using oligonucleotide microarrays, we analyzed whole genome
expression profiles in cultured amniocytes (AC) and chorionic villus cells (CV)
from pregcies with a normal karyotype and with trisomies of human chromosomes
13, 18 and 21. We observed a low to moderate up-regulation for a subset of genes
of the trisomic chromosomes. Transcriptional levels of most of the genes on the
supernumerary chromosome appeared similar to the respective chromosomal pair in
normal karyotypes. A subset of chromosome 21 genes including the DSCR1 gene
involved in fetal heart development was consistently up-regulated in different
prenatal tissues (AC, CV) of trisomy 21 fetuses whereas only minor changes were
found for genes of all other chromosomes. In contrast, in trisomy 18 vigorous
downstream transcriptional changes were found. Global transcriptome analysis for
autosomal trisomies 13, 18, and 21 supported a combination of the two major
hypotheses. AIM OF STUDY: An analysis of prenatal diagnostics efficiency of selected types
of chromosomal aberrations in the Czech Republic in 2007. Update of 1994-2007
data according to particular selected diagnoses.
TYPE OF STUDY: Retrospective epidemiological analysis of pre- and postnatal
chromosomal aberrations diagnostics and its efficiency.
MATERIAL AND METHODS: Data on pre- and postnatally diagnosed birth defects in
the Czech Republic during 1994-2007 were used. Data on prenatally diagnosed
birth defects (and for terminated pregcies) were collected from particular
departments of prenatal diagnostics, medical genetics and ultrasound diagnostics
in the Czech Republic, data on birth defects in births from the National Birth
Defects Register (Institute for Health Information and Statistics). Total
numbers over the period under the study, mean incidences of selected types of
chromosomal aberrations and mean prenatal diagnostics efficiencies were
analyzed. Following chromosomal aberrations were studied: Down, Edwards, Patau,
Turner and Klinefelter syndromes and syndromes 47,XXX and 47,XYY.
RESULTS: A relative proportion of Down, Edwards and Patau syndromes as well as
other autosomal and gonosomal aberration is presented in figures. Recently,
trisomies 13, 18 and 21 present around 70% of all chromosomal aberrations in
selectively aborted fetuses, in other pregcies, "other chromosomal
aberrations" category (mostly balanced reciprocal translocations and inversions)
present more than 2/3 of all diagnoses. During the period under the study,
following total numbers, mean relative incidences (per 10,000 live births, in
brackets) and mean prenatal diagnostics efficiency (in %) were found in
following chromosomal syndromes: Down syndrome 2,244 (16.58) and 63.37%, Edwards
syndrome 521 (3.85) and 79.93%, Patau syndrome 201 (1.49) and 68.87%, Turner
syndrome 380 (2.81) and 79.89%, 47,XXX syndrome 61 (0.45) and 59.74%,
Klinefelter syndrome 163 (1.20) and 73.65% and 47,XYY syndrome 22 (0.16) and
54.76%.
CONCLUSIONS: The study gives updated results of incidences analysis of both pre-
and postnatally diagnosed chromosomal birth defects in the Czech Republic during
the 1994-2007 period. Incidences found in our study correspond (in case of
trisomies 13, 18 and 21) with those published widely in literature as well as
with those found in large-scale international studies (ICBDSR, EUROCAT). In case
of gonosomal aberrations, incidences found in this study are lower that those
published, most probably due to a later registration (over 15 years of age of
the child) of these diagnoses. Patau syndrome is a chromosomal disorder associated with multiple malformations
caused by inheritance of an extra chromosome (trisomy 13). Some skin defects
have been reported in patients with Patau syndrome, such as scalp defects,
glabellar stains, deep palmar creases, rocker-bottom feet, convex soles,
hyperconvextity of the nails, and multiple hemangiomas. To our knowledge,
widespread comedonal and cystic acne have not been previously reported in Patau
syndrome. OBJECTIVES: To determine whether older paternal age increases the risk of
fathering a pregcy with Patau (trisomy 13), Edwards (trisomy 18), Klinefelter
(XXY) or XYY syndrome.
DESIGN: Case-control: cases with each of these syndromes were matched to four
controls with Down syndrome from within the same congenital anomaly register and
with maternal age within 6 months.
SETTING: Data from 22 EUROCAT congenital anomaly registers in 12 European
countries.
PARTICIPANTS: Diagnoses with observed or (for terminations) predicted year of
birth from 1980 to 2005, comprising live births, fetal deaths with gestational
age ≥ 20 weeks and terminations after prenatal diagnosis of the anomaly. Data
include 374 cases of Patau syndrome, 929 of Edwards syndrome, 295 of Klinefelter
syndrome, 28 of XYY syndrome and 5627 controls with Down syndrome.
MAIN OUTCOME MEASURES: Odds ratio (OR) associated with a 10-year increase in
paternal age for each anomaly was estimated using conditional logistic
regression. Results were adjusted to take account of the estimated association
of paternal age with Down syndrome (1.11; 95% CI 1.01 to 1.23).
RESULTS: The OR for Patau syndrome was 1.10 (95% CI 0.83 to 1.45); for Edwards
syndrome, 1.15 (0.96 to 1.38); for Klinefelter syndrome, 1.35 (1.02 to 1.79);
and for XYY syndrome, 1.99 (0.75 to 5.26).
CONCLUSIONS: There was a statistically significant increase in the odds of
Klinefelter syndrome with increasing paternal age. The larger positive
associations of Klinefelter and XYY syndromes with paternal age compared with
Patau and Edwards syndromes are consistent with the greater percentage of these
sex chromosome anomalies being of paternal origin. Syndromes caused by chromosomal aneuploidies are widely recognized genetic
disorders in humans and often lead to spontaneous miscarriage. Preimplantation
genetic screening is used to detect chromosomal aneuploidies in early embryos.
Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may
serve as models for human syndromes caused by aneuploidies. We have established
25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro
development. The hESC lines exhibited morphology and expressed markers typical
of hESCs. They demonstrated long-term proliferation capacity and pluripotent
differentiation. Karyotype analysis revealed that two-third of the cell lines
carry a normal euploid karyotype, while one-third remained aneuploid throughout
the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau
syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X
(Turner syndrome). On the basis of the level of single nucleotide polymorphism
heterozygosity in the aneuploid chromosomes, we determined whether the
aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene
expression profiles of the trisomic cell lines suggested that all three
chromosomes are actively transcribed. Our analysis allowed us to determine which
tissues are most affected by the presence of a third copy of either chromosome
13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic
development. The results presented here suggest that aneuploid embryos can serve
as an alternative source for either normal euploid or aneuploid hESC lines,
which represent an invaluable tool to study developmental aspects of chromosomal
abnormalities in humans. Trisomy 13 or Patau syndrome (PS) is a chromosomal disorder characterized by a
well known presentation of multiple congenital anomalies. Our objective was to
determine the clinical features and prognosis observed in a sample of patients
with PS. The series was composed of patients with diagnosis of PS consecutively
evaluated by a Clinical Genetics Service from a reference hospital of southern
Brazil, in the period between 1975 and 2012. Statistical analysis was performed
using PEPI program (version 4.0), with two-tailed Fisher's exact test for
comparison of frequencies (P<0.05). The sample consisted of 30 patients, 60%
male, median age at first evaluation of 9 days. Full trisomy of chromosome 13
was the main cytogenetic alteration (73%). The major clinical findings included:
cryptorchidism (78%), abnormal auricles (77%), congenital heart defects (76%),
polydactyly (63%), microphthalmia (60%) and micrognathia (50%). Four patients
(13%) simultaneously had micro/anophthalmia, oral clefts and polydactyly. Some
findings were only observed in our sample and included, among others,
preauricular tags (10%), duplication of the hallux (3%) and spots following the
lines of Blaschko (3%). Mosaicism (20% of cases) had a statistically significant
association only with absence of cryptorchidism. The median of survival was 26
days. Patients with and without mosaicism had similar median of survival. Our
findings, in agreement with the literature, show that the anomalies in patients
with PS can be quite variable, sometimes even atypical. There is no
pathognomonic finding, which may make the early identification of these patients
challenging. This chapter describes the physical characteristics, medical complications, and
cognitive and psychological profiles that are associated with chromosomal
aneuploidy conditions, a group of conditions in which individuals are born with
one or more additional chromosome. Overall, chromosomal aneuploidy conditions
occur in approximately 1 in 250 children. Information regarding autosomal
disorders including trisomy 21 (Down syndrome), trisomy 13 (Patau syndrome), and
trisomy 18 (Edward syndrome) are presented. Sex chromosome aneuploidy conditions
such as Klinefelter syndrome (47,XXY), XYY, trisomy X, and Turner syndrome
(45,X), in addition to less frequently occurring tetrasomy and pentasomy
conditions are also covered. Treatment recommendations and suggestions for
future research directions are discussed. BACKGROUND: Human aneuploidy is the leading cause of early pregcy loss,
mental retardation, and multiple congenital anomalies. Due to the high mortality
associated with aneuploidy, the pathophysiological mechanisms of aneuploidy
syndrome remain largely unknown. Previous studies focused mostly on whether
dosage compensation occurs, and the next generation transcriptomics sequencing
technology RNA-seq is expected to eventually uncover the mechanisms of gene
expression regulation and the related pathological phenotypes in human
aneuploidy.
RESULTS: Using next generation transcriptomics sequencing technology RNA-seq, we
profiled the transcriptomes of four human aneuploid induced pluripotent stem
cell (iPSC) lines generated from monosomy × (Turner syndrome), trisomy 8
(Warkany syndrome 2), trisomy 13 (Patau syndrome), and partial trisomy 11:22
(Emanuel syndrome) as well as two umbilical cord matrix iPSC lines as euploid
controls to examine how phenotypic abnormalities develop with aberrant
karyotype. A total of 466 M (50-bp) reads were obtained from the six iPSC lines,
and over 13,000 mRNAs were identified by gene annotation. Global analysis of
gene expression profiles and functional analysis of differentially expressed
(DE) genes were implemented. Over 5000 DE genes are determined between
aneuploidy and euploid iPSCs respectively while 9 KEGG pathways are overlapped
enriched in four aneuploidy samples.
CONCLUSIONS: Our results demonstrate that the extra or missing chromosome has
extensive effects on the whole transcriptome. Functional analysis of
differentially expressed genes reveals that the genes most affected in aneuploid
individuals are related to central nervous system development and tumorigenesis. OBJECTIVES: Pregcies with Edwards or Patau syndrome are often detected
through screening for Down's syndrome. We aimed to evaluate the impact of
screening for Down's syndrome on the prevalence of live births and antenatal
diagnoses of Edwards and Patau syndrome.
SETTING: England and Wales, 2005 to 2012.
METHODS: Data from the National Down Syndrome Cytogenetic Register, which
contains information on nearly all ante- or postnatally diagnosed cases of
Edwards or Patau syndrome in which a karyotype was confirmed, were analysed.
RESULTS: From 2005 to 2012, 3,941 diagnoses of Edwards syndrome and 1,567
diagnoses of Patau syndrome were recorded (prevalence of 7.0 and 2.8 per 10,000
births respectively). Only 11% (95% confidence interval [CI]: 10-12) of
diagnoses of Edwards syndrome and 13% (95% CI: 11-14) of Patau syndrome were
live births, resulting in live birth prevalences of 0.8 (95% CI: 0.7-0.8) and
0.4 (95% CI: 0.3-0.4) per 10,000 live births respectively. About 90% of
pregcies with Edwards or Patau syndrome were diagnosed antenatally, and this
proportion remained constant over time. The proportion of diagnoses detected
before 15 weeks increased from 50% in 2005 to 53% in 2012 for Edwards syndrome,
and from 41% in 2005 to 63% in 2012 for Patau syndrome.
CONCLUSIONS: Almost 700 women per year had a pregcy with Edwards or Patau
syndrome. Over 90% of these pregcies were detected antenatally, with the
increased use of first trimester screening for Down's syndrome resulting in the
reduction in the mean gestational age at diagnosis of these syndromes. INTRODUCTION: Patau syndrome, trisomy 13, is the third commonest autosomal
trisomy. It is associated with a 25-50% prevalence of epilepsy, but detailed
electroclinical descriptions are rare. The occurrence of early-onset
photosensitivity has recently been reported in single patients.
MATERIALS/PATIENTS: We collected electroclinical data on 8 infants (age range
from 2 months to 3 years and 9 months, median: 17 months) with Patau syndrome
referred for an EEG in our Clinical Neurophysiology Department between 1991 and
2011.
METHODS: All EEGs, case-notes, cytogenetic diagnosis and neuroimaging when
available were reviewed; data on the occurrence of seizures, epileptiform
discharges, photoparoxysmal response and their characteristics in terms of
positive frequencies, latencies, grade and duration were noted and analysed.
RESULTS: Two patients had been previously diagnosed with epilepsy (one with
tonic spasms and one with multiple seizure types). We found 3 patients with
photosensitive myoclonic epilepsy (37.5%), and one with non-photosensitive
myoclonic epilepsy. We also recorded non-epileptic myoclonic jerks in one
patient known to suffer from epileptic spasms. Among photosensitive patients we
found self-limited, Waltz's grade 2-4, spike-wave/polyspike-wave discharges in
low, medium and high frequency ranges in two patients and in the high frequency
range in the third patient, with latencies and duration from less than 1s to a
maximum of 9s.
CONCLUSIONS: In our cohort of Patau syndrome patients, we found a high
prevalence of spasms and photic-induced myoclonic jerks. Photosensitivity shows
an unusual early age of onset. Congenital diaphragmatic hernia (CDH) occurs in 5-10% associated with
chromosomal abnormalities like, Pallister Killian syndrome, Trisomy 18, and
certain deletions.. Association of CDH with trisomy 13 (Patau syndromes) is very
rare. Here, we report such an unusual association, where surgical repair was
done, but eventually the case succumbed as a result of multiple fatal
co-morbidities. |
Which is the main abnormality that arises with Sox9 locus duplication? | The 46,XX testicular disorder of sex development (DSD), also known as 46,XX male syndrome, is a rare form of DSD and clinical phenotype shows complete sex reversal from female to male. A complex network of genes determines sex in mammals. Differentiation of testicular tissue in 46,XX individuals is seen either in XX males, the majority of them with SRY gene, or in individuals, usually SRY(-), with ovotesticular disorder of sex development (OT-DSD). SOX9 is one of the genes that play critical roles in male sexual differentiation. | In humans, testis development depends on a regulated genetic hierarchy initiated
by the Y-linked SRY gene. Failure of testicular determination results in the
condition termed 46,XY gonadal dysgenesis (GD). Several components of the testis
determining pathway have recently been identified though it has been difficult
to articulate a cascade with the known elements of the system. It seems,
however, that early gonadal development is the result of a network of
interactions instead of the outcome of a linear cascade. Accumulating evidence
shows that testis formation in man is sensitive to gene dosage.
Haploinsufficiency of SF1, WT1 and SOX9 is responsible for 46,XY gonadal
dysgenesis. Besides, data on SRY is consistent with possible dosage anomalies in
certain cases of male to female sex reversal. 46,XY GD due to monosomy of distal
9p and 10q might also be associated with an insufficient gene dosage effect.
Duplications of the locus DSS can lead to a failure of testicular development
and a duplication of the region containing SOX9 has been implicated in XX sex
reversal. Transgenic studies in mouse have shown, however, that this mammal is
less sensitive to gene dosage than man. Here, we will try to put in place the
known pieces of the jigsaw puzzle that is sex determination in mammals, as far
as current knowledge obtained from man and animal models allows. We are certain
that from this attempt more questions than answers will arise. It is well established that testicular differentiation of the human embryonic
gonad depends on the action of the Y-chromosomal gene SRY. However, exceptional
cases such as SRY-negative cases of 46,XX testicular disorder of sexual
development (DSD), and of 46,XX ovotesticular DSD document that testicular
tissue can develop in the absence of the SRY gene. These SRY-negative XX sex
reversal cases are very rare and usually sporadic, but a few familial cases have
been reported. We present a large, consanguineous family with nine affected
individuals with phenotypes ranging from 46,XX testicular DSD to 46,XX
ovotesticular DSD, with predomice of male characteristics. Absence of SRY in
peripheral blood was documented by fluorescence in situ hybridization (FISH) and
PCR analysis in all nine affected individuals, and by FISH analysis on gonadal
sections with testicular tissue in four affected individuals. By quantitative
PCR, a duplication of the SOX9 gene was excluded. In addition, as linkage
analysis showed that the nine affected members of the family do not share a
common SOX9 haplotype, any mutation at the SOX9 locus could be ruled out.
Together, these findings implicate a mutation at a sex-determining locus other
than SRY and SOX9 as the cause for the XX sex reversal trait in this family. Differentiation of the bipotential gonad into testis is initiated by the Y
chromosome-linked gene SRY (Sex-determining Region Y) through upregulation of
its autosomal direct target gene SOX9 (Sry-related HMG box-containing gene 9).
Sequence and chromosome homology studies have shown that SRY most probably
evolved from SOX3, which in humans is located at Xq27.1. Mutations causing SOX3
loss-of-function do not affect the sex determination in mice or humans. However,
transgenic mouse studies have shown that ectopic expression of Sox3 in the
bipotential gonad results in upregulation of Sox9, resulting in testicular
induction and XX male sex reversal. However, the mechanism by which these
rearrangements cause sex reversal and the frequency with which they are
associated with disorders of sex development remains unclear. Rearrangements of
the SOX3 locus were identified recently in three cases of human XX male sex
reversal. We report on a case of XX male sex reversal associated with a novel de
novo duplication of the SOX3 gene. These data provide additional evidence that
SOX3 gain-of-function in the XX bipotential gonad causes XX male sex reversal
and further support the hypothesis that SOX3 is the evolutionary antecedent of
SRY. A complex network of genes determines sex in mammals. Here, we studied a
European roe deer with an intersex phenotype that was consistent with a XY
genotype with incomplete male-determination. Whole genome sequencing and
quantitative real-time PCR analyses revealed a triple dose of the SOX9 gene,
allowing insights into a new genetic defect in a wild animal. The 46,XX testicular disorder of sex development (DSD), also known as 46,XX male
syndrome, is a rare form of DSD and clinical phenotype shows complete sex
reversal from female to male. The sex-determining region Y (SRY) gene can be
identified in most 46,XX testicular DSD patients; however, approximately 20% of
patients with 46,XX testicular DSD are SRY-negative. The SRY-box 9 (SOX9) gene
has several important functions during testis development and differentiation in
males, and overexpression of SOX9 leads to the male development of 46,XX gonads
in the absence of SRY. In addition, SOX9 duplication has been found to be a rare
cause of 46,XX testicular DSD in humans. Here, we report a 4.2-year-old
SRY-negative 46,XX boy with complete sex reversal caused by SOX9 duplication for
the first time in Korea. He showed normal external and internal male genitalia
except for small testes. Fluorescence in situ hybridization and polymerase chain
reaction (PCR) analyses failed to detect the presence of SRY, and SOX9
intragenic mutation was not identified by direct sequencing analysis. Therefore,
we performed real-time PCR analyses with specific primer pairs, and duplication
of the SOX9 gene was revealed. Although SRY-negative 46,XX testicular DSD is a
rare condition, an effort to make an accurate diagnosis is important for the
provision of proper genetic counseling and for guiding patients in their
long-term management. |
Is Musclin a secretory peptide? | Yes, musclin has been described as a muscle-derived secretory peptide. | Musclin has been described as a muscle-derived secretory peptide, responsive to
insulin in vivo, and inducing insulin resistance in vitro. Because muscle fibers
display very different metabolic properties and insulin sensitivity, we tested
the hypothesis that musclin expression could depend on myofiber type. Musclin
mRNA was detected at high level in fast gastrocnemius and plantaris muscles, but
only as traces in soleus, a slow-twitch muscle. A single fiber analysis showed
that musclin was produced by muscle fibers themselves, almost exclusively type
IIb fibers. Slow to fast transition of soleus phenotype after hindlimb
suspension increased musclin mRNA levels, whereas fast to slow transition of
plantaris phenotype after functional overload decreased musclin mRNA levels.
This clearly suggests that musclin transcription is strongly related to
fast-glycolytic phenotype. We conclude that musclin is produced by myocytes in a
highly fiber-type specific manner and that physiological changes in type IIb MHC
lead to coordinated musclin expression. Musclin is a novel skeletal muscle-derived secretory factor found in the signal
sequence trap of mouse skeletal muscle cDNAs. Musclin possesses a region
homologous to the natriuretic peptide family. Thus, musclin is found to bind
with the natriuretic peptide clearance receptors. However, the role of musclin
in vascular regulation remains unclear. In this study, we aim to investigate the
direct effect of musclin on vascular tone and to analyze its role in
hypertension using the spontaneously hypertensive rats (SHR). In aortic strips
isolated from SHR, musclin induced contractions in a dose-dependent manner. We
found that the musclin-induced vasoconstriction was more marked in SHR than in
normal rats (WKY). Moreover, this contraction was reduced by blockade of
natriuretic peptide receptor C using the ab14355 antibody. Therefore, mediation
of the natriuretic peptide receptor in musclin-induced vasoconstriction can be
considered. In addition, similar to the natriuretic peptide receptor, expression
of the musclin gene in blood vessels was higher in SHR than in WKY. Injection of
musclin markedly increased the blood pressure in rats that can be inhibited by
anti-musclin antibodies. Musclin-induced vasoconstriction was more pronounced in
SHR than in WKY as in its expression. Taken together, these results suggest that
musclin is involved in blood pressure regulation. The higher expression of
musclin in hypertension indicates that musclin could be used as a new target for
the treatment of hypertension in the future. |
What tissue is commonly affected in Marfan's syndrome | Marfan syndrome (MS) is a connective tissue disorder that affects thousands of adolescents | BACKGROUND: Marfan's syndrome is an inherited disorder of connective tissue
associated with characteristic abnormalities of the skeletal, ocular, and
cardiovascular systems. Marked clinical variability and age dependency of all
manifestations of Marfan's syndrome may render the unequivocal diagnosis
difficult in mildly affected, young subjects.
HYPOTHESIS: The study and care of a 32-year-old woman with evidence of Marfan's
syndrome, several cardiac abnormalities, and paranoid schizophrenia led to an
investigation of her consenting relatives to verify the penetrance of Marfan's
syndrome and the degree of comorbidity between the disease and psychiatric
disorders.
METHODS: The patient and 12 subjects belonging to three generations of her
family underwent cardiovascular, skeletal, ophthalmologic, and psychiatric
examinations. Two-dimensional and Doppler echocardiography were performed.
RESULTS: One female index patient and six of her first-degree relatives were
found to be affected by Marfan's syndrome. All seven patients were found to have
mitral valve prolapse associated with other cardiac abnormalities. Four of these
patients were affected by the following psychiatric disorders: generalized
anxiety disorder, major depressive disorder, paranoid schizophrenia (two cases).
Six more relatives without Marfan's syndrome showed mitral valve prolapse in
association with other echocardiographic features. Two of these were found to be
affected by a major depressive disorder.
CONCLUSIONS: The present data support the hypothesis that a psychiatric
condition, associated with a significantly high frequency of cardiac
involvement, may be part of the phenotype of Marfan's syndrome. OBJECTIVE: Marfan's syndrome is a heritable connective tissue disorder that has
been associated with intracranial aneurysms. However, the prevalence of
intracranial aneurysms in Marfan's syndrome is unknown and pathological studies
of affected vessels have not been reported. We therefore examined the
neuropathological findings in a group of patients with Marfan's syndrome.
METHODS: We identified all patients with Marfan's syndrome in whom postmortem
examination had been performed at the Mayo Clinic between 1969 and 1993.
RESULTS: Autopsy included examination of the brain in seven patients with
Marfan's syndrome (five men and two women with a mean age of 28 yr). Each of two
patients had one or more intracranial aneurysms. The first patient, a
32-year-old man who died as a result of aortic dissection, was observed to have
an incidental saccular supraclinoid carotid artery aneurysm (7 mm). Microscopic
examination of the remainder of the cerebral arteries revealed duplication and
fragmentation of the internal elastic lamina. The second patient, a 20-year-old
man who died as a result of a subarachnoid hemorrhage, had ruptured saccular
supraclinoid carotid artery (3 mm) and anterior cerebral artery (20 mm)
aneurysms as well as unruptured fusiform middle cerebral artery (18 mm) and
posterior cerebral artery (13 mm) aneurysms. Microscopic examination of the
cerebral arteries revealed widespread changes consisting of intimal
proliferation, medial degeneration, and fragmentation of the internal elastic
lamina.
CONCLUSION: These findings confirm an association between Marfan's syndrome and
intracranial aneurysms. Microscopic involvement of cerebral arteries in Marfan's
syndrome may be variable, even among those with intracranial aneurysms. Marfan's syndrome is an autosomal domit disorder of connective tissue,
commonly involving the cardiovascular, ocular, and skeletal systems. Revised
criteria for the clinical diagnosis of Marfan's syndrome regard skeletal
involvement as a major criterion if at least four of eight typical skeletal
manifestations are present, one of which is protrusio acetabuli. Using Kulman's
method to determine the presence of protrusio, we analysed the pelvic X-rays of
15 patients with Marfan's syndrome and 15 controls. Protrusio was present in 47%
(7/15) of Marfan patients, compared with 7% (1/15) of controls (P = 0.035).
Using the revised criteria, the presence of protrusio would have affected the
final diagnosis of Marfan's syndrome in only one patient out of 15. Therefore,
we recommend that a pelvic X-ray is reserved for those cases in which the
presence of protrusio will alter the final diagnosis. With regard to the
radiological assessment of protrusio, in our opinion this can be performed
simply and reliably using the position of the acetabular line alone. A patient with the Marfan syndrome died suddenly from aortic rupture and
dissection in the early puerperium of her second pregcy. Although the
association of the Marfan syndrome and pregcy is extremely rare, the case
reported here being only the fifth on record, the concurrence of dissecting
aneurysm or aortic dissection with pregcy is more frequent. Furthermore it is
accepted that aortic dissection in young women below the age of 40 is more
common in the pregt than those not pregt. The cause of the enhancing
effect of pregcy is unknown but is thought to be endocrine since the
stability of connective tissue can be influenced by hormones, particularly the
sex steroids. An unusual feature of the present case is the florid inflammatory
reaction in the adventitia of the aorta, not specifically related to pregcy
or to the Marfan syndrome, and it is assumed that in this patient the congenital
defect of connective tissue assumed to be the basis of the Marfan syndrome is
associated with an acute collagen change or necrosis, possibly illustrating a
link between the heritable disorders of connective tissue and the diffuse
collagen disease. Marfans syndrome is an Autosomal domit disorder of the connective tissues
resulting in abnormalities of the musculoskeletal system, cardiovascular system
and eyes. It has a prevalence of 1 in 100,000 population1 and occurs in all
ethnic groups. It may be familial or due to new mutation (30%), in the fibrillin
gene on arm of chromosome 15. It is estimated that one person in every 3000-5000
has Marfans syndrome may have cardiovascular abnormalities and may be
complicated by infective endocartditis. About 90% of Marfan patients will
develop cardiac complications2. The patient under discussion has musculoskeletal
(Tall stature, reduced upper-lower segment ratio, arm-span to height ratio >
1.05, high arched palate) and Cardiovascular features (Severe aortic
regurgitation complicated with infective endocarditis). Marfan syndrome is an inherited multisystemic connective-tissue disease that is
caused by a mutation of the fibrillin-1 gene. The syndrome is characterized by a
wide range of clinical manifestations. Common cardiovascular manifestations,
most of which are substantial contributors to mortality, include annuloaortic
ectasia with or without aortic valve insufficiency, aortic dissection, aortic
aneurysm, pulmonary artery dilatation, and mitral valve prolapse. Scoliosis,
pectus excavatum and carinatum, arachnodactyly, and acetabular protrusion are
common musculoskeletal manifestations. Dural ectasia is a characteristic central
nervous system manifestation. In some patients with Marfan syndrome, there is
also pulmonary and ocular involvement. Early identification and treatment of
these conditions contribute to an improved quality of life and a life expectancy
close to the average for the general population in the United States.
Radiologists play a key role in the diagnosis of Marfan syndrome. Knowledge
about the various manifestations of Marfan syndrome and awareness of their
radiologic appearances permit a comprehensive diagnostic approach that allows
better patient care. The Marfan syndrome is an inherited disorder of the connective tissue which is
mainly caused by a mutation in the fibrillin-1 gene. The defect in the
connective tissue protein can lead to several organ dysfunctions. For the life
expectancy, the cardiovascular aspect is of paramount importance. Patients with
Marfan syndrome may develop aortic aneurysms and valvular heart defects. The
risk of aortic aneurysms consists in the development of aortic dissection or
rupture with their fatal consequences. Besides the cardiovascular manifestation,
the skeletal and ocular system can also be affected. The skeletal manifestation
is often characterised by long limbs, arachnodactyly, and abnormal joint
flexibility along with other signs. Patients may also have dislocated lenses,
ectasia of the dural sac, stretch marks, spontaneous pneumothorax, recurrent
hernia, or a family history suspicious for Marfan. During the past years, other
related connective tissue disorders with analogous organ manifestation have been
described (e.g., Loeys-Dietz syndrome). In this article we present the basic
knowledge about these connective tissue disorders, and we mention new insights
in the recently explored pathophysiology of the disorder which is a possible
target for future medical treatment options. Furthermore, recent new concepts
for the prophylactic treatment of the aortic manifestation are explained. Marfan's syndrome is an autosomal domit condition with an estimated
prevalence of one in 10,000 to 20,000 individuals. This rare hereditary
connective tissue disorder affects many parts of the body. The diagnosis of
Marfan's syndrome is established in accordance with a review of the diagnostic
criteria, known as the Ghent nosology, through a comprehensive assessment
largely based on a combination of major and minor clinical manifestations in
various organ systems and the family history. Aortic root dilation and mitral
valve prolapse are the main presentations among the cardiovascular malformations
of Marfan's syndrome. The pathogenesis of Marfan's syndrome has not been fully
elucidated. However, fibrillin-1 gene mutations are believed to exert a domit
negative effect. Therefore, Marfan's syndrome is termed a fibrillinopathy, along
with other connective tissue disorders with subtle differences in clinical
manifestations. The treatment may include prophylactic β-blockers and
angiotensin II-receptor blockers in order to slow down the dilation of the
ascending aorta, and prophylactic aortic surgery. Importantly, β-blocker therapy
may reduce TGF-β activation, which has been recognized as a contributory factor
in Marfan's syndrome. The present article aims to provide an overview of this
rare hereditary disorder. Marfan syndrome (MS) is a connective tissue disorder that affects thousands of
adolescents [Population Reference Bureau, 2013]. Some adolescent patients with
MS may use social media to express their experiences and emotions, but little is
known about what patients choose to share online. To investigate social media
content related to Marfan syndrome we used search terms "Marfan syndrome" and
"Marfans" on six different social media sites. The top five recent and popular
posts for each site were collected and coded weekly for five weeks. Posts were
excluded if they were reshared content or not in English. A codebook was
developed using an iterative process to categorize posts and comments. Out of
300 posts collected 147 posts (49.0%) were included for evaluation. Categories
of displayed content included personal pictures, memes and pictures featuring
symptoms of MS (41.5%) and personal MS experiences (27.1% of posts). One quarter
of the posts specifically mentioned a positive experience or how thankful the
profile owner was for their life. A unique category of posts (13.7%) referenced
Austin Carlile, a celebrity singer with MS, as a role model. Physicians and
healthcare providers may consider using social media to understand common MS
concerns and to place future health education materials. Fibrillins are the major components of microfibrils in the extracellular matrix
of elastic and non-elastic tissues. They are multi-domain proteins, containing
primarily calcium binding epidermal growth factor-like (cbEGF) domains and
8-cysteine/transforming growth factor-beta binding protein-like (TB) domains.
Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective
tissue disorder with clinical complications in the cardiovascular, skeletal,
ocular and other organ systems. Here, we review the consequences of engineered
Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal
levels. Representative point mutations associated with Marfan syndrome in
affected individuals have been introduced and analyzed in recombit
fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and
functional level. Mutations which impair folding of cbEGF domains can affect
protein trafficking. Protein folding disrupted by some mutations can lead to
defective secretion in mutant fibrillin-1 fragments, whereas fragments with
other Marfan mutations are secreted normally. Many Marfan mutations render
fibrillin-1 more susceptible to proteolysis. There is also evidence that some
mutations affect heparin binding. Few mutations have been further analyzed in
mouse models. An extensively studied mouse model of Marfan syndrome expresses
mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar
characteristics to human patients with Marfan syndrome. Overall, the analyses of
engineered mutations leading to Marfan syndrome provide important insights into
the pathogenic molecular mechanisms exerted by mutated fibrillin-1. Marfan syndrome (MFS) is an autosomal domit disorder of connective tissue,
caused by mutations of the microfibrillar protein fibrillin-1, that predisposes
affected individuals to aortic aneurysm and rupture and is associated with
increased TGFβ signaling. TGFβ is secreted from cells as a latent complex
consisting of TGFβ, the TGFβ propeptide, and a molecule of latent TGFβ binding
protein (LTBP). Improper extracellular localization of the latent complex can
alter active TGFβ levels, and has been hypothesized as an explanation for
enhanced TGFβ signaling observed in MFS. We previously reported the absence of
LTBP-3 in matrices lacking fibrillin-1, suggesting that perturbed TGFβ signaling
in MFS might be due to defective interaction of latent TGFβ complexes containing
LTBP-3 with mutant fibrillin-1 microfibrils. To test this hypothesis, we
genetically suppressed Ltbp3 expression in a mouse model of progressively severe
MFS. Here, we present evidence that MFS mice lacking LTBP-3 have improved
survival, essentially no aneurysms, reduced disruption and fragmentation of
medial elastic fibers, and decreased Smad2/3 and Erk1/2 activation in their
aortas. These data suggest that, in MFS, improper localization of latent TGFβ
complexes composed of LTBP-3 and TGFβ contributes to aortic disease progression. |
To which disease does the loss of CD28 expression by liver-infiltrating T cells contribute? | Loss of CD28 expression by liver-infiltrating T cells contributes to pathogenesis of primary sclerosing cholangitis. | BACKGROUND & AIMS: T-cell-mediated biliary injury is a feature of primary
sclerosing cholangitis (PSC). We studied the roles of CD28(-) T cells in PSC and
their regulation by vitamin D.
METHODS: Peripheral and liver-infiltrating mononuclear cells were isolated from
blood or fresh liver tissue. We analyzed numbers, phenotypes, functions, and
localization patterns of CD28(-) T cells, along with their ability to activate
biliary epithelial cells. We measured levels of tumor necrosis factor (TNF)α in
liver tissues from patients with PSC and the effects of exposure to active
vitamin D (1,25[OH]2D3) on expression of CD28.
RESULTS: A significantly greater proportion of CD4(+) and CD8(+) T cells that
infiltrated liver tissues of patients with PSC were CD28(-), compared with
control liver tissue (CD4(+): 30.3% vs 2.5%, P < .0001; and CD8(+): 68.5% vs
31.9%, P < .05). The mean percentage of CD4(+)CD28(-) T cells in liver tissues
from patients with PSC was significantly higher than from patients with primary
biliary cirrhosis or nonalcoholic steatohepatitis (P < .05). CD28(-) T cells
were activated CD69(+)CD45RA(-) C-C chemokine receptor (CCR)7(-) effector memory
and perforin(+) granzyme B(+) cytotoxic cells, which express CD11a, CX3CR1,
C-X3-C motif receptor 6 (CXCR6), and CCR10-consistent with their infiltration of
liver and localization around bile ducts. Compared with CD28(+) T cells,
activated CD28(-) T cells produced significantly higher levels of interferon γ
and TNFα (P < .05), and induced up-regulation of intercellular cell adhesion
molecule-1, HLA-DR, and CD40 by primary epithelial cells (3.6-fold, 1.5-fold,
and 1.2-fold, respectively). Liver tissue from patients with PSC contained high
levels of TNFα; TNFα down-regulated the expression of CD28 by T cells in vitro
(P < .01); this effect was prevented by administration of 1,25(OH)2D3 (P < .05).
CONCLUSIONS: Inflammatory CD28(-) T cells accumulate in livers of patients with
PSC and localize around bile ducts. The TNFα-rich microenvironment of this
tissue promotes inflammation; these effects are reversed by vitamin D in vitro. |
Are cutaneous porphyrias inherited with a recessive pattern? | No, cutaneous porphyrias are inherited in a dominant (not recessive) pattern. | The acute porphyrias constitute a group of metabolic disorders engaging enzymes
in the haem synthetic chain and generally following domit inheritance
patterns. Some gene carriers are vulnerable to a range of exogenous and
endogenous factors, which may trigger neuropsychiatric symptoms. Early diagnosis
is of prime importance since it makes way for counselling with the aim to block
the development of acute, as well as late, disease. The medical and
psycho-social consequences of a porphyria diagnosis are considerable and the
freedom for maldiagnosis correspondingly small. The strain imposed upon the
diagnostic process makes management in specialized laboratories necessary.
Inadvertent handling of the diagnostic procedures in laboratories lacking in
knowledge, experience and technical competence is repeatedly the reason for
harmful underdiagnosis and overdiagnosis. Gene diagnosis of the carrier
condition, principally within reach in all types of acute porphyria, is of
incomparable versatility and accuracy. However, despite recent great
achievements in the molecular biology of porphyric disease, genomic procedures
cannot replace biochemical methods in monitoring the activity and progress of
the disease, or the effects of therapy. The classical methods are also useful
when it comes to screening for the associated disease states. In these tasks,
professional handling of the methods and skillful interpretation of the results
are of paramount importance. Knowledge of the limitations and pitfalls of the
procedures is a guard against maldiagnosis, which may be fatal. In the article
the main diagnostic challenges are discussed; the strategy for early detection
of the gene carrier state, the recognition and surveillance of the acute
porphyric crisis, the evaluation of subacute/subchronic symptoms, the
differential diagnoses of the cutaneous porphyrias and the monitoring of late
complications. Variegate porphyria (VP) is an autosomal-domit disorder that is caused by
inheritance of a partial deficiency of the enzyme protoporphyrinogen oxidase (EC
1.3.3.4). It is characterized by cutaneous photosensitivity and/or various
neurological manifestations. Protoporphyrinogen oxidase catalyses the
penultimate step of haem biosynthesis, and mutations in the PPOX gene have been
coupled to VP. In the present study, sequencing analysis revealed 10 different
mutations in the PPOX gene in 14 out of 17 apparently unrelated Swedish VP
families. Six of the identified mutations, 3G > A (exon 2), 454C > T (exon 5),
472G > C (exon 6), 614C > T (exon 6), 988G > C (exon 10) and IVS12 + 2T > G
(intron 12), are single nucleotide substitutions, while 604delC (exon 6),
916-17delCT (exon 9) and 1330-31delCT (exon 13) are small deletions, and IVS12 +
2-3insT (intron 12) is a small insertion. Only one of these 10 mutations has
been reported previously. Three of the mutations were each identified in two or
more families, while the remaining mutations were specific for an individual
family. In addition to the 10 mutations, one previously unreported single
nucleotide polymorphism was identified. Mutation analysis of family members
revealed two adults and four children who were silent carriers of the VP trait.
Genetic analysis can now be added to the conventional biochemical analyses and
used in investigation of putative carriers of a VP trait in these families. Partial deficiency of enzymes in the haem synthetic pathway gives rise to a
group of seven inherited metabolic disorders, the porphyrias. Each deficiency is
associated with a characteristic increase in haem precursors that correlates
with the symptoms associated with individual porphyrias and allows accurate
diagnosis. Two types of clinical presentation occur separately or in
combination; acute life-threatening neurovisceral attacks and/or cutaneous
symptoms. Five of the porphyrias are low-penetrance autosomal domit
conditions in which clinical expression results from additional factors that act
by increasing demand for haem or by causing an additional decrease in enzyme
activity or by a combination of these effects. These include both genetic and
environmental factors. In familial porphyria cutanea tarda (PCTF), environmental
factors that include alcohol, exogenous oestrogens and hepatotropic viruses
result in inhibition of hepatic enzyme activity via a mechanism that involves
excess iron accumulation. In erythropoietic protoporphyria (EPP), co-inheritance
of a functional polymorphism in trans to a null ferrochelatase allele accounts
for most clinically overt cases. In the autosomal domit acute hepatic
porphyrias (acute intermittent porphyria, variegate porphyria, hereditary
coproporphyria), acute neurovisceral attacks occur in a minority of those who
inherit one of these disorders. Although various exogenous (e.g. drugs, alcohol)
and endogenous factors (e.g. hormones) have been identified as provoking acute
attacks, these do not provide a full explanation for the low penetrance of these
disorders. It seems probable that genetic background influences susceptibility
to acute attacks, but the genes that are involved have not yet been identified. |
Which disease is treated with ZMapp? | ZMapp is a combination of antibodies for treatment of Ebola virus disease. | Author information:
(1)National Laboratory for Zoonotic Diseases and Special Pathogens, Public
Health Agency of Canada, Winnipeg, Manitoba R3E 3R2, Canada.
(2)1] National Laboratory for Zoonotic Diseases and Special Pathogens, Public
Health Agency of Canada, Winnipeg, Manitoba R3E 3R2, Canada [2] Department of
Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 0J9,
Canada.
(3)1] National Laboratory for Zoonotic Diseases and Special Pathogens, Public
Health Agency of Canada, Winnipeg, Manitoba R3E 3R2, Canada [2] Institute of
Infectious Disease, He Centre for Disease Control and Prevention, Zhengzhou,
450012 He, China.
(4)Kentucky BioProcessing, Owensboro, Kentucky 42301, USA.
(5)Mapp Biopharmaceutical Inc., San Diego, California 92121, USA.
(6)1] United States Army Medical Research Institute of Infectious Diseases
(USAMRIID), Frederick, Maryland 21702, USA [2] Integrated Research Facility,
National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Frederick, Maryland 21702, USA.
(7)Institute of Infectious Disease, He Centre for Disease Control and
Prevention, Zhengzhou, 450012 He, China.
(8)1] National Laboratory for Zoonotic Diseases and Special Pathogens, Public
Health Agency of Canada, Winnipeg, Manitoba R3E 3R2, Canada [2] Department of
Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada
[3] Department of Pediatrics and Child Health, University of Manitoba, Winnipeg,
Manitoba R3A 1S1, Canada.
(9)1] National Laboratory for Zoonotic Diseases and Special Pathogens, Public
Health Agency of Canada, Winnipeg, Manitoba R3E 3R2, Canada [2] Department of
Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada
[3] Department of Immunology, University of Manitoba, Winnipeg, Manitoba R3E
0T5, Canada [4] Department of Pathology and Laboratory Medicine, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. The reports for Ebola virus Zaire (EBOV), Nipah virus, and Machupo virus (MACV)
pathogenesis, in this issue of Veterinary Pathology, are timely considering
recent events, both nationally and internationally. EBOV, Nipah virus, and MACV
cause highly lethal infections for which no Food and Drug Administration (FDA)
licensed vaccines or therapies exist. Not only are there concerns that these
agents could be used by those with malicious intent, but shifts in ecological
distribution of viral reservoirs due to climate change or globalization could
lead to more frequent infections within remote regions than previously seen as
well as outbreaks in more populous areas. The current EBOV epidemic shows no
sign of abating across 3 West African nations (as of October 2014), including
densely populated areas, far outpacing infection rates of previous outbreaks. A
limited number of cases have also arisen in the United States and Europe. With
few treatment options for these deadly viruses, development of animal models
reflective of human disease is paramount to combat these diseases. As an example
of this potential, a new treatment compound, ZMapp, that had demonstrated
efficacy against EBOV infection in nonhuman primates (NHPs) received an
emergency compassionate use exception from the FDA for the treatment of 2
American medical workers infected with EBOV, and they are currently virus free
and recovering. This paper examines how people in West Africa are reacting to the Ebola virus
disease, an epidemic presently prevalent in the region. Certain lifestyle
changes are suggested. Additionally, the heart of the paper focuses on the
request by governments to be allowed access to experimental drugs, such as Zmapp
and TKM-Ebola, for their infected populations. The author argues that granting
such a request would circumvent research ethics procedures, which could
potentially constitute significant risk to users of the drugs. The Pfizer Kano
meningitis trial of 1996 is cited as an example to buttress how unapproved drugs
could prove fatal. Plant-made or "biofarmed" viral vaccines are some of the earliest products of
the technology of plant molecular farming, and remain some of the brightest
prospects for the success of this field. Proofs of principle and of efficacy
exist for many candidate viral veterinary vaccines; the use of plant-made viral
antigens and of monoclonal antibodies for therapy of animal and even human viral
disease is also well established. This review explores some of the more
prominent recent advances in the biofarming of viral vaccines and therapies,
including the recent use of ZMapp for Ebolavirus infection, and explores some
possible future applications of the technology. Since the first case of Ebola virus disease (EVD) in Guinea was reported in
March 2014 by World Health Organization (WHO), the outbreak has continued
through the year and the total number of 19,065 patients was reported as the
confirmed or suspected in the EVD-affected countries. Among the cases, 7,388
patients were reported death by 19 December. Currently, available therapeutics
to treat the infected patients or vaccines to prevent people from infection is
not developed yet while viral diagnostic methods were already developed and
firmly established in a lot of countries as a first step for the preparedness of
Ebola outbreak. Some potential therapeutic materials including ZMapp were
supplied and the treated people got over the EVD. Several candidates of vaccines
also were investigated their efficacy in animal models by National Institute of
Health (NIH) and Department of Defense, and they are processing of clinical
tests in West Africa aiming to finish the development by the 2015. Vaccine and
therapeutic development is essential to stop the EVD outbreak in West Africa,
also to protect the world from the risk which can be generated by potential
spread of Ebola virus. The 2014 outbreak of Ebola virus disease (EVD) in West Africa, caused by Ebola
virus (Zaire Ebola virus species), is the largest outbreak of EVD in history. It
cause hemorrhagic fever in human and nonhuman primates with high mortality rate
up to 90% and can be transmitted by direct contact with blood, body fluids, skin
of EVD patients or persons who have died of EVD. As of December 17, 2014, 450
healthcare personnel are known to have been infected with Ebola, of whom 244
died. For development of Ebola vaccine and treatment are highly difficult due to
its dangerous and accessibility that requires biosafety level 4 (BSL-4) to
conduct experiment. Also there is no specific vaccine and treatment for Ebola
virus; however, many candidate vaccines and antiviral-drugs such as ZMapp and
TKM-Ebola are being developed for Ebola virus disease. In this review, we focus
on the epidemiology of 2014 outbreak of Ebola virus and candidate agent for
preventing and curing from Ebola virus. The 2014 outbreak clearly showed that Ebola viruses (EBOV) remain a substantial
threat for public health. The mainstay of management of patients with Ebola
disease is isolation of patients and use of strict barrier nursing procedures;
the present treatment strategies are mainly symptomatic and supportive (fluid
resuscitation, antypyretics, antidiarrheal drugs). Currently, there is no
approved therapy for Ebola hemorrhagic fever (EHF), however several advanced
treatment options were tested in animal models (on non-human primates or
rodents). They include use of both symptomatic (e.g. use of tissue factor
inhibitors - rhNAPc2, rhAPC - to abolish coagulopathy) and specific antiviral
approaches: e.g. monoclonal anti EBOV antibodies (ZMapp, MB-003),
phosphorodiamidate morpholino oligomers (PMOs), liposomes containing siRNA
(LNP-siRNA:TKM-Ebola) and small molecule inhibitors (e.g. BCX4430, favipiravir).
The scope of this article is to briefly review the most promising therapeutics
for EHF, based on the data coming from rare clinical reports, studies on animals
and results from in vitro models. Publisher: TITLE: Enfermedad por el virus del Ebola: epidemiologia y
manifestaciones clinicas en un contexto de emergencia de salud publica
internacional.
Introduccion. La epidemia causada por el virus del Ebola en Africa occidental
afecta a Guinea, Liberia, Sierra Leona, Nigeria, Mali y Senegal, y es la mas
grave desde que se tiene noticia de este filovirus causante de fiebre
hemorragica. En este articulo se revisan las caracteristicas epidemiologicas y
las manifestaciones clinicas asociadas a la enfermedad por el virus del Ebola.
Desarrollo. Hasta el 23 de febrero de 2015 se habian contabilizado 23.729 casos
de ebola, con un 40,1% de mortalidad. En la actual epidemia, el virus se
transmite al ser humano por tres vias: contacto con fluidos y secreciones de
sujetos enfermos ya diagnosticados, contacto con cadaveres durante las
ceremonias de enterramiento, y contagio a familiares y personal sanitario por
enfermos sin diagnosticar. El Ebola causa una enfermedad grave en humanos. Tras
un periodo de incubacion variable (2-21 dias), se inicia un sindrome febril,
cefalea, mialgias, artralgias, vomitos y diarrea. La fase avanzada cursa con
hemorragias, fracaso de multiples organos, hipotension y choque. Se desconoce la
incidencia de manifestaciones neurologicas, aunque se han descrito hemorragias
cerebrales y sindromes postinfecciosos en otras fiebres hemorragicas virales.
Los cuidados de soporte son vitales. No existe un tratamiento efectivo
demostrado, aunque varios pacientes han sido tratados con un coctel de
anticuerpos monoclonales (ZMapp). Conclusiones. La identificacion y diagnostico
precoz de casos sospechosos, el aislamiento de sujetos enfermos y las medidas de
proteccion en el personal sanitario son fundamentales para contener esta
epidemia. The 2014-2015 outbreak of Ebola virus disease is the largest epidemic to date in
terms of the number of cases, deaths, and affected areas. In October 2015, no
antiviral agents had proven antiviral efficacy in patients. However, in
September 2014, the World Health Organization inventoried and has since
regularly updated a list of potential drug candidates with demonstrated
antiviral efficacy in in vitro or animal models. This includes agents belonging
to various therapeutic classes, namely direct antiviral agents (favipiravir and
BCX4430), a combination of antibodies (ZMapp), type I interferons, RNA
interference-based drugs (TKM-Ebola and AVI-7537), and anticoagulant drugs
(rNAPc2). Here, we review the pharmacokinetic and pharmacodynamic information
presently available for these drugs, using data obtained in healthy volunteers
for pharmacokinetics and data obtained in human clinical trials or animal models
for pharmacodynamics. Future studies evaluating these drugs in clinical trials
are critical to confirm their efficacy in humans, propose appropriate doses, and
evaluate the possibility of treatment combinations. The Ebola virus disease (EVD), formerly known as a hemorrhagic fever and
discovered in 1976, is dangerous, highly infectious disease with very high
mortality. There are no licensed therapeutics against EVD, although a range of
medicines and therapies are currently being evaluated. During the 2014 Ebola
outbreak, an experimental drug named ZMapp was administered on an emergency
basis to seven patients of which five were recovered. Currently, since February
2015, ZMapp is tested in clinical trials. ZMapp is a mixture (named a cocktail)
of three chimaeric monoclonal antibodies (mAbs) of IgG class, which bind to
three different epitopes on Ebola surface glycoprotein (GP). ZMapp was created
by systematic selection of antibodies from two other three-component
cocktails--MB-003 and ZMab the components of which were produced by rapid
transient expression method in tobacco species of Australian origin--Nicotiana
benthamiana. The ZMapp antibodies of pharmaceutical grade are manufactured in
green-house grown N.benthamiana according to the cGMP (current Good
Manufacturing Practice), using RAMP platform (Rapid Antibody Manufacturing
Platform) and MagnICON system, which utilizes transient expression by
magnifection method using viral vectors delivered to plant tissue by a
bacterium--Agrobacterium tumefaciens. The applied glycosylation mutant of
N.benthamiana (delta XTFT) synthesizes human-like, biantennary N-glycans, with
terminal N-acetylglucoseamine and without typical of plants, immunogenic sugar
epitopes-beta1,2-linked xylose and alpha1,3-linked fucose. Due to an absence of
fucose on N-glycans attached to the Fc domains, the plant-produced anti-Ebola
mAbs elicited significantly stronger antibody-dependent cellular cytotoxicity
(ADCC) than the analogous anti-Ebola mAbs with fucosylated (alpha1,6-linked
fucose) N-glycans produced in a mammalian CHO cell line--the basic expression
system for the industrial production of recombit therapeutical glycoproteins.
As far as a vaccine against Ebola virus disease is considered, so-called Ebola
Immunogenic Complex (EIC) consisting of assembled molecules of a humanized IgG
mAb--6D8 specific for Ebola GPI with GP1 fused to the C-terminus of the heavy
chains, was obtained by transient expression in N. benthamiana. Recent successes with monoclonal antibody cocktails ZMapp(TM) and MIL77 against
Ebola virus (EBOV) infections have reignited interest in antibody-based
therapeutics. Since the production process for monoclonal antibodies can be
prolonged and costly, alternative treatments should be investigated. We produced
purified equine antisera from horses hyperimmunized with EBOV virus-like
particles, and tested the post-exposure efficacy of the antisera in a mouse
model of infection. BALB/c mice were given up to 2 mg of purified equine
antisera per animal, at 30 minutes, 1 or 2 days post-infection (dpi), in which
all animals survived. To decrease the possibility of serum sickness, the equine
antisera was digested with pepsin to generate F(ab')2 fragments, with in vitro
neutralizing activity comparable to whole immunoglobulin. Full protection was
achieved with when treatment was initiated at 1 dpi, but the suboptimal
protection observed with the 30 minute and 2 dpi groups demonstrate that in
addition to virus neutralization, other Fc-dependent antibody mechanisms may
also contribute to survival. Guinea pigs given 20 mg of antisera or F(ab')2 at
or starting at 1 or 2 dpi were also fully protected from EBOV infection. These
results justify future efficacy studies for purified equine products in NHPs. The growing promise of plant-made biologics is highlighted by the success story
of ZMapp™ as a potentially life-saving drug during the Ebola outbreak of
2014-2016. Current plant expression platforms offer features beyond the
traditional advantages of low cost, high scalability, increased safety, and
eukaryotic protein modification. Novel transient expression vectors have been
developed that allow the production of vaccines and therapeutics at
unprecedented speed to control potential pandemics or bioterrorism attacks.
Plant-host engineering provides a method for producing proteins with unique and
uniform mammalian post-translational modifications, providing opportunities to
develop biologics with increased efficacy relative to their mammalian
cell-produced counterparts. Recent demonstrations that plant-made proteins can
function as biocontrol agents of foodborne pathogens further exemplify the
potential utility of plant-based protein production. However, resolving the
technical and regulatory challenges of commercial-scale production, garnering
acceptance from large pharmaceutical companies, and obtaining U.S. Food and Drug
Administration approval for several major classes of biologics are essential
steps to fulfilling the untapped potential of this technology. ZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6)
against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting
outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that
Fabs from these MAbs bind a soluble EBOV GP ectodomain and that MAbs c2G4 and
c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron
tomography, we extended these findings by characterizing the structures of c2G4,
c4G7, and c13C6 IgGs bound to native, full-length GP from the West African 2014
isolate embedded in filamentous viruslike particles (VLPs). As with the isolated
ectodomain, c13C6 bound to the glycan cap, whereas c2G4 and c4G7 bound to the
base region of membrane-bound GP. The tomographic data suggest that all three
MAbs bind with high occupancy and that the base-binding antibodies can
potentially bridge neighboring GP spikes. Functional studies indicated that c2G4
and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP
into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1(+)
endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block
entry mediated by the primed (19-kDa) form of GP without impeding binding of the
C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of
action of c2G4 and c4G7 is therefore by inhibiting conformational changes in
primed, NPC1-bound GP that initiate fusion between the viral and target
membranes, similar to the action of certain broadly neutralizing antibodies
against influenza hemagglutinin and HIV Env.
IMPORTANCE: The recent West African outbreak of ebolavirus caused the deaths of
more than 11,000 individuals. Hence, there is an urgent need to be prepared with
vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of
three MAbs directed against the ebolavirus glycoprotein, is a promising
anti-ebolavirus therapeutic. Using cryo-electron tomography, we provide
structural information on how each of the MAbs in this cocktail binds to the
ebolavirus glycoprotein as it is displayed-embedded in the membrane and present
at high density-on filamentous viruslike particles that recapitulate the surface
structure and entry functions of ebolavirus. Moreover, after confirming that two
of the MAbs bind to the same region in the base of the glycoprotein, we show
that they competitively block the entry function of the glycoprotein and that
they can do so after the glycoprotein is proteolytically primed and bound to its
intracellular receptor, Niemann-Pick C1. These findings should inform future
developments of ebolavirus therapeutics. Ebola virus (EBOV) is highly pathogenic, with a predisposition to cause
outbreaks in human populations accompanied by significant mortality. An ovine
polyclonal antibody therapy has been developed against EBOV, named EBOTAb. When
tested in the stringent guinea pig model of EBOV disease, EBOTAb has been shown
to confer protection at levels of 83.3%, 50% and 33.3% when treatment was first
started on days 3, 4 and 5 post-challenge, respectively. These timepoints of
when EBOTAb treatment was initiated correspond to when levels of EBOV are
detectable in the circulation and thus mimic when treatment would likely be
initiated in human infection. The effects of EBOTAb were compared with those of
a monoclonal antibody cocktail, ZMapp, when delivered on day 3 post-challenge.
Results showed ZMapp to confer complete protection against lethal EBOV challenge
in the guinea pig model at this timepoint. The data reported demonstrate that
EBOTAb is an effective treatment against EBOV disease, even when delivered late
after infection. To date, the management of patients with suspected or confirmed Ebolavirus
disease (EVD) depends on quarantine, symptomatic management and supportive care,
as there are no approved vaccines or treatments available for human use.
However, accelerated by the recent large outbreak in West Africa, significant
progress has been made towards vaccine development but also towards specific
treatment with convalescent plasma and monoclonal antibodies. Areas covered: We
describe recent developments in monoclonal antibody treatment for EVD,
encompassing mAb114 and the MB-003, ZMAb, ZMapp™ and MIL-77E cocktails. Expert
opinion: Preventive measures, are, and will remain essential to curb EVD
outbreaks; even more so with vaccine development progressing. However, research
for treatment options must not be neglected. Small-scale animal and individual
human case studies show that monoclonal antibodies (mAbs) can be effective for
EVD treatment; thus justifying exploration in clinical trials. Potential
limitations are that high doses may be needed to yield clinical efficacy;
epitope mutations might reduce efficacy; and constant evolution of
(outbreak-specific) mAb mixtures might be required. Interim advice based on the
clinical experience to date is that treatment of patients with mAbs is sensible,
provided those could be made available in the necessary amounts in time. Adeno-associated viral vectors can be used as a platform for delivering
biological countermeasures against pandemic and biological threats. We show that
vector delivery of two antibody components of the ZMapp product is effective in
mice against systemic and airway challenge with a mouse-adapted strain of Ebola
virus. This platform provides a generic manufacturing solution and overcomes
some of the delivery challenges associated with repeated administration of the
protective protein. BACKGROUND: Data from studies in nonhuman primates suggest that the triple
monoclonal antibody cocktail ZMapp is a promising immune-based treatment for
Ebola virus disease (EVD).
METHODS: Beginning in March 2015, we conducted a randomized, controlled trial of
ZMapp plus the current standard of care as compared with the current standard of
care alone in patients with EVD that was diagnosed in West Africa by
polymerase-chain-reaction (PCR) assay. Eligible patients of any age were
randomly assigned in a 1:1 ratio to receive either the current standard of care
or the current standard of care plus three intravenous infusions of ZMapp (50 mg
per kilogram of body weight, administered every third day). Patients were
stratified according to baseline PCR cycle-threshold value for the virus (≤22
vs. >22) and country of enrollment. Oral favipiravir was part of the current
standard of care in Guinea. The primary end point was mortality at 28 days.
RESULTS: A total of 72 patients were enrolled at sites in Liberia, Sierra Leone,
Guinea, and the United States. Of the 71 patients who could be evaluated, 21
died, representing an overall case fatality rate of 30%. Death occurred in 13 of
35 patients (37%) who received the current standard of care alone and in 8 of 36
patients (22%) who received the current standard of care plus ZMapp. The
observed posterior probability that ZMapp plus the current standard of care was
superior to the current standard of care alone was 91.2%, falling short of the
prespecified threshold of 97.5%. Frequentist analyses yielded similar results
(absolute difference in mortality with ZMapp, -15 percentage points; 95%
confidence interval, -36 to 7). Baseline viral load was strongly predictive of
both mortality and duration of hospitalization in all age groups.
CONCLUSIONS: In this randomized, controlled trial of a putative therapeutic
agent for EVD, although the estimated effect of ZMapp appeared to be beneficial,
the result did not meet the prespecified statistical threshold for efficacy.
(Funded by the National Institute of Allergy and Infectious Diseases and others;
PREVAIL II ClinicalTrials.gov number, NCT02363322 .). Despite the unprecedented Ebola virus outbreak response in West Africa, no Ebola
medical countermeasures have been approved by the US Food and Drug
Administration. However, multiple valuable lessons have been learned about the
conduct of clinical research in a resource-poor, high risk-pathogen setting.
Numerous therapeutics were explored or developed during the outbreak, including
repurposed drugs, nucleoside and nucleotide analogues (BCX4430, brincidofovir,
favipiravir, and GS-5734), nucleic acid-based drugs (TKM-Ebola and AVI-7537),
and immunotherapeutics (convalescent plasma and ZMapp). We review Ebola
therapeutics progress in the aftermath of the West Africa Ebola virus outbreak
and attempt to offer a glimpse of a path forward. A neonate born to an Ebola virus-positive woman was diagnosed with Ebola virus
infection on her first day of life. The patient was treated with monoclonal
antibodies (ZMapp), a buffy coat transfusion from an Ebola survivor, and the
broad-spectrum antiviral GS-5734. On day 20, a venous blood specimen tested
negative for Ebola virus by quantitative reverse-transcription polymerase chain
reaction. The patient was discharged in good health on day 33 of life. Further
follow-up consultations showed age-appropriate weight gain and neurodevelopment
at the age of 12 months. This patient is the first neonate documented to have
survived congenital infection with Ebola virus. The 2014-2016 Ebola virus outbreak in West Africa was the deadliest in history,
prompting the evaluation of various drug candidates, including antibody-based
therapeutics for the treatment of Ebola hemorrhagic fever (EHF). Prior to 2014,
only convalescent blood products from EHF survivors had been administered to
newly infected individuals as a form of treatment. However, during the recent
outbreak, monoclonal antibody cocktails such as ZMapp, ZMAb and MB-003 were
either tested in a human clinical safety and efficacy trial or provided to some
based on compassionate grounds. This review aims to discuss the evolution of
antibody-based treatments for EHF, their clinical trial efficacy and the
development of new antibody-based therapies currently advancing in preclinical
testing. Vectored delivery of the ZMapp antibody cocktail (c2G4, c4G7, and c13C6) by
using recombit adeno-associated viruses (rAAVs) could be useful for
preventive immunization against Ebola virus infections because rAAVs can
generate long-term antibody expression. Three rAAVs (serotype 9) encoding
chimeric ZMapp antibodies were produced by triple-plasmid transfection up to
10 L-scale in WAVE bioreactors using HEK293 cells grown in suspension/serum-free
conditions. Efficacy of AAV-c2G4 via intravenous (i.v.), intramuscular (i.m.),
and intranasal (i.n.) routes of administration was evaluated in mice with two
different doses of 2.7 × 1010 and 13.0 × 1010 vector genomes (vg). The best
protective efficacies after Ebola challenge were obtained with the i.v. and i.m.
routes. Serum concentrations of ZMapp antibodies positively correlated with
survivability. Efficacy of the rAAV-ZMapp cocktail was then evaluated at a
higher dose of 30.0 × 1010 vg. It conferred a more robust protection (90% i.v.
and 60% i.m.) than rAAV-c4G7 (30%) and rAAV-c13C6 (70%), both administered
separately at the same dose. Delivery of rAAV-c2G4 alone achieved up to 100%
protection (100% i.v. and 90% i.m.) at the same dose. In conclusion, the
preventive treatment was effective in mice. However, no advantage was observed
for using the rAAV-ZMapp cocktail in comparison to the utilization of the single
rAAV-c2G4. Filoviruses, including Ebola, have the potential to be transmitted via
virus-laden droplets deposited onto mucus membranes. Protecting against such
emerging pathogens will require understanding how they may transmit at mucosal
surfaces and developing strategies to reinforce the airway mucus barrier. Here,
we prepared Ebola pseudovirus (with Zaire strain glycoproteins) and used
high-resolution multiple-particle tracking to track the motions of hundreds of
individual pseudoviruses in fresh and undiluted human airway mucus isolated from
extubated endotracheal tubes. We found that Ebola pseudovirus readily penetrates
human airway mucus. Addition of ZMapp, a cocktail of Ebola-binding
immunoglobulin G antibodies, effectively reduced mobility of Ebola pseudovirus
in the same mucus secretions. Topical delivery of ZMapp to the mouse airways
also facilitated rapid elimination of Ebola pseudovirus. Our work demonstrates
that antibodies can immobilize virions in airway mucus and reduce access to the
airway epithelium, highlighting topical delivery of pathogen-specific antibodies
to the lungs as a potential prophylactic or therapeutic approach against
emerging viruses or biowarfare agents. Author information:
(1)Laboratory of Virology, Division of Intramural Research, National Institute
of Allergy and Infectious Diseases, National Institutes of Health, Hamilton,
Montana.
(2)Joint Program Executive Office Chemical-Biological Defense, Medical
Countermeasures Systems' Joint Vaccine Acquisition Program, Fort Detrick,
Maryland.
(3)Rocky Mountain Veterinary Branch, Division of Intramural Research, National
Institute of Allergy and Infectious Diseases, National Institutes of Health,
Hamilton, Montana.
(4)SAB Biotherapeutics, Sioux Falls, South Dakota.
(5)Virology Division, United States Army Medical Research Institute of
Infectious Diseases, Fort Detrick, Maryland.
(6)Zoonotic Diseases and Special Pathogens, Public Health Agency of Canada,
Winnipeg, Manitoba, Canada. |
Can methylenetetrahydrofolate reductase (MTHFR) gene mutations cause homocystinuria? | Yes, several methylenetetrahydrofolate reductase (MTHFR) gene mutations can cause homocystinuria and hyperhomocysteinemia. | A 24 day old girl with homocystinuria and hypomethioninaemia caused by
methylenetetrahydrofolate reductase deficiency presented with rapidly
progressing encephalopathy and myopathy. An almost complete recovery was
achieved by treatment with betaine. We report findings on a child presenting with neonatal homocystinuria,
hypomethioninaemia and severe neurological symptoms, including developmental
delay and seizures. Methylmalonic aciduria was not present. The activity of
methionine synthase in fibroblasts was severely deficient and formation of
methylcobalamin from 57Co labelled cyanocobalamin was very low. The patients
cells complemented with those of a cblE patient but not with those of two cblG
patients. No biochemical or clinical response to injections of hydroxycobalamin
was found. Both off treatment and on betaine and methionine supplementation the
patient, at age 8 years, has not developed megaloblastic anaemia. In addition,
the patient is homozygous for the C677T polymorphism in the 5,10
methylenetetrahydrofolate reductase (MTHFR) gene and the concomitant existence
of this mutation with the methionine synthase defect may prevent folate
<<trapping>> and thus anaemia.
CONCLUSION: We report the lack of megaloblastic anaemia in a patient with severe
methionine synthase deficiency who is also homozygous for C677T in MTHFR,
hypothesize that the MTHFR polymorphism protects the patient against anaemia and
speculate that homozygosity for MTHFR C677T could cause the dissociation between
haematological and neurological disease seen in some patients with vitamin B12
deficiency. Severe hyperhomocysteinemia in its most frequent form, is caused by a homozygous
enzymatic deficiency of cystathionine beta-synthase (CBS). A major complication
in CBS deficiency is deep venous thrombosis or pulmonary embolism. A recent
report by Mandel et al (N Engl J Med 334:763, 1996) postulated factor V Leiden
(FVL) to be an absolute prerequisite for the development of thromboembolism in
patients with severe hyperhomocysteinemia. We studied 24 patients with
homocystinuria caused by homozygous CBS deficiency from 18 unrelated kindreds
for FVL and for the 677C-->T mutation in the methylenetetrahydrofolate reductase
(MTHFR) gene and investigated their possible interaction in the risk of venous
thrombosis. Thrombotic complications were diagnosed in six patients, of whom
only one was a carrier of FVL. On the contrary, thermolabile MTHFR caused by the
677C-->T mutation, was frequently observed among homocystinuria patients,
especially among those with thromboembolic complications: three of six
homocystinuria patients who had suffered from a thromboembolic event had
thermolabile MTHFR. These data indicate that FVL is not an absolute prerequisite
and probably not even a major determit of venous thrombosis in
homocystinuria, but, interestingly, thermolabile MTHFR may constitute a
significant risk factor for thromboembolic complications in this inborn error of
methionine metabolism. Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is an inborn error
of folate metabolism, and is inherited as an autosomal recessive trait. MTHFR is
a key enzyme in folate-dependent remethylation of homocysteine, and reduces
5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Patients with this
severe enzymatic deficiency are biochemically characterised by homocystinuria
and hypomethioninaemia, and may suffer from neurological abnormalities, mental
retardation and premature vascular disease. Here we report the molecular basis
of severe MTHFR deficiency in four unrelated families from Turkish/Greek
ancestry. By use of reverse-transcriptase (RT)-PCR, subsequently followed by
direct sequencing analysis, we were able to identify four novel mutations in the
MTHFR gene: two missense (983A-->G; 1027T-->G) and two nonsense (1084C-->T;
1711C-->T) mutations. Furthermore, a splice variant containing a premature
termination codon, was observed in one patient, probably as a secondary effect
of the 1027T-->G missense mutation. The ongoing identification and
characterisation of mutations in the MTHFR gene will provide further insight
into the heterogeneity of the clinical phenotype in severe MTHFR deficiency. Severe deficiency of methylenetetrahydrofolate reductase (MTHFR) is the most
common inborn error of folate metabolism. Patients are characterized by severe
hyperhomocysteinemia, homocystinuria and a variety of neurological and vascular
problems. Eighteen rare mutations have been reported in this group of patients.
Two polymorphisms which cause mild enzyme deficiencies have been described
(677C-->T and 1298A-->C). The first sequence change encodes a thermolabile
enzyme and is associated with mild hyperhomocysteinemia. Six novel point
mutations are described in patients with severe deficiency of MTHFR, along with
their associated polymorphisms and clinical phenotypes. Of the two nonsense
mutations (1762A-->T, 1134C-->G) and four missense mutations (1727C-->T,
1172G-->A, 1768G-->A, and 358G-->A), one was identified in the N-terminal
catalytic domain, while the others were located in the regulatory C-terminal
region. All four residues affected by missense mutations are conserved in one or
more MTHFRs of other species. This report brings the total to 24 mutations
identified in severe MTHFR deficiency, with two mutations identified in each of
22 patients. Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of
5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a carbon donor for
homocysteine remethylation to methionine. Severe MTHFR deficiency is associated
with hyperhomocysteinemia and homocystinuria. These patients show a wide variety
of neurological and vascular symptoms, with variable age of onset. Residual
enzyme activity is usually less than 20% of control values, and correlates
reasonably well with age of onset of symptoms. A milder deficiency of MTHFR,
with 30%-50% residual enzyme activity and increased enzyme thermolability, has
been described as a risk factor for vascular disease and for neural tube
defects. In earlier work, we isolated the human cDNA for MTHFR, and reported 14
mutations in severe MTHFR deficiency, as well as a common 677C-->T missense
mutation (Ala-->Val) that encodes the thermolabile MTHFR. This variant has also
been observed in some patients with severe MTHFR deficiency, in cis with their
severe mutations. We report here the in vitro expression of seven severe MTHFR
mutations in a bacterial expression system; six of these were expressed in cis
with the Val allele to mimic the situation in the patients. We show that three
of these constructs have significantly reduced enzyme activity (<10% of
control); the presence of the thermolabile variant in these patients in cis is
unlikely to affect enzyme function since activity is already low. One mutation
causes a dramatic increase in activity when it is expressed in cis with the Ala
allele, but is associated with extreme lability when in cis with the Val allele.
Three mutations cause moderate decreases in enzyme activity, with a further
decrease in activity when they are in cis with the Val allele. We hypothesize
that deleterious mutations which alter stability may be compromised to a greater
degree when the thermolabile variant is present on the same allele. Hyperhomocysteinemia, a risk factor for cardiovascular disease, is caused by
nutritional and/or genetic disruptions in homocysteine metabolism. The most
common genetic cause of hyperhomocysteinemia is the 677C-->T mutation in the
methylenetetrahydrofolate reductase (MTHFR) gene. This variant, with mild
enzymatic deficiency, is associated with an increased risk for neural tube
defects and pregcy complications and with a decreased risk for colon cancer
and leukemia. Although many studies have reported that this variant is also a
risk factor for vascular disease, this area of investigation is still
controversial. Severe MTHFR deficiency results in homocystinuria, an inborn
error of metabolism with neurological and vascular complications. To investigate
the in vivo pathogenetic mechanisms of MTHFR deficiency, we generated mice with
a knockout of MTHFR: Plasma total homocysteine levels in heterozygous and
homozygous knockout mice are 1.6- and 10-fold higher than those in wild-type
littermates, respectively. Both heterozygous and homozygous knockouts have
either significantly decreased S-adenosylmethionine levels or significantly
increased S-adenosylhomocysteine levels, or both, with global DNA
hypomethylation. The heterozygous knockout mice appear normal, whereas the
homozygotes are smaller and show developmental retardation with cerebellar
pathology. Abnormal lipid deposition in the proximal portion of the aorta was
observed in older heterozygotes and homozygotes, alluding to an atherogenic
effect of hyperhomocysteinemia in these mice. Classical homocystinuria is associated with arterial vascular diseases and
venous thrombosis. In the last decade, several studies have been published
indicating that even mild hyperhomocysteinemia is a risk factor for venous
thrombosis. The 677C-->T mutation in the methylenetetrahydrofolate reductase
(MTHFR) gene is an important cause of mild hyperhomocysteinemia, but this
polymorphism does not seem to be a risk factor for venous thrombosis. Studies on
the interaction between hyperhomocysteinemia and other thrombotic risk factors
are conflicting. Little is known about the pathophysiology of venous thrombosis
in hyperhomocysteinemia. Several mechanisms proposed for vascular disease may be
applied to venous thrombosis as well. However, up to now there is no satisfying
model which might explain a thrombophilic state at plasma homocysteine
concentrations in the range of mild hyperhomocysteinemia. The results of a first
clinical intervention study are expected in 2002. As the results are pending,
clinicians could perform homocysteine measurements in patients with venous
thrombosis if screening for thrombophilia is indicated. Vitamin supplementation
could be prescribed if homocysteine levels are elevated. However, the patient
should be informed about the uncertainty of the benefits of vitamin
supplementation. Methylenetetrahydrofolate reductase (MTHFR) synthesizes
5-methyltetrahydrofolate, a major methyl donor for homocysteine remethylation to
methionine. Severe MTHFR deficiency results in marked hyperhomocysteinemia and
homocystinuria. Patients display developmental delay and a variety of
neurological and vascular symptoms. Cloning of the human cDNA and gene has
enabled the identification of 29 rare mutations in homocystinuric patients and
two common variants [677C>T (A222V) and 1298A>C (E429A)] with mild enzymatic
deficiency. Homozygosity for 677C>T or combined heterozygosity for both
polymorphisms is associated with mild hyperhomocysteinemia. In this
communication, we describe four novel mutations in patients with homocystinuria:
two missense mutations (471C>G, I153M; 1025T>C, M338T), a nonsense mutation
(1274G>A, W421X), and a 2-bp deletion (1553delAG). We expressed the 1025T>C
mutation as well as two previously reported amino acid substitutions [983A>G
(N324S) and 1027T>G (W339G)] and observed decreased enzyme activity at 10%, 36%,
and 21% of control levels, respectively, with little or no effect on affinity
for 5-methyltetrahydrofolate. One of these mutations, 983A>G (N324S), showed
flavin adenine dinucleotide (FAD) responsiveness in vitro. Expression of these
mutations in cis with the 677C>T polymorphism, as observed in the patients,
resulted in an additional 50% decrease in enzyme activity. This report brings
the total to 33 severe mutations identified in patients with severe MTHFR
deficiency. Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme in folate
and homocysteine metabolism. Research performed during the past decade has
clarified our understanding of MTHFR deficiencies that cause homocystinuria or
mild hyperhomocysteinemia. Our cloning of the MTHFR coding sequence was
initially followed by the identification of the first deleterious mutations in
MTHFR, in patients with homocystinuria and marked hyperhomocysteinemia. Shortly
thereafter, we identified the 677C-->T variant and showed that it encoded a
thermolabile enzyme with reduced activity. Currently, a total of 41 rare but
deleterious mutations in MTHFR, as well as about 60 polymorphisms have been
reported. The 677C-->T (Ala222Val) variant has been particularly noteworthy
since it has become recognized as the most common genetic cause of
hyperhomocysteinemia. The disruption of homocysteine metabolism by this
polymorphism influences risk for several complex disorders, including
cardiovascular disease, neural tube defects and some cancers. We describe here
the complex structure of the MTHFR gene, summarize the current state of
knowledge on rare and common mutations in MTHFR and discuss some relevant
findings in a mouse model for MTHFR deficiency. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the regulation of
plasma homocysteine levels. MTHFR deficiency, an autosomal recessive disorder,
results in homocystinuria and hypomethioninaemia and presents with highly
variable symptoms affecting many organs but predomitly the central nervous
system. The common polymorphism of the MTHFR gene, c.677C>T, a known risk factor
for elevated plasma homocysteine levels, occurs frequently in the caucasian
population. In this study we investigated three subjects with moderate
hyperhomocysteinaemia (total plasma homocysteine 72 micromol/L in case 1 and 90
micromol/L in case 3, total non-protein-bound homocysteine 144-186 micromol/L in
case 2) but different clinical presentation with no symptoms in case 1, muscle
weakness at 17 years of age in case 2, and syncopes and cerebral convulsions at
18 years of age in case 3. Each subject was compound heterozygous for the
c.677C>T polymorphism and a novel mutation of the MTHFR gene (case 1: c.883G>A
[p.D291N]; case 2: c.1552_c.1553delGA [p.E514fsX536]; case 3: c.616C>T
[p.P202S]). Moderately decreased fibroblast MTHFR activity was associated with
severely reduced affinity for NADPH and increased sensitivity to inhibition by
S-adenosylmethionine (AdoMet) in case 2, and with mild FAD responsiveness in
case 3. In case 1, fibroblast MTHFR activity was normal but the sensitivity to
inhibition by AdoMet was slightly reduced. This study indicates that the
sequence alteration c.677C>T combined with severe MTHFR mutations in compound
heterozygous state may lead to moderate biochemical and clinical abnormalities
exceeding those attributed to the c.677TT genotype and might require in addition
to folate substitution further therapy to normalize homocysteine levels. Methylenetetrahydrofolate reductase (MTHFR) plays a major role in folate
metabolism. Disturbed function of the enzyme results in hyperhomocysteinemia and
causes severe vascular and neurological disorders and developmental delay. Five
patients suspected of having non-classical homocystinuria due to MTHFR
deficiency were examined with respect to their symptoms, MTHFR enzyme activity
and genotypes of the MTHFR gene. All patients presented symptoms of severe
central nervous system disease. Two patients died, at the ages of 15 months and
14 years. One patient is currently 32 years old, and is being treated with
betaine and folinic acid. The other two patients, with an early diagnosis and a
severe course of the disease, are currently improving under treatment. MTHFR
enzyme activity in the fibroblasts of four of the patients was practically
undetectable. We found four novel mutations, three of which were missense
changes c.664G> T (p.V218L), c.1316T> C (p.F435S) and c.1733T> G (p.V574G), and
the fourth was the 1-bp deletion c.1780delC (p.L590CfsX72). We also found the
previously reported nonsense mutation c.1420G> T (p.E470X). All the patients
were homozygous. Molecular modelling of the double mutant allele (p.V218L;
p.A222V) revealed that affinity for FAD was not affected in this mutant. For the
p.E470X mutation, the evidence pointed to nonsense-mediated mRNA decay. In
general, genotype-phenotype analysis predicts milder outcomes for patients with
missense changes than for those in which mutations led to severe alterations of
the MTHFR protein. Severe deficiency of methylenetetrahydrofolate reductase (MTHFR) with
homocystinuria can result in early demise or later-onset neurological
impairment, including developmental delay, motor dysfunction, and seizures. We
previously characterized BALB/c Mthfr (-/-)mice as a model for this disorder and
have recently backcrossed the disrupted allele onto the C57Bl/6 background to
examine the variable phenotypes in MTHFR deficiency. Compared with BALB/c Mthfr
(-/-)mice, C57Bl/6 Mthfr (-/-)mice have enhanced survival rates (81% vs 26.5%).
Four-day-old BALB/c mutant pups had lower body, brain, and spleen weights
relative to their wild-type counterparts compared with C57Bl/6 mutants. Pregt
BALB/c Mthfr (+/-)mice had increased resorptions and embryonic delays compared
with wild-type littermates, whereas these outcomes in C57Bl/6 c Mthfr (+/-)mice
were similar to those of wild-type C57Bl/6 mice. BALB/c-mutant pups had altered
hematological profiles (higher hematocrit, hemoglobin, and white blood cell
counts, with lower platelet counts) compared with C57Bl/6 mutants. Mutants of
both strains had similar degrees of hepatic steatosis, hepatic activity of
betaine:homocysteine methyltransferase, and altered cerebellar histology.
Electroretinograms (ERG) in C57Bl/6 Mthfr (-/-)mice revealed decreased amplitude
of scotopic and photopic waves in 6-week-old mice, with normalized ERGs at
13 weeks. Plasma homocysteine was modestly higher in C57Bl/6 compared with
BALB/c mice. Our results emphasize the variable presentation of MTHFR deficiency
in different genetic backgrounds and suggest that plasma homocysteine is not a
predictor of severity. In addition, our novel findings of decreased spleen
weights, thrombocytopenia, and impaired retinal function warrant investigation
in patients with severe MTHFR deficiency or other forms of homocystinuria. BACKGROUND: Hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR)
gene mutation have been postulated as a possible cause of recurrent miscarriage
(RM). There is a wide variation in the prevalence of MTHFR polymorphisms and
homocysteine (Hcy) plasma levels among populations around the world. The present
study was undertaken to investigate the possible association between
hyperhomocysteinemia and its causative genetic or acquired factors and RM in
Catalonia, a Mediterranean region in Spain.
METHODS: Sixty consecutive patients with ≥ 3 unexplained RM and 30 healthy
control women having at least one child but no previous miscarriage were
included. Plasma Hcy levels, MTHFR gene mutation, red blood cell (RBC) folate
and vitamin B12 serum levels were measured in all subjects.
RESULTS: No significant differences were observed neither in plasma Hcy levels,
RBC folate and vitamin B12 serum levels nor in the prevalence of homozygous and
heterozygous MTHFR gene mutation between the two groups studied.
CONCLUSIONS: In the present study RM is not associated with
hyperhomocysteinemia, and/or the MTHFR gene mutation. In neurofibromatosis type-1 (NF1), cerebrovascular disorders are rarely
encountered although vasculopathy is a well-known complication. Several
mutations seen in methylenetetrahydrofolate reductase (MTHFR) give rise to the
formation of hyperhomocysteinemia and homocystinuria, a considerable risk factor
for cardiovascular and cerebrovascular disorders, by leading to enzymatic
inactivation. In the paper, a 31-year-old young stroke female patient with the
coexistence of neurofibromatosis and MTHFR C677T gene mutation was presented. Methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare autosomal
recessive disorder. It is known that MTHFR deficiency may result in
hyperhomocysteinemia, but MTHFR deficiency-induced schizophrenia has been rarely
reported. Here we present the clinical course, biochemical and genetic
characteristics of schizophrenia resulted from MTHFR deficiency in a school-age
boy. He was 13 years old. He was admitted with a two-year history of fear,
auditory hallucination, learning difficulty, sleeping problems, irascibility,
drowsing and giggling. At admission, he had significantly elevated plasma and
urine levels of total homocysteine, significantly decreased levels of folate in
serum and cerebrospinal fluid, and a normal blood concentration of methionine.
Further DNA sequencing analysis showed 665C>T homozygous mutations in the MTHFR
gene. The patient was diagnosed with MTHFR deficiency-associated schizophrenia
and treatment with calcium folinate, vitamin B12, vitamin B6, and betaine was
initiated. After the treatment for 1 week, his plasma and urine levels of
homocysteine were decreased to a normal range and the clinical symptoms were
significantly improved. After 3 months of treatment, the patient returned to
school. He is now living with normal school life. In summary, children with
late-onset MTHFR deficiency and secondary cerebral folate deficiency may lead to
schizophrenia. This rare condition can be early diagnosed through analyses of
blood and urine total homocysteine, amino acids in blood and folate in blood and
cerebral fluid and successfully treated with folinic acid, vitamin B6, vitamin
B12 and betaine. |
What happens to H2AX upon DNA bouble strand breaks? | Phosphorylated H2AX (γH2AX) is rapidly concentrated in chromatin domains around DNA double-strand breaks (DSBs) after the action of ionizing radiation or chemical agents and at stalled replication forks during replication stress The nuclear foci of phosphorylated histone H2AX (γH2AX) are frequently used as a marker for DNA double-strand breaks (DSBs) following ionizing radiation (IR) | DNA double-strand breaks (DSBs) can induce chromosomal aberrations and
carcinogenesis and their correct repair is crucial for genetic stability. The
cellular response to DSBs depends on damage signaling including the
phosphorylation of the histone H2AX (γH2AX). However, a lack of γH2AX formation
in heterochromatin (HC) is generally observed after DNA damage induction. Here,
we examine γH2AX and repair protein foci along linear ion tracks traversing
heterochromatic regions in human or murine cells and find the DSBs and damage
signal streaks bending around highly compacted DNA. Given the linear particle
path, such bending indicates a relocation of damage from the initial induction
site to the periphery of HC. Real-time imaging of the repair protein GFP-XRCC1
confirms fast recruitment to heterochromatic lesions inside murine
chromocenters. Using single-ion microirradiation to induce localized DSBs
directly within chromocenters, we demonstrate that H2AX is early phosphorylated
within HC, but the damage site is subsequently expelled from the center to the
periphery of chromocenters within ∼ 20 min. While this process can occur in the
absence of ATM kinase, the repair of DSBs bordering HC requires the protein.
Finally, we describe a local decondensation of HC at the sites of ion hits,
potentially allowing for DSB movement via physical forces. A sequence variant of histone H2A called H2AX is one of the key components of
chromatin involved in DNA damage response induced by different genotoxic
stresses. Phosphorylated H2AX (γH2AX) is rapidly concentrated in chromatin
domains around DNA double-strand breaks (DSBs) after the action of ionizing
radiation or chemical agents and at stalled replication forks during replication
stress. γH2AX foci could be easily detected in cell nuclei using
immunofluorescence microscopy that allows to use γH2AX as a quantitative marker
of DSBs in various applications. H2AX is phosphorylated in situ by ATM, ATR, and
DNA-PK kinases that have distinct roles in different pathways of DSB repair. The
γH2AX serves as a docking site for the accumulation of DNA repair proteins, and
after rejoining of DSBs, it is released from chromatin. The molecular mechanism
of γH2AX dephosphorylation is not clear. It is complicated and requires the
activity of different proteins including phosphatases and chromatin-remodeling
complexes. In this review, we summarize recently published data concerning the
mechanisms and kinetics of γH2AX loss in normal cells and tissues as well as in
those deficient in ATM, DNA-PK, and DSB repair proteins activity. The results of
the latest scientific research of the low-dose irradiation phenomenon are
presented including the bystander effect and the adaptive response estimated by
γH2AX detection in cells and tissues. The nuclear foci of phosphorylated histone H2AX (γH2AX) are frequently used as a
marker for DNA double-strand breaks (DSBs) following ionizing radiation (IR).
However, recent studies reported that γH2AX foci do not necessarily correlate
with DSBs under other conditions. We showed that γH2AX foci induced by oxidative
stress in hydrogen peroxide (H2O2)-treated cells displayed several different
features from those induced by IR. The magnitude of γH2AX induction was
heterogeneous among H2O2-treated cells. Some cells expressed small discrete
γH2AX foci, whereas others expressed a gross γH2AX signal that was distributed
throughout the nucleus. Oxidative stress-induced γH2AX was eliminated in DSB
repair-deficient mutant cells as efficiently as in wild-type cells and was not
necessarily accompanied by phosphorylated ataxia telangiectasia mutated (ATM) or
53BP1 foci. Analyses using specific inhibitors showed that ATM- and Rad3-related
(ATR), rather than ATM, was the prominent kinase mediating the oxidative stress
response. These results suggest that a major fraction of γH2AX induced by
oxidative stress is not associated with DSBs. Single-stranded DNA arisen from
stalled replication forks can cause the ATR-mediated induction of γH2AX.
However, oxidative stress appeared to induce γH2AX in both S- and non-S-phase
cells. These results suggest that there may be another pathway leading to the
ATR-mediated induction of γH2AX in non-S-phase cells without DSBs. OBJECTIVE: Defective or inefficient DNA double-strand break (DSB) repair results
in failure to preserve genomic integrity leading to apoptotic cell death, a
hallmark of systemic lupus erythematosus (SLE). Compelling evidence linked
environmental factors that increase oxidative stress with SLE risk and the
formation of DSBs. In this study, we sought to further explore genotoxic stress
sensitivity in SLE by investigating DSB accumulation as a marker linking the
effect of environmental stressors and the chromatin microenvironment.
METHODS: DSBs were quantified in peripheral blood mononuclear cell subsets from
patients with SLE, healthy controls, and patients with rheumatoid arthritis (RA)
by measuring phosphorylated H2AX (phospho-H2AX) levels with flow cytometry.
Phospho-H2AX levels were assessed in G0/G1, S and G2 cell-cycle phases using
propidium iodide staining, and after oxidative stress using 0.5 µM hydrogen
peroxide exposure for 0, 2, 5, 10, 30 and 60 min.
RESULTS: DSB levels were significantly increased in CD4+ T cells, CD8+ T cells
and monocytes in SLE compared with healthy controls (p=2.16×10(-4), 1.68×10(-3)
and 4.74×10(-3), respectively) and RA (p=1.05×10(-3), 1.78×10(-3) and
2.43×10(-2), respectively). This increase in DSBs in SLE was independent of the
cell-cycle phase, and correlated with disease activity. In CD4+ T cells, CD8+ T
cells and monocytes, oxidative stress exposure induced significantly higher DSB
accumulation in SLE compared with healthy controls (60 min; p=1.64×10(-6),
8.11×10(-7) and 2.04×10(-3), respectively).
CONCLUSIONS: Our data indicate that SLE T cells and monocytes have increased
baseline DSB levels and an increased sensitivity to acquiring DSBs in response
to oxidative stress. Although the mechanism underlying DSB sensitivity in SLE
requires further investigation, accumulation of DSB may serve a biomarker for
disease activity in SLE and help explain increased apoptotic cell accumulation
in this disease. |
Can valproic acid prolong survival of glioblastoma patients? | Yes, there is evidence to suggest that valproic acid (VPA) is associated with prolonged survival of glioblastoma patients. Several studies have indicated that VPA has radiosensitizing effects for gliomas and radioprotective influence on normal brain tissue or hippocampal neurons. | BACKGROUND: Chemotherapy has limited effects in the treatment of high-grade
gliomas (HGGs). Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, may
sensitize HGGs to radiochemotherapy. As the drug has been given frequently as an
antiepileptic drug, a retrospective analysis was conducted to ensure relevant
information was not missed before a prospective study was launched.
MATERIALS AND METHODS: An analysis of 66 pediatric patients (range, 1-19 years
of age) with glioblastoma multiforme (GBM) (n=40) or anaplastic astrocytoma (AA)
(n=26) was retrospectively conducted for predictors of survival and response and
for effects of VPA on outcome or toxicity.
RESULTS: The overall survival (OS) was better for AA (p=0.0114) and complete
resection (p<0.00005) and event-free survival (EFS) was better for complete
resection (p=0.0049). Nine patients received VPA (for seizure) plus further
oncological treatment. The most severe adverse effect was a pulmonary embolism
(n=1); no other severe side-effects were noted. The response to nonsurgical
treatment after 8 weeks was: complete response (CR): 0, partial response (PR):
3, stable disease (SD): 4, progressive disease (PD): 2. Some of the patients
with SD responded later resulting in best response: CR:0, PR:5, SD:2, PD:2.
CONCLUSION: Treatment with VPA plus radiochemotherapy is well tolerated with an
encouraging response rate. BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive type of primary
brain tumor. It is a rapidly progressive, highly recurrent, fatal intracranial
neoplasm, and the demand for novel treatment is urgent. Valproic acid (VPA) is a
potential anticancer agent that belongs to a class of histone deacetylase (HDAC)
inhibitors, targeting the epigenetic control of gene functions in cancer cells.
This drug has been administered for the prevention or treatment of seizure
disorder in GBM patients; therefore, a retrospective analysis may further our
understanding of the effect of VPA on GBM patients.
MATERIALS AND METHODS: A retrospective analysis of 102 patients with GBM was
conducted to study the effects of VPA on disease outcome. Tumor samples from
seven patients receiving VPA treatment between the first and second operations
were obtained in order to verify the HDAC inhibitory activity of VPA in these
patients.
RESULTS: In univariate analysis, administration of VPA within 2 weeks of initial
diagnosis seemed to confer a survival benefit. However, stratified analysis
according to chemotherapy showed that VPA did not have significant impact on the
GBM patients' overall survival. Analysis of tissue samples from these patients
revealed that a small subset of patients had increased histone acetylation after
VPA treatment.
CONCLUSION: VPA treatment, when administered according to a protocol targeting
seizure control, may result in HDAC inhibition in a small subset of patients,
but does not significantly affect overall patient survival. Early administration
of VPA as an adjunct to temozolomide chemotherapy may have its merits, but the
optimal dosing schedule and target serum level require further investigation. PURPOSE: Valproic acid (VA) is an antiepileptic drug (AED) and histone
deacetylase (HDAC) inhibitor taken by patients with glioblastoma (GB) to manage
seizures, and it can modulate the biologic effects of radiation therapy (RT). We
investigated whether VA use during RT for GB was associated with overall
survival (OS).
METHODS AND MATERIALS: Medical records of 544 adults with GB were
retrospectively reviewed. Analyses were performed to determine the association
of Radiation Therapy Oncology Group recursive partitioning analysis (RTOG RPA)
class, seizure history, and concurrent temozolomide (TMZ) and AED use during RT
with OS.
RESULTS: Seizures before the end of RT were noted in 217 (40%) patients, and 403
(74%) were taking an AED during RT; 29 (7%) were taking VA. Median OS in
patients taking VA was 16.9 months (vs 13.6 months taking another AED, P=.16).
Among patients taking an AED during RT, OS was associated with VA (P=.047;
hazard ratio [HR], 0.67; 95% confidence interval [CI], 0.27-1.07), and RTOG RPA
class (P<.0001; HR, 1.49; 95% CI, 1.37-1.61). Of the 5 most common AEDs, only VA
was associated with OS. Median OS of patients receiving VA and TMZ during RT was
23.9 months (vs 15.2 months for patients taking another AED, P=.26). When the
analysis was restricted to patients who received concurrent TMZ, VA use was
marginally associated with OS (P=.057; HR, 0.54; 95% CI, -0.09 to 1.17),
independently of RTOG RPA class and seizure history.
CONCLUSIONS: VA use during RT for GB was associated with improved OS,
independently of RTOG RPA, seizure history, and concurrent TMZ use. Further
studies of treatment that combines HDAC inhibitors and RT are warranted. Between 30% and 50% of patients with brain tumors first present with a seizure,
and up to 30% more will develop seizures later. Therefore, optimal management of
these patients requires a rational approach to the use of antiseizure
medications. Based on current evidence, prophylactic prescription of long-term
antiepileptic drugs (AEDs) in patients with brain tumors in patients who did not
present with seizures is not justified. Because of the high risk of recurrence,
however, AED treatment should be strongly considered after a single seizure
considered to be due to a tumor. Because of the lack of well-controlled
randomized trials, the decision on which AED provides the best risk-benefit
ratio in the individual patient is based mostly on physician's judgment rather
than sound scientific evidence. In patients who may require chemotherapy, a
non-enzyme-inducing AED is preferred for initial treatment to minimize the risk
of drug interactions that impact adversely on the outcome of anticancer
chemotherapy. Several retrospective studies in seizure patients with
glioblastoma treated with chemotherapy have provided evidence for a moderately
improved survival with the use of valproic acid, possibly due to inhibition of
histone deacetylase. However, valproic acid may also increase the hematologic
toxicity of antineoplastic drugs, presumably by inhibiting their metabolism, and
may independently impair hemostasis, which is of some concern for patients who
require surgical intervention. Among newer generation AEDs, levetiracetam has a
number of advantageous features, including availability of a parenteral
formulation, but other agents such as gabapentin, lamotrigine, oxcarbazepine,
topiramate, and zonisamide may also be considered. Potentially more effective
treatments targeting specific mechanisms of epileptogenesis and ictogenesis are
being investigated. Resection of the tumor, radiation therapy, or chemotherapy
can bring refractory seizures under control or prolong the duration of seizure
freedom, an effect that does not appear to be necessarily related to removal or
shrinkage of the tumor mass. In patients with a successfully treated tumor and
an overall good prognosis for long-term survival, gradual discontinuation of
AEDs may be considered. PURPOSE: Glioblastoma multiforme (GBM) is the most lethal type of primary brain
tumor, and patients that undergo the maximum tumor resection that is safely
possible and standard radiochemotherapy only achieve a median survival time of
14.6 months. Several clinical studies have reported that valproic acid could
prolong survival of GBM patients. However, the results of these studies are
inconsistent. We examined relevant studies and conducted a meta-analysis to
assess the effects of VPA on survival times and recurrence.
METHODS: A bibliographic search was performed in the EMBASE, MEDLINE,
ClinicalTrials.gov and Cochrane Central Register of the Controlled Trials
databases to identify potentially relevant articles or conference abstracts that
investigated the effects of VPA on the outcome of glioma patients. Five
observational studies were included.
RESULTS: Pooled estimates of the hazard ratio (HR) and 95% confidence intervals
(CI) were calculated. Our meta-analysis confirmed the benefit of using VPA (HR,
0.56; 95% CI, 0.44-0.71). Sub-group analysis shows that patients treated with
VPA had a hazard ratio of 0.74 with a 95% confidence interval of 0.59-0.94 vs.
patients treated by other-AEDs and a hazard ratio of 0.66 with a 95% confidence
interval of 0.52-0.84 vs. patients treated by administration of non-AEDs. No
heterogeneity was observed in the subset analysis.
CONCLUSION: The results of our study suggest that glioblastoma patients may
experience prolonged survival due to VPA administration. Sub-analysis confirmed
the benefit of VPA use compared to a non-AEDs group and an other-AEDs group.
Further RCTs of this subject should be performed. Glioblastoma multiforme (GBM) has nearly uniformly fatal with a median survival
of less than 2 years. While there have not been any novel anti-GBM therapeutics
approved for many years, there has been the gradual accumulation of clinical
data suggesting that the widely used anti-convulsant agent, valproic acid (VPA)
may significantly prolong survival in GBM patients. This pre-clinical study
aimed to determine the potential clinical utility of VPA in the treatment of
GBM. Primary GBM cells were treated with VPA as a monotherapy and in combination
with temozolomide and irradiation. At clinically achievable concentrations, VPA
was shown to be effective as a monotherapy agent in the five primary lines
tested. VPA was then used as a sensitizing agent to in vitro radiation and
showed significant augmentation of in vitro irradiation therapy. In addition,
when VPA, radiation and temozolomide were combined an additive, rather than
synergistic effect was noted. Gene expression profiling demonstrated close
clustering of triple treated cells with VPA mono-treated cells while untreated
cells clustered closer with TMZ-irradiation dual treated cells. These microarray
data suggest a domit role of VPA at the gene expression level when combining
these different treatment options. Moreover, in an in vivo tumor transplantation
model, we were able to demonstrate an increase in animal survival when cells
were pre-treated with irradiation-VPA and when triple treated. These findings
provide a significant rationale for the investigation of VPA in the treatment of
GBM patients. BACKGROUND: Epileptogenic glioblastomas are thought to convey a favorable
prognosis, either due to early diagnosis or potential antitumor effects of
antiepileptic drugs. We investigated the relationship between survival and
epilepsy at presentation, early diagnosis, and antiepileptic drug therapy in
glioblastoma patients.
METHODS: Multivariable Cox regression was applied to survival data of 647
consecutive patients diagnosed with de novo glioblastoma between 2005 and 2013
in order to investigate the association between epilepsy and survival in
glioblastoma patients. In addition, we quantified the association between
survival and valproic acid (VPA) treatment.
RESULTS: Epilepsy correlated positively with survival (HR: 0.75 (95% CI:
0.61-0.92), P < .01). This effect is independent of age, sex, performance
status, type of surgery, adjuvant therapy, tumor location, and tumor volume,
suggesting that this positive correlation cannot be attributed solely to early
diagnosis. For patients who presented with epilepsy, the use of the
antiepileptic drug VPA did not associate with survival when compared with
patients who did not receive VPA treatment.
CONCLUSION: Epilepsy is an independent prognostic factor for longer survival in
glioblastoma patients. This prognostic effect is not solely explained by early
diagnosis, and survival is not associated with VPA treatment. PURPOSE: Symptomatic epilepsy is a common complication of glioblastoma and
requires pharmacotherapy. Several uncontrolled retrospective case series and a
post hoc analysis of the registration trial for temozolomide indicated an
association between valproic acid (VPA) use and improved survival outcomes in
patients with newly diagnosed glioblastoma.
PATIENTS AND METHODS: To confirm the hypothesis suggested above, a combined
analysis of survival association of antiepileptic drug use at the start of
chemoradiotherapy with temozolomide was performed in the pooled patient cohort
(n = 1,869) of four contemporary randomized clinical trials in newly diagnosed
glioblastoma: AVAGlio (Avastin in Glioblastoma; NCT00943826), CENTRIC
(Cilengitide, Temozolomide, and Radiation Therapy in Treating Patients With
Newly Diagnosed Glioblastoma and Methylated Gene Promoter Status; NCT00689221),
CORE (Cilengitide, Temozolomide, and Radiation Therapy in Treating Patients With
Newly Diagnosed Glioblastoma and Unmethylated Gene Promoter Status;
NCT00813943), and Radiation Therapy Oncology Group 0825 (NCT00884741).
Progression-free survival (PFS) and overall survival (OS) were compared between:
(1) any VPA use and no VPA use at baseline or (2) VPA use both at start of and
still after chemoradiotherapy. Results of Cox regression models stratified by
trial and adjusted for baseline prognostic factors were analyzed. The same
analyses were performed with levetiracetam (LEV).
RESULTS: VPA use at start of chemoradiotherapy was not associated with improved
PFS or OS compared with all other patients pooled (PFS: hazard ratio [HR], 0.91;
95% CI, 0.77 to 1.07; P = .241; OS: HR, 0.96; 95% CI, 0.80 to 1.15; P = .633).
Furthermore, PFS and OS of patients taking VPA both at start of and still after
chemoradiotherapy were not different from those without antiepileptic drug use
at both time points (PFS: HR, 0.92; 95% CI, 0.74 to 1.15; P = .467; OS: HR,
1.10; 95% CI, 0.86 to 1.40; P = .440). Similarly, no association with improved
outcomes was observed for LEV use.
CONCLUSION: The results of this analysis do not justify the use of VPA or LEV
for reasons other than seizure control in patients with newly diagnosed
glioblastoma outside clinical trials. Glioblastoma (GBM) is the most common and aggressive type of primary brain
neoplasm. The current standard therapy for GBM consists of maximal surgical
resection within safe limits, followed by radiation therapy (RT) and
chemotherapy with temozolomide. Despite advances in treatment, the prognosis of
GBM remains poor. Epileptic seizure is one of the most common symptoms in
patients with GBM. Valproic acid (VPA), a histone deacetylase inhibitor, is
often used as an anti-epileptic drug in patients with brain neoplasms due to its
effectiveness and low toxicity profile. Several in vivo and in vitro studies
have indicated that VPA has radiosensitizing effects for gliomas and
radioprotective influence on normal brain tissue or hippocampal neurons. The
results of several retrospective studies have also indicated potential benefit
to improve survival of patients with GBM. Moreover, the promising treatment
results of a phase 2 trial of concurrent radiation therapy, temozolomide, and
VPA for patients with GBM have been recently reported. The use of VPA in
patients with GBM has thus recently receiving more attention. In this article,
we review the role of VPA in radiation therapy for GBM, focusing on the clinical
evidence. |
What is the effect of the direct interaction of Ikaros and Foxp1 in B-lymphocytes? | Direct interaction of Ikaros and Foxp1 modulates expression of the G protein-coupled receptor G2A in B-lymphocytes and acute lymphoblastic leukemia. | Ikaros and Foxp1 are transcription factors that play key roles in normal
lymphopoiesis and lymphoid maligcies. We describe a novel physical and
functional interaction between the proteins, which requires the central zinc
finger domain of Ikaros. The Ikaros-Foxp1 interaction is abolished by deletion
of this region, which corresponds to the IK6 isoform that is commonly associated
with high-risk acute lymphoblastic leukemia (ALL). We also identify the Gpr132
gene, which encodes the orphan G protein-coupled receptor G2A, as a novel target
for Foxp1. Increased expression of Foxp1 enhanced Gpr132 transcription and
caused cell cycle changes, including G2 arrest. Co-expression of wild-type
Ikaros, but not IK6, displaced Foxp1 binding from the Gpr132 gene, reversed the
increase in Gpr132 expression and inhibited G2 arrest. Analysis of primary ALL
samples revealed a significant increase in GPR132 expression in IKZF1-deleted
BCR-ABL negative patients, suggesting that levels of wild-type Ikaros may
influence the regulation of G2A in B-ALL. Our results reveal a novel effect of
Ikaros haploinsufficiency on Foxp1 functioning, and identify G2A as a potential
modulator of the cell cycle in Ikaros-deleted B-ALL. |
Which syndrome is caused by deletion of Pds5b in mice? | Mice lacking sister chromatid cohesion protein Pds5b exhibit developmental abnormalities reminiscent of Cornelia de Lange syndrome. | PDS5B is a sister chromatid cohesion protein that is crucial for faithful
segregation of duplicated chromosomes in lower organisms. Mutations in cohesion
proteins are associated with the developmental disorder Cornelia de Lange
syndrome (CdLS) in humans. To delineate the physiological roles of PDS5B in
mammals, we generated mice lacking PDS5B (APRIN). Pds5B-deficient mice died
shortly after birth. They exhibited multiple congenital anomalies, including
heart defects, cleft palate, fusion of the ribs, short limbs, distal colon
aganglionosis, abnormal migration and axonal projections of sympathetic neurons,
and germ cell depletion, many of which are similar to abnormalities found in
humans with CdLS. Unexpectedly, we found no cohesion defects in Pds5B(-/-) cells
and detected high PDS5B expression in post-mitotic neurons in the brain. These
results, along with the developmental anomalies of Pds5B(-/-) mice, the presence
of a DNA-binding domain in PDS5B in vertebrates and its nucleolar localization,
suggest that PDS5B and the cohesin complex have important functions beyond their
role in chromosomal dynamics. Cornelia de Lange syndrome (CdLS), a disorder caused by mutations in cohesion
proteins, is characterized by multisystem developmental abnormalities. PDS5, a
cohesion protein, is important for proper chromosome segregation in lower
organisms and has two homologues in vertebrates (PDS5A and PDS5B). Pds5B mutant
mice have developmental abnormalities resembling CdLS; however the role of Pds5A
in mammals and the association of PDS5 proteins with CdLS are unknown. To
delineate genetic interactions between Pds5A and Pds5B and explore mechanisms
underlying phenotypic variability, we generated Pds5A-deficient mice. Curiously,
these mice exhibit multiple abnormalities that were previously observed in
Pds5B-deficient mice, including cleft palate, skeletal patterning defects,
growth retardation, congenital heart defects and delayed migration of enteric
neuron precursors. They also frequently display renal agenesis, an abnormality
not observed in Pds5B(-/-) mice. While Pds5A(-/-) and Pds5B(-/-) mice die at
birth, embryos harboring 3 mutant Pds5 alleles die between E11.5 and E12.5 most
likely of heart failure, indicating that total Pds5 gene dosage is critical for
normal development. In addition, characterization of these compound
homozygous-heterozygous mice revealed a severe abnormality in lens formation
that does not occur in either Pds5A(-/-) or Pds5B(-/-) mice. We further
identified a functional missense mutation (R1292Q) in the PDS5B DNA-binding
domain in a familial case of CdLS, in which affected individuals also develop
megacolon. This study shows that PDS5A and PDS5B functions other than those
involving chromosomal dynamics are important for normal development, highlights
the sensitivity of key developmental processes on PDS5 signaling, and provides
mechanistic insights into how PDS5 mutations may lead to CdLS. |
What is the inheritance of Barth syndrome? | Barth syndrome (BTHS) has an X-linked recessive pattern of inheritance. | Barth syndrome is an X-linked disorder characterised by cardioskeletal myopathy
of variable severity usually fatal in childhood, and neutropenia. We ascertained
a large pedigree with affected males in 3 generations. All affected males had
dilated cardiomyopathy, with endocardial fibroelastosis (EFE) in some. The locus
for Barth syndrome in this family was found to be closely linked to DXS52 (z =
2.78, theta = 0.0). The family was nonrecombit for DXS52 in distal Xq28, but
recombit for DXS374 which maps proximal to DXS52. This localised Barth
syndrome distal to DXS374, confirming a previous localisation to distal Xq28. As
yet there is no evidence for genetic heterogeneity of Barth syndrome. Barth syndrome is an X-linked cardiomyopathy with neutropenia and
3-methylglutaconic aciduria. Recently, mutations in the G4.5 gene, located in
Xq28, have been described in four probands with Barth syndrome. We have now
evaluated 14 Barth syndrome pedigrees for mutations in G4.5 and have identified
unique mutations in all, including four splice-site mutations, three deletions,
one insertion, five missense mutations, and one nonsense mutation. Nine of the
14 mutations are predicted to significantly disrupt the protein products of
G4.5. The occurrence of missense mutations in exons 3 and 8 suggests that these
exons encode essential portions of the G4. 5 proteins, whose functions remain
unknown. We found no correlation between the location or type of mutation and
any of the clinical or laboratory abnormalities of Barth syndrome, which
suggests that additional factors modify the expression of the Barth phenotype.
The characterization of mutations of the G4.5 gene will be useful for carrier
detection, genetic counseling, and the identification of patients with Barth
syndrome who do not manifest all of the cardinal features of this disorder. BACKGROUND: Most cardiomyopathies recognizable in utero are the dilated
type-with dilated, poorly contractile left ventricle. We propose a diagnostic
criterion for the rare spongiform (noncompacted) cardiomyopathy.
CASES: Three perinatal cases with echocardiography and autopsy are presented.
The apical ventricular myocardium was thickened and markedly trabeculated. The
ventricles were not dilated in two, and the atria were enlarged in all. Hydrops
and bradycardia were present in all three despite normal or only mildly
diminished contractility. Although the cardiomyopathy was familial in two
siblings, two of three cases were female, ruling out Barth syndrome (with
sex-linked recessive inheritance). Although all three of our cases with hydrops
died, rare survivors have been reported in the eighth decade.
CONCLUSION: Although spongiform cardiomyopathy may eventually develop into a
dilated cardiomyopathy, its early characteristic is relatively diagnostic: a
restrictive cardiomyopathy with no enlargement of the ventricles and prominent
atria. Barth syndrome is an X-linked cardiac and skeletal mitochondrial myopathy. Barth
syndrome may be due to lipid alterations because the product of the mutated gene
is homologous to phospholipid acyltransferases. Here we document that a single
mitochondrial phospholipid species, tetralinoleoyl-cardiolipin, was lacking in
the skeletal muscle (n = 2), right ventricle (n = 2), left ventricle (n = 2),
and platelets (n = 6) of 8 children with Barth syndrome.
Tetralinoleoyl-cardiolipin is specifically enriched in normal skeletal muscle
and the normal heart. These findings support the notion that Barth syndrome is
caused by alterations of mitochondrial lipids. Isolated noncompaction of the ventricular myocardium (INVM, MIM 300183 and
604169) is a congenital unclassified cardiomyopathy with numerous prominent
trabeculations and deep intertrabecular recesses in a hypertrophied and
hypokinetic myocardium. Mutations in the G4.5 gene result in a wide spectrum of
severe infantile X-linked cardiomyopathic phenotypes including Barth syndrome
with dilated cardiomyopathy and INVM. Molecular genetic analysis of INVM has
only been performed in pediatric patients. Although adult INVM patients show
similar cardiac abnormalities, the influence of genetic factors, especially of
mutations in G4.5, is unknown. We analyzed 25 adult INVM patients for the
presence of mutations in the G4.5 gene and performed a pedigree analysis of
probands. Mutations were not found in the coding sequence or splice sites of
G4.5. Systematic analysis of relatives from seven of nine probands showed
multiple affected members consistent with an autosomal domit pattern of
inheritance in the majority of cases. We conclude that INVM in the adult is an
autosomal domit disorder rarely caused by mutations in G4.5 and therefore
genetically distinct from infantile X-linked cases. OBJECTIVE: Barth syndrome, an X-linked disorder that is characterized by
cardiomyopathy, neutropenia, skeletal myopathy, and growth delay, is caused by
mutations in the taffazin gene at Xq28 that result in cardiolipin deficiency and
abnormal mitochondria. The clinical phenotype in Barth syndrome has not been
characterized systematically, and the condition may be underrecognized. We
sought to evaluate extent of cardioskeletal myopathy, potential for arrhythmia,
delays in growth, and biochemical correlates of disease severity in patients
with this disorder.
METHODS: We conducted an observational, cross-sectional study of the largest
cohort of patients with Barth syndrome to date (n = 34; age range: 1.2-22.6
years). Evaluation included echocardiography, electrocardiography (standard and
signal-averaged), microvolt T wave alters analysis, biochemical and
hematologic laboratory analyses, and physical therapy evaluation of skeletal
myopathy.
RESULTS: Family history was positive for confirmed or suspected Barth syndrome
in 63%. Ninety percent of patients had a clinical history of cardiomyopathy
(mean age at diagnosis of cardiomyopathy: 5.5 months; at genetic confirmation of
Barth syndrome: 4.6 years). Echocardiography revealed a mean ejection fraction
of 50% +/- 10%, mean fractional shortening of 28% +/- 5%, and mean left
ventricular end-diastolic volume z score of 1.9 +/- 1.8. Left ventricular
morphology demonstrated increased trabeculations or true noncompaction in 53%.
Of 16 patients who were evaluated at > or = 11 years of age, 7 (43%) had
documented ventricular arrhythmia. Growth deficiency was present (mean weight
percentile: 15%; mean height percentile: 8%). Laboratory analysis revealed low
total white blood cell count (absolute count: < 4000 cells per microL) in 25% of
those who were not on granulocyte colony-stimulating factor. Hypocholesterolemia
was present in 24%, decreased low-density lipoprotein cholesterol in 56%, low
prealbumin in 79%, and mildly elevated creatine kinase in 15%.
CONCLUSIONS: Our cohort demonstrated clinical variability, but most had
cardiomyopathy and diminished growth velocity, with a propensity toward
neutropenia and low cholesterol. There was increased incidence of ventricular
arrhythmia, predomitly in adolescents and young adults. Barth syndrome should
be considered when boys present with cardiomyopathy, especially when associated
with increased left ventricular trabeculations, neutropenia, skeletal muscle
weakness, or family history indicating an X-linked pattern of inheritance. Mutations in the human TAZ gene are associated with Barth Syndrome, an often
fatal X-linked disorder that presents with cardiomyopathy and neutropenia. The
TAZ gene encodes Tafazzin, a putative phospholipid acyltranferase that is
involved in the remodeling of cardiolipin, a phospholipid unique to the inner
mitochondrial membrane. It has been shown that the disruption of the Tafazzin
gene in yeast (Taz1) affects the assembly and stability of respiratory chain
Complex IV and its supercomplex forms. However, the implications of these
results for Barth Syndrome are restricted due to the additional presence of
Complex I in humans that forms a supercomplex with Complexes III and IV. Here,
we investigated the effects of Tafazzin, and hence cardiolipin deficiency in
lymphoblasts from patients with Barth Syndrome, using blue-native polyacrylamide
gel electrophoresis. Digitonin extraction revealed a more labile Complex
I/III(2)/IV supercomplex in mitochondria from Barth Syndrome cells, with Complex
IV dissociating more readily from the supercomplex. The interaction between
Complexes I and III was also less stable, with decreased levels of the Complex
I/III(2) supercomplex. Reduction of Complex I holoenzyme levels was observed
also in the Barth Syndrome patients, with a corresponding decrease in
steady-state subunit levels. We propose that the loss of mature cardiolipin
species in Barth Syndrome results in unstable respiratory chain supercomplexes,
thereby affecting Complex I biogenesis, respiratory activities and subsequent
pathology. OBJECTIVE: Barth syndrome is a rare, X-linked recessive disorder that affects
only boys. The cardinal characteristics include growth retardation,
cardioskeletal myopathy, chronic or cyclic neutropenia, and 3-methylglutaconic
aciduria. A preliminary study of five young boys with Barth syndrome suggested a
distinct cognitive phenotype.
METHODS: The present study was designed to explore whether additional evidence
for a cognitive phenotype emerged from a larger sample. A psychoeducational
assessment battery was administered to 15 boys with Barth syndrome. Data from
these boys were compared to data from 15 typically developing boys individually
matched on age and grade in school to each of the 15 boys with Barth syndrome.
RESULTS: Although boys with Barth syndrome had age-appropriate performance on
all measures of reading-related skills, their performance on mathematics and
visual spatial tasks was significantly lower than that of boys in the comparison
group. Moreover, specific aspects of visual short-term memory also differed from
available norms.
CONCLUSION: Our findings support the validity of the preliminary findings and
reflect a higher incidence of cognitive difficulties in boys with Barth syndrome
relative to boys in the comparison group. Coupled with the fatigue regularly
experienced by boys with Barth syndrome, our findings indicate that educational
support should be implemented during the early school-age years for children
with Barth syndrome. This is a report of a child who died at 20 months from what was clinically
thought to be cardiomyopathy of unknown etiology. Barth syndrome, an X-linked
mitochondrial cardioskeletal myopathy, was diagnosed by genetic testing at
autopsy. Barth syndrome presents in infancy or childhood with cardiomyopathy,
hypotonia, growth delays, and cyclic neutropenia. Other associated laboratory
findings can include hypocholesterolemia, relative monocytosis, low prealbumin,
low plasma carnitine, and lactic acidosis. The classic echocardiogram finding is
left ventricular noncompaction, although not always present. Until recently, the
most reliable biochemical finding has been 3-methylglutaconic aciduria. However,
quantitative analysis must be specifically requested for results to be reliable.
Recently, a confirmatory tetralinoleoyl cardiolipin high-pressure liquid
chromotography-tandem mass spectrometry blood test has become available. Genetic
testing is also confirmatory and details the underlying mutation. Diagnosis is
often missed or delayed and early diagnosis improves survival. The purpose of
this case report is to encourage physicians to include Barth syndrome in the
differential for cardiomyopathy of uncertain etiology in males, especially in
the presence of growth delays, hypotonia, neutropenia, and/or family history of
pediatric male death of unknown etiology. Barth syndrome (BTHS) is an X-linked disorder characterized by skeletal
myopathy, neutropenia, growth delay, and cardiomyopathy. It is caused by
mutations in the tafazzin gene (TAZ). Although early diagnosis is critical to
prevent the progression of heart failure, this disease remains unrecognized when
heart failure is not clinically significant. Here we report on a 13-year-old boy
with no family history of BTHS who was diagnosed with the syndrome in the
subclinical stage of heart failure. The clues to the diagnosis of BTHS in this
patient were the findings of lipid storage myopathy in the skeletal muscle
biopsy, elevated plasma brain natriuretic peptide, and the diagnosis of isolated
noncompaction of the ventricular myocardium in echocardiography. Genetic studies
of TAZ revealed a disease-causing mutation (p.Gly216Arg) in this patient.
Physicians should be aware of the possibility of this disease and carry out
genetic studies when it is considered. Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the
assembly of respiratory chain complexes, favoring full-yield operation. It also
mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked
cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain
modifications and are present at abnormally low concentrations, whereas
monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth
syndrome patients, we showed that the production of abnormal cardiolipin led to
mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes
in electron transport chain stability, resulting in cellular defects. We found a
destabilization of the supercomplex (respirasome) I+III2+IVn but also decreased
amounts of individual complexes I and IV and supercomplexes I+III and III+IV. No
changes were observed in the amounts of individual complex III and complex II.
We also found decreased levels of complex V. This complex is not part of the
supercomplex suggesting that cardiolipin is required not only for the
association/stabilization of the complexes into supercomplexes but also for the
modulation of the amount of individual respiratory chain complexes. However,
these alterations were compensated by an increase in mitochondrial mass, as
demonstrated by electron microscopy and measurements of citrate synthase
activity. We suggest that this compensatory increase in mitochondrial content
prevents a decrease in mitochondrial respiration and ATP synthesis in the cells.
We also show, by extensive flow cytometry analysis, that the type II apoptosis
pathway was blocked at the mitochondrial level and that the mitochondria of
patients with Barth syndrome cannot bind active caspase-8. Signal transduction
is thus blocked before any mitochondrial event can occur. Remarkably, basal
levels of superoxide anion production were slightly higher in patients' cells
than in control cells as previously evidenced via an increased protein
carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells
in the long term. The consequences of mitochondrial dysfunction and alterations
to apoptosis signal transduction are considered in light of the potential for
the development of future treatments. Barth syndrome (BTHS) is a genetic, X-linked, rare but often fatal, pediatric
skeletal- and cardiomyopathy occurring due to mutations in the tafazzin gene
(TAZ). TAZ encodes a transacylase involved in phospholipid biosynthesis, also
called tafazzin, which is responsible for remodeling the inner mitochondrial
membrane phospholipid, cardiolipin (CL). Tafazzin mutations lead to
compositional alterations in CL molecular species, causing extensive
mitochondrial aberrations and ultrastructural muscle damage. There are no
specific treatments or cure for BTHS. Current therapy is largely palliative and
aimed at treatment of organ-specific complications during disease progression.
Polypharmacy frequently occurs during treatment and may lead to severe adverse
events. Adverse reactions may originate from exogenous factors such as the
inadvertent co-administration of contraindicated drugs. Theoretically,
endogenous factors such as polymorphic variations in genes encoding drug
metabolizing enzymes may also precipitate fatal toxicity. Investigation of the
consequences of pharmacogenomic variations on BTHS therapy is lacking. To our
knowledge, this review presents the first examination of the possible sources of
pharmacogenomic variations that may affect BTHS therapy. We also explore
BTHSspecific patents for possible treatment options. The patents discussed
suggest innovative strategies for treatment, including feeding linoleic acid to
patients to overcome compositional CL deficiency; or the use of 2S,4R
ketoconazole formulations to augment CL levels; or the delivery of mitochondrial
stabilizing cargo. Future research directions are also discussed. Barth syndrome (BTHS) is an X-linked recessive disease primarily affecting
males. Clinically, the disease is characterized by hypertrophic or dilated
cardiomyopathy, skeletal myopathy, chronic/cyclic neutropenia,
3-methylglutaconic aciduria, growth retardation and respiratory chain
dysfunction. It is caused by mutations in the TAZ gene coding for the tafazzin
protein which is responsible for cardiolipin remodeling. In this work, we
present a novel pathogenic TAZ mutation c.83T>A, p.Val28Glu, found in mosaic
form in almost all female members of a Polish family. Sanger sequencing of DNA
from peripheral blood and from epithelial cells showed female mosaicism in three
generations. This appears to be a new mechanism of inheritance and further
research is required in order to understand the mechanism of this mosaicism. We
conclude that BTHS genetic testing should include two or more tissues for women
that appear to be noncarriers when blood DNA is initially tested. The results of
our study should not only be applicable to BTHS families, but also to families
with other X-linked diseases. |
Is Cryptococcus neoformans a frequent cause of isolated skin infections in immunocompromised individuals | Primary cutaneous cryptococcosis (PCC) without systemic infection is rare. | The authors report a male patient, a seller with no detected immunosuppression,
with an extensive ulcerated skin lesion localized on the left forearm, caused by
Cryptococcus neoformans var. gattii serotype B. Oral treatment with fluconazole
was successful. A review of the literature showed the rarity of this
localization in HIV-negative patients. In contrast, skin lesions frequently
occurs in HIV-positive patients, with Cryptococcus neoformans var. neoformans
serotype A predominating as the etiological agent. In this paper, the
pathogenicity of C. neoformans to skin lesions in patients immunocompromised or
not, is discussed, showing the efficacy of fluconazole for the treatment of
these processes. Cryptococcus neoformans is an encapsulated yeast that can cause primary
pulmonary infections or disseminate and cause infections of the central nervous
system, meninges, skin, and bone in the immunocompromised host. We present here
an unusual case of an immunocompetent patient who had laryngitis due to C.
neoformans that mimicked a laryngeal carcinoma on clinical examination and
imaging studies. OBJECTIVE: Cryptococcus is an opportunistic yeast with a worldwide distribution
that primarily causes significant infections in immunocompromised individuals,
generally by affecting the respiratory tract. But primary cutaneous
cryptococcosis (PCC) without systemic infection is rare. We report a case of PCC
in a patient with nephrotic syndrome.
METHODS: The 23-year-old man developed severe necrotising cellulitis on both the
anterior and posterior of his trunk following a massage. He had been treated
with systemic corticosteroids over 20 months for nephrotic syndrome. A skin
biopsy of the wound area revealed cutaneous vasculitis and chronic inflammation
with yeast-like organisms. Periodic acid-Schiff (PAS) staining indicated that
the structures were consistent with Cryptococcus. A Cryptococcus neoformans
infection was confirmed by culture. Azole therapy was begun, and the skin ulcers
gradually stopped disseminating. However, the patient died following continuous
capillary haemorrhage on the 22 day since admission.
CONCLUSION: Cryptococcus is crucial to be considered in the differential
diagnosis of subcutaneous necrosis in any patient on immunosuppressive therapy. Cryptococcus is a ubiquitous fungus and is known for causing meningitis and
cutaneous infections in immunocompromised individuals. Disseminated cryptococcal
infection is very rare and almost always found to occur in immunocompromised
individuals especially in persons infected with HIV. This is particularly
attributed to its capsulated spores. But there are few reported cases in which
it has been found to cause disseminated infections even in immunocompetent
individuals. We report a similar case of disseminated cryptococcal infection in
an immunocompetent host. Early detection and treatment of disseminated
cryptococcosis is essential to reduce morbidity and for better outcome. |
What is the mechanism of action of Pictilisib? | Pictilisib acts by inhibiting PI3K. It is used for breast cancer treatment. | PURPOSE: This first-in-human dose-escalation trial evaluated the safety,
tolerability, maximal-tolerated dose (MTD), dose-limiting toxicities (DLT),
pharmacokinetics, pharmacodynamics, and preliminary clinical activity of
pictilisib (GDC-0941), an oral, potent, and selective inhibitor of the class I
phosphatidylinositol-3-kinases (PI3K).
PATIENTS AND METHODS: Sixty patients with solid tumors received pictilisib at 14
dose levels from 15 to 450 mg once-daily, initially on days 1 to 21 every 28
days and later, using continuous dosing for selected dose levels.
Pharmacodynamic studies incorporated (18)F-FDG-PET, and assessment of
phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and
tumor tissue.
RESULTS: Pictilisib was well tolerated. The most common toxicities were grade
1-2 nausea, rash, and fatigue, whereas the DLT was grade 3 maculopapular rash
(450 mg, 2 of 3 patients; 330 mg, 1 of 7 patients). The pharmacokinetic profile
was dose-proportional and supported once-daily dosing. Levels of phosphorylated
serine-473 AKT were suppressed >90% in PRP at 3 hours after dose at the MTD and
in tumor at pictilisib doses associated with AUC >20 h·μmol/L. Significant
increase in plasma insulin and glucose levels, and >25% decrease in (18)F-FDG
uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A
patient with V600E BRAF-mutant melanoma and another with platinum-refractory
epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification
demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively.
CONCLUSION: Pictilisib was safely administered with a dose-proportional
pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100
mg and signs of antitumor activity. The recommended phase II dose was continuous
dosing at 330 mg once-daily. The PI3K inhibitor pictilisib plus anastrozole suppresses luminal B breast
cancer proliferation. Chronic myeloid leukemia (CML) patients who relapse on imatinib due to acquired
ABL1 kinase domain mutations are successfully treated with second-generation
ABL1-tyrosine kinase inhibitors (ABL-TKIs) such as dasatinib, nilotinib or
ponatinib. However, ~40% of relapsed patients have uncharacterized BCR-ABL1
kinase-independent mechanisms of resistance. To identify these mechanisms of
resistance and potential treatment options, we generated ABL-TKI-resistant K562
cells through prolonged sequential exposure to imatinib and dasatinib.
Dual-resistant K562 cells lacked BCR-ABL1 kinase domain mutations, but acquired
other genomic aberrations that were characterized by next-generation sequencing
and copy number analyses. Proteomics showed that dual-resistant cells had
elevated levels of FOXO1, phospho-ERK and BCL-2, and that dasatinib no longer
inhibited substrates of the PI3K/AKT pathway. In contrast to parental cells,
resistant cells were sensitive to growth inhibition and apoptosis induced by the
class I PI3K inhibitor, GDC-0941 (pictilisib), which also induced FOXO1 nuclear
translocation. FOXO1 was elevated in a subset of primary specimens from relapsed
CML patients lacking BCR-ABL1 kinase domain mutations, and these samples were
responsive to GDC-0941 treatment ex vivo. We conclude that elevated FOXO1
contributes to BCR-ABL1 kinase-independent resistance experienced by these CML
patients and that PI3K inhibition coupled with BCR-ABL1 inhibition may represent
a novel therapeutic approach. PI3K plays a key role in cellular metabolism and cancer. Using a mass
spectrometry-based metabolomics platform, we discovered that plasma
concentrations of 26 metabolites, including amino acids, acylcarnitines, and
phosphatidylcholines, were decreased in mice bearing PTEN-deficient tumors
compared with non-tumor-bearing controls and in addition were increased
following dosing with class I PI3K inhibitor pictilisib (GDC-0941). These
candidate metabolomics biomarkers were evaluated in a phase I dose-escalation
clinical trial of pictilisib. Time- and dose-dependent effects were observed in
patients for 22 plasma metabolites. The changes exceeded baseline variability,
resolved after drug washout, and were recapitulated on continuous dosing. Our
study provides a link between modulation of the PI3K pathway and changes in the
plasma metabolome and demonstrates that plasma metabolomics is a feasible and
promising strategy for biomarker evaluation. Also, our findings provide
additional support for an association between insulin resistance, branched-chain
amino acids, and related metabolites following PI3K inhibition. Mol Cancer Ther;
15(6); 1412-24. ©2016 AACR. BACKGROUND: Inhibition of phosphatidylinositol 3-kinase (PI3K) is a promising
approach to overcome resistance to endocrine therapy in breast cancer.
Pictilisib is an oral inhibitor of multiple PI3K isoforms. The aim of this study
is to establish if addition of pictilisib to fulvestrant can improve
progression-free survival in oestrogen receptor-positive, endocrine-resistant
breast cancer.
METHODS: In this two-part, randomised, double-blind, placebo-controlled, phase 2
study, we recruited postmenopausal women aged 18 years or older with oestrogen
receptor-positive, HER2-negative breast cancer resistant to treatment with an
aromatase inhibitor in the adjuvant or metastatic setting, from 123 medical
centres across 21 countries. Part 1 included patients with or without PIK3CA
mutations, whereas part 2 included only patients with PIK3CA mutations. Patients
were randomly allocated (1:1 in part 1 and 2:1 in part 2) via a
computer-generated hierarchical randomisation algorithm to daily oral pictilisib
(340 mg in part 1 and 260 mg in part 2) or placebo starting on day 15 of cycle
1, plus intramuscular fulvestrant 500 mg on day 1 and day 15 of cycle 1 and day
1 of subsequent cycles in both groups. In part 1, we stratified patients by
presence or absence of PIK3CA mutation, primary or secondary aromatase inhibitor
resistance, and measurable or non-measurable disease. In part 2, we stratified
patients by previous aromatase inhibitor treatment for advanced or metastatic
disease or relapse during or within 6 months of an aromatase inhibitor treatment
in the adjuvant setting and measurable or non-measurable disease. All patients
and those administering treatment and assessing outcomes were masked to
treatment assignment. The primary endpoint was progression-free survival in the
intention-to-treat population for both parts 1 and 2 and also separately in
patients with PIK3CA-mutated tumours in part 1. Tumour assessment (physical
examination and imaging scans) was investigator-assessed and done at screening
and after 8 weeks, 16 weeks, 24 weeks, and 32 weeks of treatment from day 1 of
cycle 1 and every 12 weeks thereafter. We assessed safety in as-treated patients
who received at least one dose of study medication. This trial is registered
with ClinicalTrials.gov, number NCT01437566.
FINDINGS: In part 1, between Sept 27, 2011, and Jan 11, 2013, we randomly
allocated 168 patients to the pictilisib (89 [53%]) or placebo (79 [47%]) group.
In part 2, between March 18, 2013, and Jan 2, 2014, we randomly allocated 61
patients to the pictilisib (41 [67%]) or placebo (20 [33%]) group. In part 1, we
found no difference in median progression-free survival between the pictilisib
(6·6 months [95% CI 3·9-9·8]) and placebo (5·1 months [3·6-7·3]) group (hazard
ratio [HR] 0·74 [95% CI 0·52-1·06]; p=0·096). We also found no difference when
patients were analysed according to presence (pictilisib 6·5 months [95% CI
3·7-9·8] vs placebo 5·1 months [2·6-10·4]; HR 0·73 [95% CI 0·42-1·28]; p=0·268)
or absence (5·8 months [3·6-11·1] vs 3·6 months [2·8-7·3]; HR 0·72 [0·42-1·23];
p=0·23) of PIK3CA mutation. In part 2, we also found no difference in
progression-free survival between groups (5·4 months [95% CI 3·8-8·3] vs 10·0
months [3·6-13·0]; HR 1·07 [95% CI 0·53-2·18]; p=0·84). In part 1, grade 3 or
worse adverse events occurred in 54 (61%) of 89 patients in the pictilisib group
and 22 (28%) of 79 in the placebo group. 19 serious adverse events related to
pictilisib treatment were reported in 14 (16%) of 89 patients. Only one (1%) of
79 patients reported treatment-related serious adverse events in the placebo
group. In part 2, grade 3 or worse adverse events occurred in 15 (36%) of 42
patients in the pictilisib group and seven (37%) of 19 patients in the placebo
group. Four serious adverse events related to pictilisib treatment were reported
in two (5%) of 42 patients. One treatment-related serious adverse event occurred
in one (5%) of 19 patients in the placebo group.
INTERPRETATION: Although addition of pictilisib to fulvestrant did not
significantly improve progression-free survival, dosing of pictilisib was
limited by toxicity, potentially limiting its efficacy. For future assessment of
PI3K inhibition as an approach to overcome resistance to hormonal therapy,
inhibitors with greater selectivity than that of pictilisib might be needed to
improve tolerability and potentially increase efficacy. No further investigation
of pictilisib in this setting is ongoing.
FUNDING: F Hoffmann-La Roche. BACKGROUND: The phosphatidylinositol-3-kinase/mammalian target of rapamycin
(PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and
mTOR inhibitors have been developed and have now reached clinical trials.
Similarly, CDKs have been investigated as cancer drug targets.
METHODS: We have synthesised and characterised a series of 6-aminopyrimidines
identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2.
Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and
signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell
lines, and in vivo HT29 efficacy studies.
RESULTS: 2,6-Diaminopyrimidines with an O(4)-cyclohexylmethyl substituent and a
C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were
growth inhibitory (GI50<20 μM). Compound 1, but not 2 or 5, potently inhibits
CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory
concentrations. Consistent with kinase inhibition data, compound 1 reduced
phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2
inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic
growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29
tumour growth inhibition in vivo.
CONCLUSIONS: These studies identified a novel series of mixed CDK2/PI3K
inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in
enhanced antitumour activity. Pictilisib (GDC-0941) is an oral class I phosphatidylinositol-3-phosphate kinase
inhibitor. This phase Ia/Ib study investigated the safety, tolerability,
pharmacokinetics, and pharmacodynamics of pictilisib in monotherapy or in
combination with carboplatin-paclitaxel and bevacizumab (CP + BEV) in Japanese
patients with advanced solid tumors or non-squamous non-small cell lung cancer.
A standard 3 + 3 dose escalation design was applied. In stage 1, 140, 260, or
340 mg/day of pictilisib was administered once daily to 12 patients with
advanced solid tumors. In stage 2, 260 or 340 mg/day of pictilisib was
administered in combination with CP + BEV to 7 patients with advanced
non-squamous non-small cell lung cancer. In stage 1, 1 of 6 patients in the
340 mg/day cohort exhibited dose limiting toxicity (DLT) of grade 3
maculopapular rash. The maximum plasma concentration and area under the curve of
pictilisib were dose-dependent. A reduction in phosphorylated AKT in platelet
rich plasma was observed. No patient had an objective anti-tumor response. In
stage 2, DLT was observed in 1 of 3 patients in the 260 mg/day cohort (grade 3
febrile neutropenia), and 2 of 4 patients in the 340 mg/day cohort (1 each of
grade 3 febrile neutropenia and grade 3 febrile neutropenia/erythema
multiforme). Partial responses were observed in 3 out of 7 patients. In
conclusion, pictilisib was shown to have good safety and tolerability in
Japanese patients with advanced solid tumors. A recommended dose of pictilisib
in monotherapy was determined to be 340 mg once daily. For combination with
CP + BEV, tolerability up to 260 mg/day was confirmed. BACKGROUND: Approximately 40% of hormone receptor-positive, HER2-negative breast
cancers (BCs) are associated with activating mutations of the
phosphatidylinositol 3-kinase (PI3K) pathway. Pictilisib, a potent and highly
specific class I pan-PI3K inhibitor, demonstrated preclinical activity in BC
cell lines and may potentiate the effect of taxanes, benefiting patients with or
without aberrant activation of the PI3K pathway. PEGGY (NCT01740336), a
randomised, placebo-controlled phase II trial, examined whether pictilisib
augments the anti-tumour activity of paclitaxel in patients with hormone
receptor-positive, HER2-negative locally recurrent or metastatic BC (mBC). We
report results from the protocol-specified interim analysis.
PATIENTS AND METHODS: One hundred and eighty-three eligible patients were
randomised (1:1) to receive paclitaxel (90 mg/m2 weekly for 3 weeks in every
28-day cycle) with either 260 mg pictilisib or placebo (daily on days 1-5 every
week). The primary end point was progression-free survival (PFS) in the
intention-to-treat (ITT) population and patients with PIK3CA-mutated tumours.
Secondary end points included overall response rate (ORR), duration of response,
and safety.
RESULTS: In the ITT population, the median PFS was 8.2 months with pictilisib (n
= 91) versus 7.8 months with placebo (n = 92) [hazard ratio (HR) for progression
or death, 0.95; 95% confidence interval (CI) 0.62-1.46; P = 0.83]. In patients
with PIK3CA-mutated tumours, the median PFS was 7.3 months for pictilisib (n =
32) versus 5.8 months with placebo (n = 30) (HR, 1.06; 95% CI 0.52-2.12; P =
0.88). ORR was similar between treatment arms. The safety profile of pictilisib
was consistent with previous reports, with no new safety signals. Proportions of
patients with grade ≥3 adverse events (AEs), serious AEs, and dose
reductions/discontinuations due to AEs were higher with pictilisib.
CONCLUSIONS: PEGGY did not meet its primary end point, revealing no significant
benefit from adding pictilisib to paclitaxel for patients with hormone
receptor-positive, HER2-negative locally recurrent or mBC.
CLINICAL TRIAL NUMBER: NCT01740336. |
What are sirtuins? | Seven sirtuins have been identified in humans, and their functions currently surpass their originally identified role as histone deacetylase and chromatin silencers to encompass nutrient sensing and metabolic function. All seven sirtuins require NAD(+) in order to carry out their enzymatic activity, and thus become activated in conditions of nutrient depletion, starvation, and cellular stress. | Sirtuins are NAD-dependent lysine deacylases that play critical roles in
cellular regulation and are implicated in human diseases. Modulators of sirtuins
are needed as tools for investigating their biological functions and possible
therapeutic applications. However, the discovery of sirtuin modulators is
hampered by the lack of efficient sirtuin assays. Here we report an improved
fluorogenic assay for SIRT1, SIRT2, and SIRT3 using a new substrate, a myristoyl
peptide with a C-terminal aminocoumarin. The new assay has several advantages,
including significantly lower substrate concentration needed, increased
signal-to-background ratio, and improved Z'-factor. The novel assay thus will
expedite high-throughput screening of SIRT1, SIRT2, and SIRT3 modulators. The role of sirtuins in age-related diseases is an area of rapidly expanding
investigation. Sirtuins are NAD+ -dependent class III histone deacetylases
(HDACs) that share extensive homologies with the yeast HDAC Sir2. Class I and
class II HDACs inhibitors have been identified as potential anticancer agents
and are in clinical studies, but much less is known about class III HDAC
inhibitors. However, inhibitors of sirtuins are currently being targeted as
potential therapeutic agents for disease such as cancer, neurodegenerative
disease and other disorders as sirtuins are discovered to regulate numerous
downstream enzymes. Given the link between sirtuins and cancer, understanding
the functionality of these enzymes may ultimately have significant impact in
cancer prevention or cancer treatment. This review gives an updated overview
regarding the regulation of sirtuin enzymes, their implications in cancer,
various sirtuin inhibitor scaffolds and their insights in drug design. |
Which miRNA is targeted by SRY/Sox9? | The testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon | Metastasis is the major factor affecting patient survival in ovarian cancer.
However, its molecular mechanisms remain unclear. Our study used isogenic pairs
of low- and high-invasive ovarian cancer cell lines to demonstrate the
downregulation of miRNA-138 in the highly invasive cells, and its functioning as
an inhibitor of cell migration and invasion. An orthotopic xenograft mouse model
further demonstrated that the expression of miRNA-138 inhibited ovarian cancer
metastasis to other organs. Results indicated that miR-138 directly targeted
SRY-related high mobility group box 4 (SOX4) and hypoxia-inducible factor-1α
(HIF-1α), and overexpression of SOX4 and HIF-1α effectively reversed the
miR-138-mediated suppression of cell invasion. Epidermal growth factor receptor
acted as the downstream molecule of SOX4 by way of direct transcriptional
control, whereas Slug was the downstream molecule of HIF-1α by way of
proteasome-mediated degradation. Analysis of human ovarian tumors further
revealed downregulation of miR-138 and upregulation of SOX4 in late-stage
tumors. Patients with miR-138(low)/SOX(high) signature are predomit in late
stage and tend to have maligt phenotypes including lymph nodes metastasis,
larger ascites volume and higher tumor grade. Our study demonstrates the role
and clinical relevance of miR-138 in ovarian cancer cell invasion and
metastasis, providing a potential therapeutic strategy for suppression of
ovarian cancer metastasis by targeting SOX4 and HIF-1α pathways. Recently, the sex determining region Y ( Sry) and the cerebellar
degeneration-related protein 1 ( CDR1as) RNA transcripts have been described to
function as a new class of post-transcriptional regulatory RNAs that behave as
circular endogenous RNA sponges for the micro RNAs (miRNAs) miR-138 and miR-7,
respectively. A special feature of the Sry gene is its ability to generate
linear and circular transcripts, both transcribed in the sense orientation. Here
we remark that both sense (e.g. Sry RNA) and antisense (e.g. CDR1as) transcripts
could circularize and behave as miRNAs sponges, and importantly, that also
protein-coding segments of mRNAs could also assume this role. Thus, it is
reasonable to think that the linear Sry sense transcript could additionally act
as a miRNA sponge, or as an endogenous competing RNA for miR-138. |
Subsets and Splits
No saved queries yet
Save your SQL queries to embed, download, and access them later. Queries will appear here once saved.