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Elaborate on the association between Genomic Regulatory Blocks (GRBs) and target genes | Genomic regulatory blocks (GRBs) are chromosomal regions spanned by highly conserved non-coding elements (HCNEs), most of which serve as regulatory inputs of one target gene in the region. The target genes are most often transcription factors involved in embryonic development and differentiation. GRBs often contain extensive gene deserts, as well as additional 'bystander' genes intertwined with HCNEs but whose expression and function are unrelated to those of the target gene. GRB target genes have properties that set them apart from their bystanders as well as other genes in the genome: longer CpG islands, a higher number and wider spacing of alternative transcription start sites, and a distinct composition of transcription factor binding sites in their core/proximal promoters. Target gene expression correlates with the acetylation state of HCNEs in the region. | We report evidence for a mechanism for the maintece of long-range conserved
synteny across vertebrate genomes. We found the largest mammal-teleost conserved
chromosomal segments to be spanned by highly conserved noncoding elements
(HCNEs), their developmental regulatory target genes, and phylogenetically and
functionally unrelated "bystander" genes. Bystander genes are not specifically
under the control of the regulatory elements that drive the target genes and are
expressed in patterns that are different from those of the target genes.
Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2,
rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the
expression patterns of these genes even if located inside or beyond bystander
genes, suggesting that the regulatory domain of a developmental regulatory gene
can extend into and beyond adjacent transcriptional units. We termed these
chromosomal segments genomic regulatory blocks (GRBs). After whole genome
duplication in teleosts, GRBs, including HCNEs and target genes, were often
maintained in both copies, while bystander genes were typically lost from one
GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy
GRBs of higher vertebrates intact. We show that loss of bystander genes and
other mutational events suffered by duplicated GRBs in teleost genomes permits
target gene identification and HCNE/target gene assignment. These findings
explain the absence of evolutionary breakpoints from large vertebrate
chromosomal segments and will aid in the recognition of position effect
mutations within human GRBs. Insect genomes contain larger blocks of conserved gene order (microsynteny) than
would be expected under a random breakage model of chromosome evolution. We
present evidence that microsynteny has been retained to keep large arrays of
highly conserved noncoding elements (HCNEs) intact. These arrays span key
developmental regulatory genes, forming genomic regulatory blocks (GRBs). We
recently described GRBs in vertebrates, where most HCNEs function as enhancers
and HCNE arrays specify complex expression programs of their target genes. Here
we present a comparison of five Drosophila genomes showing that HCNE density
peaks centrally in large synteny blocks containing multiple genes. Besides
developmental regulators that are likely targets of HCNE enhancers, HCNE arrays
often span unrelated neighboring genes. We describe differences in core
promoters between the target genes and the unrelated genes that offer an
explanation for the differences in their responsiveness to enhancers. We show
examples of a striking correspondence between boundaries of synteny blocks, HCNE
arrays, and Polycomb binding regions, confirming that the synteny blocks
correspond to regulatory domains. Although few noncoding elements are highly
conserved between Drosophila and the malaria mosquito Anopheles gambiae, we find
that A. gambiae regions orthologous to Drosophila GRBs contain an equivalent
distribution of noncoding elements highly conserved in the yellow fever mosquito
Aëdes aegypti and coincide with regions of ancient microsynteny between
Drosophila and mosquitoes. The structural and functional equivalence between
insect and vertebrate GRBs marks them as an ancient feature of metazoan genomes
and as a key to future studies of development and gene regulation. Using a comparative genomics approach to reconstruct the fate of genomic
regulatory blocks (GRBs) and identify exonic remts that have survived the
disappearance of their host genes after whole-genome duplication (WGD) in
teleosts, we discover a set of 38 candidate cis-regulatory coding exons (RCEs)
with predicted target genes. These elements demonstrate evolutionary separation
of overlapping protein-coding and regulatory information after WGD in teleosts.
We present evidence that the corresponding mammalian exons are still under both
coding and non-coding selection pressure, are more conserved than other protein
coding exons in the host gene and several control sets, and share key
characteristics with highly conserved non-coding elements in the same regions.
Their dual function is corroborated by existing experimental data. Additionally,
we show examples of human exon remts stemming from the vertebrate 2R WGD. Our
findings suggest that long-range cis-regulatory inputs for developmental genes
are not limited to non-coding regions, but can also overlap the coding sequence
of unrelated genes. Thus, exonic regulatory elements in GRBs might be
functionally equivalent to those in non-coding regions, calling for a
re-evaluation of the sequence space in which to look for long-range regulatory
elements and experimentally test their activity. |
Which therapeutic interventions for sarcopenia have been applied | The main bulk of experimental pharmacological interventions addressing the clinical problem of frailty have been focused on the use of hormones, as replacement therapy in subjects with low or normal circulating basal levels of the hormone. Results have been disappointing, except for the case of testosterone that have shown some benefits. The effectiveness of other potential therapeutic interventions (antioxidants, anti-inflammatory agents, nutritional supplements) appears to be limited or has not been explored in detail until now. | Frailty is a geriatric syndrome characterized by muscle weakness, sarcopenia,
and fatigue, and is associated with several adverse health outcomes, including
disability. Design of therapeutic interventions for geriatric frailty has been
challenging and may be because of inadequate understanding of its biological
underpinnings. Carnitine is important for energy production in skeletal muscles
and there seems to be a negative correlation between advancing age and muscle
carnitine levels. Carnitine deficiency may therefore contribute to geriatric
frailty. Age-associated carnitine deficiency from a variety of etiologies,
including organic cation transporter (OCTN2) mutation and carnitine
palmitoyltransferase II (CPT) deficiency, may potentially explain the
relationship between carnitine-associated mitochondrial dysfunction and
geriatric frailty. Development of therapeutic agents capable of prevention or
reversal of carnitine deficiency in older adults may minimize the occurrence of
frailty in geriatric populations. Sarcopenia is the loss of skeletal muscle mass and function with aging. Although
the term sarcopenia was first coined in 1989, its etiology is still poorly
understood. Moreover, a consensus for defining sarcopenia continues to elude us.
Sarcopenic changes in the muscle include losses in muscle fiber quantity and
quality, alpha-motor neurons, protein synthesis rates, and anabolic and sex
hormone production. Other factors include basal metabolic rate, increased
protein dietary requirements, and chronic inflammation secondary to age-related
changes in cytokines and oxidative stress. These changes lead to decreased
overall physical functioning, increased frailty, falls risk, and ultimately the
loss of independent living. Because the intertwining relationships of these
factors are complex, effective treatment options are still under investigation.
The published data on sarcopenia are vast, and this review is not intended to be
exhaustive. The aim of this review is to provide an update on the current
knowledge of the definition, etiology, consequences, and current clinical trials
that may help address this pressing public health problem for our aging
populations. |
What is the genetic basis of progeria? | Hutchinson-Gilford progeria syndrome is a rare, sporadic, autosomal dominant syndrome that involves premature aging, generally leading to death at approximately 13 years of age due to myocardial infarction or stroke. The genetic basis of most cases of this syndrome is a change from glycine GGC to glycine GGT in codon 608 of the lamin A (LMNA) gene, which activates a cryptic splice donor site to produce abnormal lamin A; this disrupts the nuclear membrane and alters transcription. | To characterize further the genetic basis of progeria, thermolability studies
were performed on three genetically distinct enzymes in crude extracts of
cultured skin fibroblasts derived from two subjects with that syndrome. At early
passage the progeric fibroblasts, as compared to controls, contained a
significantly higher percentage of heat-labile glucose-6-phosphate dehydrogenase
(12.83 plus or minus 1.72 vs 1.11 plus or minus 0.44 [mean plus or minus
S.E.M.], p smaller than 0.001), 6-phosphogluconate dehydrogenase (9.71 plus or
minus 0.68 vs. 0.67 plus or minus 0.22, p smaller than 0.001), and
hypoxanthine-guanine phosphoribosyltransferase (31.41 plus or minus 1.89 vs 7.67
plus or minus 1.71, p smaller than 0.001), and the differences were maintained
throughout the in vitro life-span. These data, in conjunction with previous
reports of defective HL-A antigens, indicate a widespread defect in genetic
expression. The most likely cause appears to be an aberration in protein
synthesis or degradation, or both, although multiple somatic mutations cannot be
ruled out. Increased thermolability of enzymes in cultured cells may provide a
screening test for persons predisposed to progeria and other disorders of
premature aging. While it is important to search for unifying mechanisms of aging among a variety
of model systems, evolutionary arguments suggest that the pathophysiological
details of senescence may be, to some extent, species specific. Moreover, in
species that are characterized by extensive genetic heterogeneity, such as our
own, one is likely to find kindreds with both "private" and "public" markers of
aging. Crude estimates of the number of loci with the potential to modulate
aspects of the senescent phenotype of man suggest that thousands of genes could
be involved. No single locus appears to modulate all features. Some affect
predomitly a single aspect ("unimodal progeroid syndromes"); familial
Alzheimer's disease is discussed as a prototype. Linkage studies indicate
genetic heterogeneity for autosomal domit forms of the disease. Some loci
affect multiple aspects of the phenotype ("segmental progeroid disorders"); the
prototype is Werner's syndrome, an autosomal recessive. Cells from homozygotes
behave like mutator strains and undergo accelerated senescence in vitro.
Elucidation of the biochemical genetic basis of such abiotrophic disorders may
shed light on specific aging processes in man. A systematic review of the more than 2,000 genetic loci of man cataloged by
McKusick indicated that approximately 7% may play a role in modulating the rates
of development of various aspects of the senescent phenotype. Assuming an upper
limit of about 100,000 loci in man, numerous alleles at approximately 7,000 loci
could be contributing to characteristic patterns of aging in individual human
beings. Point mutations or chromosomal aberrations involving such loci may
result in various progeroid syndromes. These have been classified into two
categories: segmental progeroid syndromes, which involve multiple aspects of the
senescent phenotype, and unimodal progeroid syndromes, in which predomitly
one aspect of the phenotype is involved. Two different examples of segmental
progeroid syndromes were discussed: the Werner syndrome (an autosomal recessive)
and the Down syndrome (trisomy 21). Examples of unimodal progeroid syndromes
included familial hypercholesterolemia (accelerated atherogenesis), xeroderma
pigmentosum (acceleration of skin aging, including age-related neoplasms), and
certain forms of intestinal polyposis (acceleration of adenocarcinoma of the
colon). It is remarkable and encouraging that the biochemical genetic basis of
many progeroid syndromes, including all of those mentioned above, may be
amenable to investigation with cultured mesenchymal somatic cells from
individual subjects. For example, cells from patients with the Werner's syndrome
have a striking limitation of their in vitro replicative life-spans and undergo
extensive chromosomal rearrangements. These abnormalities are presumably related
to an enzyme deficiency which, in principle, could be identified by biochemical
studies of cultured cells. The classical arguments favouring a genetic basis of ageing are reviewed
emphasizing the questions that remain uswered. Genes cannot be the sole
genetic determits of ageing. Mendel's paradigm cannot anymore explain all the
results recently obtained. Different aspects of the organization of the genome
must also play a role in ageing. The functions of the largest part of the human
genome remain unknown. Moreover the different genetic theories of ageing are
based on natural selection. However, other paradigms are being proposed that can
complement or replace Darwin's paradigm. They are based on the spontaneous
organization of complex systems. They must be considered in the reappraisal of
the ageing phenomenon. BACKGROUND: Hutchinson-Gilford progeria syndrome is a rare, sporadic, autosomal
domit syndrome that involves premature aging, generally leading to death at
approximately 13 years of age due to myocardial infarction or stroke. The
genetic basis of most cases of this syndrome is a change from glycine GGC to
glycine GGT in codon 608 of the lamin A (LMNA) gene, which activates a cryptic
splice donor site to produce abnormal lamin A; this disrupts the nuclear
membrane and alters transcription.
METHODS: We enrolled 15 children between 1 and 17 years of age, representing
nearly half of the world's known patients with Hutchinson-Gilford progeria
syndrome, in a comprehensive clinical protocol between February 2005 and May
2006.
RESULTS: Clinical investigations confirmed sclerotic skin, joint contractures,
bone abnormalities, alopecia, and growth impairment in all 15 patients;
cardiovascular and central nervous system sequelae were also documented.
Previously unrecognized findings included prolonged prothrombin times, elevated
platelet counts and serum phosphorus levels, measured reductions in joint range
of motion, low-frequency conductive hearing loss, and functional oral deficits.
Growth impairment was not related to inadequate nutrition, insulin
unresponsiveness, or growth hormone deficiency. Growth hormone treatment in a
few patients increased height growth by 10% and weight growth by 50%.
Cardiovascular studies revealed diminishing vascular function with age,
including elevated blood pressure, reduced vascular compliance, decreased
ankle-brachial indexes, and adventitial thickening.
CONCLUSIONS: Establishing the detailed phenotype of Hutchinson-Gilford progeria
syndrome is important because advances in understanding this syndrome may offer
insight into normal aging. Abnormal lamin A (progerin) appears to accumulate
with aging in normal cells. (ClinicalTrials.gov number, NCT00094393.) Hutchinson-Gilford Progeria Syndrome and Werner syndrome, also known as
childhood- and adulthood-progeria, respectively, represent two of the best
characterized human progeroid diseases with clinical features mimicking
physiological aging at an early age. The discovery of their genetic basis has
led to the identification of several gene mutations leading to a spectrum of
progeroid phenotypes ranging from moderate and mild-severe to very aggressive
forms. In parallel, the creation of disease registers and databases provided
available data for the design of relatively large-scale epidemiological studies,
thereby allowing a better understanding of the nature and frequency of the
premature aging-associated signs and symptoms. The aim of this article is to
review the most recent findings concerning the epidemiology of premature aging
disorders, their genetic basis, and the most recent reports on the frequency of
associated diseases. |
What is the function of cryptochrome-1 in mouse? | Cryptochrome-1 (Cry1) is an essential component of the central and peripheral circadian clocks for generation of circadian rhythms in mice. | The mammalian master clock driving circadian rhythmicity in physiology and
behavior resides within the suprachiasmatic nuclei (SCN) of the anterior
hypothalamus. Circadian rhythms are generated by a set of clock genes via
intertwined negative and positive autoregulatory transcription-translation
feedback loops. The Cryptochrome 1 and 2 genes are indispensable for molecular
core oscillator function, as evident from the arrhythmic wheel-running behavior
and lack of rhythmic clock gene expression in mCry1/mCry2 double-mutant mice in
constant darkness. In the present study, using real-time multiunit electrode
activity recordings in hypothalamic slices, we show that SCN neurons from
mCry-deficient mice kept in constant darkness lack circadian oscillations in
firing patterns. This proves that cryptochromes, and thus an intact circadian
clockwork, are prerequisites for circadian electrical activity in SCN neurons.
Interestingly, when mCry-deficient mice were kept in normal light-dark
conditions and SCN slices were prepared 2 hr after the beginning of the day, a
single noncircadian peak in neuronal activity was detected. This light-induced
rise in electrical activity of the SCN may explain why mCry-deficient mice lack
the arrhythmic short bouts of wheel-running activity and instead show apparently
normal behavior in normal day-night cycles. Many biochemical, physiological, and behavioral processes display daily rhythms
generated by an internal timekeeping mechanism referred to as the circadian
clock. The core oscillator driving this clock is located in the ventral part of
the hypothalamus, the so called suprachiasmatic nuclei (SCN). At the molecular
level, this oscillator is thought to be composed of interlocking autoregulatory
feedback loops involving a set of clock genes. Among the components driving the
mammalian circadian clock are the Period 1 and 2 (mPer1 and mPer2) and
Cryptochrome 1 and 2 (mCry1 and mCry2) genes. A mutation in the mPer2 gene leads
to a gradual loss of circadian rhythmicity in mice kept in constant darkness
(DD). Here we show that inactivation of the mCry2 gene in mPer2 mutant mice
restores circadian rhythmicity and normal clock gene expression patterns. Thus,
mCry2 can act as a nonallelic suppressor of mPer2, which points to direct or
indirect interactions of PER2 and CRY2 proteins. In marked contrast,
inactivation of mCry1 in mPer2 mutant mice does not restore circadian
rhythmicity but instead results in complete behavioral arrhythmicity in DD,
indicating different effects of mCry1 and mCry2 in the clock mechanism BACKGROUND: The cryptochrome 1 and 2 genes (cry1 and cry2) are necessary for the
generation of circadian rhythms, as mice lacking both of these genes (cry1,2-/-)
lack circadian rhythms. We studied sleep in cry1,2-/- mice under baseline
conditions as well as under conditions of constant darkness and enforced
wakefulness to determine whether cryptochromes influence sleep regulatory
processes.
RESULTS: Under all three conditions, cry1,2-/- mice exhibit the hallmarks of
high non-REM sleep (NREMS) drive (i.e., increases in NREMS time, NREMS
consolidation, and EEG delta power during NREMS). This unexpected phenotype was
associated with elevated brain mRNA levels of period 1 and 2 (per1,2), and
albumin d-binding protein (dbp), which are known to be transcriptionally
inhibited by CRY1,2. To further examine the relationship between circadian genes
and sleep homeostasis, we examined wild type mice and rats following sleep
deprivation and found increased levels of per1,2 mRNA and decreased levels of
dbp mRNA specifically in the cerebral cortex; these changes subsided with
recovery sleep. The expression of per3, cry1,2, clock, npas2, bmal1, and
casein-kinase-1epsilon did not change with sleep deprivation.
CONCLUSIONS: These results indicate that mice lacking cryptochromes are not
simply a genetic model of circadian arrhythmicity in rodents and functionally
implicate cryptochromes in the homeostatic regulation of sleep. The mammalian master clock driving circadian rhythmicity in physiology,
metabolism, and behaviour resides within the suprachiasmatic nuclei (SCN) of the
anterior hypothalamus and is composed of intertwined negative and positive
autoregulatory transcription-translation feedback loops. The Cryptochrome 1 and
2 gene products act in the negative feedback loop and are indispensable for
molecular core oscillator function, as evident from the arrhythmic wheel running
behaviour and absence of cyclic clock gene expression in mCry1/mCry2 double
mutant mice in constant darkness. Recently, we have measured real-time
multi-unit electrode activity recordings in hypothalamic slices from
mCry-deficient mice kept in constant darkness and observed a complete lack of
circadian oscillations in firing patterns. This proves that CRY proteins, and
thus an intact circadian clock, are prerequisite for circadian rhythmicity in
membrane excitability in SCN neurons. Strikingly, when mCry-deficient mice are
housed in normal light-dark cycles, a single non-circadian peak in neuronal
activity can be detected in SCN slices prepared two hours after the beginning of
the day. This light-induced increase in electric activity of the SCN suggests
that deletion of the mCry genes converts the core oscillator in an
hour-glass-like timekeeper and may explain why in normal day-night cycles
mCry-deficient mice show apparently normal behaviour. It has been reported that disruption of the circadian clock may lead to
increased risk of breast cancer in humans and to a high rate or ionizing
radiation-induced tumors and mortality in mice. Cryptochrome 1 and cryptochrome
2 proteins are core components of the mammalian circadian clock and mice mutated
in both genes are arrhythmic. We tested Cry1-/- Cry2-/- mice and fibroblasts
derived from these mice for radiation-induced cancer and killing and DNA damage
checkpoints and killing, respectively. We find that the mutant mice are
indistinguishable from the wild-type controls with respect to radiation-induced
morbidity and mortality. Similarly, the Cry1-/- Cry2-/-mutant fibroblasts are
indistinguishable from the wild-type controls with respect to their sensitivity
to ionizing radiation and UV radiation and ionizing radiation-induced DNA damage
checkpoint response. Our data suggest that disruption of the circadian clock in
itself does not compromise mammalian DNA repair and DNA damage checkpoints and
does not predispose mice to spontaneous and ionizing radiation-induced cancers.
We conclude that the effect of circadian clock disruption on cellular response
to DNA damage and cancer predisposition in mice may depend on the mechanism by
which the clock is disrupted. In mammals, circadian rhythms in behavior and physiology are controlled by a
central pacemaker, the SCN, and subordinated clocks throughout the body. On the
molecular level, these clocks are based on transcriptional/translational
feedback loops involving a set of clock genes that regulate their own
transcription. Among the components driving the mammalian circadian clock are
the Period 1 and 2 (Per1 and Per2) and Cryptochrome 1 and 2 (Cry1 and Cry2)
genes. In the present study, the authors characterize the behavioral and
molecular rhythms of Per2/Cry1 double mutant mice under 3 different lighting
conditions. In an LD cycle, the activity of these animals is masked by light,
while in DD, the mutants lose circadian rhythmicity but exhibit strong ultradian
rhythms. In LL of higher intensity, circadian rhythms are restored on the
behavioral level with a drastically shortened endogenous period. Furthermore,
both in the SCN and in the periphery, clock gene rhythms are restored. Based on
these observations and also on the fact that light-mediated induction of Per
gene expression is preserved in these mutants, the authors propose a mechanism
by which endogenous ultradian rhythms may relay timed light exposure to the SCN,
leading to a reinitiation of self-sustained circadian rhythms in LL. The circadian clock is driven by cell-autonomous transcription/translation
feedback loops. The BMAL1 transcription factor is an indispensable component of
the positive arm of this molecular oscillator in mammals. Here, we present a
molecular genetic screening assay for mutant circadian clock proteins that is
based on real-time circadian rhythm monitoring in cultured fibroblasts. By using
this assay, we identified a domain in the extreme C terminus of BMAL1 that plays
an essential role in the rhythmic control of E-box-mediated circadian
transcription. Remarkably, the last 43 aa of BMAL1 are required for
transcriptional activation, as well as for association with the circadian
transcriptional repressor CRYPTOCHROME 1 (CRY1), depending on the coexistence of
CLOCK protein. C-terminally truncated BMAL1 mutant proteins still associate with
mPER2 (another protein of the negative feedback loop), suggesting that an
additional repression mechanism may converge on the N terminus. Taken together,
these results suggest that the C-terminal region of BMAL1 is involved in
determining the balance between circadian transcriptional activation and
suppression. BACKGROUND DATA AND OBJECTIVE: Mesenchymal stem cells (MSCs) are multipotent
cells present in adult bone marrow that replicate as undifferentiated cells and
can differentiate to lineages of mesenchymal tissues. Homeostatic control of
bone remodeling maintains bone mass by ensuring that bone resorption and bone
formation occur sequentially and in a balanced manner. As most homeostatic
functions occur in a circadian manner, a circadian clock could control bone
mass. Here we show that laser irradiation can direct the extracellular
calcification of mouse MSCs by altering the intracellular localization of the
circadian rhythm protein cryptochrome 1 (CRY1).
MATERIALS AND METHODS: MSCs were irradiated with a blue laser (wavelength 405
nm) for 180 sec via a fiber attached to the bottom of the culture dish. After
laser irradiation, the MSCs were incubated in osteogenic differentiation medium
for 5 d. After laser irradiation, circadian rhythm protein CRY1 was
immunostained and histochemical staining for extracellular calcification was
observed.
RESULTS: Laser irradiation promoted extracellular calcification of MSCs, induced
the translocation of CRY1 protein from the cytoplasm to the nucleus, and
decreased CRY1 mRNA levels quantified by real-time PCR. Since the timing of
nuclear accumulation of clock proteins constitutes an important step in the
transcription-translation feedback loop driving the circadian core oscillator,
laser irradiation could provide a simple and effective technology for clock
protein localization and turnover. Our results also indicate that CRY1 is a
master regulator of circadian rhythm that regulates the extracellular
calcification of MSCs.
CONCLUSION: Laser irradiation could provide a simple and effective means of
controlling the fate of MSCs as a therapeutic strategy, and act as a "molecular
switch" of regulatory proteins by suppressing CRY transcription. Furthermore,
this model system may be useful for exploring the cross-talk between circadian
rhythm and cell function. Circadian clocks coordinate behavioral and physiological processes with daily
light-dark cycles by driving rhythmic transcription of thousands of genes.
Whereas the master clock in the brain is set by light, pacemakers in peripheral
organs, such as the liver, are reset by food availability, although the setting,
or "entrainment," mechanisms remain mysterious. Studying mouse fibroblasts, we
demonstrated that the nutrient-responsive adenosine monophosphate-activated
protein kinase (AMPK) phosphorylates and destabilizes the clock component
cryptochrome 1 (CRY1). In mouse livers, AMPK activity and nuclear localization
were rhythmic and inversely correlated with CRY1 nuclear protein abundance.
Stimulation of AMPK destabilized cryptochromes and altered circadian rhythms,
and mice in which the AMPK pathway was genetically disrupted showed alterations
in peripheral clocks. Thus, phosphorylation by AMPK enables cryptochrome to
transduce nutrient signals to circadian clocks in mammalian peripheral organs. The mammalian circadian rhythm is observed not only at the suprachiasmatic
nucleus, a master pacemaker, but also throughout the peripheral tissues. Its
conserved molecular basis has been thought to consist of intracellular
transcriptional feedback loops of key clock genes. However, little is known
about posttranscriptional regulation of these genes. In the present study, we
investigated the role of the 3'-untranslated region (3'UTR) of the mouse
cryptochrome 1 (mcry1) gene at the posttranscriptional level. Mature mcry1 mRNA
has a 610-nucleotide 3'UTR and mediates its own degradation. The middle part of
the 3'UTR contains a destabilizing cis-acting element. The deletion of this
element led to a dramatic increase in mRNA stability, and heterogeneous nuclear
ribonucleoprotein D (hnRNP D) was identified as an RNA binding protein
responsible for this effect. Cytoplasmic hnRNP D levels displayed a pattern that
was reciprocal to the mcry1 oscillation. Knockdown of hnRNP D stabilized mcry1
mRNA and resulted in enhancement of the oscillation amplitude and a slight delay
of the phase. Our results suggest that hnRNP D plays a role as a fine regulator
contributing to the mcry1 mRNA turnover rate and the modulation of circadian
rhythm. We investigated the amino acid sequences of rat PERIOD2 (rPER2) that are
required for interaction with CRYPTOCHROME1 (CRY1) to understand the molecular
mechanism of the circadian clock. Co-immunoprecipitation assays using various
C-terminal fragments of rPER2 with internal deletions revealed that amino acid
residues 1179-1198 are necessary for interaction with CRY1. To identify
precisely which amino acid residues are responsible for the interaction, we
substituted alanine for residues conserved among PER isoforms and species. We
found that more than three mutations of conserved PER2 residues impaired not
only binding to CRY1 but also subsequent nuclear translocation, although
mutations of non-conserved residues did not affect interaction with CRY1. Thus,
the conserved amino acid residues of 1179-1198 in PER2 are apparently
responsible for binding to CRY1. Direct evidence for the requirement of delay in feedback repression in the
mammalian circadian clock has been elusive. Cryptochrome 1 (Cry1), an essential
clock component, displays evening-time expression and serves as a strong
repressor at morning-time elements (E box/E' box). In this study, we reveal that
a combination of day-time elements (D box) within the Cry1-proximal promoter and
night-time elements (RREs) within its intronic enhancer gives rise to
evening-time expression. A synthetic composite promoter produced evening-time
expression, which was further recapitulated by a simple phase-vector model. Of
note, coordination of day-time with night-time elements can modulate the extent
of phase delay. A genetic complementation assay in Cry1(-/-):Cry2(-/-) cells
revealed that substantial delay of Cry1 expression is required to restore
circadian rhythmicity, and its prolonged delay slows circadian oscillation.
Taken together, our data suggest that phase delay in Cry1 transcription is
required for mammalian clock function. Mammalian metabolism is highly circadian and major hormonal circuits involving
nuclear hormone receptors display interlinked diurnal cycling. However,
mechanisms that logically explain the coordination of nuclear hormone receptors
and the clock are poorly understood. Here we show that two circadian
co-regulators, cryptochromes 1 and 2, interact with the glucocorticoid receptor
in a ligand-dependent fashion and globally alter the transcriptional response to
glucocorticoids in mouse embryonic fibroblasts: cryptochrome deficiency vastly
decreases gene repression and approximately doubles the number of
dexamethasone-induced genes, suggesting that cryptochromes broadly oppose
glucocorticoid receptor activation and promote repression. In mice, genetic loss
of cryptochrome 1 and/or 2 results in glucose intolerance and constitutively
high levels of circulating corticosterone, suggesting reduced suppression of the
hypothalamic-pituitary-adrenal axis coupled with increased glucocorticoid
transactivation in the liver. Genomically, cryptochromes 1 and 2 associate with
a glucocorticoid response element in the phosphoenolpyruvate carboxykinase 1
promoter in a hormone-dependent manner, and dexamethasone-induced transcription
of the phosphoenolpyruvate carboxykinase 1 gene was strikingly increased in
cryptochrome-deficient livers. These results reveal a specific mechanism through
which cryptochromes couple the activity of clock and receptor target genes to
complex genomic circuits underpinning normal metabolic homeostasis. BACKGROUND: This study was aimed to examine circadian variations of hepatic
antioxidant components, including the Nrf2- pathway, the glutathione (GSH)
system, antioxidant enzymes and metallothionein in mouse liver.
METHODS AND RESULTS: Adult mice were housed in light- and temperature-controlled
facilities for 2 weeks, and livers were collected every 4 h during the 24 h
period. Total RNA was isolated, purified, and subjected to real-time RT-PCR
analysis. Hepatic mRNA levels of Nrf2, Keap1, Nqo1 and Gclc were higher in the
light-phase than the dark-phase, and were female-predomit. Hepatic GSH
presented marked circadian fluctuations, along with glutathione S-transferases
(GST-α1, GST-µ, GST-π) and glutathione peroxidase (GPx1). The expressions of
GPx1, GST-µ and GST-π mRNA were also higher in females. Antioxidant enzymes
Cu/Zn superoxide dismutase (Sod1), catalase (CAT), cyclooxygenase-2 (Cox-2) and
heme oxygenase-1 (Ho-1) showed circadian rhythms, with higher expressions of
Cox-2 and CAT in females. Metallothionein, a small non-enzymatic antioxidant
protein, showed dramatic circadian variation in males, but higher expression in
females. The circadian variations of the clock gene Brain and Muscle Arnt-like
Protein-1(Bmal1), albumin site D-binding protein (Dbp), nuclear receptor
Rev-Erbα (Nr1d1), period protein (Per1 and Per2) and cryptochrome 1(Cry1) were
in agreement with the literature. Furthermore, acetaminophen hepatotoxicity is
more severe when administered in the afternoon when hepatic GSH was lowest.
CONCLUSIONS: Circadian variations and gender differences in transcript levels of
antioxidant genes exist in mouse liver, which could affect body responses to
oxidative stress at different times of the day. The mammalian circadian clock is composed of interlocking feedback loops.
Cryptochrome is a central component in the core negative feedback loop, whereas
Rev-Erbα, a member of the nuclear receptor family, is an essential component of
the interlocking loop. To understand the roles of different clock genes, we
conducted a genetic interaction screen by generating single- and double-mutant
mice. We found that the deletion of Rev-erbα in F-box/leucine rich-repeat
protein (Fbxl3)-deficient mice rescued its long-circadian period phenotype, and
our results further revealed that FBXL3 regulates Rev-Erb/retinoic acid
receptor-related orphan receptor-binding element (RRE)-mediated transcription by
inactivating the Rev-Erbα:histone deacetylase 3 corepressor complex. By
analyzing the Fbxl3 and Cryptochrome 1 double-mutant mice, we found that FBXL3
also regulates the amplitudes of E-box-driven gene expression. These two
separate roles of FBXL3 in circadian feedback loops provide a mechanism that
contributes to the period determination and robustness of the clock. |
Describe July Effect. | The July effect is the hypothetical increase in morbidity and mortality thought to be associated with the influx of new (or newly promoted) trainees during the first portion of the academic year (in July). | BACKGROUND: Each July thousands begin medical residencies and acquire increased
responsibility for patient care. Many have suggested that these new medical
residents may produce errors and worsen patient outcomes-the so-called "July
Effect;" however, we have found no U.S. evidence documenting this effect.
OBJECTIVE: Determine whether fatal medication errors spike in July.
DESIGN: We examined all U.S. death certificates, 1979-2006 (n = 62,338,584),
focusing on medication errors (n = 244,388). We compared the observed number of
deaths in July with the number expected, determined by least-squares regression
techniques. We compared the July Effect inside versus outside medical
institutions. We also compared the July Effect in counties with versus without
teaching hospitals.
OUTCOME MEASURE: JR = Observed number of July deaths / Expected number of July
deaths.
RESULTS: Inside medical institutions, in counties containing teaching hospitals,
fatal medication errors spiked by 10% in July and in no other month [JR = 1.10
(1.06-1.14)]. In contrast, there was no July spike in counties without teaching
hospitals. The greater the concentration of teaching hospitals in a region, the
greater the July spike (r = .80; P = .005). These findings held only for
medication errors, not for other causes of death.
CONCLUSIONS: We found a significant July spike in fatal medication errors inside
medical institutions. After assessing competing explanations, we concluded that
the July mortality spike results at least partly from changes associated with
the arrival of new medical residents. OBJECTIVES/HYPOTHESIS: A "July effect" of increased complications when new
trainees begin residency has been reported widely by the media. We sought to
determine the effect of admission month on in-hospital mortality, complications,
length of hospitalization, and costs for patients undergoing head and neck
cancer (HNCA) surgery.
STUDY DESIGN: Retrospective cross-sectional study.
METHODS: Discharge data from the Nationwide Inpatient Sample for 48,263 patients
who underwent an ablative procedure for a maligt oral cavity, laryngeal,
hypopharyngeal, or oropharyngeal neoplasm in 2005 to 2008 were analyzed using
cross-tabulations and multivariate regression modeling.
RESULTS: There were 3,812 cases admitted in July (8%). July admission was
significantly associated with Medicaid (RRR 1.40, P = 0.011) or self-pay payor
status (RRR 1.40, P = 0.022), medium hospital bed size (RRR 1.63, P = 0.033) and
large hospital bed size (RRR 1.73, P = 0.013). There was no association between
July admission and other patient or hospital demographic characteristics. Major
procedures and comorbidity were significantly associated with in-hospital death,
surgical and medical complications, length of hospitalization, and costs, but no
association was found for July admission, July through September discharge, or
teaching hospital status and short-term morbidity or mortality. Teaching
hospitals and large hospital bed size were predictors of increased length of
hospitalization and costs; and private, for profit hospitals were additionally
associated with increased costs. No interaction between July admission and
teaching hospitals was found for any of the outcome variables studied.
CONCLUSIONS: These data do not support evidence of a "July effect" or an
increase in morbidity or mortality at teaching hospitals providing HNCA surgical
care. BACKGROUND: The commencement of new academic cycle in July is presumed to be
associated with poor patient outcomes, although supportive evidence is limited
for cardiac surgery patients. We sought to determine if the new academic cycle
affected the outcomes of patients undergoing Coronary Artery Bypass Grafting.
METHODS: A retrospective analysis was performed on 10-year nationwide
in-hospital data from 1998-2007. Only patients who underwent CABG in the first
and final academic 3-month calendar quarter were included. Generalized
multivariate regression was used to assess indicators of hospital quality of
care such as risk-adjusted mortality, total complications and "failure to
rescue" (FTOR) - defined as death after a complication.
RESULTS: Of the 1,056,865 CABG operations performed in the selected calendar
quarters, 698,942 were at teaching hospitals. The risk-adjusted mortality,
complications and FTOR were higher in the beginning of the academic year [Odds
ratio = 1.14, 1.04 and 1.19 respectively; p < 0.001 for all] irrespective of
teaching status. However, teaching status was associated with lower mortality
(OR 0.9) despite a higher complication rate (OR 1.02); [p < 0.05 for both]. The
July Effect thus contributed to only a 2.4% higher FTOR in teaching hospitals
compared to 19% in non teaching hospitals.
CONCLUSIONS: The July Effect is reflective of an overall increase in morbidity
in all hospitals at the beginning of the academic cycle and it had a pronounced
effect in non-teaching hospitals. Teaching hospitals were associated with lower
mortality despite higher complication rates in the beginning of the academic
cycle compared to non-teaching hospitals. The July effect thus cannot be
attributed to presence of trainees alone.
ULTRAMINI ABSTRACT: This study compares the July effect in teaching and
non-teaching hospitals and demonstrates that this effect is not unique to
teaching hospitals for CABG patients. In fact, teaching hospitals have somewhat
better outcomes at the beginning of the academic cycle and the July effect is a
much broader seasonal variation. BACKGROUND: Studies of whether inpatient mortality in US teaching hospitals
rises in July as a result of organizational disruption and relative inexperience
of new physicians (July effect) find small and mixed results, perhaps because
study populations primarily include low-risk inpatients whose mortality outcomes
are unlikely to exhibit a July effect.
METHODS AND RESULTS: Using the US Nationwide Inpatient sample, we estimated
difference-in-difference models of mortality, percutaneous coronary intervention
rates, and bleeding complication rates, for high- and low-risk patients with
acute myocardial infarction admitted to 98 teaching-intensive and 1353
non-teaching-intensive hospitals during May and July 2002 to 2008. Among
patients in the top quartile of predicted acute myocardial infarction mortality
(high risk), adjusted mortality was lower in May than July in teaching-intensive
hospitals (18.8% in May, 22.7% in July, P<0.01), but similar in
non-teaching-intensive hospitals (22.5% in May, 22.8% in July, P=0.70). Among
patients in the lowest three quartiles of predicted acute myocardial infarction
mortality (low risk), adjusted mortality was similar in May and July in both
teaching-intensive hospitals (2.1% in May, 1.9% in July, P=0.45) and
non-teaching-intensive hospitals (2.7% in May, 2.8% in July, P=0.21).
Differences in percutaneous coronary intervention and bleeding complication
rates could not explain the observed July mortality effect among high risk
patients.
CONCLUSIONS: High-risk acute myocardial infarction patients experience similar
mortality in teaching- and non-teaching-intensive hospitals in July, but lower
mortality in teaching-intensive hospitals in May. Low-risk patients experience
no such July effect in teaching-intensive hospitals. STUDY DESIGN: Retrospective cohort.
OBJECTIVE: To evaluate for the presence and magnitude of the "July effect"
within elective spine surgery.
SUMMARY OF BACKGROUND DATA: The July effect is the hypothetical increase in
morbidity and mortality thought to be associated with the influx of new (or
newly promoted) trainees during the first portion of the academic year. Studies
evaluating for the presence and magnitude of the July effect have demonstrated
conflicting results.
METHODS: We accessed the American College of Surgeons National Surgical Quality
Improvement Program database from 2005-2010. Statistical analyses were conducted
using bivariate and multivariate logistic regression.
RESULTS: A total of 14,986 cases met inclusion criteria and constitute the study
population. Of these, 26.5% occurred in the first academic quarter and 25.3% had
resident involvement. The rate of serious adverse events was 1.9 times higher
and the rate of any adverse events was 1.6 times higher among cases with
resident involvement than among those without (P < 0.001 for both). Among cases
without resident involvement, the rates of serious adverse events and any
adverse events did not differ by academic quarter. Similarly, among cases with
resident involvement, the rates of serious adverse events and any adverse events
did not differ by academic quarter.
CONCLUSION: We could not demonstrate that the training of new (or newly
promoted) residents is associated with an increase in the adverse events of
spine surgery. Safeguards that have been put in place to ensure patient safety
during this training period seem to be effective. Although adverse events were
more common among cases with resident involvement than among cases without
resident involvement, our data suggest that this association is more likely a
product of the riskier population of cases in which residents participate than
of the resident involvement itself. INTRODUCTION: There has been concern of increased emergency department (ED)
length of stay (LOS) during the months when new residents are orienting to their
roles. This so-called "July Effect" has long been thought to increase LOS, and
potentially contribute to hospital overcrowding and increased waiting time for
patients. The objective of this study is to determine if the average ED LOS at
the beginning of the hospital academic year differs for teaching hospitals with
residents in the ED, when compared to other months of the year, and as compared
to non-teaching hospitals without residents.
METHODS: We performed a retrospective analysis of a nationally representative
sample of 283,621 ED visits from the National Hospital Ambulatory Medical Care
Survey (NHAMCS), from 2001 to 2008. We stratified the sample by proportion of
visits seen by a resident, and compared July to the rest of the year, July to
June, and July and August to the remainder of the year. We compared LOS for
teaching hospitals to non-teaching hospitals. We used bivariate statistics, and
multivariable regression modeling to adjust for covariates.
RESULTS: Our findings show that at teaching hospitals with residents, there is
no significant difference in mean LOS for the month of July (275 minutes) versus
the rest of the year (259 min), July and August versus the rest of the year, or
July versus June. Non-teaching hospital control samples yielded similar results
with no significant difference in LOS for the same time periods. There was a
significant difference found in mean LOS at teaching hospitals (260 minutes) as
compared to non-teaching hospitals (185 minutes) throughout the year (p<0.0001).
CONCLUSION: Teaching hospitals with residents in the ED have slower throughput
of patients, no matter what time of year. Thus, the "July Effect" does not
appear to a factor in ED LOS. This has implications as overcrowding and patient
boarding become more of a concern in our increasingly busy EDs. These results
question the need for additional staffing early in the academic year. Teaching
hospitals may already institute more robust staffing during this time,
preventing any significant increase in LOS. Multiple factors contribute to long
stays in the ED. While patients seen by residents stay longer in the ED, there
is little variability throughout the academic year. PURPOSE: Researchers have found mixed results about the risk to patient safety
in July, when newly minted physicians enter U.S. hospitals to begin their
clinical training, the so-called "July effect." However, patient and family
satisfaction and perception of physician competence during summer months remain
unknown.
METHOD: The authors conducted a retrospective observational cohort study of 815
family members of adult intensive care unit (ICU) patients who completed the
Family Satisfaction with Care in the Intensive Care Unit instrument from eight
ICUs at Beth Israel Deaconess Medical Center, Boston, Massachusetts, between
April 2008 and June 2011. The association of ICU care in the summer months
(July-September) versus other seasons and family perception of physician
competence was examined in univariable and multivariable analyses.
RESULTS: A greater proportion of family members described physicians as
competent in summer months as compared with winter months (odds ratio [OR] 1.9;
95% confidence interval [CI] 1.2-3.0; P = .003). After adjustment for patient
and proxy demographics, severity of illness, comorbidities, and features of the
admission in a multivariable model, seasonal variation of family perception of
physician competence persisted (summer versus winter, OR of judging physicians
competent 2.4; 95% CI 1.3-4.4; P = .004).
CONCLUSIONS: Seasonal variation exists in family perception of physician
competence in the ICU, but opposite to the "July effect." The reasons for this
variation are not well understood. Further research is necessary to explore the
role of senior provider involvement, trainee factors, system factors such as
handoffs, or other possible contributors. BACKGROUND: The transition from student to intern can be challenging. The
"August" or "July effect" describes increased errors and reduced patient safety
during this transition. The study objectives were to develop, pilot, and
evaluate clinical performance after an immersive simulation course for incoming
interns.
METHODS: Graduating students were recruited for a 1-week immersive simulation
course. Controls received no simulation training. Primary outcome (at baseline,
and 1 and 6 months) was clinical performance on Objective Structured Clinical
Examinations (OSCE) of clinical procedures and surgical technical skills.
Secondary outcomes were self-reported confidence and clinical procedure logbook
data.
RESULTS: Nineteen students were recruited. Sixteen completed the 6-month
follow-up, 10 in the intervention group and 6 in the control group. No
differences were demonstrated between interventions and controls at baseline
(OSCE [median, 66 vs. 78; P = .181], technical skills [48 vs. 52.5; P = .381],
and confidence [101 vs 96; P = .368]). Interventions outperformed controls at 1
month (OSCE [111 vs 82; P = .001], technical skills [78.5 vs 63; P = .030], and
confidence [142 vs. 119; P < .001]), and 6 months (OSCE [107 vs. 93; P = .007],
technical skills [92.5 vs. 69; P = .044], and confidence [148 vs. 129; P =
.022]). No differences were observed in numbers of clinical procedures performed
at 1 (P = .958), 4 (P = .093), or 6 months (P = .713).
CONCLUSION: The immersive simulation course objectively improved subjects'
clinical skills, technical skills, and confidence. Despite similar clinical
experience as controls, the intervention group's improved performance persisted
at 6 months follow-up. This feasible and effective intervention to ease
transition from student to intern could reduce errors and enhance patient
safety. OBJECT: The relationship between time of year and surgical site infection (SSI)
following neurosurgical procedures is poorly understood. Authors of previous
reports have demonstrated that rates of SSI following neurosurgical procedures
performed during the summer months were higher compared with rates during other
seasons. It is unclear, however, if this difference was related to
climatological changes or inexperienced medical trainees (the July effect). The
aim of this study was to evaluate for seasonal variation of SSI following spine
surgery in a network of nonteaching community hospitals.
METHODS: The authors analyzed 6 years of prospectively collected surveillance
data (January 1, 2007, to December 31, 2012) from all laminectomies and spinal
fusions from 20 hospitals in the Duke Infection Control Outreach Network of
community hospitals. Surgical site infections were defined using National
Healthcare Safety Network criteria and identified using standardized methods
across study hospitals. Regression models were then constructed using Poisson
distribution to evaluate for seasonal trends by month. Each analysis was first
performed for all SSIs and then for SSIs caused by specific organisms or classes
of organisms. Categorical analysis was performed using two separate definitions
of summer: June through September (definition 1), and July through September
(definition 2). The prevalence rate of SSIs during the summer was compared with
the prevalence rate during the remainder of the year by calculating prevalence
rate ratios and 95% confidence intervals.
RESULTS: The authors identified 642 SSIs following 57,559 neurosurgical
procedures (overall prevalence rate = 1.11/100 procedures); 215 occurred
following 24,466 laminectomies (prevalence rate = 0.88/100 procedures), and 427
following 33,093 spinal fusions (prevalence rate = 1.29/100 procedures). Common
causes of SSI were Staphylococcus aureus (n = 380; 59%), coagulase-negative
staphylococci (n = 90; 14%), and Escherichia coli (n = 41; 6.4%). Poisson
regression models demonstrated increases in the rates of SSI during each of the
summer months for all SSIs and SSIs due to gram-positive cocci, S. aureus, and
methicillin-sensitive S. aureus. Categorical analysis confirmed that the rate of
SSI during the 4-month summer period was higher than the rate during the
remainder of the year, regardless of which definition for summer was used
(definition 1, p = 0.008; definition 2, p = 0.003). Similarly, the rates of SSI
due to grampositive cocci and S. aureus were higher during the summer months
than the remainder of the year regardless of which definition of summer was
used. However, the rate of SSI due to gram-negative bacilli was not.
CONCLUSIONS: The rate of SSI following fusion or spinal laminectomy/laminoplasty
was higher during the summer in this network of community hospitals. The
increase appears to be related to increases in SSIs caused by gram-positive
cocci and, more specifically, S. aureus. Given the nonteaching nature of these
hospitals, the findings demonstrate that increases in the rate of SSI during the
summer are more likely related to ecological and/or environmental factors than
the July effect. |
Which intermediate filament (IF) protein can be used as a non-specific marker of the neuronal precursor cells of the subventricular zone? | Nestin can be used as a nonspecific marker protein for precursor cells in the subventricular zone (SVZ). Nestin is a unique intermediate filament protein. While it is robustly expressed in developing brain, postnatal expression is limited to the brain's SVZ. | The subventricular zone (SVZ) is an embryonic remt that persists and remains
mitotically active throughout adulthood. The rodent SVZ harbors neuronal
precursors, principally in its anterior part, and generates neuroblasts that
migrate tangentially into the olfactory bulb, thus forming the so-called rostral
migratory stream. This study aimed at characterizing the SVZ in the human brain.
Antibodies raised against the widely used SVZ molecular markers nestin, glial
fibrillary acidic protein, beta-tubulin-III and polysialylated neural cell
adhesion molecule, have allowed us to characterize in detail a zone similar to
the rodent SVZ in humans. Virtually all portions of the lateral ventricle, as
well as the ventral (hypothalamic) sector of the third ventricle, displayed
immunoreactivity for most of the molecular markers. The midline region of the
septum (septal recess) and the ventral portion of the SVZ displayed a
particularly intense immunostaining for all SVZ markers. These two regions may
represent zones of adult neurogenesis that are unique to primates. Furthermore,
the anti-apoptotic protein Bcl-2 was found to be actively synthesized and
co-expressed with all the other markers throughout the entire SVZ. This study
reveals that a well-developed SVZ exists in the adult human brain and suggests
that Bcl-2 might play an important role in the functional organization of such a
system. The mature central nervous system contains precursor cells in the subventricular
zone of the lateral ventricle. In this study we examined the possibility to
affect fate of precursor cells through exogenous manipulations. The results
indicate that administration of thyroid hormone and retinoic acid increases the
expression of Ki67, a nuclear antigen associated with cell proliferation, and of
nestin, a marker protein for precursor cells in the subventricular zone of adult
male rats. Moreover, retinoic acid increases polysialated-neural cell adhesion
molecules (PSA-NCAM)-immunoreactivity. These data suggest that nuclear receptor
ligands are potential candidates for fate determination of precursor cells in
the subventricular zone also in the adult brain. We transplanted adult whole bone marrow prelabeled with bromodeoxyuridine (BrdU)
into the ischemic boundary zone of the adult rat brain at 1 day after 2 h of
middle cerebral artery occlusion (MCAo). Approximately 3.3% of 10(6)
transplanted bone marrow cells were BrdU reactive at 14 days after MCAo.
BrdU-reactive cells expressed neuronal and astrocytic proteins, neuronal nuclei
protein (NeuN, 1%), and glial fibrillary acidic protein (GFAP, 5%)
immunoreactivities, respectively. In addition, bone marrow transplantation
promoted proliferation of ependymal and subependymal cells, identified by nestin
(a neuroepithelial stem cell marker), within the ventricular zone and
subventricular zone (VZ/SVZ). These data suggest that intracerebral
transplantation of bone marrow could potentially be used to induce plasticity in
ischemic brain. The subventricular zone of the rodent brain retains the capacity of generating
new neurons in adulthood. The newly formed neuroblasts migrate rostrally toward
the olfactory bulb, where they differentiate as granular and periglomerular
interneurons. The reported presence of differentiated neurons expressing the
neuronal isoform of nitric oxide synthase (NOS) in the periphery of the
neurogenic region and the organization of their varicose axons as a network in
which the precursors are immersed raised the hypothesis that endogenous nitric
oxide (NO) may participate in the control of neurogenesis in the subventricular
zone. Systemic administration of the NOS inhibitors N(omega)-nitro-L-arginine
methyl ester or 7-nitroindazole to adult mice produced a dose- and
time-dependent increase in the number of mitotic cells in the subventricular
zone, rostral migratory stream, and olfactory bulb, but not in the dentate gyrus
of the hippocampus, without affecting apoptosis. In the subventricular zone,
this effect was exerted selectively on a precursor subpopulation expressing
nestin but not neuronal or glial cell-specific proteins. In addition, in the
olfactory bulb, analysis of maturation markers in the newly generated neurons
indicated that chronic NOS inhibition caused a delay in neuronal
differentiation. Postmitotic cell survival and migration were not affected when
NO production was impaired. Our results suggest that NO, produced by nitrergic
neurons in the adult mouse subventricular zone and olfactory bulb, exerts a
negative control on the size of the undifferentiated precursor pool and promotes
neuronal differentiation. Precursor cells have been shown to be affected by oxidative stress, in vivo and
vitro, but little is known about the expression of antioxidant mechanisms in
neuronal/glial differentiation. We have characterized the expression of Cu/Zn
superoxide dismutase (Cu/Zn SOD), one of the main antioxidant proteins involved
in the breakdown of superoxide, in the immature rat dorsolateral subventricular
zone (SVZ), rostral migratory stream (RMS) and hippocampal subgranular zone
(SGZ). Progenitor cells were identified immunohistochemically on cryostat
sections by 5'Bromodeoxyuridine (BrdU) incorporation and expressing cells were
further characterized using double labeling for progenitor markers. In the SVZ,
only a subpopulation of BrdU+ cells, mostly found in the medial SVZ, expressed
Cu/Zn SOD. These cells were mostly nestin+ and some were also vimentin+. In
contrast, in the lateral SVZ few Cu/Zn SOD+/BrdU+ cells were found. These were
primarily nestin+, vimentin-, showed some PSA-NCAM expression, but only a few
were NG2+. In the RMS and SGZ virtually all BrdU+ progenitors were Cu/Zn SOD+
and expressed nestin and vimentin. Some RMS cells were also PSA-NCAM+. These
findings show a heterogeneous expression of Cu/Zn SOD in restricted cell types
in the germinative zones and suggest a role for antioxidant Cu/Zn SOD in
progenitor cells of the immature rat brain. Because the neural differentiation capacity of bone marrow stromal cells (BMSCs)
is still a matter of controversial debate, we performed a thorough investigation
into the differentiation capacity of human BMSCs and examined their therapeutic
potency. BMSCs were isolated from the femur and kept in cell cultures with
various cultivation protocols being applied. In standard culture conditions
using a fetal calf serum-enriched medium, while not exhibiting a neural
phenotype, the majority of cells expressed a variety of neuronal marker proteins
as well as the astrocyte marker GFAP. Only a minority of stem cells expressed
nestin, a marker for neural precursor cells. Cultivation in serum-free medium
supplemented with specific growth factors resulted in a markedly higher
percentage of nestin-positive cells. To establish the therapeutic potency of
bone marrow-derived cells, the synthesis of neurotrophic factors such as NGF,
BDNF and GDNF was analyzed under non-stimulating standard culture conditions as
well as after a neural selection procedure. The therapeutic potency of BMSCs was
further examined with regard to their migratory potential in vitro and after
transplantation in vivo. After stereotactic engraftment into the lateral
ventricle of adult rats, mesenchymal stem cells were seen to adhere to the
ependymocytes and cells of the choroids plexus. Afterwards grafted cells passed
through the ependymal barrier, locating in the subventricular space. Their BMSCs
took up a close host graft interaction without any degenerative influence on the
host cells. Furthermore, there was morphological as well as immunohistochemical
evidence for a transdifferentiation within the host tissue. In addition, BMSCs
could be efficiently transduced using a third-generation adenoviral vector,
indicating their potential feasibility for a gene-therapeutic option. Since reports that precursor cells in the adult subventricular zone (SVZ)
contribute to regenerative neuro- and gliogenesis in CA1, we wondered whether a
similar route of migration might also exist under physiological conditions.
Permanent labeling of SVZ precursor cells with a lentiviral vector for green
fluorescent protein did not reveal any migration from the SVZ into CA1 in the
intact murine brain. However, in a nestin-GFP reporter mouse we found
proliferating cells within the corpus callosum/alveus region expressing nestin
and glial fibrillary acidic protein similar to precursor cells in the
neighboring neurogenic region of the adult dentate gyrus. Within 3 weeks of BrdU
administration, BrdU-positive nestin-GFP-expressing protoplasmic astrocytes
emerged in CA1. Similar to precursor cells isolated from the dentate gyrus and
the SVZ, nestin-GFP-expressing cells from corpus callosum/alveus were
self-renewing and multipotent in vitro, whereas cells isolated from CA1 were
not. Nestin-GFP-expressing cells in CA1 differentiated into postmitotic
astrocytes characterized by S100beta expression. No new neurons were found in
CA1. The number of nestin-GFP-expressing astrocytes in CA1 was increased by
environmental enrichment. We conclude that astrogenesis in CA1 is influenced by
environmental conditions. However, SVZ precursor cells do not contribute to
physiological cellular plasticity in CA1. Embryonic neuroepithelia and adult subventricular zone (SVZ) stem and progenitor
cells express nestin. We characterized a transgenic line that expresses enhanced
green fluorescent protein (eGFP) specified to neural tissue by the second
intronic enhancer of the nestin promoter that had several novel features. During
embryogenesis, the dorsal telencephalon contained many and the ventral
telencephalon few eGFP+ cells. eGFP+ cells were found in postnatal and adult
neurogenic regions. eGFP+ cells in the SVZ expressed multiple phenotype markers,
glial fibrillary acidic protein, Dlx, and neuroblast-specific molecules
suggesting the transgene is expressed through the lineage. eGFP+ cell numbers
increased in the SVZ after cortical injury, suggesting this line will be useful
in probing postinjury neurogenesis. In non-neurogenic regions, eGFP was strongly
expressed in oligodendrocyte progenitors, but not in astrocytes, even when they
were reactive. This eGFP+ mouse will facilitate studies of proliferative
neuroepithelia and adult neurogenesis, as well as of parenchymal
oligodendrocytes. Neural stem/progenitor cells (NSCs) reside in the subventricular zone (SVZ) and
subgranular zone of the hippocampal dentate gyrus in adult mammals. The
ubiquitin ligase HRD1 is associated with degradation of amyloid precursor
protein and believed to be specifically expressed in neurons and not in
astrocytes. We investigated expression of HRD1 using immunohistochemistry and
found colocalization of HRD1 with the NSC marker protein nestin and glial
fibrillary acidic protein in the NSCs of the SVZ (the SVZ astrocytes) but not in
the hippocampus. In the hippocampal dentate gyrus, HRD1 is localized in the
nucleus of nestin-positive cells. Nestin is an intermediate filament protein expressed in neuroepithelial stem
cells during development and it is later replaced by cell specific neuronal or
glial filaments. Nevertheless, nestin⁺ cells remain within adult tissues and
they can be regarded as potential neural stem cell (NSC). Nestin⁺ cells have
been detected in Schwann cells related with sensory corpuscles of rodent and
they have been demonstrated to be NSC. We have investigated the existence of
nestin⁺ in human cutaneous cells Meissner and Pacinian corpuscles through the
use of immunohistochemistry techniques and in situ hybridization. S100 protein
(also regarded as a marker for NSC) and vimentin (the intermediate filament of
mature Schwann cells in sensory corpuscles) were also investigated. The results
show that the adult human cutaneous sensory Meissner and Pacinian corpuscles
contains a small population of Schwann-related cells (vimentin⁺) which on the
basis of their basic immunohistochemical characteristics (S100 protein⁺,
nestin⁺) can be potential NSCs. Cells sharing identical immunohistochemical
profile were also found in the close vicinity of Meissner corpuscles. Because
their localization they are easily accessible and may represent a peripheral
niche of NSC to be used for therapeutic goals. Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from
the developing and adult nervous system and to identify cancer stem cells in
brain tumors. However, despite extensive characterization of Prominin-1(+)
precursor cells from the adult subventricular zone, no information about the
expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the
adult hippocampus has been available. We show here that Prominin-1 is expressed
by a significant number of cells in the SGZ of adult mice in vivo and ex vivo,
including postmitotic astrocytes. A small subset of Prominin-1(+) cells
coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and
Sox2. Upon fluorescence-activated cell sorting, only Prominin-1/Nestin
double-positive cells fulfilled the defining stem cell criteria of
proliferation, self-renewal, and multipotentiality as assessed by a neurosphere
assay. In addition, isolated primary Prominin-1(+) cells preferentially migrated
to the neurogenic niche in the SGZ upon transplantation in vivo. Finally,
despite its expression by various stem and progenitor cells, Prominin-1 turned
out to be dispensable for precursor cell proliferation in vitro and in vivo.
Nevertheless, a net decrease in hippocampal neurogenesis, by ∼30% was found in
Prominin-1 knock-out mice, suggesting other roles in controlling adult
hippocampal neurogenesis. Remarkably, an upregulation of Prominin-2 was detected
in Prominin-1-deficient mice highlighting a potential compensatory mechanism,
which might explain the lack of severe symptoms in individuals carrying
mutations in the Prom1 gene. |
Which enzyme is involved in the maintenance of DNA (cytosine-5-)-methylation? | The mammalian DNA (cytosine-5) methyltransferase 1, DNMT1 is the major enzyme responsible for the maintenance of the DNA methylation patterns on the newly synthesized strand after DNA replication. DNMT1 prefers hemimethylated DNA and during DNA replication methylates hemimethylated CpG sites by copying methylation patterns from the parental DNA strand to the newly synthesized daughter strand. The equivalent of DNMT1 in plants is MET1. | Methylation of cytosine residues in DNA plays an important role in regulating
gene expression during vertebrate embryonic development. Conversely, disruption
of normal patterns of methylation is common in tumors and occurs early in
progression of some human cancers. In vertebrates, it appears that the same DNA
methyltransferase maintains preexisting patterns of methylation during DNA
replication and carries out de novo methylation to create new methylation
patterns. There are several indications that inherent signals in DNA structure
can act in vivo to initiate or block de novo methylation in adjacent DNA
regions. To identify sequences capable of enhancing de novo methylation of DNA
in vitro, we designed a series of oligodeoxyribonucleotide substrates with
substrate cytosine residues in different sequence contexts. We obtained evidence
that some 5-methylcytosine residues in these single-stranded DNAs can stimulate
de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase
as effectively as 5-methylcytosine residues in double-stranded DNA stimulate
maintece methylation. This suggests that double-stranded DNA may not be the
primary natural substrate for de novo methylation and that looped
single-stranded structures formed during the normal course of DNA replication or
repair serve as "nucleation" sites for de novo methylation of adjacent DNA
regions. BACKGROUND: Though Dnmt1 is considered the primary maintece methyltransferase
and Dnmt3a and Dnmt3b are considered de novo methyltransferases in mammals,
these three enzymes may work together in maintaining as well as establishing DNA
methylation patterns. It has been proposed that Dnmt1 may carry out de novo
methylation at sites in the genome with transient single-stranded regions, such
as replication origins, and then spread methylation from these nucleation sites
in vivo, even though such activity has not been reported.
RESULTS: In this study, we show that Dnmt3a does not act on single-stranded
substrates in vitro, indicating that Dnmt3a is not likely to initiate DNA
methylation at such proposed nucleation sites. Dnmt3a shows similar methylation
activity on unmethylated and hemimethylated duplex DNA, though with some
substrate preference. Unlike Dnmt1, pre-existing cytosine methylation at CpG
sites or non-CpG sites does not stimulate Dnmt3a activity in vitro and in vivo.
CONCLUSION: The fact that Dnmt3a does not act on single stranded DNA and is not
stimulated by pre-existing cytosine methylation indicates that the de novo
methylation activity of Dnmt3a is quite different from that of Dnmt1. These
findings are consistent with a model in which Dnmt3a initiates methylation on
one of the DNA strands of duplex DNA, and these hemimethylated sites then
stimulate Dnmt1 activity for further methylation. The Cdc25C phosphatase mediates cellular entry into mitosis in mammalian cells.
Cdc25C activates Cdc2 for entry into mitosis by dephosphorylating Thr and Tyr at
the site of inhibitory phosphorylation. The Cdc25C gene contains tumor
suppressor p53 binding sites and is demonstrated to contribute to the
p53-dependent cell cycle arrest upon DNA damage. Here we show that both Cdc25C
and Cdc2 were down-regulated in wild-type HCT116 cells but not in p53-null,
DNMT1-null or DNMT1and DNMT3b-null cells, upon p53 stabilization following
doxorubicin-mediated DNA damage. Furthermore, zebularine, a drug that
selectively traps and depletes nuclear DNMT1 and DNMT3b, relieved p53-mediated
repression of endogenous Cdc25C and Cdc2. Methylation analysis of the Cdc25C and
Cdc2 promoter displayed internal CG methylation proximal to the p53 binding site
upon DNA damage in a p53-dependent manner. Chromatin immunoprecipitation of
doxorubicin treated wild-type HCT116 cells showed the presence of DNMT1, p53,
H3K9me2, and the transcriptional repressor HDAC1 on the Cdc25C and Cdc2
promoters, suggesting their involvement as repressive complexes in Cdc25C and
Cdc2 gene silencing. Thus, the general mechanism of p53-mediated gene repression
may involve recruitment of other repressive factors. Previous studies have shown that DNA methyltransferase (Dnmt) 1 is required for
maintece of bulk DNA methylation and is essential for mouse development.
However, somatic disruption of DNMT1 in the human cancer cell line HCT116 was
not lethal and caused only minor decreases in methylation. Here, we report the
identification of a truncated DNMT1 protein, which was generated by the
disruption of DNMT1 in HCT116 cells. The truncated protein, which had parts of
the regulatory N-terminal domain deleted but preserved the catalytic C-terminal
domain, was present at different levels in all DNMT1 single-knockout and
DNMT1/DNMT3b double-knockout cell lines tested and retained hemimethylase
activity. DNMT1 RNAi resulted in decreased cell viability in WT and knockout
cells and further loss of DNA methylation in DNMT1 knockout cells. Furthermore,
we observed a delay in methylation after replication and an increase in
hemimethylation of specific CpG sites in cells expressing the truncated protein.
Remethylation studies after drug-induced hypomethylation suggest a putative role
of DNMT1 in the de novo methylation of a subtelomeric repeat, D4Z4, which is
lost in cells lacking full-length DNMT1. Our data suggest that DNMT1 might be
essential for maintece of DNA methylation, proliferation, and survival of
cancer cells. DNA methylation plays a central role in the epigenetic regulation of gene
expression in vertebrates. Genetic and biochemical data indicated that DNA
methyltransferase 1 (Dnmt1) is indispensable for the maintece of DNA
methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116
tumor cells had only minor effects on genomic methylation and cell viability. In
this study, we identified an alternative splicing in these cells that bypasses
the disrupting selective marker and results in a catalytically active DNMT1
protein lacking the proliferating cell nuclear antigen-binding domain required
for association with the replication machinery. Using a mechanism-based trapping
assay, we show that this truncated DNMT1 protein displays only twofold reduced
postreplicative DNA methylation maintece activity in vivo. RNA
interference-mediated knockdown of this truncated DNMT1 results in global
genomic hypomethylation and cell death. These results indicate that DNMT1 is
essential in mouse and human cells, but direct coupling of the replication of
genetic and epigenetic information is not strictly required. Postreplicative maintece of genomic methylation patterns was proposed to
depend largely on the binding of DNA methyltransferase 1 (Dnmt1) to PCNA, a core
component of the replication machinery. We investigated how the slow and
discontinuous DNA methylation could be mechanistically linked with fast and
processive DNA replication. Using photobleaching and quantitative live cell
imaging we show that Dnmt1 binding to PCNA is highly dynamic. Activity
measurements of a PCNA-binding-deficient mutant with an enzyme-trapping assay in
living cells showed that this interaction accounts for a 2-fold increase in
methylation efficiency. Expression of this mutant in mouse dnmt1-/- embryonic
stem (ES) cells restored CpG island methylation. Thus association of Dnmt1 with
the replication machinery enhances methylation efficiency, but is not strictly
required for maintaining global methylation. The transient nature of this
interaction accommodates the different kinetics of DNA replication and
methylation while contributing to faithful propagation of epigenetic
information. DNA methyltransferase 1 methylates hemi-methylated CG sites generated during DNA
replication. Serine 515 of this enzyme has been shown to be phosphorylated. To
explore the importance of S515 phosphorylation, we generated mutants of Dnmt1
which removed the phosphorylation potential (S515A) or mimic phosphoserine
(S515E), purified the proteins from insect cells and analyzed their DNA
methylation activity in vitro. The S515E mutant was found to be active, while
S515A mutant had severe loss in activity when compared to the wild type protein.
The loss of activity of the S515A variant was not due to loss of DNA binding
capacity. Furthermore, we show that a phosphorylated peptide whose sequence
mimics the surrounding of Ser515 (EKIYIS(P)KIVVE) inhibited the activity of wild
type Dnmt1 ten-fold more than the non-phosphorylated peptide. The inhibition was
specific for Dnmt1 and for the particular peptide sequence. Our data suggest
that phosphorylation of Ser515 is important for an interaction between the
N-terminal domain of Dnmt1 and its catalytic domain that is necessary for
activity and that this interaction is specifically disrupted by the
phosphorylated peptide. We conclude that phosphorylation of Dnmt1 at Ser515
could be an important regulator of Dnmt1 activity during cell cycle and after
proliferative stimuli. Site-specific methylation of cytosines is a key epigenetic mark of vertebrate
DNA. While a majority of the methylated residues are in the symmetrical
(meC)pG:Gp(meC) configuration, a smaller, but significant fraction is found in
the CpA, CpT and CpC asymmetric (non-CpG) dinucleotides. CpG methylation is
reproducibly maintained by the activity of the DNA methyltransferase 1 (Dnmt1)
on the newly replicated hemimethylated substrates (meC)pG:GpC. On the other
hand, establishment and hereditary maintece of non-CpG methylation patterns
have not been analyzed in detail. We previously reported the occurrence of site-
and allele-specific methylation at both CpG and non-CpG sites. Here we
characterize a hereditary complex of non-CpG methylation, with the
transgenerational maintece of three distinct profiles in a constant ratio,
associated with extensive CpG methylation. These observations raised the
question of the signal leading to the maintece of the pattern of asymmetric
methylation. The complete non-CpG pattern was reinstated at each generation in
spite of the fact that the majority of the sperm genomes contained either none
or only one methylated non-CpG site. This observation led us to the hypothesis
that the stable CpG patterns might act as blueprints for the maintece of
non-CpG DNA methylation. As predicted, non-CpG DNA methylation profiles were
abrogated in a mutant lacking Dnmt1, the enzymes responsible for CpG
methylation, but not in mutants defective for either Dnmt3a or Dnmt2. Methylation of specific CG residues in the mammalian genome results in
tissue-specific patterns of gene expression, which are critical for cell
differentiation. Embryos that fail to establish and maintain proper DNA
methylation patterns show severe developmental abnormalities as is the case of
DNA methyltransferase 1 (Dnmt1) -deficient embryos. Dnmt1 is the main
maintece methyltransferase in the mouse and its expression is regulated by a
splicing mechanism that dictates the expression of stage-specific isoforms.
Little is known about Dnmt1 expression in the cow and isoforms of Dnmt1 are yet
unknown in this species. Here we demonstrate that the previously described
bovine Dnmt1 transcript is ubiquitously expressed in embryos and fetal tissue.
In addition, we report the identification of a splice variant of the bovine
Dnmt1, which shows a ubiquitous expression pattern. This new transcript was
detected using 5'RACE and genomic mapping and its expression pattern was shown
to be consistent with a tissue-specific mode of regulation. Furthermore, our
analysis shows that the expression of an oocyte-specific isoform of Dnmt1 is
unlikely to occur in cattle. The newly reported isoform of Dnmt1 was
demonstrated to be, similarly to Dnmt1a, polyadenylated and if translated
possess the functional domains necessary for maintece and de novo
methyltransferase activity. DNA methyltransferase-1 (DNMT1) has a higher specific activity on hemimethylated
DNA than on unmethylated DNA, but this preference is too small to explain the
faithful mitotic inheritance of genomic methylation patterns. New genetic
studies in plants and mammals have identified a novel factor that increases the
fidelity of maintece methylation. DNA methylation in post-mitotic neurons is reported to serve a variety of
functions from survival during development to the consolidation of memory. Of
particular interest with regards neuronal functioning is the change in
site-specific methylation of a variety of gene promoters in the context of
neuronal depolarization and the coding of new information. We examined the
expression of DNMT1 and DNMT3a, representative of a maintece and de novo
methyltransferase respectively, in response to in-vitro depolarization of
cortical neurons, using standard techniques such as high potassium (KCl) or the
sodium channel agonist veratridine. KCl and veratridine mediated depolarization
caused a modest but significant and replicable reduction in the mRNA and protein
expression of both DNMTs that was time and dose dependent. These effects were
supported by parallel increases in the mRNA expression of BDNF exon-1 and exon-4
as a typical response of neurons to depolarization and to rule out the
possibility of impaired transcriptional activity as a trivial explanation. In
addition to effects on mRNA and protein expression, functional DNA
methyltransferase activity was reduced in nuclear protein extracts from cells
exposed to a depolarization condition. Also, these changes could not be
explained by differential neuronal loss as measured by cell viability
cytochemistry. Our results support the idea that a reduction in DNA
methyltransferase activity in the activated and depolarized neuron could
contribute to the enhanced intensity and multiplicity of gene expression
frequently reported. Maintece of cytosine methylation in plants is controlled by three DNA
methyltransferases. MET1 maintains CG methylation, and DRM1/2 and CMT3 act
redundantly to enforce non-CG methylation. RPS, a repetitive hypermethylated DNA
fragment from Petunia hybrida, attracts DNA methylation when transferred into
Petunia or other species. In Arabidopsis thaliana, which does not contain any
RPS homologues, RPS transgenes are efficiently methylated in all sequence
contexts. To test which DNA methylation pathways regulate RPS methylation, we
examined maintece of RPS methylation in various mutant backgrounds.
Surprisingly, CG methylation was lost in a drm1/2/cmt3 mutant, and non-CG
methylation was almost completely eliminated in a met1 mutant. An unusual
cooperative activity of all three DNA methyltransferases is therefore required
for maintece of both CG and non-CG methylation in RPS. Other unusual features
of RPS methylation are the independence of its non-CG methylation from the
RNA-directed DNA methylation (RdDM) pathway and the exceptional maintece of
methylation at a CC(m)TGG site in some epigenetic mutants. This is indicative of
activity of a methylation system in plants that may have evolved from the DCM
methylation system that controls CC(m)WGG methylation in bacteria. Our data
suggest that strict separation of CG and non-CG methylation pathways does not
apply to all target regions, and that caution is required in generalizing
methylation data obtained for individual genomic regions. DNA methylation at promoter CpG islands (CGI) is an epigenetic modification
associated with inappropriate gene silencing in multiple tumor types. In the
absence of a human pituitary tumor cell line, small interfering RNA-mediated
knockdown of the maintece methyltransferase DNA methyltransferase (cytosine
5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20.
Sustained knockdown induced reexpression of the fully methylated and normally
imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite
restriction analysis (COBRA) revealed that reexpression of Nnat was associated
with partial CGI demethylation, which was also observed at the H19
differentially methylated region. Subsequent genome-wide microarray analysis
identified 91 genes that were significantly differentially expressed in Dnmt1
knockdown cells (10% false discovery rate). The analysis showed that genes
associated with the induction of apoptosis, signal transduction, and
developmental processes were significantly overrepresented in this list (P <
0.05). Following validation by reverse transcription-PCR and detection of
inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and
S100A10) were analyzed in primary human pituitary tumors, each displaying
significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For
two of these genes, NNAT and S100A10, decreased expression was associated with
increased promoter CGI methylation. Induced expression of Nnat in stable
transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is
the first report of array-based "epigenetic unmasking" in combination with Dnmt1
knockdown and reveals the potential of this strategy toward identifying genes
silenced by epigenetic mechanisms across species boundaries. In contrast to normal cells, cancer cells exhibit both genetic and epigenetic
instability. These unique properties give rise to genetic and epigenetic
heterogeneity in a given population of cancer cells and provide a means for the
population to undergo phenotypic progression by clonal selection. DNA
methylation at CpG dinucleotides is one of the epigenetic marks that are
frequently disturbed in cancer cells. To understand how the CpG methylation
pattern is changeable in cancer cells, it is necessary to know how it is
faithfully maintained in normal cell proliferation. Toward this goal, we have
developed a novel in vitro system that is based on the well-established SV40 in
vitro replication system and functions to reconstitute concurrent DNA
replication and DNA maintece methylation reactions. We found that DNA
methylation was maintained only when exogenous DNA methyltransferase 1 (DNMT1)
and S-adenosyl methionine (SAM) were added to the reaction. We demonstrated that
DNMT1 associates with replicating and/or replicated chromatin irrespective of
the DNA methylation status of template DNA. Moreover, the PCNA-binding domain
(PBD) of DNMT1 is not required for the association. Taken together, we suggest
that DNMT1 is recruited to replicating and/or replicated chromatin in a
constitutive manner independent of the DNA methylation reaction. The in vitro
system described in this report is very useful for analyzing the molecular
mechanism underlying the DNA maintece methylation reaction. DNA methylation is a major epigenetic modification and plays a crucial role in
the regulation of gene expression. Within the family of DNA methyltransferases
(Dnmts), Dnmt3a and 3b establish methylation marks during early development,
while Dnmt1 maintains methylation patterns after DNA replication. The
maintece function of Dnmt1 is regulated by its large regulatory N-terminal
domain that interacts with other chromatin factors and is essential for the
recognition of hemi-methylated DNA. Gelfiltration analysis showed that purified
Dnmt1 elutes at an apparent molecular weight corresponding to the size of a
dimer. With protein interaction assays we could show that Dnmt1 interacts with
itself through its N-terminal regulatory domain. By deletion analysis and
co-immunoprecipitations we mapped the dimerization domain to the targeting
sequence TS that is located in the center of the N-terminal domain (amino acids
310-629) and was previously shown to mediate replication independent association
with heterochromatin at chromocenters. Further mutational analyses suggested
that the dimeric complex has a bipartite interaction interface and is formed in
a head-to-head orientation. Dnmt1 dimer formation could facilitate the
discrimination of hemi-methylated target sites as has been found for other
palindromic DNA sequence recognizing enzymes. These results assign an additional
function to the TS domain and raise the interesting question how these functions
are spatially and temporarily co-ordinated. Inheritance of epigenetic information encoded by cytosine DNA methylation
patterns is crucial for mammalian cell survival, in large part through the
activity of the maintece DNA methyltransferase (DNMT1). Here, we show that
SET7, a known histone methyltransferase, is involved in the regulation of
protein stability of DNMT1. SET7 colocalizes and directly interacts with DNMT1
and specifically monomethylates Lys-142 of DNMT1. Methylated DNMT1 peaks during
the S and G(2) phases of the cell cycle and is prone to proteasome-mediated
degradation. Overexpression of SET7 leads to decreased DNMT1 levels, and
siRNA-mediated knockdown of SET7 stabilizes DNMT1. These results demonstrate
that signaling through SET7 represents a means of DNMT1 enzyme turnover. Reactivation of silenced tumor suppressor genes by 5-azacytidine (Vidaza) and
its congener 5-aza-2'-deoxycytidine (decitabine) has provided an alternate
approach to cancer therapy. We have shown previously that these drugs
selectively and rapidly induce degradation of the maintece DNA
methyltransferase (DNMT) 1 by a proteasomal pathway. Because the toxicity of
these compounds is largely due to their incorporation into DNA, it is critical
to explore novel, nonnucleoside compounds that can effectively reactivate the
silenced genes. Here, we report that a quinoline-based compound, designated
SGI-1027, inhibits the activity of DNMT1, DNMT3A, and DNMT3B as well M. SssI
with comparable IC(50) (6-13 micromol/L) by competing with S-adenosylmethionine
in the methylation reaction. Treatment of different cancer cell lines with
SGI-1027 resulted in selective degradation of DNMT1 with minimal or no effects
on DNMT3A and DNMT3B. At a concentration of 2.5 to 5 micromol/L (similar to that
of decitabine), complete degradation of DNMT1 protein was achieved within 24 h
without significantly affecting its mRNA level. MG132 blocked SGI-1027-induced
depletion of DNMT1, indicating the involvement of proteasomal pathway. Prolonged
treatment of RKO cells with SGI-1027 led to demethylation and reexpression of
the silenced tumor suppressor genes P16, MLH1, and TIMP3. Further, this compound
did not exhibit significant toxicity in a rat hepatoma (H4IIE) cell line. This
study provides a novel class of DNA hypomethylating agents that have the
potential for use in epigenetic cancer therapy. DNA methylation regulates gene expression through a complex network of
protein-protein and protein-DNA interactions in chromatin. The maintece
methylase, DNMT1 (DNA methyltransferase 1), is a prominent enzyme in the process
that is linked to DNA replication and drives the heritable nature of epigenetic
modifications. The mechanistic details that explain how DNMT1 catalytic action
is directed and regulated in chromatin are important in our overall
understanding of gene control. In this work, we show that DNMT1 is modified by
SUMOylation and we have mapped these SUMOylation sites by defined mutations.
SUMOylated DNMT1 is catalytically active on genomic DNA in vivo and we find that
SUMOylation significantly enhances the methylase activity of DNMT1 both in vitro
and in chromatin. These data suggest that SUMOylation modulates the endogenous
activity of a prominent epigenetic maintece pathway in somatic cells. OBJECTIVE: Salivary gland neoplasms pathogenesis has not been well established.
DNA methylation occurs when methyl groups are added to cytosine nucleotides in
specific areas of the gene by the enzyme DNA methyltransferase (DNMT). This
chemical modification can alter gene expression without altering DNA sequence.
While DNMT3a is mostly involved in de novo methylation, DNMT1 acts as a
maintece methyltransferase. We aimed to investigate the immunoexpression of
DNMT3a and DNMT1 in minor salivary gland neoplasms, comparing it with normal
tissue.
MATERIAL: Forty-four formalin-fixed and paraffin-embedded samples of pleomorphic
adenoma, adenoid cystic carcinoma, mucoepidermoid carcinoma and polymorphous
low-grade adenocarcinoma were included in the study. The DNMT1 and DNMT3a
proteins were identified by using a highly sensitive polymer-based system.
RESULTS: Positive nuclear and cytoplasmic labeling for DNMT1 was observed in all
samples, including the controls. Positive nuclear labeling for DNMT3a was found
only in few neoplasms: 1 pleomorphic adenoma (9.0%), 2 adenoid cystic carcinoma
(16.6%) and 1 mucoepidermoid (9.0%) cases.
CONCLUSION: Our results were not able to demonstrate a clear correlation between
DNMT1 and DNMT3a immunoexpression and salivary gland neoplasms development. DNA methylation is a postreplicative modification occurred in most prokaryotic
and eukaryotic genomes, which has a variety of important biological functions
including regulation of gene expression, gene imprinting, preservation of
chromosomal integrity, and X-chromosome inactivation. According to their
structure and functions, DNA methyltransferases (Dnmts) are divided into two
major families in mammalian cells: maintece methyltransferase (Dnmt1) and de
novo methyltransferases (Dnmt3a, Dnmt3b, and Dnmt3L). In addition, Dnmt2 also
displays weak DNA methyltransferase catalytic activity, but newly founded
function is to methylate cytosine 38 in the anti-codon loop of tRNAAsp. These
Dnmts are crucial for mammalian growth and development. Dnmts deficiency will
lead to embryonic development defects, cancer, and other diseases. Therefore,
Dnmts could be important therapeutical targets. This article summarizes the
classification, function, and recent research progress in DNA
methyltransferases. DNA methylation is an epigenetic mark essential for mammalian development,
genomic stability, and imprinting. DNA methylation patterns are established and
maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B.
Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has
nearly 40 known splice variants expressed in a tissue- and disease-specific
manner, but very little is known about the role of these splice variants in
modulating DNMT3B function. We describe here the identification and
characterization of a novel alternatively spliced form of DNMT3B lacking exon 5
within the NH(2)-terminal regulatory domain. This variant, which we term
DNMT3B3Delta5 because it is closely related in structure to the ubiquitously
expressed DNMT3B3 isoform, is highly expressed in pluripotent cells and brain
tissue, is downregulated during differentiation, and is conserved in the mouse.
Creation of pluripotent iPS cells from fibroblasts results in marked induction
of DNMT3B3Delta5. DNMT3B3Delta5 expression is also altered in human disease,
with tumor cell lines displaying elevated or reduced expression depending on
their tissue of origin. We then compared the DNA binding and subcellular
localization of DNMT3B3Delta5 versus DNMT3B3, revealing that DNMT3B3Delta5
possessed significantly enhanced DNA binding affinity and displayed an altered
nuclear distribution. Finally, ectopic overexpression of DNMT3B3Delta5 resulted
in repetitive element hypomethylation and enhanced cell growth in a colony
formation assay. Taken together, these results show that DNMT3B3Delta5 may play
an important role in stem cell maintece or differentiation and suggest that
sequences encoded by exon 5 influence the functional properties of DNMT3B. Reprogramming of DNA methylation patterns during mammalian preimplantation
development involves the concurrent maintece of methylation on differentially
methylated domains (DMDs) of imprinted genes and a marked reduction of global
(non-DMD) genomic methylation. In the developing mammalian embryo, one allele of
a DMD is unmethylated, and the opposite parental allele is methylated, having
inherited this methylation from the parental gamete. The maintece of DMDs is
important for monoallelic imprinted gene expression and normal development of
the embryo. Because the DNMT1 cytosine methyltransferase governs maintece
methylation in mammals, rearrangements of non-DMD, but not DMD methylation in
preimplantation embryos suggest that the preimplantation DNMT1-dependent
maintece mechanism specifically targets DMD sequences. We explored this
possibility using an engineered mouse ES cell line to screen for mutant DNMT1
proteins that protect against the loss of DMD and/or global (non-DMD)
methylation in the absence of the wild-type endogenous DNMT1 methyltransferase.
We identified DNMT1 mutants that were defective in maintece of either DMD
and/or non-DMD methylation. Among these, one mutant maintained non-DMD
methylation but not imprinted DMD methylation and another mutant maintained just
DMD methylation. The mutated amino acids of these mutants reside in a
mammal-specific, disordered region near the amino terminus of DNMT1. These
findings suggest that DNMT1 participates in epigenetic reprogramming through its
ability to distinguish different categories of methylated sequences. The effect of DNA methylation on CXCR4 expression has been demonstrated in
pancreatic cancer and melanoma cells, but little is known about the effect of
DNA methyltransferases 1 and 3 (DNMT1 and DNMT3B) on CXCR4 expression. Employing
lentiviral vectors, we created stable RNA interference-mediated knockdown of
DNMT1 and DNMT3B in AsPC1 pancreatic cancer cells. Using reverse transcription
real-time quantitative PCR and flow cytometric analysis, we evaluated the
increase in the expression of CXCR4 transcript and protein levels in these
cells. Bisulfite sequencing analysis showed that the level of promoter
demethylation appeared more effective in cells with knockdown of DNMT1 than in
those with DNMT3B knockdown. Furthermore, the combined RNA interference
knockdown of both DNMT1 and DNMT3B increased promoter demethylation, leading to
a slight increase in CXCR4 expression. However, the demethylating agent
5-Aza-2'-deoxycytidine exhibited the strongest effect on promoter demethylation,
which correlated with the highest production of CXCR4 transcript and protein in
AsPC1 cells. Our results indicate that DNMT1 plays the main role in maintece
of methylation of CXCR4 promoter, while DNMT3B may function as an accessory DNA
methyltransferase to modulate CXCR4 expression in AsPC1 cells. Estrogens play a critical role in brain development by acting on areas that
express estrogen receptors. In the rodent cortex, estrogen receptor alpha (ER
alpha) mRNA expression is high early in postnatal development but declines
starting at postnatal day (PND) 10 and is virtually absent in the adult cortex.
The mechanisms controlling this regulation are largely unknown. Methylation is
important for gene silencing during development in many tissues, including the
brain. In the present study, we examined the methylation status of ER alpha 5'
untranslated exons during early postnatal development in male and female mice
using methylation-specific PCR and pyrosequencing. Several regions of ER alpha
promoter displayed a significant increase in methylation at PND 18 and 25
compared with PND 4. DNA methyltransferases (DNMT) are important for the
initiation and maintece of methylation. Real-time PCR showed that DNMT3A, the
de novo DNMT peaked at PND 10 and was decreased by PND 25. DNMT1, which is
important for maintece of methylation, increased across development and
stayed high in adult cortex. The methyl-CpG-binding protein 2 (MeCP2) is also
important for stabilization of methylation. A chromatin immunoprecipitation
assay showed a correlation between association of MeCP2 with ER alpha promoter
and the increase in methylation and decrease in ER alpha expression after PND
10. In mice containing a mutant MeCP2 protein, ER alpha mRNA expression and
promoter methylation patterns across development were different compared with
wild-type mice. These data suggest that methylation of ER alpha promoters
regulates ER alpha mRNA expression in the cortex during postnatal development in
a MeCP2-dependent fashion. 1. The genome of extraembryonic tissue, such as the placenta, is hypomethylated
relative to that in somatic tissues. However, the origin and role of this
hypomethylation remains unclear. The DNA methyltransferases DNMT1, -3A, and -3B
are the primary mediators of the establishment and maintece of DNA
methylation in mammals. In this study, we investigated promoter
methylation-mediated epigenetic down-regulation of DNMT genes as a potential
regulator of global methylation levels in placental tissue. Although DNMT3A and
-3B promoters lack methylation in all somatic and extraembryonic tissues tested,
we found specific hypermethylation of the maintece DNA methyltransferase
(DNMT1) gene and found hypomethylation of the DNMT3L gene in full term and first
trimester placental tissues. Bisulfite DNA sequencing revealed monoallelic
methylation of DNMT1, with no evidence of imprinting (parent of origin effect).
In vitro reporter experiments confirmed that DNMT1 promoter methylation
attenuates transcriptional activity in trophoblast cells. However, global
hypomethylation in the absence of DNMT1 down-regulation is apparent in
non-primate placentas and in vitro derived human cytotrophoblast stem cells,
suggesting that DNMT1 down-regulation is not an absolute requirement for genomic
hypomethylation in all instances. These data represent the first demonstration
of methylation-mediated regulation of the DNMT1 gene in any system and
demonstrate that the unique epigenome of the human placenta includes
down-regulation of DNMT1 with concomitant hypomethylation of the DNMT3L gene.
This strongly implicates epigenetic regulation of the DNMT gene family in the
establishment of the unique epigenetic profile of extraembryonic tissue in
humans. A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has
been developed. The assay applies a break light oligonucleotide in which the
methylation of an unmethylated 5'-CG-3' site is enzymatically coupled to the
development of a fluorescent signal. This sensitive assay can measure rates of
DNA methylation down to 0.34 +/- 0.06 fmol/s. The assay is reproducible, with a
coefficient of variation over six independent measurements of 4.5%. Product
concentration was accurately measured from fluorescence signals using a linear
calibration curve, which achieved a goodness of fit (R(2)) above 0.98. The
oligonucleotide substrate contains three C5-methylated cytosine residues and one
unmethylated 5'-CG-3' site. Methylation yields an oligonucleotide containing the
optimal substrate for the restriction enzyme GlaI. Cleavage of the fully
methylated oligonucleotide leads to separation of fluorophore from quencher,
giving a proportional increase in fluorescence. This method has been used to
assay activity of DNMT1, the principle maintece methyltransferase in human
cells, and for the kinetic characterization of the bacterial cytosine-C5
methyltransferase M.SssI. The assay has been shown to be suitable for the
real-time monitoring of DNMT1 activity in a high-throughput format, with low
background signal and the ability to obtain linear rates of methylation over
long periods, making this a promising method of high-throughput screening for
inhibitors. 5-Aza-2'-deoxycytidine (5-aza-dC) is a nucleoside analogue with cytotoxic and
DNA demethylating effects. Here we show that 5-aza-dC induces the proteasomal
degradation of free (non-chromatin bound) DNMT1 through a mechanism which is
dependent on DNA synthesis and the targeting of incorporated 5-aza-dC residues
by DNMT1 itself. Thus, 5-aza-dC induces Dnmt1 degradation in wild-type mouse ES
cells, but not in Dnmt [3a(-/-), 3b(-/-)] mouse ES cells which express Dnmt1 but
lack DNA methylation (<0.7% of CpG methylated) and contain few hemi-methylated
CpG sites, these being the preferred substrates for Dnmt1. We suggest that
adducts formed between DNMT1 and 5-aza-dC molecules in DNA induce a ubiquitin-E3
ligase activity which preferentially targets free DNMT1 molecules for
degradation by the proteasome. The proteasome inhibitor MG132 prevents DNMT1
degradation and reduces hypomethylation induced by 5-aza-dC. Methylation of cytosine residues in the context of CpG dinucleotides within
mammalian DNA is an epigenetic modification with profound effects on
transcriptional regulation. A group of enzymes, the DNA methyltransferases
(DNMTs) tightly regulate both the initiation and maintece of these methyl
marks. Loss of critical components of this enzymatic machinery results in
growth, viability, and differentiation defects in both mice and humans,
supporting the notion that this epigenetic modification is essential for proper
development. Beyond this, DNA methylation also provides a potent epigenetic
mechanism for cellular memory needed to silence repetitive elements and preserve
lineage specificity over repeated cell divisions throughout adulthood. Recent
work highlighting the specialized roles of DNA methylation and
methyltransferases in maintaining adult somatic stem cell function suggests that
further dissection of these mechanisms will shed new light on the complex nature
of self-renewal. DNA methyltransferases (DNMTs) are essential for maintece of aberrant
methylation in cancer cells and play important roles in the development of
cancers. Unregulated activation of PI3K/Akt pathway is a prominent feature of
many human cancers including human hepatocellular carcinoma (HCC). In present
study, we found that DNMT3B mRNA and protein levels were decreased in a dose-
and time-dependent manner in HCC cell lines with LY294002 treatment. However, we
detected that LY294002 treatment did not induce increase of the degradation of
DNMT3B protein using protein decay assay. Moreover we found that Akt induced
alteration of the expression of DNMT3B in cells transfected with myristylated
variants of Akt2 or cells transfected with small interfering RNA respectively.
Based on DNMT3B promoter dual-luciferase reporter assay, we found PI3K pathway
regulates DNMT3B expression at transcriptional level. And DNMT3B mRNA decay
analysis suggested that down-regulation of DNMT3B by LY294002 is also
post-transcriptional control. Furthermore, we demonstrated that LY294002
down-regulated HuR expression in a time-dependent manner in BEL-7404. In
summary, we have, for the first time, demonstrate that PI3K/Akt pathway
regulates the expression of DNMT3B at transcriptional and post-transcriptional
levels, which is particularly important to understand the effects of PI3K/Akt
and DNMT3B on hepatocarcinogenesis. Correction of double strand DNA breaks proceeds in an error-free pathway of
homologous recombination (HR), which can result in gene silencing of half of the
DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C.,
Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S.,
Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L.,
Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore
the mechanism that leads to HR-induced silencing, a genetic screen was carried
out based on the silencing of a GFP reporter to identify potential partners.
DMAP1, a DNMT1 interacting protein, was identified as a mediator of this
process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting
that DMAP1 is a co-repressor that supports the maintece and de novo action of
DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to
conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew
poorly and eventually ceased their growth. Analysis of the tumor suppressor gene
p16 methylation status revealed a clear reduction in methylated CpGs in the
shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway
caused the slow growth phenotype. Analysis of HR, using a fluorescence-based
reporter, revealed that knocking down DMAP1 also caused hypomethylation of the
DNA repair products following gene conversion. DMAP1 was selectively enriched in
recombit GFP chromatin based on chromatin immunoprecipitation analysis. The
picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR
repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it
has additional roles in genomic stability. Maintece of genomic methylation patterns is mediated primarily by DNA
methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1
composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase
domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically
binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker
between the DNA and the active site of DNMT1, preventing de novo methylation. In
addition, a loop projecting from BAH2 interacts with the target recognition
domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted
position and preventing it from inserting into the DNA major groove. Our studies
identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides
are occluded from the active site to ensure that only hemimethylated CpG
dinucleotides undergo methylation. In mammals Dnmt1 is the DNA methyltransferase chiefly responsible for
maintaining genomic methylation patterns through DNA replication cycles, but how
its maintece activity is controlled is still not well understood.
Interestingly, Uhrf1, a crucial cofactor for maintece of DNA methylation by
Dnmt1, is endowed with E3 ubiquitin ligase activity. Here, we show that both
Dnmt1 and Uhrf1 coprecipitate with ubiquitin specific peptidase 7 (Usp7), a
de-ubiquitinating enzyme. Overexpression of Uhrf1 and Usp7 resulted in opposite
changes in the ubiquitination status and stability of Dnmt1. Our findings
suggest that, by balancing Dnmt1 ubiquitination, Usp7 and Uhrf1 fine tune Dnmt1
stability. Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for
maintece of cytosine methylation at CpG dinucleotides in the mammalian
genome. The N-terminal replication focus targeting sequence (RFTS) domain of
Dnmt1 has been implicated in subcellular localization, protein association, and
catalytic function. However, progress in understanding its function has been
limited by the lack of assays for and a structure of this domain. Here, we show
that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited
by the RFTS domain, which functions by virtue of binding the catalytic domain to
the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate
established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity.
The crystal structure of the RFTS domain reveals a novel fold and supports a
mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may
activate Dnmt1 for DNA binding. Dnmt1, the principal DNA methyltransferase in mammalian cells, is a large and a
highly dynamic enzyme with multiple regulatory features that can control DNA
methylation in cells. This chapter highlights how insights into Dnmt1 structure
and function can advance our understanding of DNA methylation in cells. The
allosteric site(s) on Dnmt1 can regulate processes of de novo and maintece
DNA methylation in cells. Remaining open questions include which molecules, by
what mechanism, bind at the allosteric site(s) in cells? Different
phosphorylation sites on Dnmt1 can change its activity or ability to bind DNA
target sites. Thirty-one different molecules are currently known to have
physical and/or functional interaction with Dnmt1 in cells. The Dnmt1 structure
and enzymatic mechanism offer unique insights into those interactions. The
interacting molecules are involved in chromatin organization, DNA repair, cell
cycle regulation, and apoptosis and also include RNA polymerase II, some
RNA-binding proteins, and some specific Dnmt1-inhibitory RNA molecules. Combined
insights from studies of different enzymatic features of Dnmt1 offer novel ideas
for development of drug candidates, and can be used in selection of promising
drug candidates from more than 15 different compounds that have been identified
as possible inhibitors of DNA methylation in cells. Methylation of cytosine in DNA plays a crucial role in development through
inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for
the propagation of methylation patterns to the next generation via its
preferential methylation of hemimethylated CpG sites in the genome; however, how
Dnmt1 maintains methylation patterns is not fully understood. Here we report the
crystal structure of the large fragment (291-1620) of mouse Dnmt1 and its
complexes with cofactor S-adenosyl-L-methionine and its product
S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain
responsible for targeting Dnmt1 to replication foci is inserted into the
DNA-binding pocket, indicating that this domain must be removed for methylation
to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine
residue undergoes a conformation transition to a catalytically competent
position. For the recognition of hemimethylated DNA, Dnmt1 is expected to
utilize a target recognition domain that overhangs the putative DNA-binding
pocket. Taking into considerations the recent report of a shorter fragment
structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket
and prevents aberrant de novo methylation, we propose that maintece
methylation is a multistep process accompanied by structural changes. DNA methyltransferase 1 (DNMT1) is crucial for maintece of methylation, gene
regulation and chromatin stability. DNA mismatch repair, cell cycle regulation
in post-mitotic neurons and neurogenesis are influenced by DNA methylation. Here
we show that mutations in DNMT1 cause both central and peripheral
neurodegeneration in one form of hereditary sensory and autonomic neuropathy
with dementia and hearing loss. Exome sequencing led to the identification of
DNMT1 mutation c.1484A>G (p.Tyr495Cys) in two American kindreds and one Japanese
kindred and a triple nucleotide change, c.1470-1472TCC>ATA
(p.Asp490Glu-Pro491Tyr), in one European kindred. All mutations are within the
targeting-sequence domain of DNMT1. These mutations cause premature degradation
of mutant proteins, reduced methyltransferase activity and impaired
heterochromatin binding during the G2 cell cycle phase leading to global
hypomethylation and site-specific hypermethylation. Our study shows that DNMT1
mutations cause the aberrant methylation implicated in complex pathogenesis. The
discovered DNMT1 mutations provide a new framework for the study of
neurodegenerative diseases. Studies carried out in cultured cells have implicated modifiers of epigenetic
reprogramming in the regulation of telomere length, reporting elongation in
cells that were null for DNA methyltransferase DNA methyltransferase 1 (Dnmt1),
both de novo DNA methyltransferases, Dnmt3a and Dnmt3b or various histone
methyltransferases. To investigate this further, we assayed telomere length in
whole embryos or adult tissue from mice carrying mutations in four different
modifiers of epigenetic reprogramming: Dnmt1, DNA methyltransferase 3-like,
structural maintece of chromosomes hinge domain containing 1, and forkhead
box O3a. Terminal restriction fragment analysis was used to compare telomere
length in homozygous mutants, heterozygous mutants and wild-type littermates.
Contrary to expectation, we did not detect overall lengthening in the mutants,
raising questions about the role of epigenetic processes in telomere length in
vivo. DNA methylation is widespread in most species, from bacteria to mammals, and is
crucial for genomic imprinting, gene expression, and embryogenesis. DNA
methylation occurs via two major classes of enzymatic reactions:
maintece-type methylation catalyzed by DNA (cytosine-5-)-methyltransferase
(DNMT) 1, and de novo methylation catalyzed by DNMT 3 alpha (DNMT3A) and -beta
(DNMT3B). The expression pattern and regulation of DNMT genes in primordial germ
cells (PGCs) and germ line cells has not been sufficiently established in birds.
Therefore, we employed bioinformatics, RT-PCR, real-time PCR, and in situ
hybridization analyses to examine the structural conservation and conserved
expression patterns of chicken DNMT family genes. We further examined the
regulation of a candidate de novo DNA methyltransferase gene, cDNMT3B by
cotransfection of cDNMT3B 3'UTR- and cDNMT3B 3'UTR-specific miRNAs through a
dual fluorescence reporter assay. All cDNMT family members were differentially
detected during early embryonic development. Of interest, cDNMT3B expression was
highly detected in early embryos and in PGCs. During germ line development and
sexual maturation, cDNMT3B expression was reestablished in a female germ
cell-specific manner. In the dual fluorescence reporter assay, cDNMT3B
expression was significantly downregulated by four miRNAs: gga-miR-15c (25.82%),
gga-miR-29b (30.01%), gga-miR-383 (30.0%), and gga-miR-222 (31.28%). Our data
highlight the structural conservation and conserved expression patterns of
chicken DNMTs. The miRNAs investigated in this study may induce downregulation
of gene expression in chicken PGCs and germ cells. DNA methylation plays a significant role in the expression of the genetic code
and affects early growth and development through its influence on gene
expression. DNA methyltransferase 1 (Dnmt1) is the enzyme responsible for
maintaining the methylation marks through cell division. However, the de novo
methyltransferases, Dnmt3a and Dnmt3b, can also contribute to the maintece of
the methylation pattern. Manipulation of these enzymes, especially Dnmt1,
provides a means to alter DNA methylation levels. Manipulation of the DNA
methylation pattern of somatic cells will allow a better understanding of the
different molecular process associated with chromatin structure and gene
expression. Different approaches to artificially manipulate the expression of
Dnmt1 in somatic cells include the addition of 5-azacytidine, culture of cells
for an extended period of time, and the use of small interfering RNA
technologies. DNA methylation mostly occurs within the context of CpG dinucleotides and is
essential for embryonic development and gene repression. It is generally
accepted that DNA methyltransferases carry out specific and non-overlapping
functions, Dnmt3a and Dnmt3b being responsible for the establishment of
methylation around the time of implantation and Dnmt1 ensuring that methylation
is faithfully copied to daughter cells via what has come to be known as
"maintece methylation." This longstanding view has been challenged over the
years with the observation that Dnmt1 alone is incapable of perfect maintece
methylation. A new model is emerging that takes into account a contribution of
the de novo enzymes Dnmt3a and Dnmt3b in the maintece of the DNA methylation.
We recently showed that certain germ line genes are specific targets of Dnmt3b,
and that Dnmt3b remains bound to their promoter regions in somatic cells via
interaction with the transcriptional repressor E2F6. It is tempting to consider
an ongoing role for Dnmt3b in the methylation of germ line genes in somatic
cells. We propose here observations in support of the hypothesis that the
maintece of methylation and subsequent silencing of a handful of germ line
genes requires Dnmt3b but not Dnmt1. In addition to suggesting a new role for
Dnmt3b in the protection of somatic cells against the promiscuous expression of
the germ line program, these observations are of particular interest in the
field of carcinogenesis, given that the expression of catalytically inactive
Dnmt3b isoforms and aberrant expression of germ line genes are commonly observed
in cancer cells. Recent studies demonstrate that UHRF1 is required for DNA methylation
maintece by targeting DNMT1 to DNA replication foci, presumably through its
unique hemi-methylated DNA-binding activity and interaction with DNMT1. UHRF2,
another member of the UHRF family proteins, is highly similar to UHRF1 in both
sequence and structure, raising questions about its role in DNA methylation. In
this study, we demonstrate that, like UHRF1, UHRF2 also binds preferentially to
methylated histone H3 lysine 9 (H3K9) through its conserved tudor domain and
hemi-methylated DNA through the SET and Ring associated domain. Like UHRF1,
UHRF2 is enriched in pericentric heterochromatin. The heterochromatin
localization depends to large extent on its methylated H3K9-binding activity and
to less extent on its methylated DNA-binding activity. Coimmunoprecipitation
experiments demonstrate that both UHRF1 and UHRF2 interact with DNMT1, DNMT3a,
DNMT3b and G9a. Despite all these conserved functions, we find that UHRF2 is not
able to rescue the DNA methylation defect in Uhrf1 null mouse embryonic stem
cells. This can be attributed to the inability for UHRF2 to recruit DNMT1 to
replication foci during S phase of the cell cycle. Indeed, we find that while
UHRF1 interacts with DNMT1 in an S phase-dependent manner in cells, UHRF2 does
not. Thus, our study demonstrates that UHRF2 and UHRF1 are not functionally
redundant in DNA methylation maintece and reveals the cell-cycle-dependent
interaction between UHRF1 and DNMT1 as a key regulatory mechanism targeting
DNMT1 for DNA methylation. Establishment of persistent Epstein-Barr virus (EBV) infection requires
transition from a program of full viral latency gene expression (latency III) to
one that is highly restricted (latency I and 0) within memory B lymphocytes. It
is well established that DNA methylation plays a critical role in EBV gene
silencing, and recently the chromatin boundary protein CTCF has been implicated
as a pivotal regulator of latency via its binding to several loci within the EBV
genome. One notable site is upstream of the common EBNA gene promoter Cp, at
which CTCF may act as an enhancer-blocking factor to initiate and maintain
silencing of EBNA gene transcription. It was previously suggested that increased
expression of CTCF may underlie its potential to promote restricted latency, and
here we also noted elevated levels of DNA methyltransferase 1 (DNMT1) and DNMT3B
associated with latency I. Within B-cell lines that maintain latency I, however,
stable knockdown of CTCF, DNMT1, or DNMT3B or of DNMT1 and DNMT3B in combination
did not result in activation of latency III protein expression or EBNA gene
transcription, nor did knockdown of DNMTs significantly alter CpG methylation
within Cp. Thus, differential expression of CTCF and DNMT1 and -3B is not
critical for maintece of restricted latency. Finally, mutant EBV lacking the
Cp CTCF binding site exhibited sustained Cp activity relative to wild-type EBV
in a recently developed B-cell superinfection model but ultimately was able to
transition to latency I, suggesting that CTCF contributes to but is not
necessarily essential for the establishment of restricted latency. DNA methyltransferase-1 (Dnmt1) is involved in the maintece of DNA
methylation patterns and is crucial for normal mammalian development. The aim of
the present study was to assess the localization of Dnmt1 in cow, during the
latest phases of oocyte differentiation and during the early stages of
segmentation. Dnmt1 expression and localization were assessed in oocytes
according to the chromatin configuration, which in turn provides an important
epigenetic mechanism for the control of global gene expression and represents a
morphological marker of oocyte differentiation. We found that the initial
chromatin condensation was accompanied by a slight increase in the level of
global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed
by laser scanning confocal microscopy analysis (LSCM). RT-PCR confirmed the
presence of Dnmt1 transcripts throughout this phase of oocyte differentiation.
Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was
always present and localized in the cytoplasm, regardless the chromatin
configuration and the level of global DNA methylation. Moreover, our data
indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage
oocytes and zygotes, it enters the nuclei of 8-16 cell stage embryos. As
suggested in mouse, the functional meaning of the presence of Dnmt1 in the
bovine embryo nuclei could be the maintainement of the methylation pattern of
imprinted genes. In conclusion, the present work provides useful elements for
the study of Dnmt1 function during the late stage of oocyte differentiation,
maturation and early embryonic development in mammals. A subset of genes, known as imprinted genes, is present in the mammalian genome.
Genomic imprinting governs the monoallelic expression of these genes, depending
on whether the gene was inherited from the sperm or the egg. This
parent-of-origin specific gene expression is generally dependent on the
epigenetic modification, DNA methylation, and the DNA methylation status of CpG
dinucleotides residing in loci known as differentially methylated regions
(DMRs). The enzymatic machinery responsible for the addition of methyl (-CH(3))
groups to the cytosine residue in the CpG dinucleotides are known as DNA
methyltransferases (DNMTs). Correct establishment and maintece of methylation
patterns at imprinted genes has been associated with placental function and
regulation of embryonic/fetal development. Much work has been carried out on
imprinted genes in mouse and human; however, little is known about the
methylation dynamics in the bovine oocyte. The primary objective of the present
study was to characterize the establishment of methylation at maternally
imprinted genes in bovine growing oocytes and to determine if the expression of
the bovine DNMTs-DNMT3A, DNMT3B, and DNMT3L-was coordinated with DNA methylation
during oocyte development. To this end, a panel of maternally imprinted genes
was selected (SNRPN, MEST, IGF2R, PEG10, and PLAGL1) and putative DMRs for MEST,
IGF2R, PEG10, and PLAGL1 were identified within the 5' regions for each gene;
the SNRPN DMR has been reported previously. Conventional bisulfite sequencing
revealed that methylation marks were acquired at all five DMRs investigated in
an oocyte size-dependent fashion. This was confirmed for a selection of genes
using pyrosequencing analysis. Furthermore, mRNA expression and protein analysis
revealed that DNMT3A, DNMT3B, and DNMT3L are also present in the bovine oocyte
during its growth phase. This study demonstrates for the first time that an
increase in bovine imprinted gene DMR methylation occurs during oocyte growth,
as is observed in mouse. BACKGROUND: DNA methylation, histone modifications and nucleosome occupancy act
in concert for regulation of gene expression patterns in mammalian cells.
Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in
establishment of DNA methylation at embryonic gene targets in ES cells through
recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar
role in maintece of DNA methylation in somatic cells is still unclear.
RESULTS: Here we show that G9a is not essential for maintece of DNA
methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA
methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to
nucleosomes containing methylated DNA even in the absence of G9a, ensuring
faithful propagation of methylated states in cooperation with DNMT1 through
somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B
and DNA methylation-independent manner. However, G9a knockdown synergizes with
pharmacologic inhibition of DNMTs resulting in increased hypomethylation and
inhibition of cell proliferation.
CONCLUSIONS: Taken together, these data suggest that G9a is not involved in
maintece of DNA methylation in somatic cells but might play a role in
re-initiation of de novo methylation after treatment with hypomethylating drugs,
thus serving as a potential target for combinatorial treatments strategies
involving DNMTs inhibitors. DNMT1, the major maintece DNA methyltransferase in animals, helps to regulate
gene expression, genome imprinting, and X-chromosome inactivation. We report on
the crystal structure of a productive covalent mouse DNMT1(731-1602)-DNA complex
containing a central hemimethylated CpG site. The methyl group of methylcytosine
is positioned within a shallow hydrophobic concave surface, whereas the cytosine
on the target strand is looped out and covalently anchored within the catalytic
pocket. The DNA is distorted at the hemimethylated CpG step, with side chains
from catalytic and recognition loops inserting through both grooves to fill an
intercalation-type cavity associated with a dual base flip-out on partner
strands. Structural and biochemical data establish how a combination of active
and autoinhibitory mechanisms ensures the high fidelity of DNMT1-mediated
maintece DNA methylation. BACKGROUND: Early pregcy loss (EPL) is a frustrating clinical problem, whose
mechanisms are not completely understood. DNA methylation, which includes
maintece methylation and de novo methylation directed by DNA
methyltransferases (DNMTs), is important for embryo development. Abnormal
function of these DNMTs may have serious consequences for embryonic development.
METHODS: To evaluate the possible involvement of DNA methylation in human EPL,
the expression of DNMT proteins and global methylation of DNA were assessed in
villous or decidua from EPL patients. The association of maintece methylation
with embryo implantation and development was also examined.
RESULTS: We found that DNMT1 and DNMT3A were both expressed in normal human
villous and decidua. DNMT1 expression and DNA global methylation levels were
significantly down-regulated in villous of EPL. DNMT3A expression was not
significantly changed in the EPL group compared to controls in either villous or
decidua. We also found that disturbance of maintece methylation with a DNMT1
inhibitor may result in a decreased global DNA methylation level and impaired
embryonic development in the mouse model, and inhibit in vitro embryo attachment
to endometrial cells.
CONCLUSIONS: Our results demonstrate that defects in DNA maintece methylation
in the embryo, not in the mother, are associated with abnormal embryonic
implantation and development. The findings of the current study provide new
insights into the etiology of EPL. Silencing of genes by hypermethylation contributes to cancer progression and has
been shown to occur with increased frequency at specific genomic loci. However,
the precise mechanisms underlying the establishment and maintece of aberrant
methylation marks are still elusive. The de novo DNA methyltransferase 3B
(DNMT3B) has been suggested to play an important role in the generation of
cancer-specific methylation patterns. Previous studies have shown that a
reduction of DNMT3B protein levels induces antiproliferative effects in cancer
cells that were attributed to the demethylation and reactivation of tumor
suppressor genes. However, methylation changes have not been analyzed in detail
yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer
cell lines. Our results confirm that depletion of DNMT3B specifically reduced
the proliferation rate of DNMT3B-overexpressing colon cancer cell lines.
However, genome-scale DNA methylation profiling failed to reveal methylation
changes at putative DNMT3B target genes, even in the complete absence of DNMT3B.
These results show that DNMT3B is dispensable for the maintece of aberrant
DNA methylation patterns in human colon cancer cells and they have important
implications for the development of targeted DNA methyltransferase inhibitors as
epigenetic cancer drugs. The maintece methylation of hemimethylated CpG sites by the DNA
methyltransferase Dnmt1 is the molecular basis of the inheritance of DNA
methylation patterns. Based on structural data and kinetics obtained with a
truncated form of Dnmt1, an autoinhibition model for the specificity of Dnmt1
was proposed in which unmethylated DNA binds to Dnmt1's CXXC domain, which
prevents its methylation. We have prepared CXXC domain variants that lost DNA
binding. Corresponding full-length Dnmt1 variants did not display a reduction in
specificity, indicating that the autoinhibition model does not apply in
full-length Dnmt1. Furthermore, we show that the Dnmt1 M1235S variant, which
carries an exchange in the catalytic domain of the enzyme, has a marked
reduction in specificity, indicating that the recognition of the hemimethylated
state of target sites resides within the catalytic domain. Aberrant promoter DNA hypermethylation of tumor suppressor genes is a hallmark
of cancer. This alteration is largely dependent on the action of de novo DNA
methyltransferases (DNMTs) early during tumor progression, which supports the
oncogenic role for these enzymes. However, recent research has identified
several inactivating mutations of de novo DNMTs in various types of tumor. In
addition, it has been shown that loss of de novo DNA methylation activity at
advanced tumor stages leads to the promoter DNA demethylation-dependent
expression of specific oncogenes. These new data support the notion that de novo
DNMTs also have an important role in the maintece of DNA methylation and
suggest that, in addition to acting as oncogenes, they also behave as tumor
suppressors. This potential dual role might have clinical implications, as DNMTs
are currently considered bona fide targets in cancer therapy. The enzymatic control of the setting and maintece of symmetric and
non-symmetric DNA methylation patterns in a particular genome context is not
well understood. Here, we describe a comprehensive analysis of DNA methylation
patterns generated by high resolution sequencing of hairpin-bisulfite amplicons
of selected single copy genes and repetitive elements (LINE1, B1,
IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously
identifies a substantial amount of regional incomplete methylation maintece,
i.e. hemimethylated CpG positions, with variant degrees among cell types.
Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively
catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and
region-dependent non-CpG methylation is strongly linked to neighboring CpG
methylation and requires the presence of Dnmt3L. The generation of a
comprehensive data set of 146,000 CpG dyads was used to apply and develop
parameter estimated hidden Markov models (HMM) to calculate the relative
contribution of DNA methyltransferases (Dnmts) for de novo and maintece DNA
methylation. The comparative modelling included wild-type ESCs and mutant ESCs
deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM
analysis identifies a considerable de novo methylation activity for Dnmt1 at
certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b
contribute de novo function. However, both enzymes are also essential to
maintain symmetrical CpG methylation at distinct repetitive and single copy
sequences in ESCs. DNA methylation and DNA methyltransferases are essential for spermatogenesis.
Mutations in the DNA methyltransferase Dnmt1 gene exert a paternal effect on
epigenetic states and phenotypes of offspring, suggesting that DNMT1 is
important for the epigenetic remodeling of the genome that takes place during
spermatogenesis. However, the specific role of DNMT1 in spermatogenesis and the
establishment of genomic imprints in the male germ line remains elusive. To
further characterize the effect of DNMT1 deficiency on the resetting of
methylation imprints during spermatogenesis, we analyzed the methylation
profiles of imprinted regions in the spermatozoa of mice that were heterozygous
for a Dnmt1 loss-of-function mutation. The mutation did not affect the H19 or IG
differentially methylated regions (DMRs) that are usually highly methylated but
led to a partial hypermethylation of the Snrpn DMR, a region that should
normally be unmethylated in mature spermatozoa. This defect does not appear in
mouse models with mutations in Dnmt3a and Mthfr genes and, therefore, it is
specific for the Dnmt1 gene and is suggestive of a role of DNMT1 in imprint
resetting or maintece in the male germ line. Methylation at the 5-position of DNA cytosine on the vertebrate genomes is
accomplished by the combined catalytic actions of three DNA methyltransferases
(DNMTs), the de novo enzymes DNMT3A and DNMT3B and the maintece enzyme DNMT1.
Although several metabolic routes have been suggested for demethylation of the
vertebrate DNA, whether active DNA demethylase(s) exist has remained elusive.
Surprisingly, we have found that the mammalian DNMTs, and likely the vertebrates
DNMTs in general, can also act as Ca(2+) ion- and redox state-dependent active
DNA demethylases. This finding suggests new directions for reinvestigation of
the structures and functions of these DNMTs, in particular their roles in Ca(2+)
ion-dependent biological processes, including the genome-wide/local DNA
demethylation during early embryogenesis, cell differentiation, neuronal
activity-regulated gene expression, and carcinogenesis. |
Is transcapillary albumin escape altered in diabetic patients? | An altered TERalb is present in type 2 diabetic patients, both with normal and altered patterns of AER.
TERalb is increased also in normo-albuminuric type 1 diabetic patients. | BACKGROUND: Atherosclerosis is a major cause of morbidity and mortality in
insulin-dependent diabetes mellitus. Recent studies have shown that albuminuria
is a predictor of cardiovascular disease in these patients and there is a
particularly high incidence of coronary heart disease in the early stages of
albuminuria.
AVAILABLE EVIDENCE: A number of established cardiovascular risk factors, such as
elevated blood pressure, atherogenic changes in the plasma concentrations of
lipids and lipoproteins, elevated plasma levels of fibrinogen and, probably,
hyper-reactivity of platelets are present in patients with diabetic nephropathy.
Further, albuminuria may be a marker of generalized disease in the vascular wall
of small and large blood vessels. Findings of an elevated rate of transcapillary
albumin escape, an elevated plasma concentration of von Willebrand's factor and
impaired fibrinolytic capacity in early diabetic nephropathy support this
hypothesis.
CONCLUSION: The initial pathophysiological mechanisms involved in diabetic
nephropathy are still hypothetical and largely unknown. The effect of hypotensive therapy on the transcapillary escape rate of albumin
(TERalb) was studied in eight hypertensive insulin-dependent diabetic patients
(mean age 29, range 19-42 years) with nephropathy and retinopathy.
Transcapillary escape rate of albumin (initial disappearance of intravenously
injected 125I-labelled human serum albumin), urinary albumin excretion rate
(radial immunodiffusion), and glomerular filtrate rate (single bolus 51-Cr-EDTA
technique) were measured. After hypotensive treatment (mean duration, 23 months,
range 7-39 months) with combinations of metoprolol, hydralazine, and frusemide
or thiazide diuretics, arterial blood pressure fell from 152/103 +/- 18/6 mmHg
(mean +/- SD) to 133/81 +/- 12/10 mmHg (p less than 0.01), transcapillary escape
rate of albumin from 10.2 +/- 1.8 to 8.1 +/- 1.8% of intravascular mass of
albumin/h (p less than 0.01), albuminuria from 1803 (370-5066) micrograms/min to
940 (101-2676) micrograms/min (median and range, p less than 0.05), and
glomerular filtration rate from 103 +/- 23 to 84 +/- 22 ml/min/1.73 m2 (p less
than 0.01). Our study suggests that effective hypotensive treatment reduces the
abnormally elevated albumin leakage characteristically found in
insulin-dependent diabetic patients with clinical microangiopathy. This may be
due to a reduction in the hydrostatic pressure in the microcirculation. Three patients with insulin-dependent diabetes mellitus are described in whom
generalized oedema and weight gain followed the administration of excessive
monocomponent insulins, in two cases associated with symptomatic hypoglycaemia.
Serial measurements of plasma volume and transcapillary escape rate of albumin
(TERA) using 125I-labelled albumin, serum colloid osmotic pressure (COP) using a
membrane colloid osmometer, packed cell volume (PCV), and serum proteins, showed
that oedema was associated with an increased plasma volume and TERA, while serum
albumin and total protein concentration and serum COP were reduced. A reduction
in daily insulin dose abolished hypoglycaemia and resulted in weight loss,
natriuresis, diuresis, a reduction in plasma volume and TERA, and an increase in
serum albumin, total protein, and COP. Strict metabolic control in previously
poorly controlled patients may cause insulin-induced increments in plasma volume
and albumin escape rate. Lower limb venous compliance and transcapillary escape rate of transferrin were
measured in eight normotensive, insulin-dependent male diabetic patients and
eight control subjects using a dual isotope technique. Technetium-99m labelled
autologous erythrocytes were used to measure venous compliance and to correct
for local changes in blood volume, whilst Indium-113m labelled transferrin was
used to measure transcapillary escape of protein. The diabetic patients were
found to have reduced venous compliance 1.5 (0.7 to 3.4) x 10(-2) mmHg-1
compared with controls 3.2 (2.4 to 4.1) x 10(-2) mmHg-1 (p less than 0.01). The
diabetic patients were also found to have greater transcapillary escape of
transferrin -2.7 (-1.5 to -5.3) x 10(-3), compared with control subjects -5.2
(-4.1 to -8.1) x 10(-3) (p less than 0.02) in response to increasing hydrostatic
pressure. These results show reduced venous compliance in patients with a mean
duration of diabetes of 15 years and with only at most, early complications of
diabetes, and confirm previous observations showing increased transcapillary
escape of protein. The transcapillary escape rate and relative plasma disappearance of glycated and
non-glycated albumin were measured in 25 male Type 1 (insulin-dependent)
diabetic patients using a double tracer technique. The patients were divided
into three groups on the basis of their urinary albumin excretion: group 1,
normal albumin excretion (less than 30 mg/24 h) (n = 8); group 2,
microalbuminuria (30-300 mg/24 h) (n = 9); and group 3, clinical nephropathy
(greater than 300 mg/24 h) (n = 8). Six male age-matched non-diabetic persons
served as control subjects. The transcapillary escape rate of glycated albumin
was similar in group 1 and control subjects (4.7 +/- 2.1 versus 5.1 +/- 1.7%),
but significantly increased in group 2 (7.0 +/- 1.7%, p less than 0.05) and in
group 3 (7.9 +/- 3.1%, p less than 0.05). The transcapillary escape rate of
glycated albumin was slightly lower than that of non-glycated albumin in all
groups, but significant only in normal control subjects. No difference in the
catabolic rate of glycated and non-glycated albumin was found. We conclude that
the in vivo effects of glycation on the clearance and transcapillary passage of
albumin are small and not likely to play any significant role in the development
of late diabetic microvascular complications. Available evidence indicates that poor metabolic control and raised blood
pressure each accelerate the development of diabetic microangiopathy.
Microangiopathy is associated with excess albumin deposition in capillary
basement membranes and it has been suggested that increased extravasation of
plasma constituents may lead to basement membrane thickening. We measured the
transcapillary escape rate of albumin, an indicator of the rate of extravasation
of intravascular albumin from the circulation per unit time, following
intravenous injection of 125I-human serum albumin. We examined the independent
effects on the transcapillary escape rate of albumin of non-ketotic poor
metabolic control, hypertension and microangiopathy. We studied non-diabetic
control subjects and diabetic patients, initially when in non-ketotic poor
metabolic control and again when control had been improved. We also studied
normotensive well-controlled diabetic patients without microangiopathy,
normotensive well-controlled diabetic patients with microangiopathy,
hypertensive well-controlled diabetic patients without microangiopathy and
hypertensive well-controlled diabetic patients with microangiopathy. The
transcapillary escape rate of albumin was similar in non-diabetic control
subjects (5.5 +/- 0.7%/h) and in both Type 1 (5.3 +/- 1.2%/h) and Type 2 (5.1
+/- 0.6%/h) normotensive diabetic patients without long-term complications.
During poor metabolic control the transcapillary escape rate of albumin was
significantly higher than in non-diabetic subjects (8.8 +/- 0.8%/h and 5.5 +/-
0.7%/h respectively, p less than 0.01). With improved control values fell
significantly to 6.3 +/- 0.9%/h (p less than 0.02), not significantly different
from control subjects.(ABSTRACT TRUNCATED AT 250 WORDS) The transcapillary escape rate of albumin (TERalb) is often elevated in patients
with diabetic microangiopathy. The objective of this study was to examine the
effect of enalapril on the TERalb of diabetic patients with albuminuria and
normal or mildly elevated blood pressure. Seventeen diabetic patients with
diabetic retinopathy, albuminuria, a diastolic blood pressure below 100 mmHg and
increased TERalb participated in the study. Blood pressure and TERalb were
measured before and after 14 days of therapy with enalapril, 20 mg daily for 14
days. Systolic and diastolic blood pressure fell from 168 +/- 6 to 155 +/- 6 (p
< 0.001) and from 87 +/- 2 to 81 +/- 2 mmHg (p < 0.005) respectively. Mean
arterial pressure declined from 114 +/- 3 to 105 +/- 3 mmHg (p < 0.0001). The
elevated TERalb decreased from 9.5 +/- 0.5 to 7.2 +/- 0.5%/hr (p < 0.005). In
the hypertensive subset, systolic, diastolic and mean arterial pressure
decreased significantly by 15, 7 and 10 mmHg (p < 0.005, p < 0.005 and p < 0.005
respectively); TERalb decreased from 9.5 +/- 0.6 to 7.3 +/- 0.6 (p < 0.03). In
the normotensive subset, arterial pressure remained unchanged and TERalb
decreased from 9.0 +/- 0.8 to 6.8 +/- 1.0%/hr (p < 0.03). Plasma fructosamine
decreased from 373 +/- 23 to 347 +/- 20 (p < 0.05) in the hypertensive group and
remained unchanged in the normotensive patients. No correlation could be
demonstrated between variation in TERalb and changes in blood pressure. In
conclusion, enalapril decreases microvascular albumin leakage in patients with
diabetic microangiopathy and normal or mildly elevated blood pressure. Diabetic patients with elevated urinary albumin excretion rate (incipient or
clinical nephropathy) also have an increased transcapillary escape rate of
albumin. This study was designed to clarify whether this is caused by a general
vascular dysfunction or by elevated systemic blood pressure. The systemic blood
pressure and the transcapillary escape rate of albumin were measured in the
following groups after 4 weeks without antihypertensive treatment: Group
1--eleven healthy control subjects. Group 2--ten Type 1 (insulin-dependent)
diabetic patients with incipient nephropathy (urinary albumin excretion rate:
30-300 mg/24 h) and normal blood pressure. Group 3--eleven non-diabetic patients
with essential hypertension. Group 4--nine Type 1 diabetic patients with
hypertension but normal urinary albumin excretion (< 30 mg/24 h). Group
5--eleven Type 1 diabetic patients with nephropathy (urinary albumin excretion
rate > 300 mg/24 h) and hypertension. Systolic and diastolic blood pressure were
similar in the three hypertensive groups: group 3, 148 +/- 8/95 +/- 6; group 4,
150 +/- 12/94 +/- 8 and group 5; 152 +/- 12/92 +/- 7 mmHg, but significantly
elevated (p < 0.001) compared to control group 1, 117 +/- 12/74 +/- 9 and group
2, 128 +/- 7/82 +/- 4 mmHg. The transcapillary escape rate of albumin was
similar in the control subjects (5.2 +/- 2.7%) and the subjects in the
normoalbuminuric groups 3 and 4 (6.2 +/- 1.9 and 5.1 +/- 1.4%, respectively) and
significantly lower (p < 0.001) than in patients with elevated urinary albumin
excretion without or with hypertension group 2, 10.1 +/- 2.8 and group 5, 11.4
+/- 5.7%.(ABSTRACT TRUNCATED AT 250 WORDS) In light of the growing understanding of the toxic effects of glycated albumin
and of the preferential excretion of this substance, the excretion of glycated
albumin could be considered a physiologic function of the kidney. Furthermore,
if the increased load of glycated albumin in diabetic patients results in
glycated albumin excretion rates in the range of 20 to 200 microg/min, might
this not be considered "physiologic microalbuminuria"? The hypothesis is
presented that microalbuminuria composed of glycated albumin is a homeostatic
renal function. Although some proteins are glycosylated for their normal
physiologic function, many proteins are glycated nonenzymatically according to
ambient blood glucose. Albumin is subject to nonenzymatic glycation in all
humans, but at increased rates in diabetic patients. Glycated albumin induces
changes in the microvasculature and glomerulus that may lead to endothelial
dysfunction and diabetic nephropathy, respectively. Renal excretion of glycated
albumin is enhanced compared with native albumin. To explore this potential
homeostatic function of the kidney, patients with impaired renal function were
studied to determine whether glycated albumin accumulates. Plasma levels of
glycated albumin were determined in diabetic and nondiabetic patients on
hemodialysis. Hemoglobin A1c was used as an index of the rate of nonenzymatic
glycation of proteins. Hemoglobin A1c was increased in the diabetic subjects but
was normal in the nondiabetic group (7.9% +/- 0.5% v 6.2% +/- 0.2%,
respectively; P < 0.01). On the other hand, the glycated albumin was elevated in
both groups and was not significantly different between them (1.95% +/- 0.15% in
the diabetic patients v 1.75% +/- 0.14% in the nondiabetic patients; P = NS).
The results of this study provide the first clinical evidence supporting the
hypothesis that the excretion of glycated albumin is a homeostatic renal
function. The Steno hypothesis suggests that albuminuria reflects widespread vascular
damage (proliferative retinopathy and severe macroangiopathy) due to a
generalized vascular (endothelial) dysfunction. We assessed this concept in
NIDDM (non-insulin-dependent diabetic) patients with (13 female/ 39 male, age 60
+/- 7 years, group 1) and without (12 female /41 male, age 61 +/- 7 years, group
2) diabetic nephropathy compared to matched non-diabetic subjects (7 female/15
male, age 58 +/- 8 years, group 3). A 12-lead ECG was recorded and coded blindly
using the Minnesota Rating Scale; the World Health Organization cardiovascular
questionnaire was used to assess past and present evidence of myocardial
infarction, angina pectoris, stroke, and peripheral vascular disease (digital
systolic blood pressure determination). The degree of diabetic retinopathy was
scored from fundus photography. The following variables were measured:
transcapillary escape rate of albumin (initial disappearance of intravenously
injected 125I-labelled human serum albumin), plasma concentrations of prorenin
(radioimmunoassay) and serum concentrations of von Willebrand factor
(enzyme-linked immunoadsorbent assay). Prevalence of ischaemic heart disease
(ECG reading) (49/20/5)% and peripheral vascular disease as indicated by reduced
systolic blood pressure on big toe (69/30/ 14)% was significantly higher in
group 1 vs group 2 (p < 0.01) and in group 2 vs group 3 (p < 0.01),
respectively. The prevalence and severity of retinopathy was higher in group 1
vs 2 (p < 0.01). Transcapillary escape rate of albumin (%/h) was elevated in
group 1 and 2 as compared to control subjects: 7.9 (4.3-13.7); 7.4 (3.7-16.4) vs
6.0 (3.4-8.7), (p < 0.005), respectively. Plasma prorenin activity (IU/ml) was
raised in group 1 and group 2 as compared to group 3: 272 (59-2405); 192
(18-813), and 85 (28-246), p < 0.001, respectively. Serum von Willebrand factor
(IU/ ml) was elevated in group 1 as compared to group 2 and 3: 2.07 (0.83-4.34);
1.60 (0.30-2.99) and 1.50 (1.00-2.38), p < 0.001, respectively. Our study
demonstrated that NIDDM patients with and without albuminuria had increased
transcapillary escape of albumin and raised prorenin activity, whereas only
those with albuminuria had increased von Willebrand factor. Patients with NIDDM
may have abnormal endothelial function in the absence of albuminuria. The relation between urinary albumin excretion rate (UAE), transcapillary escape
rate of albumin (TERalb), haemostatic factors, ambulatory blood pressure, and
metabolic variables was investigated in 45 Type II (non-insulin-dependent)
diabetic patients without overt nephropathy or uncontrolled blood pressure. We
enrolled 44 patients in a placebo controlled study to test the effects of 3 week
long treatment with low-molecular weight heparin (tinzaparin) on the same
variables. BMI, 24 h systolic and diastolic blood pressure, plasma
concentrations of triglycerides, fasting glucose, factor VIII, von Willebrand
factor (vWf), fibrinogen, alpha-2 macroglobulin, and fibronectin were notably
higher in patients with increased albuminuria compared with normoalbuminuric
patients, whereas the TERalb was similar in the two groups. TERalb correlated
with fasting plasma glucose. UAE correlated more closely than TERalb with 24 h
ambulatory blood pressure, vWf, and factor VIII. Urinary albumin excretion rate
was unchanged during tinzaparin [28.9+/-5.6 vs 28.1+/-6.0 microg/min (geometric
mean (antilog SD)] vs placebo (18.0+/-5.4 vs 17.6+/-5.3 microg/min), and no
change was found in TERalb [6.3+/-1.6 vs 6.0+/-1.5%/h (means +/- SD), and
6.3+/-1.5 vs 5.6+/-1.8%/h; tinzaparin versus placebo, respectively]. Only minor
changes were observed in blood pressure, lipids, glycaemic control and
haemostatic factors. This study shows no correlation between albuminuria and
transcapillary escape rate in Type II diabetic patients without overt
nephropathy or uncontrolled-blood pressure. UAE is related to markers of
atherosclerosis, endothelial injury and dysfunction, and haemostatic factors.
Moreover, UAE correlates much more than TERalb with 24 h ambulatory blood
pressure, von Willebrand factor, and factor VIII. Finally, short-term treatment
with tinzaparin does not change the transvascular or glomerular leakage of
albumin. These results indicate that TERalb is not a sensitive marker of
microvascular dysfunction in such patients and that factors other than abnormal
glycosaminoglycan metabolism may contribute to the vascular damage of these
patients. Approximately 30% of diabetic patients develop nephropathy, the appearance of
which is partially under genetic control. Atrial natriuretic peptide (ANP) has
associated physiologic effects on the kidney. This study was conducted to
examine the relationship between a newly identified and known polymorphism at
the pronatriodilatin (PND) gene locus and renal involvement in type 1 diabetic
subjects. Of 454 type 1 diabetic patients (219 men, 235 women), 323 showed no
sign of nephropathy, 79 had incipient renal involvement, and 52 established
nephropathy; 58 healthy control subjects were examined for comparison. Allele
frequencies (C708 versus T708) were: 0.95 and 0.05 in normoalbuminuric patients,
respectively; 0.88 and 0.12 in microalbuminuric patients; 0.96 and 0.04 both in
those with overt nephropathy and in healthy control subjects (P = 0.011).
Patients with incipient nephropathy were in disequilibrium compared with the
total diabetic cohort (P = 0.02). In the same populations, an additional
genotype for ScaI polymorphism of the PND gene was tested. The A1 and A2 allele
frequencies were: 0.21 and 0.79 in normoalbuminuric patients; 0. 13 and 0.87 in
microalbuminuric patients; 0.06 and 0.94 in type 1 diabetic subjects with overt
nephropathy; and 0.20 and 0.80 in healthy control subjects, respectively (P <
0.0001). A subset of 55 normotensive patients with type 1 diabetes, well matched
for clinical features, plasma ANP levels, and microvascular permeability to
macromolecules, was investigated on the basis of the C708/T and A2/A1
polymorphisms. Both transcapillary escape rate of albumin (TERalb) and plasma
ANP levels were significantly lower in patients with the T708 than with C708
allele, as well as in the A1 than in A2 allele (TERalb: T708 versus C708:
5.5+/-1.7 versus 7.8+/-2.0%/h, P = 0.0001; plasma ANP levels: 8.3+/-3.9 versus
15.3+/-7.7 pg/ml, P = 0.0003; A1 versus A2: 6.05+/-2.2 versus 7.3+/-2.1%/h, P =
0.044; 8.53+/-4.6 versus 14.5+/-7.4 pg/ml, P = 0.0024, respectively). Thus, in a
large ethnically homogeneous cohort of diabetic subjects, our data show: (1) a
significant association of C708/T polymorphism with microalbuminuria in
long-term diabetes and with both lower plasma ANP levels and widespread albumin
leakage; and (2) a strong association between ScaI polymorphism and both
diabetic nephropathy and plasma ANP concentrations. These results suggest a
possible role of PND gene in conferring protection from nephropathy and
microvascular damage in type 1 diabetes. OBJECTIVE: An increase in urinary albumin excretion (UAE) in type 1 diabetic
patients might reflect changes in vascular permeability and/or local
haemodynamic factors. Indeed, transcapillary escape of albumin (TERalb), a
measure of systemic capillary efflux, is increased in diabetic patients, even in
those with a modest increase of albuminuria. In normo-albuminuric type 1
diabetic patients, systemic capillary and glomerular flow is increased. We
hypothesized that these haemodynamic changes contribute to an elevated TERalb,
even in the phase preceding micro-albuminuria.
METHODS: We measured TERalb in 39 normo-albuminuric type 1 diabetic patients and
46 healthy controls. TERalb was calculated from the disappearance curve of
125I-albumin. Renal and systemic haemodynamics were measured by standard
techniques. Forearm blood flow (FBF) was measured by plethysmography.
Endothelial function was assessed by intra-arterial infusion of acetylcholine.
The structural integrity of the vessel wall was determined by the post-occlusive
reactive hyperaemia test.
RESULTS: TERalb was increased in diabetic patients (5.53+/-0.40 versus
4.39+/-0.21 %/h, P = 0.01). Patients were divided into tertiles with respect to
their TERalb. There were no differences in UAE, blood pressure, metabolic
parameters, endothelial function or maximal vasodilatation after occlusion
between the groups. However, filtration fraction and FBF were significantly
increased in the group of diabetic patients with the highest levels of TERalb.
Overall, in diabetic patients, FBF was significantly correlated with TERalb.
CONCLUSIONS: TERalb is increased in normo-albuminuric type 1 diabetic patients.
In these patients with an increased capillary permeability, there is no evidence
of endothelial dysfunction or vessel wall damage. However, both FBF and
filtration fraction are increased. Therefore, the increased vascular
permeability in the early phase of type 1 diabetes is associated with general
haemodynamic alterations. Notably, such an increase in vascular permeability is
not necessarily reflected by abnormal UAE. This could be due to either a lack of
change in glomerular permeability or due to the fact that the threshold for
tubular reabsorption of albumin has not been exceeded. Microalbuminuria was originally considered to be an important new risk factor
for diabetic nephropathy. More recently, it has been convincingly shown that
microalbuminuria is also an independent risk factor for cardiovascular morbidity
and mortality in Type 1 and Type 2 diabetic patients. Even in the non-diabetic
background population, microalbuminuria is a risk factor for cardiovascular
mortality. What is the link between increased loss of albumin in urine and
cardiovascular disease and mortality? As microalbuminuria is apparently
associated with increased universal vascular sieving of albumin in terms of the
transcapillary escape rate of albumin (TER-alb), microalbuminuria may reflect
this universal sieving. The pathophysiology of increased TER-alb is unknown, but
could be caused by haemodynamics or damage to the functional properties of the
vascular wall. A number of studies have provided evidence of endothelial
dysfunction in patients with microalbuminuria, which may be the common link
accounting for the associations mentioned above. In this context, a number of
markers of endothelial cell dysfunction have been found to be increased in
patients with microalbuminuria. In addition, a number of functional in vivo
tests of endothelial dysfunction have been performed in Type 1 and Type 2
diabetic patients as well as in normal controls. Overall, these studies indicate
the existence of a functional vascular dysfunction in Type 1 diabetic patients
and normal controls with microalbuminuria, which may be related to dysfunction
of endothelial cells. PURPOSE: Metabolic Syndrome as defined by ATP III criteria, a constellation of
risk factors associated with insulin resistance, predisposes to premature
atherosclerosis and early coronary events. Whether that negative risk profile is
associated with endothelial dysfunction remains to be established.
MATERIALS AND METHODS: Transcapillary escape rate of albumin (TERalb), a measure
of capillary permeability and integrity of systemic capillary endothelium, and
forearm vasodilation to intra-arterial acetylcholine (ACH), an index of nitric
oxide (NO)-mediated vasomotor dysfunction, were assessed in 24 non-diabetic,
uncomplicated hypertensive men with Metabolic Syndrome according to ATP III
criteria (hypertension with at least two additional traits such as high
triglycerides, low HDL, abdominal obesity, impaired fasting or post-load plasma
glucose). Twelve age-matched lean normal hypertensive patients with normal lipid
and glucose profile and nine normotensive subjects were the controls. All
patients underwent lipids determination and fasting and post-OGTT insulin
assessment; HOMA-IR was the index of insulin resistance.
RESULTS: TERalb was higher in hypertensive patients with Metabolic Syndrome,
without differences between hypertensive and normotensive controls. Blood
pressure (BP), lipids, plasma glucose, insulin levels and HOMA-IR were unrelated
to TERalb. Responses to acetylcholine were selectively attenuated in metabolic
patients and, on an individual basis, related only to HDL cholesterol levels,
independent of LDL cholesterol, BP, body size, triglycerides, and HOMA-IR
values. No relationship existed between responses to acetylcholine and TERalb.
CONCLUSIONS: Altered systemic capillary permeability characterizes
insulin-resistant hypertensive patients with Metabolic Syndrome. That defect,
which may promote early atherosclerosis development, coexists with blunted
endothelial-mediated vasodilation, indicating a pervasive abnormality of
endothelial function. We studied the following in normo- and microalbuminuric hypertensive type 2
diabetic patients: 1) transcapillary escape rate of albumin (TERalb) and 2)
expression of mRNA slit diaphragm and podocyte proteins in renal biopsies.
Normoalbuminuric subjects had renal cancer, and kidney biopsy was performed
during surgery. TERalb was evaluated by clearance of (125)I-albumin. Real-time
PCR of mRNA slit diaphragm was measured in kidney specimens. Albumin excretion
rate (AER) was by definition lower in normoalbuminuric subjects than in
microalbuminuric subjects with typical diabetic glomerulopathy (group 1), in
microalbuminuric subjects with normal or near-normal glomerular structure (group
2), and in microalbuminuric subjects with atypical diabetic nephropathy (group
3). This classification was based on light microscopy analysis of renal tissue.
TERalb (%/h) was similar in normoalbuminuric and microalbuminuric group 1, 2,
and 3 diabetic patients (medians: 14.1 vs. 14.4 vs. 15.7 vs. 14.9, respectively)
(ANOVA, NS). mRNA expression of slit diaphragm proteins CD2AP, FAT, Actn 4,
NPHS1, and NPHS2 was higher in normoalbuminuric patients than in
microalbuminuric patients (groups 1, 2, and 3) (ANOVA, P < 0.001). All diabetic
patients had greater carotid artery intimal thickness than normal control
subjects using ultrasound technique (ANOVA, P < 0.01). In conclusion, the
present study suggests that microalbuminuria identifies a subgroup of
hypertensive type 2 diabetic patients who have altered mRNA expression of slit
diaphragm and podocyte proteins, even before glomerular structure shows
abnormalities using light microscopy analysis. On the contrary, altered TERalb
and increased carotid artery intimal thickness are shown by all hypertensive
type 2 diabetic patients, both with normal and altered patterns of AER. BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a promising
biomarker of acute kidney injury. There is a growing body of evidence suggesting
that NGAL is also a marker of kidney disease and severity in chronic kidney
disease (CKD). We studied the utility of urinary NGAL in more accurately
predicting renal function in patients with diabetic CKD.
METHODS: We studied possible relationships between urinary NGAL, estimated
glomerular filtration rate (eGFR), and proteinuria in diabetic CKD patients and
in healthy populations.
RESULTS: Urinary NGAL levels were significantly higher in CKD patients than in
healthy controls (96.0 [2.7 to 975.2] ng/mL vs. 18.8 [1.3 to 81.9] ng/mL,
P=0.02), and the GFR was lower among CKD patients (49.3 [13.1 to 78.3]
mL/min/1.73 m(2) vs. 85.6 [72 to 106.7] mL/min/1.73 m(2), P<0.0001). The urinary
NGAL level showed a significant inverse correlation with GFR (r=-0.5634,
P<0.0001). The correlation analyses between urinary protein level and urinary
NGAL levels and GFR were as follows: urine protein and urinary NGAL (r=0.3009,
P=0.0256), urine protein and GFR (r=-0.6245, P<0.0001), urine microalbumin and
urinary NGAL (r=0.1794, P=0.2275), and urine microalbumin and GFR (r=-0.5190,
P=0.0002).
CONCLUSION: From these results, we concluded that urinary NGAL is a reliable
marker of renal function in diabetic CKD patients. However, urinary NGAL did not
provide more accurate information regarding renal function than GFR. |
Which genes does thyroid hormone receptor beta1 regulate in the liver? | LDL receptor"//
"ChREBP"//
"ME", "malic enzyme"//
"cytochrome P450 oxidoreductase"// | The current study demonstrates that T3-activated transcription of the
NADPH:cytochrome P450 oxidoreductase (P450R) gene is dependent on the thyroid
hormonal status of the animal, with both transcriptional and
post-transcriptional pathways being important in regulating the cellular P450R
mRNA level. The region required for transcriptional activation of the P450R gene
by T3 has been identified. Nuclear run-on experiments demonstrated that the
effects of T3 on P450R transcription are dependent on thyroid status, with a
transcriptional enhancement obtained in T3-treated hypothyroid rat liver
(1.8-fold increase) but not in T3-treated euthyroid animals. Transient
cotransfection of P450R promoter/chloramphenicol acetyl transferase (CAT)
constructs and the thyroid hormone receptor beta1 (TR beta1) expression plasmid
into rat hepatoma H4IIE cells resulted in a 2.4-fold induction of promoter
activity that was both T3 and TR beta1 dependent. Analysis of promoter deletion
constructs identified a P450R-thyroid response region (P450R-TRE; bases, -564 to
-536) containing three imperfect direct repeats of the thyroid response motif,
AGGTCA. Mutational analysis further established that T3 induction was dependent
only on the upstream direct repeat, having the sequence AGGTGAgctgAGGCCA.
Footprint analysis showed that all three motifs were protected by proteins
present in rat liver nuclear extracts, and a direct interaction between
P450R-TRE and T3 receptors TR alpha1 and TR beta1 was demonstrated by gel-shift
analysis. In vitro binding studies with P450R-TRE revealed the formation of
heterodimeric complexes when TR alpha1 was coincubated with either the retinoic
X receptor alpha or nuclear extract from rat liver, COS, or H4IIE cells. In
addition, placement of the P450R-TRE upstream of the T3-nonresponsive
heterologous thymidine kinase promoter resulted in a 2.7-fold transcriptional
enhancement that was both T3 and TR beta1 dependent. Previous studies have
demonstrated that T3 augments P450R mRNA levels approximately 20-30-fold and
approximately 12-fold, respectively, in hypothyroid and euthyroid rats. Hence,
for the hypothyroid state, transcriptional and post-transcriptional events
contribute to the T3-induced mRNA increases; however, the marked increase in
message level in T3-treated euthyroid animals depends primarily on
post-transcriptional pathways. Thyroid hormone (TH) responsive genes can be both positively and negatively
regulated by TH through receptors (TR) alpha and beta expressed in most body
tissues. However, their relative roles in the regulation of specific gene
expression remain unknown. The TR beta knockout mouse, which lacks both TR beta1
and TR beta2 isoforms, provides a model to examine the role of these receptors
in mediating TH action. TR beta deficient (TR beta-/-) mice that show no
compensatory increase in TR alpha, and wild-type (TR beta+/+) mice of the same
strain were deprived of TH by feeding them a low iodine diet containing
propylthiouracil, and were then treated with supraphysiological doses of L-T3
(0.5, 5.5, and 25 microg/day/mouse). TH deprivation alone increased the serum
cholesterol concentration by 25% in TR beta+/+ mice and reduced it paradoxically
by 23% in TR beta-/- mice. TH deprivation reduced the serum alkaline phosphatase
(AP) concentration by 31% in TR beta+/+ mice but showed no change in the TR
beta-/- mice. Treatment with L-T3 (0.5 to 25 microg/mouse/day) caused a 57%
decrease in serum cholesterol and a 231% increase in serum AP in the TR beta+/+
mice. The TR beta-/- mice were resistant to the L-T3 induced changes in serum
cholesterol and showed increase in AP only with the highest L-T3 dose. Basal
heart rate (HR) in TR beta-/- mice was higher than that of TR beta+/+ mice by
11%. HR and energy expenditure (EE) in both TR beta+/+ and TR beta-/- mice
showed similar decreases (49 and 46%) and increases (49 and 41%) in response to
TH deprivation and L-T3 treatment, respectively. The effect of TH on the
accumulation of messenger RNA (mRNA) of TH regulated liver genes was also
examined. TH deprivation down regulated spot 14 (S14) mRNA and showed no change
in malic enzyme (ME) mRNA in both TR beta+/+ and TR beta-/- mice. In contrast
treatment with L-T3 produced an increase in S14 and ME but no change in TR
beta-/- mice. From these results, it can be concluded that regulation of HR and
EE are independent of TR beta. With the exception of serum cholesterol
concentration and liver ME mRNA accumulation, all other markers of TH action
examined during TH deprivation exhibited the expected responses in the absence
of TR beta. Thus, as previously shown for serum TSH, TR beta is not absolutely
necessary for some changes typical of hypothyroidism to occur. In contrast,
except for HR and EE, the full manifestation of TH-mediated action required the
presence of TR beta. The molecular mechanism of thyroid hormone (TH) effects to fatty acid metabolism
in liver is yet to be clear. The carbohydrate response element-binding protein
(ChREBP) as well as sterol response element-binding protein (SREBP)-1c plays a
pivotal role in hepatic lipogenesis. Both SREBP-1c and ChREBP are target genes
of liver X receptors (LXRs). Because LXRs and TH receptors (TRs) cross talk
mutually in many aspects of transcription, we examined whether TRs regulate the
mouse ChREBP gene expression. In the current study, we demonstrated that TH
up-regulated mouse ChREBP mRNA and protein expression in liver. Run-on and
luciferase assays showed that TH and TR-beta1 positively regulated the ChREBP
gene transcription. The mouse ChREBP gene promoter contains two direct repeat-4
sites (LXRE1 and LXRE2) and EMSAs demonstrated that LXR-alpha and TR-beta1
prefer to bind LXRE1 and LXRE2, respectively. The direct repeat-4 deletion and
LXRE2 mutants of the promoter deteriorate the positive regulation by TR-beta1,
indicating that LXRE2 is functionally important for the regulation. We also
showed that human ChREBP gene expression and promoter activities were
up-regulated by TH. These data suggest that ChREBP mRNA expression is positively
regulated by TR-beta1 and TH at the transcriptional level in mammals. This novel
observation indicates that TH fine-tunes hepatic lipogenesis via regulating
SREBP-1c and ChREBP gene expression reciprocally. |
Are conserved noncoding elements associated with developmental genes? | Yes. Numerous studies suggest that conserved noncoding elements span developmental regulatory genes and define regulatory domains. | Fish-mammal genomic comparisons have proved powerful in identifying conserved
noncoding elements likely to be cis-regulatory in nature, and the majority of
those tested in vivo have been shown to act as tissue-specific enhancers
associated with genes involved in transcriptional regulation of development.
Although most of these elements share little sequence identity to each other, a
small number are remarkably similar and appear to be the product of duplication
events. Here, we searched for duplicated conserved noncoding elements in the
human genome, using comparisons with Fugu to select putative cis-regulatory
sequences. We identified 124 families of duplicated elements, each containing
between two and five members, that are highly conserved within and between
vertebrate genomes. In 74% of cases, we were able to assign a specific set of
paralogous genes with annotation relating to transcriptional regulation and/or
development to each family, thus removing much of the ambiguity in identifying
associated genes. We find that duplicate elements have the potential to
up-regulate reporter gene expression in a tissue-specific manner and that
expression domains often overlap, but are not necessarily identical, between
family members. Over two thirds of the families are conserved in duplicate in
fish and appear to predate the large-scale duplication events thought to have
occurred at the origin of vertebrates. We propose a model whereby gene
duplication and the evolution of cis-regulatory elements can be considered in
the context of increased morphological diversity and the emergence of the modern
vertebrate body plan. BACKGROUND: All vertebrates share a remarkable degree of similarity in their
development as well as in the basic functions of their cells. Despite this,
attempts at unearthing genome-wide regulatory elements conserved throughout the
vertebrate lineage using BLAST-like approaches have thus far detected noncoding
conservation in only a few hundred genes, mostly associated with regulation of
transcription and development.
RESULTS: We used a unique combination of tools to obtain regional global-local
alignments of orthologous loci. This approach takes into account shuffling of
regulatory regions that are likely to occur over evolutionary distances greater
than those separating mammalian genomes. This approach revealed one order of
magnitude more vertebrate conserved elements than was previously reported in
over 2,000 genes, including a high number of genes found in the membrane and
extracellular regions. Our analysis revealed that 72% of the elements identified
have undergone shuffling. We tested the ability of the elements identified to
enhance transcription in zebrafish embryos and compared their activity with a
set of control fragments. We found that more than 80% of the elements tested
were able to enhance transcription significantly, prevalently in a
tissue-restricted manner corresponding to the expression domain of the
neighboring gene.
CONCLUSION: Our work elucidates the importance of shuffling in the detection of
cis-regulatory elements. It also elucidates how similarities across the
vertebrate lineage, which go well beyond development, can be explained not only
within the realm of coding genes but also in that of the sequences that
ultimately govern their expression. We report evidence for a mechanism for the maintece of long-range conserved
synteny across vertebrate genomes. We found the largest mammal-teleost conserved
chromosomal segments to be spanned by highly conserved noncoding elements
(HCNEs), their developmental regulatory target genes, and phylogenetically and
functionally unrelated "bystander" genes. Bystander genes are not specifically
under the control of the regulatory elements that drive the target genes and are
expressed in patterns that are different from those of the target genes.
Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2,
rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the
expression patterns of these genes even if located inside or beyond bystander
genes, suggesting that the regulatory domain of a developmental regulatory gene
can extend into and beyond adjacent transcriptional units. We termed these
chromosomal segments genomic regulatory blocks (GRBs). After whole genome
duplication in teleosts, GRBs, including HCNEs and target genes, were often
maintained in both copies, while bystander genes were typically lost from one
GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy
GRBs of higher vertebrates intact. We show that loss of bystander genes and
other mutational events suffered by duplicated GRBs in teleost genomes permits
target gene identification and HCNE/target gene assignment. These findings
explain the absence of evolutionary breakpoints from large vertebrate
chromosomal segments and will aid in the recognition of position effect
mutations within human GRBs. Metazoan genomes contain arrays of highly conserved noncoding elements (HCNEs)
that span developmental regulatory genes and define regulatory domains. We
describe Ancora http://ancora.genereg.net, a web resource that provides data and
tools for exploring genomic organization of HCNEs for multiple genomes. Ancora
includes a genome browser that shows HCNE locations and features novel HCNE
density plots as a powerful tool to discover developmental regulatory genes and
distinguish their regulatory elements and domains. Sequence conservation has traditionally been used as a means to target
functional regions of complex genomes. In addition to its use in identifying
coding regions of genes, the recent availability of whole genome data for a
number of vertebrates has permitted high-resolution analyses of the noncoding
"dark matter" of the genome. This has resulted in the identification of a large
number of highly conserved sequence elements that appear to be preserved in all
bony vertebrates. Further positional analysis of these conserved noncoding
elements (CNEs) in the genome demonstrates that they cluster around genes
involved in developmental regulation. This chapter describes the identification
and characterization of these elements, with particular reference to their
composition and organization. Pan-vertebrate developmental cis-regulatory elements are discernible as highly
conserved noncoding elements (HCNEs) and are often dispersed over large areas
around the pleiotropic genes whose expression they control. On the loci of two
developmental transcription factor genes, SOX3 and PAX6, we demonstrate that
HCNEs conserved between human and zebrafish can be systematically and reliably
tested for their regulatory function in multiple stable transgenes in zebrafish,
and their genomic reach estimated with confidence using synteny conservation and
HCNE density along these loci. HCNEs of both human and zebrafish function as
specific developmental enhancers in zebrafish. We show that human HCNEs result
in expression patterns in zebrafish equivalent to those in mouse, establishing
zebrafish as a suitable model for large-scale testing of human developmental
enhancers. Orthologous human and zebrafish enhancers underwent functional
evolution within their sequence and often directed related but non-identical
expression patterns. Despite an evolutionary distance of 450 million years, one
pax6 HCNE drove expression in identical areas when comparing zebrafish vs. human
HCNEs. HCNEs from the same area often drive overlapping patterns, suggesting
that multiple regulatory inputs are required to achieve robust and precise
complex expression patterns exhibited by developmental genes. Fibroblast growth factors (Fgfs) encode small signaling proteins that help
regulate embryo patterning. Fgfs fall into seven families, including FgfD.
Nonvertebrate chordates have a single FgfD gene; mammals have three (Fgf8,
Fgf17, and Fgf18); and teleosts have six (fgf8a, fgf8b, fgf17, fgf18a, fgf18b,
and fgf24). What are the evolutionary processes that led to the structural
duplication and functional diversification of FgfD genes during vertebrate
phylogeny? To study this question, we investigated conserved syntenies, patterns
of gene expression, and the distribution of conserved noncoding elements (CNEs)
in FgfD genes of stickleback and zebrafish, and compared them with data from
cephalochordates, urochordates, and mammals. Genomic analysis suggests that
Fgf8, Fgf17, Fgf18, and Fgf24 arose in two rounds of whole genome duplication at
the base of the vertebrate radiation; that fgf8 and fgf18 duplications occurred
at the base of the teleost radiation; and that Fgf24 is an ohnolog that was lost
in the mammalian lineage. Expression analysis suggests that ancestral
subfunctions partitioned between gene duplicates and points to the evolution of
novel expression domains. Analysis of CNEs, at least some of which are candidate
regulatory elements, suggests that ancestral CNEs partitioned between gene
duplicates. These results help explain the evolutionary pathways by which the
developmentally important family of FgfD molecules arose and the deduced
principles that guided FgfD evolution are likely applicable to the evolution of
developmental regulation in many vertebrate multigene families. Genomic regulatory blocks are chromosomal regions spanned by long clusters of
highly conserved noncoding elements devoted to long-range regulation of
developmental genes, often immobilizing other, unrelated genes into long-lasting
syntenic arrangements. Synorth http://synorth.genereg.net/ is a web resource for
exploring and categorizing the syntenic relationships in genomic regulatory
blocks across multiple genomes, tracing their evolutionary fate after teleost
whole genome duplication at the level of genomic regulatory block loci,
individual genes, and their phylogenetic context. One of the key discoveries of vertebrate genome sequencing projects has been the
identification of highly conserved noncoding elements (CNEs). Some
characteristics of CNEs include their high frequency in mammalian genomes, their
potential regulatory role in gene expression, and their enrichment in gene
deserts nearby master developmental genes. The abnormal development of neural
crest cells (NCCs) leads to a broad spectrum of congenital malformation(s),
termed neurocristopathies, and/or tumor predisposition. Here we review recent
findings that disruptions of CNEs, within or at long distance from the coding
sequences of key genes involved in NCC development, result in neurocristopathies
via the alteration of tissue- or stage-specific long-distance regulation of gene
expression. While most studies on human genetic disorders have focused on
protein-coding sequences, these examples suggest that investigation of genomic
alterations of CNEs will provide a broader understanding of the molecular
etiology of both rare and common human congenital malformations. |
In what proportion of children with heart failure has Enalapril been shown to be safe and effective? | In children with heart failure evidence of the effect of enalapril is empirical. Enalapril was clinically safe and effective in 50% to 80% of for children with cardiac failure secondary to congenital heart malformations before and after cardiac surgery, impaired ventricular function , valvar regurgitation, congestive cardiomyopathy, , arterial hypertension, life-threatening arrhythmias coexisting with circulatory insufficiency.
ACE inhibitors have shown a transient beneficial effect on heart failure due to anticancer drugs and possibly a beneficial effect in muscular dystrophy-associated cardiomyopathy, which deserves further studies. | The short-term hemodynamic effects of intravenous enalaprilat were assessed in
26 infants and children, aged 6 months to 15 years, with intracardiac shunts
undergoing cardiac catheterization. Pulmonary and systemic pressure, flow, and
resistance indices were measured by the direct Fick method before and 30 min
after enalaprilat at 0.06 mg/kg. Aortic and pulmonary artery pressure decreased
15 and 20%, respectively, by 10 min, with little further change at 30 min. The
heart rate did not change significantly and there was no reduction in systemic
flow. In those with a large ventricular septal defect and normal or near-normal
pulmonary resistance (less than 3.5 u.m2, n = 8), the mean pulmonary-systemic
flow ratio decreased from 2.9 +/- 0.3 to 2.4 +/- 0.3 (p less than 0.05) and the
mean left-to-right shunt from 7.4 +/- 0.8 to 5.9 +/- 0.7 L/min/m2 (p less than
0.02). Those with an elevated pulmonary vascular resistance (greater than 5
u.m2, n = 8) showed a varied response. Two children, both with Down's syndrome,
an atrioventricular canal defect, and reversible pulmonary hypertension (as
assessed by an infusion of isoproterenol), had no decrease in pulmonary vascular
resistance with enalaprilat. There were no adverse effects. Converting enzyme
inhibitors may benefit "heart failure" associated with large ventricular septal
defects and normal or mildly elevated pulmonary resistance. BACKGROUND: Angiotensin-converting enzyme inhibitors improve exercise capacity
in adults with congestive heart failure by decreasing systemic vascular
resistance and improving ventricular diastolic function. Patients who have
undergone the Fontan procedure have decreased cardiac output, increased systemic
vascular resistance, abnormal diastolic function, and decreased exercise
capacity compared with normal people.
METHODS AND RESULTS: To test the hypothesis that afterload reduction therapy
alters hemodynamic variables and augments exercise capacity in patients after a
Fontan procedure, we compared the results of graded exercise with maximal effort
from 18 subjects (14.5+/-6.2 years of age, 4 to 19 years after Fontan procedure)
in a randomized, double-blind, placebo-controlled crossover trial using
enalapril (0.2 to 0.3 mg x kg[-1] x d[-1], maximum 15 mg). Each treatment was
administered for 10 weeks. Diastolic filling patterns at rest were assessed by
Doppler determination of the systemic atrioventricular valve flow velocity at
the conclusion of each therapy. No difference was detected in resting heart
rate, blood pressure, or cardiac index. Diastolic filling patterns were also
similar. Exercise duration was not different (6.4+/-2.6 [enalapril] versus
6.7+/-2.6 minutes [placebo]). The mean percent increase in cardiac index from
rest to maximum exercise was slightly but significantly decreased in subjects
after 10 weeks of enalapril therapy (102+/-34% [enalapril] versus 125+/-34%
[placebo]; P<.02). At maximal exercise, cardiac index (3.5+/-0.9 [enalapril]
versus 3.8+/-0.9 L x min[-1] x m2 [placebo]), oxygen consumption (18.3+/-9
[enalapril] versus 20.5+/-7 mL x min[-1] x kg[-1] [placebo]), minute ventilation
(57.5+/-17 [enalapril] versus 55.4+/-19 L/min [placebo]), and total work
(247+/-181 [enalapril] versus 261+/-197 W [placebo]) were not different.
CONCLUSIONS: We conclude that enalapril administration for 10 weeks does not
alter abnormal systemic vascular resistance, resting cardiac index, diastolic
function, or exercise capacity in patients who have undergone a Fontan
procedure. Angiotensin convertase inhibitor (Enalapril) was used in 51 children aged 4 days
up to 18 years (mean 4.3 +/- 5.5, years). As many as 27 subjects were newborns
(4) and infants (23). The patients suffered from circulatory insufficiency due
to congestive cardiomyopathy (13 cases). 6 treated subjects suffered from
circulatory insufficiency due to congenital heart malformations before cardiac
surgery and 22 after it (including complex malformations operated according to
Fontan method). 10 children were treated because of arterial hypertension. 4
subjects suffered form life-threatening arrhythmias coexisting with circulatory
insufficiency. (These subjects were already mentioned among the patients
suffering from circulatory insufficiency). Enalapril (mainly as a drug named
Benalapril) was used in the mean dose of 0.21 mg/kg of body mass daily. 4
patients (8%) died during treatment but their deaths can not be related to
angiotensin convertase inhibitor therapy. In the other children (82%) the
beneficial influence of angiotensin convertase inhibitor use was found
(improvement in comparison with the state before convertase inhibitor
introduction). In 10% of subjects enalapril did not show any significant
therapeutic effect. According to authors' opinion enalapril use is exceptionally
profitable in the subjects surgically treated for complex heart malformations
(Fontan operation). The beneficial effect was also found in majority of children
suffering from congestive cardiomyopathy. Convertase inhibitor was always
successfully used as the unique antihypertensive drug in children suffering from
arterial hypertension. In the other cases treatment was combined (mainly with
digitalis). This combination seems to be exceptionally useful in children
suffering from congestive cardiomyopathy. Only in 1 case unserious side effect
was found (persistent cough). PURPOSE: A common late effect of doxorubicin therapy for childhood cancer is
reduced left-ventricular (LV) wall thickness resulting in elevated LV afterload
and depressed LV function. Many children are given angiotensin-converting enzyme
inhibitors, which have been studied primarily in adults. We document the
long-term effects of angiotensin-converting enzyme inhibitors in
doxorubicin-treated survivors of childhood cancer.
PATIENTS AND METHODS: In this retrospective study, we reviewed records of 18
children who had regular echocardiographic examinations during enalapril therapy
(mean age at cancer diagnosis, 8 years; mean time between completion of
doxorubicin therapy and start of enalapril, 7 years; median follow-up since the
start of enalapril, 10 years).
RESULTS: Over the first 6 years of enalapril therapy, there was progressive
improvement toward normal values in LV dimension, afterload, fractional
shortening, and mass, but all these parameters deteriorated between 6 and 10
years. LV wall thickness deteriorated throughout the study period, as did LV
contractility and systolic blood pressure. Diastolic blood pressure fell
slightly. By 6 years on enalapril, all six patients who had had congestive heart
failure at the start of enalapril therapy had either died or undergone cardiac
transplantation, compared with three of the 12 asymptomatic patients.
CONCLUSION: In doxorubicin-treated long-term survivors of childhood cancer,
enalapril-induced improvement in LV structure and function is transient. The
primary defect, which is LV wall thinning, continues to deteriorate, and thus
the short-term improvement was mostly related to lowered diastolic blood
pressure. Patients with intraatrial baffle procedure for transposition of the great
arteries (TGA) have diastolic dysfunction, decreased exercise capacity, stroke
volume response and elevated systemic vascular resistance (SVR) during exercise.
Angiotensin-converting enzyme (ACE) inhibitors improve exercise capacity in
adults with congestive heart failure by improving diastolic function and
decreasing SVR. We tested the hypothesis that ACE inhibitors decrease SVR and
improve exercise capacity in patients after intraatrial baffle procedure for
TGA. We studied the effects of enalapril in nine patients with TGA s/p
intraatrial switch (mean age, 13.8 +/- 3 years) 7 to 21 years (mean, 12 +/- 4
years) after intraatrial baffle procedure. Enalapril (0.5 mg/kg/day, maximum
dosage 20 mg bid) was administered for 12 months. Patients exercised using a
cycle ergometer ramp protocol (0.25 W/kg/min) before enalapril (baseline), 1
month, 6 months, and 12 months after treatment initiation. Heart rate, blood
pressure, cardiac output, respiratory rate, minute ventilation, oxygen
consumption (VO2), total exercise time, work, and power were measured. SVR,
cardiac index, and stroke volume index (SVI) were calculated. Two-tailed paired
Student's t-test was used to compare data to those of normal control patients
and the patients' baseline data. Patients had lower resting heart rate, cardiac
index, maximum heart rate, cardiac index (CI), SVI, VO2, exercise time, work,
and power and higher maximal SVR at baseline compared to normal control
patients. There was no significant difference in total exercise time, work,
power, VO2 (rest/peak), SVR, SVI, and CI after 12 months of therapy compared to
patients' baseline values. We conclude that short-term (<1 year) use of
enalapril does not improve exercise performance in patients with TGA in whom the
intraatrial baffle procedure has been performed. PURPOSE: To determine whether an angiotensin-converting enzyme (ACE) inhibitor,
enalapril, prevents cardiac function deterioration (defined using maximal
cardiac index [MCI] on exercise testing or increase in left ventricular
end-systolic wall stress [LVESWS]) in long-term survivors of pediatric cancer.
PATIENTS AND METHODS: This was a randomized, double-blind, controlled clinical
trial comparing enalapril to placebo in 135 long-term survivors of pediatric
cancer who had at least one cardiac abnormality identified at any time after
anthracycline exposure.
RESULTS: There was no difference in the rate of change in MCI per year between
enalapril and placebo groups (0.30 v 0.18 L/min/m(2); P =.55). However, during
the first year of treatment, the rate of change in LVESWS was greater in the
enalapril group than in the placebo group (-8.59 v 1.85 g/cm(2); P =.033) and
this difference was maintained over the study period, resulting in a 9%
reduction in estimated LVESWS by year 5 in the enalapril group. Six of seven
patients removed from random assignment to treatment because of cardiac
deterioration were initially treated with placebo (P =.11), and one has died as
a result of heart failure. Side effects from enalapril included dizziness or
hypotension (22% v 3% in the placebo group; P =.0003) and fatigue (10% v 0%; P
=.013).
CONCLUSION: Enalapril treatment did not influence exercise performance, but did
reduce LVESWS in the first year; this reduction was maintained over the study
period. Any theoretical benefits of LVESWS reduction in this
anthracycline-exposed population must be weighed against potential side effects
from ACE inhibitors when making treatment decisions. Potassium chloride (KCl) supplementation is common among critically ill
children. Intravenous (IV) KCl supplementation for pediatric patients is poorly
characterized. This study aimed to examine the efficacy and safety of IV KCL and
to determine factors affecting patient responses to IV KCL in the pediatric
cardiac intensive care unit (CICU). A retrospective review of 211 children (794
KCl doses) undergoing cardiac surgery or a hospital stay for heart failure in
the CICU of a tertiary care teaching and referral children's hospital in 2011
was performed. Demographic data, weight, height, creatinine, and concomitant
medications during each KCl dose were recorded and analyzed. Body surface area
(BSA), creatinine clearance, and change in [K(+)] were calculated. The median
age of the children was 4 months (range, 10 days-18 years). In this study, 151
KCl doses were administered to neonates (19 %), 307 doses (39 %) to females, and
510 doses (64 %) to patients with a BSA smaller than 0.33 m(2) (a group with
relative renal insufficiency). The mean KCl dose was 0.97 ± 0.006 mEq/kg. No
adverse events were associated with IV KCl administration. Blood/plasma [K(+)]
increased 0.8 ± 0.02 mEq/L. The responses to KCl did not differ significantly
between males and females, between neonates and children, or between patients
with a BSA smaller than 0.33 m(2) and those with a BSA of 0.33 m(2) or larger.
The responses to IV KCl were attenuated by concomitant furosemide (p = 0.01),
amphotericin B (p < 0.01), and KCl in parenteral nutrition (p < 0.01). The
responses were augmented by concomitant enalapril (p = 0.03), ethacrynic acid (p
< 0.001), and hemodialysis (p < 0.01). Intravenous KCl can be administered
safely for CICU patients. Responses to KCl are altered when it is given with
certain medications. Intravenous KCl should be used cautiously in children
receiving angiotensin-converting enzyme inhibitors. Future studies are needed
for further characterization of factors affecting responses to IV KCl in
children. |
Is myasthenia gravis associated with osteoporosis? | Myasthenia gravis (MG) is a neuromuscular disease which has been associated with an increased risk of glucocorticoid-induced osteoporosis. Thymectomy can also increase risk for osteoporosis. Appropriate osteoporosis preventive measures can reduce osteoporosis risk in MG patients. | Myasthenia gravis is an autoimmune disease where corticoids are the basis of
therapy. They are taken for short periods in large amounts, as well as for
prolonged periods in medium or small doses. The authors investigated in the
described groups the effect of corticoids on bone tissue. They provided evidence
of a significant effect on the diffraction pattern, geometrical arrangement of
the apatite grid and ion changes in relation to the period of corticoid
administration without significant clinical manifestations of osteoporosis, when
respecting therapeutic principles. OBJECTIVES: Consensus guidelines for bone management of patients taking
corticosteroids suggest two main interventions: Dual energy X-ray absorptiometry
(DEXA) scanning in those taking prednisolone > or =7.5 mg daily for > or =6
months (repeated every 1-3 years as indicated). Bisphosphonate therapy for those
taking prednisolone > or =15 mg daily for > or =6 months regardless of DEXA
result, and also for patients with known or high risk of developing osteoporosis
(including those aged >65 years).
MATERIAL AND METHODS: We audited adherence to these guidelines in all adults
with myasthenia gravis (MG) attending our neurology service.
RESULTS: Of 80 patients with MG (47 male, mean age 63.3 years), 34 (43%) had
received corticosteroids for > or =6 months. Eighteen were taking prednisolone >
or =7.5 mg daily (mean dose 16.6 mg) yet only 4 of these (22%) had undergone
DEXA scanning. Of the 13 patients meeting the guideline criteria to receive
bisphosphonate therapy, this was prescribed to only 7 (54%). Two others were
prescribed vitamin D, 2 a calcium supplement and 2 were receiving no
prophylaxis.
CONCLUSION: In these MG patients the guidelines were followed in only a
minority. Neurologists need greater awareness of the bone health consequences of
prescribing long-term corticosteroids. Osteoporosis is an adverse effect of prednisolone therapy, although no study has
been conducted on myasthenia gravis patients receiving high-dose prednisolone.
We measured bone density in 36 patients (26 females and 10 males) who had
undergone long-term prednisolone administration, and found a decrease in bone
density in 31% of female patients and osteoporosis in only 11.5% (three cases).
This frequency is lower than the presumptive rate of the general population in
Japan (22.6%). No osteoporosis was detected in male patients. In conclusion,
prednisolone-treated patients with myasthenia gravis have an acceptable risk of
bone loss if prophylactic medication is administered. Recent studies of animal models have suggested that an increase in the number of
T cells due to both peripheral expansion and increased thymic T cell output
plays a key role in the regulation of bone loss after ovariectomy.
Osteoclastogenic cytokines which are either produced by T cells or activate T
cells have also been implicated in ovx induced bone loss. Among them are TNF
alpha and IL-7. The present study investigates the role of thymectomy (THX) and
IL-7 in bone metabolism in humans. We studied T cells subsets, cytokine
production and bone metabolism in 13 women thymectomized for Myasthenia gravis
as compared to healthy controls. Our data demonstrate that the number of CD4+
and TNF-producing T cells is lower in THX women as compared to euthymic
controls. However in THX women the residual T cells produce higher levels of
IL-7 and RANKL. Furthermore, flow cytometry shows that IL-7 is produced by T and
B cells. Serum levels of TNF alpha were unaffected by THX and both serum TNF
alpha and the RANKL/OPG correlated inversely with BMD. There were no differences
in bone turnover and bone mineral density between THX women and the controls.
These data suggest that THX decreases the number of TNF-producing CD4+ T cells
but does not alters serum TNF levels. The RANKL/OPG ratio and indices of bone
metabolisms are also not affected by THX, although THX increases the levels of
IL-7 and RANKL. Further studies are needed to clarify the role of thymus in bone
metabolism and osteoclastogenesis in postmenopausal women. Over the past several years, tacrolimus has attracted attention as a new
therapeutic drug for myasthenia gravis (MG), but few reports have considered its
use for MG in pediatric patients, and most of these have focused on severe
systemic MG. In this case report, we used tacrolimus to successfully treat a
13-year-old boy with ocular MG who had suffered from severe steroid
complications, including a failure of thrive and osteoporosis. He first showed
symptoms of ocular MG at age 2 years 3 months. At age 13 years, he was receiving
PSL (3.75 mg/day), but the symptoms of ocular MG recurred. We increased the
dosage of oral PSL up to 30 mg/day, and three courses of mPSL pulse therapy were
applied, but these therapies had only limited effect, and his symptoms worsened.
Tacrolimus was started at 0.4 mg/day (0.011 mg/kg/day), and every 2 weeks the
dose was gradually increased by 0.2 mg/day. His symptoms of MG began to improve
3 weeks after the initial administration of tacrolimus. Approximately 3 months
after the start of tacrolimus administration, PSL was discontinued. Currently,
at 1 year and 4 months after the start of tacrolimus administration, while
slight ptosis is observed in the evening, it does not influence his daily life,
and his condition remains comparable to that when he stopped taking PSL. No
adverse effects of tacrolimus have been recognized. In pediatric patients with
steroid-dependent ocular MG without thymectomy, tacrolimus may be a safe and
effective alternative to steroid and thymectomy. Vertebral compression fractures (VFs) are observed in 30-50 % of patients
affected by steroid-induced osteoporosis, with consequentially severe back pain
and functional limitation. An alternative treatment to medical therapy for pain
caused by recent VFs is percutaneous vertebroplasty (PVP). Patients were treated
by PVP after careful selection, based on the presence of persistent pain not
resolved by standard medical therapy, correlation between pain and level of the
VF, and neuroradiological features. We performed PVP in 4 patients with
generalized MG associated with recent steroid-induced symptomatic VFs. Relief
from pain was very rapid, usually within 24 h, and retained at a 3-month
evaluation. No severe complication or MG worsening were observed in the
post-operative period. Although clinical indication for PVP is still
controversial, in our experience PVP is a useful and safe tool to be considered
in the management of recent steroid-induced symptomatic VFs in selected MG
patients. Myasthenia gravis is an important indication for the long-term prescription of
corticosteroids. We present a patient with myasthenia gravis who had worsening
of symptoms associated with the use of alendronate. A 24-year-old patient with
myasthenia gravis had been administered oral systemic corticosteroid
(deflazacort 40 mg/day) for 3 years in order to control his myasthenic symptoms.
One year earlier, his lumbar spine bone mineral density was decreased. He was
started on oral calcium/vitamin D3 and alendronate (70-mg tablets once a week)
for osteoporosis. He reported an exacerbation of muscle weakness and extreme
fatigue on days when he took alendronate. He could not work on these days and
has to be on leave. Alendronate was stopped, and he was started on intravenous
ibandronate injections given every 3 months. He did not experience muscle
weakness and fatigue with ibandronate therapy. Alendronate should be used with
caution in patients with myasthenia gravis who have corticosteroid-induced
osteoporosis. OBJECTIVE: To determine the risk of osteoporosis in patients with myasthenia
gravis (MG) in a large cohort representing 99% of the population of Taiwan.
METHODS: Data from the Taiwan National Health Insurance Research Database were
used to conduct retrospective cohort analyses. The study cohort consisted of
2,073 patients with MG who were 3-fold frequency-matched by age and sex and
assigned the same index year as a comparison cohort without MG. Cox proportional
hazard regression analysis was conducted to estimate the risk of osteoporosis.
RESULTS: The MG cohort had a 1.96-fold increased risk of developing osteoporosis
compared with the comparison cohort (hazard ratio [HR] = 1.96, 95% confidence
interval [CI] = 1.57-2.44). Patients with MG older than 30 years developed an
increased risk of osteoporosis, with the highest risk in the age group from 30
to 44 years, compared with the control cohort. Corticosteroid-naïve patients
with MG had a 1.52-fold increased risk of developing osteoporosis (HR = 1.52,
95% CI = 1.11-2.08), and the corticosteroid-treated cohort had a 2.37-fold
increased risk of developing osteoporosis (HR = 2.37, 95% CI = 1.82-3.07).
CONCLUSION: This population-based retrospective cohort study provides evidence
that MG is associated with a high risk of osteoporosis regardless of
corticosteroid use. |
Which cell type has the protein Chromogranin A as marker? | Chromogranin A is a marker for neuroendocrine cells | BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) are a group of rare tumors.
Chromogranin A (CgA) was considered as the most practical and useful serum tumor
marker in PNET patients. But peripheral blood levels of CgA are not routinely
tested in Chinese patients with PNETs. This study was to assess the diagnostic
value of CgA in Chinese patients with PNETs especially in patients with
insulinomas.
METHODS: Eighty-nine patients with PNETs including 57 insulinomas and 32
non-insulinoma PNETs as well as 86 healthy participants were enrolled in this
study between September 2003 and June 2013. Serum levels of CgA were measured by
ELISA method. Expression of CgA protein was detected in 26 PNET tissues
including 14 insulinomas by immunohistochemical staining.
RESULTS: Serum levels of CgA in 89 PNET patients were significantly higher than
that in healthy controls (P = 7.2 × 10-9). Serum levels of CgA in 57 patients
with insulinomas (median 64.8 ng/ml, range 25-164) were slightly higher than the
levels in healthy controls (median 53.4 ng/ml, range 39-94) but much lower than
the levels in 32 patients with non-insulinoma PNETs (median 193 ng/ml, range
27-9021), P = 0.001. The serum CgA levels were reduced in 16 of 17 patients with
insulinomas after tumor resection. ROC curve showed that CgA values at 60 ng/ml
distinguished patients with insulinomas from healthy controls but its
sensitivity and specificity were 66.7% and 73.3%, respectively. In contrast, CgA
values at 74 ng/ml distinguished patients with non-insulinoma PNETs from healthy
controls, and the sensitivity and specificity were 65.6% and 91.9%,
respectively. Except for two insulinomas with negative staining of CgA, 12
insulinoma tissues showed positive staining of CgA.
CONCLUSION: CgA is a reliable serum diagnostic biomarker for PNETs but not for
insulinomas. Together with Chromogranin B and Secretogranins, Chromogranin A (CGA) is stored
in secretory (chromaffin) granules of the diffuse neuroendocrine system and
released with noradrenalin and adrenalin. Co-stored within the granule together
with neuropeptideY, cardiac natriuretic peptide hormones, several prohormones
and their proteolytic enzymes, CGA is a multifunctional protein and a major
marker of the sympatho-adrenal neuroendocrine activity. Due to its partial
processing to several biologically active peptides, CGA appears an important
pro-hormone implicated in relevant modulatory actions on endocrine,
cardiovascular, metabolic, and immune systems through both direct and indirect
sympatho-adrenergic interactions. As a part of this scenario, we here illustrate
the emerging role exerted by the full-length CGA and its three derived
fragments, i.e., Vasostatin 1, catestatin and serpinin, in the control of
circulatory homeostasis with particular emphasis on their cardio-vascular
actions under both physiological and physio-pathological conditions. The
Vasostatin 1- and catestatin-induced cardiodepressive influences are achieved
through anti-beta-adrenergic-NO-cGMP signaling, while serpinin acts like
beta1-adrenergic agonist through AD-cAMP-independent NO signaling. On the whole,
these actions contribute to widen our knowledge regarding the
sympatho-chromaffin control of the cardiovascular system and its highly
integrated "whip-brake" networks. Although chromogranin A (CGA) is a useful marker for pancreatic neuroendocrine
tumors (pNET) in the West, its usefulness in Japanese populations is unclear. To
assess this, we evaluated the serum CGA levels in 189 patients with various
pancreatic diseases, including proven pNET (n = 69), pancreatic cancer (PC) (n =
50), chronic pancreatitis (CP) (n = 50) and autoimmune pancreatitis (AIP) (n =
20), and 112 normal controls (controls) using an ELISA kit. The mean CGA level
of patients with pNET was significantly higher than any of the other groups
(407.8 ± 984.6 ng/mL [pNET] vs 91.8 ± 101.8 ng/mL [PC], 93.6 ± 57.5 ng/mL [CP],
69.9 ± 52.4 ng/mL [AIP] and 62.5 ± 48.3 ng/mL [controls]). Limiting the analysis
to patients not using proton pump inhibitors (PPI), the CGA level of patients
with PC or CP was not significantly different compared with the controls.
Discrimit analysis revealed that the best cut-off value of CGA to distinguish
patients with pNET from the controls was 78.7 ng/mL, with a sensitivity and
specificity of 53.6% and 78.6%, respectively. In patients with pNET, significant
factors associating with elevated CGA levels were tumor classification, tumor
size, and the presence of liver metastases in univariate analysis as well as PPI
use and the presence of liver metastases in multivariate analysis. We show that
CGA is a useful marker for diagnosing pNET in Japanese populations and for
distinguishing patients with pNET from patients with other pancreatic diseases.
The increased use of CGA in Japan will likely be a helpful tool in managing
these patients, as found in the West. Chromogranin A (CgA) not only plays an important role in pathologic diagnosis,
but is also used as a circulating biomarker in patients with
gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN). However, the
relationship between immunohistochemistry (IHC) expression and serum levels of
CgA has not been investigated. The value of CgA for evaluating treatment
response and prognosis is still not well understood. We conducted this study to
assess the significance of CgA in GEP-NEN in terms of diagnosis, curative
effects evaluation and prognosis. One hundred forty-five patients comprising 88
patients with active disease and 57 disease-free patients were enrolled in this
study from January 2011 to November 2013. The expression of CgA was assessed by
IHC, and serial serum CgA levels were measured by enzyme linked immunosorbent
assay. The overall expression rate of CgA was 69.0% (100/145). CgA expression
was associated with tumor site and stage (P < 0.05), but not correlated with
prognosis (P = 0.07). Serum CgA levels were significantly higher in GEP-NEN
patients with active disease when compared with disease-free patients (P =
0.001) or healthy participants (P < 0.001). A CgA cutoff value of 95 ng/ml
discriminated between healthy subjects or disease-free patients and patients
with active disease (sensitivity 51.2% and specificity 87.5%, respectively).
There was a correlation between the CgA IHC expression and high serum CgA levels
(R = 0.320, P = 0.002). Serum CgA levels were much higher in patients who
classified as neuroendocrine carcinoma, mixed adenoendocrine carcinoma (P =
0.035) and who were on stage IV (P = 0.041). Changes in CgA levels normalization
or ≥ 30% decrease suggested that patients had tumor response. Furthermore,
patients with serum CgA levels higher than 95 ng/ml had a significantly shorter
survival compared with patients with levels lower than 95 ng/ml (P < 0.001). CgA
is a reliable pathologic and circulating maker for diagnosis of GEP-NEN. We
further confirmed that serial measurement of CgA may be useful for evaluating
the efficacy of different kinds of therapies in patients during follow-up, and
serum CgA level ≥ 95 ng/ml may serve as a predictor of overall survial. |
Does a selective sweep increase genetic variation? | Selective sweep is a phenomenon in which the fixation of strongly beneficial alleles within a population reduces genetic diversity at partially linked neutral loci. Reduced variation or deviations from neutrality, along with an excess of fixed replacement sites, are indicative of selective sweep. | Drosophila melanogaster originated in tropical Africa but has achieved a
cosmopolitan distribution in association with human habitation. Cosmopolitan
populations of D. melanogaster are known to have reduced genetic variation,
particularly on the X chromosome. However, the relative importance of population
bottlenecks and selective sweeps in explaining this reduction is uncertain. We
surveyed variation at 31 microsatellites across a 330-kb section of the X
chromosome located between the white and kirre genes. Two linked clusters of
loci were observed with reduced variation and a skew toward rare alleles in both
an Ecuador and a Zimbabwe population sample. Examining Zimbabwe DNA sequence
polymorphism within one of these regions allowed us to localize a selective
sweep to a 361-bp window within the 5' regulatory region of the roughest gene,
with one nucleotide substitution representing the best candidate for the target
of selection. Estimates of sweep age suggested that this fixation event occurred
prior to the expansion of D. melanogaster from sub-Saharan Africa. For both
putative sweep regions in our data set, cosmopolitan populations showed wider
footprints of selection compared to those in Zimbabwe. This pattern appears
consistent with the demographic amplification of preexisting sweep signals due
to one or more population bottlenecks. Many domestic dog breeds have originated through fixation of discrete mutations
by intense artificial selection. As a result of this process, markers in the
proximity of genes influencing breed-defining traits will have reduced variation
(a selective sweep) and will show divergence in allele frequency. Consequently,
low-resolution genomic scans can potentially be used to identify regions
containing genes that have a major influence on breed-defining traits. We model
the process of breed formation and show that the probability of two or three
adjacent marker loci showing a spurious signal of selection within at least one
breed (i.e., Type I error or false-positive rate) is low if highly variable and
moderately spaced markers are utilized. We also use simulations with selection
to demonstrate that even a moderately spaced set of highly polymorphic markers
(e.g., one every 0.8 cM) has high power to detect regions targeted by strong
artificial selection in dogs. Further, we show that a gene responsible for black
coat color in the Large Munsterlander has a 40-Mb region surrounding the gene
that is very low in heterozygosity for microsatellite markers. Similarly, we
survey 302 microsatellite markers in the Dachshund and find three linked
monomorphic microsatellite markers all within a 10-Mb region on chromosome 3.
This region contains the FGFR3 gene, which is responsible for achondroplasia in
humans, but not in dogs. Consequently, our results suggest that the causative
mutation is a gene or regulatory region closely linked to FGFR3. We investigated DNA sequence diversity for loci on chromosomes 1 and 2 in six
natural populations of Arabidopsis lyrata and tested for the role of natural
selection in structuring genomewide patterns of variability, specifically
examining the effects of recombination rate on levels of silent polymorphism. In
contrast with theoretical predictions from models of genetic hitchhiking,
maximum-likelihood-based analyses of diversity and divergence do not suggest
reduction of diversity in the region of suppressed recombination near the
centromere of chromosome 1, except in a single population from Russia, in which
the pericentromeric region may have undergone a local selective sweep or
demographic process that reduced variability. We discuss various possibilities
that might explain why nucleotide diversity in most A. lyrata populations is not
related to recombination rate, including genic recombination hotspots, and low
gene density in the low recombination rate region. Wing patterning in Heliconius butterflies is a longstanding example of both
Müllerian mimicry and phenotypic radiation under strong natural selection. The
loci controlling such patterns are "hotspots" for adaptive evolution with great
allelic diversity across different species in the genus. We characterise
nucleotide variation, genotype-by-phenotype associations, linkage
disequilibrium, and candidate gene expression at two loci and across multiple
hybrid zones in Heliconius melpomene and relatives. Alleles at HmB control the
presence or absence of the red forewing band, while alleles at HmYb control the
yellow hindwing bar. Across HmYb two regions, separated by approximately 100 kb,
show significant genotype-by-phenotype associations that are replicated across
independent hybrid zones. In contrast, at HmB a single peak of association
indicates the likely position of functional sites at three genes, encoding a
kinesin, a G-protein coupled receptor, and an mRNA splicing factor. At both HmYb
and HmB there is evidence for enhanced linkage disequilibrium (LD) between
associated sites separated by up to 14 kb, suggesting that multiple sites are
under selection. However, there was no evidence for reduced variation or
deviations from neutrality that might indicate a recent selective sweep,
consistent with these alleles being relatively old. Of the three genes showing
an association with the HmB locus, the kinesin shows differences in wing disc
expression between races that are replicated in the co-mimic, Heliconius erato,
providing striking evidence for parallel changes in gene expression between
Müllerian co-mimics. Wing patterning loci in Heliconius melpomene therefore show
a haplotype structure maintained by selection, but no evidence for a recent
selective sweep. The complex genetic pattern contrasts with the simple genetic
basis of many adaptive traits studied previously, but may provide a better model
for most adaptation in natural populations that has arisen over millions rather
than tens of years. BACKGROUND: Glucose is an important source of energy for living organisms. In
vertebrates it is ingested with the diet and transported into the cells by
conserved mechanisms and molecules, such as the trans-membrane Glucose
Transporters (GLUTs). Members of this family have tissue specific expression,
biochemical properties and physiologic functions that together regulate glucose
levels and distribution. GLUT4 -coded by SLC2A4 (17p13) is an insulin-sensitive
transporter with a critical role in glucose homeostasis and diabetes
pathogenesis, preferentially expressed in the adipose tissue, heart muscle and
skeletal muscle. We tested the hypothesis that natural selection acted on
SLC2A4.
METHODOLOGY/PRINCIPAL FINDINGS: We re-sequenced SLC2A4 and genotyped 104 SNPs
along a approximately 1 Mb region flanking this gene in 102 ethnically diverse
individuals. Across the studied populations (African, European, Asian and
Latin-American), all the eight common SNPs are concentrated in the N-terminal
region upstream of exon 7 ( approximately 3700 bp), while the C-terminal region
downstream of intron 6 ( approximately 2600 bp) harbors only 6 singletons, a
pattern that is not compatible with neutrality for this part of the gene. Tests
of neutrality based on comparative genomics suggest that: (1) episodes of
natural selection (likely a selective sweep) predating the coalescent of human
lineages, within the last 25 million years, account for the observed reduced
diversity downstream of intron 6 and, (2) the target of natural selection may
not be in the SLC2A4 coding sequence.
CONCLUSIONS: We propose that the contrast in the pattern of genetic variation
between the N-terminal and C-terminal regions are signatures of the action of
natural selection and thus follow-up studies should investigate the functional
importance of different regions of the SLC2A4 gene. Antagonistic host-parasite interactions can drive rapid adaptive evolution in
genes of the immune system, and such arms races may be an important force
shaping polymorphism in the genome. The RNA interference pathway gene
Argonaute-2 (AGO2) is a key component of antiviral defense in Drosophila, and we
have previously shown that genes in this pathway experience unusually high rates
of adaptive substitution. Here we study patterns of genetic variation in a
100-kbp region around AGO2 in three different species of Drosophila. Our data
suggest that recent independent selective sweeps in AGO2 have reduced genetic
variation across a region of more than 50 kbp in Drosophila melanogaster, D.
simulans, and D. yakuba, and we estimate that selection has fixed adaptive
substitutions in this gene every 30-100 thousand years. The strongest signal of
recent selection is evident in D. simulans, where we estimate that the most
recent selective sweep involved an allele with a selective advantage of the
order of 0.5-1% and occurred roughly 13-60 Kya. To evaluate the potential
consequences of the recent substitutions on the structure and function of AGO2,
we used fold-recognition and homology-based modeling to derive a structural
model for the Drosophila protein, and this suggests that recent substitutions in
D. simulans are overrepresented at the protein surface. In summary, our results
show that selection by parasites can consistently target the same genes in
multiple species, resulting in areas of the genome that have markedly reduced
genetic diversity. BACKGROUND: Ammonium is one of the major forms in which nitrogen is available
for plant growth. OsAMT1;1 is a high-affinity ammonium transporter in rice
(Oryza sativa L.), responsible for ammonium uptake at low nitrogen
concentration. The expression pattern of the gene has been reported. However,
variations in its nucleotides and the evolutionary pathway of its descent from
wild progenitors are yet to be elucidated. In this study, nucleotide diversity
of the gene OsAMT1;1 and the diversity pattern of seven gene fragments spanning
a genomic region approximately 150 kb long surrounding the gene were surveyed by
sequencing a panel of 216 rice accessions including both cultivated rice and
wild relatives.
RESULTS: Nucleotide polymorphism (Pi) of OsAMT1;1 was as low as 0.00004 in
cultivated rice (Oryza sativa), only 2.3% of that in the common wild rice (O.
rufipogon). A single domit haplotype was fixed at the locus in O. sativa. The
test values for neutrality were significantly negative in the entire region
stretching 5' upstream and 3' downstream of the gene in all accessions. The
value of linkage disequilibrium remained high across a 100 kb genomic region
around OsAMT1;1 in O. sativa, but fell rapidly in O. rufipogon on either side of
the promoter of OsAMT1;1, demonstrating a strong natural selection within or
nearby the ammonium transporter.
CONCLUSIONS: The severe reduction in nucleotide variation at OsAMT1;1 in rice
was caused by a selective sweep around OsAMT1;1, which may reflect the nitrogen
uptake system under strong selection by the paddy soil during the domestication
of rice. Purifying selection also occurred before the wild rice diverged into
its two subspecies, namely indica and japonica. These findings would provide
useful insights into the processes of evolution and domestication of nitrogen
uptake genes in rice. A central problem in population genetics is to detect and analyze positive
natural selection by which beneficial mutations are driven to fixation. The
hitchhiking effect of a rapidly spreading beneficial mutation, which results in
local removal of standing genetic variation, allows such an analysis using DNA
sequence polymorphism. However, the current mathematical theory that predicts
the pattern of genetic hitchhiking relies on the assumption that a beneficial
mutation increases to a high frequency in a single random-mating population,
which is certainly violated in reality. Individuals in natural populations are
distributed over a geographic space. The spread of a beneficial allele can be
delayed by limited migration of individuals over the space and its hitchhiking
effect can also be affected. To study this effect of geographic structure on
genetic hitchhiking, we analyze a simple model of directional selection in a
subdivided population. In contrast to previous studies on hitchhiking in
subdivided populations, we mainly investigate the range of sufficiently high
migration rates that would homogenize genetic variation at neutral loci. We
provide a heuristic mathematical analysis that describes how the genealogical
structure at a neutral locus linked to the locus under selection is expected to
change in a population divided into two demes. Our results indicate that the
overall strength of genetic hitchhiking--the degree to which expected
heterozygosity decreases--is diminished by population subdivision, mainly
because opportunity for the breakdown of hitchhiking by recombination increases
as the spread of the beneficial mutation across demes is delayed when migration
rate is much smaller than the strength of selection. Furthermore, the amount of
genetic variation after a selective sweep is expected to be unequal over demes:
a greater reduction in expected heterozygosity occurs in the subpopulation from
which the beneficial mutation originates than in its neighboring subpopulations.
This raises a possibility of detecting a "hidden" geographic structure of
population by carefully analyzing the pattern of a selective sweep. Sex chromosome meiotic drive has been suggested as a cause of several
evolutionary genetic phenomena, including genomic conflicts that give rise to
reproductive isolation between new species. In this paper we present a
population genetic analysis of X chromosome drive in the stalk-eyed fly,
Teleopsis dalmanni, to determine how this natural polymorphism influences
genetic diversity. We analyzed patterns of DNA sequence variation at two
X-linked regions (comprising 1325 bp) approximately 50 cM apart and one
autosomal region (comprising 921 bp) for 50 males, half of which were collected
in the field from one of two allopatric locations and the other half were
derived from lab-reared individuals with known brood sex ratios. These two
populations are recently diverged but exhibit partial postzygotic reproductive
isolation, i.e. crosses produce sterile hybrid males and fertile females. We
find no nucleotide or microsatellite variation on the drive X chromosome,
whereas the same individuals show levels of variation at autosomal regions that
are similar to field-collected flies. Furthermore, one field-caught individual
collected 10 years previously had a nearly identical X haplotype to the drive X,
and is over 2% divergent from other haplotypes sampled from the field. These
results are consistent with a selective sweep that has removed genetic variation
from much of the drive X chromosome. We discuss how this finding may relate to
the rapid evolution of postzygotic reproductive isolation that has been
documented for these flies. Selective sweeps are typically associated with a local reduction of genetic
diversity around the adaptive site. However, selective sweeps can also quickly
carry neutral mutations to observable population frequencies if they arise early
in a sweep and hitchhike with the adaptive allele. We show that the interplay
between mutation and exponential amplification through hitchhiking results in a
characteristic frequency spectrum of the resulting novel haplotype variation
that depends only on the ratio of the mutation rate and the selection
coefficient of the sweep. On the basis of this result, we develop an estimator
for the selection coefficient driving a sweep. Since this estimator utilizes the
novel variation arising from mutations during a sweep, it does not rely on
preexisting variation and can also be applied to loci that lack recombination.
Compared with standard approaches that infer selection coefficients from the
size of dips in genetic diversity around the adaptive site, our estimator
requires much shorter sequences but sampled at high population depth to capture
low-frequency variants; given such data, it consistently outperforms standard
approaches. We investigate analytically and numerically how the accuracy of our
estimator is affected by the decay of the sweep pattern over time as a
consequence of random genetic drift and discuss potential effects of
recombination, soft sweeps, and demography. As an example for its use, we apply
our estimator to deep sequencing data from human immunodeficiency virus
populations. Organisms can often adapt surprisingly quickly to evolutionary challenges, such
as the application of pesticides or antibiotics, suggesting an abundant supply
of adaptive genetic variation. In these situations, adaptation should commonly
produce 'soft' selective sweeps, where multiple adaptive alleles sweep through
the population at the same time, either because the alleles were already present
as standing genetic variation or arose independently by recurrent de novo
mutations. Most well-known examples of rapid molecular adaptation indeed show
signatures of such soft selective sweeps. Here, we review the current
understanding of the mechanisms that produce soft sweeps and the approaches used
for their identification in population genomic data. We argue that soft sweeps
might be the domit mode of adaptation in many species. The positive selection of a nucleotide substitution in exon 2 of Plasmodium
falciparum chloroquine resistance transporter (pfcrt) gene (mutation responsible
for chloroquine resistance) causes a reduction in variation of neutral loci
close to the gene. This reduction in allelic diversity around flanking regions
of pfcrt gene was reported in worldwide chloroquine resistant isolates and
referred as selective sweep. In Plasmodium falciparum isolates of India, the
selective sweep in flanking loci of pfcrt gene is well established, however,
high allelic diversity observed in intragenic microsatellites of pfcrt gene
implied an ongoing genetic recombination. To understand, if molecular evolution
of chloroquine-resistant P. falciparum isolates in India follow a selective
sweep model, we analyzed genetic diversity at both seven intragenic and seven
flanking microsatellites of pfcrt (-24 to +106kb) gene in chloroquine sensitive
and resistant parasites originating from high and low transmission areas. We
observed low expected heterozygosity at all loci of resistant pfcrt-haplotypes
(He=0-0.77) compared to the wild-type (He=0.38-0.96). Resistant SVMNT from high
transmission areas showed significantly higher mean He (P=0.03, t-test) at both
intragenic and pfcrt-flanking loci (-24 to +22 kb) in comparison to low
transmission areas. Our observation of reduction in variation at both intragenic
and flanking loci of mutant pfcrt gene confirmed the selective sweep model of
natural selection in chloroquine resistant P. falciparum isolates in India. The genetic diversity within an 11 kb segment of the MTMR8 gene in a sample of
111 sub-Saharan and 49 non-African X chromosomes was investigated to assess the
early evolutionary history of sub-Saharan Africans and the out-of-Africa
expansion. The analyses revealed a complex genetic structure of the Africans
that contributed to the emergence of modern humans. We observed partitioning of
two thirds of old lineages among southern, west/central and east African
populations indicating ancient population stratification predating the out of
Africa migration. Age estimates of these lineages, older than coalescence times
of uniparentally inherited markers, raise the question whether contemporary
humans originated from a single population or as an amalgamation of different
populations separated by years of independent evolution, thus suggesting a
greater antiquity of our species than generally assumed. While the oldest
sub-Saharan lineages, ~500 thousand years, are found among Khoe-San from
southern-Africa, a distinct haplotype found among Biaka is likely due to
admixture from an even older population. An East African population that gave
rise to non-Africans underwent a selective sweep affecting the subcentromeric
region where MTMR8 is located. This and similar sweeps in four other regions of
the X chromosome, documented in the literature, effectively reduced genetic
diversity of non-African chromosomes and therefore may have exacerbated the
effect of the demographic bottleneck usually ascribed to the out of Africa
migration. Our data is suggestive, however, that a bottleneck, occurred in
Africa before range expansion. The evolution of drug resistance in HIV occurs by the fixation of specific,
well-known, drug-resistance mutations, but the underlying population genetic
processes are not well understood. By analyzing within-patient longitudinal
sequence data, we make four observations that shed a light on the underlying
processes and allow us to infer the short-term effective population size of the
viral population in a patient. Our first observation is that the evolution of
drug resistance usually occurs by the fixation of one drug-resistance mutation
at a time, as opposed to several changes simultaneously. Second, we find that
these fixation events are accompanied by a reduction in genetic diversity in the
region surrounding the fixed drug-resistance mutation, due to the hitchhiking
effect. Third, we observe that the fixation of drug-resistance mutations
involves both hard and soft selective sweeps. In a hard sweep, a resistance
mutation arises in a single viral particle and drives all linked mutations with
it when it spreads in the viral population, which dramatically reduces genetic
diversity. On the other hand, in a soft sweep, a resistance mutation occurs
multiple times on different genetic backgrounds, and the reduction of diversity
is weak. Using the frequency of occurrence of hard and soft sweeps we estimate
the effective population size of HIV to be 1.5 x 10(5) (95% confidence interval
[0.8 x 10(5),4.8 x 10(5)]). This number is much lower than the actual number of
infected cells, but much larger than previous population size estimates based on
synonymous diversity. We propose several explanations for the observed
discrepancies. Finally, our fourth observation is that genetic diversity at
non-synonymous sites recovers to its pre-fixation value within 18 months,
whereas diversity at synonymous sites remains depressed after this time period.
These results improve our understanding of HIV evolution and have potential
implications for treatment strategies. |
Which disease phenotypes are associated to PRPS1 mutations? | X-linked Charcot-Marie-Tooth disease type 5 (CMTX5), Arts syndrome, and non-syndromic sensorineural deafness (DFN2) are allelic syndromes, caused by reduced activity of phosphoribosylpyrophosphate synthetase 1 (PRS-I) due to loss-of-function mutations in PRPS1. | Phosphoribosylpyrophosphate synthetases (PRSs) catalyze the first step of
nucleotide synthesis. Nucleotides are central to cell function, being the
building blocks of nucleic acids and serving as cofactors in cellular signaling
and metabolism. With this in mind, it is remarkable that mutations in
phosphoribosylpyrophosphate synthetase 1 (PRPS1), which is the most ubiquitously
expressed gene of the three PRS genes, are compatible with life. Mutations
described thus far in PRPS1 are all missense mutations that result in PRS-I
superactivity or in variable levels of decreased activity, resulting in X-linked
Charcot-Marie-Tooth disease-5 (CMTX5), Arts syndrome, and X-linked nonsyndromic
sensorineural deafness (DFN2). Patients with PRS-I superactivity primarily
present with uric acid overproduction, mental retardation, ataxia, hypotonia,
and hearing impairment. Postlingual progressive hearing loss is found as an
isolated feature in DFN2 patients. Patients with CMTX5 and Arts syndrome have
peripheral neuropathy, including hearing impairment and optic atrophy. However,
patients with Arts syndrome are more severely affected because they also have
central neuropathy and an impaired immune system. The neurological phenotype in
all four PRPS1-related disorders seems to result primarily from reduced levels
of GTP and possibly other purine nucleotides including ATP, suggesting that
these disorders belong to the same disease spectrum. Preliminary results of
S-adenosylmethionine (SAM) supplementation in two Arts syndrome patients show
improvement of their condition, indicating that SAM supplementation in the diet
could alleviate some of the symptoms of patients with PRPS1 spectrum diseases by
replenishing purine nucleotides (J.C., unpublished data). BACKGROUND: X-linked Charcot-Marie-Tooth disease type 5 (CMTX5) is caused by
mutations in the gene encoding phosphoribosyl pyrophosphate synthetase I
(PRPS1). There has been only one case report of CMTX5 patients. The aim of this
study was to identify the causative gene in a family with CMTX with peripheral
neuropathy and deafness.
CASE REPORT: A Korean family with X-linked recessive CMT was enrolled. The age
at the onset of hearing loss of the male proband was 5 months, and that of
steppage gait was 6 years; he underwent cochlear surgery at the age of 12 years.
In contrast to what was reported for the first patients with CMTX5, this patient
did not exhibit optic atrophy. Furthermore, there was no cognitive impairment,
respiratory dysfunction, or visual disturbance. Assessment of his family history
revealed two male relatives with very similar clinical manifestations.
Electrophysiological evaluations disclosed sensorineural hearing loss and
peripheral neuropathy. Whole-exome sequencing identified a novel p.Ala121Gly
(c.362C>G) PRPS1 mutation as the underlying genetic cause of the clinical
phenotype.
CONCLUSIONS: A novel mutation of PRPS1 was identified in a CMTX5 family in which
the proband had a phenotype of peripheral neuropathy with early-onset hearing
loss, but no optic atrophy. The findings of this study will expand the clinical
spectrum of X-linked recessive CMT and will be useful for the molecular
diagnosis of clinically heterogeneous peripheral neuropathies. BACKGROUND: X-linked Charcot-Marie-Tooth disease type 5 (CMTX5), Arts syndrome,
and non-syndromic sensorineural deafness (DFN2) are allelic syndromes, caused by
reduced activity of phosphoribosylpyrophosphate synthetase 1 (PRS-I) due to
loss-of-function mutations in PRPS1. As only few families have been described,
knowledge about the relation between these syndromes, the phenotypic spectrum in
patients and female carriers, and the relation to underlying PRS-I activity is
limited.
METHODS: We investigated a family with a novel PRPS1 mutation (c.830A > C,
p.Gln277Pro) by extensive phenotyping, MRI, and genetic and enzymatic tests.
RESULTS: The male index subject presented with an overlap of CMTX5 and Arts
syndrome features, whereas his sister presented with prelingual DFN2. Both
showed mild parietal and cerebellar atrophy on MRI. Enzymatically, PRS-I
activity was undetectable in the index subject, reduced in his less affected
sister, and normal in his unaffected mother.
CONCLUSIONS: Our findings demonstrate that CMTX5, Arts syndrome and DFN2 are
phenotypic clusters on an intrafamilial continuum, including overlapping
phenotypes even within individuals. The respective phenotypic presentation seems
to be determined by the exact PRPS1 mutation and the residual enzyme activity,
the latter being largely influenced by the degree of skewed X-inactivation.
Finally, our findings show that brain atrophy might be more common in
PRPS1-disorders than previously thought. PRPS1 codes for the enzyme phosphoribosyl pyrophosphate synthetase-1 (PRS-1).
The spectrum of PRPS1-related disorders associated with reduced activity
includes Arts syndrome, Charcot-Marie-Tooth disease-5 (CMTX5) and X-linked
non-syndromic sensorineural deafness (DFN2). We describe a novel phenotype
associated with decreased PRS-1 function in two affected male siblings. Using
whole exome and Sanger sequencing techniques, we identified a novel missense
mutation in PRPS1. The clinical phenotype in our patients is characterized by
high prenatal maternal α-fetoprotein, intrauterine growth restriction,
dysmorphic facial features, severe intellectual disability and spastic
quadraparesis. Additional phenotypic features include macular coloboma-like
lesions with retinal dystrophy, severe short stature and diabetes insipidus.
Exome sequencing of the two affected male siblings identified a shared putative
pathogenic mutation c.586C>T p.(Arg196Trp) in the PRPS1 gene that was maternally
inherited. Follow-up testing showed normal levels of hypoxanthine in urine
samples and uric acid levels in blood serum. The PRS activity was significantly
reduced in erythrocytes of the two patients. Nucleotide analysis in erythrocytes
revealed abnormally low guanosine triphosphate and guanosine diphosphate. This
presentation is the most severe form of PRPS1-deficiency syndrome described to
date and expands the spectrum of PRPS1-related disorders. |
Is indicated the use of antioxidant supplements in patients at risk for coronary artery disease? | antioxidant supplementation
However there are no clear evidencies on the clinical and prognostic benefit of this supplementation.
Currently there areno recommendation for the antioxidant therapy in patients with coronary artery disease.
Currently the American Heart Association recommends consumption of a balanced diet with emphasis on antioxidant-rich fruits and vegetables but does not recommend antioxidant supplementation for the general population. | Hypercholesterolemia attributable to increased plasma concentrations of low
density lipoproteins is a well recognized risk factor for the premature
development of coronary atherosclerosis in both experimental animals and humans.
Recent studies have indicated that modifications to low density lipoprotein
result in enhanced uptake of the modified lipoproteins by macrophages and lead
to accelerated rates of lipid deposition and the creation of foam cells.
Oxidation of low density lipoprotein has been shown to be one of the
modifications which leads to uptake of this lipoprotein by scavenger receptors
present on macrophages and results in intracellular lipid accumulation.
Treatment of hypercholesterolemic animals with antioxidant drugs, including
probucol, has been shown to reduce the development of atherosclerosis and
xanthoma regression has been observed in patients with severe
hypercholesterolemia treated with this drug. Epidemiologic studies support the
view that low plasma concentrations of antioxidant vitamins, including vitamin E
are associated with higher rates of coronary atherosclerosis in humans and that
supplementation with vitamin E is associated with a decreased incidence of
coronary artery disease. Prospective clinical trials to assess the potential
benefit of antioxidant supplementation in high risk patients are currently in
progress and these trials, when completed, should provide definitive information
concerning the potential benefits to be derived from supplementation with
antioxidant vitamins as an adjunctive therapy to prevent the premature
development of atherosclerosis. One in three Americans will eventually die of cardiovascular disease.
Antioxidant vitamins, which are postulated to reduce risk by about 20-30%, could
have substantial clinical and public health impact. Basic research, clinical
observation, and epidemiology have contributed to an emerging body of evidence
on the atherogenicity of oxidized low-density lipoprotein, which could be an
important mechanism to explain why antioxidant vitamins may decrease risk of
coronary disease. The antioxidant-vitamin/cardiovascular-disease hypothesis has
recently been explored in several large prospective cohort studies, but the
findings were not all consistent. In several randomized, small-scale trials
using subjects with existing vascular disease, data indicate benefits associated
with vitamin E and beta carotene. Over the next several years, data from a
number of ongoing primary prevention trials and proposed secondary prevention
trials should determine whether antioxidant vitamins decrease risk of vascular
disease. BACKGROUND: Interest in the antioxidant vitamin E as a possible protective
nutrient against coronary disease has intensified with the recognition that
oxidized low-density lipoprotein may be involved in atherogenesis.
METHODS: In 1980, 87,245 female nurses 34 to 59 years of age who were free of
diagnosed cardiovascular disease and cancer completed dietary questionnaires
that assessed their consumption of a wide range of nutrients, including vitamin
E. During follow-up of up to eight years (679,485 person-years) that was 97
percent complete, we documented 552 cases of major coronary disease (437
nonfatal myocardial infarctions and 115 deaths due to coronary disease).
RESULTS: As compared with women in the lowest fifth of the cohort with respect
to vitamin E intake, those in the top fifth had a relative risk of major
coronary disease of 0.66 (95 percent confidence interval, 0.50 to 0.87) after
adjustment for age and smoking. Further adjustment for a variety of other
coronary risk factors and nutrients, including other antioxidants, had little
effect on the results. Most of the variability in intake and reduction in risk
was attributable to vitamin E consumed as supplements. Women who took vitamin E
supplements for short periods had little apparent benefit, but those who took
them for more than two years had a relative risk of major coronary disease of
0.59 (95 percent confidence interval, 0.38 to 0.91) after adjustment for age,
smoking status, risk factors for coronary disease, and use of other antioxidant
nutrients (including multi-vitamins).
CONCLUSIONS: Although these prospective data do not prove a cause-and-effect
relation, they suggest that among middle-aged women the use of vitamin E
supplements is associated with a reduced risk of coronary heart disease.
Randomized trials of vitamin E in the primary and secondary prevention of
coronary disease are being conducted; public policy recommendations about the
widespread use of vitamin E should await the results of these trials. BACKGROUND: The oxidative modification of low-density lipoproteins increases
their incorporation into the arterial intima, an essential step in
atherogenesis. Although dietary antioxidants, such as vitamin C, carotene, and
vitamin E, have been hypothesized to prevent coronary heart disease, prospective
epidemiologic data are sparse.
METHODS: In 1986, 39,910 U.S. male health professionals 40 to 75 years of age
who were free of diagnosed coronary heart disease, diabetes, and
hypercholesterolemia completed detailed dietary questionnaires that assessed
their usual intake of vitamin C, carotene, and vitamin E in addition to other
nutrients. During four years of follow-up, we documented 667 cases of coronary
disease.
RESULTS: After controlling for age and several coronary risk factors, we
observed a lower risk of coronary disease among men with higher intakes of
vitamin E (P for trend = 0.003). For men consuming more than 60 IU per day of
vitamin E, the multivariate relative risk was 0.64 (95 percent confidence
interval, 0.49 to 0.83) as compared with those consuming less than 7.5 IU per
day. As compared with men who did not take vitamin E supplements, men who took
at least 100 IU per day for at least two years had a multivariate relative risk
of coronary disease of 0.63 (95 percent confidence interval, 0.47 to 0.84).
Carotene intake was not associated with a lower risk of coronary disease among
those who had never smoked, but it was inversely associated with the risk among
current smokers (relative risk, 0.30; 95 percent confidence interval, 0.11 to
0.82) and former smokers (relative risk, 0.60; 95 percent confidence interval,
0.38 to 0.94). In contrast, a high intake of vitamin C was not associated with a
lower risk of coronary disease.
CONCLUSIONS: These data do not prove a causal relation, but they provide
evidence of an association between a high intake of vitamin E and a lower risk
of coronary heart disease in men. Public policy recommendations with regard to
the use of vitamin E supplements should await the results of additional studies. Hypercholesterolemia, cigarette smoking, hypertension, and obesity are known
contributing risk factors for the development of atherosclerotic coronary artery
disease (CAD). However, they account for only half of all cases of CAD, and the
complete pathologic process underlying atherosclerosis remains unknown. Growing
evidence suggests that oxidative modification of low-density lipoprotein (LDL)
may be of particular importance in the pathogenesis. Oxidized LDL exhibits
proatherogenic effects. Therefore, current research has focused on inhibiting
the oxidation of LDL as a means of inhibiting the atherosclerotic process. One
such approach is to enhance the endogenous antioxidant defense systems within
the LDL particle with lipophilic antioxidants such as alpha-tocopherol and
beta-carotene, or by supplementing the aqueous-phase antioxidant capacity with
ascorbic acid. Observational data suggest a protective effect of antioxidant
supplementation on the incidence of CAD; however, specific doses cannot be
recommended since the data are inconclusive. BACKGROUND: The role of dietary antioxidant vitamins in preventing coronary
heart disease has aroused considerable interest because of the knowledge that
oxidative modification of low-density lipoprotein may promote atherosclerosis.
METHODS: We studied 34,486 postmenopausal women with no cardiovascular disease
who in early 1986 completed a questionnaire that assessed, among other factors,
their intake of vitamins A, E, and C from food sources and supplements. During
approximately seven years of follow-up (ending December 31, 1992), 242 of the
women died of coronary heart disease.
RESULTS: In analyses adjusted for age and dietary energy intake, vitamin E
consumption appeared to be inversely associated with the risk of death from
coronary heart disease. This association was particularly striking in the
subgroup of 21,809 women who did not consume vitamin supplements (relative risks
from lowest to highest quintile of vitamin E intake, 1.0, 0.68, 0.71, 0.42, and
0.42; P for trend 0.008). After adjustment for possible confounding variables,
this inverse association remained (relative risks from lowest to highest
quintile, 1.0, 0.70, 0.76, 0.32, and 0.38; P for trend, 0.004). There was little
evidence that the intake of vitamin E from supplements was associated with a
decreased risk of death from coronary heart disease, but the effects of
high-dose supplementation and the duration of supplement use could not be
definitely addressed. Intake of vitamins A and C did not appear to be associated
with the risk of death form coronary heart disease.
CONCLUSIONS: These results suggest that in postmenopausal women the intake of
vitamin E from food is inversely associated with the risk of death from coronary
heart disease and that such women can lower their risk without using vitamin
supplements. By contrast, the intake of vitamins A and C was not associated with
lower risks of dying from coronary disease. Clinical as well as basic research in the field of atherogenesis indicates that
the progression of this disease process can be slowed down or even reversed. It
is well established that nutrition plays an important role in the prevention and
treatment of the classical atherogenic risk factors such as obesity, diabetes
mellitus and hyperlipidemia. In addition, some nutrients such as the
polyunsaturated n-3-fatty-acids or antioxidative vitamins can intervene directly
by influencing one or more steps of the atherogenetic and/or thrombogenetic
process. A comprehensive understanding of the pathogenesis of this disease as
well as of the mechanisms of nutrient action are essential to the planning of
successful nutritional prevention strategies. Because most nutrients influence
mainly the slow and long-standing development of the atherosclerotic lesion,
their inclusion in primary nutritional prevention should be started at an early
age. Few nutrients such as the n-3 fatty acids, which also reduce the
thrombogenetic risk factors, have demonstrated some success in the secondary
prevention of CHD. Given the complexity with which nutrients intervene in the
atherosclerotic process and their interactions with each other, nutritional
prevention strategies should be based on well-grounded dietary modifications
rather than supplementation with individual nutrients. Observational studies have found that persons who consume large amounts of fruit
and vegetables have lower rates of coronary heart disease. The strength of
epidemiologic evidence, however, differs for each of the antioxidant vitamins.
High intake of vitamin E from food or supplements has generally been associated
with a lower incidence of coronary heart disease. The evidence for beta-carotene
intake is inconsistent, with several studies finding a modest reduction in risk
among persons with high intake, and others failing to find an association.
Although many studies have examined the relationship between vitamin C intake
and cardiovascular disease, no significant benefit was seen in any of the large
studies that were able to control for other antioxidant intake or multivitamin
use. Observational studies cannot discern whether the decreased risk observed is
caused by the antioxidants themselves or other characteristics of the
individuals who consume them. The epidemiologic associations may be due to other
nutrients in antioxidant-rich foods, or other dietary or lifestyle factors.
Randomized controlled trials are necessary to confirm or refute the
observational data. On the basis of available evidence, we should recommend a
healthy diet, rich in fruit and vegetables, but should not endorse vitamin
supplementation unless conclusive evidence of benefit is demonstrated in
clinical trials. BACKGROUND: Epidemiological data suggest that the intake of antioxidants such as
alpha-tocopherol (vitamin E) and beta-carotene has an inverse correlation with
the incidence of coronary heart disease. The results from clinical trials of
antioxidant supplementation in people with known coronary heart disease are
inconclusive.
METHODS: We studied the frequency of major coronary events in 1862 men enrolled
in the alpha-tocopherol beta-carotene Cancer Prevention Study (smokers aged
between 50 and 69 years) who had a previous myocardial infarction. In this
randomised, double-blind. placebo-controlled study, men had received dietary
supplements of alpha-tocopherol (50 mg/day), beta-carotene (20 mg/day), both, or
placebo. The median follow-up was 5.3 years. The endpoint of this substudy was
the first major coronary event after randomisation. Analyses were by intention
to treat.
FINDINGS: 424 major coronary events (non-fatal myocardial infarction and fatal
coronary heart disease) occurred during follow-up. There were no significant
differences in the number of major coronary events between any supplementation
group and the placebo group (alpha-tocopherol 94/466; beta-carotene 113/461;
alpha-tocopherol and beta-carotene 123/497; placebo 94/438 [log-rank test, p =
0.25]). There were significantly more deaths from fatal coronary heart disease
in the beta-carotene (74/461, multivariate-adjusted relative risk 1.75 [95% CI
1.16-2.64], p = 0.007) and combined alpha-tocopherol and beta-carotene groups
(67/497, relative risk 1.58 [1.05-2.40], p = 0.03) than in the placebo group
(39/438), but there was no significant increase in the alpha-tocopherol
supplementation group (54/466, relative risk 1.33 [0.86-2.05], p = 0.20).
INTERPRETATION: The proportion of major coronary events in men with a previous
myocardial infarction who smoke was not decreased with either alpha-tocopherol
or beta-carotene supplements. In fact, the risk of fatal coronary heart disease
increased in the groups that received either beta-carotene or the combination of
alpha-tocopherol and beta-carotene; there was a non-significant trend of
increased deaths in the alpha-tocopherol group. We do not recommend the use of
alpha-tocopherol or beta-carotene supplements in this group of patients. OBJECTIVE: To evaluate the effects of alpha tocopherol and beta carotene
supplements on recurrence and progression of angina symptoms, and incidence of
major coronary events in men with angina pectoris.
DESIGN: Placebo controlled clinical trial.
SETTING: The Finnish alpha tocopherol beta carotene cancer prevention study
primarily undertaken to examine the effects of alpha tocopherol and beta
carotene on cancer.
SUBJECTS: Male smokers aged 50-69 years who had angina pectoris in the Rose
chest pain questionnaire at baseline (n = 1795).
INTERVENTIONS: alpha tocopherol (vitamin E) 50 mg/day, beta carotene 20 mg/day
or both, or placebo in 2 x 2 factorial design.
MAIN OUTCOME MEASURES: Recurrence of angina pectoris at annual follow up visits
when the questionnaire was readministered; progression from mild to severe
angina; incidence of major coronary events (non-fatal myocardial infarction and
fatal coronary heart disease).
RESULTS: There were 2513 recurrences of angina pectoris during follow up (median
4 years). Compared to placebo, the odds ratios for recurrence in the active
treatment groups were: alpha tocopherol only 1.06 (95% confidence interval (CI)
0.85 to 1.33), alpha tocopherol and beta carotene 1.02 (0.82 to 1.27), beta
carotene only 1.06 (0.84 to 1.33). There were no significant differences in
progression to severe angina among the groups given supplements or placebo.
Altogether 314 major coronary events were observed during follow up (median 5.5
years) and the risk for them did not differ significantly among the groups given
supplements or placebo.
CONCLUSIONS: There was no evidence of beneficial effects for alpha tocopherol or
beta carotene supplements in male smokers with angina pectoris, indicating no
basis for therapeutic or preventive use of these agents in such patients. Epidemiologic studies have suggested that vitamin E (alpha-tocopherol) may play
a preventive role in reducing the incidence of atherosclerosis. The aim of this
paper was to conduct a cost-effectiveness analysis of vitamin E supplementation
in patients with coronary artery disease using data from the Cambridge Heart
Antioxidant Study (CHAOS). The study compared cost-effectiveness in the context
of Australian and United States (US) health care utilization. The main clinical
outcome used in the economic evaluation was the incidence of acute myocardial
infarction (AMI) which was nonfatal. Utilization of health care resources was
estimated by conducting a survey of Australian clinicians and published
Australian and US cost data. Cost savings of $127 (A$181) and $578/patient
randomized to vitamin E therapy compared with patients receiving placebo were
found for Australian and US settings, respectively. Savings in the vitamin E
group were due primarily to reduction in hospital admissions for AMI. This
occurred because the vitamin E group had a 4.4% lower absolute risk of AMI than
did the placebo group. Less than 10% of health care costs in the Australian
evaluation was due to vitamin E ($150 [A$214/patient]). Our economic evaluation
indicates that vitamin E therapy in patients with angiographically proven
atherosclerosis is cost-effective in the Australian and US settings. Hyperhomocysteinemia is now regarded as an established risk factor for coronary
artery disease and is present frequently in the general population. However, the
diagnostic value of this risk factor relative to others has only occasionally
been investigated. We compared the diagnostic value of classic risk factors and
of homocysteine in a retrospective case-control study in 191 cases with
angiographically established coronary artery disease and 231 healthy controls.
Life style habits were assessed by a detailed questionnaire. Laboratory
parameters including lipoproteins and blood lipids, homocysteine, folate, and
vitamin B12 were measured and their diagnostic value compared with each other by
use of receiver-operator characteristic analysis. Comparison of the
receiver-operator characteristic curves revealed that homocysteine significantly
discriminated between cases and control subjects. High-density-lipoprotein
cholesterol, triglycerides and non-esterified fatty acids also had an area under
the curve significantly different from 0.5 (the area under the curve
representing no discrimination). Homocysteine was weakly related to folate,
vitamin B12, age and serum creatinine concentration. We conclude that
hyperhomocysteinemia is at least as important as conventional risk factors for
coronary artery disease and that receiver operator characteristic analysis of
homocysteine is suitable to determine patients at the highest risk for coronary
artery disease. Clinical trials testing the effect of homocysteine lowering by
vitamin supplementation in the prevention of coronary artery disease are needed. Cardiovascular disease has a multifactorial aetiology, as is illustrated by the
existence of numerous risk indicators, many of which can be influenced by
dietary means. It should be recalled, however, that only after a
cause-and-effect relationship has been established between the disease and a
given risk indicator (called a risk factor in that case), can modifying this
factor be expected to affect disease morbidity and mortality. In this paper,
effects of diet on cardiovascular risk are reviewed, with special emphasis on
modification of the plasma lipoprotein profile and of hypertension. In addition,
dietary influences on arterial thrombotic processes, immunological interactions,
insulin resistance and hyperhomocysteinaemia are discussed. Dietary lipids are
able to affect lipoprotein metabolism in a significant way, thereby modifying
the risk of cardiovascular disease. However, more research is required
concerning the possible interactions between the various dietary fatty acids,
and between fatty acids and dietary cholesterol. In addition, more studies are
needed with respect to the possible importance of the postprandial state.
Although in the aetiology of hypertension the genetic component is definitely
stronger than environmental factors, some benefit in terms of the development
and coronary complications of atherosclerosis in hypertensive patients can be
expected from fatty acids such as alpha-linolenic acid, eicosapentaenoic acid
and docosahexaenoic acid. This particularly holds for those subjects where the
hypertensive mechanism involves the formation of thromboxane A2 and/or alpha
1-adrenergic activities. However, large-scale trials are required to test this
contention. Certain aspects of blood platelet function, blood coagulability, and
fibrinolytic activity are associated with cardiovascular risk, but causality has
been insufficiently proven. Nonetheless, well-designed intervention studies
should be initiated to further evaluate such promising dietary components as the
various n-3 and n-6 fatty acids and their combination, antioxidants, fibre, etc.
for their effect on processes participating in arterial thrombus formation.
Long-chain polyenes of the n-3 family and antioxidants can modify the activity
of immunocompetent cells, but we are at an early stage of examining the role of
immune function on the development of atherosclerotic plaques. Actually, there
is little, if any, evidence that dietary modulation of immune system responses
of cells participating in atherogenesis exerts beneficial effects. Although it
seems feasible to modulate insulin sensitivity and subsequent cardiovascular
risk factors by decreasing the total amount of dietary fat and increasing the
proportion of polyunsaturated fatty acids, additional studies on the efficacy of
specific fatty acids, dietary fibre, and low-energy diets, as well as on the
mechanisms involved are required to understand the real function of these
dietary components. Finally, dietary supplements containing folate and vitamins
B6 and/or B12 should be tested for their potential to reduce cardiovascular risk
by lowering the plasma level of homocysteine. Decreased antioxidant-vitamin nutritional status may increase lipid peroxidation
and susceptibility of low-density lipoprotein (LDL) to oxidative modification.
The aim of this study was to evaluate the vitamin nutritional status of coronary
artery disease (CAD) patients and to assess the risk of CAD related to each
individual antioxidant vitamin. The study was performed as a case-control study
with 41 patients with angiographically demonstrated CAD and 41 apparently
healthy age- and smoking status-matched controls. Plasma vitamin E, C and A
concentrations were significantly decreased in CAD patients compared with
controls (p < 0.001) after correcting for significant covariates. Per quartile
decrease in vitamin A and E concentrations was associated with increased risk of
CAD, even after adjusting for CAD risk factors, while per quartile decrease in
vitamin C concentrations was not associated with significant CAD risk after
adjusting for CAD risk factors. Decreased vitamin A and E concentrations are
independently associated with increased risk of CAD independent from other CAD
risk factors in white male South Africans and dietary intervention strategies
are advocated. Various studies have evaluated the antioxidant effects of vitamin E in the
prevention or treatment of coronary artery disease (CAD). In vitro data suggest
that vitamin E protects against oxidation of low-density lipoprotein and
decreases the deposition of atherogenic oxidized low-density lipoprotein in
arterial walls. Various observational and epidemiological studies also suggest a
relationship between vitamin E serum concentrations or intake and CAD. One
prospective, randomized trial suggested that low-dosage vitamin E
supplementation (50 IU/d) decreases the risk of angina in patients without
previously diagnosed CAD. Another study, using high-dosage vitamin E
supplementation (400 or 800 IU/d), demonstrated a decrease in the combined end
point of nonfatal myocardial infarction and cardiovascular death in patients
with established CAD. Discordant data, however, have been published that imply
no cardiovascular benefit of low-dosage vitamin E supplementation (50 IU/d) and
detrimental effects if vitamin E is combined with beta carotene. At this point,
clinicians should emphasize a low-fat diet with high intake of fruits and
vegetable sources containing vitamin E. Supplemental vitamin E may be considered
in patients at high risk for CAD or with documented CAD, but the potential
beneficial effects should be weighed against possible long-term adverse effects.
If vitamin E supplementation is initiated, the literature suggests dosages of
100 to 400 IU/d, with the higher dosage considered in patients with documented
CAD. Additional investigation is warranted to further define the role of vitamin
E supplementation in CAD and to critically evaluate the optimal dosage, duration
of use, and method of consumption (dietary vs supplemental). Clinical use of antioxidant vitamin supplementation may help to prevent coronary
heart disease (CHD). Epidemiologic studies find lower CHD morbidity and
mortality in persons who consume larger quantities of antioxidants in foods or
supplements. Clinical trials indicate that supplementation with certain
nutrients is beneficial in reducing the incidence of CHD events. Recent studies
show that supplementation with antioxidant vitamins E and C have benefits in CHD
prevention; however, supplementation with beta-carotene may have deleterious
effects and is not recommended. Current evidence suggests that patients with CHD
would probably benefit from taking vitamin E in a dosage of 400 IU per day and
vitamin C in a dosage of 500 to 1,000 mg per day. Clinicians may also want to
consider vitamin supplementation for CHD prevention in high-risk patients.
Folate lowers elevated homocysteine levels, but evidence for routine
supplemental use does not yet exist. Other nutritional supplements are currently
under investigation. Data from the 1970s first suggested that vitamin E may be effective in
decreasing mortality from cardiovascular disease. As the understanding of the
antioxidant effect of this vitamin evolved, researchers began to further study
the biologic effects of vitamin E. In vitro studies have shown vitamin E to have
several potentially cardioprotective effects, including antagonizing the
oxidation of low-density lipoproteins, inhibiting platelet aggregation and
adhesion, preventing smooth muscle proliferation, and preserving normal coronary
dilation. Several prospective studies, including the US Nurses' Health Study and
the US Health Professionals' Follow-up Study, found a 34% and 39% reduction,
respectively, in the risk of having a cardiac event for those taking vitamin E
supplements. The Iowa Women's Health Study found a 47% reduction in cardiac
mortality. Results of randomized, controlled clinical trials have not found
consistent benefit, however. The best known of these trials, the Cambridge Heart
Antioxidant Study, found a 47% reduction in fatal and nonfatal myocardial
infarction in patients with proven coronary atherosclerosis who were given 400
or 800 IU of vitamin E daily. There was, however, no effect on mortality. While
emerging and promising data suggest the potential benefit of vitamin E for
high-risk cardiac patients, physicians should be alert to the results of
randomized, controlled clinical trials already in progress. BACKGROUND: Observational and experimental studies suggest that the amount of
vitamin E ingested in food and in supplements is associated with a lower risk of
coronary heart disease and atherosclerosis.
METHODS: We enrolled a total of 2545 women and 6996 men 55 years of age or older
who were at high risk for cardiovascular events because they had cardiovascular
disease or diabetes in addition to one other risk factor. These patients were
randomly assigned according to a two-by-two factorial design to receive either
400 IU of vitamin E daily from natural sources or matching placebo and either an
angiotensin-converting-enzyme inhibitor (ramipril) or matching placebo for a
mean of 4.5 years (the results of the comparison of ramipril and placebo are
reported in a companion article). The primary outcome was a composite of
myocardial infarction, stroke, and death from cardiovascular causes. The
secondary outcomes included unstable angina, congestive heart failure,
revascularization or amputation, death from any cause, complications of
diabetes, and cancer.
RESULTS: A total of 772 of the 4761 patients assigned to vitamin E (16.2
percent) and 739 of the 4780 assigned to placebo (15.5 percent) had a primary
outcome event (relative risk, 1.05; 95 percent confidence interval, 0.95 to
1.16; P=0.33). There were no significant differences in the numbers of deaths
from cardiovascular causes (342 of those assigned to vitamin E vs. 328 of those
assigned to placebo; relative risk, 1.05; 95 percent confidence interval, 0.90
to 1.22), myocardial infarction (532 vs. 524; relative risk, 1.02; 95 percent
confidence interval, 0.90 to 1.15), or stroke (209 vs. 180; relative risk, 1.17;
95 percent confidence interval, 0.95 to 1.42). There were also no significant
differences in the incidence of secondary cardiovascular outcomes or in death
from any cause. There were no significant adverse effects of vitamin E.
CONCLUSIONS: In patients at high risk for cardiovascular events, treatment with
vitamin E for a mean of 4.5 years had no apparent effect on cardiovascular
outcomes. A review is presented of studies on the effects of vitamin E on heart disease,
studies encompassing basic science, animal studies, epidemiological and
observational studies, and four intervention trials. The in vitro, cellular, and
animal studies, which are impressive both in quantity and quality, leave no
doubt that vitamin E, the most important fat-soluble antioxidant, protects
animals against a variety of types of oxidative stress. The hypothesis that
links vitamin E to the prevention of cardiovascular disease (CVD) postulates
that the oxidation of unsaturated lipids in the low-density lipoprotein (LDL)
particle initiates a complex sequence of events that leads to the development of
atherosclerotic plaque. This hypothesis is supported by numerous studies in
vitro, in animals, and in humans. There is some evidence that the ex vivo
oxidizability of a subject's LDL is predictive of future heart events. This
background in basic science and observational studies, coupled with the safety
of vitamin E, led to the initiation of clinical intervention trials. The three
trials that have been reported in detail are, on balance, supportive of the
proposal that supplemental vitamin E can reduce the risk for heart disease, and
the fourth trial, which has just been reported, showed small, but not
statistically significant, benefits. Subgroup analyses of cohorts from the older
three trials, as well as evidence from smaller trials, indicate that vitamin E
provides protection against a number of medical conditions, including some that
are indicative of atherosclerosis (such as intermittent claudication). Vitamin E
supplementation also produces an improvement in the immune system and protection
against diseases other than cardiovascular disease (such as prostate cancer).
Vitamin E at the supplemental levels being used in the current trials, 100 to
800 IU/d, is safe, and there is little likelihood that increased risk will be
found for those taking supplements. About one half of American cardiologists
take supplemental vitamin E, about the same number as take aspirin. In fact, one
study suggests that aspirin plus vitamin E is more effective than aspirin alone.
There are a substantial number of trials involving vitamin E that are in
progress. However, it is possible, or even likely, that each condition for which
vitamin E provides benefit will have a unique dose-effect curve. Furthermore,
different antioxidants appear to act synergistically, so supplementation with
vitamin E might be more effective if combined with other micronutrients. It will
be extremely difficult to do trials that adequately probe the dose-effect curve
for vitamin E for each condition that it might affect, or to do studies of all
the possible combinations of other micronutrients that might act with vitamin E
to improve its effectiveness. Therefore, the scientific community must recognize
that there never will be a time when the science is "complete." At some point,
the weight of the scientific evidence must be judged adequate; although some may
regard it as early to that judgement now, clearly we are very close. In view of
the very low risk of reasonable supplementation with vitamin E, and the
difficulty in obtaining more than about 30 IU/day from a balanced diet, some
supplementation appears prudent now. Hyperhomocysteinemia is an important cardiovascular risk factor. Serum
homocysteine levels are specially dependent on folate nutritional status. In
addition, the oxidative modification of low-density lipoproteins (LDLs) in the
endothelial microenvironment is a damaging factor that can be modified with
fat-soluble antioxidant vitamins. The present study was done to assess the
effect of a supplementation of folic acid and antioxidant vitamins on
homocysteine levels and in vitro LDL oxidation in patients with coronary artery
disease. Twenty-three patients with angiographically proven coronary artery
disease were given supplements for 15 d consisting of one capsule twice a day of
a multivitamin preparation containing 0.65 mg folic acid, 150 mg
alpha-tocopherol, 150 mg ascorbic acid, 12.5 mg beta-carotene, and 0.4 microgram
vitamin B12. Serum lipids, vitamin and homocysteine levels, and in vitro LDL
oxidation were measured before and after the supplementation period. During the
supplementation period, serum folate levels increased from 5.0 +/- 1.5 to 10.8
+/- 3.8 ng/mL (P < 0.001), vitamin B12 increased from 317.4 +/- 130.4 to 334.5
+/- 123.8 pg/mL (P < 0.05), and alpha-tocopherol increased from 8.2 +/- 5.1 to
13.7 +/- 7.9 mg/L (P < 0.001). Serum homocysteine levels decreased from 8.7 +/-
4.3 to 6.3 +/- 2.2 mumol/L (P < 0.001). In vitro LDL oxidation decreased from
2.6 +/- 1.1 to 1.6 +/- 1.1 nmol malondialdehyde/mg protein (P < 0.001). In
comparing patients with healthy controls, basal levels of folate were lower in
the patients, whereas vitamin B12, alpha-tocopherol, and homocysteine levels
were similar. No changes in serum lipid levels or body weight were observed. In
conclusion, a short-term supplementation with folic acid and antioxidant
vitamins can reduce serum homocysteine levels and in vitro LDL oxidation in
patients with coronary artery disease. The generation of reactive oxygen species (ROS) is associated with life in
aerobic conditions. ROS are thought to be implicated in the pathogenesis of
various human diseases since they are capable of damaging biological
macromolecules such as DNA, carbohydrates and proteins. The organism maintains
defense against ROS, including enzymes and low molecular-weight antioxidants. An
important source of antioxidants is diet which contains numerous compounds
exhibiting antioxidant activity. A shortage of antioxidants in the diet might
promote coronary heart disease through accumulation of oxidized LDL in
macrophages. However, antioxidants may also influence endothelial functions,
smooth muscle cell proliferation, thrombosis and plaque rupture. Consumption of
fruits and vegetables, olive oil, red wine and tea is inversely correlated with
heart disease rates. These foods are particularly rich in natural antioxidant
nutrients, including ascorbate (vitamin C), the tocopherols (vitamin E) and
carotenoids. More than 600 naturally occurring carotenoids have been identified.
These compounds are plant pigments that provide the bright color of various
fruits and vegetables; lycopene, which gives tomatoes their red color, is under
active research. Flavonoids are > 4,000 naturally occurring substances which
provide color, texture and taste for plant foods. As free radical scavengers,
flavonoids inhibit lipid peroxidation, promote vascular relaxation and help
prevent atherosclerosis. A sufficient supply with antioxidants from diet might
help prevent or delay the occurrence of pathological changes associated with
oxidative stress. When diet fails to meet the antioxidant requirement, dietary
supplements might be indicated. The recently coined term nutriceuticals
describes a variety of nonprescription products that are used to enhance health.
The best known are vitamin E, vitamin C, carotenoids, coenzyme Q10, flavonoids
and the amino acid L-arginine. Rigorous clinical trials, particularly among
high-risk groups, are needed before they can be recommended routinely to
patients. Lipid peroxidation is thought to be one of the major factors involved in
atherogenesis. There is an increasing evidence is increasing that oxidation of
LDL cholesterol may be instrumental in atherogenesis. Diabetics are known to be
at increased risk of cardiovascular diseases, a phenomenon which has previously
been linked to the lipid peroxidation process. As a result, a number of studies
have been undertaken to evaluate the effects of antioxidant vitamins on coronary
heart disease and risks factors of ischaemic heart disease such as diabetes
mellitus. Lipid peroxidation and antioxidant status were studied in 51 patients
with ischaemic heart disease and some of with having diabetes mellitus (18%).
Results were compared before and after supplementation of 450 mg of tocopherol
acetate for three months. SOD were found to be elevated in patients with
diabetes and in whole groups of patients after supplementation of tocopherol
acetate. Also, TAS was found to be elevated in a subgroup of patients without
diabetes and no significant changes were found in glutathion-peroxidase after
supplementation. We found statistically significantly decreased mean values of
glucose after supplementation in all groups of patients. The monitoring of
antioxidant parameters in diabetic patients could be of vital importance in the
study of the disease. Traditional risk factors for coronary artery disease (CAD) can only explain
approximately two thirds of the observed clinical events. This has maintained
interest in other nutritional and biochemical factors that might contribute to
the underlying pathophysiology of vascular disease. Two such factors are dietary
antioxidants and plasma homocysteine. Established risk factors such as
hypertension, smoking and diabetes mellitus are all associated with increased
oxidative stresses due to excess free radical activity in the vascular wall.
This may facilitate the development of vascular disease because of (i) increased
oxidation of low-density lipoprotein (LDL) particles which increases their
propensity to deposition in the vascular wall, (ii) inactivation of
endothelium-derived nitric oxide, and (iii) direct cytotoxicity to endothelial
cells. Protective antioxidant molecules include vitamin C and vitamin E of which
the latter is lipid soluble and is the primary antioxidant defence in
circulating LDL particles. Epidemiological studies have suggested strongly that
individuals who have high circulating concentrations or dietary intake of
natural antioxidant vitamins are protected against vascular disease events (18).
Furthermore, many studies have demonstrated a beneficial effect of natural and
synthetic antioxidants on surrogate markers of vascular disease such as
endothelial function and lipoprotein oxidation. However, large prospective
randomized controlled intervention trials, mostly involving vitamin E (e.g.
CHAOS, HOPE (22)), have failed to demonstrate any beneficial effect upon
vascular mortality in high risk individuals. Possible reasons for these
disappointing results include the pro-oxidant effects of high dose antioxidant
supplements, particularly in patients with established vascular disease.
Homocysteine is a sulphydryl-containing amino acid derived from the
demethylation of dietary methionine. Epidemiological studies over 30 years have
shown that increased concentrations of homocysteine are associated with vascular
disease. This link is independent of other risk factors, is consistent across
many studies and is strongly dose-related. Recently, evidence has accumulated to
suggest that this link is also biologically plausible because homocysteine
promotes oxidant injury to the vascular endothelium, impairs
endothelium-dependent vasomotor regulation and may also alter the coagulant
properties of the blood. Plasma homocysteine levels can be reduced by dietary
supplements of folic acid and B vitamins. Studies are currently being undertaken
to examine the impact of these vitamins in high risk patients and, thereby,
establish a causative role for homocysteine in promoting vascular events. Vitamin E consists of a number of compounds, tocopherols and tocotrienols, that
function as lipid-soluble antioxidants. A hypothesis is that vitamin E may slow
the progression of atherosclerosis by blocking the oxidative modification of
low-density lipoprotein cholesterol and thus decrease its uptake into the
arterial lumen. Basic science and animal studies have generally supported this
hypothesis. Observational studies have primarily assessed patients with no
established coronary heart disease (CHD), and results have generally supported a
protective role of vitamin E in CHD. Early primary and secondary prevention
clinical trials (Alpha-Tocopherol, Beta-Carotene Cancer Protection study and
Cambridge Heart Antioxidant Study) showed mixed results. Despite years of
encouraging evidence from basic science and observational studies, 3 large
randomized clinical trials (Gruppo Italiano per lo Studio della Sopravvivenza
nell'Infarto miocardico, Heart Outcomes Prevention Evaluation, and Primary
Prevention Project) with a combined total of more than 25,000 patients failed to
show a significant benefit with vitamin E taken as a dietary supplement for the
prevention of CHD. Four large randomized primary prevention trials currently
under way should add to our knowledge. The American Heart Association has
recommended consumption of a balanced diet with emphasis on antioxidant-rich
fruits and vegetables but has made no recommendations regarding vitamin E
supplementation for the general population. Although vitamin E supplementation
seems to be safe for most people, recommendations from health care professionals
should reflect the uncertainty of established benefit as demonstrated in
clinical trials. Epidemiologic studies have shown a correlation between antioxidant intake and
coronary artery disease (CAD); however, the results of clinical trials have been
inconsistent. We evaluated the effect of combined antioxidant supplementation on
endothelial function and its correlation with change in low-density lipoprotein
cholesterol (LDLC) oxidation in patients with established CAD. In a
double-blind, placebo-controlled 12-week trial, 18 nonsmoking, nondiabetic
patients (mean age 62.4 +/- 8.1 years) were randomized to receive placebo or
antioxidant supplementation consisting of (a) 400 IU of vitamin E, 500 mg of
vitamin C, and 12 mg of beta-carotene; or (b) 800 IU of vitamin E, 1000 mg of
vitamin C, and 24 mg of beta-carotene daily. Endothelial function was evaluated
on the basis of percent and absolute changes in brachial artery diameter in
response to reactive hyperemia induced by occlusion-release. Baseline and
12-week values of LDL oxidation (measured on the basis of lag phase),
endothelial function, dietary composition, serum antioxidants, and lipids were
measured. We noted a significant between-group difference at 12 weeks for change
in plasma concentrations of alpha-tocopherol, vitamin C, and beta-carotene
between the placebo and antioxidant groups (p <.05). Both placebo and treatment
groups demonstrated a significant improvement in lag phase; however, the
treatment group achieved a greater, although nonsignificant, magnitude of change
compared with the placebo group (181.3 +/- 177.8 minutes vs 80.6 +/- 63.0
minutes, P =.06). Within-group change in brachial reactivity from baseline to
follow-up in the treatment group did not reach statistical significance (1.7%
+/- 3.2% and 0.07 mm +/- 0.13 mm, P =.08 and P =.09, respectively), whereas an
improved change in brachial reactivity was observed in the placebo group (2.2%
+/- 1.9%, 0.09 mm +/- 0.06 mm, P <.05). No significant correlation was found
between change in lag phase and change in endothelial function. On adjustment
for confounders, antioxidant supplementation was found not to be a significant
predictor of brachial reactivity. We conclude that antioxidant supplementation
did not significantly alter brachial reactivity, despite significantly increased
plasma levels of antioxidants and improved lag phase. These data should be
confirmed in larger-scale trials and examined in studies evaluating individual
dietary antioxidant supplementation. CONTEXT: Although vitamin deficiency is encountered infrequently in developed
countries, inadequate intake of several vitamins is associated with chronic
disease.
OBJECTIVE: To review the clinically important vitamins with regard to their
biological effects, food sources, deficiency syndromes, potential for toxicity,
and relationship to chronic disease.
DATA SOURCES AND STUDY SELECTION: We searched MEDLINE for English-language
articles about vitamins in relation to chronic diseases and their references
published from 1966 through January 11, 2002.
DATA EXTRACTION: We reviewed articles jointly for the most clinically important
information, emphasizing randomized trials where available.
DATA SYNTHESIS: Our review of 9 vitamins showed that elderly people, vegans,
alcohol-dependent individuals, and patients with malabsorption are at higher
risk of inadequate intake or absorption of several vitamins. Excessive doses of
vitamin A during early pregcy and fat-soluble vitamins taken anytime may
result in adverse outcomes. Inadequate folate status is associated with neural
tube defect and some cancers. Folate and vitamins B(6) and B(12) are required
for homocysteine metabolism and are associated with coronary heart disease risk.
Vitamin E and lycopene may decrease the risk of prostate cancer. Vitamin D is
associated with decreased occurrence of fractures when taken with calcium.
CONCLUSIONS: Some groups of patients are at higher risk for vitamin deficiency
and suboptimal vitamin status. Many physicians may be unaware of common food
sources of vitamins or unsure which vitamins they should recommend for their
patients. Vitamin excess is possible with supplementation, particularly for
fat-soluble vitamins. Inadequate intake of several vitamins has been linked to
chronic diseases, including coronary heart disease, cancer, and osteoporosis BACKGROUND: Although basic research suggests that vitamins may have an important
role in the prevention of cardiovascular diseases (CVD), the data from cohort
studies and clinical trials are inconclusive.
METHODS: This prospective cohort study was conducted among 83 639 male
physicians residing in the United States who had no history of CVD or cancer. At
baseline, data on use of vitamin E, ascorbic acid (vitamin C), and multivitamin
supplements were provided by a self-administered questionnaire. Mortality from
CVD and coronary heart disease (CHD) was assessed by death certificate review.
RESULTS: Use of supplements was reported by 29% of the participants. During a
mean follow-up of 5.5 years, 1037 CVD deaths occurred, including 608 CHD deaths.
After adjustment for several cardiovascular risk factors, supplement use was not
significantly associated with total CVD or CHD mortality. For vitamin E use, the
relative risks (RRs) were 0.92 (95% confidence interval [CI], 0.70-1.21) for
total CVD mortality and 0.88 (95% CI, 0.61-1.27) for CHD mortality; for use of
vitamin C, the RRs were 0.88 (95% CI, 0.70-1.12) for total CVD mortality and
0.86 (95% CI, 0.63-1.18) for CHD mortality; and for use of multivitamin
supplements, the RRs were 1.07 (95% CI, 0.91-1.25) for total CVD mortality and
1.02 (95% CI, 0.83-1.25) for CHD mortality.
CONCLUSIONS: In this large cohort of apparently healthy US male physicians,
self-selected supplementation with vitamin E, vitamin C, or multivitamins was
not associated with a significant decrease in total CVD or CHD mortality. Data
from ongoing large randomized trials will be necessary to definitely establish
small potential benefits of vitamin supplements on subsequent cardiovascular
risk. There is clear evidence of lipoprotein oxidation in atherosclerotic lesions.
Animal studies and observational prospective human cohort studies have been
interpreted as supporting a role for antioxidants in the prevention of coronary
heart disease (CHD). However, firm recommendations to take antioxidant
supplements to treat or prevent CHD require evidence derived from randomised
controlled studies. In primary prevention studies, low dose alpha-tocopherol
does not reduce the incidence of coronary events (ATBC study), and beta-carotene
either has no effect or increases the incidence of coronary events and cancer
death (ATBC, CARET, Physician's Health studies). Secondary preventions, those
with smaller populations and shorter duration of follow up have shown some
benefit from alpha-tocopherol (CHAOS, SPACE), but larger randomised studies
indicate no benefit from treatment with alpha-tocopherol (HOPE, GISSI, PPP).
Recent studies with antioxidant combinations also show no benefit (HATS, MPS).
On the basis of these data, supplements of alpha-tocopherol and beta-carotene
cannot be recommended for the treatment or prevention of CHD. Fundamental and
applied research may yet find a role for antioxidant supplements in the
treatment of coronary disease. However, this will require positive results from
combined antioxidant studies currently in progress, and the targeting of
oxidative processes that operate in the artery wall and cause or contribute to
disease. Cardiovascular disease, in particular coronary artery disease (CAD), remains the
most important cause of morbidity and mortality in developed countries and, in
the near future, more so in the developing world. Atherosclerotic plaque
formation is the underlying basis for CAD. Growth of the plaque leads to
coronary stenosis, causing a progressive decrease in blood flow that results in
angina pectoris. Acute myocardial infarction and unstable angina were recently
recognised as related to plaque rupture, not progressive coronary stenosis.
Acute thrombus formation causes an abrupt coronary occlusion. The
characteristics of the fibrin cap, contents of the plaque, rheological factors
and active inflammation within the plaque contribute to plaque rupture.
Oxidative processes are important in plaque formation. Oxidized low density
lipoproteins (LDL) but not unoxidized LDL is engulfed by resident intimal
macrophages, transforming them into foam cells which develop into fatty streaks,
the precursors of the atherosclerotic plaque. Inflammation is important both in
plaque formation and rupture. Animal studies have shown that antioxidants reduce
plaque formation and lead to plaque stabilisation. In humans, high intakes of
antioxidants are associated with lower incidence of CAD, despite high serum
cholesterol levels. This observation suggests a role for inflammation in CAD and
that reducing inflammation using antioxidants may ameliorate these processes.
Men and women with high intakes of vitamin E were found to have less CAD.
Vitamin E supplementation was associated with a significant reduction in
myocardial infarction and cardiovascular events in the incidence of recurrent
myocardial infarction. In the hierarchy of evidence in evidence-based medicine,
data from large placebo-controlled clinical trials is considered necessary.
Results from various mega-trials have not shown benefits (nor adverse effects)
conferred by vitamin E supplementation, suggesting that vitamin E has no role in
the treatment of CAD. These results do not seem to confirm, at the clinical
level, the effect of antioxidants against active inflammation during plaque
rupture. However, a closer examination of these studies showed a number of
limitations, rendering them inconclusive in addressing the role of vitamin E in
CAD prevention and treatment. Further studies that specifically address the
issue of vitamin E in the pathogenesis of atherosclerosis and in the treatment
of CAD need be performed. These studies should use the more potent antioxidant
property of alpha-tocotrienol vitamin E. Oxidative stress appears to be of fundamental relevance to diseases as diverse
as atherosclerosis, cancer, and Alzheimer's disease. Observational data in
humans have suggested that antioxidant vitamin intake is associated with reduced
cardiovascular disease. Animal studies are largely consistent with the concept
that dietary supplementation with antioxidant vitamins reduces the progression
of atherosclerosis. However, recent prospective, controlled clinical trials of
vitamin E, including the Cardiovascular Disease, Hypertension and
Hyperlipidemia, Adult-Onset Diabetes, Obesity, and Stroke (CHAOS) study, the
Heart Outcomes Prevention Evaluation (HOPE) trial, Gruppo Italiano per lo Studio
della Sopravvivvenza nell'Infarto Miocardico (GISSI)-Prevenzione trial, the
Secondary Prevention with Antioxidants of Cardiovascular Disease in End Stage
Renal Disease (SPACE) trial, and the Heart Protection Study (HPS) present a
confused picture. The various possibilities that have been advanced to explain
this discrepancy are discussed in this review. A striking feature of these and
other trials of antioxidants is the absence of a biochemical basis for patient
inclusion or, indeed, dose selection. Patients with high levels of oxidant
stress or depletion of natural antioxidant defense systems may be the most
likely to benefit from antioxidant therapy. If this is the case, then reliable,
quantitative indices of in vivo oxidant stress such as urinary isoprostane
levels should be considered as an inclusion criterion for patient selection.
Future trials of antioxidant therapy in cardiovascular disease should then be
targeted toward such patients with high levels of oxidant stress or patients
with depletion of natural antioxidant defense systems. Furthermore, the dose of
antioxidant should be chosen based on a surrogate readout that is a reliable,
reproducible, and easily obtainable in vivo measure of oxidant stress. In the
interim, although the safety of vitamin E up to doses of 800 IU/day has been
determined, the conflicting nature of the results published to date encourages
us to avoid making premature recommendations with respect to vitamin E
supplementation in the prevention and treatment of cardiovascular disease. In recent years, vitamin E has been investigated as a cardioprotective agent.
Experimental studies have identified potential mechanisms by which vitamin E may
inhibit the development of atherosclerosis, and observational studies of
individuals without coronary disease suggest that vitamin E intake may prevent
future cardiovascular events. Secondary prevention trials to date have
demonstrated little benefit from vitamin E supplementation. It remains possible,
however, that supplementation may be useful among certain high-risk groups,
including those with nutritional deficiencies. Limited data from completed
primary prevention trials also indicate minimal cardioprotection from vitamin E,
but large-scale trials now in progress may yet show benefit. Results from
ongoing trials will contribute powerfully to the totality of evidence on which
to formulate both appropriate clinical recommendations for individual patients
and a rational public health policy for the population as a whole. At this time,
there is insufficient evidence for issuing a public health recommendation to use
vitamin E supplements to prevent cardiovascular disease (CVD). Rather, increased
intake of fruits, vegetables, and other antioxidant-rich foods should be
promoted as part of a healthy diet because they provide nutritional benefits
beyond any potential antioxidant effect. Moreover, even if found to reduce CVD
risk, vitamin supplement use should be considered an adjunct, not an
alternative, to established cardioprotective measures, such as smoking
abstention, avoidance of obesity, adequate physical activity, and control of
high blood pressure and hyperlipidemia. The current evidence does not support the indiscriminate use of vitamins A, C,
or E or beta carotene to prevent or reduce cardiovascular disease. Despite a
plausible theory that antioxidants can prevent diseases caused by oxidative
damage, trials thus far have not proven this. In fact, some studies found
antioxidants may be harmful in some people. We review important studies of the
effects of four antioxidants (vitamins A, C, and E, and beta carotene) and
analyze whether the current evidence supports or confirms or rejects the
presumed protective role. OBJECTIVE: The British Nutrition Foundation was recently commissioned by the
Food Standards Agency to conduct a review of the government's research programme
on Antioxidants in Food. Part of this work involved an independent review of the
scientific literature on the role of antioxidants in chronic disease prevention,
which is presented in this paper.
BACKGROUND: There is consistent evidence that diets rich in fruit and vegetables
and other plant foods are associated with moderately lower overall mortality
rates and lower death rates from cardiovascular disease and some types of
cancer. The 'antioxidant hypothesis' proposes that vitamin C, vitamin E,
carotenoids and other antioxidant nutrients afford protection against chronic
diseases by decreasing oxidative damage.
RESULTS: Although scientific rationale and observational studies have been
convincing, randomised primary and secondary intervention trials have failed to
show any consistent benefit from the use of antioxidant supplements on
cardiovascular disease or cancer risk, with some trials even suggesting possible
harm in certain subgroups. These trials have usually involved the administration
of single antioxidant nutrients given at relatively high doses. The results of
trials investigating the effect of a balanced combination of antioxidants at
levels achievable by diet are awaited.
CONCLUSION: The suggestion that antioxidant supplements can prevent chronic
diseases has not been proved or consistently supported by the findings of
published intervention trials. Further evidence regarding the efficacy, safety
and appropriate dosage of antioxidants in relation to chronic disease is needed.
The most prudent public health advice remains to increase the consumption of
plant foods, as such dietary patterns are associated with reduced risk of
chronic disease. BACKGROUND: Vitamin C acts as a potent antioxidant; however, it can also be a
prooxidant and glycate protein under certain circumstances in vitro. These
observations led us to hypothesize that a high intake of vitamin C in diabetic
persons might promote atherosclerosis.
OBJECTIVE: The objective was to examine the relation between vitamin C intake
and mortality from cardiovascular disease.
DESIGN: We studied the relation between vitamin C intake and mortality from
total cardiovascular disease (n = 281), coronary artery disease (n = 175), and
stroke (n = 57) in 1923 postmenopausal women who reported being diabetic at
baseline. Diet was assessed with a food-frequency questionnaire at baseline, and
subjects initially free of coronary artery disease were prospectively followed
for 15 y.
RESULTS: After adjustment for cardiovascular disease risk factors, type of
diabetes medication used, duration of diabetes, and intakes of folate, vitamin
E, and beta-carotene, the adjusted relative risks of total cardiovascular
disease mortality were 1.0, 0.97, 1.11, 1.47, and 1.84 (P for trend < 0.01)
across quintiles of total vitamin C intake from food and supplements. Adjusted
relative risks of coronary artery disease were 1.0, 0.81, 0.99, 1.26, and 1.91
(P for trend = 0.01) and of stroke were 1.0, 0.52, 1.23, 2.22, and 2.57 (P for
trend < 0.01). When dietary and supplemental vitamin C were analyzed separately,
only supplemental vitamin C showed a positive association with mortality
endpoints. Vitamin C intake was unrelated to mortality from cardiovascular
disease in the nondiabetic subjects at baseline.
CONCLUSION: A high vitamin C intake from supplements is associated with an
increased risk of cardiovascular disease mortality in postmenopausal women with
diabetes. Numerous studies have evaluated the association between antioxidants and
coronary atherosclerosis but have been limited by its study among individuals
with advanced atherosclerosis. The authors studied 865 consecutive patients,
39-45 years of age, without known coronary artery disease and presenting for a
periodic physical examination. Antioxidant intake was assessed with the Block
Dietary Questionnaire, and coronary atherosclerosis was identified by measuring
coronary artery calcification using electron beam computed tomography. The mean
age was 42 (+/-2), 83% were male, and the prevalence of coronary artery
calcification was 20%. Vitamin supplements were used by 56% of the participants,
and the mean (+/-SD) daily intake (dietary plus supplemental) of vitamins A, C,
and E were 1683 mg (+/-1245), 371 mg (+/-375), and 97 mg (+/-165), respectively.
There was no significant correlation between coronary artery calcification score
and individual vitamin or total antioxidant vitamin intake, even after adjusting
for traditional cardiac risk factors. The highest quartile of vitamin E was
positively associated with calcification (odds ratio=1.77; 95% confidence
interval, 1.02-3.06). Antioxidant vitamin intake is not significantly related to
coronary artery calcification, implying that there is no effect on the
development of early coronary atherosclerosis. High doses of vitamin E may
confer an increased risk of calcified atherosclerosis. BACKGROUND: Many epidemiological studies have reported that antioxidant vitamin
intake from diet or supplements are associated with a lower risk of coronary
heart disease (CHD), the findings are, however, inconsistent. We undertook a
meta-analysis of cohort studies to examine the relations between antioxidant
vitamins (vitamins C, E, and beta-carotene) and CHD risk.
METHODS AND RESULTS: We included all the relevant cohort studies if they
provided a relative risk and corresponding 95% confidence interval (CI) of CHD
in relation to antioxidant vitamins intake from diet or supplement. Fifteen
cohort studies were identified involving a total of 7415 incident CHD cases and
374,488 participants with a median follow-up of approximately 10, 8.5, and 15
years for vitamins C, E, and beta-carotene, respectively. Pooled estimates
across studies were obtained by random-effects model. The potential sources of
heterogeneity and publication bias were also estimated. For vitamins C, E, and
beta-carotene, a comparison of individuals in the top third with those in the
bottom third of baseline value yielded a combined relative risk of 0.84 (95% CI,
0.73-0.95), 0.76 (95% CI, 0.63-0.89), and 0.78 (95% CI, 0.53-1.04),
respectively. Subgroup analyses show that dietary intake of vitamins C and E and
supplement use of vitamin E have an inverse association with CHD risk, but
supplement use of vitamin C has no significant association with CHD risk. In the
dose-response meta-analysis, each 30 mg/day increase in vitamin C, 30 IU/day
increase in vitamin E, and 1 mg/day increase in beta-carotene yielded the
estimated overall relative risk for CHD of 1.01 (95% CI, 0.99-1.02), 0.96 (95%
CI, 0.94-0.99), and 1.00 (95% CI, 0.88-1.14), respectively.
CONCLUSIONS: Our findings in this meta-analysis suggest that an increase in
dietary intake of antioxidant vitamins has encouraging prospects for possible
CHD prevention. The evidence that specific vitamins may be beneficial in the prevention of
cardiovascular disease (CVD) is supported by mechanistic models of
atherogenesis. We and others have published observational epidemiologic studies
in support of vitamins in the primary prevention of CVD, but the results from
intervention studies are mixed. This article summarizes the recent results for
vitamin E, vitamin D, and the B vitamins, comparing study populations, study
designs, and potential methodologic reasons for differences in findings. For
vitamin E, observational data suggest benefit at doses of 100 to 400 IU/d.
Results from recent large-scale trials are mixed, with some showing modest
benefit but others suggesting no benefit, especially for secondary prevention.
Results for B vitamins are also mixed and further complicated by the recent
folate fortification of the flour supply. If greater B vitamin intake does
reduce CVD, the benefits are likely to be greatest for primary prevention and in
populations with intake below dietary reference standards. Research on vitamin D
and CVD is just beginning to emerge, but current data suggest that if there is
benefit it likely needs to be at intake levels much higher than the current
reference intakes of 200 to 600 IU/d for American adults. OBJECTIVE: To evaluate the effect of 3-month kale (Brassica oleracea acephala)
juice supplementation on coronary artery disease risk factors among
hypercholesterolemic men.
METHODS: Thirty-two men with hypercholesterolemia (> 200 mg/dL) were recruited
after annual health examinations among the faculty and staff at university. The
subjects consumed 150 mL of kale juice per day for a 12-week intervention
period. Dietary and anthropometric assessments were performed and blood samples
were collected to evaluate biochemical profiles before and after
supplementation.
RESULTS: Serum concentrations of HDL-cholesterol, and HDL- to LDL-cholesterol
ratio were significantly increased by 27% (P<0.0001) and 52% (P<0.0001),
respectively. The LDL-cholesterol concentration and the atherogenic index were
significantly reduced by 10% (P=0.0007) and 24.2% (P<0.0001), respectively
without affecting body mass index, waist and hip circumferences, or nutrient
intakes after three months of supplementation. While there was no difference in
the concentration of malondialdehyde, significant increase in glutathione
peroxidase activity (P=0.0005) were accompanied by a significant increase in the
serum selenium level (P=0.0132). It was also found that the responses of these
risk factors to kale juice administration were dependent on smoking status.
CONCLUSION: Regular meals supplementation with kale juice can favorably
influence serum lipid profiles and antioxidant systems, and hence contribute to
reduce the risks of coronary artery disease in male subjects with
hyperlipidemia. BACKGROUND: Selenium is a central determit of antioxidative glutathione
peroxidase 1 (GPx-1) expression and activity. The relevance of selenium
supplementation on GPx-1 in coronary artery disease (CAD) needs to be
established. We assessed the effect of selenium supplementation on GPx-1 in cell
culture and on endothelial function in a prospective clinical trial.
METHODS: Human coronary artery endothelial cells were incubated with 5.78 to 578
nmol/L sodium selenite, Se-methyl-selenocysteine hydrochloride, or
seleno-l-methionine. Glutathione peroxidase 1 mRNA and protein expression and
activity were measured. Coronary artery disease patients (n = 465) with impaired
endothelial function (flow-mediated dilation [FMD] <8%) were randomly assigned
to receive 200 or 500 microg sodium selenite daily or matching placebo during a
12-week period. We tested the effect on red blood cell GPx-1 activity and
brachial artery FMD. Furthermore, differences in biomarkers of oxidative stress
and inflammation were measured.
RESULTS: Sodium selenite and Se-methyl-selenocysteine hydrochloride increased
GPx-1 protein and activity in a dose-dependent manner (P < .0001). The
intention-to-treat groups comprised 433 CAD patients. Glutathione peroxidase 1
activity increased from 37.0 U/gHb (31.3-41.7) to 41.1 U/gHb (35.2-48.4) (P <
.0001) in the 200 microg and from 38.1 U/gHb (33.2-43.8) to 42.6 U/gHb
(35.0-49.1) (P < .0001) in the 500 microg sodium selenite group treated for
12-weeks. No relevant changes were observed for FMD or biomarkers of oxidative
stress and inflammation.
CONCLUSIONS: Sodium selenite supplementation increases GPx-1 activity in
endothelial cells and in CAD patients. Future studies have to demonstrate
whether long-term CAD outcome can be improved. The evidence for the cardioprotective nature of omega-3 fatty acids is abundant,
and currently available data indicate that patients with known coronary heart
disease should consume at least 1 g daily of long-chain omega-3 fatty acids from
either oily fish or fish-oil supplements, and that individuals without disease
should consume at least 250-500 mg daily. However, this area of research poses
two questions. Firstly, which is the best source of omega-3 fatty acids-fish or
fish-oil supplements? Secondly, are recommendations for omega-3 supplementation
warranted in view of the rapid depletion of world fish stocks? The argument that
eating fish is better than taking fish-oil supplements stems from the fact that
several important nutrients, such as vitamin D, selenium, and antioxidants, are
missing from the supplements. However, three major prevention trials have
clearly indicated that omega-3 fatty acid capsules confer cardiovascular
benefits and, therefore, that both are cardioprotective. Sustainable sources of
omega-3 fatty acids will need to be identified if long-term cardiovascular risk
reduction is to be achieved at the population level. AIM: To determine whether alpha-tocopherol or beta-carotene supplementation
affects diabetic macrovascular complications and total mortality.
METHODS: This study was carried out as part of the Alpha-Tocopherol,
Beta-Carotene Cancer Prevention Study, a double-blind, randomized trial with a
2x2 factorial design. A total of 29,133 middle-aged male smokers received either
vitamin E 50 mg/day or beta-carotene 20 mg/day, or both, or placebo for a median
of 6.1 years. At base-line, 1700 men had type 2 diabetes. Of these men, 662 were
diagnosed with first-ever macrovascular complication, and 1142 died during the
19-year follow-up.
RESULTS: Neither supplementation affected the risk of macrovascular complication
or total mortality during the intervention period. For the
alpha-tocopherol-supplemented versus no alpha-tocopherol-supplemented, and
beta-carotene-supplemented versus no beta-carotene-supplemented we found
relative risk (RR) 0.84 (95% confidence interval (CI) 0.65-1.10) and RR 1.15
(95% CI 0.89-1.50) for macrovascular complication, respectively, and RR 1.00
(95% CI 0.80-1.25) and RR 1.06 (95% CI 0.85-1.33) for total mortality,
respectively. No essential changes were found in these effects when the
follow-up was extended up to 19 years.
CONCLUSION: Alpha-tocopherol or beta-carotene supplementation has no protective
effect on macrovascular outcomes or total mortality of diabetic male smokers. Macronutrient and micronutrient deficiencies are very common in the general
population and may be even more common in patients with hypertension and
cardiovascular disease due to genetic, environmental causes and prescription
drug use. The Hypertension Institute in Nashville, TN, has evaluated
micronutrient deficiencies and oxidation status, in a group of hypertensive
versus normotensive patients. There are significant differences in numerous
intracellular micronutrients and oxidation status between these two groups.
Replacement of the micronutrient deficiencies, as well as high-dose therapy of
selected nutraceuticals in combination with optimal diet, exercise and weight
management resulted in control of blood pressure to goal levels in 62% of the
hypertensive population (as defined by JNC 7) over a period of 6 months with
complete tapering and discontinuation of antihypertensive drugs. These
deficiencies will have an enormous impact on present and future cardiovascular
health and outcomes such as hypertension, myocardial infarction, stroke and
renal disease and overall health costs. It is estimated that the annual savings
in drug costs alone for the treatment of hypertension could be as much as US$10
billion. Diagnosis and treatment of these nutrient deficiencies and improvement
in oxidation status using functional intracellular assessments will reduce blood
pressure, improve vascular health, endothelial dysfunction, vascular biology and
cardiovascular events. Vascular biology assumes a pivotal role in the initiation
and perpetuation of hypertension and target organ damage sequelae. Endothelial
activation, oxidative stress, inflammation and vascular smooth muscle
dysfunction are initial events that start hypertension. Nutrient-gene
interactions determine a broad array of phenotypic consequences such as vascular
problems and hypertension. Optimal nutrition, nutraceuticals, vitamins,
antioxidants, minerals, weight loss, exercise, smoking cessation and moderate
restriction of alcohol and caffeine in addition to other lifestyle modifications
can prevent and control hypertension in many patients. An integrative approach
combining these lifestyle suggestions with the correct pharmacologic treatment
will best achieve new goal blood pressure levels, reduce cardiovascular risk
factors, improve vascular biology and vascular health, reduce cardiovascular
target organ damage and reduce healthcare expenditure. The expanded scientific
roles for nutraceutical supplements are discussed in relation to the prevention
and treatment of essential hypertension and cardiovascular diseases with
emphasis on mechanisms of action and clinical integration with drug therapy with
hypertension guidelines. It is the purpose of this paper to review only the
hypertension clinical trials that have evaluated the clinical use and efficacy
of nutrition, weight loss, exercise and selected nutritional supplements,
vitamins, minerals and antioxidants. Numerous clinical trials have evaluated the
use of nutritional supplements such as beta carotene, selenium, vitamin C and
vitamin E in the prevention of coronary heart disease and stroke yielding
conflicting results (positive, neutral and negative). In many of these clinical
trials there are enormous clinical design problems, methodologic flaws, varied
patient population, variable dose and type of vitamin use, improper selection of
vitamin used and many other issues that make the studies difficult to interpret.
It is beyond the scope of this paper to review these trials. The reader is
referred to the vast literature on this subject. OBJECTIVE: To investigate whether dietary supplementation with B vitamins or
omega 3 fatty acids, or both, could prevent major cardiovascular events in
patients with a history of ischaemic heart disease or stroke.
DESIGN: Double blind, randomised, placebo controlled trial; factorial design.
SETTING: Recruitment throughout France via a network of 417 cardiologists,
neurologists, and other physicians.
PARTICIPANTS: 2501 patients with a history of myocardial infarction, unstable
angina, or ischaemic stroke.
INTERVENTION: Daily dietary supplement containing 5-methyltetrahydrofolate (560
μg), vitamin B-6 (3 mg), and vitamin B-12 (20 μg) or placebo; and containing
omega 3 fatty acids (600 mg of eicosapentanoic acid and docosahexaenoic acid at
a ratio of 2:1) or placebo. Median duration of supplementation was 4.7 years.
MAIN OUTCOME MEASURES: Major cardiovascular events, defined as a composite of
non-fatal myocardial infarction, stroke, or death from cardiovascular disease.
RESULTS: Allocation to B vitamins lowered plasma homocysteine concentrations by
19% compared with placebo, but had no significant effects on major vascular
events (75 v 82 patients, hazard ratio, 0.90 (95% confidence interval 0.66 to
1.23, P=0.50)). Allocation to omega 3 fatty acids increased plasma
concentrations of omega 3 fatty acids by 37% compared with placebo, but also had
no significant effect on major vascular events (81 v 76 patients, hazard ratio
1.08 (0.79 to 1.47, P=0.64)).
CONCLUSION: This study does not support the routine use of dietary supplements
containing B vitamins or omega 3 fatty acids for prevention of cardiovascular
disease in people with a history of ischaemic heart disease or ischaemic stroke,
at least when supplementation is introduced after the acute phase of the initial
event.
TRIAL REGISTRATION: Current Controlled Trials ISRCTN41926726. Oxidant stress plays an important role in the pathogenesis of atherosclerosis.
In the late 1980s, biological studies demonstrated that oxygen-free radicals
oxidize low-density lipoprotein-cholesterol, resulting in the creation of foam
cells and inciting the cascade of biological events that ultimately result in
the formation of atherosclerosis. In vitro studies showed the ability of
antioxidant vitamins to scavenge free radicals and block the oxidation of
low-density lipoprotein. This data was supported in vivo by early observational
studies suggesting the benefit of antioxidants, particularly vitamin E, in the
prevention of coronary artery disease. On the basis of these studies, the use of
antioxidant supplements by the general population increased substantially and
became a multibillion dollar industry. Despite strong biological evidence and
promising observational data, more rigorous scientific evaluation did not
support a causational relationship between vitamin supplements and lowering
coronary artery disease risk. Several prospective, double-blind,
placebo-controlled trials showed no benefit and possibly harmful effects.
Therapies such as angiotensin-converting enzyme inhibitors, angiotensin receptor
blockers, and statins, which are known to have benefit in preventing and
treating atherosclerosis by reducing blood pressure and cholesterol, also have a
"pleiotropic" effect in reducing the formation of reactive oxygen species (ROS).
Advances in molecular biology and the study of ROS led to a better understanding
of the mechanisms that govern their production and role in atherogenesis. This
progress identified unforeseen pathways by which these drugs favorably alter the
balance in ROS production, and have raised possibilities for future targeted
therapies in the prevention of atherosclerosis. OBJECTIVE: The purpose of this study was to investigate the effects of coenzyme
Q10 supplementation on inflammatory markers (high-sensitivity C-reactive protein
[hs-CRP], interleukin-6 [IL-6], and homocysteine) in patients with coronary
artery disease (CAD).
METHODS: Patients with CAD (n = 51) were randomly assigned to a placebo group (n
= 14) or one of two coenzyme Q10-supplemented groups (60 mg/d, Q10-60 group, n =
19; 150 mg/d, Q10-150 group, n = 18). The intervention was administered for 12
wk. Plasma coenzyme Q10 concentration, inflammatory markers (hs-CRP, IL-6, and
homocysteine), malondialdehyde, and superoxide dismutase activities were
measured.
RESULTS: Forty subjects with CAD completed the intervention study. The plasma
coenzyme Q10 concentration increased significantly in the Q10-60 and Q10-150
groups (P < 0.01). After 12 wk of intervention, the inflammatory marker IL-6 (P
= 0.03) was decreased significantly in the Q10-150 group. Subjects in the
Q10-150 group had significantly lower malondialdehyde levels and those in the
Q10-60 (P = 0.05) and Q10-150 (P = 0.06) groups had greater superoxide dismutase
activities. Plasma coenzyme Q10 was inversely correlated with hs-CRP (r = -0.20,
P = 0.07) and IL-6 (r = -0.25, P = 0.03) at baseline. After supplementation,
plasma coenzyme Q10 was significantly correlated with malondialdehyde (r =
-0.35, P < 0.01) and superoxide dismutase activities (r = 0.52, P < 0.01).
However, there was no correlation between coenzyme Q10 and homocysteine.
CONCLUSION: Coenzyme Q10 supplementation at a dosage of 150 mg appears to
decrease the inflammatory marker IL-6 in patients with CAD. BACKGROUND: Atrial fibrillation occurs after approximately 25% to 45% of
coronary artery bypass graft (CABG) surgeries. Oxidative stress and related
electrophysiological remodeling has been proposed as a potential cause of this
atrial fibrillation. Perioperative supplementation of the antioxidant ascorbic
acid has been evaluated as a preventive agent. The current investigation was
conducted to evaluate the efficacy of ascorbic acid in reducing atrial
fibrillation in CABG patients.
METHODS: A prospective, randomized, placebo-controlled, triple-blind,
single-institution study was conducted in nonemergency CABG patients. Subjects
were monitored for episodes of arrhythmia and other complications.
RESULTS: Eighty-nine treatment and 96 control subjects completed the study
protocol. Demographics, comorbidities, and preoperative drugs were similar
between groups. Surgical characteristics and postoperative medication use also
were similar. The incidence of atrial fibrillation was 30.3% in the treatment
group and 30.2% in the control group (P = .985). No difference was found in
postoperative complications or mortality.
CONCLUSIONS: Our data indicate that supplementation of ascorbic acid in addition
to routine postoperative care does not reduce atrial fibrillation after coronary
artery bypass grafting. OBJECTIVE: To assess the efficacy of vitamin and antioxidant supplements in the
prevention of cardiovascular diseases.
DESIGN: Meta-analysis of randomised controlled trials.
DATA SOURCES AND STUDY SELECTION: PubMed, EMBASE, the Cochrane Library, Scopus,
CINAHL, and ClinicalTrials.gov searched in June and November 2012. Two authors
independently reviewed and selected eligible randomised controlled trials, based
on predetermined selection criteria.
RESULTS: Out of 2240 articles retrieved from databases and relevant
bibliographies, 50 randomised controlled trials with 294,478 participants
(156,663 in intervention groups and 137,815 in control groups) were included in
the final analyses. In a fixed effect meta-analysis of the 50 trials,
supplementation with vitamins and antioxidants was not associated with
reductions in the risk of major cardiovascular events (relative risk 1.00, 95%
confidence interval 0.98 to 1.02; I(2)=42%). Overall, there was no beneficial
effect of these supplements in the subgroup meta-analyses by type of prevention,
type of vitamins and antioxidants, type of cardiovascular outcomes, study
design, methodological quality, duration of treatment, funding source, provider
of supplements, type of control, number of participants in each trial, and
supplements given singly or in combination with other supplements. Among the
subgroup meta-analyses by type of cardiovascular outcomes, vitamin and
antioxidant supplementation was associated with a marginally increased risk of
angina pectoris, while low dose vitamin B(6) supplementation was associated with
a slightly decreased risk of major cardiovascular events. Those beneficial or
harmful effects disappeared in subgroup meta-analysis of high quality randomised
controlled trials within each category. Also, even though supplementation with
vitamin B(6) was associated with a decreased risk of cardiovascular death in
high quality trials, and vitamin E supplementation with a decreased risk of
myocardial infarction, those beneficial effects were seen only in randomised
controlled trials in which the supplements were supplied by the pharmaceutical
industry.
CONCLUSION: There is no evidence to support the use of vitamin and antioxidant
supplements for prevention of cardiovascular diseases. BACKGROUND: Inverse associations between micronutrient intake and cardiovascular
outcomes have been previously shown, but did not focus on diabetic patients.
OBJECTIVE: To systematically review the role of micronutrients in the
development/presence of cardiovascular outcomes in patients with diabetes.
METHODS: We searched Medline, Embase, and Scopus (January/1949-March/2012) for
observational studies that evaluated micronutrients and cardiovascular outcomes
in patients with diabetes, and then selected and extracted the data (two
independent reviewers).
RESULTS: From the 15 658 studies identified, five were included, comprising
three case-control and two cohorts, with a follow-up of 7-15 years. A
meta-analysis was not performed due to the different antioxidant micronutrients
(types and measurement methods) and outcomes evaluated. The micronutrients
assessed were vitamin C intake in diet and/or supplementation, chromium and
selenium in toenail samples, and α-tocopherol and zinc in serum levels. Intake
of >300 mg of vitamin C through supplementation was associated with increased
risk of cardiovascular disease, coronary artery disease (CAD), and stroke (RR
1.69-2.37). High levels of α-tocopherol in serum were associated with 30% lower
CAD risk in another study (HR 0.71; 95%CI 0.53-0.94). Among minerals (zinc,
selenium, and chromium), an inverse association between zinc and CAD was
observed; levels lower than 14.1 µmol/L were associated with an increased risk
for CAD (RR 1.70; 95%CI 1.21-2.38).
CONCLUSION: The information available on this issue is scarce. Further
prospective studies are needed to elucidate the role of these nutrients in the
cardiovascular risk of patients with diabetes. |
Which are the main functions of the human HuR (ELAVL1) protein in fibroblasts? | HuR is an RNA-binding protein that can stabilize labile mRNAs containing AU-rich elements in their 3' untranslated regions and has been shown to shuttle between the nucleus and cytoplasm. HuR function was previously shown to be implicated in the maintenance of a "young cell" phenotype in models of replicative cellular senescence. Loss of HuR is linked to reduced expression of proliferative genes during replicative senescence. Importantly, overexpression of HuR in senescent cells restored a "younger" phenotype, while a reduction in HuR expression accentuated the senescent phenotype. HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. In mesenchymal cells HuR plays a dominant role in lung development and as a key post-transcriptional regulator of networks guiding tissue remodeling during branching morphogenesis. In fibroblasts knockdown of HuR decreased the endogenous expression of TGFβ1 under exogenous TGFβ1 treatment, simultaneously with the decrease of Col1a, Col3a and fibronectin expression. HuR (human antigen R), represses ARF mRNA translation, thereby maintaining the replicative life span of mouse embryonic fibroblasts (MEFs). HuR is considered a global regulator of cell-cycle progression and tumorigenesis. Through its post-transcriptional influence on specific target mRNAs, HuR can alter the cellular response to proliferative, stress, apoptotic, differentiation, senescence, inflammatory and immune stimuli. | An important paradigm for post-transcriptional regulation is the control of
cytoplasmic mRNA stability mediated by AU-rich elements (AREs) in the 3'
untranslated region of transcripts encoding oncoproteins, cytokines and
transcription factors. While many RNA-binding proteins have been shown to bind
to AREs in vitro, neither the functional consequences nor the physiological
significance of their interactions are known. Here we demonstrate a role for the
embryonic lethal abnormal visual (ELAV) RNA-binding protein HuR in mRNA turnover
in vivo. The ELAV family of RNA-binding proteins is highly conserved in
vertebrates. In humans, there are four members; HuR is expressed in all
proliferating cells, whereas Hel-N1, HuC and HuD are expressed in terminally
differentiated neurons. We show that elevation of cytoplasmic HuR levels
inhibits c-fos ARE-mediated RNA decay but has little effect on rapid decay
directed by c-jun ARE. It appears that HuR has little effect on deadenylation
but delays onset of decay of the RNA body and slows down its subsequent decay.
We also show that HuR can be induced to redistribute from the nucleus to the
cytoplasm and that this redistribution is associated with an altered function.
Modulation of the ARE-mediated decay pathway through controlling distribution of
the ELAV proteins between nucleus and cytoplasm may be a mechanism by which cell
growth and differentiation is regulated. ELAV proteins are implicated in regulating the stability and translation of
cytokine and growth regulatory mRNAs such as GM-CSF, IL-2, c-myc, c-fos and
GLUT1 by binding to their AU-rich 3'UTRs. The tissue-specific ELAV protein HuB
(aka. Hel-N1) is predomitly cytoplasmic and has been shown to stabilize GLUT1
and c-myc mRNAs and to increase their translation following ectopic expression
in 3T3-L1 cells. We report that the most widely expressed mouse ELAV protein,
mHuA, is predominately nuclear in cultured NIH-3T3 cells, but is localized in
the cytoplasm during early G1 of the cell cycle. Therefore, much like the
primarily cytoplasmic HuB, HuA becomes temporally localized in the cytoplasm
where it can potentially regulate the stability or translation of bound mRNAs.
Moreover, we report that stimulation of mouse spleen cells using either
mitogenic or sub-mitogenic levels of anti-CD3/CD28 resulted in a dramatic
increase in the level of HuA. Upregulation of HuA corresponds to previously
documented increases in cytokine expression which are due to increased mRNA
stability following T cell activation. Consistent with these findings, HuA was
down regulated in quiescent cells and upregulated in 3T3 cells following serum
stimulation. The increase of murine HuA during the cell cycle closely resembles
that of cyclin B1 which peaks in G2/M. Together with our earlier studies, these
data indicate that mammalian ELAV proteins function during cell growth and
differentiation due in part to their effects on posttranscriptional stability
and translation of multiple growth regulatory mRNAs. This supports the
hypothesis that ELAV proteins can function as transacting factors which affect a
default pathway of mRNA degradation involved in the expression of growth
regulatory proteins. Proteins are transported into and out of the cell nucleus via specific signals.
The two best-studied nuclear transport processes are mediated either by
classical nuclear localization signals or nuclear export signals. There also are
shuttling sequences that direct the bidirectional transport of RNA-binding
proteins. Two examples are the M9 sequence in heterogeneous nuclear
ribonucleoprotein A1 and the heterogeneous nuclear ribonucleoprotein K shuttling
domain (KNS) sequence in heterogeneous nuclear ribonucleoprotein K, both of
which appear to contribute importantly to the export of mRNA to the cytoplasm.
HuR is an RNA-binding protein that can stabilize labile mRNAs containing AU-rich
elements in their 3' untranslated regions and has been shown to shuttle between
the nucleus and cytoplasm (18, 19). We have identified in HuR a shuttling
sequence that also possess transcription-dependent nuclear localization signal
activity. We propose that HuR first may bind AU-rich element-containing mRNAs in
the nucleus and then escort them through the nuclear pore, providing protection
during and after export to the cytoplasmic compartment. 3'-untranslated regions of various mRNAs have been shown to contain sequence
motifs which control mRNA stability, translatability, and efficiency of
translation as well as intracellular localization. We aimed to identify protein
binding regions of the long and highly conserved 3'UTR of the mRNA coding for
neurofibromin, a well-known tumor suppressor protein, whose genetic deficiency
causes the autosomal domit disease neurofibromatosis type 1 (NF1). We
discovered five RNA fragments that were able to undergo specific binding to
proteins from cell lysates (NF1-PBRs, NF1-protein-binding regions). Additionally
we identified the Elav-like protein HuR binding to NF1-PBR1. HuR interacts with
AU-rich elements in the 3'UTR of many protooncogenes, cytokines, and
transcription factors, thereby regulating the expression of these mRNAs on the
posttranscriptional level. Transfection assays with a CAT reporter construct
revealed reduced expression of the reporter, suggesting that HuR may be involved
in the fine-tuning of the expression of the NF1 gene. Cellular aging is accompanied by alterations in gene expression patterns. Here,
using two models of replicative senescence, we describe the influence of the
RNA-binding protein HuR in regulating the expression of several genes whose
expression decreases during senescence. We demonstrate that HuR levels, HuR
binding to target mRNAs encoding proliferative genes, and the half-lives of such
mRNAs are lower in senescent cells. Importantly, overexpression of HuR in
senescent cells restored a "younger" phenotype, while a reduction in HuR
expression accentuated the senescent phenotype. Our studies highlight a critical
role for HuR during the process of replicative senescence. Human RNA-binding protein HuR, a nucleocytoplasmic shuttling protein, is a
ubiquitously expressed member of the family of Hu proteins, which consist of two
N-terminal RNA recognition motifs (RRM1 and RRM2), a hinge region, and a
C-terminal RRM (RRM3). Although in vitro experiments showed indiscriminate
binding of Hu proteins synthesized in bacterial systems to many different
AU-rich elements (AREs), in vivo studies have pointed to a cytoplasmic role for
HuR protein in antagonizing the rapid decay of some specific ARE-containing
mRNAs, depending on physiological situations. By ectopically overexpressing HuR
and its mutant derivatives in NIH 3T3 cells to mimic HuR upregulation of
specific ARE-containing mRNAs in other systems, we have examined the in vivo
ARE-binding specificity of HuR and dissected its functionally critical domains.
We show that in NIH 3T3 cells, HuR stabilizes reporter messages containing only
the c-fos ARE and not other AREs. Two distinct binding sites were identified
within the c-fos ARE, the 5' AUUUA-containing domain and the 3'
U-stretch-containing domain. These actions of HuR are markedly different from
those of another ARE-binding protein, hnRNP D (also termed AUF1), which in vivo
recognizes AUUUA repeats found in cytokine AREs and can exert both stabilizing
and destabilizing effects. Further experiments showed that any combination of
two of the three RRM domains of HuR is sufficient for strong binding to the
c-fos ARE in vitro and to exert an RNA stabilization effect in vivo comparable
to that of intact HuR and that the hinge region containing nucleocytoplasmic
shuttling signals is dispensable for the stabilization effect of HuR. Our data
suggest that the ARE-binding specificity of HuR in vivo is modulated to interact
only with and thus regulate specific AREs in a cell type- and physiological
state-dependent manner. The expression of the major protein kinase C substrate MARCKS (myristoylated
alanine-rich C kinase substrate) is controlled by the stability of its mRNA.
While the MARCKS mRNA is long living in quiescent fibroblasts (t1/2 = 14 h), its
half-life time is drastically reduced (t1/2 = 2 h) in cells treated with phorbol
esters to activate protein kinase C (PKC) or treated with growth factors. In a
first step to study the underlying mechanism we identified both a cis-element on
the MARCKS mRNA and the corresponding trans-acting factors. Fusing the complete
3'-UTR or specific regions of the 3'-UTR of the MARCKS gene to a luciferase
reporter gene caused a drastic decrease in luciferase expression to as low as
5-10% of controls. This down-regulation was a result of destabilization of the
chimeric transcript as shown by RNA run-off and Northern blot-assays. By
RNase/EMSA and UV-cross-linking experiments, we identified a stretch of 52
nucleotides [(CUUU)11(U)8] in the 3'-UTR of the MARCKS mRNA specifically
recognized by two RNA-binding proteins, HuD and HuR. These trans-acting factors
are members of the ELAV gene family and bind the MARCKS CU-rich sequence with
high affinity. Overexpression of HuD and HuR in murine fibroblasts caused a
striking stabilization of the endogenous MARCKS mRNA even under conditions when
the MARCKS mRNA is normally actively degraded, i.e. after treating cells with
phorbol ester. These data imply, that the identified CU-rich cis-element of the
MARCKS 3'-UTR is involved in conferring instability to mRNAs and that members of
the ELAV gene family oppose this effect. Based on its structural and functional
properties, the (CUUU)11(U)8 sequence described here can be grouped into class
III of AU-rich elements. Cytoplasmic export of the RNA-binding protein HuR, a process that critically
regulates its function, was recently shown to be inhibited by the AMP-activated
protein kinase (AMPK). In the present investigation, treatment of human
fibroblasts with AMPK activators such as 5-amino-imidazole-4-carboxamide
riboside, antimycin A, and sodium azide inhibited cell growth and lowered the
expression of proliferative genes. As anticipated, AMPK activation also
decreased both the cytoplasmic HuR levels and the association of HuR with target
radiolabeled transcripts encoding such proliferative genes. HuR function was
previously shown to be implicated in the maintece of a "young cell" phenotype
in models of replicative cellular senescence. We therefore postulated that AMPK
activation in human fibroblasts might contribute to the implementation of the
senescence phenotype through mechanisms that included a reduction in HuR
cytoplasmic presence. Indeed, AMP:ATP ratios were 2-3-fold higher in senescent
fibroblasts compared with young fibroblasts. Accordingly, in vitro senescence
was accompanied by a marked elevation in AMPK activity. Evidence that increased
AMPK activity directly contributed to the implementation of the senescent
phenotype was obtained through two experimental approaches. First, use of AMPK
activators triggered senescence characteristics in fibroblasts, such as the
acquisition of senescence-associated beta-galactosidase (beta-gal) activity and
increased p16INK4a expression. Second, infection of cells with an adenoviral
vector that expresses active AMPK increased senescence-associated beta-gal
activity, whereas infection with an adenovirus that expresses domit-negative
AMPK decreased senescence-associated beta-gal activity. Together, our results
indicate that AMPK activation can cause premature fibroblast senescence through
mechanisms that likely involve reduced HuR function. RhoB is a small GTP-binding protein that is involved in apoptotic signal
transduction. We have cloned the mouse RhoB mRNA including a 1377 nucleotide
3'-untranslated region (UTR) that contains six AU-rich elements (AREs) as well
as several uridine-rich stretches. There is 94% homology overall between the
mouse and rat RhoB genes and 92% homology between the mouse and a putative human
clone. Ultraviolet light (UVL) induces RhoB production through regulated changes
in gene transcription and mRNA stabilization although the latter mechanism is
unknown. We observed that UVL increased the half-life of RhoB mRNA from 63 min
to 3.3 h in NIH/3T3 cells and from 87 min to 2.7 h in normal human keratinocyte
cells. In vitro mobility shift assays demonstrated that HuR bound the 3'-UTR of
RhoB at three distinct locations (nucleotides 1342-1696, 1765-1920 and
1897-1977) suggesting a regulatory role for this RNA-binding protein. HuR
immunoprecipitations were positive for RhoB mRNA indicating an in vivo
association, and Western blot analysis and immunofluorescence demonstrated that
HuR rapidly partitions from the nucleus to the cytoplasm after UVL. Therefore,
we propose a model in which UVL induces stress-activated signal transduction
leading to nuclear/cytoplasmic shuttling of HuR and subsequent stabilization of
RhoB mRNA. We report the antiapoptotic effect of RNA-binding protein HuR, a critical
regulator of the post-transcriptional fate of target transcripts. Among the most
prominent mRNAs complexing with HuR is that encoding prothymosin alpha
(ProTalpha), an inhibitor of the apoptosome. In HeLa cells, treatment with the
apoptotic stimulus ultraviolet light (UVC) triggered the mobilization of
ProTalpha mRNA to the cytoplasm and onto heavier polysomes, where its
association with HuR increased dramatically. Analysis of a chimeric ProTalpha
mRNA directly implicated HuR in regulating ProTalpha production: ProTalpha
translation and cytoplasmic concentration increased in HuR-overexpressing cells
and declined in cells in which HuR levels were lowered by RNA interference.
Importantly, the antiapoptotic influence engendered by HuR was vitally dependent
on ProTalpha expression, since use of oligomers that blocked ProTalpha
translation abrogated the protective effect of HuR. Together, our data support a
regulatory scheme whereby HuR binds the ProTalpha mRNA, elevates its cytoplasmic
abundance and translation, and thereby elicits an antiapoptotic program. HuR is a ligand for nuclear mRNAs containing adenylate-uridylate-rich elements
in the 3'-untranslated region. Once bound to the mRNA, HuR is recognized by
adapter proteins that then facilitate nuclear export of the complex. In the
cytosol, HuR is thought to function to control stability and translation of its
ligand message. In the 3T3-L1 cells HuR is constitutively expressed and
localized predomitly to the nucleus in the preadipocytes. However, within 30
min of exposure to the differentiation stimulus the HuR content in the cytosol
increases, consistent with HuR regulating the availability of relevant mRNAs for
translation. Using in vitro RNA gel shifts, we have demonstrated that the CCAAT
enhancer-binding protein beta (C/EBPbeta) message is a ligand for HuR. Within 2
h of initiation of the differentiation process, HuR complexes containing
C/EBPbeta mRNA could be isolated from the cytosolic compartment. Importantly,
the process appears to be highly selective, as cyclin D1, which contains a
putative HuR binding site and is expressed on the same time frame as C/EBPbeta,
was not found in the immunoprecipitated messenger ribonucleoprotein complexes.
The proximity of this event to adipogenic stimuli and the importance of
C/EBPbeta to the differentiation process have led us to hypothesize a role for
HuR in the regulation of the onset of adipogenesis. In support of this
hypothesis, small interfering RNA suppression of HuR protein content resulted in
an inhibition of C/EBPbeta protein expression and an attenuation of the
differentiation process. The RNA binding protein HuR regulates the stability of many target mRNAs. Here,
we report that HuR associated with the 3' untranslated region of the mRNA
encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1
mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress
triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting
SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell
cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and
was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of
these residues revealed a complex pattern of HuR binding, with S100 appearing to
be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings
demonstrate that HuR regulates SIRT1 expression, underscore functional links
between the two stress-response proteins, and implicate Chk2 in these processes. In this study, we investigated the molecular mechanisms underlying the ATP
analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling
of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using
synthetic protein kinase C (PKC) inhibitors and small interfering RNA
approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the
ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of
PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content
detected in hMC stably overexpressing PKC alpha compared with mock-transfected
cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a
direct interaction of PKC alpha with nuclear HuR and accompanied by increased
Ser phosphorylation as demonstrated by coimmunoprecipitation experiments.
Mapping of putative PKC target sites identified serines 158 and 221 as being
indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA
electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP
is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA.
Physiologically, the ATP-dependent increase in RNA binding is linked with an
augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin
E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation
emphasizes the importance of posttranslational modification for
stimulus-dependent HuR shuttling. We previously observed that nitric oxide (NO) exposure increases the stability
of mRNAs encoding heme oxygenase 1 (HO-1) and TIEG-1 in human and mouse
fibroblasts. Here, we have used microarrays to look broadly for changes in mRNA
stability in response to NO treatment. Using human IMR-90 and mouse NIH 3T3
fibroblasts treated with actinomycin D to block de novo transcription,
microarray analysis suggested that the stability of the majority of mRNAs was
unaffected. Among the mRNAs that were stabilized by NO treatment, seven
transcripts were found in both IMR-90 and NIH 3T3 cells (CHIC2, GADD45B, HO-1,
PTGS2, RGS2, TIEG, and ID3) and were chosen for further analysis. All seven
mRNAs showed at least one hit of a signature motif for the stabilizing
RNA-binding protein (RBP) HuR; accordingly, ribonucleoprotein
immunoprecipitation analysis revealed that all seven mRNAs associated with HuR.
In keeping with a functional role of HuR in the response to NO, a measurable
fraction of HuR increased in the cytoplasm following NO treatment. However,
among the seven transcripts, only HO-1 mRNA showed a robust increase in the
level of its association with HuR following NO treatment. In turn, HO-1 mRNA and
protein levels were significantly reduced when HuR levels were silenced in
IMR-90 cells, and they were elevated when HuR was overexpressed. In sum, our
results indicate that NO stabilizes mRNA subsets in fibroblasts, identify HuR as
an RBP implicated in the NO response, reveal that HuR alone is insufficient for
stabilizing several mRNAs by NO, and show that HO-1 induction by NO is regulated
by HuR. In the nucleus HuR binds to mRNAs containing adenylate-uridylate rich elements
in the 3'-untranslated region. HuR may influence expression of its ligand mRNA
through regulation of polyadenylation, translocation of the message to the
cytosol, stabilization of the mRNA and/or altering its translational efficiency.
Suppression of HuR using siRNA resulted in an attenuation of the 3T3-L1
differentiation program, consistent with HuR control of the expression of mRNA
ligand(s) critical to the differentiation process. In the present study, we
begin to identify mRNA ligands of HuR whose regulated expression is necessary
for adipogenesis. The RNA-binding protein, HuR, associates with the HuR mRNA, but the consequences
of this interaction are unknown. Here, we use human diploid fibroblasts (HDFs)
and cervical carcinoma cells to study this regulatory paradigm. Ectopic
overexpression of HuR potently enhanced the translation and cytoplasmic levels
of endogenous HuR, but did not affect HuR mRNA levels. Inhibition of CRM1
function by Lemptomycin B or by knockdown of CRM1 greatly diminished the
cytoplasmic levels of endogenous HuR mRNA and hence blocked the induction of
endogenous HuR by exogenous HuR. Further studies showed that HuR interacted with
the 3'-untranslated region (UTR) of HuR and that overexpression of HuR increased
the cytoplasmic levels of a chimeric luciferase-HuR 3'-UTR reporter transcript,
as well as luciferase activity; conversely, HuR knockdown reduced both
parameters. Moreover, the loss of HuR in senescent, late-passage HDFs was
accompanied by a reduced cytoplasmic presence of endogenous HuR mRNA, ectopic
Luc-HuR-3'UTR reporter transcript, and luciferase activity relative to what was
observed in young, early-passage cells. Our results reveal a positive feedback
mechanism for the regulation of HuR, which may play an important role in the
regulation of HuR during replicative senescence. OBJECTIVE: Hydrogen peroxide (H(2)O(2)) is an important mediator in the
vasculature, but its role in the regulation of soluble guanylate cyclase (sGC)
activity and expression is not completely understood. The aim of this study was
to test the effect of H(2)O(2) on sGC expression and function and to explore the
molecular mechanism involved.
METHODS AND RESULTS: H(2)O(2) increased sGCβ1 protein steady-state levels in rat
aorta and aortic smooth muscle cells (RASMCs) in a time- and dose-dependent
manner, and this effect was blocked by catalase. sGCα2 expression increased
along with β1 subunit, whereas α1 subunit remained unchanged. Vascular
relaxation to an NO donor (sodium nitroprusside) was enhanced by H(2)O(2), and
it was prevented by ODQ (sGC inhibitor). cGMP production in both freshly
isolated vessels and RASMCs exposed to H(2)O(2) was greatly increased after
sodium nitroprusside treatment. The H(2)O(2)-dependent sGCβ1 upregulation was
attributable to sGCβ1 mRNA stabilization, conditioned by the translocation of
the mRNA-binding protein HuR from the nucleus to the cytosol, and the increased
mRNA binding of HuR to the sGCβ1 3' untranslated region. HuR silencing reversed
the effects of H(2)O(2) on sGCβ1 levels and cGMP synthesis.
CONCLUSIONS: Our results identify H(2)O(2) as an endogenous mediator
contributing to the regulation of vascular tone and point to a key role of HuR
in sGCβ1 mRNA stabilization. Lung development is controlled by regulatory networks governing
mesenchymal-epithelial interactions. Transcription factors and signaling
molecules are known to participate in this process, yet little is known about
the post-transcriptional regulation of these networks. Here we demonstrate that
the RNA-binding protein (RBP) HuR is an essential regulator of mesenchymal
responses during lung branching. Its epiblast-induced deletion blocked the
morphogenesis of distal bronchial branches at the initiation of the
pseudoglandular stage. The phenotype originated from defective mesenchymal
responses since the conditional restriction of HuR deletion in epithelial
progenitors did not affect distal branching or the completion of lung
maturation. The loss of HuR resulted in the reduction of the key inducer of bud
outgrowth and endodermal branching, FGF10 and one of its putative
transcriptional regulators, Tbx4. Furthermore, exogenous FGF10 could rescue the
branching defect of affected lung buds. HuR was found to bind and control the
Fgf10 and Tbx4 mRNAs; as a result its deletion abolished their inducible
post-transcriptional regulation by the mesenchymal regulator FGF9. Our data
reveals HuR as the first RBP identified to play a domit role in lung
development and as a key post-transcriptional regulator of networks guiding
tissue remodeling during branching morphogenesis. The RNA-binding protein HuR, while known to stabilize cytoplasmic mRNAs, is
largely nuclear. In this issue of Molecular Cell, Mukherjee et al. (2011) and
Lebedeva et al. (2011) identify transcriptome-wide HuR-RNA interactions using
PAR-CLIP, unveiling HuR's nuclear role in pre-mRNA processing. The cytoplasmic events that control mammalian gene expression, primarily mRNA
stability and translation, potently influence the cellular response to internal
and external signals. The ubiquitous RNA-binding protein (RBP) HuR is one of the
best-studied regulators of cytoplasmic mRNA fate. Through its
post-transcriptional influence on specific target mRNAs, HuR can alter the
cellular response to proliferative, stress, apoptotic, differentiation,
senescence, inflammatory and immune stimuli. In light of its central role in
important cellular functions, HuR's role in diseases in which these responses
are aberrant is increasingly appreciated. Here, we review the mechanisms that
control HuR function, its influence on target mRNAs, and how impairment in
HuR-governed gene expression programs impact upon different disease processes.
We focus on HuR's well-recognized implication in cancer and chronic
inflammation, and discuss emerging studies linking HuR to cardiovascular,
neurological, and muscular pathologies. We also discuss the progress, potential,
and challenges of targeting HuR therapeutically. MicroRNAs are important regulators of gene expression in normal development and
disease. miR-9 is overexpressed in several cancer forms, including brain
tumours, hepatocellular carcinomas, breast cancer and Hodgkin lymphoma (HL).
Here we demonstrated a relevance for miR-9 in HL pathogenesis and identified two
new targets Dicer1 and HuR. HL is characterized by a massive infiltration of
immune cells and fibroblasts in the tumour, whereas maligt cells represent
only 1% of the tumour mass. These infiltrates provide important survival and
growth signals to the tumour cells, and several lines of evidence indicate that
they are essential for the persistence of HL. We show that inhibition of miR-9
leads to derepression of DICER and HuR, which in turn results in a decrease in
cytokine production by HL cells followed by an impaired ability to attract
normal inflammatory cells. Finally, inhibition of miR-9 by a systemically
delivered antimiR-9 in a xenograft model of HL increases the protein levels of
HuR and DICER1 and results in decreased tumour outgrowth, confirming that miR-9
actively participates in HL pathogenesis and points to miR-9 as a potential
therapeutic target. Persistent fibroblast activation in wound repair is believed to be the key
reason for fibrosis and transforming growth factor (TGF)β is considered as one
of the key mediators for the fibrogenic response, with the detailed mechanism
largely unknown. Here we found that TGFβ1 treatment could induce a significant
increase of endogenous TGFβ1 expression by enhancing the mRNA stability in
cardiac fibroblasts. Further study revealed that TGFβ1 treatment translocated
the nuclear HuR into cytoplasm, which in turn bound the ARE in the 3'UTR of
TGFβ1 and increased the mRNA stability as seen from the RNA-IP and reporter
assay. Knockdown of HuR decreased the endogenous expression of TGFβ1 under
exogenous TGFβ1 treatment, simultaneously with the decrease of Col1a, Col3a and
fibronectin expression. Our study here established a TGFβ1/HuR feedback circuit
regulating the fibrogenic response in fibroblasts, and targeting this feedback
loop is of great potential to control fibrosis. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HuR are highly expressed
in epithelial tissues and modulate the stability and translation of target
mRNAs. Here we present evidence that CUGBP1 and HuR jointly regulate the
translation of occludin and play a crucial role in the maintece of tight
junction (TJ) integrity in the intestinal epithelial cell monolayer. CUGBP1 and
HuR competed for association with the same occludin 3'-untranslated region
element and regulated occludin translation competitively and in opposite
directions. CUGBP1 overexpression decreased HuR binding to occludin mRNA,
repressed occludin translation, and compromised the TJ barrier function, whereas
HuR overexpression inhibited CUGBP1 association with occludin mRNA and promoted
occludin translation, thereby enhancing the barrier integrity. Repression of
occludin translation by CUGBP1 was due to the colocalization of CUGBP1 and
tagged occludin RNA in processing bodies (P-bodies), and this colocalization was
prevented by HuR overexpression. These findings indicate that CUGBP1 represses
occludin translation by increasing occludin mRNA recruitment to P-bodies,
whereas HuR promotes occludin translation by blocking occludin mRNA
translocation to P-bodies via the displacement of CUGBP1. HuR, also known as Elavl1, is an RNA-binding protein that regulates embryonic
development, progenitor cell survival, and cell stress responses. The role of
HuR in angiogenesis is not known. Using a myeloid-specific HuR knock-out mouse
model (Elavl1Mø KO), we show that HuR expression in bone marrow-derived
macrophages (BMDMs) is needed to maintain the expression of genes enriched in
AU-rich elements and U-rich elements in the 3'-UTR. In addition, BMDMs from
Elavl1Mø KO mice also showed alterations in expression of several miRNAs.
Interestingly, computational analysis suggested that miR-200b, which is
up-regulated in Elavl1Mø KO BMDMs, interacts with myeloid mRNAs very close to
the HuR binding sites, suggesting competitive regulation of gene expression. One
such mRNA encodes vascular endothelial growth factor (VEGF)-A, a major regulator
of angiogenesis. Immunoprecipitation of RNA-protein complexes and luciferase
reporter assays indicate that HuR antagonizes the suppressive activity of
miR-200b, down-regulates miR-200b expression, and promotes VEGF-A expression.
Indeed, Vegf-a and other angiogenic regulatory transcripts were down-regulated
in Elavl1Mø KO BMDMs. Interestingly, tumor growth, angiogenesis, vascular
sprouting, branching, and permeability were significantly attenuated in Elavl1Mø
KO mice, suggesting that HuR-regulated myeloid-derived factors modulate tumor
angiogenesis in trans. Zebrafish embryos injected with an elavl1 morpholino
oligomer or miR-200b mimic showed angiogenesis defects in the subintestinal vein
plexus, and elavl1 mRNA rescued the repressive effect of miR-200b. In addition,
miR-200b and HuR morpholino oligomer suppressed the activity of a zVEGF 3'-UTR
luciferase reporter construct. Together, these studies reveal an evolutionarily
conserved post-transcriptional mechanism involving competitive interactions
between HuR and miR-200b that controls angiogenesis. The activities of RNA-binding proteins are perturbed in several pathological
conditions, including cancer. These proteins include tristetraprolin (TTP,
ZFP36) and HuR (ELAVL1), which respectively promote the decay or stability of
adenylate-uridylate-rich (AU-rich) mRNAs. Here, we demonstrated that increased
stabilization and subsequent over-expression of HuR mRNA were coupled to TTP
deficiency. These findings were observed in breast cancer cell lines with an
invasive phenotype and were further confirmed in ZFP36-knockout mouse
fibroblasts. We show that TTP-HuR imbalance correlated with increased expression
of AU-rich element (ARE) mRNAs that code for cancer invasion genes. The microRNA
miR-29a was abundant in invasive breast cancer cells when compared to
non-tumourigenic cell types. When normal breast cells were treated with miR-29a,
HuR mRNA and protein expression were up-regulated. MiR-29a recognized a seed
target in the TTP 3' UTR and a cell-permeable miR-29a inhibitor increased TTP
activity towards HuR 3' UTR. This led to HuR mRNA destabilization and
restoration of the aberrant TTP-HuR axis. Subsequently, the cancer invasion
factors uPA, MMP-1 and MMP-13, and cell invasiveness, were decreased. The
TTP:HuR mRNA ratios were also perturbed in samples from invasive breast cancer
patients when compared with normal tissues, and were associated with invasion
gene expression. This study demonstrates that an aberrant ARE-mediated pathway
in invasive cancer can be normalized by targeting the aberrant and functionally
coupled TTP-HuR axis, indicating a potential therapeutic approach. p19(ARF) plays an essential role in the senescence of mouse cells, and its
expression is lost by methylation or deletion of the ARF locus; otherwise, p53
is inactivated to bypass senescence. ARF expression is tightly regulated, but
little is known about its posttranscriptional regulation. Here, we show that an
RNA-binding protein, HuR (human antigen R), represses ARF mRNA translation,
thereby maintaining the replicative life span of mouse embryonic fibroblasts
(MEFs). Loss of HuR results in premature senescence, with concomitant increases
in p19(ARF) but not p16(Ink4a) levels, and this senescence is not observed in
ARF-null MEFs that retain an intact Ink4a locus. HuR depletion does not alter
ARF transcription or stability but enhances ribosome association with ARF mRNA.
Under these conditions, ARF mRNA accumulates in nucleoli, where it associates
with nucleolin. Furthermore, adipose-specific deletion of the HuR gene results
in increased p19(ARF) expression in aged animals, which is accompanied by
decreased insulin sensitivity. Together, our findings demonstrate that p19(ARF)
is also regulated at the translational level, and this translational regulation
restrains the cellular life span and tissue functions in vivo. Post-transcriptional regulatory networks are dependent on the interplay of many
RNA-binding proteins having a major role in mRNA processing events in mammals.
We have been interested in the concerted action of the two RNA-binding proteins
hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and
having a major nuclear localization. Specifically, we present here the
application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a
population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from
the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic
fibroblast (MEF) cells. The outcome of this analysis was a list of target genes
regulated via HuR for their association (either increased or reduced) with the
nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a
selected number of nuclear mRNA transcripts, as well as to identify pre-spliced
transcripts (in addition to their mature mRNA counterpart) within the isolated
nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to
GO categories relevant to biological processes anticipated for hnRNP A1 and HuR
(such as transport, transcription, translation, apoptosis and cell cycle)
indicating their concerted function in mRNA metabolism. |
Which kinases does baricitinib inhibit? | Baricitinib is an inhibitor of Janus kinase family of enzymes (JAKs) with selectivity for JAK1 and JAK2. | PURPOSE OF REVIEW: To provide an update on the development of small molecular
compounds as novel therapeutics for the treatment of rheumatoid arthritis. The
development of such orally available agents has long been hoped for in
rheumatology; in the past year, it has become clear that the expectations are
becoming fulfilled.
RECENT FINDINGS: Over the past year, a large number of clinical trials have been
published or presented reporting positive therapeutic results with tyrosine
kinase inhibitors, a large class of orally available drugs that are also being
developed in other medical fields. This class of drugs includes the Janus kinase
(JAK) inhibitors, and data on tofacitinib published during the past year have
attested to the biologic-like efficacy of this drug and supported its subsequent
U.S. Food and Drugs Administration (FDA) approval. Positive clinical trial
results have also been reported for several other JAK inhibitors including
baricitinib. Several other JAK inhibitors and other small molecular entities are
also being developed in studies ranging from preclinical models to large
clinical trials.
SUMMARY: Tyrosine kinase inhibition has emerged as a major new direction in
rheumatoid arthritis therapy. INTRODUCTION: The JAK kinases are a family of four tyrosine receptor kinases
that play a pivotal role in cytokine receptor signalling pathways via their
interaction with signal transducers and activators of transcription proteins.
Selective inhibitors of JAK kinases are viewed as of considerable potential as
disease-modifying anti-inflammatory drugs for the treatment of rheumatoid
arthritis.
AREAS COVERED: This article provides a review of the clinical development and
available clinical results for those JAK inhibitors currently under
investigation. Phase II data for four JAK inhibitors (baricitinib, decernotinib,
filgotinib and INCB-039110) are contrasted with that reported for the recently
approved JAK inhibitor tofacitinib. The preclinical data on these, in addition
to peficitinib, ABT-494, INCB-047986 and AC-410 are also discussed, as are some
of the inhibitors in preclinical development.
EXPERT OPINION: JAK inhibitors are effective in the treatment of rheumatoid
arthritis as evidenced by several inhibitors enabling the majority of treated
patients to achieve ACR20 responses, with baricitinib and INCB-039110 both
effective when administered once daily. JAK inhibitors differ in isoform
specificity profiles, with good efficacy achievable by selective inhibition of
either JAK1 (filgotinib or INCB-039110) or JAK3 (decernotinib). It remains to be
seen what selectivity provides the optimal side-effect profile and to what
extent inhibition of JAK2 should be avoided. Baricitinib (also known as LY3009104 or INCB028050), a novel and potent small
molecule inhibitor of Janus kinase family of enzymes (JAKs) with selectivity for
JAK1 and JAK2, is currently in clinical development for the treatment of
rheumatoid arthritis (RA) and other inflammatory disorders. Two double-blind,
randomized, and placebo-controlled studies were conducted to evaluate single
ascending doses of 1-20 mg and multiple ascending doses of 2-20 mg QD and 5 mg
BID for 10 or 28 days in healthy volunteers. Following oral administration,
baricitinib plasma concentration typically attains its peak value within
1.5 hours postdose and subsequently declines in a bi-exponential fashion.
Baricitinib demonstrates dose-linear and time-invariant pharmacokinetics, with
low oral-dose clearance (17 L/h) and minimal systemic accumulation observed
following repeat dosing. The mean renal clearance of baricitinib was determined
to be ∼2 L/h. The effect of a high-fat meal on baricitinib pharmacokinetics was
insignificant. The pharmacodynamics of baricitinib, evaluated by the inhibition
of STAT3 phosphorylation following cytokine stimulation in the whole blood ex
vivo, was well correlated with baricitinib plasma concentrations. Baricitinib
was generally safe and well tolerated, with no serious treatment-related adverse
events (AEs) reported from either of the studies. An expected rapidly
reversible, dose-related decline in absolute neutrophil count was seen with
baricitinib. OBJECTIVES: To investigate baricitinib (LY3009104, formerly INCB028050), a
novel, oral inhibitor of JAK1/JAK2 in patients with moderate to severe
rheumatoid arthritis (RA) despite treatment with methotrexate.
METHODS: In this phase IIb study, 301 patients were randomised 2:1:1:1:1 to
receive once daily doses of placebo or 1, 2, 4 or 8 mg baricitinib for 12 weeks.
Patients assigned to 2, 4 and 8 mg baricitinib continued blinded treatment for
an additional 12 weeks. Patients assigned to placebo or 1 mg baricitinib were
reassigned to 2 mg twice daily or 4 mg once daily baricitinib between weeks
12-24. The primary endpoint was the proportion of patients in the combined 4 and
8 mg groups achieving an American College of Rheumatology 20% (ACR20) response
versus placebo at week 12.
RESULTS: Significantly more patients in the combined baricitinib 4 and 8 mg
groups compared with placebo achieved an ACR20 response at week 12 (76% vs 41%,
p<0.001). At week 12, significant differences versus placebo were also observed
in patients achieving ACR50, ACR70 and remission as measured by Disease Activity
Score for 28-joint counts, Clinical Disease Activity Index and Simplified
Disease Activity Index. Patients receiving 2, 4, or 8 mg baricitinib maintained
or improved in all measures through 24 weeks. Similar proportions of patients
experienced at least one adverse event in the placebo and baricitinib groups.
Serious infections developed in three patients receiving baricitinib. No cases
of tuberculosis, herpes zoster, opportunistic infections or deaths were
reported. Dose-dependent decreases in haemoglobin were observed with
baricitinib.
CONCLUSIONS: Baricitinib improved the signs and symptoms of RA in methotrexate
inadequate responders with active disease. Baricitinib was well tolerated with
no unexpected safety findings through week 24.
TRIAL REGISTRATION NUMBER: NCT01185353. BACKGROUND: Alopecia areata (AA) is an autoimmune disease resulting in hair loss
with devastating psychosocial consequences. Despite its high prevalence, there
are no FDA-approved treatments for AA. Prior studies have identified a prominent
interferon signature in AA, which signals through JAK molecules.
METHODS: A patient with AA was enrolled in a clinical trial to examine the
efficacy of baricitinib, a JAK1/2 inhibitor, to treat concomitant CANDLE
syndrome. In vivo, preclinical studies were conducted using the C3H/HeJ AA mouse
model to assess the mechanism of clinical improvement by baricitinib.
FINDINGS: The patient exhibited a striking improvement of his AA on baricitinib
over several months. In vivo studies using the C3H/HeJ mouse model demonstrated
a strong correlation between resolution of the interferon signature and clinical
improvement during baricitinib treatment.
INTERPRETATION: Baricitinib may be an effective treatment for AA and warrants
further investigation in clinical trials. |
Is there evidence to suggest that triiodothyronine has neuroprotective properties in traumatic brain injury? | Yes, it has been demonstrated that triiodothyronine exerts neuroprotective properties in traumatic brain injury setting. | Rats with parasagittal incised wounds of the telencephalon were treated for 1,7,
21 or 56 days with triiodothyronine (T3), 0.5 mug/100 g body weight, injected
once daily. Controls were injected with the aqueous solution in which the T3 was
dissolved. The brains were examined histologically and quantitative assessments
were made of the regeneration of axons into and across the lesions and of the
healing of the wounds. T3, when administered over an 8 week period, stimulated
axonal regeneration in the dorsal cortex and corpus callosum and promoted
healing of the wound in the corpus callosum. The results of this investigation
suggest that the use of T3 in the clinical treatment of injury to the central
nervous system may be of less value than the work of earlier authors had
indicated. The neuroendocrine response (NER) is an essential component of the adaptive
process to trauma, brain injury, and major surgery. While receiving additive
humoral and neural afferent inputs, the brain nuclei responsible for the NER act
mainly by efferent pathways to the hypothalamic-pituitary-adrenal (HPA) axis and
the sympathoadrenal system, the activations of which induce subsequent
circulatory and metabolic responses. The NER to brain injury is similar to the
response observed in patients with extracerebral injury, even if the response
after brain injury is extremely variable. Generally, there is a biphasic
pattern, with a sympathoadrenal storm associated with variable and altered
stimulation of the HPA during the ebb phase. The first phase is followed by a
decrease in both responses while other endocrine changes develop, involving
mainly the counter-regulatory, gonadal, and thyroid hormones. The outcome after
brain injury is closely correlated with the intensity of these changes,
particularly with catecholamine plasma levels and the severity of the low
triiodothyronine syndrome. Alterations of the thyroid hormones are largely
related to a reduction in peripheral deiodination of thyroxin. Recent research
shows that increased free-radical production and decreased selenium (an
antioxidant) serum levels play an important role in thyroid metabolism. Two
major issues remain unsolved: a) the precise definition of cerebral death, since
endocrine brain function is not abolished in the state currently defined as
brain death; and b) the question of whether substitutive hormone therapy should
be applied in severe brain injury. Thyroid hormones are essential for the development and function of the brain and
also for the maturation and repair of the peripheral nervous system. In the
brain, most of the 3,5,3'-triiodothyronine is locally produced by
5'-deiodination of thyroxine catalyzed by the type 2 deiodinase. The absence of
any information about thyroid hormone metabolism in the peripheral nervous
system prompted us to study the expression of type 2 deiodinase (mRNA and
activity) in the peripheral nervous system. Expression of type 2 deiodinase mRNA
was very low in the sciatic nerve of rats until day 5 after birth, then
increased from day 10 to 35-45 and gradually decreased afterwards, down to the
low basal levels observed in the adult. A lesion of the sciatic nerve in the
adult induced an increase in type 2 deiodinase mRNA and activity. After a
cryolesion, the stimulation was observed as early as 4 h and mRNA levels
increased until 24-48 h, then gradually declined down to basal levels around 28
days, when regeneration and functional recovery were completed. After a
permanent transection, up-regulation of type 2 deiodinase persisted in both
proximal and distal segments until the end of the experiment (28 days).
Transection and cryolesion were also followed by increased type 2 deiodinase
mRNA expression in the ipsilateral L4/L6 dorsal root ganglia within 24 h. Both
mRNA and activity were found in the peripheral nerve sheaths but not in the
internal compartment of the intact or injured nerve. Cultured fibroblasts from
the sciatic nerve expressed type 2 deiodinase 4 h after stimulation by 10 microM
forskolin, whereas purified Schwann cells did not. The present study provides
evidence that the peripheral nervous system has its own system responsible for
the local production of 3,5,3'-triiodothyronine, which may play a key role
during the regeneration process. OBJECT: The aim of the study was to evaluate the early changes in pituitary
hormone levels after severe traumatic brain injury (sTBI) and compare hormone
levels to basic neuro-intensive care data, a systematic scoring of the
CT-findings and to evaluate whether hormone changes are related to outcome.
METHODS: Prospective study, including consecutive patients, 15-70 years, with
sTBI, Glasgow Coma Scale (GCS) score ≤ 8, initial cerebral perfusion pressure >
10 mm Hg, and arrival to our level one trauma university hospital within 24
hours after head trauma (n = 48). Serum samples were collected in the morning
(08-10 am) day 1 and day 4 after sTBI for analysis of cortisol, growth hormone
(GH), prolactin, insulin-like growth factor 1 (IGF-1), thyroid-stimulating
hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4), follicular
stimulating hormone (FSH), luteinizing hormone (LH), testosterone and sex
hormone-binding globulin (SHBG) (men). Serum for cortisol and GH was also
obtained in the evening (17-19 pm) at day 1 and day 4. The first CT of the brain
was classified according to Marshall. Independent staff evaluated outcome at 3
months using GOS-E.
RESULTS: Profound changes were found for most pituitary-dependent hormones in
the acute phase after sTBI, i.e. low levels of thyroid hormones, strong
suppression of the pituitary-gonadal axis and increased levels of prolactin. The
main findings of this study were: 1) A large proportion (54% day 1 and 70% day
4) of the patients showed morning s-cortisol levels below the proposed cut-off
levels for critical illness related corticosteroid insufficiency (CIRCI), i.e.
<276 nmol/L (=10 ug/dL), 2) Low s-cortisol was not associated with higher
mortality or worse outcome at 3 months, 3) There was a significant association
between early (day 1) and strong suppression of the pituitary-gonadal axis and
improved survival and favorable functional outcome 3 months after sTBI, 4)
Significantly lower levels of fT3 and TSH at day 4 in patients with a poor
outcome at 3 months. 5) A higher Marshall CT score was associated with higher
day 1 LH/FSH- and lower day 4 TSH levels 6) In general no significant
correlation between GCS, ICP or CPP and hormone levels were detected. Only
ICPmax and LH day 1 in men was significantly correlated.
CONCLUSION: Profound dynamic changes in hormone levels are found in the acute
phase of sTBI. This is consistent with previous findings in different groups of
critically ill patients, most of which are likely to be attributed to
physiological adaptation to acute illness. Low cortisol levels were a common
finding, and not associated with unfavorable outcome. A retained ability to a
dynamic hormonal response, i.e. fast and strong suppression of the
pituitary-gonadal axis (day 1) and ability to restore activity in the
pituitary-thyroid axis (day 4) was associated with less severe injury according
to CT-findings and favorable outcome. |
What is the role played by mTOR in hypertrophic response and heart failure? | When subjected to pressure overload, mTOR-ablated mice demonstrated an impaired hypertrophic response and accelerated heart failure progression. Thus, mTOR complex 1 signaling plays an important role in myocardial response to stress, to regulate cardiomyocyte viability and heart failure. | Mechanistic target of rapamycin (MTOR) plays a critical role in the regulation
of cell growth and in the response to energy state changes. Drugs inhibiting
MTOR are increasingly used in antineoplastic therapies. Myocardial MTOR activity
changes during hypertrophy and heart failure (HF). However, whether MTOR exerts
a positive or a negative effect on myocardial function remains to be fully
elucidated. Here, we show that ablation of Mtor in the adult mouse myocardium
results in a fatal, dilated cardiomyopathy that is characterized by apoptosis,
autophagy, altered mitochondrial structure, and accumulation of eukaryotic
translation initiation factor 4E-binding protein 1 (4E-BP1). 4E-BP1 is an
MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits
translation initiation. When subjected to pressure overload, Mtor-ablated mice
demonstrated an impaired hypertrophic response and accelerated HF progression.
When the gene encoding 4E-BP1 was ablated together with Mtor, marked
improvements were observed in apoptosis, heart function, and survival. Our
results demonstrate a role for the MTORC1 signaling network in the myocardial
response to stress. In particular, they highlight the role of 4E-BP1 in
regulating cardiomyocyte viability and in HF. Because the effects of reduced
MTOR activity were mediated through increased 4E-BP1 inhibitory activity,
blunting this mechanism may represent a novel therapeutic strategy for improving
cardiac function in clinical HF. |
Is Lysine-specific demethylase 1 (LSD1) a critical regulator of hematopoiesis? | Yes. Lysine-specific demethylase 1 restricts hematopoietic progenitor proliferation and is essential for terminal differentiation of erythroid, granulomonocytic and megakaryocytic progenitors. | Gfi-1 and Gfi-1b are homologous transcriptional repressors involved in diverse
developmental contexts, including hematopoiesis and oncogenesis. Transcriptional
repression by Gfi proteins requires the conserved SNAG domain. To elucidate the
function of Gfi proteins, we purified Gfi-1b complexes and identified
interacting proteins. Prominent among these is the corepressor CoREST, the
histone demethylase LSD1, and HDACs 1 and 2. CoREST and LSD1 associate with
Gfi-1/1b via the SNAG repression domain. Gfi-1b further recruits these cofactors
to the majority of target gene promoters in vivo. Inhibition of CoREST and LSD1
perturbs differentiation of erythroid, megakaryocytic, and granulocytic cells as
well as primary erythroid progenitors. LSD1 depletion derepresses Gfi targets in
lineage-specific patterns, accompanied by enhanced histone 3 lysine 4
methylation at the respective promoters. Overall, we show that chromatin
regulatory proteins CoREST and LSD1 mediate transcriptional repression by Gfi
proteins. Lineage-restricted deployment of these cofactors through interaction
with Gfi proteins controls hematopoietic differentiation. TAL1/SCL (hereafter referred to as TAL1) is a critical transcription factor
required for hematopoiesis in which hematopoietic stem cells commit and
differentiate to different lineages. During this process, transcription of many
genes is turned on and off in part by epigenetic mechanisms. TAL1 has recently
been shown to differentially recruit LSD1 and other histone modifying complexes
to regulate its target genes. Here, we focus primarily on epigenetic mechanisms
that are regulated by TAL1 during normal and maligt hematopoiesis. We discuss
how different histone modifying enzymes are recruited by TAL1 and how these
enzymatic activities mediate the activating or repressive function of TAL1.
Finally, we further explore the possible mechanisms by which dysregulation of
the recruitment and activity of histone modifying enzymes contribute to
leukemogenesis. TAL1/SCL is a hematopoietic-specific oncogene and its activity is regulated by
associated transcriptional co-activators and corepressors. Dysregulation of TAL1
activity has been associated with T-cell leukemogenesis. However, it remains
unclear how the interactions between TAL1 and corepressors versus co-activators
are properly regulated. Here, we reported that protein kinase A (PKA)-mediated
phosphorylation regulates TAL1 interaction with the lysine-specific demethylase
(LSD1) that removes methyl group from methylated Lys 4 on histone H3 tails.
Phosphorylation of serine 172 in TAL1 specifically destabilizes the TAL1-LSD1
interaction leading to promoter H3K4 hypermethylation and activation of target
genes that have been suppressed in normal and maligt hematopoiesis. Knockdown
of TAL1 or LSD1 led to a derepression of the TAL1 target genes in T-cell acute
lymphoblast leukemia (T-ALL) Jurkat cells, which is accompanied by elevating
promoter H3K4 methylation. Similarly, treatment of PKA activator forskolin
resulted in derepression of target genes by reducing its interaction with LSD1
while PKA inhibitor H89 represses them by suppressing H3K4 methylation levels.
Consistent with the dual roles of TAL1 in transcription, TAL1-associated LSD1 is
decreased while recruitment of hSET1 is increased at the TAL1 targets during
erythroid differentiation. This process is accompanied by a dramatic increase in
H3K4 methylation. Thus, our data revealed a novel interplay between PKA
phosphorylation and TAL1-mediated epigenetic regulation that regulates
hematopoietic transcription and differentiation programs during hematopoiesis
and leukemogenesis. Lysine (K)-specific demethylase 1A (LSD1/KDM1A) has been identified as a
potential therapeutic target in solid cancers and more recently in acute myeloid
leukemia. However, the potential side effects of a LSD1-inhibitory therapy
remain elusive. Here, we show, with a newly established conditional in vivo
knockdown model, that LSD1 represents a central regulator of hematopoietic stem
and progenitor cells. LSD1 knockdown (LSD1-kd) expanded progenitor numbers by
enhancing their proliferative behavior. LSD1-kd led to an extensive expansion of
granulomonocytic, erythroid and megakaryocytic progenitors. In contrast,
terminal granulopoiesis, erythropoiesis and platelet production were severely
inhibited. The only exception was monopoiesis, which was promoted by LSD1
deficiency. Importantly, we showed that peripheral blood granulocytopenia,
monocytosis, anemia and thrombocytopenia were reversible after LSD1-kd
termination. Extramedullary splenic hematopoiesis contributed to the phenotypic
reversion, and progenitor populations remained expanded. LSD1-kd was associated
with the upregulation of key hematopoietic genes, including Gfi1b, Hoxa9 and
Meis1, which are known regulators of the HSC/progenitor compartment. We also
demonstrated that LSD1-kd abrogated Gfi1b-negative autoregulation by crossing
LSD1-kd with Gfi1b:GFP mice. Taken together, our findings distinguish LSD1 as a
critical regulator of hematopoiesis and point to severe, but reversible, side
effects of a LSD1-targeted therapy. The recent identification of germline and somatic mutations in BAP1 as well as
in multiple members of the ASXL (additional sex combs-like) family of genes has
highlighted the role of these proteins in a diverse array of biological
functions. A diverse number of possible functions have previously been ascribed
to ASXL1 in non-hematopoietic contexts, including physical co-operativity with
HP1a and LSD1. Here we discuss new evidence for a BAP1-independent function of
ASXL1 in regulating histone H3 lysine 27 methylation through interactions with
the Polycomb-repressive complex 2 (PRC2). BAP1, a nuclear-localized
deubiquitinase, has been shown to interact with a number of proteins, including
ASXL1 and/or ASXL2, but the functional importance of this interaction has
remained elusive. Here, we highlight recent work revealing the critical function
of BAP1 in restricting myelopoiesis and in regulating hematopoietic stem cell
function. These data provide evidence that BAP1 and ASXL1 function as a novel
class of tumor suppressors in myeloid maligcies. BAP1 functions through
effects on stability of host cell factor-1, and O-GlcNAcylation, and ASXL1
impacts histone post-translational modifications through interaction with PRC2.
Future studies investigating the mechanism of transformation by loss of BAP1 and
ASXL1 may result in new therapeutic approaches to treat hematological
maligcies. Here, we describe that lysine-specific demethylase 1 (Lsd1/KDM1a), which
demethylates histone H3 on Lys4 or Lys9 (H3K4/K9), is an indispensible
epigenetic governor of hematopoietic differentiation. Integrative genomic
analysis, combining global occupancy of Lsd1, genome-wide analysis of its
substrates H3K4 monomethylation and dimethylation, and gene expression
profiling, reveals that Lsd1 represses hematopoietic stem and progenitor cell
(HSPC) gene expression programs during hematopoietic differentiation. We found
that Lsd1 acts at transcription start sites, as well as enhancer regions. Loss
of Lsd1 was associated with increased H3K4me1 and H3K4me2 methylation on HSPC
genes and gene derepression. Failure to fully silence HSPC genes compromised
differentiation of hematopoietic stem cells as well as mature blood cell
lineages. Collectively, our data indicate that Lsd1-mediated concurrent
repression of enhancer and promoter activity of stem and progenitor cell genes
is a pivotal epigenetic mechanism required for proper hematopoietic maturation.
DOI:http://dx.doi.org/10.7554/eLife.00633.001. The stem cell protein SALL4 plays a critical role in hematopoiesis by regulating
the cell fate. In primitive hematopoietic precursors, it activates or represses
important genes via recruitment of various epigenetic factors such as DNA
methyltransferases, and histone deacylases. Here, we demonstrate that LSD1, a
histone lysine demethylase, also participates in the trans-repressive effects of
SALL4. Based on luciferase assays, the amine oxidase domain of LSD1 is important
in suppressing SALL4-mediated reporter transcription. In freshly isolated adult
mouse bone marrows, both SALL4 and LSD1 proteins are preferentially expressed in
undifferentiated progenitor cells and co-localize in the nuclei. Further
sequential chromatin immunoprecipitation assay confirmed that these two factors
share the same binding sites at the promoter regions of important hematopoietic
regulatory genes including EBF1, GATA1, and TNF. In addition, studies from both
gain- and loss-of-function models revealed that SALL4 dynamically controls the
binding levels of LSD1, which is accompanied by a reversely changed histone 3
dimethylated lysine 4 at the same promoter regions. Finally, shRNA-mediated
knockdown of LSD1 in hematopoietic precursor cells resulted in altered SALL4
downstream gene expression and increased cellular activity. Thus, our data
revealed that histone demethylase LSD1 may negatively regulate SALL4-mediated
transcription, and the dynamic regulation of SALL4-associated epigenetic factors
cooperatively modulates early hematopoietic precursor proliferation. |
Is K-63 linked protein ubiquitination related to proteasomal degradation? | Ubiquitination is best known for its role in targeting proteins for degradation by the proteasome, but evidence of the nonproteolytic functions of ubiquitin is also rapidly accumulating. One example of the regulatory, rather than proteolytic, function of ubiquitin is provided by study of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins, which function as ubiquitin ligases to synthesize lysine 63 (K(63))-linked polyubiquitin chains to mediate protein kinase activation through a proteasome-independent mechanism. Some TRAF proteins, such as TRAF2 and TRAF3, have recently been shown to have a positive role in the canonical pathway that activates nuclear factor kappaB (NF-kappaB) through IkappaB kinase beta (IKKbeta), but a negative role in the noncanonical pathway that activates NF-kappaB through IKKalpha. These opposing roles of TRAF proteins may be linked to their ability to synthesize distinct forms of polyubiquitin chains. Indeed, the TRAF2-interacting protein RIP can mediate IKK activation when it is modified by K(63) polyubiquitin chains, but is targeted to degradation by the proteasome when it is K(48)-polyubiquitinted by the NF-kappaB inhibitor A20. Thus, ubiquitin chains are dynamic switches that can influence signaling outputs in dramatically different ways.In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains function in nonproteasomal biological processes. | RING (really interesting new gene) and U-box E3 ligases bridge E2
ubiquitin-conjugating enzymes and substrates to enable the transfer of ubiquitin
to a lysine residue on the substrate or to one of the seven lysine residues of
ubiquitin for polyubiquitin chain elongation. Different polyubiquitin chains
have different functions. Lys(48)-linked chains target proteins for proteasomal
degradation, and Lys(63)-linked chains function in signal transduction,
endocytosis and DNA repair. For this reason, chain topology must be tightly
controlled. Using the U-box E3 ligase CHIP [C-terminus of the Hsc (heat-shock
cognate) 70-interacting protein] and the RING E3 ligase TRAF6
(tumour-necrosis-factor-receptor-associated factor 6) with the E2s Ubc13
(ubiquitin-conjugating enzyme 13)-Uev1a (ubiquitin E2 variant 1a) and UbcH5a, in
the present study we demonstrate that Ubc13-Uev1a supports the formation of free
Lys(63)-linked polyubiquitin chains not attached to CHIP or TRAF6, whereas
UbcH5a catalyses the formation of polyubiquitin chains linked to CHIP and TRAF6
that lack specificity for any lysine residue of ubiquitin. Therefore the
abilities of these E2s to ubiquitinate a substrate and to elongate polyubiquitin
chains of a specific topology appear to be mutually exclusive. Thus two
different classes of E2 may be required to attach a polyubiquitin chain of a
particular topology to a substrate: the properties of one E2 are designed to
mono-ubiquitinate a substrate with no or little inherent specificity for an
acceptor lysine residue, whereas the properties of the second E2 are tailored to
the elongation of a polyubiquitin chain using a defined lysine residue of
ubiquitin. Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin
(polyUb) chains is suggested to play important roles in a variety of cellular
events, including DNA repair, signal transduction, and receptor endocytosis.
However, identifying such modifications in living cells is complex and
cumbersome. We have generated a monoclonal antibody (mAb) that specifically
recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27,
K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and
specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell
lysates by Western blotting and in cells by immunofluorescence, and
K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will
facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a
strategy for the generation of similar reagents against other forms of polyUb. The E2 polyubiquitin-conjugating enzyme Ubc13 is a mediator of innate immune
reactions. Ubc13 mediates the conjugation of keratin (K)63-linked polyubiquitin
chains onto TNF receptor-associated factor 6 and IKKγ during NF-κB activation.
In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains
function in nonproteasomal biological processes. Although Ubc13 has been shown
to be critical for Toll-like receptor (TLR) and IL-1 receptor signaling, the
function of Ubc13 in the epidermis has not been studied. We generated
keratinocyte-specific Ubc13-deficient mice (Ubc13(flox/flox)K5-Cre). At birth,
the skin of the Ubc13(flox/flox)K5-Cre mice was abnormally shiny and smooth; in
addition, the mice did not grow and died by postnatal day 2. Histological
analysis showed atrophy of the epidermis with keratinocyte apoptosis.
Immunohistochemical analyses revealed reduced proliferation, abnormal
differentiation, and apoptosis of keratinocytes in the Ubc13(flox/flox)K5-Cre
mouse epidermis. In culture, Ubc13(flox/flox)K5-Cre keratinocyte growth was
impaired, and spontaneous cell death occurred. Moreover, the deletion of Ubc13
from cultured Ubc13(flox/flox) keratinocytes by means of an adenoviral vector
carrying Cre recombinase also resulted in spontaneous cell death. Therefore,
Ubc13 is essential for keratinocyte growth, differentiation, and survival.
Analyses of intracellular signaling revealed that the IL-1 and TNF-induced
activation of JNK, p38, and NF-κB pathways was impaired in
Ubc13(flox/flox)K5-Cre keratinocytes. In conclusion, Ubc13 appears to be
essential for epidermal integrity in mice. |
Could transcription factors act as cell-cell signalling molecules? | Yes. Recent data support the view that transcription factors - in particular, homeoproteins - can be transferred from cell to cell and have direct non-cell-autonomous (and therefore paracrine) activities. | Pax6 is a transcription factor essential for the development of tissues
including the eyes, central nervous system and endocrine glands of vertebrates
and invertebrates. It regulates the expression of a broad range of molecules,
including transcription factors, cell adhesion and short-range cell-cell
signalling molecules, hormones and structural proteins. It has been implicated
in a number of key biological processes including cell proliferation, migration,
adhesion and signalling both in normal development and in oncogenesis. The
mechanisms by which Pax6 regulates its downstream targets likely involve the use
of different splice variants and interactions with multiple proteins, allowing
it to generate different effects in different cells. Extrapolation to
developmental transcription factors in general suggests that variation in the
nature of individual factors is likely to contribute to the emergence of
differences between tissues. Recent data support the view that transcription factors - in particular,
homeoproteins - can be transferred from cell to cell and have direct
non-cell-autonomous (and therefore paracrine) activities. This intercellular
transfer, based on atypical internalization and secretion, has important
biotechnological consequences. But the real excitement stems from the
physiological and developmental implications of this mode of signal
transduction. |
Magnetic beads has been used in numerous applications. List some coatings used. | aptamers
enzymes
streptavidin
concanavalin A
carboxylic-modified
TiO2
antibodies
SELEX library
synthesized DNA
C18
C8
oligo(dT) | An electrochemical magneto biosensor for the rapid determination of biotin in
food samples is reported. The affinity reaction was performed on
streptavidin-modified magnetic microbeads as a solid support in a direct
competitive format. The biotinylated horseradish peroxidase enzyme (biotin-HRP)
competes with free biotin in the sample for the binding sites of streptavidin on
the magnetic microbeads. The modified magnetic beads were then easily captured
by a magneto graphite-epoxy composite electrode and the electrochemical signal
was based on the enzymatic activity of the HRP enzyme under the addition of
H(2)O(2) as the substrate and o-phenilendiamine as cosubstrate. The response was
electrochemically detected by square wave voltammetry. The limit of detection
was 8.4×10(-8) mol L(--1) of biotin (20 μg L(--1)) with a dynamic range from
0.94 to 2.4×10(-7) mol L(--1). Biotin-fortified commercial dietary supplement
and infant formula samples were evaluated obtaining good performances in the
results. Total time of analysis was 40 min per 20 assays. The low therapeutic index of digoxin necessitates careful monitoring of its
serum levels. Most of digoxin immunoassays suffer from interferences with
digoxin-like immunoreactive substances. Since aptamers have been shown to be
highly specific for their targets, the aim of this study was to develop DNA
aptamers for this widely used cardiac glycoside. Digoxin was coated onto the
surface of streptavidin magnetic beads. DNA aptamers against digoxin were
designed using Systematic Evolution of Ligands by Exponential enrichment method
(SELEX) by 11 iterative rounds of incubation of digoxin-coated streptavidin
magnetic beads with synthetic DNA library, DNA elution, electrophoresis and PCR
amplification. The PCR product was cloned and sequenced. Binding affinity was
determined using digoxin-BSA conjugate, coated onto ELISA plate. Inhibitory
effect of anti-digoxin aptamer was conducted using isolated guinea-pig atrium.
Three aptamers (D1, D2 and D3) were identified. Binding studies of
fluorescein-labeled truncated (without primer binding region) D1 and D2 and full
length D1 anti-digoxin aptamers were performed and their corresponding
dissociation constants values were 8.2×10(-9), 44.0×10(-9) and 17.8×10(-9) M,
respectively. This is comparable to what other workers have obtained for
interaction of monoclonal antibodies raised against digoxin. There was little
difference in binding affinity between full length and truncated anti-digoxin D1
aptamer. D1 anti-digoxin aptamer also inhibited the effects of digoxin on the
isolated guinea-pig atrium. D1 anti-digoxin aptamer distinguished between
digoxin and ouabain in both tissue study and binding experiments. Our finding
indicated that D1 anti-digoxin aptamer can selectively bind to digoxin. Further
studies might show its suitability for use in digoxin assays and as a
therapeutic agent in life-threatening digoxin toxicity. Interactions between DNA and transcription factors (TFs) guide cellular function
and development, yet the complexities of gene regulation are still far from
being understood. Such understanding is limited by a paucity of techniques with
which to probe DNA-protein interactions. We have devised magnetic protein
immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay
using flow cytometric immunofluorescence to reveal interactions among TFs,
chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic
beads, which are then incubated with nuclear lysate, permitting
sequence-specific binding by TFs, histones and methylation by native lysate
factors that can be optionally inhibited with small molecules. Lysate
protein-DNA binding is monitored by flow cytometric immunofluorescence, which
allows for accurate comparative measurement of TF-DNA affinity. Combinatorial
fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA
interaction and chromatin modification. MagPIE provides a simple and robust
method to analyze complex epigenetic interactions in vitro. In this paper, we demonstrate a novel pull-down method that dramatically reduces
the cost and preparation time of a bait protein by cell-free translation with a
puromycin linker. With the C-terminus of the bait protein linked to biotin
through a puromycin molecule after the translation reaction and subsequent mRNA
degradation by RNase, the prey protein was easily pulled down by
streptavidin-coated magnetic beads in a test tube. Three fluorescent prey
protein types were tested and confirmed by gel electrophoresis to be pulled down
easily and rapidly, depending on their affinity. We have developed a method that enriches for methylated cytosines by capturing
the fraction of bisulfite-treated DNA with unconverted cytosines. The method,
called streptavidin bisulfite ligand methylation enrichment (SuBLiME), involves
the specific labeling (using a biotin-labeled nucleotide ligand) of methylated
cytosines in bisulfite-converted DNA. This step is then followed by affinity
capture, using streptavidin-coupled magnetic beads. SuBLiME is highly adaptable
and can be combined with deep sequencing library generation and/or genomic
complexity-reduction. In this pilot study, we enriched methylated DNA from
Csp6I-cut complexity-reduced genomes of colorectal cancer cell lines (HCT-116,
HT-29 and SW-480) and normal blood leukocytes with the aim of discovering
colorectal cancer biomarkers. Enriched libraries were sequenced with SOLiD-3
technology. In pairwise comparisons, we scored a total of 1,769 gene loci and 33
miRNA loci as differentially methylated between the cell lines and leukocytes.
Of these, 516 loci were differently methylated in at least two promoter-proximal
CpG sites over two discrete Csp6I fragments. Identified methylated gene loci
were associated with anatomical development, differentiation and cell signaling.
The data correlated with good agreement to a number of published colorectal
cancer DNA methylation biomarkers and genomic data sets. SuBLiME is effective in
the enrichment of methylated nucleic acid and in the detection of known and
novel biomarkers. Cell isolation via antibody-targeted magnetic beads is a powerful tool for
research and clinical applications, most recently for isolating circulating
tumor cells (CTC). Nonetheless fundamental features of the cell-bead interface
are still unknown. Here we apply a clinically relevant antibody against the
cancer target HER2 (ErbB2) for magnetic cell isolation. We investigate how many
target proteins per cell are sufficient for a cell to be isolated. To understand
the importance of primary antibody affinity, we compared a series of point
mutants with known affinities and show that even starting with subomolar
affinity, improving antibody affinity improved cell isolation. To test the
importance of the connection between the primary antibody and the magnetic bead,
we compared bridging the antibody to the beads with Protein L, secondary
antibody, or streptavidin: the high-stability streptavidin-biotin linkage
improved sensitivity by an order of magnitude. Cytoskeletal polymerization did
not have a major effect on cell isolation, but isolation was inhibited by
cholesterol depletion and enhanced by cholesterol loading of cells. Analyzing a
panel of human cancer cell lines spanning a wide range of expression showed that
the standard approach could only isolate the highest expressing cells. However,
our optimization of cholesterol level, primary antibody affinity, and
antibody-bead linkage allowed efficient and specific isolation of cells
expressing low levels of HER2 or epithelial cell adhesion molecule. These
insights should guide future approaches to cell isolation, either magnetically
or using other means, and extend the range of cellular antigens and biomarkers
that can be targeted for CTC isolation in cancer research and diagnosis. Protein kinase signaling regulates human hematopoietic stem/progenitor cell
(HSPC) fate, yet little is known about critical pathway substrates. To address
this, we have developed and applied a large-scale, empirically optimized
phosphopeptide affinity enrichment strategy with high-throughput 2D LC-MS/MS
screening to evaluate the phosphoproteome of an isolated human CD34(+) HSPC
population. We first used hydrophilic interaction chromatography as a first
dimension separation to separate and simplify protein digest mixtures into
discrete fractions. Phosphopeptides were then enriched off-line using TiO2
-coated magnetic beads and subsequently detected online by C18 RP oflow HPLC
using data-dependent MS/MS high-energy collision-activated dissociation
fragmentation on a high-performance Orbitrap hybrid tandem mass spectrometer. We
identified 15 533 unique phosphopeptides in 3574 putative phosphoproteins.
Systematic computational analysis revealed biological pathways and
phosphopeptide motifs enriched in CD34(+) HSPC that are markedly different from
those observed in an analogous parallel analysis of isolated human T cells,
pointing to the possible involvement of specific kinase-substrate relationships
within activated cascades driving hematopoietic renewal, commitment, and
differentiation. Owing to their preeminent biological functions, the repertoire of expressed
RNA-binding proteins (RBPs) and their activity states are highly informative
about cellular systems. We have developed a novel and unbiased technique, called
interactome capture, for identifying the active RBPs of cultured cells. By
making use of in vivo UV cross-linking of RBPs to polyadenylated RNAs,
covalently bound proteins are captured with oligo(dT) magnetic beads. After
stringent washes, the mRNA interactome is determined by quantitative mass
spectrometry (MS). The protocol takes 3 working days for analysis of single
proteins by western blotting, and about 2 weeks for the determination of
complete cellular mRNA interactomes by MS. The most important advantage of
interactome capture over other in vitro and in silico approaches is that only
RBPs bound to RNA in a physiological environment are identified. When applied to
HeLa cells, interactome capture revealed hundreds of novel RBPs. Interactome
capture can also be broadly used to compare different biological states,
including metabolic stress, cell cycle, differentiation, development or the
response to drugs. We developed an approach for screening bioactive compounds from botanical drug
using multiple target-immobilized magnetic beads coupled with high performance
liquid chromatography-mass spectrometry. This novel approach was called magnetic
beads based multi-target affinity selection-mass spectrometry (MT-ASMS). It can
enrich and identify different types of ligands from mixture extracts. Multiple
targets (maltase, invertase, lipase) were immobilized on the magnetic beads by
covalent linkage using 1-(3-dimethyl-aminopropyl)-3-ethyl-carbodiimide (EDC) and
N-hydroxysuccinimide (NHS) as reaction reagents, respectively. The properties of
enzyme conjugated magnetic beads were characterized using transmission electron
microscopy, X-ray diffractometer and vibration sample magnetometer. Several
factors including pH, ion strength, incubation time and temperature were
optimized using three known ligands (caffeic acid, ferulic acid, and
hesperidin). The established MT-ASMS approach was applied to screening for
ligands from a Chinese medicine "Tang-Zhi-Qing", which was used to treat type II
diabetes in China. Seven bound compounds were identified via liquid
chromatography-mass spectrometry (LC/MS). Five active compounds including
2,3,4,6-tetra-O-galloyl-D-glucose, 1,2,3,4-tetra-O-galloyl-D-glucose,
1,2,3,4,6-penta-O-galloyl-d-glucose, quercetin-3-O-β-D-glucuronide and
quercetin-3-O-β-D-glucoside were identified and their activities were validated
by conventional inhibitory assay. Our findings suggested that the proposed
approach is efficient in screening compounds with multiple activities from
extracts of botanical drugs. The electrochemical detection of cell lines of MCF-7 (human breast cancer) has
been reported, using magnetic beads for the separation tool and high-affinity
DNA aptamers for signal recognition. The high specificity was obtained by using
the magnetic beads and aptamers, and the good sensitivity was realized with the
signal amplification of DNA capped CdS or PbS ocrystals. The ASV (anodic
stripping voltammetry) technology was employed for the detection of cadmic
cation and lead ions, for electrochemical assay of the amount of the target
cells and biomarkers on the membrane of target cells, respectively. This
electrochemical method could respond to as low as 100 cells mL(-1) of cancer
cells with a linear calibration range from 1.0×10(2) to 1.0×10(6) cells mL(-1),
showing very high sensitivity. Moreover, the amounts of HER-3 which were
overexpressed on MCF-7 cells were calculated correspond to be 3.56×10(4)
anti-HER-3 antibody molecules. In addition, the assay was able to differentiate
between different types of target and control cells based on the aptamers and
magnetic beads used in the assay, indicating the wide applicability of the assay
for early and accurate diagnose of cancers. Human neutrophil elastase (HNE) is a multifunctional serine protease, involved
in infection defense, inflammatory process regulation, and physiopathological
processes of several diseases. We developed aptamer-capture based assays for
human neutrophil elastase with different substrates and solid supports to meet
different demands, such as simplicity, sensitivity, and high throughput.
Aptamers against HNE were immobilized on magnetic beads or microplates as
affinity ligands to capture HNE, and then the enriched HNE catalyzed the
conversion of chromogenic substrates or fluorogenic substrates to products. The
measurement of the generated enzymatic products enabled the final detection of
HNE. In the assay using chromogenic substrates and aptamer modified magnetic
beads, 0.4 pM HNE could be successfully detected. The sensitivity of the assay
was further improved by using fluorogenic substrates, and a detection limit of
HNE at 20 fM was achieved. The use of aptamer-coated microplates instead of
aptamer modified magnetic beads in the assays also allowed the sensitive
detection of HNE, offering advantages in fast sample handling and measurement.
The established assays for HNE displayed good specificity, and proteins
including serum albumin, transferrin, immunoglobulin G, thrombin, porcine
pancreatic elastase, trypsin, proteinase K, chymotrypsin, lysozyme, cathepsin G,
and proteinase 3 did not cause interference in the detection of HNE. A novel amperometric magnetoimmunosensor using an indirect competitive format is
developed for the sensitive detection of the amino-terminal pro-B-type
natriuretic peptide (NT-proBNP). The immunosensor design involves the covalent
immobilization of the antigen onto carboxylic-modified magnetic beads (HOOC-MBs)
activated with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and
N-hydroxysulfosuccinimide (sulfo-NHS), and further incubation in a mixture
solution containing variable concentrations of the antigen and a fixed
concentration of an HRP-labeled detection antibody. Accordingly, the target
NT-proBNP in the sample and that immobilized on the MBs compete for binding to a
fixed amount of the specific HRP-labeled secondary antibody. The
immunoconjugate-bearing MBs are captured by a magnet placed under the surface of
a disposable gold screen-printed electrode (Au/SPE). The amperometric responses
measured at -0.10 V (vs. a Ag pseudo-reference electrode), upon addition of
3,3',5,5'-tetramethylbenzidine (TMB) as electron transfer mediator and H2O2 as
the enzyme substrate, are used to monitor the affinity reaction. The developed
magnetoimmunosensor provides attractive analytical characteristics in 10-times
diluted human serum samples, exhibiting a linear range of clinical usefulness
(0.12-42.9 ng mL(-1)) and a detection limit of 0.02 ng mL(-1), which can be used
in clinical diagnosis of chronic heart failure in the elderly and for
classifying patients at risk of death after heart transplantation. The
magnetoimmunosensor was successfully applied to the analysis of spiked human
serum samples. Here we present a sensitive and specific assay for thrombin through the affinity
capture of thrombin with mRNA display generated peptide aptamers on magnetic
beads and the subsequent thrombin-involved enzymatic reaction. Thrombin at 50 fM
can be detected. Many proteins involved in DNA repair systems interact with DNA that has
structure altered from the typical B-form helix. Using magnetic beads to
immobilize DNAs containing various types of structures, we evaluated the in
vitro binding activities of two well-characterized DNA repair proteins,
Escherichia coli MutS and human p53. E. coli MutS bound to double-stranded DNAs,
with higher affinity for a G/T mismatch compared to a G/A mismatch and highest
affinity for larger non-B-DNA structures. E. coli MutS bound best to DNA between
pH 6 and 9. Experiments discriminated between modes of p53-DNA binding, and
increasing ionic strength reduced p53 binding to nonspecific double-stranded
DNA, but had minor effects on binding to consensus response sequences or
single-stranded DNA. Compared to nonspecific DNA sequences, p53 bound with a
higher affinity to mismatches and base insertions, while binding to various
hairpin structures was similar to that observed to its consensus DNA sequence.
For hairpins containing CTG repeats, the extent of p53 binding was proportional
to the size of the repeat. In summary, using the flexibility of the magnetic
bead separation assay we demonstrate that pH and ionic strength influence the
binding of two DNA repair proteins to a variety of DNA structures. The development of enzyme immobilization techniques that will not affect
catalytic activity and conformation is an important research task. Affinity tags
that are present or added at a specific position far from the active site in the
structure of the native enzyme could be used to create strong affinity bonds
between the protein structure and a surface functionalized with the
complementary affinity ligand. These immobilization techniques are based on
affinity interactions between biotin and (strept)avidin molecules, lectins and
sugars, or metal chelate and histidine tag. Recent developments involve
immobilization of tagged enzymes onto magnetic oparticles. These supports can
improve the performance of immobilized biomolecules in analytical assay because
magnetic beads provide a relative large numbers of binding sites for biochemical
reactions resulting in faster assay kinetics. This chapter describes
immobilization procedures of tagged enzymes onto various magnetic beads. The use of immobilized capillary enzyme reactors (ICERs) and enzymes coated to
magnetic beads ((NT or CT)-MB) for ligand screening has been adopted as a new
technique of high throughput screening (HTS). In this work the selected target
was the enzyme acetylcholinesterase (AChE), which acts on the central nervous
system and is a validated target for the treatment of Alzheimer's disease, as
well as for new insecticides. A new approach for the screening of plant extracts
was developed based on the ligand fishing experiments and zonal chromatography.
For that, the magnetic beads were used for the ligand fishing experiments and
capillary bioreactors for the activity assays. The latter was employed also
under non-linear conditions to determine the affinity constants of known
ligands, for the first time, as well as for the active fished ligand. The presence of pathogenic bacteria is a major health risk factor in food
samples and the commercial food supply chain is susceptible to bacterial
contamination. Thus, rapid and sensitive identification methods are in demand
for the food industry. Quantitative polymerase chain reaction (PCR) is one of
the reliable specific methods with reasonably fast assay times. However, many
constituents in food samples interfere with PCR, resulting in false results and
thus hindering the usability of the method. Therefore, we aimed to develop an
aptamer-based magnetic separation system as a sample preparation method for
subsequent identification and quantification of the contamit bacteria by
real-time PCR. To achieve this goal, magnetic beads were prepared via suspension
polymerization and grafted with glycidylmethacrylate (GMA) brushes that were
modified into high quantities of amino groups. The magnetic beads were decorated
with two different aptamer sequences binding specifically to Escherichia coli or
Salmonella typhimurium. The results showed that even 1.0% milk inhibited PCR,
but our magnetic affinity system capture of bacteria from 100% milk samples
allowed accurate determination of bacterial contamination at less than 2.0 h
with limit of detection around 100 CFU/mL for both bacteria in spiked-milk
samples. |
is pharmacological treatment of subclinical hypothyroidism effective in reducing cardiovascular events? | whether SH confers a high risk for cardiovascular disease, and whether LT4 therapy has a long-term benefit that clearly outweighs the risks of overzealous treatment in these individuals, remain topics of controversy. | Subclinical hypothyroidism (SH), defined by elevated serum levels of thyroid
stimulating hormone (TSH) with normal levels of free thyroid hormones, is common
in adults, especially in women over 60 years of age. Among individuals with this
condition, up to two-thirds have serum TSH levels between 5-10 mU/L and thyroid
autoantibodies; almost half of them may progress to overt thyroid failure, the
annual percent risk increasing with serum TSH level. There is evidence that
elevated TSH levels in patients with SH do not reflect pituitary compensation to
maintain euthyroidism, but a mild tissue hypothyroidism sensu strictu. When
lasting more than 6-12 months, SH may be associated with an atherogenic lipid
profile, a hypercoagulable state, a subtle cardiac defect with mainly diastolic
dysfunction, impaired vascular function, and reduced submaximal exercise
capacity. The deviation from normality usually increases with serum TSH level
('dosage effect' phenomenon). Restoration of euthyroidism by levothyroxine (LT4)
treatment may correct the lipid profile and cardiac abnormalities, especially in
patients with an initially higher deviation from normality and higher serum TSH
levels. Importantly, a strong association between SH and atherosclerotic
cardiovascular disease, independent of the traditional risk factors, has been
recently reported in a large cross-sectional survey (the Rotterdam Study).
However, whether SH confers a high risk for cardiovascular disease, and whether
LT4 therapy has a long-term benefit that clearly outweighs the risks of
overzealous treatment in these individuals, remain topics of controversy.
Therefore, until randomized, controlled, prospective, and adequately powered
trials provide unequivocal answers to these critical questions, it is advisable
to prescribe LT4 therapy on a case-by-case basis, taking into account the risk
of progressive thyroid failure and the risk of cardiovascular events. Cardiovascular consequences of thyroid diseases, their prevalence and treatment,
particularly in cases with subclinical hyper- and hypothyroidism. The aim of the
study was to draw attention to an association between the thyroid and
cardiovascular diseases. The main topic was to lay emphasis on the importance of
cardiovascular diseases caused by subclinical hyper- and hypothyroidism in the
relation to the practice. The subclinical states precede the overt hyper- and
hypothyroidism, and they are often present during their treatments. The changes
in the levels of thyrotropin demonstrating subclinical thyroid diseases, could
be detected in different non-thyroid diseases and resulted from the effect of
some drugs. Particularly, the subclinical thyroid diseases become more frequent
in older age. In the background of the high prevalences of atrial fibrillation,
hypertension and psychosomatic events subclinical hyperthyroidism could be
revealed. Subclinical hypothyroidism is characterized by an increased prevalence
of elevated serum lipid levels, atherosclerosis, ischemic heart disease and
hypertension often associating with the presence of anti-thyroid antibodies. It
is important to reveal subclinical thyroid diseases in time for the effective
treatment and for stopping of the cardiovascular damages before manifestations
of cardiovascular diseases. The paper gives advice for the practice and the
rational management. At the end, a survey of the association between thyroid and
heart diseases in own department of internal medicine at the last three years is
given. Subclinical hypothyroidism is defined as an elevated serum thyroid-stimulating
hormone (TSH) level in the face of normal free thyroid hormone values. The
overall prevalence of subclinical hypothyroidism is 4-10% in the general
population and up to 20% in women aged >60 years. The potential benefits and
risks of therapy for subclinical hypothyroidism have been debated for 2 decades,
and a consensus is still lacking. Besides avoiding the progression to overt
hypothyroidism, the decision to treat patients with subclinical hypothyroidism
relies mainly on the risk of metabolic and cardiovascular alterations.
Subclinical hypothyroidism causes changes in cardiovascular function similar to,
but less marked than, those occurring in patients with overt hypothyroidism.
Diastolic dysfunction both at rest and upon effort is the most consistent
cardiac abnormality in patients with subclinical hypothyroidism, and also in
those with slightly elevated TSH levels (>6 mIU/L). Moreover, mild thyroid
failure may increase diastolic blood pressure as a result of increased systemic
vascular resistance. Restoration of euthyroidism by levothyroxine replacement is
generally able to improve all these abnormalities. Early clinical and autopsy
studies had suggested an association between subclinical hypothyroidism and
coronary heart disease, which has been subsequently confirmed by some, but not
all, large cross-sectional and prospective studies. Altered coagulation
parameters, elevated lipoprotein (a) levels, and low-grade chronic inflammation
are regarded to coalesce with the hypercholesterolemia of untreated patients
with subclinical hypothyroidism to enhance the ischemic cardiovascular risk.
Although a consensus is still lacking, the strongest evidence for a beneficial
effect of levothyroxine replacement on markers of cardiovascular risk is the
substantial demonstration that restoration of euthyroidism can lower both total
and low-density lipoprotein-cholesterol levels in most patients with subclinical
hypothyroidism. However, the actual effectiveness of thyroid hormone
substitution in reducing the risk of cardiovascular events remains to be
elucidated. In conclusion, the multiplicity and the possible reversibility of
subclinical hypothyroidism-associated cardiovascular abnormalities suggest that
the decision to treat a patient should depend on the presence of risk factors,
rather than on a TSH threshold. On the other hand, levothyroxine replacement
therapy can always be discontinued if there is no apparent benefit.
Levothyroxine replacement therapy is usually safe providing that excessive
administration is avoided by monitoring serum TSH levels. However, the
possibility that restoring euthyroidism may be harmful in the oldest of the
elderly population of hypothyroid patients has been recently raised, and should
be taken into account in making the decision to treat patients with subclinical
hypothyroidism who are aged >85 years. BACKGROUND: Subclinical hypothyroidism is defined as an elevated serum
thyroid-stimulating hormone (TSH) level with normal free thyroid hormones
values. The prevalence of subclinical hypothyroidism is 4% to 8% in the general
population, and up to 15% to 18% in women who are over 60 years of age. There is
considerable controversy regarding the morbidity, the clinical significance of
subclinical hypothyroidism and if these patients should be treated.
OBJECTIVES: To assess the effects of thyroid hormone replacement for subclinical
hypothyroidism.
SEARCH STRATEGY: We searched The Cochrane Library, MEDLINE, EMBASE and LILACS.
Ongoing trials databases, reference lists and abstracts of congresses were
scrutinized as well.
SELECTION CRITERIA: All studies had to be randomised controlled trials comparing
thyroid hormone replacement with placebo or no treatment in adults with
subclinical hypothyroidism. Minimum duration of follow-up was one month.
DATA COLLECTION AND ANALYSIS: Two authors independently assessed trial quality
and extracted data. We contacted study authors for missing or additional
information.
MAIN RESULTS: Twelve trials of six to 14 months duration involving 350 people
were included. Eleven trials investigated levothyroxine replacement with
placebo, one study compared levothyroxine replacement with no treatment. We did
not identify any trial that assessed (cardiovascular) mortality or morbidity.
Seven studies evaluated symptoms, mood and quality of life with no statistically
significant improvement. One study showed a statistically significant
improvement in cognitive function. Six studies assessed serum lipids, there was
a trend for reduction in some parameters following levothyroxine replacement.
Some echocardiographic parameters improved after levothyroxine replacement
therapy, like myocardial relaxation, as indicated by a significant prolongation
of the isovolumic relaxation time as well as diastolic dysfunction. Only four
studies reported adverse events with no statistically significant differences
between groups.
AUTHORS' CONCLUSIONS: In current RCTs, levothyroxine replacement therapy for
subclinical hypothyroidism did not result in improved survival or decreased
cardiovascular morbidity. Data on health-related quality of life and symptoms
did not demonstrate significant differences between intervention groups. Some
evidence indicates that levothyroxine replacement improves some parameters of
lipid profiles and left ventricular function. BACKGROUND: It has been suggested that low thyroid hormones levels may be
associated with increased mortality in patients with cardiovascular disease.
AIM: To evaluate the prognostic role of thyroid function deficiency in patients
with chronic heart failure (CHF).
METHODS: We evaluated 338 consecutive outpatients with stable CHF receiving
conventional therapy, all of whom underwent a physical examination,
electrocardiography and echocardiography. Blood samples were drawn to assess
renal function, and Na+, hemoglobin, NT-proBNPs, fT3, fT4 and TSH levels.
Patients with hyperthyroidism were excluded.
RESULTS: During the follow-up (15+/-8 months), heart failure progression was
observed in 79 patients (including 18 who died of heart failure after
hospitalisation and six who underwent transplantation). Univariate regression
analysis showed that TSH (p<0.0001), fT3 (p<0.0001), fT4 (p=0.016) and fT3/fT4
(p<0.0001) were associated with heart failure progression but multivariate
analysis showed that only TSH considered as a continuous variable (p = 0.001) as
well as subclinical hypothyroidism (TSH > 5.5 mUI/l; p=0.014) remained
significantly associated with the events.
CONCLUSIONS: In CHF patients TSH levels even slightly above normal range are
independently associated with a greater likelihood of heart failure progression.
This supports the need for prospective studies aimed at clarifying the most
appropriate therapeutic approach to sub-clinical hypothyroidism in such
patients. BACKGROUND: Some studies have proposed that subclinical hypothyroidism (SCH) has
adverse effects on the cardiovascular system, but little is known about the
effect on patients undergoing cardiovascular operations. We examined the
influence of preoperative SCH on postoperative outcome in patients undergoing
coronary artery bypass grafting (CABG).
METHODS: Among patients who underwent CABG between July 2005 and June 2007 at
Seoul National University Bundang Hospital, 224 with normal thyroid function and
36 with SCH were enrolled. Preoperative risks and postoperative outcomes were
evaluated prospectively without thyroid hormone replacement.
RESULTS: There were no significant differences in primary outcomes (major
adverse cardiovascular events) and secondary outcomes such as wound problems,
mediastinitis, leg infection, respiratory complications, delirium, or
reoperation during the same hospitalization. However, patients with SCH had a
higher incidence of postoperative atrial fibrillation than those with normal
thyroid function after adjustment for age, gender, body mass index, and other
independent variables such as emergency operation, the use of cardiopulmonary
bypass, combined valvular operation, preoperative creatinine levels, left
ventricular systolic dysfunction, and nonuse of beta-blockers (45.5% vs 29%;
odds ratio, 2.552; 95% confidence interval, 1.117 to 5.830; p = 0.026).
CONCLUSIONS: SCH appears to influence the postoperative outcome for patients by
increasing the development of postoperative atrial fibrillation. However, it is
still unproven whether preoperative thyroxine replacement therapy for patients
with SCH might prevent postoperative atrial fibrillation after CABG. No consensus exists whether subclinical thyroid disease should be treated or
just observed. Untreated overt thyroid disease is associated with increased risk
of cardiovascular disease, and this study was conducted to assess the risk of
cardiovascular events in subclinical thyroid disease. The population-based
prospective study was conducted in Denmark. A total of 609 subjects from general
practice aged 50 years or above with normal left ventricular function were
examined. During a median of 5 years of follow-up, major cardiovascular events
were documented. In subjects with abnormal TSH at baseline, information about
potential thyroid treatment during follow-up was obtained from case reports and
mailings. At baseline, 549 (90.7%) were euthyroid (TSH 0.40-4.00 mU/l), 31
(5.1%) were subclinical hypothyroid (TSH>4.00 mU/l), and 25 (4.1%) were
subclinical hyperthyroid (TSH<0.40 mU/l). 1 overt hyperthyroid and 3 overt
hypothyroid participants were excluded from the analyses. At baseline, the
levels of NT-proBNP were inversely associated with the levels of TSH; the lower
the levels of TSH, the higher the NT-proBNP concentration. During follow-up, 88
participants died, 81 had a major cardiovascular event, and 28 had a stroke. The
incidence of stroke was increased among subjects with subclinical
hyperthyroidism, HR 3.39 (95% CI 1.15-10.00, p=0.027) after adjusting for sex,
age, and atrial fibrillation. Subclinical hypothyroidism was not related with
any of the outcome measurements. Subclinical hyperthyroidism seems to be a risk
factor of developing major cardiovascular events, especially stroke in older
adults from the general population with normal left ventricular function. AIM: To study the time course changes in the parameters of endothelial
dysfunction in patients with type 2 diabetes mellitus (DM) and subclinical
hypothyroidism (SH) in the natural course of SH and during replacement therapy.
SUBJECTS AND METHODS: All the examined patients were divided into 2 groups: 1)
67 patients received replacement therapy with euthyrox in a dose of 25 to 100
microg/day; 2) 60 patients were followed up.
RESULTS: At the moment of study inclusion, there was a close direct correlation
between the levels of cholesterol and the aggregate intima-media thickness (IMT)
(r = 0.7) and between IMT and the levels of sICAM-1 (r = 0.71) and sVCAM-1 (r =
0.8). In the dynamics of the disease (following a year), the above correlations
became weaker due to the performed treatment. A weak positive correlation was
found between the aggregate IMT and the levels of sICAM-1 (r = 0.2) and sVCAM-1
(r = 0.3).
CONCLUSION: In patients with type 2 DM, the presence of SH serves as an
additional risk factor for endothelial dysfunction. Replacement therapy will be
able to considerably retard the progression of the disease and to reduce the
incidence of vascular events. CONTEXT: Although the negative impact of subclinical hypothyroidism (sHT) in
terms of cardiovascular risk in young adults is mostly acknowledged it remains
to be established in the elderly, especially in the oldest old.
EVIDENCE ACQUISITION: We searched Medline for reports published with the
following search words: hypothyroidism, sHT, ageing, elderly, L-thyroxin,
thyroid, guidelines, treatment, quality of life, cardiovascular risk, heart
failure (HF), ischemic heart disease (IHD), endothelial dysfunction. The search
was restricted to reports published in English since 1980, but some reports
published before 1980 were also incorporated. We supplemented the search with
records from personal files and references of relevant articles and textbooks.
Parameters analyzed included epidemiology of sHT and thyroid failure the effect
of thyroid hormone on ageing process and cardiovascular function as well as the
potential benefits of L-thyroxin therapy on quality of life, HF progression and
events.
EVIDENCE SYNTHESIS: TSH levels increase with age, even in older patients without
thyroid disease, in whom higher TSH value might favor longevity; better quality
of life and lower IHD mortality in the oldest old population has been reported
yet. However, at odds with the relationship between sHT and IHD risk and
mortality, which shows a clear age dependent feature, vanishing in the last
decades of life, the detrimental effect of sHT on HF progression and events
remains evident also in older patients, although no data are available in the
oldest old population.
CONCLUSIONS: The lack of specific randomized trials enrolling either old or very
old subjects, aimed at evaluate the efficacy of hormonal replacement on overall
survival and cardiovascular risk reduction along with the negative effects of
possible over-treatment, makes the decision to treat older people a still
unresolved clinical challenge. Moreover, the possibility that restoring
euthyroidism may be harmful in the elderly should be always taken into account. |
Could Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)
cause sudden cardiac death? | Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) can cause sudden cardiac death. | The cardiac ryanodine receptor (RyR2), the major calcium release channel on the
sarcoplasmic reticulum (SR) in cardiomyocytes, has recently been shown to be
involved in at least two forms of sudden cardiac death (SCD): (1)
Catecholaminergic polymorphic ventricular tachycardia (CPVT) or familial
polymorphic VT (FPVT); and (2) Arrhythmogenic right ventricular dysplasia type 2
(ARVD2). Eleven RyR2 missense mutations have been linked to these diseases. All
eleven RyR2 mutations cluster into 3 regions of RyR2 that are homologous to the
three maligt hyperthermia (MH)/central core disease (CCD) mutation regions of
the skeletal muscle ryanodine receptor/calcium release channel RyR1. MH/CCD RyR1
mutations have been shown to alter calcium-induced calcium release. Sympathetic
nervous system stimulation leads to phosphorylation of RyR2 by protein kinase A
(PKA). PKA phosphorylation of RyR2 activates the channel. In conditions
associated with high rates of SCD such as heart failure RyR2 is PKA
hyperphosphorylated resulting in "leaky" channels. SR calcium leak during
diastole can generate "delayed after depolarizations" that can trigger fatal
cardiac arrhythmias (e.g., VT). We propose that RyR2 mutations linked to genetic
forms of catecholaminergic-induced SCD may alter the regulation of the channel
resulting in increased SR calcium leak during sympathetic stimulation. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare
arrhythmogenic disorder characterized by syncopal events and sudden cardiac
death at a young age during physical stress or emotion, in the absence of
structural heart disease. We report the first nonsense mutations in the cardiac
calsequestrin gene, CASQ2, in three CPVT families. The three mutations, a
nonsense R33X, a splicing 532+1 G>A, and a 1-bp deletion, 62delA, are thought to
induce premature stop codons. Two patients who experienced syncopes before the
age of 7 years were homozygous carriers, suggesting a complete absence of
calsequestrin 2. One patient was heterozygous for the stop codon and experienced
syncopes from the age of 11 years. Despite the different mutations, there is
little phenotypic variation of CPVT for the CASQ2 mutations. Of the 16
heterozygous carriers of these various mutations, 14 were devoid of clinical
symptoms or ECG anomalies, whereas 2 of them had ventricular arrhythmias at ECG
on exercise tests. In line with this, the diagnosis of the probands was
difficult because of the absence of a positive family history. In conclusion,
these additional three CASQ2 CPVT families suggest that CASQ2 mutations are more
common than previously thought and produce a severe form of CPVT. The full text
of this article is available at http://www.circresaha.org. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an autosomal
domit inherited disorder characterized by adrenergic induced polymorphic
ventricular tachycardias and associated with sudden cardiac death. The human
cardiac ryanodine receptor gene (RyR2) was linked to CPVT. A 20-year-old male
was referred to our hospital because of recurrent syncope after physical and
emotional stress. Routine cardiac examinations including catheterization
revealed no structural abnormality. Exercise on treadmill induced premature
ventricular contraction in bigeminy and bidirectional ventricular tachycardia
was induced during isoproterenol infusion. Beta-blocking drug was effective in
suppressing the arrhythmias. We performed genetic screening by PCR-SSCP method
followed by DNA sequencing, and a novel missense mutation R2401H in RyR2 located
in FKBP12.6 binding region was identified. This mutation was not detected in 190
healthy controls. Since FKBP12.6 plays a critical role in Ca channel gating, the
R2401H mutation can be expected to alter Ca-induced Ca release and E-C coupling
resulting in CPVT. This is the first report of RyR2 mutation in CPVT patient
from Asia including Japan. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
disease characterized by adrenergically mediated polymorphic ventricular
tachycardia leading to syncope and sudden cardiac death. The autosomal domit
form of CPVT is caused by mutations in the RyR2 gene encoding the cardiac
isoform of the ryanodine receptor. In vitro functional characterization of
mutant RyR2 channels showed altered behavior on adrenergic stimulation and
caffeine administration with enhanced calcium release from the sarcoplasmic
reticulum. As of today no experimental evidence is available to demonstrate that
RyR2 mutations can reproduce the arrhythmias observed in CPVT patients. We
developed a conditional knock-in mouse model carrier of the R4496C mutation, the
mouse equivalent to the R4497C mutations identified in CPVT families, to
evaluate if the animals would develop a CPVT phenotype and if beta blockers
would prevent arrhythmias. Twenty-six mice (12 wild-type (WT) and 14RyR(R4496C))
underwent exercise stress testing followed by epinephrine administration: none
of the WT developed ventricular tachycardia (VT) versus 5/14 RyR(R4496C) mice
(P=0.02). Twenty-one mice (8 WT, 8 RyR(R4496C), and 5 RyR(R4496C) pretreated
with beta-blockers) received epinephrine and caffeine: 4/8 (50%) RyR(R4496C)
mice but none of the WT developed VT (P=0.02); 4/5 RyR(R4496C) mice pretreated
with propranolol developed VT (P=0.56 nonsignificant versus RyR(R4496C) mice).
These data provide the first experimental demonstration that the R4496C RyR2
mutation predisposes the murine heart to VT and VF in response caffeine and/or
adrenergic stimulation. Furthermore, the results show that analogous to what is
observed in patients, beta adrenergic stimulation seems ineffective in
preventing life-threatening arrhythmias. The RyR (ryanodine receptor) mediates rapid Ca2+ efflux from the ER (endoplasmic
reticulum) and is responsible for triggering numerous Ca2+-activated
physiological processes. The most studied RyR-mediated process is
excitation-contraction coupling in striated muscle, where plasma membrane
excitation is transmitted to the cell interior and results in Ca2+ efflux that
triggers myocyte contraction. Recently, single-residue mutations in the cardiac
RyR (RyR2) have been identified in families that exhibit CPVT (catecholaminergic
polymorphic ventricular tachycardia), a condition in which physical or emotional
stress can trigger severe tachyarrhythmias that can lead to sudden cardiac
death. The RyR2 mutations in CPVT are clustered in the N- and C-terminal
domains, as well as in a central domain. Further, a critical signalling role for
dysfunctional RyR2 has also been implicated in the generation of arrhythmias in
the common condition of HF (heart failure). We have prepared cardiac RyR2
plasmids with various CPVT mutations to enable expression and analysis of Ca2+
release mediated by the wild-type and mutated RyR2. These studies suggest that
the mutational locus may be important in the mechanism of Ca2+ channel
dysfunction. Understanding the causes of aberrant Ca2+ release via RyR2 may
assist in the development of effective treatments for the ventricular
arrhythmias that often leads to sudden death in HF and in CPVT. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a heritable
arrhythmia unmasked by exertion or stress, characterized by triggered activity
and sudden cardiac death in affected patients. In this study we used a
mathematical model to simulate two mutations linked toCPVT, in cardiac
calsequestrin (CSQN2) and the ryanodine receptor (RyR2). The aim of the present
study is to characterize the mutations responsible for CPVT and establish the
mechanistic basis for spontaneous Ca2+ release events that lead to delayed
afterdepolarizations (DADs) and triggered arrhythmias. Simulated calcium
transients in the mutant CSQN2 model recapitulated the smaller amplitude and
time to peak, as well as accelerated recovery from inactivation seen in
experiments. When simulated CSQN2-mutant myocytes were paced in current-clamp
mode, DADs were observed, suggesting that accelerated recovery of RyR2 induced
by impaired luminal Ca2(+) sensing can lead to the triggered activity observed
in the mutant CSQN2. Simulations of mutant RyR2 suggest that the hyperactive,
"leaky" receptors characteristic of reduced FKBP12.6 function may be centrally
involved in triggering DADs. These results provide plausible mechanisms by which
defects in RyR2 gating may lead to the cellular triggers of CPVT, with
implications for the development of targeted therapies. The Ca2+ release channel ryanodine receptor 2 (RyR2) is required for
excitation-contraction coupling in the heart and is also present in the brain.
Mutations in RyR2 have been linked to exercise-induced sudden cardiac death
(catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated
RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of
the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the
channel. We found that mice heterozygous for the R2474S mutation in Ryr2
(Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures
(which occurred in the absence of cardiac arrhythmias), exercise-induced
ventricular arrhythmias, and sudden cardiac death. Treatment with a novel
RyR2-specific compound (S107) that enhances the binding of calstabin2 to the
mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac
arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky
Ryr2-R2474S channels in the brain can cause seizures in mice, independent of
cardiac arrhythmias. Based on these data, we propose that CPVT is a combined
neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy,
and the same leaky channels in the heart cause exercise-induced sudden cardiac
death. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an uncommon
heritable disease presenting with syncope or sudden cardiac death. Two genes
involved in calcium homeostasis, the ryanodine receptor gene and the
calsequestrin 2 (CASQ2) gene, have been implicated in this disease. We describe
a young man presenting with exercise-induced syncope, clinically diagnosed as
CPVT. Genetic analysis revealed two mutations, p.Y55C (c.164A>G) and p.P308L
(c.923C>T), in the CASQ2 gene. Subsequent familial analysis indicates a compound
heterozygous form of inheritance. The cardiac ryanodine receptor-Ca2+ release channel (RyR2) is an essential
sarcoplasmic reticulum (SR) transmembrane protein that plays a central role in
excitation-contraction coupling (ECC) in cardiomyocytes. Aberrant spontaneous,
diastolic Ca2+ leak from the SR due to dysfunctional RyR2 contributes to the
formation of delayed after-depolarisations, which are thought to underlie the
fatal arrhythmia that occurs in both heart failure (HF) and in catecholaminergic
polymorphic ventricular tachycardia (CPVT). CPVT is an inherited disorder
associated with mutations in either the RyR2 or a SR luminal protein,
calsequestrin. RyR2 shows normal function at rest in CPVT but the RyR2
dysfunction is unmasked by physical exercise or emotional stress, suggesting
abnormal RyR2 activation as an underlying mechanism. Several potential
mechanisms have been advanced to explain the dysfunctional RyR2 observed in HF
and CPVT, including enhanced RyR2 phosphorylation status, altered RyR2
regulation at luminal/cytoplasmic sites and perturbed RyR2 intra/inter-molecular
interactions. This review considers RyR2 dysfunction in the context of the
structural and functional modulation of the channel, and potential therapeutic
strategies to stabilise RyR2 function in cardiac pathology. BACKGROUND: In Europe, sudden cardiac death (SCD) is one of the most common
causes of death. Although sudden cardiac death usually happens in older people,
5% to 10% of the affected individuals are young and apparently healthy. Sudden
death in infants, children, and young adults is relatively rare, with an
incidence of 1 to 5 per 100 000 persons per year. Nonetheless, up to 7000
asymptomatic children die in the USA each year, almost half of them without any
warning signs or symptoms.
METHOD: Selective literature review.
RESULTS: Although structural cardiovascular abnormalities explain most cases of
sudden cardiac death in young people, the cause of death remains unexplained
after autopsy in 10% to 30% of cases. Potentially lethal ion channel disorders
(channelopathies) such as the long QT syndromes (LQTS), catecholaminergic
polymorphic ventricular tachycardia (CPVT), and the Brugada syndrome (BrS) may
account for at least one-third of these unexplained cases. Most of these
diseases are hereditary with autosomal-domit transmission, i.e., there is a
50% chance that the children of affected individuals will be affected
themselves.
CONCLUSIONS: Post-mortem genetic screening for sequence variations in cardiac
ion channel genes has become an important forensic tool for elucidating the
cause of sudden cardiac death. Moreover, it allows the identification of other
family members bearing the previously undiagnosed gene defect, who can then
undergo a cardiological evaluation if indicated by their clinical history. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial
cardiac arrhythmia that is related to RYR2 or CASQ2 gene mutation. It occurs in
patients with structurally normal heart and causes exercise-emotion-triggered
syncope and sudden cardiac death. We experienced a case of CPVT in an 11
year-old female patient who was admitted for sudden cardiovascular collapse. The
initial electrocardiogram (ECG) on emergency department revealed ventricular
fibrillation. After multiple defibrillations, sinus rhythm was restored.
However, recurrent ventricular fibrillation occurred during insertion of
nasogastric tube without sedation in coronary care unit. On ECG monitoring,
bidirectional ventricular tachycardia occurred with sinus tachycardia and then
degenerated into ventricular fibrillation. To our knowledge, there has been no
previous case report of CPVT triggered by sinus tachycardia in Korea. Therefore,
we report the case as well as a review of the literature. In approximately 10-20% of all sudden deaths, no structural cardiac
abnormalities can be identified. Important potential causes of sudden cardiac
deaths in the absence of heart disease are primary electrical diseases such as
Brugada syndrome, long QT syndrome (LQTS), short QT syndrome (SQTS), and
catecholaminergic polymorphic ventricular tachyarrhythmias (CPVT). The resting
ECG and the ECG under exercise are pivotal for the diagnosis of ion channel
diseases. Molecular genetic screening can reveal underlying mutations in a
variable degree among the cardiac ion channel diseases in up to 70% (LQTS) and
may identify individuals with incomplete penetration of the disease. In patients
with primary electrical diseases, specific clinical triggers for arrhythmic
events such as syncope or sudden cardiac death have been identified including
exercise, strenuous activity, auditory stimuli, or increased vagal tone. Young,
otherwise healthy individuals are likely to be involved in sports activity.
Therefore, special attention has to be given to advise these patients.
Competitive sports and vigorous exercise are contraindications in almost all
patients. Even recreational exercise may have to be avoided in phenotypically
overt patients or silent gene carriers depending on the underlying disease. In the last decade there have been considerable advances in the understanding of
the pathophysiology of maligt ventricular tachyarrhythmias (VA) and Sudden
Cardiac Death (SCD). Over 80% of SCD occurs in patients with organic heart
disease. However, approximately 10-15% of SCD occurs in the presence of
structurally normal heart and the majority of those patients are young. In this
group of patients, changes in genes encoding cardiac ion channels produce
modification of the function of the channel resulting in an electrophysiological
substrate of VA and SCD. Collectively these disorders are referred to as Cardiac
Ion Channelopathies. The 4 major syndromes in this group are: The Long QT
Syndrome (LQTS), the Brugada Syndrome (BrS), the Short QT Syndrome (SQTS), and
the Catecholaminergic Polymorphic VT (CPVT). Each of these syndromes includes
multiple subtypes with different and sometimes complex genetic abnormalities of
cardiac ion channels. Many are associated with other somatic and neurological
abnormalities besides the risk of VA and SCD. The current management of cardiac
ion channelopathy could be summarized as follows: 1) in symptomatic patients,
the implantable cardioverter defibrillator (ICD) is the only viable option; 2)
in asymptomatic patients, risk stratification is necessary followed by the ICD,
pharmacotherapy, or a combination of both. A genotype-specific approach to
pharmacotherapy requires a thorough understanding of the molecular-cellular
basis of arrhythmogenesis in cardiac ion channelopathies as well as the specific
drug profile. Electrical cardiomyopathies contain the long QT syndrome (LQTS), the short QT
syndrome (SQTS), the Brugada syndrome, and the catecholaminergic polymorphic
ventricular tachycardia (CPVT). Patients diagnosed with an electrical
cardiomyopathy have an increased risk of syncope and sudden cardiac death (SCD).
Usually, we are dealing with young patients or even children. The prevalence of
these diseases is low. No large prospective randomized studies exist with
respect to outcome based on different clinical and genetic parameters. Thus,
risk stratification in these patients is based on retrospective data from
single- or multicenter registries.The implantable cardioverter defibrillator is
the only reliable therapy in patients with Brugada syndrome and SQTS, as no
pharmacological therapy has been proven to prevent SCD. In LQTS and CPVT, the
primary therapy relies on beta-blockers. In high-risk patients, the ICD is
indicated.In all electrical diseases, risk stratification is based on the
clinical phenotype, including the electrocardiogram, the history of unexplained
or disease-related syncope, and sudden cardiac arrest. In LQTS and CPVT,
demographic data like age and gender are important factors for risk
stratification. The genotype contributes to risk stratification only in LQTS and
CPVT.Patients with electrical cardiomyopathies have to be risk-stratified
individually based on the data and the current guidelines available. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
arrhythmogenic disorder that causes syncopal episodes related with stress or
emotion and even sudden cardiac deaths. Signs and symptoms usually begin in
childhood. A suspicion of CPVT should be kept in mind when a child or an
adolescent suddenly loses consciousness, particularly if this happens upon
physical exercise or sudden mental stress. During the past decade, the knowledge
of CPVT genetics and physiology has increased. Exercise testing is essential
when suspecting arrhythmogenic origin of syncope, and in the case of CPVT, it
may be even more sensitive than Holter monitoring. Beta-antiadrenergic
medication can substantially decrease the mortality associated with CPVT.
Asymptomatic patients with known CPVT gene defects should also be treated
because sudden cardiac death may be the first manifestation of the disease. An
implantable cardioverter-defibrillator may also be required in the most severe
CPVT cases. In this review, we summarise the current knowledge on the clinical
characteristics, diagnostic, genetic and prognostic features of CPVT in
children. In all, 133 publications covering 60 years were checked, and those
written in English and containing ten or more, mainly paediatric CPVT cases,
were included. In addition, a CPVT family with three members and delayed
diagnoses until late childhood and adulthood is presented. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac
channelopathy characterized by altered intracellular calcium handling resulting
in ventricular arrhythmias and high risk of cardiac sudden death in young cases
with normal structural hearts. Patients present with exertional syncope and the
trademark dysrhythmia is polymorphic and/or bidirectional ventricular
tachycardia during exercise or adrenergic stimulation. Early detection of CPVT
is crucial because opportune medical intervention prevents sudden cardiac death.
Mutations in the ryanodine receptor RYR2 explain nearly 70% of the CPVT cases
and cause the autosomic domit form of the disease. Mutations in calsequestrin
2 causes a recessive form and explain less than 5% of all cases. Genetic
screening in CPVT, besides providing early detection of asymptomatic carriers at
risk, has provided important insights in the mechanism underlying the disease.
Mutational analysis of RYR2 has been a challenge due to the large size of the
gene, 105 exons encoded for 4,967 amino-acids. In this review we analyze general
concepts of the disease, differential diagnosis and strategies for genetic
screening. OBJECTIVES: The purpose of this study was to investigate the follow-up and
treatment of the mutation-carrying relatives of a proband with an inherited
arrhythmia syndrome.
BACKGROUND: The congenital long QT syndrome (LQTS), catecholaminergic
polymorphic ventricular tachycardia (CPVT), and Brugada syndrome (BrS) are
primary inherited arrhythmia syndromes that may cause syncope and sudden cardiac
death in young individuals. After establishing the disease-causing
deoxyribonucleic acid (DNA) mutation in probands, we actively conducted cascade
screening to identify, most often asymptomatic, relatives who are also at risk
of life-threatening arrhythmias.
METHODS: We retrospectively collected data from our cardiogenetics database and
patient records and analyzed whether the identified carriers received
prophylactic treatment.
RESULTS: From 1996 to 2008, 130 probands with a disease-causing mutation in one
of the involved genes were identified, and 509 relatives tested positive for the
disease-causing familial mutation. These subjects subsequently underwent
cardiologic investigation (electrocardiography, exercise testing, Holter
monitoring, ajmaline testing, echocardiography, where appropriate). After a mean
follow-up of 69 +/- 31 months (LQTS), 60 +/- 19 months (CPVT), and 56 +/- 21
months (BrS), treatment was initiated and ongoing in 65% (199 of 308), 71% (85
of 120), and 6% (5 of 81) of the relatives in the LQTS, CPVT, and BrS families,
respectively. Eight carriers were lost to follow-up. Treatment included drug
treatment (n = 249) or implantation of pacemakers (n = 26) or
cardioverter-defibrillators (n = 14). All mutation carriers received lifestyle
instructions and a list of drugs to be avoided.
CONCLUSIONS: Cascade screening in families with LQTS, BrS, or CPVT, which was
based on DNA mutation carrying and subsequent cardiologic investigation,
resulted in immediate prophylactic treatment in a substantial proportion of
carriers, although these proportions varied significantly between the different
diseases. In approximately 10-20% of all sudden deaths no structural cardiac abnormalities
can be identified. Important potential causes of sudden cardiac deaths in the
absence of heart disease are primary electrical diseases such as Brugada
syndrome, long QT syndrome (LQTS), short QT syndrome and catecholaminergic
polymorphic ventricular tachyarrhythmias. Each of these cardiac channelopathies
is charaterized by unique genetic and clinical features. The resting ECG and the
ECG under exercise are pivotal for the diagnosis of ion channel diseases.
Molecular genetic screening can reveal underlying mutations in a variable degree
among the cardiac ion channel diseases in up to 70% (LQTS) and may identify
individuals with incomplete penetration of the disease. In patients with primary
electrical diseases specific clinical triggers for arrhythmic events such as
syncope or sudden cardiac death have been identified including exercise,
strenuous activity, auditory stimuli or increased vagal tone. The significance
of programmed ventricular stimulation is at present unclear concerning risk
stratification in patients with Brugada syndrome and short QT syndrome and of no
significance in long QT syndrome and catecholaminergic polymorphic ventricular
tachycardias. The success of medical therapy remains modest for prevention of
sudden cardiac death and may necessitate the insertion of an implantable
cardioverter. However, side effects with inappropriate therapies in this patient
group with often young and active individuals have to be encountered. More
insights into the arrhythmogenesis is critical for future development of
effective medical treatment strategies. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
arrhythmogenic disease that can cause sudden cardiac death due to ventricular
fibrillation (VF). While pharmacological therapy with beta-blockers and/or
Ca(2)(+) antagonists is often unreliable, a recent study has demonstrated that
flecainide can effectively suppress arrhythmia in a murine model of CPVT as well
as clinically in two human subjects suffering from CPVT. We here present the
case of an 11-year-old boy suffering from CPVT-1 as well as a review of the
current relevant literature. After resuscitation due to VF at age 9, an
automated implantable cardioverter-defibrillator (ICD) was implanted in 2007.
Under beta-blocker therapy, repeated shocks were delivered due to either fast
ventricular tachycardia (VT) or VF. This persisted under additional therapy with
verapamil. Implantable cardioverter-defibrillator routine interrogations showed
frequent non-sustained VT with an average of 8.8 per day. Additionally, the
patient suffered from impaired physical performance due to decreased
chronotropic competence. In July 2009, flecainide was added to the
beta-blocker/verapamil regimen, resulting in a plasma level of 0.20 mg/L. No ICD
shock or sustained VT occurred until December 2010. Genetic testing revealed an
RyR2 receptor mutation. The case demonstrates the challenge of diagnosis and
management of CPVT. It furthermore supports recent experimental evidence that
the class 1 antiarrhythmic drug flecainide can suppress CPVT. The presented case
supports a novel strategy in treating CPVT with the class I antiarrhythmic agent
flecainide. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
arrhythmia syndrome characterized by bidirectional or polymorphic ventricular
arrhythmias under conditions of increased sympathetic activity in young patients
with structurally normal hearts. Patients with CPVT are at high risk of
developing life-threatening ventricular arrhythmias when untreated. A wide
variety of arrhythmic event rates on conventional therapy, with β-blockers as
the cornerstone, has been reported. Here, we systematically review all available
studies describing the efficacy of β-blocker therapy for prevention of
arrhythmic events in CPVT. Because of heterogeneity between the studies, a
random-effects meta-analysis model was used to assess the efficacy of β-blocker
therapy in preventing any arrhythmic event [syncope, aborted cardiac arrest
(ACA), and sudden cardiac death (SCD)], near-fatal arrhythmic events (ACA and
SCD), and fatal arrhythmic events. Eleven studies including 403 patients, of
whom 354 (88%) had a β-blocker prescribed, were identified. Mean follow-up
ranged from 20 months to 8 years. Estimated 8-year arrhythmic, near-fatal, and
fatal event rates were 37.2% [95% confidence interval (CI): 16.6-57.7], 15.3%
(95% CI: 7.4-23.3), and 6.4% (95% CI: 3.2-9.6), respectively. In addition, we
review the recent developments in alternate chronic treatment options for CPVT
patients, including calcium channel blockers, flecainide, left cardiac
sympathetic denervation, and implantable cardioverter defibrillators. A new
treatment strategy is proposed, including a stepwise addition of the alternate
treatment options to β-blockers in patients who do not respond sufficiently to
this first-line therapy. Finally, future developments in chronic treatment
options and acute treatment options of ventricular arrhythmias are discussed. Sudden cardiac death caused by ventricular arrhythmias is a disastrous event,
especially when it occurs in young individuals. Among the five major
arrhythmogenic disorders occurring in the absence of a structural heart disease
is catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a
highly lethal form of inherited arrhythmias. Our study focuses on the autosomal
recessive form of the disease caused by the missense mutation D307H in the
cardiac calsequestrin gene, CASQ2. Because CASQ2 is a key player in excitation
contraction coupling, the derangements in intracellular Ca(2+) handling may
cause delayed afterdepolarizations (DADs), which constitute the mechanism
underlying CPVT. To investigate catecholamine-induced arrhythmias in the CASQ2
mutated cells, we generated for the first time CPVT-derived induced pluripotent
stem cells (iPSCs) by reprogramming fibroblasts from skin biopsies of two
patients, and demonstrated that the iPSCs carry the CASQ2 mutation. Next, iPSCs
were differentiated to cardiomyocytes (iPSCs-CMs), which expressed the mutant
CASQ2 protein. The major findings were that the β-adrenergic agonist
isoproterenol caused in CPVT iPSCs-CMs (but not in the control cardiomyocytes)
DADs, oscillatory arrhythmic prepotentials, after-contractions and diastolic
[Ca(2+) ](i) rise. Electron microscopy analysis revealed that compared with
control iPSCs-CMs, CPVT iPSCs-CMs displayed a more immature phenotype with less
organized myofibrils, enlarged sarcoplasmic reticulum cisternae and reduced
number of caveolae. In summary, our results demonstrate that the
patient-specific mutated cardiomyocytes can be used to study the
electrophysiological mechanisms underlying CPVT. Sudden cardiac death (SCD) is one of the most common causes of death in
developed countries, with most SCDs involving the elderly, and structural heart
disease evident at autopsy. Each year, however, thousands of sudden deaths
involving individuals younger than 35 years of age remain unexplained after a
comprehensive medicolegal investigation that includes an autopsy. In fact,
several epidemiologic studies have estimated that at least 3% and up to 53% of
sudden deaths involving previously healthy children, adolescents, and young
adults show no morphologic abnormalities identifiable at autopsy. Cardiac
channelopathies associated with structurally normal hearts such as long QT
syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT),
and Brugada syndrome (BrS) yield no evidence to be found at autopsy, leaving
coroners, medical examiners, and forensic pathologists only to speculate that a
lethal arrhythmia might lie at the heart of a sudden unexplained death (SUD). In
cases of autopsy-negative SUD, continued investigation through either a
cardiologic and genetic evaluation of first- or second-degree relatives or a
molecular autopsy may elucidate the underlying mechanism contributing to the
sudden death and allow for identification of living family members with the
pathogenic substrate that renders them vulnerable, with an increased risk for
cardiac events including syncope, cardiac arrest, and sudden death. OBJECTIVES: The goal of this study was to establish a patient-specific
human-induced pluripotent stem cells (hiPSCs) model of catecholaminergic
polymorphic ventricular tachycardia (CPVT).
BACKGROUND: CPVT is a familial arrhythmogenic syndrome characterized by abnormal
calcium (Ca(2+)) handling, ventricular arrhythmias, and sudden cardiac death.
METHODS: Dermal fibroblasts were obtained from a CPVT patient due to the M4109R
heterozygous point RYR2 mutation and reprogrammed to generate the CPVT-hiPSCs.
The patient-specific hiPSCs were coaxed to differentiate into the cardiac
lineage and compared with healthy control hiPSCs-derived cardiomyocytes
(hiPSCs-CMs).
RESULTS: Intracellular electrophysiological recordings demonstrated the
development of delayed afterdepolarizations in 69% of the CPVT-hiPSCs-CMs
compared with 11% in healthy control cardiomyocytes. Adrenergic stimulation by
isoproterenol (1 μM) or forskolin (5 μM) increased the frequency and magnitude
of afterdepolarizations and also led to development of triggered activity in the
CPVT-hiPSCs-CMs. In contrast, flecainide (10 μM) and thapsigargin (10 μM)
eliminated all afterdepolarizations in these cells. The latter finding suggests
an important role for internal Ca(2+) stores in the pathogenesis of delayed
afterdepolarizations. Laser-confocal Ca(2+) imaging revealed significant
whole-cell [Ca(2+)] transient irregularities (frequent local and large-storage
Ca(2+)-release events, broad and double-humped transients, and triggered
activity) in the CPVT cardiomyocytes that worsened with adrenergic stimulation
and Ca(2+) overload and improved with beta-blockers. Store-overload-induced
Ca(2+) release was also identified in the hiPSCs-CMs and the threshold for such
events was significantly reduced in the CPVT cells.
CONCLUSIONS: This study highlights the potential of hiPSCs for studying
inherited arrhythmogenic syndromes, in general, and CPVT specifically. As such,
it represents a promising paradigm to study disease mechanisms, optimize patient
care, and aid in the development of new therapies. Annually thousands of sudden deaths involving young individuals (<35 years of
age) remain unexplained following a complete medicolegal investigation that
includes an autopsy. In fact, epidemiological studies have estimated that over
half of sudden deaths involving previously healthy young individuals have no
morphological abnormalities identifiable at autopsy. Cardiac channelopathies
associated with structurally normal hearts such as long QT syndrome (LQTS),
catecholaminergic polymorphic ventricular tachycardia (CPVT), and Brugada
syndrome (BrS), leave no evidence to be found at autopsy, leaving investigators
to only speculate that a lethal arrhythmia might lie at the heart of a sudden
unexplained death (SUD). In cases of autopsy-negative SUD, continued
investigation, through the use of a cardiological and genetic evaluation of
first- or second-degree relatives and/or a molecular autopsy, may pinpoint the
underlying mechanism attributing to the sudden death and allow for the
identification of living family members with the pathogenic substrate that
renders them vulnerable to an increased risk for cardiac events, including
sudden death. Mutations in genes encoding ion channel pore-forming α-subunits and accessory
β-subunits as well as intracellular calcium-handling proteins that collectively
maintain the electromechanical function of the human heart serve as the
underlying pathogenic substrate for a spectrum of sudden cardiac death
(SCD)-predisposing heritable cardiac arrhythmia syndromes, including long QT
syndrome (LQTS), short QT syndrome (SQTS), Brugada syndrome (BrS), and
catecholaminergic polymorphic ventricular tachycardia (CPVT). Similar to many
Mendelian disorders, the cardiac "channelopathies" exhibit incomplete
penetrance, variable expressivity, and phenotypic overlap, whereby
genotype-positive individuals within the same genetic lineage assume vastly
different clinical courses as objectively assessed by phenotypic features such
electrocardiographic abnormalities and number/type of cardiac events. In this
Review, we summarize the current understanding of the global architecture of
complex electrocardiographic traits such as the QT interval, focusing on the
role of common genetic variants in the modulation of ECG parameters in health
and the environmental and genetic determits of incomplete penetrance and
variable expressivity in the heritable cardiac arrhythmia syndromes most likely
to be encountered in clinical practice. BACKGROUND: Sudden cardiac death of a child is a devastating event for the
family and an enormous challenge for the attending physician.
METHODS AND RESULTS: We report a family with repeat events of sudden cardiac
death and recurrent ventricular fibrillation in a teenage girl, where autopsy
data and clinical investigations were inconclusive. The diagnosis of
catecholaminergic polymorphic ventricular tachycardia (CPVT) was established
only following finding a gene mutation in the cardiac ryanodine receptor.
CONCLUSIONS: Interpretation of autopsy data, provocation testing and genetic
testing in victims of sudden death and family members are discussed to correctly
identify the cause and properly manage asymptomatic carriers in such families. BACKGROUND: Conventional therapy with beta-blockers is incompletely effective in
preventing arrhythmic events in patients with catecholaminergic polymorphic
ventricular tachycardia (CPVT). We have previously discovered that flecainide in
addition to conventional drug therapy prevents ventricular arrhythmias in
patients with genotype-positive CPVT.
OBJECTIVE: To study the efficacy of flecainide in patients with
genotype-negative CPVT.
METHODS: We studied the efficacy of flecainide for reducing ventricular
arrhythmias during exercise testing and preventing arrhythmia events during
long-term follow-up.
RESULTS: Twelve patients with genotype-negative CPVT were treated with
flecainide. Conventional therapy failed to control ventricular arrhythmias in
all patients. Flecainide was initiated because of significant ventricular
arrhythmias (n = 8), syncope (n = 3), or cardiac arrest (n = 1). At the baseline
exercise test before flecainide, 6 patients had ventricular tachycardia and 5
patients had bigeminal or frequent ventricular premature beats. Flecainide
reduced ventricular arrhythmias at the exercise test in 8 patients compared to
conventional therapy, similar to that in patients with genotype-positive CPVT in
our previous report. Notably, flecainide completely prevented ventricular
arrhythmias in 7 patients. Flecainide was continued in all patients except for
one who had ventricular tachycardia at the exercise test on flecainide. During a
follow-up of 48±94 months, arrhythmia events (sudden cardiac death and aborted
cardiac arrest) associated with noncompliance occurred in 2 patients. Flecainide
was not discontinued owing to side effects in any of the patients.
CONCLUSIONS: Flecainide was effective in patients with genotype-negative CPVT,
suggesting that spontaneous Ca(2+) release from ryanodine channels plays a role
in arrhythmia susceptibility, similar to that in patients with genotype-positive
CPVT. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a type of
cardiac arrhythmia that occurs in people with a structurally normal heart.
Stress or anxiety-induced release of endogenous catecholamines causes a
dysfunction in the myocytic calcium-ion channel, leading to ventricular
arrhythmias that can cause dizziness, syncope, or sudden cardiac death. Since
dental procedures can be anxiety-provoking, the main purpose of this paper is to
report the dental management of a young patient with dental fear and CPVT.
Several other issues are also discussed, such as the importance of continual
collaboration with medical colleagues, the risk-benefit of using
epinephrine-containing local anesthesia for dental treatment for patients with
arrythmias, the potential risk of repeated general anesthesia in a patient with
a cardiac arrhythmia, and the challenges of providing comprehensive dental
treatment in a high caries-risk patient with extreme dental anxiety. OBJECTIVES: This study aimed to identify the genetic defect in a family with
idiopathic ventricular fibrillation (IVF) manifesting in childhood and
adolescence.
BACKGROUND: Although sudden cardiac death in the young is rare, it frequently
presents as the first clinical manifestation of an underlying inherited
arrhythmia syndrome. Gene discovery for IVF is important as it enables the
identification of individuals at risk, because except for arrhythmia, IVF does
not manifest with identifiable clinical abnormalities.
METHODS: Exome sequencing was carried out on 2 family members who were both
successfully resuscitated from a cardiac arrest.
RESULTS: We characterized a family presenting with a history of ventricular
fibrillation (VF) and sudden death without electrocardiographic or
echocardiographic abnormalities at rest. Two siblings died suddenly at the ages
of 9 and 10 years, and another 2 were resuscitated from out-of-hospital cardiac
arrest with documented VF at ages 10 and 16 years, respectively. Exome
sequencing identified a missense mutation affecting a highly conserved residue
(p.F90L) in the CALM1 gene encoding calmodulin. This mutation was also carried
by 1 of the siblings who died suddenly, from whom DNA was available. The
mutation was present in the mother and in another sibling, both asymptomatic but
displaying a marginally prolonged QT interval during exercise.
CONCLUSIONS: We identified a mutation in CALM1 underlying IVF manifesting in
childhood and adolescence. The causality of the mutation is supported by
previous studies demonstrating that F90 mediates the direct interaction of CaM
with target peptides. Our approach highlights the utility of exome sequencing in
uncovering the genetic defect even in families with a small number of affected
individuals. |
What is the mechanism of action of DNA topoisomerase II inhibitors? | DNA topoisomerase II inhibitors eliminate cancer cells by causing DNA double-strand breaks, finally leading to apoptotic cell death. Moreover, drug-induced histone eviction was also shown to be associated with attenuated DNA repair, epigenetic changes and transcpription deregulation. | We have analyzed the interference of antitumoral drugs acting through the
inhibition of DNA topoisomerase II on the human HeLa cell metabolism. Different
compounds characterized by a diverse mechanism of action have been used, namely
m-amsacrine, an intercalative drug, etoposide, which does not intercalate DNA,
and suramin, which exerts its effect through an unknown mechanism. In HeLa cells
treated with increasing doses of these drugs, we have examined cell viability
and DNA synthesis capacity, and we have evaluated topoisomerase II activity.
Cellular morphology and DNA integrity have been studied in order to characterize
the mechanism of cell death. The results we have obtained clearly indicate that
topoisomerase II poisons induce cell death by apoptosis. These observations
suggest a role of the inhibition of topoisomerase II activity in the apoptotic
program. |
Are histone deacetylase (HDAC) inhibitors good candidates to control metastasis of solid tumors? | Yes, some HDAC inhibitors, such as Vorinostat, are on trial for human treatment after proving successful in animal models. Combination with other drugs is likely to be needed to control metastasis. | Cancer metastasis represents the most important cause of cancer death and agents
that may inhibit tumor cell invasion have been extensively pursued. In the
present study, we have examined the anti-invasive effect of apicidin
[cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl -L-2-amin
o-8-oxodecanoyl)], a fungal metabolite that was identified as an antiprotozoal
agent known to inhibit parasite histone deacetylase (HDAC). We show that
apicidin significantly inhibits H-ras-induced invasive phenotype of MCF10A human
breast epithelial cells in parallel with a specific downregulation of matrix
metalloproteinase (MMP)-2, but not MMP-9. We also show that apicidin induces a
morphological reversal and growth inhibition of H-ras MCF10A cells similar to
that induced by other HDAC inhibitors. Taken in conjunction with the fact that
uncontrolled ras activation is probably the most common genetic defect in human
cancer cells, our data showing the anti-invasive and detransforming activities
of apicidin in H-ras-transformed MCF10A cells may suggest a potential use of
HDAC inhibitors for treatment of cancer. AN-7, a prodrug of butyric acid, induced histone hyperacetylation and
differentiation and inhibited proliferation of human prostate 22Rv1 cancer cells
in vitro and in vivo. In nude mice implanted with these cells, 50 mg/kg AN-7
given orally thrice a week led to inhibition of tumor growth and metastasis,
tumor regression in >25% of animals and increased survival. Median time to the
experimental end point (tumor volume 2 cm3 or death) in the untreated was 52
days, and average tumor volume was 0.8 +/- 0.18 cm3. At the same time, 94.4% of
AN-7-treated mice survived and had average tumor volumes of 0.37 +/- 0.1 cm3.
PSA expression was a useful marker for 22Rv1 lung metastasis detection. Sizeable
metastases positively stained for PSA and limited air gaps were found in lungs
of untreated mice. In animals treated with AN-7, lung morphology appeared
normal. Primary tumors of treated animals were highly positive for PSA and had
an elevated level of p21 and the proapoptotic protein Bax. Sections taken from
AN-7-treated animals, examined under an electron microscope, exhibited condensed
chromatin and apoptotic bodies. PSA serum levels were higher in untreated
compared to treated animals and correlated with tumor volume. Since prolonged
oral administration with 50 mg/kg or a single oral dose of 1.2 g/kg AN-7 did not
cause adverse effects and the former exhibited significant anticancer activity,
AN-7 is likely to display a high therapeutic index and may be beneficial for
prostate cancer patients. Histone deacetylase inhibitory prodrugs that are metabolized to butyric acid and
formaldehyde possess antineoplastic properties and low toxicity. We sought to
characterize the antiangiogenic and antimetastatic activities of two lead
prodrugs, pivaloyloxymethyl butyrate (AN-9) and butyroyloxymethyl-diethyl
phosphate (AN-7) in murine cancer models. In the sc implanted human colon
carcinoma HT-29 xenograft model AN-7, exhibited superior anticancer activity
compared to AN-9, as was evident by the significantly greater inhibition of
tumor growth and reduction of serum CEA. AN-7 was also more effective in
reducing mean vessel density (MVD) by 7-fold, bFGF, Ki-67 (7-fold) and
HIF-1alpha in immunohistochemically stained tumor sections. Semi-quantitative
evaluation of the levels of bFGF, HDAC1 and HIF-1alpha by Western blot analysis
showed a decrease in expression only in the tumors of mice treated with AN-7.
The level of bFGF was reduced 3-fold in the tumor and that of TIMP1 was elevated
(by 3-fold) in the serum of AN-7 treated mice. In a 4T1 metastatic breast
carcinoma model, AN-7 inhibited the formation of lung lesions by 76% and AN-9 by
47%, further demonstrating the greater efficacy of AN-7 compared to AN-9
(P<0.02). Both AN-7 and AN-9 exhibited antimetastatic and antiangiogenic
activities by reducing vascularization, bFGF expression and HIF-1alpha. Yet,
AN-7 was more potent than AN-9. PURPOSE: Histone deactylase inhibitors (HDACi) are a promising new class of
anticancer therapeutics; however, little is known about HDACi activity in soft
tissue sarcoma (STS), a heterogeneous cohort of mesenchymal origin maligcies.
Consequently, we investigated the novel HDACi PCI-24781, alone/in combination
with conventional chemotherapy, to determine its potential anti-STS-related
effects and the underlying mechanisms involved.
EXPERIMENTAL DESIGN: Immunoblotting was used to evaluate the effects of
PCI-24781 on histone and nonhistone protein acetylation and expression of
potential downstream targets. Cell culture-based assays were utilized to assess
the effects of PCI-24781 on STS cell growth, cell cycle progression, apoptosis,
and chemosensitivity. Quantitative reverse transcription-PCR, chromatin
immunoprecipitation, and reporter assays helped elucidate molecular mechanisms
resulting in PCI-24781-induced Rad51 repression. The effect of PCI-24781, alone
or with chemotherapy, on tumor and metastatic growth was tested in vivo using
human STS xenograft models.
RESULTS: PCI-24781 exhibited significant anti-STS proliferative activity in
vitro, inducing S phase depletion, G(2)/M cell cycle arrest, and increasing
apoptosis. Superior effects were seen when combined with chemotherapy. A
PCI-24781-induced reduction in Rad51, a major mediator of DNA double-strand
break homologous recombination repair, was shown and may be a mechanism
underlying PCI-24781 chemosensitization. We showed that PCI-24781
transcriptionally represses Rad51 through an E2F binding-site on the Rad51
proximal promoter. Although single-agent PCI-24781 had modest effects on STS
growth and metastasis, marked inhibition was observed when combined with
chemotherapy.
CONCLUSIONS: In light of these findings, this novel molecular-based combination
may be applicable to multiple STS histologic subtypes, and potentially merits
rigorous evaluation in human STS clinical trials. PURPOSE: Histone deacetylase (HDAC) inhibitors have shown promising clinical
activity in the treatment of hematologic maligcies, but their activity in
solid tumor indications has been limited. Most HDAC inhibitors in clinical
development only transiently induce histone acetylation in tumor tissue. Here,
we sought to identify a "second-generation" class I HDAC inhibitor with
prolonged pharmacodynamic response in vivo, to assess whether this results in
superior antitumoral efficacy.
EXPERIMENTAL DESIGN: To identify novel HDAC inhibitors with superior
pharmacodynamic properties, we developed a preclinical in vivo tumor model, in
which tumor cells have been engineered to express fluorescent protein dependent
on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the
tumor response to HDAC inhibitors.
RESULTS: In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic
acid analogues resulted in the identification of JNJ-26481585. Once daily oral
administration of JNJ-26481585 induced continuous histone H3 acetylation. The
prolonged pharmacodynamic response translated into complete tumor growth
inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas
5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of
C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin
showed modest activity. Further characterization revealed that JNJ-26481585 is a
pan-HDAC inhibitor with marked potency toward HDAC1 (IC(50), 0.16 nmol/L).
CONCLUSIONS: The potent antitumor activity as a single agent in preclinical
models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a
promising "second-generation" HDAC inhibitor. The compound is currently in
clinical studies, to evaluate its potential applicability in a broad spectrum of
both solid and hematologic maligcies. PURPOSE: Although histone deacetylase inhibitors (HDACi) are emerging as a new
class of anticancer agents, one of the most significant concerns is that
interactions with a wide array of substrates using these agents might initiate
both therapeutic and undesired protective responses. Here, we sought to identify
the potential protective reactions initiated by HDACi and determine whether
targeting these reactions would enhance the antitumoral activity of HDACi.
EXPERIMENTAL DESIGN: Gene expression profiles were analyzed by cDNA microarray
in Molt-4 cells before and after treatment of vorinostat. Induction of CD146 by
vorinostat was examined in a wide range of tumors and nonmaligt cells. AA98,
an anti-CD146 monoclonal antibody, was used to target CD146 function.
Synergistic antitumoral and antiangiogenic effects between AA98 and vorinostat
were examined both in vitro and in vivo. The potential effect of combined AA98
and vorinostat treatment on the AKT pathway was determined by Western blotting.
RESULTS: The induction of CD146 is a common phenomenon in vorinostat-treated
cancer but not in nonmaligt cells. Targeting of CD146 with AA98 substantially
enhanced vorinostat-induced killing via the suppression of activation of AKT
pathways in cancer cells. Moreover, AA98 in combination with vorinostat
significantly inhibited angiogenesis. In vivo, AA98 synergized with vorinostat
to inhibit tumor growth and metastasis.
CONCLUSION: The present study provided the first evidence that an undesired
induction of CD146 could serve as a protective response to offset the antitumor
efficacy of vorinostat. On the other hand, targeting CD146 in combination with
vorinostat could be exploited as a novel strategy to more effectively kill
cancer cells. Histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) show promise for the treatment of cancers. The
purpose of this study was to examine the molecular mechanisms by which HDAC
inhibitor MS-275 sensitizes TRAIL-resistant breast cancer cells in vivo,
inhibits angiogenesis and metastasis, and reverses epithelial-mesenchymal
transition (EMT). BALB/c nude mice were orthotopically implanted with
TRAIL-resistant invasive breast cancer MDA-MB-468 cells and treated
intravenously with MS-275, TRAIL, or MS-275 followed by TRAIL, 4 times during
first 3 weeks. Treatment of mice with TRAIL alone had no effect on tumor growth,
metastasis, angiogenesis, and EMT. In comparison, MS-275 sensitized
TRAIL-resistant xenografts by inducing apoptosis, inhibiting tumor cell
proliferation, angiogenesis, metastasis, and reversing EMT. Treatment of nude
mice with MS-275 resulted in downregulation of NF-κB and its gene products
(cyclin D1, Bcl-2, Bcl-X(L), VEGF, HIF-1α, IL-6, IL-8, MMP-2, and MMP-9) and
upregulation of DR4, DR5, Bax, Bak, and p21(/CIP1) in tumor cells. Furthermore,
MS-275-treated mice showed significantly reduced tumor growth and decreased
circulating vascular VEGFR2-positive endothelial cells, CD31-positive or von
Willebrand factor-positive blood vessels, and lung metastasis compared with
control mice. Interestingly, MS-275 caused "cadherin switch" and reversed EMT as
shown by the upregulation of E-cadherin and downregulation of N-cadherin and
transcription factors Snail, Slug, and ZEB1. In conclusion, sequential
treatments of mice with MS-275 followed by TRAIL may target multiple pathways to
reverse EMT and inhibit tumor progression, angiogenesis, and metastasis and
represent a novel therapeutic approach to treat cancer. The concept of molecular tumor targeting might provide new hope in the treatment
of advanced prostate cancer. We evaluated metastasis blocking properties of the
histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian
target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell lines.
RAD001 or VPA were applied to PC-3 or LNCaP cells, either separately or in
combination. Adhesion to vascular endothelium or to immobilized collagen,
fibronectin or laminin was quantified. Migration and invasion were explored by a
modified Boyden chamber assay. Integrin α and β subtypes were analyzed by flow
cytometry, western blotting and RT-PCR. Effects of drug treatment on integrin
related signaling, Akt and p70S6kinase activation, histone H3 and H4 acetylation
were also determined. Separate application of RAD001 or VPA distinctly reduced
tumor cell adhesion, migration and invasion, accompanied by elevated Akt
activation and p70S6kinase de-activation. Integrin subtype expression was
altered significantly by both compounds (VPA > RAD001). When both drugs were
used in concert additive effects were observed on the migratory and invasive
behavior but not on tumor-endothelium and tumor-matrix interaction. Separate
mTOR or HDAC inhibition slows processes related to tumor metastasis. The
RAD001-VPA combination showed advantage over VPA monotreatment with particular
respect to migration and invasion. Ongoing studies are required to assess the
relevance of VPA monotherapy versus VPA-RAD001 combination on tumor cell
motility. PURPOSE: Metastasis is responsible for the death of most cancer patients, yet
few therapeutic agents are available which specifically target the molecular
events that lead to metastasis. We recently showed that inactivating mutations
in the tumor suppressor gene BAP1 are closely associated with loss of
melanocytic differentiation in uveal melanoma (UM) and metastasis. The purpose
of this study was to identify therapeutic agents that reverse the phenotypic
effects of BAP1 loss in UM.
EXPERIMENTAL DESIGN: In silico screens were done to identify therapeutic
compounds predicted to differentiate UM cells using Gene Set Enrichment Analysis
and Connectivity Map databases. Valproic acid (VPA), trichostatin A, LBH-589,
and suberoylanilide hydroxamic acid were evaluated for their effects on UM cells
using morphologic evaluation, MTS viability assays, bromodeoxyuridine
incorporation, flow cytometry, clonogenic assays, gene expression profiling,
histone acetylation and ubiquitination assays, and a murine xenograft
tumorigenicity model.
RESULTS: Histone deacetylase (HDAC) inhibitors induced morphologic
differentiation, cell-cycle exit, and a shift to a differentiated, melanocytic
gene expression profile in cultured UM cells. VPA inhibited the growth of UM
tumors in vivo.
CONCLUSIONS: These findings suggest that HDAC inhibitors may have therapeutic
potential for inducing differentiation and prolonged dormancy of micrometastatic
disease in UM. Atorvastatin and suberoylanilide hydroxamic acid (SAHA) were evaluated for
chemoprevention of mouse lung tumors. In Experiment 1, lung tumors were induced
by vinyl carbamate in strain A/J mice followed by 500 mg/kg SAHA, 60 or 180
mg/kg atorvastatin, and combinations containing SAHA and atorvastatin
administered in their diet. SAHA and both combinations, but not atorvastatin,
decreased the multiplicity of lung tumors, including large adenomas and
adenocarcinomas with the combinations demonstrating the greatest efficacy. In
Experiment 2, lung tumors were induced by
4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol in strain A/J mice followed by
180 mg/kg atorvastatin, 500 mg/kg SAHA, or both drugs administered in the diet.
SAHA and the combination of both drugs, but not atorvastatin alone, decreased
the multiplicity of lung tumors and large tumors, with the combination
demonstrating greater efficacy. In Experiment 3, lung tumors were induced by
1,2-dimethylhydrazine in Swiss-Webster mice followed by 160 mg/kg atorvastatin,
400 mg/kg SAHA, or a combination of both drugs administered in the diet. SAHA
and the combination, but not atorvastatin, decreased the multiplicity of lung
tumors with the combination demonstrating greater efficacy. The multiplicity of
colon tumors was decreased by SAHA, atorvastatin, and the combination, without
any significant difference in their efficacy. mRNA expression analysis of lung
tumor bearing mice suggested that the enhanced chemopreventive activity of the
combination is related to atorvastatin modulation of DNA repair, SAHA modulation
of angiogenesis, and both drugs modulating invasion and metastasis pathways.
Atorvastatin demonstrated chemoprevention activity as indicated by the
enhancement of the efficacy of SAHA to prevent mouse lung tumors. PURPOSE: Histone deacetylase inhibitors (HDACi) are actively explored as
new-generation epigenetic drugs but have low efficacy in cancer monotherapy. To
reveal new mechanism for combination therapy, we show that HDACi induce cell
death but simultaneously activate tumor-progressive genes to ruin therapeutic
efficacy. Combined treatments to target tumorigenesis and HDACi-activated
metastasis with low toxic modalities could develop new strategies for long-term
cancer therapy.
EXPERIMENTAL DESIGN: Because metastasis is the major cause of cancer mortality,
we measured cell migration activity and profiled metastasis-related gene
expressions in HDACi-treated cancer cells. We developed low toxic combination
modalities targeting tumorigenesis and HDACi-activated metastasis for
preclinical therapies in mice.
RESULTS: We showed that cell migration activity was dramatically and dose
dependently enhanced by various classes of HDACi treatments in 13 of 30 examined
human breast, gastric, liver, and lung cancer cell lines. Tumor metastasis was
also enhanced in HDACi-treated mice. HDACi treatments activated multiple PKCs
and downstream substrates along with upregulated proapoptotic p21. For targeting
tumorigenesis and metastasis with immediate clinical impact, we showed that new
modalities of HDACi combined drugs with PKC inhibitory agent, curcumin or
tamoxifen, not only suppressed HDACi-activated tumor progressive proteins and
cell migration in vitro but also inhibited tumor growth and metastasis in vivo.
CONCLUSION: Treatments of different structural classes of HDACi simultaneously
induced cell death and promoted cell migration and metastasis in multiple cancer
cell types. Suppression of HDACi-induced PKCs leads to development of low toxic
and long-term therapeutic strategies to potentially treat cancer as a chronic
disease. |
Is muscle lim protein (MLP) involved in cardiomyopathies? | The skeletal muscle LIM protein 1 (SLIM1) is highly expressed in skeletal and cardiac muscle, and its expression is downregulated significantly in dilated human cardiomyopathy. Loss of murine MLP results in dilated cardiomyopathy, and mutations in human MLP lead to cardiac hypertrophy, indicating a critical role for MLP in maintaining normal cardiac function. | The skeletal muscle LIM protein 1 (SLIM1) is highly expressed in skeletal and
cardiac muscle, and its expression is downregulated significantly in dilated
human cardiomyopathy. However, the function of SLIM1 is unknown. In this study,
we investigated the intracellular localization of SLIM1. Endogenous and
recombit SLIM1 localized to the nucleus, stress fibers, and focal adhesions
in skeletal myoblasts plated on fibronectin, collagen, or laminin. However,
after inhibition of integrin signaling either by plating on poly-l-lysine or by
soluble RGD peptide, SLIM1 localized diffusely in the cytosol, with decreased
nuclear expression. Disruption of the actin cytoskeleton by cytochalasin D did
not inhibit nuclear localization of SLIM1 in integrin-activated cells. Green
fluorescent protein-tagged SLIM1 shuttled in the nucleus of untransfected NIH
3T3 cells, in a heterokaryon fusion assay. Overexpression of SLIM1 in Sol8
myoblasts inhibited cell adhesion and promoted cell spreading and migration.
These studies show SLIM1 localizes in an integrin-dependent manner to the
nucleus and focal adhesions where it functions downstream of integrin activation
to promote cell spreading and migration. BACKGROUND: Muscle LIM protein (MLP) is an essential nuclear regulator of
myogenic differentiation. Additionally, it may act as an integrator of protein
assembly of the actin-based cytoskeleton. MLP-knockout mice develop a marked
cardiac hypertrophy reaction and dilated cardiomyopathy (DCM). MLP is therefore
a candidate gene for heritable forms of hypertrophic cardiomyopathy (HCM) and
DCM in humans.
METHODS AND RESULTS: We analyzed 1100 unrelated individuals (400 patients with
DCM, 200 patients with HCM, and 500 controls) for mutations in the human CRP3
gene that encodes MLP. We found 3 different missense mutations in 3 unrelated
patients with familial HCM but detected no mutation in the DCM group or the
controls. All mutations predicted an amino acid exchange at highly conserved
residues in the functionally important LIM1 domain, which is responsible for
interaction with alpha-actinin and with certain muscle-specific transcription
factors. Protein-binding studies indicate that mutations in the CRP3 gene lead
to a decreased binding activity of MLP to alpha-actinin. All 3 index patients
were characterized by typical asymmetrical septal hypertrophy. Family studies
revealed cosegregation of clinically affected individuals with the respective
mutations in MLP.
CONCLUSION: Here, we present evidence that mutations in the CRP3/MLP gene can
cause HCM. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two
genes have been identified for the X-linked forms (dystrophin and tafazzin),
while mutations in multiple genes cause autosomal domit DCM. Muscle LIM
protein (MLP) is a member of the cysteine-rich protein (CRP) family and has been
implicated in both myogenesis and sarcomere assembly. In the latter role, it
binds zyxin and alpha-actinin, both of which are involved in actin organization.
An MLP-deficient mouse has been described; these mice develop dilated
cardiomyopathy and heart failure. Based upon these data, and the recent
descriptions of mutations in MLP in patients with DCM or hypertrophic
cardiomyopathy, we screened patients for mutations in the MLP and
alpha-actinin-2 genes. We identified a patient with DCM and EFE, having a
mutation in MLP with the residue lysine 69 substituted by arginine (K69R). This
is within a highly conserved region adjacent to the first LIM domain involved in
alpha-actinin binding. Analysis in cell culture systems demonstrated that the
mutation abolishes the interaction between MLP and alpha-actinin-2 and the
cellular localization of MLP was altered. In another individual with DCM, a W4R
mutation was identified. However, this mutation did not segregate with disease
in this family. In another patient with DCM, a Q9R mutation was identified in
alpha-actinin-2. This mutation also disrupted the interaction with MLP and
appeared to inhibit alpha-actinin function in cultured cells, in respect to the
nuclear localization of actinin and the initiation of cellular differentiation. Previous work has shown that mutations in muscle LIM protein (MLP) can cause
hypertrophic cardiomyopathy (HCM). In order to gain an insight into the
molecular basis of the disease phenotype, we analysed the binding
characteristics of wild-type MLP and of the (C58G) mutant MLP that causes
hypertrophic cardiomyopathy. We show that MLP can form a ternary complex with
two of its previously documented myofibrillar ligand proteins, N-RAP and
alpha-actinin, which indicates the presence of distinct, non-overlapping binding
sites. Our data also show that, in comparison to wild-type MLP, the capacity of
the mutated MLP protein to bind both N-RAP and alpha-actinin is significantly
decreased. In addition, this single point mutation prevents zinc coordination
and proper folding of the second zinc-finger in the first LIM domain, which
consequently renders the protein less stable and more susceptible to
proteolysis. The molecular basis for HCM-causing mutations in the MLP gene might
therefore be an alteration in the equilibrium of interactions of the ternary
complex MLP-N-RAP-alpha-actinin. This assumption is supported by the previous
observation that in the pathological situation accompanied by MLP down
regulation, cardiomyocytes try to compensate for the decreased stability of MLP
protein by increasing the expression of its ligand N-RAP, which might finally
result in the development of myocyte disarray that is characteristic of this
disease. OBJECTIVE: Defects in myocardial mitochondrial structure and function have been
associated with heart failure in humans and animal models. Mice lacking the
muscle LIM protein (MLP) develop morphological and clinical signs resembling
human dilated cardiomyopathy and heart failure. We tested the hypothesis that
defects in the cytoskeleton lead to dilated cardiomyopathy through mitochondrial
dysfunction in the MLP mouse model.
METHODS AND RESULTS: Oxidative phosphorylation activity was determined in left
ventricles of MLP knockout (KO) mice and control littermates by measuring
complex activities of the electron transport chain (I-IV) and ATP synthase
(complex V). All complexes and citrate synthase (CS) showed decreased activities
in the KO mice, although activity per amount of CS, a measure for mitochondrial
density, was normal. Light and electron microscopy revealed a disorganization of
mitochondria and a dramatic decrease in mitochondrial density, even revealing
regions completely lacking mitochondria in the KO hearts. Real-time PCR analysis
showed decreased transcript levels of mtDNA and nuclear encoded mitochondrial
genes and of peroxisome proliferator activated receptor gamma co-activator
1alpha (PGC-1alpha), a key regulator of mitochondrial biogenesis. MtDNA copy
number (ratio mtDNA/nuclear DNA) was slightly increased in the MLP KO mice.
CONCLUSION: Our results show that the absence of MLP causes a local loss of
mitochondria. We hypothesize that this is caused by a disturbed interaction
between cytoskeleton and mitochondria, which interferes with energy sensing and
energy transfer. Recovery of energy depletion by stimulating mitochondrial
biogenesis might be a useful therapeutic strategy for improving the energy
imbalance in heart failure. Hypertrophic cardiomyopathy (HCM) is a frequent genetic cardiac disease and the
most common cause of sudden cardiac death in young individuals. Most of the
currently known HCM disease genes encode sarcomeric proteins. Previous studies
have shown an association between CSRP3 missense mutations and either dilated
cardiomyopathy (DCM) or HCM, but all these studies were unable to provide
comprehensive genetic evidence for a causative role of CSRP3 mutations. We used
linkage analysis and identified a CSRP3 missense mutation in a large German
family affected by HCM. We confirmed CSRP3 as an HCM disease gene. Furthermore,
CSRP3 missense mutations segregating with HCM were identified in four other
families. We used a newly designed monoclonal antibody to show that muscle LIM
protein (MLP), the protein encoded by CSRP3, is mainly a cytosolic component of
cardiomyocytes and not tightly anchored to sarcomeric structures. Our functional
data from both in vitro and in vivo analyses suggest that at least one of MLP's
mutated forms seems to be destabilized in the heart of HCM patients harbouring a
CSRP3 missense mutation. We also present evidence for mild skeletal muscle
disease in affected persons. Our results support the view that HCM is not
exclusively a sarcomeric disease and also suggest that impaired mechano-sensory
stress signalling might be involved in the pathogenesis of HCM. Muscle LIM protein (MLP) can be found at the Z-disk of sarcomeres where it is
hypothesized to be involved in sensing muscle stretch. Loss of murine MLP
results in dilated cardiomyopathy, and mutations in human MLP lead to cardiac
hypertrophy, indicating a critical role for MLP in maintaining normal cardiac
function. Loss of MLP in Drosophila (mlp84B) also leads to muscle dysfunction,
providing a model system to examine MLP's mechanism of action. Mlp84B-null flies
that survive to adulthood are not able to fly or beat their wings. Transgenic
expression of the mlp84B gene in the Mlp84B-null background rescues flight
ability and restores wing beating ability. Mechanical analysis of skinned flight
muscle fibers showed a 30% decrease in oscillatory power production and a slight
increase in the frequency at which maximum power is generated for fibers lacking
Mlp84B compared with rescued fibers. Mlp84B-null muscle fibers displayed a 25%
decrease in passive, active, and rigor stiffness compared with rescued fibers,
but no significant decrease in isometric tension generation was observed. Muscle
ultrastructure of Mlp84B-null muscle fibers is grossly normal; however, the null
fibers have a slight decrease, 11%, in thick filament number per unit
cross-sectional area. Our data indicate that MLP contributes to muscle stiffness
and is necessary for maximum work and power generation. AIMS: Muscle LIM protein (MLP) null mice are often used as a model for human
dilated cardiomyopathy. So far, little is known about the time course and
pathomechanisms leading to the development of the adult phenotype.
METHODS AND RESULTS: We systematically analysed the contractile phenotype,
myofilament calcium (Ca(2)(+)) responsiveness, passive myocardial mechanics,
histology, and mRNA expression in mice aged 4 and 12 weeks. In 4-week-old
animals, there was no significant difference in the force-frequency relationship
(FFR) and catecholamine response of intact isolated papillary muscles between
wild-type (WT) and MLP null myocardium. In 12-week-old animals, WT myocardium
exhibited a significantly positive FFR, while that of MLP null mice was
significantly negative, and the inotropic response to catecholamines was
significantly reduced in MLP null mice. This time course of decline in
contractile function was confirmed in vivo by echocardiography. Whereas at 4
weeks of age MLP null mice and WT littermates showed similar levels of SERCA2a
(sarcoplasmic reticulum Ca(2+) ATPase) expression, the expression was
significantly lower in 12-week-old MLP null mice compared with littermate
controls. Myofilament Ca(2)(+) responsiveness was not affected by the lack of
MLP, irrespective of age. Whereas in 4-week-old animals MLP null myocardium
showed a trend to an increased compliance compared with the WT, myocardium of
12-week-old MLP null mice was significantly less compliant than WT myocardium.
Parallel to the decrease in compliance there was an increase in fibrosis in the
MLP null animals.
CONCLUSION: Our data suggest that MLP deficiency does not primarily influence
myocardial contractility. A lack of MLP leads to an age-dependent impairment of
excitation-contraction coupling with resulting contractile dysfunction and
secondary fibrosis. Muscle LIM protein (MLP) has been proposed to be a central player in the
pathogenesis of heart muscle disease. In line with this notion, the homozygous
loss of MLP results in cardiac hypertrophy and dilated cardiomyopathy. Moreover,
MLP is induced in several models of cardiac hypertrophy such as aortic banding
and myocardial infarction. We thus hypothesized that overexpression of MLP might
change the hypertrophic response to cardiac stress. In order to answer the
question whether MLP modulates cardiac hypertrophy in vivo, we generated a novel
transgenic mouse model with cardiac-specific overexpression of MLP. Three
independent transgenic lines did not show a pathological phenotype under
baseline conditions. Specifically, contractile function and heart weight to body
weight ratios at different ages were normal. Next, the transgenic animals were
challenged with pressure overload due to aortic constriction. Surprisingly,
transgenic mice developed cardiac hypertrophy to the same extent as their
wild-type littermates. Moreover, neither contractile dysfunction nor
pathological gene expression in response to pressure overload were
differentially affected by MLP overexpression. Finally, in a milder in vivo
model of hypertrophy induced by chronic infusion of angiotensin-II, cardiac mass
and hypertrophic gene expression were again identical in MLP transgenic mice and
controls. Taken together, we provide evidence that cardiac overexpression of MLP
does not modulate the heart's response to various forms of pathological stress. Muscle lim protein (MLP) has emerged as a critical regulator of striated muscle
physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3
(CSRP3), the gene encoding MLP, have been directly associated with human
cardiomyopathies, whereas aberrant expression patterns are reported in human
cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has
an important role in both myogenic differentiation and myocyte cytoarchitecture,
although the full spectrum of its intracellular roles has not been delineated.
We report the discovery of an alternative splice variant of MLP, designated as
MLP-b, showing distinct expression in neuromuscular disease and direct roles in
actin dynamics and muscle differentiation. This novel isoform originates by
alternative splicing of exons 3 and 4. At the protein level, it contains the
N-terminus first half LIM domain of MLP and a unique sequence of 22 amino acids.
Physiologically, it is expressed during early differentiation, whereas its
overexpression reduces C2C12 differentiation and myotube formation. This may be
mediated through its inhibition of MLP/cofilin-2-mediated F-actin dynamics. In
differentiated striated muscles, MLP-b localizes to the sarcomeres and binds
directly to Z-disc components, including α-actinin, T-cap and MLP. The findings
of the present study unveil a novel player in muscle physiology and
pathophysiology that is implicated in myogenesis as a negative regulator of
myotube formation, as well as in differentiated striated muscles as a
contributor to sarcomeric integrity. |
Which gene harbors the mutation T790M? | The T790M mutation refers to the mutation in exon 20 of the EGFR gene | PURPOSE: Acquired resistance to erlotinib in patients with EGFR-mutant non-small
cell lung cancer can result from aberrant activation of alternative receptor
tyrosine kinases, such as the HGF-driven c-MET receptor. We sought to determine
whether inhibition of AKT signaling could augment erlotinib activity and
abrogate HGF-mediated resistance.
METHODS: The effects of MK-2206, a selective AKT inhibitor, were evaluated in
combination with erlotinib on a large panel of 13 lung cancer cell lines
containing different EGFR or KRAS abnormalities. The activity of the combination
was assessed using proliferation assays, flow cytometry and immunoblotting. The
MEK inhibitor PD0325901 was used to determine the role of the MAP kinase pathway
in erlotinib resistance.
RESULTS: The combination of MK-2206 and erlotinib resulted in synergistic growth
inhibition independent of EGFR mutation status. In cell lines where HGF blocked
the anti-proliferative and cytotoxic effects of erlotinib, MK-2206 could restore
cell cycle arrest, but MEK inhibition was required for erlotinib-dependent
apoptosis. Both AKT and MEK inhibition contributed to cell death independent of
erlotinib in the T790M-containing H1975 and the EGFR-WT cell lines tested.
CONCLUSIONS: These findings illustrate the potential advantages and challenges
of combined signal transduction inhibition as a generalized strategy to
circumvent acquired erlotinib resistance. To overcome T790M-mediated acquired resistance of lung cancer cells to epidermal
growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), second generation
TKIs such as BIBW2992 (afatinib) and third generation TKIs including WZ4002 have
been developed. However, clinical data on their efficacy in treating T790M
mutant tumors are lacking. Histone deacetylase (HDAC) inhibitors have been
reported to arrest cell growth and to lead to differentiation and apoptosis of
various cancer cells, both in vitro and in vivo. In the present study, we
assessed whether the combination of suberoylanilide hydroxamic acid (SAHA,
vorinostat), a potent HDAC inhibitor, and BIBW2992 or WZ4002 could overcome EGFR
TKI resistance associated with T790M mutation in lung cancer cells. While
treatment with BIBW2992 or WZ4002 alone slightly reduced the viability of PC-9G
and H1975 cells, which possess T790M mutation, combining them with SAHA resulted
in significantly decreased cell viability through the activation of the
apoptotic pathway. This combination also enhanced autophagy occurrence and
inhibition of autophagy significantly reduced the apoptosis induced by the
combination treatment, showing that autophagy is required for the enhanced
apoptosis. Caspase-independent autophagic cell death was also induced by the
combination treatment with SAHA and either BIBW2992 or WZ4002. Finally, the
combined treatment with SAHA and either BIBW2992 or WZ4002 showed an enhanced
anti-tumor effect on xenografts of H1975 cells in vivo. In conclusion, the
combination of new generation EGFR TKIs and SAHA may be a new strategy to
overcome the acquired resistance to EGFR TKIs in T790M mutant lung cancer. INTRODUCTION: Patients with non-small-cell lung cancer (NSCLC) with somatic
activating mutations of the epidermal growth factor receptor gene (EGFR
mutations) generally respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs).
β-Catenin is a key component of the Wnt/β-Catenin signal and is an important
oncogene that is involved in the pathogenesis and progression of maligt
tumors, especially cancer stem cells.
METHODS AND RESULTS: We found that EGFR-mutated NSCLC cell lines exhibited a
high expression level of β-Catenin, compared with cell lines with the wild-type
EGFR gene, and XAV939 (a β-Catenin inhibitor) enhanced the sensitivities to
EGFR-TKI in EGFR-mutated NSCLC cell lines. In EGFR-mutated NSCLC cell lines with
the acquired resistance threonine-to-methionine mutation in codon 790 (T790M)
mutation, XAV939 enhanced the sensitivity of the cells to an irreversible
EGFR-TKI but not a reversible EGFR-TKI. The combination of XAV939 and EGFR-TKIs
strongly inhibited the β-Catenin signal and strongly decreased the
phosphorylation of EGFR, compared with the use of EGFR-TKIs alone, suggesting an
interaction between EGFR and the β-Catenin signal. The stem cell-like properties
of the EGFR-mutated cell line carrying the T790M mutation were inhibited by
XAV939 and BIBW2992 (an irreversible EGFR-TKI). Furthermore, the stem cell-like
properties were strongly inhibited by a combination of both the agents. A
xenograft study demonstrated that β-Catenin knockdown enhanced the antitumor
effect of BIBW2992 in the EGFR-mutated NSCLC cell line carrying the T790M
mutation.
CONCLUSION: Our findings indicate that β-Catenin might be a novel therapeutic
target in EGFR-mutated NSCLC carrying the T790M mutation. Non‑small cell lung cancer (NSCLC) cells harboring mutations in the epidermal
growth factor receptor (EGFR) gene initially respond well to EGFR tyrosine
kinase inhibitors (TKI), including gefitinib. However the tumor cells will
invariably develop acquired resistance to the drug. The EGFR T790M mutation is
generally considered to be the molecular genetic basis of acquired TKI
resistance. The present study aimed to explore how the T790M mutation induces
tumor cells to escape inhibition by TKI treatment. An acquired
gefitinib‑resistant cell line (NCI‑H1975/GR) was generated from the NCI‑H1975
human NSCLC cell line, which harbors the sensitive L858R and resistant T790M
mutations of EGFR. The resistant cell line was established by exposing the cells
intermittently to increasing concentrations of gefitinib. The mechanisms by
which NSCLC acquires resistance to TKIs based on the T790M mutation, were
investigated by detecting the protein expression levels of the EGFR/Kirsten rat
sarcoma viral oncogene homolog (KRAS)/v‑Raf murine sarcoma viral oncogene
homolog B (BRAF) transduction pathway, and epithelial‑mesenchymal transition
(EMT) with immunocytochemistry. The resistance of the NCI‑H1975/GR cells to
gefitinib was 2.009‑fold, as compared with the parent cells; however, the
protein expression levels of EGFR, KRAS and BRAF were lower in the resistant
cells. Some mesenchymal morphology was observed in the NCI‑H1975/GR cells,
alongside a decreasing E‑cadherin expression and increasing vimentin expression.
These results suggest that the reactivation of the EGFR/KRAS/BRAF transduction
pathway was not detected in the NCI‑H1975/GR cells. EMT may have an important
role in the development of acquired resistance to EGFR‑TKIs in NSCLC cells with
sensitivity and resistance mutations. BACKGROUND: Two types of epidermal growth factor receptor (EGFR) mutations in
exon 19 and exon 21 (ex19del and L858R) are prevalent in lung cancer patients
and sensitive to targeted EGFR inhibition. A resistance mutation in exon 20
(T790M) has been found to accompany drug treatment when patients relapse. These
three mutations are valuable companion diagnostic biomarkers for guiding
personalized treatment. Quantitative polymerase chain reaction (qPCR)-based
methods have been widely used in the clinic by physicians to guide treatment
decisions. The aim of this study was to evaluate the technical and clinical
sensitivity and specificity of the droplet digital polymerase chain reaction
(ddPCR) method in detecting the three EGFR mutations in patients with lung
cancer.
METHODS: Genomic DNA from H1975 and PC-9 cells, as well as 92 normal human blood
specimens, was used to determine the technical sensitivity and specificity of
the ddPCR assays. Genomic DNA of formalin-fixed, paraffin-embedded specimens
from 78 Chinese patients with lung adenocarcinoma were assayed using both qPCR
and ddPCR.
RESULTS: The three ddPCR assays had a limit of detection of 0.02% and a wide
dynamic range from 1 to 20,000 copies measurement. The L858R and ex19del assays
had a 0% background level in the technical and clinical settings. The T790M
assay appeared to have a 0.03% technical background. The ddPCR assays were
robust for correct determination of EGFR mutation status in patients, and the
dynamic range appeared to be better than qPCR methods. The ddPCR assay for T790M
could detect patient samples that the qPCR method failed to detect. About 49% of
this patient cohort had EGFR mutations (L858R, 15.4%; ex19del, 29.5%; T790M,
6.4%). Two patients with the ex19del mutation also had a naïve T790M mutation.
CONCLUSION: These data suggest that the ddPCR method could be useful in the
personalized treatment of patients with lung cancer. In nonsmall cell lung cancer (NSCLC), the threonine(790)-methionine(790) (T790M)
point mutation of EGFR kinase is one of the leading causes of acquired
resistance to the first generation tyrosine kinase inhibitors (TKIs), such as
gefitinib and erlotinib. Herein, we describe the optimization of a series of
7-oxopyrido[2,3-d]pyrimidinyl-derived irreversible inhibitors of EGFR kinase.
This led to the discovery of compound 24 which potently inhibits
gefitinib-resistant EGFR(L858R,T790M) with 100-fold selectivity over wild-type
EGFR. Compound 24 displays strong antiproliferative activity against the H1975
nonsmall cell lung cancer cell line, the first line mutant HCC827 cell line, and
promising antitumor activity in an EGFR(L858R,T790M) driven H1975 xenograft
model sparing the side effects associated with the inhibition of wild-type EGFR. |
what is the role of FGF-2 in cardiac regeneration after myocardial infarction? | Exogenous FGF-2 was shown to increase angiogenesis and myocardial perfusion, promote myocardial regeneration by activating the SDF-1α/CXCR4 axis, and thereby improve the cardiac function after myocardial infarction. Furthermore, prevascularization with basic FGF-incorporated microspheres enhances the benefits of cardiomyocyte transplantation. In another study, transmyocardial drilling revascularization combined with heparinized basic fibroblast growth factor (bFGF)-incorporating degradable stent implantation (TMDRSI) was shown to promote Cardiac Progenitor Cells proliferation and differentiation into cardiomyocytes through activating the SDF-1/CXCR4 axis, while inhibiting myocardial apoptosis, thereby enhancing myocardial regeneration following myocardial infarction and improving cardiac function. | OBJECTIVE: The effects of cell transplantation on the ischemic failing heart
have already been documented. However, the area in and around infarct regions is
not a good environment for cells to survive in because they are exposed to poor
conditions in which certain requirements cannot be adequately supplied. We
therefore designed a study to investigate the efficacy of prevascularization in
ischemic regions before cell transplantation.
METHODS: Rats with myocardial infarction were randomized into 4 groups: 11 rats
received a culture medium injection to the left ventricular wall (control
group), 11 received fetal cardiomyocyte transplantation (TX group), 11 received
gelatin hydrogel microspheres incorporating basic fibroblast growth factor (FGF
group), and 11 received basic fibroblast growth factor pretreatment
sequentially, followed by cardiomyocyte transplantation (FGF-TX group). Four
weeks later, left ventricular function was assessed by means of echocardiography
and cardiac catheterization.
RESULTS: In the FGF and FGF-TX groups neovascularization was found in the scar
tissue 1 week later. The TX, FGF, and FGF-TX groups showed better fractional
shortening than the control group (TX, FGF, FGF-TX, and control: 28% +/- 4.4%,
24% +/- 8.6%, 27% +/- 7.3%, and 17% +/- 4.6%, respectively; P <.01). Left
ventricular maximum time-varying elastance was higher in the FGF-TX group than
in the TX and FGF groups (FGF-TX, TX, and FGF: 0.52 +/- 0.23, 0.30 +/- 0.08, and
0.27 +/- 0.20 mm Hg/microL, respectively; P <.01). Histologically, more
transplanted cells survived in the FGF-TX group than in the TX group.
CONCLUSIONS: Prevascularization with basic fibroblast growth factor-incorporated
microspheres enhances the benefits of cardiomyocyte transplantation. We expect
that this system will contribute to regeneration medicine through its extensive
application to other growth factors. We previously reported that intramyocardial injection of bone marrow-derived
mesenchymal stem cells overexpressing Akt (Akt-MSCs) inhibits ventricular
remodeling and restores cardiac function measured 2 wk after myocardial
infarction. Here, we report that the functional improvement occurs in < 72 h.
This early remarkable effect cannot be readily attributed to myocardial
regeneration from the donor cells. Thus, we hypothesized that paracrine actions
exerted by the cells through the release of soluble factors might be important
mechanisms of tissue repair and functional improvement after injection of the
Akt-MSCs. Indeed, in the current study we demonstrate that conditioned medium
from hypoxic Akt-MSCs markedly inhibits hypoxia-induced apoptosis and triggers
vigorous spontaneous contraction of adult rat cardiomyocytes in vitro. When
injected into infarcted hearts, the Akt-MSC conditioned medium significantly
limits infarct size and improves ventricular function relative to controls.
Support to the paracrine hypothesis is provided by data showing that several
genes, coding for factors (VEGF, FGF-2, HGF, IGF-I, and TB4) that are potential
mediators of the effects exerted by the Akt-MSC conditioned medium, are
significantly up-regulated in the Akt-MSCs, particularly in response to hypoxia.
Taken together, our data support Akt-MSC-mediated paracrine mechanisms of
myocardial protection and functional improvement. OBJECTIVE: To investigate the effects of exogenous basic fibroblast growth
factor (bFGF) on myocardial regeneration after acute myocardial infarction
(AMI).
METHODS: AMI models were established by ligating the mid-third of left anterior
descending artery, thereafter, miniswines were randomly divided into control
(none treatment, n = 6) and bFGF groups (n = 6). For the bFGF group, bFGF (100
μg) was injected with a sterile microinjection at five sites within the ischemic
region. 5-Bromo-2-deoxyuridine (250 mg) was administrated intravenously twice a
week after the operation, to label cells undergoing DNA replication. The
expression of stromal cell-derived factor-1α (SDF-1α) and CXC chemokine receptor
4 (CXCR4), cardiac stem cell-mediated myocardial regeneration, myocardial
apoptosis, histological and immunohistochemical analyses, and cardiac function
were evaluated at different time points.
RESULTS: Four weeks after bFGF therapy, it showed an increased vessel density
and myocardial perfusion (P < 0.001), upregulative expression of SDF-1α and
CXCR4 (P < 0.001), increased c-kit and 5-bromo-2-deoxyuridine-positive cells (P
< 0.001), enhanced myocardial viability (P < 0.001), and improved left
ventricular ejection fraction (P = 0.007), compared with the control.
CONCLUSION: Exogenous bFGF was shown to have increased angiogenesis and
myocardial perfusion, promoted myocardial regeneration by activating the
SDF-1α/CXCR4 axis, and thereby improved the cardiac function, which may provide
a new therapeutic strategy for AMI. |
Have 5q35 microdeletions been implicated in Sotos syndrome development? | Loss-of-function mutations of NSD1 and 5q35 microdeletions encompassing NSD1 are a major cause of Sotos syndrome (Sos), which is characterized by overgrowth, macrocephaly, characteristic facies, and variable intellectual disability (ID). | We observed a novel 3.5 Mb 5q subtelomeric deletion in a 3-year-old girl with
developmental delay, hypotonia and multiple minor anomalies. Comparison of her
phenotype with the few published patients with terminal 5q35 deletions revealed
several overlapping features, but also showed remarkable differences such as
shortness of stature versus macrosomia. After the report of 5q35.3
microdeletions in Sotos syndrome we integrated the published BACs into the
public draft sequence and exactly mapped the deletion size in our patient by
FISH analysis with 15 BAC probes. We demonstrated that the deletion in our
patient is immediately adjacent to the reported Sotos syndrome deletion site.
Subtracting the symptoms of Sotos syndrome from the published patients with
larger 5q35.3 deletions allowed us to delineate a distinct phenotype of prenatal
lymphedema with increased nuchal translucency, pronounced muscular hypotonia and
delay of reaching motor milestones, but speech development within normal limits,
wide fontanels, failure to thrive with postnatal short stature, and multiple
minor anomalies such as mildly bell-shaped chest, minor congenital heart
disease, and a distinct facial gestalt, associated with the novel 3.5 Mb cryptic
deletion. We further showed in our patient that the deletion of the LCT(4)
synthase gene results in a reduction of cysteinyl leukotriene synthesis to about
65% compared to normal values. The prenatal nuchal lymphedema associated with
this deletion syndrome my be related to the deletion of the FLT4 gene causing
autosomal domit primary lymphedema and contributes to the differential
diagnosis of increased fetal nuchal translucency. BACKGROUND: Sotos syndrome is characterised by learning difficulties,
overgrowth, and a typical facial appearance. Microdeletions at 5q35.3,
encompassing NSD1, are responsible for approximately 10% of non-Japanese cases
of Sotos. In contrast, a recurrent approximately 2 Mb microdeletion has been
reported as responsible for approximately 50% of Japanese cases of Sotos.
METHODS: We screened 471 cases for NSD1 mutations and deletions and identified
23 with 5q35 microdeletions. We investigated the deletion size, parent of
origin, and mechanism of generation in these and a further 10 cases identified
from published reports. We used "in silico" analyses to investigate whether
repetitive elements that could generate microdeletions flank NSD1.
RESULTS: Three repetitive elements flanking NSD1, designated REPcen, REPmid, and
REPtel, were identified. Up to 18 cases may have the same sized deletion, but at
least eight unique deletion sizes were identified, ranging from 0.4 to 5 Mb. In
most instances, the microdeletion arose through interchromosomal rearrangements
of the paternally inherited chromosome.
CONCLUSIONS: Frequency, size, and mechanism of generation of 5q35 microdeletions
differ between Japanese and non-Japanese cases of Sotos. Our microdeletions were
identified from a large case series with a broad range of phenotypes, suggesting
that sample selection variability is unlikely as a sole explanation for these
differences and that variation in genomic architecture might be a contributory
factor. Non-allelic homologous recombination between REPcen and REPtel may have
generated up to 18 microdeletion cases in our series. However, at least 15
cannot be mediated by these repeats, including at least seven deletions of
different sizes, implicating multiple mechanisms in the generation of 5q35
microdeletions. BACKGROUND: Most cases of Sotos syndrome are caused by intragenic NSD1 mutations
or 5q35 microdeletions. It is uncertain whether allelic or genetic heterogeneity
underlies the residual cases and it has been proposed that other mechanisms,
such as 11p15 defects, might be responsible for Sotos cases without NSD1
mutations or 5q35 microdeletions.
OBJECTIVE: To develop a multiplex ligation dependent probe amplification (MLPA)
assay to screen NSD1 for exonic deletions/duplications.
METHODS: Analysis was undertaken of 18 classic Sotos syndrome cases in which
NSD1 mutations and 5q35 microdeletions were excluded. Long range polymerase
chain reaction (PCR) was used to characterise the mechanism of generation of the
partial NSD1 deletions.
RESULTS: Eight unique partial NSD1 deletions were identified: exons 1-2 (n = 4),
exons 3-5, exons 9-13, exons 19-21, and exon 22. Using long range PCR six of the
deletions were confirmed and the precise breakpoints in five cases
characterised. This showed that three had arisen through Alu-Alu recombination
and two from non-homologous end joining.
CONCLUSIONS: MLPA is a robust, inexpensive, simple technique that reliably
detects both 5q35 microdeletions and partial NSD1 deletions that together
account for approximately 15% of Sotos syndrome. Haploinsufficiency of the NSD1 gene due to 5q35 microdeletions or intragenic
mutations causes Sotos syndrome (SoS). In 46 of the 49 Japanese deletion cases,
common deletion breakpoints were located at two flanking low copy repeats
(LCRs), implying that non-allelic homologous recombination (NAHR) between LCRs
is the major mechanism for the common deletion. In the other three cases of
atypical deletions, the mechanism(s) of deletions remains uswered. We
characterized the atypical microdeletions using fluorescence in situ
hybridization (FISH), quantitative real-time polymerase chain reaction (qPCR),
and Southern blot hybridization. All the deletion breakpoints in the three cases
were successfully determined at the nucleotide level. Two deletions are 1.07 Mb
(SoS19) and 1.23 Mb (SoS109) in size, and another consisted of two deletions
with sizes of 28 kb and 0.72 Mb, intervened by an intact 29-kb segment (SoS44).
All deletions were smaller than a typical 1.9-Mb common deletion. Alu elements
were identified in both deletion breakpoints in SoS19, one of deletion
breakpoints in SoS109, and both deletion breakpoints of the proximal 28-kb
deletion in SoS44. Alu-mediated NAHR is strongly suggested at least in two of
atypical deletions. Finally, qPCR is a very useful method to determine deletion
breakpoints even in repeat-related regions. Loss-of-function mutations of NSD1 and 5q35 microdeletions encompassing NSD1 are
a major cause of Sotos syndrome (Sos), which is characterized by overgrowth,
macrocephaly, characteristic facies, and variable intellectual disability (ID).
Microduplications of 5q35.2-q35.3 including NSD1 have been reported in only five
patients so far and described clinically as a reversed Sos resulting from a
hypothetical gene dosage effect of NSD1. Here, we report on nine patients from
five families with interstitial duplication 5q35 including NSD1 detected by
molecular karyotyping. The clinical features of all 14 individuals are reviewed.
Patients with microduplications including NSD1 appear to have a consistent
phenotype consisting of short stature, microcephaly, learning disability or mild
to moderate ID, and distinctive facial features comprising periorbital fullness,
short palpebral fissures, a long nose with broad or long nasal tip, a smooth
philtrum and a thin upper lip vermilion. Behavioral problems, ocular and minor
hand anomalies may be associated. Based on our findings, we discuss the possible
etiology and conclude that it is possible, but so far unproven, that a gene
dosage effect of NSD1 may be the major cause. Sotos syndrome is a multiple anomaly syndrome characterized by pre- and
postnatal overgrowth with advanced bone age, macrocephaly, developmental delay,
and distinctive facial phenotype. Autosomal domit mutations and deletions of
the nuclear receptor set domain gene (NSD1), which is located at chromosome
5q35, are responsible for most of the cases. We describe a six-year old boy who
had tall stature, macrocephaly, typical facial appearance, learning disability,
megaloencephaly, corpus callosum dysgenesis, and colpocephaly. Although he had
normal bone age, the diagnosis of Sotos syndrome was suspected with these
clinical findings, and fluorescence in situ hybridization analysis of the
patient showed a heterozygous deletion covering the NSD1 region in the 5q35
locus. A brief overview of the syndrome is presented. CONTEXT: Sotos syndrome is a rare genetic disorder with a distinct phenotypic
spectrum including overgrowth and learning difficulties. Here we describe a new
case of Sotos syndrome with a 5q35 microdeletion, affecting the fibroblast
growth factor receptor 4 (FGFR4) gene, presenting with infantile hypercalcemia.
OBJECTIVE: We strove to elucidate the evanescent nature of the observed
hypercalcemia by studying the ontogenesis of FGFR3 and FGFR4, which are both
associated with fibroblast growth factor (FGF) 23-mediated mineral homeostasis,
in the developing human kidney.
DESIGN: Quantitative RT-PCR and immunohistochemical analyses were used on
archival human kidney samples to investigate the expression of the FGFR
signaling pathway during renal development.
RESULTS: We demonstrated that renal gene and protein expression of both FGFRs
increased during fetal development between the gestational ages (GAs) of 14-40
weeks. Yet FGFR4 expression increased more rapidly as compared with FGFR3 (slope
0.047 vs 0.0075, P = .0018). Moreover, gene and protein expression of the
essential FGFR coreceptor, Klotho, also increased with a significant positive
correlation between FGFR and Klotho mRNA expression during renal development.
Interestingly, we found that perinatal FGFR4 expression (GA 38-40 wk) was 7-fold
higher as compared with FGFR3 (P = .0035), whereas in adult kidney tissues,
FGFR4 gene expression level was more than 2-fold lower compared with FGFR3 (P =
.0029), thus identifying a molecular developmental switch of FGFR isoforms.
CONCLUSION: We propose that the heterozygous FGFR4 deletion, as observed in the
Sotos syndrome patient, leads to a compromised FGF23 signaling during infancy
accounting for transient hypercalcemia. These findings represent a novel and
intriguing view on FGF23 mediated calcium homeostasis. OBJECTIVE: To present prenatal diagnosis and molecular cytogenetic
characterization of a de novo 5q35 microdeletion associated with Sotos syndrome.
METHODS: This was the first pregcy of a 29-year-old woman. The pregcy was
uneventful until 27 weeks of gestation when left ventriculomegaly was first
noted. At 31 weeks of gestation, polyhydramnios, macrocephaly, and
ventriculomegaly were prominent on ultrasound, and left pyelectasis and
bilateral ventriculomegaly were diagnosed on magnetic resoce imaging. The
woman underwent amniocentesis and cordocentesis at 32 weeks of gestation.
Conventional cytogenetic analysis was performed using cultured amniocytes and
cord blood lymphocytes. Array comparative genomic hybridization (aCGH) was
performed on uncultured amniocytes and parental blood. Metaphase fluorescence in
situ hybridization (FISH) was performed on cultured lymphocytes.
RESULTS: Conventional cytogenetics revealed a karyotype of 46,XX. aCGH on
uncultured amniocytes revealed a de novo 1.07-Mb microdeletion at 5q35.2-q35.3
encompassing NSD1. Metaphase FISH analysis on the cord blood lymphocytes
confirmed the deletion at 5q35.2. The postnatal phenotype was consistent with
Sotos syndrome.
CONCLUSION: Fetuses with Sotos syndrome may present macrocephaly,
polyhydramnios, ventriculomegaly, and pyelectasis in the third trimester. aCGH
and metaphase FISH are useful for rapid diagnosis of 5q35 microdeletion
associated with Sotos syndrome. |
Tumors of which three organs are classically associated with the multiple endocrine neoplasia type 1 syndrome? | Multiple endocrine neoplasia type 1 syndrome is an inherited cancer syndrome defined by occurrence of multiple neuro-endocrine tumors and is classically associated with the combined occurrence of two or more tumors involving parathyroid gland, pancreas and pituitary gland. Other tumors, including but not limited to adrenal cortical tumor, carcinoid tumors lipoma, leiomyoma, duodenal gastrinoma, hepatic focal nodular hyperplasia, and renal angiomyolipoma, angiofibroma, colagenoma, thyroid tumor and meningioma, may also be present. | OBJECTIVES: To evaluate patients with multiple endocrine neoplasia type 1 (MEN
1) for cutaneous manifestations.
DESIGN: Survey during a 3-year period.
SETTING: The National Institutes of Health, a tertiary referral research
hospital in Bethesda Md.
PATIENTS: A consecutive sample of 32 individuals with previously diagnosed MEN1
who were not preselected for the presence of skin lesions were examined for
cutaneous abnormalities. None of the patients or family members were diagnosed
as having tuberous sclerosis.
INTERVENTIONS: Lesions were identified by clinical appearance, photographed, and
confirmed histologically.
MAIN OUTCOME MEASURE: To determine the frequency of skin lesions in patients
with MEN1.
RESULTS: Multiple facial angiofibromas were observed in 28 (88%) of the patients
with MEN1, with 16 patients (50%) having 5 or more. Angiofibromas were
clinically and histologically identical to those in individuals with tuberous
sclerosis. Collagenomas were observed in 23 patients (72%). Also observed were
cafe au lait macules in 12 patients (38%), lipomas in 11 patients (34%),
confetti-like hypopigmented macules in 2 patients (6%), and multiple gingival
papules in 2 patients (6%).
CONCLUSIONS: Multiple angiofibromas, collagenomas, lipomas, confetti-like
hypopigmented macules and multiple gingival papules are cutaneous manifestations
of MEN1 and should be looked for in both family members of patients with MEN1
and individuals with hyperparathyroidism of other MEN1-associated tumors.
Multiple angiofibromas can no longer be considered pathognomonic for tuberous
sclerosis. The observation of angiofibromas in individuals without tuberous
sclerosis necessitates further biochemical testing for MEN1. Multiple endocrine neoplasia type 1 (MEN1) consists of benign, and sometimes
maligt, tumors (often multiple in a tissue) of the parathyroids,
enteropancreatic neuroendocrine system, anterior pituitary, and other tissues.
Skin angiofibromas and skin collagenomas are common. Typically, MEN1 tumors
begin two decades earlier than sporadic tumors. Because of tumor multiplicity
and the tendency for postoperative tumor recurrence, specialized methods have
been developed for preoperative and intraoperative localization of many
MEN1-associated tumors. The MEN1 gene was recently isolated by positional
cloning. This strategy progressively narrows the size of the candidate MEN1 gene
interval on the chromosome and then finds and tests many or, if needed, all
genes within that interval. The MEN1 gene was finally identified because it was
the one gene that contained mutations in most DNAs from a test panel of MEN1
cases. It has been suggested that MEN1, like many hereditary cancer syndromes,
is caused by mutation in a tumor suppressor gene that contributes to neoplasia
when both gene copies in a tumor precursor cell have been sequentially
inactivated ("two-hit" oncogenesis mechanism). Germline MEN1 mutations were
found in most families with MEN1 and in most cases of sporadic MEN1. In
addition, the MEN1 gene was the gene most likely to show acquired mutation in
several sporadic or nonhereditary tumors-parathyroid adenomas, gastrinomas,
insulinomas, and bronchial carcinoids. Most germline or acquired MEN1 mutations
predicted truncation (and thus likely inactivation) of the encoded protein,
supporting expectations for the "first hit" to a tumor suppressor gene. Testing
for MEN1 germline mutation is possible in a research setting. Candidates for
MEN1 mutation testing include patients with MEN1 or its phenocopies and
first-degree relatives of persons with MEN1. Multiple endocrine neoplasia type 1 (MEN1) is characterized by the development
of endocrine tumors of the parathyroid and pituitary glands, pancreas, and
duodenum. Less frequently occurring tumors associated with MEN1 include
non-endocrine tumors such as lipomas and angiofibromas. An increased incidence
of thyroid neoplasms, leiomyomas, adrenal cortical hyperplasia, hepatic focal
nodular hyperplasia, and renal angiomyolipoma has been noted in the MEN1
population. The pathogenesis of non-neuroendocrine tumors in MEN1 is unknown. We
report a complex clinical course and a detailed morphologic and genetic analysis
of a series of tumors that developed in a patient with MEN1. All tumors were
microdissected and analyzed for loss of heterozygosity of the MEN1 gene. A
germline mutation of the MEN1 gene was detected, and deletions of the MEN1 gene
were consistently detected in multiple neuroendocrine tumors involving the
parathyroid glands and the pancreas and a hepatic neuroendocrine tumor
metastasis, as predicted by Knudson's "two hit" hypothesis. Two hits of the MEN1
gene were also detected in esophageal leiomyoma tissue, suggesting that
tumorigenesis was directly related to the patient's underlying MEN1. In
contrast, follicular thyroid adenoma, papillary thyroid carcinoma, hepatic focal
nodular hyperplasia, and adrenal cortical hyperplasia consistently showed
retained heterozygosity of the MEN1 gene with flanking markers and an intragenic
marker. Therefore, these tumors appear to develop along pathogenetic pathways
that are different from classical MEN1-associated tumors. Pancreatic endocrine tumors occur sporadically and as part of the multiple
endocrine neoplasia type 1 (MEN 1) and von Hippel-Lindau (VHL) syndromes. The
MEN1 locus on 11q13 and a candidate tumor suppressor locus on 3p are known to be
hemi- or homozygously mutated in a subset of these tumors. Chromosome arm 18q
harbors the SMAD4/DPC4 tumor suppressor gene that is frequently deleted and
inactivated in tumors of the exocrine pancreas. We have analyzed 22 nonfamilial
and 16 MEN 1-associated pancreatic endocrine tumors for loss of heterozygosity
(LOH) at 3p, 11q13, and 18q. LOH at 3p was revealed in 45% and 36% of tumors
from 31 patients with nonfamilial and MEN 1-associated disease, respectively.
The corresponding proportions for 11q13 were 55% and 91%, and for 18q 27% and
25%, respectively. A striking relation between LOH at 11q13 and 3p and a
maligt phenotype was found for the nonfamilial tumors. None of the six benign
tumors (all of them insulinomas) had allelic loss at 3p or 11q13, whereas 92% (P
< 0.01) of the maligt tumors (including maligt insulinomas) had such
deletions. Besides the 11q13 abnormality, more than half of the MEN 1-associated
tumors had additional genetic lesions affecting 3p or 18q. LOH analysis of
several tumors from two MEN 1 patients suggested different clonal origin of the
lesions. Sequencing of the SMAD4/DPC4 gene did not identify mutations in coding
regions or at exon/intron boundaries in tumors with LOH at 18q. The data
indicate involvement of tumor suppressor genes on 3p and 18q, in addition to the
MEN1 gene at 11q13, in the tumorigenesis of both nonfamilial and MEN
1-associated pancreatic endocrine tumors. Multiple endocrine neoplasia type 1 (MEN 1) is a familial syndrome characterized
by parathyroid, enteropancreatic and pituitary tumors. The gene responsible for
this syndrome is localized at chromosomal 11q13 region and DNA markers from this
region cosegregate with the disease. The recent identification of the MEN1 gene,
encoding for a protein termed menin of 610 amino acids, allowed mutational
screening to be performed both in affected families and sporadic cases. To date
many different heterozygous mutations, spreading across all the encoding
sequence, have been identified in MEN 1 patients with no apparent mutational hot
spots or genotype-phenotype correlation. To analyze the genetic alterations of
the MEN1 gene occurring in Italian patients we performed mutational screening by
Denaturant Gradient Gel Electrophoresis followed by sequencing of exons 2-10 of
the MEN1 gene in 27 Italian MEN 1 families and in five sporadic cases. We
identified 17 different heterozygous mutations in 60% of analyzed cases. Twelve
of these mutations are novel. Two mutations each occurred twice in unrelated
families but no evidence of genotype-phenotype correlation can be established
for these families. The extension of genetic diagnosis to asymptomatic family
members allowed the identification of 10 MEN1 mutant gene carriers, one newly
described and nine previously detected by linkage analysis with DNA markers from
the 11q13 region. Our findings add new information to the diversity of mutations
occurring in the MEN1 gene and confirm that the mutational screening of MEN 1 is
a useful approach to detect individuals at higher risk of developing MEN
1-associated tumors. Although the identification of menin-interacting partners and other evidence
support a role for menin, the multiple endocrine neoplasia type 1 gene (MEN1)
product, in regulating gene expression, little is known about the cellular
pathways dysregulated by menin loss during tumorigenesis. The mouse models of
MEN1 accurately mimic the human syndrome and provide an opportunity to assess
the transcriptional effects of Men1 deletion in different endocrine tumor types
to identify common pathway aberrations underlying tumorigenesis in MEN1-affected
tissues. We compared the global gene expression profiles of pituitary adenomas
and pancreatic islet tumors with control tissues from wild-type littermates.
Amongst the 551 differentially expressed genes was significant
over-representation of genes associated with chromatin remodelling,
transcription and cell cycling, including some genes known to encode
menin-binding partners, e.g., Rhox5 and Mll1. Consistent with increased
cell-cycle transition from G1 to S phase was an elevation of Cdc7 expression in
the tumors, which was confirmed by qRT-PCR using independent samples. In support
of previous findings in islet tumors, we found down-regulation of the cell-cycle
regulator, p18, in both the pancreatic islet and pituitary adenomas, suggesting
that reduced p18 levels may be important for Men1-related tumorigenesis in
multiple tissues. Surprisingly, we identified increased p16 transcript in
pancreatic islet and pituitary tumors. This was accompanied by increased
cytoplasmic localization p16 protein in tumor cells. The specific genes and
general pathways we have found to be commonly dysregulated in MEN1 tumors,
provide a platform for determining their roles in endocrine tumorigenesis. PURPOSE: Multiple endocrine neoplasia type 1 (MEN1) is defined clinically by the
combined occurrence of multiple tumors, typically of the parathyroid glands,
pancreatic islet cells, and anterior pituitary gland. A mouse model with a
heterozygous deletion of the Men1 gene recapitulates the tumorigenesis of MEN1.
We wished to determine the role of vascular endothelial growth factor (VEGF)-A
in the vascularization and growth of MEN1-associated tumors, with an emphasis on
pituitary adenomas.
EXPERIMENTAL DESIGN: To investigate whether tumor growth in Men1(+/-) mice is
mediated by VEGF-A dependent angiogenesis, we carried out a monotherapy with the
anti-VEGF-A monoclonal antibody (mAb) G6-31. We evaluated tumor growth by
magnetic resoce imaging and assessed vascular density in tissue sections. We
also measured hormone levels in the serum.
RESULTS: During the treatment with mAb G6-31, a significant inhibition of the
pituitary adenoma growth was observed, leading to an increased mean tumor
doubling-free survival compared with mice treated with a control antibody.
Similarly, the growth of s.c. pituitary adenoma transplants was effectively
inhibited by administration of anti-VEGF-A mAb. Serum prolactin was lowered by
mAb G6-31 treatment but not by control antibody, potentially providing a new
therapeutic approach for treating the hormonal excess in MEN1 patients.
Additionally, the vascular density in pancreatic islet tumors was significantly
reduced by the treatment.
CONCLUSIONS: These results suggest that VEGF-A blockade may represent a
nonsurgical treatment for benign tumors of the endocrine system. MEN1 and MEN2 are rare inherited cancer syndromes which express a variety of
endocrine and nonendocrine tumors. The improved knowledge of molecular and
clinical physiopathology of MEN syndromes, together with the availability of
genetic testing, have led to earlier detection and intervention, with consequent
reduction of mortality and morbidity for MEN-associated tumors. Genetic testing
has gained a key role in the detection of asymptomatic patients harbouring
mutations responsible for these syndrome, and allows institution of early and
tailored intervention with a positive impact on the course of disease. Multiple endocrine neoplasia type 1 (MEN1) is an autosomal domit disorder
characterized by the combined occurrence of parathyroid and adrenocortical
tumors, and neuroendocrine tumors (NETs) of the pancreas and pituitary. The
pancreatic NETs are predomitly gastrinomas and insulinomas, and the pituitary
NETs are mostly prolactinomas and somatotrophinomas. We postulated that the
different types of pancreatic and pituitary NETs may be partly due to
differences in their proliferation rates, and we therefore assessed these in
MEN1-associated tumors and gonadal tumors that developed in mice deleted for an
Men1 allele (Men1(+/-)). To label proliferating cells in vivo, Men1(+/-) and
wild-type (Men1(+/+)) mice were given 5-bromo-2-deoxyuridine (BrdU) in drinking
water from 1-12 wk, and tissue sections were immunostained using anti-BrdU and
hormone-specific antibodies. Proliferation in the tumors of Men1(+/-) mice was
significantly (P < 0.001) increased when compared with the corresponding normal
Men1(+/+) tissues. Pancreatic, pituitary and adrenocortical proliferation fitted
first- and second-order regression lines in Men1(+/+) tissues and Men1(+/-)
tumors, respectively, R(2) = 0.999. Apoptosis was similar in Men1(+/-)
pancreatic, pituitary, and parathyroid tumors when compared with corresponding
normal tissues, decreased in Men1(+/-) adrenocortical tumors, but increased in
Men1(+/-) gonadal tumors. Mathematical modeling of NET growth rates
(proliferation minus apoptosis rates) predicted that in Men1(+/-) mice, only
pancreatic β-cells, pituitary lactotrophs and somatotrophs could develop into
tumors within a murine lifespan. Thus, our studies demonstrate that Men1(+/-)
tumors have low proliferation rates (<2%), second-order kinetics, and the higher
occurrence of insulinomas, prolactinomas, and somatotrophinomas in MEN1 is
consistent with a mathematical model for NET proliferation. Multiple endocrine neoplasia type 1 (MEN1) is inherited in an autosomal domit
fashion and predisposes to the development of hyperplastic or neoplastic changes
in the parathyroid and pituitary glands and the endocrine pancreas, along with
numerous other characteristic tumors and features. The management of each entity
differs to some degree from their sporadic counterparts, while the lack of a
genotype-phenotype correlation requires lifelong clinical, biochemical and
radiological screening for the development of new tumors. While the syndrome
itself is relatively rare (a prevalence of 1-10/100 000), it is likely that
health-care practitioners from numerous specialities will occasionally encounter
a patient with MEN1 and therefore a basic knowledge of the syndrome is
important. In addition, many of the associated tumors are seen commonly in
sporadic form, and a judicious policy is therefore required in deciding how
thoroughly patients who develop these tumors should be screened for MEN1. The
current literature on MEN1 is reviewed and key learning points are suggested for
the clinician. |
Which is the protein implicated in Spinocerebellar ataxia type 3? | Ataxin-3 is a ubiquitously expressed deubiqutinating enzyme with important functions in the proteasomal protein degradation pathway and regulation of transcription. The C-terminus of the ataxin-3 protein contains a polyglutamine (PolyQ) region that, when mutationally expanded to over 52 glutamines, causes the neurodegenerative disease spinocerebellar ataxia 3 (SCA3). | Machado-Joseph disease (MJD), also called spinocerebellar ataxia type 3, is
caused by mutant ataxin-3 with a polyglutamine expansion. Although there is no
treatment available at present to cure or delay the onset of MJD, mouse models
have been generated to facilitate the development of a therapy. In this review,
the published reports on mouse models of MJD and other polyglutamine
spinocerebellar ataxias are compared. Based on these studies, the following
approaches will be discussed as candidate treatments for MJD: 1) interfering
with the formation of the mutant ataxin-3 cleavage fragment and possibly
aggregate or inclusions, 2) reducing the disease protein nuclear localization,
and 3) decreasing mutant ataxin-3 expression in neurons. Spinocerebellar ataxia type 3 is a neurodegenerative disorder caused by the
expansion of the polyglutamine repeat region within the ataxin-3 protein. The
mutant protein forms intracellular aggregates in the brain. However, the
cellular mechanisms causing toxicity are still poorly understood and there are
currently no effective treatments. In this study we show that administration of
a rapamycin ester (cell cycle inhibitor-779, temsirolimus) improves motor
performance in a transgenic mouse model of spinocerebellar ataxia type 3.
Temsirolimus inhibits mammalian target of rapamycin and hence upregulates
protein degradation by autophagy. Temsirolimus reduces the number of aggregates
seen in the brains of transgenic mice and decreases levels of cytosolic soluble
mutant ataxin-3, while endogenous wild-type protein levels remain unaffected.
Temsirolimus is designed for long-term use in patients and therefore represents
a possible therapeutic strategy for the treatment of spinocerebellar ataxia type
3. Using this disease model and treatment paradigm, we employed a microarray
approach to investigate transcriptional changes that might be important in the
pathogenesis of spinocerebellar ataxia type 3. This identified ubiquitin
specific peptidase-15, which showed expression changes at both the messenger
ribonucleic acid and protein level. Ubiquitin specific peptidase-15 levels were
also changed in mice expressing another mutant polyglutamine protein,
huntingtin. In total we identified 16 transcripts that were decreased in
transgenic ataxin-3 mice that were normalized following temsirolimus treatment.
In this mouse model with relatively mild disease progression, the number of
transcripts changed was low and the magnitude of these changes was small.
However, the importance of these transcriptional alterations in the pathogenesis
of spinocerebellar ataxia type 3 remains unclear. Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar
Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in
the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites
through which it anchors polyubiquitin chains of different linkages that are
then cleaved by the N-terminal catalytic (Josephin) domain. The properties of
the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well
established. Very little is known, however, about how two recently identified
ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain
binding and cleavage. In the current study, we sought to define the specific
contribution of the Josephin domain to the catalytic properties of ataxin-3 and
assess how the topology and affinity of these binding sites modulate ataxin-3
activity. Using NMR we modeled the structure of diUb/Josephin complexes and
showed that linkage preferences are imposed by the topology of the two binding
sites. Enzymatic studies further helped us to determine a precise hierarchy
between the sites. We establish that the structure of Josephin dictates
specificity for K48-linked chains. Site 1, which is close to the active site, is
indispensable for cleavage. Our studies open the way to understand better the
cellular function of ataxin-3 and its link to pathology. Mutant ataxin-3 is aberrantly folded and proteolytically cleaved in
spinocerebellar ataxia type 3. The C-terminal region of the protein includes a
polyglutamine stretch that is expanded in spinocerebellar ataxia type 3. Here,
we report on the analysis of an ataxin-3 mutant mouse that has been obtained by
gene trap integration. The ataxin-3 fusion protein encompasses 259 N-terminal
amino acids including the Josephin domain and an ubiquitin-interacting motif but
lacks the C-terminus with the polyglutamine stretch, the valosin-containing
protein binding region and part of the ubiquitin-interacting motif 2. Homozygous
ataxin-3 mutant mice were viable and showed no apparent anatomical defects at
birth. However, at the age of 9 months, homozygous and heterozygous mutant mice
revealed significantly altered behaviour and progressing deficits of motor
coordination followed by premature death at ∼12 months. At this time, prominent
extranuclear protein aggregates and neuronal cell death was found in mutant
mice. This was associated with disturbances of the endoplasmic
reticulum-mediated unfolded protein response, consistent with the normal role of
ataxin-3 in endoplasmic reticulum homeostasis. Thus, the ataxin-3 gene trap
model provides evidence for a contribution of the non-polyglutamine containing
ataxin-3 N-terminus, which mimics a calpain fragment that has been observed in
spinocerebellar ataxia type 3. Consistent with the disease in humans, gene trap
mice develop cytoplasmic inclusion bodies and implicate impaired unfolded
protein response in the pathogenesis of spinocerebellar ataxia type 3. Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3
(SCA3), may be the most common domitly inherited ataxia in the world. Here I
will review historical, clinical, neuropathological, genetic, and pathogenic
features of MJD, and finish with a brief discussion of present, and possible
future, treatment for this currently incurable disorder. Like many other
domitly inherited ataxias, MJD/SCA3 shows remarkable clinical heterogeneity,
reflecting the underlying genetic defect: an unstable CAG trinucleotide repeat
that varies in size among affected persons. This pathogenic repeat in MJD/SCA3
encodes an expanded tract of the amino acid glutamine in the disease protein,
which is known as ataxin-3. MJD/SCA3 is one of nine identified polyglutamine
neurodegenerative diseases which share features of pathogenesis centered on
protein misfolding and accumulation. The specific properties of MJD/SCA3 and its
disease protein are discussed in light of what is known about the entire class
of polyglutamine diseases. AIMS: Spinocerebellar ataxia type 3 (SCA3) is an inherited spinocerebellar
ataxia caused by the expansion of trinucleotide CAG repeats in the gene encoding
ataxin-3. The clinical manifestations of SCA3 include peripheral neuropathy,
which is an important cause of disability in a subset of patients. Although the
loss of neurones in the dorsal root ganglion (DRG) has been postulated to be the
cause of this neuropathy, the precise mechanism remains to be elucidated.
METHODS: To clarify the clinicopathological characteristics of SCA3-associated
peripheral neuropathy, we performed nerve conduction studies and
histopathological analyses. Nerve conduction studies were carried out in 18 SCA3
patients. Immunohistochemical analyses of the anterior and posterior roots of
the spinal cord and peripheral nerves were performed in five SCA3 patients. We
also employed immunohistochemistry and immunoelectron microscopy analyses with
an anti-polyglutamine antibody.
RESULTS: The mean sensory nerve action potentials of the SCA3 patients were half
of the normal values. The motor conduction velocities were decreased, and the
distal latencies were also significantly prolonged in the nerves studied
relative to the those in normal controls. Histopathological analyses detected
axonal sprouting and myelin thinning in all cases. Ataxin-3 aggregates were
found in the cytoplasm of Schwann cells in all of the SCA3 patients examined but
not in control subjects.
CONCLUSIONS: In addition to the previously reported neuronopathy, the results of
the present study indicate that Schwann cells are involved in the formation of
the pathogenic intracytoplasmic ataxin-3 protein aggregates in patients with
SCA3-associated neuropathy. Spinocerebellar ataxia type 3 is caused by a polyglutamine expansion in the
ataxin-3 protein, resulting in gain of toxic function of the mutant protein. The
expanded glutamine stretch in the protein is the result of a CAG triplet repeat
expansion in the penultimate exon of the ATXN3 gene. Several gene silencing
approaches to reduce mutant ataxin-3 toxicity in this disease aim to lower
ataxin-3 protein levels, but since this protein is involved in deubiquitination
and proteasomal protein degradation, its long-term silencing might not be
desirable. Here, we propose a novel protein modification approach to reduce
mutant ataxin-3 toxicity by removing the toxic polyglutamine repeat from the
ataxin-3 protein through antisense oligonucleotide-mediated exon skipping while
maintaining important wild type functions of the protein. In vitro studies
showed that exon skipping did not negatively impact the ubiquitin binding
capacity of ataxin-3. Our in vivo studies showed no toxic properties of the
novel truncated ataxin-3 protein. These results suggest that exon skipping may
be a novel therapeutic approach to reduce polyglutamine-induced toxicity in
spinocerebellar ataxia type 3. Spinocerebellar ataxia type 3 (SCA3) is the most frequent inherited cerebellar
ataxia in Europe, the US and Japan, leading to disability and death through
motor complications. Although the affected protein ataxin-3 is found
ubiquitously in the brain, grey matter atrophy is predomit in the cerebellum
and the brainstem. White matter pathology is generally less severe and thought
to occur in the brainstem, spinal cord, and cerebellar white matter. Here, we
investigated both grey and white matter pathology in a group of 12 SCA3 patients
and matched controls. We used voxel-based morphometry for analysis of tissue
loss, and tract-based spatial statistics (TBSS) on diffusion magnetic resoce
imaging to investigate microstructural pathology. We analysed correlations
between microstructural properties of the brain and ataxia severity, as measured
by the Scale for the Assessment and Rating of Ataxia (SARA) score. SCA3 patients
exhibited significant loss of both grey and white matter in the cerebellar
hemispheres, brainstem including pons and in lateral thalamus. On between-group
analysis, TBSS detected widespread microstructural white matter pathology in the
cerebellum, brainstem, and bilaterally in thalamus and the cerebral hemispheres.
Furthermore, fractional anisotropy in a white matter network comprising frontal,
thalamic, brainstem and left cerebellar white matter strongly and negatively
correlated with SARA ataxia scores. Tractography identified the thalamic white
matter thus implicated as belonging to ventrolateral thalamus. Disruption of
white matter integrity in patients suffering from SCA3 is more widespread than
previously thought. Moreover, our data provide evidence that microstructural
white matter changes in SCA3 are strongly related to the clinical severity of
ataxia symptoms. Ataxin-3 is a ubiquitously expressed deubiqutinating enzyme with important
functions in the proteasomal protein degradation pathway and regulation of
transcription. The C-terminus of the ataxin-3 protein contains a polyglutamine
(PolyQ) region that, when mutationally expanded to over 52 glutamines, causes
the neurodegenerative disease spinocerebellar ataxia 3 (SCA3). In spite of
extensive research, the molecular mechanisms underlying the cellular toxicity
resulting from mutant ataxin-3 remain elusive and no preventive treatment is
currently available. It has become clear over the last decade that the hallmark
intracellular ataxin-3 aggregates are likely not the main toxic entity in SCA3.
Instead, the soluble PolyQ containing fragments arising from proteolytic
cleavage of ataxin-3 by caspases and calpains are now regarded to be of greater
influence in pathogenesis. In addition, recent evidence suggests potential
involvement of a RNA toxicity component in SCA3 and other PolyQ expansion
disorders, increasing the pathogenic complexity. Herein, we review the
functioning of ataxin-3 and the involvement of known protein and RNA toxicity
mechanisms of mutant ataxin-3 that have been discovered, as well as future
opportunities for therapeutic intervention. Ataxin-3 (AT3) is the protein that triggers the inherited neurodegenerative
disorder spinocerebellar ataxia type 3 when its polyglutamine (polyQ) stretch
close to the C-terminus exceeds a critical length. AT3 consists of the
N-terminal globular Josephin domain (JD) and the C-terminal disordered one. It
cleaves isopeptide bonds between ubiquitin monomers, an event involved in
protein quality control mechanisms. AT3 has been implicated in the pathway that
sorts aggregated protein to aggresomes via microtubules, in which dynein and
histone deacetylase 6 (HDAC6) also seem to be involved. By taking advantage of
small angle X-ray scattering (SAXS) and surface plasmon resoce (SPR), we have
investigated the interaction of AT3 with tubulin and HDAC6. Based on SAXS
results, the AT3 oligomer, consisting of 6-7 subunits, tightly binds to the
tubulin hexameric oligomer in a "parallel" fashion. By SPR analysis we have
demonstrated that AT3 binds to tubulin dimer with a 50nM affinity. Binding fits
with a Langmuir 1:1 model and involves a single binding interface. Nevertheless,
the interaction surface consists of three distinct, discontinuous
tubulin-binding regions (TBR), one located in the JD, and the two others in the
disordered domain, upstream and downstream of the polyQ stretch. In the absence
of any of the three TBRs, the affinity is drastically reduced. By SPR we have
also provided the first evidence of direct binding of AT3 to HDAC6, with
affinity in the range 0.1-1μM. These results shed light on the interactions
among the components of the transport machinery that sorts aggregate protein to
the aggresome, and pave the way to in vivo studies aimed at further clarifying
their roles. |
What is Tn-seq? | The transposon mutagenesis and high-throughput sequencing (Tn-seq) is a technique that allows for quantitative assessment of individual mutants within a transposon mutant library by sequencing the transposon-genome junctions and then compiling mutant presence by mapping to a base genome. Using Tn-seq, it is possible to quickly define all the insertional mutants in a library and thus identify nonessential genes under the conditions in which the library was produced. Identification of fitness of individual mutants under specific conditions can be performed by exposing the library to selective pressures. | The lagging annotation of bacterial genomes and the inherent genetic complexity
of many phenotypes is hindering the discovery of new drug targets and the
development of new antimicrobials and vaccines. Here we present the method
Tn-seq, with which it has become possible to quantitatively determine fitness
for most genes in a microorganism and to screen for quantitative genetic
interactions on a genome-wide scale and in a high-throughput fashion. Tn-seq can
thus direct studies in the annotation of genes and untangle complex phenotypes.
The method is based on the construction of a saturated Mariner transposon
insertion library. After library selection, changes in frequency of each
insertion mutant are determined by sequencing of the flanking regions en masse.
These changes are used to calculate each mutant's fitness. The method has been
developed for the Gram-positive bacterium Streptococcus pneumoniae, a causative
agent of pneumonia and meningitis; however, due to the wide activity of the
Mariner transposon, Tn-seq can be applied to many different microbial species. We describe a deep-sequencing procedure for tracking large numbers of transposon
mutants of Pseudomonas aeruginosa. The procedure employs a new Tn-seq
methodology based on the generation and amplification of single-strand circles
carrying transposon junction sequences (the Tn-seq circle method), a method
which can be used with virtually any transposon. The procedure reliably
identified more than 100,000 transposon insertions in a single experiment,
providing near-saturation coverage of the genome. To test the effectiveness of
the procedure for mutant identification, we screened for mutations reducing
intrinsic resistance to the aminoglycoside antibiotic tobramycin. Intrinsic
tobramycin resistance had been previously analyzed at genome scale using
mutant-by-mutant screening and thus provided a benchmark for evaluating the new
method. The new Tn-seq procedure identified 117 tobramycin resistance genes, the
majority of which were then verified with individual mutants. The group of genes
with the strongest mutant phenotypes included nearly all (13 of 14) of those
with strong mutant phenotypes identified in the previous screening, as well as a
nearly equal number of new genes. The results thus show the effectiveness of the
Tn-seq method in defining the genetic basis of a complex resistance trait of P.
aeruginosa and indicate that it can be used to analyze a variety of
growth-related processes. Strains of Extraintestinal Pathogenic Escherichia c oli (ExPEC) exhibit an array
of virulence strategies and are a major cause of urinary tract infections,
sepsis and meningitis. Efforts to understand ExPEC pathogenesis are challenged
by the high degree of genetic and phenotypic variation that exists among
isolates. Determining which virulence traits are widespread and which are
strain-specific will greatly benefit the design of more effective therapies.
Towards this goal, we utilized a quantitative genetic footprinting technique
known as transposon insertion sequencing (Tn-seq) in conjunction with
comparative pathogenomics to functionally dissect the genetic repertoire of a
reference ExPEC isolate. Using Tn-seq and high-throughput zebrafish infection
models, we tracked changes in the abundance of ExPEC variants within saturated
transposon mutant libraries following selection within distinct host niches.
Nine hundred and seventy bacterial genes (18% of the genome) were found to
promote pathogen fitness in either a niche-dependent or independent manner. To
identify genes with the highest therapeutic and diagnostic potential, a novel
Trait Enrichment Analysis (TEA) algorithm was developed to ascertain the
phylogenetic distribution of candidate genes. TEA revealed that a significant
portion of the 970 genes identified by Tn-seq have homologues more often
contained within the genomes of ExPEC and other known pathogens, which, as
suggested by the first axiom of molecular Koch's postulates, is considered to be
a key feature of true virulence determits. Three of these Tn-seq-derived
pathogen-associated genes--a transcriptional repressor, a putative
metalloendopeptidase toxin and a hypothetical DNA binding protein--were deleted
and shown to independently affect ExPEC fitness in zebrafish and mouse models of
infection. Together, the approaches and observations reported herein provide a
resource for future pathogenomics-based research and highlight the diversity of
factors required by a single ExPEC isolate to survive within varying host
environments. Whole-genome fitness analysis in microbes that uses saturating transposon
mutagenesis combined with massively parallel sequencing (Tn-seq) is providing a
measure of the contribution of each gene to a given growth condition. With this
technique, gene fitness profiles and essential genes are discovered by
simultaneous analyses of whether the absence of each gene product alters the
growth kinetics of the bacterium. Here we modify the standard Tn-seq procedure
to simplify and shorten the process by including delivery of the transposon
through conjugation and liquid culture enrichment of the mutant pool, creating
transposon liquid enrichment sequencing (TnLE-seq). To illustrate the success of
these modifications and the robustness of the procedure, analyses of gene
fitness of two cultures of the strictly anaerobic bacterium Desulfovibrio
vulgaris Hildenborough were performed, with growth on lactate as the electron
donor and sulfate as the electron acceptor. These data demonstrate
reproducibility and provide a base condition for analysis of fitness changes in
deletion mutants and in various growth conditions. The procedural modifications
will facilitate the application of this powerful genetic analysis to microbes
lacking a facile genetic system. Pilot studies produced 2.5×10(5) and 3.4×10(5)
unique insertion mutants in the anaerobe Desulfovibrio vulgaris Hildenborough
grown under typical laboratory conditions in rich medium. These analyses
provided two similar high-resolution maps of gene fitness across the genome, and
the method was also applied to growth in minimal medium. These results were also
compared to the coverage obtained with a ca. 13,000-member cataloged transposon
library constructed by sequencing transposon insertion sites in individual
mutants. Porphyromonas gingivalis is a keystone pathogen in the development and
progression of periodontal disease. Obstacles to the development of saturated
transposon libraries have previously limited transposon mutant-based screens as
well as essential gene studies. We have developed a system for efficient
transposon mutagenesis of P. gingivalis using a modified mariner transposon.
Tn-seq is a technique that allows for quantitative assessment of individual
mutants within a transposon mutant library by sequencing the transposon-genome
junctions and then compiling mutant presence by mapping to a base genome. Using
Tn-seq, it is possible to quickly define all the insertional mutants in a
library and thus identify nonessential genes under the conditions in which the
library was produced. Identification of fitness of individual mutants under
specific conditions can be performed by exposing the library to selective
pressures. |
Is metabolic syndrome related with cardiovascular disease? | The metabolic syndrome (MetS) is characterized by a cluster of risk factors including central obesity, hypertension, dyslipidemia and insulin resistance, The MetS is associated with an increased risk for cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM). | OBJECTIVE: Associations between metabolic syndrome and its individual components
with risk of incident dementia and its different subtypes are inconsistent.
RESEARCH DESIGN AND METHODS: The 7,087 community-dwelling subjects aged > or =65
years were recruited from the French Three-City (3C) cohort. Hazard ratios (over
4 years) of incident dementia and its subtypes (vascular dementia and
Alzheimer's disease) and association with metabolic syndrome (defined according
to the National Cholesterol Education Program Adult Treatment Panel III
criteria) and its individual components (hypertension, large waist
circumference, high triglycerides, low HDL cholesterol, and elevated fasting
glycemia) were estimated in separate Cox proportional hazard models.
RESULTS: Metabolic syndrome was present in 15.8% of the study participants. The
presence of metabolic syndrome increased the risk of incident vascular dementia
but not Alzheimer's disease over 4 years, independent of sociodemographic
characteristics and the apolipoprotein (apo) Eepsilon4 allele. High triglyceride
level was the only component of metabolic syndrome that was significantly
associated with the incidence of all-cause (hazard ratio 1.45 [95% CI
1.05-2.00]; P = 0.02) and vascular (2.27 [1.16-4.42]; P = 0.02) dementia, even
after adjustment of the apoE genotype. Diabetes, but not impaired fasting
glycemia, was significantly associated with all-cause (1.58 [1.05-2.38]; P =
0.03) and vascular (2.53 [1.15-5.66]; P = 0.03) dementia.
CONCLUSIONS: The observed relation between high triglycerides, diabetes, and
vascular dementia emphasizes the need for detection and treatment of vascular
risk factors in older individuals in order to prevent the likelihood of clinical
dementia. OBJECTIVE: To assess the predictive value of the metabolic syndrome in patients
with type 1 diabetes.
RESEARCH DESIGN AND METHODS: Patients were from the prospective Finnish Diabetic
Nephropathy (FinnDiane) Study (n = 3,783): mean age 37 +/- 12 years and diabetes
duration 23 +/- 12 years. Metabolic syndrome was defined according to World
Health Organization (WHO), National Cholesterol Education Program (NCEP), and
International Diabetes Federation (IDF) definitions. Follow-up time was median
5.5 years (interquartile range 3.7-6.7). Mortality data were complete, whereas
morbidity data were available in 69% of the patients.
RESULTS: The WHO definition was associated with a 2.1-fold increased risk of
cardiovascular events and a 2.5-fold increased risk of cardiovascular- and
diabetes-related mortality, after adjustment for traditional risk factors and
diabetic nephropathy. The NCEP definition did not predict outcomes when adjusted
for nephropathy but markedly added to the risk associated with elevated
albuminuria alone (P < 0.001). The IDF definition did not predict outcomes.
CONCLUSIONS: The metabolic syndrome is a risk factor, beyond albuminuria, for
cardiovascular morbidity and diabetes-related mortality in type 1 diabetes. Metabolic syndrome significantly increases the risk for cardiovascular disease
and chronic kidney disease. The increased risk for cardiovascular diseases can
partly be caused by a prothrombotic state that exists because of abdominal
obesity. Multiple observational studies have consistently shown that increased
body mass index as well as insulin resistance and increased fasting insulin
levels is associated with chronic kidney disease, even after adjustment for
related disorders. Metabolic syndrome appears to be a risk factor for chronic
kidney disease, likely due to the combination of dysglycemia and high blood
pressure. Metabolic syndrome is associated with markedly reduced renal clinical
benefit and increased progression to hemodialysis following endovascular
intervention for atherosclerotic renal artery stenosis. Metabolic syndrome is
associated with inferior early outcomes for dialysis access procedures. Cardiometabolic disorders have been associated with primary hyperparathyroidism
(PHPT), while the relationship of cardiovascular risk score (CRS) and metabolic
syndrome (MS) with different clinical presentation of PHPT remains undefined.
Our aim was to evaluate CRS, MS and its components in PHPT looking for their
correlation to different clinical forms. In 68 consecutive PHPT patients and 68
matched controls, CRS, MS and its components were assessed to perform an
observational case-control study at an ambulatory referral center for Bone
Metabolism Diseases. Patients were stratified in symptomatic and asymptomatic
PHPT; these latter were divided in high-risk and low-risk subgroups for
end-organ damage. An increased proportion of PHPT patients had intermediate-high
CRS and MS (mean, 95 % Confidence Interval (CI) 51.5 %, 39.6-63.3 and 20.6 %,
11.0-30.2, respectively, p < 0.02 vs. controls). Intermediate-high CRS was
prevalent both in symptomatic and low-risk asymptomatic PHPT while MS resulted
prevalent in low-risk asymptomatic but not in symptomatic PHPT. Type 2 DM, IFG,
mixed dyslipidemia, hypertriglyceridemia, HDL-hypocholesterolemia, and
LDL-hypercholesterolemia predominated in low-risk asymptomatic, while only
LDL-hypercholesterolemia prevailed also in symptomatic PHPT. In patients and
controls without cardiometabolic risk factors, HOMA-IR index was significantly
increased in PHPT vs. controls (p < 0.03) and associated to total calcium (R =
0.73; p < 0.001). By multivariate analysis low-risk asymptomatic PHPT predicted
MS after adjusting for age, sex, and BMI. Our data show an increased frequency
of intermediate-high CRS both in symptomatic and low-risk asymptomatic PHPT
while MS prevails in low-risk asymptomatic PHPT, supporting the potential for
cardiovascular morbidity and mortality also in this form. The concept of Sasang Constitutional Medicine (SCM) has been in existence in
Traditional Korean Medicine for more than 100 years. SCM consists of 4 different
types; So-Eum (SE), So-Yang (SY), Tae-Eum (TE), and Tae-Yang (TY). In Western
medicine, it is more like stratifying individuals according to phenotypic
expression. It is of great importance that the Sasang constitution type be
evaluated accurately and recognized by the medical communities for prevention,
early diagnosis and treatment of cardiovascular diseases (CVD).
SUBJECTS AND METHODS: From the Ansung-Ansan prospective cohort study, 10,038
participants were recruited from years 2001-2002. Of 10,038 original
participants, 3022 subjects underwent Sasang Constitutional Type (SCT)
evaluation. The Cox proportional hazard model was used to predict CVD during the
ten year follow-up period.
RESULTS: Of 3022 participants, SCT classified into 364 (12%) SE, 1053 (34.8%)
SY, 1605 (53.1%) TE, and no TY. Three hundred seventy nine (16%) newly developed
CVD during the following period, yielding 10-year cumulative incidence of
160/1000 person. The frequency of CVD within three SCT without metabolic
syndrome (MetS) shows 13.4% in SE, 13.6% in SY, and 14.3% in TE, respectively
(p=NS). The CVD events were significantly different among the types when MetS
was present. The demographic and clinical characteristics revealed the TE group
was significantly older, more obese, higher blood pressure, glucose values, and
lipid profiles levels. The frequency of MetS and type 2 diabetes mellitus (T2DM)
was also higher in TE type than either SE and SY types (all p<0.001). The Cox
proportional hazard analysis revealed age, female gender, rural residence,
higher ALT level, and lower beta-cell function remain as an independent risk
factor, as well as SY with MetS (RR=1.838 (95% CI 1.23-2.74), p=0.003).
Furthermore, 10 year CVD survival rate was 86.4% in no MetS group, 83.4% in TE,
79.6% in SE, and 76.4% in SY all with MetS (p<0.001).
CONCLUSIONS: The findings from this study suggest MetS increases risk for CVD in
certain physical conditions like SY type. Therefore, we would like to suggest
that SCT is a strong indicator for CVD. The metabolic syndrome (MetS) is associated with a higher risk for both, type 2
diabetes mellitus and cardiovascular disease. The cornerstone of treatment is
lifestyle modification, encompassing weight reduction and physical exercise.
However, pharmacotherapy is usually also required to achieve the recommended
target values for the various components of the MetS, such as hypertension,
dysglycemia and dyslipidemia. Regarding lipid treatment, statins are the main
therapeutic agents while in blood pressure control a significant amount of
pathophysiological and clinical evidence would suggest the use, as first line
agents, of ACE inhibitors or angiotensin receptor blockers. Metformin seems to
be the drug of choice for dysglycemia, specially since recent evidence questions
the safety of thiazolidinediones. New drugs, targeting multiple components of
the MetS, are under development but no data are currently available regarding
their long-term efficacy and safety profile. In general, a multifactorial
approach is recommended to decrease cardiovascular risk in patients with the
MetS. Resistin is an adipocyte- and monocyte-derived cytokine which has been
implicated in the modulation of insulin action, energy, glucose and lipid
homeostasis. Resistin has been associated with insulin resistance and many of
its known complications. As a molecular link between metabolic signals,
inflammation, and vascular dysfunction, resistin can be proposed as playing a
significant role in the heightened inflammatory state induced by metabolic
stress linked to excessive caloric intake, thus contributing to the risk for
metabolic syndrome (MetS), type 2 diabetes (T2DM), and cardiovascular diseases
(CVD). In this review, we highlighted the role of resistin, as an inflammatory
cytokine, in the development of CVD, T2DM and the MetS. The metabolic syndrome (MetS) is characterized by a cluster of risk factors
including central obesity, hypertension, dyslipidemia and insulin resistance,
The MetS is associated with an increased risk for cardiovascular disease (CVD)
and type 2 diabetes mellitus (T2DM). Several international organizations have
defined MetS using different diagnostic criteria that produced discrepancies in
the results of previous studies, thus leading to the latest Joint Interim
Societies (JIS) MetS definition. Other risk factors than the diagnostic criteria
that have been associated with MetS include lipid abnormalities, uric acid,
liver function, prothrombotic factors, cytokines, adipokines, vitamin D,
arterial stiffness, polycystic ovary syndrome and obstructive sleep apnea. Apart
from CVD and T2DM, MetS has been related to non-cardiac vascular diseases and in
particular to stroke, carotid artery disease, peripheral artery disease, chronic
kidney disease, atherosclerotic renal artery stenosis and abdominal aortic
aneurysms. In this narrative review, the associations of these diseases with
MetS and its components will be discussed. These associations may further
increase CVD risk in MetS patients, highlighting the importance of treating such
high-risk individuals early and "to target". In this context, multifactorial
treatment including a statin has been proven beneficial, and thus should be
considered, in MetS patients. OBJECTIVE: Metabolic disturbances are well-recognized clinical features of
polycystic ovary syndrome (PCOS). Carotid intima-media thickness (CIMT) has been
widely used as a surrogate marker of atherosclerosis and cardiovascular disease
(CVD). CIMT in women with PCOS has been investigated in many studies, but there
has been only one report in the Korean population. The aim of the present study
was to compare the presence of subclinical atherosclerosis in young untreated
Korean women with PCOS and age-matched controls, specifically by measuring their
CIMT.
METHODS: CIMT was measured by one radiologist in 56 PCOS patients and 56
controls. To compare the CIMT according to PCOS phenotypes, women with PCOS were
divided into two subgroups according to the presence of hyperandrogenism.
RESULTS: Although PCOS patients were more obese and had higher blood pressure
and insulin resistance index than the age-matched controls, the CIMT was not
different between the two groups (0.49 ± 0.09 mm in PCOS patients vs. 0.50 ±
0.11 mm in controls, respectively, p = 0.562). When the CIMT in the control
group was compared with hyperandrogenic and non-hyperandrogenic PCOS groups,
also no significant differences were found.
CONCLUSION: Despite the significant differences in some vascular risk factors
between women with PCOS and controls, PCOS patients did not have a significantly
higher CIMT (even in the hyperandrogenic subgroups). Although our study did not
show the increased risk of subclinical atherosclerosis in PCOS patients, the
role of CIMT continues to be investigated considering the importance of
screening and monitoring CVD risk factors in women with PCOS. |
Is there any functional association during viral replication between flaviviridae viral RNA depended RNA polymerase and viral helicase? | Several labs have obtained evidence for a protein complex that involves many of the nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. The interaction between the helicase, NS3, and the RNA polymerase, NS5B play a key role in viral replication. Pull-down experiments and surface plasmon resonance data indicate a direct interaction between NS3 and NS5B that is primarily mediated through the protease domain of NS3. This interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates product formation in RNA unwinding experiments under conditions of excess nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under pre-steady-state conditions, in which NS3 and substrate concentrations are similar, product formation increased in the presence of NS5B. The increase was consistent with 1:1 complex formed between the two proteins. | Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a
re-emerging human pathogen of global significance. DEN2 nonstructural protein 3
(NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain
of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside
triphosphatase (NTPase)/RNA helicase and a 5'-RNA triphosphatase (RTPase). In
this study the first biochemical and kinetic properties of full-length NS3
(NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL
showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3,
which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active
protease catalytic triad is not required for the stimulatory effect, suggesting
that the overall folding of the N-terminal protease domain contributes to this
enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5'-RNA
methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5
on the NS3 NTPase activity was examined. The results show that NS5 stimulated
the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was
dose-dependent until an equimolar ratio was reached. Moreover, the conserved
motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and
modulating the NTPase/RNA helicase and RTPase activities of NS3. Flaviviruses are enveloped viruses with a single-stranded, 10.7kb positive-sense
RNA genome. The genomic RNA, which has a 5' cap but no poly(A) tail, is
translated as a single polyprotein that is then cleaved into three structural
proteins and seven non-structural (NS) proteins by both viral and host
proteases. The NS proteins include an RNA-dependent RNA polymerase (NS5), a
helicase/protease (NS3), and other proteins that form part of the viral
replication complex. Sequences and structures in the 5' and 3' untranslated
regions (UTR) and capsid gene, including the cyclization sequences, the upstream
AUG region, and the terminal 3' stem-loop, regulate translation, RNA synthesis
and viral replication. We have also found that an RNA hairpin structure in the
capsid coding region (cHP) influences start codon selection and viral
replication of the flavivirus dengue virus (DENV). Peptide-conjugated
phosphorodiamidate morpholino oligomers (P-PMOs) were used to further dissect
the role of conserved regions of the 5' and 3' UTRs; several P-PMOs were shown
to specifically inhibit DENV translation and/or RNA synthesis and, hence, are
potentially useful as antiviral agents. Regarding the mechanism of DENV
translation, we have shown that DENV undergoes canonical cap-dependent
translation initiation as well as a non-canonical mechanism when cap-dependent
translation is suppressed. Although much remains to be elucidated about the
molecular biology of flavivirus infection, progress is being made towards
defining the cis and trans factors that regulate flavivirus translation and
replication. Hepatitis C virus (HCV) infects over 170 million persons worldwide. It is the
leading cause of liver disease in the U.S. and is responsible for most liver
transplants. Current treatments for this infectious disease are inadequate;
therefore, new therapies must be developed. Several labs have obtained evidence
for a protein complex that involves many of the nonstructural (NS) proteins
encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact
structurally and functionally. In this study, we investigated the interaction
between the helicase, NS3, and the RNA polymerase, NS5B. Pull-down experiments
and surface plasmon resoce data indicate a direct interaction between NS3 and
NS5B that is primarily mediated through the protease domain of NS3. This
interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates
product formation in RNA unwinding experiments under conditions of excess
nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of
nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under
pre-steady-state conditions, in which NS3 and substrate concentrations are
similar, product formation increased in the presence of NS5B. The increase was
consistent with 1:1 complex formed between the two proteins. A fluorescently
labeled form of NS3 was used to investigate this interaction through
fluorescence polarization binding assays. Results from this assay support
interactions that include a 1:1 complex formed between NS3 and NS5B. The
modulation of NS3 by NS5B suggests that these proteins may function together
during replication of the HCV genome. NS3 of pestiviruses contains a protease domain and a RNA helicase/NTPase domain.
Contradictory results have been reported regarding NS3 in RNA synthesis. To
investigate the effect of NS3 on classical swine fever virus (CSFV) NS5B
RNA-dependent RNA polymerase activity (RdRp) activity and NS3-NS5B interaction,
RdRp reactions, GST-pull-down assays and co-immunoprecipitation analyses
containing NS5B and either of NS3 protein and the different truncated NS3
mutants were performed, respectively. We found that NS3 stimulated NS5B RdRp
activity in a dose-dependent manner by binding to NS5 through a NS3 protease
domain. Furthermore, mapping important regions of the NS3 protease domain was
carried out by deletion mutagenesis, associated with RdRp reactions,
GST-pull-down assays and co-immunoprecipitation analyses. Results showed that
stimulation of CSFV NS5B RdRp activity was obtained by NS3 binding to NS5B
through a 31-amino acid fragment at the N-terminal end of NS3 protease domain,
which mediated a specific NS3-NS5B interaction. Production of high-quality, well-characterized recombit proteins facilitates
screening of compound libraries. The protocols detailed in this unit are used to
purify three recombit enzymes that are widely used in HCV research: the HCV
NS3 protease domain, the helicase domain as an NS3+NS4A complex, and the NS5B
RNA-dependent RNA polymerase. The active enzymes are purified to homogeneity by
two-column chromatography to support a screening program for HCV inhibitors. The ultimate goal of hepatitis C virus (HCV) treatment is the eradication of the
virus. Ongoing research continues to add to knowledge of the HCV life cycle,
revealing new potential viral and host targets for the development of therapy.
Understanding of HCV was initially hampered by the inability to achieve viral
replication in cell culture. Advances such as the HCV replicon and complete cell
culture systems, however, have permitted rapid growth in knowledge and
accelerated testing of candidate antiviral agents. Among potential targets are
viral entry factors, including scavenger receptor type B1 (SR-B1) and CD81, as
well as neutralizing antibodies against the viral glycoproteins. Popular targets
related to translation and replication are the NS3/4A protease (inhibited by
telaprevir and boceprevir) and the NS5B polymerase, as well as the NS2/3
autoprotease, the NS3 helicase, and nonenzymatic targets such as NS4B and NS5A
proteins. Host targets are also available, including microRNAs and cyclophilins.
This article summarizes a presentation by Charles M. Rice, PhD, at the IAS-USA
live continuing medical education course, Management of Hepatitis C Virus in the
New Era: Small Molecules Bring Big Changes, held in New York City in April 2011. The replication of dengue virus (DENV) RNA requires at least two viral
non-structural (NS) proteins, NS3 and NS5. To facilitate the study of the DENV
replication complex, human monoclonal IgG that are specific for NS proteins have
been generated and characterised. The anti-NS3 IgG, 3F8, binds a conserved
epitope (aa526-531) in the NS3 helicase domain, and cross-reacts with NS3 from
all four DENV serotypes and the related yellow fever virus. The anti-NS2B IgG,
3F10, binds aa49-66 of NS2B (CF18), which forms part of the 47 aa hydrophilic
cofactor region required for NS3 protease activity. The specificity of the IgG
for their respective non-structural proteins has been demonstrated by
immunofluorescence of cells infected with DENV and Western blotting. 3F8 is able
to co-immunoprecipitate NS3 and NS5 from BHK-21 cells infected with DENV2, and
3F10 is able to detect an interaction between recombit NS2B(CF18)NS3
full-length protein and the NS5 RNA-dependent RNA polymerase (RdRp) domain in an
ELISA-based binding assay. The assay is specific and highly reproducible, with a
clear binding curve seen when RdRp is incubated with increasing amounts of
full-length NS3, but not the NS3 protease domain. The NS3 helicase domain
competes with NS3 full-length for NS5 RdRp binding, with a K(d.) of 2.5μM. Since
NS3 and NS5 are required for DENV replication, this fascile assay could be used
to screen for non-nucleoside, allosteric inhibitors that disrupt the interaction
between the two proteins. Influence of the biogenic polyamines spermine, spermidine, and putrescine as
well as their derivatives on the replication enzymes of hepatitis C virus (HCV)
was investigated. It was found that spermine and spermidine activate HCV
RNA-dependent RNA polymerase (NS5B protein). This effect was not caused by the
stabilization of the enzyme or by competition with template-primer complex, but
rather it was due to achievement of true maximum velocity V(max). Natural
polyamines and their derivatives effectively inhibited the helicase reaction
catalyzed by another enzyme of HCV replication - helicase/NTPase (NS3 protein).
However, these compounds affected neither the NTPase reaction nor its activation
by polynucleotides. Activation of the HCV RNA polymerase and inhibition of the
viral helicase were shown at physiological concentrations of the polyamines.
These data suggest that biogenic polyamines may cause differently directed
effects on the replication of the HCV genome in an infected cell. Great progress has been made over the past years in elucidating the structure
and function of the hepatitis C virus (HCV) proteins, most of which are now
actively being pursued as antiviral targets. The structural proteins, which form
the viral particle, include the core protein and the envelope glycoproteins E1
and E2. The nonstructural proteins include the p7 viroporin, the NS2 protease,
the NS3-4A complex harboring protease and NTPase/RNA helicase activities, the
NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. NS4B is a
master organizer of replication complex formation while NS5A is a
zinc-containing phosphoprotein involved in the regulation of HCV RNA replication
versus particle production. Core to NS2 make up the assembly module while NS3 to
NS5B represent the replication module (replicase). However, HCV proteins exert
multiple functions during the viral life cycle, and these may be governed by
different structural conformations and/or interactions with viral and/or
cellular partners. Remarkably, each viral protein is anchored to intracellular
membranes via specific determits that are essential to protein function in
the cell. This review summarizes current knowledge of the structure and function
of the HCV proteins and highlights recent advances in the field. |
Are defects in recombination repair involved in carcinogenesis? | Yes. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in recombination repair. | Human cells can process DNA double-strand breaks (DSBs) by either homology
directed or non-homologous repair pathways. Defects in components of DSB repair
pathways are associated with a predisposition to cancer. The products of the
BRCA1 and BRCA2 genes, which normally confer protection against breast cancer,
are involved in homology-directed DSB repair. Defects in another
homology-directed pathway, single-strand annealing, are associated with genome
instability and cancer predisposition in the Nijmegen breakage syndrome and a
radiation-sensitive ataxia-telangiectasia-like syndrome. Many DSB repair
proteins also participate in the signaling pathways which underlie the cell's
response to DSBs. Repair of DNA double-strand breaks (DSB) is essential for cell viability and
genome stability. Homologous recombination repair plays an important role in DSB
repair and impairment of this repair mechanism may lead to loss of genomic
integrity, which is one of the hallmarks of cancer. Recent research has shown
that the tumor suppressor genes p53 and BRCA1 and -2 are involved in the proper
control of homologous recombination, suggesting a role of this type of repair in
human cancer. We developed a novel assay based on recombination between two
Green Fluorescent Protein (GFP) sequences in transiently transfected plasmid
DNA. The plasmid construct contains an intact, emission-shifted, "blue" variant
of GFP (BFP), with a 300 nucleotide stretch of homology to a nonfunctional copy
of GFP. In the absence of homologous recombination only BFP is present, but
homologous recombination can create a functional GFP. The homologous regions in
the plasmid were constructed in both the direct and the inverted orientation of
transcription to detect possible differences in the recombination mechanisms
involved. A panel of human tumor cell lines was chosen on the basis of genetic
background and chromosome integrity and tested for homologous recombination
using this assay. The panel included cell lines with varying levels of
karyotypic abnormalities, isogenic cell lines with normal and mutant p53,
isogenic cell lines with or without DNA mismatch repair, BRCA1 and -2 mutant
cell lines, and the lymphoma cell line DT40. With this assay, the observed
differences between cell lines with the lowest and highest levels of
recombination were about 100-fold. Increased levels of recombination were
associated with mutant p53, whereas a low level of recombination was found in
the BRCA1 mutant cell line. In the cell line HT1080TG, a mutagenized derivative
of HT1080 with two mutant alleles of p53, high levels of recombination were
found with the direct orientation but not with the inverted orientation plasmid.
No difference in recombination was detected between two isogenic cell lines that
only differed in DNA mismatch repair capability. We conclude that this assay can
detect differences in homologous recombination capacity in cultured cell lines
and that these differences follow the patterns that would be expected from the
different genotypes of these cell lines. Future application in normal cells may
be useful to identify genetic determits controlling genomic integrity or to
detect differences in DNA repair capacity in individuals. The DNA double-strand break (DSB) is the principle cytotoxic lesion for ionizing
radiation and radio-mimetic chemicals but can also be caused by mechanical
stress on chromosomes or when a replicative DNA polymerase encounters a DNA
single-strand break or other type of DNA lesion. DSBs also occur as
intermediates in various biological events, such as V(D)J recombination in
developing lymphoid cells. Inaccurate repair or lack of repair of a DSB can lead
to mutations or to larger-scale genomic instability through the generation of
dicentric or acentric chromosomal fragments. Such genome changes may have
tumourigenic potential. In other instances, DSBs can be sufficient to induce
apoptosis. Because of the threats posed by DSBs, eukaryotic cells have evolved
complex and highly conserved systems to rapidly and efficiently detect these
lesions, signal their presence and bring about their repair. Here, I provide an
overview of these systems, with particular emphasis on the two major pathways of
DSB repair: non-homologous end-joining and homologous recombination. Inherited
or acquired defects in these pathways may lead to cancer or to other human
diseases, and may affect the sensitivity of patients or tumour cells to
radiotherapy and certain chemotherapies. An increased knowledge of DSB repair
and of other DNA DSB responses may therefore provide opportunities for
developing more effective treatments for cancer. XRCC3 is a RAD51 paralog that functions in the repair of DNA double-strand
breaks (DSBs) by homologous recombination (HR). XRCC3 mutation causes severe
chromosome instability. We find that XRCC3 mutant cells display radically
altered HR product spectra, with increased gene conversion tract lengths,
increased frequencies of discontinuous tracts, and frequent local rearrangements
associated with HR. These results indicate that XRCC3 function is not limited to
HR initiation, but extends to later stages in formation and resolution of HR
intermediates, possibly by stabilizing heteroduplex DNA. The results further
demonstrate that HR defects can promote genomic instability not only through
failure to initiate HR (leading to nonhomologous repair) but also through
aberrant processing of HR intermediates. Both mechanisms may contribute to
carcinogenesis in HR-deficient cells. Cancer develops when cells no longer follow their normal pattern of controlled
growth. In the absence or disregard of such regulation, resulting from changes
in their genetic makeup, these errant cells acquire a growth advantage,
expanding into precancerous clones. Over the last decade, many studies have
revealed the relevance of genomic mutation in this process, be it by
misreplication, environmental damage, or a deficiency in repairing endogenous
and exogenous damage. Here, we discuss homologous recombination as another
mechanism that can result in a loss of heterozygosity or genetic rearrangements.
Some of these genetic alterations may play a primary role in carcinogenesis, but
they are more likely to be involved in secondary and subsequent steps of
carcinogenesis by which recessive oncogenic mutations are revealed. Patients,
whose cells display an increased frequency of recombination, also have an
elevated frequency of cancer, further supporting the link between recombination
and carcinogenesis. The breast- and ovarian-specific tumor suppressor, BRCA1, has been implicated to
function in many nuclear processes, including DNA damage repair, recombination,
transcription, ubiquitination, cell cycle checkpoint enforcement, and centrosome
regulation. Utilizing a previously described interaction between BRCA1 and RNA
helicase A (RHA), we have developed a domit-negative approach to block BRCA1
function in human breast epithelial cells. Overexpression of a truncated RHA
peptide that can bind to the BRCA1 carboxy-terminus prevents normal BRCA1
function, such as BRCA1 association with nuclear foci following DNA damage.
Overexpression of this domit-negative protein induces pleomorphic nuclei,
aberrant mitoses with extra centrosomes, and tetraploidy. This model system
allows us to observe changes to mammary epithelial cells that occur acutely
following loss of BRCA1 function. Furthermore, inhibition of BRCA1 via
overexpressing the RHA fragment coincides with a reduction in PARP-1 protein
expression, suggesting a possible mechanism for BRCA1 in the maintece of
genomic integrity. DNA repair has an essential role in protecting the genome from damage by
endogenous and environmental agents. Polymorphisms in DNA repair genes and
differences in repair capacity between individuals have been widely documented.
For colorectal cancer, the loss of mismatch repair gene activity is a key
genetic determit. Nucleotide excision repair (NER), recombination repair (RR)
and base excision repair (BER) pathways have critical roles in protection
against other cancers, and we wished to investigate their role in colorectal
cancer. We have compared the frequency of polymorphisms in the NER genes, XPD,
XPF, XPG, ERCC1; in the BER gene, XRCC1; and in the RR gene, XRCC3; in
colorectal cancer patients and in a control group. No significant associations
were found for any of the NER gene polymorphisms or for the XRCC1 polymorphism.
The C allele (position 18067) of the XRCC3 gene was weakly but significantly
associated with colorectal cancer (odds ratio 1.52, 95% confidence interval
1.04-2.22, P=0.03). For all patients who were heterozygous for any of the repair
genes studied, tumour tissue was investigated for loss of heterozygosity (LOH).
Only one example of LOH was found for all the genes examined. From the
association and LOH data, we conclude that these genes do not have an important
role in protection against colorectal carcinogenesis. Cellular DNA is under constant challenge by exogenous and endogenous genotoxic
stress, which results in both transient and accumulated DNA damage and genomic
instability. All cells are equipped with DNA damage response pathways that
trigger DNA repair, cell cycle arrest, and, if need be, apoptosis, to eliminate
DNA damage or damaged cells. The consequences of these processes for stem cells
can be profound: diminution in stem cell pools, or, because of altered gene
expression, an increased chance for stem cell differentiation or maligt
transformation. Furthermore, a number of DNA repair abnormalities are linked to
premature aging syndromes, and these are associated with defects in the stem
cell population. The specific DNA repair systems for which there are data
regarding the impact of repair defects on stem cell function include
O(6)-alkylguanine DNA alkyltransferase, nucleotide excision repair, base
excision repair, mismatch repair, non-homologous DNA end-joining Fanconi's
anemia protein complex, and homologous recombination. It has recently become
clear that deficiencies of these processes are associated not only with cancer
and/or aging but also with stem cell defects. This discovery raises the
possibility of a link between aging and stem cell dysfunction. In this review,
we provide evidence for a link between DNA repair systems and the maintece
and longevity of stem cells. Mitomycin C (MMC) induces various types of DNA damages that cause significant
cytotoxicity to cells. Accordingly, repair of MMC-induced damages involves
multiple repair pathways such as nucleotide excision repair, homologous
recombination repair and translesion bypass repair pathways. Nonetheless, repair
of the MMC-induced DNA damages in mammals have not been fully delineated. In
this study, we investigated potential roles for Xeroderma pigmentosum (XP)
proteins in the repair of MMC-induced DNA damages using an assay that detects
the ssDNA patches generated following treatment with MMC or 8'-methoxy-psoralen
(8-MOP) + UVA (ultraviolet light A). Human wild-type cells formed distinctive
ssDNA foci following treatment with MMC or 8-MOP + UVA, but not with those
inducing alkylation damage, oxidative damage or strand-break damage, suggesting
that the foci represent ssDNA patches formed during the crosslink repair. In
contrast to wild-type cells, mutant defective in XPE orXPG did not form the
ssDNA foci following MMC treatment, while XPF mutant cells showed a
significantly delayed response in forming the foci. A positive role for XPG in
the repair of MMC-induced DNA damages was further supported by observations that
cells treated with MMC induced a tight association of XPG with chromatin, and a
targeted inhibition of XPG abolished MMC-induced ssDNA foci formation, rendering
cells hypersensitive to MMC. Together, our results suggest that XPG along with
XPE and XPF play unique role(s) in the repair of MMC-induced DNA damages. DNA repair mechanisms are essential for cellular survival in mammals. A rapid
repair of DNA breaks ensures faster growth of normal cells as well as cancer
cells, making DNA repair machinery, a potential therapeutic target. Although
efficiency of these repair processes substantially decrease the efficacy of
cancer chemotherapies that target DNA, compromised DNA repair contributes to
mutagenesis and genomic instability leading to carcinogenesis. Thus, an ideal
target in DNA repair mechanisms would be one that specifically kills the rapidly
dividing cancer cells without further mutagenesis and does not affect normal
cells. Endo-exonucleases play a pivotal role in nucleolytic processing of DNA
ends in different DNA repair mechanisms especially in homologous recombination
repair (HRR) which mainly repairs damaged DNA in S and G2 phases of the cell
cycle in rapidly dividing cells. HRR machinery has also been implicated in cell
signaling and regulatory functions in response to DNA damage that is essential
for cell viability in mammalian cells where as the predomit nonhomologous
end-joining pathway is constitutive. Although HRR is thought to be involved at
other stages of the cell cycle, it is predomit in growing phases (S and G2)
of the cell cycle. The faster growing cells are believed to carryout more HRR in
replicative stages of the cell cycle where homologous DNA is available for HRR.
Targeting endo-exonucleases specifically involved in HRR will make the normal
cells less prone to mutagenesis, rendering the fast growing tumor cells more
susceptible to DNA-damaging agents, used in cancer chemotherapy. Accurate repair of DNA double-strand breaks is essential to life. Indeed,
defective DNA double-strand break repair can lead to toxicity and large scale
sequence rearrangements that cause cancer and promote premature aging. Here, we
highlight the two major repair systems for handling DNA double-strand breaks:
homologous recombination and non-homologous end joining. To clarify
recombination mechanisms, we present animations that illustrate DNA strand
movements. In addition to describing how these pathways operate, we also
describe why appropriate pathway choice is critical to genomic stability, and we
summarize key pathway control features related to cell cycle checkpoint and
apoptosis signaling. Importantly, recent progress in delineating the effects of
specific defects in repair and checkpoint control has helped to explain several
disease phenotypes, including cancer and premature aging. Improved understanding
of these pathways has also sparked development of novel chemotherapeutic
strategies that kill tumors with increased specificity and efficacy. This review
aims to provide a foundational understanding of how the homologous recombination
and non-homologous end joining pathways operate, and to demonstrate how a better
understanding of these processes has advanced both our understanding of the
underlying causes of cancer and our ability to innovate novel cancer treatment
strategies. The efficient repair of double-strand breaks (DSBs) is crucial in maintaining
genomic integrity. Sister chromatid cohesion is important for not only faithful
chromosome segregation but also for proper DSB repair. During DSB repair, the
Smc1-Smc3 cohesin complex is loaded onto chromatin around the DSB to support
recombination-mediated DSB repair. In this study, we investigated whether Ctf18,
a factor implicated in the establishment of sister chromatid cohesion, is
involved in DSB repair in budding yeast. Ctf18 was recruited to HO-endonuclease
induced DSB sites in an Mre11-dependent manner and to damaged chromatin in G2/M
phase-arrested cells. The ctf18 mutant cells showed high sensitivity to
DSB-inducible genotoxic agents and defects in DSB repair, as well as defects in
damage-induced recombination between sister chromatids and between homologous
chromosomes. These results suggest that Ctf18 is involved in damage-induced
homologous recombination. The maintece of the stability of genetic material is an essential feature of
every living organism. Organisms across all kingdoms have evolved diverse and
highly efficient repair mechanisms to protect the genome from deleterious
consequences of various genotoxic factors that might tend to destabilize the
integrity of the genome in each generation. One such group of proteins that is
actively involved in genome surveillance is the RecQ helicase family. These
proteins are highly conserved DNA helicases, which have diverse roles in
multiple DNA metabolic processes such as DNA replication, recombination and DNA
repair. In humans, five RecQ helicases have been identified and three of them
namely, WRN, BLM and RecQL4 have been linked to genetic diseases characterized
by genome instability, premature aging and cancer predisposition. This helicase
family plays important roles in various DNA repair pathways including protecting
the genome from illegitimate recombination during chromosome segregation in
mitosis and assuring genome stability. This review mainly focuses on various
roles of human RecQ helicases in the process of recombination-based DNA repair
to maintain genome stability and physiological consequences of their defects in
the development of cancer and premature aging. BACKGROUND: Werner syndrome (WS) results from defects in the RecQ helicase (WRN)
and is characterized by premature aging and accelerated tumorigenesis.
Contradictorily, WRN deficient human fibroblasts derived from WS patients show a
characteristically slower cell proliferation rate, as do primary fibroblasts and
human cancer cell lines with WRN depletion. Previous studies reported that WRN
silencing in combination with deficiency in other genes led to significantly
accelerated cellular proliferation and tumorigenesis. The aim of the present
study was to examine the effects of silencing WRN in p53 deficient HL60 and p53
wild-type TK6 hematopoietic cells, in order to further the understanding of
WRN-associated tumorigenesis.
METHODOLOGY/PRINCIPAL FINDINGS: We found that silencing WRN accelerated the
proliferation of HL60 cells and decreased the cell growth rate of TK6 cells.
Loss of WRN increased DNA damage in both cell types as measured by COMET assay,
but elicited different responses in each cell line. In HL60 cells, but not in
TK6 cells, the loss of WRN led to significant increases in levels of
phosphorylated RB and numbers of cells progressing from G1 phase to S phase as
shown by cell cycle analysis. Moreover, WRN depletion in HL60 cells led to the
hyper-activation of homologous recombination repair via up-regulation of RAD51
and BLM protein levels. This resulted in DNA damage disrepair, apparent by the
increased frequencies of both spontaneous and chemically induced structural
chromosomal aberrations and sister chromatid exchanges.
CONCLUSIONS/SIGNIFICANCE: Together, our data suggest that the effects of WRN
silencing on cell proliferation and genomic instability are modulated probably
by other genetic factors, including p53, which might play a role in the
carcinogenesis induced by WRN deficiency. PALB2 interacts with BRCA1 and BRCA2 in supercomplexes involved in DNA repair
via homologous recombination. Heterozygous germline mutations in PALB2 confer a
moderate risk of breast cancer, while biallelic PALB2 mutations are linked to a
severe form of Fanconi anaemia characterized by early childhood solid tumours
and severe chromosomal instability. In contrast to BRCA1- or BRCA2-associated
cancers, breast tumours in heterozygous PALB2 mutation carriers do not show loss
of the wild-type allele, suggesting PALB2 might be haploinsufficient for tumour
suppression. To study the role of PALB2 in development and tumourigenesis, we
have generated Palb2(GT) mouse mutants using a gene trap approach. Whereas
Palb2(GT/GT) homozygous mutant embryos died at mid-gestation due to massive
apoptosis, Palb2(GT/+) heterozygous mice were viable and did not show any
obvious abnormalities. Deletion of p53 alleviated the phenotype of Palb2(GT/GT)
embryos, but did not rescue embryonic lethality. In addition, loss of p53 did
not significantly collaborate with Palb2 heterozygosity in tumourigenesis in
heterozygous or homozygous p53 knockout mice. Tumours arising in Palb2(GT/+)
;p53(+/-) or Palb2(GT/+) ;p53(-/-) compound mutant mice retained the wild-type
Palb2 allele and did not display increased genomic instability. Whereas oncogenic retroviruses are common in animals, human T-lymphotropic virus
1 (HTLV-1) is the only transmissible retrovirus associated with cancer in humans
and is etiologically linked to adult T-cell leukemia. The leukemogenesis process
is still largely unknown, but relies on extended survival and clonal expansion
of infected cells, which in turn accumulate genetic defects. A common feature of
human tumor viruses is their ability to stimulate proliferation and survival of
infected pretumoral cells and then hide by establishing latency in cells that
have acquired a transformed phenotype. Whereas disruption of the DNA repair is
one of the major processes responsible for the accumulation of genomic
abnormalities and carcinogenesis, the absence of DNA repair also poses the
threat of cell-cycle arrest or apoptosis of virus-infected cells. This study
describes how the HTLV-1 p30 viral protein inhibits conservative homologous
recombination (HR) DNA repair by targeting the MRE11/RAD50/NBS1 complex and
favors the error-prone nonhomologous-end-joining (NHEJ) DNA-repair pathway
instead. As a result, HTLV-1 p30 may facilitate the accumulation of mutations in
the host genome and the cumulative risk of transformation. Our results provide
new insights into how human tumor viruses may manipulate cellular DNA-damage
responses to promote cancer. Although alcohol consumption is related to increased cancer risk, its molecular
mechanism remains unclear. Here, we demonstrate that an intake of 10% alcohol
for 4 weeks in rats is genotoxic due to induction of micronuclei. Acetaldehyde
(AA), the first product of ethanol metabolism, is believed to be responsible for
DNA damage induced by alcohol. Here, we observe that AA effectively blocks DNA
replication elongation in mammalian cells, resulting in DNA double-strand breaks
associated with replication. AA-induced DNA damage sites colocalize with the
homologous recombination (HR) repair protein RAD51. HR measured in the
hypoxhantineguaninefosforibosyltransferase (HPRT) gene is effectively induced by
AA and recombination defective mammalian cells are hypersensitive to AA, clearly
demonstrating that HR is essential in the repair of AA-induced DNA damage.
Altogether, our data indicate that alcohol genotoxicity related to AA produces
replication lesions on DNA triggering HR repair. Copy number variations (CNVs) encompass a variety of genetic alterations
including deletions and amplifications and cluster in regions of the human
genome with intrinsic instability. Small-sized CNVs can act as initial genetic
changes giving rise to larger CNVs such as acquired somatic copy number
aberrations (CNAs) promoting cancer formation. Previous studies provided
evidence for CNVs as an underlying cause of elevated breast cancer risk when
targeting breast cancer susceptibility genes and of accelerated breast cancer
progression when targeting oncogenes. With the development of novel techniques
for genome-wide detection of CNVs at increasingly higher resolution, it became
possible to qualitatively and quantitatively analyse manifestation of DNA damage
resulting from defects in any of the large variety of DNA double-strand break
(DSB) repair mechanisms. Breast carcinogenesis, particularly in familial cases,
has been linked with a defect in the homologous recombination (HR) pathway,
which in turn switches damage removal towards alternative, more error-prone DSB
repair pathways such as microhomology-mediated non-homologous end joining
(mmNHEJ). Indeed, increased error-prone DSB repair activities were detected in
peripheral blood lymphocytes from individuals with familial breast cancer risk
independently of specific gene mutations. Intriguingly, sequence analysis of
breakpoint regions revealed that the majority of genome aberrations found in
breast cancer specimens are formed by mmNHEJ. Detection of pathway-specific
error-prone DSB repair activities by functional testing was proposed to serve as
biomarker for hereditary breast cancer risk and responsiveness to therapies
targeting HR dysfunction. Identification of specific error-prone DSB repair
mechanisms underlying CNAs and ultimately mammary tumour formation highlights
potential targets for future breast cancer prevention regimens. DNA damage response and repair pathways are important barriers to
carcinogenesis. Here, we show that promyelocytic leukaemia (PML, also known as
TRIM19), involved in sensing DNA damage and executing homologous recombination
repair, is down-regulated in non-tumour liver cells surrounding hepatitis B
virus (HBV)-related hepatocellular carcinoma (HCC). No PML mutation or deletion
was found in HBV-infected liver or HCC cells. Immunohistochemical analysis of
liver biopsies from patients with breast or liver cancer and HBV reactivation
after chemotherapy revealed PML up-regulation and HBV exacerbation in normal
liver tissue in response to DNA damage (functional PML), PML down-regulation in
HCC peritumour cells associated with high HBsAg accumulation and low HBV
replication activity (suppressive PML), and heterogeneous nuclear PML expression
in HCC cells that lost HBV DNA and HBsAg and were non-reactive to DNA damage
(dysregulated PML). Loss of PML in HBsAg-transgenic mice promoted chromosome
breaks in liver cells and accelerated the accumulation of body and liver fat and
the development of a liver steatosis-dysplasia-adenoma-carcinoma sequence in an
inflammation-independent and male-predomit manner, compared to PML knock-out
or HBsAg-transgenic mice during the same time period. These results indicate
that PML deficiency facilitates genomic instability and promotes HBsAg-related
hepatocarcinogenesis, which also involves androgen and lipid metabolism. These
findings uncover a novel PML link between HBV-related tumourigenesis, DNA
repair, and metabolism. Hexavalent chromium [Cr(VI)] is a well known mutagen and carcinogen. Since
genomic instability due to generation of double strand breaks (DSBs) is causally
linked to carcinogenesis, we tested a hypothesis that Cr(VI) causes in vivo
generation of DSBs and elicits DNA damage response. We fed repair proficient
Drosophila melanogaster (Oregon R(+)) larvae Cr(VI) (20.0μg/ml) mixed food for
24 and 48h and observed a significant (p<0.05) induction of DSBs in their midgut
cells after 48h using neutral Comet assay. Global gene expression profiling in
Cr(VI)-exposed Oregon R(+) larvae unveiled mis-regulation of DSBs responsive
repair genes both after 24 and 48h. In vivo generation of DSBs in exposed
Drosophila was confirmed by an increased pH2Av immunostaining along with the
activation of cell cycle regulation genes. Analysis of mis-regulated genes
grouped under DSB response by GOEAST indicated the participation of
non-homologous end joining (NHEJ) DSB repair pathway. We selected two strains,
one mutant (ligIV) and another ku80-RNAi (knockdown of ku80), whose functions
are essentially linked to NHEJ-DSB repair pathway. As a proof of principle, we
compared the DSBs generation in larvae of these two strains with that of repair
proficient Oregon R(+). Along with this, DSBs generation in spn-A and okr
[essential genes in homologous recombination repair (HR) pathway] mutants was
also tested for the possible involvement of HR-DSB repair. A significantly
increased DSBs generation in the exposed ku80-RNAi and ligIV (mutant) larvae
because of impaired repair, concomitant with an insignificant DSBs generation in
okr and spn-A mutant larvae indicates an active participation of NHEJ repair
pathway. The study, first of its kind to our knowledge, while providing
evidences for in vivo generation of DSBs in Cr(VI) exposed Drosophila larvae,
assumes significance for its relevance to higher organisms due to causal link
between DSB generation and Cr(VI)-induced carcinogenesis. Radiation therapy plays an important role in the management of a wide range of
cancers. Besides innovations in the physical application of radiation dose,
radiation therapy is likely to benefit from novel approaches exploiting
differences in radiation response between normal and tumor cells. While ionizing
radiation induces a variety of DNA lesions, including base damages and
single-strand breaks, the DNA double-strand break (DSB) is widely considered as
the lesion responsible not only for the aimed cell killing of tumor cells, but
also for the general genomic instability that leads to the development of
secondary cancers among normal cells. Homologous recombination repair (HRR),
non-homologous end-joining (NHEJ), and alternative NHEJ, operating as a backup,
are the major pathways utilized by cells for the processing of DSBs. Therefore,
their function represents a major mechanism of radiation resistance in tumor
cells. HRR is also required to overcome replication stress - a potent
contributor to genomic instability that fuels cancer development. HRR and
alternative NHEJ show strong cell-cycle dependency and are likely to benefit
from radiation therapy mediated redistribution of tumor cells throughout the
cell-cycle. Moreover, the synthetic lethality phenotype documented between HRR
deficiency and PARP inhibition has opened new avenues for targeted therapies.
These observations make HRR a particularly intriguing target for treatments
aiming to improve the efficacy of radiation therapy. Here, we briefly describe
the major pathways of DSB repair and review their possible contribution to
cancer cell radioresistance. Finally, we discuss promising alternatives for
targeting DSB repair to improve radiation therapy and cancer treatment. In cells of higher eukaryotes, repair of DNA double strand breaks (DSBs)
utilizes different forms of potentially error-prone non-homologous end joining
(NHEJ): canonical DNA-PK-dependent (C-NHEJ) and alternative backup pathways
(A-NHEJ). In contrast to C-NHEJ, A-NHEJ shows pronounced efficiency fluctuations
throughout the cell cycle and is severely compromised as cells cease
proliferating and enter the plateau phase (Windhofer et al., 2007 [23]). The
molecular mechanisms underpinning this response remain unknown but changes in
chromatin structure are prime candidate-A-NHEJ-modulators. Since parameters
beyond chromatin acetylation appear to determine A-NHEJ efficiency (Manova et
al., 2012 [42,76]), we study here the role of chromatin decondensation mediated
either by treatment with 5'-aza-2'-deoxycytidine (AzadC) or growth in hypotonic
conditions, on A-NHEJ. We report that both treatments have no detectable effect
on C-NHEJ but provoke, specifically for A-NHEJ, cell-growth-dependent effects.
These results uncover for the first time a link between A-NHEJ and chromatin
organization and provide means for understanding the regulatory mechanisms
underpinning the growth-state dependency of A-NHEJ. A-NHEJ is implicated in the
formation of chromosomal translocations and in chromosome fusions that underlie
genomic instability and carcinogenesis. The observations reported here may
therefore contribute to the development of drug-based A-NHEJ
suppression-strategies aiming at optimizing cancer treatment outcomes and
possibly also at suppressing carcinogenesis. |
Which is the prevalence of cystic fibrosis in the human population? | Prevalence of Cystic Fibrosis varies according to the population. A theoretical estimate of the prevalence of cystic fibrosis based on anthropological data suggested a frequency of 25 affected individuals/100,000 inhabitants. However, real data indicated that the true prevalence in the population was considerably lower (6.9 cases/100,000 inhabitants). Results of literature reviews, surveys, and registry analyses revealed a mean prevalence of 0.737/10,000 in the 27 EU countries, which is similar to the value of 0.797 in the United States, and only one outlier, namely the Republic of Ireland at 2.98. | The aim of this study was to evaluate the screening policies of cystic fibrosis
(CF) in the Jewish population. The prevalence of mutations that account for CF
in Israel have been defined in the past by determining the frequency of CF
mutations in affected individuals. This study is a population-based study and
is, therefore, different from previous patient-based studies. We found that the
CF mutations D1152H, W1089X, and 405 + IG-->A were present in some ethnic groups
in which no CF patients carrying these mutations were reported. These facts
necessitate a reevaluation of the screening policy regarding the ethnic groups
in Israel. We studied 9,430 healthy Jewish Israeli individuals of 36 countries
of origin. The prevalence of CF mutations was 1:19, 1:19, 1:28, and 1:42 for the
Ashkenazi, Sephardi, North African, and Eastern Jews, respectively. CF mutations
were identified in 374 (4.0%) individuals. These included 173 (46.3%) carriers
of the W1282X mutation; 110 (29.4%) found to carry delF508; 23 (6.1%) who
carried G542X; 22 (5.9%) who carried 3849 + 10Kb (C-->T; 20 (5.3%) who carried
D1152H; 10 (2.7%) who carried N1303K; 11 (2.9%) who carried 405 + IG-->A; 4
(1.1%) who carried W1089X; and one (0.3%) who carried S549R. No carriers were
detected for the 1717-1G-->A, G85E, and T360K mutations, which were tested for
in 7,383, 1,558, and 41 individuals, respectively. Cystic fibrosis is the most common hereditary disease in populations of European
descent, with its prevalence depending on the populations and ethnic groups
studied. In contrast to Europe and North America, there is little information
about this disease in Latin America. Uruguay currently has a human population of
3,000,000, with a low rate of miscegenation and no remaining isolated Amerindian
groups. In the present study, we estimated the prevalence of cystic fibrosis in
this country based on the detection of DeltaF508 mutation carriers in 500
unrelated individuals and on the frequency of individuals homozygous for this
mutation within the affected population. The latter was calculated from the
frequency of the different mutations and genotypes observed in a sample of 52
previously described patients with confirmed cystic fibrosis. A theoretical
estimate of the prevalence of cystic fibrosis based on anthropological data
suggested a frequency of 25 affected individuals/100,000 inhabitants. However,
our data indicated that the true prevalence in the population was considerably
lower (6.9 cases/100,000 inhabitants). BACKGROUND: The epidemiology of cystic fibrosis (CF) is poorly known in North
African populations, in particular in Morocco and the CF carrier frequency in
the general Moroccan population has never been evaluated.
METHODS: To estimate the prevalence of CF mutations in Morocco, blood samples
from 150 healthy Moroccans were tested for frequent CFTR mutations and the
intron 8 polyT variant.
RESULTS: Two subjects were heterozygous for F508del and eight others for the
(T)5 variant.
CONCLUSION: These findings indicate that the Moroccan population is at risk for
CF and CFTR-related disorders. CF prevalence could be in the range of that found
in European populations. Wider studies are necessary to identify the clinical
pattern and accurately determine the prevalence and molecular basis of CF in
Morocco. PURPOSE: This study reports on the phenotype of cystic fibrosis patients
identified to be carriers of the p.Ser489X (p.Ser489*; c.1466C>A) cystic
fibrosis transmembrane conductance regulator (CFTR) mutation, a variant rarely
described in the cystic fibrosis literature, as well as on its allelic frequency
in a French-Canadian cystic fibrosis patient cohort.
METHODS: Reported phenotypes and allelic frequency of this variant were
collected based on the data from a large French-Canadian cystic fibrosis patient
cohort.
RESULTS: Cystic fibrosis patients found to carry the p.Ser489X variant generally
presented with classic gastrointestinal manifestations of this condition in
infancy. The allelic frequency of this variant was calculated to be 0.7% for
this population.
CONCLUSION: The p.Ser489X CFTR variant is a severe disease-causing CFTR allele
that is relatively frequent in the French-Canadian cystic fibrosis patient
population, warranting its inclusion into CFTR molecular testing panel for this
population. |
Are seizures among the neurological symptoms of incontinentia pigmenti? | Incontinentia pigmenti is an X-linked dominant disorder resulting from a mutation of IKBKG. This disorder has a classic dermatologic presentation, but neurologic involvement, with seizures and cortical infarction, can arise shortly after birth. | Incontinentia Pigmenti is an X-linked domit neurocutaneous disorder with
central nervous system manifestations in 30% of cases, including seizures and
mental retardation. Ischemic or hemorrhagic cerebrovascular accidents have been
reported rarely in incontinentia pigmenti. Chart review and literature search
was performed following identification of the index case. We describe a patient
with incontinentia pigmenti who developed bilateral cerebrovascular accidents in
the neonatal period, with resultant severe neurologic sequelae. This is the
second reported case of bilateral cerebrovascular accidents in a patient with
incontinentia pigmenti. This finding may be secondary to cerebrovascular
anomalies, similar to those observed in the retina. Recognition of
cerebrovascular accidents as a complication of incontinentia pigmenti will
hopefully lead to earlier recognition and treatment. Familial incontinentia pigmenti (IP) (OMIM #308300) is a rare genetic disorder
which segregates in an X-linked domit way. The female-to-male ratio ranges
from 20 to 37 : 1. In affected females IP causes highly variable abnormalities
of the skin, hair, nails, teeth, eyes, and central nervous system (CNS).
Cardiovascular anomalies, cerebral infarction, and immune dysfunction are rare
complications of IP. The pathogenesis of cerebral changes in IP remains elusive.
We report the case of two IP-affected sisters who presented in each case with
neonatal seizures on the fifth day of life. Via cranial magnetic resoce
tomographic imaging (MRI) different types of lesions in both hemispheres were
demonstrable in both patients. To date the pathogenetic mechanisms for the
cerebral lesions are not fully understood. However, multiple microscopic
infarcts could serve as a possible explanation. The clinical course and the
neurological development of the older child are favorable and so far the younger
sibling appears to be developing normally, which is uncommon for patients with
early onset of neurological symptoms. Symptomatic seizures in IP are an
important differential diagnosis in benign non-familial and familial neonatal
seizures. Incontinentia pigmenti (IP) is a rare genetic multisystem disorder that may
affect many organs including the skin, bone, eyes and the central nervous
system. Central nervous system manifestations are seen in 30% of cases with
seizures and mental retardation. Seizures occurring as the presenting sign of IP
are rarely reported. We report a case of a female newborn with IP who had
seizures on day 4 of life, which were followed in her second month by the
development of the characteristic cutaneous changes for IP. With this case
report, we would like to emphasize the need for inclusion of IP in the
differential diagnosis of neonatal seizures. Incontinentia Pigmenti is a rare X-linked multisystem disorder with well
described and pathognomonic skin manifestations. Neurological manifestations are
found in 30% of IP patients, forming one of the major causes of morbidity and
mortality of the condition. In this review, clinical and brain imaging data of
45 IP patients with a neurological phenotype are reviewed. Several clinical
presentations could be identified, comprising seizures, infantile
encephalopathy, acute disseminated encephalomyelitis and ischemic stroke. Most
neurological features presented during the neonatal period. No patients
presented during adolescence or at adult age. Seizures of different type are
reported in about 20% of the patients at young age and seem to correlate with
the degree of cerebrovascular damage. Brain MRI findings include periventricular
and subcortical white matter disease, haemorrhagic changes, corpus callosum
hypoplasia, cerebral atrophy and cerebellar hypoplasia. Ocular findings comprise
a range of retinal vascular changes and optic atrophy, but also developmental
defects like microphthalmia and cataract. Most findings may reflect changes
following brain injury. Both (ischemic) vascular and inflammatory components may
play a role in the cerebral and ocular phenotype. However, a role of disturbed
apoptosis during development may also be a contributing factor. Incontinentia pigmenti (IP) is a rare X-linked domit neurocutaneous disorder
affecting ectodermal tissue: skin, eyes, central nervous system, hair, nails,
and teeth. It is usually lethal for males in utero. The involved gene is NEMO,
an essential component of the nuclear factor-kappa B (NF-κB) signaling pathway.
Skin lesions are highly diagnostic, occurring in neonates, with a particular
distribution on Blaschko lines. The severity of the disease is related to ocular
and neurological impairment. The hallmark of ocular IP is retinal vasculopathy
including peripheral retinal vascular nonperfusion, macular infarction and
neovascularization, and preretinal neovascularization. CNS involvement consists
of seizures, mental retardation, hemiparesis, spasticity, microcephaly,
cerebellar ataxia, and coma. It often occurs in neonates. Some patients have
persistent pharmacoresistant seizures throughout life. MRI findings consist
essentially in: white-matter lesions; scattered cortical neuronal necrosis;
multiple cerebral infarctions; cerebral atrophy, hypoplasia of the corpus
callosum, encephalomalacia and neuronal heterotopia. A predomit role of
vascular occlusive phenomena in small vessels is highly suspected. In fact
several intricate mechanisms could be discussed: vascular, inflammatory,
developmental mechanisms. Their role and predictive factors of IP CNS
involvement in neonatal IP need to be better understood to identify effective
innovative therapies. Hypomelanosis of Ito can occur in the neonate, infancy, or
childhood, be isolated or diffuse, often following the Blaschko lines, and can
fade in childhood or adulthood. It is due to reduced melanin in the epidermis.
Eye, central nervous (mental retardation, epilepsy, language disabilities, motor
system dysfunction, psychiatric symptoms including autism - with frequent
cortical malformations including hemimegalencephaly and white matter
involvement), and musculoskeletal systems can also be affected. Mosaicism with
various chromosomal rearrangements has been reported. Author information:
(1)Department of Neurology, Johns Hopkins University School of Medicine,
Baltimore, MD, USA Division of Pediatric Neurology, Emory University/Children's
Healthcare of Atlanta, Atlanta, GA, USA [email protected].
(2)Department of Pediatrics, Eudowood Neonatal Pulmonary Division, Johns Hopkins
University School of Medicine, Baltimore, MD, USA.
(3)McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University
School of Medicine, Baltimore, MD, USA.
(4)Division of Pediatric Dermatology, Johns Hopkins University School of
Medicine, Baltimore, MD, USA.
(5)McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University
School of Medicine, Baltimore, MD, USA Kennedy Krieger Institute, Baltimore, MD,
USA Division of Pediatric Endocrinology, Johns Hopkins University School of
Medicine, Baltimore, MD, USA.
(6)The Wilmer Eye Institute, Johns Hopkins University School of Medicine,
Baltimore, MD, USA.
(7)Department of Neurology, Johns Hopkins University School of Medicine,
Baltimore, MD, USA. BACKGROUND: Some children with incontinentia pigmenti exhibit encephalopathic
features with severe seizures and disturbed consciousness, from the neonatal
through the early infantile period. However, the pathological mechanism of brain
lesion development is not fully understood.
METHODS: We measured the cerebrospinal fluid levels of cytokines and oxidative
stress markers (8-hydroxy-2-deoxyguanosine and the hexanoyl-lysine adduct) in a
young girl with incontinentia pigmenti complicated by an encephalopathic event
that occurred on her first day of life. Magnetic resoce imaging revealed
widespread reduction of water diffusion in the basal ganglia, the
periventricular and subcortical white matter, and the corpus callosum.
RESULTS: Oxidative stress markers were elevated at 4 days of age but decreased
mildly by 25 days of age. Elevated levels of soluble tumor necrosis factor
receptor 1 were observed at both 4 and 25 days of age, although tumor necrosis
factor-α levels were below the limit of detection. No other cytokine levels were
elevated, except for those of interleukin-10 at 25 days of age.
CONCLUSIONS: Tumor necrosis factor-α expression and oxidative stress are
involved in the pathogenesis of brain lesions in children with incontinentia
pigmenti, and elevated cerebrospinal fluid cytokine levels may not be apparent
during encephalopathic events. |
Which are the different homologs or family members of the hedgehog proteins in mammals? | Hedgehog (Hh) signaling proteins stimulate cell proliferation, differentiation, and tissue patterning at multiple points in animal development. A single Hh homolog is present in Drosophila, but three Hh homologs, Sonic hedgehog (Shh), Indian hedgehog (Ihh) , and Desert hedgehog (Dhh), are present in mammals. | Pancreas organogenesis is regulated by the interaction of distinct signaling
pathways that promote or restrict morphogenesis and cell differentiation.
Previous work has shown that activin, a TGF(beta+) signaling molecule, permits
pancreas development by repressing expression of Sonic hedgehog (Shh), a member
of the hedgehog family of signaling molecules that antagonize pancreas
development. Here we show that Indian hedgehog (Ihh), another hedgehog family
member, and Patched 1 (Ptc1), a receptor and negative regulator of hedgehog
activity, are expressed in pancreatic tissue. Targeted inactivation of Ihh in
mice allows ectopic branching of ventral pancreatic tissue resulting in an
annulus that encircles the duodenum, a phenotype frequently observed in humans
suffering from a rare disorder known as annular pancreas. Shh(-)(/)(-) and
Shh(-)(/)(-) Ihh(+/)(-) mutants have a threefold increase in pancreas mass, and
a fourfold increase in pancreatic endocrine cell numbers. In contrast, mutations
in Ptc1 reduce pancreas gene expression and impair glucose homeostasis. Thus,
islet cell, pancreatic mass and pancreatic morphogenesis are regulated by
hedgehog signaling molecules expressed within and adjacent to the embryonic
pancreas. Defects in hedgehog signaling may lead to congenital pancreatic
malformations and glucose intolerance. Sonic hedgehog (SHH), Desert hedgehog (DHH) and Indian hedgehog (IHH) bind to
Patched family receptors (PTCH1 and PTCH2) to transduce signals to GLI1, GLI2
and GLI3. GLI family transcription factors then activate transcription of
Hedgehog target genes, such as FOXE1 and FOXM1 encoding Forkhead-box
transcription factors. Hedgehog signaling pathway plays a pivotal role in a
variety of human tumors, such as gastric cancer, pancreatic cancer, colorectal
cancer, breast cancer, prostate cancer, basal cell carcinoma and brain tumors.
Rat orthologs for human DHH and IHH remain to be identified. Here, we identified
and characterized rat Dhh and Ihh genes by using bioinformatics. Rat Dhh
complete coding sequence (CDS) was determined by assembling nucleotide positions
426397-426963, 429715-429976 and 430244-430898 of the AC114446.3 genome
sequence. Rat Ihh complete CDS was determined by assembling nucleotide positions
63433-64033, 66432-66693 and 68242-69169 of AC095777.6 genome sequence. Rat Dhh
mRNA was expressed in prostate, duodenum and dorsal root ganglia, while rat Ihh
mRNA was expressed in cartilage. Rat Dhh showed 99.7% total-amino-acid identity
with mouse Dhh, and 96.5% total-amino-acid identity with human DHH. Rat Ihh and
human IHH were shorter than mouse Ihh by 38 amino acids. Rat Ihh showed 97.6%
total-amino-acid identity with mouse Ihh and 94.4% total-amino-acid identity
with human IHH. Hedgehog family proteins consist of signal peptide, Hedgehog
ligand peptide and C-terminal peptide. Hedgehog ligand peptides derived from
mammalian Hedgehog family proteins were conserved well, while C-terminal
peptides were relatively divergent. The HPLGMXXXXS motif in the C-terminus was
conserved in Shh orthologs and Ihh orthologs, but not in Dhh orthologs. Hedgehog (Hh) signaling proteins stimulate cell proliferation, differentiation,
and tissue patterning at multiple points in animal development. A single Hh
homolog is present in Drosophila, but three Hh homologs, Sonic Hh, Indian Hh,
and Desert Hh, are present in mammals. Distribution, movement, and reception of
Hh signals are tightly regulated, and abnormal Hh signaling is associated with
developmental defects and cancer. In addition to the integral membrane proteins
Patched and Smoothened, members of the Drosophila Ihog family of adhesion-like
molecules have recently been shown to bind Hh proteins with micromolar affinity
and positively regulate Hh signaling. Cell adhesion molecule-related,
down-regulated by oncogenes (CDO) and Brother of CDO (BOC) are the closest
mammalian relatives of Drosophila Ihog, and CDO binds Sonic Hh with micromolar
affinity and positively regulates Hh signaling. Despite these similarities,
structural and biochemical studies have shown that Ihog and CDO utilize
nonorthologous domains and completely different binding modes to interact with
cognate Hh proteins. We report here biochemical and x-ray structural studies of
Sonic, Indian, and Desert Hh proteins both alone and complexed with active
domains of CDO and BOC. These results show that all mammalian Hh proteins bind
CDO and BOC in the same manner. We also show that interactions between Hh
proteins and CDO are weakened at low pH. Formation of Hh-mediated Hh oligomers
is thought to be an important feature of normal Hh signaling, but no conserved
self-interaction between Hh proteins is apparent from inspection of 14
independent Hh-containing crystal lattices. Indian hedgehog (IHH) is a secreted signaling molecule of the hedgehog family
known to play important roles in the regulation of chondrocyte differentiation,
cortical bone formation, and the development of joints. Here, we describe that
copy-number variations of the IHH locus involving conserved noncoding elements
(CNEs) are associated with syndactyly and craniosynostosis. These CNEs are able
to drive reporter gene expression in a pattern highly similar to wild-type Ihh
expression. We postulate that the observed duplications lead to a misexpression
and/or overexpression of IHH and by this affect the complex regulatory signaling
network during digit and skull development. |
What does mTOR stands for? | mTOR stands for: mammalian target of rapamycin. | BACKGROUND: Enhanced proliferation, resistance to apoptosis, and metabolic shift
to glycolysis of pulmonary arterial vascular smooth muscle cells (PAVSMCs) are
key pathophysiological components of pulmonary vascular remodeling in idiopathic
pulmonary arterial hypertension (PAH). The role of the distinct mammalian target
of rapamycin (mTOR) complexes mTORC1 (mTOR-Raptor) and mTORC2 (mTOR-Rictor) in
PAVSMC proliferation and survival in PAH and their therapeutic relevance are
unknown.
METHODS AND RESULTS: Immunohistochemical and immunoblot analyses revealed that
mTORC1 and mTORC2 pathways are markedly upregulated in small remodeled pulmonary
arteries and isolated distal PAVSMCs from subjects with idiopathic PAH that have
increased ATP levels, proliferation, and survival that depend on glycolytic
metabolism. Small interfering RNA- and pharmacology-based analysis showed that
although both mTORC1 and mTORC2 contribute to proliferation, only mTORC2 is
required for ATP generation and survival of idiopathic PAH PAVSMCs. mTORC2
downregulated the energy sensor AMP-activated protein kinase, which led to
activation of mTORC1-S6 and increased proliferation, as well as a deficiency of
the proapoptotic protein Bim and idiopathic PAH PAVSMC survival. NADPH oxidase 4
(Nox4) protein levels were increased in idiopathic PAH PAVSMCs, which was
necessary for mTORC2 activation, proliferation, and survival. Nox4 levels and
mTORC2 signaling were significantly upregulated in small pulmonary arteries from
hypoxia-exposed rats at days 2 to 28 of hypoxia. Treatment with the mTOR kinase
inhibitor PP242 at days 15 to 28 suppressed mTORC2 but not Nox4, induced smooth
muscle-specific apoptosis in small pulmonary arteries, and reversed
hypoxia-induced pulmonary vascular remodeling in rats.
CONCLUSIONS: These data provide a novel mechanistic link of Nox4-dependent
activation of mTORC2 via the energy sensor AMP-activated protein kinase to
increased proliferation and survival of PAVSMCs in PAH, which suggests a new
potential pathway for therapeutic interventions. The proliferation and migration of vascular smooth muscle cells (VSMCs) in the
intima of an artery, known as intimal hyperplasia, is an important component of
cardiovascular diseases. This is seen most clearly in the case of in-stent
restenosis, where drug eluting stents are used to deliver agents that prevent
VSMC proliferation and migration. One class of agents that are highly effective
in the prevention of in-stent restenosis is the mammalian Target of Rapamycin
(mTOR) inhibitors. Inhibition of mTOR blocks protein synthesis, cell cycle
progression, and cell migration. Key to the effects on cell cycle progression
and cell migration is the inhibition of mTOR-mediated degradation of p27Kip1
protein. p27Kip1 is a cyclin dependent kinase inhibitor that is elevated in
quiescent VSMCs and inhibits the G1 to S phase transition and cell migration.
Under normal conditions, vascular injury promotes degradation of p27Kip1 protein
in an mTOR dependent manner. Recent reports from our lab suggest that in the
presence of diabetes mellitus, elevation of extracellular signal response kinase
activity may promote decreased p27Kip1 mRNA and produce a relative resistance to
mTOR inhibition. Here we review these findings and their relevance to designing
treatments for cardiovascular disease in the presence of diabetes mellitus. Ghrelin exhibits its biological effect through binding to the growth hormone
secretagogue 1a receptor (GHS-R1a). Recently, it has been reported that ghrelin
has an anti-apoptotic effect in several cell types. However, the molecule
mechanisms underlying the anti-apoptotic effect of ghrelin remain poorly
understood. In this study, we investigated the intracellular mechanisms
responsible for anti-apoptotic effect of ghrelin on human umbilical vein
endothelial cells (HUVEC). Treatment of HUVEC with ghrelin inhibited high
glucose-induced cell apoptosis. Ghrelin stimulated the rapid phosphorylation of
mammalian target of rapamycin (mTOR), P70S6K and S6. The GHS-R1a-specific
antagonist [D-Lys3]-GHRP-6 abolished the anti-apoptotic effect and inhibited the
activation of mTOR, P70S6K, S6 induced by ghrelin. Pretreatment of cells with
specific inhibitor of mTOR blocked the anti-apoptotic effect of ghrelin. In
addition, ghrelin protected HUVECs against high glucose induced apoptosis by
increasing Bcl-2/Bax ratio. Taken together, our results demonstrate that ghrelin
produces a protective effect on HUVECs through activating GHS-R1a and
mTOR/P70S6K signaling pathway mediates the effect of ghrelin. These observations
suggest that ghrelin may act as a survival factor in preventing HUVECs apoptosis
caused by high glucose. OBJECTIVE: This study aimed to examine the relationship between expression of
mammal target of rapamycin (mTOR) and phosphorylation of mTOR (p-mTOR) protein
in the PI3K/Akt/mTOR signaling pathways in gastrointestinal stromal tumors and
relatiuonships with clinical factors.
METHODS: Immunohistochemistry was used to detect the expression of the
associated proteins mTOR, p-mTOR, and phosphorylation of the tumor suppressor
genes PTEN, P27, VEGF, and EGFR in 40 cases of gastrointestinal stromal tumors,
with division into a very low and low risk group as well as a moderate and high
risk group.
RESULTS: The positive rate of mTOR and p-mTOR was significantly increased in the
moderate and high risk group compared with the very low and low risk group. The
difference was statistically significant (P<0.05). When grouped according to
size, the positive mTOR expression rate exhibited a statistical difference
(P<0.05), which was significantly increased in the group of tumors larger than 5
cm. The difference in the positive mTOR and p-mTOR expression rate exhibit no
statistical significance among the PTEN, P27, VEGF, and EGFR expression
subgroups (P>0.05).
CONCLUSION: The different expressions of mTOR and p-mTOR in the signal
transduction pathway of gastrointestinal stromal tumor in the different
degree-of-risk groups suggested that the mTOR and p-mTOR of the signal
transduction pathway serve an important function in the occurrence and
development of gastrointestinal stromal tumors. The chronic myeloproliferative neoplasms (MPNs), are characterized by a Janus
Kinase (JAK)-2 V617F point mutation but this molecular abnormality does not
explain by itself the pathogenesis of these disorders, or the phenotypic
diversity associated with essential thrombocythemia, polycythemia vera (PV), and
myelofibrosis. Beyond the JAK/signal transducer and activator of transcription
network, a wide number of molecular alterations were described in MPN including
the fosfatidilinositolo-3-chinasi (PI3K)/protein kinase B (Akt)/mammalian target
of rapamycin (mTOR) pathway constitutive activation. Several pathway inhibitors
were developed, including everolimus, up to the latest class of catalytic
inhibitors such as BKM120 and BEZ235. In this review, we present some clinical
and experimental evidence showing that the PI3K/Akt/mTOR pathway could represent
a therapeutic target in MPNs. In in vitro studies, everolimus has been shown to
inhibit cell proliferation and clonogenic potential in human and murine JAK2
V617F mutated cell lines. Patients with PV and primary myelofibrosis
hematopoietic progenitors were significantly more sensitive to everolimus
compared with healthy control subjects. Of much interest, a combination of
everolimus and the JAK1/2 inhibitor, ruxolitinib, showed strong synergism in
inducing cell cycle arrest and blockade of cell proliferation. Similar data were
obtained using a dual PI3K/mTOR inhibitor, BEZ235, with activity that was also
shown in preclinical murine models. A multicenter phase I/II trial with
everolimus in myelofibrosis documented a well tolerated clinical efficiency in
terms of spleen size reduction and resolution of systemic symptoms and pruritus.
These observations indicate that the PI3K/Akt/mTOR pathway might represent a
novel target for treatment in MPN. The synergism demonstrated in vitro with JAK2
inhibitors could open additional therapeutic possibilities based on concurrent
targeting of different pathways that might optimize efficacy and reduce toxicity
in patients. With the recent introduction of inhibitors of mammalian target of rapamycin
(mTOR) in oncology, distinct cutaneous and oral adverse events have been
identified. In fact, stomatitis and rash are documented as the most frequent and
potentially dose-limiting side effects. Clinically, mTOR inhibitor-associated
stomatitis (mIAS) more closely resembles aphthous stomatitis than oral mucositis
due to conventional anticancer therapies. While most cases of mIAS are mild to
moderate and self-limiting, more severe and persistent mIAS can become a
dose-limiting toxicity. Small ulcerations may cause significant pain and mucosal
sensitivity may occur in the absence of clinical changes. Use of clinical
assessment tools that are primarily driven by ulceration size may underestimate
mIAS, and assessment should include patient-reported outcomes. This article
provides an up-to-date review of the clinical presentation, terminology,
pathogenesis, assessment and management of mIAS and other mTOR
inhibitor-associated oral adverse events. In addition, areas of future research
are considered. The objective of this study was to analyze the expression levels of multiple
components in the mammalian target of rapamycin (mTOR) signaling pathway in
radical nephrectomy specimens from patients with metastatic renal-cell carcinoma
(RCC) treated with mTOR inhibitors in order to identify factors predicting
susceptibility to these agents. This study retrospectively included a total of
48 consecutive patients undergoing radical nephrectomy, who were diagnosed with
metastatic RCC and subsequently treated with an mTOR inhibitor (everolimus or
temsirolimus) as either first- or second-line systemic therapy. Expression
levels of 5 molecular markers involved in the signaling pathway associated with
mTOR, including PTEN, phosphorylated (p)-Akt, p-mTOR, p-p70 ribosomal S6 kinase,
and p-4E-binding protein 1 (4E-BP1), were measured by immunohistochemical
staining of primary RCC specimens. Of several factors examined, bone metastasis,
liver metastasis, and the expression level of p-4E-BP1 were shown to have
significant impacts on the response to the mTOR inhibitors. Progression-free
survival (PFS) was significantly correlated with the expression levels of PTEN
and p-4E-BP1 in addition to the presence of bone metastasis on univariate
analysis. Of these significant factors, p-4E-BP1 expression and bone metastasis
appeared to be independently associated with PFS on multivariate analysis. These
findings suggest that it would be useful to consider the expression levels of
potential molecular markers in the mTOR signaling pathway, particularly
p-4E-BP1, as well as conventional clinical parameters when selecting patients
with metastatic RCC who are likely to benefit from treatment with mTOR
inhibitors. Colorectal cancer is a major contributor of cancer-related mortality. The
mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in
colorectal cancers, promoting cancer progression and chemo-resistance. In the
current study, we investigated the anti-colorectal cancer effect of a novel mTOR
complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal
cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without
inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1
(S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and
activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition
by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking
down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the
apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration
significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the
mice survival was dramatically improved. At the same time, in xenografted tumors
administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely
inhibited, and autophagic markers were significantly increased. Thus, AZD-2014
inhibits colorectal cancer cell growth both in vivo and in vitro. Our results
suggest that AZD-2014 may be further investigated for colorectal cancer therapy
in clinical trials. BACKGROUND: The mammalian target of rapamycin (mTOR) has been suggested as a
target for radiosensitization. Given that radiotherapy is a primary treatment
modality for glioblastoma (GBM) and that mTOR is often dysregulated in GBM, the
goal of this study was to determine the effects of AZD2014, a dual mTORC1/2
inhibitor, on the radiosensitivity of GBM stem-like cells (GSCs).
METHODS: mTORC1 and mTORC2 activities were defined by immunoblot analysis. The
effects of this mTOR inhibitor on the in vitro radiosensitivity of GSCs were
determined using a clonogenic assay. DNA double strand breaks were evaluated
according to γH2AX foci. Orthotopic xenografts initiated from GSCs were used to
define the in vivo response to AZD2014 and radiation.
RESULTS: Exposure of GSCs to AZD2014 resulted in the inhibition of mTORC1 and 2
activities. Based on clonogenic survival analysis, addition of AZD2014 to
culture media 1 hour before irradiation enhanced the radiosensitivity of CD133+
and CD15+ GSC cell lines. Whereas AZD2014 treatment had no effect on the initial
level of γH2AX foci, the dispersal of radiation-induced γH2AX foci was
significantly delayed. Finally, the combination of AZD2014 and radiation
delivered to mice bearing GSC-initiated orthotopic xenografts significantly
prolonged survival as compared with the individual treatments.
CONCLUSIONS: These data indicate that AZD2014 enhances the radiosensitivity of
GSCs both in vitro and under orthotopic in vivo conditions and suggest that this
effect involves an inhibition of DNA repair. Moreover, these results suggest
that this dual mTORC1/2 inhibitor may be a radiosensitizer applicable to GBM
therapy. Vascular endothelial growth factor (VEGF) is supposed to contribute to the
pathogenesis of allergic airway disease. VEGF expression is regulated by a
variety of stimuli such as nitric oxide, growth factors, and hypoxia-inducible
factor-1 alpha (HIF-1α). Recently, inhibition of the mammalian target of
rapamycin (mTOR) has been shown to alleviate cardinal asthmatic features,
including airway hyperresponsiveness, eosinophilic inflammation, and increased
vascular permeability in asthma models. Based on these observations, we have
investigated whether mTOR is associated with HIF-1α-mediated VEGF expression in
allergic asthma. In studies with the mTOR inhibitor rapamycin, we have
elucidated the stimulatory role of a mTOR-HIF-1α-VEGF axis in allergic response.
Next, the mechanisms by which mTOR is activated to modulate this response have
been evaluated. mTOR is known to be regulated by phosphoinositide 3-kinase
(PI3K)/Akt or protein kinase C-delta (PKC δ) in various cell types. Consistent
with these, our results have revealed that suppression of PKC δ by rottlerin
leads to the inhibition of PI3K/Akt activity and the subsequent blockade of a
mTOR-HIF-1α-VEGF module, thereby attenuating typical asthmatic attack in a
murine model. Thus, the present data indicate that PKC δ is necessary for the
modulation of the PI3K/Akt/mTOR signaling cascade, resulting in a tight
regulation of HIF-1α activity and VEGF expression. In conclusion, PKC δ may
represent a valuable target for innovative therapeutic treatment of allergic
airway disease. Differentiation of cancer stem cells (CSCs) into cancer cells causes increased
sensitivity to chemotherapeutic agents. Although inhibition of mammalian target
of rapamycin (mTOR) leads to CSC survival, the effect of branched chain amino
acids (BCAAs), an mTOR complex 1 (mTORC1) activator remains unknown. In this
study, we examined the effects of BCAA on hepatocellular carcinoma (HCC) cells
expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or
5-fluorouracil (FU) on expression of EpCAM and other CSC-related markers, as
well as cell proliferation in HCC cells and in a xenograft mouse model. We also
characterized CSC-related and mTOR signal-related molecule expression and
tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or
overexpression of constitutively active rheb (caRheb). mTOR signal-related
molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA
reduced the frequency of EpCAM-positive cells and improved sensitivity to the
anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better
antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high
doses of BCAA activated mTORC1. Knockdown and overexpression experiments
revealed that inhibition of mTOR complex 2 (mTORC2) or activation of mTORC1 led
to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance
the sensitivity to chemotherapy by reducing the population of cscs via the mTOR
pathway. This result suggests the utility of BCAA in liver cancer therapy. |
Which is the most common genetic lesion among patients with Joubert Syndrome and a cerebello-oculo-renal phenotype? | Joubert syndrome (JBTS) is an inherited ciliopathy giving rise to NPHP with cerebellar vermis aplasia and retinal degeneration. Among patients with JBTS and a cerebello-oculo-renal phenotype, mutations in CEP290 (NPHP6) are the most common genetic lesion. | Nephronophthisis (NPHP) is the major cause of pediatric renal failure, yet the
disease remains poorly understood, partly due to the lack of appropriate animal
models. Joubert syndrome (JBTS) is an inherited ciliopathy giving rise to NPHP
with cerebellar vermis aplasia and retinal degeneration. Among patients with
JBTS and a cerebello-oculo-renal phenotype, mutations in CEP290 (NPHP6) are the
most common genetic lesion. We present a Cep290 gene trap mouse model of JBTS
that displays the kidney, eye, and brain abnormalities that define the syndrome.
Mutant mice present with cystic kidney disease as neonates. Newborn kidneys
contain normal amounts of lymphoid enhancer-binding factor 1 (Lef1) and
transcription factor 1 (Tcf1) protein, indicating normal function of the Wnt
signaling pathway; however, an increase in the protein Gli3 repressor reveals
abnormal Hedgehog (Hh) signaling evident in newborn kidneys. Collecting duct
cells from mutant mice have abnormal primary cilia and are unable to form
spheroid structures in vitro. Treatment of mutant cells with the Hh agonist
purmorphamine restored normal spheroid formation. Renal epithelial cells from a
JBTS patient with CEP290 mutations showed similar impairments to spheroid
formation that could also be partially rescued by exogenous stimulation of Hh
signaling. These data implicate abnormal Hh signaling as the cause of NPHP and
suggest that Hh agonists may be exploited therapeutically. |
Which scales are recommended by the American Heart Association for depression screening in cardiovascular patients? | Patient Health Questionnaire-2 (PHQ-2) and Patient Health Questionnaire-9 (PHQ-9) are recommended by the American Heart Association for depression screening in cardiovascular patients. | Depression is commonly present in patients with coronary heart disease (CHD) and
is independently associated with increased cardiovascular morbidity and
mortality. Screening tests for depressive symptoms should be applied to identify
patients who may require further assessment and treatment. This multispecialty
consensus document reviews the evidence linking depression with CHD and provides
recommendations for healthcare providers for the assessment, referral, and
treatment of depression. The American Heart Association (AHA) and the American Psychiatric Association
jointly recommend screening for depression in cardiology clinics. This includes
screening for suicidality. It is not known how frequently patients disclose
suicidal thinking (ideation) in this setting, and what proportion of those will
turn out to have suicidal intent. Patients were screened for depression using a
protocol identical to the one endorsed by the AHA in a cardiology community
clinic in Elmhurst (Queens, New York). Depression was assessed using the Patient
Health Questionnaire. Reports of suicidal ideation were immediately evaluated by
a mental health professional. We determined the degree to which suicidal
ideation was identified, the proportion of patients with suicidal intent of
those reporting suicidal ideation, and the relation between depression and
suicidal ideation in this setting. One thousand three patients were screened;
886 had complete Patient Health Questionnaire data. Of those, 12% (109 patients)
expressed suicidal ideation. Four of those were hospitalized for suicidal intent
(0.45% of all screened patients). Suicidal ideation and depression were
correlated (point biserial correlation coefficient 0.478). In conclusion,
suicidal ideation can and will be identified using the AHA depression screening
recommendations, but only a very small fraction (0.45%) of screened patients
will turn out to have suicidal intent. Discovery and stabilization of suicidal
patients is an important benefit of the screening, but the fact that >12% of all
screened patients will need to be immediately evaluated for suicidal intent has
important implications for resource allocation to screening programs. BACKGROUND: The American Heart Association (AHA) statement has recommended
routine screening for depression in coronary artery disease with a 2-stage
implementation of the Patient Health Questionnaire (PHQ). Because there is
little evidence on feasibility, accuracy, and impact of such a program on
depression recognition in coronary patients, the AHA recommendation has met
substantial debate and criticism.
METHODS AND RESULTS: Before the AHA statement was released, the Mid America
Heart and Vascular Institute (MAHVI) had implemented a depression screening
protocol for patients with acute myocardial infarction that was virtually
identical to the AHA recommendations. To (1) evaluate this MAHVI quality
improvement initiative, (2) compare MAHVI depression recognition rates with
those of other hospitals, and (3) examine health care providers' implementation
feedback, we compared the results of the MAHVI screening program with data from
a parallel prospective acute myocardial infarction registry and interviewed
MAHVI providers. Depressive symptoms (PHQ-2, PHQ-9) were assessed among 503
MAHVI acute myocardial infarction patients and compared with concurrent
depression assessments among 3533 patients at 23 US centers without a screening
protocol. A qualitative summary of providers' suggestions for improvement was
also generated. A total of 135 (26.8%) eligible MAHVI patients did not get
screened. Among screened patients, 90.9% depressed (PHQ-9 ≥10) patients were
recognized. The agreement between the screening and registry data using the full
PHQ-9 was 61.5% for positive cases (PHQ-9 ≥10) but only 35.6% for the PHQ-2
alone. Although MAHVI had a slightly higher overall depression recognition rate
(38.3%) than other centers not using a depression screening protocol (31.5%),
the difference was not statistically significant (P=0.31). Staff feedback
suggested that a single-stage screening protocol with continuous feedback could
improve compliance.
CONCLUSIONS: In this early effort to implement a depression screening protocol,
a large proportion of patients did not get screened, and only a modest impact on
depression recognition rates was realized. Simplifying the protocol by using the
PHQ-9 alone and providing more support and feedback may improve the rates of
depression detection and treatment. BACKGROUND: Depression screening in cardiac patients has been recommended by the
American Heart Association, but the best approach remains unclear.
OBJECTIVES: To evaluate nurse-administered versions of the Patient Health
Questionnaire for depression screening in patients hospitalized for acute
coronary syndrome.
METHODS: Staff nurses in an urban cardiac care unit administered versions 2, 9,
and 10 of the questionnaire to 100 patients with acute coronary syndrome. The
Depression Interview and Structured Hamilton was administered by advanced
practice nurses blinded to the results of the Patient Health Questionnaire. With
the results of the Depression Interview and Structured Hamilton as a criterion,
receiver operating characteristic analyses were done for each version of the
Patient Health Questionnaire. The Delong method was used for pairwise
comparisons. Cutoff scores balancing false-negatives and false-positives were
determined by using the Youden Index.
RESULTS: Each version of the questionnaire had excellent area-under- the-curve
statistics: 91.2%, 92.6%, and 93.4% for versions 2, 9, and 10, respectively.
Differences among the 3 versions were not significant. Each version yielded
higher symptom scores in depressed patients than in nondepressed patients:
version 2 scores, 3.4 vs 0.6, P = .001; version 9 scores, 13 vs 3.4, P < .001;
and version 10 scores, 14.5 vs 3.6, P < .001.
CONCLUSIONS: For depression screening in hospitalized patients with acute
coronary syndrome, the Patient Health Questionnaire 2 is as accurate as longer
versions when administered by nurses. Further study is needed to determine if
screening with this tool changes clinical decision making or improves outcomes
in these patients. |
Is there a genome-wide technique for the detection of R-loop formation? | Genome-wide analysis of fragile sites by chromatin immunoprecipitation (ChIP) and microarray (ChIP-chip) of phosphorylated H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of R loops. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. The latter pathway operates on nascent transcripts that are not simultaneously translated and requires factors Rho, NusG, and NusA, each of which is essential for viability of WT Escherichia coli. DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. | Two pathways of transcription termination, factor-independent and -dependent,
exist in bacteria. The latter pathway operates on nascent transcripts that are
not simultaneously translated and requires factors Rho, NusG, and NusA, each of
which is essential for viability of WT Escherichia coli. NusG and NusA are also
involved in antitermination of transcription at the ribosomal RNA operons, as
well as in regulating the rates of transcription elongation of all genes. We
have used a bisulfite-sensitivity assay to demonstrate genome-wide increase in
the occurrence of RNA-DNA hybrids (R-loops), including from antisense and
read-through transcripts, in a nusG missense mutant defective for Rho-dependent
termination. Lethality associated with complete deficiency of Rho and NusG (but
not NusA) was rescued by ectopic expression of an R-loop-helicase UvsW,
especially so on defined growth media. Our results suggest that factor-dependent
transcription termination subserves a surveillance function to prevent
translation-uncoupled transcription from generating R-loops, which would block
replication fork progression and therefore be lethal, and that NusA performs
additional essential functions as well in E. coli. Prevention of R-loop-mediated
transcription-replication conflicts by cotranscriptional protein engagement of
nascent RNA is emerging as a unifying theme among both prokaryotes and
eukaryotes. Strand asymmetry in the distribution of guanines and cytosines, measured by GC
skew, predisposes DNA sequences toward R-loop formation upon transcription.
Previous work revealed that GC skew and R-loop formation associate with a core
set of unmethylated CpG island (CGI) promoters in the human genome. Here, we
show that GC skew can distinguish four classes of promoters, including three
types of CGI promoters, each associated with unique epigenetic and gene ontology
signatures. In particular, we identify a strong and a weak class of CGI
promoters and show that these loci are enriched in distinct chromosomal
territories reflecting the intrinsic strength of their protection against DNA
methylation. Interestingly, we show that strong CGI promoters are depleted from
the X chromosome while weak CGIs are enriched, a property consistent with the
acquisition of DNA methylation during dosage compensation. Furthermore, we
identify a third class of CGI promoters based on its unique GC skew profile and
show that this gene set is enriched for Polycomb group targets. Lastly, we show
that nearly 2000 genes harbor GC skew at their 3' ends and that these genes are
preferentially located in gene-dense regions and tend to be closely arranged.
Genomic profiling of R-loops accordingly showed that a large proportion of genes
with terminal GC skew form R-loops at their 3' ends, consistent with a role for
these structures in permitting efficient transcription termination. Altogether,
we show that GC skew and R-loop formation offer significant insights into the
epigenetic regulation, genomic organization, and function of human genes. DNA replication in Escherichia coli is normally initiated at a single origin,
oriC, dependent on initiation protein DnaA. However, replication can be
initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic
evidence indicates that initiation from oriK depends on RNA-DNA hybrids
(R-loops), which are normally removed by enzymes such as RNase HI to prevent
oriK from misfiring during normal growth. Initiation from oriK sites occurs in
RNase HI-deficient mutants, and possibly in wild-type cells under certain
unusual conditions. Despite previous work, the locations of oriK and their
impact on genome stability remain unclear. We combined 2D gel electrophoresis
and whole genome approaches to map genome-wide oriK locations. The DNA copy
number profiles of various RNase HI-deficient strains contained multiple peaks,
often in consistent locations, identifying candidate oriK sites. Removal of
RNase HI protein also leads to global alterations of replication fork migration
patterns, often opposite to normal replication directions, and presumably
eukaryote-like replication fork merging. Our results have implications for
genome stability, offering a new understanding of how RNase HI deficiency
results in R-loop-mediated transcription-replication conflict, as well as
inappropriate replication stalling or blockage at Ter sites outside of the
terminus trap region and at ribosomal operons. Author information:
(1)Department of Genetics, Harvard Medical School, Boston, MA 02215, USA;
Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline
Avenue, Boston, MA 02215, USA. Electronic address:
[email protected].
(2)Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE,
UK.
(3)Department of Biostatistics and Computational Biology, Dana-Farber Cancer
Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Department of
Biostatistics, Harvard School of Public Health, Boston, MA 02115, USA.
(4)Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Computer
Science and Artificial Intelligence Laboratory (CSAIL), MIT, Cambridge, MA
02139, USA.
(5)Department of Genetics, Harvard Medical School, Boston, MA 02215, USA;
Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline
Avenue, Boston, MA 02215, USA.
(6)Department of Genetics, Harvard Medical School, Boston, MA 02215, USA;
Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline
Avenue, Boston, MA 02215, USA. Electronic address:
[email protected]. |
How is CBX1/M31 related to position-effect variegation? | M31 is a heterochromatin component, that is concentrated in the XY body during spermatogenesis. M31 overexpression has two contrasting effects which are dependent on chromosomal context: (i) it enhanced PEV in those lines with centromeric or pericentromeric transgene locations; and (ii) it suppressed PEV when the transgene was non-centromeric. | The formation of the sex vesicle, or XY body, during male meiosis and pairing of
the sex chromosomes are thought to be essential for successful spermatogenesis.
Despite its cytological discovery a century ago, the mechanism of XY body
formation, particularly heterochromatinization of the sex chromosomes, has
remained unclear. The HP1 class of chromobox genes are thought to encode
proteins involved in the packaging of chromosomal DNA into repressive
heterochromatin domains, as seen, for example, in position-effect variegation.
Study of the distribution of a murine HP1-like chromodomain protein, M31, during
spermatogenesis revealed spreading from the tip of the XY body in mid-stage
pachytene spermatocytes to include the whole of the XY body in late-pachytene
spermatocytes. We also demonstrate that the formation of the XY body during
spermatogenic progression in neonatal mice coincides with the expression of a
novel nuclear isoform of M31, M31(p21). These results support the view that a
common mechanistic basis exists for heterochromatin-induced repression, homeotic
gene silencing, and sex-chromosome inactivation during mammalian
spermatogenesis. Centromeres of eukaryotes are frequently associated with constitutive
heterochromatin and their activity appears to be coregulated by epigenetic
modification of higher order chromatin. Recently, we isolated murine (Suv39h1)
and human (SUV39H1) homologues of the domit Drosophila suppressor of position
effect variegation Su(var)3-9, which is also related to the S. pombe silencing
factor Clr4. We have shown that mammalian Su(var)3-9 homologues encode novel
centromeric proteins on metaphase-arrested chromosomes. Here, we describe a
detailed analysis of the chromatin distribution of human SUV39H1 during the cell
cycle. Although there is significant heterochromatic overlap between SUV39H1 and
M31 (HP1(beta)) during interphase, mitotic SUV39H1 displays a more restricted
spatial and temporal association pattern with metaphase chromosomes than M31
(HP1(beta)), or the related HP1(&agr;) gene product. SUV39H1 specifically
accumulates at the centromere during prometaphase but dissociates from
centromeric positions at the meta- to anaphase transition. In addition, SUV39H1
selectively associates with the active centromere of a dicentric chromosome and
also with a neocentromere. Interestingly, SUV39H1 is shown to be a
phosphoprotein with modifications at serine and, to a lesser degree, also at
threonine residues. Whereas SUV39H1 steady-state protein levels appear constant
during the cell cycle, two additional phosphorylated isoforms are detected in
mitotic extracts. This intriguing localisation and modification pattern would be
consistent with a regulatory role(s) for SUV39H1 in participating in higher
order chromatin organisation at mammalian centromeres. SUV39H1, a human homologue of the Drosophila position effect variegation
modifier Su(var)3-9 and of the Schizosaccharomyces pombe silencing factor clr4,
encodes a novel heterochromatic protein that transiently accumulates at
centromeric positions during mitosis. Using a detailed structure-function
analysis of SUV39H1 mutant proteins in transfected cells, we now show that
deregulated SUV39H1 interferes at multiple levels with mammalian higher-order
chromatin organization. First, forced expression of full-length SUV39H1 (412
amino acids) redistributes endogenous M31 (HP1beta) and induces abundant
associations with inter- and metaphase chromatin. These properties depend on the
C-terminal SET domain, although the major portion of the SUV39H1 protein (amino
acids 89 to 412) does not display affinity for nuclear chromatin. By contrast,
the M31 interaction surface, which was mapped to the first 44 N-terminal amino
acids, together with the immediately adjacent chromo domain, directs specific
accumulation at heterochromatin. Second, cells overexpressing full-length
SUV39H1 display severe defects in mitotic progression and chromosome
segregation. Surprisingly, whereas localization of centromere proteins is
unaltered, the focal, G(2)-specific distribution of phosphorylated histone H3 at
serine 10 (phosH3) is dispersed in these cells. This phosH3 shift is not
observed with C-terminally truncated mutant SUV39H1 proteins or with deregulated
M31. Together, our data reveal a domit role(s) for the SET domain of SUV39H1
in the distribution of prominent heterochromatic proteins and suggest a possible
link between a chromosomal SU(VAR) protein and histone H3. |
Can bioprinting use human cells? | Yes, human cells can be used for bioprinting | Advanced solid freeform fabrication (SFF) techniques have been an interest for
constructing tissue engineered polymeric scaffolds because of its repeatability
and capability of high accuracy in fabrication resolution at the scaffold macro-
and microscales. Among many important scaffold applications, hydrogel scaffolds
have been utilized in tissue engineering as a technique to confide the desired
proliferation of seeded cells in vitro and in vivo into its architecturally
porous three-dimensional structures. Such fabrication techniques not only enable
the reconstruction of scaffolds with accurate anatomical architectures but also
enable the ability to incorporate bioactive species such as growth factors,
proteins, and living cells. This paper presents a bioprinting system designed
for the freeform fabrication of porous alginate scaffolds with encapsulated
endothelial cells. The bioprinting fabrication system includes a multinozzle
deposition system that utilizes SFF techniques and a computer-aided modeling
system capable of creating heterogeneous tissue scaffolds. The manufacturing
process is biologically compatible and is capable of functioning at room
temperature and relatively low pressures to reduce the fluidic shear forces that
could deteriorate biologically active species. The deposition system resolution
is 10 microm in the three orthogonal directions XYZ and has minimum velocity of
100 microm/s. The ideal concentrations of sodium alginate and calcium chloride
were investigated to determine a viable bioprinting process. The results
indicated that the suitable fabrication parameters were 1.5% (w/v) sodium
alginate and 0.5% (w/v) calcium chloride. Degradation studies via mechanical
testing showed a decrease in the elastic modulus by 35% after 3 weeks. Cell
viability studies were conducted on the cell encapsulated scaffolds for
validating the bioprinting process and determining cell viability of 83%. This
work exhibits the potential use of accurate cell placement for engineering
complex tissue regeneration using computer-aided design systems. Regenerative medicine seeks to repair or replace dysfunctional tissues with
engineered biological or biohybrid systems. Current clinical regenerative models
utilize simple uniform tissue constructs formed with cells cultured onto
biocompatible scaffolds. Future regenerative therapies will require the
fabrication of complex three-dimensional constructs containing multiple cell
types and extracellular matrices. We believe bioprinting technologies will
provide a key role in the design and construction of future engineered tissues
for cell-based and regenerative therapies. This review describes the current
state-of-the-art bioprinting technologies, focusing on direct-write bioprinting.
We describe a number of process and device considerations for successful
bioprinting of composite biohybrid constructs. In addition, we have provided
baseline direct-write printing parameters for a hydrogel system (Pluronic F127)
often used in cardiovascular applications. Direct-write dispensed lines (gels
with viscosities ranging from 30 mPa s to greater than 600 × 10⁶ mPa s) were
measured following mechanical and pneumatic printing via three commercially
available needle sizes (20 ga, 25 ga, and 30 ga). Example patterns containing
microvascular cells and isolated microvessel fragments were also bioprinted into
composite 3D structures. Cells and vessel fragments remained viable and
maintained in vitro behavior after incorporation into biohybrid structures.
Direct-write bioprinting of biologicals provides a unique method to design and
fabricate complex, multicomponent 3D structures for experimental use. We hope
our design insights and baseline parameter descriptions of direct-write
bioprinting will provide a useful foundation for colleagues to incorporate this
3D fabrication method into future regenerative therapies. Developing tools to reproduce and manipulate the cell micro-environment,
including the location and shape of cell patterns, is essential for tissue
engineering. Parallel to inkjet printing and pressure-operated mechanical
extruders, laser-assisted bioprinting (LAB) has emerged as an alternative
technology to fabricate two- and three-dimensional tissue engineering products.
The objective of this work was to determine laser printing parameters for
patterning and assembling o-hydroxyapatite (nHA) and human osteoprogenitors
(HOPs) in two and three dimensions with LAB. The LAB workstation used in this
study comprised an infrared laser focused on a quartz ribbon that was coated
with a thin absorbing layer of titanium and a layer of bioink. The scanning
system, quartz ribbon and substrate were piloted by dedicated software, allowing
the sequential printing of different biological materials into two and/or three
dimensions. nHA printing material (bioink) was synthesized by chemical
precipitation and was characterized prior and following printing using
transmission electron microscopy, Fourier transformed infrared spectroscopy and
x-ray diffraction. HOP bioink was prepared using a 30 million cells ml(-1)
suspension in culture medium and cells were characterized after printing using a
Live/Dead assay and osteoblastic phenotype markers (alcaline phosphatase and
osteocalcin). The results revealed that LAB allows printing and organizing nHA
and HOPs in two and three dimensions. LAB did not alter the physico-chemical
properties of nHA, nor the viability, proliferation and phenotype of HOPs over
time (up to 15 days). This study has demonstrated that LAB is a relevant method
for patterning nHA and osteoblastic cells in 2D, and is also adapted to the
bio-fabrication of 3D composite materials. With the advantages of high throughput, digital control, and highly accurate
placement of cells and biomaterial scaffold to the desired 2D and 3D locations,
bioprinting has great potential to develop promising approaches in translational
medicine and organ replacement. The most recent advances in organ and tissue
bioprinting based on the thermal inkjet printing technology are described in
this review. Bioprinting has no or little side effect to the printed mammalian
cells and it can conveniently combine with gene transfection or drug delivery to
the ejected living systems during the precise placement for tissue construction.
With layer-by-layer assembly, 3D tissues with complex structures can be printed
using scanned CT or MRI images. Vascular or nerve systems can be enabled
simultaneously during the organ construction with digital control. Therefore,
bioprinting is the only solution to solve this critical issue in thick and
complex tissues fabrication with vascular system. Collectively, bioprinting
based on thermal inkjet has great potential and broad applications in tissue
engineering and regenerative medicine. This review article introduces some
important patents related to bioprinting of living systems and the applications
of bioprinting in tissue engineering field. Heart valve disease is a serious and growing public health problem for which
prosthetic replacement is most commonly indicated. Current prosthetic devices
are inadequate for younger adults and growing children. Tissue engineered living
aortic valve conduits have potential for remodeling, regeneration, and growth,
but fabricating natural anatomical complexity with cellular heterogeneity remain
challenging. In the current study, we implement 3D bioprinting to fabricate
living alginate/gelatin hydrogel valve conduits with anatomical architecture and
direct incorporation of dual cell types in a regionally constrained manner.
Encapsulated aortic root sinus smooth muscle cells (SMC) and aortic valve
leaflet interstitial cells (VIC) were viable within alginate/gelatin hydrogel
discs over 7 days in culture. Acellular 3D printed hydrogels exhibited reduced
modulus, ultimate strength, and peak strain reducing slightly over 7-day
culture, while the tensile biomechanics of cell-laden hydrogels were maintained.
Aortic valve conduits were successfully bioprinted with direct encapsulation of
SMC in the valve root and VIC in the leaflets. Both cell types were viable (81.4
± 3.4% for SMC and 83.2 ± 4.0% for VIC) within 3D printed tissues. Encapsulated
SMC expressed elevated alpha-smooth muscle actin, while VIC expressed elevated
vimentin. These results demonstrate that anatomically complex, heterogeneously
encapsulated aortic valve hydrogel conduits can be fabricated with 3D
bioprinting. Recently, there has been growing interest in applying bioprinting techniques to
stem cell research. Several bioprinting methods have been developed utilizing
acoustics, piezoelectricity, and lasers to deposit living cells onto receiving
substrates. Using these technologies, spatially defined gradients of immobilized
biomolecules can be engineered to direct stem cell differentiation into multiple
subpopulations of different lineages. Stem cells can also be patterned in a
high-throughput manner onto flexible implementation patches for tissue
regeneration or onto substrates with the goal of accessing encapsulated stem
cells of interest for genomic analysis. Here, we review recent achievements with
bioprinting technologies in stem cell research, and identify future challenges
and potential applications including tissue engineering and regenerative
medicine, wound healing, and genomics. In recent years, the use of a simple inkjet technology for cell printing has
triggered tremendous interest and established the field of biofabrication. A key
challenge has been the development of printing processes which are both
controllable and less harmful, in order to preserve cell and tissue viability
and functions. Here, we report on the development of a valve-based cell printer
that has been validated to print highly viable cells in programmable patterns
from two different bio-inks with independent control of the volume of each
droplet (with a lower limit of 2 nL or fewer than five cells per droplet). Human
ESCs were used to make spheroids by overprinting two opposing gradients of
bio-ink; one of hESCs in medium and the other of medium alone. The resulting
array of uniform sized droplets with a gradient of cell concentrations was
inverted to allow cells to aggregate and form spheroids via gravity. The
resulting aggregates have controllable and repeatable sizes, and consequently
they can be made to order for specific applications. Spheroids with between 5
and 140 dissociated cells resulted in spheroids of 0.25-0.6 mm diameter. This
work demonstrates that the valve-based printing process is gentle enough to
maintain stem cell viability, accurate enough to produce spheroids of uniform
size, and that printed cells maintain their pluripotency. This study includes
the first analysis of the response of human embryonic stem cells to the printing
process using this valve-based printing setup. OBJECTIVE: To explore the three dimensional(3D)bioprinting technology, using
human dental pulp cells (hDPCs) mixture as bioink and to lay initial foundations
for the application of the 3D bioprinting technology in tooth regeneration.
METHODS: Imageware 11.0 computer software was used to aid the design of the 3D
biological printing blueprint. Sodium alginate-gelatin hydrosol was prepared and
mixed with in vitro isolated hDPCs. The mixture contained 20 g/L sodium alginate
and 80 g/L gelatin with cell density of 1×10(6)/mL. The bioprinting of hDPCs
mixture was carried out according to certain parameters; the 3D constructs
obtained by printing were examined; the viability of hDPCs after printing by
staining the constructs with calcein-AM and propidium iodide dye and scanning of
laser scanning confocal microscope was evaluated. The in vitro constructs
obtained by the bioprinting were cultured, and the proliferation of hDPCs in the
constructs detected.
RESULTS: By using Imageware 11.0 software, the 3D constructs with the grid
structure composed of the accumulation of staggered cylindrical microfilament
layers were obtained. According to certain parameters, the hDPCs-sodium
alginate-gelatin blends were printed by the 3D bioprinting technology. The
self-defined shape and dimension of 3D constructs with the cell survival rate of
87%± 2% were constructed. The hDPCs could proliferate in 3D constructs after
printing.
CONCLUSION: In this study, the 3D bioprinting of hDPCs mixtures was realized,
thus laying initial foundations for the application of the 3D bioprinting
technology in tooth regeneration. Three-dimensional (3-D) bioprinting is the layer-by-layer deposition of
biological material with the aim of achieving stable 3-D constructs for
application in tissue engineering. It is a powerful tool for the spatially
directed placement of multiple materials and/or cells within the 3-D sample.
Encapsulated cells are protected by the bioink during the printing process. Very
few materials are available that fulfill requirements for bioprinting as well as
provide adequate properties for cell encapsulation during and after the printing
process. A hydrogel composite including alginate and gelatin precursors was
tuned with different concentrations of hydroxyapatite (HA) and characterized in
terms of rheology, swelling behavior and mechanical properties to assess the
versatility of the system. Instantaneous as well as long-term structural
integrity of the printed hydrogel was achieved with a two-step mechanism
combining the thermosensitive properties of gelatin with chemical crosslinking
of alginate. Novel syringe tip heaters were developed for improved temperature
control of the bioink to avoid clogging. Human mesenchymal stem cells mixed into
the hydrogel precursor survived the printing process and showed high cell
viability of 85% living cells after 3 days of subsequent in vitro culture. HA
enabled the visualization of the printed structures with micro-computed
tomography. The inclusion of HA also favors the use of the bioink for bone
tissue engineering applications. By adding factors other than HA, the composite
could be used as a bioink for applications in drug delivery, microsphere
deposition or soft tissue engineering. Three-dimensional (3D) bioprinting, a flexible automated on-demand platform for
the free-form fabrication of complex living architectures, is a novel approach
for the design and engineering of human organs and tissues. Here, we demonstrate
the potential of 3D bioprinting for tissue engineering using human skin as a
prototypical example. Keratinocytes and fibroblasts were used as constituent
cells to represent the epidermis and dermis, and collagen was used to represent
the dermal matrix of the skin. Preliminary studies were conducted to optimize
printing parameters for maximum cell viability as well as for the optimization
of cell densities in the epidermis and dermis to mimic physiologically relevant
attributes of human skin. Printed 3D constructs were cultured in submerged media
conditions followed by exposure of the epidermal layer to the air-liquid
interface to promote maturation and stratification. Histology and
immunofluorescence characterization demonstrated that 3D printed skin tissue was
morphologically and biologically representative of in vivo human skin tissue. In
comparison with traditional methods for skin engineering, 3D bioprinting offers
several advantages in terms of shape- and form retention, flexibility,
reproducibility, and high culture throughput. It has a broad range of
applications in transdermal and topical formulation discovery, dermal toxicity
studies, and in designing autologous grafts for wound healing. The
proof-of-concept studies presented here can be further extended for enhancing
the complexity of the skin model via the incorporation of secondary and adnexal
structures or the inclusion of diseased cells to serve as a model for studying
the pathophysiology of skin diseases. Tissue engineering has great potential to provide a functional de novo living
valve replacement, capable of integration with host tissue and growth. Among
various valve conduit fabrication techniques, three-dimensional (3-D)
bioprinting enables deposition of cells and hydrogels into 3-D constructs with
anatomical geometry and heterogeneous mechanical properties. Successful
translation of this approach, however, is constrained by the dearth of printable
and biocompatible hydrogel materials. Furthermore, it is not known how human
valve cells respond to these printed environments. In this study, 3-D printable
formulations of hybrid hydrogels are developed, based on methacrylated
hyaluronic acid (Me-HA) and methacrylated gelatin (Me-Gel), and used to bioprint
heart valve conduits containing encapsulated human aortic valvular interstitial
cells (HAVIC). Increasing Me-Gel concentration resulted in lower stiffness and
higher viscosity, facilitated cell spreading, and better maintained HAVIC
fibroblastic phenotype. Bioprinting accuracy was dependent upon the relative
concentrations of Me-Gel and Me-HA, but when optimized enabled the fabrication
of a trileaflet valve shape accurate to the original design. HAVIC encapsulated
within bioprinted heart valves maintained high viability, and remodeled the
initial matrix by depositing collagen and glyosaminoglycans. These findings
represent the first rational design of bioprinted trileaflet valve hydrogels
that regulate encapsulated human VIC behavior. The use of anatomically accurate
living valve scaffolds through bioprinting may accelerate understanding of
physiological valve cell interactions and progress towards de novo living valve
replacements. The aim of tissue engineering is to produce functional three-dimensional (3D)
tissue substitutes. Regarding native organ and tissue complexity, cell density
and cell spatial 3D organization, which influence cell behavior and fate, are
key parameters in tissue engineering. Laser-Assisted Bioprinting (LAB) allows
one to print cells and liquid materials with a cell- or picoliter-level
resolution. Thus, LAB seems to be an emerging and promising technology to
fabricate tissue-like structures that have the physiological functionality of
their native counterparts. This technology has additional advantages such as
automation, reproducibility, and high throughput. It makes LAB compatible with
the (industrial) fabrication of 3D constructs of physiologically relevant sizes.
Here we present exhaustively the numerous steps that allow printing of viable
cells with a well-preserved micrometer pattern. To facilitate the understanding
of the whole cell patterning experiment using LAB, it is discussed in two parts:
(1) preprocessing: laser set-up, bio-ink cartridge and bio-paper preparation,
and pattern design; and (2) processing: bio-ink printing on the bio-paper. Fabrication of three dimensional (3D) organoids with controlled
microarchitectures has been shown to enhance tissue functionality. Bioprinting
can be used to precisely position cells and cell-laden materials to generate
controlled tissue architecture. Therefore, it represents an exciting alternative
for organ fabrication. Despite the rapid progress in the field, the development
of printing processes that can be used to fabricate macroscale tissue constructs
from ECM-derived hydrogels has remained a challenge. Here we report a strategy
for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA)
hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to
15% with varying cell densities and found a direct correlation between
printability and the hydrogel mechanical properties. Furthermore, encapsulated
HepG2 cells preserved cell viability for at least eight days following the
bioprinting process. In summary, this work presents a strategy for direct-write
bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find
widespread application for tissue engineering, organ printing and the
development of 3D drug discovery platforms. In this work we present a bioprinting technique that exploits the
electrohydrodynamic process to obtain a jet of liquid alginate beads containing
cells. A printer is used to microfabricate hydrogels block by block following a
bottom-up approach. Alginate beads constitute the building blocks of the
microfabricated structures. The beads are placed at predefined position on a
target substrate made of calcium-enriched gelatin, where they crosslink upon
contact without the need of further postprocessing. The printed sample can be
easily removed from the substrate at physiological temperature.
Three-dimensional printing is accomplished by the deposition of multiple layers
of hydrogel. We have investigated the parameters influencing the process, the
compatibility of the printing procedure with cells, and their survival after
printing. Bioprinting, which is based on thermal inkjet printing, is one of the most
attractive enabling technologies in the field of tissue engineering and
regenerative medicine. With digital control cells, scaffolds, and growth factors
can be precisely deposited to the desired two-dimensional (2D) and
three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal
approach to fabricate tissues mimicking their native anatomic structures. In
order to engineer cartilage with native zonal organization, extracellular matrix
composition (ECM), and mechanical properties, we developed a bioprinting
platform using a commercial inkjet printer with simultaneous photopolymerization
capable for 3D cartilage tissue engineering. Human chondrocytes suspended in
poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage
construction via layer-by-layer assembly. The printed cells were fixed at their
original deposited positions, supported by the surrounding scaffold in
simultaneous photopolymerization. The mechanical properties of the printed
tissue were similar to the native cartilage. Compared to conventional tissue
fabrication, which requires longer UV exposure, the viability of the printed
cells with simultaneous photopolymerization was significantly higher. Printed
neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II
production, which was consistent with gene expression. Therefore, this platform
is ideal for accurate cell distribution and arrangement for anatomic tissue
engineering. Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically
inert matrix that can be used for encapsulating and 3D bioprinting of
bone-related SaOS-2 cells. However, the cells, embedded in this matrix, remain
in a non-proliferating state. Here we show that addition of an overlay onto the
bioprinted alginate/gelatine/SaOS-2 cell scaffold, consisting of agarose and the
calcium salt of polyphosphate [polyP·Ca(2+)-complex], resulted in a marked
increase in cell proliferation. In the presence of 100 μm polyP·Ca(2+)-complex,
the cells proliferate with a generation time of approximately 47-55 h. In
addition, the hardness of the alginate/gelatin hydrogel substantially increases
in the presence of the polymer. The reduced Young's modulus for the
alginate/gelatin hydrogel is approximately 13-14 kPa, and this value drops to
approximately 0.5 kPa after incubation of the cell containing scaffolds for 5 d.
In the presence of 100 μm polyP·Ca(2+)-complex, the reduced Young's modulus
increases to about 22 kPa. The hardness of the polyP·Ca(2+)-complex containing
hydrogel remains essentially constant if cells are absent in the matrix, but it
drops to 3.2 kPa after a 5 d incubation period in the presence of SaOS-2 cells,
indicating that polyP·Ca(2+)-complex becomes metabolized, degraded, by the
cells. The alginate/gelatine-agarose system with polyP·Ca(2+)-complex cause a
significant increase in the mineralization of the cells. SEM analyses revealed
that the morphology of the mineral nodules formed on the surface of the cells
embedded in the alginate/gelatin hydrogel do not significantly differ from the
nodules on cells growing in monolayer cultures. The newly developed technique,
using cells encapsulated into an alginate/gelatin hydrogel and a secondary layer
containing the morphogenetically active, growth promoting polymer
polyP·Ca(2+)-complex opens new possibilities for the application of 3D
bioprinting in bone tissue engineering. Bioprinting allows the fabrication of living constructs with custom-made
architectures by spatially controlled deposition of multiple bioinks. This is
important for the generation of tissue, such as osteochondral tissue, which
displays a zonal composition in the cartilage domain supported by the underlying
subchondral bone. Challenges in fabricating functional grafts of clinically
relevant size include the incorporation of cues to guide specific cell
differentiation and the generation of sufficient cells, which is hard to obtain
with conventional cell culture techniques. A novel strategy to address these
demands is to combine bioprinting with microcarrier technology. This technology
allows for the extensive expansion of cells, while they form multi-cellular
aggregates, and their phenotype can be controlled. In this work, living
constructs were fabricated via bioprinting of cell-laden microcarriers.
Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via
static culture or spinner flask expansion, were encapsulated in gelatin
methacrylamide-gellan gum bioinks, and the printability of the composite
material was studied. This bioprinting approach allowed for the fabrication of
constructs with high cell concentration and viability. Microcarrier
encapsulation improved the compressive modulus of the hydrogel constructs,
facilitated cell adhesion, and supported osteogenic differentiation and bone
matrix deposition by MSCs. Bilayered osteochondral models were fabricated using
microcarrier-laden bioink for the bone compartment. These findings underscore
the potential of this new microcarrier-based biofabrication approach for bone
and osteochondral constructs. Additive manufacturing, otherwise known as three-dimensional (3D) printing, is
driving major innovations in many areas, such as engineering, manufacturing,
art, education and medicine. Recent advances have enabled 3D printing of
biocompatible materials, cells and supporting components into complex 3D
functional living tissues. 3D bioprinting is being applied to regenerative
medicine to address the need for tissues and organs suitable for
transplantation. Compared with non-biological printing, 3D bioprinting involves
additional complexities, such as the choice of materials, cell types, growth and
differentiation factors, and technical challenges related to the sensitivities
of living cells and the construction of tissues. Addressing these complexities
requires the integration of technologies from the fields of engineering,
biomaterials science, cell biology, physics and medicine. 3D bioprinting has
already been used for the generation and transplantation of several tissues,
including multilayered skin, bone, vascular grafts, tracheal splints, heart
tissue and cartilaginous structures. Other applications include developing
high-throughput 3D-bioprinted tissue models for research, drug discovery and
toxicology. Bioprinting has exciting prospects for printing three-dimensional (3-D) tissue
constructs by delivering living cells with appropriate matrix materials.
However, progress in this field is currently extremely slow due to limited
choices of bioink for cell encapsulation and cytocompatible gelation mechanisms.
Here we report the development of clinically relevant sized tissue analogs by
3-D bioprinting, delivering human nasal inferior turbinate tissue-derived
mesenchymal progenitor cells encapsulated in silk fibroin-gelatin (SF-G) bioink.
Gelation in this bioink was induced via in situ cytocompatible gelation
mechanisms, namely enzymatic crosslinking by mushroom tyrosinase and physical
crosslinking via sonication. Mechanistically, tyrosinases oxidize the accessible
tyrosine residues of silk and/or gelatin into reactive o-quinone moieties that
can either condense with each other or undergo nonenzymatic reactions with
available amines of both silk and gelatin. Sonication alters the hydrophobic
interaction and accelerates self-assembly of silk fibroin macromolecules to form
β-sheet crystals, which physically crosslink the hydrogel. However, sonication
has no effect on the conformation of gelatin. The effect of optimized rheology,
secondary conformations of silk-gelatin bioink, temporally controllable gelation
strategies and printing parameters were assessed to achieve maximum cell
viability and multilineage differentiation of the encapsulated human nasal
inferior turbinate tissue-derived mesenchymal progenitor cells. This strategy
offers a unique path forward in the direction of direct printing of spatially
customized anatomical architecture in a patient-specific manner. Cardiovascular diseases are the leading cause of deaths throughout the world.
Vascular diseases are mostly treated with autografts and blood vessel
transplantations. However, traditional grafting methods have several problems
including lack of suitable harvest sites, additional surgical costs for
harvesting procedure, pain, infection, lack of donors, and even no substitutes
at all. Recently, tissue engineering and regenerative medicine approaches are
used to regenerate damaged or diseased tissues. Most of the tissue engineering
investigations have been based on the cell seeding into scaffolds by providing a
suitable environment for cell attachment, proliferation, and differentiation.
Because of the challenges such as difficulties in seeding cells spatially,
rejection, and inflammation of biomaterials used, the recent tissue engineering
studies focus on scaffold-free techniques. In this paper, the development of
novel computer aided algorithms and methods are developed for 3D bioprinting of
scaffold-free biomimetic macrovascular structures. Computer model mimicking a
real human aorta is generated using imaging techniques and the proposed
computational algorithms. An optimized three-dimensional bioprinting path
planning are developed with the proposed self-supported model. Mouse embryonic
fibroblast (MEF) cell aggregates and support structures (hydrogels) are 3D
bioprinted layer-by-layer according to the proposed self-supported method to
form an aortic tissue construct. 3D printing, or solid freeform fabrication, applied to regenerative medicine
brings technologies from several industries together to help solve unique
challenges in both basic science and tissue engineering. By more finely
organizing cells and supporting structures precisely in 3D space, we will gain
critical knowledge of cell-cell communications and cell-environment
interactions. As we increase the scale, we will move toward complex tissue and
organ structures where several cell phenotypes will functionally and
structurally interact, thus recapitulating the form and function of native
tissues and organs. |
Is transcription-associated mutagenesis (TAM) related to gene expression levels? | Spontaneous point mutation rate in a gene increases with its transcription level, suggesting that movement of RNA polymerase through the target initiates a mutagenic process(es). This phenomenon is termed transcription-associated mutation (TAM). Transcription-associated mutagenesis is directly proportional to the level of gene expression. | High levels of transcription are associated with elevated mutation rates in
yeast, a phenomenon referred to as transcription-associated mutation (TAM). The
transcription-associated increase in mutation rates was previously shown to be
partially dependent on the Rev3p translesion bypass pathway, thus implicating
DNA damage in TAM. In this study, we use reversion of a pGAL-driven lys2DeltaBgl
allele to further examine the genetic requirements of TAM. We find that TAM is
increased by disruption of the nucleotide excision repair or recombination
pathways. In contrast, elimination of base excision repair components has only
modest effects on TAM. In addition to the genetic studies, the lys2DeltaBgl
reversion spectra of repair-proficient low and high transcription strains were
obtained. In the low transcription spectrum, most of the frameshift events
correspond to deletions of AT base pairs whereas in the high transcription
strain, deletions of GC base pairs predominate. These results are discussed in
terms of transcription and its role in DNA damage and repair. High levels of transcription are associated with increased mutation rates in
Saccharomyces cerevisiae, a phenomenon termed transcription-associated mutation
(TAM). To obtain insight into the mechanism of TAM, we obtained LYS2 forward
mutation spectra under low- versus high-transcription conditions in which LYS2
was expressed from either the low-level pLYS2 promoter or the strong pGAL1-10
promoter, respectively. Because of the large size of the LYS2 locus, forward
mutations first were mapped to specific LYS2 subregions, and then those
mutations that occurred within a defined 736-bp target region were sequenced. In
the low-transcription strain base substitutions comprised the majority (64%) of
mutations, whereas short insertion-deletion mutations predominated (56%) in the
high-transcription strain. Most notably, deletions of 2 nucleotides (nt)
comprised 21% of the mutations in the high-transcription strain, and these
events occurred predomitly at 5'-(G/C)AAA-3' sites. No -2 events were present
in the low-transcription spectrum, thus identifying 2-nt deletions as a unique
mutational signature for TAM. High levels of transcription in Saccharomyces cerevisiae are associated with
increased genetic instability, which has been linked to DNA damage. Here, we
describe a pGAL-CAN1 forward mutation assay for studying
transcription-associated mutagenesis (TAM) in yeast. In a wild-type background
with no alterations in DNA repair capacity, ≈50% of forward mutations that arise
in the CAN1 gene under high-transcription conditions are deletions of 2-5 bp.
Furthermore, the deletions characteristic of TAM localize to discrete hotspots
that coincide with 2-4 copies of a tandem repeat. Although the signature
deletions of TAM are not affected by the loss of error-free or error-prone
lesion bypass pathways, they are completely eliminated by deletion of the TOP1
gene, which encodes the yeast type IB topoisomerase. Hotspots can be transposed
into the context of a frameshift reversion assay, which is sensitive enough to
detect Top1-dependent deletions even in the absence of high transcription. We
suggest that the accumulation of Top1 cleavage complexes is related to the level
of transcription and that their removal leads to the signature deletions. Given
the high degree of conservation between DNA metabolic processes, the links
established here among transcription, Top1, and mutagenesis are likely to extend
beyond the yeast system. High transcription is associated with genetic instability, notably increased
spontaneous mutation rates, which is a phenomenon termed
Transcription-Associated-Mutagenesis (TAM). In this study, we investigated TAM
using the chromosomal CAN1 gene under the transcriptional control of two strong
and inducible promoters (pGAL1 and pTET) in Saccharomyces cerevisiae. Both pTET-
and pGAL1-driven high transcription at the CAN1 gene result in enhanced
spontaneous mutation rates. Comparison of both promoters reveals differences in
the type of mutagenesis, except for short (-2 and -3 nt) deletions, which depend
only on the level of transcription. This mutation type, characteristic of TAM,
is sequence dependent, occurring prefentially at di- and trinucleotides repeats,
notably at two mutational hotspots encompassing the same 5'-ACATAT-3' sequence.
To explore the mechanisms underlying the formation of short deletions in the
course of TAM, we have determined Can(R) mutation spectra in yeast mutants
affected in DNA metabolism. We identified topoisomerase 1-deficient strains
(top1Δ) that specifically abolish the formation of short deletions under high
transcription. The rate of the formation of (-2/-3nt) deletions is also reduced
in the absence of RAD1 and MUS81 genes, involved in the repair of Top1p-DNA
covalent complex. Furthermore ChIP analysis reveals an enrichment of trapped
Top1p in the CAN1 ORF under high transcription. We propose a model, in which the
repair of trapped Top1p-DNA complexes provokes the formation of short deletion
in S. cerevisiae. This study reveals unavoidable conflicts between Top1p and the
transcriptional machinery and their potential impact on genome stability. Reporter gene assays have demonstrated both transcription-associated mutagenesis
(TAM) and transcription-coupled repair, but the net impact of transcription on
mutation rate remains unclear, especially at the genomic scale. Using
comparative genomics of related species as well as mutation accumulation lines,
we show in yeast that the rate of point mutation in a gene increases with the
expression level of the gene. Transcription induces mutagenesis on both DNA
strands, indicating simultaneous actions of several TAM mechanisms. A
significant positive correlation is also detected between the human germline
mutation rate and expression level. These results indicate that transcription is
overall mutagenic. |
Is there a pharmacogenetic test for trastuzumab? | Yes. HER2 testing is performed in breast cancer patients to determine suitability for trastuzumab (Herceptin therapy). | Pharmacogenomic analysis aspires to identify individuals with specific genetic
characteristics in order to predict a positive response or reduce a negative
response to a therapeutic modality. While the search continues for the many
single nucleotide polymorphisms which will be used in such genetic analyses,
other genetic alterations in specific cell types have proven useful in
determining the potential for response to therapy. One such genetic alteration
is amplification of entire gene sequences which results in overexpression of a
gene product or protein. Amplification of the HER2 (neu, erbB-2) oncogene is
found in up to 35% of human breast cancers and is associated with a poor
prognosis. In addition, this genetic alteration may predict response to various
therapeutic modalities. Assays are available to detect the HER2 protein receptor
or copies of the HER2 gene sequence to determine eligibility for Herceptin
treatment or adriamycin treatment in node positive patients, respectively. This
model represents a somatic event used in the functional determination of a
therapeutic strategy. BACKGROUND: Laboratory testing of HER2/neu in breast carcinoma has become vital
to patient care following the approval of trastuzumab as the first therapy to
target the HER2/neu oncoprotein. Initial clinical trials used
immunohistochemistry (IHC) to test for HER2/neu overexpression in order to
select patients for therapy. Fluorescence in situ hybridization (FISH), which
tests for gene amplification, is more specific and sensitive than IHC when
either assay is compared with HER2/neu overexpression as determined by Northern
or Western blot analysis. Many weak overexpressors on IHC testing are not gene
amplified on FISH analysis. Such weak overexpressors may be considered
false-positives and raise the question of how best to test for HER2/neu.
METHODS: The literature was surveyed regarding testing for HER2/neu
overexpression in breast carcinomas and alternative testing strategies.
RESULTS: False-positive results are a significant problem when IHC is
exclusively used to test for HER2/neu overexpression. The false-positives are
overwhelmingly confined to the group of 2+ positives and do not respond to
targeted therapy. In contrast, concordance between IHC and FISH is high when
immunostaining is interpreted as either negative or strongly positive (3+).
Whereas some recent studies have suggested that FISH may better predict response
to anti-HER2/neu therapy than IHC, others have indicated that IHC is as
effective a predictor as FISH. IHC is less technically demanding and costly than
FISH.
CONCLUSIONS: IHC analysis of HER2/neu in breast carcinoma is a useful predictor
of response to therapy with trastuzumab when strongly positive. Negative
immunostaining is highly concordant with a lack of gene amplification by FISH.
Most weakly positive overexpressors are false-positives on testing with FISH.
Thus, screening of breast carcinomas with IHC and confirmation of weakly
positive IHC results by FISH is an effective evolving strategy for testing
HER2/neu as a predictor of response to targeted therapy. OBJECTIVE: To evaluate amplification of the HER-2/neu gene by fluorescence ir
situ hybridization (FISH) in tumors with weakly positive (2+)
immunohistochemical staining.
METHODS: A total of 1556 breast tumor biopsy specimens were referred to Mayo
Medical Laboratories, Rochester, Minn, for HER2 testing between August and
December 2000. Immunohistochemical (IHC) analysis was performed with use of a
diagnostic test for the assessment of HER2 overexpression, the HercepTest. The
IHC-stained slides were interpreted and scored on a scale ranging from 0 to 3+
according to Food and Drug Administration-approved guidelines. All specimens
scored as 2+ were also routinely evaluated by FISH with use of a HER-2/neu DNA
probe kit (PathVysion). Specimens were determined to be amplified if the ratio
of HER-2/neu signals to chromosome 17 centromere (CEP17) signals was higher than
2.0.
RESULTS: Thirty-eight percent of the specimens evaluated with the HercepTest
were scored 0, 35% were 1+, 14% were 2+, and 13% were 3+. Of the 216 tumor
specimens scored as 2+, 26 (12%) had a high level of HER-2/neu gene
amplification, 54 (25%) demonstrated duplication of HER2, 4 (2%) deleted
HER-2/neu and/or CEP17, and 123 (57%) had no apparent HER-2/neu anomaly, no
apparent CEP17 anomaly, nor apparent single gain (aneusomy) of CEP17.
CONCLUSION: We recommend that all specimens with a 2+ HercepTest result be
evaluated by FISH for HER-2/neu gene amplification. The results of both assays
should be considered before making a decision to recommend anti-HER2 therapy. The 4th European Breast Cancer Conference, organized under the auspices of the
European Organization for Research and Treatment of Cancer Breast Cancer Group,
of the European Breast Cancer Coalition (Europa Donna) and of the European
Society of Mastology (EUSOMA), was held in Hamburg, Germany on 16-20 March 2004.
The leading theme of the conference was partnership among scientists,
clinicians, carers, advocates and patients. The present article provides a brief
description of the most important conference presentations on molecular biology,
epidemiology, prevention, pathology, diagnosis and treatment at all stages of
breast cancer. OBJECTIVES: The HER-2/neu oncogene is overexpressed in many types of cancer and
especially in 25% to 30% of breast cancers. Single reports mention HER-2/neu
positivity in hematological maligcies like Hodgkins's disease and even
diffuse large-cell lymphoma.
OBJECTIVE: To test for HER-2/neu overexpression in patients with non-Hodgkin's
lymphoma and the possible role of the recombit monoclonal anti-HER-2/neu
antibody trastuzumab (Herceptin) in the treatment of non-Hodgkin's lymphoma.
MATERIALS AND METHODS: Serum samples from 87 consecutive unselected patients
with non-Hodgkin's lymphoma were retrospectively retrieved from a serum bank and
tested for the shed antigen of HER-2/neu using the Oncogene Sciences ELISA assay
(Cambridge, MA, USA). From those lymphoma patients, the paraffin-embedded
lymph-node specimens of 25 cases with diffuse large-cell lymphoma were stained
with the HER-2/neu DAKO HercepTest.
RESULTS: In 87 lymphoma patients, the serum level of HER-2/neu ranged from 3.6
to 244.1 ng/ml (median 8.0 ng/ml). Only 2 patients showed a marginal or
increased HER-2/neu level with 15 ng/ml (which is the upper limit of normal) and
244.1 ng/ml, respectively. No patient with diffuse large-cell lymphoma showed
HER-2/neu overexpression by immunhistochemistry of the lymph node. The paraffin
block of the one patient with a very high HER-2/neu serum level was also stained
for HER-2/neu overexpression. In this patient, suffering from a high-grade
T-cell non-Hodgkin's lymphoma, no staining could be found.
CONCLUSION: HER-2/neu is not overexpressed in non-Hodgkin's lymphoma and
especially not in diffuse large-cell lymphoma, using a standardized
immunochemistry technique with complementary serum testing. Thus, specific
anti-HER-2/neu-targeted therapy should play no role in the treatment of
non-Hodgkin's lymphoma. PURPOSE: To evaluate concordance between local and central laboratory HER2
testing results in patients from the North Central Cancer Treatment Group
(NCCTG) N9831 adjuvant trial of trastuzumab.
PATIENTS AND METHODS: NCCTG N9831 is a randomized, phase III clinical trial
comparing three drug regimens: doxorubicin/cyclophosphamide followed by
paclitaxel with trastuzumab added concurrently, sequentially, or not at all as
adjuvant therapy for women with HER2-positive resected breast cancer.
Originally, patients were eligible if their tumors were HER2 positive by either
local laboratory immunohistochemistry (IHC) or fluorescence in situ
hybridization (FISH). A protocol modification in 2002 made central laboratory
testing mandatory, with additional testing of discordant cases conducted by a
reference laboratory. Concordance between local and central laboratory, and
level of agreement between central and reference laboratory HER2 findings in
discordant cases were examined.
RESULTS: HER2 positivity was confirmed in 85.8% of 2,535 patients registered
since March 2002. When local and central evaluation used the same methodology,
concordance was 88.1% for FISH and 81.6% for a diagnostic test for presence of
the HER2 protein. Among discordant cases examined at the reference laboratory,
there was 94.3% agreement for IHC (0, 1+, 2+) and 95.2% agreement for FISH (not
gene amplified).
CONCLUSION: There was a high degree of discordance between local and central
testing for IHC and FISH, but a high degree of agreement between central and
reference laboratories. These findings support the importance of using
high-volume, experienced laboratories for HER2 testing to improve the process of
selecting patients likely to benefit from trastuzumab therapy. While the importance of patient autonomy is widely acknowledged and discussed in
the bioethics literature, clinicians' autonomy, their ability to make the best
choices about patients' care free from outside interference, is far less
debated. This paper takes one form of external influence over clinical decisions
- the cost of drugs - and applies it to a specific case, that of HER2 positive
breast cancer and the use of the drug Herceptin in the UK. Drawing on interviews
with clinicians, researchers and policymakers, I explore the way ficial
decisions about Herceptin shape clinicians' autonomy, and how clinicians as
individuals and as professional groups respond to these limits and seek to
provide treatment to the highest number of the most deserving patients they can.
The point of this paper is not to castigate bioethicists for misguidedly
focusing on patient autonomy but point out that clinicians' autonomy may be so
circumscribed by external factors that it may make no sense to speak of their
actions as stemming from ethical decisions. At the same time, I suggest that
ficial constraints create areas at the margin of clinical practice which are
deserving of bioethical consideration. BACKGROUND: Biotin-labeled trastuzumab (BiotHER) can be used to test for HER2 by
immunohistochemistry. We previously showed that BiotHER immunoreactivity is
highly correlated with HER2 amplification and indicated that it could be
associated with better clinical outcome in advanced breast cancer patients
receiving trastuzumab.
PATIENTS AND METHODS: Tumor specimens and clinical information from 234 patients
who received trastuzumab-based treatments were collected from 10 institutions.
HER2 amplification and BiotHER immunoreactivity were assessed centrally. The
effect of BiotHER positivity on response rate (RR), time to progression and
survival were studied by univariate and multivariate analysis in patients
presenting HER2-amplified breast cancer. The pathologic reviews of the assays
were blinded to patient outcomes.
RESULTS: BiotHER was positive in 109/194 (56%) HER2-amplified breast cancers and
in one not amplified tumor. RRs were 74% [95% (confidence interval) CI 64%-81%]
and 47% (95% CI 36%-58%) in BiotHER-positive and -negative tumors, respectively
(P < 0.001). BiotHER immunoreactivity was independently associated with
increased probability of tumor response (odds ratio 3.848; 95% CI 1.952-7.582),
with reduced risk of disease progression [hazard ratio (HR) 0.438; 95% CI
0.303-0.633] and with reduced risk of death (HR 0.566; 95% CI 0.368-0.870) by
multivariate analysis.
CONCLUSION: The results support a role for BiotHER testing in better tailoring
trastuzumab-based treatments in patients with advanced HER2-amplified breast
cancers. Evaluation of HER2 status is critical in determining appropriate treatment for
breast cancer. Currently, Fluorescence In Situ Hybridization (FISH) and
Immunohistochemistry (IHC) are the FDA-approved tests. Studies show, FISH is
superior to IHC in accuracy and prediction of clinical outcomes with respect to
response to anti-human epidermal growth factor receptor 2 (HER2) therapy
trastuzumab. The impact of factors on choice of test for HER2 detection was
determined using logistic regression. We show that geographic location, cancer
stage, and diagnosis date have significant effects on choice of test. These
findings indicate that disparities may be present in breast cancer care, and
highlight the importance of testing guidelines. Trastuzumab is standard of care in the treatment of human epidermal growth
factor receptor (HER)-2⁺ early and advanced breast cancer. Recently, it has been
approved for the treatment of HER-2⁺ advanced gastric cancer. Trastuzumab is an
IgG1 humanized monoclonal antibody administered by intravenous infusion on a
weekly or three weekly schedule. In all registered indications, trastuzumab is
almost always given in combination with chemotherapy. In hormonal
receptor-positive breast cancer in postmenopausal women, trastuzumab can be
combined with an aromatase inhibitor. Main toxicity is reduction in the left
ventricular ejection fraction, which in a minority of patients can become
symptomatic, but in many patients is at least partly reversible. Long-term
safety needs to be further determined. There have been several success stories in the field of pharmacogenetics in
recent years, including the analysis of HER2 amplification for trastuzumab
selection in breast cancer and VKORC1 genotyping for warfarin dosing in
thrombosis. Encouraging results from these studies suggest that genetic factors
may indeed be important determits of drug response and toxicity for at least
some drugs. However, to apply pharmacogenetics appropriately, a thorough
understanding of the scope and limitations of this field is required. The
challenges include an appreciation of biological variability, logistical issues
pertaining to the proper management of information, the need for robust methods
and adequate sample quality with well-designed workflows. At the same time, the
economics of pharmacogenetic testing from the perspective of clinicians,
patients, governments, insurance companies and pharmaceutical companies will
play an important role in determining its future use. Ethical considerations
such as informed consent and patient privacy, as well as the role of regulatory
bodies in addressing these issues, must be fully understood. Only once these
issues are properly dealt with can the full benefits of pharmacogenetics begin
to be realised. Pharmacogenetics and pharmacogenomics have been widely recognized as fundamental
steps toward personalized medicine. They deal with genetically determined
variants in how individuals respond to drugs, and hold the promise to
revolutionize drug therapy by tailoring it according to individual genotypes.The
clinical need for novel approaches to improve drug therapy derives from the high
rate of adverse reactions to drugs and their lack of efficacy in many
individuals that may be predicted by pharmacogenetic testing.Significant
advances in pharmacogenetic research have been made since inherited differences
in response to drugs such as isoniazid and succinylcholine were explored in the
1950s. The clinical utility and applications of pharmacogenetics and
pharmacogenomics are at present particularly evident in some therapeutic areas
(anticancer, psycotrophic, and anticoagulant drugs).Recent evidence derived from
several studies includes screening for thiopurine methyl transferase or uridine
5'-diphosphoglucuronosyl-transferase 1A1 gene polymorphisms to prevent
mercaptopurine and azathioprine or irinotecan induced myelosuppression,
respectively. Also there is a large body of information concerning cytochrome
P450 gene polymorphisms and their relationship to drug toxicity and response.
Further examples include screening the presence of the HLA-B*5701 allele to
prevent the hypersensitivity reactions to abacavir and the assessment of the
human epidermal growth factor receptor (HER-2) expression for trastuzumab
therapy of breast cancer or that of KRAS mutation status for cetuximab or
panitumumab therapy in colorectal cancer.Moreover, the application of
pharmacogenetics and pharmacogenomics to therapies used in the treatment of
osteoarticular diseases (e.g. rheumatoid arthritis, osteoporosis) holds great
promise for tailoring therapy with clinically relevant drugs (e.g.
disease-modifying antirheumatic drugs, vitamin D, and estrogens). Although the
classical candidate gene approach has helped unravel genetic variants that
influence clinical drug responsiveness, gene-wide association studies have
recently gained attention as they enable to associate specific genetic variants
or quantitative differences in gene expression with drug response.Although
research findings are accumulating, most of the potential of pharmacogenetics
and pharmacogenomics remains to be explored and must be validated in prospective
randomized clinical trials.The genetic and molecular foundations of personalized
medicine appear solid and evidence indicates its growing importance in
healthcare. |
Which calcium channels does ethosuximide target? | Ethosuximide blocks the T-type calcium channels. | Briefly after withdrawal of the (T-type) calcium channel blocker mibefradil from
the market, four cases of life-threatening interaction of mibefradil with
dihydropyridines were reported. We investigated in vitro whether mibefradil
interacts with a dihydropyridine, as described for other non-dihydropyridine
compounds. Rat working hearts were used to examine functional interactions
between amlodipine and mibefradil. Gallopamil and another T-type-channel
blocker, ethosuximide, were included for comparison. Effects of mibefradil, (+)-
and (-)-gallopamil on [3H](+)-isradipine binding were studied in membranes from
tsA201-cells transfected with alpha(1c)-, alpha(2)delta-, and beta(1a)- or
beta(2a)-calcium channel subunits. Mibefradil increased negative inotropic
effect of amlodipine, but not of gallopamil. Gallopamil and ethosuximide showed
no influence on contractile effects of amlodipine. Furthermore, mibefradil
concentration-dependently caused bradycardic rhythm disturbance. The same type
of arrhythmia was observed combining low concentrations of mibefradil with
amlodipine, or with gallopamil, respectively. Amlodipine alone, or the
combination of gallopamil or ethosuximide with amlodipine did not cause any
arrhythmia. Binding studies showed a concentration-dependent positive allosteric
interaction between [3H](+)-isradipine and mibefradil, but not with
[3H](+)-isradipine and gallopamil etiomers. Molecular and functional evidence
points to an interaction between a dihydropyridine and mibefradil. Mibefradil
caused rhythm disturbances and potentiation of negative inotropy when combined
with amlodipine. Several types of voltage- or ligand-activated calcium channels contribute to the
excitability of neuronal cells. Low-voltage-activated (LVA), T-type calcium
channels are characterised by relatively negative threshold of activation and
therefore they can generate low-threshold spikes, which are essential for burst
firing. At least three different proteins form T-type calcium current in
neurons: Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3. Expression of these proteins in
various brain regions is complementary. Individual channel types could be
distinguished by different sensitivity towards inorganic cations. This
inhibition can contribute to the toxicity of some heavy metals. Selective
inhibition of T-type calcium channels by organic blockers may have clinical
importance in some forms of epilepsy. Mibefradil inhibits the expressed
Ca(v2)3.1, Ca(v)3.2 and Ca(v)3.3 channels in omolar concentrations with
Ca(v)3.3 channel having lowest affinity. The sensitivity of the expressed
Ca(v)3.1 channel to the antiepileptic drugs, valproate and ethosuximide, is low.
Ca(v)3.1 channel is moderately sensitive to phenytoin. The Ca(v)3.2 channel is
sensitive to ethosuximide, amlodipine and amiloride. All three LVA calcium
channels are moderately sensitive to active metabolites of methosuximide, i.e.
alpha-methyl-alpha-phenylsuccinimide. Several neuroleptics inhibit all three LVA
channels in clinically relevant concentrations. All three channels are also
inhibited by the endogenous cannabinoid adamide. A high affinity peptide
blocker for these Ca channels is the scorpion toxin kurtoxin which inhibits the
Ca(v)3.1 and Ca(v)3.2, but not the Ca(v)3.3 channel in omolar concentrations.
Nitrous oxide selectively inhibits the Ca(v)3.2, but not the Ca(v)3.1 channel.
The Ca(v)3.2, but not the Ca(v)3.1 channel is potentiated by stimulation of
Ca(2+)/CaM-dependent protein kinase. Gamma-aminobutyric acid (GABA), one of the main inhibitory neurotransmitters in
the brain, interacts with three types of receptors for GABA--GABA(A), GABA(B)
and GABA(C). GABA(A) receptors, associated with binding sites for
benzodiazepines and barbiturates in the form of a receptor complex, control
opening of the chloride channel. When GABA binds to the receptor complex, the
channel is opened and chloride anions enter the neuron, which is finally
hyperpolarized. GABA(B) receptors are metabotropic, linked to a cascade of
second messengers whilst the physiological meaning of ionotropic GABA(C)
receptors, mainly located in the retina, is generally unknown. Novel
antiepileptic drugs acting selectively through the GABA-ergic system are
tiagabine and vigabatrin. The former inhibits neuronal and glial uptake of GABA
whilst the latter increases the synaptic concentration of GABA by inhibition of
GABA-aminotransferase. Gabapentin, designed as a precursor of GABA easily
entering the brain, was shown to increase brain synaptic GABA. This
antiepileptic drug also decreases influx of calcium ions into neurons via a
specific subunit of voltage-dependent calcium channels. Conventional
antiepileptics generally inhibit sodium currents (carbamazepine, phenobarbital,
phenytoin, valproate) or enhance GABA-ergic inhibition (benzodiazepines,
phenobarbital, valproate). Ethosuximide, mainly controlling absences, reduces
calcium currents via T-type calcium channels. Novel antiepileptic drugs, mainly
associated with an inhibition of voltage-dependent sodium channels are
lamotrigine and oxcarbazepine. Since glutamate-mediated excitation is involved
in the generation of seizure activity, some antiepileptics are targeting
glutamatergic receptors--for instance, felbamate, phenobarbital, and topiramate.
Besides, they also inhibit sodium currents. Zonisamide, apparently sharing this
common mechanism, also reduces the concentration of free radicals. Novel
antiepileptic drugs are better tolerated by epileptic patients and practically
are devoid of important pharmacokinetic drug interactions. The purpose of the present study was to explore the analgesic effects of the low
voltage-activated T-type Ca2+ channel blockers ethosuximide, trimethadione, and
mibefradil in persistent and acute nociceptive tests. The anticonvulsant effects
of the compounds were also determined in the intravenous pentylenetetrazol
seizure model. Following intraperitoneal administration, ethosuximide and
trimethadione dose-dependently reversed capsaicin-induced mechanical
hyperalgesia. Similarly, the highest dose of mibefradil tested (30 microg,
intracisternal) reversed capsaicin-induced mechanical hyperalgesia. Ethosuximide
and mibefradil produced statistically significant analgesic effects in both
early and late phase formalin-induced behaviors and trimethadione reduced late
phase behaviors. Additionally, ethosuximide and trimethadione produced
antinociceptive effects in the rat-tail flick reflex test. In contrast,
following intracisternal administration, mibefradil had no effect in the tail
flick reflex test. In addition, the anticonvulsants ethosuximide and
trimethadione increased the doses of pentylenetetrazol required to produce both
first twitch and clonic seizures. In contrast however, mibefradil had no
anticonvulsant effect. The present results demonstrate that the clinically used
anticonvulsants ethosuximide and trimethadione provide analgesic effects at
doses, which are anticonvulsant. Furthermore, the data further supports the idea
that T-type Ca2+ channels may be important targets for treating persistent pain
syndromes. T-type calcium channels are strongly associated with the generation of rhythmic
firing patterns in the CNS. Blockers of these channels may have therapeutic
potential for treating various types of tremor. The present study aimed to study
the effects of a range of T-type calcium channel blockers in a parkinsonian
tremor model in rats. We tested the effects of several T-type calcium channel
blockers, including zonisamide (ZNS), ethosuximide, lomerizine, amiloride,
mibefradil, and NCC 55-0396, a mibefradil derivative, on tacrine-induced
tremulous jaw movements (TJMs), an animal model of parkinsonian tremor. Among
the tested drugs, only ZNS and NCC 55-0396 significantly suppressed TJMs when
given at a non-sedating dose. The transitivity of drugs to the central nervous
system (CNS) may at least partially explain their differential anti-TJM effects.
However, further studies are necessary to reveal other factors, since
ethosuximide failed to show anti-TJM effects despite being known to cross the
blood brain barrier. The present results suggest that T-type calcium channels in
the CNS may be a suitable target for developing new therapeutic strategies for
treating parkinsonian tremor. Chronic ethanol exposure produces profound disruptions in both brain rhythms and
diurnal behaviors. The thalamus has been identified as a neural pacemaker of
both normal and abnormal rhythms with low-threshold, transient (T-type) Ca(2+)
channels participating in this activity. We therefore examined T-type channel
gene expression and physiology in the thalamus of C57Bl/6 mice during a 4-wk
schedule of chronic intermittent ethanol exposures in a vapor chamber. We found
that chronic ethanol disrupts the normal daily variations of both thalamic
T-type channel mRNA levels and alters thalamic T-type channel gating properties.
The changes measured in channel expression and function were associated with an
increase in low-threshold bursts of action potentials during acute withdrawal
periods. Additionally, the observed molecular and physiological alterations in
the channel properties in wild-type mice occurred in parallel with a progressive
disruption in the normal daily variations in theta (4-9 Hz) power recorded in
the cortical electroencephalogram. Theta rhythms remained disrupted during a
subsequent week of withdrawal but were restored with the T-type channel blocker
ethosuximide. Our results demonstrate that a key ion channel underlying the
generation of thalamic rhythms is altered during chronic ethanol exposure and
withdrawal and may be a novel target in the management of abnormal network
activity due to chronic alcoholism. Chronic alcohol abuse depresses the nervous system and, upon cessation, rebound
hyperexcitability can result in withdrawal seizure. Withdrawal symptoms,
including seizures, may drive individuals to relapse, thus representing a
significant barrier to recovery. Our lab previously identified an upregulation
of the thalamic T-type calcium (T channel) isoform CaV3.2 as a potential
contributor to the generation and propagation of seizures in a model of
withdrawal. In the present study, we examined whether ethosuximide (ETX), a
T-channel antagonist, could decrease the severity of ethanol withdrawal seizures
by evaluating electrographical and behavioral correlates of seizure activity.
DBA/2J mice were exposed to an intermittent ethanol exposure paradigm. Mice were
treated with saline or ETX in each withdrawal period, and cortical EEG activity
was recorded to determine seizure severity. We observed a progression in seizure
activity with each successive withdrawal period. Treatment with ETX reduced
ethanol withdrawal-induced spike and wave discharges (SWDs), in terms of
absolute number, duration of events, and contribution to EEG power in the 6-10
Hz frequency range. We also evaluated the effects of ETX on handling-induced
convulsions. Overall, we observed a decrease in handling-induced convulsion
severity in mice treated with ETX. Our findings suggest that ETX may be a useful
pharmacological agent for studies of alcohol withdrawal and treatment of
resulting seizures. BACKGROUND: T-type Ca(2+) channels (TCC) are important for pain transmission,
especially the Ca(V)3.2 subtype. In this study, we examined the effects of
intrathecal TCC blockers in the L5/6 spinal nerve ligation pain rat model.
METHODS: Under isoflurane anaesthesia, rats received right L5/6 spinal nerve
ligation and intrathecal catheters (attached to an infusion pump) were sited.
After surgery, saline, mibefradil, ethosuximide or NiCl2 were given
intrathecally for seven days. The right hindpaw withdrawal thresholds to von
Frey hair stimuli and withdrawal latencies to radiant heat were measured before
and once daily for seven days after surgery. Double immunofluorescence and
western blotting were used to examine the expression of Ca(V)3.2 in dorsal root
ganglion (DRG) and spinal cord.
RESULTS: On post-ligation day seven, rats receiving mibefradil, ethosuximide or
NiCl2 had significant higher median withdrawal thresholds (15.0, 10.2, and 10.9
g) and latencies (8.0, 7.6 and 7.6 s) than saline-treated rats (1.6 g and 4.3 s,
respectively). Ca(V)3.2 was expressed in parvalbumin(+), IB4(+), CGRP(+) and
VR1(+) neurones in DRG and most neurones in spinal dorsal horn. Ca(V)3.2 was
up-regulated in the right L5/6 DRG and spinal cord seven days after nerve
ligation.
CONCLUSIONS: In this study, we demonstrated that intrathecal TCC blockers
attenuate the development of nerve injury-induced mechanical allodynia and
thermal hyperalgesia. Our data suggest that continuous intrathecal infusion of
TCC or Ca(V)3.2 blockers may be a promising alternative for the management of
nerve injury-induced pain. |
What is the sedimentation coefficient of the mammalian mitoribosome? | The mammalian mitochondrial ribosome (mitoribosome) has a highly protein-rich composition with a small sedimentation coefficient of 55 S, consisting of 39 S large and 28 S small subunits. | Mitochondrial ribosomes comprise the most diverse group of ribosomes known. The
mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S)
and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial
ribosomes. The bovine mitochondrial ribosome has been developed as a model
system for the study of human mitochondrial ribosomes to address several
questions related to the structure, function, biosynthesis and evolution of
these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from
each subunit have been identified and characterized with respect to
individuality and electrophoretic properties, amino acid sequence, topographic
disposition, RNA binding properties, evolutionary relationships and reaction
with affinity probes of ribosomal functional domains. Several distinctive
properties of these ribosomes are being elucidated, including their antibiotic
susceptibility and composition. Mammalian mitochondrial ribosomes lack several
of the major RNA stem structures of bacterial ribosomes but they contain a
correspondingly higher protein content (as many as 80 proteins), suggesting a
model where proteins have replaced RNA structural elements during the evolution
of these ribosomes. Despite their lower RNA content they are physically larger
than bacterial ribosomes, because of the 'extra' proteins they contain. The
extra proteins in mitochondrial ribosomes are 'new' in the sense that they are
not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the
new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in
nuclear genes (a separate set from those encoding cytoplasmic ribosomal
proteins) which are evolving more rapidly than those encoding cytoplasmic
ribosomal proteins. The MRPs are imported into mitochondria where they assemble
coordinately with mitochondrially transcribed rRNAs into ribosomes that are
responsible for translating the 13 mRNAs for essential proteins of the oxidative
phosphorylation system. Interest is growing in the structure, organization,
chromosomal location and expression of genes for human MRPs. Proteins which are
essential for mitoribosome function are candidates for involvement in human
genetic disease. The mammalian mitochondrial (mt) ribosome (mitoribosome) is a bacterial-type
ribosome but has a highly protein-rich composition. Almost half of the rRNA
contained in the bacterial ribosome is replaced with proteins in the
mitoribosome. Escherichia coli elongation factor G (EF-G Ec) has no translocase
activity on the mitoribosome but EF-G mt is functional on the E.coli ribosome.
To investigate the functional equivalency of the mt and E.coli ribosomes, we
prepared hybrid mt and E.coli ribosomes. The hybrid mitoribosome containing
E.coli L7/12 (L7/12 Ec) instead of L7/12 mt clearly activated the GTPase of EF-G
Ec and efficiently promoted its translocase activity in an in vitro translation
system. Thus, the mitoribosome is functionally equivalent to the E.coli ribosome
despite their distinct compositions. The mt EF-Tu-dependent translation activity
of the E.coli ribosome was also clearly enhanced by replacing the C-terminal
domain (CTD) of L7/12 Ec with the mt counterpart (the hybrid E.coli ribosome).
This strongly indicates that the CTD of L7/12 is responsible for EF-Tu function.
These results demonstrate that functional compatibility between elongation
factors and the L7/12 protein in the ribosome governs its translational
specificity. Mitochondria can be isolated from skeletal muscle in a manner that preserves
tightly coupled bioenergetic function in vitro. The purpose of this study was to
characterize the composition of such preparations using a proteomics approach.
Mitochondria isolated from human vastus lateralis biopsies were functional as
evidenced by their response to carbohydrate and fat-derived fuels. Using
one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were
detected, and 487 of these were assigned to the mitochondrion, including the
newly characterized SIRT5, MitoNEET and RDH13. Proteins detected included 9 of
the 13 mitochondrial DNA-encoded proteins and 86 of 104 electron transport chain
(ETC) and ETC-related proteins. In addition, 59 of 78 proteins of the 55S
mitoribosome, several TIM and TOM proteins and cell death proteins were present.
This study presents an efficient method for future qualitative assessments of
proteins from functional isolated mitochondria from small samples of healthy and
diseased skeletal muscle. |
Describe the mechanism of action of aliskiren. | Aliskiren is a low-molecular-weight, orally active, hydrophilic non-peptide molecule that blocks renin and consequential angiotensin I generation. Renin inhibition interrupts the renin-angiotensin-aldosterone system (RAAS). | The first oral direct renin inhibitor, aliskiren, recently received approval for
the treatment of hypertension. This article addresses the premise, promise, and
potential limitations of this new class of renin-angiotensin system inhibitor.
Although aliskiren adds to a list of more than 100 drugs approved for the
treatment of hypertension, its introduction into clinical medicine is of
particular interest because of the novel mechanism of action: inhibition of
renin's catalytic activity, the most proximal and rate-limiting step in
renin-angiotensin system activation. By producing more complete
renin-angiotensin system inhibition than with existing agents, direct renin
inhibitors may afford greater protection from hypertensive complications. Other
potential advantages include additional blood pressure reduction when used in
combination therapy, a placebo-like side-effect profile, avid renal
concentration, and long duration of action. Potential limitations include modest
levels of blood pressure reduction that are equivalent to but not greater than
angiotensin receptor blockers, reduced gastrointestinal absorption with a
high-fat meal, and large reactive increases in renin secretion--the functional
importance of which is under intense investigation. The results of outcomes
trials are eagerly awaited. Intensive efforts have been spent to discover therapeutic, non-peptide and
orally effective hypertensive drugs. One drug that emerged from this effort is
aliskiren, a direct human renin inhibitor that blocks the conversion of
angiotensinogen to angiotensin I (Ang I). In contrast to other antihypertensive
agents, aliskiren decreases plasma renin activity (PRA). In healthy human
subjects, doses of between 40 and 640 mg of aliskiren exert a dose-dependent
reduction in PRA and Ang I and Ang II levels. The bioavailability of aliskiren
is low (2%), peak plasma concentrations are reached within one to three hours
and the binding with plasma proteins achieves approximately 47-51%. Aliskiren is
slightly metabolized (20%) by CYP3A4. The most common adverse events include
diarrhea, headache, back pain and gastrointestinal disorders. Aliskiren is well
tolerated, and may be used alone or in combination with other antihypertensive
agents. Aliskiren belongs to a new class of agents that effectively and
specifically inhibit the RAS. This drug functions through a novel mechanism of
action and has the potential to become a true alternative to angiotensin
converting enzyme inhibitors and angiotensin receptor blockers in the therapy of
hypertension and other cardiovascular and renal disorders. An association has been shown between plasma renin activity (PRA) and the risk
of cardiovascular disease. There is also evidence that angiotensin II exerts
detrimental effects on progression and instabilization of atherosclerotic
plaque. The renin-angiotensin system (RAS) can be inhibited through inhibition
of angiotensin I (Ang I) generation from angiotensinogen by direct renin
inhibitors, inhibition of angiotensin II (Ang II) generation from angiotensin I
by angiotensin-converting enzyme inhibitors and finally by direct inhibition of
the action of Ang II receptor level. Aliskiren, the first direct renin inhibitor
to reach the market, is a low-molecular-weight, orally active, hydrophilic
nonpeptide. Aliskiren blocks Ang I generation, while plasma renin concentration
increases because the drugs blocks the negative feed-back exerted by Ang II on
renin synthesis. Because of its long pharmacological half-life, aliskiren is
suitable for once-daily administration. Its through-to-peak ratio approximates
98% for the 300 mg/day dose. Because of its mechanism of action, aliskiren might
offer the additional opportunity to inhibit progression of atherosclerosis at
tissue level. Hypertension is an approved indication for this drug, which is
also promising for the treatment of heart failure. The efficacy of this drug in
reducing major clinical events is being tested in large ongoing clinical trials. Aliskiren gained FDA approval for the treatment of hypertension in 2007. It is
the first approved pharmaceutical to manage hypertension by direct renin
inhibition. With the introduction of novel drugs and mechanisms of action comes
the challenge of monitoring for new unreported adverse events. The side effect
profile for aliskiren has not yet been fully described. We describe the first
apparent report of aliskiren-induced QT prolongation resulting in torsades de
pointes. Hypertension is a major risk factor for the development of cardiovascular and
renal disease. The incidence of hypertension is increasing globally and the rate
of blood pressure control remains inadequate. Renin-angiotensin-aldosterone
system (RAAS) plays a crucial role in volume regulation and maintece of blood
pressure. Pathological activation of RAAS results in chronic hypertension and
consequent end organ damage. Most patients with hypertension require combination
therapy using agents with complimentary mechanisms of action.
Hydrochlorothiazide (HCTZ) together with an agent blocking the RAAS such as an
angiotensin converting enzyme (ACE) inhibitor or angiotensin receptor blocker
(ARB) are widely used effective anti-hypertensive therapy. Aliskiren is an
orally effective direct renin inhibitor that blocks the generation of
angiotensin I from angiotensinogen, the rate limiting step of RAAS activation.
Studies have shown equivalent antihypertensive efficacy of aliskiren when
compared to existing medications such as HCTZ, ACE inhibitors and ARBs.
Aliskiren has also been tested in combination therapies. The current review aims
to look at the efficacy of aliskiren therapy in hypertension and the evidence
for using aliskiren in combination with HCTZ. The renin-angiotensin-aldosterone system (RAAS) has a central function in the
regulation of blood pressure. Aliskiren, the first direct renin inhibitor to be
approved for the treatment of hypertension, blocks the RAAS at its point of
activation. As renin inhibition acts at the top of the RAAS cascade, this
mechanism has been proposed to offer advantages over existing modes of RAAS
blockade. The RAAS is also considered to be a major factor in the pathogenesis
of many renal diseases, especially diabetic nephropathy (DN), the main cause of
end-stage renal disease. Existing therapies to block the RAAS slow the
progression of DN, but they do not halt the disease. Therefore, more effective
modes of interventions are needed. Studies to determine the efficacy of
aliskiren in human renal disease are in progress. This review summarizes in vivo
studies in which the efficacy of aliskiren was tested in experimental models of
renal disease, and presents in vitro studies that provide insights into the
possible mechanisms by which aliskiren confers renoprotection in animals. These
works are discussed in the framework of the intrarenal RAAS and suggest that
aliskiren may act by unique renoprotective mechanisms. IMPORTANCE OF THE FIELD: The renin-angiotensin-aldosterone system (RAAS) is a
key regulator of blood pressure (BP), as well as volume and electrolytes, in
both hypertensive and normotensive individuals. Inappropriate activation of the
RAAS is important in hypertension-induced cardiovascular disease (CVD) and
chronic kidney disease (CKD). Renin is the rate-limiting step in the RAAS
cascade, which makes direct renin inhibitors (DRIs) an attractive target for
RAAS suppression and treatment of hypertension. Current regimens using either
angiotensin-converting enzyme inhibitor (ACEi) or angiotensin receptor blocker
(ARB) result in feedback upregulation of renin and aldosterone breakthrough,
which contribute to incomplete suppression of the RAAS. Thereby, aliskiren -
alone or in combination - might offer a novel therapeutic intervention to
improve suppression of the RAAS, with potential to translate to improved CVD and
CKD outcomes.
AREAS COVERED IN THIS REVIEW: Herein, we present the current state of knowledge
of DRIs in the preclinical and clinical realm and their antihypertensive
efficacy in relation to cardiovascular and renal risk. Recent clinical trials
(2007 - 2009) support the efficacy of aliskiren, and studies suggest the
potential for improved CVD and CKD outcomes.
WHAT THE READER WILL GAIN: An understanding of the mechanism of action of DRIs
and a perspective of recent clinical trials.
TAKE HOME MESSAGE: The DRI aliskiren is an effective antihypertensive agent that
preliminary data suggests has a beneficial effect in CVD and CKD. Combination of
aliskiren with an ACEi or ARB may be better tolerated than the ACEi-ARB
combination. Future work is needed to further quantify aliskiren's impact on
hard CVD and CKD end points. The renin-angiotensin-aldosterone system (RAAS) is a major factor for the
development and maintece of hypertension and a major cause for cardiovascular
remodeling and cardiovascular complications through its active peptide
angiotensin (Ang) II. Blockade of RAAS with ACE inhibitors (ACEIs) results in
suppression of Ang II levels, which eventually return to baseline levels after
prolonged ACEI administration. This leads to an escape phenomenon through
generation of Ang II from enzymes other than ACE and led to the hypothesis that
dual blockade of RAAS with an ACEI/Ang receptor blocker (ARB) combination could
lead to total blockade of RAAS, since ARBs block the action of Ang II at the AT1
receptor level, irrespective of the mechanism of Ang II generation and will have
an additive blood pressure (BP)-lowering effect. However, this hypothesis has
not materialized clinically, as the ACEI/ARB combination produces modest BP
reductions that are not significantly greater than monotherapy with the
component drugs, and is frequently associated with higher incidence of side
effects. A new dual RAAS blockade with the direct renin inhibitor aliskiren and
the ARB valsartan produces greater BP reductions than monotherapy with the
component drugs and is safe and well tolerated. The combination of aliskiren
with valsartan, and with other antihypertensive drugs is discussed. There is now clear evidence that reducing blood pressure (BP) with a broad range
of agents, including angiotensin converting enzyme inhibitors and angiotensin
receptor blockers, improves cardiovascular and renal outcomes. There is also
evidence suggesting that these drugs have beneficial effects that are
independent of BP lowering. Aliskiren is a direct renin inhibitor that
interrupts the renin-angiotensin-aldosterone system (RAAS) at its rate-limiting
step. Unlike angiotensin-converting enzyme inhibitors and angiotensin receptor
blockers, aliskiren produces a sustained reduction in plasma renin activity and
reduces plasma levels of angiotensin II and aldosterone. Preclinical data and
clinical trials in high-risk patients using surrogate markers increasingly
suggest that aliskiren can reduce the progression of end-organ damage beyond
that afforded by BP control. With its unique mechanism of action, combining
aliskiren with another RAAS-blocking agent that has a different mechanism of
action may provide more comprehensive blockade of the RAAS, potentially
conferring additional clinical benefits. Evaluation of these end-organ effects
in humans is underway in clinical trials designed to assess the effects of
aliskiren alone and in combination with other antihypertensive agents on
cardiovascular and renal outcomes. INTRODUCTION: The recent discovery of a specific receptor for renin/prorenin
(PRR) has added new interest to the potential pharmacological actions of
aliskiren, the first direct renin inhibitor.
MATERIALS AND METHODS: In the present study, to gain new insights into the
pharmacological properties of aliskiren, we investigated the effect of aliskiren
on PRR expression and activity in cultured human smooth muscle cells (HSMCs).
RESULTS: Co-incubation of HSMCs with angiotensinogen (ANG) (1.5 × 10(-7)M) and
prorenin (10(-8)-10(-7)M) resulted in an efficient production (within 4h) of
angiotensin I, almost completely inhibited by 10(-5)M aliskiren (-86.0 ± 14.0%).
In HSMCs stimulated with both ANG and prorenin, a 24h incubation with aliskiren
(10(-6)-10(-5)M) resulted in a concentration-dependent reduction of PRR mRNA
levels (IC(50) 4.6 × 10(-6)M). The cell surface expression of PRR determined by
flow cytometry analysis was also reduced after incubation with aliskiren in a
concentration-dependent manner. The lower levels of PRR were associated with a
reduced expression of TGF-β, PAI-1 and type I collagen mRNA.
CONCLUSIONS: These results suggest a direct pharmacological action of aliskiren
on PRR expression and its signalling pathway in HSMCs. This reported action of
aliskiren may reveal a new scenario of the pharmacological properties of
aliskiren. Renin inhibitors are antihypertensive drugs that block the first step in the
renin-angiotensin system. Their mechanism of action differs from that of the
angiotensin-converting enzyme inhibitors and angiotensin-receptor antagonists,
but like these drugs, renin inhibitors interrupt the negative feedback effects
of angiotensin II on renin secretion. The renin-angiotensin-aldosterone system
(RAAS) has long been recognized to play a significant role in hypertension
pathophysiology. Certain agents that modify the RAAS can control blood pressure
and improve cardiovascular outcomes. Optimization of this compound by Novartis
led to the development of aliskiren - the only direct renin inhibitor which is
clinically used as an antihypertensive drug. Aliskiren is the first of a new
class of antihypertensive agents. Aliskiren is a new renin inhibitor of a novel
structural class that has recently been shown to be efficacious in hypertensive
patients after once-daily oral dosing. In short-term studies, it was effective
in lowering blood pressure either alone or in combination with valsartan and
hydrochlorothiazide, and had a low incidence of serious adverse effects. It was
approved by the Food and Drug Administration in 2007 for the use as a
monotherapy or in combination with other antihypertensives. Greater reductions
in blood pressure have been achieved when aliskiren was used in combination with
hydrochlorothiazide or an angiotensin-receptor blocker. The most common adverse
effects reported in clinical trials were headache, fatigue, dizziness, diarrhea,
and nasopharyngitis. Aliskiren has not been studied in patients with moderate
renal dysfunction; as an RAAS-acting drug, it should be prescribed for such
patients only with caution. Activation of the atrial renin-angiotensin system plays an important role in the
pathophysiology of atrial fibrillation (AF). The pulmonary vein (PV) and left
atrium (LA) are important trigger and substrate for the genesis of AF. We
investigate the effects of a direct renin inhibitor, aliskiren, on the PV and LA
arrhythmogenic activity and the underlying electromechanical mechanisms.
Conventional microelectrodes were used to record action potentials and
contractility in isolated rabbit PVs and LA tissues before and after the
administration of aliskiren (0.1, 1, 3 and 10 μM). By the whole-cell patch clamp
and indo-1 fluorimetric ratio techniques, ionic currents and intracellular
calcium transient were studied in isolated single PV and LA cardiomyocyte before
and after the administration of aliskiren (3 μM). Aliskiren (0.1, 1, 3 and
10 μM) reduced PV firing rate in a concentration-dependent manner (6, 10, 14 and
17%) and decreased PV diastolic tension, which could be attenuated in the
presence of 100 μM L-N(G)-Nitroarginine Methyl Ester (L-NAME). Aliskiren induced
PV automatic rhythm exit block causing slow and irregular PV activity with
variable pauses. Aliskiren increased PV and LA contractility, which could be
abolished by pre-treating with 0.1 μM ryanodine. Aliskiren (3 μM) decreased
L-type calcium currents, but increased reverse-mode of Na( + )/Ca(2+ ) exchanger
currents, intracellular calcium transients, and sarcoplasmic reticulum calcium
content in PV and LA cardiomyocytes. Pretreatment with renin, losartan or
angiotensin II did not alter the effect of aliskiren on sarcolemmal calcium
flux. In conclusion, aliskiren reduces PV arrhythmogenic activity with a direct
vasodilatory property and has a positive inotropic effect on cardiomyocytes.
These findings may reveal the anti-arrhythmic and anti-heart failure potentials
of aliskiren. The present study was designed to investigate the potential of aliskiren, a
direct renin inhibitor, in chronic constriction injury (CCI)-induced neuropathic
pain in rats. Neuropathic pain was induced by placing four loose ligatures
around the sciatic nerve. Acetone drop, von Frey hair, pin-prick and hot plate
tests were performed to assess cold allodynia, mechanical allodynia, mechanical
and heat hyperalgesia, respectively. The levels of Tumor necrosis factor-alpha
(TNF-α) were measured in the sciatic nerve as an inflammatory marker. CCI was
associated with the development of cold allodynia, mechanical allodynia,
mechanical and heat hyperalgesia along with a rise in the levels of Tumor
necrosis factor-alpha (TNF-α). Administration of aliskiren (25 or 50 mg/kg
intraperitoneal (i.p.)) for 14 days in CCI-subjected rats significantly
attenuated CCI-induced pain-related behavior and rise in TNF-α level. It may be
concluded that aliskiren-mediated anti-inflammatory actions may be responsible
for its beneficial effects in neuropathic pain in rats. The burden of cardiovascular (CV) disease remains high nowadays, despite the
tremendous development in the therapeutic strategies that has occurred during
the last 30 years. The growing focus on pharmacological strategies capable of
interacting with key pathophysiological mechanisms has resulted in a better
control of CV disease. The renin-angiotensin system (RAS) is involved in many
pathophysiological processes underlying the development of major CV and renal
diseases and, thus, it represents an 'ideal' target for the pharmacological
treatment of these clinical conditions. Recently, in addition to the traditional
therapeutic approaches, mostly based on the use of ACE inhibitors and/or
angiotensin II type 1 receptor antagonists (angiotensin receptor blockers
[ARBs]), the scientific and medical community have focused on a new therapeutic
possibility to interfere with RAS activity. Indeed, the first compound of the
new drug class of direct renin inhibitors, aliskiren, has been made available
for clinical use. The therapeutic properties of aliskiren are of particular
interest, since it interferes with the enzymatic activities of renin, the key
rate-limiting step of the RAS cascade. This unique mechanism of action, e.g.
occupation of the renin active site, provides an 'upstream' modulation in the
RAS enzymatic-proteic cascade. This results in an inhibition of the biological
properties of renin to promote the cleavage of the angiotensin I peptide from
the angiotensinogen substrate.Clinical studies have demonstrated that the use of
aliskiren provides antihypertensive efficacy comparable with, or even superior
to, that observed with other classes of antihypertensive drugs. Also, the
addition of aliskiren to different antihypertensive strategies results in a
further significant increase in blood pressure (BP) reduction than those
achieved with monotherapy based on diuretics, calcium-channel antagonists, ACE
inhibitors and ARBs. The overall safety and tolerability of aliskiren are
comparable with other classes of antihypertensive drugs and almost overlap with
placebo. Recent evidence also demonstrates that aliskiren is effective in
promoting the regression of organ damage (e.g. microalbuminuria and left
ventricular hypertrophy), beyond its BP-lowering properties and in addition to
standard therapies, including ARBs. An ambitious programme of clinical
development of this modern antihypertensive drug will investigate whether
aliskiren may further limit the incidence of major CV and renal events in
hypertensive patients with co-morbidities, treated with the available
therapeutic strategies, including ACE inhibitors and ARBs. Hypotension and syncopal attacks have been reported in association with drugs
which block the renin-angiotensin-aldosterone system (RAAS). It has been
proposed that the underlying mechanism is due to sensitisation of the
Bezold-Jarisch reflex leading to withdrawal of sympathetic tone, profound and
prolonged bradycardia, and hypotension. Sensitisation of this reflex occurs in
the presence of blockade of the RAAS. In the ALTITUDE trial the use of the
direct renin inhibitor, aliskiren, was associated with hypotensive episodes and
an excess of ischaemic stroke. It is hypothesised that this is best explained by
activation of the Bezold-Jarisch reflex, which may be particularly important in
circumstances where there is dual blockade of the RAAS. INTRODUCTION: Angiotensin-converting enzyme (ACE) inhibitors cause angioedema
due to diminished degradation of bradykinin. Angiotensin receptor blockers may
occasionally cause angioedema but the mechanism is unknown, and are generally
considered safe, even in those who have reacted to ACE inhibitors. We determined
whether aliskiren, a renin inhibitor, has an effect on the rate of bradykinin
degradation.
METHODS: The ability of renin to metabolize bradykinin was studied and the rate
of bradykinin degradation compared in the presence or absence of aliskiren.
Enalapril, a known ACE inhibitor that causes angioedema served as positive
control.
RESULTS: Renin was unable to digest bradykinin, indicating that a renin
inhibitor is unlikely to affect the rate of bradykinin degradation. In a plasma
system, aliskiren had no effect on the rate of bradykinin degradation while
enalapril inhibited it appreciably. An inhibitory effect of aliskiren on the
rate of bradykinin degradation by human pulmonary endothelial cells was
observed, estimated to be about 5% of that of enalapril.
CONCLUSION: Aliskiren has no effect upon the rate of bradykinin degradation in
plasma and a minimal effect employing vascular endothelial cells. The latter
suggests inhibition of a non-renin enzyme that is a minor contributor to
bradykinin degradation. Dopamine causes in anesthetized rats expressed diuretic response that is
accompanied by an increase in GRF and a significant enhance of sodium and
potassium excretion. Pretreatment the animals in diclofenac sodium or contrical
in doses, that inhibit respectively activity of renal PG-system and
kallikrein-kinin system, don't prevent of renal effects of dopamine. Preliminary
assignment a direct renin inhibitor aliskiren enhances the diuretic, natriuretic
and kaliyuretic effects of the drug. It is concluded that renal PG-system and
kallikrein-kinin system are not involved in the formation of renal effects of
dopamine. Renal tissue RAS directly included in the mechanism of action of
dopamine in the kidney, acting as a modulator, preventing excessive loss of
water and electrolytes with urine. |
Which proteins act as histone-like molecules in prokaryotes? | Prokaryotic histone-like proteins (Hlps) or nucleoid-associated proteins (NAPs) are abundant proteins found in bacterial and plastid nucleoids. HU protein is a small, basic, heat-stable DNA-binding protein that is well-conserved in prokaryotes and is associated with the bacterial nucleoid. HU is well conserved in all prokaryotes but surprisingly, it is also homologous to another E. coli DNA-binding protein, IHF. In prokaryotes, IHF and HU are key architectural proteins present at high concentrations. Histone-like Nucleoid Structuring (H-NS) protein can facilitate correct recognition of a promoter by RNA polymerase in AT-rich gene regulatory regions | Thermoplasma acidophilum, a thermophilic mycoplasma, has several unusual
features suggesting a possible relationship to eukaryotic cells. One feature is
a histone-like protein that is associated with the DNA, condensing it into
subunits similar to those in eukaryotic chromatin. A second feature is an
association of cytoplasmic proteins that resembles eukaryotic actin and myosin.
These two components are widely distributed in different groups of eukaryotic
cells, but are typically lacking in prokaryotic cells. Furthermore, T.
acidophilum lacks cytochromes and respires by enzymes that apparently are not
coupled to oxidative phosphorylation. This primitive type of respiration
resembles that of microbodies, another feature which is represented in the
cytoplasm of all groups of eukaryotic cells. Furthermore, since T. acidophilum
lacks a cell wall and appears to have a primitive correlate of endocytosis, it
would appear to be mechanically capable of acquiring a symbiotic mitochondrion.
Thus, our observations are consistent with the symbiotic hypothesis for the
origin of eukaryotic cells. We suggest that an organism similar to T.
acidophilum was the host cell for the original symbiosis, becoming the nucleus
and cytoplasm of modern eukaryotic cells. There are many procaryotic and eucaryotic organisms in plant kingdom. It is
hoped that the study of plant histones will be useful in evolutionary studies.
The histones of great variety of animal species have been studied and well
characterized. Less information is available concerning plant histones. The
general conclusion drawn from these investigations is that most organisms of
eucaryotic plant and animal species contain the same five major histone
fractions. Recently the histone-like proteins were found in some primitive
eucaryotes and procaryotes. Data on histones from higher and lower eucaryotes
and histone-like proteins of procaryotes are reviewed. Evolution of histones and
their appearance prior to that of eucaryotic cell is postulated. The role of
histones in evolution of nucleosomes is discussed. HU is one of the most abundant DNA binding proteins in Escherichia coli. Like
the histones, HU is able to condense DNA in vitro and to introduce negative
super-coiling in covalently closed circular, relaxed DNA molecules in the
presence of topoisomerase I. HU is well conserved in all prokaryotes but
surprisingly, it is also homologous to another E. coli DNA-binding protein, IHF.
Contrary to HU, IHF shows sequence specificity and is much less abundant that
HU. Both are heterodimers and all four polypeptide chains probably arose from a
common ancestor. The question we raised was whether IHF could supply the main
functions of HU in its absence. The answer seems to be negative for the
following reasons. We did not observe any significant regulation of expression
of the genes coding for HU by IHF, or vice-versa. The structures that these two
proteins form with double-stranded or single-stranded DNA are completely
different. Finally, overexpression of IHF does not relieve the growth defects
observed in HU-less mutants. However, it can be speculated from our results that
even if the two proteins are not equivalent and cannot replace one another, both
could stimulate (or inhibit) some specific protein-DNA interactions or affect
the DNA-binding properties of one another. Experiments are reviewed that allow one to assign naturally occurring
DNA-containing plasmas to either of two classes by virtue of their sensitivity
to aggregation upon dehydration in organic solvents. The interphase nuclei of
higher cells are relatively insensitive, while the DNA plasmas represented by
bacterial nucleoids, vegetative bacteriophage and the chromosomes of
dinoflagellates are sensitive. In higher cells the bulk of DNA is organised with
histones in the form of nucleosomes. In prokaryotes and in the pool of
vegetative phage DNA the most abundant histone-like protein HU is not associated
with the bulk DNA, but localised in the border region with ribosomes where
transcription and translation occur. These experimental results strongly suggest
that the two classes of DNA plasmas are distinguishable by a low (1:10) or high
(1:1) protein-to-DNA ratio. The hypothesis is formulated that the vegetative DNA
(replicating and transcribing), throughout the living world, is nucleosome-free;
during evolution, nucleosomes would have been introduced as a simple and
adequate means for compacting the resting DNA. Condensation of DNA does not
occur with prokaryotic nucleoids, but does take place when DNA is withdrawn from
the vegetative phage pool to become packaged into phage heads. Dinoflagellate
chromosomes are rather condensed although structurally different from eukaryotic
chromosomes (e.g., those from Euglena) and are much more aggregation-sensitive. From the cells of an Escherichia coli K-12 strain, a 22,000-dalton protein which
has an affinity for the superhelical DNA molecule was purified to apparent
homogeneity by monitoring the DNA-binding activity using the filter binding
assay. In the sedimentation analysis of the DNA-protein complex, the protein has
an affinity for the superhelical or single-stranded DNA molecule but neither for
the open-circular nor for the linear DNA molecule. The amino acid composition of
the protein resembled those of the other prokaryotic histone-like proteins and
also to eukaryotic histones H2A and H2B. The protein precipitated upon heating,
which is in contrast to the heat-stable feature of the other histone-like
proteins. Furthermore, DNA and RNA syntheses in vitro were not affected by the
presence of the protein. In view of these characteristics, this protein may play
a role in maintaining the bacterial nucleoid structure. The prokaryotic protein HU functions as an accessory factor in many different
biochemical reactions. We have characterized the role of HU in assembling the
invertasome, an intermediate nucleoprotein complex involved in Hin-mediated
site-specific recombination. Formation of this complex requires the looping of
intervening DNA segments between sites bound by the Hin recombinase and the Fis
protein. HU stimulates this process on substrates containing intervening
segments of length < 100 bp. Characterization of the activity of HU in
Hin-mediated recombination in vitro and in vivo yields evidence that its role in
this reaction is primarily to facilitate the looping of the intervening DNA
segment. By using this reaction as an assay, we identify proteins from mammals,
yeast, trypanosomes, and wheat which can fulfill the same function in vitro.
Using ligase-mediated circularization of short DNA fragments we also show that
HU, the high mobility group (HMG) 1 and 2 proteins from mammals, and a protein
from yeast can bend DNA extremely efficiently. These results support the view
that this ubiquitous class of proteins enhance the assembly of nucleoprotein
complexes under conditions of limited DNA flexibility. The mammalian high mobility group proteins HMG1 and HMG2 are abundant,
chromatin-associated proteins whose cellular function is not known. In this
study we show that these proteins can substitute for the prokaryotic DNA-bending
protein HU in promoting the assembly of the Hin invertasome, an intermediate
structure in Hin-mediated site-specific DNA inversion. Formation of this complex
requires the assembly of the Hin recombinase, the Fis protein, and three
cis-acting DNA sites, necessitating the looping of intervening DNA segments.
Invertasome assembly is strongly stimulated by HU or HMG proteins when one of
these segments is shorter than 104 bp. By use of ligase-mediated circularization
assays, we demonstrate that HMG1 and HMG2 can bend DNA extremely efficiently,
forming circles as small as 66 bp, and even 59-bp circles at high HMG protein
concentrations. In both invertasome assembly and circularization assays,
substrates active in the presence of HMG1 contain one less helical turn of DNA
compared with substrates active in the presence of HU protein. Analysis of
different domains of HMG1 generated by partial proteolytic digestion indicate
that DNA-binding domain B is sufficient for both bending and invertasome
assembly. We suggest that an important biological function of HMG1 and HMG2 is
to facilitate cooperative interactions between cis-acting proteins by promoting
DNA flexibility. A general role for HMG1 and HMG2 in chromatin structure is also
suggested by their ability to wrap DNA duplexes into highly compact forms. We report the identification of the first histone-like protein of Mycobacterium
tuberculosis (MTB) (HLPMt). The T cell blot assay was used to identify antigens
of MTB associated with human immune response in healthy contacts. Fraction 21
corresponding to proteins in the molecular weight range of approximately 30 kDa
were found to be immunogenic in tuberculin reactors. None of the fractions were
found to be immunogenic by this assay in non-reactors to tuberculin. All sera,
irrespective of the source, showed reactivity with MTB antigen(s) over a wide
molecular weight range (205-->16 kDa). In the present study fraction 21 was
processed for the generation of murine polyclonal sera and amino acid
sequencing. The sequence of a 16-amino acid long peptide showed a 100% homology
with an open reading frame (ORF) in the translated sequence of cosmid cY349
(Sanger Centre, Cambridge, UK). The ORF was predicted to code for a protein of
214 amino acids. Oligonucleotide primers were synthesized based on the
nucleotide sequence located at the 5' and 3' regions of the gene. The gene
encoding the predicted protein was PCR-amplified, cloned, sequenced and
expressed in Escherichia coli as a protein of 28 kDa. The expressed HLPMt
protein was shown to react with the polyclonal murine sera originally raised
against fraction 21. Human immune response to the recombit HLPMt protein was
demonstrated by its ability to induce lymphoproliferation in peripheral blood
derived mononuclear cells, and the presence of anti-HLPMt antibodies in pooled
patient sera by immunoblot. The recombit HLPMt protein elicited a vigorous
lymphoproliferative response especially in healthy tuberculin reactors compared
to non-reactors and patients of tuberculosis, (P < 0.05). The protein has unique
dual domains with homology to both bacterial histone-like proteins (HU) and
eukaryotic histone H1. Homology to prokaryotic and eukaryotic deoxyribonucleic
acid (DNA)-binding proteins suggested that HLPMt could bind DNA. DNA-binding
properties were confirmed by South-Western analysis strongly suggesting an
interaction between HLPMt and the MTB chromosome. Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play
important roles in signal transduction required for cell proliferation,
differentiation, and stress response and adaptation. It is also known that some
prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in
the characterization of these kinases is how to identify their specific
substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2
from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly
expressed in E. coli, cells are unable to form colonies on agar plates. This
lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the
highly conserved Lys residue was changed to Asn, indicating that phosphorylation
of a cellular protein(s) in E. coli resulted in growth arrest. Several clones
from an E. coli genomic library were found to suppress the lethal effect when
co-expressed with pkn2. Four out of seven multi-copy suppressors were identified
to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein.
Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59,
and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the
phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part
to the lethal effect in E. coli. The present method termed the STEK method
(Suppressors of Toxic Effects of Kinases) may be widely used for the substrate
identification not only for prokaryotic protein Ser/Thr kinases but also for
eukaryotic kinases. The HU protein is a small, basic, heat-stable DNA-binding protein that is
well-conserved in prokaryotes and is associated with the bacterial nucleoid. In
enterobacteria, including Escherichia coli, HU is a heterotypic dimer,
HUalphabeta, composed of two closely related sub-units encoded by the hupA and
hupB genes, respectively. HU was shown to participate in vitro in the initiation
of DNA replication as an accessory factor to assist the action of DnaA protein
in the unwinding of oriC DNA. To further elucidate the role of HU in the
regulation of the DNA replication initiation process, we tested the synchrony
phenotype in the absence of either one or both HU sub-units. The hupAB mutant
exhibits an asynchronous initiation, the hupA mutant shows a similar reduced
synchrony, whereas the hupB mutant shows a normal phenotype. Using a
thermosensitive dnaA46 strain (dnaA46ts), an initiation mutant, we reveal a
special role of HUbeta. The presence of a plasmid overproducing HUbeta in a
dnaA46ts lacking HU (hupAB background) compensates for the thermosensitivity of
this initiation mutant. Moreover, the overproduction of HUbeta confers to
dnaA46ts a pattern of asynchrony similar to that of a dnaAcos, the intragenic
suppressor of dnaA46ts. We show that the relative ratio of HUalpha versus HUbeta
is greatly perturbed in dnaA46ts which accumulates little, if any, HUbeta.
Therefore, the suppression of thermosensitivity in dnaA46hupAB by HUbeta may be
caused by an unexpected absence of HUbeta in the dnaA46ts mutant. Visibly the HU
composition is sensitive to the different states of DnaA, and may play a role
during the regulation of the initiation process of the DNA replication by
affecting subsequent events along the cell cycle. Short-chain poly-(R)-3-hydroxybutyrate (cPHB), a highly flexible, amphiphilic
molecule with salt-solvating properties, is a ubiquitous constituent of
prokaryotic and eukaryotic cells, wherein it is mainly conjugated to proteins.
The solvating properties and cellular distribution of cPHB suggest it may be
associated with proteins that bind and/or transfer DNA. Here we examine
Escherichia coli protein H-NS and calf thymus histones, H1, H2A, H2B, H3, and
H4, for the presence of cPHB. The proteins are related in that all bind to DNA
and are implicated in the compact organization of the chromosome. The presence
of cPHB in E. coli H-NS was first detected in Western blots of two-dimensional
sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of total cell
proteins, probed with anti-cPHB IgG, and then by Western blot analysis of the
purified protein. Western blot analysis of the calf thymus histones indicated
that each contained cPHB. The presence of cPHB in H-NS and histones was
confirmed by chemical assay. The in vivo size of conjugated cPHB could not be
established due to the lack of standards and degradation of cPHB during protein
purification and storage. The molecular characteristics of cPHB and its presence
in histone-like and histone proteins of diverse organisms suggest it may play a
role in DNA binding and/or DNA organization. DNA looping is often involved in positive and negative regulation of gene
transcription in both prokaryotes and eukaryotes. The transcription of the gal
operon of Escherichia coli from two overlapping promoters P1 and P2 is
negatively regulated via Gal repressosome assembly. It involves binding of two
dimeric Gal repressor proteins (GalR) to two operators, O(E) and O(I), flanking
the two promoters, and formation of 113 bp DNA loop due to tetramerization of
the two bound GalR dimers. The process requires negatively supercoiled DNA and
the presence of the histone-like protein HU. Previous modeling of the
repressosome based on evaluation of DNA elastic energy suggested a mutual
antiparallel, rather than parallel, orientation of the two gal operators in an
under-twisted DNA loop. To visualize the Gal loop by atomic force microscopy
(AFM), plasmid DNA molecules were constructed with increased distance between
the two operators. The AFM results demonstrated the formation of an antiparallel
DNA loop in the Gal repressosome consistent with our earlier hypothesis.
Importantly, the overall shape of the GalR mediated loop proved to be
indistinguishable from that in the chimerical loop of the same size containing
two lac operators (instead of two gal operators) and formed by LacI. In
addition, a possibility of the gal operon repression mediated by GalR in the
absence of HU was shown in the new DNA constructs. Implications of these
findings for the DNA structural organization in bacterial nucleoid are
discussed. The plastid is a semiautonomous organelle essential in photosynthesis and other
metabolic activities of plants and algae. Plastid DNA is organized into the
nucleoid with various proteins and RNA, and the nucleoid is subject to dynamic
changes during the development of plant cells. Characterization of the major
DNA-binding proteins of nucleoids revealed essential differences in the two
lineages of photosynthetic eukaryotes, namely nucleoids of green plants contain
sulfite reductase as a major DNA-binding protein that represses the genomic
activity, whereas the prokaryotic DNA-binding protein HU is abundant in plastid
nucleoids of the rhodophyte lineage. In addition, current knowledge on
DNA-binding proteins, as well as the replication and transcription systems of
plastids, is reviewed from comparative and evolutionary points of view. A
revised hypothesis on the discontinuous evolution of plastid genomic machinery
is presented: despite the cyanobacterial origin of plastids, the genomic
machinery of the plastid genome is fundamentally different from its counterpart
in cyanobacteria. The energetic cost of bending short segments of DNA is very high. This bending
is critical for the packaging of DNA and is exploited to regulate many cellular
processes. In prokaryotes, IHF and HU are key architectural proteins present at
high concentrations. New protein-DNA co-crystal structures, and the adaptation
of advanced biophysical and biochemical techniques have led to an improved
understanding of how these proteins interact with DNA. These techniques include
time-resolved synchrotron X-ray footprinting, differential scanning calorimetry,
isothermal titration calorimetry and single-molecule experiments. We have addressed the distribution and abundance of 75 transcription factor (TF)
families in complete genomes from 90 different bacterial and archaeal species.
We found that the proportion of TFs increases with genome size. The deficit of
TFs in some genomes might be compensated by the presence of proteins organizing
and compacting DNA, such as histone-like proteins. Nine families are represented
in all the bacteria and archaea we analyzed, whereas 17 families are specific to
bacteria, providing evidence for regulon specialization at an early stage of
evolution between the bacterial and archeal lineages. Ten of the 17 families
identified in bacteria belong exclusively to the proteobacteria defining a
specific signature for this taxonomical group. In bacteria, 10 families are lost
mostly in intracellular pathogens and endosymbionts, while 9 families seem to
have been horizontally transferred to archaea. The winged helix-turn-helix (HTH)
is by far the most abundant structure (motif) in prokaryotes, and might have
been the earliest HTH motif to appear as shown by its distribution and abundance
in both bacterial and archaeal cellular domains. Horizontal gene transfer and
lineage-specific gene losses suggest a progressive elimination of TFs in the
course of archaeal and bacterial evolution. This analysis provides a framework
for discussing the selective forces directing the evolution of the
transcriptional machinery in prokaryotes. Prokaryotic histone-like proteins (Hlps) are abundant proteins found in
bacterial and plastid nucleoids. Hlps are also found in the eukaryotic
dinoflagellates and the apicomplexans, two major lineages of the Alveolata. It
may be expected that Hlps of both groups were derived from the same ancestral
Alveolates. However, our phylogenetic analyses suggest different origins for the
dinoflagellate and the apicomplexan Hlps. The apicomplexan Hlps are affiliated
with the cyanobacteria and probably originated from Hlps of the plastid genome.
The dinoflagellate Hlps and the proteobacterial long Hlps form a clade that
branch off from the node with the proteobacterial short Hlps. In mesophilic prokaryotes, the DNA-binding protein HU participates in nucleoid
organization as well as in regulation of DNA-dependent processes. Little is
known about nucleoid organization in thermophilic eubacteria. We show here that
HU from the hyperthermophilic eubacterium Thermotoga maritima HU bends DNA and
constrains negative DNA supercoils in the presence of topoisomerase I. However,
while binding to a single site occludes approximately 35 bp, association of T.
maritima HU with DNA of sufficient length to accommodate multiple protomers
results in an apparent shorter occluded site size. Such complexes consist of
ordered arrays of protomers, as revealed by the periodicity of DNase I cleavage.
Association of TmHU with plasmid DNA yields a complex that is remarkably
resistant to DNase I-mediated degradation. TmHU is the only member of this
protein family capable of occluding a 35 bp nonspecific site in duplex DNA; we
propose that this property allows TmHU to form exceedingly stable associations
in which DNA flanking the kinks is sandwiched between adjacent proteins. We
suggest that T. maritima HU serves an architectural function when associating
with a single 35 bp site, but generates a very stable and compact aggregate at
higher protein concentrations that organizes and protects the genomic DNA. |
Which receptor is targeted by telcagepant? | Telcagepant (MK-0974) is a novel calcitonin gene-related peptide (CGRP) receptor antagonist currently undergoing clinical trials for migraine. | OBJECTIVE: To determine an effective and tolerable dose of a novel oral
calcitonin gene-related peptide (CGRP) receptor antagonist, MK-0974, for the
acute treatment of migraine.
METHODS: Randomized, double-blind, parallel-group, clinical trial with a
two-stage, adaptive, dose-ranging design. Patients were allocated to treat a
moderate or severe migraine attack with MK-0974 (25, 50, 100, 200, 300, 400, or
600 mg), rizatriptan 10 mg, or placebo taken orally. The primary endpoint was
pain relief (reduction to mild or none) 2 hours after dosing. Secondary
endpoints included pain freedom at 2 hours and sustained pain relief at 24
hours. A prespecified, blinded, automated interim analysis was used to
discontinue randomization to less effective doses.
RESULTS: Per the adaptive study design, the four lowest MK-0974 groups (25, 50,
100, 200 mg) were discontinued due to insufficient efficacy. For the remaining
treatment groups, the estimated pain relief proportions at 2 hours were 300 mg
(n = 38) 68.1%, 400 mg (n = 45) 48.2%, 600 mg (n = 40) 67.5%, rizatriptan 10 mg
(n = 34) 69.5%, and placebo (n = 115) 46.3%. The prespecified primary efficacy
hypothesis test, which compared the average 2-hour pain relief response
proportion of the combined 300, 400, and 600 mg MK-0974 groups to placebo, was
significant (P = 0.015). A generally similar efficacy pattern was seen for other
endpoints. MK-0974 was generally well tolerated and there did not appear to be
an increase in adverse events with increasing dose.
CONCLUSIONS: The novel, orally administered calcitonin gene-related peptide
(CGRP) receptor antagonist, MK-0974, was effective and generally well tolerated
for the acute treatment of migraine. Calcitonin gene-related peptide (CGRP) is a potent neuropeptide that plays a key
role in the pathophysiology of migraine headache. CGRP levels in the cranial
circulation are increased during a migraine attack, and CGRP itself has been
shown to trigger migraine-like headache. The correlation between CGRP release
and migraine headache points to the potential utility of CGRP receptor
antagonists as novel therapeutics in the treatment of migraine. Indeed, clinical
proof-of-concept in the acute treatment of migraine was demonstrated with an
intravenous formulation of the CGRP receptor antagonist BIBN4096BS (olcegepant).
Here we report on the pharmacological characterization of the first orally
bioavailable CGRP receptor antagonist in clinical development, MK-0974
[N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide].
In vitro, MK-0974 is a potent antagonist of the human (K(i) = 0.77 nM) and
rhesus (K(i) = 1.2 nM) CGRP receptors but displays >1500-fold lower affinity for
the canine and rat receptors as determined via (125)I-human CGRP competition
binding assays. A rhesus pharmacodynamic assay measuring capsaicin-induced
changes in forearm dermal blood flow via laser Doppler imaging was utilized to
determine the in vivo activity of CGRP receptor antagonism. MK-0974 produced a
concentration-dependent inhibition of dermal vasodilation, generated by
capsaicin-induced release of endogenous CGRP, with plasma concentrations of 127
and 994 nM required to block 50 and 90% of the blood flow increase,
respectively. In conclusion, MK-0974 is a highly potent, selective, and orally
bioavailable CGRP receptor antagonist, which may be valuable in the acute
treatment of migraine. MK-0974 (1a),
N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifuoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo-[4,5-B]
pyridine-1-yl)piperidine-1-carboxamide, is a novel calcitonin gene-related
peptide (CGRP) receptor antagonist with two chiral centers. Direct separation of
its four stereoisomers (1a-d) was achieved using a cellulose chiral stationary
phase, a Chiralcel OJ-RH column (150 mm x 4.6 mm), under reversed-phase
condition, following the extraction of 0.2 mL plasma on Oasis muElution HLB
96-well solid-phase-extraction (SPE) plate. The tandem mass spectrometric
detection was conducted in the positive-ion mode with a turbo-ion-spray (TIS)
interface using multiple-reaction-monitoring on a Sciex API3000. Addition of
ammonium trifluoroacetate to low-organic mobile phase improved detection
sensitivity by more than 30-fold. The simultaneous quantification of the four
stereoisomers in human plasma was validated over the ranges of 0.5-5000 nM for
1a and 0.5-500 nM for its three isomers (1b-d). Intraday validation, conducted
with five lots of human control plasma, resulted in <12.4% (% coefficient of
variation, CV) precision and 96.3-105.4% accuracy for all four stereoisomers.
Further evaluation indicated that the assay was specific, the samples were
stable after three freeze/thaw cycles, the recovery was reasonable (above 65%)
and no matrix effect was observed for all four isomers. Investigation on the
chiral integrity of 1a indicated that the diastereomer 1c, inversion at
azepinone-3 carbon, was the only isomer observed in the post-dose clinical
samples and accounted for 2.4-5.2% of MK-0974 exposure in the circulatory
system. The possibility of inversion during blood collection, plasma storage and
sample preparation was ruled out, while inversion was observed in the clinical
formulation accounting for approximately 0.12% of 1a in a 100-mg capsule. Calcitonin gene-related peptide (CGRP) is linked to migraine and other primary
headache disorders. It is found in every location described in migraine genesis
and processing, including meninges, trigeminal ganglion, trigeminocervical
complex, brainstem nuclei, and cortex. It is released in animal models following
stimulation of the CNS similar to that seen in migraine, and triptans inhibit
this release. Injection of CGRP into migraineurs results in delayed headache
similar to migraine. Elevation of CGRP occurs during migraine, resolving
following migraine-specific treatment. Finally, and most importantly, CGRP
receptor antagonists terminate migraine with efficacy similar to triptans. Both
intravenous olcegepant (BIBN 4096 BS) and oral telcagepant (MK-0974) have been
effective, safe, and well tolerated in phase I and II studies. Telcagepant is
currently in phase III trials, and preliminary results are favorable. The
potential for a migraine-specific medication without vasoconstrictive or
vascular side effects is enormous. CGRP receptor blockade may also have
applications in other pathologic and pain syndromes. BACKGROUND: Calcitonin gene-related peptide (CGRP) probably has a role in
migraine pathophysiology, and antagonism of its receptors might provide
treatment without the vasoconstrictor effects of triptans. We aimed to assess
the clinical profile of MK-0974 (telcagepant), an orally bioavailable antagonist
of CGRP receptor.
METHODS: In a randomised, parallel-treatment, placebo-controlled, double-blind,
trial at 81 sites in the Europe and the USA, adults with migraine diagnosed by
International Headache Society criteria treated moderate or severe attacks with
either oral telcagepant 150 mg or 300 mg, zolmitriptan 5 mg, or placebo. The
five co-primary endpoints were pain freedom, pain relief, or absence of
photophobia, phonophobia, or nausea at 2 h after treatment. Analysis was by the
full analysis set and multiplicity was controlled for with a step-down
closed-testing procedure. This trial is registered with ClinicalTrials.gov,
number NCT00442936.
FINDINGS: 1380 patients were randomly assigned to receive telcagepant 150 mg
(n=333) or 300 mg (354), zolmitriptan (345), or placebo (348). Telcagepant 300
mg was more effective than placebo for pain freedom (95 [27%] of 353 patients vs
33 [10%] of 343 [p<0.0001]), pain relief (194 [55%] of 353 vs 95 [28%] of 343
[p<0.0001]), and absences of phonophobia (204 [58%] of 353 vs 126 [37%] of 342
[p<0.0001]), photophobia (180 [51%] of 353 vs 99 [29%] of 342 [p<0.0001]), and
nausea (229 [65%] of 352 vs 189 [55%] of 342 [p=0.0061]). Efficacy of
telcagepant 300 mg and zolmitriptan 5 mg were much the same, and both were more
effective than telcagepant 150 mg. Adverse events were recorded for 31% taking
telcagepant 150 mg, 37% taking telcagepant 300 mg, 51% taking zolmitriptan 5 mg,
and 32% taking placebo.
INTERPRETATION: Telcagepant 300 mg is effective as an acute treatment for
migraine with efficacy comparable to that of zolmitriptan 5 mg, but with fewer
associated adverse effects.
FUNDING: Merck Research Laboratories. Calcitonin gene-related peptide (CGRP) is a neuropeptide that plays a key role
in the pathophysiology of migraine headache. MK-0974 (telcagepant) is a potent
and selective antagonist of the human and rhesus CGRP receptors and is currently
in Phase III clinical studies for the acute treatment of migraine. The
pharmacology of MK-0974 has been studied extensively, but there has not been a
thorough characterization of its binding properties. Here, we characterize the
binding of a tritiated analog of MK-0974 on human neuroblastoma (SK-N-MC)
membranes and rhesus cerebellum. [(3)H]MK-0974 displayed reversible and
saturable binding to both SK-N-MC membranes and rhesus cerebellum with a K(D) of
1.9 nM and 1.3 nM, respectively. Agonists and antagonists of the CGRP receptor
displaced [(3)H]MK-0974 in a concentration-dependent manner in competition
binding experiments. Both CGRP and adrenomedullin demonstrated biphasic
competition while MK-0974 and the peptide antagonist CGRP(8-37) displaced
[(3)H]MK-0974 in a monophasic fashion. In competitive binding studies with
[(3)H]MK-0974 and CGRP, the fraction of high-affinity binding was reduced
significantly by incubating the membranes with GTPgammaS. In kinetic binding
experiments, the off-rate of [(3)H]MK-0974 was determined to be 0.51 min(-1)
with a half-life of 1.3 min. In conclusion, the radioligand [(3)H]MK-0974 has
proven to be a useful tool for studying the binding characteristics of MK-0974
and has broadened our understanding of this promising molecule. Calcitonin gene related peptide (CGRP) has been proposed to contribute to pain
transmission and inflammation and for these reasons to the mechanism of
migraine. CGRP is, in fact, expressed in and released from a subset of polymodal
primary sensory neurons of the trigeminal ganglion. Release of CGRP in the
dorsal spinal cord has been associated to nociceptive transmission, and release
from perivascular nerve endings causes neurogenic vasodilatation. CGRP levels
increase in the cranial circulation during migraine attacks, and GRP injection
in migraineurs results in migraine-like attacks. Most importantly, two
chemically unrelated CGRP-receptor antagonists, the parenteral agent,
olcegepant, and the orally available telcagepant demonstrated efficacy in the
treatment of migraine attacks, thus supporting CGRP as an important mediator in
migraine. The underlying mechanism for low oral bioavailability of MK-0974, a potent
calcitonin-gene related peptide (CGRP)-receptor antagonist, in monkeys and for
species-dependent non-linear pharmacokinetics in monkeys and rats were
investigated. In monkeys, MK-0974 displayed moderate clearance (14-20 ml min(-1)
kg(-1)), while oral bioavailability was 6%. The pharmacokinetics of MK-0974
remained linear across 0.5-10 mg kg(-1) intravenous dose in monkeys, but the
oral area under the plasma concentration-time curve (AUC) increase (5-30 mg
kg(-1)) was 15-fold over dose-proportional. Based on a comparison of AUC
following hepatic portal vein administration and cephalic vein infusion, MK-0974
exhibited a low-to-moderate hepatic extraction ratio (36%) in monkeys. Following
oral dose of [14C]MK-0974 to monkeys, the hepatic portal AUC ratio of MK-0974
versus total radioactivity was 0.32, and the total radioactivity recovered in
bile and urine was 45-83%. MK-0974 undergoes significant oxidative metabolism
(cytochrome P450 (CYP) 3A) in monkey intestinal microsomes. In contrast, oral
AUC of MK-0974 in rats was near dose-proportional (15-100 mg kg(-1)). Following
oral administration of [14C]MK-0974 to rats, the hepatic portal AUC ratio of
MK-0974 to total radioactivity (0.67) was higher than in monkeys. Additionally,
the metabolic rate of MK-0974 was slower in rat than in monkey intestinal
microsomes. Collectively, intestinal first-pass metabolism played a significant
role in the low oral bioavailability in monkeys and contributed to the
species-dependent non-linear oral pharmacokinetics in rats and monkeys of
MK-0974. Telcagepant (MK-0974) is a novel oral calcitonin gene-related peptide (CGRP)
receptor antagonist and is currently under clinical development. Results from
phases II and III clinical trials have suggested that telcagepant is effective
for migraine treatment. A reliable and high throughput protein precipitation
(PPT) method for determination of telcagepant in human plasma using liquid
chromatography coupled with atmospheric pressure chemical ionization (APCI)
tandem mass spectrometry has been developed. Clinical samples, internal standard
(IS) and acetonitrile are transferred into 96-well plates using a robotic liquid
handling system. An aliquot of 10 microL supernatant is directly injected into
the LC-MS/MS system where separation is performed on a FluoPhase RP (150 x
2.1mm, 5 microm) column with an isocratic mobile phase (60% acetonitrile with
0.1% formic acid and 40% water with 0.1% formic acid) at 0.2 mL/min. The
interfering 3S-diastereomer of telcagepant, which is observed in clinical
samples, is chromatographically resolved from telcagepant. The PPT procedure
significantly reduces the time required for sample processing and the assay is
sufficiently sensitive for detection using both API 4000 and API 3000 mass
spectrometers. The linear calibration range is 5-5000 nM using 200 microL of
plasma. Assay intraday validation was conducted using six calibration curves
derived from six lots of human control plasma. Calibration standard accuracy did
not deviate by more than 3% and 6% of nominal values, and precision did not
exceed 4% coefficient of variation (CV) and 10% CV, respectively on the API 4000
and API 3000. Several clinical phases IIb and III studies have been successfully
supported with this assay. Migraine is a complex neurological disorder that affects a significant
proportion of the adult population worldwide. It is associated with an increase
in plasma calcitonin gene-related peptide (CGRP) level. CGRP, a neuropeptide
released from activated trigeminal sensory nerves, dilates intracranial blood
vessels and transmits vascular nociception. Therefore, it is proposed that CGRP
may have an important role in migraine pathophysiology and inhibition of
trigeminal CGRP release or CGRP-induced cranial vasodilatation may abort
migraine. The mode of action of CGRP antagonists is inhibition of neurogenic
vasodilatation and inflammation, an additional neuronal action cannot be ruled
out. Both intravenous applied olcegepant (BIBN 4096) and the oral telcagepant
(MK-0974) were tested in clinical trials and showed clinical benefit and
excellent tolerability, with recent presentation of phase III data on
telcagepant suggesting that proof of concept is positive. CGRP receptor
antagonists terminate migraine with efficacy similar to triptans. The results
from clinical trials suggest that this new type of drug might offer advantages
over existing therapies without vasoconstrictive or vascular side effects. Migraine is a complex, neurovascular disorder in which genetic and environmental
factors interact. At present, frontline therapies in the acute treatment of
migraine include the use of non-steroidal anti-inflammatory drugs and triptans.
Evidence indicates that calcitonin gene-related peptide (CGRP) plays a
fundamental role in the mechanism of migraine. CGRP is a strong vasodilatatory
neuropeptide that is released from activated trigeminal sensory nerves. The
development of CGRP antagonists has also been driven by the fact that triptans
are vasoconstrictive and cannot be safely used in patients with cardiovascular
risk factors. Olcegepant (BIBN4096) is the first CGRP antagonist for the
treatment of migraine that has been tested in clinical trials, but because of
its poor oral bioavailability, only the intravenous formulation has been tested.
The first oral non-peptide CGRP antagonist, telcagepant, has been shown recently
to be highly effective in the treatment of migraine attacks. This development
can be considered as the most important pharmacological breakthrough for
migraine treatment since the introduction of sumatriptan in the early 1990s.
These results are also of importance, since they support an interesting
pathophysiological hypothesis of migraine. The pipeline of future compounds for
the treatment of acute migraine headaches include TPRV1 antagonists,
prostaglandin E receptor 4 (EP(4)) receptor antagonists, serotonin 5HT1(F)
receptor agonists and nitric oxide synthase inhibitors. The immediate future of
a preventative treatment for migraine headaches is well represented by botulinum
toxin type-A, glutamate NMDA receptor antagonists, gap-junction blocker
tonabersat and an angiotensin type 1 blocker candesartan. Telcagepant is an oral calcitonin-gene related peptide (CGRP) receptor
antagonist that is being developed by Merck & Co Inc for the treatment of
migraine. This compound blocks CGRP receptors and may block the dilation of
dural vessels and reduce neurotransmission in the CNS, resulting in pain relief.
Telcagepant does not cause vasoconstriction, one of the major limitations in the
use of triptans, which are considered to be the standard of care for migraine.
Data from phase II and III clinical trials suggest that the use of telcagepant
for the acute treatment of migraine was comparable with the use of triptan
compounds and was superior to placebo in all primary endpoints, including pain
relief and freedom from pain at 2 h. However, recent data reported elevated
transaminase levels when telcagepant was dosed daily rather than acutely. It was
concluded that, if these hepatic toxicities are not observed in ongoing/future
trials of the acute use of telcagepant, then this agent may offer an alternative
to triptan therapy for the treatment of migraine. Several lines of evidence suggest a major role of calcitonin gene-related
peptide (CGRP) in the pathogenesis of migraine and other primary headaches.
Inhibition of CGRP receptors by olcegepant and telcagepant has been successfully
used to treat acute migraine and to reduce the activity of spinal trigeminal
neurons involved in meningeal nociception in rodents. The site of CGRP receptor
inhibition is unclear, however. In adult Wistar rats anaesthetized with
isofluorane systemic intravenous infusion (0.9 mg/kg) or unilateral facial
injection (1 mM in 100 microl) of capsaicin was used to induce activity in the
trigeminal nociceptive system. Animals were pre-treated either by saline or
olcegepant. In comparison with vehicle infusion or the non-injected side of the
face, capsaicin significantly increased the expression of the activation markers
Fos in the spinal trigeminal nucleus and phosphorylated extracellular
signal-regulated kinase in the trigeminal ganglion. Pre-treatment with
olcegepant (900 microg/kg) inhibited the capsaicin-induced expression of Fos
throughout the spinal trigeminal nucleus by 57%. In contrast, the expression of
phosphorylated extracellular signal-regulated kinase in the trigeminal ganglion
was not changed by olcegepant pre-treatment. CGRP receptor inhibition, which has
been shown to decrease spinal trigeminal activity, is likely to occur in the
central nervous system rather than in the periphery including the trigeminal
ganglion. This may be important for future therapeutic interventions with CGRP
receptor antagonists in migraine. After the triptans, a calcitonin gene-related peptide blocker (telcagepant) is
the first acute medicine that has been developed primarily for treatment of
acute migraine. Otherwise, the new drugs have been developed first for other
purposes, like anticonvulsants, antihypertensives and antidepressants used for
migraine prophylaxis. For acute attacks, a new way to administer a traditional
drug like dihydroergotamine is under way, and documentation of efficacy in
migraine has been gained for some commonly used painkillers and
anti-inflammatory drugs, and for some herbal extracts. Based on insights into
the basic pathophysiological mechanisms of the disorder, some drugs have been
developed which seem promising in early phase II studies (NOS inhibitors and
5HT1F-receptor agonists). In the future, development and enhancements of
existing medicines must be accompanied by increased efforts to develop truly new
migraine drugs based on knowledge of the pathophysiology if one wishes to reduce
substantially the great burden migraine poses on patients and society. Migraine is a highly prevalent neurovascular disorder that can be provoked by
infusion of calcitonin gene-related peptide (CGRP). CGRP, a neuropeptide
released from activated trigeminal sensory nerves, dilates intracranial and
extracranial blood vessels and centrally modulates vascular nociception. On this
basis, it has been proposed that: (i) CGRP may play an important role in the
pathophysiology of migraine; and (ii) blockade of CGRP receptors may abort
migraine. With the advent of potent and selective CGRP receptor antagonists, the
importance of CGRP in the pathophysiology of migraine and the therapeutic
principle of CGRP receptor antagonism were clearly established. Indeed, both
olcegepant (BIBN4096BS, given intravenously) and telcagepant (MK-0974, given
orally) have been shown to be safe, well tolerated and effective acute
antimigraine agents in phase I, phase II, and for telcagepant phase III,
studies. However, recent data reported elevated liver transaminases when
telcagepant was dosed twice daily for three months for the prevention of
migraine rather than acutely. The potential for a specific acute antimigraine
drug, without producing vasoconstriction or vascular side effects and with an
efficacy comparable to triptans, is enormous. The present review will discuss
the role of CGRP in the pathophysiology of migraine and the various treatment
modalities that are currently available to target this neuropeptide. BACKGROUND: Migraine is a debilitating headache disorder which affects
approximately 12% of the general population and is the cause of significant loss
of productivity (i.e., lost time from work or school) for those afflicted. The
current standard of care, the 5-HT(1B/1D) agonists known as triptans, is
contraindicated in patients with cardiovascular disease due to their inherent
vasoconstrictive activity; thus, there is a need to develop an alternative
therapy for the treatment of the disorder.
OBJECTIVE: This article reviews patent publications related to the use of small
molecule calcitonin gene-related peptide (CGRP) receptor antagonists for the
treatment of migraine that have appeared in the literature within the past
decade. The commentary is supplemented by information presented in journal
articles and focuses on the activity of several major pharmaceutical companies
in the field.
CONCLUSION: Two small molecule CGRP receptor antagonists, olcegepant and
telcagepant, have been shown to be clinically efficacious in the treatment of
migraine, and thus provide validation of this novel therapeutic mechanism. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: * Calcitonin gene-related peptide
(CGRP) was first described as a potent vasodilator. * CGRP is also increasingly
recognized as a key player in the pathophysiology of migraine, and CGRP receptor
antagonists potentially offer a new approach for treating migraine. * A novel
pharmacodynamic assay to measure CGRP receptor antagonist activity
non-invasively in humans has been developed, which involves measuring the
increase in dermal blood flow induced by topical application of capsaicin on the
forearm.
WHAT THIS STUDY ADDS: * This study shows that the novel oral CGRP receptor
antagonist, telcagepant, inhibits the increases in dermal blood flow induced by
the topical application of capsaicin on the human forearm. * This experimental
medicine model may have utility to assist in dose selection for the development
of CGRP receptor antagonists.
AIMS: To evaluate inhibition of capsaicin-induced increase in dermal blood flow
(DBF) following telcagepant (MK-0974), a potent and selective orally
bioavailable calcitonin gene-related peptide (CGRP) receptor antagonist being
developed for the acute treatment of migraine.
METHODS: A three-period crossover study in 12 healthy adult men. Each subject
received a single oral dose of telcagepant 300 mg, telcagepant 800 mg or placebo
at 0 h, followed 0.5 and 3.5 h later by two topical doses of 300 and 1000 microg
capsaicin per 20 microl water-ethanol mixture. Capsaicin was applied at two
sites on the volar surface of the subjects' left and right forearms. DBF was
assessed by laser Doppler perfusion imaging immediately before ('baseline'), and
0.5 h after each capsaicin application at 1 and 4 h. Plasma samples to determine
telcagepant concentrations were collected immediately after laser Doppler
perfusion imaging. A pharmacodynamic model was developed to explore the
relationship between plasma concentration and inhibition of capsaicin-induced
increase in DBF.
RESULTS: Geometric mean plasma concentrations after dosing with 300 mg and 800
mg telcagepant were 720 and 1146 nm, respectively, at 1 h, vs. 582 and 2548 nm,
respectively, at 4 h. The pharmacodynamic model suggested that the EC(90) for
telcagepant inhibition of capsaicin-induced increases in DBF was 909 nm.
CONCLUSIONS: Telcagepant inhibits the increases in DBF induced by the topical
application of capsaicin on the human forearm. This experimental medicine model
may have utility to assist in dose selection for the development of CGRP
receptor antagonists. The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of two
membrane proteins: calcitonin receptor-like receptor (CLR) and receptor
activity-modifying protein 1 (RAMP1). CLR is a class B G-protein-coupled
receptor (GPCR), possessing a characteristic large amino-terminal extracellular
domain (ECD) important for ligand recognition and binding. Dimerization of CLR
with RAMP1 provides specificity for CGRP versus related agonists. Here we report
the expression, purification, and refolding of a soluble form of the CGRP
receptor comprising a heterodimer of the CLR and RAMP1 ECDs. The extracellular
protein domains corresponding to residues 23-133 of CLR and residues 26-117 of
RAMP1 were shown to be sufficient for formation of a stable, monodisperse
complex. The binding affinity of the purified ECD complex for the CGRP peptide
was significantly lower than that of the native receptor (IC(50) of 12 microM
for the purified ECD complex vs 233 pM for membrane-bound CGRP receptor),
indicating that other regions of CLR and/or RAMP1 are important for peptide
agonist binding. However, high-affinity binding to known potent and specific
nonpeptide antagonists of the CGRP receptor, including olcegepant and
telcagepant (K(D) < 0.02 muM), as well as N-terminally truncated peptides and
peptide analogues (140 nM to 1.62 microM) was observed. Calcitonin gene-related peptide (CGRP) has a widespread distribution throughout
the trigeminovascular system and other brain areas involved in migraine
pathogenesis. Serum levels of CGRP are elevated during the migraine attack and
return to normal with alleviation of pain. Intravenous injection of CGRP in
migraineurs results in delayed headache similar to migraine. Since CGRP receptor
antagonists lack direct vasoconstrictor activity, this therapeutic approach may
offer advantages over the current mainstay of specific acute migraine treatment
with 5-HT1B/1D receptor agonists (triptans), contra-indicated in patients with
underlying cardiovascular disease. Intravenous BIBN4096BS (olcegepant) and oral
MK-0974 (telcagepant), two CGRP-receptor antagonists, were safe and effective in
the treatment of migraine attacks in Phase I and II trials. In a Phase III
clinical trial, the efficacy of telcagepant 300 mg was comparable to that of
zolmitriptan 5 mg. We intend to review the rationale for the use of
CGRP-receptor antagonists, and to outline current developments and future
perspectives. The calcitonin receptor-like receptor (CLR) associates with the accessory
protein RAMP1 to form a receptor for the neuropeptide calcitonin gene-related
peptide (CGRP). Multiple lines of evidence have implicated CGRP in the
pathophysiology of migraine headache making the CGRP receptor an attractive
target for development of small-molecule antagonists as a novel treatment for
this debilitating condition. The CGRP receptor antagonists telcagepant and
olcegepant (BIBN4096BS) have demonstrated clinical efficacy in the treatment of
migraine and there is now a need to better understand how these molecules
interact with the receptor. Previous work has shown the extracellular portion of
RAMP1 to be important for binding of these antagonists, with tryptophan-74 being
a key interaction site. The crystal structure of the extracellular portion of
human RAMP1 placed tryptophan-74 in a hydrophobic patch hypothesized to interact
with CGRP receptor ligands and also identified nearby residues that may be
important for ligand binding. In this study we explored the role played by these
residues of RAMP1 using an alanine replacement strategy. We confirmed a role for
tryptophan-74 in antagonist binding and also identified arginine-67 as being
important for binding of telcagepant but not compound 3, a close analog of
BIBN4096BS. We also identified tryptophan-84 as being critical for both
high-affinity binding of the non-peptide antagonists as well as the peptides
CGRP and CGRP(8-37). These data for the first time pinpoint a specific RAMP1
residue important for both antagonist and agonist potency and are consistent
with the N-terminal domain of RAMP1 forming the binding pocket interface with
CLR. INTRODUCTION: Calcitonin gene-related peptide (CGRP) is a neuronal messenger in
intracranial sensory nerves and is considered to play a significant role in
migraine pathophysiology.
MATERIALS AND METHODS: We investigated the effect of the CGRP receptor
antagonist, telcagepant, on CGRP-induced cranial vasodilatation in human
isolated cerebral and middle meningeal arteries. We also studied the expression
of the CGRP receptor components in cranial arteries with immunocytochemistry.
Concentration response curves to αCGRP were performed in human isolated cerebral
and middle meningeal arteries in the absence or presence of telcagepant.
Arterial slices were stained for RAMP1, CLR and actin in a double
immunofluorescence staining.
RESULTS: In both arteries, we found that: (i) telcagepant was devoid of any
contractile or relaxant effects per se; (ii) pretreatment with telcagepant
antagonised the αCGRP-induced relaxation in a competitive manner; and (iii)
immunohistochemistry revealed expression and co-localisation of CLR and RAMP1 in
the smooth muscle cells in the media layer of both arteries.
CONCLUSIONS: Our findings provide morphological and functional data on the
presence of CGRP receptors in cerebral and meningeal arteries, which illustrates
a possible site of action of telcagepant in the treatment of migraine. METHODS: This study evaluated the calcitonin gene-related peptide (CGRP)
receptor antagonist telcagepant (tablet formulation) for treatment of a migraine
attack and across four attacks. Adults with migraine were randomized,
double-blind, to telcagepant 140 mg, telcagepant 280 mg, or control treatment
sequences to treat four moderate-to-severe migraine attacks. Control patients
received placebo for three attacks and telcagepant 140 mg for one attack.
Efficacy for the first attack (Attack 1) and consistency of efficacy over
multiple attacks were assessed. For an individual patient, consistent efficacy
was defined as ≥ 3 successes, and lack of consistent efficacy was defined as ≥ 2
failures, in treatment response. A total of 1677 patients treated ≥ 1 attack and
1263 treated all four attacks.
RESULTS: Based on Attack 1 data, telcagepant 140 mg and 280 mg were
significantly (p < .001) more effective than placebo for 2-hour pain freedom,
2-hour pain relief, 2-hour absence of migraine-associated symptoms (phonophobia,
photophobia, nausea), and 2-24 hours sustained pain freedom. The percentage of
patients with 2-hour pain freedom consistency and 2-hour pain relief consistency
was significantly (p < .001) higher for both telcagepant treatment sequences
versus control. Adverse events within 48 hours for telcagepant with an incidence
≥ 2% and twice that of placebo were somnolence (placebo = 2.3%, 140 mg = 5.9%,
280 mg = 5.7%) and vomiting (placebo = 1.4%, 140 mg = 1.0%, 280 mg = 2.9%).
CONCLUSION: Telcagepant 140 mg and 280 mg were effective for treatment of a
migraine attack and were more consistently effective than control for
intermittent treatment of up to four migraine attacks. Telcagepant was generally
well tolerated. (Clinicaltrials.gov; NCT00483704). OBJECTIVE: To evaluate whether the same or different patients respond to
triptans and telcagepant.
BACKGROUND: Telcagepant is an oral calcitonin gene-related peptide receptor
antagonist with acute antimigraine efficacy comparable to oral triptans. It is
currently unknown whether migraine patients who cannot be adequately helped with
triptans might benefit from treatment with telcagepant.
METHODS: Post-hoc analysis of data from a randomized, controlled trial of
telcagepant (150 mg, 300 mg) zolmitriptan 5 mg, or placebo for a moderate/severe
migraine. Responder rates were analyzed according to patients' self-reported
historical triptan response (HTR): (1) good HTR (N = 660): response in 75-100%
of attacks; (2) intermediate HTR (N = 248): response in 25-74% of attacks; (3)
poor HTR/no use (N = 407): response in < 25% of attacks, or patient did not take
triptans. A limitation of the analysis is that the last subgroup comprised
mainly (91%) patients who reported that they did not take triptans, but it was
not known whether these patients were triptan-naïve or had previously used
triptans and stopped taking them.
RESULTS: For zolmitriptan, 2-hour pain relief rates were higher in the good HTR
subgroup (116/162, 72%) than in the intermediate (29/62, 47%) and poor/no use
(44/111, 40%) HTR subgroups. The 2-hour pain relief rates were similar across
HTR subgroups for telcagepant 150 mg (48-58%), 300 mg (52-58%), and placebo
(26-31%). In the poor/no use HTR subgroup, more patients receiving telcagepant
300 mg (56/98, 57.1%) had 2-hour pain relief than those receiving zolmitriptan
(44/111, 39.6%; odds ratio = 2.11 [95% CI: 1.20,3.71], P = .009); the percentage
for telcagepant 150 mg (57/119, 47.9%) was not significantly different from
zolmitriptan (odds ratio = 1.41 [95% CI: 0.82, 2.40], P = .211).
CONCLUSIONS: This suggests that different patients may respond to triptans or
telcagepant 300 mg. Caution should be exercised in interpreting the results
because of the post-hoc nature of the analysis (clinical trial registry:
NCT00442936). The conceptual shift of our understanding of migraine from a vascular disorder
to a brain disorder has dramatically altered the approach to the development of
new medicines in the field. Current pharmacologic treatments of acute migraine
consist of nonspecific and relatively specific agents. Migraine-specific drugs
comprise two classes, the ergot alkaloid derivatives and the triptans, serotonin
5-HT(1B/1D) receptor agonists. The ergots, consisting of ergotamine and
dihydroergotamine (DHE), are the oldest specific antimigraine drugs available
and are considered relatively safe and effective. Ergotamine has been used less
extensively because of its adverse effects; DHE is better tolerated. The triptan
era, beginning in the 1990s, was a period of considerable change, although these
medicines retained vasoconstrictor actions. New methods of delivering older
drugs include orally inhaled DHE and the transdermal formulation of sumatriptan,
both currently under study. Novel medicines being developed are targeted at
neural sites of action. Serotonin 5-HT(1F) receptor agonists have proven
effective in phase II studies and have no vascular actions. Calcitonin
gene-related peptide (CGRP) receptor antagonists are another promising
nonvasoconstrictor approach to treating acute migraine. Olcegepant (BIBN4096BS)
and telcagepant (MK-0974) have been shown to be safe and effective in phase I,
II, and (for telcagepant) phase III clinical trials. Other targets under
investigation include glutamate (AMPA/kainate), TRPV1, prostanoid EP4, and
nitric oxide synthase. With new neural targets and the potential for therapeutic
advances, the next era of antimigraine medications is near. BACKGROUND: The calcitonin gene-related peptide (CGRP) receptor antagonists
olcegepant and telcagepant are very potent drugs. Both are effective in migraine
but in doses much higher than would be predicted from receptor binding and other
in vitro results. This could perhaps suggest an effect of CGRP antagonists
behind the blood-brain barrier (BBB), i.e. in the central nervous system (CNS).
METHODS: Comparison of doses needed for CGRP blocking effect in vitro with dose
needed in vivo in man and monkeys. Discussion of these doses in relation to
doses needed for anti-migraine activity.
RESULTS: In vivo studies in monkeys and man showed that high doses compared to
doses needed in vitro are needed to block capsaicin-induced in skin blood flow,
a CGRP-mediated reaction. These doses are close to those needed for
anti-migraine activity.
CONCLUSION: The apparently high doses of CGRP receptor antagonists, olcegepant
and telcagepant needed for anti-migraine effect are not so high after all. They
do not allow a conclusion as to whether CGRP antagonists act on peripheral sites
or central sites in migraine. OBJECTIVE: The primary purpose of the study was to explore the safety and
tolerability of telcagepant in patients with stable coronary artery disease.
BACKGROUND: Triptans are effective acute anti-migraine drugs whose
vasoconstrictive effects limit their use in patients at risk for adverse
cardiovascular events. Telcagepant, a calcitonin gene-related peptide receptor
antagonist, is being developed for the acute treatment of migraine. Antagonism
of calcitonin gene-related peptide, which does not appear to cause
vasoconstriction, may allow for treatment of migraine in patients with coronary
artery disease.
METHODS: In this randomized, double-blind, placebo-controlled, crossover study,
patients with documented stable coronary artery disease were assigned to 1 of 2
treatment sequences: telcagepant then placebo, or placebo then telcagepant. In
each treatment period, patients received 2 doses of telcagepant 300-mg or
placebo 2 hours apart. They remained in the research center for 24 hours after
receiving the first dose of each period, during which time continuous 12-lead
ambulatory electrocardiographic (Holter) monitoring was performed.
RESULTS: Twenty-eight patients were enrolled; all patients completed the study
and were included in all analyses. Telcagepant was generally well tolerated. No
laboratory or serious adverse experiences were reported, and no patient
discontinued due to an adverse experience. There were no consistent
treatment-related changes in laboratory, vital signs or electrocardiogram safety
parameters. Three patients (2 after receiving placebo and 1 after receiving
telcagepant) experienced ST segment depression during the study; none of these
patients reported chest pain.
CONCLUSIONS: Two doses of 300-mg telcagepant, administered 2 hours apart, did
not appear to exacerbate spontaneous ischemia and were generally well tolerated
in a small cohort of patients with stable coronary artery disease. Results of
this study support further evaluation of telcagepant in patients with stable
coronary artery disease. The treatment of migraine was advanced dramatically with the introduction of
triptans in the early 1990s. Despite the substantial improvement in the quality
of life that triptans have brought to many migraineurs, a substantial cohort of
patients remain highly disabled by attacks and need new therapeutic approaches,
which ideally should be quick-acting, have no vasoconstrictor activity, and have
a longer duration of action and be better tolerated than current therapies. The
calcitonin gene-related peptide (CGRP) receptor antagonists (gepants)-olcegepant
(BIBN 4096 BS), telcagepant (MK-0974), MK3207, and BI 44370 TA-are effective in
treating acute migraine. They have no vasoconstrictive properties, fewer adverse
effects, and may act longer than triptans. Their development has been
complicated by liver toxicity issues when used as preventives. Results from
studies with BI 44370 TA do not support broad concern about a class effect, and
further studies are ongoing in this respect. Many experimental studies and
clinical trials suggest that nitric oxide may have a role in the pathophysiology
of migraine. Therefore, the inhibition of nitric oxide synthase (NOS) for the
acute or prophylactic treatment of migraine offered a feasible approach; as
inducible NOS (iNOS) is involved in several pain states, such as inflammatory
pain, it appeared to be an attractive target. However, despite high selectivity
and potency, the iNOS inhibitor GW274150 was not effective for acute treatment
or prophylaxis of migraine, suggesting that iNOS is very unlikely to be a
promising target. OBJECTIVE: To evaluate the efficacy of telcagepant in patients with migraine and
coronary artery disease.
BACKGROUND: Calcitonin gene-related peptide receptor antagonists, such as
telcagepant, may be useful for acute migraine treatment in patients with
cardiovascular disease, a population for whom triptans are contraindicated.
METHODS: Randomized, double-blind, two-period (6 weeks per period) crossover
study in patients with stable coronary artery disease and migraine. Patients
were randomized 1:1 to either: (1) Period 1: telcagepant (280-mg tablet/300-mg
capsule), Period 2: acetaminophen (1000-mg); or (2) Period 1: placebo for attack
1 then acetaminophen for subsequent attacks, Period 2: telcagepant. Patients
could treat up to 12 migraine attacks per period to assess the tolerability of
telcagepant. The primary efficacy analysis evaluated telcagepant vs placebo on
2-hour pain freedom during the first attack of Period 1.
RESULTS: One hundred and sixty-five of the planned 400 patients were enrolled,
and 114 took at least one dose of treatment. Telcagepant was not statistically
different from placebo for 2-hour pain freedom (25.0% vs 18.9%, odds ratio =
1.62 [95% confidence interval: 0.62, 4.25]). The median number of attacks
treated per period was 3. No cardiovascular thrombotic adverse events occurred
within 14 days of dosing.
CONCLUSION: The study was underpowered due to enrollment difficulties and did
not demonstrate a significant efficacy difference between telcagepant and
placebo for the treatment of a migraine attack in patients with stable coronary
artery disease. Telcagepant was generally well tolerated for acute intermittent
migraine treatment in these patients. Telcagepant (MK-0974) is a novel calcitonin gene-related peptide (CGRP) receptor
antagonist currently undergoing clinical trials for migraine
(http://www.merck.com/research/pipeline/home.html). MK-0974 is currently being
studied in phase III clinical trials. IMPORTANCE OF THE FIELD: Calcitonin gene-related peptide (CGRP) receptor
antagonists have recently come to attention with the development of olcegepant
and telcagepant for the treatment of migraine. The availability of
high-affinity, non-peptide antagonists opens the way for trials of these
compounds in other conditions where CGRP antagonism might be useful, such as
septic shock and inhibition of angiogenesis.
AREAS COVERED IN THIS REVIEW: This review summarises knowledge about the
structure and signalling properties of the CGRP receptor. The clinical
ramifications of targeting the CGRP receptor, the profiles of existing
antagonists and the requirements for screening new compounds will be discussed.
WHAT THE READER WILL GAIN: Readers will gain an overview of how current
non-peptide antagonists seem to bind similar epitopes contributed by both
calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein
1 (RAMP1), the main CGRP receptor subunits. We will discuss how current
antagonists have low bioavailability, limiting their use. For selectivity at
CGRP receptors, it will be necessary to target parts of the receptor influenced
by both RAMP1 and CLR.
TAKE HOME MESSAGE: For the design of radically new antagonists, more structural
information on the receptor is needed. Current screens are largely based on
measuring CGRP-mediated changes in cAMP. CGRP receptors can influence other
signalling pathways and pathway-selective allosteric antagonists may be useful,
but more information is needed about the mechanism of action of CGRP to assess
the value of this. OBJECTIVE: The objective of this article is to assess the effects of sumatriptan
monotherapy, telcagepant monotherapy, and their combination on blood pressure
(BP) in migraine patients during a headache-free period.
METHODS: A double-blind, placebo-controlled, four-period, single-dose,
randomized crossover study in 24 migraine patients was conducted. In each
period, patients received a single oral dose of sumatriptan 100 mg alone,
telcagepant 600 mg alone, sumatriptan 100 mg coadministered with telcagepant 600
mg, or placebo. Semi-recumbent BP was measured pre-dose and at seven post-dose
time points over a period of six hours. Individual time-weighted averages in
mean arterial pressure (MAP) were evaluated using a linear mixed-effects model.
The pharmacokinetics of sumatriptan alone and in the presence of telcagepant
were also evaluated using limited sampling times.
RESULTS: The mean difference in time-weighted (0-2.5 h) MAP (90% confidence
interval) was 1.2 mmHg (-0.2, 2.7) between telcagepant and placebo, 4.0 mmHg
(2.5, 5.5) between sumatriptan and placebo, and 1.5 mmHg (0.0, 3.0) between
telcagepant with sumatriptan vs sumatriptan alone. When coadministered with
telcagepant, the AUC0-6h and C(max) of sumatriptan were increased by 23% and
24%, respectively. The small MAP increases observed after coadministration could
possibly be associated with the slight elevations in sumatriptan levels.
CONCLUSION: Telcagepant does not elevate mean MAP, and coadministration of
telcagepant with sumatriptan results in elevations in MAP similar to those
observed following administration of sumatriptan alone in migraineurs during the
interictal period. When coadministered, telcagepant slightly increases the
plasma levels of sumatriptan, but without an apparent clinically meaningful
effect. OBJECTIVE: To evaluate whether the calcitonin gene-related peptide (CGRP)
receptor antagonist telcagepant might be effective for migraine prevention.
METHODS: In this randomized, double-blind, placebo-controlled, multicenter trial
(ClinicalTrials.gov NCT00797667), patients experiencing 3-14 migraine days
during a 4-week baseline were randomized to telcagepant 140 mg, telcagepant 280
mg, or placebo twice daily for 12 weeks. Efficacy was assessed by mean monthly
headache days and migraine/probable migraine days (headache plus ≥ 1 associated
symptom).
RESULTS: The trial was terminated following a recommendation from the Safety
Monitoring Board due to hepatotoxicity concerns. At termination, the planned 660
patients had been randomized, 656 had been treated with ≥ 1 dose of study
medication, and 14 had completed the trial. The mean treatment duration was
48-50 days. Thirteen patients, all in the telcagepant groups, had an alanine
aminotransferase (ALT) elevation ≥ 3 × the upper limit of normal and 7 of these
also had an aspartate aminotransferase elevation ≥ 3 × the upper limit of
normal. Two patients had very high symptomatic transaminase elevations that
occurred within 2-6 weeks of treatment initiation and resolved after treatment
discontinuation. The originally planned efficacy analysis over 12 weeks was not
performed due to limited data at later time points, but there was evidence that
telcagepant resulted in a larger reduction from baseline than placebo for mean
monthly headache days (month 1: 140 mg = -2.9, 280 mg = -3.1, placebo = -1.7; p
< 0.05) and migraine/probable migraine days (month 1: 140 mg = -2.7, 280 mg =
-3.0, placebo = -1.6; p < 0.05).
CONCLUSIONS: These data suggest a potential role for CGRP receptor antagonism in
migraine prophylaxis. However, the observed aminotransferase elevations do not
support the use of telcagepant for daily administration.
CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that in
patients with migraine, telcagepant taken daily reduces headache days by 1.4
days per month compared to placebo and causes 2.5% of patients to have
elevations of serum ALT levels. |
What is the role of photodynamic therapy for meningioma treatment? | Photodynamic therapy was shown to have activity againt meningioma treatment. Gefitinib and ciprofloxacin enhance efficacy of photodynamic therapy. | Despite being benign CNS tumours, meningiomas are not always curable and the
likelihood of recurrence depends upon the completeness of initial removal.
Adjuvant therapy for incompletely resected meningiomas is generally
unsatisfactory and such lesions continue to pose difficult management problems.
Photodynamic therapy (PDT) has been employed in the management of recurrent
cerebral gliomas but its activity against meningiomas has not been specifically
studied. An in vitro study of the effects of PDT against a variety of
meningiomas was therefore conducted. It was found that PDT using
haematoporphyrin derivative as a photosensitizing drug showed dose-dependent
activity against a variety of histological subtypes of meningioma. The activity
of PDT against meningiomas should be investigated further and may eventually
provide a useful form of adjuvant therapy for incompletely resected lesions. Photodynamic therapy is a promising treatment for human brain tumors because of
the selective retention of certain compounds by tumor cells. Certain lipophilic
cationic compounds, such as tetraphenylphosphonium (TPP), are selectively taken
up by a variety of carcinomas. Although preferential retention of TPP has been
demonstrated for the breast carcinoma cell line MCF-7, this compound had not
been tested previously on cells derived from nervous system tumors. In the
present study, tritiated-TPP (3H-TPP) uptake and retention for eight different
cell cultures of three histologically different types of nervous system tumors
was measured and the data were compared to a positive control (MCF-7) and
negative controls (normal African Green monkey kidney epithelium (CV-1) and the
normal human fibroblast (WI-38) cell lines). Uptake and retention
characteristics could be grouped by specific pathological tumor types, but
individual tumor variability was notable. Maligt astrocytoma (grade III/III
glioblastoma) and maligt neurofibrosarcoma cells showed preferential uptake
and retention of 3H-TPP relative to meningioma cells and normal controls. A
clonogenic assay utilizing the cytotoxic lipophilic cationic compound
dequalinium showed strong retainers of 3H-TPP to be more susceptible to the
effects of dequalinium than weak retainers. These data demonstrate that certain
human and experimental animal nervous system tumor cell lines retain lipophilic
compounds possessing a delocalized positive charge. Lipophilic cationic
compounds may be useful in the intraoperative delineation of tumor margins and
in the photodynamic therapy of certain nervous system tumors. 30 patients with primary or recurrent maligt brain tumors (9 primary, 18
recurrent maligt gliomas, 1 maligt meningioma, 2 melanomas) were treated
altogether 37 times by photodynamic therapy (PDT) whether after intravenous,
intraarterial or direct intratumoral sensitisation by hematoporphyrin (HPD)
after conventional surgical removal of the tumor mass. The light was produced by
an Argon pumped dye laser at doses varying from 40-220 J/cm2. A single dose of
radiation of 4 Gy was administered to 18 patients immediately after PDT. The 9
patients with primary glioblastomas received in addition a full course of
radiation therapy. The histological specimens taken during PDT demonstrated
tumor necrosis, with oedema of normal brain tissue adjacent to the tumorbed. The
median survival of patients with multiple recurrences and various radio- and
chemotherapeutic modalities was 6 months (range 4-13 months). 9 patients with
primary manifestation of a glioblastoma had a median survival of 19 months
(0.5-29 months). Increased phototoxicity of the skin was the only side effect of
PDT and did not reduce the quality of life of the patients. The photocytotoxicity characteristics of aluminium phthalocyanine chloride
(AIPc), aluminium phthalocyanine disulphonate (AlS2Pc), aluminium phthalocyanine
tetrasulphonate (AlS4PC) and haematoporphyrin derivative (HpD) were compared
using primary cultures of human meningioma cells. Cells were preincubated with
the photosensitising agent for 16 h, then illuminated for 15 min with broad band
red light (5 OW/cm2). The resultant cytotoxicity was assessed by tetrazolium
(MTT) reduction 24 h later. AlPc was found to be 400, 10,000 and 250 times more
potent that AlS2Pc, AlS4Pc and HpD, respectively, as an in vitro
photosensitizing agent for meningioma cells. The subcellular localisation of
AlPc, AlS2Pc, AlS4Pc and HpD in meningioma cells was determined by confocal
laser scanning microscopy. None of the agents localized to the nucleus. The
distribution of ALPc was quite diffuse through the cytoplasm. In contrast,
AlS2Pc and AlS4Pc were localized vesicles suggestive of lysosomes, and HpD in
membranous organelles distinct from mitochondria. AlPc and HpD were tested with
five different meningioma samples and provided a range of IC50 values from 0.009
to 0.022 OM and from 3.5 to 6.5 OM, respectively. When the MTT assay with AlPc
was performed 0, 24, 48 and 72 h after illumination, the mean IC50 values were
0.25, 0.037, 0.019 and 0.012 OM, respectively, indicating that the cytotoxic
effect continued to increase up to 72 h. Cells were incubated with AlPc and HpD
for different times up to 24 h before exposure to light. AlPc cytotoxicity was
half-maximal with an incubation time of 8 h, whereas HpD cytotoxicity was
half-maximal with an incubation time of 2 h, implying slower uptake kinetics for
AlPc than for HpD. These data indicate unique features of AlPc which suggests
its application as a potent, non-toxic photosensitizer in the photodynamic
therapy of human meningiomas. BACKGROUND AND OBJECTIVES: PDT has been used in the treatment of maligt brain
tumors. This communication updates our series of unselected maligt gliomas
treated with Photofrin-PDT.
STUDY DESIGN AND METHODS: We examined the records of 112 patients with maligt
gliomas, metastatic brain tumors and meningiomas treated with Photofrin-PDT at
St. Michael's Hospital, Toronto. These patients were treated prior to the onset
of ongoing randomized trials in Photofrin PDT or had pathology that excluded
them from such trials.
RESULTS: The overall post-PDT survival of 96 patients with supratentorial
gliomas was 42 weeks, with a 40 and 22% 1- and 2-year survival, respectively.
The greater the degree of oligodendroglial involvement the longer was the
survival. Seventy-five percent of patients had no significant post-operative
complications.
CONCLUSIONS: Photofrin-PDT was safe. However, higher light doses than were used
in these patients may be required for improved responses. BACKGROUND: Since the ATP-binding cassette transporter ABCG2 plays a
physiologically significant role of porphyrin efflux from living cells, the
expression of ABCG2 may influences the efficacy of photodynamic therapy (PDT).
In this study, we evaluated the effect of gefitinib, a potent ABCG2 inhibitor,
on 5-aminolevulinic acid (5-ALA)-PDT in brain tumor cell lines in vitro.
MATERIALS AND METHODS: Four human glioma cell lines (U87MG, U118MG, A172, and
T98G) and a maligt meningioma cell line (IOMM-Lee) were incubated with
gefitinib (0.01-1.0μM) before incubation with 5-ALA (1mM). The effects gefitinib
on intracellular protoporphyrin IX (PpIX), mRNA and protein expression of ABCG2,
and PDT were evaluated, in vitro.
RESULTS: At concentrations of 0.1μM or higher, gefitinib enhanced intracellular
levels of PpIX in a dose-dependent manner in all those cell lines. Gefitinib
decreased mRNA and plasma membrane protein expression of ABCG2, and efficiently
enhanced the effect of 5-ALA-PDT in maligt brain tumor cells.
CONCLUSION: Gefitinib can inhibit ABCG2-mediated PpIX efflux from maligt
brain tumor cells to increase the intracellular PpIX and thereby enhance the PDT
effect. |
Do carmustine wafers improve survival of glioblastoma patients? | Yes, it has been documented that implantation of carmustine wafers improves survival of newly diagnosed and recurrent glioblastoma patients. | OBJECTIVE: To determine the risks and survival benefit associated with
implantation of an absorbable, 1,3-bis(2chloroethyl)-1-nitrosourea-impregnated
polymer wafer, we prospectively studied patients with recurrent glioblastoma
multiforme and compared them with a demographically matched cohort group.
METHODS: Over a 29-month period, 62 patients underwent operations. All had tumor
growth despite standard treatment, a Karnofsky performance score of > or =70,
and histopathological confirmation of glioblastoma. Seventeen patients underwent
gross total resection with placement of 1,3-bis(2-chloroethyl)-1-nitrosourea
wafers (wafer group) at a median 44 weeks from diagnosis (6 women, 11 men;
median age, 56 years). A cohort group of 45 patients undergoing surgery for
recurrent glioblastoma during the same time period, but not receiving wafers,
was identified. Surgery was performed at a median 47 weeks from diagnosis (14
women, 31 men; median age, 54 years).
RESULTS: Within 6 weeks of surgery, 13 complications were identified in 8
patients in the wafer group. In the cohort group, 6 patients sustained 8
complications. We were unable to identify any survival advantage using
Kaplan-Meier analysis. In the wafer group, median survival was 58 weeks from
diagnosis and 14 weeks from wafer implantation. In the cohort group, median
survival was 97 weeks from diagnosis and 50 weeks from operation.
CONCLUSION: 1,3-bis(2-chloroethyl)-1-Nitrosourea wafer implantation for
recurrent glioblastoma was associated with a higher risk of postoperative
complications, particularly those related to infection and wound healing. No
clear survival benefit associated with wafer implantation was identified. Controlled release delivery of carmustine from biodegradable polymer wafers was
approved as an adjunct to surgical resection in the treatment of recurrent
glioblastoma multiforme after it was shown in clinical trials to be well
tolerated and effective. Given the localised nature of the drug in the brain
tissue, no direct pharmacokinetic measurements have been made in humans after
implantation of a carmustine wafer. However, drug distribution and clearance
have been extensively studied in both rodent and non-human primate brains at
various times after implantation. In addition, studies to characterise the
degradation of the polymer matrix, the release kinetics of carmustine and the
metabolic fate of the drug and polymer degradation products have been conducted
both in vitro and in vivo. GLIADEL wafers have been shown to release carmustine
in vivo over a period of approximately 5 days; when in continuous contact with
interstitial fluid, wafers should degrade completely over a period of 6 to 8
weeks. Metabolic elimination studies of the polymer degradation products have
demonstrated that sebacic acid monomers are excreted from the body in the form
of expired CO(2), whereas 1,3-bis-(p-carboxyphenoxy)propane monomers are
excreted primarily through the urine. Carmustine degradation products are also
excreted primarily through the urine. Pharmacokinetic studies in animals and
associated modelling have demonstrated the capability of this modality to
produce high dose-delivery (millimolar concentrations) within millimetres of the
polymer implant, with a limited penetration distance of carmustine from the site
of delivery. The limited spread of drug is presumably due to the high
transcapillary permeability of this lipophilic molecule. However, the presence
of significant convective flows due to postsurgical oedema may augment the
diffusive transport of drug in the hours immediately after wafer implantation,
leading to a larger short-term spread of drug. Additionally, in non-human
primates, the presence of significant doses in more distant regions of the brain
(centimetres away from the implant) has been shown to persist over the course of
a week. The drug in this region was presumed to be transported from the implant
site by either cerebral blood flow or cerebrospinal fluid flow, suggesting that
although drug is able to penetrate the blood-brain barrier at the site of
delivery, it may re-enter within the confines of the brain tissue. A previous placebo-controlled trial has shown that biodegradable 1,3-bis
(2-chloroethyl)-1-nitrosourea (BCNU) wafers (Gliadel wafers) prolong survival in
patients with recurrent glioblastoma multiforme. A previously completed phase 3
trial, also placebo controlled, in 32 patients with newly diagnosed maligt
glioma also demonstrated a survival benefit in those patients treated with BCNU
wafers. Because of the small number of patients in that trial, a larger phase 3
trial was performed to confirm these results. Two hundred forty patients were
randomized to receive either BCNU or placebo wafers at the time of primary
surgical resection; both groups were treated with external beam radiation
postoperatively. The two groups were similar for age, sex, Karnofsky performance
status (KPS), and tumor histology. Median survival in the intent-to-treat group
was 13.9 months for the BCNU wafer-treated group and 11.6 months for the
placebo-treated group (log-rank P -value stratified by country = 0.03), with a
29% reduction in the risk of death in the treatment group. When adjusted for
factors affecting survival, the treatment effect remained positive with a risk
reduction of 28% ( P = 0.03). Time to decline in KPS and in 10/11
neuroperformance measures was statistically significantly prolonged in the BCNU
wafer-treated group ( P </= 0.05). Adverse events were comparable for the 2
groups, except for CSF leak (5% in the BCNU wafer-treated group vs. 0.8% in the
placebo-treated group) and intracranial hypertension (9.1% in the BCNU
wafer-treated group vs. 1.7% in the placebo group). This study confirms that
local chemotherapy with BCNU wafers is well tolerated and offers a survival
benefit to patients with newly diagnosed maligt glioma. OBJECTIVE: Recently a randomized placebo-controlled phase III trial of
biodegradable polymers containing carmustine has demonstrated a significant
survival benefit for patients treated with local chemotherapy. A local
chemotherapy applied directly to the resection cavity may act directly on
residual tumor cells in adjacent brain possibly leading to a local control of
the tumor and increased survival.
METHODS: We have analyzed the pattern of recurrence using serial MRI studies of
24 patients treated with GLIADEL Wafers or placebo wafers following resection of
glioblastomas.
RESULTS: Of 24 patients 11 received carmustine wafers and 13 placebo. The age
distribution and Karnowsky performance scores of the two populations were not
different. However, the median survival (14.7 versus 9.5 months; P = 0.007) and
the time to neurological deterioration (12.9 +/- 4.85 vs. 9.4 +/- 2.73 months; P
= 0.035) was significantly longer in the treatment group versus the placebo
treated control. Preoperative and follow up MRI studies were evaluated in a
blinded fashion. Out of 24 patients that entered the analysis 11 showed
clearance of all contrast enhancement following resection of glioblastomas.
Seventeen tumors progressed locally and 7 showed different patterns of distant
failure. Within the carmustine treated group 8 patients showed a local treatment
failure with recurrent tumors immediately adjacent to the resection cavity or
progression form a residual tumor. Three patients showed a multifocal distant
and local pattern of failure after complete or subtotal removal. In no case the
local chemotherapy resulted in a distant recurrence only. However, the time to
radiographic progression was 165.1 +/- 80.75 days for the GLIADEL Wafer group
and 101.9 +/- 43.06 days for the placebo group (P = 0.023).
CONCLUSION: In this subgroup analysis of a phase III trial population both the
clinical progression and radiological progression were significantly delayed in
patients treated with local chemotherapy, resulting in an increased survival
time. Local chemotherapy with carmustine containing wafer implants did not
result in an altered pattern of recurrence and did not promote multifocal
patterns of recurrence. Two types of chemotherapy used in the treatment of patients with maligt
glioma are carboplatin and Gliadel wafer [(3.85%
1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU)]. To date there have been no
published data examining their concurrent use in this disease. The purpose of
this study was to evaluate combination chemotherapy with Gliadel wafer and
carboplatin in patients with high-grade, maligt glioma. In this prospective
phase I study, 16 patients underwent surgery, Gliadel wafer implantation (up to
8 wafers), intravenous carboplatin given postoperatively (day 3 or 4) at a dose
escalation range of area under the curve (AUC)=2-6, and external beam radiation.
Median age was 55 years (range 27-66 years). Fourteen (88%) patients had
glioblastoma multiforme and 2 (12%) had anaplastic astrocytoma. Performance
status was as follows: Eastern Cooperative Oncology Group (ECOG)=0 (2 patients),
ECOG=1 (13 patients), and ECOG=2 (2 patients). Three patients were treated at
each dosing level (AUC=2-6), and 4 patients were treated at an AUC=5.
Carboplatin was administered to all patients by postoperative day 4. Radiation
was begun on day 14-36. No grade 3 or 4 toxicities were noted in this study.
Median progression-free and overall survival was 266 and 679 days, respectively.
We conclude that administering systemic carboplatin is safe and well tolerated
in the postoperative period immediately following resection and implantation of
Gliadel wafer for the treatment of maligt glioma. Further evaluation in a
phase II setting, at maximal carboplatin dose to establish potential efficacy,
with this combination is warranted. Following the resection of newly diagnosed or recurrent glioblastomas, local
implantation of carmustine-impregnated biodegradable wafers (Gliadel) in the
resection cavity constitutes an adjuvant therapy that can improve the
possibilities of survival. However, some precautions should be taken regarding
Gliadel implantation. We report three cases in whom patients with glioblastoma
multiforme were implanted with fibrin glue-secured Gliadel after the lateral
ventricles had been opened, and who later developed severe hydrocephalus leading
to death. Although Gliadel may be an important adjunct to treatment, opening of
the ventricles during surgery as part of its application should be considered a
contra-indication. Carmustine wafers (Gliadel) and temozolomide (Temodal) were recently approved
for initial management of glioblastoma. Gliadel) is a polymer wafer containing
carmustine. These wafers are designed to be placed in the surgical cavity after
glioblastoma resection to deliver local chemotherapy. This treatment is intended
for tumors for which gross total resection is possible. Temozolomide is
administered concomitantly with radiotherapy for six weeks followed by six
cycles of adjuvant temozolomide (EORTC 26981, also known as "Stupp's protocol").
Temozolomide administered according to this protocol produced a median survival
benefit of 2 months in glioblastomas, and carmustine a similar benefit in
high-grade gliomas. The two-year survival rate was 26.5% with radiotherapy plus
temozolomide compared with 10.4% with radiotherapy alone. In patients with
complete resection, two-year survival reached 38%. These two new treatments are
essentially intended for patients younger than 70 years and with a Karnofsky
index>70. Ongoing studies are evaluating the possible value of combining these
two treatments. Glioblastoma multiforme (GBM) are among the most devastating neoplasms claiming
the lives of patients within a median of 1 year after diagnosis. Treatment of
GBM requires a multidisciplinary approach. Treatments include surgery,
radiotherapy, chemotherapy and so on. Temozolomide (TMZ) has emerged as an
active agent against maligt gliomas. On the basis of the work by the European
Organisation for Research and Treatment of Cancer/National Cancer Institute of
Canada, concurrent radiotherapy and the oral alkylating agent TMZ followed by
adjuvant TMZ has become the standard of care for patients with newly diagnosed
GBM, although the methylation status of the O(6)-mehylguanine-DNA
methyltransferase promoter is predictive for survival of GBM patients. Gliadel
is a biodegradable polymer wafer impregnated with carmustine. Gliadel has been
one of the few treatment modalities to demonstrate a statistical benefit in
patients with maligt glioma. These new FDA approved drugs advanced the
treatment of maligt glioma, but more progress is needed. Patients require
improvements in chemotherapy, surgery, radiotherapy, molecular targeted therapy,
immunotoxin using the convection-enhanced delivery and more. OBJECT: Effective treatment options are limited for patients with recurrent
glioblastoma multiforme (GBM), and survival is usually <1 year. Novel treatment
approaches are needed. Localized adjunct treatment with carmustine (BCNU) wafers
or permanent, low-activity 125I seed implants has been shown to be effective for
GBM. This study assessed the efficacy and safety of these therapies in
combination following tumor resection.
METHODS: Thirty-four patients with recurrent GBM were treated with maximal tumor
resection followed by implantation of BCNU wafers and permanent 125I seeds into
the tumor cavity. Patients were followed up with clinical evaluations and
magnetic resoce imaging studies once every 3 months. Survival and
progression-free survival (PFS) were evaluated.
RESULTS: During follow-up, local disease progression was observed in 27
patients, and 23 of them died. The median survival period was 69 weeks, and the
median PFS was 47 weeks. The 12-month survival and PFS rates were 66 and 32%,
respectively. Baseline factors associated with prolonged survival included
Karnofsky Performance Scale score>or=70, 125I seed activity>or=0.8 mCi/cm3 of
tumor cavity, and age<60 years. Brain necrosis developed in 8 patients (24%) and
was successfully treated with surgery or hyperbaric oxygen therapy.
CONCLUSIONS: The use of adjunct therapy combining BCNU wafers and permanent 125I
seeds resulted in survival that compares favorably with data from similar
studies performed in patients with recurrent GBM. The incidence of brain
necrosis appeared to be higher than that expected with either treatment alone,
although the necrosis was manageable and did not affect survival. This novel
approach warrants further investigation in recurrent and newly diagnosed GBM. High-grade glioma is a devastating disease that leaves the majority of its
victims dead within 2 years. To meaningfully increase survival, a trimodality
approach of surgery, radiation, and chemotherapy is needed. Carmustine (1,3-bis
(2-chloroethyl)-1-nitrosourea) is a nitrosourea alkylating agent that exerts its
antitumor effect by akylating DNA and RNA. Systemic administration of
nitrosoureas as a single agent or as part of
procarbazine/3-cyclohexyl-1-nitroso-urea/vincristine has demonstrated little
efficacy in the treatment of high-grade glioma. The development of carmustine
wafers (Gliadel((R)) Wafer) as a method for controlled released delivery of
carmustine from biodegradable polymer wafers enhances the therapeutic ratio by
fully containing the drug within the confines of the brain tumor environment
while minimizing systemic toxicities. Preclinical and clinical studies have
proven the safety and efficacy of Gliadel in the management of glioblastoma.
From these results, Gliadel is currently approved for use in patients with
recurrent glioblastoma as an adjunct to surgery and in newly diagnosed patients
with high-grade glioma as an adjunct to surgery and radiation. Other promising
advances in the use of locally delivered chemotherapy for CNS maligcies,
including Gliadel for brain metastases and combination therapies with systemic
or biologic agents, are discussed. BACKGROUND: Gliadel (polifeprosan 20 with carmustine [BCNU] implant) is commonly
used for local delivery of BCNU to high-grade gliomas after resection and is
associated with increased survival. Various complications of Gliadel wafers have
been reported but not consistently reproduced. We set out to characterize
Gliadel-associated morbidity in our 10-year experience with Gliadel wafers for
treatment of maligt glioma.
METHODS: We retrospectively reviewed records of 1013 patients undergoing
craniotomy for resection of maligt brain astrocytoma (World Health
Organization grade III/IV disease). Perioperative morbidity occurring within 3
months of surgery was assessed for patients and compared between patients
receiving versus not receiving Gliadel wafer. Overall survival was assessed for
all patients.
RESULTS: A total of 1013 craniotomies were performed for maligt brain
astrocytoma. A total of 288 (28%) received Gliadel wafer (250 glioblastoma
multiforme (GBM), 38 anaplastic astrocytoma/anaplastic oligodendroglioma
(AA/AO), 166 primary resection, 122 revision resection). Compared with the
non-Gliadel cohort, patients receiving Gliadel were older (55 +/- 14 vs. 50 +/-
17, P = .001) and more frequently underwent gross total resection (75% vs 36%, P
< .01) but otherwise similar. Patients in Gliadel versus non-Gliadel cohorts had
similar incidences of perioperative surgical site infection (2.8% vs. 1.8%, P =
.33), cerebrospinal fluid leak (2.8% vs. 1.8%, P = .33), meninigitis (.3% vs.
.3%, P = 1.00), incisional wound healing difficulty (.7% vs. .4%, P = .63),
symptomatic maligt edema (2.1% vs. 2.3%, P = 1.00), 3-month seizure incidence
(14.6% vs. 15.7%, P = .65), deep-vein thrombosis (6.3% vs. 5.2%, P = .53), and
pulmonary embolism (PE) (4.9% vs. 3.7%, P = .41). For patients receiving Gliadel
for GBM, median survival was 13.5 months after primary resection (20% alive at 2
years) and 11.3 months after revision resection (13% alive at 2 years). For
patients receiving Gliadel for AA/AO, median survival was 57 months after
primary resection (66% alive at 2 years) and 23.6 months after revision
resection (47% alive at 2 years).
CONCLUSION: In our experience, use of Gliadel wafer was not associated with an
increase in perioperative morbidity after surgical treatment of maligt
astrocytoma. OBJECT: Gliadel (BCNU) wafer and concomitant temozolomide (TMZ) therapy, when
used individually as adjuvant therapies, extend survival from that achieved by
resection and radiation therapy (XRT) for glioblastoma multiforme (GBM). It
remains unstudied whether combining Gliadel and TMZ therapy is safe or further
improves survival in patients with newly diagnosed GBM. The authors reviewed
their initial experience utilizing combined Gliadel, TMZ, and radiation therapy
for the treatment of GBM.
METHODS: All cases involving patients undergoing primary resection of GBM with
or without Gliadel wafer (3.85% BCNU) implantation and adjuvant XRT over a
10-year period (1997-2006) were retrospectively reviewed. Beginning in 2004,
concomitant TMZ became the standard of care at the authors' institution and all
patients with Gliadel implantation also received concomitant TMZ (Stupp
protocol). Overall survival and treatment-related morbidity were assessed for
all patients treated with Gliadel plus concomitant TMZ (XRT + Gliadel + TMZ).
Age-matched (<or= 70 years) comparison of survival and morbidity was performed
between the XRT + Gliadel + TMZ (post-2003) and XRT + Gliadel (pre-2004)
cohorts.
RESULTS: Thirty-three patients were treated with XRT + Gliadel + TMZ. The median
survival in this group was 20.7 months, with a 2-year survival rate of 36%.
Six-month morbidity included surgical site infection in 1 case (3%),
perioperative seizures in 2 cases (6%), deep-vein thrombus in 1 (3%), pulmonary
embolism in 3 (9%), and cerebral edema requiring admission for intravenous
dexamethasone in 1 case (3%). Myelosuppression required premature termination of
TMZ in 7 patients (21%) (thrombocytopenia in 5, neutropenia in 2 cases). In
patients <or= 70 years of age, XRT + Gliadel + TMZ (30 patients, post-2003) was
independently associated with improved median survival (21.3 vs 12.4 months, p =
0.005) versus XRT + Gliadel (78 patients, pre-2004), with 2-year survival of 39
versus 18%, respectively. In these patients, XRT + Gliadel + TMZ was not
associated with an increase in perioperative morbidity in comparison with XRT +
Gliadel.
CONCLUSIONS: In this experience, concomitant TMZ therapy in addition to Gliadel
wafer implantation was associated with a median survival of nearly 21 months
without increased perioperative morbidity. Temozolomide can be safely
administered to patients receiving Gliadel wafers after resection of GBM. Convection-enhanced delivery (CED) of cintredekin besudotox (CB) was compared
with Gliadel wafers (GW) in adult patients with glioblastoma multiforme (GBM) at
first recurrence. Patients were randomized 2:1 to receive CB or GW. CB (0.5
microg/mL; total flow rate 0.75 mL/h) was administered over 96 hours via 2-4
intraparenchymal catheters placed after tumor resection. GW (3.85%/7.7 mg
carmustine per wafer; maximum 8 wafers) were placed immediately after tumor
resection. The primary endpoint was overall survival from the time of
randomization. Prestated interim analyses were built into the study design.
Secondary and tertiary endpoints were safety and health-related quality-of-life
assessments. From March 2004 to December 2005, 296 patients were enrolled at 52
centers. Demographic and baseline characteristics were balanced between the 2
treatment arms. Median survival was 36.4 weeks (9.1 months) for CB and 35.3
weeks (8.8 months) for GW (P = .476). For the efficacy evaluable population, the
median survival was 45.3 weeks (11.3 months) for CB and 39.8 weeks (10 months)
for GW (P = .310). The adverse-events profile was similar in both arms, except
that pulmonary embolism was higher in the CB arm (8% vs 1%, P = .014). This is
the first randomized phase III evaluation of an agent administered via CED and
the first with an active comparator in GBM patients. There was no survival
difference between CB administered via CED and GW. Drug distribution was not
assessed and may be crucial for evaluating future CED-based therapeutics. PURPOSE: Retrospective study of patients treated for high-grade glioma, with or
without biodegradable carmustine wafers and according to the Stupp protocol.
METHODS AND MATERIALS: Between May 2007 and June 2008, 65 patients underwent
surgery for high-grade glioma, 28 had implantation of Gliadel and 37 patients
did not. Patients received radiotherapy with concomitant temozolomide followed
by 5 consecutive days of temozolomide every month for 6 months.
RESULTS: Overall median follow-up was 17.1 months; the median relapse-free
survival (RFS) was 14 months with a RFS of 54% at 12 months, and 38% at 24
months. For patient with and without Gliadel, median and 1-year RFS were 12.9
months and 52% vs. 14 months and 42%, respectively (p = 0.89). According to
pathology, Gliadel did not influence RFS of patients with Grade III or
glioblastoma. However, for all patients, in multivariate analysis,
non-methylated methylguanine methyltransferase (MGMT) was the only unfavorable
prognostic factor of RFS (p = 0.017; HR 2.8; CI [1.2-7]). Median overall
survival (OS) was 20.8 months; the OS rate at 12 months was 78.5%, and at 24
months 35.4%. For patients treated with and without Gliadel, median and 1-year
OS were 20.6 months and 78.6% vs. 20.8 months and 78.4%, respectively. According
to pathology, Gliadel did not influence OS of patients with Grade III or
glioblastoma. For all patients, in multivariate analysis, unfavorable
prognosticators for OS were non-methylated MGMT (p = 0.001; HR: 6.5; CI [2-20])
and irradiation dose <60 Gy (p = 0.02; HR: 6.3; CI [2-20]). With carmustine
wafers, before irradiation, median gross tumor volume plus edema was 84 mL
(27-229), whereas it was 68 mL (10-362) without carmustine (p = nonsignificant).
Four cases of Grade 3 thrombopenia occurred, all in the carmustine wafer group.
CONCLUSION: In patients with high-grade gliomas, adding Gliadel before
performing a Stupp protocol did not improve survival. Despite a high effort in the research of maligt brain tumors, the clinical
results in treatment of maligt brain tumors are still very poor. Brain tumors
are a major cause of morbidity and mortality in the population. New primary
brain tumors develop in 2-4 of 100,000 adults each year (1). Recent evidence
indicates that the prevalence of primary brain tumors is increasing, especially
in the elderly (2). The astroglial brain tumors, including the highly maligt
glioblastoma multiforme (GBM), are the most common primary brain tumors. For
these tumors, the first line of treatment is surgery and almost always
radiotherapy as an adjuvant. A variety of patient-management strategies are
currently used for GBM, from supportive care to aggressive multimodality
approaches. The principal reason for this wide spectrum of approaches is that,
despite aggressive therapy, which includes surgical removal of the tumor,
postoperative high-dose radiation (60 gy), chemotherapy, and other adjuvant
treatments, the prognosis of patients with GBM is very poor (3 -6). In a series
of NCOG protocols on glioblastoma multiforme patients with Karnofsky performance
scores of 60 or higher, who were treated with postsurgical radiation therapy and
adjuvant chemotherapy with nitrosourea-based drug combinations, the median
survival and time of tumor progression were consistently above 50 and 34 wk,
respectively (7 -9). The nitrosoureas (BCNU and CCNU), alone and in combination,
are the most active cytotoxic drugs for recurrent and progressive tumors,
although most of these responses are transient and in patients with
well-differentiated gliomas. When glioblastoma multiforme recurs, which happens
in nearly 100% of all cases, however, the median survival from the start of
treatment is about 6 mo, with only 22% of patients surviving longer than 1 yr
(11). Therefore, there is a great interest in local treatment modalities. A
wafer impregnated with carmustine, for use as an implant after surgical removal
of recurrent GBM showed a prolongation in the median survival time of only 2 mo,
from 20 to 28 wk in a study with a total of 222 patients. In another study, a
median survival of 9 mo was found in a selected group of patients with recurrent
GBM who underwent a second operation, but a reasonable quality of life in those
patients was limited to 10 wk (12). OBJECTIVE: The prognosis of high-grade glioma (HGG) is poor with a median
survival of about 1 year for glioblastoma. In 2007, NICE published a technology
appraisal (TA121) recommending the use of carmustine wafers (Gliadel) and
systemic therapy with temozolomide for selected patients with HGG. Outcomes for
HGG surgery in the United Kingdom with these combined treatments have not been
published.
DESIGN: Retrospective audit of consecutive patients in a single unit with
carmustine wafer implantation.
SUBJECTS: Fifty-nine patients had carmustine wafers implanted at primary
surgery, between October 2005 and October 2010 at Wessex Neurological Centre,
Southampton, UK.
METHODS: Patients were given chemotherapeutic treatments strictly according to
NICE TA121. Survival was calculated using Kaplan-Meier method.
RESULTS: Fifty-five patients had WHO grade IV tumours and four had grade III.
Median age was 61 years. At follow-up, 39 patients had died. Median survival was
15.3 months. Eight patients (13.5%) experienced post-operative complications
(including five infections) for which four had the carmustine wafers removed.
Forty-seven (80%) patients were treated with radical radiotherapy (55-60 Gy) and
six (10%) patients received palliative radiotherapy (30 Gy). Thirty-seven
patients (63%) received concomitant temozolomide chemotherapy. In the subset of
37 patients receiving multimodal treatment with radical radiotherapy and
concomitant temozolomide, median survival was 15.8 months compared with 7.4
months in those not receiving multimodal treatment.
DISCUSSION: Carmustine wafers for primary HGG surgery in accordance with the
NICE TA121 were associated with a median survival of 15.3 months; this is
improved compared with previously reported randomised trials. Multimodal
treatment with carmustine wafers, radical radiotherapy and concomitant
temozolomide was associated with improved survival. Increased incidence of
infections was observed in cases receiving carmustine wafers. BACKGROUND: Combining Gliadel wafers and radiochemotherapy with TMZ may carry
the risk of increased adverse events (AE). We analyzed the efficacy and safety
in patients with glioblastoma who underwent multimodal treatment with
implantation of Gliadel wafers.
METHODS: One hundred sixty-five consecutive patients with newly diagnosed (77
patients) or recurrent (88 patients) glioblastoma were studied. Forty-seven
patients underwent surgery + Gliadel. The impact of age (≥65 vs. <65), resection
extent (gross total vs. partial), use of Gliadel and adjuvant treatment (TMZ vs.
other schemes/no adjuvant therapy) on overall survival (OS, for patients with
newly diagnosed glioblastoma) and on recurrence-survival (for patients with
recurrent glioblastoma) was analyzed with Cox regression. The impact of age,
history (newly diagnosed vs. recurrent glioblastoma), number of Gliadel wafers
implanted (0 vs. <8 vs. 8), resection extent (gross-total vs. partial) and
adjuvant treatment (TMZ vs. other schemes/no adjuvant therapy) on the occurrence
of AE and on the occurrence of implantation site-related AE (ISAE) was analyzed
with the logistic regression model. Significance was set at p < 0.05.
RESULTS: Multivariate analysis showed the only factor associated with longer
survival, both for newly diagnosed and for recurrent GBM, was resection extent.
Both patients with a higher number of wafers implanted and patients with
recurrent tumors were significantly at risk for AE and ISAE. Patients with eight
Gliadel wafers implanted had a 3-fold increased risk of AE and a 5.6-fold
increased risk of ISAE, and patients with recurrent tumor had a 2.8-fold
increased risk of AE and a 9.3-fold increased risk of ISAE.
CONCLUSIONS: Adding Gliadel to standard treatment did not significantly improve
the outcome. The toxicity after Gliadel use was significantly higher, both for
patients with newly diagnosed and patients with recurrent glioblastoma. The Polifeprosan 20 with carmustine (BCNU, bis-chloroethylnitrosourea,
Gliadel(®)) polymer implant wafer is a biodegradable compound containing 3.85%
carmustine which slowly degrades to release carmustine and protects it from
exposure to water with resultant hydrolysis until the time of release. The
carmustine implant wafer was demonstrated to improve survival in blinded
placebo-controlled trials in selected patients with newly diagnosed or recurrent
maligt glioma, with little increased risk of adverse events. Based on these
trials and other supporting data, US and European regulatory authorities granted
approval for its use in recurrent and newly diagnosed maligt glioma, and it
remains the only approved local treatment. The preclinical and clinical data
suggest that it is optimally utilized primarily in the proportion of patients
who may have total or near total removal of gross tumor. The aim of this work
was to review the evidence for the use of carmustine implants in the management
of maligt astrocytoma (World Health Organization grades III and IV),
including newly diagnosed and recurrent disease, especially in the setting of a
standard of care that has changed since the randomized trials were completed.
Therapy has evolved such that patients now generally receive temozolomide
chemotherapy during and after radiotherapy treatment. For patients undergoing
repeat resection for maligt glioma, a randomized, blinded, placebo-controlled
trial demonstrated a median survival for 110 patients who received carmustine
polymers of 31 weeks compared with 23 weeks for 122 patients who only received
placebo polymers. The benefit achieved statistical significance only on analysis
adjusting for prognostic factors rather than for the randomized groups as a
whole (hazard ratio = 0.67, P = 0.006). A blinded, placebo-controlled trial has
also been performed for carmustine implant placement in newly diagnosed patients
prior to standard radiotherapy. Median survival was improved from 11.6 to 13.9
months (P = 0.03), with a 29% reduction in the risk of death. When patients with
glioblastoma multiforme alone were analyzed, the median survival improved from
11.4 to 13.5 months, but this improvement was not statistically significant.
When a Cox's proportional hazard model was utilized to account for other
potential prognostic factors, there was a significant 31% reduction in the risk
of death (P = 0.04) in this subgroup. Data from other small reports support
these results and confirm that the incidence of adverse events does not appear
to be increased meaningfully. Given the poor prognosis without possibility of
cure, these benefits from a treatment with a favorable safety profile were
considered meaningful. There is randomized evidence to support the use of
carmustine wafers placed during resection of recurrent disease. Therefore,
although there is limited specific evidence, this treatment is likely to be
efficacious in an environment when nearly all patients receive temozolomide as
part of initial management. Given that half of the patients in the randomized
trial assessing the value of carmustine implants in recurrent disease had
received prior chemotherapy, it is likely that this remains a valuable treatment
at the time of repeat resection, even after temozolomide. There are data from
multiple reports to support safety. Although there is randomized evidence to
support the use of this therapy in newly diagnosed patients who will receive
radiotherapy alone, it is now standard to administer both adjuvant temozolomide
and radiotherapy. There are survival outcome reports for small cohorts of
patients receiving temozolomide with radiotherapy, but this information is not
sufficient to support firm recommendations. Based on the rationale and evidence
of safety, this approach appears to be a reasonable option as more information
is acquired. Available data support the safety of using carmustine wafers in
this circumstance, although special attention to surgical guidelines for
implanting the wafers is warranted. OBJECT: Locoregional chemotherapy with carmustine wafers, positioned at surgery
and followed by radiation therapy, has been shown to prolong survival in
patients with newly diagnosed glioblastoma, as has concomitant radiochemotherapy
with temozolomide. A combination of carmustine wafers with the Stupp treatment
regimen has only been investigated in retrospective studies.
METHODS: In a single-institution prospective study, the authors assessed
12-month progression-free survival (PFS), toxicity, and overall survival in
patients with glioblastoma treated with surgery, carmustine wafers,
radiotherapy, and 6-month metronomic temozolomide chemotherapy. Thirty-five
patients with de novo glioblastoma, between the ages of 18 and 70 years, and
with Karnofsky Performance Scale scores of at least 70, were included in the
study. Patients were followed monthly and assessed using MRI every 2 months.
RESULTS: After a median follow-up of 15 months, the median time to tumor
progression was 12.5 months and median survival was 17.8 months. Due to toxicity
(mostly hematological), 7 patients had to prematurely stop temozolomide
treatment. Twenty-two patients developed Grade 3 CD4(+) lymphocytopenia. Three
patients developed oral-esophageal candidiasis, 2 developed pneumonia, and 1
developed a dorsolumbar zoster. Early intracranial hypertension was observed in
1 patient, and 1 was treated empirically for suspected brain abscess. One
patient died of Legionella pneumonia soon after repeat surgery.
CONCLUSIONS: Overall, this treatment schedule produced promising results in
terms of PFS without a marked increase in toxicities as compared with the Stupp
regimen. However, the gain in median survival using this schedule was less
clear. Only prospective comparative trials will determine whether these
preliminary results will translate into a long-term survival advantage with an
acceptable toxicity profile. Current treatment strategies in patients with newly-diagnosed glioblastoma
include surgical resection with post-operative radiotherapy and
concomitant/adjuvant temozolomide (the "Stupp protocol") or resection with
implantation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) wafers in the
surgical cavity followed by radiotherapy. In clinical practice, patients with
maligt glioma treated with BCNU wafer often also receive adjuvant
temozolomide. However, current treatment guidelines are unclear on whether and
how these treatment practices can be combined, and no prospective phase 3 study
has assessed the safety and efficacy of combining BCNU wafers with temozolomide
and radiation in high-grade maligt glioma. The rationale for multimodal
therapy comprising surgical resection with adjunct local BCNU wafers followed by
radiotherapy and temozolomide is based on complementary and synergistic
mechanisms of action between BCNU and temozolomide in preclinical studies; a
shared primary resistance pathway, methylguanine-DNA methyltransferase (MGMT);
and the opportunity to overcome resistance through MGMT depletion to boost
cytotoxic activity. A comprehensive review of the literature identified 19
retrospective and prospective studies investigating the use of this multimodal
strategy. Median overall survival in 14 studies of newly-diagnosed patients
suggested a modest improvement versus resection followed by Stupp protocol or
resection with BCNU wafers, with an acceptable and manageable safety profile. The prognostic role of O(6)-methylguanine-DNA methyltransferase (MGMT) promoter
methylation in glioblastoma patients treated with carmustine (BCNU) wafer
implantation is unclear. Here, we report on a retrospective study of 47 patients
with either newly diagnosed (30 patients) or recurrent (17 patients)
glioblastoma (WHO grade IV) treated with BCNU (bis-chloroethylnitrosourea)
wafers. Thirteen of the newly diagnosed patients received local BCNU and
irradiation only (first-line BCNU), while 17 patients additionally received
concomitant and adjuvant temozolomide (TMZ) radiochemotherapy (first-line BCNU +
TMZ). Of the 17 patients treated for recurrent glioblastoma (second-line BCNU),
16 had received radiotherapy with concomitant and adjuvant TMZ as an initial
treatment. Median overall survival (OS) did not significantly differ between 19
patients with MGMT promoter methylated tumors when compared to 28 patients with
unmethylated tumors (18.9 vs 15.0 months; p = 0.1054). In the first-line BCNU +
TMZ group, MGMT promoter methylation was associated with longer OS (21.0 vs 11.1
months, p = 0.0127), while no significant survival differences were detected in
the other two subgroups. Progression-free survival did not significantly differ
between patients with and without MGMT promoter methylated tumors in the entire
patient cohort or any of the three subgroups. The first-line BCNU + TMZ group
showed no significant difference in OS when compared to the first-line BCNU
group (18.9 vs 14.7 months), but tended to have more therapy-related adverse
effects (53% vs 24%, p = 0.105). In summary, MGMT promoter methylation showed a
non-significant trend toward longer survival in our patient cohort. The
combination of TMZ radiochemotherapy with local delivery of BCNU did not provide
a significant survival benefit compared to local BCNU alone, but was associated
with a higher rate of adverse effects. Owing to the small number of patients
investigated, however, these findings would need to be corroborated in larger
patient cohorts. Glioblastoma is the most common maligt primary brain tumor. Cures are rare
and median survival varies from several to 22 months. Standard treatment for
good performance patients consists of maximal safe surgical resection followed
by radiotherapy with concurrent temozolomide (TMZ) chemotherapy and six cycles
of postradiotherapy TMZ. At recurrence, treatment options include repeat surgery
(with or without Gliadel wafer placement), reirradiation or systemic therapy.
Most patients with good performance status are treated with cytotoxic
chemotherapy or targeted biologic therapy following or in lieu of repeat
surgery. Cytotoxic chemotherapy options include nitrosoureas, rechallenge with
TMZ, platins, phophoramides and topoisomerase inhibitors, although efficacy is
limited. Despite the intense effort of developing biologic agents that target
angiogenesis and growth and proliferative pathways, bevacizumab is the only
agent that has shown efficacy in clinical trials. It was awarded accelerated
approval in the USA after demonstrating an impressive radiographic response in
two open-label, prospective Phase II studies. Two randomized, Phase III trials
of upfront bevacizumab have completed and may demonstrate survival benefit;
however, results are pending at this time. Given the limited treatment options
at tumor recurrence, consideration for enrollment on a clinical trial is
encouraged. Grade IV glioma (glioblastoma) is one of the most common brain/central nervous
system cancers. In 2005, the standard of care for adjuvant treatment was
significantly changed with the approval of temozolomide. Carmustine wafers have
also gained some popularity. Phase III trials are currently evaluating
bevacizumab in conjunction with the standard temozolomide regimen. Despite these
recent advances in pharmacotherapy, roughly two-thirds of patients do not
survive longer than 2 years after diagnosis. Meanwhile, the costs of treatment
are substantial. The goal of this study is to review the clinical,
cost-of-illness, and cost-effectiveness literature relevant to treating
glioblastoma. Estimates of the economic burden of glioblastoma within different
healthcare systems were converted to 2013 US dollars. Temozolomide has
demonstrated a 2.5-month increase in overall survival and a 1.9-month increase
in progression-free survival, relative to radiotherapy alone. Carmustine wafers
have also been shown to increase overall survival by 2.3 months, compared with
placebo wafers. Cost-effectiveness studies of temozolomide have produced
incremental cost-effectiveness ratios, adjusted to 2013 US dollars, with a range
from US$73,586 per quality-adjusted life-year (QALY) (UK National Health Service
perspective) to US$105,234 per QALY (US societal perspective). More research is
needed to quantify the full societal burden of illness. |
Is Dicer part of the RISC loading complex? | Yes, Dicer is part of the RISC loading complex. | RNA interference (RNAi)-based sequence-specific gene silencing is applied to
identify gene function and also possesses great potential for inhibiting virus
replication both in animals and plants. Small interfering RNA (siRNA) molecules
are the inducers of gene silencing in the RNAi pathway but may also display
immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21
nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex
when introduced into the cells, while longer siRNA molecules are first processed
by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have
applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality
double-stranded RNA molecules that are specific for the UL29 gene of herpes
simplex virus (HSV). The 653 nt long double-stranded RNA molecules were
converted to siRNA and DsiRNA pools using Dicer enzymes originating from human
or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in
length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA
targeting the UL29 were used as references. The impact of these siRNAs on cell
viability, inflammatory responses, gene silencing, and anti-HSV activity were
assayed in cells derived from human nervous system and skin. Both pools and the
canonical single-site siRNAs displayed substantial antiviral activity resulting
in four orders of magnitude reduction in virus titer. Notably, the pool of
DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs,
whereas the immunostimulation effect was in relation to the length with the
single-site siRNAs. Our results also propose differences in the processivity of
the two Dicers. One of the most exciting recent developments in RNA biology has been the
discovery of small non-coding RNAs that affect gene expression through the RNA
interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are
small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme,
plays a central role in the RNAi pathway by cleaving precursors of both of these
classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the
RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite
structurally distinct; miRNA precursors are short, imperfect hairpins while
siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to
process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA
binding domain (dsRBD), but the data are sparse on the exact role this domain
plays in the mechanism of Dicer binding and cleavage. To further explore the
role of human Dicer-dsRBD in the RNAi pathway, we determined its binding
affinity to various RNAs modeling both miRNA and siRNA precursors. Our study
shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only
minimally influenced by a single-stranded - double-stranded junction caused by
large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD
contributes directly to substrate binding but not to the mechanism of
differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin
relaxation and MD simulations provide an overview of the role that dynamics
contribute to the binding mechanism. We compare this current study with our
previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile
of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs
in general. Despite progress in mechanistic understanding of the RNA interference (RNAi)
pathways, the subcellular sites of RNA silencing remain under debate. Here we
show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA)
into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and
Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough
endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found
in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations
co-sediment almost exclusively with the rER membranes, together with the RISC
loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein
activator of the interferon-induced protein kinase (PACT). Fractionation and
membrane co-immune precipitations further confirm that siRNA-loaded Ago2
physically associates with the cytosolic side of the rER membrane. Additionally,
RLC-associated double-stranded siRNA, diagnostic of RISC loading, and
RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally,
we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an
RNA-independent manner. Together, our findings demonstrate that the outer rER
membrane is a central nucleation site of siRNA-mediated RNA silencing. The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding
proteins, including protein kinase RNA activator (PACT), transactivation
response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into
mature microRNAs (miRNAs) that target specific mRNA species for regulation.
There is increasing evidence for important functional interactions between the
miRNA and nuclear receptor (NR) signaling networks, with recent data showing
that estrogen, acting through the estrogen receptor, can modulate initial
aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC
proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding
NR coregulators that target steroid-responsive promoters and regulate NR
activity and downstream gene expression. Furthermore, each of the RISC proteins,
together with Argonaute 2, associates with SRA and specific pre-microRNAs in
both the nucleus and cytoplasm, providing evidence for links between NR-mediated
transcription and some of the factors involved in miRNA processing. Dicer is an enzyme of the RNase III endoribonuclease family, which is crucial
for RNA interference (RNAi) in eukaryotes. Dicer is a component of the protein
machinery (the RNA Induced Silencing Complex [RISC]) which is involved in
catalyzing the formation of mature microRNAs from their precursors in the
process of microRNA biogenesis. RISC-associated microRNAs bind to specific
sequences in the 3' untranslated region of cognate mRNAs largely through
complementary base pairing, resulting in either translational inhibition and/or
the degradation of a specific mRNA pool. MicroRNAs epigenetically regulate the
cellular levels of receptors, transcription factors and signaling proteins that
govern the developmental pathways and functions of multiple cellular processes.
The pivotal role played by Dicer in microRNA formation has also piqued the
interest of molecular immunologists who have sought to understand the biological
relevance of microRNAs in the development and function of the immune system.
Here, we review the major findings of these studies and provide an overview of
the role of Dicer and microRNAs in immune cell development and function.
Additionally, we highlight deficiencies in our knowledge and new research areas
that may enhance our understanding of the role of Dicer and microRNAs in
immunity. |
What is the Arnold-Chiari syndrome? | Arnold Chiari syndrome is a condition characterized by herniation of the cerebellar tonsils through the foramen magnum, manifesting usually with downbeat nystagmus, palsy of the caudal cerebral nerves, headache, and vertigo. | BACKGROUND: Arnold-Chiari Syndrome I is a malformation of the cervicomedullary
junction, manifesting usually with downbeat nystagmus, palsy of the caudal
cerebral nerves, headache, and vertigo.
PATIENTS AND METHODS: We present three patients with unusual symptomatology.
RESULTS: A two-year-old child with isolated non-ocular torticollis, a
52-year-old male patient, and a 42-year-old female patient, both with
gaze-evoked nystagmus, underwent a cerebral MRI examination. The findings of the
first two patients were typical for an Arnold-Chiari syndrome. The malformation
in the third patient was found only by reviewing the initial MRI.
CONCLUSIONS: Arnold-Chiari malformation may manifest atypically. An important
step in the work-up of these patients is to ask the neuroradiologist to include
the cervicomedullary junction in his evaluation. Many hypotheses concerning pathogenesis of syringomyelia were abandoned because
of evidence found in more recent investigations. We should rank among them the
"classical" theories of Gardner and Williams based on the assumption that
syringomyelic cavities result from directing the fluid from the fourth ventricle
to the central canal of the spine in the case of disturbances of circulation of
the cerebrospinal fluid in the region of the cranio-spinal junction. The theory
of intraspinal pulsation pressure of Greitz may explain the pathogenesis of
syringomyelia in the case of obstacles to fluid flow from the cranial cavity to
the spinal canal as in patients with Arnold-Chiari syndrome. The origin of
Arnold-Chiari syndrome is connected with narrowness in the posterior fossa,
particularly with narrowing of the arachnoid spaces. Improvement of clinical
condition after surgical restoration of the fluid spaces within the posterior
cranial cavity and improvement of cerebrospinal fluid flow in the region of the
cranio-cervical junction are factors supporting this opinion. The Arnold-Chiari malformation is very rare hindbrain abnormalities
characterized by herniation of the hindbrain through the foramen magnum. It
usually does not present until adulthood, and then its symptoms may be varied
and subtle. Patient 49 years was diagnosed in Foniatrics and Audiology Clinic
because of tinnitus in left ear lasting 3 months. She underwent audiological
diagnostic, that did not reveal any abnormalities, except for electrogustometry
and hyporeflexia of stapedial reflex and labyrinth on the left side. In MRI scan
we have noticed hindbrain abnormalities characteristic for Chiari type I
malformation. Treatment consisted of immediate supportive care as needed but
without surgical decompression, which was found unnecessary. HTLV-I is associated with a broad spectrum of manifestations, including tropical
spastic paraparesis and adult T-cell leukemia/lymphoma. Arnold Chiari syndrome
is a condition characterized by herniation of the cerebellar tonsils through the
foramen magnum. This condition should be suspected in all patients with headache
and impaired motor coordination. Syringomyelia is a developmental anomaly that
leads to the formation of an intramedullary cavity. Its clinical presentation is
classically characterized by syringomyelic dissociation of sensation, with
suspended distribution in the proximal portion of the trunk and upper limbs and
preservation in other regions. We report here a case of association of the three
diseases, which is rare in clinical practice, illustrating the difficulty in the
diagnosis and therapeutic management of these conditions. |
What is the biological role of SERCA2 SUMOylation in cardiac physiology and pathophysiology, such as in heart failure? | Recently, the impact of small ubiquitin-related modifier 1 (SUMO-1) on the regulation and preservation of sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA2a) function was discovered. The small ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target proteins, and is involved in many cellular processes. SERCA2a is SUMOylated at lysines 480 and 585 and this SUMOylation is essential for preserving SERCA2a ATPase activity and stability in mouse and human cells. The levels of SUMO1 and the SUMOylation of SERCA2a itself were greatly reduced in failing hearts. SUMO-1 gene transfer improved cardiac function supporting the critical role of SUMO-1 in SERCA2a function and underlining the therapeutic potential of SUMO-1 for HF patients. | The calcium-transporting ATPase ATP2A2, also known as SERCA2a, is a critical
ATPase responsible for Ca(2+) re-uptake during excitation-contraction coupling.
Impaired Ca(2+) uptake resulting from decreased expression and reduced activity
of SERCA2a is a hallmark of heart failure. Accordingly, restoration of SERCA2a
expression by gene transfer has proved to be effective in improving cardiac
function in heart-failure patients, as well as in animal models. The small
ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target
proteins, and is involved in many cellular processes. Here we show that SERCA2a
is SUMOylated at lysines 480 and 585 and that this SUMOylation is essential for
preserving SERCA2a ATPase activity and stability in mouse and human cells. The
levels of SUMO1 and the SUMOylation of SERCA2a itself were greatly reduced in
failing hearts. SUMO1 restitution by adeno-associated-virus-mediated gene
delivery maintained the protein abundance of SERCA2a and markedly improved
cardiac function in mice with heart failure. This effect was comparable to
SERCA2A gene delivery. Moreover, SUMO1 overexpression in isolated cardiomyocytes
augmented contractility and accelerated Ca(2+) decay. Transgene-mediated SUMO1
overexpression rescued cardiac dysfunction induced by pressure overload
concomitantly with increased SERCA2a function. By contrast, downregulation of
SUMO1 using small hairpin RNA (shRNA) accelerated pressure-overload-induced
deterioration of cardiac function and was accompanied by decreased SERCA2a
function. However, knockdown of SERCA2a resulted in severe contractile
dysfunction both in vitro and in vivo, which was not rescued by overexpression
of SUMO1. Taken together, our data show that SUMOylation is a critical
post-translational modification that regulates SERCA2a function, and provide a
platform for the design of novel therapeutic strategies for heart failure. Heart disease remains the leading cause of death and disability in the Western
world. Current therapies aim at treating the symptoms rather than the
subcellular mechanisms, underlying the etiology and pathological remodeling in
heart failure. A universal characteristic, contributing to the decreased
contractile performance in human and experimental failing hearts, is impaired
calcium sequestration into the sarcoplasmic reticulum (SR). SR calcium uptake is
mediated by a Ca(2+)-ATPase (SERCA2), whose activity is reversibly regulated by
phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA and
phosphorylation of PLN relieves this inhibition. However, the initial simple
view of a PLN/SERCA regulatory complex has been modified by our recent
identification of SUMO, S100 and the histidine-rich Ca-binding protein as
regulators of SERCA activity. In addition, PLN activity is regulated by 2
phosphoproteins, the inhibitor-1 of protein phosphatase 1 and the small heat
shock protein 20, which affect the overall SERCA-mediated Ca-transport. This
review will highlight the regulatory mechanisms of cardiac contractility by the
multimeric SERCA/PLN-ensemble and the potential for new therapeutic avenues
targeting this complex by using small molecules and gene transfer methods. |
Which trinucleotide repeat disorders are affecting the nervous system? | At least six neudegenerative disorders result from trinucleotide repeat expansion: X-linked spinal and bulbar muscular atrophy (SBMA), two fragile X syndromes of mental retardation (FRAXA and FRAXE), Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), and dentatorubral-pallidoluysian atrophy (DRPLA). | Expansion of trinucleotide repeats is now recognized as a major cause of
neurological disease. At least seven disorders result from trinucleotide repeat
expansion: X-linked spinal and bulbar muscular atrophy (SBMA), two fragile X
syndromes of mental retardation (FRAXA and FRAXE), myotonic dystrophy,
Huntington's disease, spinocerebellar ataxia type 1 (SCA1), and
dentatorubral-pallidoluysian atrophy (DRPLA). The expanded trinucleotide repeats
are unstable, and the phenomenon of anticipation, i.e., worsening of disease
phenotype over successive generations, correlates with increasing expansion
size. In this review, we compare the clinical and molecular features of the
trinucleotide repeat diseases, which may be classified into two types. Fragile X
and myotonic dystrophy are multisystem disorders usually associated with large
expansions of untranslated repeats, while the four neurodegenerative disorders,
SBMA, Huntington's disease, SCA1, and DRPLA, are caused by smaller expansions of
CAG repeats within the protein coding portion of the gene. CAG repeats encode
polyglutamine tracts. Polyglutamine tract expansion thus appears to be a common
mechanism of inherited neurodegenerative disease. Although polyglutamine tract
lengthening presumably has a toxic gain of function effect in the CAG
trinucleotide repeat disorders, the basis of this neuronal toxicity remains
unknown. |
Is hypersensitivity to DNA crosslinking agents a hallmark of Fanconi anemia? | Yes, hypersensitivity to DNA crosslinking agents is one of the hallmarks of Fanconi anemia, the others being defective FANCD2 monoubiquitination, and genomic instability. | Features of chromosomal aberrations, hypersensitivity to DNA crosslinking
agents, and predisposition to maligcy have suggested a fundamental anomaly of
DNA repair in Fanconi anemia. The function of the recently isolated FACC
(Fanconi anemia group C complementing) gene for a subset of this disorder is not
yet known. The notion that FACC plays a direct role in DNA repair would predict
that the polypeptide should reside in the nucleus. In this study, a polyclonal
antiserum raised against FACC was used to determine the subcellular location of
the polypeptide. Immunofluorescence and subcellular fractionation studies of
human cell lines as well as COS-7 cells transiently expressing human FACC showed
that the protein was localized primarily to the cytoplasm under steady-state
conditions, transit through the cell cycle, and exposure to crosslinking or
cytotoxic agents. However, placement of a nuclear localization signal from the
simian virus 40 large tumor antigen at the amino terminus of FACC directed the
hybrid protein to the nuclei of transfected COS-7 cells. These observations
suggest an indirect role for FACC in regulating DNA repair in this group of
Fanconi anemia. Fanconi anemia (FA) is one of several genetic diseases with characteristic
cellular hypersensitivity to DNA crosslinking agents which suggest that FA
proteins may function as part of DNA repair processes. At the clinical level, FA
is characterized by bone marrow failure that affects children at an early age.
The clinical phenotype is heterogeneous and includes various congenital
malformations as well as cancer predisposition. FA patients are distributed into
eight complementation groups suggesting a complex molecular pathway. Three of
the eight possible FA genes have been cloned, although their function(s) have
not been identified. FA cells are highly sensitive to DNA crosslinking agents
(mitomycin C (MMC) and diepoxybutane), with some variability between cell lines.
Sensitivity to monofunctional alkylating agents has been reported in some cases,
although these studies were performed with genetically unclassified FA cells. To
further analyse and characterize the newly identified FA complementation groups,
we tested their sensitivity to UV radiation, monofunctional and bifunctional
alkylating agents and to the X-ray mimetic drug bleomycin. We found that FA
complementation groups D to H show increased sensitivity to the X-ray mimetic
drug bleomycin. Furthermore, the single known FA-H cell line shows increased
sensitivity to ethylethane sulfonate (EMS), methylmethane sulfonate (MMS) in
addition to the characteristic sensitivity to crosslinking agents, suggesting a
broader spectrum of drug sensitivities in FA cells. Fanconi anemia (FA) is a human autosomal disorder characterized by cancer
susceptibility and cellular sensitivity to DNA crosslinking agents such as
mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene
designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a
mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and
subsequently found to be identical to FANCG. A nuclear complex containing the
FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a
sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes
with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci.
In this study, we have assigned NM3, a nitrogen mustard-hypersensitive Chinese
hamster mutant to the same genetic complementation group as UV40. NM3, like
human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of
sister chromatid exchange. We show that both NM3 and UV40 are also
hypersensitive to other DNA crosslinking agents (including diepoxybutane and
chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin,
streptonigrin and EMS), and that all these sensitivities are all corrected upon
transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40
were found not to express the active monoubiquitinated isoform of the FANCD2
protein, although expression of the FANCD-L isoform was restored in the FANCG
cDNA transformants, correlating with the correction of mutagen-sensitivity.
These data indicate that cellular resistance to these DNA damaging agents
requires FANCG and that the FA gene pathway, via its activation of FANCD2 and
that protein's subsequent interaction with BRCA1, is involved in maintaining
genomic stability in response not only to DNA interstrand crosslinks but also a
range of other DNA damages including DNA strand breaks. NM3 and other "FA-like"
Chinese hamster mutants should provide an important resource for the study of
these processes in mammalian cells. Biallelic BRCA2-mutations can cause Fanconi anemia and are found in
approximately 7% of pancreatic cancers. Recently, several sequence changes in
FANCC and FANCG were reported in pancreatic cancer. Functional defects in the
Fanconi pathway can result in a marked hypersensitivity to interstrand
crosslinking agents, such as mitomycin C. The functional implications of
mutations in the Fanconi pathway in cancer have not been fully studied yet;
these studies are needed to pave the way for clinical trials of treatment with
crosslinking agents of Fanconi-defective cancers. The competence of the proximal
Fanconi pathway was screened in 21 pancreatic cancer cell lines by an assay of
Fancd2 monoubiquitination using a Fancd2 immunoblot. The pancreatic cancer cell
lines Hs766T and PL11 were defective in Fancd2 monoubiquitination. In PL11, this
defect led to the identification of a large homozygous deletion in FANCC, the
first cancer cell line found to be FANCC-null. The Fanconi-defective cell lines
Hs766T, PL11, and CAPAN1 were hypersensitive to the crosslinking agent mitomycin
C and some to cis-platin, as measured by cell survival assays and G(2)/M
cell-cycle arrest. These results support the practical exploration of
crosslinking agents for non-Fanconi anemia patients that have tumors defective
in the Fanconi pathway. Fanconi anemia (FA) is a rare genetic disorder characterized by aplastic anemia,
cancer/leukemia susceptibility and cellular hypersensitivity to DNA crosslinking
agents, such as cisplatin. To date, 12 FA gene products have been identified,
which cooperate in a common DNA damage-activated signaling pathway regulating
DNA repair (the FA pathway). Eight FA proteins form a nuclear complex harboring
E3 ubiquitin ligase activity (the FA core complex) that, in response to DNA
damage, mediates the monoubiquitylation of the FA protein FANCD2.
Monoubiquitylated FANCD2 colocalizes in nuclear foci with proteins involved in
DNA repair, including BRCA1, FANCD1/BRCA2, FANCN/PALB2 and RAD51. All these
factors are required for cellular resistance to DNA crosslinking agents. The
inactivation of the FA pathway has also been observed in a wide variety of human
cancers and is implicated in the sensitivity of cancer cells to DNA crosslinking
agents. Drugs that inhibit the FA pathway may be useful chemosensitizers in the
treatment of cancer. Publication history: Republished from Current BioData's
Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com). Fanconi anemia (FA) is an inherited chromosomal recessive syndrome characterized
by cellular hypersensitivity to DNA crosslinking agents and bone marrow failure,
which cause aplastic anemia, and an increased incidence of maligcy. 13
complementation groups are currently discovered, and 13 distinct genes have been
cloned (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FNACI, FANCJ,
FANCL, FANCM, FANCN). Stem cells can theoretically divide to other cells without
limit as long as a person is still alive. The stem cells that form blood and
immune cells are known as hematopoietic stem cells. Hematopoietic stem cells can
be acquired from a Fanconi anemia patient, whereas genomic DNA can be obtained
easily from blood cells of a normal person. Normal genes also can be synthesised
by PCR method. Normal genomic DNA will be delivered into a patient's stem cells
via microinjection or transfection after enzyme digestion; the defective genes
might be repaired by homologous genetic recombination. The gene-corrected stem
cells can be transplanted into the same patient finally. It is possible that
human genomic DNA to be considered as materials for homologous genetic
recombination to repair defective genes in vivo. This might be an efficient
method for gene therapy, which has no or less immunological rejection for
Fanconi anemia and some genetic diseases. Several related observations and
experiments are discussed to support this possible means of stem cell gene
therapy of Fanconi anemia. The Bloom protein (BLM) and Topoisomerase IIIalpha are found in association with
proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased
cellular sensitivity to DNA crosslinking agents. In order to determine if the
association reflects a functional interaction for the maintece of genome
stability, we have analyzed the effects of siRNA-mediated depletion of the
proteins in human cells. Depletion of Topoisomerase IIIalpha or BLM leads to
increased radial formation, as is seen in FA. BLM and Topoisomerase IIIalpha are
epistatic to the FA pathway for suppression of radial formation in response to
DNA interstrand crosslinks since depletion of either of them in FA cells does
not increase radial formation. Depletion of Topoisomerase IIIalpha or BLM also
causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome
cells. Human Fanconi anemia cells, however, do not demonstrate increased sister
chromatid exchanges, separating this response from radial formation. Primary
cell lines from mice defective in both Blm and Fancd2 have the same interstrand
crosslink-induced genome instability as cells from mice deficient in the Fancd2
protein alone. These observations demonstrate that the association of BLM and
Topoisomerase IIIalpha with Fanconi proteins is a functional one, delineating a
BLM-Topoisomerase IIIalpha-Fanconi pathway that is critical for suppression of
chromosome radial formation. Fanconi anemia (FA) is characterized by cellular hypersensitivity to DNA
crosslinking agents, but how the Fanconi pathway protects cells from DNA
crosslinks and whether FA proteins act directly on crosslinks remain unclear. We
developed a chromatin-IP-based strategy termed eChIP and detected association of
multiple FA proteins with DNA crosslinks in vivo. Interdependence analyses
revealed that crosslink-specific enrichment of various FA proteins is controlled
by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and
FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their
crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core
complex for its recruitment. FA core complex requires nucleotide excision repair
proteins XPA and XPC for its association. Consistent with the distinct
recruitment mechanism, recombination-independent crosslink repair was inversely
affected in cells deficient of FANC-core versus BRCA-related FA proteins. Thus,
FA proteins participate in distinct DNA damage response mechanisms governed by
DNA replication status. Fanconi anemia (FA) is a recessive cancer prone syndrome featuring bone marrow
failure and hypersensitivity to DNA interstrand crosslinks (ICLs) and, to a
milder extension, to ionizing radiation and oxidative stress. Recently, we
reported that human oxidative DNA glycosylase, NEIL1 excises with high
efficiency the unhooked crosslinked oligomer within three-stranded DNA repair
intermediate induced by photoactivated psoralen exposure. Complete
reconstitution of repair of the ICL within a three-stranded DNA structure shows
that it is processed in the short-patch base excision repair (BER) pathway. To
examine whether the DNA damage hypersensitivity in FA cells follows impaired BER
activities, we measured DNA glycosylase and AP endonuclease activities in
cell-free extracts from wild-type, FA, and FA-corrected cells. We showed that
immortalized lymphoid cells of FA complementation Groups A, C, and D and from
control cells from normal donors contain similar BER activities. Intriguingly,
the cellular level of NEIL1 protein strongly depends on the intact FA pathway
suggesting that the hypersensitivity of FA cells to ICLs may, at least in part,
arise from downregulation or degradation of NEIL1. Consistent with this result,
plasmid-based expression of the FLAG-tagged NEIL1 protein partially complements
the hypersensitivity FA cells to the crosslinking agents exposures, suggesting
that NEIL1 specifically complements impaired capability of FA cells to repair
ICLs and oxidative DNA damage. These findings shed light to how the FA pathway
may regulate DNA repair proteins and bring explanation for the long-time
disputed problem of the oxidative stress sensitive phenotype of FA cells. Fanconi anemia (FA) is an inherited disease characterized by bone marrow
failure, increased cancer risk and hypersensitivity to DNA cross-linking agents,
implying a role for this pathway in the maintece of genomic stability. The
central player of the FA pathway is the multi-subunit E3 ubiquitin ligase
complex activated through a replication- and DNA damage-dependent mechanism. A
consequence of the activation of the complex is the monoubiquitylation of FANCD2
and FANCI, late term effectors in the maintece of genome integrity. The
details regarding the coordination of the FA-dependent response and the DNA
replication process are still mostly unknown. We found, by yeast two-hybrid
assay and co-immunoprecipitation in human cells, that the core complex subunit
FANCF physically interacts with PSF2, a member of the GINS complex essential for
both the initiation and elongation steps of DNA replication. In HeLa cells
depleted for PSF2, we observed a decreased binding to chromatin of the FA core
complex, suggesting that the GINS complex may have a role in either loading or
stabilizing the FA core complex onto chromatin. Consistently, GINS and core
complex bind chromatin contemporarily upon origin firing and PSF2 depletion
sensitizes cells to DNA cross-linking agents. However, depletion of PSF2 is not
sufficient to reduce monoubiquitylation of FANCD2 or its localization to nuclear
foci following DNA damage. Our results suggest a novel crosstalk between DNA
replication and the FA pathway. Fanconi anemia is a rare recessive disorder characterized by genome instability,
congenital malformations, progressive bone marrow failure and predisposition to
hematologic maligcies and solid tumors. At the cellular level,
hypersensitivity to DNA interstrand crosslinks is the defining feature in
Fanconi anemia. Mutations in thirteen distinct Fanconi anemia genes have been
shown to interfere with the DNA-replication-dependent repair of lesions
involving crosslinked DNA at stalled replication forks. Depletion of SLX4, which
interacts with multiple nucleases and has been recently identified as a Holliday
junction resolvase, results in increased sensitivity of the cells to DNA
crosslinking agents. Here we report the identification of biallelic SLX4
mutations in two individuals with typical clinical features of Fanconi anemia
and show that the cellular defects in these individuals' cells are complemented
by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here
as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P. Fanconi anemia (FA) is an autosomal disorder that causes genome instability. FA
patients suffer developmental abnormalities, early-onset bone marrow failure,
and a predisposition to cancer. The disease is manifested by defects in DNA
repair, hypersensitivity to DNA crosslinking agents, and a high degree of
chromosomal aberrations. The FA pathway comprises 13 disease-causing genes
involved in maintaining genomic stability. The fast pace of study of the novel
DNA damage network has led to the constant discovery of new FA-like genes
involved in the pathway that when mutated lead to similar disorders. A majority
of the FA proteins act as signal transducers and scaffolding proteins to employ
other pathways to repair DNA. This review discusses what is known about the FA
proteins and other recently linked FA-like proteins. The goal is to clarify how
the proteins work together to carry out interstrand crosslink repair and
homologous recombination-mediated repair of damaged DNA. Fanconi anemia (FA) is an inherited disorder characterized by defective DNA
repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is
associated with high risk for marrow failure, leukemia and head and neck
squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the
use of total-body irradiation for hematopoietic stem cell transplantation and
radiation therapy for HNSCC. A radioprotector for the surrounding tissue would
therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation
survival curves were determined for pre- or postirradiation treatment with the
parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell
lines and two transgene-corrected subclonal lines. FancG(-/-) (PD326) and
FancD2(-/-) (PD20F) patient lines were more sensitive to the DNA crosslinking
agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both
P < 0.0001). FancD2(-/-) cells were more radiosensitive than the transgene
restored subclonal cell line (ñ = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P =
0.03). In contrast, FancG(-/-) cells were radioresistant relative to the
transgene-restored subclonal cell line (ñ = 9.4 ± 1.5 and 2.2 ± 05,
respectively, P = 0.001). DNA strand breaks measured by the comet assay
correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients
showed radiosensitivity similar to that of Fanc-D2(-/-) cells. A
fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the
cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower
concentration than Tempol significantly increased the radioresistance and
stabilized the antioxidant stores of all cell lines. Tempol increased the
toxicity of MMC in FancD2(-/-) cells. These data provide support for the
potential clinical use of JP4-039 for normal tissue radioprotection during
chemoradiotherapy in FA patients. Fanconi Anemia (FA) is a rare autosomic recessive and X-linked disease with
chromosomal instability after exposure to crosslinking agents as the hallmark.
Clinical features of FA are somatic malformations, progressive bone marrow
failure and cancer proneness, however there is wide clinical heterogeneity. The
symptom most frequently and early associated with morbidity and mortality is
progressive pancytopenia in the first decade of life although acute myelogenous
leukemia (AML) or myelodysplastic syndrome (MDS) can appear before aplastic
anemia. Squamous cell carcinoma (SCC) of the head-neck, intestinal or genital
tract has a very high incidence in FA and can appear at young age. This paper
will focus on treatment of bone marrow failure in FA. C. elegans provides an excellent model system for the study of the Fanconi
Anemia (FA), one of the hallmarks of which is sensitivity to interstrand
crosslinking agents. Central to our understanding of FA has been the
investigation of DOG-1, the functional ortholog of the deadbox helicase FANCJ.
Here we review the current understanding of the unique role of DOG-1 in
maintaining stability of G-rich DNA in C. elegans and explore the question of
why DOG-1 animals are crosslink sensitive. We propose a dynamic model in which
noncovalently linked G-rich structures form and un-form in the presence of
DOG-1. When DOG-1 is absent but crosslinking agents are present the G-rich
structures are readily covalently crosslinked, resulting in increased crosslinks
formation and thus giving increased crosslink sensitivity. In this
interpretation DOG-1 is neither upstream nor downstream in the FA pathway, but
works alongside it to limit the availability of crosslink substrates. This model
reconciles the crosslink sensitivity observed in the absence of DOG-1 function
with its unique role in maintaining G-Rich DNA and will help to formulate
experiments to test this hypothesis. |
Which are the cellular targets of imatinib mesylate? | The cellular targets of imatinib mesylate are BCR-ABL, platelet-derived growth factor receptor (PDGFR) and c-kit kinases. | Imatinib mesylate is a tyrosine kinase inhibitor of the ABL, platelet-derived
growth factor receptor (PDGFR), and c-kit kinases. Inhibition of BCR-ABL and
c-kit accounts for its clinical activity in leukemia and sarcoma, respectively.
In this report, we describe other cellular targets for imatinib. Treatment of
head and neck squamous carcinoma cells with clinically relevant concentrations
of imatinib-induced changes in cell morphology and growth similar to changes
associated with epidermal growth factor receptor (EGFR) activation.
Imatinib-induced changes were blocked with the EGFR antagonist cetuximab, which
suggested direct involvement of EGFR in this process. Western blot analysis of
cells incubated with imatinib demonstrated activation of EGFR and downstream
signaling that was reduced by inhibition of mitogen-activated
protein/extracellular signal-regulated kinase kinase 1 (MEK1) and EGFR, but not
Her2/ErbB2. An in vitro kinase assay showed that imatinib did not directly
affect EGFR kinase activity, suggesting involvement of EGFR-activating
molecules. Inhibitors and neutralizing antibodies against heparin-binding
epidermal growth factor-like growth factor (HB-EGF), and to a lesser extent
transforming growth factor-alpha, reduced imatinib-mediated mitogen activated
protein kinase (MAPK) activation. Imatinib stimulated the rapid release of
soluble HB-EGF and the subsequent induction of membrane-bound HB-EGF, which
correlated with biphasic MAPK activation. Together, these results suggested that
imatinib affects EGFR activation and signaling pathways through rapid release
and increased expression of endogenous EGFR-activating ligands. Although,
imatinib primarily inhibits tyrosine kinases, it also stimulates the activity of
EGFR tyrosine kinase in head and neck squamous tumors. This finding demonstrates
the need for careful use of this drug in cancer patients. |
Which are the clinical characteristics of Tuberous Sclerosis? | The clinical characteristics of Tuberous Sclerosis include epilepsy, subependymal giant cell astrocytomas, lymphangioleiomyomatosis, rhabdomyoma, renal angiomyolipomas, cortical tubers, neurofibromas, angiofibromas, mental retardation, and behavioral disorders. | Tuberous sclerosis (TS) is a genetic disorder affecting multiple body systems,
and resulting from alterations in cell differentiation and proliferation. The
disease is characterized by the development of benign hamartomatous tumors:
neurofibromas and angiofibromas, located in the skin, central nervous system,
mucosas and other organs. Abnormal neural cell migration plays an important role
in the neurological dysfunctions found in TS, the predomit features being
mental retardation, seizures and behavioral disorders. The condition is produced
by mutations in genes TSC1 of chromosome 9q34 and TSC2 of chromosome 16p13.3,
and exhibits a domit autosomal hereditary trait--though 60-70% of cases are
sporadic and represent new mutations. The phenotype is highly variable. The
prevalence of TS varies between 1/6000 and 1/10,000 live births. The present
study reports the case of a 21-year-old male with TS and oral manifestations of
the disease. The clinical characteristics are described, along with the
diagnostic criteria and the management strategies, with a review of the
literature on the disease. BACKGROUND: Pulmonary lymphangioleiomyomatosis is a rare disease that occurs
mainly in women of reproductive age. The clinical characteristics include
recurrent spontaneous pneumothorax and progressive dyspnea. The features of
chest computed tomography are nearly pathognomonic with the detection of
bilateral thin-walled cysts.
CASE REPORT: A 32-year-old female presented with sudden-onset right-sided chest
pain, which aggravated during inspiration, dyspnea at exertion, and cough. She
had a history of bilateral recurrent spontaneous pneumothorax. Physical
examination showed reduced pulmonary sounds on the right side. Chest X-ray
confirmed the diagnosis of recurrent right-sided pneumothorax. Computed
tomography showed multiple bilateral bullae. Video-assisted pleurectomy, bulla
resection, and bulla coagulation were performed. The diagnosis of pulmonary
lymphangioleiomyomatosis was confirmed by pulmonary biopsy. After pleurectomy,
the patient remained symptom-free without recurrent pneumothorax.
CONCLUSION: Pulmonary lymphangioleiomyomatosis is a rare cause of recurrent
pneumothorax and should be considered a differential diagnosis, especially in
young women with diffuse bilateral bullous emphysema or tuberous sclerosis. Balloon cells are histopathological hallmarks of various cortical malformations,
i.e., focal cortical dysplasia (Taylor's type, FCD IIb), hemimegalencephaly
(HME) or cortical tubers (tuberous sclerosis, TSC). Whether this intriguing cell
type results from similar pathogenetic pathways remains to be shown. Here, we
analyzed the immunohistochemical distribution pattern of the CD34 epitope in
surgical specimens from 34 patients with FCD IIb, compared to that of 6 patients
with TSC and 3 patients with HME. In normal brain, CD34 occurs only transiently
during neurulation, but cannot be detected in mature neuroectodermal cell
progenies. In contrast, 58% of our patients showed CD34 immunoreactivity within
a subpopulation of balloon cells. Interestingly, CD34-positive balloon cells
were confined to the white matter, but never observed in neocortical layers.
Furthermore, balloon cells expressing neurofilament protein were also restricted
to white matter, whereas GFAP-positive balloon cells were observed either in
white or gray matter location. Clinical characteristics did not significantly
differ between patients with CD34-positive versus CD34-negative lesions. No
significant correlation was found between CD34 expression and genetic
alterations of the TSC1 gene, which is affected in many FCD and TSC patients and
which plays a role in the regulation of cell size. Further studies are warranted
to clarify the restricted expression of CD34 in balloon cells of the white
matter. RATIONALE: Pulmonary lymphangioleiomyomatosis is a progressive cystic lung
disease that is associated with infiltration of atypical smooth muscle-like
cells. Previous descriptions of clinical characteristics of subjects with
lymphangioleiomyomatosis have been based on a limited number of patients.
OBJECTIVES: To describe the clinical characteristics of subjects with pulmonary
lymphangioleiomyomatosis, both sporadic and tuberous sclerosis-related forms.
METHODS: Over a 3-yr period, from 1998 to 2001, 243 subjects with pulmonary
lymphangioleiomyomatosis were enrolled into a national registry; 13 subjects who
had already undergone lung transplantation were excluded for the purposes of
this report.
MEASUREMENTS AND MAIN RESULTS: All 230 subjects were women, aged 18 to 76 yr
(mean +/- SE, 44.5 +/- 0.65 yr). The average age at onset of symptoms was 38.9
+/- 0.73 yr and at diagnosis was 41.0 +/- 0.65 yr. Tuberous sclerosis complex
was present in 14.8% of subjects. Pulmonary manifestations, most commonly
spontaneous pneumothorax, were the primary events leading to the diagnosis in
86.5% of cases. Nearly 55% of the subjects were being treated with a
progesterone derivative. An obstructive pattern on pulmonary function testing
was observed in 57.3% of the subjects, whereas 33.9% had normal spirometric
results. Women with tuberous sclerosis-related lymphangioleiomyomatosis were
younger and had less impaired lung function compared with those with the
sporadic form.
CONCLUSIONS: The age range of women afflicted with pulmonary
lymphangioleiomyomatosis is broader than previously appreciated and the degree
of pulmonary function can be quite variable, with one-third of subjects having
normal spirometry at enrollment into this registry. PURPOSE: Lymphangioleiomyomatosis (LAM) is a rare, progressive, frequently
lethal cystic lung disease that almost exclusively affects women. Prognostic
information in LAM has been limited by small numbers and heterogeneous study
methodology. Early retrospective cohorts cited 5- and 10-year mortality of 40
and 80 %, respectively. More recently, mortality at 10 years has been estimated
to be approximately 10-20 % from the onset of symptoms and 30 % at 10 years from
the time of lung biopsy but varies widely in individual patients. Given the
heterogeneous disease course, it would be useful to establish which clinical
characteristics are associated with survival to develop prediction models for
disease outcome.
METHODS: The LAM Foundation maintains a population-based registry of 1,149
registered self-identified LAM patients. Of these, 590 have completed a "General
Information/Clinical History Questionnaire" with limited demographic and
clinical data, 410 of whom were identified as U.S. residents and provided date
of birth. Vital status was obtained on all 410 participants through December 31,
2007 by linking patient identifiers and the National Death Index. Survival time
was calculated as the time since first lung-related symptom or physician
diagnosis until censoring (still alive, received lung transplant, or died). Cox
proportional hazard analysis evaluated the association of demographic and
clinical features with survival.
RESULTS: Among the 410 subjects, there were 50 deaths and 55 lung
transplantations during a median of 10.4 years of observation time. The
estimated median transplant-free survival time for LAM patients in the United
States is 29 years from symptom onset and 23 years from diagnosis. The estimated
10-year survival transplant-free was 86 %. Age at disease onset, smoking status,
race, presence of tuberous sclerosis, occurrence of pneumothorax, and pregcy
did not demonstrate an association with survival or transplant. Greater age at
presentation and presence of angiomyolipoma were associated with less risk of
mortality. Treatment with hormonal therapy was associated with an increased risk
of death/transplant (hazard ratio (HR) 2.93; 95 % confidence interval (CI),
1.54-5.58; p = 0.001), particularly progesterone therapy (HR 2.17; 95 % CI
1.26-3.75, p = 0.005), and may represent confounding by indication. Patients who
required oxygen therapy had a worse outcome (HR 4.53; 95 % CI 2.76-7.42; p <
0.001).
CONCLUSIONS: Our population-based study showed that the median survival in
patients with LAM from the onset of symptoms or diagnosis is much longer than
previously described. This has important implications for life choices and
treatment decisions regarding medication use and lung transplantation for
patients with LAM. Clobazam (CLB) was recently approved by the FDA, but has not been evaluated in
tuberous sclerosis complex (TSC). We retrospectively reviewed a cohort of
patients with TSC and refractory epilepsy who started CLB over a 5-year period.
Clinical characteristics and number of tubers on MRI were assessed. Duration of
therapy, therapeutic response and adverse events were recorded. CLB was
prescribed in 29 adults and children of whom 72% were cognitively impaired, with
a median age at seizure onset of 5 months. Mean duration of CLB therapy was 17.3
months with a 12 and 24-month estimated retention rate of 82% and 68%,
respectively. Twenty patients (69%) reported a good response (>50% seizure
reduction) at the end of the titration, and six patients (21%) remained good
responders after 12 months of CLB therapy. Adverse events occurred in 13
patients, predomitly somnolence and behavioral disorders. One quarter of the
responders reported improvement in behavior. No predictive factor for a good
response could be identified. CLB appears to be a well-tolerated and valuable
option for treatment of refractory epilepsy in TSC. OBJECT: Subependymal giant cell astrocytomas (SEGAs) are benign tumors, most
commonly associated with tuberous sclerosis complex (TSC). The vast majority of
these tumors arise from the lateral ependymal surface adjacent to the foramen of
Monro, therefore potentially encroaching on one or both foramina, and resulting
in obstructive hydrocephalus that necessitates surgical decompression. The
indications for surgery, intraoperative considerations, and evolution of the
authors' management paradigm are presented.
METHODS: Patients with TSC who underwent craniotomy for SEGA resection at New
York University Langone Medical Center between January 1997 and March 2011 were
identified. Preoperative imaging, clinical characteristics, management
decisions, operative procedures, and outcomes were reviewed.
RESULTS: Eighteen patients with TSC underwent 22 primary tumor resections for
SEGAs. The indication for surgery was meaningful radiographic tumor progression
in 16 of 21 cases. The average age at the time of operation was 10.3 years.
Average follow-up duration was 52 months (range 12-124 months). The operative
approach was intrahemispheric-transcallosal in 16 cases,
transcortical-transventricular in 5, and neuroendoscopic in 1. Nine tumors were
on the right, 9 on the left, and 3 were bilateral. Gross-total resection was
documented in 16 of 22 cases in our series, with radical subtotal resection
achieved in 4 cases, and subtotal resection (STR) in 2 cases. Two patients had
undergone ventriculoperitoneal shunt placement preoperatively and 7 patients
required shunt placement after surgery for moderate to severe ventriculomegaly.
Two patients experienced tumor progression requiring reoperation; both of these
patients had initially undergone STR.
CONCLUSIONS: The authors present their management strategy for TSC patients with
SEGAs. Select patients underwent microsurgical resection of SEGAs with
acceptable morbidity. Gross-total resection or radical STR was achieved in 90.9%
of our series (20 of 22 primary tumor resections), with no recurrences in this
group. Approximately half of our patient series required CSF diversionary
procedures. There were no instances of permanent neurological morbidity
associated with surgery. PURPOSE: Prevalence and long-term outcome of epilepsy in tuberous sclerosis
complex (TSC) is reported to be variable, and the reasons for this variability
are still controversial.
METHODS: We reviewed the clinical characteristics of patients with TSC who were
regularly followed since 2000 at the San Paolo Multidisciplinary Tuberous
Sclerosis Centre in Milan, Italy. From patient charts we collected data about
age at epilepsy onset, seizure frequency and seizure type, history of infantile
spasms (IS), epileptic syndrome, evolution to refractory epilepsy or to seizure
freedom and/or medication freedom, electroencephalography (EEG) features,
magnetic resoce imaging (MRI) findings, cognitive outcome, and genetic
background.
KEY FINDINGS: Among the 160 subjects (120 adults and 40 children), 116 (72.5%)
had epilepsy: 57 (35.6%) were seizure-free, and 59 (36.9%) had drug-resistant
epilepsy. Most seizure-free patients had a focal epilepsy (89.5%), with 54.4% of
them drug resistant for a period of their lives. Epilepsy onset in the first
year of life with IS and/or focal seizures was characteristic of the
drug-resistant group of patients, as well as cognitive impairment and TSC2
mutation (p < 0.05). A small group of patients (7 patients, 4.4%) experienced a
seizure only once; all of them had normal cognition.
SIGNIFICANCE: Although epilepsy management can be challenging in TSC, more than
one third of patients had their seizures controlled: through monotherapy in 56%
and by polytherapy in 32%. Moreover, 12% of the patients became seizure-free and
were off medication. Identifying predictive features of epilepsy and cognitive
outcome can ensure better management for patients with TSC and delineate
genotype-phenotype correlations. |
What is known about MER41 repeat sequences? | We report eleven new families of MEdium Reiteration frequency (MER) interspersed repeats in the genomes of Primates, Rodentia, and Lagomorpha. Two families of the human repeats, MER 46 and MER 47, represent non-autonomous DNA transposons. These sequences are flanked by TA target site duplications and have terminal inverted repeats (TIRs) similar to TIRs of DNA transposons. The sequences of five other families of repeats, MER41, MER48, MER50, MER51, and RMER3, resemble long terminal repeats of retroviruses. A potential involvement of some of the reported MER repeats in the regulation of transcription and genetic rearrangements is suggested. Age estimations place the origin of most MER repeats at the time of decline in MIR (Mammalian-wide Interspersed Repeats) retroposition and before the origin of the Alu familyMER41 repeat sequences contain inducible STAT1 binding sites | We report eleven new families of MEdium Reiteration frequency (MER) interspersed
repeats in the genomes of Primates, Rodentia, and Lagomorpha. Two families of
the human repeats, MER 46 and MER 47, represent non-autonomous DNA transposons.
These sequences are flanked by TA target site duplications and have terminal
inverted repeats (TIRs) similar to TIRs of DNA transposons. The sequences of
five other families of repeats, MER41, MER48, MER50, MER51, and RMER3, resemble
long terminal repeats of retroviruses. A potential involvement of some of the
reported MER repeats in the regulation of transcription and genetic
rearrangements is suggested. Age estimations place the origin of most MER
repeats at the time of decline in MIR (Mammalian-wide Interspersed Repeats)
retroposition and before the origin of the Alu family. Chromatin immunoprecipitation combined with massively parallel sequencing
methods (ChIP-seq) is becoming the standard approach to study interactions of
transcription factors (TF) with genomic sequences. At the example of public
STAT1 ChIP-seq data sets, we present novel approaches for the interpretation of
ChIP-seq data.We compare recently developed approaches to determine STAT1
binding sites from ChIP-seq data. Assessing the content of the established
consensus sequence for STAT1 binding sites, we find that the usage of "negative
control" ChIP-seq data fails to provide substantial advantages. We derive a
single refined probabilistic model of STAT1 binding sequences from these
ChIP-seq data. Contrary to previous claims, we find no evidence that STAT1 binds
to multiple distinct motifs upon interferon-gamma stimulation in vivo. While a
large majority of genomic sites with high ChIP-seq signal is associated with a
nucleotide sequence resembling a STAT1 binding site, only a very small subset of
the over 5 million potential STAT1 binding sites in the human genome is covered
by ChIP-seq data. Furthermore a surprisingly large fraction of the ChIP-seq
signal (5%) is absorbed by a small family of repetitive sequences (MER41). The
observation of the binding of activated STAT1 protein to a specific repetitive
element bolsters similar reports concerning p53 and other TFs, and strengthens
the notion of an involvement of repeats in gene regulation. Incidentally MER41
are specific to primates, consequently, regulatory mechanisms in the IFN-STAT
pathway might fundamentally differ between primates and rodents. On a
methodological aspect, the presence of large numbers of nearly identical binding
sites in repetitive sequences may lead to wrong conclusions about intrinsic
binding preferences of TF as illustrated by the spacing analysis STAT1 tandem
motifs. Therefore, ChIP-seq data should be analyzed independently within
repetitive and non-repetitive sequences. |
How functional connectivity of the default mode network changes in patients with disorders of consciousness? | Functional connectivity in the default mode network (DMN) is reduced in patients with different disorders of consciousness, and correlates with the level of consciousness.
Specifically, functional connectivity in the default mode network was shown to be absent in brain death patients, extremely low in vegetative state patients and slightly decreased in minimally conscious state patients when compared to healthy subjects. Therefore, functional connectivity in the default mode network was suggested to be valuable in differentiating patients with different disorders of consciousness. Clinically, functional connectivity in the default mode network was also shown to be an indicator of the extent of cortical disruption and predict reversible impairments in consciousness. | It is debatable as to whether the spontaneous blood-oxygen-level dependent
fluctuations that are observed in the resting brain in turn reflect consciously
directed mental activity or, alternatively, constitute an intrinsic property of
functional brain organisation persisting in the absence of consciousness. This
report shows for the first time, in three patients, that the persistent
vegetative state (PVS) is marked by a dysfunctional default mode network, with
decreased connectivity in several brain regions, including the dorsolateral
prefrontal cortex and anterior cingulated cortex, especially in the right
hemisphere. This finding supports the view that the resting state is involved in
self-consciousness, and that the right-hemisphere default state may play a major
role in conscious processes. It is speculated that the default state may act as
a surrogate marker of PVS with awareness contents and, therefore, could replace
a more complex activation paradigm. Recent studies in patients with disorders of consciousness (DOC) tend to support
the view that awareness is not related to activity in a single brain region but
to thalamo-cortical connectivity in the frontoparietal network. Functional
neuroimaging studies have shown preserved albeit disconnected low-level cortical
activation in response to external stimulation in patients in a "vegetative
state" or unresponsive wakefulness syndrome. While activation of these "primary"
sensory cortices does not necessarily reflect conscious awareness, activation in
higher-order associative cortices in minimally conscious state patients seems to
herald some residual perceptual awareness. PET studies have identified a
metabolic dysfunction in a widespread frontoparietal "global neuronal workspace"
in DOC patients including the midline default mode network ("intrinsic" system)
and the lateral frontoparietal cortices or "extrinsic system." Recent studies
have investigated the relation of awareness to the functional connectivity
within intrinsic and extrinsic networks, and with the thalami in both
pathological and pharmacological coma. In brain damaged patients, connectivity
in all default network areas was found to be non-linearly correlated with the
degree of clinical consciousness impairment, ranging from healthy controls and
locked-in syndrome to minimally conscious, vegetative, coma, and brain dead
patients. Anesthesia-induced loss of consciousness was also shown to correlate
with a global decrease in cortico-cortical and thalamo-cortical connectivity in
both intrinsic and extrinsic networks, but not in auditory, or visual networks.
In anesthesia, unconsciousness was also associated with a loss of cross-modal
interactions between networks. These results suggest that conscious awareness
critically depends on the functional integrity of thalamo-cortical and
cortico-cortical frontoparietal connectivity within and between "intrinsic" and
"extrinsic" brain networks. Recent advances in the study of spontaneous brain activity have demonstrated
activity patterns that emerge with no task performance or sensory stimulation;
these discoveries hold promise for the study of higher-order associative network
functionality. Additionally, such advances are argued to be relevant in
pathological states, such as disorders of consciousness (DOC), i.e., coma,
vegetative and minimally conscious states. Recent studies on resting state
activity in DOC, measured with functional magnetic resoce imaging (fMRI)
techniques, show that functional connectivity is disrupted in the task-negative
or the default mode network. However, the two main approaches employed in the
analysis of resting state functional connectivity data (i.e., hypothesis-driven
seed-voxel and data-driven independent component analysis) present multiple
methodological difficulties, especially in non-collaborative DOC patients.
Improvements in motion artifact removal and spatial normalization are needed
before fMRI resting state data can be used as proper biomarkers in severe brain
injury. However, we anticipate that such developments will boost clinical
resting state fMRI studies, allowing for easy and fast acquisitions and
ultimately improve the diagnosis and prognosis in the absence of DOC patients'
active collaboration in data acquisition. Diagnosis of patients with a disorder of consciousness is very challenging.
Previous studies investigating resting state networks demonstrate that 2 main
features of the so-called default mode network (DMN), metabolism and functional
connectivity, are impaired in patients with a disorder of consciousness.
However, task-induced deactivation--a third main feature of the DMN--has not
been explored in a group of patients. Deactivation of the DMN is supposed to
reflect interruptions of introspective processes. Seventeen patients with
unresponsive wakefulness syndrome (UWS, former vegetative state), 8 patients in
minimally conscious state (MCS), and 25 healthy controls were investigated with
functional magnetic resoce imaging during a passive sentence listening task.
Results show that deactivation in medial regions is reduced in MCS and absent in
UWS patients compared to healthy controls. Moreover, behavioral scores assessing
the level of consciousness correlate with deactivation in patients. On
single-subject level, all control subjects but only 2 patients in MCS and 6 with
UWS exposed deactivation. Interestingly, all patients who deactivated during
speech processing (except for one) showed activation in left frontal regions
which are associated with conscious processing. Our results indicate that
deactivation of the DMN can be associated with the level of consciousness by
selecting those who are able to interrupt ongoing introspective processes. In
consequence, deactivation of the DMN may function as a marker of consciousness. OBJECTIVE: To evaluate the possible role of the default mode network (DMN) in
consciousness and assess the diagnostic or prognostic potential of DMN
connectivity measures in the assessment of a patient group lacking cognitive
awareness.
METHODS: DMN connectivity was established using independent component analysis
of resting-state fMRI data in patients with reversible (n = 2) and irreversible
(n = 11) coma following cardiac arrest and compared to healthy controls (n =
12).
RESULTS: A present and intact DMN was observed in controls and those patients
who subsequently regained consciousness, but was disrupted in all patients who
failed to regain consciousness.
CONCLUSIONS: The results suggest that the DMN is necessary but not sufficient to
support consciousness. Clinically, DMN connectivity may serve as an indicator of
the extent of cortical disruption and predict reversible impairments in
consciousness. Increasing attention has been paid recently to the study of spontaneous brain
activity; moreover, particular attention has been paid to the concept of a
default-mode network of brain function. Although the functional significance of
the default-mode network remains a matter of debate, it has been suggested to be
a candidate for the network subserving basic functions related to consciousness.
We report the case of a 29-year-old man with encephalopathy and a reversible
lesion of the entire corpus callosum. Despite resolution of corpus callosum
lesion on magnetic resoce imaging (MRI) within 1 week, the patient
persistently presented disturbance of consciousness. Resting-state functional
MRI revealed that the posterior cingulate cortex/precuneus was functionally
disconnected from other brain regions within the default-mode network. Our case
report suggests that assessment of the functional connectivity in the
resting-state default-mode network could be a useful marker of consciousness
disturbance even in the presence of a reversible brain lesion. OBJECTIVE: Resting-state networks (RSNs), including the default mode network
(DMN), have been considered as markers of brain status such as consciousness,
developmental change, and treatment effects. The consistency of functional
connectivity among RSNs has not been fully explored, especially among
resting-state-related independent components (RSICs).
MATERIALS AND METHODS: This resting-state fMRI study addressed the consistency
of functional connectivity among RSICs as well as their spatial consistency
between 'at day 1' and 'after 4 weeks' in 13 healthy volunteers.
RESULTS: We found that most RSICs, especially the DMN, are reproducible across
time, whereas some RSICs were variable in either their spatial characteristics
or their functional connectivity. Relatively low spatial consistency was found
in the basal ganglia, a parietal region of left frontoparietal network, and the
supplementary motor area. The functional connectivity between two independent
components, the bilateral angular/supramarginal gyri/intraparietal lobule and
bilateral middle temporal/occipital gyri, was decreased across time regardless
of the correlation analysis method employed, (Pearson's or partial correlation).
CONCLUSION: RSICs showing variable consistency are different between spatial
characteristics and functional connectivity. To understand the brain as a
dynamic network, we recommend further investigation of both changes in the
activation of specific regions and the modulation of functional connectivity in
the brain network. Cerebral edema, a well-known feature of acute liver disease, can occur in
cirrhotic patients regardless of hepatic encephalopathy (HE) and adversely
affect prognosis. This study characterized and correlated functional HE
abnormalities in the brain to cerebral edema using resting-state functional
magnetic resoce imaging (rs-fMRI) and diffusion tensor imaging (DTI).
Forty-one cirrhotic patients (16 without HE, 14 minimal HE, 11 overt HE) and 32
healthy controls were assessed. The HE grade in cirrhotic patients was evaluated
by the West Haven criteria and neuro-psychological examinations. Functional
connectivity correlation coefficient (fc-CC) of the default mode network (DMN)
was determined by rs-fMRI, while the corresponding mean diffusivity (MD) was
obtained from DTI. Correlations among inter-cortical fc-CC, DTI indices,
Cognitive Ability Screening Instrument scores, and laboratory tests were also
analyzed. Results showed that gradual reductions of HE-related consciousness
levels, from "without HE" or "minimal HE" to "overt HE", correlated with
decreased anterior-posterior fc-CC in DMN [F(4.415), p = 0.000)]. The MD values
from regions with anterior-posterior fc-CC differences in DMN revealed
significant differences between the overt HE group and other groups. Increased
MD in this network was inversely associated with decreased fc-CC in DMN and
linearly correlated with poor cognitive performance. In conclusion, cerebral
edema can be linked to altered cerebral temporal architecture that modifies both
within- and between-network connectivity in HE. Reduced fc-CC in DMN is
associated with behavior and consciousness deterioration. Through appropriate
targets, rs-fMRI technology may provide relevant supplemental information for
monitoring HE and serve as a new biomarker for clinical diagnosis. |
Have C12orf65 mutations been associated with axonal neuropathy and optic atrophy? | Novel C12orf65 mutations in patients with axonal neuropathy and optic atrophy | OBJECTIVE: Charcot-Marie Tooth disease (CMT) forms a clinically and genetically
heterogeneous group of disorders. Although a number of disease genes have been
identified for CMT, the gene discovery for some complex form of CMT has lagged
behind. The association of neuropathy and optic atrophy (also known as CMT type
6) has been described with autosomaldomit, recessive and X-linked modes of
inheritance. Mutations in Mitofusin 2 have been found to cause domit forms of
CMT6. Phosphoribosylpyrophosphate synthetase-I mutations cause X-linked CMT6,
but until now, mutations in the recessive forms of disease have never been
identified.
METHODS: We here describe a family with three affected individuals who inherited
in an autosomal recessive fashion a childhood onset neuropathy and optic
atrophy. Using homozygosity mapping in the family and exome sequencing in two
affected individuals we identified a novel protein-truncating mutation in the
C12orf65 gene, which encodes for a protein involved in mitochondrial
translation. Using a variety of methods we investigated the possibility of
mitochondrial impairment in the patients cell lines.
RESULTS: We described a large consanguineous family with neuropathy and optic
atrophy carrying a loss of function mutation in the C12orf65 gene. We report
mitochondrial impairment in the patients cell lines, followed by multiple lines
of evidence which include decrease of complex V activity and stability (blue
native gel assay), decrease in mitochondrial respiration rate and reduction of
mitochondrial membrane potential.
CONCLUSIONS: This work describes a mutation in the C12orf65 gene that causes
recessive form of CMT6 and confirms the role of mitochondrial dysfunction in
this complex axonal neuropathy. Combined oxidative phosphorylation deficiency type 7 (COXPD7) is a rare disorder
of mitochondrial metabolism that results in optic atrophy and Leigh
syndrome-like disease. We describe 2 siblings with compound heterozygous
mutations in the recently identified C12orf65 gene who presented with optic
atrophy and mild developmental delays and subsequently developed bilateral,
symmetric lesions in the brainstem reminiscent of Leigh syndrome. Repeat
neuroimaging demonstrated reversibility of the findings in 1 sibling and
persistent metabolic stroke in the other. This article highlights the phenotypic
manifestations from a novel mutation in the C12orf65 gene and reviews the
clinical presentation of the 5 other individuals reported to date who carry
mutations in this gene. C12orf65 participates in the process of mitochondrial translation and has been
shown to be associated with a spectrum of phenotypes, including early onset
optic atrophy, progressive encephalomyopathy, peripheral neuropathy, and spastic
paraparesis.We used whole-genome homozygosity mapping as well as exome
sequencing and targeted gene sequencing to identify novel C12orf65
disease-causing mutations in seven affected individuals originating from two
consanguineous families. In four family members affected with childhood-onset
optic atrophy accompanied by slowly progressive peripheral neuropathy and
spastic paraparesis, we identified a homozygous frame shift mutation c.413_417
delAACAA, which predicts a truncated protein lacking the C-terminal portion. In
the second family, we studied three affected individuals who presented with
early onset optic atrophy, peripheral neuropathy, and spastic gait in addition
to moderate intellectual disability. Muscle biopsy in two of the patients
revealed decreased activities of the mitochondrial respiratory chain complexes I
and IV. In these patients, we identified a homozygous splice mutation,
g.21043 T>A (c.282+2 T>A) which leads to skipping of exon 2. Our study broadens
the phenotypic spectrum of C12orf65 defects and highlights the triad of optic
atrophy, axonal neuropathy and spastic paraparesis as its key clinical features.
In addition, a clear genotype-phenotype correlation is anticipated in which
deleterious mutations which disrupt the GGQ-containing domain in the first
coding exon are expected to result in a more severe phenotype, whereas
down-stream C-terminal mutations may result in a more favorable phenotype,
typically lacking cognitive impairment. |
List drugs included in the DHAP-R chemotherapy regiment. | Dexamethasone (a steroid hormone), cytarabine (ara-C), cisplatin (platinum agent) and rituximab are included in the DHAP-R chemotherapy protocol. | BACKGROUND AND OBJECTIVE: The treatment of the patients with relapsed and
refractory non-Hodgkin's lymphoma(NHL) remains difficult. It was reported that
DHAP regimen(cisplatin + Ara-C + dexamthsone) was an effective salvage therapy,
but there was no report about it in China. The current study was designed to
observe the efficacy and toxicity of DHAP regimen for the patients with relapsed
and refractory NHL.
METHOD: Seventeen patients with relapsed and 10 with refractory intermediate or
high grade NHL was involved in this study. These patients were treated with
cytarabine 1 g/m2 intravenous(i.v.) every 12 hours on day 1 to 2, cisplatin 20
mg/m2 i.v. on day 1 to 4, dexamethone 40 mg i.v. on day 1 to 4. Four patients
with CR after DHAP were followed by autologous peripheral blood stem cell
transplantation(APBPCT).
RESULTS: Overt responses to DHAP were seen in 12 patients (44.4%) including 8
complete responses(CR) (29.6%) and 4 partial responses (PR) (14.8%). The
effective release time lasted a median of 4.8 months. Median survival time was
8.3 months. 1-year and 2-year survival rate were 30.8% and 19.3%, respectively.
Myelosuppression was the major toxicity; 15 patients(57.7%) had grade III-IV
neutropenia and 21 patients (80.8%) had grade III-IV thromcytopenia, but there
was no treatment-related death.
CONCLUSION: The authors conclude that DHAP regimen is an effective salvage
therapy for the patients with relapsed and refractory NHL, but the remission
duration time is short and long-term prognosis remains poor. High dose
chemotherapy supported by APBPCT is necessary for improvement in long-term
survival. The multicentre phase III CORAL study aims to guide choice of salvage
chemotherapy in diffuse large B-cell lymphoma (DLBCL) and assess the role of
rituximab maintece after autologous stem cell transplantation (ASCT).
Patients are first randomised between ICE (ifosfamide, carboplatin, etoposide)
and DHAP (dexamethasone, ara-C and cisplatin), both combined with rituximab
(R-ICE or R-DHAP). After three courses, responders are treated by ASCT with
BEAM. A second randomisation then allocates patients to maintece treatment
with rituximab 375 mg/m(2), one injection every 2 months six times, or
observation. Accrual to the study is now proceeding well and the planned 400
patients are likely to be enrolled within the next 1.5 years. Results to date
are very preliminary but suggest encouraging rates of response. However, they
also indicate that initial exposure to rituximab may increase the difficulty of
salvaging patients who fail first-line therapy. We evaluated the role of rituximab during remission induction chemotherapy in
relapsed aggressive CD20+ non-Hodgkin lymphoma. Of 239 patients, 225 were
evaluable for analysis. Randomized to DHAP
(cisplatin-cytarabine-dexamethasone)-VIM
(etoposide-ifosfamide-methotrexate)-DHAP (cisplatin-cytarabine-dexamethasone)
chemotherapy with rituximab (R; R-DHAP arm) were 119 patients (113 evaluable)
and to chemotherapy without rituximab (DHAP arm) 120 patients (112 evaluable).
Patients in complete remission (CR) and partial remission (PR) after 2
chemotherapy courses were eligible for autologous stem-cell transplantation.
After the second chemotherapy cycle, 75% of the patients in the R-DHAP arm had
responsive disease (CR or PR) versus 54% in the DHAP arm (P=.01). With a median
follow-up of 24 months, there was a significant difference in failure-free
survival (FFS24; 50% vs 24% vs, P<.001), and progression free survival (PFS24;
52% vs 31% P<.002) in favor of the R-DHAP arm. Cox-regression analysis
demonstrated a significant effect of rituximab treatment on FFS24 (HR 0.41, 95%
confidence interval [CI] 0.29-0.57 versus 0.51, 95% CI 0.37-0.70) and
overall-survival (OS24: HR 0.60 [0.41-0.89] vs 0.76 [0.52-1.10]) when adjusted
for time since upfront treatment, age, World Health Organization performance
status, and secondary age-adjusted international prognostic index. These results
demonstrate improved FFS and PFS for relapsed aggressive B-cell NHL if rituximab
is added to the re-induction chemotherapy regimen. Anthracycline-based chemotherapy is the cornerstone of modern curative regimens
for aggressive lymphomas; however, these drugs cannot be safely administered in
the setting of severe hepatic dysfunction. Alternative regimens for this setting
are required. We describe 2 patients with newly diagnosed advanced aggressive
lymphoma (diffuse large B-cell lymphoma [DLBCL] and classic Hodgkin lymphoma
[HL]) presenting with severe hepatic dysfunction with hyperbilirubinemia
(4.3-13.2 mg/dL). Because of the inability to safely administer unattenuated
doses of anthracycline-based regimens, dexamethasone, high-dose cytarabine, and
cisplatin (DHAP) was used at full doses (along with rituximab for the DLBCL
patient) until hepatic function normalized (1-5 cycles). The treatment was then
converted to R-CHOP (rituximab/cyclophosphamide/
doxorubicin/vincristine/prednisone) and ABVD
(doxorubicin/bleomycin/vinblastine/dacarbazine) for the DLBCL and HL patient,
respectively, to complete therapy. The patients had a partial remission and
complete remission, respectively. These data suggest that DHAP is a safe and
effective regimen that can be used without dose modification as part of initial
therapy in the setting of aggressive lymphoma and hepatic failure. The
literature on the use of treatment regimens for aggressive lymphoma in the
setting of hepatic dysfunction is reviewed. PURPOSE: Salvage chemotherapy followed by high-dose therapy and autologous
stem-cell transplantation (ASCT) is the standard treatment for relapsed diffuse
large B-cell lymphoma (DLBCL). Salvage regimens have never been compared; their
efficacy in the rituximab era is unknown.
PATIENTS AND METHODS: Patients with CD20(+) DLBCL in first relapse or who were
refractory after first-line therapy were randomly assigned to either rituximab,
ifosfamide, etoposide, and carboplatin (R-ICE) or rituximab, dexamethasone,
high-dose cytarabine, and cisplatin (R-DHAP). Responding patients received
high-dose chemotherapy and ASCT.
RESULTS: The median age of the 396 patients enrolled (R-ICE, n = 202; R-DHAP, n
= 194) was 55 years. Similar response rates were observed after three cycles of
R-ICE (63.5%; 95% CI, 56% to 70%) and R-DHAP (62.8%; 95 CI, 55% to 69%). Factors
affecting response rates (P < .001) were refractory disease/relapse less than
versus more than 12 months after diagnosis (46% v 88%, respectively),
International Prognostic Index (IPI) of more than 1 versus 0 to 1 (52% v 71%,
respectively), and prior rituximab treatment versus no prior rituximab (51% v
83%, respectively). There was no significant difference between R-ICE and R-DHAP
for 3-year event-free survival (EFS) or overall survival. Three-year EFS was
affected by prior rituximab treatment versus no rituximab (21% v 47%,
respectively), relapse less than versus more than 12 months after diagnosis (20%
v 45%, respectively), and IPI of 2 to 3 versus 0 to 1 (18% v 40%, respectively).
In the Cox model, these parameters were significant (P < .001).
CONCLUSION: In patients who experience relapse more than 12 months after
diagnosis, prior rituximab treatment does not affect EFS. Patients with early
relapses after rituximab-containing first-line therapy have a poor prognosis,
with no difference between the effects of R-ICE and R-DHAP. BACKGROUND: Salvage therapy for patients with refractory/relapsed B-cell
non-Hodgkin lymphoma (NHL) is based on polychemotherapy, followed by high-dose
therapy and autologous stem cell transplantation in eligible patients
(HDT/ASCT). R-DHAP combines rituximab with cisplatin, cytarabine, and
dexamethasone.
PATIENTS AND METHODS: We substituted cisplatin with oxaliplatin to avoid
nephrotoxicity and retrospectively analyzed a large series of 91 patients with
refractory/relapsed B-cell NHL to evaluate toxicities, response rates (RRs), and
survival. Median age at R-DHAX (rituximab/dexamethasone/cytarabine/oxaliplatin)
treatment was 60 years (range, 28-82 years). Renal insufficiency was present in
18 patients. The most frequent histologic subtypes were diffuse large B-cell
lymphoma (n = 42) and follicular lymphoma (n = 30). Seventeen patients (19%)
were naive to rituximab at time of R-DHAX.
RESULTS: Grade III/IV toxicities were mainly hematologic, including anemia (n =
9), neutropenia (n = 44), and thrombocytopenia (n = 47). Grade I/II neurologic
toxicities, sensitive or motor, were observed, and these were mainly transient
except for 3 cases of motor neuropathy associated with previous exposure to
vincristine. Neither renal toxicities nor degradation of previous renal
insufficiency were observed. The overall RR was 75%, with a complete RR of 57%,
with no statistical difference between patients previously treated with
rituximab versus without rituximab. At a median follow-up of 23 months, 2-year
probability rates of overall survival and progression-free survival were 75% and
43%, respectively, with a significant difference between patients treated with
HDT/ASCT and patients not eligible for HDT/ASCT.
CONCLUSION: R-DHAX is an efficient regimen in patients with relapsed/refractory
B-cell NHL even in elderly patients if hematologic toxicities are closely
managed. The aim of this study was to evaluate the efficacy and toxicity of two
chemotherapy regimens based on platinum and cytarabine in association with
etoposide and methylprednisolone (ESHAP) or with dexamethasone (DHAP) with or
without Rituximab (± R) in patients with refractory or a relapsed Primary
Central Nervous System Lymphoma (PCNSL). All consecutive patients from two
French centers with refractory or relapsed PCNSL treated with ESHAP/DHAP ± R
were included. We analyzed the overall response rate (ORR), toxicity and overall
survival (OS) after salvage chemotherapy. Intensive chemotherapy and
hematopoietic stem cell rescue (IC + HCR) was offered to patients less than 65
years of age and consisted of high-dose thiotepa, busulfan and cyclophosphamide.
These results were compared with two previously reported series of PCNSL
patients treated with the CYVE (high-dose cytarabine and etoposide) regimen at
relapse. Twenty-two patients received a total of 60 DHAP/ESHAP cycles (median 3;
range 1-5). The median age was 59 years. The ORR after salvage chemotherapy was
59%. Toxicity was mainly hematological, 18% of patients showing febrile
neutropenia. There was no treatment-related death. ESHAP or DHAP regimens led to
similar ORRs compared to the CYVE regimen in relapsed or refractory PCNSL,
although they seemed less toxic. The therapeutic results of the ESHAP/DHAP
regimens in relapsed or refractory PCNSL were also similar to those for relapsed
systemic non-Hodgkin's lymphomas (sNHL). Both chemotherapies, CYVE regimen and
ESHAP/DHAP are treatment options to be considered in relapsed or refractory
PCNSL, especially when IC + HCR is planned as a consolidation treatment. More
efforts are still needed to improve the ORR at relapse. PURPOSE: To evaluate the prognostic value of the cell of origin (COO) in
patients with relapsed/refractory diffuse large B-cell lymphoma (DLBLC),
prospectively treated by rituximab, dexamethasone, high-dose cytarabine, and
cisplatin (R-DHAP) versus rituximab, ifosfamide, carboplatin, and etoposide and
followed by intensive therapy plus autologous stem-cell transplantation on the
Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) trial.
PATIENTS AND METHODS: Among the 396 patients included on the trial, histologic
material was available for a total of 249 patients at diagnosis (n = 189
patients) and/or at relapse (n = 147 patients), which included 87 matched pairs.
The patient data were analyzed by immunochemistry for CD10, BCL6, MUM1, FOXP1,
and BCL2 expression and by fluorescent in situ hybridization for BCL2, BCL6 and
c-MYC breakpoints. The correlation with survival data was performed by using the
log-rank test and the Cox model.
RESULTS: Characteristics of immunophenotype and chromosomal abnormalities were
statistically highly concordant in the matched biopsies. In univariate analysis,
the presence of c-MYC gene rearrangement was the only parameter to be
significantly correlated with a worse progression-free survival (PFS; P = .02)
and a worse overall survival (P = .04). When treatment interaction was tested,
the germinal center B (GCB) -like DLBCL that was based on the algorithm by Hans
was significantly associated with a better PFS in the R-DHAP arm. In
multivariate analysis, independent prognostic relevance was found for the
GCB/non-GCB the Hans phenotype interaction treatment (P = .04), prior rituximab
exposure (P = .0052), secondary age-adjusted International Prognostic Index (P =
.039), and FoxP1 expression (P = .047). Confirmation was obtained by gene
expression profiling in a subset of 39 patients.
CONCLUSION: COO remains a major and independent factor in relapsed/refractory
DLBCL, with a better response to R-DHAP in GCB-like DLBCL. This needs
confirmation by a prospective study. Salvage chemotherapy followed by high-dose therapy and autologous stem cell
transplantation is the standard of treatment for chemosensitive relapses in
diffuse large B-cell lymphoma. The addition of rituximab to chemotherapy has
improved the response rate and failure-free survival after first-line treatment
and relapses. Fewer relapses are expected, although there is no consensus on the
best salvage regimen. The intergroup Collaborative Trial in Relapsed Aggressive
Lymphoma (CORAL) set the limits for this standard of treatment after first
comparing 2 salvage regimens: rituximab, ifosfamide, etoposide, and carboplatin
(R-ICE) and rituximab, dexamethasone, aracytine, and cisplatin (R-DHAP). There
was no difference in response rates or survivals between these salvage regimens.
Several factors affected survival: prior treatment with rituximab, early relapse
(< 12 months), and a secondary International Prognostic Index score of 2-3. For
patients with 2 factors, the response rate to salvage was only 46%, which
identified easily a group with poor outcome. Moreover, patients with an ABC
subtype or c-MYC translocation responded poorly to treatment. More than 70% of
patients will not benefit from standard salvage therapy, and continued progress
is needed. Studies evaluating immunotherapy after transplantation, including
allotransplantation, new conditioning regimens with radioimmunotherapy and other
combinations of chemotherapy based on diffuse large B-cell lymphoma subtype, are
discussed herein. Early relapses and/or patients refractory to upfront
rituximab-based chemotherapy have a poor response rate and prognosis. A better
biological understanding of these patients and new approaches are warranted. |
Are psammoma bodies characteristic to meningiomas? | Yes, psammoma bodies are commonly seen and are characteristic to meningiomas. However, they can be also present in other types of tumors or non-neoplastic tissues. | In this study we analyzed the morphologic and ultrastructural characteristics of
the psammoma bodies in ten meningiomas of different histologic subtypes,
characterizing the components of the psammoma body and the elements of the
tumor, such as the capillaries and degenerative cells that have been classically
considered as initiators of the formation of these calcareous is structures. The
discussion considers the diagnostic interest of psammoma bodies in central
nervous system tumors, as well as its histogenesis. The different points of view
explaining biological mineralization in other territories of the organism are
analyzed, as well as the formation of psammoma bodies, comparing the morphologic
data obtained in this study. It is concluded that the mineralization of the
psammoma bodies is induced principally by the collagen fibers synthesized by the
meningocytes and that the form of mineralization is spherical and growth is
radial, controlled by the tumoral cells. A case of the cytologically verified intrathecal dissemination of benign
meningiomas was reported. A 36-year-old man was admitted to our hospital because
trans-sphenoidal surgery was planned for the repair of CSF rhinorrhea caused by
a recurrent pituitary adenoma. The CT scan demonstrated incidental multiple
tumors spread throughout in the pontomedullary and ambient cisterns. The head
and spinal MRI showed more than fifteen small tumors in the posterior fossa and
the thoracic spinal canal. CSF cytology revealed benign fibroblastic or
meningotheliomatous meningioma with whorl formation and psammoma body. Several
comments were made on the mechanism of the intrathecal dissemination of the
meningiomas. Forty-four previously diagnosed surgical biopsy specimens of oligodendroglioma
with calcification from the file of Veterans General Hospital-Taipei between
1977-1985 form the base of this study. Thirty-two patients (72.8%) had varied
degree of calcification deposits in tumor parenchyma related to blood vessels
while 12 patients (27.2%) in tumor parenchyma unrelated to blood vessels. Ten
patients (22.7%) disclosed calcification at the vicinity of tumor tissue.
Histochemical studies of the calcified deposits revealed no trace minerals other
than calcium and iron. Electron microscopic examination of the calculi showed
membrane-bound vesicles and radially precipitated crystals that simulated
hydroxyapatite of psammoma body in meningioma. There was no statistically
significant correlation between the demise or recurrence of patients and the
calcification of tumor when Chi-squared test was applied. Psammoma bodies in meningocytic whorls were investigated by electron microscopy.
In some whorls, connective tissue fibers were seen and membrane-bound vesicles
were contiguous to degenerated cells. Some small vesicles, 0.1 to 0.5 micron in
diameter, were outlined by plasma membrane (matrix vesicles), other larger ones,
about 1 to several micron in diameter, were invested by a thick wall (matrix
giant bodies). Mineralized deposits were frequent in these vesicles and
occasionally large masses of mineralized connective tissue fibers (psammoma
bodies) were seen. Analysis of the material in the mineralized vesicles and
fibers, using an energy dispersive X-ray microanalyzer, showed that both calcium
and phosphorous were evident and hydroxyapatite was substantiated using an X-ray
diffractometer. Psammoma body formation in the meningocytic whorls may represent
degeneration in some whorls of the central cells which contain connective tissue
fibers, producing cell debris such as membrane invested vesicles. Subsequently,
calcification occurs in these vesicles, and the mineralization process extends
to neighboring connective tissue fibers. The calcified mass forms a psammoma
body. Calcification in the stroma of adult telencephalic choroid plexus was studied of
20 postmortem brains and one biopsy by light microscopy, transmission and
scanning electron microscopy and compared with calcification in psammoma bodies
in normal arachnoid, five spinal meningiomas and in calcospherites of six pineal
glands. Fifteen fetal and newborn choroid plexuses were also examined by light
microscopy. Calcium deposition was observed in the subepithelial regions of the
adult choroid plexus, in the walls of blood vessels but was mostly seen in
spherical psammoma bodies. Collagen whorls 20-60 microns in diameter and
surrounded by arachnoid cells, were observed in the stroma of the choroid
plexus; calcium, phosphorous and iron were deposited in the collagen whorls to
form psammoma bodies. Matrix vesicles and spicules resembling hydroxyapatite
were associated with the arachnoid cells surrounding the collagen whorls and
with the collagen fibres within the whorls. The dense amorphous calcified core
of each psammoma body was surrounded by an outer coating of entwined collagen
fibres readily visible by scanning electron microscopy. Similar psammoma bodies
were occasionally observed in normal arachnoid. Psammoma bodies in meningiomas
resembled those in the choroid plexus stroma. Calcospherites in the pineal
differed from psammoma bodies; they were lobulated, more irregular in shape and
did not have a collagen base. The results of this study suggest that psammoma
bodies in the choroid plexus, as in meningiomas, form by a process of dystrophic
calcification associated with arachnoid cells and collagen fibres. The presence
of iron in the choroid plexus psammoma bodies may be a result of haemorrhage
into the stroma. The mechanism of calcification in pineal remains unclear. The fine structure of psammoma bodies was examined in four cases of fibroblastic
meningioma. In general, large numbers of various-sized calcified bodies
(psammoma bodies) were scattered among the interstitial fibers. In these bodies,
the smallest calcific site was found in the extracellular membrane-bound matrix
vesicles, which measured approximately 0.1 to 0.2 mu in diameter. In addition,
extracellular "matrix giant bodies," with or without hydroxyapatite aggregates
and measuring up to 3 mu in diameter, were frequently encountered. These bodies
were apparently invested with single, double, or multiple concentric walls
averaging nearly 0.1 mu thick. The presumably originated from the neoplastic
cells as a consequence of cytoplasmic residuals associated with cellular
degeneration or necrotic cell processes. Hydroxyapatite crystals precipitated
repeatedly within the bodies. The precipitate may gradually aggregate within the
bodies, and gather in clusters, resulting in a large psammoma body. Finally,
collagen fibers around the calcified giant bodies accrued deposits of apatite
crystals to make a huge psammoma body. These findings suggest that both matrix
giant bodies and matrix vesicles may serve as initial nidus of calcification of
psammoma bodies in fibroblastic meningioma. Consequently, this mineralization
process may represent a certain dystrophic calcification of meningocytic cells. Twenty human meningiomas were examined for IgG and IgM by the direct
immunofluorescence of immunoperoxidase methods, or both. IgG was conspicuously
found in and around the blood vessels, whorls, and psammoma bodies. It was also
clearly present on the cytoplasmic membranes of the tumour cells. On the other
hand, IgM was seen only within the blood vessels. Significance of these findings
is briefly discussed including possible humoral immune reactions in regard to
whorl and psammoma body formation in meningioma. OBJECTIVE: To examine the etiology of and clinicopathologic findings associated
with meningioangiomatosis and to evaluate MIB1 immunoreactivity (marker of cell
proliferation) in this lesion.
DESIGN: Retrospective surgical pathology series of three patients.
SETTING: Tertiary referral center with a high volume of epilepsy surgery.
PATIENTS: Individuals with meningioangiomatosis who underwent surgical resection
of the lesion.
RESULTS: Three patients aged 11, 14, and 32 years (two females, one male)
comprise the study. All patients presented with a history of intractable
seizures (2, 3, and 30 years duration). None of the patients had von
Recklinghausen's disease. All three patients underwent gross total resection of
the lesion, and postoperative seizure-free intervals range from 7 to 15 months.
Histologically, the lesion is characterized by a proliferation of blood vessels
and meningothelial cells arranged around vessels in the meninges, cortex, and
underlying white matter. Psammoma body formation or dystrophic mineralization
and gliosis of the intervening parenchyma was observed in all three cases.
Bodian stains failed to demonstrate neurofibrillary tangles within intervening
parenchymal neurons. Focal epithelial membrane antigen positivity was observed
in two lesions and was absent in one. MIB1 staining was observed in two lesions
(MIB1 index = 0.8 and 0.6) within the meningothelial cell nuclei and was absent
in one case.
CONCLUSIONS: Meningioangiomatosis is a rare malformative or hamartomatous lesion
of the central nervous system characterized by a proliferation of vessels and
perivascular cuffs of meningothelial cells. Absent MIB1 immunoreactivity or low
MIB1 indices in the lesion support the clinical impression of a slow-growing
lesion and further support a malformative or hamartomatous etiology. Calcification such as psammoma body is sometimes found especially in spinal cord
meningioma but ossification of the meningeal tumor was rarely observed. Two
cases of ossificated spinal cord meningiomas, located extramedullary
intradurally apart from spinal bone, were clinicopathologically analyzed. The
patients were 75-year-old female with meningioma at T9-10 level and 60-year-old
female with one at T6-8 level. With pathological examination, concentration of
psammoma bodies is not related to the formation of bone tissue. Metaplasia of
arachnoid cell is seemed to be the origin of the bone structure. Intraosseous tumor was found in a 63 year old male patient with lung cancer
during metastatic work-up study. Plain skull X-ray film showed a large
osteolytic lesion in the left temporo-parietal bone. Bone and Ga scintigrams
revealed an increased activity in this lesion. CT scan demonstrated an isodense
lentiform configuration, which was homogeneously enhanced with contrast medium.
It was iso-intense in T1, and high-intense in T2 and proton weighted MR images.
The tumor was removed en bloc with surrounding normal bone. The attached dura
was markedly thickened, but there was no tumor infiltration. Histological
diagnosis was transitional meningioma with psammoma body. Postoperatively he was
transferred back to Okinawa Hospital and right lower lobectomy was performed for
the lung tumor. Histological diagnosis was well-differentiated adenocarcinoma.
We examined immunohistochemically p53 protein expression, but p53 immunoactivity
was observed in neither tumor. Multiple neoplasms associated with brain tumor
are relatively rare, but the incidence will increase in the future as the
diagnostic work-up develops. Pathological diagnosis will give us the decisive
opportunity for proper treatment. It is to be stressed that, in multiple
neoplasms, care should be taken to avoid misdiagnosis of metastasis. Seven solitary fibrous tumors (SFTs) of the meninges are presented and their
clinicopathologic features are compared with those of 64 fibrous meningiomas
(FM). Patients with SFT included 5 females and 2 males age 47 to 73 years. The
dura-based tumors involved the parasagittal region (1), tentorium (2),
cerebellopontine angle (2), and spinal region (2). One each showed invasion of
brain and of a spinal nerve root. Of four SFTs with at least 1-year follow-up,
one subtotally resected example recurred. No tumors metastasized. All consisted
of spindle cells disposed in fascicles between prominent, eosinophilic bands of
collagen. Whorls and storiform cell arrangements were lacking. Mitoses ranged
from 1 to 7/10 400 x fields. MIB-1 labeling indices ranged from 1% to 18% (mean
4%). All were PAS negative and showed strong immunoreactivity for vimentin and
CD34. Of cases studied, half were estrogen and all were progesterone receptor
immunopositive. The majority (72%) of FMs occurred in females and most (72%)
were supratentorial. Recurrence was noted in 15%. Mitotic activity varied from 0
to 3 mitoses per 10 400 x fields (mean < 1). MIB-1 labeling indices ranged from
1% to 5% (mean 1.5%). Unlike SFT, FMs were glycogen-containing and variously
exhibited a storiform pattern (13 of 20), psammoma body formation (9 of 20), and
calcification of collagen (4 of 20). Immunoreactivities included vimentin
(100%), focal to patchy EMA (80%), S-100 protein (80%), collagen IV (25%), and
patchy, mild-to-moderate CD34 staining (60%). Of cases studied, nearly half were
estrogen and all were progesterone receptor staining positive. Meningeal SFTs
represent a distinct morphologic entity, the morphologic and immunohistochemical
features of which differ from those of FM and suggest a histogenetic
relationship to pleural SFT. Although a minority histologically appear to be low
grade maligt, our limited experience suggests that they behave in a benign
fashion. The classification of mesenchymal tumors affecting the central nervous
system must be expanded to include SFT. In contrast to the inner structure, three-dimensional structure of psammona
bodies in meningiomas is not well defined. This study examined three cultured
meningiomas, in which surface observation of psammoma bodies might be easier
than in the tumor tissues since influence of interposing connective tissue is
minimized in tissue culture. Early culture revealed that psammoma bodies with
frank calcification were suspended in the tissue culture medium, and so were
they collected, centrifuged, and then processed for electron microscopy.
Ultrastructurally, psammoma bodies were mostly spherical in shape and composed
of a core of dense calcification and surrounding collagen fiber bundles. Apart
from psammoma bodies, round bodies with concentric lamination like a
transversely cut onion were frequently noted by light microscopy. These bodies
were composed mainly of tangles of collagen fibers emerged from surrounding
tumor cell processes. The results suggest that psammoma bodies in meningiomas
arise in part from meningothelial whorls due to collagen production by tumor
cells followed by obliteration and disappearance of tumor cell processes,
although some of the alternative pathways for psammoma body formation proposed
by other investigators cannot be ruled out by this study. We report a rare case of microcystic meningioma presenting with atypical
neuroradiological features. A 76-year-old woman was admitted to our hospital in
October, 1996 because of head heaviness without obvious neurological deficits.
Previous CT scan in 1989 revealed no abnormality, but a subsequent scan in 1992
showed a low-density area in the right frontal region. Since then the patient
has been followed up as a case of cerebral infarction. At the time of the
current admission, CT scan disclosed the low-density area as having grown to the
size of 4 cm and heterogeneously enhanced after injection of contrast medium. On
MRI the mass lesion was depicted as low-intensity on T1-weighted image and
high-intensity on T2-weighted image. The mass was heterogeneously enhanced after
administration of Gd-DTPA. Right internal and external carotid angiograms
revealed neither tumor feeder nor tumor stain. Craniogram showed neither
hyperostosis nor bone erosion, and yet bone scintigram demonstrated markedly
increased uptake involving the right frontal bone near the tumor. At surgery the
tumor was found to have originated from the dura in the right frontal convexity,
which was well demarcated and separated from the surrounding brain tissue. Total
removal designated as Simpson grade I procedure was accomplished. Light
microscopy revealed abundant microcysts of varied size throughout the tumor
tissue with the presence of whorl formation and psammoma body, but no maligcy
was indicated. Electron microscopy further demonstrated interdigitation of the
neighboring cell membranes, desmosomes, and intracytoplasmic filaments, which
are pathognomonic findings of meningiomas. The microcysts were seen to reside in
the extracellular spaces rather than in the cytoplasm. With these pathological
findings, the tumor was finally diagnosed as microcystic meningioma. |
Which are the most common symptoms in Lambert-Eaton Myasthenic Syndrome? | Lambert-Eaton myasthenic syndrome is a neuromuscular junction disorder characterized by proximal limb muscle weakness, fatigability, decreased deep-tendon reflexes, and autonomic symptoms. There are 2 forms of Lambert-Eaton myasthenic syndrome: one most frequently associated with small-cell lung cancer (P-Lambert-Eaton myasthenic syndrome) and the other that is a pure autoimmune form (NP-Lambert-Eaton myasthenic syndrome) | INTRODUCTION: Paraneoplastic neurological syndromes are rare non-metastatic
complications of cancer that have an immune-mediated etiology. The Lambert-Eaton
myasthenic syndrome (LEMS) is a neuromuscular disorder, often associated with
small cell lung carcinoma (SCLC), which is characterized by reduced quantal
release of acetylcholine from the motor nerve terminals. LAMBERT-EATON
MYASTHENIC SYNDROME: The Lambert-Eaton Myasthenic Syndrome (LEMS) is
characterized by proximal muscle weakness initially affecting gait, autonomic
symptoms (dry mouth, constipation, erectile failure) and augmentation of
strength during initial voluntary activation. Symptomatic treatment of the
junctional disorder is based on cholinergic drugs, immunosuppression,
immunomodulation and physical therapy useful in case of unsuccessful
antineoplastic therapy.
CASE REPORT: A rare case of ovarian cancer with Eaton-Lambert syndrome is
reported. A 50-year-old woman was admitted to the gynecologic department,
complaining of weakness and pain in her arms and shoulders. Physical therapy
resulted in partial improvement. Treatment of paraneoplastic syndrome markedly
improves the quality of life of cancer patients. Patients presenting with this
syndrome should undergo a careful evaluation for the presence of an occult
maligcy. The Lambert-Eaton myasthenic syndrome (LEMS) is a disease of neuromuscular
transmission in which autoantibodies against the P/Q-type voltage-gated calcium
channel (VGCC) at the presynaptic nerve terminal play a major role in decreasing
quantal release of acetylcholine (ACh), resulting in skeletal muscle weakness
and autonomic symptoms. It is associated with cancer, particularly small-cell
lung carcinoma (SCLC), in 50-60% of LEMS patients; the nerve terminal and
carcinoma cells apparently share a common antigen (VGCC), suggesting an
immunological cross-reactivity that may lead to the neurological abnormality.
Non-tumor LEMS has a strong association with HLA-DR3-B8. In approximately 15% of
LEMS patients, no anti-P/Q-type VGCC antibodies are found, suggesting
recognition of other targets(s). The VGCC-associated protein synaptotagmin could
be one candidate, because it acts as an exocytotic calcium receptor, is
implicated in fast ACh release; its N-terminus is exposed extracellularly during
exocytosis and it is expressed in SCLC. Antibodies against synaptotagmin-1 were
detected in both anti-VGCC-positive and -negative LEMS patients (20%), and it
can be immunogenic, allowing induction of an animal model of LEMS. Another
candidate target is the M1-type presynaptic muscarinic ACh receptor (M1 mAChR),
also expressed extracellularly on motor nerve terminals; it modulates
cholinergic transmission, linking to P/Q-type VGCC. In our series of 25 LEMS
patients with and without SCLC, anti-M1 mAChR antibodies were prevalent in both
anti-VGCC-positive and -negative LEMS patients. Autonomic symptoms seemed more
frequent in the latter; serum from one of them passively transferred LEMS-type
electrophysiological defects to mice. As a compensatory mechanism, researchers
in Oxford suggested a shift in the dependence of ACh release from the P/Q-type
to other types of VGCC. We have also focused on G protein-coupled mAChRs and
neurotrophins, which may affect both P/Q-type VGCC and clathrin-independent
"kiss-and-run" synaptic vesicle recycling (fast-mode of endocytosis) via protein
kinase C activation. We hypothesize that these signaling cascades help to
compensate for the immune-mediated defects in calcium entry in LEMS,
compensation that may frequently be restricted by the coincident anti-M1 mAChR
antibodies in this disease. In Lambert-Eaton myasthenic syndrome (LEMS), antibodies against presynaptic
voltage-gated calcium channels reduce the quantal release of acetylcholine,
causing muscle weakness and autonomic dysfunction. More than half of the
affected patients have associated small cell lung cancer, and thorough screening
for an underlying maligcy is crucial. The mainstay of treatment for LEMS is
symptomatic but immunotherapy is needed in more severely affected patients.
Symptomatic therapies aim at increasing the concentration of acetylcholine at
the muscle endplate. While acetylcholinesterase inhibitors were the first drugs
to be used for the amelioration of symptoms, 3,4-diaminopyridine (3,4-DAP,
amifampridine) has been shown to be more effective. 3,4-DAP blocks presynaptic
potassium channels, thereby prolonging the action potential and increasing
presynaptic calcium concentrations. This then results in increased quantal
release of acetylcholine. The efficacy of 3,4-DAP for increasing muscle strength
and resting compound muscle action potentials has been demonstrated by four
placebo-controlled trials. Side effects are usually mild, and the most
frequently reported are paresthesias. The most common serious adverse events are
epileptic seizures. 3,4-DAP is currently the treatment of choice in patients
with Lambert-Eaton myasthenic syndrome. |
What is the pyroptotic pathway? | Pyroptosis is an inflammasome-mediated programmed cell death pathway. | Macrophages mediate crucial innate immune responses via caspase-1-dependent
processing and secretion of interleukin 1β (IL-1β) and IL-18. Although infection
with wild-type Salmonella typhimurium is lethal to mice, we show here that a
strain that persistently expresses flagellin was cleared by the cytosolic
flagellin-detection pathway through the activation of caspase-1 by the NLRC4
inflammasome; however, this clearance was independent of IL-1β and IL-18.
Instead, caspase-1-induced pyroptotic cell death released bacteria from
macrophages and exposed the bacteria to uptake and killing by reactive oxygen
species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated
Legionella pneumophila and Burkholderia thailandensis by cytokine-independent
mechanisms. This demonstrates that activation of caspase-1 clears intracellular
bacteria in vivo independently of IL-1β and IL-18 and establishes pyroptosis as
an efficient mechanism of bacterial clearance by the innate immune system. Burkholderia cenocepacia infections in CF patients involve heightened
inflammation, fatal sepsis, and high antibiotic resistance. Proinflammatory
IL-1β secretion is important in airway inflammation and tissue damage. However,
little is known about this pathway in macrophages upon B. cenocepacia infection.
We report here that murine macrophages infected with B. cenocepacia K56-2
produce proinflammatory cytokine IL-1β in a TLR4 and caspase-1-mediated manner.
We also determined that the OPS (O antigen) of B. cenocepacia LPS contributes to
IL-1β production and pyroptotic cell death. Furthermore, we showed that the
malfunction of the CFTR channel augmented IL-1β production upon B. cenocepacia
infection of murine macrophages. Taken together, we identified eukaryotic and
bacterial factors that contribute to inflammation during B. cenocepacia
infection, which may aid in the design of novel approaches to control pulmonary
inflammation. BACKGROUND: Macrophage death in advanced lesion has been confirmed to play an
important role in plaque instability. However, the mechanism underlying lesion
macrophage death still remains largely unknown.
METHODS AND RESULTS: Immunohistochemistry showed that caspase-1 activated in
advanced lesion and co-located with macrophages and TUNEL positive reaction. In
in-vitro experiments showed that ox-LDL induced caspase-1 activation and this
activation was required for ox-LDL induced macrophages lysis, IL-1β and IL-18
production as well as DNA fragmentation. Mechanism experiments showed that CD36
and NLRP3/caspase-1/pathway involved in ox-LDL induced macrophage pyroptosis.
CONCLUSION: Our study here identified a novel cell death, pyroptosis in ox-LDL
induced human macrophage, which may be implicated in lesion macrophages death
and play an important role in lesion instability. The fungal pathogen Candida albicans causes macrophage death and escapes, but
the molecular mechanisms remained unknown. Here we used live-cell imaging to
monitor the interaction of C. albicans with macrophages and show that C.
albicans kills macrophages in two temporally and mechanistically distinct
phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a
proinflammatory macrophage death. Pyroptosis is controlled by the developmental
yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type
C. albicans hyphae cause significantly less macrophage killing for up to 8 h
postphagocytosis. After the first 8 h, a second macrophage-killing phase is
initiated. This second phase depends on robust hyphal formation but is
mechanistically distinct from pyroptosis. The transcriptional regulator Mediator
is necessary for morphogenesis of C. albicans in macrophages and the
establishment of the wild-type surface architecture of hyphae that together
mediate activation of macrophage cell death. Our data suggest that the defects
of the Mediator mutants in causing macrophage death are caused, at least in
part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae
of apparently wild-type morphology but is defective in triggering early
macrophage death shows a breakdown of cell surface architecture and reduced
exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and
pathogen pathways for macrophage killing. The current model of mechanical
piercing of macrophages by C. albicans hyphae should be revised to include
activation of pyroptosis by hyphae as an important mechanism mediating
macrophage cell death upon C. albicans infection. IMPORTANCE Upon phagocytosis
by macrophages, Candida albicans can transition to the hyphal form, which causes
macrophage death and enables fungal escape. The current model is that the highly
polarized growth of hyphae results in macrophage piercing. This model is
challenged by recent reports of C. albicans mutants that form hyphae of
wild-type morphology but are defective in killing macrophages. We show that C.
albicans causes macrophage cell death by at least two mechanisms. Phase 1
killing (first 6 to 8 h) depends on the activation of the pyroptotic programmed
host cell death by fungal hyphae. Phase 2 (up to 24 h) is rapid and depends on
robust hyphal formation but is independent of pyroptosis. Our data provide a new
model for how the interplay between fungal morphogenesis and activation of a
host cell death pathway mediates macrophage killing by C. albicans hyphae. The macrophage NLRC4 inflammasome drives potent innate immune responses against
Salmonella by eliciting caspase-1-dependent proinflammatory cytokine production
(e.g., interleukin-1β [IL-1β]) and pyroptotic cell death. However, the potential
contribution of other cell types to inflammasome-mediated host defense against
Salmonella was unclear. Here, we demonstrate that neutrophils, typically viewed
as cellular targets of IL-1β, themselves activate the NLRC4 inflammasome during
acute Salmonella infection and are a major cell compartment for IL-1β production
during acute peritoneal challenge in vivo. Importantly, unlike macrophages,
neutrophils do not undergo pyroptosis upon NLRC4 inflammasome activation. The
resistance of neutrophils to pyroptotic death is unique among
inflammasome-signaling cells so far described and allows neutrophils to sustain
IL-1β production at a site of infection without compromising the crucial
inflammasome-independent antimicrobial effector functions that would be lost if
neutrophils rapidly lysed upon caspase-1 activation. Inflammasome pathway
modification in neutrophils thus maximizes host proinflammatory and
antimicrobial responses during pathogen challenge. Diabetic cardiomyopathy is a common cardiac condition in patients with diabetes
mellitus, which can result in cardiac hypertrophy and subsequent heart failure,
associated with pyroptosis, the pro-inflammatory programmed cell death.
MicroRNAs (miRNAs), small endogenous non-coding RNAs, have been shown to be
involved in diabetic cardiomyopathy. However, whether miRNAs regulate pyroptosis
in diabetic cardiomyopathy remains unknown. Our study revealed that mir-30d
expression was substantially increased in streptozotocin (STZ)-induced diabetic
rats and in high-glucose-treated cardiomyocytes as well. Upregulation of mir-30d
promoted cardiomyocyte pyroptosis in diabetic cardiomyopathy; conversely,
knockdown of mir-30d attenuated it. In an effort to understand the signaling
mechanisms underlying the pro-pyroptotic property of mir-30d, we found that
forced expression of mir-30d upregulated caspase-1 and pro-inflammatory
cytokines IL-1β and IL-18. Moreover, mir-30d directly repressed foxo3a
expression and its downstream protein, apoptosis repressor with caspase
recruitment domain (ARC). Furthermore, silencing ARC by siRNA mimicked the
action of mir-30d: upregulating caspase-1 and inducing pyroptosis. These
findings promoted us to propose a new signaling pathway leading to cardiomyocyte
pyroptosis under hyperglycemic conditions: mir-30d↑→foxo3a↓→
ARC↓→caspase-1↑→IL-1β, IL-18↑→pyroptosis↑. Therefore, mir-30d may be a promising
therapeutic target for the management of diabetic cardiomyopathy. |
Which types of bacterial microflora are associated with the progression of peri-implantitis? | Bacteria such as A. actinomycetemcomitans, P. gingivalis, T. forsythensis, T. denticola, P. intermedia and F. nucleatum are associated with the progression of peri-implantitis. | This study examines the microbiota associated with the progression of
experimental peri-implantitis and periodontitis induced concurrently in
partially edentulous adult monkeys. Root-form and plate-form implants with fixed
prosthesis in place for at least 12 months and their corresponding opposite
molar teeth were ligated for 6 months. The microbiota in plaque around these
ligated dental implants and molars were studied at 0, 1, 2, 3, and 6 months
post-ligation. Plaque samples were analyzed by dark-field microscopy and
selective and non-selective culture. Putative periodontal pathogens were
detected as a major component of the microbiota cultured from plaque samples
obtained from experimental peri-implantitis sites. Overall, the types and
relative proportions of putative periodontal pathogens in plaque associated with
ligature-induced peri-implantitis and ligature-induced periodontitis were
similar. Only levels of anaerobic Actinomyces and spirochetes were significantly
different between both sites. Spirochete levels were significantly higher at
peri-implantitis sites when compared with levels at periodontitis sites after 6
months, and spirochete levels increased significantly between 0 and 6 months
post-ligation at implant sites. Levels of spirochetes correlated significantly
with probing depth and bone loss at peri-implantitis sites. Overall, Actinomyces
levels were higher at periodontitis sites. Porphyromonas species were not
detected continuously as part of the peri-implantitis microbiota. In conclusion,
this study finds that the microbiota associated with the progression of
experimental peri-implantitis and periodontitis occurring concurrently in
partially edentulous mouths are similar. Many oral pathologies, such as dental caries, periodontal disease and
peri-implantitis are plaque-related. Dental plaque is a microbial biofilm formed
by organisms tightly bound to a solid substrate and each other by means of an
exopolymer matrix. Bacteria exhibit different properties when contained within a
biofilm. Knowing the mechanisms controlling the formation and development of
biofilms can help to understand the emergence and progression of such
pathologies and to plan effective treatment. Most periodontal pathogens are
common saprophytes of the oral cavity, expressing their virulence only in a
susceptible host or when some changes come about in the oral environment.
Physical, metabolic and physiological interactions may cause positive or
negative effects among the various microbiota present. Such mechanisms of
antagonism/synergy select the bacterial population and alterations of its
composition affect the balance with the host and may lead to pathology. The
effectiveness of antimicrobial agents, as measured through in vitro tests, is
dramatically reduced in vivo due to the properties of the microbial community:
mature, intact biofilms are less sensitive to such agents, as the exopolymer
matrix, bacterial enzymes and slow growth rate hinder the action of
chemotherapeutic agents. The present literature review aims to examine the most
representative studies, focusing on the characteristics of bacterial communities
and the crucial shift from oral health to plaque-related diseases. The search for the etiologic agents of periodontal diseases started in the
Golden Era of medical bacteriology, when the etiologic agents of many bacterial
infections were isolated and characterized. After the initial enthusiasm in
establishing the infectious nature and the true agents of periodontal diseases,
this concept was virtually ignored for the next four decades. Until the early
1970s treatment regimens based on the non-specific plaque hypothesis were
directed towards a non-specific reduction in plaque amount. Later, the specific
plaque hypothesis established the role of some microorganisms such as A.
actinomycetemcomitans, P. gingivalis, T. forsythensis, T. denticola, P.
intermedia and F. nucleatum in different forms of periodontal diseases. It was
recently suggested that these suspected periodontal pathogens seem to not act
alone and interactions between species, especially the balance between
pathogenic and beneficial species affect both progression of disease and
response of tissues to periodontal therapy. Nowadays it is well established that
one of the goals of therapy is to control such periodontal pathogens. Among the
most commonly used therapies to treat periodontal infections are scaling and
root planing (SRP), supragingival plaque control and periodontal surgeries. Many
studies confirmed the reduction of "red complex" species by SRP, and apically
repositioned flap can lead to an additional beneficial effect in the subgingival
microbiota by decreasing levels of "red" and "orange complexes" species.
Furthermore, the level of plaque control maintained by the patients has been
considered a crucial step in preventing recurrence of destructive periodontitis. AIM: To analyze the microbial profile around teeth and implants following
ligature removal in experimental periodontitis and peri-implantitis in dogs.
MATERIAL AND METHODS: Four implants with similar geometry and with two different
surface characteristics (implant A: turned/implant B: TiUnite; NobelBiocare AB)
were placed pairwise in the right side of the mandible 3 months after tooth
extraction in five dogs. Experimental periodontitis and peri-implantitis were
initiated 3 months later by ligature placement around implants and mandibular
premolars and plaque formation. The ligatures were removed after 10 weeks.
Microbial samples were obtained using paper points immediately after ligature
removal, at 10 and 25 weeks after ligature removal. The microbiological analysis
was performed by "checkerboard" DNA-DNA hybridization, including a panel of 16
bacterial species.
RESULTS: The amount of bone loss that occurred during the period following
ligature removal was significantly larger at implants with a modified surface
than at implants with a turned surface and at teeth. The microbiological
analysis revealed that the total bacterial load increased during the period
following ligature removal and established an anaerobic Gram-negative
microflora.
CONCLUSION: It is suggested that the large variation in regard to the microbial
profiles makes interpretation of a correlation between disease progression and
microbial profiles difficult. |
What are the known families of deadenylases? | All known deadenylases belong to either the DEDD or the exonuclease–endonuclease–phosphatase (EEP) superfamily. Members of DEDD include the POP2, CAF1Z, PARN and PAN2 families. Members of EEP include the CCR4, Nocturnin, ANGEL and 2'-PDE families. | BACKGROUND: The yeast yCCR4 factor belongs to the CCR4-NOT transcriptional
regulatory complex, in which it interacts, through its leucine-rich repeat (LRR)
motif with yPOP2. Recently, yCCR4 was shown to be a component of the major
cytoplasmic mRNA deadenylase complex, and to contain a fold related to the
Mg2+-dependent endonuclease core.
RESULTS: Here, we report the identification of nineteen yCCR4-related proteins
in eukaryotes (including yeast, plants and animals), which all contain the yCCR4
endonuclease-like fold, with highly conserved CCR4-specific residues.
Phylogenetic and genomic analyses show that they form four distinct families,
one of which contains the yCCR4 orthologs. The orthologs in animals possess a
leucine-rich repeat domain. We show, using two-hybrid and far-Western assays,
that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2,
in a LRR-dependent manner.
CONCLUSIONS: We have identified the mammalian orthologs of yCCR4 and have shown
that the human member binds to the human yPOP2 homologs, thus strongly
suggesting conservation of the CCR4-NOT complex from yeast to human. All members
of the four identified yCCR4-related protein families show stricking
conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal
domain and therefore constitute a new family of potential deadenylases in
mammals. The CCR4 family proteins are 3'-5'-deadenylases that function in the first step
of the degradation of poly(A) mRNA. Here we report the purification to
homogeneity of the yeast CCR4 protein and the analysis of its substrate
specificities. CCR4 deadenylated a 7N+23A substrate (seven nucleotides followed
by 23 A residues) in a distributive manner. Only small differences in CCR4
activity for different A length substrates were observed until only 1 A residue
remained. Correspondingly, the K(m) for a 25N+2A substrate was found to be at
least 20-fold lower than that for a 26N+1A substrate, although their V(max)
values differed by only 2-fold. In addition, the total length of the RNA was
found to contribute to CCR4 activity: up to 17 nucleotides (not necessarily
poly(A)) could be recognized by CCR4. Poly(U), poly(C), and poly(G) were also
found to be 12-30-fold better inhibitors of CCR4 compared with poly(A),
supporting the observation that CCR4 contains a non-poly(A)-specific binding
site. Surprisingly, even longer substrates (>/=45 nucleotides) stimulated CCR4
to become a processive enzyme, suggesting that CCR4 undergoes an additional
transition in the presence of such substrates. CCR4 also displayed no difference
in its activity with capped or uncapped RNA substrates. These results indicate
that CCR4 recognition of its RNA substrates involves several features of the RNA
that could be sites in vivo for controlling the rate of specific mRNA
deadenylation. In Saccharomyces cerevisiae, a large complex, known as the Ccr4-Not complex,
containing two nucleases, is responsible for mRNA deadenylation. One of these
nucleases is called Pop2 and has been identified by similarity with PARN, a
human poly(A) nuclease. Here, we present the crystal structure of the nuclease
domain of Pop2 at 2.3 A resolution. The domain has the fold of the DnaQ family
and represents the first structure of an RNase from the DEDD superfamily.
Despite the presence of two non-canonical residues in the active site, the
domain displays RNase activity on a broad range of RNA substrates. Site-directed
mutagenesis of active-site residues demonstrates the intrinsic ability of the
Pop2 RNase D domain to digest RNA. This first structure of a nuclease involved
in the 3'-5' deadenylation of mRNA in yeast provides information for the
understanding of the mechanism by which the Ccr4-Not complex achieves its
functions. The yeast Pop2 protein, belonging to the eukaryotic Caf1 family, is required for
mRNA deadenylation in vivo. It also catalyzes poly(A) degradation in vitro, even
though this property has been questioned. Caf1 proteins are related to RNase D,
a feature supported by the recently published structure of Pop2. Yeast Pop2
contains, however, a divergent active site while its human homologs harbor
consensus catalytic residues. Given these differences, we tested whether its
deadenylase activity is conserved in the human homologs Caf1 and Pop2. Our data
demonstrate that both human factors degrade poly(A) tails indicating their
involvement in mRNA metabolism. mRNA poly(A) tails affect translation, mRNA export and mRNA stability, with
translation initiation involving a direct interaction between eIF4G and the
poly(A)-binding protein Pab1. The latter factor contains four RNA recognition
motifs followed by a C-terminal region composed of a linker and a PABC domain.
We show here that yeast mutants lacking the C-terminal domains of Pab1 display
specific synthetic interactions with mutants in the 5'-3' mRNA decay pathway.
Moreover, these mutations impair mRNA decay in vivo without significantly
affecting mRNA export or translation. Inhibition of mRNA decay occurs through
slowed deadenylation. In vitro analyses demonstrate that removal of the Pab1
linker domain directly interferes with the ability of the Pop2-Ccr4 complex to
deadenylate the Pab1-bound poly(A). Binding assays demonstrate that this results
from a modulation of poly(A) packaging by the Pab1 linker region. Overall, our
results demonstrate a direct involvement of Pab1 in mRNA decay and reveal the
modular nature of this factor, with different domains affecting various cellular
processes. These data suggest new models involving the modulation of poly(A)
packaging by Pab1 to control mRNA decay. The Tob/BTG family is a group of antiproliferative proteins containing two
highly homologous regions, Box A and Box B. These proteins all associate with
CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D)
family of deadenylases and is a component of the CCR4-Not deadenylase complex.
Here we determined the crystal structure of the complex of the N-terminal region
of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas hCaf1 most
closely resembled the catalytic domain of yeast Pop2 and human poly(A)-specific
ribonuclease. Interestingly, the association of hCaf1 was mediated by both Box A
and Box B of Tob. Cell growth assays using both wild-type and mutant proteins
revealed that deadenylase activity of Caf1 is not critical but complex formation
is crucial to cell growth inhibition. Caf1 tethers Tob to the CCR4-Not
deadenylase complex, and thereby Tob gathers several factors at its C-terminal
region, such as poly(A)-binding proteins, to exert antiproliferative activity. Accurate gene expression requires the precise control of mRNA levels, which are
determined by the relative rates of nuclear (pre-)mRNA synthesis and processing,
and cytoplasmic mRNA turnover. A key step in mRNA degradation is the removal of
the poly(A) tail, which involves several deadenylases including components of
the Ccr4-Not complex. Here, we focused on the role of the human paralogues CNOT7
(hCaf1/Caf1a) and CNOT8 (hPop2/Caf1b/Calif), which possess deadenylase activity
mediated by DEDD nuclease domains. We show that efficient proliferation requires
both subunits, although combined knockdown of CNOT7 and CNOT8 further reduces
cell proliferation indicating partial redundancy between these proteins.
Interestingly, the function of CNOT7 in cell proliferation partly depends on its
catalytic activity. On the other hand, the interaction between CNOT7 and BTG2, a
member of the antiproliferative BTG/Tob family involved in transcription and
mRNA decay appears less important for proliferation of MCF7 cells, suggesting
that CNOT7 does not function solely in conjunction with BTG2. Further analysis
of gene expression profiles of CNOT7 and/or CNOT8 knockdown cells underscores
the partial redundancy between these subunits and suggests that regulation of
several genes, including repression of the antiproliferative genes MSMB and
PMP22, by the Ccr4-Not complex contributes to cell proliferation. To maintain homeostasis in an ever-changing environment organisms have evolved
mechanisms to reprogram gene expression. One central mechanism regulating gene
expression is messenger RNA (mRNA) degradation, which is initiated by poly(A)
tail shortening (deadenylation). The carbon catabolite repressor 4-CCR4
associated factor1 (CCR4-CAF1) complex is the major enzyme complex that
catalyzes mRNA deadenylation and is conserved among eukaryotes. However, the
components and functions of this global regulatory complex have not been well
characterized in plants. Here we investigate the CAF1 family in Arabidopsis
(Arabidopsis thaliana). We identify 11 AtCAF1 homologs and show that a subset of
these genes are responsive to mechanical wounding, among them are AtCAF1a and
AtCAF1b whose expression levels are rapidly and transiently induced by wounding.
The differential expression profiles of the various AtCAF1s suggest that not all
AtCAF1 genes are involved in stress-responsive regulation of transcript levels.
Comparison of misexpressed genes identified via transcript profiling of Atcaf1a
and Atcaf1b mutants at different time points before and after wounding suggests
that AtCAF1a and AtCAF1b target shared and unique transcripts for deadenylation
with temporal specificity. Consistent with the AtPI4Kgamma3 transcript
exhibiting the largest increase in abundance in Atcaf1b, AtCAF1b targets
AtPI4Kgamma3 mRNA for deadenylation. Stress-tolerance assays demonstrate that
AtCAF1a and AtCAF1b are involved in mediating abiotic stress responses. However,
AtCAF1a and AtCAF1b are not functionally redundant in all cases, nor are they
essential for all environmental stresses. These findings demonstrate that these
closely related proteins exhibit overlapping and distinct roles with respect to
mRNA deadenylation and mediation of stress responses. CCR4, an evolutionarily conserved member of the CCR4-NOT complex, is the main
cytoplasmic deadenylase. It contains a C-terminal nuclease domain with homology
to the endonuclease-exonuclease-phosphatase (EEP) family of enzymes. We have
determined the high-resolution three-dimensional structure of the nuclease
domain of CNOT6L, a human homologue of CCR4, by X-ray crystallography using the
single-wavelength anomalous dispersion method. This first structure of a
deadenylase belonging to the EEP family adopts a complete alpha/beta sandwich
fold typical of hydrolases with highly conserved active site residues similar to
APE1. The active site of CNOT6L should recognize the RNA substrate through its
negatively charged surface. In vitro deadenylase assays confirm the critical
active site residues and show that the nuclease domain of CNOT6L exhibits full
Mg(2+)-dependent deadenylase activity with strict poly(A) RNA substrate
specificity. To understand the structural basis for poly(A) RNA substrate
binding, crystal structures of the CNOT6L nuclease domain have also been
determined in complex with AMP and poly(A) DNA. The resulting structures suggest
a molecular deadenylase mechanism involving a pentacovalent phosphate
transition. The vertebrate 2-5A system is part of the innate immune system and central to
cellular antiviral defense. Upon activation by viral double-stranded RNA,
5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As)
are synthesized by one of several 2'-5'-oligoadenylate synthetases. These
unusual oligonucleotides activate RNase L, an unspecific endoribonuclease that
mediates viral and cellular RNA breakdown. Subsequently, the 2-5As are removed
by a 2'-phosphodiesterase (2'-PDE), an enzyme that apart from breaking 2'-5'
bonds also degrades regular, 3'-5'-linked oligoadenylates. Interestingly, 2'-PDE
shares both functionally and structurally characteristics with the CCR4-type
exonuclease-endonuclease-phosphatase family of deadenylases. Here we show that
2'-PDE locates to the mitochondrial matrix of human cells, and comprise an
active 3'-5' exoribonuclease exhibiting a preference for oligo-adenosine RNA
like canonical cytoplasmic deadenylases. Furthermore, we document a marked
negative association between 2'-PDE and mitochondrial mRNA levels following
siRNA-directed knockdown and plasmid-mediated overexpression, respectively. The
results indicate that 2'-PDE, apart from playing a role in the cellular immune
system, may also function in mitochondrial RNA turnover. Deadenylation is the first and rate-limiting step during turnover of mRNAs in
eukaryotes. In the yeast, Saccharomyces cerevisiae, two distinct 3'-5'
exonucleases, Pop2p and Ccr4p, have been identified within the Ccr4-NOT
deadenylase complex, belonging to the DEDD and
Exonuclease-Endonuclease-Phosphatase (EEP) families, respectively. Ngl3p has
been identified as a new member of the EEP family of exonucleases based on
sequence homology, but its activity and biological roles are presently unknown.
Here, we show using in vitro deadenylation assays on defined RNA species
mimicking poly-A containing mRNAs that yeast Ngl3p is a functional 3'-5'
exonuclease most active at slightly acidic conditions. We further show that the
enzyme depends on divalent metal ions for activity and possesses specificity
towards poly-A RNA similar to what has been observed for cellular deadenylases.
The results suggest that Ngl3p is naturally involved in processing of
poly-adenylated RNA and provide insights into the mechanistic variations
observed among the redundant set of EEP enzymes found in yeast and higher
eukaryotes. Nocturnin is a member of the CCR4 deadenylase family, and its expression is
under circadian control with peak levels at night. Because it can remove poly(A)
tails from mRNAs, it is presumed to play a role in post-transcriptional control
of circadian gene expression, but its target mRNAs are not known. Here we
demonstrate that Nocturnin expression is acutely induced by the endotoxin
lipopolysaccharide (LPS). Mouse embryo fibroblasts (MEFs) lacking Nocturnin
exhibit normal patterns of acute induction of TNFα and iNOS mRNAs during the
first three hours following LPS treatment, but by 24 hours, while TNFα mRNA
levels are indistinguishable from WT cells, iNOS message is significantly
reduced 20-fold. Accordingly, analysis of the stability of the mRNAs showed that
loss of Nocturnin causes a significant decrease in the half-life of the iNOS
mRNA (t(1/2) = 3.3 hours in Nocturnin knockout MEFs vs. 12.4 hours in wild type
MEFs), while having no effect on the TNFα message. Furthermore, mice lacking
Nocturnin lose the normal nighttime peak of hepatic iNOS mRNA, and have improved
survival following LPS injection. These data suggest that Nocturnin has a novel
stabilizing activity that plays an important role in the circadian response to
inflammatory signals. The shortening of the 3'-end poly(A) tail, also called deadenylation, is crucial
to the regulation of mRNA processing, transportation, translation and
degradation. The deadenylation process is achieved by deadenylases, which
specifically catalyze the removal of the poly(A) tail at the 3'-end of
eukaryotic mRNAs and release 5'-AMP as the product. To achieve their
physiological functions, all deadenylases have numerous binding partners that
may regulate their catalytic properties or recruit them into various protein
complexes. To study the effects of various partners, it is important to develop
new deadenylase assay that can be applied either in vivo or in vitro. In this
research, we developed the deadenylase assay by the size-exclusion
chromatography (SEC) method. The SEC analysis indicated that the poly(A) or
oligo(A) substrate and the product AMP could be successfully separated and
quantified. The enzymatic parameters of deadenylase could be obtained by
quantifying the AMP generation. When using the commercial poly(A) as the
substrate, a biphasic catalytic process was observed, which might correlate to
the two distinct states of poly(A) in the commercial samples. Different lots of
commercial poly(A) had dissimilar size distributions and were dissimilar in
response to the degradation of deadenylase. The deadenylation pattern,
processive or distributive, could also be investigated using the SEC assay by
monitoring the status of the substrate and the generation kinetics of AMP and
A2. The SEC assay was applicable to both simple samples using the purified
enzyme and complex enzyme reaction conditions such as using protein mixtures or
crude cell extracts as samples. The influence of solutes with absorption at 254
nm could be successfully eliminated by constructing the different SEC profiles. The function of cytoplasmic PABPs [poly(A)-binding proteins] in promoting mRNA
translation has been intensively studied. However, PABPs also have less clearly
defined functions in mRNA turnover including roles in default deadenylation, a
major rate-limiting step in mRNA decay, as well as roles in the regulation of
mRNA turnover by cis-acting control elements and in the detection of aberrant
mRNA transcripts. In the present paper, we review our current understanding of
the complex roles of PABP1 in mRNA turnover, focusing on recent progress in
mammals and highlighting some of the major questions that remain to be
addressed. PARN, Nocturnin and Angel are three of the multiple deadenylases that have been
described in eukaryotic cells. While each of these enzymes appear to target
poly(A) tails for shortening and influence RNA gene expression levels and
quality control, the enzymes differ in terms of enzymatic mechanisms, regulation
and biological impact. The goal of this review is to provide an in depth
biochemical and biological perspective of the PARN, Nocturnin and Angel
deadenylases. Understanding the shared and unique roles of these enzymes in cell
biology will provide important insights into numerous aspects of the
post-transcriptional control of gene expression. This article is part of a
Special Issue entitled: RNA Decay mechanisms. Shortening and removal of the 3' poly(A) tail of mature mRNA by poly(A)-specific
3' exonucleases (deadenylases) is the initial and often rate-limiting step in
mRNA degradation. The majority of cytoplasmic deadenylase activity is associated
with the Ccr4-Not and Pan2-Pan3 complexes. Two distinct catalytic subunits,
Caf1/Pop2 and Ccr4, are associated with the Ccr4-Not complex, whereas the Pan2
enzymatic subunit forms a stable complex with Pan3. In this review, we discuss
the composition and activity of these two deadenylases. In addition, we comment
on generic and specific mechanisms of recruitment of Ccr4-Not and Pan2-Pan3 to
mRNAs. Finally, we discuss specialised and redundant functions of the
deadenylases and review the importance of Ccr4-Not subunits in the regulation of
physiological processes. This article is part of a Special Issue entitled: RNA
Decay mechanisms. Deadenylases specifically catalyze the degradation of eukaryotic mRNA poly(A)
tail in the 3'- to 5'-end direction with the release of 5'-AMP as the product.
Among the deadenylase family, poly(A)-specific ribonuclease (PARN) is unique in
its domain composition, which contains three potential RNA-binding domains: the
catalytic nuclease domain, the R3H domain and the RRM domain. In this research,
we investigated the roles of these RNA-binding domains by comparing the
structural features and enzymatic properties of mutants lacking either one or
two of the three RNA-binding domains. The results showed that the R3H domain had
the ability to bind various oligonucleotides at the micromolar level with no
oligo(A) specificity. The removal of the R3H domain dissociated PARN into
monomers, which still possessed the RNA-binding ability and catalytic functions.
Unlike the critical role of the RRM domain in PARN processivity, the removal of
the R3H domain did not affect the catalytic pattern of PARN. Our results
suggested that both R3H and RRM domains were essential for the high affinity of
long poly(A) substrate, but the R3H domain did not contribute to the substrate
recognition of PARN. Compared to the RRM domain, the R3H domain played a more
important role in the structural integrity of the dimeric PARN. The multiple
RNA-binding domain architecture endows PARN the property of highly efficient
catalysis in a highly processive mode. |
Which translocation is harbored in the Askin tumor cells? | The Askin tumor is a primitive malignant small-cell tumor of the chest wall mostly seen among children and adolescents. It is closely related to Ewing's sarcoma of the same location, with both tumors harboring reciprocal translocation t(11;22) (q24;q12). | The Askin tumor, a primitive maligt small-cell tumor of the chest wall, is
mostly seen among children and adolescents. It is closely related to Ewing's
sarcoma of the same location, both tumors showing a chromosomal translocation
t(11;22). Its origin from neuroectodermal cells is deducted from several
ultrastructural details and from the expression of specific markers like NSE.
Pain and deformation of the chest wall are the cardinal clinical signs of the
tumor. Chest X-rays will frequently show destruction of ribs and pleural
effusions. Effective therapy consists of radical surgery, local radiation and
adjuvant chemotherapy. This multimodal concept allows minority of patients to
remain disease-free but the overall outcome is rather unfortunate. A patient previously reported to have Ewing's sarcoma showed a t(11;22)
(q23;q11) and spontaneous expression of fra(11)(q23) [1]. Subsequent review of
pathologic specimen indicated, however, that it was an Askin's tumor. Reciprocal
translocations of chromosomes 11 and 22 are the most common cytogenetic
abnormalities in Ewing's sarcoma and the related Askin's tumor. After radiation
therapy of a residual metastatic brain lesion, subsequent studies of the
recurrent brain tumor indicated the presence of the original translocations as
well as five new reciprocal translocations and two deleted segments (9p-, 10p-).
The new chromosome abnormalities were consistently found, indicating that
progression of the tumor was clonal. The newly observed clonal aberrations were
considered secondary in nature. A relationship between craniocerebral
irradiation and development of brain tumors has been reported in several
studies, but the mechanism for tumor induction has not yet been elucidated. It
is important that the role of radiation therapy in the evolutionary process of
chromosomal changes be studied in a large group of similar cases. A rare case of peripheral primitive neuroectodermal tumor (PNET) is reported. A
68-year-old woman complaining of lumbago was admitted to our hospital. Diagnosis
was made based on pathological findings characterized by Homer Wright-type
rosettes. Ultrastructural examination showed the presence of neurosecretory
granules and short cytoplasmic processes, which were highly suggestive of neural
differentiation. Chromosomal analysis of the neoplastic cells revealed
translocation (11;22)(q24;q12), which is often found in Ewing's sarcoma and
Askin tumor. These results strengthen the hypothesis of a common histogenesis
for these small round cell tumors, and suggest common oncogenesis for these
neoplasms. The Ewing sarcoma family of tumors includes osseous Ewing sarcoma, extraskeletal
Ewing sarcoma, primitive neuroectodermal tumor, and Askin tumor. They share a
karyotype abnormality with translocation involving chromosomes 11 and 22.
Histologically, these lesions demonstrate crowded sheets of small round blue
cells. Imaging features of osseous Ewing sarcoma often suggest the diagnosis,
with aggressive long-bone destruction in the metadiaphysis of an adolescent or
young adult and an associated soft-tissue mass. Focal areas of cortical
destruction are frequent, allowing continuity between the intraosseous and
extraosseous components. This continuity is also commonly seen as subtle
channels extending through the cortex at computed tomography or magnetic
resoce (MR) imaging, a finding that reflects the underlying pathologic
appearance. Extraskeletal Ewing sarcoma commonly demonstrates a nonspecific
radiologic appearance of a large soft-tissue mass affecting the paraspinal
region or lower extremity. Askin tumor represents extraskeletal Ewing sarcoma
involving the chest wall. Imaging typically reveals a large pleural-based mass
and associated pleural effusion. Treatment of these tumors is usually a
combination of neoadjuvant chemotherapy followed by surgical resection, which
may be supplemented with radiation therapy. Imaging, particularly MR, is also
vital to evaluate response to neoadjuvant therapy, direct surgical resection,
and detect local recurrence or metastatic disease. |
What is the function of the AtxA pleiotropic regulator? | AtxA is the gene encoding the trans-activator of anthrax toxin synthesis and is essential for virulence of B. anthracis. It is located on the resident 185-kb plasmid pXO1 and its activation is stimulated by bicarbonate. AtxA controls the expression of more than a hundred genes belonging to all genetic elements, the chromosome and both virulence plasmids, including those encoding the major virulence factors. AtxA can activate or repress gene expression. In atxA+ strains, toxin gene expression is increased 5- to 20-fold in cells grown in 5% CO2 relative to cells grown in air. Dual promoters control expression of AtxA. Transcription of the atxA gene occurs from two independent promoters, P1 and P2, whose transcription start sites are separated by 650 bp. | Bacillus anthracis plasmid pXO1 carries the structural genes for the three
anthrax toxin proteins, cya (edema factor), lef (lethal factor), and pag
(protective antigen). Expression of the toxin genes by B. anthracis is enhanced
during growth under elevated levels of CO2. This CO2 effect is observed only in
the presence of another pXO1 gene, atxA, which encodes a transactivator of
anthrax toxin synthesis. Here we show that transcription of atxA does not appear
to differ in cells grown in 5% CO2 compared with cells grown in air. Using a new
efficient method for gene replacement in B. anthracis, we constructed an
atxA-null mutant in which the atxA-coding sequence on pXO1 is replaced with an
omega km-2 cassette. Transcription of all three toxin genes is decreased in the
absence of atxA. The pag gene possesses two apparent transcription start sites,
P1 and P2; only transcripts with 5' ends mapping to P1 are decreased in the
atxA-null mutant. Deletion analysis of the pag promoter region indicates that
the 111 bp region upstream of the P1 site is sufficient for atxA-mediated
activation of this transcript. The cya and lef genes each have one apparent
start site for transcription. Transcripts with 5' ends mapping to these sites
are not detected in the atxA-null mutant. The atxA-null mutant is avirulent in
mice. Moreover, the antibody response to all three toxin proteins is decreased
significantly in atxA-null mutant-infected mice. These data suggest that the
atxA gene product also regulates toxin gene expression during infection. The two major virulence factors of Bacillus anthracis are the tripartite toxin
and the polyglutamate capsule, which are encoded by genes on the large plasmids,
pXO1 and pXO2, respectively. The genes atxA, located on pXO1, and acpA, located
on pXO2, encode positive trans-acting proteins that are involved in
bicarbonate-mediated regulation of toxin and capsule production, respectively. A
derivative strain cured of pXO1 produced less capsular substance than the parent
strain harbouring both pXO1 and pXO2, and electroporation of the strain cured of
pXO1 with a plasmid containing the cloned atxA gene resulted in an increased
level of capsule production. An acpA-null mutant was complemented by not only
acpA but also the atxA gene. The cap region, which is essential for
encapsulation, contains three genes capB, capC, and capA, arranged in that
order. The atxA gene stimulated capsule synthesis from the cloned cap region.
Transcriptional analysis of cap by RNA slot-blot hybridization and
primer-extension analysis revealed that atxA activated expression of cap in
trans at the transcriptional level. These results indicate that cross-talk
occurs, in which the pXO1-located gene, atxA, activates transcription of the cap
region genes located on pXO2. We identified two major apparent transcriptional
start sites, designated P1 and P2, located at positions 731 bp and 625 bp,
respectively, upstream of the translation-initiation codon of capB.
Transcription initiated from P1 and P2 was activated by both atxA and acpA, and
activation appeared to be stimulated by bicarbonate. Deletion analysis of the
upstream region of the cap promoter revealed that activation by both atxA and
acpA required a DNA segment of 70 bp extending upstream of the P1 site. These
results suggest that cross-talk by atxA to the genes encoding capsule synthesis
is caused by the interaction of the atxA gene product with a regulatory sequence
upstream of cap. Fully virulent Bacillus anthracis bacilli are encapsulated and toxinogenic.
These bacteria carry two plasmids, pXO1, and pXO2, encoding toxins and capsule
synthetic-enzymes (capB, C, A, dep), respectively. The PXO1 plasmid strongly
enhances capsule formation. This influence was studied by analysing the
expression of a capB-lacZ fusion in various backgrounds. The beta-galactosidase
activities were similar in a delta atxA strain and a pXO1 cured strain.
Moreover, the capB-lacZ expression level could be restored, in a pXO1 cured
strain, by addition of atxA in trans. Thus, we conclude that the pX01 influence
on capsule synthesis is mediated by AtxA, the pXO1-encoded trans-activator of
the toxin gene expression. Anthrax toxin gene expression in Bacillus anthracis is dependent on the presence
of atxA, a trans-acting regulatory gene located on the resident 185-kb plasmid
pXO1. In atxA+ strains, expression of the toxin genes (pag, lef, and cya) is
enhanced by two physiologically significant signals: elevated CO2/bicarbonate
and temperature. To determine whether increased toxin gene expression in
response to these signals is associated with increased atxA expression, we
monitored steady-state levels of atxA mRNA and AtxA protein in cells cultured in
different conditions. We purified histidine-tagged AtxA [AtxA(His)] from
Escherichia coli and used anti-AtxA(His) serum to detect AtxA in protein
preparations from B. anthracis cells. AtxA was identified as a protein with an
apparent size of 56 kDa in cytoplasmic fractions of B. anthracis cells. Our data
indicate that atxA expression is not influenced by CO2/bicarbonate levels.
However, the steady-state level of atxA mRNA in cells grown in elevated
CO2/bicarbonate at 37 degrees C is five- to sixfold higher than that observed in
cells grown in the same conditions at 28 degrees C. A corresponding difference
in AtxA protein was also seen at the different growth temperatures. When atxA
was cloned on a multicopy plasmid in B. anthracis, AtxA levels corresponding to
the atxA gene copy number were observed. However, this strain produced
significantly less pag mRNA and protective antigen protein than the parental
strain harboring atxA in single copy on pXO1. These results indicate that
increased AtxA expression does not lead to a corresponding increase in pag
expression. Our data strongly suggest that an additional factor(s) is involved
in regulation of pag and that the relative amounts of such a factor(s) and AtxA
are important for optimal toxin gene expression. The Bacillus anthracis toxin genes, cya, lef, and pag, can be viewed as a
regulon, in which transcription of all three genes is activated in trans by the
same regulatory gene, atxA, in response to the same signal, CO2. In atxA+
strains, toxin gene expression is increased 5- to 20-fold in cells grown in 5%
CO2 relative to cells grown in air. CO2-enhanced toxin gene transcription is not
observed in atx4-null mutants. Here, we used two independent techniques to
obtain evidence for additional CO2-induced atxA-regulated genes. First, total
protein preparations from atxA4+ and atxA isolates grown in 5% CO2 and in air
were examined by two-dimensional electrophoresis. Comparison of the resulting
protein patterns indicated that synthesis of non-toxin proteins is influenced by
growth in elevated CO2 and the toxin gene regulator, atxA. Second, we generated
random transcriptional lacZ fusions in B. anthracis with transposon Tn917-LTV3.
Transposon-insertion libraries were screened for mutants expressing CO2-enhanced
atxA-dependent beta-galactosidase activity. DNA sequence analysis of transposon
insertion sites in 17 mutants carrying CO2- and atxA-regulated fusions revealed
10 mutants carrying independent insertions on the 185-kb toxin plasmid pXO1
which did not map to the toxin genes. The tcr-lacZ fusion mutants (tcr for toxin
coregulated) were Tox+, indicating that these genes may not be involved in
anthrax toxin gene activation. Our data indicate a clear association of atxA
with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA
regulates genes other than the structural genes for the anthrax toxin proteins. Members of the Bacillus cereus group (B. anthracis, B. cereus, B. mycoides and
B. thuringiensis) are well-known pathogens of mammals (B. anthracis and B.
cereus) and insects (B. thuringiensis). The specific diseases they cause depend
on their capacity to produce specific virulence factors, such as the lethal
toxin of B. anthracis and the Cry toxins of B. thuringiensis. However, these
Bacillus spp. also produce a variety of proteins, such as phospholipases C,
which are known to act as virulence factors in various pathogenic bacteria. Few
genes encoding these virulence factors have been characterized in pathogenic
Bacillus spp. and little is known about the regulation of their expression. We
had previously reported that in B. thuringiensis expression of the
phosphatidylinositol-specific phospholipase C gene is regulated by the
transcriptional activator PlcR. Here we report the identification of several
extracellular virulence factor genes by the virtue of their PlcR-regulated
expression. These PlcR-regulated genes encode degradative enzymes, cell-surface
proteins and enterotoxins. The PlcR-regulated genes are widely dispersed on the
chromosome and therefore do not constitute a pathogenic island. Analysis of the
promoter region of the PlcR-regulated genes revealed the presence of a highly
conserved palindromic region (TATGNAN4TNCATA), which is presumably the specific
recognition target for PlcR activation. We found that the plcR gene is also
present in and probably restricted to all the members of the B. cereus group.
However, although the polypeptide encoded by the B. cereus PlcR gene is
functionally equivalent to the B. thuringiensis regulator, the polypeptide
encoded by the B. anthracis gene is truncated and not active as a
transcriptional activator. PlcR is the first example described of a pleiotropic
regulator involved in the control of extracellular virulence factor expression
in pathogenic Bacillus spp. These results have implications for the taxonomic
relationships among members of the B. cereus group, the virulence properties of
these bacteria and the safety of B. thuringiensis-based biopesticides. Two regulatory genes, acpA and atxA, have been reported to control expression of
the Bacillus anthracis capsule biosynthesis operon capBCAD. The atxA gene is
located on the virulence plasmid pXO1, while pXO2 carries acpA and the cap
genes. acpA has been viewed as the major regulator of the cap operon because it
is essential for capsule gene expression in a pXO1(-) pXO2(+) strain. atxA is
essential for toxin gene transcription but has also been implicated in control
of the cap genes. The molecular functions of the regulatory proteins are
unknown. We examined cap gene expression in a genetically complete pXO1(+)
pXO2(+) strain. Our results indicate that another pXO2 gene, acpB (previously
called pXO2-53; accession no. NC002146.1:49418-50866), has a role in cap
expression. The predicted amino acid sequence of AcpB is 62% similar to that of
AcpA and 50% similar to that of AtxA. Assessment of cap gene transcription
revealed that cap expression was not affected in a pXO1(+) pXO2(+) acpB-null
mutant and was slightly reduced in an isogenic acpA mutant. However, cap gene
expression was abolished in an acpA acpB double mutant. Microscopic examination
of capsule synthesis by the mutants corroborated these findings. acpA and acpB
expression is controlled by atxA; capsule synthesis and transcription of acpA
and acpB were markedly reduced in an atxA mutant. The data suggest that, in a
strain containing both virulence plasmids, atxA is the major regulator of
capsule synthesis and controls capBCAD expression indirectly, via positive
regulation of acpA and acpB. Bacillus anthracis causes host damage by producing two toxins, the lethal factor
and the oedema factor. Their production and that of other virulence factors
depend on the activity of the AtxA transcription factor. The mechanisms that
control AtxA activity are reported in this issue of Molecular Microbiology. The
protein can be phosphorylated at two distinct sites by components of the
phosphotransferase system (PTS). One phosphorylation event stimulates
transcription activation by AtxA, whereas the second prevents AtxA activity.
This mode of regulation and the predicted domain structures are highly similar
to transcription regulators controlled by PTS regulation domains (PRDs). Thus,
the phosphorylated domains of AtxA represent a novel form of PRDs. As a similar
organization is found in transcription regulators in many other pathogens, AtxA
might become the paradigm of a new class of virulence regulators. Expression of genes for Bacillus anthracis toxin and capsule virulence factors
are dependent upon the AtxA transcription factor. The mechanism by which AtxA
regulates the transcription of its target genes is unknown. Here we report that
bioinformatic analyses suggested the presence in AtxA of two PTS
(phosphenolpyruvate : sugar phosphotransferase system) regulation domains (PRD)
generally regulated by phosphorylation/dephosphorylation at conserved histidine
residues. By means of amino acid substitutions that mimic the phosphorylated (H
to D) or the unphosphorylated (H to A) state of the protein, we showed that
phosphorylation of H199 of PRD1 is likely to be necessary for AtxA activation
while phosphorylation of H379 in PRD2 is inhibitory to toxin gene transcription.
In vivo labelling experiments with radioactive phosphate allowed us to propose
that H199 and H379 are AtxA residues subject to regulated phosphorylation. In
support to these notions, we also show that deletion of ptsHI, encoding the HPr
intermediate and the EI enzymes of PTS, or growth in the presence of glucose
affect positively and negatively, respectively, the activity of AtxA. Our
results link virulence factor production in B. anthracis to carbohydrate
metabolism and, for the first time, provide a mechanistic explanation for AtxA
transcriptional activity. The AtxA virulence regulator of Bacillus anthracis is required for toxin and
capsule gene expression. AtxA is a phosphotransferase system regulatory
domain-containing protein whose activity is regulated by
phosphorylation/dephosphorylation of conserved histidine residues. Here we
report that transcription of the atxA gene occurs from two independent
promoters, P1 (previously described by Dai et al. [Z. Dai, J. C. Sirard, M.
Mock, and T. M. Koehler, Mol. Microbiol. 16:1171-1181, 1995]) and P2, whose
transcription start sites are separated by 650 bp. Both promoters have -10 and
-35 consensus sequences compatible with recognition by sigma(A)-containing RNA
polymerase, and neither promoter depends on the sporulation sigma factor SigH.
The dual promoter activity and the extended untranslated mRNA suggest that
as-yet-unknown regulatory mechanisms may act on this region to influence the
level of AtxA in the cell. Fifteen years ago, AtxA was isolated as a toxin gene activator and five years
later it was shown to be a Bacillus anthracis master regulator. AtxA controls
the expression of more than a hundred genes belonging to all genetic elements,
the chromosome and both virulence plasmids, including those encoding the major
virulence factors. AtxA can activate or repress gene expression. The mechanism
by which AtxA exerts its control is unknown; it is indirect on some genes but
may be direct on others. The expression of many AtxA-controlled genes is induced
by the presence of bicarbonate/CO(2). AtxA links the metabolic state and
virulence gene expression. Capsule and toxin are the major virulence factors of Bacillus anthracis. The B.
anthracis pleiotropic regulator CodY activates toxin gene expression by
post-translationally regulating the accumulation of the global regulator AtxA.
However, the role of CodY on B. anthracis capsulation and virulence of
encapsulated strains has been unknown. The role of CodY in B. anthracis
virulence was studied in mouse and guinea pig models. Spore outgrowth and
dissemination of the vegetative cells was followed in mice by bioluminescent
imaging. We also determined the state of capsulation and the iron requirement
for growth of the codY mutant. In all models tested, the codY mutant strain was
strongly attenuated compared to the wild-type strain and, in mice, also compared
to the atxA strain. The disruption of codY did not affect either ex vivo or in
vivo capsulation, whereas atxA deletion affected ex vivo capsulation only. The
disruption of codY led to a delayed initiation of dissemination but similar
kinetics of subsequent spread of the bacilli. The codY mutant cannot grow on
heme iron as sole iron source, whereas the parental and complemented strains
can. The lack of CodY-mediated transcription weakens virulence by controlling
iron acquisition and synthesis of toxin, but without modifying capsulation. AtxA, a unique regulatory protein of unknown molecular function, positively
controls expression of the major virulence genes of Bacillus anthracis. The 475
amino acid sequence of AtxA reveals DNA binding motifs and regions similar to
proteins associated with the phosphoenolpyruvate: carbohydrate
phosphotransferase system (PTS). We used strains producing native and functional
epitope-tagged AtxA proteins to examine protein-protein interactions in cell
lysates and in solutions of purified protein. Co-affinity purification,
non-denaturing polyacrylamide gel electrophoresis and bis(maleimido)hexane (BMH)
cross-linking experiments revealed AtxA homo-multimers. Dimers were the most
abundant species. BMH cross-links available cysteines within 13 Å. To localize
interaction sites, six AtxA mutants containing distinct Cys→Ser substitutions
were tested for multimerization and cross-linking. All mutants multimerized, but
one mutation, C402S, prevented cross-linking. Thus, BMH uses C402 to make the
inter-molecular bond between AtxA proteins, but C402 is not required for
protein-protein interaction. C402 is in a region bearing amino acid similarity
to Enzyme IIB proteins of the PTS. The AtxA EIIB motif may function in protein
oligomerization. Finally, cultures grown with elevated CO(2) /bicarbonate
exhibited increased AtxA dimer/monomer ratios and increased AtxA activity,
relative to cultures grown without added CO(2) /bicarbonate, suggesting that
this host-associated signal enhances AtxA function by shifting the dimer/monomer
equilibrium towards the dimeric state. BACKGROUND: Upon infection of a mammalian host, Bacillus anthracis responds to
host cues, and particularly to elevated temperature (37°C) and bicarbonate/CO2
concentrations, with increased expression of virulence factors that include the
anthrax toxins and extracellular capsular layer. This response requires the
presence of the pXO1 virulence plasmid-encoded pleiotropic regulator AtxA. To
better understand the genetic basis of this response, we utilized a controlled
in vitro system and Next Generation sequencing to determine and compare RNA
expression profiles of the parental strain and an isogenic AtxA-deficient strain
in a 2 × 2 factorial design with growth environments containing or lacking
carbon dioxide.
RESULTS: We found 15 pXO1-encoded genes and 3 chromosomal genes that were
strongly regulated by the separate or synergistic actions of AtxA and carbon
dioxide. The majority of the regulated genes responded to both AtxA and carbon
dioxide rather than to just one of these factors. Interestingly, we identified
two previously unrecognized small RNAs that are highly expressed under
physiological carbon dioxide concentrations in an AtxA-dependent manner.
Expression levels of the two small RNAs were found to be higher than that of any
other gene differentially expressed in response to these conditions. Secondary
structure and small RNA-mRNA binding predictions for the two small RNAs suggest
that they may perform important functions in regulating B. anthracis virulence.
CONCLUSIONS: A majority of genes on the virulence plasmid pXO1 that are
regulated by the presence of either CO2 or AtxA separately are also regulated
synergistically in the presence of both. These results also elucidate novel
pXO1-encoded small RNAs that are associated with virulence conditions. |
Is cystatin C or cystatin 3 used as a biomarker of kidney function? | Yes, cystatin C (CysC) is a novel biomarker of renal function. | BACKGROUND: Cystatin C could improve chronic kidney disease (CKD) classification
in HIV-infected women relative to serum creatinine.
DESIGN: Retrospective cohort analysis.
METHODS: Cystatin C and creatinine were measured from specimens taken and stored
during the 1999-2000 examination among 908 HIV-infected participants in the
Women's Interagency HIV study (WIHS). Mean follow-up was 10.2 years. Predictors
of differential glomerular filtration rate (GFR) estimates were evaluated with
multivariable linear regression. The associations of baseline categories (<60,
60-90, and >90 ml/min per 1.73 m) of creatinine estimated GFR (eGFRcr), cystatin
C eGFR (eGFRcys), and combined creatinine-cystatin C eGFR (eGFRcr-cys) with
all-cause mortality were evaluated using multivariable Cox regression. The net
reclassification index (NRI) was calculated to evaluate the effect of cystatin C
on reclassification of CKD staging.
RESULTS: CKD risk factors were associated with lower eGFRcys and eGFRcr-cys
values compared with eGFRcr. Relative to eGFR more than 90, the eGFR less than
60 category by eGFRcys (Adjusted hazard ratio: 2.56; 95% confidence interval:
1.63-4.02), eGFRcr-cys (3.11; 1.94-5.00), and eGFRcr (2.34; 1.44-3.79) was
associated with increased mortality risk. However, the eGFR 60-90 category was
associated with increased mortality risk for eGFRcys (1.80; 1.28-2.53) and
eGFRcr-cys (1.91; 1.38-2.66) but not eGFRcr (1.20; 0.85-1.67). The overall NRI
for mortality was 26% when reclassifying from eGFRcr to eGFRcys (P < 0.001) and
was 20% when reclassifying from eGFRcr to eGFRcr-cys (P < 0.001).
CONCLUSION: The addition of cystatin C may improve mortality risk prediction by
stages of kidney function relative to creatinine. CKD risk factors are
associated with an overestimate of GFR by serum creatinine relative to cystatin
C. Conventional creatinine-based glomerular filtration rate (GFR) equations are
insufficiently accurate for estimating GFR in cirrhosis. The Chronic Kidney
Disease Epidemiology Collaboration (CKD-EPI) recently proposed an equation to
estimate GFR in subjects without cirrhosis using both serum creatinine and
cystatin C levels. Performance of the new CKD-EPI creatinine-cystatin C equation
(2012) was superior to previous creatinine- or cystatin C-based GFR equations.
To evaluate the performance of the CKD-EPI creatinine-cystatin C equation in
subjects with cirrhosis, we compared it to GFR measured by nonradiolabeled
iothalamate plasma clearance (mGFR) in 72 subjects with cirrhosis. We compared
the "bias," "precision," and "accuracy" of the new CKD-EPI creatinine-cystatin C
equation to that of 24-hour urinary creatinine clearance (CrCl), Cockcroft-Gault
(CG), and previously reported creatinine- and/or cystatin C-based GFR-estimating
equations. Accuracy of CKD-EPI creatinine-cystatin C equation as quantified by
root mean squared error of difference scores (differences between mGFR and
estimated GFR [eGFR] or between mGFR and CrCl, or between mGFR and CG equation
for each subject) (RMSE = 23.56) was significantly better than that of CrCl
(37.69, P = 0.001), CG (RMSE = 36.12, P = 0.002), and GFR-estimating equations
based on cystatin C only. Its accuracy as quantified by percentage of eGFRs that
differed by greater than 30% with respect to mGFR was significantly better
compared to CrCl (P = 0.024), CG (P = 0.0001), 4-variable MDRD (P = 0.027), and
CKD-EPI creatinine 2009 (P = 0.012) equations. However, for 23.61% of the
subjects, GFR estimated by CKD-EPI creatinine-cystatin C equation differed from
the mGFR by more than 30%.
CONCLUSION: The diagnostic performance of CKD-EPI creatinine-cystatin C equation
(2012) in patients with cirrhosis was superior to conventional equations in
clinical practice for estimating GFR. However, its diagnostic performance was
substantially worse than reported in subjects without cirrhosis. BACKGROUND/AIMS: Several formulas for glomerular filtration rate (GFR)
estimation, based on serum creatinine or cystatin C, have been proposed. We
assessed the impact of some of these equations on estimated GFR (eGFR) and
chronic kidney disease (CKD) prevalence, and on the association with
cardiovascular risk factors, in a general population sample characterized by a
young mean age.
METHODS: We studied 1,199 individuals from three Alpine villages enrolled into
the MICROS study. eGFR was obtained with the 4- and 6-parameter MDRD study
equations, the Virga equation, and with the three CKD-EPI formulas for
creatinine, cystatin C, and the combination of creatinine and cystatin C. We
assessed the concordance between quantitative eGFR levels, CKD prevalence, and
in terms of association with total, LDL, and HDL cholesterol.
RESULTS: The highest and lowest eGFR levels corresponded to the cystatin C-based
and MDRD-4 equations, respectively. CKD prevalence varied from 1.8% (Virga) to
5.8% (MDRD-4). The CKD-EPI based on creatinine showed the highest agreement with
all other equations. Agreement between methods was higher at lower eGFR levels,
older age, and in the presence of diabetes and hypertension. Creatinine-based
estimates of eGFR were associated with total and low-density lipoprotein but not
high-density lipoprotein cholesterol. The opposite was observed for the cystatin
C-based GFR.
CONCLUSION: GFR estimation is strongly affected by the chosen equation.
Differences are more pronounced in healthy and younger individuals. To identify
CKD risk factors, the choice of the equation is of secondary importance to the
choice of the biomarker used in the formula. If eGFR is not calibrated to a gold
standard GFR in the general population, reports about CKD prevalence should be
considered with caution. Whether kidney dysfunction is associated with coronary artery calcium (CAC) in
young and middle-aged adults who have a cystatin C-derived estimated glomerular
filtration rate (eGFRcys) greater than 60 mL/min/1.73 m(2) is unknown. In the
Coronary Artery Risk Development in Young Adults (CARDIA) cohort (recruited in
1985 and 1986 in Birmingham, Alabama; Chicago, Illinois; Minneapolis, Minnesota;
and Oakland, California), we examined 1) the association of eGFRcys at years 10
and 15 and detectable CAC over the subsequent 5 years and 2) the association of
change in eGFRcys and subsequent CAC, comparing those with stable eGFRcys to
those whose eGFRcys increased (>3% annually over 5 years), declined moderately
(3%-5%), or declined rapidly (>5%). Generalized estimating equation Poisson
models were used, with adjustment for age, sex, race, educational level, income,
family history of coronary artery disease, diabetes, body mass index,
low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and
tobacco use. Among 3,070 participants (mean age 35.6 (standard deviation, 4.1)
years and mean eGFRcys 106.7 (standard deviation, 18.5) mL/min/1.73 m(2)), 529
had detectable CAC. Baseline eGFRcys was not associated with CAC. Moderate
eGFRcys decline was associated with a 33% greater relative risk of subsequent
CAC (95% confidence interval: 5, 68; P = 0.02), whereas rapid decline was
associated with a 51% higher relative risk (95% confidence interval: 10, 208; P
= 0.01) in adjusted models. In conclusion, among young and middle-aged adults
with eGFRcys greater than 60 mL/min/1.73 m(2), annual decline in eGFRcys is an
independent risk factor for subsequent CAC. AIM: To study an association between clinical and morphological evidence and the
serum and daily urinary levels of cystatin C (CysC) and neutrophil
gelatinase-associated lipocalin (NGAL) in patients with primary
glomerulopathies.
SUBJECTS AND METHODS: The investigation included 104 patients; morphological
examination showed minimal change disease in 15 (14.4%) patients, focal
segmental glomerulosclerosis in 24 (23.1%), membrane nephropathy in 32 (30.8%),
and IgA nephropathy (mesangioproliferative glomerulonephritis) in 33 (31.7%).
The investigators analyzed the clinical type of nephropathy, performed
conventional laboratory and instrumental examinations, and determined the level
of CysC (by immunoturbodimetry) and NGAL (by enzyme immunoassay) in the serum
and daily urine taken before kidney biopsy. The degree of glomerulosclerosis,
tubulointerstititial sclerosis, and tubular atrophy was semiquantitatively
estimated.
RESULTS: Urinary CysC and NGAL excretion correlated with the degree of
glomerulosclerosis and proteinuria and the reduced glomerular filtration rate
(GFR) regardless of the method of its determination. The urinary level of NGAL
positively correlated with the degree of tubular atrophy. The GFR value
determined from serum CysC and creatinine levels more precisely reflected the
degree of glomerulosclerosis.
CONCLUSION: The tubulointerstitial compartment in primary glomerulopathies
should be determined not only by morphological changes, but also by tubular
function parameters by estimating the urinary excretion of biomarkers. The
urinary content of CysC reflects tubular epithelial dysfunction whereas that of
NGAL also characterizes tubular atrophy. To estimate the degree of
glomerulosclerosis, it is more preferable to use the GFR calculated from the
blood concentrations of CysC and creatinine, by keeping in mind clinical
findings (using Chronic Kidney Disease Epidemiology Collaboration formula,
2012). Diastolic dysfunction of the heart is correlated with cardiac mortality. Serum
cystatin C (CysC) is an endogenous marker of kidney function. It is not clear
whether serum CysC is associated with diastolic dysfunction in patients with
varying cardiac conditions with concomitant diastolic abnormalities and
preserved ejection fraction (EF). The authors measured serum CysC levels in
patients with cardiac diseases and examined the relationships between serum CysC
levels and diastolic function. Serum CysC was measured and echocardiography was
performed in 124 consecutive patients with cardiac diseases. Transmitral flow
(TMF) patterns surrogating diastolic function were categorized into two groups:
a normal group and an abnormal group. Serum CysC and BNP showed a significant
positive correlation. There were no significant differences in serum CysC among
those cardiac diseases. Seventy-eight patients with cardiac disease and
preserved EF (left ventricular EF ≥50%) and without renal dysfunction (estimated
glomerular filtration rate ≥60 mL/minute/1.73 m(2) ) were examined. Multivariate
linear regression analysis demonstrated that left atrium diameter and abnormal
TMF patterns were independent determits of serum CysC. Furthermore, patients
with elevated serum CysC levels had poor prognosis. Serum CysC is associated
with diastolic dysfunction in patients with various cardiac diseases and
preserved EF. Serum CysC might be a biomarker of cardiac diastolic dysfunction
in patients with preserved EF. OBJECTIVE: To evaluate the precision of methods used to assess renal function in
patients with neurogenic voiding dysfunction.
MATERIALS AND METHODS: This multicenter prospective study, which was set in
Toulouse and Lyon, France, included 60 patients (mean age, 48.9 ± 15.2 years)
with neurogenic bladder and sphincter dysfunction. The correlation and the
concordance with the inulin clearance of each method of renal function
evaluation were assessed.
RESULTS: The correlation of serum creatinine with inulin clearance was low when
using serum creatinine-based equations such as the Modification of Diet in Renal
Disease (simplified and complete) and Cockcroft-Gault equations. The r and r(2)
coefficients were higher for creatinine-based methods, such as 24-hour (r =
0.72) and 3-hour creatinine clearance (r = 0.78). The strongest correlation was
found for serum cystatin C-based equations: the Chronic Kidney Disease
Epidemiology Collaboration (CKD-EPI) creatinine/cystatin C combined equation (r
= 0.78) and the CKD-EPI cystatin C equation (r = 0.80). Mean bias of serum
creatinine-based equations estimating glomerular filtration rate, the
Cockcroft-Gault, and the simplified and complete Modification of Diet in Renal
Disease equations, was 27.5 ± 28.6, 17.48 ± 29.40, and 21.98 ± 30.40 mL/min,
respectively. Mean bias of creatinine clearance was 19.89 ± 15.30 mL/min at 3
hours and 19.00 ± 31.08 mL/min at 24 hours. Mean bias of the CKD-EPI cystatin C
and the CKD-EPI creatinine/cystatin C combined equations was 11.98 ± 17.68
mL/min and 18.62 ± 17.85 mL/min, respectively. Limitations are the numerous
types of neurologic diseases.
CONCLUSION: The CKD-EPI equation using cystatin C was the most precise method of
renal function evaluation in patients with neurogenic bladder. BACKGROUND AND OBJECTIVES: Kidney disease is associated with physiologic changes
that may predispose to frailty. This study sought to investigate whether lower
levels of kidney function were associated with prevalent or incident frailty in
Cardiovascular Health Study (CHS) participants.
DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: CHS enrolled community-dwelling
adults age ≥65 years between 1989-1990 and 1992-1993. To examine prevalent
frailty, included were 4150 participants without stroke, Parkinson disease,
prescribed medications for Alzheimer disease or depression, or severely impaired
cognition. To examine incident frailty, included were a subset of 3459
participants without baseline frailty or development of exclusion criteria
during follow-up. The primary predictor was estimated GFR (eGFR) calculated
using serum cystatin C (eGFR(cys)). Secondary analyses examined eGFR using serum
creatinine (eGFR(SCr)). Outcomes were prevalent frailty and incident frailty at
4 years of follow-up. Frailty was ascertained on the basis of weight loss,
exhaustion, weakness, slowness, and low physical activity.
RESULTS: The mean age was 75 years and the median eGFR(cys) was 73 ml/min per
1.73 m(2). Among participants with an eGFR(cys) <45 ml/min per 1.73 m(2), 24%
had prevalent frailty. In multivariable analysis and compared with eGFR(cys) ≥90
ml/min per 1.73 m(2), eGFR(cys) categories of 45-59 (odds ratio [OR], 1.80; 95%
confidence interval [CI], 1.17 to 2.75) and 15-44 (OR, 2.87; 95% CI, 1.72 to
4.77) were associated with higher odds of frailty, whereas 60-75 (OR, 1.14; 95%
CI, 0.76 to 1.70) was not. In multivariable analysis, eGFR(cys) categories of
60-75 (incidence rate ratio [IRR], 1.72; 95% CI, 1.07 to 2.75) and 15-44 (IRR,
2.28; 95% CI, 1.23 to 4.22) were associated with higher incidence of frailty
whereas 45-59 (IRR, 1.53; 95% CI, 0.90 to 2.60) was not. Lower levels of
eGFR(SCr) were not associated with higher risk of prevalent or incident frailty.
CONCLUSIONS: In community-dwelling elders, lower eGFR(cys) was associated with a
higher risk of prevalent and incident frailty whereas lower eGFR(SCr) was not.
These findings highlight the importance of considering non-GFR determits of
kidney function. BACKGROUND AND OBJECTIVES: AKI occurs frequently in older persons. Elevated
circulating fibroblast growth factor-23 (FGF-23), a known marker of impaired
mineral metabolism, may also reflect tubular dysfunction and risk of AKI. This
study evaluated FGF-23 as well as traditional markers of kidney disease, namely
urine albumin-to-creatinine ratio (UACR) and creatinine-cystatin C estimated GFR
(eGFRCrCyC), as risk factors for AKI in elderly individuals.
DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Plasma FGF-23, UACR, and
eGFRCrCyC were measured in 3241 community-dwelling elderly individuals in the
Cardiovascular Health Study. Hospitalization for AKI was defined by
International Classification of Diseases, Ninth Revision, Clinical Modification
codes. Associations of each biomarker with AKI were evaluated using Cox
proportional hazards models adjusted for demographics, cardiovascular risk
factors, and biomarkers of kidney function.
RESULTS: The mean participant age was 78 years; 60% of participants were women
and 16% were African American. The median (interquartile range) values of
biomarkers were as follows: FGF-23, 70 RU/ml (53, 99); UACR, 8.88 mg/g (4.71,
20.47); and eGFRCrCyC, 71 ml/min per 1.73 m(2) (59, 83). Hospitalized AKI
occurred in 119 participants over 10.0 years of median follow-up. In fully
adjusted analyses, compared with the lowest quartiles, the highest quartiles of
FGF-23 (≥100 RU/ml) and UACR (≥20.9 mg/g) were associated with AKI (FGF-23:
hazard ratio [HR], 1.99; 95% confidence interval [95% CI], 1.04 to 3.80; and
UACR: HR, 3.35; 95% CI, 1.83 to 6.13). Compared with the highest quartile, the
lowest quartile of eGFRCrCyC (<57 ml/min per 1.73 m(2)) was associated with AKI
with an HR of 2.15 (95% CI, 1.21 to 3.82).
CONCLUSIONS: FGF-23 adjusted for albuminuria, cardiovascular disease risk
factors, and baseline eGFR is independently associated with a higher risk of AKI
hospitalizations in community-dwelling elderly individuals. Further studies to
understand the nature of this association are warranted. Multiple organ failure in sepsis substantially increases mortality. This study
examined if there was greater hepatic, pancreatic, splenic, or renal injury in
mice that would die during sepsis induced by cecal ligation and puncture (CLP)
compared with that of those that would survive. Mice were stratified into groups
predicted to die (Die-P) or predicted to live (Live-P) in the first 5 days after
CLP based on plasma interleukin 6 levels. Groups were sacrificed to harvest
organs for histology. Separate animals were followed for survival with daily
blood sampling to examine renal function. No significant histological evidence
of organ injury was observed in either the Live-P or Die-P mice. Minimal hepatic
injury occurred as plasma aspartate transaminase demonstrated less than a 2-fold
increase over normal in both groups. In addition, pancreatic injury was minimal
as there was also less than a 2-fold increase in plasma amylase levels. In
contrast, blood urea nitrogen levels were nearly five times higher within 24 h
in Die-P mice compared with those of mice predicted to live. Mice with blood
urea nitrogen levels higher than 44 mg/dL had a 17.6 higher relative risk of
dying (95% confidence interval, 4.5-69.4). Cystatin C, a more specific kidney
function biomarker, was also elevated at 24 h after CLP. When the cystatin C
levels were analyzed relative to the hours before death, rather than hours after
CLP, they were also significantly increased in mice Dead by day 5 compared with
those Alive after day 5. We conclude that limited liver, pancreas, and spleen
injury develops during murine CLP-induced sepsis while significant kidney injury
is present. The renal injury becomes worse closer to death. BACKGROUND/OBJECTIVES: to explore the effect of ageing on renal function with
cystatin C as the marker of glomerular filtration rate (GFR) in the general
population without vascular disease or diabetes.
DESIGN: a cross-sectional analysis of a healthy subset from the Good Aging in
Skåne-cohort study representative of the Swedish general population.
SUBJECTS: 1252 participants without vascular disease and diabetes (43.9% men) of
whom 203 were over 80 years old were included from the original cohort of 2931.
METHODS: plasma cystatin C and plasma creatinine were used as markers for GFR.
Estimated GFR (eGFR) was calculated with three chronic kidney disease
epidemiology collaboration (CKD-EPI) formulas involving cystatin C, creatinine
or both.
RESULTS: the median for plasma cystatin C was 0.93 mg/l (60-69 years old), 1.04
(70-79 years old) and 1.24 (80+ years old). The difference in mg/l between the
5th and 95th percentile was 0.46, 0.62 and 0.90 for these age groups. Male sex
increased the age effect on plasma cystatin C levels with 0.004 mg/l/year (P =
0.03), adjusted for vascular risk factors. Smoking, lower HDL and higher
diastolic blood pressure were associated with higher cystatin C levels. 54.7%
(CKD-EPI creatinine) to 73.9% (CKD-EPI cystatin C) of the 80+ had an eGFR < 60
ml/min/1.73 m2.
CONCLUSION: non-diabetics without overt vascular disease exhibit an age related
but heterogeneous decline in renal function. The ageing effect is more
pronounced in men. At least half of healthy 80+ years old could be expected to
have at least CKD Stage 3 with eGFR < 60 ml/min/1.73 m2. |
Which R/bioconductor package utilizes the Hilbert curve in order to visualize genomic data? | The so-called Hilbert curve visualization can complement genome browsers and help to get further insights into the structure of one's data. An open-source application, called HilbertVis, has been developed for R/bioconductor that allows the user to produce and interactively explore such plots. | In many genomic studies, one works with genome-position-dependent data, e.g.
ChIP-chip or ChIP-Seq scores. Using conventional tools, it can be difficult to
get a good feel for the data, especially the distribution of features. This
article argues that the so-called Hilbert curve visualization can complement
genome browsers and help to get further insights into the structure of one's
data. This is demonstrated with examples from different use cases. An
open-source application, called HilbertVis, is presented that allows the user to
produce and interactively explore such plots.
AVAILABILITY: http://www.ebi.ac.uk/huber-srv/hilbert/. |
Can fetal aneuploidy be detected with non-invasive prenatal testing? | Yes, the non-invasive preanatal test of cell-free fetal DNA is being used for fetal aneuploidy screening. | A pregt woman who was a carrier for a balanced chromosome translocation
[46,XX, t(1;6) (p31;q14)] and who had had six miscarriages, declined invasive
testing but agreed to non-invasive prenatal diagnosis by analysis of fetal cells
in maternal blood. Monoclonal antibody (Mab) against the zeta (z) and gamma
(gamma) chains of embryonic and fetal haemoglobin were used to identify fetal
nucleated erythrocytes (FNRBC). There were no FNRBC detected at 7 weeks, one
anti-z-positive FNRBC was detected at 11 weeks, and 12 anti-gamma-positive FNRBC
were detected at 20 weeks. Fluorescent in-situ hybridization was performed using
probes for chromosomes X, Y, 1 and 6 to identify fetal gender and the presence
of an unbalanced chromosomal translocation. A tentative prenatal diagnosis was
made of a female fetus disomic for chromosomes 1 and 6. A female infant with a
46,XX karyotype was born at term. This is the first attempt of exclusion of a
chromosome translocation using fetal cells isolated from maternal blood. There
is an advantage of using fetal cells isolated from maternal blood for
non-invasive prenatal diagnosis in couples who have a history of multiple
miscarriages due to a parental translocation, and who decline invasive testing
in a pregcy that continues to the second trimester. BACKGROUND: Identification of fetal nucleated red blood cells (NRBCs) in
maternal circulation can facilitate non-invasive prenatal diagnosis, but
technical difficulties still exist. An increase in the number of circulating
NRBCs, however, could indicate fetal aneuploidies or pregcy complications.
MATERIALS AND METHODS: The number of NRBCs was determined from 20 mL peripheral
blood in 351 women in the second trimester of pregcy after isolation by
magnetic cell sorting (MACS) with anti-CD71 antibody and identification with
May-Grunwald/Giemsa staining.
RESULTS: An average of eight NRBCs (range 1-12) were identified among 282 women
with chromosomally normal fetuses. In cases known to carry aneuploid fetuses the
mean number was 35 (range 7-113), but when the fetus had trisomy 21 (n = 17) an
average of 71 NRBCs were identified. Among 26 carriers of beta-thalassemia, 42
NRBCs (range 22-158) were isolated. In pregcies with abnormal Doppler
findings in both uterine arteries (n = 20), 15 NRBCs (range 2-75) were isolated.
CONCLUSION: Determining the number of NRBCs in maternal circulation could
represent an additional screening step for fetal aneuploidies, as long as the
anemic status of the mother is taken into consideration. However, more cases
with abnormal Doppler results must be investigated before this test is used for
in the prediction of pregcy complications. The incidence of numerical chromosome aberrations is about 5% during the entire
pregcy and about 0.2% in live-born infants. Most commonly observed numerical
aberrations in live births are trisomies of chromosomes 13, 18, 21, X and Y and
monosomy of the X chromosome. It is estimated that approximately 70-80% of
newborns with aneuploidies are born by women who did not present obvious risk
factors, therefore, according to a recent recommendation by PTG, prenatal
diagnosis increasing the detection of fetal aneuploidy should be offered to the
entire population of women. Due to the risk of complications associated with
invasive tests and a large number of unnecessarily performed tests of this type,
it is postulated that invasive diagnostics should be used in very specific
cases, and a non-invasive diagnostics should have a screening character
Non-invasive diagnostics include: 1) detailed ultrasonography performed in 11-13
(+6 days) hbd and in 18-24 hbd; 2) biochemical tests: PAPP-A (first-trimester
test) and the triple test (second-trimester test) and less frequently performed:
double, quadruple, and integrated tests. High detection rate of chromosomal
aberrations in non-invasive tests (at least 75%, with no more than 5% risk of
obtaining false positive results) and lack of procedure-related pregcy losses
constitute the advantage of noninvasive prenatal diagnosis. Blood plasma of pregt women contains circulating cell-free fetal DNA
(ccffDNA), originating from the placenta. The use of this DNA for non-invasive
detection of fetal aneuploidies using massively parallel sequencing
(MPS)-by-synthesis has been proven previously. Sequence performance may,
however, depend on the MPS platform and therefore we have explored the
possibility for multiplex MPS-by-ligation, using the Applied Biosystems SOLiD(™)
4 system. DNA isolated from plasma samples from 52 pregt women, carrying
normal or aneuploid fetuses, was sequenced in multiplex runs of 4, 8 or 16
samples simultaneously. The sequence reads were mapped to the human reference
genome and quantified according to their genomic location. In case of a fetal
aneuploidy, the number of reads of the aberrant chromosome is expected to be
higher or lower than in normal reference samples. To statistically determine
this, Z-scores per chromosome were calculated as described previously, with
thresholds for aneuploidies set at > +3.0 and < -3.0 for chromosomal over- or
underrepresentation, respectively. All samples from fetal aneuploidies yielded
Z-scores outside the thresholds for the aberrant chromosomes, with no false
negative or positive results. Full-blown fetal aneuploidies can thus be reliably
detected in maternal plasma using a multiplex MPS-by-ligation approach.
Furthermore, the results obtained with a sample from a pregcy with 45,X in
the cytotrophoblastic cell layer and 46,XX in the mesenchymal core cells show
that ccffDNA originates from the cytotrophoblastic cell layer. Discrepancies
between the genetic constitution of this cell layer and the fetus itself are
well known, and therefore, care should be taken when translating results to the
fetus itself. The recent release of new, non-invasive prenatal tests for fetal aneuploidy
using cell-free fetal DNA (cffDNA) has been hailed as a revolution in prenatal
testing and has triggered significant commercial interest in the field. Ongoing
research portends the arrival of a wide range of cffDNA tests. However, it is
not yet clear how these tests will be integrated into well-established prenatal
testing strategies in the USA, as the timing of such testing and the degree to
which new non-invasive tests will supplement or replace existing screening and
diagnostic tools remain uncertain. We argue that there is an urgent need for
policy-makers, regulators and professional societies to provide guidance on the
most efficient and ethical manner for such tests to be introduced into clinical
practice in the USA. Prenatal detection of fetal aneuploidies is one of the main goals of the
prenatal diagnostic approach. As a benefit of the development of advanced
ultrasound equipment and advances in molecular biology in the last decade, there
is a significant progress in screening methods for fetal aneuploidies, although
invasive methods remain the gold standard for aneuploidy detection. Non-invasive
prenatal diagnosis has substantial medical impact as it targets the development
of safer and more effective methods to avoid the risk of fetal loss associated
with currently used invasive methods. Identification of fetal-specific messenger
ribonucleic acids, digital polymerase chain reaction and next-generation
sequencing give the real chance for non-invasive prenatal diagnosis of fetal
aneuploidies. Although all these methods have both advantages and limitations,
some of them are moving closer to clinical implementation. In this review the
authors highlight the most recent advances in methods for non-invasive prenatal
diagnosis of aneuploidies. This document has been archived because it contains outdated information. It
should not be consulted for clinical use, but for historical research only.
Please visit the journal website for the most recent guidelines. OBJECTIVE: The aim of this study was to assess awareness, potential adoption,
and current utilization of non-invasive prenatal testing (NIPT) analysis for
common fetal aneuploidies among obstetricians.
METHODS: A 36-item web-based survey was designed to assess the current practice
of fetal aneuploidy screening and knowledge and utilization of NIPT for fetal
trisomy. Practicing obstetricians in the United States were invited via email to
participate in the survey.
RESULTS: Of the 101 obstetricians that completed the survey (27% academic-based,
73% private practice), 97% offer screening to high-risk patients and 91% offer
screening to average-risk patients. With regard to current screening tests, the
top three advantages were as follows: recommendation by professional societies,
no risk to the pregcy, and long history/experience with the test, whereas the
top three limitations were as follows: patient anxiety, risks of follow-up
invasive testing, and high false positives. NIPT had been used by 32% of
respondents and 22% were familiar with NIPT and the associated clinical data.
The majority of physicians predicted that they would offer NIPT to high-risk
women (86.1%) and average-risk women (76.2%) within 12 months.
CONCLUSION: Obstetricians plan to increase their utilization of NIPT and expect
that the majority of both high-risk and average-risk patients will be offered
NIPT as an option. OBJECTIVE: To report secondary or additional findings arising from introduction
of non-invasive prenatal testing (NIPT) for aneuploidy by whole genome
sequencing as a clinical service.
METHODS: Five cases with secondary findings were reviewed.
RESULTS: In Case 1, NIPT revealed a large duplication in chromosome 18p, which
was supported by arrayCGH of amniocyte DNA, with final karyotype showing mosaic
tetrasomy 18p. In Case 2, a deletion in the proximal long arm of chromosome 18
of maternal origin was suspected and confirmed by arrayCGH of maternal white
cell DNA. In Case 3, NIPT was negative for trisomies 21 and 18. In-depth
analysis for deletions/duplications was requested when fetal structural
anomalies were detected at routine scan. A deletion in the proximal long arm of
chromosome 3 was found and confirmed by karyotyping. In Case 4, NIPT correctly
predicted confined placental mosaicism with triple trisomy involving chromosomes
X, 7 and 21. In Case 5, NIPT correctly detected a previously unknown maternal
mosaicism for 45X.
CONCLUSION: Non-invasive prenatal testing is able to detect a wide range of
fetal, placental and maternal chromosomal abnormalities. This has important
implications on patient counseling when an abnormality is detected by NIPT. Non-invasive prenatal testing (NIPT) for aneuploidy using cell-free DNA in
maternal plasma is revolutionizing prenatal screening and diagnosis. We review
NIPT in the context of established screening and invasive technologies, the
range of cytogenetic abnormalities detectable, cost, counseling and ethical
issues. Current NIPT approaches involve whole-genome sequencing, targeted
sequencing and assessment of single nucleotide polymorphism (SNP) differences
between mother and fetus. Clinical trials have demonstrated the efficacy of NIPT
for Down and Edwards syndromes, and possibly Patau syndrome, in high-risk women.
Universal NIPT is not cost-effective, but using NIPT contingently in women found
at moderate or high risk by conventional screening is cost-effective. Positive
NIPT results must be confirmed using invasive techniques. Established screening,
fetal ultrasound and invasive procedures with microarray testing allow the
detection of a broad range of additional abnormalities not yet detectable by
NIPT. NIPT approaches that take advantage of SNP information potentially allow
the identification of parent of origin for imbalances, triploidy, uniparental
disomy and consanguinity, and separate evaluation of dizygotic twins. Fetal
fraction enrichment, improved sequencing and selected analysis of the most
informative sequences should result in tests for additional chromosomal
abnormalities. Providing adequate prenatal counseling poses a substantial
challenge given the broad range of prenatal testing options now available. BACKGROUND: A newly introduced cell-free fetal DNA sequencing based non-invasive
prenatal testing (DNA-NIPT) detects Down syndrome with sensitivity of 99% at
early gestational stage without risk of miscarriage. Attention has been given to
its public health implications; little is known from consumer perspectives. This
qualitative study aimed to explore women's motivations for using, and
perceptions of, DNA-NIPT in Hong Kong.
METHODS AND FINDINGS: In-depth interviews were conducted with 45 women who had
undertaken DNA-NIPT recruited by purposive sampling based on socio-demographic
and clinical characteristics. The sample included 31 women identified as
high-risk from serum and ultrasound based Down syndrome screening (SU-DSS).
Thematic narrative analysis examined informed-decision making of the test and
identified the benefits and needs. Women outlined a number of reasons for
accessing DNA-NIPT: reducing the uncertainty associated with risk
probability-based results from SU-DSS, undertaking DNA-NIPT as a comprehensive
measure to counteract risk from childbearing especially at advanced age,
perceived predictive accuracy and absence of risk of harm to fetus. Accounts of
women deemed high-risk or not high-risk are distinctive in a number of respects.
High-risk women accessed DNA-NIPT to get a clearer idea of their risk. This
group perceived SU-DSS as an unnecessary and confusing procedure because of its
varying, protocol-dependent detection rates. Those women not deemed high-risk,
in contrast, undertook DNA-NIPT for psychological assurance and to reduce
anxiety even after receiving the negative result from SU-DSS.
CONCLUSIONS: DNA-NIPT was regarded positively by women who chose this method of
screening over the routine, less expensive testing options. Given its perceived
utility, health providers need to consider whether DNA-NIPT should be offered as
part of universal routine care to women at high-risk for fetal aneuploidy. If
this is the case, then further development of guidelines and quality assurance
will be needed to provide a service suited to patients' needs. BACKGROUND: Non-invasive prenatal testing (NIPT) by massively parallel
sequencing is a useful clinical test for the detection of common fetal
aneuploidies. While the accuracy of aneuploidy detection can approach 100%,
results discordant with the fetus are occasionally reported. In this study we
investigated the basis of a discordant T21 positive and T18 negative NIPT result
associated with a T18 fetus confirmed by karyotyping.
METHODS: Massively parallel sequencing was used to detect fetal DNA in maternal
circulating plasma. The parental origin and nature of the fetal and placental
aneuploidies were investigated by quantitative fluorescent PCR of short tandem
repeat (STR) sequences and by copy number variation (CNV) sequencing.
RESULTS: There was no evidence of T21 maternal mosaicism, T21 microchimerism or
a vanishing twin to explain the discordant NIPT result. However, examination of
multiple placental biopsies showed both T21 and T18 mosaicism, including one
confined region with a significantly higher proportion of T21 cells. Based on
fetal DNA fractions and average mosaicism levels, the effective T21 and T18
fetal DNA fractions should have been sufficient for the detection of both
trisomies.
CONCLUSIONS: In this pregcy, we speculate that confined placental region(s)
with higher proportions of T21 cells were preferentially releasing fetal DNAs
into the maternal circulation. This study highlights placental mosaicism as a
significant risk factor for discordant NIPT results. OBJECTIVES: The aim of the study was to present initial results of non-invasive
prenatal diagnosis of common aneuploidies of chromosomes 21, 18 and 13 based on
cell-free fetal DNA in maternal serum in high-risk patients, and to compare the
results with routine karyotyping.
MATERIAL AND METHODS: Before the invasive procedure, 10 ml of peripheral blood
from 10 patients was collected to isolate cell-free fetal DNA and to perform a
non-invasive fetal trisomy test (NIFTY provided by Beijing Genomics Institute,
BGI, Shenzen, China).
RESULTS: Three out of 10 samples showed an abnormal karyotype in traditional
karyotyping. There were 9 conclusive NIFTY results. NIFTY detected 1 out of 2
trisomies 18. The quantity of cell-free fetal DNA in maternal plasma in the
second probe with trisomy 18 was unsatisfactory fora conclusive NIFTY result. In
1 case traditional karyotyping revealed mosaicism impossible to detect with
NIFTY Non-invasive prenatal testing (NIPT) of fetal aneuploidy using cell-free fetal
DNA is becoming part of routine clinical practice. RAPIDR (Reliable Accurate
Prenatal non-Invasive Diagnosis R package) is an easy-to-use open-source R
package that implements several published NIPT analysis methods. The input to
RAPIDR is a set of sequence alignment files in the BAM format, and the outputs
are calls for aneuploidy, including trisomies 13, 18, 21 and monosomy X as well
as fetal sex. RAPIDR has been extensively tested with a large sample set as part
of the RAPID project in the UK. The package contains quality control steps to
make it robust for use in the clinical setting.
AVAILABILITY AND IMPLEMENTATION: RAPIDR is implemented in R and can be freely
downloaded via CRAN from here:
http://cran.r-project.org/web/packages/RAPIDR/index.html.
CONTACT: [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. OBJECTIVES: To evaluate the feasibility of non-invasive prenatal testing (NIPT)
of maternal plasma samples collected from pregt Chinese women in early
gestation, between 8 + 0 and 12 + 6 weeks' gestation.
METHODS: In this pilot study, 212 women with high-risk pregcies were
recruited at a single Chinese Hospital. Fetal aneuploidies associated with
chromosomes 21, 18, 13, X and Y were detected by massively parallel sequencing
of maternal plasma DNA samples. Invasive prenatal diagnosis by either chorionic
villus sampling or amniocentesis and then karyotyping was offered to all women
to confirm both positive and negative NIPT results. Fetal DNA fraction was also
determined in male pregcies, by the relative percentage of Y-chromosome
sequences. All confirmed NIPT-negative pregcies were followed up to birth and
neonates were clinically evaluated for any symptoms of chromosomal disease.
RESULTS: Autosomal aneuploidies trisomy 21 (n = 2), 18 (n = 1) and 13 (n = 1)
were detected by NIPT and confirmed by amniocentesis and karyotyping. There were
one false-positive 45,X sample and two false-negative samples associated with
fetal karyotypes 47,XXY and 45,X[16]/47,XXX[14]. In the 100 male pregcies,
the median fetal DNA fraction was 8.54% and there was a trend towards an
increasing fetal fraction from 8 + 0 to 12 + 6 weeks' gestation. The majority
(95%) of pregcies had a fetal DNA fraction > 4%, which is generally the limit
for accurate aneuploidy detection by NIPT. Across this early gestational time
period, there was a weak inverse relationship (R(2) = 0.186) between fetal DNA
fraction and maternal weight.
CONCLUSIONS: NIPT is highly reliable and accurate when applied to maternal DNA
samples collected from pregt women in the first trimester between 8 + 0 and
12 + 6 weeks. OBJECTIVE: Non-invasive prenatal testing (NIPT) using cell-free fetal DNA in
maternal plasma has been developed for the detection of fetal aneuploidy.
Clinical trials have shown high sensitivity and specificity for trisomy 21 (T21)
in both high-risk and average-risk populations. Although its great potential for
prenatal medicine is evident, more information regarding the consequences of
implementing NIPT in a national programme for prenatal screening is required.
STUDY DESIGN: A decision-analytic model was developed to compare costs and
outcomes of current clinical practice in The Netherlands using conventional
screening only, with two alternatives: implementing NIPT as an optional
secondary screening test for those pregcies complicated by a high risk for
T21, and implementing NIPT as primary screening test, replacing conventional
screening. Probability estimates were derived from a systematic review of
international literature. Costs were determined from a health-care perspective.
Data were analysed to obtain outcomes, total costs, relative costs and
incremental cost-effectiveness ratios (ICERs) for the different strategies.
Sensitivity analysis was used to assess the impact of assumptions on model
results.
RESULTS: Implementing NIPT as an optional secondary, or as primary screening
test will increase T21 detection rate by 36% (from 46.8% to 63.5%) and 54% (from
46.8% to 72.0%), simultaneously decreasing the average risk of procedure-related
miscarriage by 44% (from 0.0168% to 0.0094% per pregt woman) and 62% (from
0.0168% to 0.0064% per pregt woman), respectively. None of the strategies
clearly dominated: current clinical practice is the least costly, whereas
implementing NIPT will cause total costs of the programme to increase by 21%
(from €257.09 to €311.74 per pregt woman), leading to an ICER of k€94 per
detected case of T21, when utilised as an optional secondary screening test and
by 157% (from €257.09 to €660.94 per pregt woman), leading to an ICER of
k€460 per detected case of T21, when utilised as primary screening test.
However, implementing NIPT as triage test did result in the lowest expected
relative costs per case of T21 diagnosed (k€141).
CONCLUSION: NIPT should be implemented in national health care as an optional
secondary screening test for those pregcies complicated by a high risk for
T21. OBJECTIVE: To track and analyze two false positive cases from non-invasive
prenatal testing for potential fetal aneuploidy.
METHODS: The two cases, respectively reported to have XO (+++) and T18 (1/20)
XO(+), were analyzed with conventional karyotyping, fluorescence in situ
hybridization (FISH) and massively parallel genomic sequencing (MPS).
RESULTS: The first fetus, who was suspected for XO(+++), was verified to have
super female syndrome (47,XXX/46,XX) due to confined placental mosaicism by
karyotyping of amniotic fluid cells, FISH analysis of placenta and massively
parallel sequencing (MPS) of fetal tissue. The second fetus, suspected to have
trisomy 18 (1/20) XO(+), was verified to have Turner syndrome by karyotyping,
FISH and MPS analyses of umbilical cord blood cells. And the karyotype was
45,X[48]/46, X, der(X) del(X) (p11.21) del(X) (q13.3)[62].
CONCLUSION: Non-invasive prenatal testing carries a risk for false positive
diagnosis of fetal sex chromosome and trisomy 18. Combined cytogenetic and
molecular techniques are required to ensure an accurate diagnosis. At 17(+4) week, non-invasive prenatal testing (NIPT) results of a 24-years-old
mother showed high risk of monosomy X (45, X). Abnormally shaped head and
cardiac defects were observed in prenatal ultrasound scan at 19(+3) week.
Amniocentesis conducted at 19(+3) week identified karyotype 47, XX, +18, which
suggested that the NIPT failed to detect trisomy 18 (T18) in this case. With a
further massively parallel sequencing (MPS) of maternal blood, fetal and
placental tissues, we found a confined placental mosaicism (CPM) with non-mosaic
T18 fetus and multiclonal placenta with high prevalence of 45, X and low level
of T18 cells. FISH and SNP-array evidence from the placental tissue confirmed
genetic discrepancy between the fetus and placenta. Because the primary source
of the fetal cell-free DNA that NIPT assesses is mostly originated from
trophoblast cells, the level of T18 placental mosaicism may cause false negative
NIPT result in this rare case of double aneuploidy. Obesity is a worldwide epidemic and can have a profound effect on pregcy
risks. Obese patients tend to be older and are at increased risk for structural
fetal anomalies and aneuploidy, making screening options critically important
for these women. Failure rates for first-trimester nuchal translucency (NT)
screening increase with obesity, while the ability to detect soft-markers
declines, limiting ultrasound-based screening options. Obesity also decreases
the chances of completing the anatomy survey and increases the residual risk of
undetected anomalies. Additionally, non-invasive prenatal testing (NIPT) is less
likely to provide an informative result in obese patients. Understanding the
limitations and diagnostic accuracy of aneuploidy and anomaly screening in obese
patients can help guide clinicians in counseling patients on the screening
options. OBJECTIVE: To explore the value of next-generation sequencing for the
non-invasive prenatal testing of fetal chromosomal aneuploidies.
METHODS: Plasma from 4004 women with singleton pregcy at a gestational age
between 12-35(+5) weeks was collected prior to amniocentesis between April 19th
2011 and December 31st 2013. The samples were divided into three groups: (1)
High risk for Down syndrome by biochemical screening; (2) Advanced maternal age;
(3) Abnormalities by ultrasound or other methods. Plasma DNA extracted from
above samples was sequenced at low coverage. Positive results were verified
against the karyotypes of the fetuses. For those with negative results, the
fetuses were followed up by telephone call for at least six months after birth.
RESULTS: Among 4003 samples subjected to non-invasive prenatal diagnosis, 66
(1.65%) had a positive result. In group 1, 22 cases of trisomy 21 (T21), 3 cases
of trisomy 18 (T18), 1 case of 13 trisomy (T13), 8 cases of 45,X and 2 cases of
other chromosomal abnormality were detected. In group 2, 13 cases of T21, 2
cases of T18, 1 case of T13, 5 cases of 45,X, 2 cases of 47,XXN and 1 case of
other chromosomal abnormality were detected. In group 3, 1 case of T21, 1 case
of T18, 1 case of T13, and 3 cases of 47,XXN were detected. For 55 samples
underwent prenatal diagnosis, 30 cases of T21 and 4 cases of T18 were
discovered, which was consistent with the results of non-invasive prenatal
diagnosis. For the 13 cases indicated as 45,X, 3 were verified by karyotype
analysis, 2 were verified as mosaicism (45,X/46,XN), 8 were 46,XN (false
positives). For the 5 cases indicated as 47,XXN, 2 were verified by karyotype
analysis, the other 3 were 46,XN (false positives). Karyotypes of 3 cases
suspected for other chromosomal abnormalities were all verified as 46,XN (false
positive). Until May 1st 2014, telephone follow-up for those with negative
screening results only identified a boy with facial abnormalities and
developmental delay, which was similar to his older sister, combined karyotyping
and fluorescence in situ hybridization analysis has verified the karyotype of
the boy as 46,XY,rec(14)dup(14q)inv(14)(p12q14)pat.
CONCLUSION: Our results indicated that sequencing of plasma free DNA can rapidly
detect fetal chromosomal aneuploidies. The method is non-invasive, and the
results are highly consistent with karyotype analysis in terms of accuracy and
specificity. Non-invasive testing can be used as an effective adjunct to
conventional prenatal diagnostic methods, which can greatly reduce unnecessary
invasive prenatal diagnosis. However, the sensitivity and accuracy for
aneuploidy detection other than chromosome 13/18/21 still need to be improved. |
Which packages are used for performing overlap analysis of genomic regions in R/bioconductor? | IRanges, GenomicRanges, and GenomicFeatures provide scalable data structures for representing annotated ranges on the genome, with special support for transcript structures, read alignments and coverage vectors. Computational facilities include efficient algorithms for overlap and nearest neighbor detection, coverage calculation and other range operations. This infrastructure directly supports more than 80 other Bioconductor packages, including those for sequence analysis, differential expression analysis and visualization. | |
Is dichlorphenamide effective for periodic paralysis? | Yes, dichlorphenamide is effective for periodic paralysis. Dichlorphenamide--a carbonic anhydrase inhibitor--has been shown in a controlled trial to prevent attacks for many patients with both hypokalemic and hypokalemic periodic paralysis. | Three patients with Hypokalemic Periodic Paralysis (HOPP)-associated progressive
interattack muscle weakness, who became unresponsive or worsened by
acetazolamide, responded favorably to dichlorophenamide, a more potent carbonic
anhydrase inhibitor. Dichlorophenamide in single-blind placebo-controlled
trials, considerably improved functional strength in two of the patients and had
a moderate but definite effect in the third. Muscle groups graded 4/5 (MRC
scale)returned to normal; very weak (0-3/5) atrophic muscles, improved to a
minor degree. In one patient with acetazolamide-resistant paralytic attacks,
dichlorophenamide also diminished the frequency and severity of the acute
attacks. Dichlorophenamide had, in the present study, less effect than
acetazolamide in reducing serum HCO3(-) and elevating Cl-. Its effectiveness may
be related to the degree of sensitivity of certain HOPP patients to alterations
of Cl- and/or HCO3(-) serum levels or to a different action of the drug
unrelated to carbonic anhydrase inhibition or acidosis. Dichlorophenamide should
be considered as an alternate to acetazolamide in the treatment of patients with
HOPP-associated interattack muscle weakness who have become unresponsive or
worsened by acetazolamide. Although the carbonic anhydrase inhibitors have been used in the treatment of
the primary periodic paralyses (PPs), their efficacy has not been demonstrated
in double-blind, placebo-controlled trials. Therefore, we tested the efficacy of
dichlorphenamide (DCP; Daranide), a potent carbonic anhydrase inhibitor, in the
treatment of episodic weakness in the primary PPs. We performed two multicenter,
randomized, double-blind, placebo-controlled crossover trials, one involving 42
subjects with hypokalemic periodic paralysis (HypoPP) and the other involving 31
subjects with potassium-sensitive periodic paralysis (PSPP). In each trial, two
8-week treatment periods were separated by an active washout period of at least
9 weeks. The primary outcome variable in the HypoPP trial was the occurrence of
an intolerable increase in attack severity or frequency (end point). The primary
outcome variable in the PSPP trial was the number of attacks per week. In the
HypoPP trial, there were 13 subjects who exhibited a preference (in terms of the
end point) for either DCP or placebo, and 11 of these preferred DCP. In the PSPP
trial, DCP significantly reduced attack rates relative to placebo. DCP also
significantly reduced attack rates relative to placebo in the HypoPP subjects.
We conclude that DCP is effective in the prevention of episodic weakness in both
HypoPP and PSPP. BACKGROUND: Primary periodic paralyses are rare inherited muscle diseases
characterised by episodes of flaccid weakness affecting one or more limbs,
lasting several hours to several days, caused by mutations in skeletal muscle
channel genes.
OBJECTIVES: The objective of this review was to systematically review treatment
of periodic paralyses.
SEARCH STRATEGY: We searched the Cochrane Neuromuscular Disease Group Trials
Register, MEDLINE (from January 1966 to July 2007), and EMBASE (from January
1980 to July 2007) and any other available international medical library sources
from the University of Milan for randomised trials.
SELECTION CRITERIA: We included randomised (including cross-over studies) and
quasi-randomised trials in participants with primary periodic paralyses, in
which any form of treatment, including physical therapy and alternative
therapies, was compared to placebo or another treatment.
DATA COLLECTION AND ANALYSIS: Our primary outcome measure was the change in
attack severity or frequency by eight weeks from the start of treatment. Our
secondary outcome measures were: change in muscle strength and mass; change in
Quality of Life, using Short Form 36 (SF36) or similar; preference of treatment
strategy; adverse effects at eight weeks.
MAIN RESULTS: Three studies met our inclusion criteria. In one study
dichlorphenamide (DCP) vs placebo was tested in two groups of participants: 42
with hypokalemic periodic paralysis (HypoPP) and 31 with hyperkalemic periodic
paralysis (HyperPP), based on clinical criteria. Thirty-four of 42 participants
with hypokalemic periodic paralysis completed both treatment phases. For the 34
participants having attack rate data for both treatment phases, the mean
improvement in attack rate (P = 0.02) and severity-weighted attack rate (P =
0.01) on DCP relative to placebo were statistically significant. Fifteen
preferred DCP, three placebo and six their baseline medication. Twenty-four of
31 participants with hyperkalemic periodic paralysis completed both treatment
phases: for the 16 participants who had attack rate data for both treatment
phases, the mean improvement in attack rate (P = 0.006) and in severity-weighted
attack rate (P = 0.02) on DCP relative to placebo were significant. Fifteen
preferred DCP, one placebo and five their baseline medication. Acetazolamide
proved to improve muscle strength in eight participants with HypoPP in one other
study and pinacidil, a potassium channel opener, also improved muscle strength
in 2/4 participants with HypoPP in a third study.
AUTHORS' CONCLUSIONS: The largest included study that met our inclusion criteria
suggested that DCP was effective in the prevention of episodic weakness in both
hypokalemic and hyperkalemic periodic paralyses. The other two studies provide
some evidence that either acetazolamide or pinacidil may improve muscle
strength. However we still lack sufficient evidence to provide full guidelines
for the treatment of people with periodic paralysis. Management considerations in hypokalemic periodic paralysis include accurate
diagnosis, potassium dosage for acute attacks, choice of diuretic for
prophylaxis, identification of triggers, creating a safe physical environment,
peri-operative measures, and issues in pregcy. A positive genetic test in the
context of symptoms is the gold standard for diagnosis. Potassium chloride is
the favored potassium salt given at 0.5-1.0 mEq/kg for acute attacks. The oral
route is favored, but if necessary, a mannitol solvent can be used for
intravenous administration. Avoidance of or potassium prophylaxis for common
triggers, such as rest after exercise, high carbohydrate meals, and sodium, can
prevent attacks. Chronically, acetazolamide, dichlorphenamide, or
potassium-sparing diuretics decrease attack frequency and severity but are of
little value acutely. Potassium, water, and a telephone should always be at a
patient's bedside, regardless of the presence of weakness. Perioperatively, the
patient's clinical status should be checked frequently. Firm data on the
management of periodic paralysis during pregcy is lacking. Patient support
can be found at http://www.periodicparalysis.org. Our understanding of the molecular pathogenesis of the neuromuscular ion
channelopathies has increased rapidly over the past two decades due to the
identification of many of the genes whose mutation causes these diseases. These
molecular discoveries have facilitated identification and classification of the
hereditary periodic paralyses and the myotonias, and are likely to shed light on
acquired ion channelopathies as well. Despite our better understanding of the
pathogenesis of these disorders, current treatments are largely empirical and
the evidence in favor of specific therapy largely anecdotal. For periodic
paralysis, dichlorphenamide--a carbonic anhydrase inhibitor--has been shown in a
controlled trial to prevent attacks for many patients with both hypokalemic and
hypokalemic periodic paralysis. A second trial, comparing dichlorphenamide with
acetazolamide versus placebo, is currently in progress. For myotonia, there is
only anecdotal evidence for treatment, but a controlled trial of mexiletine
versus placebo is currently being funded by a Food and Drug
Administration-orphan products grant and is scheduled to begin in late 2008. In
the future, mechanism-based approaches are likely to be developed. For example,
exciting advances have already been made in one disorder, myotonic dystrophy-1
(DM-1). In a mouse model of DM-1, a morpholino antisense oligonucleuotide
targeting the 3' splice site of CLCN1 exon 7a repaired the RNA splicing defect
by promoting the production of full-length chloride channel transcripts.
Abnormal chloride conductance was restored, and myotonia was abolished. Similar
strategies hold potential for DM-2. The era of molecularly-based treatments is
about to begin. |
What is evaluated with the Hydrocephalus Outcome Questionnaire? | The Hydrocephalus Outcome Questionnaire (HOQ) is a simple, reliable, and valid measure of health status in children with hydrocephalus. | OBJECTIVE: To compare three separate methods for establishing interpretability
for a health status measure, the Hydrocephalus Outcome Questionnaire (HOQ).
STUDY DESIGN AND SETTING: The mothers of children with hydrocephalus attending
the outpatient clinics at a pediatric hospital completed the HOQ (for which
scores can range from 0 to 1.0 with the smallest possible incremental change
being .005), the Health Utilities Index-2 (HUI-2), and a global rating of their
child's health. The surgeon for the child also provided a global rating of the
child's health following their visit. These data were used to calculate (i) the
minimal important difference (MID) based on global health ratings, (ii) the MID
based on an effect size approach, and (iii) the conversion of numerical HOQ
scores into health utility scores obtained from the HUI-2.
RESULTS: Based on mothers' responses (n = 79) and surgeons' responses (n = 61),
respectively, the MID for the HOQ was estimated to be .12 and .10. Using the
effect size approach, the MID was estimated to be much lower at .03. HOQ scores
were found to be readily translatable to HUI-2 utility scores using a simple
linear transformation. The mean utility score for this sample of patients was
.77.
CONCLUSIONS: Two methods for determining the MID yielded quantitatively
different results. Conversion of numerical health status scores to utility
scores was done successfully and providing another element of interpretability. OBJECT: The objectives of this study were to assess, in a cohort of children
with recently treated hydrocephalus, the correlation between scores on the
Hydrocephalus Outcome Questionnaire (HOQ) and the children's type of schooling
and motor functioning, and to assess the overall outcome of the children.
METHODS: The health status of 142 pediatric patients (85 boys) with previous
hydrocephalus, born between 1995 and 1999, was assessed. Outcomes were
determined using the HOQ, type of schooling, and motor functioning. Data were
obtained from parental interviews and patient medical records. RESULTS. Twelve
patients died (8.5%). Responses to the HOQ were obtained from 107 patients (65
boys). The mean age of the patients was 7 years and 9 months +/- 1.42 years
(range 6-10 years). The Physical Health score of the HOQ correlated well with
the motor functioning score (r = 0.652) as did the Cognitive Health score with
the type of schooling (r = 0.672). Fifty-nine percent of the patients were able
to attend a school for students with normal intelligence. Disabling motor
functioning was found in only 30% of patients. Epilepsy was present in 14%.
CONCLUSIONS: The results show a good correlation between the type of schooling
and the Cognitive HOQ score and between the Physical HOQ score and the motor
functioning score. The HOQ is a simple and very useful measurement for
determining outcome in pediatric hydrocephalus. This study exampled the properties of a child-completed version of the
Hydrocephalus Outcome Questionnaire (cHOQ) and compared these with parental
responses to the HOQ (parent version). This was a cross-sectional study in the
outpatient clinics at three Canadian paediatric hospitals (Toronto, Vancouver,
and Halifax). All cognitively-capable children with previously treated
hydrocephalus who were aged between 6 and 19 years were eligible. Parents
completed the HOQ and the Health Utilities Index Mark 3; children completed the
cHOQ. A total of 273 children participated (146 males, 127 females; mean age 14
y 1 mo, SD 2 y 7 mo). Internal consistency of the cHOQ was 0.93 and test-retest
reliability was 0.86 (95% confidence interval 0.78-0.92). Mother-child agreement
and father-child agreement were 0.57 (0.40-0.68) and 0.62 (0.48-0.73)
respectively. Agreement was higher for assessments of physical health, but lower
for assessments of cognitive health and social-emotional health. There was
greater parent-child agreement for older children. When there was disagreement,
it seemed that children tended to rate their health better than their parents
did. In older children with hydrocephalus, the cHOQ appears to be a
scientifically reliable means of assessing long-term outcome. The differences in
child and parent perceptions of health need to be appreciated when conducting
outcome studies in this population. PURPOSE: In the current literature, there are essentially no comparisons of
quality of life (QOL) outcome after endoscopic third ventriculostomy (ETV) and
shunt in childhood hydrocephalus. Our objective was to compare QOL in children
with obstructive hydrocephalus, treated with either ETV or shunt.
METHODS: A cross-sectional survey was conducted at SickKids, Toronto of children
between ages five and 18 years, with obstructive hydrocephalus due to aqueductal
obstruction and no other brain abnormalities. Measures of QOL were the
Hydrocephalus Outcome Questionnaire and the Health Utilities Index Mark 3. A
subset of patients was given the Wechsler Intelligence Scales for Children
(WISC-IV).
RESULTS: A total of 47 of 59 (80%) eligible patients participated (24 had ETV as
primary treatment, 23 had shunt as primary treatment), with a mean age of 12.1
years (standard deviation 3.9) at assessment. The ETV group was older at initial
surgery (p < 0.001) and had larger ventricle size at last follow-up (p = 0.047).
In all QOL measures, there were no significant differences between the ETV group
and shunt group (all p > or = 0.09). Treatment failure, hydrocephalus
complications, and the presence of a functioning ETV at assessment were not
associated with QOL differences. Among the 11 children (six ETV, five shunt) who
were given the WISC-IV, there were no significant differences between the scores
of the ETV group and shunt group (all p > or = 0.11).
CONCLUSIONS: This is the first study to provide a meaningful comparison of QOL
after ETV and shunt in children. These preliminary results suggest that there is
no obvious difference in QOL after ETV and shunt. OBJECT: Quality of life (QOL) studies comparing treatment with endoscopic third
ventriculostomy (ETV) and CSF shunting are very limited. The authors compared
QOL outcomes following these 2 treatments in a large cohort of children with
hydrocephalus by using multivariable statistical techniques to adjust for
possible confounder variables.
METHODS: The families of children between 5 and 18 years of age with previously
treated hydrocephalus at 3 Canadian pediatric neurosurgery centers completed
measures of QOL: the Hydrocephalus Outcome Questionnaire (HOQ) and the Health
Utilities Index Mark 3 (HUI3). Medical records and recent brain imaging studies
were reviewed. A linear regression analysis was performed with the QOL measures
as the dependent variable. In multivariable analyses, the authors assessed the
independent effect of initial hydrocephalus treatment (ETV vs shunting) while
adjusting for the treatment center, current patient age, age at initial
treatment, etiology of hydrocephalus, total number of days spent in the hospital
for initial treatment, total number of days spent in the hospital for subsequent
hydrocephalus complications, functioning ETV at follow-up assessment, frequency
of seizures, and current ventricle size.
RESULTS: Data from 603 patients were available for analysis. Fifty-eight
patients had undergone ETV as their primary treatment and 545 had undergone CSF
shunting. Endoscopic third ventriculostomy patients were slightly younger at the
follow-up assessment, were older at the first surgery, and spent fewer days in
the hospital for hydrocephalus complications. Without adjustment for any
confounders, treatment with ETV was associated with significantly higher HOQ
physical scores and HUI3 scores. After multivariable adjustment, however, there
was no significant difference in any outcome measure. A functioning ETV at the
time of the follow-up assessment was not significant in any model.
CONCLUSIONS: Treatment with either ETV or CSF shunting does not appear to be
associated with any substantial difference in QOL outcome after adjusting for
prognostic factors. Further study is needed to definitively determine the
relative QOL benefit of either procedure, if any. PURPOSE: The long-term outcome in spina bifida-Chiari II-hydrocephalus complex
is poorly understood. Traditional neurosurgical outcome measures are crude.
Neuropsychological testing is increasingly important in outcome assessment. We
investigated the health, disability, lifestyle and cognitive function in adults
who had myelomeningocoele closure at birth.
METHODS: Adult patients under routine follow-up were assessed in a joint
neurosurgery/neuropsychology clinic. Patients completed lifestyle
questionnaires, the hydrocephalus outcome questionnaire (HOQ) and underwent
cognitive testing. Clinical variables including number of shunt revisions, shunt
infection and surgical decompression of foramen magnum, which may influence
outcome, were investigated.
RESULTS: Twenty-one adults with a median age of 35 years were investigated. All
had treated hydrocephalus, and eight had foramen magnum decompression for
headache or progressive brainstem symptoms with stabilisation of symptoms in
seven and improvement in one. Only eight patients were living independently,
five were in paid employment and five work voluntarily. HOQ scores for cognitive
function were lower (0.56 ± 0.20; mean ± standard deviation (SD)) than those for
physical (0.64 ± 0.15) and social-emotional (0.65 ± 0.17) health. Cognitive
function varied across the cohort with attention most severely affected (73.9 ±
17.0; mean ± SD). Repeated episodes of shunt malfunction or foramen magnum
decompression were not associated with a worse cognitive function.
CONCLUSIONS: Despite intervention in childhood and adequate cerebrospinal fluid
diversion the prognosis for independent living into adulthood remains poor. All
patients have elements of cognitive impairment. Structural brain abnormalities
may be more important determits of cognitive outcome than shunt malfunction. PURPOSE: Benign external hydrocephalus (BEH) is characterized by excess
cerebrospinal fluid in the frontal subarachnoid spaces in infants evaluated for
macrocephaly. We sought to describe the natural history of this disorder,
specifically its clinical presentation, disease course and long-term health
status impact using the validated, disease-specific Hydrocephalus Outcome
Questionnaire (HOQ).
METHODS: An inception cohort of children >5 and <18 years old, with a history of
BEH was assembled by ICD-9 and a key word search of medical and radiology
records. Review confirmed the diagnosis of BEH, excluded major medical
comorbidities and assessed presentation, radiographic features and head
size/growth velocity. The HOQ was administered by mail.
RESULTS: We identified 99 patients, 5-12 years old (55% males). Twenty were born
prematurely, 12 with <33 weeks gestation. Children presented at an average age
of 9 ± 4.8 months (mean ± SD). The presenting complaint was macrocephaly in 65
cases. Other presenting findings were positional head shape deformity and
torticollis; 10% had a family history of macrocephaly. Developmental delay was
present in 21% of patients (4% verbal, 20% gross motor, 4% fine motor delay).
Four patients had small subdural hematomas, none with suspicion of a
non-accidental trauma. During clinical reassessment over a mean follow-up of 13
months, the average head percentile was stable and none of the patients
developed new subdural hematomas. Gross motor delay resolved in 15/20 and fine
motor delay in 4/4 patients. Verbal delay resolved in 2/4 patients, but
interestingly, was newly detected in 6 other children. None of the patients
required cerebrospinal fluid shunting. The response rate to the HOQ was 25%
(median age 7 years, 74% females). The average overall HOQ score was 0.75 ± 0.24
versus 0.68 ± 0.19 for a previously published cohort of shunted hydrocephalic
children.
CONCLUSIONS: BEH patients in this series generally saw resolution of presenting
motor developmental delays; however, new verbal delays were detected in a
non-trivial number of patients. Quality of life measurements suggest some
reduction in health status, but less so than is seen with shunted hydrocephalus. OBJECT: The Hydrocephalus Outcome Questionnaire (HOQ) is an established means of
measuring quality of life, but the cognitive component of this questionnaire has
never been formally compared with gold-standard neuropsychological test scores.
The authors hypothesized that the HOQ Cognitive Health score would demonstrate a
relatively strong correlation with neuropsychological test scores, whereas much
weaker correlations would be seen for HOQ Physical and Social-Emotional Health
scores.
METHODS: A cross-sectional study of children with long-standing hydrocephalus
presenting to The Hospital for Sick Children's Neurosurgery Clinic was performed
between July 2006 and September 2008. Participating children and families
completed the HOQ and a battery of 21 standard neuropsychological tests and
questionnaires. Pearson correlation analysis was then performed.
RESULTS: A total of 83 patients (81% participation) was accrued; the mean age
was 11.5 ± 3.4 years (mean ± SD) at the time of assessment. The mean age at
hydrocephalus treatment was 1.3 ± 2.6 years. The mean overall HOQ score was 0.69
± 0.21. The HOQ Cognitive score had a moderate or strong correlation with 19
(90%) of 21 neuropsychological test scores, much more so than the HOQ
Social-Emotional score (5 moderate or strong correlations, 24%) and the HOQ
Physical score (1 moderate correlation, 5%). For 19 neuropsychological tests
(90%), the HOQ Cognitive score had a stronger correlation than the other scores.
The HOQ Cognitive score had particularly strong correlations with the Verbal IQ,
List Learning, Behavior Problems, and Metacognitive Abilities components.
CONCLUSIONS: Data from a wide-ranging representative sample of children with
long-standing hydrocephalus provide added evidence of the validity of the HOQ
Cognitive score and the overall domain structure of the HOQ itself. |
What is known about maternal smoking and brain tumor risk? | Findings regarding association of maternal smoking and brain tumor risk are mixed. It was shown that children of women who smoked during pregnancy had an increased incidence of brain tumors (hazard ratio = 1.24; 95% confidence interval: 1.01-1.53). The increase in risk was similar for benign and malignant tumors, and was most apparent for astrocytoma. However, other authors did not find association between maternal smoking and brain tumor risk. | We questioned mothers of 209 young brain tumor patients and mothers of 209
controls about experiences of possible etiological relevance which they had
during pregcy or which their children had while growing up. Long-suspected
brain tumor risk factors such as head trauma and X-rays appeared to be factors
for relatively few cases. Increased risk was associated with maternal contact
with nitrosamine-containing substances such as burning incense (odds ratio, 3.3;
p = 0.005), sidestream cigarette smoke (odds ratio, 1.5; p = 0.03), and face
makeup (odds ratio, 1.6; p = 0.02); with maternal use of diuretics (odds ratio,
2.0; p = 0.03) and antihistamines (odds ratio, 3.4; p = 0.002); and with the
level of maternal consumption of cured meats (p = 0.008). These drugs contain
nitrosatable amines and amides, and the cured meats contain nitrites, chemicals
which are precursors of N-nitroso compounds. We propose a hypothesis that brain
tumors in these young people are related to in utero exposure to N-nitroso
compounds and their precursors, the most potent nervous system carcinogens known
in experimental animals. Data from a large, population-based, case-control study were analyzed to assess
the role of parental smoking in childhood brain tumors. Parents of 361 cases,
newly diagnosed between January 1, 1977 and December 31, 1981 and ascertained
from eight Surveillance, Epidemiology, and End Results (SEER) program
registries, and 1,083 controls had been interviewed. No significant differences
in risks were found to be associated with maternal or paternal smoking at any
time (odds ratio (OR) = 0.92 for mothers and 1.06 for fathers), during the year
of birth of the child (which included both the prenatal and postnatal periods)
(ORs = 0.84 for < 1 pack/day and 1.0 for > or = 1 pack/day for mothers, and 0.68
for < 1 pack/day and 1.07 for > or = 1 pack/day for fathers), or 2 years before
the child was born, i.e., the pre-conception period (ORs = 0.75 for < 1 pack/day
and 1.01 for > or = 1 pack/day for mothers, and 0.90 for < 1 pack/day and 1.15
for > or = 1 pack/day for fathers). Mothers were also specifically asked if they
smoked during the pregcy, and no association was found compared with never
smokers (OR = 1.08, 95% confidence interval (CI) 0.80-1.45) or for ever-smokers
who continued to smoke during pregcy compared with those who stopped smoking
during pregcy (OR = 1.15, 95% CI 0.75-1.78). Finally, no significant increase
in risk of brain tumors was found for the child's passive exposure to parental
smoking during the period from birth to diagnosis of the brain tumor in the
case. The lack of an effect of parental smoking was observed for both the major
histologic types and locations of brain tumors. These findings and those from
earlier studies provide no support for the hypothesis that parental cigarette
smoking influences the risk of brain tumors in children. Data from a large, population-based case-control study were analyzed to
investigate the relationship between prenatal exposure to tobacco smoke and
childhood brain tumors (CBTs). A total of 540 CBT patients, diagnosed between
1984 and 1991, were identified from population-based tumor registries in 19 West
Coast counties that included Seattle, WA (13 counties), San Francisco, CA (5
counties), and Los Angeles, CA (1 county). Random digit dial was used to select
801 control subjects from the three geographical regions to obtain a
case:control ratio of 1:2 in San Francisco and Seattle and 1:1 in Los Angeles.
The data first were analyzed separately by geographical site and then were
combined with adjustments made for gender, age at the time of diagnosis (or
reference date of control subjects), birth year of the index child, and maternal
race. No association was found between the risk of CBTs and maternal or paternal
smoking before pregcy and there was no association between CBTs and maternal
smoking during pregcy [odds ratio (OR) = 0.98; 95% confidence interval (CI) =
0.72-1.3]. A slightly increased OR for CBTs was found for paternal smoking
during pregcy in the absence of maternal smoking (OR = 1.2; 95% CI =
0.90-1.5) and for maternal exposure to passive smoke from any source (OR = 1.2;
95% CI = 0.95-1.6). The results of this analysis are consistent with results
from several prior epidemiological studies that showed no significant
association between CBTs and maternal smoking before or during pregcy or
maternal exposure to passive smoke during pregcy. We identified more than 30 studies on the association between exposure to
maternal tobacco smoke during pregcy and cancer in childhood. We combined
their results in meta-analyses based on a random effects model. The results of
the meta-analyses suggest a small increase in risk of all neoplasms [relative
risk (RR) 1.10; 95% confidence interval (CI), 1.03-1.19; based on 12 studies],
but not of specific neoplasms such as leukemia (RR 1.05; CI, 0.82-1.34; 8
studies) and central nervous system tumors (RR 1.04; CI, 0.92-1. 18; 12
studies). Results for other specific neoplasms were sparse, but the available
data did not suggest a strong association for any type of tumor. No clear
evidence of dose response was present in the studies that addressed this issue.
The results on exposure to maternal tobacco smoke before or after pregcy are
too sparse to allow a conclusion. The results on exposure to paternal tobacco
smoke suggest an association with brain tumors (RR 1.22; CI, 1.05-1. 40; based
on 10 studies) and lymphomas (RR 2.08; CI, 1.08-3.98; 4 studies). The data are
too sparse for the other neoplasms, although the results of a few recent large
studies are compatible with a weak carcinogenic effect of paternal smoke. For
exposure from either maternal or paternal smoke, bias and confounding cannot yet
be ruled out. Further studies are needed to confirm the hypothesis that parental
tobacco smoke, from the father in particular, is a risk factor of childhood
cancer. Results on the risk of lung cancer in adulthood and childhood passive
smoking exposure are available from 11 studies: they do not provide evidence of
an increased risk (summary RR 0.91; CI, 0.80-1.05). BACKGROUND: From 1993 to 1997 we conducted two population-based case-control
studies on childhood cancer and a variety of potential risk factors in Germany.
One case group involved children under the age of 15 years having a tumor of the
central nervous system (CNS).
PROCEDURE: For both studies, one conducted in the northwestern area of Germany,
the other covering the whole of West Germany, incident cases were identified
from the nationwide German Childhood Cancer Registry, and controls were randomly
selected from complete population registration files.
RESULTS: In total 466 pediatric CNS tumor cases and 2,458 controls were
available for analyses. We observed only few positive associations, namely,
between CNS tumors and low birth weight [<2,500 g; odds ratio (OR), 1.73; 95%
confidence interval (CI), 1.06-2.84], between ependymoma and maternal smoking
during pregcy (>10 cigarettes per day: OR, 4.71; 95% CI, 1.69-13.1), and
between astrocytoma and exposure to wood preservatives (OR, 1.91; 95% CI,
1.22-3.01). CNS tumors were not associated with high birth weight, duration of
breast feeding, maternal age at time of delivery, duration of gestation,
previous fetal losses, paternal smoking during pregcy, maternal alcohol
consumption, the child's exposure to pesticides, maternal diagnostic X-ray
examinations during pregcy, X-ray examinations of the child, or exposure to
residential magnetic fields.
CONCLUSIONS: Despite the large study population, we found only few factors that
were associated with CNS tumors or one of the morphological subgroups.
Therefore, our results suggest that aspects of the prenatal and neonatal period
play only a minor role in the etiology of pediatric CNS tumors. The etiology of childhood brain tumors (CBTs) remains unknown. Tobacco smoke
contains several known carcinogens and can induce DNA adducts in human placenta
and hemoglobin adducts in fetuses. We present the results of an international
case-control study to evaluate the association between CBTs and exposure of
parents and children to cigarette smoke. The study was undertaken as part of the
SEARCH program of the IARC. Nine centers in 7 countries were involved. The
studies mainly covered the 1980s and early 1990s. Cases (1,218, ages 0-19 years)
were children newly diagnosed with a primary brain tumor; there were 2,223
population-based controls. Most mothers who agreed to participate were
interviewed in person at home. Odds ratios (ORs) were calculated by
unconditional logistic regression, adjusted for age, sex and center, for all
types of CBT combined, 4 CBT histotypes, 5 age groups and each center. There was
no association between the risk of brain tumors in the child and parental
smoking prior to pregcy, maternal smoking or regular exposure to others'
cigarette smoke during pregcy at home or at work, or passive smoking by the
child during the first year of life. These results did not change considering
the child's age at diagnosis, the histologic type of tumor or center. OBJECTIVE: Prior epidemiological studies suggest a possible association between
maternal smoking during pregcy and risk of childhood brain tumors. A
meta-analysis was performed statistically pooling all available observational
studies on this topic in order to evaluate this suspected association.
METHODS: Using previously described methods, a protocol was developed for a
meta-analysis examining the association between maternal smoking during
pregcy and subsequent development of primary brain tumors in their offspring.
Literature search techniques, study inclusion criteria and statistical
procedures were prospectively defined. Data from epidemiological studies were
pooled using a general variance-based meta-analytic method employing confidence
intervals previously described by Greenland. The outcome of interest was a
summary relative risk (RRs) reflecting the risk of childhood brain tumor
development associated with mother's smoking during the index pregcy.
Sensitivity analyses were performed when necessary to explain any observed
statistical heterogeneity and/or to evaluate the impact of demographic or study
characteristics on the summary estimate of effect.
RESULTS: Twelve observational studies meeting protocol specified inclusion
criteria were obtained via a comprehensive literature search. These studies
enrolled a total of 6566 patients. Analysis for homogeneity demonstrated that
the data were homogeneous (P > 0.50) and could be statistically combined.
Pooling all twelve reports yielded an RRs of 1.05 (0.90-1.21), a
non-statistically significant result suggesting no clear association between
maternal smoking during pregcy and risk of childhood brain tumor development.
Numerous sensitivity analyses examining the possible effect of study design and
various patient characteristics failed to show any influence on the RRs further
supporting the observed lack of association.
CONCLUSION: The available epidemiological data do not support a clear
association between maternal smoking during pregcy and pediatric brain tumor
development. Although it appears likely that no association exists, limitations
in study designs limit definitive conclusions based on available data. OBJECTIVE: Studies of the effect of maternal smoking during pregcy on
development of brain tumors in the offspring generally have found no increase in
risk but most have mainly relied on retrospective exposure assessment. We
conducted a prospective study on a large birth cohort in Sweden.
METHODS: Women giving birth during 1983-1997 were classified as smokers or
non-smokers based on information ascertained at the first prenatal visit and
recorded in the Swedish Birth Register. Follow-up of brain tumor incidence among
offspring through 1997 was achieved by linkage with the Swedish Cancer Register.
Hazard ratios were estimated using Cox proportional hazard regression, adjusting
for demographic characteristics available in the Birth Register.
RESULTS: Brain tumors (n=480) occurred at a rate of 4.5 cases per 100,000
person-years. Children of women who smoked during pregcy had an increased
incidence of brain tumors (hazard ratio = 1.24; 95% confidence interval:
1.01-1.53). The increase in risk was similar for benign and maligt tumors,
and was most apparent for astrocytoma. The effect of smoking on the occurrence
of brain tumors was seen most strongly among 2-4 year-old children.
CONCLUSIONS: These results support a role for maternal smoking during pregcy
in the etiology of childhood brain tumors. Our findings should be confirmed in
other prospective studies. BACKGROUND: A recent meta-analysis suggested an association between exposure to
paternal smoking during pregcy and childhood brain tumor risk, but no studies
have evaluated whether this association differs by polymorphisms in genes that
metabolize tobacco-smoke chemicals.
METHODS: We assessed 9 functional polymorphisms in 6 genes that affect the
metabolism of polycyclic aromatic hydrocarbons (PAH) to evaluate potential
interactions with parental smoking during pregcy in a population-based
case-control study of childhood brain tumors. Cases (N = 202) were ≤10 years
old, diagnosed from 1984-1991 and identified in three Surveillance,
Epidemiology, and End Results (SEER) registries in the western U.S. Controls in
the same regions (N = 286) were frequency matched by age, sex, and study center.
DNA for genotyping was obtained from archived newborn dried blood spots.
RESULTS: We found positive interaction odds ratios (ORs) for both maternal and
paternal smoking during pregcy, EPHX1 H139R, and childhood brain tumors
(P(interaction) = 0.02; 0.10), such that children with the high-risk (greater
PAH activation) genotype were at a higher risk of brain tumors relative to
children with the low-risk genotype when exposed to tobacco smoke during
pregcy. A dose-response pattern for paternal smoking was observed among
children with the EPHX1 H139R high-risk genotype only (OR(no exposure) = 1.0;
OR(≤3 hours/day) = 1.32, 95% CI: 0.52-3.34; OR(>3 hours/day )= 3.18, 95% CI:
0.92-11.0; P(trend )= 0.07).
CONCLUSION: Parental smoking during pregcy may be a risk factor for childhood
brain tumors among genetically susceptible children who more rapidly activate
PAH in tobacco smoke. |
Which pathway is activated by ficolin-3? | Ficolin-3 activates lectin complement pathway. | The ficolins and mannose-binding lectin (MBL) are collagen-like defence proteins
that serve as recognition molecules in lectin complement pathway. Differential
features that may indicate diverse functions of these proteins are poorly
understood. In this study we compared important biological features of the
ficolins and MBL. We investigated the tissue distribution of the FCN1-3 and the
MBL2 genes encoding the ficolins and MBL by quantitative PCR. Recombit
proteins were produced and structural and biological characteristics were
investigated and compared. Our main findings were that FCN3 mRNA was highly
expressed in the liver and lung compared with the other genes revealing the lung
as the tissue with the highest FCN3 expression pattern. Ficolin-3 revealed
higher complement activating capacity compared with Ficolin-2, MBL and Ficolin-1
and was highly resistant to bacterial collagenase treatment, which is different
from the other ficolins and MBL. We discovered several unique properties of
Ficolin-3 showing that FCN3 is the most highly expressed gene in liver and lung
among the lectin complement pathway initiators. Moreover, Ficolin-3 has a high
complement activating potential and is the only collagenase proteolytic
resistant molecule among the lectin complement pathway initiators. Ficolin-3 (Hakata antigen or H-ficolin) is a soluble pattern recognition
molecule in the lectin complement pathway. We speculated whether common genetic
variations in the FCN3 gene contribute to deficiency of Ficolin-3. The FCN3 gene
was sequenced in 237 healthy Danish Caucasians. The relevance of polymorphisms
was assessed with antibodies against Ficolin-3 in a novel ELISA system and by
production of recombit Ficolin-3 variants. Ficolin-3 serum profiles were
analyzed by SDS-PAGE and western blotting. Ficolin-3 serum concentration varied
10-fold (median, 24microg/ml; range, 3-54microg/ml). Out of several
polymorphisms one FCN3+1637delC causing a reading frame shift and a distortion
of the C-terminal end of the molecule with an allele frequency of 0.011 was
particularly interesting. In individuals heterozygous for the FCN3+1637delC
deletion lowered Ficolin-3 concentration was observed (P=0.025). SDS-PAGE and
western blotting of serum revealed a weak band corresponding to the truncated
molecule in addition to the normal Ficolin-3 pattern. Characterization of
recombit Ficolin-3 derived from FCN3+1637delC showed that in the homozygous
situation this allelic variant would lead to Ficolin-3 deficiency. In conclusion
an FCN3+1637delC deletion variant disrupting the possibility for pattern
recognition was detected. Characterization of recombit variant Ficolin-3
shows that homozygosity for the FCN3+1637delC deletion may lead to Ficolin-3
deficiency and may thus be the basis for a novel complement deficiency state. Ficolin-3, encoded by the FCN3 gene and expressed in the lung and liver, is a
recognition molecule in the lectin pathway of the complement system.
Heterozygosity for an FCN3 frameshift mutation (rs28357092), leading to a
distortion of the C-terminal end of the molecule, occurs in people without
disease (allele frequency among whites, 0.01). We describe a patient with
recurrent infections who was homozygous for this mutation, who had undetectable
serum levels of ficolin-3, and who had a deficiency in ficolin-3-dependent
complement activation. BACKGROUND: The human lectin complement pathway (LCP) involves circulating
complexes consisting of mannose-binding lectin (MBL) or ficolins in association
with serine proteases named MASP-1, -2 and -3 and a non-enzymatic protein, sMAP.
MASP-3 originates from the MASP1 gene through differential splicing and little
is known about its biological characteristics. For this reason we expressed
recombit MASP-3 and generated specific monoclonal antibodies to establish
biochemical characteristics and to determine the serum levels, the interactions
with the LCP recognition molecules and the influence on complement activation of
MASP-3.
METHODS: We expressed rMASP-3 in CHO-DG44 cells and used SDS-PAGE and Western
blotting for biochemical characterization. We generated monoclonal antibodies
against MASP-3 and developed a quantitative MASP-3 assay to establish the serum
levels in 100 Danish blood donors. In addition we assessed the association
levels between MASP-3 and Ficolin-2, -3 and MBL using both ELISA and
immunoprecipitation techniques. Moreover, we assessed the influence on
complement factor C4 deposition.
RESULTS: We found the mean serum MASP-3 concentration to be 6.4mg/l (range:
2-12.9mg/l) and that MASP-3 in serum is primarily found in complex with
Ficolin-3. In contrast to this the MASP-3 association with Ficolin-2 and
especially with MBL seems to be less evident. rMASP-3 significantly inhibited
Ficolin-3 mediated C4 deposition, while the opposite was the case for rMASP-1.
CONCLUSION: Our results show that MASP-3 is present in relatively high serum
concentrations. Moreover, Ficolin-3 is the primary acceptor molecule of MASP-3
among the LCP activator molecules, but MASP-3 appears to down-regulate Ficolin-3
mediated complement activation through the lectin pathway. Ficolin-1 (M), ficolin-2 (L), ficolin-3 (H) and man-binding lectin (MBL)
activate the complement system and have opsonic activity. The specificity of
ficolin-3 is poorly characterized and currently limited to a few ligands only.
We present new specific targets for human ficolin-3, identified among
lipopolysaccharides (LPSs, endotoxin) of Hafnia alvei. The interaction was
restricted to LPSs of four strains: 23, Polish Collection of Microorganisms
(PCM) 1200, PCM 1203 and PCM 1205 and limited to their O-specific
polysaccharides (O-specific PSs) composed of different numbers of
oligosaccharide (OS) repeating units (RUs). Moreover, these LPS/ficolin-3
complexes activated the lectin pathway of complement in a C4b-deposition assay
in a calcium- and magnesium-dependent way. A neoglycoconjugate of the O-specific
PS fraction of H. alvei 1200 LPS with bovine serum albumin (BSA) was prepared
and used as a tool for the determination of ficolin-3 concentration and activity
in serum. To confirm a structure of the O-specific PS 1200 selected for the
conjugate preparation, structural analysis was performed on a series of
O-specific PSs released by the mild acid hydrolysis of the LPS. The isolated
O-specific PSs, showing the different length distributions, were devoid of a
major part of the core OS region and had Hep-Kdo disaccharide at a reducing end.
The neoglycoconjugate was a highly selective tool for the determination of
ficolin-3 concentration and activity in serum (lectin pathway activation in the
C4b deposition assay) and was not affected by MBL, ficolin-1 and ficolin-2 or
natural antibodies. Ficolin-1, -2 and -3 are recognition molecules in the lectin complement pathway
and form complexes with serine proteases named MASP-1, -2 and -3 and two
nonenzymatic proteins. MASP-2 is the main initiator of lectin pathway
activation, while ficolin-3 is the most abundant ficolin molecule in the
circulation. The significance of lectin pathway complexes in the circulation is
unknown. Thus, we established an assay for the measurement of circulating
MASP-2/ficolin-3 complexes. A quantitative sandwich ELISA was developed for the
measurement of the MASP-2/ficolin-3 complexes in serum based on monoclonal
antibodies against MASP-2 for coating and anti-ficolin-3 for detection. In
addition, we assessed the serum concentrations of ficolin-3 and MASP-2 and the
extent of ficolin-3 mediated C4 deposition on acetylated BSA in samples from 97
healthy donors. The median concentration of MASP-2/ficolin-3 complexes was found
to be 119.7 AU/ml (range: 2.9-615.5 AU/ml). Significant correlations were found
between the level of MASP-2/ficolin-3 complexes and the concentration of
ficolin-3 (Spearman r=0.2532, p=0.0124), and MASP-2 (Spearman r=0.4505,
p<0.0001), as well as the degree of C4 deposition (Spearman r=0.671, p<0.0001).
When ficolin-3 deficient (homozygous for the rs28357092 polymorphism) and MASP-2
deficient (homozygous for the rs72550870 polymorphism) sera were incubated
together, complex formation was induced between MASP-2 and ficolin-3. The
complex formation disappeared in the presence of EDTA. An assay allowing
quantitative measurement exclusively of MASP-2/ficolin-3 complexes in serum is
described. This method may add further insight into the pathophysiology of
disorders associated with the deficiency or abnormal activities of MASP-2 and
ficolin-3. OBJECTIVES: To assess the involvement of ficolin-3, the main initiator of the
lectin complement pathway (LCP), in subarachnoid hemorrhage (SAH) pathology and
outcome.
METHODS: In this preliminary exploratory study, plasma concentration of
ficolin-3 and of ficolin-3-mediated functional LCP activity was measured, along
with that of other LCP initiators (mannose-binding lectin, ficolin-2, and
ficolin-1), C3 activation products, and soluble C5b-9 terminal complex, in a
prospective cohort of 39 patients with SAH and 20 healthy controls. The
following parameters were recorded: SAH severity, assessed using the World
Federation of Neurosurgical Societies grading scale; vasospasm, defined as
neuro-worsening with angiographic confirmation of vessel narrowing; cerebral
ischemia, defined as hypodense lesion on CT scan performed before discharge; and
6-month outcome, assessed using the Glasgow Outcome Scale.
RESULTS: In patients, no changes were detected for ficolin-3 compared with
controls. Notably, however, ficolin-3-mediated functional LCP activity was
reduced. Low levels of plasma ficolin-3 and ficolin-3-mediated functional LCP
activity were related to SAH severity, vasospasm, and cerebral ischemia.
Moreover, ficolin-3 functional LCP activity was decreased in patients with
unfavorable outcome.
CONCLUSION: Our data provide evidence that LCP is activated after SAH and that
the actual plasma concentrations of ficolin-3 reflect the severity of brain
injury as evaluated by clinical and structural parameters. These results support
the idea that ficolin-3-mediated functional LCP activity may be targeted to
control injury progression in SAH. The complement system plays a pathophysiological role in systemic lupus
erythematosus (SLE). This study aims to investigate whether an association
exists between the ficolins that are part of the lectin complement pathway and
SLE. EDTA plasma samples from 68 Danish SLE patients and 29 healthy donors were
included in the study. Plasma concentrations of Ficolin-1, -2, and -3 were
determined in specific sandwich ELISAs. Lectin pathway activity via Ficolin-3
was measured in ELISA on acetylated bovine serum albumin (acBSA) and measured as
Ficolin-3 binding and deposition of C4, C3 and the terminal complement complex
(TCC). SLE patients had increased levels of Ficolin-3, 21.6μg/ml as compared to
17.0μg/ml in healthy controls (P=0.0098). The Ficolin-1 plasma concentration was
negatively correlated with SLE Disease Activity Index (SLEDAI) (Rho=-0.29,
P=0.015) and positively correlated to the [Systemic Lupus International
Collaborating Clinics (SLICC)/American College of Rheumatology (ACR) Damage
Index] (SDI) (Rho=0.27, P=0.026). The Ficolin-1 concentration was also
associated with the occurrence of arterial (P=0.0053) but not venous thrombosis
(P=0.42). Finally, deposition of C4, C3 and TCC in the Ficolin-3 pathway were
all correlated to SLEDAI, respectively (P<0.0076). The Ficolin-1 association to
SLEDAI and SDI as well as arterial thrombosis shown in this study suggests that
Ficolin-1 may be a potential new biomarker for patients with SLE. Furthermore,
Ficolin-3 mediated complement activation may be valuable in monitoring disease
activity in SLE patients due to the high sensitivity for complement consumption
in the assay independent of the Ficolin-3 concentration. Ficolin-3 (also called H-ficolin or Hakata antigen) is a complement-activating
pattern recognition molecule, possessing a fibrinogen-like domain involved in
carbohydrate binding. Amongst human ficolins, Ficolin-3 has the highest
concentration in serum and is the most potent lectin pathway activator in vitro.
Evidence for its physiological function is sparse, although its deficiency has
been suggested to increase susceptibility to infections. The specificity of
Ficolin-3 is poorly characterized and currently few ligands are known. Here we
report agglutination of Hafnia alvei, a Gram-negative enteric commensal
bacterium and opportunist pathogen, in the presence of recombit Ficolin-3 and
calcium. Ficolin-3 also augmented phagocytosis of H. alvei by macrophages and
displayed bactericidal activity. Additionally, Ficolin-3 inhibited host cells'
response to TLR4/MD-2/CD14-LPS dependent NF-κB activation. This is the first
demonstration of protective activity of Ficolin-3 against a human bacterial
pathogen. Although human Ficolin-3 does not recognise and bind to common
pathogenic bacteria, it could be an important component of innate immunity
providing protection, for example, from commensal flora that can cause
extraintestinal, opportunistic infections. |
Is nivolumab used for treatment of Non–Small-Cell Lung Cancer? | Yes, nivolumab used for treatment of Non–Small-Cell Lung Cancer. | Two PD-1 inhibitors, Bristol-Myers Squibb's nivolumab and Merck's MK-3475, both
demonstrated positive results in phase I trials of previously treated patients
with non-small cell lung cancer, reported at the World Conference on Lung Cancer
in Sydney, Australia. Immune checkpoint inhibition as a new treatment approach is undergoing extensive
investigation in non-small cell lung cancer (NSCLC) and other maligcies.
Unlike standard chemotherapy or targeted agents, which act directly on the tumor
cells, immune checkpoint inhibitors work by restoring the immune system's
capacity to eradicate tumors. Agents currently in active clinical development
for lung cancer include ipilimumab, which modulates the cytotoxic
T-lymphocyte-associated antigen 4 pathway, and multiple agents targeting the
programmed death protein 1 (PD-1) pathway, both anti-PD-1 compounds (nivolumab,
pembrolizumab [MK-3475]) and those that target programmed death ligand 1
(PD-L1), a key ligand for PD-1 (BMS-936559, MPDL3280A). Preliminary evidence
shows activity for these agents in NSCLC as monotherapy or in combination with
chemotherapy. This article reviews the immune checkpoint inhibitors and the
available data to date on their use in lung cancer. Clinical implications for
the use of these therapies in NSCLC are discussed as they relate to their novel
mechanisms of action, response patterns, and safety profiles. Non-small-cell lung cancer (NSCLC) is a highly prevalent and aggressive disease.
In the metastatic setting, major advances include the incorporation of
immunotherapy and targeted therapies into the clinician's therapeutic
armamentarium. Standard chemotherapeutic regimens have long been reported to
interfere with the immune response to the tumor; conversely, antitumor immunity
may add to the effects of those therapies. The aim of immunotherapy is to
specifically enhance the immune response directed to the tumor. Recently, many
trials addressed the role of such therapies for metastatic NSCLC treatment:
ipilimumab, tremelimumab, nivolumab and lambrolizumab are immunotherapeutic
agents of main interest in this field. In addition, anti-tumor vaccines, such as
MAGE-A3, Tecetomide, TG4010, CIMAvax, ganglioside vaccines, tumor cell vaccines
and dendritic cell vaccines, emerged as potent inducers of immune response
against the tumor. The current work aims to address the most recent developments
regarding these innovative immunotherapies and their implementation in the
treatment of metastatic NSCLC. Conflict of interest statement: Declaration of interests NAR has received
personal fees from Bristol-Myers Squibb, Genentech, Roche, MedImmune,
AstraZeneca, and Merck. TES has received research grant support from
Bristol-Myers Squibb and personal fees from Genentech, Eli Lilly, Celgene, and
Boehringer Ingelheim. SJA has received research grant support from MedImmune,
and personal fees from Bristol-Myers Squibb, MedImmune, and AstraZeneca. LH has
received research grant support from Astellas, has served as a non-paid
consultant to Bayer and Xcovery, and has received personal fees from
Bristol-Myers Squibb, Merck, Clovis, Helix Bio, and Genentech. HL has received
personal fees from Bristol-Myers Squibb and Merck, and non-ficial support
from Bristol-Myers Squibb and Roche. BM has served as a consultant to
Bristol-Myers Squibb and on advisory boards for Merck Sharp & Dohme. GAO has
received research grant support from Bristol-Myers Squibb, Pfizer,
GlaxoSmithKline, New Link Genetics, Genentech, and Boehringer Ingelheim, and has
received personal fees from Genentech and Boehringer Ingelheim. DRG has received
research grant support from Bristol-Myers Squibb. BPL has received personal fees
from Eli Lilly, Genentech, Pfizer, Biodesix, and Boehringer Ingelheim. GZ has
received personal fees from Bristol-Myers Squibb. JW has received research
grants from Boehringer Ingelheim, Novartis, Pfizer, Roche, and Bayer, and has
served on advisory boards for and received lecture fees from Bristol-Myers
Squibb, AstraZeneca, Boehringer Ingelheim, Clovis, Novartis, Pfizer, Roche, and
Merck Sharp and Dohme. PJS has received personal fees, non-ficial support,
and support for clinical studies from Roche. FC has received personal fees from
Bristol-Myers Squibb. CC has served as a consultant to Bristol-Myers Squibb,
Merck, and Roche. RS has received research grant support and honoraria from and
has served on advisory boards for Bristol-Myers Squibb. RMH has served on
advisory boards for Bristol-Myers Squibb. CTH, CB, and BJL are employed by and
own stock in Bristol-Myers Squibb. SSR has received research grant support and
personal fees from Bristol-Myers Squibb and has received personal fees from
Amgen, AstraZeneca, Aveo, Boehringer Ingelheim, Celgene, Genentech, Eli Lilly,
and Novartis. SJA, DRG, AD, and SSR have received funding from the National
Institutes of Health (USA). The other authors declare no competing interests. Author information:
(1)Scott N. Gettinger and Mario Sznol, Yale Cancer Center, New Haven, CT; Leora
Horn, David P. Carbone, and Jeffrey A. Sosman, Vanderbilt University Medical
Center; David R. Spigel, Sarah Cannon Research Institute/Tennessee Oncology,
Nashville, TN; Leena Gandhi, David M. Jackman, and F. Stephen Hodi, Dana-Farber
Cancer Institute; Rebecca S. Heist and Lecia V. Sequist, Massachusetts General
Hospital Cancer Center; David F. McDermott, Beth Israel Deaconess Medical
Center, Boston, MA; Scott J. Antonia and Mary C. Pinder-Schenck, H. Lee Moffitt
Cancer Center and Research Institute, Tampa, FL; Naiyer A. Rizvi, Richard D.
Carvajal, and Matthew D. Hellmann, Memorial Sloan Kettering Cancer Center, New
York, NY; John D. Powderly, Carolina BioOncology Institute, Huntersville, NC;
David C. Smith, University of Michigan, Ann Arbor, MI; Philip Leming, Christ
Hospital Cancer Center, Cincinnati, OH; Suzanne L. Topalian, Drew M. Pardoll,
and Julie R. Brahmer, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins,
Baltimore, MD; and Vindira Sankar, Christoph M. Ahlers, Mark Salvati, Jon M.
Wigginton, Georgia D. Kollia, and Ashok K. Gupta, Bristol-Myers Squibb,
Princeton, NJ. [email protected].
(2)Scott N. Gettinger and Mario Sznol, Yale Cancer Center, New Haven, CT; Leora
Horn, David P. Carbone, and Jeffrey A. Sosman, Vanderbilt University Medical
Center; David R. Spigel, Sarah Cannon Research Institute/Tennessee Oncology,
Nashville, TN; Leena Gandhi, David M. Jackman, and F. Stephen Hodi, Dana-Farber
Cancer Institute; Rebecca S. Heist and Lecia V. Sequist, Massachusetts General
Hospital Cancer Center; David F. McDermott, Beth Israel Deaconess Medical
Center, Boston, MA; Scott J. Antonia and Mary C. Pinder-Schenck, H. Lee Moffitt
Cancer Center and Research Institute, Tampa, FL; Naiyer A. Rizvi, Richard D.
Carvajal, and Matthew D. Hellmann, Memorial Sloan Kettering Cancer Center, New
York, NY; John D. Powderly, Carolina BioOncology Institute, Huntersville, NC;
David C. Smith, University of Michigan, Ann Arbor, MI; Philip Leming, Christ
Hospital Cancer Center, Cincinnati, OH; Suzanne L. Topalian, Drew M. Pardoll,
and Julie R. Brahmer, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins,
Baltimore, MD; and Vindira Sankar, Christoph M. Ahlers, Mark Salvati, Jon M.
Wigginton, Georgia D. Kollia, and Ashok K. Gupta, Bristol-Myers Squibb,
Princeton, NJ. Immune-regulatory mechanisms are used by cancer to hide from the immune system.
Advances and in-depth understanding of the biology of melanoma and its
interaction with the immune system have led to the development of some of
antagonistic antibodies to the programmed death 1 pathway (PD-1) and one of its
ligands, programmed death ligand 1 (PD-L1), which are demonstrating high
clinical benefit rates and tolerability. Blocking the immune-regulatory
checkpoints that limit T-cell responses to melanoma upon PD-1/PD-L1 modulation
has provided clinically validated targets for cancer immunotherapy. Combinations
with other anti-melanoma agents may result in additional benefits. Nivolumab,
pembrolizumab (formerly known as MK-3475 and lambrolizumab), and pidilizumab are
anti-PD-1 antibodies in clinical development for melanoma, non-small cell lung
cancer, renal cell carcinoma, head and neck cancers, lymphoma, and several other
cancers. Long-term survivors already have been reported with these therapies. In
this review, we discuss the current state of anti-PD-1 agents, the evidence in
the literature to support the combination of anti-PD-1 antibodies with other
anti-cancer agents and discuss the future directions for rational design of
clinical trials that keep on increasing the number of long-term survivors. BACKGROUND: Patients with advanced squamous-cell non-small-cell lung cancer
(NSCLC) who have disease progression during or after first-line chemotherapy
have limited treatment options. This randomized, open-label, international,
phase 3 study evaluated the efficacy and safety of nivolumab, a fully human IgG4
programmed death 1 (PD-1) immune-checkpoint-inhibitor antibody, as compared with
docetaxel in this patient population.
METHODS: We randomly assigned 272 patients to receive nivolumab, at a dose of 3
mg per kilogram of body weight every 2 weeks, or docetaxel, at a dose of 75 mg
per square meter of body-surface area every 3 weeks. The primary end point was
overall survival.
RESULTS: The median overall survival was 9.2 months (95% confidence interval
[CI], 7.3 to 13.3) with nivolumab versus 6.0 months (95% CI, 5.1 to 7.3) with
docetaxel. The risk of death was 41% lower with nivolumab than with docetaxel
(hazard ratio, 0.59; 95% CI, 0.44 to 0.79; P<0.001). At 1 year, the overall
survival rate was 42% (95% CI, 34 to 50) with nivolumab versus 24% (95% CI, 17
to 31) with docetaxel. The response rate was 20% with nivolumab versus 9% with
docetaxel (P=0.008). The median progression-free survival was 3.5 months with
nivolumab versus 2.8 months with docetaxel (hazard ratio for death or disease
progression, 0.62; 95% CI, 0.47 to 0.81; P<0.001). The expression of the PD-1
ligand (PD-L1) was neither prognostic nor predictive of benefit.
Treatment-related adverse events of grade 3 or 4 were reported in 7% of the
patients in the nivolumab group as compared with 55% of those in the docetaxel
group.
CONCLUSIONS: Among patients with advanced, previously treated squamous-cell
NSCLC, overall survival, response rate, and progression-free survival were
significantly better with nivolumab than with docetaxel, regardless of PD-L1
expression level. (Funded by Bristol-Myers Squibb; CheckMate 017
ClinicalTrials.gov number, NCT01642004.). |
Global quantitative phosphoproteomic analyses are emerging. List the preferred technologies for the enrichment for phosphorylated peptides? | There are many different approaches to enrich for phosphorylated peptides: titanium dioxide, IMAC, simple derivatization through phosphoramidate chemistry and antibodies. | Current methods for phosphoproteome analysis have several limitations. First,
most methods for phosphopeptide enrichment lack the specificity to truly purify
phosphopeptides. Second, fragmentation spectra of phosphopeptides, in particular
those of phosphoserine and phosphothreonine containing peptides, are often
dominated by the loss of the phosphate group(s) and therefore lack the
information required to identify the peptide sequence and the site of
phosphorylation, and third, sequence database search engines and statistical
models for data validation are not optimized for the specific fragmentation
properties of phosphorylated peptides. Consequently, phosphoproteomic data are
characterized by large and unknown rates of false positive and false negative
phosphorylation sites. Here we present an integrated chemical, mass
spectrometric and computational strategy to improve the efficiency, specificity
and confidence in the identification of phosphopeptides and their site(s) of
phosphorylation. Phosphopeptides were isolated with high specificity through a
simple derivatization procedure based on phosphoramidate chemistry.
Identification of phosphopeptides, their site(s) of phosphorylation and the
corresponding phosphoproteins was achieved by the optimization of the mass
spectrometric data acquisition procedure, the computational tools for database
searching and the data post processing. The strategy was applied to the mapping
of phosphorylation sites of a purified transcription factor, dFOXO and for the
global analysis of protein phosphorylation of Drosophila melanogaster Kc167
cells. Recent advances in MS instrumentation and progresses in phosphopeptide
enrichment, in conjunction with more powerful data analysis tools, have
facilitated unbiased characterization of thousands of site-specific
phosphorylation events. Combined with stable isotope labeling by amino acids in
cell culture metabolic labeling, these techniques have made it possible to
quantitatively evaluate phosphorylation changes in various physiological states
in stable cell lines. However, quantitative phosphoproteomics in primary cells
and tissues remains a major technical challenge due to the lack of adequate
techniques for accurate quantification. Here, we describe an integrated strategy
allowing for large scale quantitative profiling of phosphopeptides in complex
biological mixtures. In this technique, the mixture of proteolytic peptides was
subjected to phosphopeptide enrichment using a titania affinity column, and the
purified phosphopeptides were subsequently labeled with iTRAQ reagents. After
further fractionation by strong-cation exchange, the peptides were analyzed by
LC-MS/MS on an Orbitrap mass spectrometer, which collects CID and high-energy
collisional dissociation (HCD) spectra sequentially for peptide identification
and quantitation. We demonstrate that direct phosphopeptide enrichment of
protein digests by titania affinity chromatography substantially improves the
efficiency and reproducibility of phosphopeptide proteomic analysis and is
compatible with downstream iTRAQ labeling. Conditions were optimized for HCD
normalized collision energy to balance the overall peptide identification and
quantitation using the relative abundances of iTRAQ reporter ions. Using this
approach, we were able to identify 3557 distinct phosphopeptides from HeLa cell
lysates, of which 2709 were also quantified from HCD scans. BACKGROUND: The proteasome inhibitor bortezomib represents an important advance
in the treatment of multiple myeloma (MM). Bortezomib inhibits the activity of
the 26S proteasome and induces cell death in a variety of tumor cells; however,
the mechanism of cytotoxicity is not well understood.
METHODOLOGY/PRINCIPAL FINDINGS: We investigated the differential phosphoproteome
upon proteasome inhibition by using stable isotope labeling by amino acids in
cell culture (SILAC) in combination with phosphoprotein enrichment and LC-MS/MS
analysis. In total 233 phosphoproteins were identified and 72 phosphoproteins
showed a 1.5-fold or greater change upon bortezomib treatment. The
phosphoproteins with expression alterations encompass all major protein classes,
including a large number of nucleic acid binding proteins. Site-specific
phosphopeptide quantitation revealed that Ser38 phosphorylation on stathmin
increased upon bortezomib treatment, suggesting new mechanisms associated to
bortezomib-induced apoptosis in MM cells. Further studies demonstrated that
stathmin phosphorylation profile was modified in response to bortezomib
treatment and the regulation of stathmin by phosphorylation at specific Ser/Thr
residues participated in the cellular response induced by bortezomib.
CONCLUSIONS/SIGNIFICANCE: Our systematic profiling of phosphorylation changes in
response to bortezomib treatment not only advanced the global mechanistic
understanding of the action of bortezomib on myeloma cells but also identified
previously uncharacterized signaling proteins in myeloma cells. Understanding the signaling pathways governing pluripotency and self-renewal is
a prerequisite for better controlling stem cell differentiation to specific
fates. Reversible protein phosphorylation is one of the most important
posttranslational modifications regulating signaling pathways in biological
processes. Global analysis of dynamic changes in protein phosphorylation is,
therefore, key to understanding signaling at the system level. Here, we describe
a generic mass spectrometry (MS)-based phosphoproteomics strategy applied to
monitor phosphorylation dynamics after bone morphogenetic protein 4
(BMP4)-induced differentiation of human embryonic stem cells (hESCs). Our method
combines the use of strong cation exchange (SCX) and titanium dioxide (TiO(2))
for phosphopeptide enrichment, high-resolution MS for peptide and protein
identification, and stable isotope labeling by amino acids in cell culture
(SILAC) for quantification. This approach allows us to identify thousands of
phosphorylation sites and profile their relative abundance during
differentiation. This systems-biology-based approach provides new insights into
how human pluripotent stem cells exit the pluripotent state. Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in
the regulation of immune responses. TSLP requires a heterodimeric receptor
complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene
symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g.
overexpression of TSLP or its unique receptor TSLPR) contributes to the
development of a number of diseases including asthma and leukemia. However, a
detailed understanding of the signaling pathways activated by TSLP remains
elusive. In this study, we performed a global quantitative phosphoproteomic
analysis of the TSLP signaling network using stable isotope labeling by amino
acids in cell culture. By employing titanium dioxide in addition to
antiphosphotyrosine antibodies as enrichment methods, we identified 4164
phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino
acids in cell culture-based quantitation, we determined that the phosphorylation
status of 226 proteins was modulated by TSLP stimulation. Our analysis
identified activation of several members of the Src and Tec families of kinases
including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report
TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and
Ptpn11 (Shp2), which has also not been reported previously.
Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2
in a TSLP-dependent manner. This is the first demonstration of an inducible
protein complex in TSLP signaling. A kinase inhibitor screen revealed that
pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family
kinases or Btk suppressed TSLP-dependent cellular proliferation making them
candidate therapeutic targets in diseases resulting from aberrant TSLP
signaling. Our study is the first phosphoproteomic analysis of the TSLP
signaling pathway that greatly expands our understanding of TSLP signaling and
provides novel therapeutic targets for TSLP/TSLPR-associated diseases in humans. Even though protein phosphatases are key regulators of signal transduction,
their cellular mechanisms of action are poorly understood. Here, we undertook a
large-scale proteomics survey to identify cellular protein targets of a
serine/threonine phosphatase. We used SILAC-based quantitative MS to measure
differences in protein expression and phosphorylation upon ablation of the
serine/threonine phosphatase Ppt1 in Saccharomyces cerevisiae. Phosphopeptide
fractionation by strong cation exchange chromatography combined with immobilized
metal affinity chromatography (IMAC) enrichment enabled quantification of more
than 8000 distinct phosphorylation sites in Ppt1 wild-type versus Ppt1-deficient
yeast cells. We further quantified the relative expression of 1897 yeast
proteins and detected no major protein changes accompanying Ppt1 deficiency.
Notably, we found 33 phosphorylation sites to be significantly and reproducibly
up-regulated while no phosphorylation events were repressed in cells lacking
Ppt1. Ppt1 acted on its cellular target proteins in a sequence- and
site-specific fashion. Several of the regulated phosphoproteins were involved in
the response to heat stress in agreement with known Ppt1 functions.
Additionally, biosynthetic enzymes were particularly prominent among
Ppt1-regulated phosphoproteins, pointing to unappreciated roles of Ppt1 in the
control of various metabolic functions. These results demonstrate the utility of
large-scale and quantitative phosphoproteomics to identify cellular sites of
serine/threonine phosphatase action in an unbiased manner. Protein phosphorylation-dephosphorylation events play a primary role in
regulation of almost all aspects of cell function including signal transduction,
cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on
analysis of phosphotyrosine residues, and little is known about the role of
serine/threonine phosphorylation in early activation of the T cell receptor
(TCR). Therefore, we performed a quantitative mass spectrometry-based analysis
of the global phosphoproteome of human primary T cells in response to 5 min of
TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an
antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment,
isobaric tag for the relative and absolute quantitation methodology, and strong
cation exchange separation, we were able to identify 2814 phosphopeptides. These
unique sites were employed to investigate the site-specific phosphorylation
dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive
changes. We found that upon 5 min of stimulation of the TCR, specific serine and
threonine kinase motifs are overrepresented in the set of responsive
phosphorylation sites. These phosphorylation events targeted proteins with many
different activities and are present in different subcellular locations. Many of
these proteins are involved in intracellular signaling cascades related mainly
to cytoskeletal reorganization and regulation of small GTPase-mediated signal
transduction, probably involved in the formation of the immune synapse. The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in
cell growth and cytoskeletal regulation. Despite being dysregulated in a variety
of human cancers, its precise functions are not fully understood. Identification
of the substrates of c-Src remains a major challenge, because there is no simple
way to directly stimulate its activity. Here we combine the chemical rescue of
mutant c-Src and global quantitative phosphoproteomics to obtain the first high
resolution snapshot of the range of tyrosine phosphorylation events that occur
in the cell immediately after specific c-Src stimulation. After enrichment by
anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src
substrate proteins. Tyrosine phosphopeptide mapping allowed the identification
of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable
isotope labeling of amino acids in cell culture-based quantitation allowed the
detection of 97 nonredundant tyrosine phosphopeptides whose level of
phosphorylation is increased by c-Src. A large number of previously
uncharacterized c-Src putative protein targets and phosphorylation sites are
presented here, a majority of which play key roles in signaling and cytoskeletal
networks, particularly in cell adhesion. Integrin signaling and focal adhesion
kinase signaling pathway are two of the most altered pathways upon c-Src
activation through chemical rescue. In this context, our study revealed the
temporal connection between c-Src activation and the GTPase Rap1, known to
stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool
to dissect the spatiotemporal mechanism of activation of the Rap1 guanine
exchange factor, C3G, one of the identified potential c-Src substrates that
plays a role in focal adhesion signaling. In addition to unveiling the role of
c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results
exemplify a strategy for obtaining a comprehensive understanding of the
functions of nonreceptor tyrosine kinases with high specificity and kinetic
resolution. We report a global quantitative phosphoproteomic study of bloodstream and
procyclic form Trypanosoma brucei using SILAC labeling of each lifecycle stage.
Phosphopeptide enrichment by SCX and TiO2 led to the identification of a total
of 10096 phosphorylation sites on 2551 protein groups and quantified the ratios
of 8275 phosphorylation sites between the two lifecycle stages. More than 9300
of these sites (92%) have not previously been reported. Model-based gene
enrichment analysis identified over representation of Gene Ontology terms
relating to the flagella, protein kinase activity, and the regulation of gene
expression. The quantitative data reveal that differential protein
phosphorylation is widespread between bloodstream and procyclic form
trypanosomes, with significant intraprotein differential phosphorylation.
Despite a lack of dedicated tyrosine kinases, 234 phosphotyrosine residues were
identified, and these were 3-4 fold over-represented among site changing
>10-fold between the two lifecycle stages. A significant proportion of the T.
brucei kinome was phosphorylated, with evidence that MAPK pathways are
functional in both lifecycle stages. Regulation of gene expression in T. brucei
is exclusively post-transcriptional, and the extensive phosphorylation of RNA
binding proteins observed may be relevant to the control of mRNA stability in
this organism. |
Which histone modifications are correlated with transcription elongation? | This is accompanied by reductions in the level of H3K36 trimethylation, a posttranslational histone modification associated with efficient transcriptional elongation, and the number of full-length transcripts from these genes. The 3' ZNF exons contain H3K36me3, a mark of transcriptional elongation. | Set2-mediated H3 Lys(36) methylation is a histone modification that has been
demonstrated to function in transcriptional elongation by recruiting the Rpd3S
histone deacetylase complex to repress intragenic cryptic transcription.
Recently, we identified a trans-histone pathway in which the interaction between
the N terminus of Set2 and histone H4 Lys(44) is needed to mediate trans-histone
H3 Lys(36) di- and trimethylation. In the current study, we demonstrate that
mutation of the lysine 44 residue in histone H4 or the Set2 mutant lacking the
histone H4 interaction motif leads to intragenic cryptic transcripts, indicating
that the Set2 and histone H4 interaction is important to repress intragenic
cryptic transcription. We also determine that histone H2A residues (Leu(116) and
Leu(117)), which are in close proximity to histone H4 Lys(44), are needed for
proper trans-histone H3 Lys(36) methylation. Similar to H4 Lys(44) mutants,
histone H2A Leu(116) and Leu(117) mutations exhibited decreased H3 Lys(36) di-
and trimethylation, increased histone H4 acetylation, increased resistance to
6-azauracil, and cryptic transcription. Interestingly, the combined histone H4
Lys(44) and H2A mutations have more severe methylation defects and increased H4
acetylation levels. Furthermore, we identify that additional histone H2A and H3
core residues are also needed for H3 Lys(36) di- and trimethylation. Overall,
our results show and suggest that multiple H4, H2A, and H3 residues contribute
to and form a Set2 docking/recognition site on the nucleosomal surface so that
proper Set2-mediated H3 Lys(36) di- and trimethylation, histone acetylation, and
transcriptional elongation can occur. Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved
patterns of histone modification. Whereas histone modifications have established
roles in transcription initiation, their functions during elongation are not
understood. Mono-ubiquitylation of histone H2B (H2Bub1) plays a key role in
coordinating co-transcriptional histone modification by promoting site-specific
methylation of histone H3. H2Bub1 also regulates gene expression through an
unidentified, methylation-independent mechanism. Here we reveal bidirectional
communication between H2Bub1 and Cdk9, the ortholog of metazoan positive
transcription elongation factor b (P-TEFb), in the fission yeast
Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that
lowering Cdk9 activity or preventing phosphorylation of its substrate, the
transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely,
mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and
decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation,
combined with deletion of the histone H3 Lys4 methyltransferase Set1,
phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting
independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1.
Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the
Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology
defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin
immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on
distribution of transcribing RNAPII. Therefore, whereas mutual dependence of
H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression
by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must
be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance
and leads to deranged gene expression and aberrant cell morphologies, revealing
a novel function of a conserved, co-transcriptional histone modification. Like that of many protein-coding genes, expression of the p21(CIP1) cell cycle
inhibitor is regulated at the level of transcription elongation. While many
transcriptional activators have been shown to stimulate elongation, the
mechanisms by which promoter-specific repressors regulate pausing and elongation
by RNA polymerase II (RNA PolII) are not well described. Here we report that the
transcription factor Sp3 inhibits basal p21(CIP1) gene expression by
promoter-bound RNA PolII. Knockdown of Sp3 led to increased p21(CIP1) mRNA
levels and reduced occupancy of the negative elongation factor (NELF) at the
p21(CIP1) promoter, although the level of binding of the positive transcription
elongation factor b (P-TEFb) kinase was not increased. Sp3 depletion correlated
with increased H3K36me3 and H2Bub1, two histone modifications associated with
transcription elongation. Further, Sp3 was shown to promote the binding of
protein phosphatase 1 (PP1) to the p21(CIP1) promoter, leading to reduced H3S10
phosphorylation, a finding consistent with Sp3-dependent regulation of the local
balance between kinase and phosphatase activities. Analysis of other targets of
Sp3-mediated repression suggests that, in addition to previously described SUMO
modification-dependent chromatin-silencing mechanisms, inhibition of the
transition of paused RNA PolII to productive elongation, described here for
p21(CIP1), is a general mechanism by which transcription factor Sp3 fine-tunes
gene expression. During infection by human adenovirus (HAdV), the proteins encoded by the early
region 1A (E1A) gene bind and appropriate components of the cellular
transcriptional machinery to activate the transcription of viral early genes.
Previously, we identified roles for the human Bre1 (hBre1) and hPaf1 complexes
in E1A-mediated transcriptional activation of HAdV early genes. Here we show
that E1A binds hBre1 directly and that this complex targets the hPaf1 complex
via the Rtf1 subunit. Depletion of hPaf1 reduces E1A-dependent activation of
transcription from the E2e, E3, and E4 viral transcription units, and this does
not result from a reduced ability of RNA polymerase II to be recruited to the
promoter-proximal regions of these genes. In contrast, depletion of hPaf1
reduces the occupancy of RNA polymerase II across these transcription units.
This is accompanied by reductions in the level of H3K36 trimethylation, a
posttranslational histone modification associated with efficient transcriptional
elongation, and the number of full-length transcripts from these genes.
Together, these results indicate that E1A uses hBre1 to recruit the hPaf1
complex in order to optimally activate viral early transcription by enhancing
transcriptional elongation.
IMPORTANCE: This work provides the mechanism by which the hPaf1 complex
contributes to E1A-dependent activation of early gene transcription. The work
also demonstrates that E1A induces gene expression by stimulating
transcriptional elongation, in addition to its better-characterized effects on
transcriptional initiation. It is widely accepted that transcriptional regulation of eukaryotic genes is
intimately coupled to covalent modifications of the underlying chromatin
template, and in certain cases the functional consequences of these
modifications have been characterized. Here we present evidence that gene
activation in the silent heterochromatin of the yeast Saccharomyces cerevisiae
can occur in the context of little, if any, covalent histone modification. Using
a SIR-regulated heat shock-inducible transgene, hsp82-2001, and a natural
drug-inducible subtelomeric gene, YFR057w, as models we demonstrate that
substantial transcriptional induction (>200-fold) can occur in the context of
restricted histone loss and negligible levels of H3K4 trimethylation, H3K36
trimethylation and H3K79 dimethylation, modifications commonly linked to
transcription initiation and elongation. Heterochromatic gene activation can
also occur with minimal H3 and H4 lysine acetylation and without replacement of
H2A with the transcription-linked variant H2A.Z. Importantly, absence of histone
modification does not stem from reduced transcriptional output, since
hsp82-ΔTATA, a euchromatic promoter mutant lacking a TATA box and with threefold
lower induced transcription than heterochromatic hsp82-2001, is strongly
hyperacetylated in response to heat shock. Consistent with negligible H3K79
dimethylation, dot1Δ cells lacking H3K79 methylase activity show unimpeded
occupancy of RNA polymerase II within activated heterochromatic promoter and
coding regions. Our results indicate that large increases in transcription can
be observed in the virtual absence of histone modifications often thought
necessary for gene activation. |
Does thyroid hormone receptor beta1 affect insulin secretion? | No | |
List disorders that have been associated to the polymorphism rs2535629. | schizophrenia and major depressive disorder. | Prior genome-wide association studies (GWAS) of major depressive disorder (MDD)
have met with limited success. We sought to increase statistical power to detect
disease loci by conducting a GWAS mega-analysis for MDD. In the MDD discovery
phase, we analyzed more than 1.2 million autosomal and X chromosome
single-nucleotide polymorphisms (SNPs) in 18 759 independent and unrelated
subjects of recent European ancestry (9240 MDD cases and 9519 controls). In the
MDD replication phase, we evaluated 554 SNPs in independent samples (6783 MDD
cases and 50 695 controls). We also conducted a cross-disorder meta-analysis
using 819 autosomal SNPs with P<0.0001 for either MDD or the Psychiatric GWAS
Consortium bipolar disorder (BIP) mega-analysis (9238 MDD cases/8039 controls
and 6998 BIP cases/7775 controls). No SNPs achieved genome-wide significance in
the MDD discovery phase, the MDD replication phase or in pre-planned secondary
analyses (by sex, recurrent MDD, recurrent early-onset MDD, age of onset,
pre-pubertal onset MDD or typical-like MDD from a latent class analyses of the
MDD criteria). In the MDD-bipolar cross-disorder analysis, 15 SNPs exceeded
genome-wide significance (P<5 × 10(-8)), and all were in a 248 kb interval of
high LD on 3p21.1 (chr3:52 425 083-53 822 102, minimum P=5.9 × 10(-9) at
rs2535629). Although this is the largest genome-wide analysis of MDD yet
conducted, its high prevalence means that the sample is still underpowered to
detect genetic effects typical for complex traits. Therefore, we were unable to
identify robust and replicable findings. We discuss what this means for genetic
research for MDD. The 3p21.1 MDD-BIP finding should be interpreted with caution
as the most significant SNP did not replicate in MDD samples, and genotyping in
independent samples will be needed to resolve its status. We examined the association between the selected polymorphisms in two candidate
genes, the arsenite methyltransferase gene (AS3MT, rs11191454) and the
inter-α-trypsin inhibitors heavy chain-3 gene (ITIH3, rs2535629), and
attention-deficit hyperactivity disorder (ADHD) in a Korean population. A total
of 238 patients with ADHD, along with both of their biological parents, were
recruited. The children were administered intelligence quotient tests, whereas
their parents completed the Child Behavior Checklist. In the transmission
disequilibrium test on 181 trios, we found overtransmission of the A allele at
the AS3MT rs11191454 polymorphism in children with ADHD (χ²=8.81, P=0.003).
However, there was no preferential transmission at the ITIH3 rs52535629
polymorphism (χ²=0.14, P=0.707). Our results provide preliminary evidence for
the overtransmission of the A allele at the AS3MT rs11191454 polymorphism in
ADHD. |
What is targeted by monoclonal antibody Pembrolizumab? | Pembrolizumab inhibits the programmed cell death 1 (PD-1) immune checkpoint and has antitumor activity in patients with advanced melanoma. Pembrolizumab is approved by the US Food and Drug Administration for the treatment of advanced melanoma, and additional regulatory approvals are expected across the oncologic spectrum for a variety of other agents that target these pathways. | Immune checkpoint inhibition as a new treatment approach is undergoing extensive
investigation in non-small cell lung cancer (NSCLC) and other maligcies.
Unlike standard chemotherapy or targeted agents, which act directly on the tumor
cells, immune checkpoint inhibitors work by restoring the immune system's
capacity to eradicate tumors. Agents currently in active clinical development
for lung cancer include ipilimumab, which modulates the cytotoxic
T-lymphocyte-associated antigen 4 pathway, and multiple agents targeting the
programmed death protein 1 (PD-1) pathway, both anti-PD-1 compounds (nivolumab,
pembrolizumab [MK-3475]) and those that target programmed death ligand 1
(PD-L1), a key ligand for PD-1 (BMS-936559, MPDL3280A). Preliminary evidence
shows activity for these agents in NSCLC as monotherapy or in combination with
chemotherapy. This article reviews the immune checkpoint inhibitors and the
available data to date on their use in lung cancer. Clinical implications for
the use of these therapies in NSCLC are discussed as they relate to their novel
mechanisms of action, response patterns, and safety profiles. BACKGROUND: The anti-programmed-death-receptor-1 (PD-1) antibody pembrolizumab
has shown potent antitumour activity at different doses and schedules in
patients with melanoma. We compared the efficacy and safety of pembrolizumab at
doses of 2 mg/kg and 10 mg/kg every 3 weeks in patients with
ipilimumab-refractory advanced melanoma.
METHODS: In an open-label, international, multicentre expansion cohort of a
phase 1 trial, patients (aged ≥18 years) with advanced melanoma whose disease
had progressed after at least two ipilimumab doses were randomly assigned with a
computer-generated allocation schedule (1:1 final ratio) to intravenous
pembrolizumab at 2 mg/kg every 3 weeks or 10 mg/kg every 3 weeks until disease
progression, intolerable toxicity, or consent withdrawal. Primary endpoint was
overall response rate (ORR) assessed with the Response Evaluation Criteria In
Solid Tumors (RECIST, version 1.1) by independent central review. Analysis was
done on the full-analysis set (all treated patients with measurable disease at
baseline). This study is registered with ClinicalTrials.gov, number NCT01295827.
FINDINGS: 173 patients received pembrolizumab 2 mg/kg (n=89) or 10 mg/kg (n=84).
Median follow-up duration was 8 months. ORR was 26% at both doses--21 of 81
patients in the 2 mg/kg group and 20 of 76 in the 10 mg/kg group (difference 0%,
95% CI -14 to 13; p=0·96). Treatment was well tolerated, with similar safety
profiles in the 2 mg/kg and 10 mg/kg groups and no drug-related deaths. The most
common drug-related adverse events of any grade in the 2 mg/kg and 10 mg/kg
groups were fatigue (29 [33%] vs 31 [37%]), pruritus (23 [26%] vs 16 [19%]), and
rash (16 [18%] vs 15 [18%]). Grade 3 fatigue, reported in five (3%) patients in
the 2 mg/kg pembrolizumab group, was the only drug-related grade 3 to 4 adverse
event reported in more than one patient.
INTERPRETATION: The results suggest that pembrolizumab at a dose of 2 mg/kg or
10 mg/kg every 3 weeks might be an effective treatment in patients for whom
there are few effective treatment options.
FUNDING: Merck Sharp and Dohme. Primary mucosal melanoma of the oral cavity is an exceedingly rare neoplasm
which is estimated to comprise 1-2% of all oral maligcies. In contrast to
cutaneous melanomas, the risk factors and pathogenesis are poorly understood.
The predominate localization of primary oral melanoma is hard palate and
maxillary alveolus. Dermoscopy may be utilized as an adjunctive tool in the
clinical differential diagnosis of oral mucosal melanoma whenever the lesion is
accessible with a dermoscope. Surgery is the mainstay of treatment, but it may
be challenging depending on the location of the tumor within the oral cavity and
its size. Adjuvant therapy with dacarbazine, platinum analogs, nitrosoureas and
interleukin-2 have been utilized with low response rates. Imatinib may be
effective for patients with with c-Kit gene mutations. Sunitinib and dasatinib
have been reported effective in selected cases. Vemurafenib and dabrafenib are
targeted agents for patients with BRAF mutation-positive melanoma. Ipilimumab,
an anti-cytotoxic T-lymphocyte antigen 4 antibody and pembrolizumab, a
monoclonal antibody targeting programmed death 1 receptor may be a feasible
treatment option in patients with metastatic mucosal melanoma. Immunologic checkpoint blockade with antibodies that target cytotoxic T
lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell death protein 1
pathway (PD-1/PD-L1) have demonstrated promise in a variety of maligcies.
Ipilimumab (CTLA-4) and pembrolizumab (PD-1) are approved by the US Food and
Drug Administration for the treatment of advanced melanoma, and additional
regulatory approvals are expected across the oncologic spectrum for a variety of
other agents that target these pathways. Treatment with both CTLA-4 and
PD-1/PD-L1 blockade is associated with a unique pattern of adverse events called
immune-related adverse events, and occasionally, unusual kinetics of tumor
response are seen. Combination approaches involving CTLA-4 and PD-1/PD-L1
blockade are being investigated to determine whether they enhance the efficacy
of either approach alone. Principles learned during the development of CTLA-4
and PD-1/PD-L1 approaches will likely be used as new immunologic checkpoint
blocking antibodies begin clinical investigation. The anti programmed cell death-1 (PD-1) antibodies pembrolizumab and nivolumab
have been recently licensed by the Food and Drug Administration for the
treatment of advanced melanoma. Immune checkpoint inhibitors such as these can
induce endocrine adverse events but autoimmune diabetes has not been described
to date. However, there is a strong preclinical rationale that supports this
autoimmune toxicity. We describe for the first time the case of an adult patient
who developed autoimmune diabetes likely as a consequence of PD-1 inhibition
with pembrolizumab. The presence of high serum titres of anti-glutamic acid
decarboxylase antibodies together with a suggestive clinical presentation, age
of the patient and preclinical data strongly support an autoimmune aetiology of
the diabetes. Moreover, the patient was found to have a well-known high-risk
human leucocyte antigen type for the development of type 1 diabetes in children,
so the PD-1 inhibition is very likely to have triggered the autoimmune
phenomenon. Our case suggests that insulin-dependent diabetes might be a rare
but important anti-PD-1 immune-related adverse event. BACKGROUND: The immune checkpoint inhibitor ipilimumab is the standard-of-care
treatment for patients with advanced melanoma. Pembrolizumab inhibits the
programmed cell death 1 (PD-1) immune checkpoint and has antitumor activity in
patients with advanced melanoma.
METHODS: In this randomized, controlled, phase 3 study, we assigned 834 patients
with advanced melanoma in a 1:1:1 ratio to receive pembrolizumab (at a dose of
10 mg per kilogram of body weight) every 2 weeks or every 3 weeks or four doses
of ipilimumab (at 3 mg per kilogram) every 3 weeks. Primary end points were
progression-free and overall survival.
RESULTS: The estimated 6-month progression-free-survival rates were 47.3% for
pembrolizumab every 2 weeks, 46.4% for pembrolizumab every 3 weeks, and 26.5%
for ipilimumab (hazard ratio for disease progression, 0.58; P<0.001 for both
pembrolizumab regimens versus ipilimumab; 95% confidence intervals [CIs], 0.46
to 0.72 and 0.47 to 0.72, respectively). Estimated 12-month survival rates were
74.1%, 68.4%, and 58.2%, respectively (hazard ratio for death for pembrolizumab
every 2 weeks, 0.63; 95% CI, 0.47 to 0.83; P=0.0005; hazard ratio for
pembrolizumab every 3 weeks, 0.69; 95% CI, 0.52 to 0.90; P=0.0036). The response
rate was improved with pembrolizumab administered every 2 weeks (33.7%) and
every 3 weeks (32.9%), as compared with ipilimumab (11.9%) (P<0.001 for both
comparisons). Responses were ongoing in 89.4%, 96.7%, and 87.9% of patients,
respectively, after a median follow-up of 7.9 months. Efficacy was similar in
the two pembrolizumab groups. Rates of treatment-related adverse events of grade
3 to 5 severity were lower in the pembrolizumab groups (13.3% and 10.1%) than in
the ipilimumab group (19.9%).
CONCLUSIONS: The anti-PD-1 antibody pembrolizumab prolonged progression-free
survival and overall survival and had less high-grade toxicity than did
ipilimumab in patients with advanced melanoma. (Funded by Merck Sharp & Dohme;
KEYNOTE-006 ClinicalTrials.gov number, NCT01866319.). Preclinical work has led to an increased understanding of the immunomodulatory
mechanisms involved in the regulation of the antitumor response in a variety of
tumor types. PD-1 (programmed death 1) appears to be a key checkpoint involved
in immune suppression in the tumor microenvironment, even in diseases not
previously thought to be sensitive to immune manipulation. More recently, the
subsequent clinical development of PD-1-based therapy has resulted in a major
breakthrough in the field of oncology. Pembrolizumab, a humanized highly
selective IgG4 anti-PD-1 monoclonal antibody, was recently approved for the
treatment of advanced melanoma based on promising early-phase clinical data.
Encouraging results have also been seen in other maligcies, and PD-1-targeted
therapies are likely to markedly change the treatment landscape. Future work
will center on rationally designed combination strategies in order to potentiate
the antitumor immune response and overcome mechanisms of resistance. BACKGROUND: Somatic mutations have the potential to encode "non-self"
immunogenic antigens. We hypothesized that tumors with a large number of somatic
mutations due to mismatch-repair defects may be susceptible to immune checkpoint
blockade.
METHODS: We conducted a phase 2 study to evaluate the clinical activity of
pembrolizumab, an anti-programmed death 1 immune checkpoint inhibitor, in 41
patients with progressive metastatic carcinoma with or without mismatch-repair
deficiency. Pembrolizumab was administered intravenously at a dose of 10 mg per
kilogram of body weight every 14 days in patients with mismatch repair-deficient
colorectal cancers, patients with mismatch repair-proficient colorectal cancers,
and patients with mismatch repair-deficient cancers that were not colorectal.
The coprimary end points were the immune-related objective response rate and the
20-week immune-related progression-free survival rate.
RESULTS: The immune-related objective response rate and immune-related
progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9
patients), respectively, for mismatch repair-deficient colorectal cancers and 0%
(0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair-proficient
colorectal cancers. The median progression-free survival and overall survival
were not reached in the cohort with mismatch repair-deficient colorectal cancer
but were 2.2 and 5.0 months, respectively, in the cohort with mismatch
repair-proficient colorectal cancer (hazard ratio for disease progression or
death, 0.10 [P<0.001], and hazard ratio for death, 0.22 [P=0.05]). Patients with
mismatch repair-deficient noncolorectal cancer had responses similar to those of
patients with mismatch repair-deficient colorectal cancer (immune-related
objective response rate, 71% [5 of 7 patients]; immune-related progression-free
survival rate, 67% [4 of 6 patients]). Whole-exome sequencing revealed a mean of
1782 somatic mutations per tumor in mismatch repair-deficient tumors, as
compared with 73 in mismatch repair-proficient tumors (P=0.007), and high
somatic mutation loads were associated with prolonged progression-free survival
(P=0.02).
CONCLUSIONS: This study showed that mismatch-repair status predicted clinical
benefit of immune checkpoint blockade with pembrolizumab. (Funded by Johns
Hopkins University and others; ClinicalTrials.gov number, NCT01876511.). |
Does replication timing affect the rate of somatic mutations? | Mutation rate, as reflected in recent evolutionary divergence and human nucleotide diversity, is markedly increased in later-replicating regions of the human genome. All classes of substitutions are affected, suggesting a generalized mechanism involving replication time-dependent DNA damage. DNA repair systems, in general, are less efficient in late replicating heterochromatic regions compared to early replicating euchromatic regions of the genome. In yeast the mutation rate increases with the replication timing by more than 30% between the earliest and the latest replicating regions. Limited evidence from one chromosome arm in Drosophila melanogaster suggests the opposite pattern, with regions overlapping early-firing origins showing increased levels of diversity and divergence. In humans DNA replication patterns help shape the mutational landscapes of normal and cancer genomes. | Eukaryotic DNA replication is highly stratified, with different genomic regions
shown to replicate at characteristic times during S phase. Here we observe that
mutation rate, as reflected in recent evolutionary divergence and human
nucleotide diversity, is markedly increased in later-replicating regions of the
human genome. All classes of substitutions are affected, suggesting a
generalized mechanism involving replication time-dependent DNA damage. This
correlation between mutation rate and regionally stratified replication timing
may have substantial evolutionary implications. Previous experimental studies suggest that the mutation rate is nonuniform
across the yeast genome. To characterize this variation across the genome more
precisely, we measured the mutation rate of the URA3 gene integrated at 43
different locations tiled across Chromosome VI. We show that mutation rate
varies 6-fold across a single chromosome, that this variation is correlated with
replication timing, and we propose a model to explain this variation that relies
on the temporal separation of two processes for replicating past damaged DNA:
error-free DNA damage tolerance and translesion synthesis. This model is
supported by the observation that eliminating translesion synthesis decreases
this variation. Mutation rates vary significantly within the genome and across species. Recent
studies revealed a long suspected replication-timing effect on mutation rate,
but the mechanisms that regulate the increase in mutation rate as the genome is
replicated remain unclear. Evidence is emerging, however, that DNA repair
systems, in general, are less efficient in late replicating heterochromatic
regions compared to early replicating euchromatic regions of the genome. At the
same time, mutation rates in both vertebrates and invertebrates have been shown
to vary with generation time (GT). GT is correlated with genome size, which
suggests a possible nucleotypic effect on species-specific mutation rates. These
and other observations all converge on a role for DNA replication checkpoints in
modulating generation times and mutation rates during the DNA synthetic phase (S
phase) of the cell cycle. The following will examine the potential role of the
intra-S checkpoint in regulating cell cycle times (GT) and mutation rates in
eukaryotes. This article was published online on August 5, 2011. An error was
subsequently identified. This notice is included in the online and print
versions to indicate that both have been corrected October 4, 2011. Several reports from mammals indicate that an increase in the mutation rate in
late-replicating regions may, in part, be responsible for the observed genomic
heterogeneity in neutral substitution rates and levels of diversity, although
the mechanisms for this remain poorly understood. Recent evidence also suggests
that late replication is associated with high mutability in yeast. This then
raises the question as to whether a similar effect is operating across all
eukaryotes. Limited evidence from one chromosome arm in Drosophila melanogaster
suggests the opposite pattern, with regions overlapping early-firing origins
showing increased levels of diversity and divergence. Given the availability of
genome-wide replication timing profiles for D. melanogaster, we now return to
this issue. Consistent with what is seen in other taxa, we find that divergence
at synonymous sites in exon cores, as well as divergence at putatively
unconstrained intronic sites, is elevated in late-replicating regions. Analysis
of genes with low codon usage bias suggests a ∼30% difference in mutation rate
between the earliest and the latest replicating sequence. Intronic sequence
suggests a more modest difference. We additionally show that an increase in
diversity in late-replicating sequences is not owing to replication timing
covarying with the local recombination rate. If anything, the effects of
recombination mask the impact of replication timing. We conclude that, contrary
to prior reports and consistent with what is seen in mammals and yeast, there is
indeed a relationship between rates of nucleotide divergence and diversity and
replication timing that is consistent with an increase in the mutation rate
during late S-phase in D. melanogaster. It is therefore plausible that such an
effect might be common among eukaryotes. The result may have implications for
the inference of positive selection. Mammalian DNA replication initiates at multiple sites along chromosomes at
different times during S phase, following a temporal replication program. The
specification of replication timing is thought to be a dynamic process regulated
by tissue-specific and developmental cues that are responsive to epigenetic
modifications. However, the mechanisms regulating where and when DNA replication
initiates along chromosomes remains poorly understood. Homologous chromosomes
usually replicate synchronously, however there are notable exceptions to this
rule. For example, in female mammalian cells one of the two X chromosomes
becomes late replicating through a process known as X inactivation(1). Along
with this delay in replication timing, estimated to be 2-3 hr, the majority of
genes become transcriptionally silenced on one X chromosome. In addition, a
discrete cis-acting locus, known as the X inactivation center, regulates this X
inactivation process, including the induction of delayed replication timing on
the entire inactive X chromosome. In addition, certain chromosome rearrangements
found in cancer cells and in cells exposed to ionizing radiation display a
significant delay in replication timing of >3 hours that affects the entire
chromosome(2,3). Recent work from our lab indicates that disruption of discrete
cis-acting autosomal loci result in an extremely late replicating phenotype that
affects the entire chromosome(4). Additional 'chromosome engineering' studies
indicate that certain chromosome rearrangements affecting many different
chromosomes result in this abnormal replication-timing phenotype, suggesting
that all mammalian chromosomes contain discrete cis-acting loci that control
proper replication timing of individual chromosomes(5). Here, we present a
method for the quantitative analysis of chromosome replication timing combined
with fluorescent in situ hybridization. This method allows for a direct
comparison of replication timing between homologous chromosomes within the same
cell, and was adapted from(6). In addition, this method allows for the
unambiguous identification of chromosomal rearrangements that correlate with
changes in replication timing that affect the entire chromosome. This method has
advantages over recently developed high throughput micro-array or sequencing
protocols that cannot distinguish between homologous alleles present on
rearranged and un-rearranged chromosomes. In addition, because the method
described here evaluates single cells, it can detect changes in chromosome
replication timing on chromosomal rearrangements that are present in only a
fraction of the cells in a population. A recent flurry of reports correlates replication timing (RT) with mutation
rates during both evolution and cancer. Specifically, point mutations and copy
number losses correlate with late replication, while copy number gains and other
rearrangements correlate with early replication. In some cases, plausible
mechanisms have been proposed. Point mutation rates may reflect temporal
variation in repair mechanisms. Transcription-induced double-strand breaks are
expected to occur in transcriptionally active early replicating chromatin.
Fusion partners are generally in close proximity, and chromatin in close
proximity replicates at similar times. However, temporal enrichment of copy
number gains and losses remains an enigma. Moreover, many conclusions are
compromised by a lack of matched RT and sequence datasets, the filtering out of
developmental variation in RT, and the use of somatic cell lines to make
inferences about germline evolution. |
Which metazaon species or taxa are known to lack selenoproteins | Some insect genomes have lost the capacity of synthesizing selenoproteins. Several insect species without selenoproteins have been identified. | While the genome sequence and gene content are available for an increasing
number of organisms, eukaryotic selenoproteins remain poorly characterized. The
dual role of the UGA codon confounds the identification of novel selenoprotein
genes. Here, we describe a comparative genomics approach that relies on the
genome-wide prediction of genes with in-frame TGA codons, and the subsequent
comparison of predictions from different genomes, wherein conservation in
regions flanking the TGA codon suggests selenocysteine coding function.
Application of this method to human and fugu genomes identified a novel
selenoprotein family, named SelU, in the puffer fish. The
selenocysteine-containing form also occurred in other fish, chicken, sea urchin,
green algae and diatoms. In contrast, mammals, worms and land plants contained
cysteine homologues. We demonstrated selenium incorporation into chicken SelU
and characterized the SelU expression pattern in zebrafish embryos. Our data
indicate a scattered evolutionary distribution of selenoproteins in eukaryotes,
and suggest that, contrary to the picture emerging from data available so far,
other taxa-specific selenoproteins probably exist. Selenoproteins are a diverse group of proteins that contain selenocysteine
(Sec), the 21st amino acid. In the genetic code, UGA serves as a termination
signal and a Sec codon. This dual role has precluded the automatic annotation of
selenoproteins. Recent advances in the computational identification of
selenoprotein genes have provided a first glimpse of the size, functions, and
phylogenetic diversity of eukaryotic selenoproteomes. Here, we describe the
identification of a selenoprotein family named SelJ. In contrast to known
selenoproteins, SelJ appears to be restricted to actinopterygian fishes and sea
urchin, with Cys homologues only found in cnidarians. SelJ shows significant
similarity to the jellyfish J1-crystallins and with them constitutes a distinct
subfamily within the large family of ADP-ribosylation enzymes. Consistent with
its potential role as a structural crystallin, SelJ has preferential and
homogeneous expression in the eye lens in early stages of zebrafish development.
A structural role for SelJ would be in contrast to the majority of known
selenoenzymes. The unusually highly restricted phylogenetic distribution of
SelJ, its specialization, and the comparative analysis of eukaryotic
selenoproteomes reveal the diversity and functional plasticity of selenoproteins
and point to a mosaic evolution of the use of Sec in proteins. BACKGROUND: Selenoproteins are a diverse family of proteins notable for the
presence of the 21st amino acid, selenocysteine. Until very recently, all
metazoan genomes investigated encoded selenoproteins, and these proteins had
therefore been believed to be essential for animal life. Challenging this
assumption, recent comparative analyses of insect genomes have revealed that
some insect genomes appear to have lost selenoprotein genes.
METHODOLOGY/PRINCIPAL FINDINGS: In this paper we investigate in detail the fate
of selenoproteins, and that of selenoprotein factors, in all available arthropod
genomes. We use a variety of in silico comparative genomics approaches to look
for known selenoprotein genes and factors involved in selenoprotein
biosynthesis. We have found that five insect species have completely lost the
ability to encode selenoproteins and that selenoprotein loss in these species,
although so far confined to the Endopterygota infraclass, cannot be attributed
to a single evolutionary event, but rather to multiple, independent events. Loss
of selenoproteins and selenoprotein factors is usually coupled to the deletion
of the entire no-longer functional genomic region, rather than to sequence
degradation and consequent pseudogenisation. Such dynamics of gene extinction
are consistent with the high rate of genome rearrangements observed in
Drosophila. We have also found that, while many selenoprotein factors are
concomitantly lost with the selenoproteins, others are present and conserved in
all investigated genomes, irrespective of whether they code for selenoproteins
or not, suggesting that they are involved in additional, non-selenoprotein
related functions.
CONCLUSIONS/SIGNIFICANCE: Selenoproteins have been independently lost in several
insect species, possibly as a consequence of the relaxation in insects of the
selective constraints acting across metazoans to maintain selenoproteins. The
dispensability of selenoproteins in insects may be related to the fundamental
differences in antioxidant defense between these animals and other metazoans. Selenocysteine (Sec), the 21st amino acid, is incorporated into proteins through
the recoding of a termination codon, an inefficient translational process
mediated by a complex molecular machinery. Sec is a rare amino acid in extant
proteins, chemically similar to cysteine (Cys), found in homologous position to
Cys of nonselenoprotein families. Selenoproteins account for the dependence of
vertebrates on environmental selenium (Se) and have an important role in several
Se-deficiency diseases. Selenoproteins are poorly characterized enzymes and
reports on the functional exchangeability of Sec with Cys are limited and
controversial. Whether the unique role of Sec in some selenoenzymes illustrates
the broader contribution of Se to protein function is unknown (Gromer S,
Johansson L, Bauer H, Arscott LD, Rauch S, Ballou DP, Williams CH Jr, Schirmer
RH, Arnér ES. 2003. Active sites of thioredoxin reductases: why selenoproteins?
Proc Natl Acad Sci USA. 100:12618-12623). Here, we address this question from an
evolutionary perspective by the simultaneous identification of the patterns of
divergence in almost half a billion years of vertebrate evolution and diversity
within the human lineage for the full complement of enzymatic Sec residues in
these proteomes. We complete this analysis with data for the homologous Cys
residues in the same genomes. Our results indicate concerted purifying selection
across Sec and Cys sites in all selenoproteomes, consistent with a unique role
of Sec in protein function, low exchangeability, and an unknown degree of
functional divergence with Cys homologs. The distinct biochemical properties of
Sec, rather than the geographical distribution of Se, global O(2) levels or Sec
metabolic cost, appear to play a major role in driving adaptive changes in
vertebrate selenoproteomes. A better understanding of the selenoproteomes and
neutral evolutionary patterns in other taxa will be necessary to fully assess
the generality of this conclusion. BACKGROUND: The co-translational incorporation of selenocysteine into nascent
polypeptides by recoding the UGA stop codon occurs in all domains of life. In
eukaryotes, this event requires at least three specific factors: SECIS binding
protein 2 (SBP2), a specific translation elongation factor (eEFSec),
selenocysteinyl tRNA, and a cis-acting selenocysteine insertion sequence (SECIS)
element in selenoprotein mRNAs. While the phylogenetic relationships of
selenoprotein families and the evolution of selenocysteine usage are well
documented, the evolutionary history of SECIS binding proteins has not been
explored.
RESULTS: In this report we present a phylogeny of the eukaryotic SECIS binding
protein family which includes SBP2 and a related protein we herein term SBP2L.
Here we show that SBP2L is an SBP2 paralogue in vertebrates and is the only form
of SECIS binding protein in invertebrate deuterostomes, suggesting a key role in
Sec incorporation in these organisms, but an SBP2/SBP2L fusion protein is unable
to support Sec incorporation in vitro. An in-depth phylogenetic analysis of the
conserved L7Ae RNA binding domain suggests an ancestral relationship with
ribosomal protein L30. In addition, we describe the emergence of a motif
upstream of the SBP2 RNA binding domain that shares significant similarity with
a motif within the pseudouridine synthase Cbf5.
CONCLUSION: Our analysis suggests that SECIS binding proteins arose once in
evolution but diverged significantly in multiple lineages. In addition, likely
due to a gene duplication event in the early vertebrate lineage, SBP2 and SBP2L
are paralogous in vertebrates. BACKGROUND: The selenocysteine (Sec) containing proteins, selenoproteins, are an
important group of proteins present throughout all 3 kingdoms of life. With the
rapid progression of selenoprotein research in the post-genomic era, application
of bioinformatics methods to the identification of selenoproteins in newly
sequenced species has become increasingly important. Although selenoproteins in
human and other vertebrates have been investigated, studies of primitive
invertebrate selenoproteomes are rarely reported outside of insects and
nematodes.
RESULT: A more integrated view of selenoprotein evolution was constructed using
several representative species from different evolutionary eras. Using a
SelGenAmic-based selenoprotein identification method, 178 selenoprotein genes
were identified in 6 invertebrates: Amphimedon queenslandica, Trichoplax
adhaerens, Nematostella vectensis, Lottia gigantean, Capitella teleta, and
Branchiostoma floridae. Amphioxus was found to have the most abundant and
variant selenoproteins of any animal currently characterized, including a
special selenoprotein P (SelP) possessing 3 repeated Trx-like domains and Sec
residues in the N-terminal and 2 Sec residues in the C-terminal. This gene
structure suggests the existence of two different strategies for extension of
Sec numbers in SelP for the preservation and transportation of selenium. In
addition, novel eukaryotic AphC-like selenoproteins were identified in sponges.
CONCLUSION: Comparison of various animal species suggests that even the most
primitive animals possess a selenoproteome range and variety similar to humans.
During evolutionary history, only a few new selenoproteins have emerged and few
were lost. Furthermore, the massive loss of selenoproteins in nematodes and
insects likely occurred independently in isolated partial evolutionary branches. |
What is the mode of inheritance of Facioscapulohumeral muscular
dystrophy (FSHD)? | The mode of inheritance of Facioscapulohumeral muscular dystrophy is autosomal dominant. | Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disease,
characterized by an autosomal domit mode of inheritance, facial involvement,
and selectivity and asymmetry of muscle involvement. In general, FSHD typically
presents before age 20 years. Usually, FSHD muscle involvement starts in the
face and then progresses to the shoulder girdle, the humeral muscles and the
abdominal muscles, and then the anterolateral compartment of the leg. Disease
severity is highly variable and progression is very slow. About 20% of FSHD
patients become wheelchair-bound. Lifespan is not shortened. The diagnosis of
FSHD is based on a genetic test by which a deletion of 3.3kb DNA repeats (named
D4Z4 and mapping to the subtelomeric region of chromosome 4q35) is identified.
The progressive pattern of FSHD requires that the severity of symptoms as well
as their physical, social and psychological impact be evaluated on a regular
basis. A yearly assessment is recommended. Multidisciplinary management of
FSHD--consisting of a combination of genetic counselling, functional assessment,
an assessment by a physical therapist, prescription of symptomatic therapies and
prevention of known complications of this disease--is required. Prescription of
physical therapy sessions and orthopedic appliances are to be adapted to the
patient's deficiencies and contractures. |
Is NOD1 activated in inflammation? | Nod proteins fight off bacterial infections by stimulating proinflammatory signaling and cytokine networks and by inducing antimicrobial effectors, such as nitric oxide and antimicrobial peptides. NOD1 engagement generates an inflammatory response via activation of NFκB and MAPK pathways and several inflammatory disorders, such as Crohn's disease and asthma, are linked to genetic changes in either Nod1 or Nod2. Nod1 is also critically implicated in shaping adaptive immune responses towards bacterial-derived constituents. Together, Nod1 and Nod2 represent central players in the control of immune responses to bacterial infections and inflammation. Mice deficient for both Nod1 and Nod2 had attenuated inflammatory pathology, reduced levels of inflammatory cytokines, and increased colonization of the mucosal tissue | PURPOSE: NOD1 plays an important role in host defense and recognizes the minimal
component of bacterial cell walls, meso-diaminopimelic acid (iE-DAP).
Polymorphisms in NOD1 are associated with autoinflammatory diseases
characterized by uveitis such as Crohn's disease and sarcoidosis. NOD1 is
homologous to NOD2, which is responsible for an autosomal domit form of
uveitis. Nonetheless, the role of NOD1 in intraocular inflammation has not been
explored. The induction of uveitis by iE-DAP in mice and the potential
contribution of interleukin (IL)-1beta were investigated.
METHODS: BALB/c mice or mice deficient in caspase-1 or IL-1R1 and their congenic
controls were injected intravitreally with iE-DAP or saline. The time course,
dose response, and contribution of IL-1beta to ocular inflammation were
quantified by intravital video microscopy, histology, and immunohistochemistry.
NOD1 and IL-1beta were measured in eye tissue by immunoblotting and ELISA.
RESULTS: NOD1 protein is expressed in the eye and promotes ocular inflammation
in a dose- and time-dependent fashion. The authors previously defined the role
of IL-1beta in NOD2 uveitis and tested whether NOD1 and NOD2 used similar
mechanisms. Treatment with iE-DAP significantly increased IL-1beta, which was
caspase-1 dependent. However, in contrast to NOD2, caspase-1 and IL-1R1 were
essential mediators of iE-DAP-induced uveitis, suggesting that NOD1 and NOD2
induce ocular inflammation by distinct mechanisms involving IL-1beta.
CONCLUSIONS: These findings demonstrate that NOD1 is expressed within the eye
and that its activation results in uveitis in an IL-1beta-dependent mechanism.
Characterizing the differences between NOD1 and NOD2 responses may provide
insight into the pathogenesis of uveitis. There is growing evidence that the host innate immune system has a critical role
in regulating carcinogenesis, but the specific receptors involved and the
importance of their interaction with commensal bacteria need to be elucidated.
Two major classes of innate immune receptors, the Toll-like receptors and
Nod-like receptors, many of which are upstream of nuclear factor-kappaB, are
involved in the detection of intestinal bacteria. The Toll-like receptors have
been implicated in promoting colon tumorigenesis, but the role of Nod-like
receptors in regulating tumorigenesis remains unclear. Using an established
mouse model system of colitis-associated colon tumorigenesis, we show that Nod1
deficiency results in the increased development of both colitis-associated and
Apc tumor suppressor-related colon tumors. In the absence of Nod1 signaling,
there is a greater disruption of the intestinal epithelial cell barrier due to
chemically induced injury as manifested by increased surface epithelial
apoptosis early on during chemically induced colitis and increased intestinal
permeability. The increased intestinal permeability is associated with enhanced
inflammatory cytokine production and epithelial cell proliferation in
Nod1-deficient mice as compared with wild-type mice. Depletion of the gut
microbiota suppressed tumor development in Nod1-deficient mice, thus
highlighting a link between the commensal bacteria within the intestine and the
host innate immune Nod1 signaling pathway in the regulation
inflammation-mediated colon cancer development. The pattern recognition molecules Nod1 and Nod2 play important roles in
intestinal homeostasis; however, how these proteins impact on the development of
inflammation during bacterial colitis has not been examined. In the
streptomycin-treated mouse model of Salmonella colitis, we found that mice
deficient for both Nod1 and Nod2 had attenuated inflammatory pathology, reduced
levels of inflammatory cytokines, and increased colonization of the mucosal
tissue. Nod1 and Nod2 from both hematopoietic and nonhematopoietic sources
contributed to the pathology, and all phenotypes were recapitulated in mice
deficient for the signaling adaptor protein Rip2. However, the influence of Rip2
was strictly dependent on infection conditions that favored expression of the
Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS), as
Rip2 was dispensable for inflammation when mice were infected with bacteria
grown under conditions that promoted expression of the SPI-1 TTSS. Thus, Nod1
and Nod2 can modulate inflammation and mediate efficient clearance of bacteria
from the mucosal tissue during Salmonella colitis, but their role is dependent
on the expression of the SPI-2 TTSS. There is a strong association between infection and prematurity; however, the
underlying mechanisms remain largely unknown. Nod1 and Nod2 are intracellular
pattern recognition receptors that are activated by bacterial peptides and
mediate innate immunity. We previously demonstrated that human first-trimester
trophoblasts express Nod1 and Nod2, which trigger inflammation upon stimulation.
This study sought to determine the expression and function of Nod1 and Nod2 in
third-trimester trophoblasts, and to characterize the in vivo effects of Nod1
activation on pregcy outcome. Human term placental tissues and isolated term
trophoblast expressed Nod1, but not Nod2. Activation of Nod1 by its agonist,
bacterial γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), in term trophoblast
cultures induced a proinflammatory cytokine profile, characterized by elevated
levels of secreted IL-6, GRO-α, and MCP-1, when compared with the control.
However, these cytokines were not upregulated in response to Nod2 stimulation
with bacterial MDP. Administration of high-dose bacterial iE-DAP to pregt
C57BL/6J mice on embryonic day 14.5 triggered preterm delivery within 24 h.
iE-DAP at a lower dose that did not induce prematurity, reduced fetal weight,
altered the cytokine profile at the maternal-fetal interface, and induced fetal
inflammation. Thus, functional Nod1 is expressed by trophoblast cells across
gestation and may have a role in mediating infection-associated inflammation and
prematurity. This study demonstrates that pattern recognition receptors, other
than the TLRs, may be implicated or involved in infection-associated preterm
labor. OBJECTIVE: Insulin resistance associates with chronic inflammation, and
participatory elements of the immune system are emerging. We hypothesized that
bacterial elements acting on distinct intracellular pattern recognition
receptors of the innate immune system, such as bacterial peptidoglycan (PGN)
acting on nucleotide oligomerization domain (NOD) proteins, contribute to
insulin resistance.
RESEARCH DESIGN AND METHODS: Metabolic and inflammatory properties were assessed
in wild-type (WT) and NOD1/2(-/-) double knockout mice fed a high-fat diet (HFD)
for 16 weeks. Insulin resistance was measured by hyperinsulinemic euglycemic
clamps in mice injected with mimetics of meso-diaminopimelic acid-containing PGN
or the minimal bioactive PGN motif, which activate NOD1 and NOD2, respectively.
Systemic and tissue-specific inflammation was assessed using enzyme-linked
immunosorbent assays in NOD ligand-injected mice. Cytokine secretion, glucose
uptake, and insulin signaling were assessed in adipocytes and primary
hepatocytes exposed to NOD ligands in vitro.
RESULTS: NOD1/2(-/-) mice were protected from HFD-induced inflammation, lipid
accumulation, and peripheral insulin intolerance. Conversely, direct activation
of NOD1 protein caused insulin resistance. NOD1 ligands induced peripheral and
hepatic insulin resistance within 6 h in WT, but not NOD1(-/-), mice. NOD2
ligands only modestly reduced peripheral glucose disposal. NOD1 ligand elicited
minor changes in circulating proinflammatory mediators, yet caused adipose
tissue inflammation and insulin resistance of muscle AS160 and liver FOXO1. Ex
vivo, NOD1 ligand caused proinflammatory cytokine secretion and impaired
insulin-stimulated glucose uptake directly in adipocytes. NOD1 ligand also
caused inflammation and insulin resistance directly in primary hepatocytes from
WT, but not NOD1(-/-), mice.
CONCLUSIONS: We identify NOD proteins as innate immune components that are
involved in diet-induced inflammation and insulin intolerance. Acute activation
of NOD proteins by mimetics of bacterial PGNs causes whole-body insulin
resistance, bolstering the concept that innate immune responses to distinctive
bacterial cues directly lead to insulin resistance. Hence, NOD1 is a plausible,
new link between innate immunity and metabolism. Understanding the mechanisms by which pathogens induce vascular inflammation and
dysfunction may reveal novel therapeutic targets in sepsis and related
conditions. The intracellular receptor NOD1 recognises peptidoglycan which
features in the cell wall of gram negative and some gram positive bacteria. NOD1
engagement generates an inflammatory response via activation of NFκB and MAPK
pathways. We have previously shown that stimulation of NOD1 directly activates
blood vessels and causes experimental shock in vivo. In this study we have used
an ex vivo vessel-organ culture model to characterise the relative contribution
of the endothelium in the response of blood vessels to NOD1 agonists. In
addition we present the novel finding that NOD1 directly activates human blood
vessels. Using human cultured cells we confirm that endothelial cells respond
more avidly to NOD1 agonists than vascular smooth muscle cells. Accordingly we
have sought to pharmacologically differentiate NOD1 and TLR4 mediated signalling
pathways in human endothelial cells, focussing on TAK1, NFκB and p38 MAPK. In
addition we profile novel inhibitors of RIP2 and NOD1 itself, which specifically
inhibit NOD1 ligand induced inflammatory signalling in the vasculature. This
paper is the first to demonstrate activation of whole human artery by NOD1
stimulation and the relative importance of the endothelium in the sensing of
NOD1 ligands by vessels. This data supports the potential utility of NOD1 and
RIP2 as therapeutic targets in human disease where vascular inflammation is a
clinical feature, such as in sepsis and septic shock. It is indispensable to thoroughly characterize each animal model in order to
distinguish between primary and secondary effects of genetic changes. The
present study analyzed Nod1 and Nod2 double deficient (Nod1/2 DKO) mice under
physiological and inflammatory conditions. Nod1 and Nod2 are members of the
Nucleotide-binding domain and Leucine-rich repeat containing Receptor (NLR)
family. Several inflammatory disorders, such as Crohn's disease and asthma, are
linked to genetic changes in either Nod1 or Nod2. These associations suggest
that Nod1 and Nod2 play important roles in regulating the immune
system.Three-month-old wildtype (Wt) and Nod1/2 DKO mice were sacrificed, body
and organ weight were determined, and blood was drawn. Except for lower liver
weight in Nod1/2 DKO mice, no differences were found in body/organ weight
between both strains. Leukocyte count and composition was comparable. No
significant changes in analyzed plasma biochemical markers were found.
Additionally, intestinal and vascular permeability was determined. Nod1/2 DKO
mice show increased susceptibility for intestinal permeability while vascular
permeability was not affected. Next we induced septic shock and organ damage by
administering LPS+PGN intraperitoneally to Wt and Nod1/2 DKO mice and sacrificed
animals after 2 and 24 hours. The systemic inflammatory and metabolic response
was comparable between both strains. However, renal response was different as
indicated by partly preserved kidney function and tubular epithelial cell damage
in Nod1/2 DKO at 24 hours. Remarkably, renal inflammatory mediators Tnfα, KC and
Il-10 were significantly increased in Nod1/2 DKO compared with Wt mice at
2 hours.Systematic analysis of Nod1/2 DKO mice revealed a possible role of
Nod1/2 in the development of renal disease during systemic inflammation. Preterm birth remains one of the most important issues facing perinatal medicine
today, with chronic inflammation and/or infection being the biggest etiological
factor. The nucleotide oligomerization domain (NOD) intracellular molecules
recognize a wide range of microbial products as well as other intracellular
danger signals, thereby initiating inflammation through activation of nuclear
factor KB (NFKB), a central regulator of the terminal processes of human labor
and delivery. The aims of this study were to determine the effect of 1) human
labor, proinflammatory cytokines, and bacterial endotoxin LPS on NOD1 and NOD2
expression and 2) NOD1 and NOD2 activation on the expression of prolabor
mediators in human fetal membranes and myometrium. NOD1 and NOD2 expression was
significantly higher in fetal membranes and myometrium after spontaneous labor
when compared to nonlaboring tissues. Bacterial endotoxin LPS and the
proinflammatory cytokines TNF and IL1B significantly increased NOD2, but not
NOD1, expression. Furthermore, LPS-induced NOD2 expression was decreased by the
NFKB inhibitor BAY 11-7082. In both fetal membranes and myometrium, the NOD1
ligand bacterial iE-DAP and the NOD2 ligand bacterial MDP significantly
increased the expression and secretion of proinflammatory cytokines (IL6 and
IL8), cyclooxygenase (PTGS2) expression and subsequent release of prostaglandins
PGE2 and PGF2alpha, and the expression and activity of MMP9. The effects of
these NOD1 and NOD2 ligands were mediated via NFKB, as 1) both iE-DAP and MDP
significantly increased NFKB activation and 2) the NFKB inhibitor BAY 11-7082
attenuated iE-DAP- and MDP-induced expression and secretion of prolabor
mediators. In conclusion, NOD1 and NOD2 are increased in laboring fetal
membranes and myometrium and with bacterial infection. Agonist activation of
NOD1 and NOD2 by bacterial products leads to NFKB activation and transcription
of NFKB induced prolabor genes. NOD1 and NOD2 may thus represent therapeutic
targets for the treatment and/or management of preterm birth. OBJECTIVES: The underlying mechanisms of protective immunity of placental
trophoblast cells against bacterial infection remain largely unknown. NOD1 are
intracellular pattern recognition receptors that are activated by bacterial
peptides and mediate innate immunity. This study aimed to investigate the
expression and function of NOD1 in first trimester trophoblast cells, and
evaluate the potential role of trophoblast cells in infection-associated
inflammation.
STUDY DESIGN: Human extravillous trophoblast cell line HTR8 cells were
stimulated with various concentrations of iE-DAP for various periods of time.
NOD1 expression was detected by immunofluorescence, and the changes in NOD1 and
RICK mRNA and protein in H8 cells were determined by real-time polymerase chain
reaction and Western blot analysis. The concentrations of interleukin (IL)-8 and
IL-6 secreted by H8 cells were examined by enzyme-linked immunosorbent assay.
NF-κB transcription activity and P65 expression were detected by electrophoretic
mobility shift assay and Western blot analysis.
RESULTS: H8 cells expressed NOD1, and the effects of iE-DAP on NOD1 were dose-
and time-dependent. The concentration of IL-8 increased gradually with
increasing concentration of iE-DAP, and the levels of IL-8 and IL-6 were
associated with the duration of exposure to iE-DAP. The dose of iE-DAP was
significantly associated with expression of RICK and P65, and stimulation of H8
cells by iE-DAP altered NF-κB transcription activity.
CONCLUSIONS: NOD1 may have a role in mediating infection-associated
inflammation. Once iE-DAP is recognized by NOD1, the inflammatory response may
be induced via NOD1-RICK-NF-κB-mediated pathways. The cytosolic nucleotide oligomerization domain (NOD)-like receptors NOD1 and
NOD2 are important contributors to the intracellular recognition of pathogens
including Chlamydophila pneumoniae, but little is known about their influence on
allergen-induced airway inflammation. In BALB/c mice, we observed that infection
with C. pneumoniae before systemic sensitization with ovalbumin (OVA) and local
OVA airway exposure diminished airway hyperresponsiveness (AHR). Thus, the
impact of the NOD1 agonist FK156 and the NOD2 agonist muramyl dipeptide given 6
hours before each sensitization or airway challenge was evaluated regarding AHR,
OVA-specific plasma immunoglobulins, bronchoalveolar lavage fluid differentials,
and cytokines. Spleen dendritic cells of FK156-treated mice were isolated and
cocultured with OVA-specific T cells isolated from DO11.10 mice, and T-cell
proliferation was quantified after OVA restimulation. T-cell proliferation was
investigated in vivo in lungs and lymph nodes of FK156-treated and OVA-exposed
DO11.10 mice. FK156, but not muramyl dipeptide, reduced AHR and pulmonary
eosinophilic infiltration if given before OVA sensitization or challenge,
whereas T-helper (Th)2 cytokines were not diminished. Dendritic cells from
FK156-treated mice evoked less OVA-specific T-cell proliferation as compared
with solvent-treated controls. Similarly, antigen-specific T-cell activation in
lung tissue was diminished after FK156 treatment. We conclude that NOD1
activation reduced AHR in allergen-induced lung inflammation, which was
accompanied by a reduction of allergen-specific T-cell proliferation. Innate immune responses provoke the accumulation of leukocytes at sites of
inflammation. In addition to monocytes and granulocytes, B cells also
participate in antimicrobial innate immune responses; however, the mechanisms
for accumulation of B cells to sites of inflammation are not well understood. To
study B cell accumulation following systemic inflammation, we used a model
synthetic ligand that stimulates a specific pattern recognition molecule,
nucleotide-binding oligomerization domain-containing protein 1 (Nod1). Upon
exposure to Nod1 agonists, both B cells and neutrophils rapidly accumulate
within the spleen, and dendritic cells migrate into the periarterial lymphoid
sheath. Nod1 stimulation led to a marked increase in several chemokines within
the spleen, including CXCL13, CCL2, and CCL20. Whereas the lymphotoxin pathway
was critical for the induction of the B cell chemoattractant CXCL13 in response
to Nod1 agonists, B cell accumulation within the spleen following Nod1-induced
systemic inflammation was independent of the lymphotoxin pathway. In contrast, a
CCR6/CCL20 chemokine loop instructed rapid increase of B cells in the spleen in
response to systemic administration of Nod1 agonists in a TNF-α-dependent
manner. Moreover, CCR6 was required to regulate Nod1-mediated B cell responses.
These results reveal a novel mechanism of B cells during inflammation and shed
light on how B cells participate in innate immune responses to microbial
stimulation. Obesity is associated with inflammation that can drive metabolic defects such as
hyperlipidemia and insulin resistance. Specific metabolites can contribute to
inflammation, but nutrient intake and obesity are also associated with altered
bacterial load in metabolic tissues (i.e. metabolic endotoxemia). These
bacterial cues can contribute to obesity-induced inflammation. The specific
bacterial components and host receptors that underpin altered metabolic
responses are emerging. We previously showed that Nucleotide-binding
oligomerization domain-containing protein 1 (NOD1) activation with bacterial
peptidoglycan (PGN) caused insulin resistance in mice. We now show that PGN
induces cell-autonomous lipolysis in adipocytes via NOD1. Specific bacterial PGN
motifs stimulated lipolysis in white adipose tissue (WAT) explants from WT, but
not NOD1⁻/⁻mice. NOD1-activating PGN stimulated mitogen activated protein
kinases (MAPK),protein kinase A (PKA), and NF-κB in 3T3-L1 adipocytes. The
NOD1-mediated lipolysis response was partially reduced by inhibition of ERK1/2
or PKA alone, but not c-Jun N-terminal kinase (JNK). NOD1-stimulated lipolysis
was partially dependent on NF-κB and was completely suppressed by inhibiting
ERK1/2 and PKA simultaneously or hormone sensitive lipase (HSL). Our results
demonstrate that bacterial PGN stimulates lipolysis in adipocytes by engaging a
stress kinase, PKA, NF-κB-dependent lipolytic program. Bacterial NOD1 activation
is positioned as a component of metabolic endotoxemia that can contribute to
hyperlipidemia, systemic inflammation and insulin resistance by acting directly
on adipocytes. Maternal peripheral insulin resistance and increased inflammation are two
features of pregcies, complicated by gestational diabetes mellitus (GDM). The
nucleotide-binding oligomerisation domain (NOD) intracellular molecules
recognise a wide range of microbial products, as well as other intracellular
danger signals, thereby initiating inflammation through activation of nuclear
factor κB (NFκB). The aim of this study was to determine whether levels of NOD1
and NOD2 are increased in adipose tissue of women with GDM. The effect of NOD1
and NOD2 activation on inflammation and the insulin signalling pathway was also
assessed. NOD1, but not NOD2, expression was higher in omental and subcutaneous
adipose tissues obtained from women with GDM when compared with those from women
with normal glucose tolerance (NGT). In both omental and subcutaneous adipose
tissues from NGT and GDM women, the NOD1 ligand g-d-glutamyl-meso-diaminopimelic
acid (iE-DAP) significantly induced the expression and secretion of the
pro-inflammatory cytokine interleukin 6 (IL6) and chemokine IL8; COX2 (PTGS2)
gene expression and subsequent prostaglandin production; the expression and
secretion of the extracellular matrix remodelling enzyme matrix
metalloproteinase 9 (MMP9) and the gene expression and secretion of the adhesion
molecules ICAM1 and VCAM1. There was no effect of the NOD2 ligand muramyl
dipeptide on any of the endpoints tested. The effects of the NOD1 ligand iE-DAP
were mediated via NFκB, as the NFκB inhibitor BAY 11-7082 significantly
attenuated iE-DAP-induced expression and secretion of pro-inflammatory
cytokines, COX2 gene expression and subsequent prostaglandin production, MMP9
expression and secretion and ICAM1 and VCAM1 gene expression and secretion. In
conclusion, the present findings describe an important role for NOD1 in the
development of insulin resistance and inflammation in pregcies complicated by
GDM. BACKGROUND: Nucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic
receptor involved in recognizing bacterial peptidoglycan fragments that localize
to the cytosol. NOD1 activation triggers inflammation, antimicrobial mechanisms
and autophagy in both epithelial cells and murine macrophages. NOD1 mediates
intracellular pathogen clearance in the lungs of mice; however, little is known
about NOD1's role in human alveolar macrophages (AMs) or its involvement in
Mycobacterium tuberculosis (Mtb) infection.
METHODS: AMs, monocytes (MNs), and monocyte-derived macrophages (MDMs) from
healthy subjects were assayed for NOD1 expression. Cells were stimulated with
the NOD1 ligand Tri-DAP and cytokine production and autophagy were assessed.
Cells were infected with Mtb and treated with Tri-DAP post-infection. CFUs
counting determined growth control, and autophagy protein recruitment to
pathogen localization sites was analyzed by immunoelectron microscopy.
RESULTS: NOD1 was expressed in AMs, MDMs and to a lesser extent MNs. Tri-DAP
stimulation induced NOD1 up-regulation and a significant production of IL1β,
IL6, IL8, and TNFα in AMs and MDMs; however, the level of NOD1-dependent
response in MNs was limited. Autophagy activity determined by expression of
proteins Atg9, LC3, IRGM and p62 degradation was induced in a NOD1-dependent
manner in AMs and MDMs but not in MNs. Infected AMs could be activated by
stimulation with Tri-DAP to control the intracellular growth of Mtb. In
addition, recruitment of NOD1 and the autophagy proteins IRGM and LC3 to the Mtb
localization site was observed in infected AMs after treatment with Tri-DAP.
CONCLUSIONS: NOD1 is involved in AM and MDM innate responses, which include
proinflammatory cytokines and autophagy, with potential implications in the
killing of Mtb in humans. Helicobacter pylori (H. pylori) is the strongest known risk factor for gastric
carcinogenesis. One cancer-linked locus is the cag pathogenicity island, which
translocates components of peptidoglycan into host cells. NOD1 is an
intracellular immune receptor that senses peptidoglycan from Gram-negative
bacteria and responds by inducing autophagy and activating NF-κB, leading to
inflammation-mediated bacterial clearance; however chronic pathogens can evade
NOD1-mediated clearance by altering peptidoglycan structure. We previously
demonstrated that the H. pylori cag(+) strain 7.13 rapidly induces gastric
cancer in Mongolian gerbils. Using 2D-DIGE and mass spectrometry, we identified
a novel mutation within the gene encoding the peptidoglycan deacetylase PgdA;
therefore, we sought to define the role of H. pylori PgdA in NOD1-dependent
activation of NF-κB, inflammation, and cancer. Coculture of H. pylori strain
7.13 or its pgdA(-) isogenic mutant with AGS gastric epithelial cells or HEK293
epithelial cells expressing a NF-κB reporter revealed that pgdA inactivation
significantly decreased NOD1-dependent NF-κB activation and autophagy. Infection
of Mongolian gerbils with an H. pylori pgdA(-) mutant strain led to
significantly decreased levels of inflammation and maligt lesions in the
stomach; however, preactivation of NOD1 before bacterial challenge reciprocally
suppressed inflammation and cancer in response to wild-type H. pylori.
Expression of NOD1 differs in human gastric cancer specimens compared with
noncancer samples harvested from the same patients. These results indicate that
peptidoglycan deacetylation plays an important role in modulating host
inflammatory responses to H. pylori, allowing the bacteria to persist and induce
carcinogenic consequences in the gastric niche. |
List diseases where protein citrullination plays an important role. | Rheumatoid arthritis, multiple sclerosis, prion diseases, cancer and Alzheimer's disease are examples of diseases where protein citrullination involvement has been demonstrated. | Posttranslational modifications are chemical changes to proteins that take place
after synthesis. One such modification, peptidylarginine to peptidylcitrulline
conversion, catalysed by peptidylarginine deiminases, has recently received
significant interest in biomedicine. Introduction of citrulline dramatically
changes the structure and function of proteins. It has been implicated in
several physiological and pathological processes. Physiological processes
include epithelial terminal differentiation, gene expression regulation, and
apoptosis. Rheumatoid arthritis, multiple sclerosis, and Alzheimer's disease are
examples of human diseases where protein citrullination involvement has been
demonstrated. In this review, we discuss our current understanding on the
importance of protein deimination in these processes. We describe the enzymes
catalyzing the reaction, as well as their known protein substrates. We review
the citrullinated peptide epitopes that are proposed as disease markers,
specifically recognized in certain human autoimmune disorders. The potential
autopathogenic role of citrullinated epitopes is also discussed. During the development of multiple sclerosis the destruction of the myelin
sheath surrounding the neurites is accompanied by citrullination of several
central nervous system (CNS) proteins, including myelin basic protein and glial
fibrillary acidic protein. In experimental autoimmune encephalomyelitis (EAE), a
disease induced in animals by immunization with proteins or peptides from the
CNS, the animals develop symptoms similar to multiple sclerosis (MS). The
increased levels of citrullinated CNS proteins associated with MS are also
observed during the development of EAE. To study the role of CNS protein
citrullination in EAE development, we induced EAE with a peptide derived from
myelin oligodendrocyte glycoprotein (MOG(35-55)) in mice lacking the
peptidylarginine deiminase 2 (PAD2) protein, because this enzyme was the most
likely candidate to be involved in catalyzing CNS protein citrullination in the
diseased state. Even though the PAD2 knockout mice displayed a dramatic
reduction in the amount of citrullination present in the CNS, indicating that
PAD2 is indeed responsible for the majority of detectable citrullination
observed in EAE, the development of EAE was not impaired by genetic deletion of
PAD2, suggesting that PAD2 catalyzed citrullination is not essential to the
development of EAE. Peptidylarginine deiminases (PADs), which are a group of posttranslational
modification enzymes, are involved in protein citrullination (deimination) by
the conversion of peptidylarginine to peptidylcitrulline in a calcium
concentration-dependent manner. Among the PADs, PAD2 is widely distributed in
various tissues and is the only type that is expressed in brain. To elucidate
the involvement of protein citrullination by PAD2 in the pathogenesis of
brain-specific prion diseases, we examined the profiles of citrullinated
proteins using the brains of scrapie-infected mice as a prion disease model. We
found that, compared with controls, increased levels of citrullinated proteins
of various molecular weights were detected in different brain sections of
scrapie-infected mice. In support of this data, expression levels of PAD2
protein as well as its enzyme activity were significantly increased in brain
sections of scrapie-infected mice, including hippocampus, brain stem, and
striatum. Additionally, the expression levels of PAD2 mRNA were increased during
scrapie infection. Moreover, PAD2 immunoreactivity was increased in
scrapie-infected brains, with staining detected primarily in reactive
astrocytes. Using two-dimensional electrophoresis and matrix-assisted laser
desorption/ionization-time of flight mass spectrometry, various citrullinated
proteins were identified in the brains of scrapie-infected mice, including glial
fibrillary acidic protein, myelin basic protein, enolases, and aldolases. This
study suggests that accumulated citrullinated proteins and abnormal activation
of PAD2 may function in the pathogenesis of prion diseases and serve as
potential therapeutic targets. Peptidylarginine deiminases (PADs) are a group of posttranslational modification
enzymes that citrullinate (deiminate) protein arginine residues in a
Ca(2+)-dependent manner. Enzymatic citrullination abolishes positive charges of
native protein molecules, inevitably causing significant alterations in their
structure and functions. Among the five isoforms of PADs, PAD2 and PAD4 are
proved occupants of the central nervous system (CNS), and especially PAD2 is a
main PAD enzyme expressed in the CNS. We previously reported that abnormal
protein citrullination by PAD2 has been closely associated with the pathogenesis
of neurodegenerative disorders such as Alzheimer's disease and prion disease.
Protein citrullination in these patients is thought to play a role during the
initiation and/or progression of disease. However, the contribution of changes
in PAD2 levels, and consequent citrullination, during developmental and aging
processes remained unclear. Therefore, we used quantitative real-time RT-PCR,
Western blot analysis, and immunohistochemical methods to measure PAD2
expression and localization in the brain during those processes. PAD2 mRNA
expression was detected in the brains of mice as early as embryonic day 15, and
its expression in cerebral cortex, hippocampus, and cerebellum increased
significantly as the animals aged from 3 to 30 months old. No citrullinated
proteins were detected during that period. Moreover, we found here, for the
first time, that PAD2 localized specifically in the neuronal cells of the
cerebral cortex and Purkinje cells of the cerebellum. These findings indicate
that, despite PAD2's normally inactive status, it becomes active and
citrullinates cellular proteins, but only when the intracellular Ca(2+) balance
is upset during neurodegenerative changes. Peptidylarginine deiminases (PADs)-mediated post-translational citrullination
processes play key roles in protein functions and structural stability through
the conversion of arginine to citrulline in the presence of excessive calcium
concentrations. In brain, PAD2 is abundantly expressed and can be involved in
citrullination in disease. Recently, we have reported pathological
characterization of PAD2 and citrullinated proteins in scrapie-infected mice,
but the implication of protein citrullination in the pathophysiology in human
prion disease is not clear. In the present study, we explored the molecular and
biological involvement of PAD2 and the pathogenesis of citrullinated proteins in
frontal cortex of patients with sporadic Creutzfeldt-Jakob disease (sCJD). We
found increased expression of PAD2 in reactive astrocytes that also contained
increased levels of citrullinated proteins. In addition, PAD activity was
significantly elevated in patients with sCJD compared to controls. From
two-dimensional gel electrophoresis and MALDI-TOF mass analysis, we found
various citrullinated candidates, including cytoskeletal and energy
metabolism-associated proteins such as vimentin, glial fibrillary acidic
protein, enolase, and phosphoglycerate kinase. Based on these findings, our
investigations suggest that PAD2 activation and aberrant citrullinated proteins
could play a role in pathogenesis and have value as a marker for the postmortem
classification of neurodegenerative diseases. Protein citrullination results from enzymatic deimination of peptidylarginine
and plays an important role in health and disease. Despite increasing scientific
interest, the identity and function of citrullinated proteins in vivo remain
widely unknown. Thorough proteomic studies could contribute to a better
understanding of the role of this posttranslational modification but will
require tools for enrichment of citrullinated polypeptides. This study presents
a simple technique for a highly specific enrichment of citrullinated peptides
that is based on the specific reaction of glyoxal derivatives with the
citrulline ureido group under acidic conditions. Beads were functionalized with
4-hydroxyphenylglyoxal attached via a base-labile linker. Incubation of these
"citrulline reactive beads" with peptide mixtures at low pH resulted in
selective immobilization of citrullinated peptides. Unbound noncitrullinated
peptides were removed by extensive washing. Finally, citrullinated peptides
carrying a modified ureido group were cleaved off at high pH and were analyzed
by mass spectrometry. The procedure was validated by enrichment of synthetic
citrulline-containing peptides from a tryptic digest of bovine serum albumin and
from an endoproteinase LysC digest of a cytosolic fraction of a cell line. The
technique was further applied to enrich citrullinated peptides from a digest of
deiminated myelin basic protein. Peptidylarginine deiminases (PAD) are a group of post-translational modification
enzymes that citrullinate (deiminate) protein arginine residues in a calcium
ion-dependent manner. Enzymatic citrullination abolishes positive charges of
native protein molecules, inevitably causing significant alterations in their
structure and functions. Citrullinated protein has an important physiological
purpose; the formation of a cornified layer of skin that covers the human body.
Despite this beneficial function, citrullinated protein also has a negative
side, because this protein's accumulation in the brain is a possible cause of
Alzheimer's disease. In the present review, we introduce PAD and their protein
citrullination function, now considered critical for advancing research on aging
and disease. We aimed to investigate potential synovial autoantigens in rheumatoid arthritis
(RA) that could trigger the induction of B-cell autoantibodies. Total protein
extract of synovial tissue obtained from seven RA patients was pooled and
separated by 1-DE and 2-DE. The corresponding blots were probed with sera from
RA (n = 30) and disease control samples (n = 30). Protein spots showing a
sensitivity of >15% were identified by MS. 1-D immunoblots revealed one protein
band with a specificity in RA of 100%, a sensitivity of 43%, which was
identified as fibrinogen β chain. 2-D analysis revealed the subunits of
fibrinogen, especially the β and γ chain, as the most prominent synovial
autoantigens. We also identified vimentin, the Sa-antigen and carbonic anhydrase
I as a potentially new synovial autoantigen. The protein patterns of these
immunoreactive spots were observed as trains. The spots showing the highest
autoimmune reactivity occurred at the acidic side of these trains and were
recognized by anticitrullinated protein/peptide antibodies positive RA sera.
Antimodified citrulline staining of these patterns confirmed protein
citrullination. Therefore, PTMs such as citrullination due to alterations of
peptidylarginine deiminase activity or generation of RA-specific epitopes,
should be considered as a trigger in tolerance break. Peptidyl arginine deiminases (PADs) catalyze a post-translational protein
modification reaction called citrullination, where arginine is converted to
citrulline. This modification has been linked to the pathogenesis of autoimmune
diseases including rheumatoid arthritis (RA). More recently, several studies
have suggested that Alzheimer's disease (AD), a devastating neurodegenerative
disorder, may have an autoimmune component. In the present study, we have
investigated the possibility that expression of PADs and protein citrullination
plays a role in the production of brain-reactive autoantibodies that may
contribute to Alzheimer's-related brain pathology. Here, we report the selective
expression of the PAD isoforms, PAD2 and PAD4, in astrocytes and neurons,
respectively, and the concomitant accumulation of citrullinated proteins within
PAD4-expressing cells, including neurons of the hippocampus and cerebral cortex.
Expression of PADs and citrullinated proteins is prominent in brain regions
engaged in neurodegenerative changes typical for AD pathology. Furthermore, we
also demonstrate that the pentatricopeptide repeat domain2 (PTCD2) protein, an
antigen target of a prominent AD diagnostic autoantibody, is present in a
citrullinated form in AD brains. Our results suggest that disease-associated
neuronal loss results in the release of cellular contents, including
citrullinated proteins, into the brain interstitium. We propose that these
citrullinated proteins and their degradation fragments enter into the blood and
lymphatic circulation, and some are capable of eliciting an immune response that
results in the production of autoantibodies. The long-term and progressive
nature of AD and other neurodegenerative diseases results in chronic exposure of
the immune system to these citrullinated products and may drive the continual
production of autoantibodies. The post-translational citrullination (deimination) process is mediated by
peptidylarginine deiminases (PADs), which convert peptidylarginine into
peptidylcitrulline in the presence of high calcium concentrations. Over the past
decade, PADs and protein citrullination have been commonly implicated as
abnormal pathological features in neurodegeneration and inflammatory responses
associated with diseases such as multiple sclerosis, Alzheimer disease and
rheumatoid arthritis. Based on this evidence, we investigated the roles of PADs
and citrullination in the pathogenesis of prion diseases. Prion diseases (also
known as transmissible spongiform encephalopathies) are fatal neurodegenerative
diseases that are pathologically well characterized as the accumulation of
disease-associated misfolded prion proteins, spongiform changes, glial cell
activation and neuronal loss. We previously demonstrated that the upregulation
of PAD2, mainly found in reactive astrocytes of infected brains, leads to
excessive citrullination, which is correlated with disease progression. Further,
we demonstrated that various cytoskeletal and energy metabolism-associated
proteins are particularly vulnerable to citrullination. Our recent in vivo and
in vitro studies elicited altered functions of enolase as the result of
citrullination; these altered functions included reduced enzyme activity,
increased protease sensitivity and enhanced plasminogen-binding affinity. These
findings suggest that PAD2 and citrullinated proteins may play a key role in the
brain pathology of prion diseases. By extension, we believe that abnormal
increases in protein citrullination may be strong evidence of neurodegeneration. Multiple sclerosis (MS) is the most common CNS-demyelinating disease of humans,
showing clinical and pathological heterogeneity and a general resistance to
therapy. We first discovered that abnormal myelin hypercitrullination, even in
normal-appearing white matter, by peptidylarginine deiminases (PADs) correlates
strongly with disease severity and might have an important role in MS
progression. Hypercitrullination is known to promote focal demyelination through
reduced myelin compaction. Here we report that 2-chloroacetamidine (2CA), a
small-molecule, PAD active-site inhibitor, dramatically attenuates disease at
any stage in independent neurodegenerative as well as autoimmune MS mouse
models. 2CA reduced PAD activity and protein citrullination to pre-disease
status. In the autoimmune models, disease induction uniformly induced
spontaneous hypercitrullination with citrulline+ epitopes targeted frequently.
2CA rapidly suppressed T cell autoreactivity, clearing brain and spinal cord
infiltrates, through selective removal of newly activated T cells. 2CA
essentially prevented disease when administered before disease onset or before
autoimmune induction, making hypercitrullination, and specifically PAD enzymes,
a therapeutic target in MS models and thus possibly in MS. While a group of oral commensals have been implicated in the aetiology of
chronic periodontitis; the asaccharolytic Gram negative anaerobe Porphyromonas
gingivalis is most commonly reported to be associated with severe forms of the
disease. Although a variety of human tissues can produce a number of
peptidylarginine deiminase (PAD), enzymes that convert peptide bound arginine
residues to citrulline, P. gingivalis is one of the few prokaryotes known to
express PAD. Protein and peptide citrullination are important in the development
of rheumatoid arthritis and in recent years a number of authors have suggested a
possible link between periodontitis and rheumatoid arthritis (RA). Indeed, some
have linked P. gingivalis directly to RA via the action of PAD. Accordingly, the
prime purpose of this study was to further characterise PAD in P. gingivalis
cells particular emphasis on substrate specificity, using arginine containing
peptides and RA relevant proteins.
METHODS: P. ginigvalis W50 was anaerobically cultured in BHI broth, cells
harvested and resuspended in assay buffer. A colourimetric assay was developed
to measure citrulline and employed to determine enzyme activity using the
substrate BAEE. The assay was employed to investigate the effects of
environmental pH and temperature on activity. Citrullination of BAEE by
sonicated cells allowed the proportion of intracellular enzyme to be estimated.
Enzyme specificity and substrate preference were investigated by using various
arginine containing peptides, proteins and arginine analogues, as substrates and
measuring the rate of citrullination. The influence of gingipains on
citrullination was assessed by measuring the rates of citrullination of bovine
serum albumin in the presence of protease inhibitors.
RESULTS: Enzyme activity decreased by 13% following exposure of cells to 60 °C
for 10 min. A comparison of intact and disrupted cells indicated that 90% of PAD
activity is cell surface associated and the remainder cytoplasmic. Optimal pH
for enzyme activity was between pH 7.5 and 8. All small arginine-containing
peptides were citrullinated with reaction rates faster than that for free
arginine with rates that varied with arginine residue position and number.
Arginine analogues exhibited minimal effect and influence when tested as either
substrates or competitive inhibitors. Cells were able to citrullinate yeast
enolase, human vimentin and fibrin at varying rates. All proteins were modified
at slower rates than those for peptide substrates. Inhibition of gingipains had
no influence on the rate of protein citrullination.
CONCLUSIONS: P. gingivalis PAD is a primarily cell surface associated, heat
stable, enzyme that exhibits optimal activity under alkaline conditions similar
to those present in the inflammatory environment. The enzyme displays high
specificity for arginine residues in peptides and modified arginine in all
positions and the gingipains did not influence the rate of protein
citrullination. The ability of the enzyme to convert arginine residues in all
proteins tested would indicate that its presence in inflamed tissue may promote
autoimmune reactions by creation of altered host epitopes. The post-translational conversion of peptidylarginine to peptidylcitrulline, a
process also known as citrullination, is catalyzed by the enzyme family of
peptidylarginine deiminases (PADs) and has been demonstrated to be involved in
many physiological processes, including the regulation of gene expression. In
addition, citrullination has been shown to be associated with several diseases,
such as cancer, multiple sclerosis, rheumatoid arthritis, and Alzheimer's
disease. To get more insight into the role of PAD enzymes and citrullination in
both health and disease, experimental strategies to study PAD activity and to
characterize citrullinated proteins in complex biological samples are crucial.
Here, we describe the chemical, proteomic and antibody-based procedures that are
currently available and discuss their applicability for the analysis of complex
samples. The methods that have been developed can be used to provide more
insight in the substrate specificity of PAD enzymes. Because the evidence that
PADs play a pathophysiological role in the diseases mentioned above is
increasing, they become attractive targets for therapeutic interventions. More
knowledge of PAD specificity and the availability of reliable, high-throughput
assays for PAD activity will facilitate the development of highly specific PAD
inhibitors. |
What is commotio cordis? | Commotio cordis is a term used to describe cases of blunt thoracic impact causing sudden death without structural damage of the heart | Nonpenetrating cardiac injuries due to direct precordial blunt impacts are a
commonly encountered phenomenon in medicolegal offices. These injuries vary from
contusions to valvular lacerations, or papillary muscle rupture to coronary
arterial injury with resulting infarction. A less commonly occurring
manifestation of nonpenetrating injury is a concussion of the heart (commotio
cordis), often with dramatic physiological consequences but no morphologic
cardiac injury. We describe four case reports of this entity. The cases were
collected over a 5-year period (1978-1983) from the Wayne County Medical
Examiner's Office in Detroit, Michigan. Cardiac concussion, previously known as commotio cordis, occurs in structurally
normal hearts without gross or microscopic injury to the myocardium, cardiac
valves, or coronary arteries, as opposed to other sports-related deaths known to
occur more frequently in structural or congenital heart disease. We describe the
sudden cardiac death of a 2-year-old child, who died as a result of a blunt
force impact to the chest. A thorough medicolegal investigation was necessary to
determine that the child died as a result of foul play. Commotio cordis due to blunt trauma to the precordium is a rare cause of death
in young athletes, occurring less frequently than all of the other
athletics-related deaths. Several measures, such as the use of safety baseballs
and the use of chest protectors, can help protect young athletes from commotio
cordis. In general, sudden cardiac death in athletes is receiving increasing
attention from the public as a result of recent deaths of high-profile athletes.
Sudden cardiac death, however, is rare, with an estimated 1 out of 200,000 high
school athletes at risk each year. However, the personal, physiological, and
cardiovascular benefits of athletics far outweigh the risks. Therefore, the
message to parents is to allow their children to participate in athletics
because the benefits far outweigh the risks. BACKGROUND: Commotio cordis is a term used to describe cases of blunt thoracic
impact causing fatality without gross structural damage of the heart and
internal organs. Death is attributed to ventricular fibrillation or cardiac
arrhythmia aggravated by traumatic apnea. The biomechanical response related to
the risk of commotio cordis has not been determined.
METHODS: Reanalysis of previously published experimental data was performed to
determine which biomechanical parameter predicts the occurrence of commotio
cordis. Logistic regression was used to determine the risk for commotio cordis
with the level of chest compression, rate of chest deformation, and viscous
criterion.
RESULTS: By using only cases without serious tissue injury (Abbreviated Injury
Scale score < 4), viscous criterion was the best predictor of commotio cordis or
ventricular fibrillation (chi2 = 7.69, p = 0.006). It was also the best
predictor of heart rupture (chi2 = 13.19,p = 0.0003) and severe cardiac injury
with Abbreviated Injury Scale score > or = 4 (chi2 = 25.03, p = 0.0001).
CONCLUSION: Based on this in-depth analysis, the viscous criterion is the
relevant biomechanical response to assess the risk of commotio cordis and more
severe thoracic injury in high-speed blunt impact. Sudden death following blunt chest trauma is a frightening occurrence known as
'commotio cordis' or 'concussion of the heart'. It is speculated that commotio
cordis could be caused by ventricular fibrillation secondary to an
impact-induced energy that was transmitted via the chest wall to the myocardium
during its vulnerable repolarization period. We describe a survivor of commotio
cordis caused by a baseball. In this patient, an initial ventricular
fibrillation was documented and converted by direct current defibrillation.
Serial electrocardiographic changes (bifascicular conduction block and T wave
inversion in precordial leads) were noticed in this patient. Our case suggested
that coronary vasospasm might also play a role in commotio cordis. Over the last few years, the recognised cardiovascular risks of sporting
activities have been extended to include cardiac arrest resulting from
low-energy precordial chest impact produced by projectiles (e.g. baseball) or
bodily contact, in the young, healthy and active athlete [also known as commotio
cordis (CC)]. However, case reports of CC in European medical literature can be
traced back for at least 130 years. CC accounts for a small, but important,
subset of sudden death during sporting activities. It is a devastating
electrophysiological event in the young athlete, and one which has generated
considerable concern, both in the medical profession as well as in the public.
The mechanism of sudden death appears to be caused by ventricular fibrillation,
which occurs when the chest impact is delivered within a narrow, electrically
vulnerable portion of the cardiac cycle, that is, during repolarisation, just
before the peak of the T wave. Resuscitation of these victims is possible with
prompt cardiopulmonary resuscitation and defibrillation. Preventive measures,
such as the use of age-appropriate safety baseballs and suitably designed chest
wall protection, may reduce the risk of sudden death and, thus, make the
athletic field a safer place for young athletes. Although thought to be rare, sudden deaths caused by nonpenetrating chest wall
impact in the absence of structural injury to the ribs, sternum, and heart
(commotio cordis) are reported with increasing frequency. This phenomenon is
described in individuals when they are struck by relatively innocent blows to
the chest wall. Young male athletes aged 5 to 18 years are particularly at risk
for this catastrophe. It has been described after blows to the chest from
baseballs, softballs, hockey pucks, and other objects. Death is usually
instantaneous, and successful resuscitation is uncommon. A recently reported
experimental model provides clues to the mechanisms and inferences for the
prevention and treatment of this devastating condition. This swine model shows
that a) ventricular fibrillation results from low-energy chest wall impacts
during a vulnerable period of repolarization, b) the risk of this event can be
decreased with softer-than-standard baseballs, and c) prompt defibrillation is
crucial for resuscitation to be successful. Moderate pre-cordial mechanical impact can cause sudden cardiac death, even in
the absence of morphological damage to the heart. This is the most severe
expression of a condition termed, in the 19th century, Commotio cordis.
Experimental studies performed in the early 1930s showed that sudden cardiac
death after chest impact is brought about by an intrinsic cardiac response to
the mechanical stimulus. The precise (sub-)cellular mechanisms of this response
are still poorly understood. This article summarises experimental findings on
the condition and relates them to the more recently established concept of
cardiac mechano-electric feedback. As a result, an explanation of the mechanisms
that give rise to sudden cardiac death by Commotio cordis and targets for
further research are suggested. Commotio cordis is the condition of sudden cardiac death or near sudden cardiac
death after blunt, low-impact chest wall trauma in the absence of structural
cardiac abnormality. Ventricular fibrillation is the most commonly reported
induced arrhythmia in commotio cordis. Blunt impact injury to the chest with a
baseball is the most common mechanism. Survival rates for commotio cordis are
low, even with prompt CPR and defibrillation. CONTEXT: Although blunt, nonpenetrating chest blows causing sudden cardiac death
(commotio cordis) are often associated with competitive sports, dangers implicit
in such blows can extend into many other life activities.
OBJECTIVE: To describe the comprehensive spectrum of commotio cordis events.
DESIGN AND SETTING: Analysis of confirmed cases from the general community
assembled in the US Commotio Cordis Registry occurring up to September 1, 2001.
MAIN OUTCOME MEASURE: Commotio cordis event.
RESULTS: Of 128 confirmed cases, 122 (95%) were in males and the mean (SD) age
was 13.6 (8.2) years (median, 14 years; range, 3 months to 45 years); only 28
(22%) cases were aged 18 years or older. Commotio cordis events occurred most
commonly during organized sporting events (79 [62%]), such as baseball, but 49
(38%) occurred as part of daily routine and recreational activities. Fatal blows
were inflicted with a wide range of velocities but often occurred inadvertently
and under circumstances not usually associated with risk for sudden death in
informal settings near the home or playground. Twenty-two (28%) participants
were wearing commercially available chest barriers, including 7 in whom the
projectile made direct contact with protective padding (baseball catchers and
lacrosse/hockey goalies), and 2 in whom the projectile was a baseball
specifically designed to reduce risk. Only 21 (16%) individuals survived their
event, with particularly prompt cardiopulmonary resuscitation/defibrillation
(most commonly reversing ventricular fibrillation) the only identifiable factor
associated with a favorable outcome.
CONCLUSIONS: The expanded spectrum of commotio cordis illustrates the potential
dangers implicit in striking the chest, regardless of the intent or force of the
blow. These findings also suggest that the safety of young athletes will be
enhanced by developing more effective preventive strategies (such as chest wall
barriers) to achieve protection from ventricular fibrillation following
precordial blows. Commotio cordis is a recognised cause of sudden death in which an apparently
minor blow to the chest causes ventricular fibrillation and cardiac arrest. It
is best known for causing death during games of youth baseball in the United
States, but individual cases have been recorded as a result of a wide range of
activities, principally sporting. The underlying biochemical and
mechano-electric causes have been well documented. However, there are few
reported cases where commotio cordis is implicated as the cause of death in
homicide cases. We present three cases from the north-east of England where an
assault caused death by this mechanism. Commotio cordis (CC), sudden death as a result of a blunt, often
innocent-appearing chest wall blow, is being reported with increasing frequency.
The clinical spectrum is diverse; however, a substantial number of cases occur
in youth athletics. In events that occur during sport, victims are struck by
projectiles regarded as standard implements of the game. Sudden death is
instantaneous and victims are most often found in ventricular fibrillation (VF).
Overall survival is poor; however, successful resuscitation can be achieved with
early defibrillation. Autopsy is notable for the absence of any significant
cardiac or thoracic injury. Development of an experimental model has allowed for
substantial insights into the underlying mechanisms of sudden death. In
anesthetized juvenile swine, induction of VF is instantaneous following chest
wall blows occurring during a vulnerable window before the T wave peak. Crucial
variables including the velocity of impact, impact location, and hardness of the
impact object have been identified. Rapid left ventricular (LV) pressure rise
following chest impact likely results in activation of ion channels via
mechano-electric coupling. The generation of inward current via
mechano-sensitive ion channels likely results in augmentation of repolarization
and nonuniform myocardial activation, and is the cause of premature ventricular
depolarizations that are triggers of VF in CC. While softer-than-standard safety
baseballs reduce the risk of CC, commercially available chest protectors are
ineffective in preventing CC. The development of more effective chest protectors
and more widespread use of automated external defibrillators at youth sporting
events are needed. Sudden arrhythmic death as a result of a blunt chest wall blow has been termed
Commotio Cordis (CC). CC is being reported with increasing frequency with more
than 180 cases now described in the United States Commotio Cordis Registry. The
clinical spectrum is diverse; however young athletes tend to be most at risk,
with victims commonly being struck by projectiles regarded as standard
implements of the sport. Sudden death is instantaneous and victims are most
often found in ventricular fibrillation (VF). Chest blows are not of sufficient
magnitude to cause any significant damage to overlying thoracic structures and
autopsy is notable for the absence of any structural cardiac injury. Development
of an experimental model has allowed for substantial insights into the
underlying mechanisms of sudden death. In anesthetized juvenile swine, induction
of VF is instantaneous following chest impacts that occur during a vulnerable
window before the T wave peak. Other critical variables, including the impact
velocity and location, and the hardness of the impact object have also been
identified. Rapid left ventricular pressure rise following chest impact likely
results in activation of ion channels via mechano-electric coupling. The
generation of inward current through mechano-sensitive ion channels results in
augmentation of repolarization and non-uniform myocardial activation, and is the
cause of premature ventricular depolarizations that are triggers of VF in CC.
Currently available chest protectors commonly used in sport are not adequately
designed to prevent CC. The development of more effective chest protectors and
the widespread availability of automated external defibrillators at youth
sporting events could improve the safety of young athletes. INTRODUCTION: Commotio cordis, sudden cardiac death secondary to blunt
nonpenetrating chest blows in sports, is reported with increasing frequency. In
a swine model, ventricular fibrillation (VF) is induced by a baseball blow to
the chest, and the initiation of VF is related to the peak left ventricular (LV)
pressure produced by the blow. LV pressure changes likely result in cell
membrane stretch and mechanical activation of ion channels. Disruption of cell
cytoskeleton that anchors the cell membrane prior to precordial blows offers the
opportunity to explore whether cell membrane deformation is critical to commotio
cordis.
METHODS AND RESULTS: Twelve juvenile swine (mean 12.7 +/- 1.6 kg) were
randomized to intravenous normal saline (control, n = 6) or 10 mg of intravenous
colchicine (n = 6), which is known to depolymerize microtubules. Animals were
given up to six blows timed to the vulnerable portion of the cardiac cycle with
a 30 mph baseball on the chest directly over the cardiac silhouette. VF was
initiated by 14 of the 29 (48%) impacts in the colchicine-treated animals
compared with only 3 of 28 (11%) in the controls (P = 0.002). The peak generated
LV pressure did not differ between colchicine animals (405 +/- 61 mmHg) and
controls (387 +/- 115) (P = 0.47). However, animals administered colchicine were
more likely to have VF generated by the chest blow at all pressures.
CONCLUSION: The initiation of VF by chest blows is significantly increased by
selective disruption of the cytoskeleton, suggesting that mechanical deformation
of the cell membrane is fundamental to the activation of ion channels and
underlies the mechanism of VF in commotio cordis. Commotio cordis is a term used for cases of sudden cardiac death due to
nonpenetrating chest trauma without evidence of underlying myocardial disease or
injury. Contusio cordis has been reserved for cases of chest trauma where there
is cardiac bruising. Three deaths due to blunt cardiac and chest trauma after
vehicle accidents are presented where the only significant injuries were
contusions of the heart and fractures of the sternum and ribs. One case had
moderate coronary artery atherosclerosis and another had a blood alcohol level
of 0.218%. Given that individuals with cardiac bruising, chest trauma, coronary
atherosclerosis, and alcohol intoxication may still die of the same mechanisms
as in classic commotio cordis, and that these entities represent a spectrum of
findings after chest impact, it may be more useful to separate cases into
related subcategories: (A) those with no evidence of injury or underlying
cardiovascular disease, (B) those with chest wall fractures, chest wall
contusions and/or cardiac contusions, and (C) those with underlying
cardiovascular disease or the presence of substances such as alcohol or drugs
that may reduce the threshold for cardiac arrhythmias. As there may be cases
with a number of these factors, a fourth category (D) includes those with a
combination of injuries and predisposing factors (ie, categories B and C).
Including cases such as these under the diagnostic umbrella of commotio cordis
may demonstrate that a wider range of individuals are at risk for death from
blunt cardiac trauma than sports-playing adolescents. Commotio cordis is arrhythmia or sudden death from low-impact, blunt trauma to
the chest without apparent heart injury. Ventricular fibrillation is the most
common associated arrhythmia, and heart block, bundle branch block, and
ST-segment elevation are also seen. Commotio cordis occurs most commonly in
baseball but has also been reported in hockey, softball, and several other
sports. Approximately two to four cases are reported each year, but the true
incidence is uncertain. Survival is low, even when resuscitation is performed.
Preventive measures include education of participants and coaches, chest
protection, and softer baseballs. Other considerations include having external
automatic defibrillators and trained personnel at youth sporting events. BACKGROUND: Sudden death due to low-energy blunt trauma to the precordium
(commotio cordis) has been described with a variety of sporting objects.
However, the risk of ventricular fibrillation (VF) relative to the shape of the
impact object is not known.
OBJECTIVE: The objective of the current experiment is to test whether the impact
object shape is a clinical variable that affects the risk for commotio cordis.
METHODS: In a juvenile swine model, impacts were given in random order with two
different spherical shapes (72 mm diameter, equivalent to a baseball; 42 mm
diameter, equivalent to a golf ball) and a flat round object 72 mm in diameter.
Objects were equal in weight (150 g), thrown at 30 mph, and gated to the
vulnerable portion of the cardiac cycle.
RESULTS: Sixteen swine received 144 impacts. The flat object did not cause VF (P
= .01 compared with the two spherical objects), nonsustained VF, ST elevation,
or bundle branch block. The smaller diameter sphere caused VF in nine of 48
impacts (19%), and the larger diameter sphere caused VF in five of 48 impacts
(10%; P = .25). The smaller diameter sphere was associated with a greater
increase in left ventricular pressure (P <.0001 and P = .001 compared with
larger sphere only) and a higher likelihood of ST segment elevations (P <.001
and P = .08 compared with larger sphere only) and bundle branch block (Fisher's
exact P = .008, and Fisher's exact P = .18 compared with larger sphere only).
CONCLUSION: The shape of the projectile markedly influences the risk of VF from
chest wall impact. This effect is likely mediated via a greater increase in left
ventricular pressure with smaller diameter objects. Spreading the impact force
over a larger area may decrease the risk of sudden death and has implications
for the design of protective athletic equipment. Blunt, non-penetrating trauma of the chest (commotio cordis) can cause sudden
death in individuals without known cardiac disease. Sudden death in commotio
cordis is due to ventricular fibrillation. The timing of the blow must be during
the electric vulnerable period of the ECG cyclus, 10-40 milliseconds before the
peak of the T-wave. The risk of arrhythmia increases progressively, until a
velocity of 64 km/h, and at higher velocities the risk of ventricular
fibrillation begins to diminish. The location of the impact must be directly
over the cardiac silhouette. Commotio cordis is a rare type of blunt cardiac injury in which low impact chest
trauma causes sudden cardiac arrest, usually occurs from being struck by a
projectile during sports. The most common arrhythmia during commotio cordis is
ventricular fibrillation, although complete heart block and an idioventricular
rhythm have also been reported. We describe a case of a young patient who
presented with a persistent third-degree atrioventricular block and a left
bundle branch block, following blunt chest trauma, as a result of blow by soccer
ball and subsequently needed a permanent pacemaker. Commotio Cordis (CC) is the second leading cause of mortality in youth sports.
Impacts occurring directly over the left ventricle (LV) during a vulnerable
period of the cardiac cycle can cause ventricular fibrillation (VF), which
results in CC. In order to better understand the pathophysiology of CC, and
develop a mechanical model for CC, appropriate injury criteria need to be
developed. This effort consisted of impacts to seventeen juvenile porcine
specimens (mass 21-45 kg). Impacts were delivered over the cardiac silhouette
during the venerable period of the cardiac cycle. Four impact speeds were used:
13.4, 17.9, 22.4, and 26.8 m/s. The impactor was a lacrosse ball on an aluminum
shaft instrumented with an accelerometer (mass 188 g-215 g). The impacts were
recorded using high-speed video. LV pressure was measured with a catheter.
Univariate binary logistic regression analyses were performed to evaluate the
predictive ability of ten injury criteria. A total of 187 impacts were used in
the analysis. The criteria were evaluated on their predictive ability based on
Somers' D (D) and Goodman-Kruskal gamma (γ). Injury risk functions were created
for all criteria using a 2-parameter Weibull distribution using survival
analysis. The best criteria for predicting CC were impact force (D=0.52, and
γ=0.52) force*compression (D=0.49, and γ=0.49), and impact power (D=0.49, and
γ=0.49). All of these criteria proved significant in predicting the probability
of CC from projectile impacts in youth sports (p<0.01). Force proved to be the
most predictive of the ten criteria evaluated. BACKGROUND: Commotio cordis events due to precordial blows triggering
ventricular fibrillation are a cause of sudden death (SD) during sports and also
daily activities. Despite the absence of structural cardiac abnormalities, these
events have been considered predomitly fatal with low survival rates.
OBJECTIVE: To determine whether expected mortality rates for commotio cordis
have changed over time, associated with greater public visibility.
METHODS: US Commotio Cordis Registry was accessed to tabulate frequency of
reported SD or resuscitated cardiac arrest over 4 decades.
RESULTS: At their commotio cordis event, 216 study patients were 0.2-51 years
old (mean age 15±9 years); 95% were males. Death occurred in 156 individuals
(72%), while the other 60 (28%) survived. Proportion of survivors increased
steadily with concomitant decrease in fatal events. For the initial years
(1970-1993), 6 of 59 cases survived (10%), while during 1994-2012, 54 of 157
(34%) survived (P = .001). The most recent 6 years, survival from commotio
cordis was 31 of 53 (58%), with survivor and nonsurvivor curves ultimately
crossing. Higher survival rates were associated with more prompt resuscitation
(40%<3 minutes vs 5%>3 minutes; P<.001) and participation in competitive sports
(39%; P<.001), but with lower rates in African Americans (1 of 24; 4%) than in
whites (54 of 166; 33%; P = .004). Independent predictors of mortality were
black race (P = .045) and participation in noncompetitive sports (P = .002),
with an on-site automated external defibrillator use protective against SD (P =
.01).
CONCLUSIONS: Survival from commotio cordis has increased, likely owing to more
rapid response times and access to defibrillation, as well as greater public
awareness of this condition. |
What is the cause of Phthiriasis Palpebrarum? | Phthiriasis palpebrarum is a rare eyelid infestation caused by phthirus pubis. | Phthiriasis palpebrarum is an unusual cause of blepharoconjunctivitis and may
easily be overlooked because of the failure of physicians to recognize Phthirus
pubis. We report a case of a 30-year-old woman with persistent itching in the
left eyelid which was unsuccessfully treated under the diagnosis of allergic
blepharoconjunctivitis. Careful ophthalmic examination revealed seven bugs with
multiple red pinpoint excretions and numerous small translucent oval eggs (nits)
coating the eyelashes. The patient was successfully treated with mechanical
removal of all the lice and nits from the eyelashes. The specimen proved
histopathologically to be the Phthirus pubis infestation. The Phthirus pubis
infestation is usually associated with poor hygiene in overcrowded or
undeveloped country. However, it may become a notable problem because of
frequent traveling and commercial activities across the different countries. Phthiriasis palpebrarum, caused by Phthirus pubis, is an uncommon cause of
blepharoconjunctivitis; therefore, this condition is easily misdiagnosed. When
diagnosed, genital involvement must be ruled out. Association with other
venereal diseases is common. Affected children must be searched for sexual
abuse. The number of diagnosed patients in our department has increased in
recent years. We review the epidemiological, clinical and diagnostic features of
phthriasis palpebrarum as well as the different treatment options to eradicate
the parasite and to prevent infestations. Phthiriasis palpebrarum (lice infestation of palpabrae) is a rarely reported
disorder and may present as blepharoconjuctivitis. It is usually seen in lower
socioeconomic groups and spreads through either sexual contacts or directly
through linen or clothing. We report a family with phthiriasis palpebrarum in
which the primary source of infestation was the paternal uncle of two children.
Mechanical removal proved to be quite effective in treating the disease and
preventing its recurrence. Phthiriasis palpebrarum (PP) is a rare eyelid infestation caused by phthirus
pubis. We report a case of PP mimicking lid eczema and blepharitis. A
68-year-old woman had moderate itching in both eyes. Her initial diagnosis was
considered to be lid eczema or blepharitis because of findings similar to
exfoliative lesions and color changes in eyelids and to excretions over
eyelashes. Careful observation revealed many lice and translucent nits,
protuberances and hyperpigmentary changes, and the buried lice in both eyelids.
No hyperemia or secretion was observed on the lids and in the conjunctiva in
both eyes. The patient was treated with pilocarpine hydrochloride 4% drops. At
the end of the first week, no louse or nit was present. Although it was known
that PP is a rare cause of blepharoconjunctivitis, it might observe as an
isolated infestation of the eyelids and this condition can easily be
misdiagnosed as lid eczema and blepharitis. We describe a case of chronic conjunctivitis related to phthiriasis palpebrarum.
A 36 year-old female presented with gradual pruritus and painless ocular
hyperaemia over the previous 3 months. On examination, nasal pterygium,
conjunctival hyperaemia, oedema, and mild hypertrophy of the palpebral margin
were observed. A slit-lamp examination revealed numerous creamy oval structures
approximately 1 mm in diameter localised in the middle area of the lashes, and
bloody crusts and a semi-transparent deposit were present in the superior
palpebral margin. Based on the observation of numerous nits at the base of the
eyelashes and the ectoparasite in the palpebral margin, a diagnosis of
phthiriasis palpebrarum was made. The patient was referred to an infectologist
for evaluation of other sexually transmitted diseases and examination of other
body areas. She was successfully treated with oral ivermectin, shampoo for
ciliary hygiene and artificial tears. Other recommendations to avoid
re-infestation were made, such as changing, washing and sterilising clothes,
towels and sheets daily. This report emphasizes the importance of the correct
diagnosis and management of this disease, considered as sexually transmitted. The similarities of the larval and nymph stages of the tick and louse (Pthirus
pubis) may lead to misdiagnosis in rare cases of infestation of the eyelashes.
The most frequent manifestations of tick in the eye are conjunctivitis, uveitis,
keratitis, and vasculitis. Tick inoculation of the skin can locally lead to
formation of granuloma and abscess. More concerning is the potential systemic
sequelae that can result from transmission of zoonoses such as Lyme disease. P.
pubis can cause pruritic eyelid margins or unusual blepharoconjunctivitis. We
present a case of phthiriasis palpebrarum in a 4-year-old boy. INTRODUCTION: Phthiriasis palpebrarum is an ectoparasitosis in which Phthirus
pubis infest the eyelashes. It is rare and it can easily be misdiagnosed as
blepharitis. The purpose of this study is to describe seven cases of phthiriasis
palpebrarum so as to discuss its mode of infestation, diagnosis and treatment.
PATIENTS AND METHODS: This is a study of all cases of phthiriasis palpebrarum
reported in our laboratory. For each patient, an ophthalmic examination and
parasitological examination of the eyelashes were performed.
RESULTS: There were five men and two women. Their ages ranged from 4 to 50years
with an average of 21.57years. There were four children and three adults. The
main symptom was itching of the eyelids. Clinical signs included reddish-brown
crusts at the base of the eyelashes in all the cases and visible lice and nits
in three cases. Biomicroscopic examination showed lice and nits anchored to the
eyelashes in three cases. In the other two cases, the initial diagnosis was felt
to be blepharitis. In all cases, the diagnosis of phthiriasis palpebrarum was
confirmed by parasitological examination of eyelashes, which revealed the
presence of adult and nit forms of Phthirus pubis. The number of adult lice
ranged from 1 to 30. In all cases, treatment was based on mechanical removal of
both the lice and nits. Outcomes were favorable without recurrence.
CONCLUSION: In conclusion, phthiriasis palpebrarum can be easily diagnosed by
close examination of the eyelashes and eyelid margins at the slit lamp and can
be managed mechanically. Parasitological examination of the eyelashes can
confirm the diagnosis. BACKGROUND: Pediculosis capitis is a common parasitic infestation, whereas
phthiriasis palpebrarum is an uncommon infection due to Phthirus pubis (pubic
lice) inoculating the eyelashes and surrounding tissues of the eye. Emergency
physicians should recognize the causes of this uncommon disease. Cases of
phthiriasis palpebrarum should trigger the clinician to consider the potential
for child abuse when suspected or when social history dictates the risk for
abuse.
OBJECTIVE: A case of a pediculosis capitis and phthiriasis palpebrarum
coinfection in a 4-year-old girl is presented, which was suspicious for child
abuse given the patient's social history. Diagnosis, treatment, and need for
vigilance when encountering cases of phthiriasis palpebrarum, especially in
young children, are discussed herein.
CASE REPORT: A 4-year-old girl presented with swelling and redness around her
eyes. The girl had recurrent head lice infestations, however, on the day of
presentation the mother noted lice appeared on the girl's eyelashes and eyelids.
Head lice typically do not infect the eyes, and given the different morphology
of the lice on the patient's head and eyes, a diagnosis of phthiriasis
palpebrarum was made. Because phthiriasis pubis infection of the eyelids may
represent sexual abuse, especially in children, child protective services was
notified to ensure patient safety.
CONCLUSIONS: Pediatric phthiriasis palpebrarum can represent child abuse, and
the origins of this infection need to be carefully discerned. A thorough history
can provide information to assess whether further action is needed and, if in
doubt, social services should be contacted to ensure child safety. Phthiriasis palpebrarum is an infestation of the eyelashes caused by the louse
Pthirus pubis (Linnaeus, 1758). We report a case of phthiriasis palpebrarum in a
6-year-old girl, which was initially misdiagnosed as allergic
blepharoconjunctivitis. Parasites and their nits were found adhering to the
eyelashes and eyelids of her right eye as well as scalp hairs. No abnormality
was found in the left eye. The histopathology exam revealed the presence of
adults and eggs of Pthirus pubis. We mechanically removed all the eyelashes of
the right eye at their base, with lice and nits. The scalp was shaved and washed
with phenothrin shampoo. No recurrence was found during 3 months of follow-up.
Removal of the eyelashes, cutting of scalp hairs, and phenothrin shampoo may be
effective in treating phthiriasis palpebrarum. In cases of
blepharoconjunctivitis, eyelids and eyelashes should be carefully examined by
slit lamp to avoid misdiagnosis. |
Does the histidine-rich Ca-binding protein (HRC) interact with triadin? | Yes. HRC may play a key role in the regulation of SR Ca cycling through its direct interactions with SERCA2 and triadin, mediating a fine cross talk between SR Ca uptake and release in the heart. A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. | The present study documents the binding interaction of skeletal muscle
sarcoplasmic reticulum (SR) transmembrane protein triadin with peripheral
histidine-rich, Ca(2+)-binding protein (HCP). In addition to providing further
evidence that HCP coenriches with RyR1, FKBP-12, triadin and calsequestrin (CS)
in sucrose-density-purified TC vesicles, using specific polyclonal antibody, we
show it to be expressed as a single protein species, both in fast-twitch and
slow-twitch fibers, and to identically localize to the I-band. Colocalization of
HCP and triadin at junctional triads is supported by the overlapping staining
pattern using monoclonal antibodies to triadin. We show a specific binding
interaction between digoxigenin-HCP and triadin, using ligand blot techniques.
The importance of this finding is strengthened by the similarities in binding
affinity and in Ca2+ dependence, (0.1-1 mM Ca2+) of the interaction of
digoxigenin-HCP with immobilized TC vesicles. Suggesting that triadin dually
interacts with HCP and with CS, at distinct sites, we have found that triadin-CS
interaction in overlays does not require the presence of Ca2+. Consistent with
the binding of CS to triadin luminal domain (Guo and Campbell, 1995), we show
that binding sites for digoxigenin-CS, although not binding sites for
digoxigenin-HCP, can be recovered in the 92 kDa triadin fragment, after
chymotryptic cleavage of the NH2-terminal end of the folded molecule in intact
TC vesicles. These differential effects form the basis for the hypothesis that
HCP anchors to the junctional membrane domain of the SR, through binding to
triadin short cytoplasmic domain at the NH2 terminus. Although the function of
this interaction, as such, is not well understood, it seems of potential
biological interest within the more general context of the structural-functional
role of triadin at the triadic junction in skeletal muscle. A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the
main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of
skeletal muscle, seems well supported. Opinions are still divided, however,
concerning the triadin domain involved, either the cytoplasmic or the lumenal
domain, and the exact role played by Ca(2+), in the protein-to-protein
interaction. Further support for colocalization of HRC with triadin cytoplasmic
domain is provided here by experiments of mild tryptic digestion of tightly
sealed TC vesicles. Accordingly, we show that HRC is preferentially
phosphorylated by endogenous CaM K II, anchored to SR membrane on the
cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate
that HRC can be isolated as a complex with triadin, following equilibrium
sucrose-density centrifugation in the presence of mM Ca(2+). Here, we
characterized the COOH-terminal portion of rabbit HRC, expressed and purified as
a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and
to the interaction with triadin on blots, as a function of the concentration of
Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as
the Ca(2+)-binding site responsible, both for the acceleration in mobility of
HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for
the enhancement by high Ca(2+) of the interaction between HRC and triadin
cytoplasmic segment. (c)2001 Elsevier Science. The histidine-rich Ca(2+) binding protein (HRC) is a high capacity Ca(2+)
binding protein in the sarcoplasmic reticulum (SR). Because HRC appears to
interact directly with triadin, HRC may play a role in the regulation of Ca(2+)
release during excitation-contraction coupling. In this study, we examined the
physiological effects of HRC overexpression in rat neonatal cardiomyocytes. Both
caffeine-induced and depolarization-induced Ca(2+) release from the SR were
increased significantly in the HRC overexpressing cardiomyocytes. Consistently,
the Ca(2+) content, normally depleted from the SR in the presence of
cyclopiazonic acid (CPA), remained elevated in these cells. In contrast, the
density and the ryanodine-binding kinetics of the ryanodine receptor
(RyR)/Ca(2+) release channel were slightly reduced or not significantly altered
in the HRC overexpressing cardiomyocytes. We suggest that HRC is involved in the
regulation of releasable Ca(2+) content into the SR. Contractile dysfunction and ventricular arrhythmias associated with heart
failure have been attributed to aberrant sarcoplasmic reticulum (SR) Ca(2+)
cycling. The study of junctin (JCN) and histidine-rich Ca(2+) binding protein
(HRC) becomes of particular importance since these proteins have been shown to
be critical regulators of Ca(2+) cycling. Specifically, JCN is a SR membrane
protein, which is part of the SR Ca(2+) release quaternary structure that also
includes the ryanodine receptor, triadin and calsequestrin. Functionally, JCN
serves as a bridge between calsequestrin and the Ca(2+) release channel,
ryanodine receptor. HRC is a SR luminal Ca(2+) binding protein known to
associate with both triadin and the sarcoplasmic reticulum Ca(2+)-ATPase, and
may thus mediate the crosstalk between SR Ca(2+) uptake and release. Indeed,
evidence from genetic models of JCN and HRC indicate that they are important in
cardiophysiology as alterations in these proteins affect SR Ca(2+) handling and
cardiac function. In addition, downregulation of JCN and HRC may contribute to
Ca(2+) cycling perturbations manifest in the failing heart, where their protein
levels are significantly reduced. This review examines the roles of JCN and HRC
in SR Ca(2+) cycling and their potential significance in heart failure. Histidine-rich calcium binding protein (HRC) is a high capacity, low affinity
Ca(2+) binding protein, specifically expressed in striated muscles of mammals.
In rabbit skeletal and cardiac muscles, HRC binds to sarcoplasmic reticulum (SR)
membranes via triadin, a junctional SR protein. Recently, a potential role in
heart failure and arrhythmogenesis has been assigned to HRC due to its activity
as regulator of SR Ca(2+) uptake and Ca(2+) release. HRC might play a
particularly relevant role in the equine heart, given its slower resting heart
rate (20-35 beats/min) and longer action potential duration (APD) (0.6-1.0 s)
than are found in other mammals. The results from this study showed for the
first time direct evidence that HRC protein in equine cardiac muscle was
expressed in association with the SR membranes and that HRC transcriptional
activity was three times higher in the ventricles compared to the atria. The
predomice of HRC mRNA up-regulation in ventricular myocardium was specific to
the horse heart, since a more even distribution between atria and ventricles was
found in animals of similar body size or species, such as cattle or domestic
donkeys. BACKGROUND: Histidine-rich calcium binding protein (HRC) is located in the lumen
of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA
affecting Ca(2+) cycling in the SR. Chronic overexpression of HRC that may
disrupt intracellular Ca(2+) homeostasis is implicated in pathogenesis of
cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under
basal condition, but exhibited a significantly increased susceptibility to
isoproterenol-induced cardiac hypertrophy. In the present study, we
characterized the functions of HRC related to Ca(2+) cycling and pathogenesis of
cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated
virus (AAV)-mediated HRC knock-down (KD) systems, respectively.
METHODOLOGY/PRINCIPAL FINDINGS: AAV-mediated HRC-KD system was used with or
without C57BL/6 mouse model of transverse aortic constriction-induced failing
heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in
failing heart (FH). Initially we expected that HRC-KD could elicit cardiac
functional recovery in failing heart (FH), since predesigned siRNA-mediated
HRC-KD enhanced Ca(2+) cycling and increased activities of RyR2 and SERCA2
without change in SR Ca(2+) load in neonatal rat ventricular cells (NRVCs) and
HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with
decreased fractional shortening and increased cardiac fibrosis compared with
control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and
phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly
increased level of cleaved caspase-3, a cardiac cell death marker was also
found, consistent with the result of TUNEL assay.
CONCLUSIONS/SIGNIFICANCE: Increased Ca(2+) leak and cytosolic Ca(2+)
concentration due to a partial KD of HRC could enhance activity of CaMKII and
phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in
TAC-FH. Our results present evidence that down-regulation of HRC could
deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca(2+)
cycling. Depressed sarcoplasmic reticulum (SR) calcium cycling, reflecting impaired SR
Ca-transport and Ca-release, is a key and universal characteristic of human and
experimental heart failure. These SR processes are regulated by multimeric
protein complexes, including protein kinases and phosphatases as well as their
anchoring and regulatory subunits that fine-tune Ca-handling in specific SR
sub-compartments. SR Ca-transport is mediated by the SR Ca-ATPase (SERCA2a) and
its regulatory phosphoprotein, phospholamban (PLN). Dephosphorylated PLN is an
inhibitor of SERCA2a and phosphorylation by protein kinase A (PKA) or
calcium-calmodulin-dependent protein kinases (CAMKII) relieves these inhibitory
effects. Recent studies identified additional regulatory proteins, associated
with PLN, that control SR Ca-transport. These include the inhibitor-1 (I-1) of
protein phosphatase 1 (PP1), the small heat shock protein 20 (Hsp20) and the
HS-1 associated protein X-1 (HAX1). In addition, the intra-luminal
histidine-rich calcium binding protein (HRC) has been shown to interact with
both SERCA2a and triadin. Notably, there is physical and direct interaction
between these protein players, mediating a fine-cross talk between SR Ca-uptake,
storage and release. Importantly, regulation of SR Ca-cycling by the PLN/SERCA
interactome does not only impact cardiomyocyte contractility, but also survival
and remodeling. Indeed, naturally occurring variants in these Ca-cycling genes
modulate their activity and interactions with other protein partners, resulting
in depressed contractility and accelerated remodeling. These genetic variants
may serve as potential prognostic or diagnostic markers in cardiac
pathophysiology. |
How are CpG island shores defined? | CpG island "shores" are defined as genomic regions up to 2kb distant to known CpG islands. Differential DNA methylation correlates with gene expression more strongly at CpG island shores than CpG islands. | For the past 25 years, it has been known that alterations in DNA methylation
(DNAm) occur in cancer, including hypomethylation of oncogenes and
hypermethylation of tumor suppressor genes. However, most studies of cancer
methylation have assumed that functionally important DNAm will occur in
promoters, and that most DNAm changes in cancer occur in CpG islands. Here we
show that most methylation alterations in colon cancer occur not in promoters,
and also not in CpG islands, but in sequences up to 2 kb distant, which we term
'CpG island shores'. CpG island shore methylation was strongly related to gene
expression, and it was highly conserved in mouse, discriminating tissue types
regardless of species of origin. There was a notable overlap (45-65%) of the
locations of colon cancer-related methylation changes with those that
distinguished normal tissues, with hypermethylation enriched closer to the
associated CpG islands, and hypomethylation enriched further from the associated
CpG island and resembling that of noncolon normal tissues. Thus, methylation
changes in cancer are at sites that vary normally in tissue differentiation,
consistent with the epigenetic progenitor model of cancer, which proposes that
epigenetic alterations affecting tissue-specific differentiation are the
predomit mechanism by which epigenetic changes cause cancer. Induced pluripotent stem (iPS) cells are derived by epigenetic reprogramming,
but their DNA methylation patterns have not yet been analyzed on a genome-wide
scale. Here, we find substantial hypermethylation and hypomethylation of
cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as
compared to their parental fibroblasts. The differentially methylated regions
(DMRs) in the reprogrammed cells (denoted R-DMRs) were significantly enriched in
tissue-specific (T-DMRs; 2.6-fold, P < 10(-4)) and cancer-specific DMRs (C-DMRs;
3.6-fold, P < 10(-4)). Notably, even though the iPS cells are derived from
fibroblasts, their R-DMRs can distinguish between normal brain, liver and spleen
cells and between colon cancer and normal colon cells. Thus, many DMRs are
broadly involved in tissue differentiation, epigenetic reprogramming and cancer.
We observed colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs
and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with
hypomethylated C-DMRs and the absence of bivalent marks, suggesting two
mechanisms for epigenetic reprogramming in iPS cells and cancer. Recent genome-wide descriptions of CpG methylation patterns in mammalian cells
identified many differentially methylated regions (DMRs) located at CpG island
"shores." Feinberg, Daley, and colleagues now report in Nature Genetics that
reconfiguration of these DMRs occurs during induced reprogramming (Doi et al.,
2009). Epigenetic modifications must underlie lineage-specific differentiation as
terminally differentiated cells express tissue-specific genes, but their DNA
sequence is unchanged. Haematopoiesis provides a well-defined model to study
epigenetic modifications during cell-fate decisions, as multipotent progenitors
(MPPs) differentiate into progressively restricted myeloid or lymphoid
progenitors. Although DNA methylation is critical for myeloid versus lymphoid
differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs,
a comprehensive DNA methylation map of haematopoietic progenitors, or of any
multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million
CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs),
common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs),
and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity
accompanied both lymphoid and myeloid restriction. Myeloid commitment involved
less global DNA methylation than lymphoid commitment, supported functionally by
myeloid skewing of progenitors following treatment with a DNA methyltransferase
inhibitor. Differential DNA methylation correlated with gene expression more
strongly at CpG island shores than CpG islands. Many examples of genes and
pathways not previously known to be involved in choice between lymphoid/myeloid
differentiation have been identified, such as Arl4c and Jdp2. Several
transcription factors, including Meis1, were methylated and silenced during
differentiation, indicating a role in maintaining an undifferentiated state.
Additionally, epigenetic modification of modifiers of the epigenome seems to be
important in haematopoietic differentiation. Our results directly demonstrate
that modulation of DNA methylation occurs during lineage-specific
differentiation and defines a comprehensive map of the methylation and
transcriptional changes that accompany myeloid versus lymphoid fate decisions. Methylated DNA immunoprecipitation followed by high-throughput sequencing
(MeDIP-seq) has the potential to identify changes in DNA methylation important
in cancer development. In order to understand the role of epigenetic modulation
in the development of acute myeloid leukemia (AML) we have applied MeDIP-seq to
the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed
leukemia-associated differentially methylated regions that included gene
promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and
DPP6) with significantly methylated promoters were of interest and further
analysis of their expression showed them to be repressed in AML. We also
demonstrated considerable cytogenetic subtype specificity in the methylomes
affecting different genomic features. Significantly distinct patterns of
hypomethylation of certain interspersed repeat elements were associated with
cytogenetic subtypes. The methylation patterns of members of the SINE family
tightly clustered all leukemic patients with an enrichment of Alu repeats with a
high CpG density (P<0.0001). We were able to demonstrate significant inverse
correlation between intragenic interspersed repeat sequence methylation and gene
expression with SINEs showing the strongest inverse correlation (R(2) = 0.7). We
conclude that the alterations in DNA methylation that accompany the development
of AML affect not only the promoters, but also the non-promoter genomic
features, with significant demethylation of certain interspersed repeat DNA
elements being associated with AML cytogenetic subtypes. MeDIP-seq data were
validated using bisulfite pyrosequencing and the Infinium array. BACKGROUND: The predomit model for regulation of gene expression through DNA
methylation is an inverse association in which increased methylation results in
decreased gene expression levels. However, recent studies suggest that the
relationship between genetic variation, DNA methylation and expression is more
complex.
RESULTS: Systems genetic approaches for examining relationships between gene
expression and methylation array data were used to find both negative and
positive associations between these levels. A weighted correlation network
analysis revealed that i) both transcriptome and methylome are organized in
modules, ii) co-expression modules are generally not preserved in the
methylation data and vice-versa, and iii) highly significant correlations exist
between co-expression and co-methylation modules, suggesting the existence of
factors that affect expression and methylation of different modules (i.e., trans
effects at the level of modules). We observed that methylation probes associated
with expression in cis were more likely to be located outside CpG islands,
whereas specificity for CpG island shores was present when methylation,
associated with expression, was under local genetic control. A structural
equation model based analysis found strong support in particular for a
traditional causal model in which gene expression is regulated by genetic
variation via DNA methylation instead of gene expression affecting DNA
methylation levels.
CONCLUSIONS: Our results provide new insights into the complex mechanisms
between genetic markers, epigenetic mechanisms and gene expression. We find
strong support for the classical model of genetic variants regulating
methylation, which in turn regulates gene expression. Moreover we show that,
although the methylation and expression modules differ, they are highly
correlated. Aberrant DNA methylation of CpG islands, CpG island shores and first exons is
known to play a key role in the altered gene expression patterns in all human
cancers. To date, a systematic study on the effect of DNA methylation on gene
expression using high resolution data has not been reported. In this study, we
conducted an integrated analysis of MethylCap-sequencing data and Affymetrix
gene expression microarray data for 30 breast cancer cell lines representing
different breast tumor phenotypes. As well-developed methods for the integrated
analysis do not currently exist, we created a series of four different analysis
methods. On the computational side, our goal is to develop methylome data
analysis protocols for the integrated analysis of DNA methylation and gene
expression data on the genome scale. On the cancer biology side, we present
comprehensive genome-wide methylome analysis results for differentially
methylated regions and their potential effect on gene expression in 30 breast
cancer cell lines representing three molecular phenotypes, luminal, basal A and
basal B. Our integrated analysis demonstrates that methylation status of
different genomic regions may play a key role in establishing transcriptional
patterns in molecular subtypes of human breast cancer. We applied Illumina Human Methylation450K array to perform a genomic-scale
single-site resolution DNA methylation analysis in neuronal and nonneuronal
(primarily glial) nuclei separated from the orbitofrontal cortex of postmortem
human brain. The findings were validated using enhanced reduced representation
bisulfite sequencing. We identified thousands of sites differentially methylated
(DM) between neuronal and nonneuronal cells. The DM sites were depleted within
CpG-island-containing promoters but enriched in predicted enhancers.
Classification of the DM sites into those undermethylated in neurons (neuronal
type) and those undermethylated in nonneuronal cells (glial type), combined with
findings of others that methylation within control elements typically negatively
correlates with gene expression, yielded large sets of predicted neuron-specific
and non-neuron-specific genes. These sets of predicted genes were in excellent
agreement with the available direct measurements of gene expression in human and
mouse. We also found a distinct set of DNA methylation patterns that were unique
for neuronal cells. In particular, neuronal-type differential methylation was
overrepresented in CpG island shores, enriched within gene bodies but not in
intergenic regions, and preferentially harbored binding motifs for a distinct
set of transcription factors, including neuron-specific activity-dependent
factors. Finally, non-CpG methylation was substantially more prevalent in
neurons than in nonneuronal cells. |
Does magnesium sulfate improve outcomes of subarachnoid hemorrhage patients? | No. Although initial studies have provided with encouraging findings regarding administration of magnesium sulphate in aneurysmal subarachnoid haemorrhage patients, but subsequent larger studies have reported that intravenous magnesium sulphate does not improve clinical outcome after aneurysmal subarachnoid haemorrhage, therefore routine administration of magnesium cannot be recommended. | Magnesium sulfate therapy, standard in preventing seizures in preeclampsia, is
under active investigation as a neuroprotective agent. The authors studied the
effect of magnesium as a cerebral vasodilator by measuring the cerebral blood
flow velocity (CBFV) response to a 5g intravenous bolus of MgSO4 compared with a
saline placebo after subarachnoid hemorrhage (SAH). Transcranial Doppler
ultrasonography of the middle cerebral artery (MCA) was measured after each
infusion. Patients were studied up to three times after SAH at prescribed time
intervals. Fourteen patients (11 women, 3 men; mean age 58 years) underwent 29
studies. All patients underwent hypertensive, hypervolemic therapy. Four
patients developed cerebral vasospasm. Doubling serum magnesium levels did not
affect MCA CBFV but slightly lowered mean arterial blood pressure and systemic
vascular resistance. Intravenous magnesium bolus did not reduce elevated CBFV in
the subset of SAH patients with clinical vasospasm. The role of magnesium
sulfate as a cerebral vasodilator in patients with SAH requires further study. OBJECT: Vasospasm remains a significant source of neurological morbidity and
mortality following aneurysmal subarachnoid hemorrhage (SAH), despite advances
in current medical, surgical, and endovascular therapies. Magnesium sulfate
therapy has been demonstrated to be both safe and effective in preventing
neurological complications in obstetrical patients with eclampsia. Evidence
obtained using experimental models of brain injury, cerebral ischemia, and SAH
indicate that Mg may also have a role as a neuroprotective agent. The authors
hypothesize that MgSO4 therapy is safe, feasible, and has a beneficial effect on
vasospasm and, ultimately, on neurological outcome following aneurysmal SAH.
METHODS: A prospective randomized single-blind clinical trial of high-dose MgSO4
therapy following aneurysmal SAH (Hunt and Hess Grades II-IV) was performed in
40 patients, who were enrolled within 72 hours following SAH and given
intravenous MgSO4 or control solution for 10 days. Serum Mg++ levels were
maintained in the 4 to 5.5 mg/dl range throughout the treatment period. Clinical
management principles were the same between groups (including early use of
surgery or endovascular treatment, followed by aggressive vasospasm prophylaxis
and treatment). Daily transcranial Doppler (TCD) ultrasonographic recordings
were obtained, and clinical outcomes were measured using the Glasgow Outcome
Scale (GOS). The patients' GOS scores and the TCD recordings were analyzed using
the independent t-test. Forty patients were enrolled in the study: 20 (15 female
and five male patients) received treatment and 20 (11 female and nine male
patients) comprised a control group. The mean ages of the patients in these
groups were 46 and 51, respectively, and the mean clinical Hunt and Hess grades
were 2.6 +/- 0.68 in the MgSO4 treatment group and 2.3 +/- 0.73 in the control
group (mean +/- standard deviation [SD], p = 0.87). Fisher grades were similar
in both groups. Mean middle cerebral artery velocities were 93 +/- 27 cm/second
in MgSO4-treated patients and 102 +/- 34 cm/second in the control group (mean
+/- SD, p = 0.41). Symptomatic vasospasm, confirmed by angiography, occurred in
six of 20 patients receiving MgSO4 and in five of 16 patients receiving placebo.
Mean GOS scores were 3.8 +/- 1.6 and 3.6 +/- 1.5 (mean +/- SD, p = 0.74) in the
treatment and control groups, respectively. Significant adverse effects from
treatment with MgSO4 did not occur.
CONCLUSIONS: Administration of high-dose MgSO4 following aneurysmal SAH is safe,
and steady Mg++ levels in the range of 4 to 5.5 mg/dl are easily maintained.
This treatment does not interfere with neurological assessment, administration
of anesthesia during surgery, or other aspects of clinical care. We observed a
trend in which a higher percentage of patients obtained GOS scores of 4 or 5 in
the group treated with MgSO4, but the trend did not reach a statistically
significant level. A larger study is needed to evaluate this trend further. BACKGROUND: Magnesium is a neuroprotective agent which might prevent or reverse
delayed cerebral ischemia (DCI) after aneurysmal subarachnoid haemorrhage (SAH).
Although the dosage for short-term magnesium therapy is well established, there
is lack of knowledge on the dosage for extended use of magnesium. Our aim was to
find a dosage schedule of magnesium sulphate to maintain a serum magnesium level
of 1.0-2.0 mmol/L for 14 days to cover the period of DCI.
METHODS: We prospectively studied 14 patients admitted within 48 hours after
aneurysmal subarachnoid haemorrhage (SAH) to our hospital. Magnesium sulphate
was administrated intravenously for 14 days, using 3 different dosage schedules.
Group A (n=3) received a bolus injection of 16 mmol magnesium sulphate followed
by a continuous infusion of 16 mmol/daily; group B (n=6) a continuous infusion
of 30 mmol/daily; and group C (n=5) a continuous infusion of 64 mmol/daily.
Serum magnesium was measured at least every two days and all patients were under
continuous observation during magnesium treatment. Renal magnesium excretion was
measured only in group C.
FINDINGS: In treatment group A the mean serum magnesium level during treatment
was 1.03+/-0.14 (range 0.82-1.34) mmol/L, in group B 1.10+/-0.15 (range
0.87-1.43) mmol/L, and in group C 1.38+/-0.18 (range 1.11-1.98) mmol/L. The
renal magnesium excretion in group C was equal to the administrated doses within
48 hours after treatment had started. All patients in group A reported a
flushing sensation during the bolus injection; no other side effects were noted.
INTERPRETATION: With a continuous intravenous dosage of 64 mmol/L per day, serum
magnesium levels maintained within the range of 1.0-2.0 mmol/L for 14 days. OBJECTIVE: Based on preclinical investigations, magnesium sulfate (MgSO4) has
gained interest as a neuroprotective agent. However, the ability of peripherally
administered MgSO4 to penetrate the blood-brain barrier is limited in normal
brain. The current study measured the passage of intravenously administered Mg
into cerebrospinal fluid in patients with brain injury requiring ventricular
drainage.
DESIGN: A prospective evaluation of the cerebrospinal fluid total and ionized
magnesium concentration, [Mg], during sustained hypermagnesemia was performed.
SETTING: Neurosciences intensive care unit at a major teaching institution.
PATIENTS: Thirty patients with acute brain injury secondary to subarachnoid
hemorrhage, traumatic brain injury, primary intracerebral hemorrhage, subdural
hematoma, brain tumor, central nervous system infection, or ischemic stroke were
studied.
INTERVENTIONS: Patients underwent 24 hrs of induced hypermagnesemia during which
total and ionized cerebrospinal fluid [Mg] was measured. Serum [Mg] was adjusted
to 2.1-2.5 mmol/L. Cerebrospinal fluid [Mg] was measured at baseline, at 12 and
24 hrs after onset of infusion, and at 12 hrs following infusion termination.
MEASUREMENTS AND MAIN RESULTS: At baseline, total (1.25 +/- 0.14 mmol/L) and
ionized (0.80 +/- 0.10 mmol/L) cerebrospinal fluid [Mg] was greater than serum
total (0.92 +/- 0.18 mmol/L) and ionized (0.63 +/- 0.07 mmol/L) [Mg] (p < .05).
Total (1.43 +/- 0.13 mmol/L) and ionized (0.89 +/- 0.12 mmol/L) cerebrospinal
fluid [Mg] was maximally increased by 15% and 11% relative to baseline,
respectively, during induced hypermagnesemia (p < .05).
CONCLUSIONS: Hypermagnesemia produced only marginal increases in total and
ionized cerebrospinal fluid [Mg]. Regulation of cerebrospinal fluid [Mg] is
largely maintained following acute brain injury and limits the brain
bioavailability of MgSO4. BACKGROUND AND PURPOSE: Magnesium reverses cerebral vasospasm and reduces
infarct volume after experimental subarachnoid hemorrhage (SAH) in rats. We
aimed to assess whether magnesium reduces the frequency of delayed cerebral
ischemia (DCI) in patients with aneurysmal SAH.
METHODS: Patients were randomized within 4 days after SAH. Magnesium sulfate
therapy consisted of a continuous intravenous dose of 64 mmol/L per day, to be
started within 4 days after SAH and continued until 14 days after occlusion of
the aneurysm. The primary outcome DCI (defined as the occurrence of a new
hypodense lesion on computed tomography compatible with clinical features of
DCI) was analyzed according to the "on-treatment" principle. For the secondary
outcome measures "poor outcome" (Rankin >3) and "excellent outcome" (Rankin 0),
we used the "intention-to-treat" principle.
RESULTS: A total of 283 patients were randomized. Magnesium treatment reduced
the risk of DCI by 34% (hazard ratio, 0.66; 95% CI, 0.38 to 1.14). After 3
months, the risk reduction for poor outcome was 23% (risk ratio, 0.77; 95% CI,
0.54 to 1.09). At that time, 18 patients in the treatment group and 6 in the
placebo group had an excellent outcome (risk ratio, 3.4; 95% CI, 1.3 to 8.9).
CONCLUSIONS: This study suggests that magnesium reduces DCI and subsequent poor
outcome, but the results are not yet definitive. A next step should be a phase
III trial to confirm the beneficial effect of magnesium therapy, with poor
outcome as primary outcome. INTRODUCTION: Cerebral vasospasm in aneurysmal subarachnoid hemorrhage (SAH) is
associated with poor outcome. The safety and feasibility of continuous high-dose
intravenous magnesium sulfate (MgSO4) for the prevention of cerebral vasospasm
and ischemic cerebral injury has not been well studied.
METHODS: Patients presenting to our center within 72 hours of aneurysmalSAH
(confirmed by computed tomography [CT] scanning and cerebral angiography)
between June 2001 and October 2002 were enrolled in a prospective pilot study in
which they received MgSO4 as an adjunct to standard SAH management. Study
patients received an intravenous infusion of 12 g of MgSO4 in a 500-mL solution
of 0.9% NaCl administered at a rate of 4.06 mM (or 0.5 g) every hour over a
24-hour period for 10 days to achieve a target predetermined serum Mg range of
more than 1.5 to less than 4.0 mM/L. The effect of MgSO4 on clinical
examination, heart rate, and blood pressure was measured every 2 hours; serum
glucose and phenytoin levels were monitored daily. Outcome measures included
evidence of vasospasm on clinical examination, transcranial Doppler study
((TCD); velocity >or=100 cm/s), or repeat cerebral angiogram obtained within 10
days of SAH; and Glasgow Outcome Scale (GOS) score assessment and CT scan
evidence of ischemic infarction at 30 days.
RESULTS: Nineteen patients (mean age: 55 years; range: 39-84 years; 11 males, 8
females) were enrolled in the study. Presenting Hunt Hess grade was II or
higher; mean Fisher grade was 3. Vasospasm was observed in nine patients (by
clinical examination in two, TCD in five, and angiogram in nine). The mean serum
Mg level was 2.7 mM/L (standard deviation: +/- 0.37) and was maintained during
the infusion period. No clinical adverse effects, hemodynamic changes, or
fluctuations in serum glucose or phenytoin levels were observed. None of the
patients died; no CT evidence of ischemic infarction was present; and most had
good outcomes (GOS 5 in 10 patients; GOS 4 in 8 patients).
CONCLUSION: Our study confirmed the safety and feasibility of a continuous
infusion of high-dose intravenous MgSO4 in patients with aneurysmal SAH.
Randomized controlled trials are required to confirm the promising results. BACKGROUND: Magnesium is a neuroprotective agent that might prevent or reverse
delayed cerebral ischaemia after aneurysmal subarachnoid haemorrhage (SAH). We
are presently running a randomized, placebo-controlled, double blind trial with
magnesium sulphate (64 mmol/day intravenously). We studied whether this
treatment regime resulted in our target serum magnesium levels of 1.0-2.0
mmol/L.
METHODS: Magnesium sulphate was administered intravenously as soon as possible
after admission and continued until 14 days after occlusion of the aneurysm.
Serum magnesium measurements were done at baseline and at least every 2 days
during administration of trial medication. For comparison we used the serum
magnesium levels of the placebo-treated patients.
RESULTS: Magnesium therapy was begun in 94 patients. The mean magnesium level in
the treatment period was 1.47 +/- 0.32 mmol/L. In 81 patients serum magnesium
stayed within target levels during the entire treatment period. One patient had
a serum magnesium level below 1.0 mmol/L (0.91 mmol/L) in a single measurement
and 10 patients had serum magnesium levels above 2.0 mmol/L at one or more
measurements. In six patients magnesium therapy was discontinued: in three
because of nausea, headache, or both in combination with serum magnesium levels
above 2.0 mmol/L and in the other three because of hypotension, phlebitis and
renal failure.
CONCLUSIONS: With an intravenous dosage schedule of 64 mmol magnesium sulphate a
day, serum magnesium levels of 1.0-2.0 mmol/L can easily be maintained without
severe side effects for an extended period in a vast majority of patients with
SAH. BACKGROUND: Among the many complications of SAH, one of the most important is
vasospasm. Several treatment alternatives have been proposed for this condition,
with far-from-ideal results being obtained. Magnesium sulfate recently returned
to the scene (with still unproven benefit) as an adjuvant in the treatment of
vasospasm.
METHODS: Seventy-two patients diagnosed with SAH by aneurysm rupture were
submitted to microsurgery craniotomy and subdivided in 2 groups. Group 1, formed
by 48 patients, received prophylactic hypervolemic and hemodilution therapy in
addition to nimodipine. Group 2, composed of 24 patients, received the same
treatment of group 1 with the addition of magnesium sulfate in continuous
infusion from 120 to 150 mg a day, keeping serum magnesium levels close to
double normal values.
RESULTS: Age was 49 +/- 12.6 years. Ratio of female to male was 3.16:1. Most
patients were admitted in a Hunt-Hess grade 2 (46.4%) and Fisher grade 3
(52.8%). Anterior communicating artery aneurysms were the most common in
location (38.8%). Both groups were compared, and there was no statistical
difference related to age, sex, and Glasgow, Fisher, or Hunt-Hess admission
grades. No statistical difference in vasospasm incidence was found between the
two groups. However, in group 1, vasospasm was correlated with a longer
hospitalization time (P = .0003), different from group 2, in which patients with
vasospasm receiving magnesium sulfate required less hospitalization time.
CONCLUSION: Magnesium did not seem to interfere in vasospasm frequency but
apparently acted favorably in decreasing morbidity and length of hospital stay. We evaluated the effects of magnesium sulfate on brain tissue oxygen (PtO2)
tension, carbon dioxide (PtCO2) tension and pH (pHt) in patients undergoing
temporary artery occlusion for clipping of cerebral aneurysm. We studied 18
patients with aneurysmal subarachnoid hemorrhage. All patients received standard
anesthetics using target controlled infusion of propofol (3 microg/ml) and
remifentanil (10 ng/ml). After craniotomy, a calibrated multiparameter sensor
(Neurotrend, Diametrics Medical, Minneapolis, MN) was inserted to measure PtO2,
PtCO2 and pHt in tissue at risk of ischemia during temporary artery occlusion.
Patients were then randomly allocated to receive either intravenous saline or
magnesium 20 mmol over 10 min followed by an infusion 4 mmol/h. Plasma magnesium
concentration, brain tissue gases and pHt were determined at baseline, 30 min
after study drug infusion and 4 min after temporary clipping. Data were analyzed
by factorial ANOVA with repeated measures. Intergroup difference was compared
with unpaired t test. P value < 0.05 was considered significant. Patient
characteristics, baseline brain tissue gases and pHt did not differ between
groups. Magnesium infusion increased PtO2 by 34%. Following temporary artery
occlusion, PtO2 and pHt decreased and PtCO2 increased in both groups. However,
tissue hypoxia was less severe and the rate of PtO2 decline was slower in the
magnesium group. Our data suggested that magnesium enhances tissue oxygenation
and attenuates hypoxia during temporary artery occlusion. OBJECTIVES: Magnesium sulfate (MgSO4) may be useful in preventing neurological
injury after subarachnoid haemorrhage (SAH). In this randomized, double-blind
study we evaluated the safety and efficacy of MgSO4 infusion to improve clinical
outcome after aneurysmal SAH.
METHODS: With ethics committee approval and informed consents, 45 patients with
SAH were randomly allocated to receive either MgSO4 80 mmol/day or saline
infusion for 14 days. All patients also received intravenous nimodipine.
Episodes of vasospasm were treated with hypertensive, hypervolemic therapy.
Neurological status was assessed 3 months after haemorrhage using Barthel index
and Glasgow outcome scale (GOS). Incidences of cardiac and pulmonary
complications were also recorded. Data were compared between groups using
Mann-Whitney or Fisher exact tests as appropriate. P < 0.05 was considered
significant.
RESULTS: Patient characteristics, severity of SAH and surgical treatment did not
differ between groups. Although the number of episodes was not reduced, MgSO4
shortened the duration of vasospasm. Patients receiving MgSO4 tended to have
fewer neurological deficits, better functional recovery and an improved score in
GOS. However, none of these outcome variables reached statistical significance.
The incidence of cardiac and pulmonary complications in the MgSO4 group (43%)
was also similar to that in the saline group (59%), P = 0.14.
CONCLUSIONS: MgSO4 infusion after aneurysmal SAH is well tolerated and may be
useful in producing better outcome. A larger study is required to confirm the
neuroprotective effect of MgSO4. OBJECTIVE: The prophylactic use of nimodipine in patients with aneurysmal
subarachnoid hemorrhage reduces the risk of ischemic brain damage. However, its
efficacy seems to be rather moderate. The question arises whether other types of
calcium antagonists offer better protection. Magnesium, nature's physiological
calcium antagonist, is neuroprotective in animal models, promotes dilatation of
cerebral arteries, and has an established safety profile. The aim of the current
pilot study is to evaluate the efficacy of magnesium versus nimodipine to
prevent delayed ischemic deficits after aneurysmal subarachnoid hemorrhage.
METHODS: One hundred and thirteen patients with aneurysmal subarachnoid
hemorrhage were enrolled in the study and were randomized to receive either
magnesium sulfate (loading 10 mg/kg followed by 30 mg/kg daily) or nimodipine
(48 mg/d) intravenously until at least postoperative Day 7. Primary outcome
parameters were incidence of clinical vasospasm and infarction. Secondary
outcome measures were the incidence of transcranial Doppler/angiographic
vasospasm, the neuronal markers (neuron-specific enolase, S-100), and the
patients' Glasgow Outcome Scale scores at discharge and after 1 year.
RESULTS: One hundred and four patients met the study requirements. In the
magnesium group (n = 53), eight patients (15%) experienced clinical vasospasm
and 20 (38%) experienced transcranial Doppler/angiographic vasospasm compared
with 14 (27%) and 17 (33%) patients in the nimodipine group (n = 51). If
clinical vasospasm occurred, 75% of the magnesium-treated versus 50% of the
nimodipine-treated patients experienced cerebral infarction resulting in fatal
outcome in 37 and 14%, respectively. Overall, the rate of infarction
attributable to vasospasm was virtually the same (19 versus 22%). There was no
difference in outcome between groups.
CONCLUSION: The efficacy of magnesium in preventing delayed ischemic
neurological deficits in patients with aneurysmal subarachnoid hemorrhage seems
to be comparable with that of nimodipine. The difference in their
pharmacological properties makes studies on the combined administration of
magnesium and nimodipine seem promising. OBJECT: Despite the application of current standard therapies, vasospasm
continues to result in death or major disability in patients treated for
ruptured aneurysms. The authors investigated the effectiveness of continous
MgSO4 infusion for vasospasm prophylaxis.
METHODS: Seventy-six adults (mean age 54.6 years; 71% women; 92% Caucasian) were
included in this comparative matched-cohort study of patients with aneurysmal
subarachnoid hemorrhage on the basis of computed tomography (CT) findings.
Thirty-eight patients who received continuous MgSO4 infusion were matched for
age, race, sex, treatment option, Fisher grade, and Hunt and Hess grade to 38
historical control individuals who did not receive MgSO4infusion. Twelve grams
of MgSO4 in 500 ml normal saline was given intravenously daily for 12 days if
the patient presented within 48 hours of aneurysm rupture. Vasospasm was
diagnosed on the basis of digital substraction angiography, CT angiography, and
transcranial Doppler ultrasonography, and evidence of neurological
deterioration. Symptomatic vasospasm was present at a significantly lower
frequency in patients who received MgSO4 infusion (18%) compared with patients
who did not receive MgSO4 (42%) (p = 0.025). There was no significant difference
in mortality rate at discharge (p = 0.328). A trend toward improved outcome as
measured by the modifed Rankin Scale (p = 0.084), but not the Glasgow Outcome
Scale (p = 1.0), was seen in the MgSO4 treated group.
CONCLUSIONS: Analysis of the results suggests that MgSO4 infusion may have a
role in cerebral vasospasm prophylaxis if therapy is initiated within 48 hours
of aneurysm rupture. BACKGROUND: Secondary ischaemia is a frequent cause of poor outcome in patients
with subarachnoid haemorrhage (SAH). Its pathogenesis has been incompletely
elucidated, but vasospasm probably is a contributing factor. Experimental
studies have suggested that calcium antagonists can prevent or reverse vasospasm
and have neuroprotective properties.
OBJECTIVES: To determine whether calcium antagonists improve outcome in patients
with aneurysmal SAH.
SEARCH STRATEGY: We searched the Cochrane Stroke Group Trials Register (last
searched April 2006), MEDLINE (1966 to March 2006) and EMBASE (1980 to March
2006). We handsearched two Russian journals (1990 to 2003), and contacted
trialists and pharmaceutical companies in 1995 and 1996.
SELECTION CRITERIA: Randomised controlled trials comparing calcium antagonists
with control, or a second calcium antagonist (magnesium sulphate) versus control
in addition to another calcium antagonist (nimodipine) in both the intervention
and control groups.
DATA COLLECTION AND ANALYSIS: Two review authors independently extracted the
data and assessed trial quality. Trialists were contacted to obtain missing
information.
MAIN RESULTS: Sixteen trials, involving 3361 patients, were included in the
review; three of the studies were of magnesium sulphate in addition to
nimodipine. Overall, calcium antagonists reduced the risk of poor outcome: the
relative risk (RR) was 0.81 (95% confidence interval (CI) 0.72 to 0.92); the
corresponding number of patients needed to treat was 19 (95% CI 1 to 51). For
oral nimodipine alone the RR was 0.67 (95% CI 0.55 to 0.81), for other calcium
antagonists or intravenous administration of nimodipine the results were not
statistically significant. Calcium antagonists reduced the occurrence of
secondary ischaemia and showed a favourable trend for case fatality. For
magnesium in addition to standard treatment with nimodipine, the RR was 0.75
(95% CI 0.57 to 1.00) for a poor outcome and 0.66 (95% CI 0.45 to 0.96) for
clinical signs of secondary ischaemia.
AUTHORS' CONCLUSIONS: Calcium antagonists reduce the risk of poor outcome and
secondary ischaemia after aneurysmal SAH. The results for 'poor outcome' depend
largely on a single large trial of oral nimodipine; the evidence for other
calcium antagonists is inconclusive. The evidence for nimodipine is not beyond
all doubt, but given the potential benefits and modest risks of this treatment,
oral nimodipine is currently indicated in patients with aneurysmal SAH.
Intravenous administration of calcium antagonists cannot be recommended for
routine practice on the basis of the present evidence. Magnesium sulphate is a
promising agent but more evidence is needed before definite conclusions can be
drawn. The vasodilatory effect of intra-cisternal infusion of magnesium sulfate
solution was evaluated in 10 patients with symptomatic vasospasm after
aneurysmal subarachnoid hemorrhage (SAH) who underwent early clipping surgery.
Cisternal drainage was installed in the prepontine and/or sylvian fissures.
Carotid angiography was performed immediately after the onset of symptomatic
vasospasm, then intra-cisternal infusion of 15 mmol/l magnesium sulfate in
Ringer solution was started at 20 ml/hr and continued until day 14. Irrigation
was performed from the cisternal tube (inlet) to the spinal drainage (outlet).
The cerebrospinal fluid magnesium ion concentration (1.2 +/- 0.2 mEq/l)
significantly increased after the infusion therapy (6.0 +/- 1.7 mEq/l, p <
0.001). Repeat angiography showed vasodilatory effect on the spastic cerebral
arteries at 3 hours after the infusion, especially in the arteries near to the
site of cisternal drainage placement. The magnesium infusion also caused
decreased mean arterial blood velocity in the spastic arteries in 6 of the 7
measured patients (162 +/- 38 cm/sec to 114 +/- 42 cm/sec, p < 0.001). Finally,
5 of the 10 patients achieved good recovery, 1 patient had moderate disability,
1 patient became severely disabled due to meningitis, and 3 patients were
vegetative or dead, due to failure of magnesium irrigation in 1 patient and
advanced age in the other 2 (more than 80 years old). This preliminary study
indicates that intra-cisternal infusion of magnesium sulfate solution has
vasodilatory effect on the spastic cerebral arteries after aneurysmal SAH. Despite the publication of several randomized controlled studies, there is still
much debate on whether magnesium sulfate improves outcome in patients with
aneurysmal subarachnoid hemorrhage. Here we present data to assess the clinical
effectiveness of magnesium sulfate in the prevention of cerebral vasospasm in
patients who have suffered from aneurysmal subarachnoid hemorrhage. The EMBASE
and PubMed databases were searched using the following terms: "magnesium
sulfate" or "MgSO(4)" with "subarachnoid hemorrhage" or "cerebral vasospasm". A
manual search of the bibliographies of relevant articles was also conducted. Two
co-authors of the present study designed the meta analysis of published
randomized clinical trials and extracted the data. Data were analysed by using
Review Manager 4.2 from the Cochrane Collaboration (Oxford, UK). Five published
manuscripts were identified according to the screening criteria. The occurrence
of poor outcome (death, vegetative state, or dependency) in patients treated
with magnesium sulfate was less likely than control group patients (odds ratio
[OR] 0.54 [95% confidence interval, CI 0.36-0.81]). Mortality rates did not
differ between magnesium sulfate (14%) and control treated (12%) patients (OR
1.16 [95% CI 0.51-2.65]). Our results indicate that although there was reduced
likelihood of a poor outcome for patients treated with magnesium sulfate after
SAH, patient mortality was not improved. BACKGROUND: A meta-analysis of current data suggests that magnesium sulfate
infusion improves the outcome after aneurysmal subarachnoid hemorrhage through a
reduction in delayed ischemic neurological deficit. Two multi-center randomized
controlled trials are currently underway to investigate this hypothesis. The
possible pharmacological basis of this hypothesis includes neuroprotection and
vasodilatation. We aim to investigate the cerebral hemodynamic effects of
magnesium sulfate infusion in aneurysmal subarachnoid hemorrhage patients.
METHOD: A total of 12 patients who had experienced aneurysmal subarachnoid
hemorrhage were randomized to magnesium sulfate infusion (n = 6) or placebo
infusion (n = 6) for 14 days. Each patient had two perfusion MRIs performed, one
in the first week after subarachnoid hemorrhage and one in the second week after
subarachnoid hemorrhage.
FINDINGS: Age, sex, and Fisher CT grade were not different between the two
groups. All but one patient were of WFNS Grade I to II on presentation. There
was no increase in rCBV, rCBF and MTT between the two perfusion scans within the
same group or between the two groups.
CONCLUSION: Magnesium sulfate infusion, in the dosage of current clinical
trials, did not increase cerebral blood volume and cerebral blood flow, as
postulated by dilation of small vessels and/or collateral pathways. BACKGROUND: In patients with aneurysmal subarachnoid haemorrhage (SAH), headache
typically is severe and often requires treatment with opioids. Magnesium has
analgesic effects in several conditions, but whether it reduces headache after
SAH is unknown.
METHODS: In a cohort of 108 SAH patients included in the randomised controlled
trial Magnesium in Aneurysmal Subarachnoid Haemorrhage-II (MASH-II), severity of
headache was regularly assessed on an 11-point scale until day 10 after SAH.
Headache was treated according to a standardised protocol with acetaminophen,
codeine, tramadol or piritramide. Serum magnesium levels were assessed every
other day. Differences in mean headache scores between patients with mean high
(>1.0 mmol/l) and normal (< or =1.0 mmol/l) magnesium levels were analysed with
a Student t test. Crude and adjusted ORs for the use of codeine, tramadol and
piritramide for patients with high versus normal magnesium levels were
calculated with logistic regression.
RESULTS: The 61 patients with high magnesium levels had lower mean headache
scores (4.1) than the 47 patients with normal magnesium levels (4.9; mean
difference, 0.8; 95% CI 0.1 to 1.6) and required less tramadol (adjusted OR,
0.3; 95% CI 0.1 to 0.7) or piritramide (adjusted OR 0.2; 95% CI 0.1 to 0.5).
There were no differences in the use of acetaminophen or codeine.
CONCLUSION: In SAH patients, elevated serum magnesium levels are associated with
slightly less severe headache and less frequent use of opioids. These data imply
that intravenous magnesium therapy, besides a supposed beneficial effect on
outcome, also provides pain relief for SAH patients, for whom it might also
improve functional outcome. OBJECTIVE: To examine whether the maintece of elevated magnesium serum
concentrations by intravenous administration of magnesium sulfate can reduce the
occurrence of cerebral ischemic events after aneurysmal subarachnoid hemorrhage.
DESIGN: Prospective, randomized, placebo-controlled study.
SETTING: Neurosurgical intensive care unit of a University hospital.
INTERVENTIONS: One hundred ten patients were randomized to receive intravenous
magnesium sulfate or to serve as controls. Magnesium treatment was started with
a bolus of 16 mmol, followed by continuous infusion of 8 mmol/hr. Serum
concentrations were measured every 8 hrs, and infusion rates were adjusted to
maintain target levels of 2.0-2.5 mmol/L. Intravenous administration was
continued for 10 days or until signs of vasospasm had resolved. Thereafter,
magnesium was administered orally and tapered over 12 days.
MEASUREMENTS AND MAIN RESULTS: Delayed ischemic infarction (primary end point)
was assessed by analyzing serial computed tomography scans. Transcranial Doppler
sonography and digital subtraction angiography were used to detect vasospasm.
Delayed ischemic neurologic deficit was determined by continuous detailed
neurologic examinations; clinical outcome after 6 months was assessed using the
Glasgow outcome scale. Good outcome was defined as Glasgow outcome scale score 4
and 5.The incidence of delayed ischemic infarction was significantly lower in
magnesium-treated patients (22% vs. 51%; p = .002); 34 of 54 magnesium patients
and 27 of 53 control patients reached good outcome (p = .209). Delayed ischemic
neurologic deficit was nonsignificantly reduced (9 of 54 vs. 15 of 53 patients;
p = .149) and transcranial Doppler-detected/angiographic vasospasm was
significantly reduced in the magnesium group (36 of 54 vs. 45 of 53 patients; p
= .028). Fewer patients with signs of vasospasm had delayed cerebral infarction.
CONCLUSION: These data indicate that high-dose intravenous magnesium can reduce
cerebral ischemic events after aneurysmal subarachnoid hemorrhage by attenuating
vasospasm and increasing the ischemic tolerance during critical hypoperfusion. PRIMARY OBJECTIVE: Experimental and clinical studies have suggested that
magnesium has neuroprotective and vasodilatation properties. A meta-analysis was
conducted to assess the effectiveness and safety of intravenous magnesium
therapy in patients with aneurysmal subarachnoid haemorrhage (SAH).
RESEARCH DESIGN: Meta-analysis.
METHODS AND PROCEDURES: Medline, EMBASE and the Cochrane Library were searched
for prospective controlled trials evaluating intravenous magnesium for treating
SAH after a ruptured aneurysm without language restrictions. Two researchers
performed the literature search and data extraction independently.
MAIN OUTCOMES AND RESULTS: Six prospective controlled trials involving 699
patients were included in this meta-analysis. Magnesium infusion reduced the
risk of poor outcome and delayed cerebral ischemia (DCI): the relative risk was
0.62 (95% confidence interval (CI) 0.46-0.83) and 0.73 (95% CI 0.53-1.00),
respectively. Sensitivity analyses were consistent with the meta-analysis. The
withdrawal rate for adverse effects was higher in the magnesium-treatment arm
compared to the placebo arm, RR 9.98 (95% CI 3.04-32.74).
CONCLUSION: The meta-analysis suggests that intravenous magnesium therapy
reduces the risk of DCI and poor outcome after aneurysmal SAH. Serum magnesium
should be routinely monitored for both effectiveness and safety considerations. BACKGROUND AND PURPOSE: Pilot clinical trials using magnesium sulfate in
patients with acute aneurysmal subarachnoid hemorrhage have reported trends
toward improvement in clinical outcomes. This Phase III study aimed to compare
intravenous magnesium sulfate infusion with saline placebo among such patients.
METHODS: We recruited patients with aneurysmal subarachnoid hemorrhage within 48
hours of onset from 10 participating centers. The patients were randomly
assigned to magnesium sulfate infusion titrated to a serum magnesium
concentration twice the baseline concentration or saline placebo for 10 to 14
days. Patients and assessors were blinded to treatment allocation. The study is
registered at www.strokecenter.org/trials (as Intravenous Magnesium Sulphate for
Aneurysmal Subarachnoid Hemorrhage [IMASH]) and www.ClinicalTrials.gov
(NCT00124150).
RESULTS: Of the 327 patients recruited, 169 were randomized to receive treatment
with intravenous magnesium sulfate and 158 to receive saline (placebo). The
proportions of patients with a favorable outcome at 6 months (Extended Glasgow
Outcome Scale 5 to 8) were similar, 64% in the magnesium sulfate group and 63%
in the saline group (OR, 1.0; 95% CI, 0.7 to 1.6). Secondary outcome analyses
(modified Rankin Scale, Barthel Index, Short Form 36, and clinical vasospasm)
also showed no significant differences between the 2 groups. Predefined
subgroups included age, admission World Federation of Neurological Surgeons
grade, pre-existing hypertension, intracerebral hematoma, intraventricular
hemorrhage, location of aneurysm, size of aneurysm, and mode of aneurysm
treatment. In none of the subgroups did the magnesium sulfate group show a
better outcome at 6 months.
CONCLUSIONS: The results do not support a clinical benefit of intravenous
magnesium sulfate infusion over placebo infusion in patients with acute
aneurysmal subarachnoid hemorrhage. BACKGROUND AND PURPOSE: Conflicting data have been obtained on optimal plasma
magnesium concentrations for clinical outcomes in patients with aneurysmal
subarachnoid hemorrhage.
METHODS: Adults (aged 18 years or older) who had acute aneurysmal subarachnoid
hemorrhage diagnosed were randomly assigned to receive either an intravenous
MgSO(4) infusion (80 mmol in 500 mL normal saline per day) or a placebo (500 mL
normal saline per day) for up to 14 days. Post hoc multivariable binary logistic
regression analyses were performed by dividing mean plasma magnesium
concentrations into 4 quartiles according to treatment group and then comparing
with the lowest quartiles.
RESULTS: The worst clinical outcomes at 6 months were seen in MgSO(4) group
patients, with mean plasma magnesium concentrations in the fourth quartile, and
in placebo group patients, with mean such concentrations in the third and fourth
quartiles.
CONCLUSIONS: No evidence was found to suggest that a higher mean plasma
magnesium concentration improves clinical outcomes. On the contrary, we found an
association between high plasma magnesium concentration and worse clinical
outcomes. Delayed ischemic neurological deficit or clinical vasospasm remained a major
cause for delayed neurological morbidity and mortality for patients with
aneurysmal subarachnoid hemorrhage (SAH). Magnesium is a cerebral vasodilator.
In experimental model of drug or SAH-induced vasospasm, magnesium blocks
voltage-dependent calcium channels and reverses cerebral vasoconstriction.
Furthermore, its antagonistic action on N-methyl-D-aspartate receptor in the
brain prevents glutamate stimulation and decreases calcium influx during
ischemic injury. Clinically, the protective effect of magnesium has also been
found useful in women with preeclampsia, a condition thought to be due to
cerebral vasospasm. Initial experimental result in human was found to safe and
effective as compared to historical data. In our pilot study, 60 patients were
randomly allocated to receive either magnesium sulfate infusion 80 mmol/day or
saline infusion for 14 days. The incidence of symptomatic vasospasm decreased
from 13/30(43%) in the saline group to 7/30(23%) in the patients receiving
magnesium sulfate infusion, p = 0.10, odds ratio 0.398, 95% CI 0.131-1.211.
Favorable outcome (Good recovery and moderate disability, as defined by Glasgow
Outcome Scale) was achieved in 20 of 30 (67%) patients receiving magnesium
sulfate infusion and 16 of 30 (53%) patients receiving placebo treatment, p =
0.292, odds ratio 1.750, 95% CI 0.616-4.974.From literature review, a total of
441 patients from four studies (including ours) were grouped for analysis. Using
random effects model (Mantel-Haenszel, Robins-Breslow-Greenland), the pooled
odds ratio for symptomatic vasospasm or delayed cerebral ischemia is, 0.620, 95%
CI 0.389-0.987, statistically significant. Similarly, the pooled odds ratio for
favorable outcome is 1.598, 95% CI 1.074-2.377, statistically significant. There
are two multi-center phase III studies (IMASH and MASH2) being carried out to
assess the clinical effects, in which IMASH has finished data collection on 30th
June 2009. INTRODUCTION: Previous meta-analyses of magnesium sulphate infusion in the
treatment of aneurysmal subarachnoid hemorrhage (SAH) have become outdated due
to recently published clinical trials. Our aim was thus to perform an up-to-date
systemic review and meta-analysis of published data on the use of magnesium
sulphate infusion in aneurysmal SAH patients.
METHODS: A systemic review and meta-analysis of the literature was carried out
on published randomized controlled clinical trials that investigated the
efficacy of magnesium sulphate infusion in aneurysmal SAH patients. The results
were analyzed with regard to delayed cerebral ischemia (DCI), delayed cerebral
infarction, and favorable neurological outcomes at three and six months. The
risks of bias were assessed using the Jadad criteria, with a Jadad score >3
indicating a lower such risk. Meta-analyses are presented in terms of relative
risk (RR) with 95% confidence intervals (CIs).
RESULTS: Six eligible studies with 875 patients were reviewed. The pooled RR for
DCI was 0.87 (95% CI, 0.36 to 2.09; P = 0.75). That for delayed cerebral
infarction was 0.58 (95% CI, 0.35 to 0.97; P = 0.04), although this result did
not persist if only randomized clinical trials with a lower risk of bias were
included (RR 0.61, 95% CI, 0.31 to 1.22; P = 0.17). The pooled RR for a
favorable outcome at three months was 1.14 (95% CI, 0.99 to 1.31; P = 0.07), and
that for a favorable outcome at six months was 1.08 (95% CI, 0.94 to 1.24; P =
0.29).
CONCLUSIONS: The present findings do not lend support to a beneficial effect of
magnesium sulphate infusion on delayed cerebral infarction. The reduction in DCI
and improvement in the clinical outcomes of aneurysmal SAH patients following
magnesium sulphate infusion observed in previous pilot studies are not
confirmed, although a beneficial effect cannot be ruled out because of sample
size limitation. BACKGROUND: There has been longstanding controversy over the use of magnesium
sulfate infusion in the medical management of aneurysmal subarachnoid hemorrhage
(SAH). Several clinical trials evaluating the beneficial effects of magnesium on
cerebral vasospasm and their poor outcome have been published. However, results
from the majority of these studies have been inconclusive. This meta-analysis
was performed to evaluate the effectiveness of magnesium on patient outcomes
after aneurysmal SAH.
MATERIALS AND METHODS: PubMed and the Cochrane library were searched for
controlled clinical trials assessing the efficacy of magnesium sulfate infusion
after aneurysmal SAH. Eight studies consisting of 936 patients were included.
RESULTS: There was a decreased risk of poor outcome at 3-6 months after SAH in
magnesium treatment groups when compared to placebo [0.78 (95% CI 0.66-0.93)].
Poor outcome was defined as severe disability, persistent vegetative state, or
death, as measured by the Glasgow outcome scale (GOS), extended Glasgow outcome
scale (GOSE) or modified Rankin scale (mRS). The risk of mortality after SAH was
unaffected by magnesium treatment [RR 0.68 (95% CI 0.58-1.27)].
CONCLUSION: Magnesium sulfate infusion decreases risk of poor outcome after
aneurysmal SAH. Current studies in the literature do not suggest a role for
magnesium sulfate in mortality reduction after SAH. Magnesium, one of the essential trace elements, plays important roles in
maintaining both normal cellular and body functions. S100 calcium-binding
protein B (S100B) has been used as a marker of glial damage in several
neurological disorders. Thirty patients with ruptured intracranial aneurysms
treated by clipping are included. The patients were randomized within 4 days
after the attack of hemorrhage. The patients were divided into two groups with
15 patients in each group. Group I received magnesium infusion within 4 days.
Group II is the control group. World Federation of Neurological Surgeons,
Fisher, and Glasgow outcome scores are evaluated at an intensive care unit in
addition to 3 months clinical follow-up evaluation. Samplings of serum S100B
were performed on admission and on postoperative days 1, 3, and 7. There is a
statistically significant difference between both groups as regards the second
reading of the S100B (day 1 postoperative; P < 0.05). There is no statistically
significant difference between both groups as regards outcome at 3 months using
clinical status and S100B values. There is a tendency in the magnesium group to
have better outcomes. Further studies with more number of patients with
subarachnoid hemorrhage are needed to determinate the accuracy of S100B protein
as a prognostic marker and of magnesium sulfate as a neuroprotector. BACKGROUND: Magnesium sulphate is a neuroprotective agent that might improve
outcome after aneurysmal subarachnoid haemorrhage by reducing the occurrence or
improving the outcome of delayed cerebral ischaemia. We did a trial to test
whether magnesium therapy improves outcome after aneurysmal subarachnoid
haemorrhage.
METHODS: We did this phase 3 randomised, placebo-controlled trial in eight
centres in Europe and South America. We randomly assigned (with
computer-generated random numbers, with permuted blocks of four, stratified by
centre) patients aged 18 years or older with an aneurysmal pattern of
subarachnoid haemorrhage on brain imaging who were admitted to hospital within 4
days of haemorrhage, to receive intravenous magnesium sulphate, 64 mmol/day, or
placebo. We excluded patients with renal failure or bodyweight lower than 50 kg.
Patients, treating physicians, and investigators assessing outcomes and
analysing data were masked to the allocation. The primary outcome was poor
outcome-defined as a score of 4-5 on the modified Rankin Scale-3 months after
subarachnoid haemorrhage, or death. We analysed results by intention to treat.
We also updated a previous meta-analysis of trials of magnesium treatment for
aneurysmal subarachnoid haemorrhage. This study is registered with
controlled-trials.com (ISRCTN 68742385) and the EU Clinical Trials Register
(EudraCT 2006-003523-36).
FINDINGS: 1204 patients were enrolled, one of whom had his treatment allocation
lost. 606 patients were assigned to the magnesium group (two lost to follow-up),
597 to the placebo (one lost to follow-up). 158 patients (26·2%) had poor
outcome in the magnesium group compared with 151 (25·3%) in the placebo group
(risk ratio [RR] 1·03, 95% CI 0·85-1·25). Our updated meta-analysis of seven
randomised trials involving 2047 patients shows that magnesium is not superior
to placebo for reduction of poor outcome after aneurysmal subarachnoid
haemorrhage (RR 0·96, 95% CI 0·86-1·08).
INTERPRETATION: Intravenous magnesium sulphate does not improve clinical outcome
after aneurysmal subarachnoid haemorrhage, therefore routine administration of
magnesium cannot be recommended.
FUNDING: Netherlands Heart Foundation, UK Medical Research Council. OBJECTIVE: We aimed to investigate the profiles and prognostic values of delayed
cerebral ischemia (DCI) and delayed cerebral infarction.
METHODS: IMASH (Intravenous Magnesium Sulphate for Aneurysmal Subarachnoid
Hemorrhage) was registered at http://www.strokecenter.org/trials , and
http://www.ClinicalTrials.gov (NCT00124150). Data of 327 patients were retrieved
for logistic regression analyses.
RESULTS: Seventy-one (22%) patients developed DCI, and 35 (11%) patients
developed delayed cerebral infarction. Only 18 (25%) patients with DCI and 7/35
(20%) patients with delayed cerebral infarction had mean middle cerebral artery
velocities (transcranial Doppler ultrasound) over 120 cm/s. Regarding the
prognostic significance of the components of DCI, delayed cerebral infarction
predicted unfavorable outcome in terms of Extended Glasgow Outcome Scale (OR
3.1, 95% [CI] 1.3-7.8), poor outcome in terms of modified Rankin Scale (odds
ratio [OR] 3.0, 95% confidence interval CI 1.2-7.7), and dependent activity of
daily living in terms of Barthel Index (OR 3.6, 95% CI 1.4-9.2) at 6 months,
after adjustments for other prognostic factors. On the other hand, clinical
deterioration predicted inpatient mortality (OR 8.8, 95% CI 1.6-48.8) after
adjustments for other prognostic factors.
CONCLUSIONS: Delayed cerebral ischemia and delayed cerebral infarction carried
different prognostic values in aneurysmal subarachnoid hemorrhage. Cerebral vasospasm and delayed cerebral ischemia remain an unsolved problem in
patients with aneurysmal subarachnoid hemorrhage (SAH). In theory, high-dose
magnesium sulfate (MgSO(4)) therapy offers vascular and neuroprotective benefits
and is therefore currently under evaluation. The intensity of the inflammatory
response after SAH is associated with the outcome. The aim of the current study
was to evaluate a possible link between the inflammatory response and MgSO(4)
therapy, since magnesium (Mg(2+)) has anti-inflammatory properties. In 15
patients with SAH, inflammatory cytokine levels in the cerebrospinal fluid (CSF)
and peripheral blood were determined daily using an enzyme-linked immunosorbent
assay between day 4 and day 12. Eight patients were treated with standard
therapy alone (group 1) and seven patients were treated with an additional,
high-dose of MgSO(4) (group 2). Serum Mg(2+) levels in group 2 were
significantly higher compared to group 1: 1.48 ± 0.04 mmol/L versus 0.90 ± 0.01
mmol/L, ρ<0.001. Interleukin-6 (IL-6) in the CSF was significantly lower in
group 2 compared to group 1: 6680 ± 989 vs.11079 ± 1277 pg/mL, ρ = 0.021. A
trend towards lower systemic IL-6 levels was found in group 2: 58 ± 7 versus 104
± 21 pg/mL, ρ = 0.052. Systemic IL-1β levels were significantly lower in group
2: 0.66 ± 0.11 and 0.15 ± 0.01 pg/mL (ρ<0.001), while the CSF levels did not
differ. Tumor necrosis factor-α levels did not differ between the two groups.
Although there were more patients with favorable outcome in group 2, the
difference was not statistically significant. This was probably due to the small
sample size. The results indicate a suppression of inflammatory cytokine
release, in particular IL-6, in patients treated with high-dose MgSO(4). These
results call for further studies of the effect of Mg(2+) on the inflammatory
signaling pathway with regard to delayed cerebral ischemia following SAH. OBJECTIVE: To elucidate the role of magnesium sulfate in patients with
subarachnoid hemorrhagic (SAH) brain injury
METHOD: Studies for the meta-analysis were identified from PubMed (1966 to
2009), Embase (1980 to 2009), and two Chinese journals (1989 to 2009). Paper
selection was based on randomized controlled trials comparing magnesium sulfate
to placebo treatment in patients with SAH. Two independent review authors
extracted the data and assessed trial quality. Meta-analysis was performed using
the Cochrane Review Manger software.
RESULTS: Five trials involving 482 patients were included in the review.
Magnesium sulfate reduced the risk of poor outcome and reduced the occurrence of
delayed cerebral ischemia. In the treatment groups, relative risk for poor
outcome was 0.73 (CI 0.57-0.93) and 0.66 (CI 0.47-0.92) for delayed cerebral
ischemia. Case fatality assessment at three to six months did not show
statistically significant data (RR 0.88; CI 0.61-1.29).
CONCLUSION: Magnesium sulfate appears to be an effective treatment option in the
management of SAH. Further clinical trials are needed before magnesium sulfate
can become a routine treatment for SAH. BACKGROUND: The effect of serum magnesium concentration on the incidence of
cerebral arterial vasospasm following aneurysmal subarachnoid haemorrhage (SAH)
is unclear.
OBJECTIVE: To test whether induced hypermagnesaemia reduces the incidence of
cerebral arterial vasospasm following aneurysmal SAH.
METHODS: The study was conducted at two tertiary hospitals in Australia and
patients were recruited between 1 April 2005 and 31 December 2009. Within 72
hours of aneurysmal SAH, patients were randomly assigned to a high or normal
target for serum magnesium concentration (1.60-2.50 mmol/L or 0.65-1.05 mmol/L,
respectively). The primary end point was cerebral arterial vasospasm diagnosed
by blinded assessment of digital subtraction angiography. Secondary outcomes
included severity of vasospasm and functional recovery at 90 days. Analysis was
by intention to treat.
RESULTS: Of 162 patients, 81 were assigned to the normal range group and 81 were
assigned to the high-range group; the primary outcome was available for 78 and
79 patients, respectively. The groups had similar baseline characteristics.
Vasospasm occurred in 40 patients (50.6%) and 50 patients (64.1%) assigned to
high-range and normal-range groups, respectively (adjusted OR, 0.51; 95% CI,
0.26-1.02; P = 0.06). At 90 days, neurological recovery between the groups was
not significantly different (adjusted OR for worse outcome, 0.71; 95% CI,
0.39-1.32; P = 0.28). Patients in the high-range group were treated with more
noradrenaline to support arterial blood pressure (79 [16- 218] mg) v 59 [14-129]
mg; P = 0.03) and had lower mean (SD) serum calcium concentration (1.9 [0.2]
mmol/L v 2.1 [0.2] mmol/L, P < 0.001).
CONCLUSION: Patients assigned a higher serum magnesium concentration had a
reduced incidence of vasospasm as seen by angiography, but the difference was
not statistically significant. Clinically significant outcomes were not
different between groups. A firm recommendation for induced hypermagnesaemia
cannot be made from this study.
TRIAL REGISTRATION NUMBER: ACTRN12605000058673. BACKGROUND: The failures of recent studies intended to prevent cerebral
vasospasm have moved the focus of research into delayed cerebral ischemia away
from cerebral artery constriction towards other mechanisms. Recent accumulating
evidence has suggested that early brain injury is also involved in the
development of delayed cerebral ischemia, and that hydrogen can prevent early
brain injury. Therefore, we have established a combination therapy of
intravenous hydrogen infusion and intra-cisternal magnesium sulfate infusion for
the treatment of both early brain injury and cerebral vasospasm. The present
randomized controlled clinical trial is designed to investigate the effects of
this novel therapeutic strategy on the occurrence of cerebral vasospasm, delayed
cerebral ischemia, and clinical outcomes after high-grade subarachnoid
hemorrhage.
METHODS: This study is a randomized, double-blind, placebo-controlled design to
be conducted in two hospitals. A total of 450 patients with high-grade
subarachnoid hemorrhage will be randomized to one of three arms: (i) Mg + H2
group, (ii) Mg group, and (iii) control group. Patients who are assigned to the
Mg + H2 group will receive intra-cisternal magnesium sulfate infusion
(2.5 mmol/L) at 20 mL/h for 14 days and intravenous hydrogen-rich fluid infusion
(200 mL) twice a day for 14 days. Patients who are assigned to the Mg group will
receive intra-cisternal magnesium sulfate infusion (2.5 mmol/L) at 20 mL/h for
14 days and intravenous normal glucose-electrolyte solution (200 mL) without
added hydrogen twice a day for 14 days. Patients who are assigned to the control
group will receive intra-cisternal Ringer solution without magnesium sulfate at
20 mL/h for 14 days and intravenous normal glucose-electrolyte solution (200 mL)
without added hydrogen twice a day for 14 days. Primary outcome measures will be
occurrence of delayed cerebral ischemia and cerebral vasospasm. Secondary
outcome measures will be modified Rankin scale score at 3, 6, and 12 months and
biochemical markers.
DISCUSSION: The present protocol for a randomized, placebo-controlled study of
intravenous hydrogen therapy with intra-cisternal magnesium infusion is expected
to establish the efficacy and safety of this therapeutic strategy.
TRIAL REGISTRATION UMIN-CTR: UMIN000014696. |
Which gene is mutated in a subtype of arrhythmogenic right ventricular cardiomyopathy known as Naxos disease? | Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease).Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder associated with arrhythmias and sudden death. A recessive mutation in the gene encoding plakoglobin has been shown to cause Naxos disease, a cardiocutaneous syndrome characterized by ARVC and abnormalities of hair and skin. | BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an
autosomal domit heart muscle disorder that causes arrhythmia, heart failure,
and sudden death. Previously we mapped the genetic locus for the triad of
autosomal recessive ARVC, palmoplantar keratoderma, and woolly hair (Naxos
disease) to chromosome 17q21, in which the gene for plakoglobin is encoded. This
protein is a key component of desmosomes and adherens junctions, and is
important for the tight adhesion of many cell types, including those in the
heart and skin.
METHODS: We studied 19 individuals with Naxos disease, as well as unaffected
family members and unrelated individuals from the neighbouring Greek islands of
Naxos and Milos. Gene sequence was determined by reverse transcriptase PCR from
RNA isolated from the skin of an affected individual and mutations in other
cases were confirmed by restriction-enzyme analysis.
FINDINGS: A homozygous 2 base pair deletion in the plakoglobin gene was
identified only in the 19 affected individuals. This deletion caused a
frameshift and premature termination of the protein, which was shown by western
blot analysis. 29 clinically unaffected family members were heterozygous for the
mutation; 20 unrelated individuals from Naxos and 43 autosomal domit ARVC
probands were homozygous for the normal allele.
INTERPRETATION: The finding of a deletion in plakoglobin in ARVC suggests that
the proteins involved in cell-cell adhesion play an important part in
maintaining myocyte integrity, and when junctions are disrupted, cell death, and
fibrofatty replacement occur. Therefore, the discovery of a mutation in a
protein with functions in maintaining cell junction integrity has important
implications for other domit forms of ARVC, related cardiomyopathies, and
other cutaneous diseases. OBJECTIVES: The purpose of this study was to examine the genotype-phenotype
relation with respect to penetrance, age and severity of expression, disease
progression and prognosis in a recessively inherited arrhythmogenic right
ventricular cardiomyopathy (ARVC).
BACKGROUND: Naxos disease is a recessively inherited ARVC caused by a mutation
in the gene encoding plakoglobin (cell adhesion protein) in which the cardiac
phenotype is associated with palmoplantar keratoderma and woolly hair.
METHODS: Twelve families with Naxos disease underwent cardiac and molecular
genetic investigation. Serial cardiac assessment with annual resting 12-lead and
24-h ambulatory electrocardiogram (ECG) and two-dimensional echocardiography was
performed during 1 to 16 years, median 7 +/- 6 years in all 78 surviving
members.
RESULTS: Twenty-eight surviving members were homozygous and 40 were heterozygous
for the mutation. All adults who were homozygous (n = 26) fulfilled the
diagnostic criteria for ARVC, the youngest by the age of 13 years. In eight who
were heterozygous, minor ECG or echocardiographic abnormalities were observed.
Of the 26 subjects who were affected homozygotes, 92% showed ECG abnormalities,
92% ventricular arrhythmias, 100% right ventricular structural alterations and
27% left ventricular involvement. During follow-up (10 +/- 6 years), 16 (62%)
developed structural progression, 12 (46%) arrhythmic events and 7 (27%) heart
failure. The annual disease-related and sudden death mortality was 3% and 2.3%,
respectively.
CONCLUSIONS: Autosomal recessive ARVC caused by a mutation in plakoglobin was
100% penetrant by adolescence. Affected subjects who were homozygous experienced
progressive disease with adverse prognosis. A minority of subjects who were
heterozygous showed minor ECG/echocardiographic changes, but clinically
significant disease did not develop. Mutations in genes encoding desmosomal proteins have been implicated in the
pathogenesis of heart and skin diseases. This has led to the hypothesis that
defective cell-cell adhesion is the underlying cause of injury in tissues that
repeatedly bear high mechanical loads. In this study, we examined the effects of
two different mutations in plakoglobin on cell migration, stiffness, and
adhesion. One is a C-terminal mutation causing Naxos disease, a recessive
syndrome of arrhythmogenic right ventricular cardiomyopathy (ARVC) and abnormal
skin and hair. The other is an N-terminal mutation causing domit inheritance
of ARVC without cutaneous abnormalities. To assess the effects of plakoglobin
mutations on a broad range of cell mechanical behavior, we characterized a model
system consisting of stably transfected HEK cells which are particularly well
suited for analyses of cell migration and adhesion. Both mutations increased the
speed of wound healing which appeared to be related to increased cell motility
rather than increased cell proliferation. However, the C-terminal mutation led
to dramatically decreased cell-cell adhesion, whereas the N-terminal mutation
caused a decrease in cell stiffness. These results indicate that different
mutations in plakoglobin have markedly disparate effects on cell mechanical
behavior, suggesting complex biomechanical roles for this protein. Two sons of a consanguineous marriage developed biventricular cardiomyopathy.
One boy died of severe heart failure at the age of 6 years, the other was
transplanted because of severe heart failure at the age of 10 years. In
addition, focal palmoplantar keratoderma and woolly hair were apparent in both
boys. As similar phenotypes have been described in Naxos disease and Carvajal
syndrome, respectively, the genes for plakoglobin (JUP) and desmoplakin (DSP)
were screened for mutations using direct genomic sequencing. A novel homozygous
2 bp deletion was identified in an alternatively spliced region of DSP. The
deletion 5208_5209delAG led to a frameshift downstream of amino acid 1,736 with
a premature truncation of the predomit cardiac isoform DSP-1. This novel
homozygous truncating mutation in the isoform-1 specific region of the DSP
C-terminus caused Carvajal syndrome comprising severe early-onset heart failure
with features of non-compaction cardiomyopathy, woolly hair and an acantholytic
form of palmoplantar keratoderma in our patient. Congenital hair abnormality and
manifestation of the cutaneous phenotype in toddler age can help to identify
children at risk for cardiac death. Loss-of-function mutation of Jup has been associated with Naxos disease, which
is characterized by arrhythmogenic cardiomyopathy and the cutaneous disorder
palmoplantar keratoderma. Previously, we have shown that genetic ablation of Jup
in cardiomyocytes in mice leads to arrhythmogenic cardiomyopathy similar to
Naxos disease in humans. Currently, to determine the pathogenesis of Naxos
disease-associated keratoderma, we generated Jup mutant mice by inactivating Jup
restrictively in keratinocytes. Jup mutant mice largely recapitulated the
clinical features of human palmoplantar keratoderma: overcornification and
thickening of the epidermis. Jup mutant mice also suffered skin ulceration and
inflammation. Cell apoptosis and proliferation were significantly elevated in
Jup mutant epidermis. Ultrastructural analyses revealed the disruption of the
assembly of desmosomes and adherens junctions in Jup mutant epidermis. We also
demonstrated the compensational increase in β-catenin at Jup mutant cell-cell
junctions without altering its signaling activities. Our findings provide
important insights for understanding the pathogenesis of human palmoplantar
keratoderma. |
What is the role of edaravone in traumatic brain injury? | Edaravone, a free radical scavenger, has been shown to have neuroprotective effects after traumatic brain injury. In animal models, edaravone has been shown to reduce neuronal damage by scavenging reactive oxygen species (ROS), maintain intact the autoregulation of the cerebral vasculature, decrease neuronal loss, reduce programmed cell death, the presence of inflammatory cytokines, cerebral edema, and blood-brain barrier (BBB) permeability. Edaravone also protects against neurological deficits and memory deficits following traumatic brain injury. | Lipid peroxidation is caused by reactive oxygen species (ROS) and is involved in
traumatic brain injury (TBI). Consequently, a therapeutic strategy for TBI may
be to control lipid peroxidation. The only drug approved to date for blocking
lipid peroxidation is edaravone (MCI-186), a novel free-radical scavenger shown
to exert neuroprotective effects in acute ischemic stroke. Although edaravone
scavenges hydroxyl and nitric oxide radicals, its effect on alkoxyl radicals
(OR-), which also contribute to lipid peroxidation, is unknown. To date, the
study of free radicals in blood has been severely hampered by technical
difficulties in their detection. We used an in vitro and ex vivo electron spin
resoce (ESR) method employing 5,5-dimethyl-1-pyrroline-N-oxide as a spin trap
to investigate whether edaravone can scavenge OR-. By mixing either
methemoglobin or human blood with tert-butyl hydroperoxide, we found that this
technique can detect OR- generated in vitro. We also found that generated OR-
can be completely absorbed by administration of edaravone in vitro (400 microM).
Analysis of jugular venous blood collected from 17 TBI patients immediately
before and 20 minutes after the administration of edaravone (30 mg, i.v.)
revealed higher OR- levels in the untreated patients blood than in normal
control blood samples. However, treatment with edaravone suppressed these OR-
levels by 24.6% (radical intensity = 71.1 +/- 5.2-53.6 +/- 5.2; p < 0.01). Thus,
edaravone can scavenge OR- and significantly reduce levels of these radicals in
TBI patients. The novel ex vivo ESR method described here provides a valuable
clinical measure of oxidative stress. Edaravone (MCI-186) is a novel synthetic free radical scavenger intended to have
neuroprotective effect against ischemic insult. It is currently used on patients
with cerebral infarction. Here, we note beneficial pharmaceutical effects of
edaravone in rat experimental traumatic brain injury. Under specific
experimental conditions, edaravone minimized traumatic brain injury by
functioning as a synthetic antioxidant. Clinical trials testing the efficacy of
edaravone are warranted. Edaravone is a novel free radical scavenger that is clinically employed in
patients with acute cerebral infarction, but has not previously been used to
treat traumatic brain injury (TBI). In this study, we investigated the effect of
edaravone administration on rat TBI. In particular, we used immunohistochemistry
to monitor neural stem cell (NSC) proliferation around the area damaged by TBI.
Two separate groups of rats were administered saline or edaravone (3 mg/kg)
after TBI and then killed chronologically. We also used ex vivo techniques to
isolate NSCs from the damaged region and observed nestin-positive cells at 1, 3,
and 7 days following TBI in both saline- and edaravone-treated groups. At 3 days
following TBI in both groups, there were many large cells that morphologically
resembled astrocytes. At 1 and 7 days following TBI in the saline group, there
were a few small nestin-positive cells. However, in the edaravone group, there
were many large nestin-positive cells at 7 days following TBI. At 3 and 7 days
following TBI, the number of nestin-positive cells in the edaravone group
increased significantly compared with the saline group. There were many
single-stranded DNA-, 8-hydroxy-2'-deoxyguanosine-, and
4-hydroxy-2-nonenal-positive cells in the saline group following TBI, but only a
few such cells in the edaravone group following TBI. Furthermore, almost all
ssDNA-positive cells in the saline group co-localized with Hu, nestin, and glial
fibrillary acidic protein (GFAP) staining, but not in the edaravone group. In
the ex vivo study, spheres could only be isolated from injured brain tissue in
the saline group at 3 days following TBI. However, in the edaravone group,
spheres could be isolated from injured brain tissue at both 3 and 7 days
following TBI. The number of spheres isolated from injured brain tissue in the
edaravone group showed a significant increase compared with the saline group.
The spheres isolated from both saline and edaravone groups were immunopositive
for nestin, but not Tuj1 or vimentin. Moreover, the spheres differentiated into
Tuj1-, GFAP-, and O4-positive cells after 4 days in culture without bFGF. This
result indicated that the spheres were neurospheres composed of NSCs that could
differentiate into neurons and glia. Edaravone administration inhibited
production of free radicals known to induce neuronal degeneration and cell death
after brain injury, and protected nestin-positive cells, including NSCs, with
the potential to differentiate into neurons and glia around the area damaged by
TBI. OBJECTIVE: To study the protective effect of edaravone on severe traumatic brain
injury (TBI) and its potential mechanism.
METHODS: Two hundred and seventy-three male Sprague-Dawley (SD) rats were
divided randomly into four groups: control group (n=45), model group (n=88),
low-dose edaravone treatment group (n=72), high-dose edaravone treatment group
(n=68). TBI rat model was reproduced by weight-dropping injury. One, 6, 24, 48
and 72 hours after injury, changes in brain tissue were observed with light and
electron microscopy. The expression of phosphorylated extracellular
signal-regulated kinase 1/2 (p-ERK1/2) was determined by Western blotting. The
rate of neuron apoptosis was observed with immunohistochemistry and
terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL)
method. Learning and memory function assessments were performed with Morris
water maze from 7th day to 10th day after injury.
RESULTS: Compared with control group, a part of neurons in hippocampus displayed
histopathologic changes denoting necrosis 6, 24, 48 and 72 hours after injury.
The p-ERK1/2 expression level (pg/unit) increased 1, 6, 24, 48 hours after
injury (2.05 + or - 0.40, 4.40 + or - 0.96, 6.70 + or - 0.87, 3.67 + or - 0.28
vs. 0.40 + or - 0.04, 0.41 + or - 0.05, 0.43 + or - 0.06, 0.40 + or - 0.03), and
the number of apoptotic cells increased 6, 24, 48, 72 hours after injury (9.60 +
or - 2.69, 12.68 + or - 2.99, 16.94 + or - 3.92, 25.82 + or - 4.61 vs. 2.42 + or
- 0.38, 2.58 + or - 0.57, 2.74 + or - 0.56, 2.61 + or - 0.58); latent period to
find the safety platform (s) was significantly prolonged (119.8 + or - 25.0,
105.6 + or - 24.5, 98.5 + or - 21.8, 92.0 + or - 19.5 vs. 49.5 + or - 7.5, 32.7
+ or - 6.3, 25.8 + or - 6.5, 24.8 + or - 5.5, all P<0.05). After treatment with
edaravone, the degree of morphological injury, p-ERK1/2 level and number of
apoptotic neurons decreased, latent period to find the safety platform was
significantly shortened (in low-dose edaravone treatment group, p-ERK1/2
expression level at 6, 24, 48 hours was 2.46 + or - 0.22, 4.00 + or - 0.84, 2.38
+ or - 0.32, and in high-dose edaravone treatment group was 1.67 + or - 0.15,
1.86 + or - 0.38, 1.27 + or - 0.28; in low-dose edaravone treatment group, the
apoptotic cells at 6, 24, 48, 72 hours was 5.20 + or - 1.23, 7.10 + or - 1.72,
9.54 + or - 1.36, 14.12 + or - 3.19, and in high-dose edaravone treatment group
was 3.40 + or - 0.49 , 4.39 + or - 0.73, 5.02 + or - 1.12, 8.78 + or - 2.16; in
low-dose edaravone treatment group, latent period to find the safety platform at
7-10 days was 94.8 + or - 22.8, 65.2 + or - 19.0, 62.0 + or - 16.7, 59.5 + or -
15.6, and in high-dose edaravone treatment group it was 81.5 + or - 20.7, 55.4 +
or - 18.5, 40.0 + or - 12.3, 32.2 + or - 11.0, all P<0.05). High-dose edaravone
showed a better effect (all P<0.05).
CONCLUSION: Edaravone gives good therapeutic effect on severe TBI, and the
molecular mechanism is related to attenuation of ERK1/2 pathway and neuronal
apoptosis following severe brain trauma. 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), an effective free radical
scavenger, provides neuroprotection in stroke models and patients. In this
study, we investigated its neuroprotective effects in a chronic rotenone rat
model for Parkinson's disease. Here we showed that a five-week treatment with
edaravone abolished rotenone's activity to induce catalepsy, damage mitochondria
and degenerate dopamine neurons in the midbrain of rotenone-treated rats. This
abolishment was attributable at least partly to edaravone's inhibition of
rotenone-induced reactive oxygen species production or apoptotic promoter Bax
expression and its up-regulation of the vesicular monoamine transporter 2
(VMAT2) expression. Collectively, edaravone may provide novel clinical
therapeutics for PD. Edaravone was originally developed as a potent free radical scavenger and has
been widely used to treat cerebral infarction in Japan since 2001. Several free
radical scavengers have been developed and some of them have progressed to
clinical trials for the treatment of cerebral infarction. One such scavenger,
edaravone, has been approved by the regulatory authority in Japan for the
treatment of patients with cerebral infarction. Of particular interest is the
ability of edaravone to diffuse into the central nervous system in various
neurologic diseases. Aside from its hydroxyl radical scavenging effect,
edaravone has been found to have beneficial effects on inflammation, matrix
metalloproteinases, nitric oxide production and apoptotic cell death.
Concordantly, edaravone has been found to have neuroprotective effects in a
number of animal models of disease, including stroke, spinal cord injury,
traumatic brain injury, neurodegenerative diseases and brain tumors. The proven
safety of edaravone following 9 years of use as a free radical scavenger
suggests that it may have potential for development into an effective treatment
of multiple neurologic conditions in humans. Traumatic axonal injury (TAI), a feature of traumatic brain injury (TBI),
progressively evolves over hours through impaired axonal transport and is
thought to be a major contributor to cognitive dysfunction. In spite of various
studies suggesting that pharmacologic or physiologic interventions might reduce
TAI, clinical neuroprotective treatments are still unavailable. Edaravone, a
free radical scavenger, has been shown to exert neuroprotective effects in
animal models of several brain disorders. In this study, to evaluate whether
edaravone suppresses TAI following TBI, mice were subjected to weight drop
injury and had either edaravone (3.0mg/kg) or saline administered intravenously
immediately after impact. Axonal injury and oxidative stress were assessed using
immunohistochemistry with antibodies against amyloid precursor protein, a marker
of impaired axonal transport, and with 8-hydroxy-2'-deoxyguanosine, a marker of
oxidative DNA damage. Edaravone significantly suppressed axonal injury and
oxidative stress in the cortex, corpus callosum, and hippocampus 24h after
injury. The neuroprotective effects of edaravone were observed in mice receiving
1.0, 3.0, or 10mg/kg of edaravone immediately after impact, but not after
0.3mg/kg of edaravone. With treatment 1h after impact, axonal injury was also
significantly suppressed and this therapeutic effect persisted up to 6h after
impact. Furthermore, behavioral tests performed 9 days after injury showed
memory deficits in saline-treated traumatized mice, which were not evident in
the edaravone-treated group. These results suggest that edaravone protects
against memory deficits following TBI and that this protection is mediated by
suppression of TAI and oxidative stress. Therapeutic hypothermia (TH) is thought to be due to the downregulation of free
radical production, although the details of this process remain unclear. Here,
we investigate changes in oxidative stress and endogenous biological antioxidant
potential during TH in patients with post-cardiac arrest syndrome (PCAS).
Nineteen PCAS patients were enrolled in the study. Brain temperature was
decreased to the target temperature of 33°C, and it was maintained for 24 h.
Patients were rewarmed slowly (0.1°C/h, <1°C/day). The generation of reactive
oxygen metabolites (ROMs) was evaluated in plasma samples by d-ROM test. Plasma
antioxidant capacity was measured by the biological antioxidant potential (BAP)
test. Levels of d-ROMs and BAP levels during the hypothermic stage (33°C) were
suppressed significantly compared with pre-TH induction levels (P < 0.05), while
both d-ROM and BAP levels increased with rewarming (33-36°C) and were correlated
with brain temperature. Clinical monitoring of oxidative stress and antioxidant
potential is useful for evaluating the redox state of patients undergoing TH
after PCAS. Additional therapy to support the antioxidant potential in the
rewarming stage following TH may reduce some of the observed side effects
associated with the use of TH. Author information:
(1)Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Rua Ramiro
Barcelos, 2350, 90035-903 Porto Alegre, RS, Brazil ; PPG Ciências Farmacêiticas,
Universidade Federal do Piauí, 64049-550 Terezina, PI, Brazil ; Genetics
Service, Hospital Universitário, Universidade Federal de Santa Catarina,
88040-900 Florianópolis, SC, Brazil. |
Subsets and Splits