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To what extent does HPV vaccination reduce the risk for cervical cancer? | The most effective strategy therein was vaccination of 12-year-olds, plus a temporary 12-24-year-old catch-up program covering both sexes; whereby HPV 6/11/16/18-related cervical cancer, high-grade cervical precancer, and genital wart incidence was reduced by 84-98% during year 50 following vaccine introduction. Vaccine efficacy in prevention of CIN 2 or higher lesions in HPV 16 or HPV 18 negative women, who received all vaccination doses, ranges between 98% and 100% | Prophylactic HPV vaccination provides an opportunity to profoundly affect
cervical cancer incidence worldwide. The quadrivalent HPV VLP 6, 11, 16, 18
vaccine (Gardasil) and the bivalent HPV VLP 16, 18 vaccine (Cervarix) are
effective for prevention of HPV infection and cervical precancerous lesions. The
quadrivalent vaccine is also effective for prevention of vulvar and vaginal
lesions and genital warts. With the introduction of the vaccines general issues
have to be raised such as optimal age for vaccination, duration of protection
after vaccination, impact on cervical cancer screening, vaccination of males and
feasibility of application to developing countries. The prospects of a vaccine
which will protect against the most common viral sexually transmitted infection
and thereby, protect against the complications of HPV infection such as cervical
cancer is extremely appealing. The success of HPV vaccination as a major public
health prevention opportunity, however, will entirely depend on efficient
infrastructures to deliver the vaccines and on the acceptance by individuals,
parents and health care providers. With the introduction of cervical screening programs, the incidence and
mortality of cervical cancer has been drastically reduced. Techniques such as
the traditional Papanicolaou test and the newer liquid-based cytology allow for
the early detection of cervical abnormalities prior to the development of
invasive cervical cancer. As oncogenic human papillomavirus (HPV) infection is
necessary for cervical cancer, HPV-DNA testing has also been proposed as a
routine screening method for the general population. Screening limitations, such
as adherence, test sensitivity and specificity, access, and cost-effectiveness
are reflected in current screening guidelines. The development of prophylactic
cervical cancer vaccines is a major milestone in cervical cancer prevention.
These vaccines protect against the initial infection of certain oncogenic HPV
types, and therefore prevent the development of cervical dysplasia, precancerous
lesions, and cervical cancer. Considering routine cervical cancer vaccination in
adolescent girls, screening guidelines must adapt in order to retain efficient
and cost-effective prevention measures. Although the true epidemiological and
economic impact of cervical cancer vaccines cannot be immediately realized,
mathematical models predict various scenarios in which vaccination, in addition
to cervical screening, will be cost-effective and further reduce cervical cancer
disease. Cervical screening has resulted in a major reduction in the incidence and
mortality of cervical cancer in the UK and other developed countries.
Nevertheless approximately 2700 women present with cervical cancer in the UK
each year with mortality in excess of 1000 cases. Prophylactic HPV vaccination
against HPV 16 and 18 has been shown to be highly effective in preventing HPV
related maligcy in clinical trials. Newly introduced HPV vaccination
programmes in the UK and elsewhere are ultimately likely to result in a further
significant reduction in the incidence and mortality of cervical cancer. These
vaccination programmes will be most effective in early adolescence when
prevalence of HPV infection is low. Consequently, vaccination programmes in the
UK have been initially targeted at 12 to 13-year olds. In Australia favourable
estimates of cost effectiveness have supported funding of a 'catch-up' programme
to 26 years. In the UK the catch up programme has for the present been
restricted to 18 years for cost effectiveness reasons. In addition the value of
HPV vaccination beyond 26 years has not yet been fully clarified. Nevertheless
women up to 45 years of age have been shown to exhibit strong immune responses
to the bivalent HPV vaccine which might be expected to reduce the risk of HPV
re-infection and address the second peak of HPV related maligcy in later
life, evident over 45 years of age. Early data from randomised trials testing
the quadrivalent HPV vaccine in women over 25 years has suggested high vaccine
efficacy comparable to younger women. This paper will explore the evidence
supporting HPV vaccination in HPV naïve and HPV exposed sexually active women up
to 26 years and beyond this age group. INTRODUCTION: Cervical cancers (CC) demonstrate the second highest incidence of
female cancers in Malaysia. The costs of chronic management have a high impact
on nation's health cost and patient's quality of life that can be avoided by
better screening and HPV vaccination.
METHODOLOGY: Respondents were interviewed from six public Gynecology-Oncology
hospitals. Methods include experts' panel discussions to estimate treatment
costs by severity and direct interviews with respondents using costing and SF-36
quality of life (QOL) questionnaires. Three options were compared i.e. screening
via Pap smear; quadrivalent HPV Vaccination and combined strategy (screening
plus vaccination). Scenario based sensitivity analysis using screening
population coverage (40-80%) and costs of vaccine (RM 300-400/dose) were
calculated.
RESULTS: 502 cervical pre invasive and invasive cervical cancer (ICC) patients
participated in the study. Mean age was 53.3 +/- 11.2 years, educated till
secondary level (39.4%), Malays (44.2%) and married for 27.73 +/- 12.1 years.
Life expectancy gained from vaccination is 13.04 years and average Quality
Adjusted Life Years saved (QALYs) is 24.4 in vaccinated vs 6.29 in unvaccinated.
Cost/QALYs for Pap smear at base case is RM 1,214.96/QALYs and RM 1,100.01 at
increased screening coverage; for HPV Vaccination base case is at RM 35,346.79
and RM 46,530.08 when vaccination price is higher. In combined strategy, base
case is RM 11,289.58; RM 7,712.74 at best case and RM 14,590.37 at worst case
scenario. Incremental cost-effectiveness ratio (ICER) showed that screening at
70% coverage or higher is highly cost effective at RM 946.74 per QALYs saved and
this is followed by combined strategy at RM 35,346.67 per QALYs saved.
CONCLUSION: Vaccination increase life expectancy with better QOL of women when
cancer can be avoided. Cost effective strategies will include increasing the Pap
smear coverage to 70% or higher. Since feasibility and long term screening
adherence is doubtful among Malaysian women, vaccination of young women is a
more cost effective strategy against cervical cancers. The discovery that the Human PapillomaVirus (HPV) is the necessary cause of
cervical cancer has led to the development of prophylactic vaccines. Cervical
cancer is the second most common cause of death from cancer among young women in
Europe: mortality is still high, despite its important reduction due to
screening programs for early detection. Besides cervical cancer, HPV is
responsible for a significant proportion of other anogenital cancers and an
increasing number of oropharyngeal cancers, representing together an at least
equal burden compared to cervical cancer. HPV is also responsible for conditions
such as condyloma acuminata (genital warts) and recurrent respiratory
papillomatosis. Organized vaccination programs against HPV have the potential to
prevent about 70% of cervical cancers and the vast majority of the other
HPV-related conditions. Recommendations for HPV vaccination of at least one
cohort of females have been issued in nearly all western European countries, and
national/regional publicly funded vaccination programs have been introduced in
most of them. Different approaches have been chosen for the implementation of
HPV vaccination, based on the organization of each country's health care system.
A brief outline of these programs in Europe is presented. As for all preventive
public health interventions, high coverage of the target population with HPV
vaccines pre-exposure is essential to achieve maximum reduction of cases:
therefore, in order to obtain the maximum and most equitable coverage and future
benefit, programs targeting adolescents before exposure to HPV should be
preferred and population-based. Catch-up programs should also be implemented
wherever possible, in order to deliver more and even earlier benefits, and
effective communication strategies need to be adopted. EINLEITUNG: Notwendige Voraussetzung für die Entstehung von Zervixkarzinomen ist
eine persistierende Infektion mit humanen Papillomaviren (HPV). Die HPV-Typen 16
und 18 verursachen mit etwa 70% den überwiegenden Teil der Zervixkarzinome. Seit
2006/2007 stehen zwei Impfstoffe gegen HPV 16 und 18 zur Verfügung.
FRAGESTELLUNG: Wie effektiv ist die HPV-Impfung hinsichtlich der Reduktion von
Zervixkarzinomen bzw. ihren Vorstufen (CIN)? Stellt die HPV-Impfung eine
kosteneffektive Ergänzung zur derzeitigen Screeningpraxis dar? Gibt es
Unterschiede bezüglich der Kosten-Effektivität zwischen den beiden verfügbaren
Impfstoffen? Sollte aus gesundheitsökonomischer Perspektive eine Empfehlung für
den Einsatz der HPV-Impfung gegeben werden? Falls ja, welche Empfehlungen
bezüglich der Ausgestaltung einer Impfstrategie lassen sich ableiten? Welche
ethischen, sozialen und juristischen Implikationen sind zu berücksichtigen?
METHODEN: Basierend auf einer systematischen Literaturrecherche werden
randomisierte kontrollierte Studien zur Wirksamkeit der HPV-Impfungen für die
Prävention von Zervixkarzinomen bzw. deren Vorstufen, den zervikalen
intraepithelialen Neoplasien, identifiziert. Gesundheitsökonomische
Modellierungen werden zur Beantwortung der ökonomischen Fragestellungen
herangezogen. Die Beurteilung der Qualität der medizinischen und ökonomischen
Studien erfolgt mittels anerkannter Standards zur systematischen Bewertung
wissenschaftlicher Studien
ERGEBNISSE: Bei zu Studienbeginn HPV 16/18 negativen Frauen, die alle Impfdosen
erhalten haben, liegt die Wirksamkeit der Impfungen gegen HPV 16/18-induzierten
CIN 2 oder höher bei 98% bis 100%. Nebenwirkungen der Impfung sind vor allem mit
der Injektion assoziierte Beschwerden (Rötungen, Schwellungen, Schmerzen). Es
gibt keine signifikanten Unterschiede für schwerwiegende unerwünschte Ereignisse
zwischen Impf- und Placebogruppe. Die Ergebnisse der Basisfallanalysen der
gesundheitsökonomischen Modellierungen reichen bei ausschließlicher
Berücksichtigung direkter Kostenkomponenten von ca. 3.000 Euro bis ca. 40.000
Euro pro QALY (QALY = Qualitätskorrigiertes Lebensjahr), bzw. von ca. 9.000 Euro
bis ca. 65.000 Euro pro LYG (LYG = Gewonnenes Lebensjahr).
DISKUSSION: Nach den Ergebnissen der eingeschlossenen Studien sind die
verfügbaren HPV-Impfstoffe wirksam zur Prävention gegen durch HPV 16/18
verursachte prämaligne Läsionen der Zervix. Unklar ist derzeit noch die Dauer
des Impfschutzes. Hinsichtlich der Nebenwirkungen ist die Impfung als sicher
einzustufen. Allerdings ist die Fallzahl der Studien nicht ausreichend groß, um
das Auftreten sehr seltener Nebenwirkungen zuverlässig zu bestimmen. Inwieweit
die HPV-Impfung zur Reduktion der Inzidenz und Mortalität des Zervixkarzinoms in
Deutschland führen wird, hängt nicht allein von der klinischen Wirksamkeit der
Impfstoffe ab, sondern wird von einer Reihe weiterer Faktoren wie der Impfquote
oder den Auswirkungen der Impfungen auf die Teilnahmerate an den bestehenden
Screeningprogrammen determiniert. Infolge der Heterogenität der methodischen
Rahmenbedingungen und Inputparameter variieren die Ergebnisse der
gesundheitsökonomischen Modellierungen erheblich. Fast alle Modellanalysen
lassen jedoch den Schluss zu, dass die Einführung einer Impfung mit lebenslanger
Schutzdauer bei Fortführung der derzeitigen Screeningpraxis als kosteneffektiv
zu bewerten ist. Eine Gegenüberstellung der beiden verschiedenen Impfstoffe
ergab, dass die Modellierung der tetravalenten Impfung bei der Berücksichtigung
von QALY als Ergebnisparameter in der Regel mit einem niedrigeren (besseren)
Kosten-Effektivitäts-Verhältnis einhergeht als die Modellierung der bivalenten
Impfung, da auch Genitalwarzen berücksichtigt werden. In Sensitivitätsanalysen
stellten sich sowohl die Schutzdauer der Impfung als auch die Höhe der
Diskontierungsrate als wesentliche Einflussparameter der Kosten-Effektivität
heraus.
SCHLUSSFOLGERUNG: Die Einführung der HPV-Impfung kann zu einem verringerten
Auftreten von Zervixkarzinomen bei geimpften Frauen führen. Jedoch sollten die
Impfprogramme von weiteren Evaluationen begleitet werden, um die langfristige
Wirksamkeit und Sicherheit beurteilen sowie die Umsetzung der Impfprogramme
optimieren zu können. Von zentraler Bedeutung sind hohe Teilnahmeraten sowohl an
den Impfprogrammen als auch - auch bei geimpften Frauen - an den
Früherkennungsuntersuchungen. Da die Kosten-Effektivität entscheidend von der
Schutzdauer, die bislang ungewiss ist, beeinflusst wird, ist eine abschließende
Beurteilung der Kosten-Effektivität der HPV-Impfung nicht möglich. Eine
langfristige Schutzdauer ist eine bedeutende Vorraussetzung für die
Kosten-Effektivität der Impfung. Der Abschluss einer Risk-Sharing-Vereinbarung
zwischen Kostenträgern und Herstellerfirmen stellt eine Option dar, um die
Auswirkungen der Unsicherheit der Schutzdauer auf die Kosten-Effektivität zu
begrenzen. It will likely be more than 20 years before there is unequivocal evidence
available that HPV vaccination decreases the incidence of invasive cervical
cancer. However, existing data strongly suggests that as many as 440,000
cervical cancer cases and 220,000 deaths due to this maligcy will be
prevented with the establishment of an effective worldwide HPV immunization
program. |
Have germline variants been associated to colorectal cancer? | Yes. Whole-genome sequencing (WGS) applied to medical research has revealed how germline variants and mutations may be associated with colorectal cancer. It is likely that this level of knowledge can be translated into predictions of predisposition. | BACKGROUND: The patient with 10 or more adenomas in the colon poses a diagnostic
challenge. Beside germline mutations in the APC and MUTYH genes, only four cases
of mosaic APC mutations have been reported.
AIM: Given the relatively high frequency of de novo APC mutations in familial
adenomatous polyposis (FAP), an investigation was carried out into whether the
proportion of somatic mosaic APC mutations is currently underestimated.
METHODS: Between 1 January 1994 and 31 December 2005 germline mutation analysis
was performed in 599 consecutive index patients with polyposis coli referred for
diagnostic APC scanning using a combination of denaturing gradient gel
electrophoresis (DGGE) and protein truncation test (PTT). Variants were analysed
by direct sequencing with primers flanking those used for DGGE and PTT, and
quantified using pyrosequencing.
RESULTS: Scrutinizing the molecular genetic results and family data of 242 index
patients with pathogenic APC mutations led to the identification of 10 mosaic
cases (4%). C>T transitions were observed in CGA sites in four of the 10 cases
with somatic mosaicism, which is significantly more than 26 of the 232
non-mosaic cases (p = 0.02). Phenotypes of patients with somatic mosaicism
ranged from an attenuated form of polyposis coli to florid polyposis with major
extracolonic manifestations.
CONCLUSIONS: Mosaicism occurs in a significant number of APC mutations and it is
estimated that one-fifth of the de novo cases of FAP are mosaic. Clinically, the
severity of manifestations in offspring and the recurrence risk for siblings of
apparently sporadic polyposis patients may be underestimated due to parental APC
mosaicism. PURPOSE: Familial adenomatous polyposis is a phenotypically heterogeneous
disease predisposing to colorectal cancer. It is domitly transmitted, when
associated with the APC gene, and recessively inherited, when associated with
MUTYH gene. We searched for APC and MUTYH germline alterations in Italian and
Greek patients with attenuated polyposis, a phenotypic variant whose genetic
cause remains unknown in many cases.
METHODS: We studied 26 unrelated patients (and 16 relatives) with multiple
colorectal adenomas (3-100, by endoscopic analysis) that had screened APC
mutation-negative by protein truncation test. We searched for APC rearrangements
by multiplex ligation-dependent probe amplification and for MUTYH mutations by
sequencing. We performed a screening of five MUTYH recurrent pathogenic
mutations in 501 Italian and 144 Greek controls.
RESULTS: One patient proved to carry an APC whole-gene deletion; 4 of 25 (16%)
patients showed biallelic and 3 of 25 (12%) monoallelic MUTYH mutations. In the
three heterozygous subjects no pathogenetic variants were found in OGG1, MTH1,
APE1, MSH2, and MSH6 genes. Frequency assessment of MUTYH mutations in healthy
subjects showed that only Y165C and G382D reach a subpolymorphic frequency.
CONCLUSION: Attenuated polyposis patients without "conventional" APC mutations
are genetically heterogeneous, and the phenotype is not directly related to the
germline defect. Therefore, the families' appropriate management requires an
accurate genetic and clinical investigation. PURPOSE: There is substantial germline genetic variability within angiogenesis
pathway genes, thereby causing interindividual differences in angiogenic
capacity and resistance to antiangiogenesis therapy. We investigated germline
polymorphisms in genes involved in VEGF-dependent and -independent angiogenesis
pathways to predict clinical outcome and tumor response in metastatic colorectal
cancer (mCRC) patients treated with bevacizumab and oxaliplatin-based
chemotherapy.
EXPERIMENTAL DESIGN: A total of 132 patients treated with first-line bevacizumab
and FOLFOX or XELOX were included in this study. Genomic DNA was isolated from
whole-blood samples by PCR-RFLP or direct DNA sequencing. The endpoints of the
study were progression-free survival (PFS), overall survival (OS), and response
rate (RR).
RESULTS: The minor alleles of EGF rs444903 A>G and IGF-1 rs6220 A>G were
associated with increased OS and remained significant in multivariate Cox
regression analysis (HR: 0.52; 95% CI: 0.31-0.87; adjusted P = 0.012 and HR:
0.60; 95% CI: 0.36-0.99; adjusted P = 0.046, respectively). The minor allele of
HIF1α rs11549465 C>T was significantly associated with increased PFS but lost
its significance in multivariate analysis. CXCR1 rs2234671 G>C, CXCR2 rs2230054
T>C, EGFR rs2227983 G>A, and VEGFR-2 rs2305948 C>T predicted tumor response,
with CXCR1 rs2234671 G>C remaining significant in multiple testing (P(act) =
0.003).
CONCLUSION: In this study, we identified common germline variants in
VEGF-dependent and -independent angiogenesis genes predicting clinical outcome
and tumor response in patients with mCRC receiving first-line bevacizumab and
oxaliplatin-based chemotherapy. PURPOSE: Recent evidence suggests that cancer stem cells (CSC) are responsible
for key elements of colon cancer progression and recurrence. Germline variants
in CSC genes may result in altered gene function and/or activity, thereby
causing interindividual differences in a patient's tumor recurrence capacity and
chemoresistance. We investigated germline polymorphisms in a comprehensive panel
of CSC genes to predict time to tumor recurrence (TTR) in patients with stage
III and high-risk stage II colon cancer.
EXPERIMENTAL DESIGN: A total of 234 patients treated with 5-fluorouracil-based
chemotherapy at the University of Southern California were included in this
study. Whole blood samples were analyzed for germline polymorphisms in genes
that have been previously associated with colon CSC (CD44, Prominin-1, DPP4,
EpCAM, ALCAM, Msi-1, ITGB1, CD24, LGR5, and ALDH1A1) by PCR-RFLP or direct
DNA-sequencing.
RESULTS: The minor alleles of CD44 rs8193 C>T, ALCAM rs1157 G>A, and LGR5
rs17109924 T>C were significantly associated with increased TTR (9.4 vs. 5.4
years; HR, 0.51; 95% CI: 0.35-0.93; P = 0.022; 11.3 vs. 5.7 years; HR, 0.56; 95%
CI: 0.33-0.94; P = 0.024, and 10.7 vs. 5.7 years; HR, 0.33; 95% CI: 0.12-0.90; P
= 0.023, respectively) and remained significant in the multivariate analysis
stratified by ethnicity. In recursive partitioning, a specific gene variant
profile including LGR5 rs17109924, CD44 rs8193, and ALDH1A1 rs1342024
represented a high-risk subgroup with a median TTR of 1.7 years (HR, 6.71, 95%
CI: 2.71-16.63, P < 0.001).
CONCLUSION: This is the first study identifying common germline variants in
colon CSC genes as independent prognostic markers for stage III and high-risk
stage II colon cancer patients. We describe an approach for targeted genome resequencing, called
oligonucleotide-selective sequencing (OS-Seq), in which we modify the
immobilized lawn of oligonucleotide primers of a next-generation DNA sequencer
to function as both a capture and sequencing substrate. We apply OS-Seq to
resequence the exons of either 10 or 344 cancer genes from human DNA samples. In
our assessment of capture performance, >87% of the captured sequence originated
from the intended target region with sequencing coverage falling within a
tenfold range for a majority of all targets. Single nucleotide variants (SNVs)
called from OS-Seq data agreed with >95% of variants obtained from whole-genome
sequencing of the same individual. We also demonstrate mutation discovery from a
colorectal cancer tumor sample matched with normal tissue. Overall, we show the
robust performance and utility of OS-Seq for the resequencing analysis of human
germline and cancer genomes. To uncover pathogenic deep intronic variants in patients with colorectal
adenomatous polyposis, in whom no germline mutation in the APC or MUTYH genes
can be identified by routine diagnostics, we performed a systematic APC
messenger RNA analysis in 125 unrelated mutation-negative cases. Overall, we
identified aberrant transcripts in 8% of the patients (familial cases 30%;
early-onset manifestation 21%). In eight of them, two different out-of-frame
pseudoexons were found consisting of a 167-bp insertion from intron 4 in five
families with a shared founder haplotype and a 83-bp insertion from intron 10 in
three patients. The pseudoexon formation was caused by three different
heterozygous germline mutations, which are supposed to activate cryptic splice
sites. In conclusion, a few deep intronic mutations contribute substantially to
the APC mutation spectrum. Complementary DNA analysis and/or target sequencing
of intronic regions should be considered as an additional mutation discovery
approach in polyposis patients. Many individuals with multiple or large colorectal adenomas or early-onset
colorectal cancer (CRC) have no detectable germline mutations in the known
cancer predisposition genes. Using whole-genome sequencing, supplemented by
linkage and association analysis, we identified specific heterozygous POLE or
POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no
controls. The variants associated with susceptibility, POLE p.Leu424Val and
POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated
with endometrial cancer predisposition. The mutations map to equivalent sites in
the proofreading (exonuclease) domain of DNA polymerases ɛ and δ and are
predicted to cause a defect in the correction of mispaired bases inserted during
DNA replication. In agreement with this prediction, the tumors from mutation
carriers were microsatellite stable but tended to acquire base substitution
mutations, as confirmed by yeast functional assays. Further analysis of
published data showed that the recently described group of hypermutant,
microsatellite-stable CRCs is likely to be caused by somatic POLE mutations
affecting the exonuclease domain. |
Can Alzheimer's disease related miRNAs be detected in patients' blood? | Yes. It has been demonstrated that blood miRNAs could be useful as biomarkers in Alzheimer's disease. | Various coding genes representing multiple functional categories are
downregulated in blood mononuclear cells (BMC) of patients with sporadic
Alzheimer disease (AD). Noncoding microRNAs (miRNA) regulate gene expression by
degrading messages or inhibiting translation. Using BMC as a paradigm for the
study of systemic alterations in AD, we investigated whether peripheral miRNA
expression is altered in this condition. MicroRNA levels were assessed using the
microRNA microarray (MMChip) containing 462 human miRNA, and the results
validated by real time PCR. Sixteen AD patients and sixteen normal elderly
controls (NEC) were matched for ethnicity, age, gender and education. The
expression of several BMC miRNAs was found to increase in AD relative to NEC
levels, and may differ between AD subjects bearing one or two APOE4 alleles. As
compared to NEC, miRNAs significantly upregulated in AD subjects and confirmed
by qPCR were miR-34a and 181b. Predicted target genes downregulated in Alzheimer
BMC that correlated with the upregulated miRNAs were largely represented in the
functional categories of Transcription/Translation and Synaptic Activity.
Several miRNAs targeting the same genes were within the functional category of
Injury response/Redox homeostasis. Taken together, induction of microRNA
expression in BMC may contribute to the aberrant systemic decline in mRNA levels
in sporadic AD. An association study of heterogeneous nuclear ribonucleoprotein (hnRNP)-A1 was
carried out in a population of 274 patients with frontotemporal lobar
degeneration (FTLD) and 287 with Alzheimer disease (AD) as compared with 344
age- and gender-matched controls. In addition, we evaluated expression levels of
hnRNP-A1 and its regulatory microRNA (miR)-590-3p in blood cells from patients
and controls. A statistically significant increased frequency of the hnRNP-A1
rs7967622 C/C genotype was observed in FTLD, but not in AD, in patients as
compared to controls (23.0 versus 15.4%; p = 0.022, odds ratio [OR] 1.64,
confidence interval [CI] 1.09-2.46). Stratifying according to gender, a
statistically significant increased frequency of the hnRNP-A1 rs7967622 C/C
genotype was observed in male patients as compared to male controls (23.1 versus
11.3%; p = 0.015, OR 2.36, CI 1.22-4.58 but not in females. Considering the
rs4016671 single-nucleotide polymorphism (SNP), all patients and controls were
wild type. Significantly increased hnRNP-A1 relative expression levels in
peripheral blood mononuclear cells (PBMCs) was observed in patients with AD, but
not with FTLD, as compared to controls (2.724 ± 0.570 versus 1.076 ± 0.187,
p = 0.021). Decreased relative expression levels of hsa-miR-590-3p was observed
in patients with AD versus controls (0.685 ± 0.080 versus 0.931 ± 0.111,
p = 0.079), and correlated negatively with hnRNP-A1 mRNA levels (r = -0.615,
p = 0.0237). According to these findings, hnRNP-A1 and its transcription
regulatory factor miR-590-3p are disregulated in patients with AD, and the
hnRNP-A1 rs7967622 C/C genotype is likely a risk factor for FTLD in male
populations. The oxidized LDL receptor 1 gene (OLR1) rs1050283 single nucleotide polymorphism
(SNP) has been previously shown to be associated with Alzheimer's disease (AD).
An association analysis of OLR1 was carried out in a population of 443 patients
with AD as compared with 393 age-matched controls. In addition, an expression
analysis of OLR1 and its regulatory hsa-miR369-3p was performed in peripheral
mononuclear blood cells (PBMC) from 20 patients and 15 controls. Logistic
regression analysis, adjusted for gender and apolipoprotein E (ApoE) status,
showed a statistically significant association of OLR1 rs1050283 under the
assumption of a domit model (CC and CT individuals versus TT: p = 0.014, OR:
1.50, 95%CI: 1.08-2.08) and a genotypic model (TC versus TT: p = 0.002, OR:
1.61, 95%CI: 1.14-2.26). No significant differences in OLR1 expression was
observed between patients and controls (p > 0.05). However, stratifying patients
according to the rs1050283 status, significantly decreased relative PBMC
expression levels of OLR1 were observed in carriers of CC+CT genotypes as
compared with TT carriers (0.13 ± 0.013 versus 0.46 ± 0.028, p = 0.022), whereas
no differences in relative expression levels of the hsa-miR369-3p were observed
(p > 0.05). The effect observed was not due to the presence of the ApoE ε4
allele. The OLR1 rs1050283 SNP likely acts as a risk factor for sporadic AD. The
presence of at least one C allele is associated with a decreased expression of
OLR1 mRNA in the absence of hsa-miR369-3p de-regulation, suggesting that the
presence of the polymorphic allele influences the binding of hsa-miR369-3p to
its 3'UTR consensus sequence. Nevertheless, the limited power of the study
requires further investigations with a larger sample size. There is an urgent need to identify non-invasive biomarkers for the detection of
sporadic Alzheimer's disease (AD). We previously studied microRNAs (miRNAs) in
AD autopsy brain samples and reported a connection between miR-137, -181c, -9,
-29a/b and AD, through the regulation of ceramides. In this study, the potential
role of these miRNAs as diagnostic markers for AD was investigated. We
identified that these miRNAs were down-regulated in the blood serum of probable
AD patients. The levels of these miRNAs were also reduced in the serum of AD
risk factor models. Although the ability of these miRNAs to conclusively
diagnose for AD is currently unknown, our findings suggest a potential use for
circulating miRNAs, along with other markers, as non-invasive and relatively
inexpensive biomarkers for the early diagnosis of AD, however, with further
research and validation. The contribution of the autosomal domit mutations to the etiology of familial
Alzheimer's disease (AD) is well characterized. However, the molecular
mechanisms contributing to sporadic AD are less well understood. Increased
ceramide levels have been evident in AD patients. We previously reported that
increased ceramide levels, regulated by increased serine palmitoyltransferase
(SPT), directly mediate amyloid β (Aβ) levels. Therefore, we inhibited SPT in an
AD mouse model (TgCRND8) through subcutaneous administration of L-cylcoserine.
The cortical Aβ₄₂ and hyperphosphorylated tau levels were down-regulated with
the inhibition of SPT/ceramide. Positive correlations were observed among
cortical SPT, ceramide, and Aβ₄₂ levels. With no evident toxic effects observed,
inhibition of SPT could be a safe therapeutic strategy to ameliorate the AD
pathology. We previously observed that miR-137, -181c, -9, and 29a/b
post-transcriptionally regulate SPT levels, and the corresponding miRNA levels
in the blood sera are potential diagnostic biomarkers for AD. Here, we observe a
negative correlation between cortical Aβ₄₂ and sera Aβ₄₂, and a positive
correlation between cortical miRNA levels and sera miRNA levels suggesting their
potential as noninvasive diagnostic biomarkers. |
Which is the clinical meaning of the presence of delayed enhancement in patients with hypertrophic cardiomyopathy? | Occurrence of myocardial fibrosis in hypertrophic cardiomyopathy is associated with left atrial and ventricular dysfunction as well as with the severity of heart failure symptoms and arrhythmic risk factors. | OBJECTIVES: Our aim was to determine whether myocardial fibrosis, detected by
cardiovascular magnetic resoce (CMR), represents an arrhythmogenic substrate
in hypertrophic cardiomyopathy (HCM).
BACKGROUND: Myocardial fibrosis is identified frequently in HCM; however, the
clinical significance of this finding is uncertain.
METHODS: We studied prevalence and frequency of tachyarrhythmias on 24-h
ambulatory Holter electrocardiogram (ECG) with regard to delayed enhancement
(DE) on contrast-enhanced CMR in 177 HCM patients (age 41 +/- 16 yrs; 95%
asymptomatic or mildly symptomatic).
RESULTS: Premature ventricular contractions (PVCs), couplets, and nonsustained
ventricular tachycardia (NSVT) were more common in patients with DE than those
without DE (PVCs: 89% vs. 72%; couplets: 40% vs. 17%; NSVT: 28% vs. 4%; p <
0.0001 to 0.007). Patients with DE also had greater numbers of PVCs (202 +/- 655
vs. 116 +/- 435), couplets (1.9 +/- 5 vs. 1.2 +/- 10), and NSVT runs (0.4 +/-
0.8 vs. 0.06 +/- 0.4) than non-DE patients (all p < 0.0001); DE was an
independent predictor of NSVT (relative risk 7.3, 95% confidence interval 2.6 to
20.4; p < 0.0001). However, extent (%) of DE was similar in patients with and
without PVCs (8.2% vs. 9.1%; p = 0.93), couplets (8.5% vs. 8.4%; p = 0.99), or
NSVT (8.3% vs. 8.5%; p = 0.35).
CONCLUSIONS: In this large HCM cohort with no or only mild symptoms, myocardial
fibrosis detected by CMR was associated with greater likelihood and increased
frequency of ventricular tachyarrhythmias (including NSVT) on ambulatory Holter
ECG. Therefore, contrast-enhanced CMR identifies HCM patients with increased
susceptibility to ventricular tachyarrhythmias. To clarify the spatial relationship between coronary microvascular dysfunction
and myocardial fibrosis in hypertrophic cardiomyopathy (HCM), we compared the
measurement of hyperemic myocardial blood flow (hMBF) by PET with the extent of
delayed contrast enhancement (DCE) detected by MRI.
METHODS: In 34 patients with HCM, PET was performed using (13)N-labeled ammonia
during hyperemia induced by intravenous dipyridamole. DCE and systolic
thickening were assessed by MRI. Left ventricular myocardial segments were
classified as with DCE, either transmural (DCE-T) or nontransmural (DCE-NT), and
without DCE, either contiguous to DCE segments (NoDCE-C) or remote from them
(NoDCE-R).
RESULTS: In the group with DCE, hMBF was significantly lower than in the group
without DCE (1.81 +/- 0.94 vs. 2.13 +/- 1.11 mL/min/g; P < 0.001). DCE-T
segments had lower hMBF than did DCE-NT segments (1.43 +/- 0.52 vs. 1.91 +/- 1
mL/min/g, P < 0.001). Similarly, NoDCE-C segments had lower hMBF than did
NoDCE-R (1.98 +/- 1.10 vs. 2.29 +/- 1.10 mL/min/g, P < 0.01) and had no
significant difference from DCE-NT segments. Severe coronary microvascular
dysfunction (hMBF in the lowest tertile of all segments) was more prevalent
among NoDCE-C than NoDCE-R segments (33% vs. 24%, P < 0.05). Systolic thickening
was inversely correlated with percentage transmurality of DCE (Spearman rho =
-0.37, P < 0.0001) and directly correlated with hMBF (Spearman rho = 0.20, P <
0.0001).
CONCLUSION: In myocardial segments exhibiting DCE, hMBF is reduced. DCE extent
is inversely correlated and hMBF directly correlated with systolic thickening.
In segments without DCE but contiguous to DCE areas, hMBF is significantly lower
than in those remote from DCE and is similar to the value obtained in
nontransmural DCE segments. These results suggest that increasing degrees of
coronary microvascular dysfunction might play a causative role for myocardial
fibrosis in HCM. BACKGROUND: The clinical, morphological, and electrocardiographical relevance of
delayed enhancement (DE) in cardiac magnetic resoce (CMR) was studied in
patients with hypertrophic cardiomyopathy (HCM).
METHODS AND RESULTS: A total of 56 patients underwent both gadolinium-enhanced
CMR and 12-lead electrocardiogram. The CMR demonstrated DE at the left
ventricular (LV) wall in 39 patients. The patients with DE included more cases
with dilated phase of HCM, higher New York Heart Association (NYHA) classes and
incidence of ventricular tachyarrhythmias (VT), lower LV ejection fraction
(LVEF) and mean LV wall thickness (WT), and a larger ratio of maximum to minimum
LVWT. The QRS duration was prolonged and the QRS axis deviated toward left with
increases in the DE volume (r = 0.58 and r = 0.41, P < .01). Abnormal Q waves
were present in 5 patients and the location coincided with the DE segments in 4
patients, but the concordance was not significant. The amplitude of T waves
correlated with the ratio of the apex to basal LVWT (r = 0.38, P < .01) and was
more negative in cases with DE at the apex.
CONCLUSIONS: In HCM, the DE was associated with higher NYHA classes and
prevalence of VT, impaired global LV function and asymmetrical hypertrophy, and
conduction disturbance, abnormal Q waves, and giant negative T waves. BACKGROUND: Contrast-enhanced cardiovascular magnetic resoce with delayed
enhancement (DE) can provide in vivo assessment of myocardial fibrosis. However,
the clinical significance of DE in hypertrophic cardiomyopathy (HCM) remains
unresolved.
METHODS AND RESULTS: Cine and cardiovascular magnetic resoce with DE were
performed in 202 HCM patients (mean age, 42+/-17 years; 71% male), DE was
compared with clinical and demographic variables, and patients were followed up
for 681+/-249 days for adverse disease events. DE was identified in 111 (55%)
HCM patients, occupying 9%+/-11% of left ventricular myocardial volume,
including >25% DE in 10% of patients. The presence of DE was related to
occurrence of heart failure symptoms (P=0.05) and left ventricular systolic
dysfunction (P=0.001). DE was present in all patients with ejection fraction <
or =50% but also in 53% (102/192) of patients with preserved ejection fraction
(P<0.001); %DE was both inversely related to (r=-0.3; P<0.001) and an
independent predictor of ejection fraction (r=-0.4; P<0.001). DE (7%+/-7% of
left ventricle) was present in 54 patients who were asymptomatic (and with
normal ejection fraction). Over the follow-up period, the annualized adverse
cardiovascular event rate in patients with DE exceeded that in patients without
DE but did not achieve statistical significance (5.5% versus 3.3%; P=0.5).
CONCLUSIONS: In a large HCM cohort, DE was an independent predictor of systolic
dysfunction but with only a modest relationship to heart failure symptoms. These
data suggest an important role for myocardial fibrosis in the clinical course of
HCM patients but are not sufficient at this time to consider DE as an
independent risk factor for adverse prognosis. The aim of the present study was to evaluate, in patients with hypertrophic
cardiomyopathy (HC), the association between late gadolinium enhancement and
clinical end points, such as nonsustained ventricular tachycardia, arrhythmic
risk factors, New York Heart Association class, symptoms, and left ventricular
functional parameters. A total of 20 normal subjects (mean age 38 years, 16 men)
and 100 patients with HC (mean age 46 years, 70 men) were enrolled in the
present study. In the late gadolinium enhancement images, the extent of
unenhanced, mildly enhanced, and higher enhanced myocardium was measured. Higher
enhancement was present in 80% of the HC population and was significantly
greater in patients with a New York Heart Association class >1. Mild enhancement
was present in all the patients with HC. Receiver operating characteristic
analysis revealed that a cutoff of >4.9% of mild enhancement had 100%
sensitivity and 86% specificity to predict the occurrence of nonsustained
ventricular tachycardia, and a cutoff of >2.4% of hyperenhancement had 77%
sensitivity and 96% specificity. In conclusion, late gadolinium enhancement was
associated with nonsustained ventricular tachycardia, arrhythmic risk factors,
and worse New York Heart Association class. BACKGROUND: Hypertrophic cardiomyopathy (HCM) is reported to show patchy midwall
myocardial hyperenhancement on delayed-enhancement magnetic resoce imaging
(DE-MRI). The intramural distribution of myocardial hyperenhancement and its
correlation with clinical symptoms, ventricular arrhythmias, and cardiac
function have not been described forsymptomatic apical HCM.
PURPOSE: To evaluate the features and significance of myocardial
hyperenhancement on DE-MRI insymptomatic apical HCM.
MATERIAL AND METHODS: Thirteen patients with symptomatic apical HCM and their 65
apical segments were investigated. Myocardial hyperenhancement and regional and
global functional parameters were determined with MRI. We investigated the
intramural distribution and frequencies of this myocardial hyperenhancement and
compared them with the patients' clinical symptoms, the presence of ventricular
arrhythmias, and cine MRI.
RESULTS: Eight (61.5%) patients with symptomatic apical HCM displayed apical
myocardial hyperenhancement, and 22 (33.8%) of the 65 apical segments examined
showed myocardial hyperenhancement. Of the myocardial hyperenhancement observed,
81.8% showed a subendocardial pattern.The hyperenhanced apical myocardium had a
lower percentage of systolic myocardial thickening, and was associated with
serious symptoms (e.g. syncope) and ventricular arrhythmias.
CONCLUSION: Patients with symptomatic apical HCMshowed myocardial
hyperenhancement involving the subendocardial layer, which might be related to
regional systolic dysfunction, serious clinical symptoms, and ventricular
arrhythmias. Hypertrophic cardiomyopathy (HCM) is a disease that typically has heterogeneous
hypertrophy and dysfunction of the myocardium. Cardiac magnetic resoce
imaging (CMR) can be used to accurately assess ventricular wall thickness and
regional fibrosis. We investigated the effects of hypertrophy and fibrosis on
the heterogeneity of regional and global myocardial function in HCM. Forty
patients who were diagnosed with HCM were consecutively enrolled.
Echocardiography and CMR with delayed hyper-enhancement imaging (DHE) was
performed for each patient. Left ventricular (LV) regional and global
longitudinal strain (SL(R) and SL(G)) were obtained by two-dimensional speckle
tracking method on echocardiography. With CMR, regional myocardial wall
thickness was measured, and the amount of DHE was calculated semi-quantitatively
in each segment. Overall, 720 segments were analyzed. SL(R) was significantly
decreased in the hypertrophied segments (thickness > 11 mm) and segments with
DHE (P < 0.001). SL(R) was correlated with myocardial wall thickness (r = 0.47,
P = 0.001) and amount of regional DHE (r = 0.39, P < 0.001). On multivariate
analysis, regional LV wall thickness and amount of DHE were the only independent
determits of SL(R). SL(G) was associated with LV diastolic functional
parameters in echocardiography, total DHE volume, and LV mass index. Total DHE
volume and LV mass index were independent determits of SL(G) on multivariate
analysis. The extent of regional myocardial fibrosis is associated with regional
myocardial function independently of morphological changes of the myocardium,
and the correlation extended to global LV function. In this context, DHE may be
a useful parameter to discover early myocardial dysfunction independently of LV
hypertrophy. |
What disease is mirtazapine predominantly used for? | Mirtazapine is predominantly used in the treatment of major depression. | Major depression is a serious disease with various systemic effects, including
dysfunction of the immune response. Taurine has been known to be related to
certain modifications of the immune system. The aim of this study was to
determine the taurine concentration in lymphocytes of patients with major
depression and to evaluate the influence of the antidepressant treatment with
mirtazapine for six weeks on the levels of taurine. Gamma-aminobutyric acid,
aspartate, glutamate and glutamine were also determined. Taurine, aspartate and
glutamine levels were increased in the lymphocytes of depressed patients before
mirtazapine treatment compared to the control group, and were normalized after
treatment. Gamma-aminobutyric acid and glutamate did not differ between patients
and controls. There was a significant and positive correlation between the
severity of the disorder, measured by the Hamilton Rating Scale, and the
concentration of taurine in the lymphocytes of depressed patients before
treatment. This correlation was not observed after treatment and neither was
there a correlation observed for the other amino acids. The present observations
could be an indication of the relevance of taurine as a protective agent in the
lymphocytes of patients with severe depression, and could be the result of
modifications of taurine transport or efflux processes. BACKGROUND: The "long/short"polymorphism (5HTTLPR) in the promoter of the
serotonin transporter gene (SLC6A4) has been proposed as a pharmacogenetic
marker for antidepressant efficacy. Some but not all studies have found that the
short form of 5HTTLPR (S allele) results in decreased efficacy of selective
serotonin reuptake inhibitors.
OBJECTIVE: To determine if the 5HTTLPR polymorphism influences the efficacy and
tolerability of mirtazapine and paroxetine hydrochloride, 2 frequently
prescribed antidepressants with differing pharmacologic profiles, in geriatric
depression.
DESIGN: Double-blind, randomized 8-week study.
SETTING: Eighteen academic and private outpatient clinics.
PATIENTS: We evaluated 246 cognitively intact patients 65 years or older with
major depression.
INTERVENTIONS: Antidepressant therapy with 15 to 45 mg/d of mirtazapine (n =
124) or 20 to 40 mg/d of paroxetine (n = 122).
MAIN OUTCOME MEASURES: The Hamilton Depression Rating Scale-17 and Geriatric
Depression Scale, severity of adverse events and dosing compliance indexes, and
discontinuations due to adverse events. Outcome measures were stratified
according to 5HTTLPR genotypes.
RESULTS: Geriatric Depression Scale scores indicated that S allele carriers
treated with paroxetine showed a small impairment in antidepressant response.
Among mirtazapine-treated patients, there was little indication that the 5HTTLPR
genotype affected antidepressant efficacy. However, the 5HTTLPR polymorphism had
a dramatic effect on adverse events. Among paroxetine-treated subjects, S allele
carriers experienced more severe adverse events during the course of the study,
achieved significantly lower final daily doses, and had more discontinuations at
days 14, 21, 28, 42, and 49. Surprisingly, among mirtazapine-treated subjects, S
allele carriers had fewer discontinuations due to adverse events, experienced
less severe adverse events, and achieved higher final daily doses.
CONCLUSIONS: These results support the hypothesis that the S allele of 5HTTLPR
at the SLC6A4 locus is associated with a poor outcome after treatment with
selective serotonin reuptake inhibitors. However, the major effect was on the
tolerability of these drugs rather than efficacy. Results from
mirtazapine-treated patients indicate that the effect of this polymorphism on
outcome may depend on the mechanism of antidepressant action. Linezolid was initially discovered as an antidepressant because of its effect on
blocking intracellular metabolism of serotonin, norepinephrine, and other
biogenic amines. As time passed, it was realized that linezolid possessed
antibacterial activity, and linezolid has been developed and marketed as such.
In medicine we are quick to categorize drugs into specific classes as a
mechanism to recall indication and use. By classifying linezolid as an
antibacterial, it is common to forget about its antidepressant roots. A case
report involving linezolid with citalopram and mirtazepine in the precipitation
of serotonin syndrome in a critically ill bone marrow transplant patient is
described in this article. CONTEXT: The US Food and Drug Administration (FDA) has issued warnings that use
of antidepressant medications poses a small but significantly increased risk of
suicidal ideation/suicide attempt for children and adolescents.
OBJECTIVE: To assess the efficacy and risk of reported suicidal ideation/suicide
attempt of antidepressants for treatment of pediatric major depressive disorder
(MDD), obsessive-compulsive disorder (OCD), and non-OCD anxiety disorders.
DATA SOURCES AND STUDY SELECTION: PubMed (1988 to July 2006), relevant US and
British regulatory agency reports, published abstracts of important scientific
meetings (1998-2006), clinical trial registries, and information from authors.
Studies were published and unpublished randomized, placebo-controlled,
parallel-group trials of second-generation antidepressants (selective serotonin
reuptake inhibitors, nefazodone, venlafaxine, and mirtazapine) in participants
younger than 19 years with MDD, OCD, or non-OCD anxiety disorders.
DATA EXTRACTION: Information was extracted on study characteristics, efficacy
outcomes, and spontaneously reported suicidal ideation/suicide attempt.
DATA SYNTHESIS: Twenty-seven trials of pediatric MDD (n = 15), OCD (n = 6), and
non-OCD anxiety disorders (n = 6) were selected, and risk differences for
response and for suicidal ideation/suicide attempt estimated by random-effects
methods. Pooled risk differences in rates of primary study-defined measures of
responder status significantly favored antidepressants for MDD (11.0%; [95%
confidence interval {CI}, 7.1% to 14.9%]), OCD (19.8% [95% CI, 13.0% to 26.6%),
and non-OCD anxiety disorders (37.1% [22.5% to 51.7%]), corresponding to a
number needed to treat (NNT) of 10 (95% CI, 7 to 15), 6 (4 to 8), and 3 (2 to
5), respectively. While there was increased risk difference of suicidal
ideation/suicide attempt across all trials and indications for drug vs placebo
(0.7%; 95% CI, 0.1% to 1.3%) (number needed to harm, 143 [95% CI, 77 to 1000]),
the pooled risk differences within each indication were not statistically
significant: 0.9% (95% CI, -0.1% to 1.9%) for MDD, 0.5% (-1.2% to 2.2%) for OCD,
and 0.7% (-0.4% to 1.8%) for non-OCD anxiety disorders. There were no completed
suicides. Age-stratified analyses showed that for children younger than 12 years
with MDD, only fluoxetine showed benefit over placebo. In MDD trials, efficacy
was moderated by age, duration of depression, and number of sites in the
treatment trial.
CONCLUSIONS: Relative to placebo, antidepressants are efficacious for pediatric
MDD, OCD, and non-OCD anxiety disorders, although the effects are strongest in
non-OCD anxiety disorders, intermediate in OCD, and more modest in MDD. Benefits
of antidepressants appear to be much greater than risks from suicidal
ideation/suicide attempt across indications, although comparison of benefit to
risk varies as a function of indication, age, chronicity, and study conditions. The popularity of antidepressants in the treatment of insomnia is not supported
by a large amount of convincing data, but rather by opinions and beliefs of the
prescribing physicians on the advantages of these agents compared with drugs
acting on the benzodiazepine receptor or other drugs used for the treatment of
insomnia. The existing data do not allow for clear-cut, evidence-based
recommendations concerning the use of antidepressants in insomnia. Our
conclusions result from a few short-term studies on single agents, clinical
experience and inferences from knowledge on the effect of antidepressants in
other indications. At present, prescribing antidepressants for short-term
treatment of insomnia can be useful if there is some amount of concomitant
depressive symptomology or a history of depression, raising the impression that
the present insomnia may be a prodromal sign for a new depressive episode. In
all other cases, benzodiazepine receptor agonists, especially the
nonbenzodiazepines among them (the so-called 'z drugs') should be the drugs of
choice. For long-term treatment, antidepressants are among the pharmacological
options, in addition to other groups of psychotropics. Off-label use of
antidepressants may be considered for chronic insomnia if there is a concomitant
depressive symptomalogy (which is not so pronounced that an antidepressant
treatment with adequate higher doses would be required) and if there is no
specific indication for one of the other groups of psychotropics (e.g.
dementia-related nocturnal agitation, in which case an antipsychotic would be
preferred, or circadian problems, in which case melatonin or a melatonin agonist
would be favoured). If antidepressants are used to treat insomnia, sedating ones
should be preferred over activating agents such as serotonin reuptake
inhibitors. In general, drugs lacking strong cholinergic activity should be
preferred. Drugs blocking serotonin 5-HT2A or 5-HT2C receptors should be
preferred over those whose sedative property is caused by histamine receptor
blockade only. The dose should be as low as possible (e.g. as an initial dose:
doxepin 25 mg, mirtazapine 15 mg, trazodone 50 mg, trimipramine 25 mg).
Regarding the lack of substantial data allowing for evidence-based
recommendations, we are facing a clear need for well designed, long-term,
comparative studies to further define the role of antidepressants versus other
agents in the management of insomnia. Even though there are many drugs for the treatment of gastric ulcers, these
drugs sometimes cannot succeed. Since the 1950s, antidepressant drugs have been
used for several non-psychiatric indications. A lot of antidepressant drugs have
been shown experimentally to produce antiulcer activity in various ulcer models.
This study aimed to investigate the antiulcer effects of mirtazapine and to
determine its relationship with antioxidant mechanisms. The antiulcer activities
of 15, 30, and 60 mg/kg mirtazapine have been investigated on
indomethacin-induced ulcers in rats, and the results have been compared with
that of the control group. Mirtazapine decreased the indomethacin-induced ulcers
significantly at all doses used. Mirtazapine significantly increased the
glutathione (GSH) level, which decreased in the control group given only
indomethacin. All doses of mirtazapine significantly decreased the catalase
(CAT) level in stomach tissue compared to the control. Additionally, all doses
of mirtazapine reversed the decrease in the superoxide dismutase (SOD) level in
the stomach tissue of control rats. And finally, all doses of mirtazapine
decreased malondialdehyde (MDA) and myeloperoxidase (MPO) levels significantly
compared to the control. In conclusion, the activation of enzymatic and
non-enzymatic antioxidant mechanisms and the inhibition of some toxic oxidant
mechanisms play a role in the antiulcer effect mechanism of mirtazapine. This
new indication of mirtazapine will make it the first-choice drug in depressive
patients with gastric ulcers. Fibromyalgia syndrome is a chronic disease of widespread and debilitating pain
whose cause is unknown and whose risk factors are poorly understood. It is often
comorbid with rheumatoid and other pain disorders as well as psychiatric
disorders such as anxiety and depression. Although they are not officially
approved for this indication, antiepileptics and antidepressants are often used
to treat fibromyalgia. The tricyclic antidepressants (TCAs), particularly
amitriptyline, are among the most common treatment strategies. Because of the
poor tolerability of the tricyclics, the newer antidepressants have been widely
tested in fibromyalgia. The selective serotonin reuptake inhibitors (SSRIs) and
the reversible monoamine oxidase inhibitors do not seem to be particularly
helpful. The serotonin and norepinephrine reuptake inhibitors (SNRIs),
duloxetine and milnacipran, on the other hand, have been shown in
placebo-controlled trials to offer significant relief to patients suffering from
fibromyalgia. Although no direct comparative studies have been performed, these
compounds appear to be as effective as the TCAs but much better tolerated. The
effectiveness of the SNRIs as well as other dual acting antidepressants, such as
mirtazapine, but not the SSRIs, implies that a dysfunction of both serotonin and
norepinephrine neurotransmission probably exists in fibromyalgia. The
effectiveness of antidepressants appears to be independent of their effect on
comorbid depression. OBJECTIVES: The aim of this study was to establish the relative safety and
balance of risks for antidepressant treatment in older people. The study
objectives were to (1) determine relative and absolute risks of predefined
adverse events in older people with depression, comparing classes of
antidepressant drugs [tricyclic and related antidepressants (TCAs), selective
serotonin reuptake inhibitors (SSRIs), monoamine oxidase inhibitors (MAOIs) and
other antidepressants] and commonly prescribed individual drugs with non-use of
antidepressant drugs; (2) directly compare the risk of adverse events for SSRIs
with TCAs; (3) determine associations with dose and duration of antidepressant
medication; (4) describe patterns of antidepressant use in older people with
depression; and (5) estimate costs of antidepressant medication and primary care
visits.
DESIGN: A cohort study of patients aged 65 years and over diagnosed with
depression.
SETTING: The study was based in 570 general practices in the UK supplying data
to the QResearch database.
PARTICIPANTS: Patients diagnosed with a new episode of depression between the
ages of 65 and 100 years, from 1 January 1996 to 31 December 2007. Participants
were followed up until 31 December 2008.
INTERVENTIONS: The exposure of interest was treatment with antidepressant
medication. Antidepressant drugs were grouped into the major classes and
commonly prescribed individual drugs were identified.
MAIN OUTCOME MEASURES: There were 13 predefined outcome measures: all-cause
mortality, sudden cardiac death, suicide, attempted suicide/self-harm,
myocardial infarction, stroke/transient ischaemic attack (TIA), falls,
fractures, upper gastrointestinal bleeding, epilepsy/seizures, road traffic
accidents, adverse drug reactions and hyponatraemia.
RESULTS: In total, 60,746 patients were included in the study cohort. Of these,
54,038 (89.0%) received at least one prescription for an antidepressant during
follow-up. The associations with the adverse outcomes were significantly
different between the classes of antidepressant drugs for seven outcomes. SSRIs
were associated with the highest adjusted hazard ratios (HRs) for falls [1.66,
95% confidence interval (CI) 1.58 to 1.73] and hyponatraemia (1.52, 95% CI 1.33
to 1.75), and the group of other antidepressants was associated with the highest
HRs for all-cause mortality (1.66, 95% CI 1.56 to 1.77), attempted
suicide/self-harm (5.16, 95% CI 3.90 to 6.83), stroke/TIA (1.37, 95% CI 1.22 to
1.55), fracture (1.63, 95% CI 1.45 to 1.83) and epilepsy/seizures (2.24, 95% CI
1.60 to 3.15) compared with when antidepressants were not being used. TCAs did
not have the highest HR for any of the outcomes. There were also significantly
different associations between the individual drugs for seven outcomes, with
trazodone, mirtazapine and venlafaxine associated with the highest rates for
several of these outcomes. The mean incremental cost (for all antidepressant
prescriptions) ranged between £51.58 (amitriptyline) and £641.18 (venlafaxine)
over the 5-year post-diagnosis period.
CONCLUSIONS: This study found associations between use of antidepressant drugs
and a number of adverse events in older people. There was no evidence that SSRIs
or drugs in the group of other antidepressants were associated with a reduced
risk of any of the adverse outcomes compared with TCAs; however, they may be
associated with an increased risk for certain outcomes. Among individual drugs
trazodone, mirtazapine and venlafaxine were associated with the highest rates
for some outcomes. Indication bias and residual confounding may explain some of
the study findings. The risks of prescribing antidepressants need to be weighed
against the potential benefits of these drugs.
FUNDING: The National Institute for Health Research Health Technology Assessment
programme. OBJECTIVES: To investigate the association between antidepressant treatment and
risk of several potential adverse outcomes in older people with depression and
to examine risks by class of antidepressant, duration of use, and dose.
DESIGN: Cohort study of people aged 65 and over diagnosed as having depression.
SETTING: 570 general practices in the United Kingdom supplying data to the
QResearch primary care database.
PARTICIPANTS: 60,746 patients diagnosed as having a new episode of depression
between the ages of 65 and 100 years from 1 January 1996 to 31 December 2007 and
followed up until 31 December 2008.
MAIN OUTCOME MEASURES: Hazard ratios associated with antidepressant use for all
cause mortality, attempted suicide/self harm, myocardial infarction,
stroke/transient ischaemic attack, falls, fractures, upper gastrointestinal
bleeding, epilepsy/seizures, road traffic accidents, adverse drug reactions, and
hyponatraemia, adjusted for a range of potential confounding variables. Hazard
ratios were calculated for antidepressant class (tricyclic and related
antidepressants, selective serotonin reuptake inhibitors, other
antidepressants), dose, and duration of use and for commonly prescribed
individual drugs.
RESULTS: 54,038 (89.0%) patients received at least one prescription for an
antidepressant during follow-up. A total of 1,398,359 antidepressant
prescriptions were issued: 764,659 (54.7%) for selective serotonin reuptake
inhibitors, 442,192 (31.6%) for tricyclic antidepressants, 2203 (0.2%) for
monoamine oxidase inhibitors, and 189,305 (13.5%) for the group of other
antidepressants. The associations with the adverse outcomes differed
significantly between the antidepressant classes for seven outcomes. Selective
serotonin reuptake inhibitors were associated with the highest adjusted hazard
ratios for falls (1.66, 95% confidence interval 1.58 to 1.73) and hyponatraemia
(1.52, 1.33 to 1.75) compared with when antidepressants were not being used. The
group of other antidepressants was associated with the highest adjusted hazard
ratios for all cause mortality (1.66, 1.56 to 1.77), attempted suicide/self harm
(5.16, 3.90 to 6.83), stroke/transient ischaemic attack (1.37, 1.22 to 1.55),
fracture (1.64, 1.46 to 1.84), and epilepsy/seizures (2.24, 1.60 to 3.15),
compared with when antidepressants were not being used. Tricyclic
antidepressants did not have the highest hazard ratio for any of the outcomes.
Significantly different associations also existed between the individual drugs
for the same seven outcomes; trazodone (tricyclic antidepressant), mirtazapine,
and venlafaxine (both in the group of other antidepressants) were associated
with the highest rates for some of these outcomes. Absolute risks over 1 year
for all cause mortality were 7.04% for patients while not taking
antidepressants, 8.12% for those taking tricyclic antidepressants, 10.61% for
selective serotonin reuptake inhibitors, and 11.43% for other antidepressants.
CONCLUSIONS: Selective serotonin reuptake inhibitors and drugs in the group of
other antidepressants were associated with an increased risk of several adverse
outcomes compared with tricyclic antidepressants. Among individual drugs,
trazodone, mirtazapine, and venlafaxine were associated with the highest risks
for some outcomes. As this is an observational study, it is susceptible to
confounding by indication, channelling bias, and residual confounding, so
differences in characteristics between patients prescribed different
antidepressant drugs that could account for some of the associations between the
drugs and the adverse outcomes may remain. Further research is needed to confirm
these findings, but the risks and benefits of different antidepressants should
be carefully evaluated when these drugs are prescribed to older people. BACKGROUND: Since antidepressants are prescribed for multiple indications, the
use of an antidepressant cannot be equated with a diagnosis of depression.
OBJECTIVE: The objective of this study was to examine the quality of
antidepressant prescribing in Belgian nursing homes, with a critical evaluation
of indications and dosages, to see whether depression was appropriately treated
in terms of drug choice, the indications for which antidepressants were being
prescribed and whether there was underdosing.
METHODS: This analysis was based on data obtained in the Prescribing in Homes
for the Elderly in Belgium (PHEBE) study, a cross-sectional, descriptive study
of a representative, stratified, random sample of 1,730 residents from 76
Belgian nursing homes. The PHEBE study investigated overall drug utilization in
Belgian nursing homes in 2006. Clinical and medication data for the present
study were obtained from this study. A 28-item checklist of clinical conditions
was designed ad hoc for the PHEBE study and sent to the residents' general
practitioners (GPs) to collect clinical information. We copied the residents'
medication charts, classified the drugs using the Anatomical Therapeutic
Chemical (ATC) classification system codes and transferred the drug names and
dosages into a database. Information on indications was retrospectively obtained
from the GPs, so that we could link the indication to each medication. Minimum
effective doses (MEDs) of antidepressants to treat major depression were
obtained from the literature to assess underdosing.
RESULTS: The overall use of antidepressants in nursing homes was 39.5 % (95 % CI
37.2, 41.8). The physicians classified 34.2 % (95 % CI 32.0, 36.4) of the
residents as having depression, and 80.9 % of these patients were treated with
an antidepressant. Indications among the single antidepressant users (n = 551)
were depression (66.2 %), insomnia (13.4 %), anxiety (6.2 %) and neuropathic
pain (1.6 %). In the indication of depression, 74.8 % used a selective serotonin
reuptake inhibitor (SSRI), predomitly citalopram, sertraline and
escitalopram. Venlafaxine was used by 10.7 % of the residents. Dosages for these
antidepressants were equal to or higher than the MED. But when trazodone,
amitriptyline or mirtazapine were used to treat depression, respectively, 92.3,
55.5 and 44.5 % of prescribed dosages were below the MED. In the indication of
insomnia, most of the time, trazodone (90.5 %) or mirtazapine (5.4 %) were used,
and in lower dosages than those required for depression treatment (<MED).
Tricyclic antidepressants were predomitly used for the treatment of
neuropathic pain and were also used at lower dosages. Of all the residents
receiving a medication for anxiety, only 13.9 % received an antidepressant
(mostly an SSRI), and the remaining received a benzodiazepine.
CONCLUSIONS: The number one indication for the use of an antidepressant was
depression. Within this indication, mostly the recommended SSRIs were used, in
dosages equal to or higher than the MED. Furthermore, we noticed that there was
substantial use of sedative antidepressants for insomnia and that the physicians
preferred to prescribe benzodiazepines over the recommended SSRIs to treat
anxiety chronically. |
Is it possible to visualize subtahalamic nucleus by using transcranial ultrasound? | Yes, it has been shown that it is possible to visualize subtahalamic nucleus by using transcranial ultrasound. Transcranial ultrasound is safe and reliable method that can be employed to monitor lead location and intraoperative visualization of deep-brain stimulation (DBS) electrodes. | BACKGROUND/AIMS: To evaluate the use of the NeuroMate stereotactic robot with a
novel ultrasound registration system for movement disorder surgery (MDS).
METHODS: Using the robot in a frameless mode, 51 patients underwent MDS.
Surgical planning was carried out using MRI data obtained more than 24 h before
surgery.
RESULTS: 37 out of 50 targets in the subthalamic nucleus were satisfactorily
identified with a single microelectrode trajectory and the final electrode
positions were at a mean distance of 1.7 mm from the calculated target. There
was a significant improvement in motor scores of the Unified Parkinson's Disease
Rating Scale III (off medication) at 6 (43%) and 18 months (51.7%) compared to
pre-operative scores (p < 0.05).
CONCLUSIONS: The frameless robot using only MRI data can be used for MDS. The
temporal separation of imaging from the surgical procedure provides additional
time for detailed image analysis and planning. BACKGROUND: In the search for a better preoperative knowledge of the position of
probes and electrodes, we assessed the feasibility and the usefulness of
transcranial sonography during surgery for the implantation of stimulation
electrodes into the subthalamic nucleus (STN) of patients with Parkinson's
disease.
METHODS: Transcranial sonography was carried out during stereotactic surgery in
8 patients with Parkinson's disease who had a suitable temporal bone window on
the side receiving the electrode. Test stimulation parameters were 130 Hz, 0.1
ms, up to 0 to 4.5 V.
RESULTS: The test probe with a diameter of 0.8 mm was visualized through the
temporal preauricular window. The correct anatomic position of the electrode tip
could be indirectly assessed thanks to the topographic relationship of the STN
with the hyperechogenic substantia nigra and the nucleus ruber. The tip position
of the final electrode was easily documented. A laterality of 10.5 to 11.5 mm,
verified by teleradiographic ventriculography and plain films, was correlated
with the best response of symptoms of Parkinson's disease to electrical impulses
delivered to the STN.
CONCLUSIONS: Transcranial sonography is easily feasible during stereotactic
surgery. In combination with the clinical effects of electrostimulation on the
symptoms of Parkinson's disease and with stereotactic x-ray images, it enables
the assessment and the documentation of the correct position of implanted STN
electrodes in real time. BACKGROUND AND PURPOSE: In Parkinson's disease (PD) subthalamic nucleus deep
brain stimulation (STN-DBS) improves motor function. Also an effect on the
neurovascular coupling in motor cortex was reported due to a parallel activation
of a subthalamic vasodilator area (SVA). To address this issue further we
analysed neurovascular coupling in a non-motor area.
METHODS: Twenty PD patients selected for bilateral STN-DBS were investigated
with functional transcranial Doppler (f-TCD) before and after surgery.
Hemodynamic responses to visual stimulation were registered in left posterior
cerebral artery (PCA) and analysed with a control-system approach (parameters
gain, rate time, attenuation and natural frequency). To exclude autonomic
effects of STN-DBS, we also addressed spectrum analysis of heart rate and of
systolic arterial blood pressure variability, and baroreceptor gain. Findings in
the PD group were compared with healthy age-matched controls.
RESULTS: PD patients showed no neurovascular coupling changes in PCA territory,
compared to controls, and STN-DBS changed neither blood flow regulatory
parameters nor autonomic function.
CONCLUSIONS: Improvement of vasoregulation in some motor cortical areas after
STN-DBS might be related to an improved neuronal functional rather than
indicating an effect on the neurovascular coupling or autonomic function. In patients with deep brain stimulation (DBS), poor postoperative outcome or
unexpected clinical change require brain imaging to check the lead location.
Here, we studied safety, reliability and prognostic value of transcranial
sonography (TCS) for DBS lead localization applying predefined TCS criteria.
After measuring thermal effects of TCS and imaging artefact sizes of DBS lead
using a skull phantom, we prospectively enrolled 34 patients with DBS of globus
pallidus internus, ventro-intermediate thalamic or subthalamic nucleus. TCS had
no influence on lead temperature, electrical parameters of DBS device or
clinical state of patients. TCS measures of lead coordinates agreed with MRI
measures in anterior-posterior and medial-lateral axis. Lead dislocation
requiring reinsertion was reliably detected. Only patients with optimal lead
position on TCS had favorable clinical 12-month outcome (>50% improvement),
whereas unfavorable outcome (<25% improvement) was associated with suboptimal
lead position. TCS may therefore become a first-choice modality to monitor lead
location. PURPOSE: Disturbances of the autonomic nervous system are common in patients
with Parkinson's disease (PD) but the effect of deep brain stimulation of the
subthalamic nucleus on cerebrovascular reactivity is not entirely known.
METHODS: Seven patients in an advanced stage of the disease and seven healthy
age-matched controls participated in the study, which took place after one night
of drug withdrawal. Cerebral blood flow velocity was continuously monitored on
both sides with transcranial Doppler ultrasound, and cerebrovascular reactivity
(CR) was evaluated with the cold pressure test. The measurements were repeated
and compared during the stimulation-on and -off phases.
RESULTS: The PD patients had significantly higher CR values in the
stimulation-on than -off conditions (15.1% ± 6.9 versus 9.4% ± 6.2; p = 0.03).
CR values were higher in controls than in patients in the stimulation-off
condition (20.4% ± 12.5 versus 9.4% ± 6.2; p = 0.007) without a significant
difference with the stimulation-on phase.
CONCLUSIONS: CR, evaluated by the response to the cold pressure test, is
impaired in patients with advanced PD and improved by subthalamic nucleus. |
Is cancer related to global DNA hypo or hypermethylation? | DNA hypermethylation and hypomethylation are independent processes and appear to play different roles in tumor progression. Cancer cells are characterized by a generalized disruption of the DNA methylation pattern involving an overall decrease in the level of 5-methylcytosine together with regional hypermethylation of particular CpG islands. Tumors have reduced levels of genomic DNA methylation and contain hypermethylated CpG islands. | NBL2 is a tandem 1.4-kb DNA repeat, whose hypomethylation in hepatocellular
carcinomas was shown previously to be an independent predictor of disease
progression. Here, we examined methylation of all cytosine residues in a 0.2-kb
subregion of NBL2 in ovarian carcinomas, Wilms' tumors, and diverse control
tissues by hairpin-bisulfite PCR. This new genomic sequencing method detects
5-methylcytosine on covalently linked complementary strands of a DNA fragment.
All DNA clones from normal somatic tissues displayed symmetrical methylation at
seven CpG positions and no methylation or only hemimethylation at two others.
Unexpectedly, 56% of cancer DNA clones had decreased methylation at some
normally methylated CpG sites as well as increased methylation at one or both of
the normally unmethylated sites. All 146 DNA clones from 10 cancers could be
distinguished from all 91 somatic control clones by assessing methylation
changes at three of these CpG sites. The special involvement of DNA
methyltransferase 3B in NBL2 methylation was indicated by analysis of cells from
immunodeficiency, centromeric region instability, and facial anomalies syndrome
patients who have mutations in the gene encoding DNA methyltransferase 3B. Blot
hybridization of 33 cancer DNAs digested with CpG methylation-sensitive enzymes
confirmed that NBL2 arrays are unusually susceptible to cancer-linked
hypermethylation and hypomethylation, consistent with our novel genomic
sequencing findings. The combined Southern blot and genomic sequencing data
indicate that some of the cancer-linked alterations in CpG methylation are
occurring with considerable sequence specificity. NBL2 is an attractive
candidate for an epigenetic cancer marker and for elucidating the nature of
epigenetic changes in cancer. Changes in DNA methylation patterns are an important characteristic of human
cancer. Tumors have reduced levels of genomic DNA methylation and contain
hypermethylated CpG islands, but the full extent and sequence context of DNA
hypomethylation and hypermethylation is unknown. Here, we used methylated CpG
island recovery assay-assisted high-resolution genomic tiling and CpG island
arrays to analyze methylation patterns in lung squamous cell carcinomas and
matched normal lung tissue. Normal tissues from different individuals showed
overall very similar DNA methylation patterns. Each tumor contained several
hundred hypermethylated CpG islands. We identified and confirmed 11 CpG islands
that were methylated in 80-100% of the SCC tumors, and many hold promise as
effective biomarkers for early detection of lung cancer. In addition, we find
that extensive DNA hypomethylation in tumors occurs specifically at repetitive
sequences, including short and long interspersed nuclear elements and LTR
elements, segmental duplications, and subtelomeric regions, but single-copy
sequences rarely become demethylated. The results are consistent with a specific
defect in methylation of repetitive DNA sequences in human cancer. We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation
patterns during four progressive stages of prostate cancer in the transgenic
adenocarcinoma of mouse prostate (TRAMP) model, including prostatic
intraepithelial neoplasia, well-differentiated tumors, early poorly
differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and
Dnmt3b protein expression were increased in all stages; however, after
normalization to cyclin A to account for cell cycle regulation, Dnmt proteins
remained overexpressed in prostatic intraepithelial neoplasia and
well-differentiated tumors, but not in poorly differentiated tumors. Restriction
landmark genomic scanning analysis of locus-specific methylation revealed a high
incidence of hypermethylation only in poorly differentiated (early and late)
tumors. Several genes identified by restriction landmark genomic scanning showed
hypermethylation of downstream regions correlating with mRNA overexpression,
including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA
methylation analyses suggests that gene overexpression precedes downstream
hypermethylation during prostate tumor progression. In contrast to gene
hypermethylation, genomic DNA hypomethylation, including hypomethylation of
repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both
early and late stages of prostate cancer. DNA hypermethylation and DNA
hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not
correlate with either variable, with the exception of a borderline significant
association between Dnmt1 expression and DNA hypermethylation. In summary, our
data reveal the relative timing of and relationship between key alterations of
the DNA methylation pathway occurring during prostate tumor progression in an in
vivo model system. |
Do thyroid hormone receptors change after brain injury? | thyroid hormone receptors increase after brain injury | The effect of cerebral ischemia and subsequent recirculation on the nuclear
thyroid hormone receptors was investigated. Ischemia was produced by occlusion
of the right common carotid artery in the Mongolian gerbil. The thyroid hormone
receptors were measured in vitro by a [125I]triiodothyrorine (T3) binding assay
with isolated nuclei and Scatchard analysis. A rapid increase of the total
number of binding sites for T3 appeared within 30 min of ischemia and reached
over 40% by 3 h. During the same 3-h period, the relative binding affinity was
reduced by 25%. Upon recirculation after 30 min or 3 h of ischemia, a rapid
reversal of measured T3 binding sites occurred, which progressed to 20-30% below
the control value by the recirculation period of 3 h. If the ischemic period was
only 30 min, the nuclear T3 binding capacity recovered toward the control level
and the affinity constant returned normal after recirculation for 24 h. When the
ischemic period was extended to 3 h, there was progressive loss of receptor
sites, and no tendency for recovery of the affinity constant was observed. These
results demonstrated a prompt alteration of a specific nuclear regulatory
component in cerebral ischemia, which may indicate the importance of such
changes within the nuclear regulatory mechanism for reversibility of cerebral
function following ischemic insult. The molecular events occurring after cerebral ischemia in hypertension may
include de novo expression of numerous genes. Receptor genes are predomitly
involved in the process of cell death, neuroprotection and reconstruction after
ischemic injury. Ischemic stroke was observed in the non-genetic, non-surgical
model of hypertension, the cold-induced hypertensive rat. In hypertensive rats
suppression subtractive hybridization analysis was used to identify
differentially expressed receptor genes in stroke-tissue compared to normal rat
brain. We found 76 genes predomitly expressed in hypertensive rat
stroke-tissue. These predomitly expressed genes included genes involved in
energy metabolism, signal transduction/cell regulation, and
replication/transcription/translation. For example, the T3 receptor alpha was
predomitly expressed in stroke-tissue, indicating that regeneration of nerves
in stroke tissue may be facilitated by increased T3 receptor alpha expression. Intraventricular hemorrhage (IVH) remains a major cause of white matter injury
in preterm infants with no viable therapeutic strategy to restore myelination.
Maturation of oligodendrocytes and myelination is influenced by thyroid hormone
(TH) signaling, which is mediated by TH receptor α (TRα) and TRβ. In the brain,
cellular levels of TH are regulated by deiodinases, with deiodinase-2 mediating
TH activation and deiodinase-3 TH inactivation. Therefore, we hypothesized that
IVH would decrease TH signaling via changes in the expression of deiodinases
and/or TRs, and normalization of TH signaling would enhance maturation of
oligodendrocytes and myelination in preterm infants with IVH. These hypotheses
were tested using both autopsy materials from human preterm infants and a rabbit
model of IVH. We found that deiodinase-2 levels were reduced, whereas
deiodinase-3 levels were increased in brain samples of both humans and rabbits
with IVH compared with controls without IVH. TRα expression was also increased
in human infants with IVH. Importantly, treatment with TH accelerated the
proliferation and maturation of oligodendrocytes, increased transcription of
Olig2 and Sox10 genes, augmented myelination, and restored neurological function
in pups with IVH. Consistent with these findings, the density of myelinating
oligodendrocytes was almost doubled in TH-treated human preterm infants compared
with controls. Thus, in infants with IVH the combined elevation in deiodinase-3
and reduction in deiodinase-2 decreases TH signaling that can be worsened by an
increase in unliganded TRα. Given that TH promotes neurological recovery in IVH,
TH treatment might improve the neurodevelopmental outcome of preterm infants
with IVH. |
What is known about efficacy of the high dose intravenous ascorbate in the treatment of cancer patients? | It was reported that ascorbate, given orally and intravenously at doses of up to 10 g/day, was effective in the treatment of cancer. However, double-blind placebo-controlled clinical trials showed no survival advantage when the same doses of ascorbate were given orally, leading the medical and scientific communities to dismiss the use of ascorbate as a potential cancer treatment. Pharmacologic actions of ascorbate against cancer cells remain to be fully understood. It is thought that high dose ascorbate is selectively cytotoxic to cancer cell lines through the generation of extracellular hydrogen peroxide. High dose intravenous ascorbate (IVC) may be able to modulate inflammation, which in turn might improve outcomes for cancer patients. IVC may serve as a safe, adjunctive therapy in clinical cancer care | In 1975, we reported the remarkable case of a 42-year-old man with
histologically proven widely disseminated reticulum cell sarcoma who, in a
remarkably short time, appeared to enjoy not one, but two, complete spontaneous
regressions of his fatal illness. Both these regressions coincided exactly in
time with intravenous high-dose ascorbate administration, and it seemed
reasonable to conclude that this unconventional therapy must have been
responsible for his excellent responses. For those interested in spontaneous
regressions of cancer and the possible mechanisms, we now report his subsequent
progress some 17 years later. Some clinicians and alternative therapy practitioners advocate megadose
intravenous and oral ascorbate treatment of cancer. Randomized control studies
using oral ascorbate showed no benefit. Recent data show that intravenous but
not oral administration of ascorbate can produce millimolar plasma
concentrations, which are toxic to many cancer cell lines. We propose that
ascorbate treatment of cancer should be reexamined by rigorous scientific
scrutiny in the light of new evidence. Case studies suggest that vitamin C, given intravenously at doses of 10-100
grams/day can improve patient well being and in some cases, reduce tumor size.
While ascorbate is generally considered safe, clinical data on high intravenous
doses is limited. Twenty-four late stage terminal cancer patients were given
continuous infusions of 150 to 710 mg/kg/day for up to eight weeks. Blood
chemistry and blood count profiles were obtained at roughly one-week intervals
while patient health, adverse events and tumor progression were monitored. The
majority of patients were vitamin C deficient prior to treatment. Intravenous
infusions increased plasma ascorbate concentrations to a mean of 1.1 mM. The
most common adverse events reported were nausea, edema, and dry mouth or skin;
and these were generally minor. Two Grade 3 adverse events 'possibly related' to
the agent were reported: one patient with a history of renal calculi developed a
kidney stone after thirteen days of treatment and another patient experienced
hypokalemia after six weeks of treatment. White blood cell counts were stable
while hemoglobin and hematocrit levels dropped slightly during treatment,
consistent with trends observed prior to therapy. Blood creatinine, BUN,
glucose, and uric acid concentrations decreased or remained stable during
therapy, suggesting that ascorbate infusions did not adversely affect renal
function. One patient had stable disease and continued the treatment for
forty-eight weeks. These data suggest that intravenous vitamin C therapy for
cancer is relatively safe, provided the patient does not have a history of
kidney stone formation. In many cancers, a chronic increase in oxidant stress - associated with elevated
levels of hydrogen peroxide - contributes to the increased proliferative rate,
diminished apoptosis, increased angiogenic and metastatic capacity, and
chemoresistance that often characterize advanced maligcies. This oxidant
stress often reflects up-regulation of expression and activity of NADPH oxidase,
and/or decreased activity of catalase, which functions as suppressor gene in
oxidant-dependent cancers. These characteristics of oxidant-dependent cancers
suggest a dual strategy for treatment of these cancers. Since ascorbate can
react spontaneously with molecular oxygen to generate hydrogen peroxide,
high-dose intravenous ascorbate should be selectively toxic to tumors that are
low in catalase activity - as suggested by numerous cell culture studies.
Measures which concurrently improve the oxygenation of hypoxic tumor regions
would be expected to boost the efficacy of such therapy; calcitriol and
high-dose selenium might also be useful in this regard. Secondly, during the
intervals between sessions of ascorbate therapy, administration of agents which
can safely inhibit NADPH oxidase would be expected to slow the proliferation and
spread of surviving tumor cells - while providing selection pressure for a
further decline in catalase activity. In effect, cancers treated in this way
would be whipsawed between lethally excessive and inadequately low oxidant
stress. An additional possibility is that ascorbate-induced oxidant stress in
tumors might potentiate the cell kill achieved with concurrently administered
cytotoxic drugs, inasmuch as oxidant mechanisms appear to play a mediating role
in the apoptosis induced by many such drugs, largely via activation of c-Jun
NH(2)-terminal kinase; cell culture studies would be useful for evaluating this
possibility. The antioxidant perhaps most widely used in complementary oncology is vitamin C,
particularly by intravenous injection. In light of the recent clinical
pharmacokinetic findings, the in vitro evidence of anti-tumour mechanisms and
some well-documented cases of advanced cancers the role of high-dose intravenous
vitamin C therapy in cancer treatment should be reassessed. High dose
intravenous vitamin C therapy may have benefits in patients with advanced
cancers, and cancers with poor prognosis and limited therapeutic options, but
further clinical studies regarding the safety and efficacy of this therapy are
necessary, especially in Germany. Ascorbate (ascorbic acid, vitamin C) is one of the early, unorthodox treatments
for cancer. The evidence upon which people base the use of ascorbate in cancer
treatment falls into two categories: clinical data on dose concentration
relationships, and laboratory data describing potential cell toxicity with high
concentrations of ascorbate in vitro. Clinical data show that when ascorbate is
given orally, fasting plasma concentrations are tightly controlled by decreased
absorption, increased urine excretion, and reduced ascorbate bioavailability. In
contrast, when ascorbate is administered intravenously, concentrations in the
millimolar level are achieved. Thus, it is clear that intravenous administration
of ascorbate can yield very high plasma levels, while oral treatment does not. Two popular complementary, alternative, and integrative medicine therapies,
high-dose intravenous ascorbic acid (AA) and intravenous glutathione (GSH), are
often coadministered to cancer patients with unclear efficacy and drug-drug
interaction. In this study we provide the first survey evidence for clinical use
of iv GSH with iv AA. To address questions of efficacy and drug-drug
interaction, we tested 10 cancer cell lines with AA, GSH, and their combination.
The results showed that pharmacologic AA induced cytotoxicity in all tested
cancer cells, with IC(50) less than 4 mM, a concentration easily achievable in
humans. GSH reduced cytotoxicity by 10-95% by attenuating AA-induced H(2)O(2)
production. Treatment in mouse pancreatic cancer xenografts showed that
intraperitoneal AA at 4 g/kg daily reduced tumor volume by 42%. Addition of
intraperitoneal GSH inhibited the AA-induced tumor volume reduction. Although
all treatments (AA, GSH, and AA+GSH) improved survival rate, AA+GSH inhibited
the cytotoxic effect of AA alone and failed to provide further survival benefit.
These data confirm the pro-oxidative anti-cancer mechanism of pharmacologic AA
and suggest that AA and GSH administered together provide no additional benefit
compared with AA alone. There is an antagonism between ascorbate and glutathione
in treating cancer, and therefore iv AA and iv GSH should not be coadministered
to cancer patients on the same day. Recent studies have revealed the scientific basis for the use of intravenous
(i.v.) vitamin C or ascorbic acid (ascorbate) in treating cancers, and raised
the possibility of using i.v. ascorbate as a prooxidant anticancer therapy.
Through the production of H2O2, pharmacologic ascorbate can induce some cancer
cell death in vitro and inhibit a number of types of tumor growth in animal
models. However, the mechanism of cell death triggered by ascorbate is not well
understood. In this study, we investigated the cytotoxicity of pharmacological
concentrations of ascorbate to human prostate cancer cells and the mechanisms
involved. The results showed that ascorbate in the millimolar range induced
cytotoxicity in five of the six tested prostate cancer cell lines. The IC50
values in the sensitive prostate cancer cells ranged from 1.9 to 3.5 mmol/l,
concentrations clinically achievable with i.v. ascorbate use. All tested
androgen-independent cells were sensitive to ascorbate treatment. The
ascorbate-insensitive cell line LaPC4 is hormonally dependent. Whereas the
reasons for sensitivity/resistance to ascorbate treatment need to be
investigated further, cell death in sensitive cells was dependent on H2O2.
Ascorbate treatment depleted ATP and induced autophagy in sensitive prostate
cancer cells, resulting in cell death. Taken together with previous studies,
high-dose ascorbate has the potential to be a novel treatment option to
hormone-refractory prostate cancer. BACKGROUND: An inflammatory component is present in the microenvironment of most
neoplastic tissues. Inflammation and elevated C-reactive protein (CRP) are
associated with poor prognosis and decreased survival in many types of
cancer.Vitamin C has been suggested as having both a preventative and
therapeutic role in a number of pathologies when administered at much
higher-than-recommended dietary allowance levels.Since in vitro studies
demonstrated inhibition of pro-inflammatory pathways by millimolar
concentrations of vitamin C, we decided to analyze the effects of high dose IVC
therapy in suppression of inflammation in cancer patients.
METHODS: 45 patients with prostate cancer, breast cancer, bladder cancer,
pancreatic cancer, lung cancer, thyroid cancer, skin cancer and B-cell lymphoma
were treated at the Riordan Clinic by high doses of vitamin C (7.5 g -50 g)
after standard treatments by conventional methods.CRP and tumor markers were
measured in serum or heparin-plasma as a routine analysis. In addition, serum
samples were collected before and after the IVCs for the cytokine kit tests.
RESULTS: According to our data positive response to treatment, which was
demonstrated by measurements of C- reactive protein, was found in 75% of
patients and progression of the inflammation in 25% of patients. IVC treatments
on all aggressive stage cancer patients showed the poor response of
treatment.There was correlation between tumor markers (PSA, CEA, CA27.29 and
CA15-3) and changes in the levels of C-reactive protein.Our test of the effect
of IVC on pro-inflammatory cytokines demonstrated that inflammation cytokines
IL-1α, IL-2, IL-8, TNF-α, chemokine eotaxin and CRP were reduced significantly
after treatments.
CONCLUSIONS: The high dose intravenous ascorbic acid therapy affects C-reactive
protein levels and pro-inflammation cytokines in cancer patients. In our study,
we found that modulation of inflammation by IVC correlated with decreases in
tumor marker levels.In summary, our data support the hypothesis that high dose
intravenous ascorbate treatments may reduce inflammation in cancer patients. Our
results suggest that further investigations into the use of IVC to reduce
inflammation in diseases where inflammation is relevant are warranted. BACKGROUND: Treatment for pancreatic cancer with pharmacological ascorbate
(ascorbic acid, vitamin C) decreases tumor progression in preclinical models. A
phase I clinical trial was performed to establish safety and tolerability of
pharmacological ascorbate combined with gemcitabine in patients with
biopsy-proven stage IV pancreatic adenocarcinoma.
DESIGN: Nine subjects received twice-weekly intravenous ascorbate (15-125 g)
employing Simon's accelerated titration design to achieve a targeted
post-infusion plasma level of ≥350 mg/dL (≥20 mM). Subjects received concurrent
gemcitabine. Disease burden, weight, performance status, hematologic and
metabolic laboratories, time to progression and overall survival were monitored.
RESULTS: Mean plasma ascorbate trough levels were significantly higher than
baseline (1.46 ± 0.02 vs. 0.78 ± 0.09 mg/dL, i.e., 83 vs. 44 μM, p < 0.001).
Adverse events attributable to the drug combination were rare and included
diarrhea (n = 4) and dry mouth (n = 6). Dose-limiting criteria were not met for
this study. Mean survival of subjects completing at least two cycles (8 weeks)
of therapy was 13 ± 2 months.
CONCLUSIONS: Data suggest pharmacologic ascorbate administered concurrently with
gemcitabine is well tolerated. Initial data from this small sampling suggest
some efficacy. Further studies powered to determine efficacy should be
conducted. SIGNIFICANCE: Ewan Cameron reported that ascorbate, given orally and
intravenously at doses of up to 10 g/day, was effective in the treatment of
cancer. Double-blind placebo-controlled clinical trials showed no survival
advantage when the same doses of ascorbate were given orally, leading the
medical and scientific communities to dismiss the use of ascorbate as a
potential cancer treatment. However, the route of administration results in
major differences in ascorbate bioavailability. Tissue and plasma concentrations
are tightly controlled in response to oral administration, but this can be
bypassed by intravenous administration. These data provide a plausible
scientific rationale for the absence of a response to orally administered
ascorbate in the Mayo clinic trials and indicate the need to reassess ascorbate
as a cancer therapeutic.
RECENT ADVANCES: High dose ascorbate is selectively cytotoxic to cancer cell
lines through the generation of extracellular hydrogen peroxide (H2O2). Murine
xenograft models confirm a growth inhibitory effect of pharmacological
concentrations. The safety of intravenous ascorbate has been verified in
encouraging pilot clinical studies.
CRITICAL ISSUES: Neither the selective toxicity of pharmacologic ascorbate
against cancer cells nor the mechanism of H2O2-mediated cytotoxicity is fully
understood. Despite promising preclinical data, the question of clinical
efficacy remains.
FUTURE DIRECTIONS: A full delineation of mechanism is of interest because it may
indicate susceptible cancer types. Effects of pharmacologic ascorbate used in
combination with standard treatments need to be defined. Most importantly, the
clinical efficacy of ascorbate needs to be reassessed using proper dosing, route
of administration, and controls. In the 1970s, Pauling and Cameron reported increased survival of patients with
advanced cancer treated with high-dose intravenous (IV) vitamin C (L-ascorbate,
ascorbic acid). These studies were criticized for their retrospective nature and
lack of standardization of key prognostic factors including performance status.
Subsequently, several well-designed randomized controlled trials failed to
demonstrate a significant survival benefit, although these trials used high-dose
oral vitamin C. Marked differences are now recognized in the pharmacokinetics of
vitamin C with oral and IV administration, opening the issue of therapeutic
efficacy to question. In vitro evidence suggests that vitamin C functions at low
concentrations as an antioxidant but may have pro-oxidant activity at high
concentrations. The mechanism of its pro-oxidant action is not fully understood,
and both intra- and extracellular mechanisms that generate hydrogen peroxide
have been proposed. It remains to be proven whether vitamin C-induced reactive
oxygen species occur in vivo and, if so, whether this will translate to a
clinical benefit. Current clinical evidence for a therapeutic effect of
high-dose IV vitamin C is ambiguous, being based on case series. The
interpretation and validation of these studies is hindered by limited
correlation of plasma vitamin C concentrations with response. The methodology
exists to determine if there is a role for high-dose IV vitamin C in the
treatment of cancer, but the limited understanding of its pharmacodynamic
properties makes this challenging. Currently, the use of high-dose IV vitamin C
cannot be recommended outside of a clinical trial. |
Alpha-spectrin and beta-spectrin subunits form parallel or antiparallel heterodimers? | Alpha and beta spectrin subunits form antiparallel spectrin heterodimers by lateral association. | The antiparallel side-to-side association of spectrin alpha and beta monomers is
a two-step process which occurs in seconds even at 0 degrees C and at low
concentrations. Assembly involves initial contact of complementary nucleation
sites on each subunit, which are located near the actin binding end of the long,
flexible heterodimer rod. The minimum nucleation sites are comprised of
approximately four contiguous 106-residue homologous segments or repeats. Three
repeats in the nucleation site contain an 8-residue insertion and have the
highest homology to the four spectrin-like repeats in alpha-actinin. The
adjacent actin binding domain on the beta subunit and the adjacent EF hand
motifs on the alpha subunit are not required for heterodimer assembly. The
nucleation sites probably have a specific lock and key structure which defines
the unique side-to-side pairing of the many homologous segments in both
subunits. Assembly of spectrin heterodimers is probably most analogous to a
zipper. After initial nucleation site binding, the remainder of the subunits
quickly associate along their full lengths to reconstitute a normal dimer by
supercoiling around each other to form a rope-like, flexible rod. Assembly is
terminated if either polypeptide is interrupted by a protease cleavage.
Heterozygotic mutations involving either nucleation site are predicted to affect
allele incorporation into the mature membrane skeleton. The actin-cross-linking protein spectrin is a prominent component of the
membrane cytoskeleton. Spectrin is a tetramer of two antiparallel
alphabeta-dimers which share a unique and ancient gene structure. The
alpha-spectrin and beta-spectrin genes are composed primarily of tandemly
repeated 106-amino-acid segments, each of which forms a triple alpha-helical
coiled coil. Both the genes and the repeats themselves are homologous. The two
genes are thought to be the result of a gene duplication event, and each gene is
the product of duplications of the 106-amino-acid repeats. In this work we
compare the process of molecular evolution across the repeated segments of the
alpha- and beta-spectrin genes. We find that the alpha-spectrin segments have,
for the most part, evolved in a homogeneous fashion, while considerable
heterogeneity is found among beta-spectrin segments. Several segments with
unique known functions are found to have evolved differently than the others. On
the basis of heterogeneity of the evolutionary process, we suggest that at least
one repeat has a unique function that has yet to be documented. We also present
new statistical methods for comparing the evolutionary process between different
regions of DNA sequences. Cytoskeletal proteins belonging to the spectrin family have an elongated
structure composed of repetitive units. The three-dimensional solution structure
of the 16th repeat from chicken brain alpha-spectrin (R16) has been determined
by NMR spectroscopy and distance geometry-simulated annealing calculations. We
used a total of 1035 distance restraints, which included 719 NOE-based values
obtained by applying the ambiguous restraints for iterative assignment (ARIA)
method. In addition, we performed a direct refinement against 1H-chemical
shifts. The final ensemble of 20 structures shows an average RMSD of 1.52 A from
the mean for the backbone atoms, excluding loops and N and C termini. R16 is
made up of three antiparallel alpha-helices separated by two loops, and folds
into a left-handed coiled-coil. The basic unit of spectrin is an antiparallel
heterodimer composed of two homologous chains, beta and alpha. These assemble a
tetramer via a mechanism that relies on the completion of a single repeat by
association of the partial repeats located at the C terminus of the beta-chain
(two helices) and at the N terminus of the alpha-chain (one helix). This
tetramer is the assemblage able to cross-link actin filaments. Model building by
homology of the "tetramerization" repeat from human erythrocyte spectrin
illuminates the possible role of point mutations which cause hemolytic anemias. The spectrin heterodimer is formed by the antiparallel lateral association of an
alpha and a beta subunit, each of which comprises largely a series of homologous
triple-helical motifs. Initiation of dimer assembly involves strong binding
between complementary motifs near the actin-binding end of the dimer. In this
study, the mechanism of lateral spectrin association at this dimer nucleation
site was investigated using the analytical ultracentrifuge to analyze
heterodimers formed from recombit peptides containing two or four homologous
motifs from each subunit (alpha20-21/beta1-2; alpha18-21/beta1-4). Both the
two-motif and four-motif dimer associations were weakened substantially with
increasing salt concentration, indicating that electrostatic interactions are
important for the dimer initiation process. Modeling of the electrostatic
potential on the surface of the alpha20 and beta2 motifs showed that the side of
the motifs comprising the A and B helices is the most favorable for association,
with an area of positive electrostatic potential on the AB face of the beta2
motif opposite negative potential on the AB face of the alpha20 motif and vise
versa. Protease protection analysis of the alpha20-21/beta1-2 dimer showed that
multiple trypsin and proteinase K sites in the A helices of the beta2 and
alpha21 motifs become buried upon dimer formation. Together, these data support
a model where complementary long range electrostatic interactions on the AB
faces of the triple-helical motifs in the dimer nucleation site initiate the
correct pairing of motifs, i.e. alpha21-beta1 and alpha20-beta2. After initial
docking of these complementary triple-helical motifs, this association is
probably stabilized by subsequent formation of stronger hydrophobic interactions
in a complex involving the A helices of both subunits and possibly most of the
AB faces. The beta subunit A helix in particular appears to be buried in the
dimer interface. The large size of spectrin, the flexible protein promoting reversible
deformation of red cells, has been an obstacle to elucidating the molecular
mechanism of its function. By studying cloned fragments of the repeating unit
domain, we have found a correspondence between positions of selected spectrin
repeats in a tetramer with their stabilities of folding. Six fragments
consisting of two spectrin repeats were selected for study primarily on the
basis of the predicted secondary structures of their linker regions. Fragments
with a putatively helical linker were more stable to urea- and heat-induced
unfolding than those with a putatively nonhelical linker. Two of the less stably
folded fragments, human erythroid alpha-spectrin repeats 13 and 14
(HEalpha13,14) and human erythroid beta-spectrin repeats 8 and 9 (HEbeta8,9),
are located opposite each other on antiparallel spectrin dimers. At least
partial unfolding of these repeats under physiological conditions indicates that
they may serve as a hinge. Also less stably folded, the fragment of human
erythroid alpha-spectrin repeats 4 and 5 (HEalpha4,5) lies opposite the site of
interaction between the partial repeats at the C- and N-terminal ends of beta-
and alpha-spectrin, respectively, on the opposing dimer. More stably folded
fragments, human erythroid alpha-spectrin repeats 1 and 2 (HEalpha1,2) and human
erythroid alpha-spectrin repeats 2 and 3 (HEalpha2,3), lie nearly opposite each
other on antiparallel spectrin dimers of a tetramer. These clusterings along the
spectrin tetramer of repeats with similar stabilities of folding may have
relevance for spectrin function, particularly for its well known flexibility. Protein extensibility appears to be based broadly on conformational changes that
can in principle be modulated by protein-protein interactions. Spectrin family
proteins, with their extensible three-helix folds, enable evaluation of
dimerization effects at the single molecule level by atomic force microscopy.
Although some spectrin family members function physiologically only as
homodimers (e.g. alpha-actinin) or are strictly monomers (e.g. dystrophin),
alpha- and beta-spectrins are stable as monomeric forms but occur
physiologically as alpha,beta-heterodimers bound laterally lengthwise. For short
constructs of alpha- and beta-spectrin, either as monomers or as
alpha,beta-dimers, sawtooth patterns in atomic force microscopy-forced extension
show that unfolding stochastically extends repeats approximately 4-5-fold
greater in length than native conformations. For both dimers and monomers,
distributions of unfolding lengths appear bimodal; major unfolding peaks reflect
single repeats, and minor unfolding peaks at twice the length reflect tandem
repeats. Cooperative unfolding thus propagates through helical linkers between
serial repeats (1, 2). With lateral heterodimers, however, the force
distribution is broad and shifted to higher forces. The associated chains in a
dimer can stay together and unfold simultaneously in addition to unfolding
independently. Weak lateral interactions do not inhibit unfolding, but strong
lateral interactions facilitate simultaneous unfolding analogous to serial
repeat coupling within spectrin family proteins. Previous X-ray crystal structures have shown that linkers of five amino acid
residues connecting pairs of chicken brain alpha-spectrin and human erythroid
beta-spectrin repeats can undergo bending without losing their alpha-helical
structure. To test whether bending at one linker can influence bending at an
adjacent linker, the structures of two and three repeat fragments of chicken
brain alpha-spectrin have been determined by X-ray crystallography. The
structure of the three-repeat fragment clearly shows that bending at one linker
can occur independently of bending at an adjacent linker. This observation
increases the possible trajectories of modeled chains of spectrin repeats.
Furthermore, the three-repeat molecule crystallized as an antiparallel dimer
with a significantly smaller buried interfacial area than that of alpha-actinin,
a spectrin-related molecule, but large enough and of a type indicating
biological specificity. Comparison of the structures of the spectrin and
alpha-actinin dimers supports weak association of the former, which could not be
detected by analytical ultracentrifugation, versus strong association of the
latter, which has been observed by others. To correlate features of the
structure with solution properties and to test a previous model of stable
spectrin and dystrophin repeats, the number of inter-helical interactions in
each repeat of several spectrin structures were counted and compared to their
thermal stabilities. Inter-helical interactions, but not all interactions,
increased in parallel with measured thermal stabilities of each repeat and in
agreement with the thermal stabilities of two and three repeats and also partial
repeats of spectrin. Questions of if and when protein structures change within cells pervade biology
and include questions of how the cytoskeleton sustains stresses on
cells--particularly in mutant versus normal cells. Cysteine shotgun labeling
with fluorophores is analyzed here with mass spectrometry of the spectrin-actin
membrane skeleton in sheared red blood cell ghosts from normal and diseased
mice. Sheared samples are compared to static samples at 37 °C in terms of cell
membrane intensity in fluorescence microscopy, separated protein fluorescence,
and tryptic peptide modification in liquid chromatography-tandem mass
spectrometry (LC-MS/MS). Spectrin labeling proves to be the most sensitive to
shear, whereas binding partners ankyrin and actin exhibit shear thresholds in
labeling and both the ankyrin-binding membrane protein band 3 and the
spectrin-actin stabilizer 4.1R show minimal differential labeling. Cells from
4.1R-null mice differ significantly from normal in the shear-dependent labeling
of spectrin, ankyrin, and band 3: Decreased labeling of spectrin reveals less
stress on the mutant network as spectrin dissociates from actin. Mapping the
stress-dependent labeling kinetics of α- and β-spectrin by LC-MS/MS identifies
Cys in these antiparallel chains that are either force-enhanced or
force-independent in labeling, with structural analyses indicating the
force-enhanced sites are sequestered either in spectrin's triple-helical domains
or in interactions with actin or ankyrin. Shear-sensitive sites identified
comprehensively here in both spectrin and ankyrin appear consistent with stress
relief through forced unfolding followed by cytoskeletal disruption. |
Is gastro esophageal reflux related to burning mouth syndrome? | No data indicate causal connection between gastro esophageal/laryngopharyngeal(LPR) reflux disease and the occurrence of intraoral burning sensations | 61 patients with symptoms suggestive for gastro-esophageal reflux (GER) disease,
with or without endoscopic evidence of esophagitis, were studied in order to
recognize any neurotic traits connected to GERD and its esophageal motility
disorders. The results were compared with those from a group of patients without
digestive diseases as well as those from a control group of the same age and
status. Psychological assessment was made by using the Middlesex Hospital
Questionnaire and esophageal motility pattern was analyzed with a low-compliance
manometric system. Patients with gastro-esophageal reflux (GER), irrespectively
or not from esophagitis, showed, after such a psychological assessment, neurotic
traits more pronounced than control subjects and patients without digestive
disease. In GER patients, it was observed a close relationship between some
psychological traits and a few esophageal manometric variable. In the two groups
of GER patients, with and without esophagitis, it was not found any significant
difference in scores referring to the evaluated psychological traits apart from
symptoms somatization, prevailing in GER patients without esophagitis. These
results support the pathogenetic role of psychological distresses in the genesis
of GER, even if other factors may be necessary to the development of organic
inflammatory lesions such as esophagitis. The burning mouth syndrome is characterized by burning and painful sensations of
the mouth in the absence of significant mucosal abnormalities. For patients in
whom no causative factor can be identified, empiric antifungal, nutritional, and
estrogen replacement therapy can be initiated. If these fail, long-term therapy
with antidepressants, benzodiazepines, and clonazepam can be considered. Topical
capsaicin and laser therapy have been reported beneficial in a few patients. Burning mouth syndrome is a common condition particularly affecting elderly
women. Numerous precipitating factors are recognized that lead to a burning
sensation in clinically normal mucosa. By taking each precipitating factor into
account, a favorable treatment outcome usually can be achieved. This article
highlights the significance of precipitating factors in burning mouth syndrome
and suggests a treatment protocol based on current scientific evidence. STATEMENT OF PROBLEM: Dental practitioners occasionally have patients present
clinically with a history of chief complaint of burning and painful sensations
in the oral cavity. Often the patient demonstrates clinically normal mucosa,
which can make formulating a diagnosis challenging. This scenario, has been
referred to as burning mouth syndrome, a multifactorial syndrome.
PURPOSE: The purpose of this article is to present a review of etiologic factors
and clinical implications related to the condition of burning mouth syndrome. Burning mouth syndrome is a complicated, poorly understood, predomitly oral
condition that affects more than 1 million people in the United States. Women
are particularly affected by the condition; they are diagnosed with symptoms
seven times more frequently than males. Burning mouth syndrome is characterized
by a burning, painful sensation of the oral mucosa that most commonly involves
the anterior tongue. Many precipitating factors to burning mouth syndrome have
been proposed, and treatment addressing these factors has had limited success.
Patients with burning mouth syndrome are more likely to be evaluated by
physicians, and therefore it is advantageous for the physician to be familiar
with this oral condition. This paper reviews burning mouth syndrome, associated
causative factors, and treatment strategies for the physician. Glossodynia (synonym: burning mouth syndrome) is thought to be a disorder with a
wide range of possible causes. Aetiologies may include haematological diseases,
vitamin deficiencies, dental work, hormonal factors, or infections. In addition,
psychological disorders such as neuroses, depression, or phobias have been
reported as playing a significant role in the initiation of burning mouth
syndrome. Typically, the oral mucosa is found to be normal in most burning mouth
syndrome patients. A multidisciplinary approach appears to be essential for
appropriate assessment of this disorder, i.e. the diagnostic procedure should
involve dentistry, neurology, and internal medicine. If possible, careful
treatment of underlying causes must be undertaken. However, the replacement of
suspected but unproven hormonal/nutritional deficiencies should be avoided. Burning mouth syndrome is characterized by a burning sensation in the tongue or
other oral sites, usually in the absence of clinical and laboratory findings.
Affected patients often present with multiple oral complaints, including
burning, dryness and taste alterations. Burning mouth complaints are reported
more often in women, especially after menopause. Typically, patients awaken
without pain but note increasing symptoms through the day and into the evening.
Conditions that have been reported in association with burning mouth syndrome
include chronic anxiety or depression, various nutritional deficiencies, type 2
diabetes (formerly known as non-insulin-dependent diabetes) and changes in
salivary function. However, these conditions have not been consistently linked
with the syndrome, and their treatment has had little impact on burning mouth
symptoms. Recent studies have pointed to dysfunction of several cranial nerves
associated with taste sensation as a possible cause of burning mouth syndrome.
Given in low dosages, benzodiazepines, tricyclic antidepressants or
anticonvulsants may be effective in patients with burning mouth syndrome.
Topical capsaicin has been used in some patients. OBJECTIVE: An assessment of oral symptoms and signs in patients with
inflammatory bowel disease (IBD).
METHODS: Fifty-four patients with IBD, 34 with Crohn's disease (CD) and 20 with
ulcerative colitis (UC) participated in the study. Forty-two patients without
gastrointestinal disease or complaints attending the orthopedic clinic served as
controls. Each patient completed a written questionnaire and was subjected to an
oral examination.
RESULTS: The main findings of this study were the higher prevalence of halitosis
(50% vs 10% P < 0.0008), nausea (30% vs 7%, P < 0.017) and reflux
(regurgitation) (45% vs 17%, P < 0.017) in patients with UC, and nausea (50% vs
7%, P < 0.026), dry mouth and halitosis (29% vs 10%, P < 0.026) and vomiting
(41% vs 5%, P = 0.01) in patients with CD, compared with controls. Patients with
active CD had a higher prevalence of dry mouth, nausea and vomiting compared
with controls (46, 69 and 54% vs 10, 7 and 5%, respectively, P < 0.001) and of
reflux compared with non-active CD (46% vs 5%, P < 0.001). Patients with active
UC had a higher prevalence of halitosis and regurgitation (50 and 60% vs 10 and
17%, P < 0.001) compared with controls.
CONCLUSIONS: The present study demonstrates increased frequency of oral signs
and symptoms in patients with IBD. Patients with active CD had more oral signs
compared with non-active CD patients. Manifestations such as nausea, vomiting,
regurgitation and dry mouth may have detrimental effects on teeth and soft
tissues of the oral cavity. Communication between gastroenterologists and
dentists is imperative for success of the overall treatment of their patients. Burning mouth syndrome (BMS) is currently described as a burning pain in the
tongue or other parts of mucous cavi oris without pathological signs of mucous
cavi oris and changes in laboratory blood tests. On the basis of the current
literature and our patients' examinations we described the incidence, etiology,
symptoms and offered model of treatment of BMS. Burning mouth syndrome (BMS) is a predomitly oral condition characterized by
the occurrence of a chronic burning that commonly involves the anterior tongue,
painful sensation, dryness and taste alterations. The syndrome is reported more
often in women, usually without any oral mucosal signs and laboratory
abnormalities. Its etiopathogenesis remains poorly understood, and there is no
consensus on diagnostic criteria and treatment strategies. Tongue burning is
though to be also one of a non-oesophageal symptom of gastro-oesophageal reflux
disease. As reported below, although this symptom may well be diagnostically
misleading, careful diagnosis based on clinical signs may distinguish patients
with BMS from those with reflux disease, and successful management of burning
mouth is often enables. Burning in the mouth in and of itself is not all that uncommon. It may result
from a variety of local or generalized oral mucosal disorders, or may be
secondary to referred phenomena from other locations. Primary burning mouth
syndrome, on the other hand, is relatively uncommon. Burning mouth syndrome is
an idiopathic pain disorder, which appears to be neuropathic in origin. Thoughts
on management of secondary and particularly primary burning mouth syndrome are
discussed. OBJECTIVE: To assess the occurrence of oral pathological changes and symptoms in
patients affected by gastro-oesophageal reflux disease (GERD).
PATIENTS AND METHODS: 200 patients with GERD and 100 matched healthy controls
were studied. Thorough visual examination of the dental and oral mucosal tissues
was performed and medical history relevant to oral symptoms was collected. The
primary outcome was defined as a statistically significant difference, between
the study group and controls, in the presence of the following indicators:
soft/hard palate and uvula erythema, tooth wear, xerostomia, oral acid/burning
sensation, subjective halitosis and dental sensitivity. Statistical analysis
included chi-squared test, and crude odds ratio with 95% CI.
RESULTS: Univariate analysis showed that xerostomia, oral acid/burning
sensation, subjective halitosis, and soft and hard palate mucosa and uvula
erythema were more common in patients with GERD than matched controls (P <
0.05).
CONCLUSIONS: This study failed to find any significant association between GERD
and dental erosions, whereas some symptoms and other objective oral mucosal
changes were found to be significantly associated with GERD. Burning mouth syndrome (BMS) is a chronic disease characterized by burning of
the oral mucosa associated with a sensation of dry mouth and/or taste
alterations. BMS occurs more frequently among postmenopausal women. The
pathophysiology of the disease is still unknown, and evidence is conflicting;
although some studies suggest a central origin, others point to a peripheral
neuropathic origin. The efficacy of some medications in the treatment of BMS
suggests that the dopaminergic system may be involved. A 45-year-old woman, with a body mass index of 41.8 kg/m2 and a medical history
of anxiety-depression syndrome, had iatrogenic hypothyroidism and degenerative
osteoarticular pathology of the spinal column and complained of a burning
sensation behind the sternum associated with an acidic taste in her mouth.
Symptoms had appeared 3 months previously and were especially prevalent when
lying down, following large meals or after drinking coffee. The patient had
started to experience symptoms on most days approximately 1 month earlier. Upper
digestive endoscopy (UDE) revealed isolated erosions of the distal third of the
oesophagus, compatible with a diagnosis of erosive reflux oesophagitis.
Lifestyle changes were recommended and 8 weeks' treatment with pantoprazole 40
mg/day taken 15-30 minutes before breakfast was prescribed. Follow-up UDE showed
resolution of oesophageal lesions with no pathological changes of the mucosa.
Mild regurgitation and pyrosis persisted; therefore the patient continued to
receive pantoprazole 40 mg for a further 3 weeks. Pain in the tongue or oral tissues described as "burning" has been referred to
by many terms including burning mouth syndrome. When a burning sensation in the
mouth is caused by local or systemic factors, it is called secondary burning
mouth syndrome and when these factors are treated the pain will resolve. When
burning mouth syndrome occurs in the absence of identified risk indicators, the
term primary burning mouth syndrome is utilized. This article focuses on
descriptions, etiologic theories, and management of primary burning mouth
syndrome, a condition for which underlying causative agents have been ruled out. Burning mouth syndrome (BMS) is a chronic condition that is characterized by
burning symptoms of the oral mucosa without obvious clinical examination
findings. This syndrome has complex characteristics, but its cause remains
largely enigmatic, making treatment and management of patients with BMS
difficult. Despite not being accompanied by evident organic changes, BMS can
significantly reduce the quality of life for such patients. Therefore, it is
incumbent on dental professionals to diagnose and manage patients with BMS as a
part of comprehensive care. |
Which is the protein (antigen) targeted by anti-Vel antibodies in the Vel blood group? | Disruption of SMIM1 causes the Vel- blood type. The protein carrying the Vel blood group antigen was biochemically purified from red blood cell membranes. Mass spectrometry-based de novo peptide sequencing identified this protein to be small integral membrane protein 1 (SMIM1), a previously uncharacterized single-pass membrane protein. Expression of SMIM1 cDNA in Vel- cultured cells generated anti-Vel cell surface reactivity, confirming that SMIM1 encoded the Vel blood group antigen. (PMID: 23505126) | BACKGROUND AND OBJECTIVES: A patient experienced a severe haemolytic transfusion
reaction. Neither the haemolytic property nor the specificity of the causative
antibody had been sufficiently recognised when performing a microcolumn gel
test.
MATERIALS AND METHODS: Subsequent to the transfusion reaction, the serological
property and specificity of the causative antibody were analysed. Tube and gel
test methods were compared, as were various reagent red cell specimens and their
constituents.
RESULTS: A haemolytic anti-Vel was detected in the tube test. In contrast, the
particular commercial gel test kit used did not reveal the haemolytic property
or specificity of the antibody. Our experiments suggest that this was apparently
due to the presence of EDTA in the low ionic strength saline solution of the
test kit.
CONCLUSIONS: In rare cases life-treatening haemolytic activity of an irregular
blood group antibody may be undetected by a commericial microcolum gel test kit
in which EDTA is a constituent. BACKGROUND: There is only little information on the transfusion support of
patients with antibodies to high-frequency RBC antigens.
STUDY DESIGN AND METHODS: In cooperation with reference laboratories and
transfusion services in Austria, Germany, and Switzerland, the transfusion
support provided to hospitalized patients identified as having such antibodies
was reviewed during a 20-month period.
RESULTS: A total of 52 patients with antibodies to high-frequency antigens were
treated in hospitals. Twenty-two of them received 104 units of antigen-negative
RBCs. In 23 cases, a deviation from the standard transfusion policy (e.g.,
transfusion of antigen-incompatible units) occurred. The use of frozen or fresh
units varied amongst the different countries but did not affect the rate of
deviation from protocol. About 20 percent of all units were supplied
internationally. Four antibody specificities, anti-Kpb, anti-Vel, anti-Lub, and
anti-Yta, were identified in two-thirds of the patients.
CONCLUSION: This survey indicated that transfusion support was unsatisfactory in
about one-third of the hospitalized patients with antibodies to high-frequency
antigens. Maintaining a rapidly accessible stock of just four types of rare
blood units would ensure adequate transfusion support for most of these
patients. Anti-Vel is an uncommon antibody to a high-prevalence antigen. Its clinical
significance and management in the prenatal setting are not well characterized.
We present a case that demonstrates the utility of serial prenatal anti-Vel
quantitative serologic monitoring with 2-ME serum treatment during pregcy.
The patient is a 23-year-old Hispanic woman with history of prior pregcy and
prior transfusion who was discovered to have an antibody to the high-prevalence
Vel antigen in the first trimester (week 7) of her second pregcy. Interval
measurements of the serologic antibody titers were performed during the next 26
weeks. The untreated serum (IgM and IgG) titer increased from a baseline of 4 to
16 during that interval, while the 2-ME (presumed IgG component) titer remained
stable at 4. Responding to ultrasound findings suspicious for fetal anemia, the
child was delivered without complications at 34 weeks' gestation. At birth, the
DAT was negative and there was no evidence of HDN. Placed in the context of
other similar reports, this case demonstrates the importance of separately
reporting the IgG fraction (after either DTT treatment or 2-ME treatment) from
the untreated (IgM and IgG) fraction and the importance of correlating the
treated serum titer with potential clinical significance. Here, we report the biochemical and genetic basis of the Vel blood group
antigen, which has been a vexing mystery for decades, especially as anti-Vel
regularly causes severe haemolytic transfusion reactions. The protein carrying
the Vel blood group antigen was biochemically purified from red blood cell
membranes. Mass spectrometry-based de novo peptide sequencing identified this
protein to be small integral membrane protein 1 (SMIM1), a previously
uncharacterized single-pass membrane protein. Expression of SMIM1 cDNA in Vel-
cultured cells generated anti-Vel cell surface reactivity, confirming that SMIM1
encoded the Vel blood group antigen. A cohort of 70 Vel- individuals was found
to be uniformly homozygous for a 17 nucleotide deletion in the coding sequence
of SMIM1. The genetic homogeneity of the Vel- blood type, likely having a common
origin, facilitated the development of two highly specific DNA-based tests for
rapid Vel genotyping, which can be easily integrated into blood group genotyping
platforms. These results answer a 60-year-old riddle and provide tools of
immediate assistance to all clinicians involved in the care of Vel- patients. |
Which are the subtypes of Pfeiffer syndrome? | Pfeiffer syndrome is divided into three clinical subtypes. | Steven Pfeiffer syndrome pedigrees (three 3 generation and four 2 generation)
have been recorded to date in addition to at least a dozen sporadic cases.
Autosomal domit inheritance with complete penetrance is characteristic of the
7 familial instances. Variable expressivity has involved mostly the presence or
absence of syndactyly and the degree of syndactyly when present. Classic
Pfeiffer syndrome is designated type I. Type 2 consists of cloverleaf skull with
Pfeiffer hands and feet together with ankylosis of the elbows. Such patients do
poorly with an early death. All reported instances to date have been sporadic.
Type 3 is similar to type 2 but without cloverleaf skull. Ocular proptosis is
severe in degree and the anterior cranial base is markedly short. These patients
also do poorly and tend to have an early death. To date all cases have occurred
sporadically. Although these 3 clinical subtypes do not have status as separate
entities, their diagnostic and prognostic implications are important. Type 1 is
commonly associated with normal intelligence, generally good outcome, and can be
found domitly inherited in some families. Types 2 and 3 generally have severe
neurological compromise, poor prognosis, early death, and sporadic occurrence.
Recognition of type 3 is particularly important because extreme ocular proptosis
in the absence of cloverleaf skull but with various visceral anomalies can
result in failure to diagnose Pfeiffer syndrome and labeling the patient as an
"unknown" or as a "newly recognized entity."(ABSTRACT TRUNCATED AT 250 WORDS) Pfeiffer syndrome (PS) is an autosomal domit condition comprising bilateral
coronal craniosynostosis, midface hypoplasia with a beaked nasal tip, and broad
and medially deviated thumbs and great toes. It is a clinically variable
disorder and has been divided into three subtypes [Cohen, 1993: Am J Med Genet
45:300-307]. Type 1 represents the less severe cases, while types 2 and 3 are
the more severe cases. These latter types tend to have a higher risk for
neurodevelopmental problems and a reduced life expectancy. Here we review the
clinical course of seven children with PS type 3. All of these children had
severe manifestations of PS; however, development was essentially normal in
three, mild delay was noted in two, and moderate delay in one. Favorable
outcomes in children with types 2 and 3 PS were also documented by Moore et al.
[1995: Cleft Pal-Craniofac J 32:62-70]. These cases illustrate that while
children with PS types 2 and 3 have an increased risk for neurodevelopmental
difficulties, a favorable outcome can be achieved in some cases with aggressive
medical and surgical management. Finally, although such management should be the
rule for PS types 2 and 3, it needs to be remembered that normal outcome is not
the rule. The prognosis for favorable neurodevelopmental outcome and/or life
expectancy remains guarded in most cases. Pfeiffer syndrome, an autosomal domit disorder, consists of craniosynostosis,
broadening of the thumbs and great toes, and partial soft tissue syndactyly of
the hands and feet. Three clinical subtypes have been classified mainly for the
purpose of genetic counseling. Mutations in FGFR1 and FGFR2 are known to be
associated with the syndrome. However, the correlation between genotype and
phenotype is not well defined. Only one patient with Pfeiffer syndrome with no
other clinical information has been reported to have had an A344P mutation of
the FGFR2. Here we report a Thai male patient with sporadic Pfeiffer syndrome
type 1 with impaired intelligence (IQ = 77). Mutation analysis revealed A344P in
FGFR2. Identification of the clinical features and molecular defects in more
patients is required to better correlate the genotype and phenotype of this
complex syndrome. Pfeiffer syndrome is clinically and genetically heterogeneous. Three clinical
subtypes have been delineated based on the severity of acrocephalysyndactyly and
associated manifestations. Severe cases are usually sporadic and caused by a
number of different mutations in exons IIIa and IIIc of the fibroblast growth
factor receptor 2 (FGFR2) gene. Mild cases are either sporadic or familial and
are caused by mutations in FGFR2 or FGFR1, respectively. We report on two
individuals with different novel de novo mutations in FGFR2. The first is a
17-year-old male who has a severe phenotype, within the spectrum of subtype 1
including severe ocular proptosis, elbow ankylosis, visceral anomalies, and
normal intelligence. This patient was found to have a novel complex mutation at
the 3' acceptor site of exon IIIc of FGFR2, denoted as C952-3 del10insACC. The
other patient, a 2-year-old female, has a mild phenotype, typical of the classic
subtype 1 including brachycephaly with coronal synostosis and hypertelorism. She
was also found to have a mutation at the 3' acceptor site (the same splice site)
of exon IIIc of FGFR2, a point mutation designated as 952-1G-->A. Speculation on
the molecular mechanisms that cause severe and mild phenotypes is presented in
relation to these two cases. BACKGROUND: Pfeiffer syndrome is rarely encountered, even at major craniofacial
centers. Published reports indicate high mortality rates (25 to 85 percent) for
severely affected subtypes. The authors reviewed their surgically treated
patients to improve outcomes.
METHODS: The authors conducted a 17-year, single-center, retrospective outcome
assessment of all children treated for Pfeiffer syndrome, with data summarized
using descriptive statistics.
RESULTS: Of 802 patients treated for craniosynostosis, 28 were identified with
Pfeiffer syndrome: 17 were classified as type I (61 percent), seven were
classified as type II (25 percent), and four were classified as type III (14
percent). The mean age was 10 years (range, 12 months to 39 years), with an
average of 9.3 operations per child (2.5 cranial vaults, 1.1 Le Fort III
procedures). Fifty-nine percent had external auditory canal atresia (100 percent
of type III patients), and 29 percent had some visual disturbance.
Tracheostomies were recommended in 100 percent of type II and III patients, and
two type II patients required tracheal stenosis repairs. Eighty-four percent had
acquired Chiari malformations (100 percent of type II and III patients), and 61
percent required treatment for hydrocephalus. Fifty percent of shunted patients
(mean age, 7 years) have required Chiari decompressions, but no patients
undergoing endoscopic third ventriculostomies (mean age, <3 years) have required
treatment. The mortality rate was 7 percent, with both deaths occurring at home
without proximity to surgery.
CONCLUSIONS: The authors' mortality rates for type II and III Pfeiffer syndrome
are lower than those previously published. The authors believe a preemptory
tarsorrhaphy strategy can prevent visual loss and that further reductions in
mortality rates are possible with aggressive airway management (early
tracheostomies) and more frequent screening (e.g., magnetic resoce imaging,
sleep studies) for Chiari malformations. |
Is thrombophilia related to increased risk of miscarriage? | Thrombophilia has been found to be considerably more common in women with pregnancy-associated complications in comparison with the general population, and most frequently in conjunction with venous thromboembolism during pregnancy and the postpartum period. In particular there is an increased risk of pregnancy-related venous thrombosis in carriers of severe inherited thrombophilia. When counseling white women with a history of preeclampsia, screening for thrombophilia can be useful for preconceptional counseling and pregnancy management. | Homozygous carriers of factor V Leiden have an approximately 80-fold increased
risk of venous thrombosis. Also double heterozygous carriers of both the factor
V Leiden and the prothrombin gene mutations are at high thrombotic risk. The
magnitude of the risk of venous thrombosis in pregt women with the two severe
thrombophilic conditions has not been estimated so far. We performed a
multicenter retrospective family study in women with homozygous factor V Leiden,
double heterozygous factor V Leiden and the prothrombin gene mutation, and women
with normal coagulation. Only relatives of index patients with thrombosis formed
the study cohort. Fifteen homozygous and 39 double heterozygous women were
compared to 182 women with normal coagulation. Venous thrombosis occurred in 3
of 19, 2 of 50 and 1 of 221 pregcies, respectively. One thrombotic episode
occurred in the third trimester, the remaining 5 in the postpartum. The
prevalence of venous thrombosis was 15.8% (95% CI 3.4-39.6) for homozygotes.
4.0% (95% CI 0.5-13.7) for double heterozygotes and 0.5% for women with normal
coagulation. The relative risk of pregcy-related venous thrombosis was 41.3
(95% CI 4.1-419.7) for homozygous and 9.2 (95% CI 0.8-103.2) for double
heterozygous carriers. In conclusion, homozygous carriers of factor V Leiden
and, to a lesser extent, double heterozygous carriers of factor V Leiden and of
the prothrombin mutation have an increased risk of venous thrombosis during
pregcy, particularly high during the postpartum period. On the basis of these
findings we recommend that these women receive anticoagulant prophylaxis at
least in the postpartum, that should perhaps be extended to the whole pregcy
in homozygous carriers. Thromboembolism in pregcy and the puerperium and inherited or acquired
thrombophilia are associated. Thrombophilia can be revealed by pregcy.
Thrombotic risk during pregcy and the puerperium is higher in asymptomatic
women with than without thrombophilia. Antithrombin deficiency, combined
deficiencies and homozygous or double-heterozygotes factor V Leiden and factor
II G 20210 A mutations are associated with a higher thrombotic risk than
heterozygote mutations or protein S and C deficiencies, whereas
hyperhomocysteinemia does not appear as a risk factor for maternal
thromboembolic disease. Antiphospholipid syndrome with lupus anticoagulant is
strongly associated with thrombotic risk in pregcy and the puerperium.
Further studies are required to assess the thrombotic risk in women with
preeclampsia as well as early or late recurrent pregcy loss. The main inherited thrombophilias (antithrombin deficiency, protein C and S
deficiency, FVL, the prothrombin gene variant, and MTHFR C677T homozygotes) have
a combined prevalence in Western European populations of 15% to 20%. One or more
of these inherited thrombophilias is usually found in approximately 50% of women
who have a personal history of VTE. Obstetricians must therefore be aware of the
interaction between thrombophilias and the procoagulant state of pregcy and
should have an understanding of additional risk factors that may act
synergistically with thrombophilias to induce VTE. Such knowledge combined with
the appropriate use of thromboprophylaxis and treatment in women who have
objectively confirmed VTE continue to improve maternal and perinatal outcomes. Pregcy in healthy women is accompanied by hypercoagulable changes that may
interact with thrombophilia risk factors and threaten pregcy. However, the
literature on this issue is conflicting. In investigating the relationship
between pregcy-associated complications and the presence of thrombophilia
risk factors, we studied the records of 414 women who had been examined for
inherited and acquired thrombophilia in the period 1996 to 2006 because of
pregcy-associated complications. Of a total of 885 pregcies among the
women, 397 were recorded as foetal loss/intrauterine foetal death during the
first (62 %), second (25 %) or third trimester (13 %). One-hundred-and-two (25
%) women had had a thromboembolic event during one of their pregcies, and 98
(24 %) had had pre-eclampsia on at least one occasion. Intrauterine growth
restriction was found in 105 (25 %) of the women, and 29 (7 %) suffered
placental abruption. We found that 120 (29 %) women had at least one
thrombophilia risk factor. Factor V Leiden heterozygosity was the most common
thrombophilia factor (n = 52), mostly linked with the risk of venous
thromboembolism during pregcy or postpartum and to foetal death during the
second or third trimester. Fifty-three (13 %) women had antiphospholipid
antibodies (lupus anticoagulant and/or anti-beta2-glycoprotein 1 antibodies)
mainly associated with the risk of spontaneous abortion during the first
trimester. In conclusion, thrombophilia was found to be considerably more common
in women with pregcy-associated complications in comparison with the general
population, and most frequently in conjunction with venous thromboembolism
during pregcy and the postpartum period. OBJECTIVE: To determine the prevalence of thrombophilic genetic variants in an
American Indian population and determine if they are associated with
preeclampsia.
METHODS: A total of 87 cases, 165 controls and an additional 75 population-based
controls were genotyped for two thrombophilic polymorphisms.
RESULTS: The allelic prevalence of the factor V Leiden and 20210 G/A prothrombin
variants in this population was 2.1% and 0.5% respectively. No statistically
significant associations between these genetic variants and preeclampsia were
found.
CONCLUSION: The prevalence of thrombophilic variants is of possible public
health significance for other morbidity; but perhaps not in relation to
preeclampsia. OBJECTIVE: Preeclampsia is associated with increased risk of cardiovascular
disease. The aim of this pilot study was to assess whether the presence of
thrombophilia results in a greater tendency to develop endothelial dysfunction
and cardiovascular diseases.
METHODS: Ten women with thrombophilia were matched with 10 women without
thrombophilia for a history of hypertensive disorders during pregcy.
Laboratory measurements: blood pressure, insulin sensitivity, and micro- and
macrovascular function were determined.
RESULTS: Women with thrombophilia had significant lower total- and low-density
cholesterol, were more insulin sensitive, and had better microvascular function.
CONCLUSION: This study suggests that thrombophilia "mediates" in lowering of
cardiovascular risk factors in women with a history of preeclampsia. Thrombophilias have been implicated in complications related to ischemic
placental disease including recurrent pregcy loss, intrauterine fetal demise,
preeclampsia, fetal growth restriction, placental abruption, and preterm
delivery. Maternal screening and treatment may lower the recurrence of these
outcomes. Our objective was to estimate if antenatal screening for
thrombophilias with the intention to offer treatment among women with a prior
adverse pregcy outcome (APO) is preferable to no screening. A
decision-analytical model was constructed for pregt women with prior APO,
comparing screening for thrombophilia with intention to treat with no screening.
Values obtained from previously published studies include probability of
positive test: 0.3 (0.1 to 0.6); good outcome with treatment: 0.9 (0.3 to 0.99);
no thrombophilia, good outcome: 0.75 (0.5 to 0.9); test negative, thrombophilia
positive: 0.05 (0.01 to 0.1); test negative, thrombophilia positive, good
outcome: 0.75 (0.5 to 0.9); thrombophilia/test negative, good outcome: 0.98 (0.5
to 0.99). Sensitivity analyses were run over a wide range of assumptions.
Thrombophilia screening with intention to treat in women with prior APO
associated with ischemic placental disease is the strategy of choice compared
with no testing over a wide range of assumptions. Sensitivity analyses support
this to be robust. Women with poor pregcy history related to placental
ischemic disease may benefit from thrombophilia screening and treatment in a
subsequent pregcy. Pregcy is an acquired state of hypercoagulation. An association has been
found between various pregcy complications and thrombophilia. Among those
complications are: preeclampsia, intrauterine fetal death, intrauterine growth
retardation and placentaL abruption. This article will present a novel scoring
system estimating pregcy complications in women with thrombophilia. The
biological and epidemiological background of the association between pregcy
complications and thrombophilia will be discussed and the therapeutic options
will be evaluated. Finally, for illustrative purposes, a patient presenting with
combined thrombophilia--both genetic and acquired--will be discussed. This
patient had suffered severe gestational complications that led to devastating
obstetrical outcome. |
Intact macromolecular assemblies are analysed by advanced mass spectrometry. How large complexes (in molecular weight) have been studied? | 2.3 megadalton | Previous studies have shown that pulmonary surfactant protein D (SP-D) is
composed of a 43-kDa polypeptide with a short NH2-terminal domain, a collagen
sequence, and a COOH-terminal C-type lectin domain. In the present studies,
ultrastructural and biochemical techniques were used to examine the quaternary
structure of native rat SP-D (rSP-D). Electron microscopy of freeze-dried
preparations demonstrated a highly homogeneous population of molecules with four
identical rod-like arms (46 nm in length), each with an 8-9-nm diameter globular
terminal expansion. The arms, which are similar in diameter to the type I
collagen helix (approximately 4 nm), emanate from the central "hub" in two pairs
that closely parallel each other for their first 10 nm. This structure is
consistent with hydrodynamic studies that predict an highly asymmetric and
extended molecule (f/f0 = 3.26) with a large Stokes radius (Rs = 18 nm). Pepsin
digestion gave glycosylated, trimeric collagenous fragments (43 +/- 4 nm, 17
kDa/chain). Trimeric subunits containing intact triple helical domains were also
liberated from SP-D dodecamers by sulfhydryl reduction under non-denaturing
conditions. Digestion of rSP-D with bacterial collagenase generated a
COOH-terminal carbohydrate binding fragment and a smaller peptide (approximately
12 kDa, unreduced) that contains interchain disulfide bonds. Electron microscopy
also demonstrated higher orders of multimerization, with as many as 8 molecules
associated at the hub. These studies demonstrate that SP-D is assembled as
homopolymers of four identical trimeric subunits, that interactions between the
amino-terminal domains of the trimers are stabilized by interchain disulfide
bonds, and that SP-D molecules can associate to form complex multimolecular
assemblies. It is established that noncovalent complexes can be maintained both during and
after electrospray and that assemblies of increasing size and complexity often
lead to broadened peaks in mass spectra. This broadening arises from the
tendency of large protein assemblies to form adducts with salts and is
compounded when complexes are isolated directly from cells, without the full
protein complement. To investigate the origins of this broadening in mass
spectral peaks and to develop the optimal method for analyzing mass spectra of
large protein complexes, we have carried out a systematic investigation of a
series of noncovalent complexes representing a range of different sizes and
architectures. We establish a positive correlation between peak width and the
increased mass observed and show that this correlation is independent of the
instrumental parameters employed. Using this relationship we show that we can
determine masses of both 30S subunits and intact 2.3 MDa 70S ribosomes from
Thermus thermophilus. The masses of both particles are consistent with multiple
populations of ribosomes. To identify these various populations we combine
simulated mass spectra of ribosomes, with and without the full protein
complement, and estimate the extent of adducts from our study of known
complexes. The results allow us to determine the contribution of the different
subpopulations to the overall mass spectrum. We confirm the existence of these
subpopulations using tandem mass spectrometry of intact 30S subunits. Overall,
the results show that, rather than uniform particles, gas-phase ribosomes
consist of a number of discrete populations. More generally, the results
establish a rigorous procedure for accurate mass measurement and spectral
analysis of heterogeneous macromolecular assemblies. In recent years mass spectrometry based techniques have emerged as structural
biology tools for the characterization of macromolecular, noncovalent
assemblies. Many of these efforts involve preservation of intact protein
complexes within the mass spectrometer, providing molecular weight measurements
that allow the determination of subunit stoichiometry and real-time monitoring
of protein interactions. Attempts have been made to further elucidate subunit
architecture through the dissociation of subunits from the intact complex by
colliding it into inert gas atoms such as argon or xenon. Unfortunately, the
amount of structural information that can be derived from such strategies is
limited by the nearly ubiquitous ejection of a single, unfolded subunit. Here,
we present results from the gas-phase dissociation of protein-protein complexes
upon collision into a surface. Dissociation of a series of tetrameric and
pentameric proteins demonstrate that alternative subunit fragments, not observed
through multiple collisions with gas atoms, can be generated through surface
collision. Evidence is presented for the retention of individual subunit
structure, and in some cases, retention of noncovalent interactions between
subunits and ligands. We attribute these differences to the rapid large energy
input of ion-surface collisions, which leads to the dissociation of subunits
prior to the unfolding of individual monomers. |
What is targeted by Palbociclib? | Necitumumab is a fully human IgG(1) monoclonal antibody directed at the epidermal growth factor receptor (EGFR). It is used for treatment of nonsmall cell lung cancer. | PURPOSE: This study aimed to determine a maximum tolerated dose (MTD) and
recommended dose for disease-directed studies of necitumumab (IMC-11F8), a fully
human IgG(1) monoclonal antibody directed at the epidermal growth factor
receptor, and to characterize the safety profile, pharmacokinetics, preliminary
antitumor activity, and immunogenicity of necitumumab.
EXPERIMENTAL DESIGN: Patients with advanced solid maligcies were treated with
100 to 1,000 mg (flat dosing) necitumumab followed by a 2-week pharmacokinetics
sampling period, before beginning 6-week cycles of therapy.
RESULTS: Sixty patients received necitumumab weekly (29 patients) or every other
week (31 patients). Two patients receiving 1,000 mg every 2 weeks experienced
dose-limiting toxicities (DLT; grade 3 headache), accompanied by grade 3 nausea
and vomiting in one patient. Occurring hours after the initial dose, these DLTs
established 800 mg as the MTD. Mild dose-related skin toxicity was the most
common drug-related toxicity (80%). One patient in each arm experienced grade 3
acneform rash, which responded to oral antibiotics and topical therapy. Toxicity
was similar on both schedules. Necitumumab exhibited saturable elimination and
nonlinear pharmacokinetics. At 800 mg (both arms), its half-life was
approximately 7 days. All patients treated with >or=600 mg necitumumab achieved
target trough concentrations (>or=40 microg/mL). Antibodies against necitumumab
were not detected. Partial response and stable disease were experienced by 2 and
16 patients, respectively.
CONCLUSION: Well tolerated, necitumumab is associated with preliminary evidence
of antitumor activity, and achieves biologically relevant concentrations
throughout the dosing period. The recommended dose of necitumumab for further
clinical development is 800 mg (flat dose) weekly or every 2 weeks based on the
clinical setting. Blockade of the epidermal growth factor receptor (EGFR) by monoclonal antibodies
is a strategy to improve outcome in patients with non-small cell lung cancer.
Cetuximab, a chimeric anti-EGFR monoclonal antibody, has been studied in
combination with different chemotherapy protocols in both phase II and phase III
trials in patients with advanced NSCLC. In the phase III FLEX trial, cetuximab
added to cisplatin/vinorelbine resulted in an absolute overall survival benefit
of 1.2 months compared to the same chemotherapy alone in patients with advanced
EGFR-expressing NSCLC. In the second phase III trial, cetuximab added to
carboplatin plus paclitaxed failed to improve progression-free survival but
suggested a survival benefit similar to that seen in the FLEX trial. However,
the benefit in survival reached statistical significance only in the FLEX trial.
A meta-analysis that included patients from four randomized trials confirmed the
efficacy of cetuximab when added to chemotherapy. Thus addition of cetuximab to
platinum-based chemotherapy represents a new treatment option for patients with
advanced NSCLC. Matuzumab and panitumumab have also been evaluated in phase II
trials. Necitumumab is currently evaluated in combination with chemotherapy in
two randomized phase III trials. INTRODUCTION: Treatment outcomes in unselected patients with advanced NSCLC
remain disappointing with platinum-based chemotherapy. The addition of
monoclonal antibodies targeting EGFR to standard first-line therapy is a
validated strategy and has been associated with statistically significant
survival advantage in advanced NSCLC. Necitumunab is a fully human IgG1
monoclonal antibody targeting EGFR, having the potential benefit of lower
hypersensitivity reaction risk as compared with cetuximab and also equivalent
antibody-dependent cell-mediated cytotoxicity.
AREAS COVERED: This paper reviews literature on preclinical and early clinical
development of necitumumab that is available in PubMed and published abstracts
from conferences, as well as ongoing trials as specified by clinicaltrials.gov.
Recently, the Phase III clinical trial evaluating the addition of necitumumab to
pemetrexed and cisplatin in non-squamous NSCLC was prematurely closed due to
concerns about the increased risk of thromboembolic events in the experimental
arm. Accrual in the Phase III trial of necitumumab in combination with
gemcitabine and cisplatin in squamous NSCLC is ongoing.
EXPERT OPINION: Results of the ongoing large randomized trials will be
instrumental in determining the drug's clinical significance and, with the
analysis of potential molecular predictive factors, are expected to bring
valuable additions to future therapeutic strategies in NSCLC. Necitumumab, a monoclonal antibody directed against EGFR, is currently under
development as a treatment for advanced NSCLC. Two Phase III randomized trials
are ongoing, testing the addition of necitumumab to first-line platinum-based
chemotherapy. In the same setting, cetuximab produced a statistically
significant but clinically modest benefit in the whole study population, and no
solid data have been produced about predictive factors of efficacy. Will the
difference in structure between the two antibodies be enough to obtain a
clinically relevant advantage, making real progress in the treatment of advanced
NSCLC? Large Phase III trials in unselected patients risk demonstrating
statistically significant results with debatable clinical relevance in the whole
population, and the study of predictive factors is often left to subgroup
analysis performed after the conduction of the trial. We do not need further
'me-too' drugs, or drugs that produce a small benefit in the unselected
population. On the contrary, the oncologic community needs drugs to be used with
a proper selection of patients, to obtain larger, relevant benefits in
molecularly characterized subgroups. Final results of randomized trials with
necitumumab in advanced NSCLC are expected in a couple of years. Joining cetuximab, sorafenib, afatinib, intedanib, and crizotinib in phase III
development for non-small cell lung cancer (NSCLC) are ramucirumab (developed by
ImClone, a subsidiary of Lilly), necitumumab (developed by ImClone and
Bristol-Myers Squibb), and tivantinib (ARQ 197, developed by ArQule and Daiichi
Sankyo). Necitumumab is a second-generation anti-EGFR monoclonal antibody (mAb)
similar to cetuximab. Enrollment has been stopped in one of two necitumumab
phase III trials because of safety concerns. Ramucirumab is an anti-VEGFR2 mAb
targeting the same pathway as bevacizumab. Although the phase II safety data for
ramucirumab appear better than the data for necitumumab, fewer phase III data
are available. Tivantinib is a highly selective, orally available MET tyrosine
kinase inhibitor. MET is overexpressed in 61% of NSCLC cases. Although
tivantinib is the last of the three agents discussed here to enter phase III,
its phase II results are the most robust. PURPOSE OF REVIEW: The epidermal growth factor receptor (EGFR) is overexpressed
in many nonsmall cell lung cancers (NSCLCs). Blockade of EGFR by monoclonal
antibodies has been studied as a strategy to improve the outcome of first-line
chemotherapy in patients with NSCLC. The present review updates the findings
from phase III trials.
RECENT FINDINGS: Cetuximab improved survival when combined with first-line
chemotherapy and this benefit was limited to patients with high EGFR expression
in their tumors. A Southwest Oncology Group study currently prospectively
evaluates the predictive biomarkers for cetuximab. In the SQUIRE phase III
trial, necitumumab added to cisplatin and gemcitabine increased the survival in
patients with advanced squamous cell NSCLC. The INSPIRE trial studied
chemotherapy with and without necitumumab in patients with nonsquamous NSCLC but
was prematurely halted because of increased thromboembolic events with
chemotherapy and necitumumab.
SUMMARY: EGFR monoclonal antibodies improved the outcome including survival in
selected patients with advanced NSCLC. Prospective validation of predictive
biomarkers is ongoing. BACKGROUND: Necitumumab is a second-generation recombit human immunoglobulin
G1 EGFR monoclonal antibody that competitively inhibits ligand binding. We aimed
to compare necitumumab plus pemetrexed and cisplatin with pemetrexed and
cisplatin alone in patients with previously untreated, stage IV, non-squamous
non-small-cell lung cancer (NSCLC).
METHODS: We did this randomised, open-label, controlled phase 3 study at 103
sites in 20 countries. Patients aged 18 years or older, with an Eastern
Cooperative Oncology Group (ECOG) performance status of 0-2 and adequate organ
function, were randomly assigned 1:1 to treatment with a block randomisation
scheme (block size of four) via a telephone-based interactive voice-response
system or interactive web-response system. Patients received either cisplatin 75
mg/m(2) and pemetrexed 500 mg/m(2) on day 1 of a 3-week cycle for a maximum of
six cycles alone, or with necitumumab 800 mg on days 1 and 8. Necitumumab was
continued after the end of chemotherapy until disease progression or
unacceptable toxic effects. Randomisation was stratified by smoking history,
ECOG performance status, disease histology, and geographical region. Patients
and study investigators were not masked to group assignment. The primary
endpoint was overall survival. Efficacy analyses were by intention to treat.
This trial is registered with ClinicalTrials.gov, number NCT00982111.
FINDINGS: Between Nov 11, 2009, and Feb 2, 2011, we randomly assigned 633
patients to receive either necitumumab plus pemetrexed and cisplatin (n=315) or
pemetrexed and cisplatin alone (n=318). Enrolment was stopped on Feb 2, 2011,
after a recommendation from the independent data monitoring committee. There was
no significant difference in overall survival between treatment groups, with a
median overall survival of 11·3 months (95% CI 9·5-13·4) in the necitumumab plus
pemetrexed and cisplatin group versus 11·5 months (10·1-13·1) in the pemetrexed
and cisplatin group (hazard ratio 1·01 [95% CI 0·84-1·21]; p=0·96). The
incidence of grade 3 or worse adverse events, including deaths, was higher in
the necitumumab plus pemetrexed and cisplatin group than in the pemetrexed and
cisplatin group; in particular, deaths regarded as related to study drug were
reported in 15 (5%) of 304 patients in the necitumumab group versus nine (3%) of
312 patients in the pemetrexed and cisplatin group. Serious adverse events were
likewise more frequent in the necitumumab plus pemetrexed and cisplatin group
than in the pemetrexed and cisplatin group (155 [51%] of 304 vs 127 [41%] of 312
patients). Patients in the necitumumab plus pemetrexed and cisplatin group had
more grade 3-4 rash (45 [15%] of 304 vs one [<1%] of 312 patients in the
pemetrexed and cisplatin alone group), hypomagnesaemia (23 [8%] vs seven [2%]
patients), and grade 3 or higher venous thromboembolic events (23 [8%] vs 11
[4%] patients) than did those in the pemetrexed and cisplatin alone group.
INTERPRETATION: Our findings show no evidence to suggest that the addition of
necitumumab to pemetrexed and cisplatin increases survival of previously
untreated patients with stage IV non-squamous NSCLC. Unless future studies
identify potentially useful predictive biomarkers, necitumumab is unlikely to
provide benefit in this patient population when combined with pemetrexed and
cisplatin.
FUNDING: Eli Lilly and Company. |
Which peptide plays a pivotal role in human cystatin C fibrillization? | Human cystatin C (HCC) is a low molecular weight member of the cystatin family (type2). HCC consists of 120 amino acids. Normally it is an inhibitor of cysteine proteases, but in pathological conditions it forms amyloid fibrils in brain arteries of young adults. An 'aggregation-prone' pentapeptide ((47)LQVVR(51)) was located within the HCC sequence using AmylPred, an 'aggregation-prone' peptide prediction algorithm developed in our lab. This peptide was synthesized and self-assembled into amyloid-like fibrils in vitro, as electron microscopy, X-ray fiber diffraction, Attenuated Total Reflectance Fourier-Transform Spectroscopy and Congo red staining studies reveal. Thus, the (47)LQVVR(51) peptide seems to have an important role in HCC fibrillization. | Human cystatin C (HCC) is a low molecular weight member of the cystatin family
(type2). HCC consists of 120 amino acids. Normally it is an inhibitor of
cysteine proteases, but in pathological conditions it forms amyloid fibrils in
brain arteries of young adults. An 'aggregation-prone' pentapeptide
((47)LQVVR(51)) was located within the HCC sequence using AmylPred, an
'aggregation-prone' peptide prediction algorithm developed in our lab. This
peptide was synthesized and self-assembled into amyloid-like fibrils in vitro,
as electron microscopy, X-ray fiber diffraction, Attenuated Total Reflectance
Fourier-Transform Spectroscopy and Congo red staining studies reveal. Thus, the
(47)LQVVR(51) peptide seems to have an important role in HCC fibrillization. |
What is ChiRP-seq (Chromatin Isolation by RNA Purification sequencing)? | ChiRP-seq (Chromatin Isolation by RNA Purification sequencing) is a method where tiling oligonucleotides retrieve specific lncRNAs with bound protein and DNA sequences, which are enumerated by deep sequencing. ChIRP-seq of three lncRNAs reveal that RNA occupancy sites in the genome are focal, sequence-specific, and numerous. ChIRP-seq is generally applicable to illuminate the intersection of RNA and chromatin with newfound precision genome wide. | Long noncoding RNAs (lncRNAs) are key regulators of chromatin state, yet the
nature and sites of RNA-chromatin interaction are mostly unknown. Here we
introduce Chromatin Isolation by RNA Purification (ChIRP), where tiling
oligonucleotides retrieve specific lncRNAs with bound protein and DNA sequences,
which are enumerated by deep sequencing. ChIRP-seq of three lncRNAs reveal that
RNA occupancy sites in the genome are focal, sequence-specific, and numerous.
Drosophila roX2 RNA occupies male X-linked gene bodies with increasing tendency
toward the 3' end, peaking at CES sites. Human telomerase RNA TERC occupies
telomeres and Wnt pathway genes. HOTAIR lncRNA preferentially occupies a GA-rich
DNA motif to nucleate broad domains of Polycomb occupancy and histone H3 lysine
27 trimethylation. HOTAIR occupancy occurs independently of EZH2, suggesting the
order of RNA guidance of Polycomb occupancy. ChIRP-seq is generally applicable
to illuminate the intersection of RNA and chromatin with newfound precision
genome wide. |
What is the use of MammaPrint and Oncotype DX? | The MammaPrint and Oncotype DX assays are used to predict breast cancer recurrence risk and guide adjuvant chemotherapy decisions. | Recently emerging diagnostic tools such as MammaPrint and oncotype-DX are
beginning to have impact on clinical practice of breast cancer. They are based
on gene expression profiling, i.e., gene expression analysis of a large number
of genes. Their unique characteristic is the use of a score calculated from
expression values of a number of genes, for which the Food and Drug
Administration (FDA) created a new diagnostic category entitled "in vitro
diagnostic multivariate index assay (IVDMIA)." In contrast to conventional
biomarkers, IVDMIA requires an algorithm to calculate the diagnostic score. The
linear classifier is the preferred algorithm. When the number of diagnostic
genes is n, each tumor is represented by a point in an n-dimensional space made
from gene expression values. Diagnostic algorithms (linear classifier) make an
(n-1)-dimensional plane in the n-dimensional space to separate two patient
groups. Calculation of the diagnostic score is achieved by dimension reduction.
Currently, IVDMIA is restricted to gene expression profiling, and will also be
applied to maligcies other than breast cancer. With the increasingly early diagnosis of breast cancer and the advent of breast
tumor subtyping, the need for determining which patients need adjuvant therapy
has become more pressing and more complex. While clinical and pathologic
features to predict benefit are valuable, the use of molecular techniques to
better determine which tumors will benefit from chemotherapy is expected to
further improve outcomes, reduce long-term complications, and provide
cost-effective care. We will review the primary tools in clinical use:
Adjuvant!, Oncotype DX, and MammaPrint as well as intrinsic subtypes and the
plans for their further assessment in the clinical trial setting. The expected
benefit from these models are that treatment recommendations for women with
early-stage breast cancer will become more individualized and thereby
appropriate by combining standard clinicopathologic and molecular features. This
concept is currently being evaluated in multiple well-designed clinical trials. BACKGROUND: Optimizing treatment through microarray-based molecular subtyping is
a promising method to address the problem of heterogeneity in breast cancer;
however, current application is restricted to prediction of distant recurrence
risk. This study investigated whether breast cancer molecular subtyping
according to its global intrinsic biology could be used for treatment
customization.
METHODS: Gene expression profiling was conducted on fresh frozen breast cancer
tissue collected from 327 patients in conjunction with thoroughly documented
clinical data. A method of molecular subtyping based on 783 probe-sets was
established and validated. Statistical analysis was performed to correlate
molecular subtypes with survival outcome and adjuvant chemotherapy regimens.
Heterogeneity of molecular subtypes within groups sharing the same distant
recurrence risk predicted by genes of the Oncotype and MammaPrint predictors was
studied.
RESULTS: We identified six molecular subtypes of breast cancer demonstrating
distinctive molecular and clinical characteristics. These six subtypes showed
similarities and significant differences from the Perou-Sørlie intrinsic types.
Subtype I breast cancer was in concordance with chemosensitive basal-like
intrinsic type. Adjuvant chemotherapy of lower intensity with CMF yielded
survival outcome similar to those of CAF in this subtype. Subtype IV breast
cancer was positive for ER with a full-range expression of HER2, responding
poorly to CMF; however, this subtype showed excellent survival when treated with
CAF. Reduced expression of a gene associated with methotrexate sensitivity in
subtype IV was the likely reason for poor response to methotrexate. All subtype
V breast cancer was positive for ER and had excellent long-term survival with
hormonal therapy alone following surgery and/or radiation therapy. Adjuvant
chemotherapy did not provide any survival benefit in early stages of subtype V
patients. Subtype V was consistent with a unique subset of luminal A intrinsic
type. When molecular subtypes were correlated with recurrence risk predicted by
genes of Oncotype and MammaPrint predictors, a significant degree of
heterogeneity within the same risk group was noted. This heterogeneity was
distributed over several subtypes, suggesting that patients in the same risk
groups require different treatment approaches.
CONCLUSIONS: Our results indicate that the molecular subtypes established in
this study can be utilized for customization of breast cancer treatment. BACKGROUND: Gene expression profiling (GEP) is being used increasingly for risk
stratification to identify women with lymph node-negative, estrogen
receptor-positive, early stage breast cancer who are most likely to benefit from
adjuvant chemotherapy. The authors of this report evaluated the cost
effectiveness of recurrence score-guided treatment using 2 commercially
available GEP tests, Oncotype DX (Genomic Health, Redwood City, Calif) and
MammaPrint (Agendia Inc., Irvine, Calif), from a third-party payer's
perspective.
METHODS: A 10-year Markov model was developed to compare the costs and
quality-adjusted life-years (QALYs) of treatment decisions guided by either
Oncotype DX or MammaPrint in a hypothetical cohort of women with early stage,
lymph node-negative, estrogen receptor-positive breast cancer who may experience
recurrence. Outcomes included no recurrence, recurrence, and death. The costs
considered included gene test costs, the costs of adjuvant chemotherapy and
other chemotherapy (including premedication, oncology visits, and monitoring for
adverse events), the cost of treating recurrence, costs associated with the
treatment of adverse events, and end-of-life care costs.
RESULTS: The model demonstrated that the patients who received the Oncotype DX
test to guide treatment spent $27,882 (in US dollars) and gained 7.364 QALYs,
whereas patients who received the MammaPrint test to guide treatment spent
$21,598 and gained 7.461 QALYs. Sensitivity analyses demonstrated that the
results were robust to changes in all parameters.
CONCLUSIONS: The model suggested that MammaPrint is a more cost-effective GEP
test compared with Oncotype DX at a threshold willingness-to-pay of $50,000 per
QALY. Because Oncotype DX is the most frequently used GEP in clinical practice
in the United States, the authors concluded that the current findings have
implications for health policy, particularly health insurance reimbursement
decisions. BACKGROUND: We critically evaluated the available evidence on genomic tests in
breast cancer to define their prognostic ability and likelihood to determine
treatment benefit.
DESIGN: Independent evaluation of six genomic tests [Oncotype Dx™,
MammaPrint(®), Genomic Grade Index, PAM50 (ROR-S), Breast Cancer Index, and
EndoPredict] was carried out by a panel of experts in three parameters:
analytical validity, clinical validity, and clinical utility based on the
principles of the EGAPP criteria. PANEL STATEMENTS: The majority of the working
group members found the available evidence on the analytical and clinical
validity of Oncotype Dx™ and MammaPrint(®) to be convincing. None of the genomic
tests demonstrated robust evidence of clinical utility: it was not clear from
the current evidence that modifying treatment decisions based on the results of
a given genomic test could result in improving clinical outcome.
CONCLUSIONS: The IMPAKT 2012 Working Group proposed the following
recommendations: (i) a need to develop models that integrate clinicopathologic
factors along with genomic tests; (ii) demonstration of clinical utility should
be made in the context of a prospective randomized trial; and (iii) the creation
of registries for patients who are subjected to genomic testing in the daily
practice. Oncotype DX, PAM50, and MammaPrint are multigene tests that are being used
clinically for early-stage breast cancer to predict recurrence risk and guide
adjuvant chemotherapy decisions. These tests have been validated in multiple
retrospective studies, and prospective clinical trials are in progress. The
TAILORx trial uses the Oncotype DX recurrence score to assign estrogen
receptor-positive (ER+), node-negative patients to chemotherapy plus hormonal
therapy versus hormonal therapy alone. The RxPONDER (SWOG S1007) trial uses
Oncotype DX in a similar approach but on node-positive patients, and it includes
the PAM50 test as a secondary analysis. The MINDACT trial uses Mamma-Print and
Adjuvant! Online for treatment arm assignments. MINDACT has very broad
eligibility criteria and 2 secondary randomizations for selecting chemotherapy
and hormonal therapy regimens. This article discusses how the latest results on
cancer genome sequencing apply to early-stage breast cancer. Several hundred
breast cancers have already undergone genome sequencing, and the somatic DNA
changes found in the tumor, compared with the patient's normal DNA, have been
identified. Higher rates of point mutations and chromosomal translocations are
found in aromatase inhibitor-resistant ER+ cancers and in the basal-like and
HER2-enriched breast cancer subtypes. Correlations of somatic mutations with
neoadjuvant aromatase inhibitor response are discussed. Genome sequencing can
potentially identify the molecular abnormalities that underlie the poor risk
identified by multigene tests and provide potential new targets for therapy, but
more clinical trials correlating clinical outcome and somatic DNA changes are
needed. Author information:
(1)Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica, Università
di Napoli Federico II, Napoli, Italy. Electronic address:
[email protected].
(2)Unità Operativa Multidisciplanare di Patologia Mammaria/Breast Unit,
Dipartmento di Chirurgia, Azienda Istituti Ospitalieri di Cremona, Italy.
(3)Department of Biomedical Sciences and Human Oncology, University of Turin,
Turin, Italy.
(4)Department of Medical Oncology, National Institute for Cancer Research,
Genoa, Italy.
(5)Department of Oncology, Hematology and Respiratory Diseases, Modena
University Hospital, Modena, Italy.
(6)Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica, Università
di Napoli Federico II, Napoli, Italy.
(7)Centro Oncologico, Università di Trieste, Trieste, Italy.
(8)Medical Oncology Unit, S. Gerardo Hospital, Monza, Italy.
(9)Department of Oncology, University Hospital of Udine, Udine, Italy; Unità
Operativa Multidisciplanare di Patologia Mammaria/Breast Unit, Dipartmento di
Chirurgia, Azienda Istituti Ospitalieri di Cremona, Italy.
(10)Department of Oncology, University Hospital of Udine, Udine, Italy.
(11)Oncologia Medica, Dipartimento di Scienze Cliniche e Biologiche, Università
di Torino, TORINO, Italy. BACKGROUND: Advances in the treatment of breast cancer have led to a reduction
in breast-cancer-related mortality. However, these therapies are known to be
associated with toxicities. Thus there is a crucial need to accurately define
the populations of women with an excellent prognosis who may safely avoid the
risks of systemic therapy. Recent developments utilizing novel technologies that
incorporate our growing understanding of the biology and pathophysiology of
breast cancer strive to better identify these patients.
OBJECTIVE: To provide a review of newer prognostic markers with a focus on the
21-gene recurrence score (Oncotype DX(™)), 70-gene prognosis profile
(Mammaprint(®)), and Adjuvant! Online.
CONCLUSION: These techniques differ in their execution and application and have
been demonstrated to provide further data on risk stratification as compared
with conventional breast-cancer-risk factors. |
Does nifedipine inhibit L-type calcium channels? | Yes, nifedipine is a typical blocker of L-type calcium channels. | We have investigated the participation of the N-type (omega-conotoxin
GVIA-sensitive) and L-type (nifedipine-sensitive) calcium channels in the alpha
2-adrenoceptor mediated autoinhibition of the release of [3H]noradrenaline from
chick sympathetic neurons in culture. Blockade of 3,4-diaminopyridine-sensitive
potassium channels resulted in tetrodotoxin-sensitive and calcium-dependent
increase of the release of [3H]noradrenaline evoked by electrical stimulation.
Nifedipine attenuated the evoked release under control conditions by 20%, but in
the presence of 3,4-diaminopyridine by 51%, while omega-conotoxin decreased the
release under control conditions by 87% and in the presence of
3,4-diaminopyridine by only 43%. The L-type calcium channel activator Bay k 8644
increased the evoked release of the transmitter both in the absence and in the
presence of 3,4-diaminopyridine. Under control conditions, the alpha
2-adrenoceptor agonist UK 14304 decreased the evoked release by 57% and the
alpha 2-adrenoceptor antagonist rauwolscine increased it by 14%. Nifedipine did
not prevent this modulation. In the presence of 3,4-diaminopyridine, UK 14304
lost its effect on the release of noradrenaline, but its inhibitory action was
restored when nifedipine, but not omega-conotoxin, was added. Changes in the
increase of intracellular calcium concentration ([Ca2+]i) evoked by electrical
stimulation, measured in the cell processes by microfluorimetry, paralleled the
changes in the release of [3H]noradrenaline. Under control conditions,
nifedipine attenuated the rise of intracellular calcium by only 16%, while
omega-conotoxin did so by 66%. 3,4-Diaminopyridine enhanced the evoked rise of
[Ca2+]i; in its presence the rise of intracellular calcium was about equally
reduced by nifedipine and omega-conotoxin (by 46 and 36%, respectively). These
effects were additive. UK 14304 diminished the peak concentration of [Ca2+]i
elicited by the standard electrical stimulation by 31% and rauwolscine
antagonised this effect. UK 14304 did not measurably inhibit the
stimulation-evoked rise of intraterminal [Ca2+]i in the presence of
3,4-diaminopyridine but it produced an inhibition by 26% if nifedipine had been
applied together with 3,4-diaminopyridine. Our observations show that, under
control conditions, the stimulated release of [3H]noradrenaline is mainly
associated with the opening of N-type channels, while in the presence of
3,4-diaminopyridine the contribution of L-type channels becomes more important.
The alpha 2-adrenoceptor stimulation by UK 14304 inhibits the release of
[3H]noradrenaline but, in the presence of 3,4-diaminopyridine, the inhibition of
release can only be observed if the massive influx through L-type calcium
channels is prevented. These data suggest that presynaptic alpha 2-adrenoceptors
of chick sympathetic neurons preferentially influence the N-type calcium
channels. Calcium channel blockers (CCBs) inhibit voltage-dependent L-type calcium
channels. This leads to vascular smooth muscle relaxation and negative inotropic
and chronotropic effects in the heart. The latter are counteracted in vivo by a
vasodilatation-triggered, baroreceptor-mediated reflex increase in sympathetic
tone, resulting in indirect cardiostimulation. The mean vascular/cardiac effect
ratios of the first-generation CCBs-verapamil, nifedipine, and diltiazem-are
relatively low and amount to approximately 3, 10, and 3, respectively. The
pharmacokinetic properties of verapamil, nifedipine, and diltiazem are similar.
The drugs are almost completely absorbed after oral administration, but their
bioavailability is reduced because of first-pass hepatic metabolism. The onset
of action of verapamil, nifedipine, and diltiazem, at least in immediate-release
formulations, is relatively fast (0.5-2 hours), and their elimination half-lives
range from 2 to 7 hours. The second-generation CCBs (e.g., amlodipine,
felodipine, and nisoldipine) have a slower onset of action (due to either
intrinsic properties of the drug or a slow-release formulation), a longer
duration of action, and greater vascular/cardiac effect ratios. These features
may provide therapeutic benefits, for example, a less pronounced increase in
sympathetic tone and reflex tachycardia, and reduced likelihood of negative
inotropic effects. These agents can therefore probably be used in patients with
left ventricular dysfunction. The effects of nifedipine, niguldipine, nimodipine and nitrendipine on the high
K+-induced intracellular Ca2+ ([Ca2+]i) transient in dibutyryl
cAMP-differentiated neuroblastoma x glioma hybrid NG 108-15 cells were studied
by using the fluorescent Ca2+ indicator fura-2. It was observed that nifedipine
at the concentration of 50 microM inhibited the high K+-induced [Ca2+]i
transient by about 60%; niguldipine at the concentration of 10 microM caused a
reduction of about 65% in the high K+-induced calcium signal and a further
increase in the concentration up to 50 microM did not result in a significant
further reduction in the high K+-induced calcium signal. However, on the other
hand, nimodipine and nitrendipine at 50 microM inhibited almost completely the
high K+-induced [Ca2+]i transient. Consequently, it was demonstrated in the
present study that nimodipine and nitrendipine inhibit both L- and N-type
calcium channels and thus seem to be unique among the dihydropyridines examined
in their effects on calcium channels in dibutyryl cAMP-differentiated
neuroblastoma x glioma hybrid NG 108-15 cells, whereas nifedipine and
niguldipine appear to block mainly L-type calcium channels. We have used the model of L-2-chloropropionic acid (L-CPA)-induced selective
cerebellar granule necrosis to study excitatory amino acid-induced necrotic cell
death in vivo produced by the activation of N-methyl-D-aspartate (NMDA)
receptors. However, the mechanism for the NMDA receptor activation and the
biochemical events which dictate the anatomical selectivity for the
L-CPA-induced lesion are as yet unknown. We examined whether blockade of sodium
and calcium channels may reduce the neurotoxicity through a reduction of
glutamate release from granule cells. None of the sodium channel antagonists
examined, i.e. phenytoin, lamotrigine or rilazole nor the mixed sodium/calcium
channel blocker, lifarazine, altered the L-CPA neurotoxicity. However, L-type
calcium channel blockers, verapamil and nifedipine enhanced the L-CPA-induced
granule cell necrosis, assessed by measuring the degree of L-CPA-induced
reductions in cerebellar aspartate concentration, increases in cerebellar
glycine concentrations and the development of cerebellar oedema. In addition,
the locomotor activity of rats receiving both L-CPA and either verapamil or
nifedipine was significantly lower than when rats received L-CPA alone,
suggesting an enhancement of the neurotoxicity of L-CPA by L-type calcium
channel blockade. The data suggest that L-CPA may interfere with non-L-type
calcium channels located on granule cell bodies and nerve terminals leading to
reduction of the calcium entry into the cells. We suggest that a combination of
L-type channel blockade and non-L-type channels which are sensitive to L-CPA
produces reductions in intracellular calcium concentrations below that required
for neuronal survival. Tottering mice inherit a recessive mutation of the calcium channel alpha1A
subunit that causes ataxia, polyspike discharges, and intermittent dystonic
episodes. The calcium channel alpha1A subunit gene encodes the pore-forming
protein of P/Q-type voltage-dependent calcium channels and is predomitly
expressed in cerebellar granule and Purkinje neurons with moderate expression in
hippocampus and inferior colliculus. Because calcium misregulation likely
underlies the tottering mouse phenotype, calcium channel blockers were tested
for their ability to block the motor episodes. Pharmacologic agents that
specifically block L-type voltage-dependent calcium channels, but not P/Q-type
calcium channels, prevented the inducible dystonia of tottering mutant mice.
Specifically, the dihydropyridines nimodipine, nifedipine, and nitrendipine, the
benzothiazepine diltiazem, and the phenylalkylamine verapamil all prevented
restraint-induced tottering mouse motor episodes. Conversely, the L-type calcium
channel agonist Bay K8644 induced stereotypic tottering mouse dystonic at
concentrations significantly below those required to induce seizures in control
mice. In situ hybridization demonstrated that L-type calcium channel alpha1C
subunit mRNA expression was up-regulated in the Purkinje cells of tottering
mice. Radioligand binding with [3H]nitrendipine also revealed a significant
increase in the density of L-type calcium channels in tottering mouse
cerebellum. These data suggest that although a P/Q-type calcium channel mutation
is the primary defect in tottering mice, L-type calcium channels may contribute
to the generation of the intermittent dystonia observed in these mice. The
susceptibility of L-type calcium channels to voltage-dependent facilitation may
promote this abnormal motor phenotype. The mechanism(s) responsible for beta2-adrenergic receptor-mediated skeletal
muscle and cardiac hypertrophy remains undefined. This study examined whether
calcium influx through L-type calcium channels contributed to the development of
cardiac and skeletal muscle (plantaris; gastrocnemius; soleus) hypertrophy
during an 8-day treatment with the beta2-adrenergic receptor agonist
clenbuterol. Concurrent blockade of L-type calcium channels with nifedipine did
not reverse the hypertrophic action of clenbuterol. Moreover, nifedipine
treatment alone resulted in both cardiac and soleus muscle hypertrophy (6% and
7%, respectively), and this effect was additive to the clenbuterol-mediated
hypertrophy in the heart and soleus muscles. The hypertrophic effects of
nifedipine were not associated with increases in total beta-adrenergic receptor
density, nor did nifedipine reverse clenbuterol-mediated beta-adrenergic
receptor downregulation in either the left ventricle or soleus muscle. Both
nifedipine and clenbuterol-induced hypertrophy increased total protein content
of the soleus and left ventricle, with no change in protein concentration. In
conclusion, our results support the hypothesis that beta2-adrenergic receptor
agonist-induced muscle hypertrophy is mediated by mechanisms other than calcium
influx through L-type calcium channels. Intrinsic membrane properties are important in the regulation of motoneuronal
output during such behaviours as locomotion. A conductance through L-type
calcium channels has been implicated as an essential component in the
transduction of motoneuronal input to output during locomotion. Given the
developmental changes in calcium currents occurring postnatally in some neurons,
and the increasing interest in the study of spinal locomotor output in neonatal
preparations, experiments were conducted to investigate the postnatal
development of L-type calcium channels in mouse motoneurons. This was assessed
both physiologically, using a chemically induced rhythmic motor output, and
anatomically, using immunohistochemical methods. The electrophysiological data
were obtained during rhythmic bursting produced by application of
N-methyl-D-aspartate (NMDA) and strychnine to the isolated spinal cord at
various postnatal ages. The L-type calcium channel blocker nifedipine has no
effect on this ventral root bursting in postnatal day (P) P2-P5 animals, but
reversibly reduced the amplitude and/or burst duration of this activity in
animals greater than P7. The immunohistochemical evidence demonstrates a
dramatic change in the cellular profile of both the alpha1C and alpha1D subunits
of L-type calcium channels during postnatal development; the labelling of both
subunits increases with age, approximating the adult pattern by P18. These
results demonstrate that in the spinal cord, the L-type calcium channel profile
develops both physiologically and anatomically in the early postnatal period.
This development parallels the development of the mature functional behaviours
of weight bearing and walking, and may be necessary for the production of
complex motor behaviour in the mature mammal. The advantages of using isolated cells have led us to develop short-term
cultures of hippocampal pyramidal cells, which retain many of the properties of
cells in acute preparations and in particular the ability to generate
afterhyperpolarizations after a train of action potentials. Using
perforated-patch recordings, both medium and slow afterhyperpolarization
currents (mI(AHP) and sI(AHP), respectively) could be obtained from pyramidal
cells that were cultured for 8-15 days. The sI(AHP) demonstrated the kinetics
and pharmacologic characteristics reported for pyramidal cells in slices. In
addition to confirming the insensitivity to 100 nM apamin and 1 mM TEA, we have
shown that the sI(AHP) is also insensitive to 100 nM charybdotoxin but is
inhibited by 100 microM D-tubocurarine. Concentrations of nifedipine (10 microM)
and nimodipine (3 microM) that maximally inhibit L-type calcium channels reduced
the sI(AHP) by 30 and 50%, respectively. However, higher concentrations of
nimodipine (10 microM) abolished the sI(AHP), which can be partially explained
by an effect on action potentials. Both nifedipine and nimodipine at maximal
concentrations were found to reduce the HVA calcium current in freshly
dissociated neurons to the same extent. The N-type calcium channel inhibitor,
omega-conotoxin GVIA (100 nM), irreversibly inhibited the sI(AHP) by 37%.
Together, omega-conotoxin (100 nM) and nifedipine (10 microM) inhibited the
sI(AHP) by 70%. 10 microM ryanodine also reduced the sI(AHP) by 30%, suggesting
a role for calcium-induced calcium release. It is concluded that activation of
the sI(AHP) in cultured hippocampal pyramidal cells is mediated by a rise in
intracellular calcium involving multiple pathways and not just entry via L-type
calcium channels. We tested the assumption that nifedipine blocks L-type calcium current
[I(Ca(L))] at +10 mV and unmasks Na(+)/Ca(2+) exchange-triggered contractions in
guinea pig isolated ventricular myocytes. Voltage-clamp pulses elicited I(Ca(L))
at +10 mV and evoked contractions in myocytes superfused with Tyrode's solution
(35 degrees C). Nifedipine blocked I(Ca(L)) with an IC(50) of 0.3 microM; this
decreased to 50 nM at a holding potential of -40 mV, indicating preferential
block of inactivated L-type Ca(2+) channels. Use-independent block of I(Ca(L))
increased with concentration (10-100 microM) and application time when
nifedipine was rapidly applied (t(1/2) = approximately 0.2 s) during rest
intervals (5-30 s). The fraction of use-dependent block of I(Ca(L)) diminished
with increasing drug concentration. Nifedipine also accelerated I(Ca(L))
inactivation on the first test pulse. The combination of 30 microM nifedipine/30
microM Cd(2+) (Nif 30/Cd 30) was as effective as 100 microM nifedipine to
suppress I(Ca(L)) on the first test pulse at +10 mV. The incidence of complete
block of contractions, as for complete block of I(Ca(L)), increased as a
function of nifedipine concentration and application time. Neither nifedipine
nor Nif 30/Cd 30 affected Na(+)/Ca(2+) exchange current at +10 to +100 mV.
Contractions at +100 mV, although as large as those at +10 mV, were delayed in
onset and resistant to nifedipine or Nif 30/Cd 30. We conclude that
nifedipine-sensitive I(Ca(L)) triggers contractions at +10 mV, whereas
nifedipine-resistant Na(+)/Ca(2+) exchange current initiates those at +100 mV. Rises in intracellular Ca2+ induced by activation of glutamate receptors are of
ultimate importance for neuronal excitability and pathophysiological processes.
In the present study, we aimed to elucidate the types of voltage-dependent Ca2+
channels involved in the NMDA-stimulated influx of Ca2+ into the isolated rat
retina by using selective blockers. Additionally, the number of binding sites
for radioligands labelling L- ([3H]nitrendipine), N- ([125I]omega-conotoxin
MVIIA) and P/Q-type ([125I]omega-conotoxin MVIIC) Ca2+ channels was assessed in
the rat retina and, for further comparison, in the rat cortex. Incubation of
isolated rat retinas with 100 microM NMDA produced a three-fold increase in the
influx of 45Ca2+ that was completely blunted by MK-801, a NMDA receptor
antagonist, and partially attenuated (approximately 20%) by tetrodotoxin, a Na+
channel blocker. The L-type Ca2+ channel blocker nifedipine reduced
NMDA-stimulated Ca2+ influx in a dose-related fashion, with a maximum reduction
of approximately 50%. Similar effects were observed with verapamil and
diltiazem. Blockers of N- and P/Q-type Ca2+ channels had no significant effect
on the influx of Ca2+ evoked by NMDA. Co2+, a non-specific Ca2+ channel blocker,
caused an inhibition of NMDA-stimulated Ca2+ influx similar to that of
nifedipine. Therefore, of all voltage-dependent Ca2+ channels, L-type channels
appear to make the greatest contribution (up to 50%) to the NMDA-stimulated
influx of Ca2+ into the isolated rat retina. This finding contrasts with
evidence obtained in brain neurones supporting a role for L-, N- and P/Q-type
channels in NMDA-evoked Ca2+ signals. A comparison of the number of radioligand
binding sites associated with L-, N- or P/Q-type Ca2+ channels in the rat cortex
and retina revealed that such a difference cannot be ascribed to a distinct
expression pattern of these channels in both tissues, although some variations
were found. Interestingly, a different affinity of [3H]nitrendipine for L-type
Ca2+ channels in the rat retina and cortex was observed which may reflect the
expression of different classes of L-type channels in these tissues. The ability
of L-type Ca2+ channel blockers to attenuate NMDA-stimulated Ca2+ influx may
underlie their neuroprotective effects in the retina. We have sought to elucidate the biochemical mechanisms that underlie the memory
enhancing properties of the neural peptide vasopressin. Toward that goal we have
investigated vasopressin induction of calcium signaling cascades, long held to
be involved in long-term memory function, in neurons derived from the cerebral
cortex, a brain region associated with long-term memory. Our previous studies
demonstrated that in cultured cortical neurons, V1a vasopressin receptor (V1aR)
activation resulted in a sustained rise in intracellular calcium concentration
that was dependent on calcium influx (Son & Brinton, 1998). To investigate the
mechanism of V1aR-induced calcium influx, we investigated V1aR activation of the
calcium channel subtype(s) in cortical neurons cultured from Sprague-Dawley rat
embryonic day 18 fetuses. The results of these analyses demonstrated that the
L-type calcium channel blocker nifedipine blocked 250 nM V1 vasopressin receptor
agonist (V1 agonist)-induced calcium influx. Intracellular calcium imaging
analyses using fura-2AM demonstrated that blockade of L-type calcium channels
prevented the 250 nM V1 agonist-induced rise in intracellular calcium
concentration. These results indicate that the influx of extracellular calcium
via L-type calcium channels is an essential step in the initiation of the V1
agonist-induced rise in intracellular calcium concentration. To determine the
mechanism of V1aR activation of L-type calcium channels, regulatory components
of the phosphatidylinositol signaling pathway were investigated. The results of
these analyses demonstrated that V1 agonist-induced calcium influx was blocked
by both a phospholipase C inhibitor (U-73122) and a protein kinase C inhibitor
(bisindolylmaleimide I). Further analysis of V1aR activation of protein kinase C
(PKC) demonstrated that V1 agonist induced PKC activity within 1 min of exposure
in cultured cortical neurons. These data indicate that in cultured cortical
neurons, V1aR activation regulates the influx of extracellular calcium via
L-type calcium channel activation through a protein kinase-C-dependent
mechanism. The results of these studies provide biochemical mechanisms by which
vasopressin could enhance memory function. Those mechanisms include a complex
cascade that is initiated by activation of the phosphatidylinositol pathway,
activation of protein kinase C, followed by phosphorylation of L-type calcium
channels to initiate the influx of extracellular calcium to activate a cascade
of calcium-dependent release of intracellular calcium. Mudpuppy parasympathetic neurons exhibit spontaneous miniature
hyperpolarizations (SMHs) that are generated by potassium currents, which are
spontaneous miniature outward currents (SMOCs), flowing through clusters of
large conductance voltage- and calcium (Ca(2+))-activated potassium (BK)
channels. The underlying SMOCs are initiated by a Ca(2+)-induced Ca(2+) release
(CICR) mechanism. Perforated-patch whole cell voltage recordings were used to
determine whether activation of SMHs contributed to action potential (AP)
repolarization or affected the latency to AP generation. Blockade of BK channels
by iberiotoxin (IBX, 100 nM) slowed AP repolarization and increased AP duration.
Treatment with omega-conotoxin GVIA (3 microM) or nifedipine (10 microM) to
inhibit Ca(2+) influx through N- or L-type voltage-dependent calcium channels
(VDCCs), respectively, also decreased the rate of AP repolarization and
increased AP duration. Elimination of CICR by treatment with either thapsigargin
(1 microM) or ryanodine (10 microM) produced no significant change in AP
repolarization or duration. Blockade of BK channels with IBX and inhibition of
N-type VDCCs with omega-conotoxin GVIA, but not inhibition of L-type VDCCs with
nifedipine, decreased the latency of AP generation. A decrease in latency to AP
generation occurred with elimination of SMHs by inhibition of CICR following
treatment with thapsigargin. Ryanodine treatment decreased AP latency in three
of six cells. Apamin (100 nM) had no affect on AP repolarization, duration, or
latency to AP generation, but did decrease the hyperpolarizing afterpotential
(HAP). Inhibition of L-type VDCCs by nifedipine also decreased HAP amplitude.
Inhibition of CICR by either thapsigargin or ryanodine treatment increased the
number of APs generated with long depolarizing current pulses, whereas exposure
to IBX or omega-conotoxin GVIA depressed excitability. We conclude that CICR,
the process responsible for SMH generation, represents a unique mechanism to
modulate the response to subthreshold depolarizing currents that drive the
membrane potential toward the threshold for AP initiation but does not
contribute to AP repolarization. Subthreshold depolarizations would not activate
sufficient numbers of VDCCs to allow Ca(2+) influx to elevate [Ca(2+)](i) to the
extent needed to directly activate nearby BK channels. However, the elevation in
[Ca(2+)](i) is sufficient to trigger CICR from ryanodine-sensitive Ca(2+)
stores. Thus CICR acts as an amplification mechanism to trigger a local
elevation of [Ca(2+)](i) near a cluster of BK channels to activate these
channels at negative levels of membrane potential. Transmitter release from nerve terminals is dependent on the entry of Ca(2+)
through neuronal voltage-gated calcium channels. In sympathetic neurones both N-
and L-type calcium channels are present. Potassium channel blockade increases
Ca(2+) entry into sympathetic neurones. We examined the participation of N- and
L-type calcium channels in the stimulation-evoked release of noradrenaline from
vascular sympathetic neurones. Rings of rabbit carotid artery were preincubated
with [3H]-noradrenaline. Electrical field stimulation was used to evoke 3H
overflow. The selective N-type calcium channel blocking agent omega-conotoxin
GVIA (single concentrations: 3 x 10(-10)-10(-8) M) caused a slowly developing
reduction of the stimulation-evoked 3H overflow. At 3 x 10(-8) M,
omega-conotoxin GVIA caused an equilibrium block with a rapid (15 min.) onset.
After 2 hr exposure to omega-conotoxin the inhibition was steady (pIC50 (-log
M): 9.43; Emax: 91%). The selective L-type calcium blocking agents nifedipine
(10(-7)-10(-5) M) and nimodipine (10(-8)-10(-5) M) had no effect on the
stimulation-evoked 3H overflow. The calcium channel opener Bay K 8644 (10-6 M)
likewise had no effect. The potassium channel blocking agent 4-aminopyridine
(10-5-10-3 M) enhanced the stimulation-evoked 3H overflow up to 5 times.
4-Aminopyridine (10(-4) M) did not alter the inhibitory effect of
omega-conotoxin GVIA (3 x 10(-8) M). In the presence of 4-aminopyridine (10(-4)
M), nifedipine (10(-5) M) and nimodipine (10(-6) M) enhanced the 3H overflow. We
conclude that the stimulation-evoked release of noradrenaline from sympathetic
neurones in rabbit carotid artery is mediated by N-type calcium channels and
that L-type channels are not involved even when potassium channels are blocked
by 4-aminopyridine. Recently, we have demonstrated that sensory neurons of rat lumbar dorsal root
ganglia (DRG) respond to hypoxia with an activation of endothelial nitric oxide
(NO) synthase (eNOS) resulting in enhanced NO production associated with
mitochondria which contributes to resistance against hypoxia. Extracellular
calcium is essential to this effect. In the present study on rat DRG slices, we
set out to determine what types of calcium channels operate under hypoxia, and
which upstream events contribute to their activation, thereby focusing upon
mitochondrial complex II. Both the metallic ions Cd2+ and Ni2+, known to inhibit
voltage-gated calcium channels and T-type channels, respectively, and verapamil
and nifedipine, typical blocker of L-type calcium channels completely prevented
the hypoxic neuronal NO generation. Inhibition of complex II by
thenoyltrifluoroacetone at the ubiquinon binding site or by 3-nitropropionic
acid at the substrate binding site largely diminished hypoxic-induced NO
production while having an opposite effect under normoxia. An additional
blockade of voltage-gated calcium channels entirely abolished the hypoxic
response. The complex II inhibitor malonate inhibited both normoxic and hypoxic
NO generation. These data show that complex II activity is required for
increased hypoxic NO production. Since succinate dehydrogenase activity of
complex II decreased at hypoxia, as measured by histochemistry and densitometry,
we propose a hypoxia-induced functional switch of complex II from succinate
dehydrogenase to fumarate reductase, which subsequently leads to activation of
voltage-gated calcium channels resulting in increased NO production by eNOS. Dihydropyridine calcium channel blockers are not uniform in terms of their
action on calcium channel. L-type calcium channel blockers dilate the resistance
arterioles. Cilnidipine is a dihydropyridine calcium channel blocker that also
acts on N-type calcium channels, and may dilate venules through its effect on
the sympathetic receptor. The influence of an L-type calcium channel blocker
(nifedipine) or this L+N type blocker at 10(-7) mol to 10(-4) mol on venular
diameter was examined by superfusion of male Syrian hamster cheek pouches.
Nifedipine dose dependently dilated the arterioles alone, whereas cilnidipine
dilated both arterioles and venules. Application of 10(-7) mol omega conotoxin,
an inhibitor of N-type channels, after nifedipine led to significant dilation of
venules, while it had no influence when administered after cilnidipine. These
findings indicate that the effects of calcium channel blockers on the venules
differ according to the action on N-type calcium channels, and that cilnidipine
(an L+N type calcium channel blocker) dilates venules through its additional
action on N-type channels. A large body of evidence supports the role of L-type calcium channels in
epileptogenesis. The aim of the present study was to study the efficacy of the
specific L-type calcium channel blocker nifedipine on seizure activity induced
by picrotoxin (PTX). Adult female Sprague-Dawley rats were used in these
experiments. The intraperitoneal administration of nifedipine (5 mg/kg) did not
significantly alter the latency to onset of clonic seizure induced by
intraperitoneal injection of PTX (4 mg/kg). Higher doses of the drug (10 and 20
mg/kg) significantly increased the latency of onset of clonic seizure in a
dose-dependent manner. Nifedipine (10 mg/kg) did not reduce the incidence of
clonic seizures in the animals injected with PTX, but inhibited tonic seizure
and the progression of clonic seizures into maximal tonic seizures in four of
eight of the animals. The drug (20 mg/kg) inhibited clonic seizure in four of
six of the animals and abolished minimal or maximal tonic seizures in all the
animals. In conclusion, our study provides further evidence on the antiepileptic
effect of L-type calcium channel blocker nifedipine by showing its protective
effect on seizure activity induced by PTX. Calcium ions are widely accepted as critically important in responses of neurons
to a stimulus. We have show previously the central involvement of angiotensin II
(ANGII) in water intake. This study determined whether voltage-dependent calcium
channels are involved in ANGII-induced behavioral drinking implicating nitrergic
mechanism. The antidipsogenic actions of L-type calcium channel antagonists
nifedipine, on ANGII-induced drinking behavior were studied when it is injected
into the median preoptic nucleus (MnPO). The influence of nitric oxide (NO) on
nifedipine antidipsogenic action was also studied by utilizing the
N(W)-nitro-L-arginine methyl ester (L-NAME) a constitutive nitric oxide synthase
inhibitor constitutive (cNOSI) and 7-nitroindazol (7-NIT) a specific neuronal
nitric oxide synthase inhibitor (nNOSI) and L-arginine a NO donor. Rats 200-250
g, with cannulae implanted into MnPO, pre-treated into MnPO with either
nifedipine, followed by ANGII, drank significantly less water than controls
during the first 15 min after injection. However, L-NAME potentiated the
dipsogenic effect of ANGII that is blocked by prior injection of nifedipine and
L-arginine. 7-NIT injected prior to ANGII into MnPO also potentiated the
dipsogenic effect of ANGII but with a less intensity than L-NAME that it is also
blocked by prior injection of nifedipine. The results described in this paper
provide evidence that calcium channels play important roles in the ANGII-induced
behavioral water intake. The structures containing NO in the brain such as MnPO
include both endothelial cells and neurons might be responsible for the
influence of nifedipine on dipsogenic effect of ANGII. These data shows the
correlation between L-type calcium channel and a free radical gas NO produced
endogenously from amino acids L-arginine by endothelial and neuronal NO synthase
in the control of ANGII-dipsogenic effect. This suggests that an L-type calcium
channel participates in both short- and longer-term neuronal actions of ANGII by
nitrergic way. To determine how acute ethanol intoxication may alter memory processing, we
examined the effects of stepwise increases in ethanol on long-term potentiation
(LTP) in rat hippocampal slices. LTP was inhibited by acute administration of 60
mM ethanol, but was readily induced if ethanol was increased gradually to 60 mM
over 75 min. Administration of 2-amino-5 phosphonovalerate (APV), an
N-methyl-D-aspartate receptor (NMDAR) antagonist, during the stepwise increase
in ethanol inhibited LTP, suggesting involvement of NMDARs in the development of
tolerance. However, APV and nifedipine, an inhibitor of L-type calcium channels,
failed to inhibit LTP when administered following the slow increase in ethanol.
Ethanol-tolerant LTP was inhibited by thapsigargin, suggesting a major role for
intracellular calcium release in this form of plasticity. The unique properties
of ethanol-tolerant LTP suggest that memories formed during binge drinking are
not acquired by standard synaptic mechanisms and that acute tolerance may
involve the induction of novel mechanisms to maintain function. BACKGROUND AND OBJECTIVES: The non-depolarizing muscle relaxant vecuronium
inhibits contraction by competitive inhibition of postsynaptic acetylcholine
receptors (AchRs), which decreases the number of quanta released per impulse in
response to 50 Hz stimulation. The specific role of calcium influx through
L-type calcium channels is the promotion of endocytosis and vesicle recycling
during high-frequency stimulation. Vecuronium also induces four pulse tetanic
fade, a proxy measure of decreased quanta release. We examined whether
vecuronium suppresses neuromuscular transmission during high-frequency
stimulation by inhibiting presynaptic L-type calcium channels.
METHODS: Fifty male Sprague-Dawley rats were divided into five treatment groups:
unstimulated control group, α-bungarotoxin (BTX) group, nifedipine group,
vecuronium group, and nifedipine plus vecuronium group. Rat phrenic
nerve-diaphragm neuromuscular juctions were stimulated at 50 Hz and field
excitatory post-synaptic potentials (fEPSPs) were recorded. Expression levels of
the presynaptic Ca(2+)-binding, protein synaptotagmin 1, and the presynaptic
plasma membrane protein, syntaxin 1, were measured by Western blots.
RESULTS: The fEPSPs evoked by 50 Hz stimulus trains were decreased by
vecuronium, nifedipine, and by vecuronium plus nifedipine. Nifedipine, an L-type
calcium channel blocker, reduced the expression of synaptogamin and syntaxin and
blocked the suppressive effect of vecuronium, suggesting that both agents
inhibit presynaptic L-type calcium channels.
CONCLUSIONS: Vecuronium which blocked L-type calcium channels may suppress
activity of the α(3)β(2) nAChR subunit, which exists in the presynaptic membrane
and enhances quantal release. This α(3)β(2) nAChR-mediated positive feedback
effect may be facilitated by L-type Ca(2+) channel activity under high-frequency
stimulation. Vecuronium may disrupt this positive feedback cycle, leading to
suppression of fEPSPs. Vercuronium may reduce neuromuscular transmission through
presynaptic and postsynaptic mechanisms. Cisplatin-like chemotherapeutics cause vomiting via release of multiple
neurotransmitters (dopamine, serotonin (5-HT), or substance P (SP)) from the
gastrointestinal enterochromaffin cells and/or the brainstem via a calcium
dependent process. Diverse channels in the plasma membrane allow extracellular
Ca(2+) entry into cells for the transmitter release process. Agonists of 5-HT3
receptors increase calcium influx through both 5-HT3 receptors and L-type Ca(2+)
channels. We envisaged that L-type calcium agonists such as FPL 64176 should
cause vomiting and corresponding antagonists such as nifedipine would behave as
broad-spectrum antiemetics. Administration of FPL 64176 did cause vomiting in
the least shrew in a dose-dependent fashion. Nifedipine and the 5-HT3 receptor
antagonist palonosetron, potently suppressed FPL 64176-induced vomiting, while a
combination of ineffective doses of these antagonists was more efficacious.
Subsequently, we investigated the broad-spectrum antiemetic potential of
nifedipine against diverse emetogens including agonists of serotonergic 5-HT3-
(e.g. 5-HT or 2-Me-5-HT), SP tachykinin NK1- (GR73632), dopamine D2-
(apomorphine or quinpirole), and cholinergic M1- (McN-A-343) receptors, as well
as the non-specific emetogen, cisplatin. Nifedipine by itself suppressed
vomiting in a potent and dose-dependent manner caused by the above emetogens
except cisplatin. Moreover, low doses of nifedipine potentiated the antiemetic
efficacy of non-effective or semi-effective doses of palonosetron against
vomiting caused by either 2-Me-5-HT or cisplatin. Thus, our findings demonstrate
that activation of L-type calcium channels causes vomiting, whereas blockade of
these ion channels by nifedipine-like antagonists not only provides
broad-spectrum antiemetic activity but can also potentiate the antiemetic
efficacy of well-established antiemetics such as palonosetron. L-type calcium
channel antagonists should also provide antiemetic activity against drug-induced
vomiting as well as other emetogens including bacterial and viral proteins. Cardiogenesis depends on a tightly regulated balance between proliferation and
differentiation of cardiac progenitor cells (CPCs) and their cardiomyocyte
descendants. While exposure of early mouse embryos to Ca(2+) channel antagonists
has been associated with abnormal cardiac morphogenesis, less is known about the
consequences of Ca(2+) channel blockade on proliferation and differentiation of
CPCs at the cellular level. Here we showed that at embryonic day (E) 11.5, the
murine ventricles express several L-type and T-type Ca(2+) channel isoforms, and
that the dihydropyridine Ca(2+) channel antagonist, nifedipine, blunts
isoproterenol induced increases in intracellular Ca(2+). Nifedipine mediated
Ca(2+) channel blockade was associated with a reduction in cell cycle activity
of E11.5 CPCs and impaired assembly of the cardiomyocyte contractile apparatus.
Furthermore, in cell transplantation experiments, systemic administration of
nifedipine to adult mice receiving transplanted E11.5 ventricular cells
(containing CPCs and cardiomyocytes) was associated with smaller graft sizes
compared to vehicle treated control animals. These data suggest that
intracellular Ca(2+) is a critical regulator of the balance between CPC
proliferation and differentiation and demonstrate that interactions between
pharmacological drugs and transplanted cells could have a significant impact on
the effectiveness of cell based therapies for myocardial repair. Catharanthus roseus is a traditional herbal medicine used in Asian and African
countries for the treatment of various diseases including hypertension. The
present study examined possible cellular mechanisms for the relaxation of rat
renal arteries induced by vindorosine extracted from C. roseus. Intrarenal
arteries were isolated from 200-300 g male Sprague-Dawley rats and treated with
different pharmacological blockers and inhibitors for the measurement of
vascular reactivity on a Multi Myograph System. Fluorescence imaging by laser
scanning confocal microscopy was utilized to determine the intracellular Ca(2+)
level in the vascular smooth muscles of the renal arteries. Vindorosine in
micromolar concentrations relaxes renal arteries precontracted by KCl,
phenylephrine, 11-dideoxy-9α,11α-epoxymethanoprostaglandin F2α, and serotonin.
Vindorosine-induced relaxations were unaffected by endothelium denudation or by
treatment with the nitric oxide synthase inhibitor N (G)-nitro-L-arginine methyl
ester hydrochloride, the guanylyl cyclase inhibitor
1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, the cyclooxygenase inhibitor
indomethacin, or K(+) channel blockers such as tetraethylammonium ions,
glibenclamide, and BaCl2. Vindorosine-induced relaxations were attenuated in the
presence of 0.1 µM nifedipine (an L-type Ca(2+) channel blocker). Vindorosine
also concentration-dependently suppressed contractions induced by CaCl2
(0.01-5 mM) in Ca-free 60 mM KCl solution. Furthermore, fluorescence imaging
using fluo-4 demonstrated that 30 min incubation with 100 µM vindorosine reduced
the 60 mM KCl-stimulated Ca(2+) influx in the smooth muscles of rat renal
arteries. The present study is probably the first report of blood vessel
relaxation by vindorosine and the possible underlying mechanisms involving the
inhibition of Ca(2+) entry via L-type Ca(2+) channels in vascular smooth
muscles. |
Is there any data to suggest that TRH (thyrotropin releasing hormone) administration can improve symptom severity of amyotrophic lateral sclerosis patients? | Yes, there are studies demonstrating that TRH (thyrotropin releasing hormone) administration can improve symptom severity of amyotrophic lateral sclerosis patients. However, some studies have failed to demonstrate symptom improvement following TRH administration. | We have studied effects of TRH analogue, TA-0910
(3-methyl-(s)-5,6-dihydroorotyl-L-histidyl-L-prolinamide) (from Tanabe, Osaka,
Japan) on explanted ventral and dorsal spinal cord cultures from 13- and
14-day-old rat embryos. TA-0910-treated cultures had significantly increased
neurite outgrowth with cultures of ventral spinal cord, but not with cultures of
dorsal spinal cord. The effect was dose-dependent. A possible role for TRH in
amyotrophic lateral sclerosis remains to be defined. Evidence that thyrotropin-releasing hormone (TRH) has prominent trophic effects
on the motor system led to several negative therapeutic trials in amyotrophic
lateral sclerosis, a disease of the motor system. Since TRH crosses the
blood-brain barrier poorly, if at all, we postulated that the negative
parenteral clinical trials could be a result of insufficient drug-receptor
interaction. We thus carried out a blinded, placebo-controlled, crossover study
of intrathecal TRH in 36 patients by delivery through an implanted, constant
infusion pump achieving a steady-state CSF level comparable with that shown to
be effective in tissue culture experiments. Utilizing a quantitative measurement
technique to assess motor unit loss, we did not observe any alteration of the
progressive course during 6 months on TRH and 6 months on saline placebo.
However, the implanted pump delivery system proved to be safe, reliable, and
well tolerated. This study presents the experience of one year of treatment in patients with
amyotrophic lateral sclerosis, with intrathecal TRH administered daily by a
subcutaneous reservoir connected to the intrathecal lumbar space by a double
catheter system as to provide continuous circulation of CSF and to avoid sac
formation since this would be a source of infection. Clinical evaluation was
carried out with a scale developed by the authors with the main aim of
evaluating the loss of vital motor abilities and not as a localized evaluation.
Secondary effects due to the implantation of the reservoir in addition to its
use are presented although data were not important. Intrathecal administration
of TRH was carried out similarly (600 micrograms/day) with secondary effects
being the same as those by other routes of administration although of a lesser
intensity. The results of the clinical evaluation at the beginning and end of
the treatment as well as after patient follow up demonstrated that beneficial
effects do not occur equally in all patients but rather are transitory and do
not improve the natural evolution of the disease. The authors conclude that,
methodologically, this series study does not enter within the frame of an
advisable statistical study since the aim is to provide data for a future
controlled study. Seven patients, six suffering from amyotrophic lateral sclerosis (ALS) and one
from Friedreich ataxia, were treated with a placebo i.v. infusion during the
first day and with TRH-T i.v. infusion at a rate of 2 mg/h for 8 h daily (total
daily dosage 16 mg) on the 2 consecutive days. Continuous blood pressure (BP)
and EKG monitorings were performed during 3 days infusion. Blood samples were
collected for endocrinological evaluations. The neurological evaluation after
acute TRH-T treatment showed an objective improvement in 3 of the 8. We found
significantly higher values of systolic (max. difference of 10.1 mm Hg) and
diastolic (max. difference of 8.8 mm Hg) BP than during placebo, beginning from
the 5th h of the infusion (p less than 0.05). A trend in progressive increase of
the heart rate (HR) reached statistical significance (p less than 0.01) at the
8th h of the second TRH-T infusion. The cardiovascular changes during the i.v.
continuous TRH-T infusions were clinically irrelevant and never required the
interruption of the treatment. A 64-year-old woman who had amyotrophic lateral sclerosis (ALS) with disturbance
of vertical ocular movement was presented. She was admitted to our hospital with
progressive dysphagia, dysarythria and weakness of the extremities. Neurological
examinations revealed disturbance of vertical ocular movement with normal doll's
eye phenomenon (supranuclear origin), bulbar palsy, muscle weakness of the
extremities, extensor plantar signs, and fasciculations of the costal and
interosseal muscles. EMG studies showed denervation potentials, and muscle
biopsy demonstrated group atrophy, fiber type grouping and small angular fibers.
TRH injections resulted in improvement of disturbance of vertical ocular
movement, but no effect was seen on the weakness of the limb. There was about 20
Japanese cases with disturbance of ocular movement in ALS, but it was rare to
see ocular movement disorder from the early stage of ALS. The pathophysiology of
ocular movement disorder in ALS has been thought to be due to supranuclear
origin, i.e., the disturbance in the pathway from the frontal cortex to the
mesencephalon. In this case, TRH might effect at some point of the
frontomesencephalic pathway. 30 subjects--23 with amyotrophic lateral sclerosis (ALS), 4 with Charcot-Marie
Tooth atrophy, 2 with progressive spinal muscle atrophy and 1 with radiation
myelopathy--were given chronic low-dose TRH therapy. The effects of treatment
were assessed on the scale of Norris et al. (1974). The outcome of the study, in
agreement with some and at variance with other studies, was that TRH induced a
statistically significant neurological improvement in 17 of the 23 ALS patients
but little or none in the other ALS patients and in patients with other
neurological diseases. In six patients suffering from amyotrophic lateral sclerosis we evaluated
changes of T4, T3, TSH, PRL, and GH during treatment by continuous iv infusion
of TRH for at least 15 days. No clinical improvement was detected. A significant
rise of thyroid hormone levels was observed, as well as an upward trend of basal
TSH levels and no change of basal PRL and GH levels. TRH acute test-induced TSH
and PRL responses became blunted. Treatment provoked also the onset of a
responsiveness of PRL to GHRH. The reduced TSH and PRL responses to acute TRH
test during treatment could be explained by a down-regulation of TRH pituitary
receptors. On the contrary, the onset of PRL responsiveness to GHRH is at
present without a satisfactory explanation. Ten consecutive patients with motor neuron disease (MND) who had bulbar symptoms
received one or two intravenous doses followed by increasing oral doses of a TRH
analogue (RX77368). Similar improvements in speech, swallowing and in tongue and
jaw movements were seen after iv and oral administration in nine, five and eight
patients respectively. The initial time course of improvement correlated with
increasing plasma levels of the drug, but most clinical effects persisted when
the levels decreased and became undetectable after 24 hours. The oral solution
was tasteless and had no, or minimal, side effects. 13 patients with amyotrophic lateral sclerosis (ALS) were treated with
intravenous infusion of thyrotropin-releasing hormone (TRH). In 6 patients 2
mg/day of TRH was i.v. given over 2 hours for 10 days. In 7 others 2 mg/day of
TRH was continuously infused by means of a pump. An increase of thyroid hormones
related to the duration of the treatment was observed. A surprising finding was
the onset of prolactin (PRL) response to growth hormone releasing hormone
(GHRH), previously absent. The blood serum TSH and PRL levels were studied in 12 ALS patients after TRH
stimulation. The TSH test was repeated after 4-weeks TRH treatment. The results
were compared with the data obtained in the control group. It was shown that
after TRH stimulation the TSH responses did not reveal greater abnormalities,
but PRL responses were significantly diminished. The results could confirm our
previous observations concerning the dysregulation of dopamine metabolism in ALS
patients. The lack of effective therapy for many of the chronic neuromuscular diseases
such as amyotrophic lateral sclerosis, hereditary motor sensory neuropathy
(Charcot-Marie-Tooth disease), spinocerebellar degenerations and idiopathic
polyneuropathy has led to a search for substances that may stimulate peripheral
nerve regeneration. Two such agents that have been proposed are gangliosides
(mixed purified bovine brain gangliosides, Cronassial) and thyrotropin releasing
factor (TRH). Studies on both of these agents were initially reported with
enthusiasm to be successful, but later double-blind controlled studies have
failed to confirm these findings. This review provides critical analysis of the
designs of studies of potentially effective agents in chronic neuromuscular
diseases, and emphasizes the power of the placebo response, and the importance
of designing placebos which are indistinguishable from the trial medication
other than in the active effect. The results of the various studies and an analysis of the methodology are
presented in TABLE 1. As can be seen, there was no "perfect" study. In five of
the studies enough information was presented with regard to the measurements and
the behavior of control patients that a statistical analysis could be performed.
Three of the studies showed a transient, statistically significant effect in at
least some muscles. The two studies that demonstrated no such effect both used
TRH in very small doses. It therefore seems reasonable to conclude that the
effect of TRH in ALS is a definite, acute, and transient response. The cause of
this response, however, has not been documented, and whether it is associated
with an effect of the drug on the disease process remains to be seen. The critical points that must be addressed in evaluating ergotropic drugs are
exemplified by the current morass of positive and negative results that have
been obtained in clinical investigations of TRH or its analogues. Appropriate
subject selection is crucial. These patients may have bulbar symptoms, and those
features of ALS should be specifically assayed for treatment effects relative to
placebo. Gender-specific effects of TRH need to be accounted for in study
design. In addition, electrophysiological techniques such as single fiber
density may help determine the responsiveness of patients to TRH or its
analogues. The clinical significance of an increase in fiber density following
TRH or other drugs should be determined, as it will provide insight into the
state of motor neurons in the spinal cord of patients with ALS and possibly
could be important in determining those who may respond to TRH if such a
response is possible. Clinical studies have quite clearly shown conflicting
results. Basic studies, however, have shown that response to TRH is state
dependent, that is, whether the patient is male or female. Clinical studies have
shown that response to TRH is state dependent, that is, it depends on whether
the patient has bulbar or nonbulbar signs and is male or female. Future studies
must take into consideration this state dependence as a specific feature of the
pharmacological action of TRH and its analogues. Oculomotor disorders have been considered to be one of the negative symptoms in
motor neuron disease (MND). However, recently, ophthalmoplegia, abnormal Bell's
phenomenon and disturbance of pursuit movement have been reported. We tried to
evaluate 32 patients with MND (19 males and 13 females; age, 35 to 77 years;
52.4 +/- 10.1 years) by bedside examination and electro-oculography (EOG) using
an eye tracking method. Twenty-three of them were classified as amyotrophic
lateral sclerosis (ALS) and seven as bulbospinal muscular atrophy, and two were
unclassified. One hundred healthy persons for bedside examination and twenty-two
for EOG were investigated as controls. Findings by bedside examinations were as
follows; 1) Slight limitations of upward only, up & downward and upward &
horizontal gaze were observed in 5 cases (15.6%), 1 case (3.1%) and 1 case
(3.1%), respectively. 2) Incomplete convergence was observed in 11 cases.
(34.4%) 3) Horizontal gaze nystagmus was observed in 6 cases. (18.8%) 4) As
regards the frequency of limitation of upward gaze and incomplete convergence,
there were no statistically significant differences from those in controls.
Electrooculographic results were: 1) square wave jerks (SWJs) were recorded in 3
cases. (9.4%) 2) Amplitude ratio of saccade was significantly higher in MND than
that in controls with the risk less than 0.1%. 3) The degree of ocular dysmetria
was significantly higher in MND than that in controls with the risk less than
0.5%. These abnormalities were not directly related to suffering period.
Although the mechanism is not known, several reports of the effectiveness of
thyrotropin releasing hormone (TRH) in ALS were recently published.(ABSTRACT
TRUNCATED AT 250 WORDS) A trial of Thyrotropin Releasing Hormone (TRH) 5.0 mg/kg body weight
subcutaneously every other day for two weeks produced transient increased tone
in muscles, along with other (side-) effects in patients with Amyotrophic
Lateral Sclerosis (ALS). One patient's extensor plantar transiently changed to a
flexor plantar reflex after injection, probably due to disproportionate increase
in tone of the calf muscles. No significant changes in F-waves or H-reflexes
were seen. No increase in useful voluntary strength, or in strength measured by
Medical Research Council (MRC) testing or strain gauge isometric strength
testing was seen. However, dyspnea was seen within 10 minutes of TRH injection. Many hormonal dysfunctions were noticed in amyotrophic lateral sclerosis (ALS).
The study aimed at measuring blood serum level of TSH and PRL after THR loading
in 10 ALS patients and in the 10 healthy individuals. Mean baseline levels of
TSH and PRL in ALS patients were with in normal range. After TRH loading, the
TSH responses in the ALS patients were with in normal range, where as PRL
responses were diminished. The obtained results could indicate some disorders on
the dopaminergic neurons level. Protirelin (thyrotropin-releasing hormone) appears to be a neuromodulator in the
extrahypothalamic nervous system and has been suggested as an adjunct in the
treatment of amyotrophic lateral sclerosis (ALS). Clinical studies have been
divided on the efficacy of protirelin (TRH) despite strong experimental findings
that are consistent with a role for the peptide in ALS. Recent findings provide
evidence of a gender-related specificity in the ability of protirelin to
potentiate the monosynaptic reflex. While castration in male neonatal rats
lowered the sensitivity to protirelin, testosterone treatment restored that
sensitivity. An examination of the clinical studies reveals a failure either to
identify patients' sex or to separate the results on the basis of sex. These
findings provide convincing evidence for the potential efficacy of protirelin in
ALS if the patient's sex and underlying hormonal status are taken into account. The purpose of this paper is to illustrate the advantages of the
chemo-morphological approach in the study of pathological material. On one hand,
the analysis of selected pathological cases (amputations, spinal transections)
is able to provide invaluable information concerning the cells of origin of
certain spinal transmitters in the human being. On the other hand, chemical
neuropathology allows a more precise identification of the neuronal nets or
types that are involved in a disease process. This advantage is underlined by
studies performed in amyotrophic lateral sclerosis. In this condition, certain
modifications, such as the reductions of acetylcholinesterase, choline
acetyltransferase, cholinergic muscarinic, glycine or TRH receptors, are
probably a consequence of motoneuron degeneration. In contradistinction, other
findings, such as specific metabolic changes of motoneurons or early
disappearance of SP-containing fibers in lamina IX, might be relevant for the
pathogenesis of the disease. A double-blind, placebo-controlled trial of single doses of thyrotropin
releasing hormone (TRH) was performed on 12 patients with amyotrophic lateral
sclerosis. Each patient was given subcutaneous injections of TRH 150 mg or
placebo, and IV infusions of TRH 500 mg or placebo at 72- to 96-hour intervals.
Eight motor and functional ratings were scored at regular intervals after each
injection. Side effects were seen in all patients and were obvious to patients
and examiners, making true blinding impossible. Nevertheless, statistically
significant improvement was seen only in dynametric strength 1 hour after
subcutaneous injection (p less than 0.05). Significant improvement occurred, in
one patient only, on subjective speech testing during IV infusion of TRH. In
none of six other ratings was there a significant difference between TRH and
placebo. Subjective improvement was noted by 11 of 12 patients. A double-blind controlled trial of thyrotropin releasing hormone (TRH) 150 mg IM
daily in 30 patients with amyotrophic lateral sclerosis is reported. The
drug/placebo was administered for 2 months, followed by a 2-month "wash-out".
Evaluation of strength, functional ability, and respiratory functions was
performed. A temporary increase in the strength of some muscles was detected
following the administration of TRH, but no change in functional performance was
noted. Neither the patients nor the investigators believed the effects were of
any marked clinical significance. The course of the illness was not altered. We performed double-blind crossover trials to assess the effects of
thyrotropin-releasing hormone (TRH) on amyotrophic lateral sclerosis patients.
For acute intravenous trials, 500 mg TRH or placebo with norepinephrine was
given at 1-week intervals (16 patients). CSF TRH concentration increased, and
clinical side effects appeared with TRH. For chronic studies, 25 mg TRH and a
saline placebo were given subcutaneously every day for 3 months (25 patients).
CSF TRH level increased 29-fold after a single TRH injection, and mild transient
side effects occurred. Vital signs, respiratory function, semiquantitative and
quantitative neurologic function, muscle strength by manual and dynamometer
testing, and EMG were studied. With daily TRH, 10 patients noted subjective
improvement without objective evidence, and 10 patients complained of worsening
of the disease with objective decline after TRH was stopped. Statistical
analysis, however, showed no beneficial effects from either acute or chronic TRH
trials. Nine patients (7 with amyotrophic lateral sclerosis, 1 with progressive spinal
amyotrophy and 1 with chronic anterior poliomyelitis) were treated by sequential
intravenous administration of 240 mg of TRH over one hour every two weeks.
Results were assessed by an analytical evaluation of muscle strength before and
24 h after each infusion and by objective and subjective evaluation of
spasticity. Significant improvement, as shown by statistical analysis, was noted
in muscle strength in the 9 patients by 5 infusions over a 4-week period and a
sub-group of 5 patients treated by 8 infusions over 10 weeks. Continued use of
this therapy is justified by the need to determine its long-term effects and the
psychological improvement noted in some patients after an even transient
improvement in motor performance. However this treatment is obviously not
curative. In a pilot therapeutic trial, four patients with amyotrophic lateral sclerosis
(ALS) were treated with long term, continuous infusions of TRH, three
intrathecally and one epidurally. They had prompt increases in serum TSH and
thyroid hormone concentrations, averaging 120% for TSH, 49% for serum T4, 68%
for the serum free T4 index, 49% for serum T3, and 67% for the serum free T3
index. These elevations were statistically significant for all but serum T3 and
persisted for the duration of treatment (4-7 months). Mean values during
treatment were near the upper limit of normal for each of these hormone
measurements. After TRH withdrawal, serum TSH fell transiently below the normal
range. A comparison group of four patients with ALS treated by twice weekly
intrathecal bolus doses of TRH had no significant changes in serum TSH, T4, or
T3. During continuous TRH treatment, the responsiveness of both TSH and PRL to a
standard iv TRH stimulation test was blunted, but not abolished. Basal serum PRL
was occasionally elevated in the two women during continuous TRH treatment, but
was normal in the men, and serum GH was normal in all patients. In the patients
receiving continuous TRH treatment, indexes of end-organ effects of thyroid
hormone were inconclusive; none had a rise in serum ferritin, one of four had a
rise in serum sex hormone-binding globulin, and three had increased creatinuria.
These results provide direct evidence in man that chronic TRH administration can
cause modest sustained increases in serum TSH and thyroid hormones, though the
metabolic consequences of these changes are uncertain, and appears to raise the
set-point of the pituitary-thyroid axis, i.e. the serum T4 and T3 concentrations
needed for a given degree of suppression of basal TSH secretion. Despite the efforts of many workers, the cause and therapy has not been
clarified. We carried out the therapeutic trial of thyrotropin releasing hormone
(TRH) for amyotrophic lateral sclerosis (ALS) from January, 1979 to January,
1983. There were 16 subjects. The patients were given a low dose (0.5-2 mg) of
TRH intravenously or intramusculary. Mild to moderate improvement was found in 9
(56%) of 16 patients. TRH has been reported to have the activating effects on
the pyramidal tract, brainstem motor nuclei, and motoneuron in the spinal cord
as a neurotransmitter or neuromodulator. We thought such action of TRH to be
useful to the therapy of ALS. Focal, small-to-moderate and transient improvement occurred in the muscle
strength and function of patients with ALS who received TRH in dose-response and
screening studies. In a small pilot study of 12 patients, 3 months
administration of TRH at 10 mg per kg on alternate days resulted in localized
increased strength of jaw muscles as well as significant improvement in lower
extremity function. Aerobic exercise capacity was particularly improved in
patients with ALS following administration of TRH. Autonomic effects of TRH on
heart rate, respiration, and blood pressure were not serious and attenuated
slightly over the course of the study. Thyrotropin releasing hormone (TRH) has potential therapeutic applications in
amyotrophic lateral sclerosis (ALS) and related diseases because of its function
as a neuroregulator of the anterior horn cell. However, its therapeutic
potential, and that of other neuropeptides, is reduced by the blood-brain
barrier that limits access to neuronal cells. We have thus explored the direct
intrathecal administration of TRH in ALS, with both short-term boluses and
infusions and chronic constant infusions. Our experience suggests that this
approach is safe, has high patient acceptance, and is worthy of more careful
evaluation. Thyrotropin-releasing hormone has been reported to increase strength in patients
with amyotrophic lateral sclerosis (ALS). DN-1417 is an analog of
thyrotropin-releasing hormone, which has less endocrinologic activity, but more
anterior horn cell stimulating effect (with no "autorefractory state"). However,
2 mg DN-1417, IM twice a day for 1 month in an open-label trial, produced no
objective improvement of strength in nine patients with ALS. No patient entered
the double-blind, placebo-controlled phase of the trial. In view of earlier reports in the literature it was tried to use TRH
(thyrotropin releasing hormone) in cases of amyotrophic lateral sclerosis. TRH
was given during 3 weeks once daily intravenously in drip infusions in a 0.4 mg
dose. For objectivization of the results the muscle power was assessed using a
five-grade scale of Lovette. TRH effect on EMG was analysed also. It was found
that in only 3 out of 14 patients with moderately progressed disease no
improvement was achieved, while in 11 cases the improvement was from 10 to 20%.
However, the improvement was transient, and TRH treatment failed to stop the
progression of the disease. Sixteen patients with motor neuron disease received RX77368, a TRH analogue, IV,
repeatedly over 1-12 weeks (median 2 weeks). Slight to moderate improvement in
bulbar function, particularly speech, was reproduced or persisted with repeated
infusions in 8 of 12 responders over a median of 18 days (range 14-90) during
the period of study. Cramps (5/9) and spasticity (5/8) improved for a median of
14 days (range 7-35) and 7 days (range 2-14) respectively. The highest
benefit/side effect ratio was seen with 0.2 mg/kg (0.15 mg/kg in those with
severe bulbar palsy) every 3-4 days. Long term studies with this analogue in MND
are indicated. Effects of high-dose TRH on the vibratory inhibition of soleus H-reflex have
been studied in 9 patients with amyotrophic lateral sclerosis. In 6 of the 9,
TRH induced a significant increase in vibratory inhibition. This suggests that
the TRH-induced reduction of spasticity might be due to an increase in
presynaptic inhibition acting on Ia fibres. Eight patients with amyotrophic lateral sclerosis received 500 mg TRH by IV
infusion, at a progressive rate during 3 hours. Only 3 patients noted subjective
improvement of strength. Clinical muscular testing and H response study failed
to show any change. Moreover modifications of the prolactin, growth hormone, TSH
and T3 serum levels raise a question concerning the tolerance with long term
utilization of TRH. A double-blind crossover trial was conducted of thyrotropin releasing hormone
treatment in six patients with amyotrophic lateral sclerosis. Patients received
4 mg of thyrotropin releasing hormone intramuscularly daily during the two-week
treatment period. Although three patients reported subjective improvement,
objective evaluation failed to demonstrate therapeutic effectiveness of
thyrotropin releasing hormone in this dosage. Motor neurone disease, or amyotrophic lateral sclerosis, is a serious
progressive neurological disorder, characterized by loss of UMN and LMN.
Pathological features include characteristic intracytoplasmic MN inclusion
bodies and appearances on ubiquitin staining. The aetiopathogenesis of the
disease remains unknown and there is, to date, no effective treatment. Several
abnormalities have been demonstrated in neurotransmitter, neuropeptide and gene
expression studies. Abnormalities in glutamate metabolism have led to the
excitotoxin hypothesis of MN destruction. Other theories include deficits in MN
trophic factors, trans-synaptic degeneration, impaired ability to detoxify
putative toxic agents and impaired DNA/RNA metabolism. The existence of familial
forms, some of which show linkage to markers in chromosome 21, allows a genetic
approach to the mechanisms of disease. Recent studies suggest that mutations in
the Cu/Zn SOD gene may be important in some of the familial forms. The atypical
forms seen in the Western Pacific have stimulated a search for environmental
agents. Agents undergoing therapeutic trials at present include CNTF, IGF1
glutamate antagonists, branched-chain amino acids and TRH analogue. F responses were recorded in 6 cases with SSP and in 22 normal controls. The
present study confirmed that frequency, number of phases, F/M amplitude ratio
and duration were significantly increased and CV of onset latencies was
significantly reduced in cases with SSP. After intravenous injection of TRH (2
mg), all the parameters were altered toward normal sides. It was suggested that
the hyperexcitability of motor neuron pool in SSP was stabilized by effects of
TRH on injured pyramidal tracts and consequently abnormalities of F responses
were improved. The Effect of TRH to correct the abnormal F responses in SSP
might be consistent with effects of TRH to reduce spasticity in amyotrophic
lateral sclerosis described previously. Heart rate, respiratory rate, systolic and diastolic blood pressure, and oral
and skin temperature changes following intravenous administration of TRH occur
at lower infusion rates in patients with ALS and bulbar involvement than in
patients with ALS without bulbar involvement. This autonomic sensitivity to TRH
infusion is characteristic of a more advanced stage of ALS with difficulties in
speaking and swallowing. Patients who will be receiving neuropeptides, such as
TRH, must be carefully studied to determine whether subclinical bulbar
involvement is present. Such patients may require a lower initial dose or dose
rate adjustment as well as continual monitoring during neuropeptide
administration. |
How can DUF families be deciphered? | The genome projects have unearthed an enormous diversity of genes of unknown function that are still awaiting biological and biochemical characterization. These genes, as most others, can be grouped into families based on sequence similarity. The PFAM database currently contains over 2,200 such families, referred to as domains of unknown function (DUF).
Critically examining domain covariation across metagenomic datasets can grant new perspectives on the roles and associations of DUFs in an ecological setting. Targeted attempts at DUF characterization in the laboratory or in silico may draw from these insights and opportunities to discover new associations and corroborate existing ones will arise as more large-scale metagenomic datasets emerge.
In a coordinated effort, the four large-scale centers of the NIH Protein Structure Initiative have determined the first three-dimensional structures for more than 250 of these DUF families. | The carbohydrate active enzyme (CAZy) database is an invaluable resource for
glycobiology and currently contains 45 glycosyltransferase families that are
represented in plants. Glycosyltransferases (GTs) have many functions in plants,
but the majority are likely to be involved in biosynthesis of polysaccharides
and glycoproteins in the plant cell wall. Bioinformatic approaches and
structural modeling suggest that a number of protein families in plants include
GTs that have not yet been identified as such and are therefore not included in
CAZy. These families include proteins with domain of unknown function (DUF)
DUF23, DUF246, and DUF266. The evidence for these proteins being GTs and their
possible roles in cell wall biosynthesis is discussed. Eukaryotic organisms have evolved a set of strategies to safeguard genome
integrity, but the underlying mechanisms remain poorly understood. Here, we
report that asymmetric leaves1/2 enhancer7 (AE7), an Arabidopsis thaliana gene
encoding a protein in the evolutionarily conserved Domain of Unknown Function 59
family, participates in the cytosolic iron-sulfur (Fe-S) cluster assembly (CIA)
pathway to maintain genome integrity. The severe ae7-2 allele is embryo lethal,
whereas plants with the weak ae7 (ae7-1) allele are viable but exhibit highly
accumulated DNA damage that activates the DNA damage response to arrest the cell
cycle. AE7 is part of a protein complex with CIA1, NAR1, and MET18, which are
highly conserved in eukaryotes and are involved in the biogenesis of cytosolic
and nuclear Fe-S proteins. ae7-1 plants have lower activities of the cytosolic
[4Fe-4S] enzyme aconitase and the nuclear [4Fe-4S] enzyme DNA glycosylase ROS1.
Additionally, mutations in the gene encoding the mitochondrial ATP binding
cassette transporter ATM3/ABCB25, which is required for the activity of
cytosolic Fe-S enzymes in Arabidopsis, also result in defective genome integrity
similar to that of ae7-1. These results indicate that AE7 is a central member of
the CIA pathway, linking plant mitochondria to nuclear genome integrity through
assembly of Fe-S proteins. BACKGROUND: The proportion of conserved DNA sequences with no clear function is
steadily growing in bioinformatics databases. Studies of sequence and structural
homology have indicated that many uncharacterized protein domain sequences are
variants of functionally described domains. If these variants promote an
organism's ecological fitness, they are likely to be conserved in the genome of
its progeny and the population at large. The genetic composition of microbial
communities in their native ecosystems is accessible through metagenomics. We
hypothesize the co-variation of protein domain sequences across metagenomes from
similar ecosystems will provide insights into their potential roles and aid
further investigation.
METHODOLOGY/PRINCIPAL FINDINGS: We calculated the correlation of Pfam protein
domain sequences across the Global Ocean Sampling metagenome collection,
employing conservative detection and correlation thresholds to limit results to
well-supported hits and associations. We then examined intercorrelations between
domains of unknown function (DUFs) and domains involved in known metabolic
pathways using network visualization and cluster-detection tools. We used a
cautious "guilty-by-association" approach, referencing knowledge-level resources
to identify and discuss associations that offer insight into DUF function. We
observed numerous DUFs associated to photobiologically active domains and
prevalent in the Cyanobacteria. Other clusters included DUFs associated with DNA
maintece and repair, inorganic nutrient metabolism, and sodium-translocating
transport domains. We also observed a number of clusters reflecting known
metabolic associations and cases that predicted functional reclassification of
DUFs.
CONCLUSION/SIGNIFICANCE: Critically examining domain covariation across
metagenomic datasets can grant new perspectives on the roles and associations of
DUFs in an ecological setting. Targeted attempts at DUF characterization in the
laboratory or in silico may draw from these insights and opportunities to
discover new associations and corroborate existing ones will arise as more
large-scale metagenomic datasets emerge. Crystal structures of three members (BACOVA_00364 from Bacteroides ovatus,
BACUNI_03039 from Bacteroides uniformis and BACEGG_00036 from Bacteroides
eggerthii) of the Pfam domain of unknown function (DUF4488) were determined to
1.95, 1.66, and 1.81 Å resolutions, respectively. The protein structures adopt
an eight-stranded, calycin-like, β-barrel fold and bind an endogenous unknown
ligand at one end of the β-barrel. The amino acids interacting with the ligand
are not conserved in any other protein of known structure with this particular
fold. The size and chemical environment of the bound ligand suggest binding or
transport of a small polar molecule(s) as a potential function for these
proteins. These are the first structural representatives of a newly defined
PF14869 (DUF4488) Pfam family. |
Which genes does thyroid hormone receptor beta1 regulate in the heart? | β-MHC, HCN4, KCND2/3, SERCA, TRbeta1, alpha-MHC | Physiological and pathological cardiac hypertrophy have directionally opposite
changes in transcription of thyroid hormone (TH)-responsive genes, including
alpha- and beta-myosin heavy chain (MyHC) and sarcoplasmic reticulum
Ca(2+)-ATPase (SERCA), and TH treatment can reverse molecular and functional
abnormalities in pathological hypertrophy, such as pressure overload. These
findings suggest relative hypothyroidism in pathological hypertrophy, but serum
levels of TH are usually normal. We studied the regulation of TH receptors (TRs)
beta1, alpha1, and alpha2 in pathological and physiological rat cardiac
hypertrophy models with hypothyroid- and hyperthyroid-like changes in the TH
target genes, alpha- and beta-MyHC and SERCA. All 3 TR subtypes in myocytes were
downregulated in 2 hypertrophy models with a hypothyroid-like mRNA phenotype,
phenylephrine in culture and pressure overload in vivo. Myocyte TRbeta1 was
upregulated in models with a hyperthyroid-like phenotype, TH (triiodothyronine,
T3), in culture and exercise in vivo. In myocyte culture, TR overexpression, or
excess T3, reversed the effects of phenylephrine on TH-responsive mRNAs and
promoters. In addition, TR cotransfection and treatment with the
TRbeta1-selective agonist GC-1 suggested different functional coupling of the TR
isoforms, TRbeta1 to transcription of beta-MyHC, SERCA, and TRbeta1, and
TRalpha1 to alpha-MyHC transcription and increased myocyte size. We conclude
that TR isoforms have distinct regulation and function in rat cardiac myocytes.
Changes in myocyte TR levels can explain in part the characteristic molecular
phenotypes in physiological and pathological cardiac hypertrophy. Pressure overload-induced cardiac hypertrophy leads to decreased contractile
performance, frequently progressing to heart failure. Cardiac hypertrophy and
heart failure can be accompanied by the so-called sick thyroid syndrome,
resulting in decreased serum T(3) levels along with decreased expression of
thyroid hormone receptors (TRalpha1 and TRbeta1) and sarco(endo)plasmic
reticulum Ca-ATPase (SERCA). Because the binding of T(3) occupied receptors to
the thyroid response elements in the SERCA promotor can increase gene
expression, we wanted to determine whether increasing TR expression in the
hypertrophied heart could also improve SERCA expression and cardiac function.
Mice subjected to aortic constriction to generate pressure overload-induced
hypertrophy were also subjected to gene therapy using adeno-associated virus
(AAV) expressing either TRalpha1 or TRbeta1, with LacZ expressing AAV serving as
control. After 8 wk of aortic constriction, a similar degree of hypertrophy was
observed in all three groups; however, mice treated with TRalpha1 or TRbeta1
showed improved contractile function. Administration of a physiological dose of
T(3) increased serum T(3) levels only into the lower range of normal. This T(3)
dose, with or without AAV TR treatment, did not result in any significant
increase in contractile performance. Calcium transients measured in isolated
myocytes also exhibited an enhanced rate of decay associated with TRalpha1 or
TRbeta1 treatment. Western blot analysis showed increased SERCA expression in
the TRalpha1- or TRbeta1-treated groups relative to the LacZ-treated control
group. These results demonstrate that increasing TR expression in the
hypertrophied heart is associated with an improvement in contractile function
and increased SERCA expression. The cardiac transient outward current I(to) is regulated by thyroid hormone
(T3). However, it remains unclear whether T3 directly modulates underlying gene
transcription and which thyroid receptor (TR) isoform might be responsible for
gene transactivation. To clarify this situation, we analysed the role of T3 and
its receptors alpha1 (TRalpha1) and beta1 (TRbeta1) in regulation of KCNA4,
KCND2, KCND3 and KCNIP2 transcription in rat cardiomyocytes. Initial results
demonstrated a T3-mediated increase of I(to) current density. T3 stimulation
enhanced KCND2 and KCND3 expression and decreased KCNA4 transcription, while
KCNIP2 remained unaffected. To dissect the role of TRalpha1 and TRbeta1 in
T3-dependent I(to) modulation, TRalpha1 and TRbeta1 were overexpressed in
cardiomyocytes by adenovirus-mediated gene transfer. TRalpha1 increased I(to),
while TRbeta1 significantly reduced I(to) in size, which was associated with
TRalpha1-mediated increase and TRbeta1-mediated reduction of KCND2/3
transcription. To further evaluate a possible direct interaction of TRalpha1 and
TRbeta1 with the KCND3 promoter, TR expression vectors were cotransfected with a
construct containing 2335 bp of the KCND3 5'-flanking sequence linked to a
luciferase reporter into ventricular myocytes. While the TRalpha1 aporeceptor
enhanced KCND3 transcription, the TRbeta1 aporeceptor suppressed KCND3
expression, with both effects exhibiting ligand-dependent amplification upon T3
stimulation. Deletion of the KCND3 5'-flanking region localized the suppressible
promoter sequence for TRbeta1 to within -293 bp and the activating promoter
sequence for TRalpha1 to within -2335 to -1654 bp of the transcription start
site. Disruption of putative TR binding sites by mutagenesis abolished the
TRalpha1- (G-1651T) and TRbeta1- (G-73T) mediated effects, indicating that
TRalpha1 and TRbeta1 response elements map to different regions of the KCND3
promoter. Thus, I(to) is modulated by diverse T3-dependent regulation of
underlying gene transcription. TRalpha1 and TRbeta1 exhibit distinct effects on
KCND3 transactivation with TRalpha1 enhancing and TRbeta1 suppressing KCND3
transcription. MicroRNAs (miRNAs), small noncoding RNAs, are negative regulators of gene
expression and play important roles in gene regulation in the heart. To examine
the role of miRNAs in the expression of the two isoforms of the cardiac myosin
heavy chain (MHC) gene, α- and β-MHC, which regulate cardiac contractility,
endogenous miRNAs were downregulated in neonatal rat ventricular myocytes
(NRVMs) using lentivirus-mediated small interfering RNA (siRNA) against Dicer,
an essential enzyme for miRNA biosynthesis, and MHC expression levels were
examined. As a result, Dicer siRNA could downregulate endogenous miRNAs
simultaneously and the β-MHC gene but not α-MHC, which implied that specific
miRNAs could upregulate the β-MHC gene. Among 19 selected miRNAs, miR-27a was
found to most strongly upregulate the β-MHC gene but not α-MHC. Moreover, β-MHC
protein was downregulated by silencing of endogenous miR-27a. Through a
bioinformatics screening using TargetScan, we identified thyroid hormone
receptor β1 (TRβ1), which negatively regulates β-MHC transcription, as a target
of miR-27a. Moreover, miR-27a was demonstrated to modulate β-MHC gene regulation
via thyroid hormone signaling and to be upregulated during the differentiation
of mouse embryonic stem (ES) cells or in hypertrophic hearts in association with
β-MHC gene upregulation. These findings suggested that miR-27a regulates β-MHC
gene expression by targeting TRβ1 in cardiomyocytes. |
Can siRNA affect response to afatinib treatment? | When afatinib was combined with an EGFR-specific siRNA there was a strong biological effect on growth inhibition and induction of apoptosis. | BACKGROUND: The epidermal growth factor receptor (EGFR) is a validated
therapeutic target in non-small cell lung cancer (NSCLC). However, current
single agent receptor targeting does not achieve a maximal therapeutic effect,
and some mutations confer resistance to current available agents. In the current
study we have examined, in different NSCLC cell lines, the combined effect of
RNA interference targeting the EGFR mRNA, and inactivation of EGFR signaling
using different receptor tyrosine kinase inhibitors (TKIs) or a monoclonal
antibody cetuximab.
METHODS: NSCLC cells (cell lines HCC827, H292, H358, H1650, and H1975) were
transfected with EGFR siRNA and/or treated with the TKIs gefitinib, erlotinib,
and afatinib, and/or with the monoclonal antibody cetuximab. The reduction of
EGFR mRNA expression was measured by real-time quantitative RT-PCR. The
down-regulation of EGFR protein expression was measured by western blot, and the
proliferation, viability, caspase3/7 activity, and apoptotic morphology were
monitored by spectrophotometry, fluorimetry, and fluorescence microscopy. The
combined effect of EGFR siRNA and different drugs was evaluated using a
combination index.
RESULTS: EGFR-specific siRNA strongly inhibited EGFR protein expression almost
equally in all cell lines and inhibited cell growth and induced cell apoptosis
in all NSCLC cell lines studied, albeit with a different magnitude. The effects
on growth obtained with siRNA was strikingly different from the effects obtained
with TKIs. The effects of siRNA probably correlate with the overall oncogenic
significance of the receptor, which is only partly inhibited by the TKIs. The
cells which showed weak response to TKIs, such as the H1975 cell line containing
the T790M resistance mutation, were found to be responsive to siRNA knockdown of
EGFR, as were cell lines with downstream TKI resistance mutations. The cell line
HCC827, harboring an exon 19 deletion mutation, was more than 10-fold more
sensitive to TKI proliferation inhibition and apoptosis induction than any of
the other cell lines. Cetuximab alone had no relevant in vitro activity at
concentrations obtainable in the clinic. The addition of EGFR siRNA to either
TKIs or cetuximab additively enhanced growth inhibition and induction of
apoptosis in all five cell lines, independent of the EGFR mutation status
(wild-type or sensitizing mutation or resistant mutation). The strongest
biological effect was observed when afatinib was combined with an EGFR-specific
siRNA.
CONCLUSIONS: EGFR knockdown by siRNA further decreases the cell growth of lung
cancer cells that are treated with TKIs or cetuximab alone, confirming that
single agent drug targeting does not achieve a maximal biological effect. The
siRNA inhibits EGFR oncogenic activity that bypasses downstream "resistance"
mutations such as KRAS and PTEN. The combined treatment of siRNA and EGFR
inhibitory agents is additive. The combination of a potent, irreversible kinase
inhibitor such as afatinib, with EGFR-specific siRNAs should be further
investigated as a new strategy in the treatment of lung cancer and other EGFR
dependent cancers, including those with downstream resistance mutations. The epidermal growth factor receptor (EGFR) is a validated therapeutic target in
non-small cell lung cancer (NSCLC). However, some mutations confer resistance to
current available agents, especially the frequently occurring T790M mutation. In
the current study, we have examined, in a NSCLC cell line H1975 containing both
L858R and T790M mutations, the effect of T790M-specific-siRNAs versus other
EGFR-specific-siRNAs. T790M-specific-siRNAs were able to inhibit T790M and EGFR
mRNA, to reduce EGFR protein expression, as well as to reduce the cell growth
and induce cell caspase activity in H1975 cells. However, this effect showed
less potency compared to the other EGFR-specific-siRNAs. EGFR-specific-siRNAs
strongly inhibited cell growth and induced apoptosis in H358, H1650, H292,
HCC827 and also in H1975 cells, which showed weak response to tyrosine kinase
inhibitors (TKIs) or cetuximab. The addition of T790M-specific-siRNAs could
rescue the sensitivity of T790M mutant H1975 cells to TKIs. The combination of
T790M-specific-siRNAs and cetuximab also additively enhanced cell growth
inhibition and induction of apoptosis in H1975 cells. Among the anti-EGFR agents
tested, the strongest biological effect was observed when afatinib was combined
with T790M-specific-siRNAs. Afatinib also offered extra effect when combined
with cetuximab in H1975 cells. In conclusion, knock-down of T790M transcript by
siRNAs further decreases the cell growth of T790M mutant lung cancer cells that
are treated with TKIs or cetuximab. The combination of a potent, irreversible
kinase inhibitor such as afatinib, with T790M-specific-siRNAs should be further
investigated as a new strategy in the treatment of lung cancer containing the
resistant T790M mutation. |
What is the gold standard treatment for Iatrogenic male incontinence? | The artificial urethral sphincter has represented, until today, the gold standard but, in the recent years, sling systems have been investigated as minimally invasive alternative options. | OBJECTIVES: To evaluate retrospectively the objective and subjective parameters
in 42 male patients who underwent bone anchored sub-urethral sling positioning
(BAUS) for SUI (stress urinary incontinence) due to ISD (intrinsic sphincter
deficiency).
METHODS: Patients with SUI due to radical retropubic prostatectomy (36
patients), transurethral resection of prostate (5 patients) and open simple
prostatectomy (1 patient) underwent BAUS positioning between July 1999 and
September 2005 (mean FU = 41 months). Before and after surgery, the patients
were evaluated by physical examination, urethrocystoscopy, urodynamics, 1 h pad
test and QoL questionnaire. Surgical technique involved perineal implantation to
the pubic rami using four anchors of a sub-urethral sling made of synthetic (26
patients), biological (4 patients) or mixed (12 patients) material. Patients
were stratified into three groups: (1) Cured: dry patients at stress test, pad
weight 0-1 g. (2) Improved: patients with mild-moderate incontinence, pad weight
2-50 g. (3) Failed: unchanged patients, pad weight > 50 g.
RESULTS: At the final follow-up visit cured, improved and failed patients were
26 (62%), 4 (8%) and 12 (30%), respectively. Mean pad weight significantly
decreased from 104.6 to 47.3 g (55%) and mean total questionnaire score
significantly increased to 50.7 (66%). Mean ALPP significantly increased to 50.4
cmH2O (44.8%). Better results were seen with synthetic slings. Main
complications were perineal pain (76%), detrusor overactivity (12%) and sling
infection (4.8%).
CONCLUSIONS: BAUS implantation is a safe, effective, minimally invasive option
for iatrogenic male incontinence due to ISD. It compares favourably with AUS. PURPOSE: We retrospectively report objective and subjective outcomes in 40 male
patients who underwent bone anchored suburethral synthetic sling positioning for
stress urinary incontinence due to intrinsic sphincter deficiency.
MATERIALS AND METHODS: Patients with stress urinary incontinence due to radical
retropubic prostatectomy (32), robot assisted laparoscopic prostatectomy (3) and
transurethral prostate resection (5) underwent bone anchored suburethral
synthetic sling positioning between December 2002 and December 2007. Mean
followup was 35.2 months (range 2 to 62). Previous anti-incontinence procedures,
radiotherapy and transurethral procedures due to urethral stricture were
performed in 5, 11 and 5 patients, respectively. Before and after surgery
patients were evaluated by physical examination, urethral cystoscopy,
urodynamics, a 1-hour pad test and a quality of life questionnaire. Patients
were stratified into 3 groups, including group 1-cured (dry with a pad weight of
0 to 1 gm), group 2-improved (mild to moderate incontinence with a pad weight of
2 to 50 gm) and group 3-failed (patient condition unchanged with a pad weight of
greater than 50 gm).
RESULTS: At the final followup visit 22 (55%), 5 (12.5%) and 13 patients (32.5%)
were cured, improved and failed, respectively. Mean pad weight significantly
decreased to 51.3 gm in 54% of cases, while the mean total questionnaire score
significantly increased to 72.9 in 65% and abdominal leak point pressure
significantly increased to 92.5 cm H(2)O in 52%. Statistical analysis showed a
significant association between preoperative radiotherapy and treatment failure
(85% of patients). Complications were perineal pain in 73% of cases, detrusor
overactivity in 5% and sling infection in 15%.
CONCLUSIONS: The bone anchored suburethral synthetic sling is a simple and
attractive procedure that can produce immediate good results with low morbidity,
especially when strictly selected patients are treated. Radiotherapy remains a
strong predictor of failure. OBJECTIVES: The increasing number of prostatectomies entails an increasing
number of patients suffering from iatrogenic incontinence despite improved
surgical techniques. The severity of this problem often requires invasive
treatments such as periurethral injection of bulking agents, artificial urinary
sphincter (AUS) implantation, and sub-urethral sling positioning. The artificial
urethral sphincter has represented, until today, the gold standard but, in the
recent years, sling systems have been investigated as minimally invasive
alternative options. Today, three different sling procedures are commonly
performed: bone-anchored, readjustable, and trans-obturator slings systems. The
aim of this review is to critically report the current status of sling systems
in the treatment of iatrogenic male incontinence.
MATERIALS AND METHODS: MEDLINE and PubMed databases were searched and all
articles between 1974 and 2009 were evaluated.
RESULTS: With regard to bone-anchored, readjustable, and trans-obturator slings
systems, cure rates ranged between 58.0% and 86.0%, 55.5% and 73.0%, and 40.0%
and 63.0%, respectively, while major complication rates ranged between 0 and
14.5%, 10.0 and 22.2%, and 0 and 10.0%, respectively.
CONCLUSIONS: Suburethral slings are the only alternative techniques which can be
favorably compared with the AUS, showing more advantages with respect to AUS
implantations which are mainly represented by a quick and less invasive
approach, low morbidity, and low costs. In spite of the difficulty in
identifying the most effective sling procedure, overall, sling systems can be
recommended for patients with persistent mild or moderate incontinence. However,
the indication can also be extended to patients with severe incontinence, after
appropriate counseling, allowing AUS implantation in the event of sling failure. Male Stress Urinary Incontinence (SUI) is an increasingly recognized problem
particularly after the treatment of prostate cancer. Postprostatectomy
incontinence is a major problem that needs to be solved, since it has great
impact on quality of life affecting the patient's physical activity and social
well-being. The initial treatment for SUI that persists after 12 months consists
of conservative measures such as pelvic floor muscle exercises and behavioral
therapy. Properly selected and informed patients can also be treated efficiently
with minimally invasive procedures such as the implantation of a male
suburethral sling, although the experience with such devices is not extensive.
However, the implantation of artificial urinary sphincter is the gold standard
therapy. |
Which is the main CHEK2 genetic variant, thought to be involved in familial breast cancer? | CHEK2 1100delC mutation is recurrently detected in the general population and is thought to confer a moderate risk for breast cancer. | We recently reported that a sequence variant in the cell-cycle-checkpoint kinase
CHEK2 (CHEK2 1100delC) is a low-penetrance breast cancer-susceptibility allele
in noncarriers of BRCA1 or BRCA2 mutations. To investigate whether other CHEK2
variants confer susceptibility to breast cancer, we screened the full CHEK2
coding sequence in BRCA1/2-negative breast cancer cases from 89 pedigrees with
three or more cases of breast cancer. We identified one novel germline variant,
R117G, in two separate families. To evaluate the possible association of R117G
and two germline variants reported elsewhere, R145W and I157T with breast
cancer, we screened 737 BRCA1/2-negative familial breast cancer cases from 605
families, 459 BRCA1/2-positive cases from 335 families, and 723 controls from
the United Kingdom, the Netherlands, and North America. All three variants were
rare in all groups, and none occurred at significantly elevated frequency in
familial breast cancer cases compared with controls. These results indicate that
1100delC may be the only CHEK2 allele that makes an appreciable contribution to
breast cancer susceptibility. Searching for low-penetrance genes involved in breast cancer susceptibility has
been a field of interest in the last few years. Recently, the CHEK 2 gene,
involved in DNA damage and replication checkpoints, has been pointed out as a
good candidate; moreover, a specific variant in this gene,1100delC, has been
found to increase breast cancer susceptibility among familial breast cancer
cases not attributable to mutations in BRCA1 or BRCA2 genes. In our present
study, we evaluated the role of the 1100delC variant as a susceptibility allele
in breast cancer in the Spanish population. However, our results suggest that
this variant is absent or very infrequent in our population, making its
screening irrelevant from the practical point of view. Checkpoint kinase 2 (CHEK2, Chk2) emerges as an important signal transducer of
cellular responses to DNA damage and a candidate tumor suppressor whose defects
contribute to molecular pathogenesis of diverse types of human maligcies,
both sporadic and hereditary. Here, we briefly outline the molecular properties,
regulation and physiological role of CHEK2, and review in more detail its
defects that predispose to tumors, with particular emphasis on familial breast
cancer. The frequency, penetrance and epidemiological as well as clinical
significance of the two most studied breast cancer-predisposing variants of the
CHEK2 gene, 1100delC and I157T, are highlighted in more depth, and additional
CHEK2 mutations and their cancer relevance are discussed as well. These recent
findings are considered also from a broader perspective of CHEK2 as the integral
component of the ataxia telangiectasia-mutated-CHEK2-p53 pathway within the
genome integrity maintece system and a barrier against tumor progression.
Finally, the potential value of information about the CHEK2 status in family
counseling and optimizition of individualized cancer treatment is discussed. The CHEK2-1100delC mutation is recurrent in the population and is a moderate
risk factor for breast cancer. To identify additional CHEK2 mutations
potentially contributing to breast cancer susceptibility, we sequenced 248 cases
with early-onset disease; functionally characterized new variants and conducted
a population-based case-control analysis to evaluate their contribution to
breast cancer risk. We identified 1 additional null mutation and 5 missense
variants in the germline of cancer patients. In vitro, the CHEK2-H143Y variant
resulted in gross protein destabilization, while others had variable suppression
of in vitro kinase activity using BRCA1 as a substrate. The germline
CHEK2-1100delC mutation was present among 8/1,646 (0.5%) sporadic, 2/400 (0.5%)
early-onset and 3/302 (1%) familial breast cancer cases, but undetectable
amongst 2,105 multiethnic controls, including 633 from the US. CHEK2-positive
breast cancer families also carried a deleterious BRCA1 mutation. 1100delC
appears to be the only recurrent CHEK2 mutation associated with a potentially
significant contribution to breast cancer risk in the general population.
Another recurrent mutation with attenuated in vitro function, CHEK2-P85L, is not
associated with increased breast cancer susceptibility, but exhibits a striking
difference in frequency across populations with different ancestral histories.
These observations illustrate the importance of genotyping ethnically diverse
groups when assessing the impact of low-penetrance susceptibility alleles on
population risk. Our findings highlight the notion that clinical testing for
rare missense mutations within CHEK2 may have limited value in predicting breast
cancer risk, but that testing for the 1100delC variant may be valuable in
phenotypically- and geographically-selected populations. |
Is there an association between presenteeism and depression? | Yes. Presenteeism is associated with depression. Remission of depression is associated with improvement of presenteeism. | OBJECTIVE: The relationship of depressive symptoms, satisfaction with health
care, and 2-year work outcomes was examined in a national cohort of employees.
METHOD: A total of 6,239 employees of three corporations completed surveys on
health and satisfaction with health care in 1993 and 1995. This study used
bivariate and multivariate analyses to examine the relationships of depressive
symptoms (a score below 43 on the Medical Outcomes Study Short-Form Health
Survey mental component summary), satisfaction with a variety of dimensions of
health care in 1993, and work outcomes (sick days and decreased effectiveness in
the workplace) in 1995.
RESULTS: The odds of missed work due to health problems in 1995 were twice as
high for employees with depressive symptoms in both 1993 and 1995 as for those
without depressive symptoms in either year. The odds of decreased effectiveness
at work in 1995 was seven times as high. Among individuals with depressive
symptoms in 1993, a report of one or more problems with clinical care in 1993
predicted a 34% increase in the odds of persistent depressive symptoms and a 66%
increased odds of decreased effectiveness at work in 1995. There was a weaker
association between problems with plan administration and outcomes.
CONCLUSIONS: Depressive disorders in the workplace persist over time and have a
major effect on work performance, most notably on "presenteeism," or reduced
effectiveness in the workplace. The study's findings suggest a potentially
important link between consumers' perceptions of clinical care and work outcomes
in this population. OBJECTIVE: To discuss the impact of major disease states, including depression,
in the loss of productivity in the workplace and how integration of health care
can decrease cost to employers.
SUMMARY: The majority of costs associated with depressive illness can be traced
to lost productivity, and the employer, therefore, bears most of the economic
burden. Efforts to improve employee health and productivity have been hampered
by the compartmentalization of medical costs, pharmacy costs, behavioral health
costs, and productivity measures. This situation can be rectified by "busting"
these silos and promoting a reintegration of prospective costs and parties.
Health risk assessments enable employers to identify illnesses that are suitable
targets for integrated health and productivity management programs. In the case
of depression, employers can act proactively to identify employees at risk,
working to minimize risk factors such as stress before these individuals become
heavy utilizers of company resources. For employees who are currently depressed,
recent research evidence has demonstrated that pharmacotherapy can have a
dramatic and positive effect on lost productivity, absenteeism, and
presenteeism. The selection of antidepressants and subsequent follow-up must be
improved, however, if the benefits of pharmacotherapy are to be optimized.
CONCLUSION: Understanding the linkage of disease management and productivity in
the workplace can result in dramatic decreases in absenteeism and presenteeism
and increased cost savings to the employers. OBJECTIVE: Employers provide most American mental health benefits and are
increasingly cost conscious. However, commonplace anxiety and depressive
disorders have enormous economic and workplace performance costs.
METHODS: We performed multiple literature searches on several areas of pertinent
research (and on key articles) covering the past 5 years.
RESULTS: Substantial research exists about anxiety and depression costs, such as
performance and productivity, absenteeism, presenteeism, disability, physical
disability exacerbation, mental health treatment, increased medical care costs,
exacerbating of physical illness, and studies of mental health care limitations
and cost-offset. Research addressing the potential value of higher quality
mental health care is limited.
CONCLUSIONS: Commonplace anxiety and depressive disorders are costly in the
workplace. Employers and researchers remain largely unaware of the value of
quality care and psychiatric skills. Effective solutions involve the increased
use of psychiatric skills and appropriate treatment. OBJECTIVE: To review the recent descriptive and social epidemiology of common
mental disorders in the workplace, including prevalence, participation, work
disability, and impact of quality of work, as well as to discuss the
implications for identifying targets for clinical and preventive interventions.
METHOD: We conducted a structured review of epidemiologic studies in community
settings (that is, in the general population or in workplaces). Evidence was
restricted to the peer-reviewed, published, English-language literature up to
the end of June 2005. We further restricted evidence to studies that used recent
classification systems; then, if evidence was insufficient, we reviewed studies
that used standardized psychiatric screening scales. To distinguish this article
from recent reviews of health and work quality, we focused on new areas of
investigation and new evidence for established areas of investigation:
underemployment, organizational justice, job control and demand, effort-reward
imbalance, and atypical (nonpermanent) employment.
RESULTS: Depression and simple phobia were found to be the most prevalent
disorders in the working population. The limited data on rates of participation
suggested higher participation among people with depression, simple phobia,
social phobia, and generalized anxiety disorder. Depression and anxiety were
more consistently associated with "presenteeism" (that is, lost productivity
while at work) than with absenteeism, whether this was measured by cutback days
or by direct questionnaires. Seven longitudinal studies, with an average sample
size of 6264, showed a strong association between aspects of low job quality and
incident depression and anxiety. There was some evidence that atypical work was
associated with poorer mental health, although the findings for fixed-term work
were mixed.
CONCLUSIONS: Mental health risk reduction in the workplace is an important
complement to clinical interventions for reducing the current and future burden
of depression and anxiety in the workplace. OBJECTIVE: To explore methodological refinements in measuring health-related
lost productivity and to assess the business implications of a full-cost
approach to managing health.
METHODS: Health-related lost productivity was measured among 10 employers with a
total of 51,648 employee respondents using the Health and Work Performance
Questionnaire combined with 1,134,281 medical and pharmacy claims. Regression
analyses were used to estimate the associations of health conditions with
absenteeism and presenteeism using a range of models.
RESULTS: Health-related productivity costs are significantly greater than
medical and pharmacy costs alone (on average 2.3 to 1). Chronic conditions such
as depression/anxiety, obesity, arthritis, and back/neck pain are especially
important causes of productivity loss. Comorbidities have significant
non-additive effects on both absenteeism and presenteeism. Executives/Managers
experience as much or more monetized productivity loss from depression and back
pain as Laborers/Operators. Testimonials are reported from participating
companies on how the study helped shape their corporate health strategies.
CONCLUSIONS: A strong link exists between health and productivity. Integrating
productivity data with health data can help employers develop effective
workplace health human capital investment strategies. More research is needed to
understand the impacts of comorbidity and to evaluate the cost effectiveness of
health and productivity interventions from an employer perspective. PURPOSE: Depressed employees are vulnerable to adverse work outcomes. We
hypothesized that work performance is impaired by depression and is worsened by
exposure to psychosocial work stressors.
DESIGN: Longitudinal cohort study with surveys administered at baseline, 6, 12,
and 18 months.
SETTING: Recruitment in primary care offices.
SUBJECTS: A total of 14,268 were screened; 286 depressed, employed adults (18-62
years) and 193 controls were enrolled.
MEASURES: At-work limitations (presenteeism) and absenteeism were measured with
the Work Limitations Questionnaire (WLQ) and WLQ Work Absence Module,
respectively. Work stressors were assessed using a modified version of the Job
Content Questionnaire.
ANALYSIS: Univariate and multivariate tests assessed the degree to which at-work
limitations were related to depression and/or stressful work.
RESULTS: Presenteeism and absenteeism were significantly worse for the
depression group at each time point (p < or = .001). In cross-sectional models,
presenteeism was associated with more severe depression symptoms, poorer general
physical health, psychologically demanding work, the interaction
ofpsychologically demanding work with depression, and less job control (r2 range
= .33-.54). Absences were explained by depression symptom severity and poorer
general physical health but not work stressors (r2 = .19). Because of minimal
change in the work stressors, their longitudinal effects on outcomes were mostly
nonsignificant.
CONCLUSION: This study found that depression symptoms are related to work
absences and impaired work performance, and results partly confirmed that work
stressors add to this impact. Results suggest that workers with depression may
benefit from care involving medical and vocational interventions. This systematic review examines the economic and human costs of depression and
the potential savings associated with improvement in patient adherence to
treatment with antidepressants through the use of enhanced-care programs. A
MEDLINE search was conducted for papers published on the health economics and
costs of depression and compliance, adherence, and persistence. Compliance data
collected through the online antidepressant compliance support website iCAN
(www.ican.co.uk) were compared with data for patients with depression from the
IMS Disease Analyzer UK database. Depression frequently causes unemployment,
absenteeism, and presenteeism, which results in significantly reduced
productivity. Indirect costs of depression accounted for more than $50 billion,
whereas direct costs resulted in expenditure of $26 billion, in the US in 2000.
Improving patients' compliance with their antidepressant medication results in
improved outcomes and prolongs remission from depression, increasing work
productivity, and thus reducing overall costs. The implementation of remote
enhanced-care programs may improve compliance and reduce overall costs. Novel
methods for delivering enhanced-care programs to assist in maintaining
compliance have the potential to further reduce costs and should be a focus of
future research. In conclusion, depression is a common disorder with a high
economic impact. Enhanced-care programs may lower costs associated with
depression and improve patients' lives. OBJECTIVES: To examine the impact of ankylosing spondylitis (AS) on patients
across the UK and to identify factors associated with unemployment, absenteeism,
and presenteeism.
METHODS: One thousand patients with AS from 10 specialist rheumatology centres
across the UK were invited to participate in a study evaluating a new outcome
measure. Patients completed a questionnaire, which included questions relating
to their work, sociodemographic and clinical characteristics.
RESULTS: The questionnaire was completed by 612 patients (438 males; 72%). The
mean age of the participants was 50.8 (SD 12.2) years, mean disease duration was
17.3 (SD 11.7) years, and mean symptom duration 22.4 (SD 12.4) years. A total of
206 (40%) patients of working age were not employed. Factors associated with not
being employed were social deprivation [odds ratio (OR) 3.52, 95% confidence
interval (CI) 2.14-5.80], poor function (OR 3.42, 95% CI 1.90-6.13), depression
(OR 2.05, 95% CI 1.12-3.78), increasing age (OR 1.05 per year, 95% CI
1.02-1.08), and longer disease duration (OR 1.03 per year, 95% CI 1.01-1.06).
Disease activity (OR 3.24, 95% CI 1.11-9.48) and depression (OR 3.22, 95% CI
1.22-8.48) were associated with absenteeism, while depression (OR 5.69, 95% CI
1.77-18.27, disease activity (OR 3.97, 95% CI 1.76-8.98), anxiety (OR 3.90, 95%
CI 1.83-8.31), self-efficacy (OR 0.71, 95% CI 0.58-0.86), and increasing age (OR
1.04 per year, 95% CI 1.00-1.08) were associated with presenteeism.
CONCLUSION: Psychological, sociodemographic, and disease-related factors were
all found to be related to work status. These factors should be taken into
account when considering early treatment and management. Depression, in
particular, appears to be associated with employment, absenteeism, and
presenteeism, and should therefore be prioritized in clinical practice. BACKGROUND: Depression is reported to be a major cause of illness-related
sub-optimal work performance (presenteeism). However, the majority of studies
examining presenteeism have relied on self-report measures of work performance.
Furthermore, employers currently face a number of practical challenges in
attempting to facilitate early identification of depression.
AIMS: To test whether a web-based screening tool for depression could be used
successfully in the workplace and whether it was possible to detect an
association between rates of depression and objective measures of impaired
workgroup performance.
METHODS: All permanent employees of a telecommunications company with UK-based
call centres were encouraged to complete a web-based psychological assessment
using the Patient Health Questionnaire depression scale (PHQ-9). In addition to
confidential individual level results, the tool was able to provide anonymized
summary statistics for each workgroup. Four objective measures of work
performance were collected for each workgroup.
RESULTS: During the study period, 1161 web-based PHQ-9 questionnaires were
completed. There was a negative linear relationship between rates of depressive
symptoms and the overall performance of a workgroup (P < 0.001). The linear
relationship between depression and workgroup performance remained after
controlling for gender balance, percent of temporary staff, employees' perceived
level of engagement and satisfaction with their line manager (P < 0.01).
CONCLUSIONS: Workgroups with high levels of depressive symptoms tend to perform
poorly. Computer-aided web-based screening for symptoms of depression is
feasible in a work setting. OBJECTIVE: Depressive disorders influence socioeconomic burden at both the
individual and organizational levels. This study estimates the lost productive
time (LPT) and its resulting cost among workers with major depressive disorder
(MDD) compared with a comparison group. It also estimates the change in
productivity after 8 weeks of outpatient psychiatric treatment with
antidepressants.
METHODS: Working patients diagnosed with MDD without other major physical or
mental disorders were recruited (n = 102), along with age- and sex-matched
healthy controls from the Seoul Metropolitan area (n = 91). The World Health
Organization's Health and Work Performance Questionnaire and the Hamilton Rating
Scale for Depression were utilized to measure productivity and severity of
depression, respectively, at baseline and at 8 weeks of treatment.
RESULTS: The LPT from absenteeism and presenteeism (reduced performance while
present at work) was significantly higher among the MDD group. Workers with MDD
averaged costs due to LPT at 33.4% of their average annual salary, whereas the
comparison group averaged costs of 2.5% of annual salary. After 8 weeks of
treatment, absenteeism and clinical symptoms of depression were significantly
reduced and associated with significant improvement in self-rated job
performance (31.8%) or cost savings of $7508 per employee per year.
CONCLUSIONS: We confirmed that significant productivity loss arises from MDD and
that this loss can be reduced with psychiatric intervention after a time period
as short as 8 weeks. Mental health professionals should work with employers to
devise a cost-effective system to provide workers with accessible quality care. The objective of this study is to identify the contribution that selected
demographic characteristics, health behaviors, physical health outcomes, and
workplace environmental factors have on presenteeism (on-the-job productivity
loss attributed to poor health and other personal issues). Analyses are based on
a cross-sectional survey administered to 3 geographically diverse US companies
in 2010. Work-related factors had the greatest influence on presenteeism (eg,
too much to do but not enough time to do it, insufficient technological
support/resources). Personal problems and ficial stress/concerns also
contributed substantially to presenteeism. Factors with less contribution to
presenteeism included physical limitations, depression or anxiety, inadequate
job training, and problems with supervisors and coworkers. Presenteeism was
greatest for those ages 30-49, women, separated/divorced/widowed employees, and
those with a high school degree or some college. Clerical/office workers and
service workers had higher presenteeism. Managers and professionals had the
highest level of presenteeism related to having too much to do but too little
time to do it, and transportation workers had the greatest presenteeism because
of physical health limitations. Lowering presenteeism will require that
employers have realistic expectations of workers, help workers prioritize, and
provide sufficient technological support. Ficial stress and concerns may
warrant ficial planning services. Health promotion interventions aimed at
improving nutrition and physical and mental health also may contribute to
reducing presenteeism. OBJECTIVES: To characterize work productivity in relapsing multiple sclerosis
(MS) by using a work productivity scale and to identify associations between
work productivity and disability, depression, fatigue, anxiety, cognition, and
health-related quality of life.
METHODS: Three hundred seventy-seven subjects with a clinically isolated
syndrome or relapsing remitting MS participated in the study. Subjects underwent
neurological examinations and completed patient-reported outcome and cognitive
measures. Subjects also completed the Work Productivity and Activity Impairment
Questionnaire: General Health to quantify absenteeism (missing work because of
health problems), presenteeism (impairment while working), overall work
impairment, and daily activity impairment attributable to health problems.
Univariate correlations and multivariate models were used to determine the
associations between each work productivity variable and clinical,
patient-reported outcome, and cognitive measures.
RESULTS: Seventy-six percent of subjects were employed. Fourteen percent of
working subjects reported absenteeism, and 47% reported presenteeism. The mean
work time lost because of absenteeism was 4%, and the mean work time lost
because of presenteeism was 12%. Absenteeism was not significantly associated
with disease or patient-reported outcome measures. Statistically significant
correlations (0.32-0.53) were found between presenteeism and increasing
disability, fatigue, depression, anxiety, and reduced quality of life. No
associations were observed between presenteeism and disease duration or
cognitive function.
CONCLUSIONS: Subjects with clinically isolated syndrome/relapsing remitting MS
reported substantial work productivity losses due to presenteesim. Presenteeism
was associated with increasing fatigue, depression, anxiety, and reduced quality
of life. It is possible that the early identification and treatment of fatigue
and mental health symptoms may improve productivity while working and extend
employment for individuals with MS. OBJECTIVE: To examine the prospective association between sickness presenteeism
(SP), that is, working while ill, and the onset of depression.
METHODS: We carried out a two-wave (2006 to 2008) questionnaire-based study
among 1271 employees from 60 Danish workplaces. Sickness presenteeism was
assessed by asking participants to report the number of days that they went to
work despite illness in the preceding year.
RESULTS: Multivariate logistic regression revealed that, after controlling for
several health-related variables and other relevant confounders, reporting 8 or
more days of SP was associated with an increased risk of depression among
initially nondepressed participants (odds ratio, 2.45; 95% confidence interval,
1.06 to 5.64). No significant sex-related differences were observed in this
relationship.
CONCLUSION: Adding to previous evidence on the health effects of SP, this study
suggests that working while ill may also be a significant risk factor for the
development of depression. PURPOSES: To assess predictors of presenteeism (reduced productivity at work)
and activity impairment outside work in patients with spondyloarthritis (SpA).
METHODS: Multivariate logistic regression analysis was used to study predictors
of presenteeism and activity impairment in 1,253 patients with SpA based on a
2.5 year follow-up questionnaire. The Work Productivity and Activity Impairment
(WPAI) questionnaire was used as main outcome. Age, gender, lifestyle factors,
subgroups, disease duration, and different patient reported outcome measures
(PROMs) were studied as possible predictors. The association between
presenteeism and WPAI activity impairment outside work was assessed.
RESULTS: Out of 1,253 patients, 757 reported being in work and of these 720
responded to the WPAI questionnaire. The mean (confidence interval, CI) reported
presenteeism was 25% (23-27%) and mean activity impairment 33% (31-35%) (0-100%,
0 = no reduction). Significant predictors of presenteeism and activity
impairment at follow-up (controlled for gender, age, spondyloarthritis subgroups
and presenteeism at baseline) were presenteeism at baseline, poor quality of
life, worse disease activity, decreased physical function, lower self-efficacy
pain and symptom, higher scores of anxiety, depression, smoking and low
education level, and for activity impairment also female sex. There was a strong
association between presenteeism and activity impairment outside work (OR 16.7;
95% CI 11.6-24.3; p < 0.001).
CONCLUSIONS: Presenteeism and activity impairment were not only predicted by
presenteeism at baseline, but also by several PROMs commonly used in clinical
rheumatology practice. Impaired activity outside work could indicate problems
also at work suggesting why both areas need to be addressed in the clinical
situation. While it is known that psychiatric illness and subclinical psychiatric illness
can be very disabling, their impact on workers' productivity has been little
appreciated or appropriately addressed. Complex variables are involved in
fashioning an appropriate policy to ameliorate the impact of mental illness on
productivity including the identification of effective treatments and potential
negative effects of controlling patients' access to them. The cost-effectiveness
of such treatments is considered from the differing perspectives and goals of
the various stakeholders involved, including employers, insurers, and workers
with psychiatric illness. Depression in workers leads to significant
absenteeism, "presenteeism" (diminished capacity due to illness while still
present at work), and significantly increased medical expenses in addition to
the costs of psychiatric care. In addition to the specific usefulness of
psychotropic medication, there are a variety of studies on the
cost-effectiveness of different psychotherapeutic treatments that improve health
and productivity in psychiatrically ill workers. Research indicates the
usefulness of approaches including employee assistance programs, specialized
cognitive-behavioral treatments, and brief and longer term psychodynamic
interventions. It is clear that substance abuse disorders and especially
depression and subsyndromal depression have a profound negative effect on work
productivity and increases in medical visits and expenses. The current system of
mental health care suffers from ignorance of the negative effects of psychiatric
illness in workers, from a lack of subtle awareness of which treatments are most
appropriate for which diagnoses and from the reluctance by payers to invest in
them. Access to evidence-based appropriate treatment can improve the negative
impact on productivity as well as workers' health. This article considers these
issues and argues for a role of psychotherapy in the treatment of mental illness
and substance abuse from the perspective of worker productivity. BACKGROUND: The purpose of this study was to assess the economic benefit of
achieving remission among outpatients with major depressive disorder (MDD) who
are currently employed in Korea.
METHODS: Cross-sectional observational study. A total of 337 outpatients with
MDD with paid jobs were recruited from 14 psychiatric clinics in Korea and were
then divided into three groups as follows: new visit group (n = 128), remitted
group (n = 100) and non-remitted group (n = 109). The 17-item Hamilton
Depression Rating Scale (HAM-D) was used to decide whether a patient should be
assigned to the remitted or non-remitted group. Direct medical and non-medical
costs were measured via interview with the subjects. The World Health
Organization Health and Work Performance Questionnaire (HPQ) were applied in
order to measure the lost productive time (LPT) and related productivity costs.
RESULTS: The three groups did not show a significant difference in direct
medical cost. However, the difference between the remitted group and
non-remitted group was statistically significant (25.49 ± 52.99 vs.
44.79 ± 126.55, χ (2) = 12.99, p = 0.0015). The remitted group demonstrated a
significant improvement in productivity (particularly presenteeism) when
compared with the new visit group (Z = -3.29, p = 0.001). Although the
non-remitted group received treatment at psychiatric clinics similar to the
remitted group, it lost 33 more working hours per month, which is compatible to
$332 per month.
CONCLUSION: These results suggest the economic importance of achieving remission
in treating depression. |
Which hormone abnormalities are common in Williams syndrome | Elevated Thyrotropin - TSH
Low FT4
Growth Hormone deficiency
Calcitonin deficiency
Elevated Prolactin
Elevated Cortisol
Elevated Oxytocin
Elevated Vasopressin | We have investigated the possibility of mutations in the calcitonin/calcitonin
gene related peptide (CGRP) gene in children with Williams syndrome. Involvement
of the calcitonin/CGRP gene in Williams syndrome is postulated on the basis that
Williams syndrome children often have infantile hypercalcemia and deficient
expression of calcitonin, a hormone that lowers serum calcium levels. To test
the hypothesis that mutations in the calcitonin/CGRP gene might be responsible
for the reduced calcitonin levels, we examined the calcitonin/CGRP gene
structure in Williams syndrome children. Analysis of white blood cell DNA by
Southern blot hybridizations in 5 individuals did not show any detectable large
deletions or rearrangements in the calcitonin/CGRP gene locus. The possibility
of small deletions or point mutations within the exon encoding the mature
calcitonin hormone is unlikely based on ribonuclease protection assays with
patient DNA amplified by the polymerase chain reaction (PCR) technique. These
findings suggest that the calcitonin deficiency might be due either to mutations
elsewhere in the gene or to defects in the cellular machinery needed for
calcitonin synthesis and/or secretion. Supravalvular aortic stenosis (SVAS) may occur as an isolated autosomal domit
trait or as a feature of Williams syndrome. It has been suggested that a defect
in calcitonin function may play a role in Williams syndrome. We have excluded
calcitonin as a candidate gene for SVAS using a gene specific probe. A 31-year-old man who had been under regular hemodialysis for 6 months was
diagnosed as Williams syndrome (WS) by fluorescence in situ hybridization (FISH)
chromosomal analysis. The association of WS and chronic renal failure (CRF) is
only rarely encountered. Endocrinological examinations revealed
hypergonadotropic hypogonadism. Prolonged and exaggerated responses of
adrenocorticotropin (ACTH) to insulin-induced hypoglycemia and corticotropin
releasing hormone (CRH) were also noted. While most of the endocrinological
abnormalities observed in this patient could be attributed to altered endocrine
circumstances in CRF, some findings stand in contrast. Furthermore, the
testicular biopsy specimen showed severe hypospermatogenesis. Endocrine
disorders observed in this patient may be at least in part, responsible for
various clinical features underlying WS. Growth retardation is a consistent finding in Williams-Beuren syndrome. The
cause of short stature in this syndrome is unknown. Endocrine studies have
failed to reveal abnormalities in the growth hormone-insulin-like growth factor
I axis. We report a boy with confirmed Williams-Beuren syndrome, who was found
to have classical growth hormone deficiency and responded well to growth hormone
therapy.
CONCLUSION: Although growth hormone deficiency is not likely to be a common
cause of short stature in Williams-Beuren syndrome, we nevertheless recommend
evaluation of the growth hormone-insulin-like growth factor I axis in all cases. A girl with Williams syndrome (WS) presented with elevated thyrotropin (TSH)
levels (7.0 microU/ml), normal free thyroid hormone concentrations, and absent
antithyroid autoantibodies. Thyroid ultrasonography and scintigraphy showed
hemiagenesis of the left lobe and no evidence of ectopic tissue. TSH response to
thyrotropin-releasing hormone (TRH) injection (200 microg/mq, i.v.) was
exaggerated and prolonged, suggesting subclinical hypothyroidism. The biological
activity of circulating TSH was slightly below the normal range [TSH bioactivity
(B) to immunoreactivity (I) ratio (TSH B/I) = 0.4, normal: 0.6-2.2]. These
abnormalities are similar to those seen in patients with hypothalamic
hypothyroidism. Thyroid function is not a recognized manifestation of WS and is
not routinely investigated. However, abnormalities of the
hypothalamic-pituitary-thyroid (HPT) axis and thyroid dysgenesis have been found
in other WS cases. Genes mapping at 7q11.23, contiguous to the chromosomal
region deleted in most WS patients, may be involved in the development of the
thyroid gland, contributing to the complex phenotype of WS. The authors report a female presenting with congenital heart defects, liver
hemangiomas, and facial dysmorphisms admitted to hospital at 3 months of age
because of feeding difficulties and poor growth. She had hypotonia and large
tongue, "coarse" face, and umbilical hernia in presence of complex congenital
cardiovascular malformations. In spite of normal neonatal screening we performed
serum levels of thyroid hormones. Thyrotropin level was very high (>50
microU/ml; normal value 0.2-4 microU/ml), while serum free T(3) (FT3) and free
T(4) (FT4) levels were normal (FT3 3.6 pg/ml, normal value 2.8-5.6 pg/ml; FT4
11.6 pg/ml, normal value 6.6-14 pg/ml); antithyroid autoantibodies were absent.
Thyroid scintigraphy with sodium 99m Tc pertechnetate showed a small ectopic
thyroid located in sublingual position, so treatment with L-thyroxine 37.5
microg/24 hr was started with rapid improvement of the clinical picture. At 17
months of age the patient developed the complete characteristic phenotype of
Williams syndrome (WS); the clinical diagnosis was proven by fluorescent in situ
hybridization (FISH) analysis which showed hemizygous deletion of the elastin
gene on chromosome 7. Recently a case of thyroid hemiagenesis in a child with WS
has been reported; our patient underscores the association of hypothyroidism and
WS. Moreover, our case shows that clinical manifestations of hypothyroidism may
be present and the treatment may be necessary as it is in isolated congenital
hypothyroidism. Pre- and postnatal growth retardation of unknown pathogenesis is a common
clinical feature in patients with Williams-Beuren syndrome (WBS). However,
growth hormone deficiency (GHD) has not been considered a major cause of growth
retardation. There is only one patient in the literature with confirmed GHD who
responded well to human growth hormone (hGH) therapy. We report a female infant
with confirmed WBS who, through provocative testing, was found to have GHD and
who responded satisfactorily to hGH therapy. Height SDS was -4.2 at the age of
12 months when hGH was initiated and increased to -0.8 at the age of 4.25 years.
The pathogenesis of GHD in our patient is unclear. Nevertheless, the elevated
levels of prolactin and the response of hGH to growth hormone releasing hormone
(GHRH) administration are indicative of a hypothalamic rather than pituitary
defect. In conclusion, GH deficiency might contribute to the growth failure in a
number of patients with WBS and in such cases hGH therapy will most likely
improve final height. OBJECTIVE: To evaluate the prevalence of abnormalities of thyroid function and
morphology in a cohort of patients with Williams syndrome (WS).
METHODS: Serum concentrations of free-T3, free-T4, TSH, thyroperoxidase
antibodies (TPOA) and thyroglobulin antibodies (TgA), as well as
ultrasonographic data, of 20 patients with WS (12 females and eight males), aged
1.7-34.9 years, were evaluated.
RESULTS: Three cases (15%) of subclinical hypothyroidism were identified. Overt
hypothyroidism was diagnosed in two cases (10%). Thyroid antibodies were
negative in all patients. Fourteen patients (70%) showed thyroid hypoplasia
involving the entire gland. In these patients, the left thyroid lobe appeared
usually, but not significantly, reduced compared with the right thyroid lobe.
One patient (5%) showed thyroid hemiagenesis. Only five patients (25%) showed a
thyroid with normal volume, and of these five, one patient showed marked thyroid
hypoplasia of the left lobe. In all WS patients with diagnosis of subclinical or
overt hypothyroidism, thyroid hypoplasia was detected. No cases of subclinical
or overt hypothyroidism were found in WS with normal thyroid volume.
CONCLUSIONS: This study confirms the presence of alterations of thyroid function
in WS and also suggests the frequent occurrence of abnormalities of thyroid
morphology in these patients. Patients with WS should be monitored for thyroid
function and a thyroid ultrasound screening should be considered, especially in
those patients with changes in thyroid function. Thyroid involvement in Williams syndrome (WS) was recently reported in two small
groups of patients, both showing an increased prevalence of elevation of TSH
serum concentration; in one of the two reports, 70% of the patients demonstrated
a hypoplasia of thyroid gland as well. In our institution, we currently follow a
large population of WS patients who periodically undergo a multispecialist
clinical evaluation that includes ultrasound evaluation of the thyroid gland,
and levels of FT3, FT4, TSH, and anti-thyroid antibodies. Here, we report on the
prevalence of thyroid structural and functional anomalies, in a population of 95
WS patients, half of them followed for more than 5 years. Our study confirms the
increased incidence of both elevated TSH serum values (37.9% in our sample) and
thyroid gland hypoplasia (74.7%). Moreover, we demonstrated that TSH elevation
declines with age. For this reason, we suggest that a complete thyroid
evaluation be performed in every patient with WS, and that this medical
complication should be periodically searched for in follow-up visits. OBJECTIVE: To verify the prevalence of morpho-volumetric and functional thyroid
abnormalities in young patients with Williams syndrome (WS).
STUDY DESIGN: Ninety-two patients with WS (49 boys and 43 girls, 0.2-17.2 years
of age) underwent evaluation of thyroid function by means of thyroid-stimulating
hormone (TSH), fT3, and fT4 measurement. Thyroid ultrasonography was performed
in 37 patients. Thyroid antibodies (thyroid peroxidase and thyroglobulin) were
measured in all patients with abnormal thyroid function tests.
RESULTS: None of our patients had overt hypothyroidism; 29 patients (31.5%) had
subclinical hypothyroidism. Thyroid antibodies were absent in all patients. The
prevalence of patients with subclinical hypothyroidism was significantly higher
in the younger patients. Ultrasonography revealed morphological or volumetric
abnormalities of the thyroid gland in 67.5% of patients; these abnormalities
were more frequently observed in the older children.
CONCLUSIONS: Subclinical hypothyroidism is a frequent but stable finding in
young children with WS. The great majority of patients with WS >10 years, either
with normal or hypoplastic thyroid, have normal thyroid function. Therefore, we
suggest yearly monitoring of thyroid function and sonographic studies at least
once in patients with WS. Treatment should be reserved for the patients with
overt hypothyroidism or for those whose thyroid function shows signs of
progressive deterioration. In the Williams-Beuren syndrome (WBS), disorders of the thyroid function and
morphology have been reported and programs of thyroid screening and surveillance
are recommended. However, the frequency of biochemical thyroid assessment,
particularly in the first year of life, is being debated. In this report we
describe an infant with WBS and congenital hypothyroidism, due to an important
thyroid hypoplasia. The patient, a 1-month-old female, negative at primary
neonatal thyroid screening, was referred to our hospital for dyspnea. Thyroid
function tests showed a raised TSH (42 mIU/l; normal range 0.5-4 mIU/l) with a
low FT(4) concentration (10.21 pmol/l; normal range: 10.29-24.45 pmol/l).
Ultrasound examination of the neck showed a significant thyroid hypoplasia,
whereas (99m)Tc-pertechnetate thyroid scintigraphy evidenced a thyroid gland in
normal position, with reduced shape and overall weak fixation. Therefore,
treatment with L-thyroxinewas started. Thyroid hypoplasia is a frequent
characteristic of WBS and abnormalities of thyroid function are common in
patients with this feature. Therefore, the possibility of congenital
hypothyroidism should always be taken into consideration too and, even if
congenital hypothyroidism neonatal screening is negative, thyroid (morphology
and function) evaluation should be regularly assessed when the diagnosis is made
and, thereafter, every year in the first years of life. The molecular and neural mechanisms regulating human social-emotional behaviors
are fundamentally important but largely unknown; unraveling these requires a
genetic systems neuroscience analysis of human models. Williams Syndrome (WS), a
condition caused by deletion of ~28 genes, is associated with a gregarious
personality, strong drive to approach strangers, difficult peer interactions,
and attraction to music. WS provides a unique opportunity to identify endogenous
human gene-behavior mechanisms. Social neuropeptides including oxytocin (OT) and
arginine vasopressin (AVP) regulate reproductive and social behaviors in
mammals, and we reasoned that these might mediate the features of WS. Here we
established blood levels of OT and AVP in WS and controls at baseline, and at
multiple timepoints following a positive emotional intervention (music), and a
negative physical stressor (cold). We also related these levels to standardized
indices of social behavior. Results revealed significantly higher median levels
of OT in WS versus controls at baseline, with a less marked increase in AVP.
Further, in WS, OT and AVP increased in response to music and to cold, with
greater variability and an amplified peak release compared to controls. In WS,
baseline OT but not AVP, was correlated positively with approach, but negatively
with adaptive social behaviors. These results indicate that WS deleted genes
perturb hypothalamic-pituitary release not only of OT but also of AVP,
implicating more complex neuropeptide circuitry for WS features and providing
evidence for their roles in endogenous regulation of human social behavior. The
data suggest a possible biological basis for amygdalar involvement, for
increased anxiety, and for the paradox of increased approach but poor social
relationships in WS. They also offer insight for translating genetic and
neuroendocrine knowledge into treatments for disorders of social behavior. Williams syndrome (WS) is a neurodevelopmental genetic disorder associated with
high rates of anxiety and social issues. We examined diurnal cortisol, a
biomarker of the stress response, in adults with WS in novel and familiar
settings, and compared these profiles to typically developing (TD) adults. WS
and TD participants had similar profiles in a familiar setting, while
participants with WS had elevated cortisol late in the day in the novel setting
when social demands were higher. The cortisol awakening response in WS was
associated with parent-reported levels of somatic complaints and social
difficulties. Results suggest that adults with WS have a typical diurnal
cortisol profile that may be sensitive to social and activity transitions
throughout the day. |
What is the treatment of choice for gastric lymphoma? | The treatment of choice for localized primary GI lymphoma is controversial. Complete surgical resection may increase the chance of complete remission, but mortality and relapse rates might be higher than those observed with combination chemotherapy alone.
In early stages of disease, H. pylori eradication alone may lead to complete lymphoma remission in up to 75% of cases. Nonresponder or locally advanced lymphoma should be treated with radiation therapy. | A review of 52 patients with gastric lymphoma at the Texas A&M University
College of Medicine Affiliated Scott and White Memorial Hospital (Temple, TX).
was performed to determine the influence of different treatment modalities.
Thirty-one patients had a potentially curative resection, while 21 underwent a
palliative procedure or biopsy alone. Overall 5-year survival was 73.4 per cent
after curative resection and 38.3 per cent for lesser operative procedures (P
less than .005). Adjuvant radiation was given to 14 patients after curative
resection with a 5-year survival rate of 71.5 per cent compared to 82.4 per cent
in the 17 patients treated by curative resection alone (nonsignificant).
Patients who underwent palliative surgery or biopsy who received postoperative
radiation therapy had a 38.0 per cent 5-year survival rate compared to a 0.0 per
cent 5-year survival rate in patients who received no therapy (P = .18). The
authors conclude that curative resection is the treatment of choice for gastric
lymphoma, but radiation therapy may offer some benefit when complete resection
is not feasible. Eighteen patients with primary gastric lymphomas and two with pseudolymphomas
treated at the University of Louisville affiliated hospitals were analyzed in
order to develop a more precise understanding of these rare diseases. Abdominal
pain and weight loss were the most common initial symptoms. Only one patient
presented with an abdominal mass. Upper GI series were helpful but failed to
show a definite abnormality in two of 18 cases. Endoscopic examinations in all
18 were compatible with maligcy on gross finding, but six out of 15
endoscopic biopsies were not conclusive. All four cases, which proved fatal in
less than two years, showed serosal invasion and diffuse histological pattern.
On the basis of our analysis, we suggest that in patients with abdominal pain
and weight loss of more than two months duration an aggressive course of
evaluation should include upper gastrointestinal x ray and repeated endoscopic
biopsy. If symptoms persist, laparotomy and biopsy may be warranted even if
endoscopic biopsy shows no neoplasm. Curative surgery is the treatment of
choice, but radiation therapy should be added in patients with serosal
involvement. Very careful histological assessment of pseudolymphomas is
necessary, because they may contain maligt lymphoma. The treatment of primary gastric lymphoma is controversial. The role of surgery
has come to be questioned with increasing knowledge about the pathogenesis of
gastric lymphoma and with new therapeutic approaches such as eradication of
Helicobacter pylori. We review published clinical trials of primary gastric
lymphoma, including preliminary results of our own prospective multicenter
trial. The results of 7 trials of H. pylori eradication and 12 prospective
therapeutic trials trial are discussed. On basis of these data it is concluded
that surgery with intention of R0 resection is the treatment of choice in stages
EI2 and EII1 of low-grade lymphoma. In high-grade lymphomas it is still unclear
whether surgery or its primary combination with radio- or chemotherapy should be
preferred. The eradication of H. pylori is a promising therapeutic approach for
localized low-grade mucosa-associated lymphoid tissue lymphoma. A randomized
trial is needed to clarify whether medical or surgical management of localized
gastric lymphoma or a combination of two is the best treatment modality. BACKGROUND & AIMS: Appropriate management of primary gastric lymphoma is
controversial. This prospective, multicenter study aimed to evaluate the
accuracy of endoscopic biopsy diagnosis and clinical staging procedures and
assess a treatment strategy based on Helicobacter pylori status and tumor stage
and grade.
METHODS: Of 266 patients with primary gastric B-cell lymphoma, 236 with stages
EI (n = 151) or EII (n = 85) were included in an intention-to-treat analysis.
Patients with H. pylori-positive stage EI low-grade lymphoma underwent
eradication therapy. Nonresponders and patients with stage EII low-grade
lymphoma underwent gastric surgery. Depending on the residual tumor status and
predefined risk factors, patients received either radiotherapy or no further
treatment. Patients with high-grade lymphoma underwent surgery and chemotherapy
at stages EI/EII, complemented by radiation in case of incomplete resection.
RESULTS: Endoscopic-bioptic typing and grading and clinical staging were
accurate to 73% and 70%, respectively, based on the histopathology of resected
specimens. The overall 2-year survival rates for low-grade lymphoma did not
differ in the risk-adjusted treatment groups, ranging from 89% to 96%. In
high-grade lymphoma, patients with complete resection or microscopic tumor
residuals had significantly better survival rates (88% for EI and 83% for EII)
than those with macroscopic tumor residues (53%; P < 0.001).
CONCLUSIONS: There is a considerable need for improvement in clinical diagnostic
and staging procedures, especially with a view toward nonsurgical treatment.
With the exception of eradication therapy in H. pylori-positive low-grade
lymphoma of stage EI and the subgroup of locally advanced high-grade lymphoma,
resection remains the treatment of choice. However, because there is an
increasing trend toward stomach-conserving therapy, a randomized trial comparing
cure of disease and quality of life with surgical and conservative treatment is
needed. Controversy remains regarding the best treatment for primary gastric lymphoma
(PGL). Recent developments in diagnosis and chemotherapy have changed strategies
for this disease. Fourteen patients with primary gastric non-Hodgkin's lymphoma
underwent surgery. Before surgery 9/14 patients underwent Helicobacter pylori
eradication, and 4/14 were treated with chemotherapy. In two patients
chemotherapy was not possible because of risk of perforation recurred. Total
gastrectomy with N2 lymphadenectomy, splenectomy, biopsy of mesenteric lymph
nodes, and hepatic biopsy were done. Then patients underwent post-operative
chemotherapy. Involved-field radiation therapy was made in four patients. The
overall survival was 64.2 percent. Surgery was the treatment of choice in cases
of gastric lymphoma non-responsive to medical therapy and to control
complications or when gastroscopy did not supply correct diagnosis. OBJECTIVE: We began a controlled clinical trial to assess efficacy and toxicity
of surgery (S), surgery + radiotherapy (SRT), surgery + chemotherapy (SCT), and
chemotherapy (CT) in the treatment of primary gastric diffuse large cell
lymphoma in early stages: IE and II1.
SUMMARY BACKGROUND DATA: Management of primary gastric lymphoma remains
controversial. No controlled clinical trials have evaluated the different
therapeutic schedules, and prognostic factors have not been identified in a
uniform population.
PATIENTS AND METHODS: Five hundred eighty-nine patients were randomized to be
treated with S (148 patients), SR (138 patients), SCT (153 patients), and CT
(150 patients). Radiotherapy was delivered at doses of 40 Gy; chemotherapy was
CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) at standard
doses. International Prognostic Index (IPI) and modified IPI (MIPI) were
assessed to determine outcome.
RESULTS: Complete response rates were similar in the 4 arms. Actuarial curves at
10 years of event-free survival (EFS) were as follows: S: 28% (95% confidence
interval [CI], 22% to 41%); SRT: 23% (95% CI, 16% to 29%); that were
statistically significant when compared with SCT: 82% (95% CI, 73% to 89%); and
CT: 92% (95% CI, 84% to 99%) (P < 0.001). Actuarial curves at 10 years showed
that overall survivals (OS) were as follows: S: 54% (95% CI, 46% to 64%); SRT:
53% (95% CI, 45% to 68%); that were statistically significant to SCT: 91% (95%
CI, 85% to 99%); CT: 96% (95% CI, 90% to 103%)(P < 0.001). Late toxicity was
more frequent and severe in patients who undergoing surgery. IPI and MIPI were
not useful in determining outcome and multivariate analysis failed to identify
other prognostic factors.
CONCLUSION: In patients with primary gastric diffuse large cell lymphoma and
aggressive histology, diffuse large cell lymphoma in early stage SCT achieved
good results, but surgery was associated with some cases of lethal
complications. Thus it appears that CT should be considered the treatment of
choice in this patient setting. Current clinical classifications of risk are not
useful in defining treatment. Gastric lymphoma and gastrointestinal stromal tumours (GISTs) are rare
maligcies of the upper gastrointestinal tract. The most common gastric
lymphoma are low-grade marginal zone B-cell lymphoma (MZBCL) of MALT type. They
develop as a consequence of chronic Helicobacter pylori infection, the
histological hallmark are lymphoepithelial lesions. In early stages of disease,
H. pylori eradication alone may lead to complete lymphoma remission in up to 75%
of cases. Nonresponder or locally advanced lymphoma should be treated with
radiation therapy. Advanced lymphoma may be treated with the nucleoside analogon
cladribine within clinical trials. Based on clinical and novel molecular markers
a risk stratification and a prediction of response to therapy might be possible
in the future. GISTs are mesenchymal tumours that characteristically express
CD-117 (c-kit). They are mostly localized in the upper gastrointestinal tract
and are frequently diagnosed in an advanced stage. Conventional chemotherapy is
ineffective. For resectable non-metastasized tumours surgical therapy is the
treatment of choice. Imatinib is the first and so far only effective systemic
therapy which is presently indicated in irresectable or metastasized GISTs. More
than 80% of patients respond to imatinib therapy either with partial remission
or stable disease. FDG-PET plays an important role in the early prediction of
response to imatinib therapy. The optimal dosage and duration of treatment and
the role of imatinib as adjuvant or neo-adjuvant therapy for GISTs remains to be
defined. Clinicopathologic information of gastrointestinal (GI) lymphoma in Southeast
Asia is lacking. A retrospective analysis of 120 cases of GI lymphoma in
Thailand diagnosed at Siriraj Hospital based on WHO classification was
performed. All were non-Hodgkin lymphoma (NHL). The peak age was in the sixth
and seventh decades; a slight male preponderance was observed. Sites of
involvement included stomach (49.2%), intestine (46.7%), and multiple sites
(4.2%). There were 104 cases of primary GI lymphoma (86.7%) and 16 cases of
secondary GI lymphoma (13.3%). Presenting GI symptoms were more common in the
former; while superficial lymphadenopathy and fever were more common in the
latter. Mass lesions were observed in both groups (72.1% vs 56.3%). Localized
and advanced diseases were found in 68.3% and 31.7% of primary GI lymphomas,
respectively. The most common type of lymphoma in both groups was diffuse large
B-cell lymphoma. Lymphoepithelial lesions (LEL) were not significantly different
between the two groups (58.2% vs 42.9%), but Helicobacterpylori infection was
significantly associated with primary gastric lymphoma (p < 0.0001). The
treatment of choice for localized primary GI lymphoma is controversial. Complete
surgical resection may increase the chance of complete remission, but mortality
and relapse rates might be higher than those observed with combination
chemotherapy alone. GI lymphomas in Thailand are mostly primary B-cell NHL. LEL
is not indicative of primary GI lymphoma, but H. pylori infection is closely
associated with primary gastric lymphoma. A prospective study to determine the
treatment of choice for localized GI lymphoma is needed. OBJECTIVE: To study the clinical diagnosis, treatment and prognosis of primary
gastric lymphoma (PGL).
METHODS: Clinical data of 200 patients with PGL who were treated at the Tianjin
Cancer Hospital were retrospectively reviewed.
RESULTS: There were 110 males and 90 females. The age ranged between 19 and 80
(median, 54) years old. Symptoms or laboratory examinations were not specific.
Before the treatment, 130 cases were diagnosed correctly, and the remaining 70
were misdiagnosed as gastric cancer (n=59), gastric ulcer (n=5) and chronic
gastritis (n=6). Twenty-four cases received chemotherapy alone, 29 cases surgery
alone, 132 cases underwent surgery plus chemotherapy, and 15 were not treated.
The 200 cases all received follow-up ranging from 1 to 246 months (median, 26
months). The 1, 3, 5, and 10-year survival rates were 65.0%, 57.5%, 56.2%, and
55.0%. Univariate analysis and multivariate analysis showed that emaciation
(P<0.01), tumor location (P<0.01), treatment modality (P<0.01), histological
type (P<0.05), and tumor staging (P<0.05) were independent factors of survival
in patients with PGL.
CONCLUSIONS: The misdiagnosis of PGL is very frequent. The treatment of choice
should be comprehensive therapy based on surgery. |
Which assays can be used for detecting DNA fragmentation resulting from programmed cell death (apoptosis)? | The biochemical hallmark of apoptosis is internucleosomal DNA cleavage into oligonucleosome-length fragments. Measurement of apoptosis-associated internucleosomal DNA fragmentation through determination of the percentage of fragmented DNA by electrophoresis or centrifugation of whole cell lysates is by far the most common means of quantifying apoptosis. DNA fragmentation due to apoptosis can also be identified using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragments (TUNEL), in situ end labeling (ISEL) of the genomic DNA in fragmented nuclei, and measurement of cytosolic histone-bound DNA fragments (cell death ELISA assays). | The ability of glucocorticoids to induce apoptosis or programmed cell death in
mouse thymocytes is well-established. Measurement of apoptosis-associated
internucleosomal DNA fragmentation through determination of the percentage of
fragmented DNA by electrophoresis or centrifugation of whole cell lysates is by
far the most common means of quantifying apoptosis. Since these methods measure
DNA fragmentation in whole cell lysates rather than intact cells, they have
severe limitations, particularly with heterogeneous cell populations. When mouse
thymocytes were incubated with glucocorticoids, fixed, stained with propidium
iodide and analysed flow cytometrically for cell cycle distribution, a distinct
subpopulation of cells was observed to form below the G0/G1 region, denoted as
the A0 region. The presence of cells in this region was consistent with the
presence of internucleosomal DNA fragments as determined by gel electrophoresis.
Inhibitors of transcription, translation and endonuclease activity, and a
glucocorticoid receptor antagonist prevented accumulation of cells in this
region. Irradiation of mouse thymocytes also produced a population in the A0
region. Cells in this region are believed to have undergone
glucocorticoid-induced DNA fragmentation. This method represents a useful
alternative to whole cell lysate assays, since apoptosis can be evaluated on an
individual cell basis. BACKGROUND: Programmed cell death is an essential event during mammalian
morphogenesis which eliminates unnecessary cells to accomplish histogenesis and
organogenesis. Cell death in interdigital spaces of the developing limb is a
classical example of morphogenetic cell death. We investigated whether classical
programmed cell death in the interdigital tissue of the developing limb in mice
is apoptosis with fragmentation of nuclear DNA and also examined sequentially
the occurrence of programmed cell death and cell proliferation in the developing
limb of mouse fetuses to analyze their interrelation.
METHODS: We examined the occurrence of apoptotic cell death in the developing
limbs of mouse fetuses by using Nile blue sulphate staining, agarose gel
electrophoresis for detecting DNA laddering, and a cytochemical labeling of DNA
fragmentation. We also labeled proliferating cells using BrdU/anti-BrdU
immunohistochemistry and examined the interrelation between apoptotic programmed
cell death and cell proliferation.
RESULTS: DNA ladders, a biochemical evidence of apoptosis, were detected in DNA
extracts from the interdigital tissue of day 13 mouse fetuses by agarose gel
electrophoresis. Programmed cell death and DNA fragmentation were detected by
Nile blue staining and cytochemical labeling of DNA fragmentation, respectively,
in the interdigital mesoderm and in the regions of presumptive joints of the
digit. BrdU/anti-BrdU immunohistochemistry for identifying proliferating S-phase
cells revealed that interdigital mesenchymal cells cease DNA synthesis before
programmed cell death and DNA fragmentation begin.
CONCLUSIONS: We confirmed that both cytological apoptotic alterations and
fragmentation of nuclear DNA occur in the interdigital tissue and presumptive
joint areas of fetal mouse limbs, and they appear to play a significant role in
the separation of digits as well as the formation of joint cavities. Ameloblasts responsible for tooth enamel formation are classified into two
different phases: secretion and maturation. At the transition between these
secretion and maturation stages, a considerable number of cells die. In this
study, we examined the morphology of degenerating ameloblasts by conventional
electron microscopy, and DNA cleavage in degenerating ameloblast nuclei by the
in situ terminal transferase assay. The results suggest that apoptosis
(programmed cell death) in ameloblasts, including DNA ligation is induced at the
transitional stage. The nuclear fragments, chromatin condensation and DNA
relocation in apoptotic nuclei were examined quantitatively by post-embedding
anti-DNA immunogold electron microscopy and the in situ terminal transferase
assay combined with electron microscopy. Numerical analysis revealed that
immunogold labeling density in the condensed chromatin of apoptotic nuclei was
comparable on the average to that in the perinuclear heterochromatin of normal
nuclei, and that individual apoptotic nuclear fragments exhibited highly
variable to that of normal heterochromatin, to fragments with densities twice as
high as that of normal heterochromatin. The in situ terminal transferase assay
combined with electron microscopy detected DNA ends exposed by ultrathin
sectioning as well as DNA cleavage by a putative endonuclease. In conclusion,
the state of the DNA, including its ligation and degeneration, changes gradually
during chromatin condensation and nuclear fragmentation of apoptosis. Exposure to silica dust can result in lung inflammation that may progress to
fibrosis, for which there is no effective clinical treatment. The mechanisms
involved in the development of pulmonary silicosis have not been well defined;
however, most current evidence implicates a central role for alveolar
macrophages (AM) in this process. We propose that the fibrotic potential of a
particulate depends upon its ability to cause apoptosis in AM. In this study,
human AM were treated with fibrogenic, poorly fibrogenic, and nonfibrogenic
model particulates, such as silica (133 micrograms/ml), amorphous silica (80
micrograms/ml), and titanium dioxide (60 micrograms/ml), respectively. Cell were
treated with these particulates in vitro for 6 and 24 hr and examined for
apoptosis by morphological analysis, DNA fragmentation, and levels of cytosolic
histone-bound DNA fragments (cell death ELISA assays). Treatment with silica
resulted in morphological changes typical of apoptotic cells, enhanced DNA
fragmentation (a characteristic feature of programmed cell death), and
significant alveolar macrophage apoptosis as observed by cell death ELISA
assays. In contrast, amorphous silica and titanium dioxide demonstrated no
significant apoptotic potential. To elucidate the possible mechanism by which
silica causes apoptosis, we investigated the role of the scavenger receptor (SR)
in silica-induced apoptosis. Cells were pretreated with and without SR ligand
binding inhibitor, polyinosinic acid (poly(I), 500 micrograms/ml), for 10 min
prior to silica treatment. Pretreatment with poly(I) resulted in complete
inhibition of silica-induced apoptosis as measured by cell death ELISA. Further,
we examined the involvement of interleukin-converting enzyme (ICE) in
silica-mediated apoptosis using an ICE inhibitor, Z-Val-Ala-Asp-fluoromethyl
ketone. Z-Val-Ala-Asp-fluoromethyl ketone inhibited silica-induced apoptosis and
IL-1 beta release. These results suggest that fibrogenic particulates, such as
silica, caused apoptosis of alveolar macrophages and that this apoptotic
potential of fibrogenic particulates may be a critical factor in initiating an
inflammatory response resulting in fibrosis. Additionally, silica-induced
apoptosis of alveolar macrophages may be due to the interaction of silica
particulates with the SR, initiating one or a number of signaling pathways
involving ICE, ultimately leading to apoptosis. BACKGROUND: Infection with Helicobacter pylori induces chronic gastritis in
virtually all infected persons, and such gastritis has been associated with an
increased risk of developing gastric cancer. This risk is further enhanced with
cagA+ (positive for cytotoxin-associated gene A) H. pylori strains and may be a
consequence of induced gastric cell proliferation and/or alteration in apoptosis
(programmed cell death) in the gastric epithelium.
PURPOSE: To determine whether the H. pylori cagA genotype and another
virulence-related characteristic, the vacA (vacuolating cytotoxin A) s1a
genotype, differentially affect epithelial cell proliferation, apoptosis, and
the histologic parameters of inflammation and injury, we quantitated these
characteristics in infected and uninfected persons.
METHODS: Fifty patients underwent upper gastrointestinal endoscopy, and biopsy
specimens were taken. Apoptotic cells in the specimens were quantitated after
terminal deoxynucleotidyl transferase labeling of DNA fragments with
digoxigenin-deoxyuridine triphosphate; epithelial cell proliferation was scored
by immunohistochemical analysis of the proliferation-associated antigen Ki-67.
Antibodies directed against H. pylori and CagA protein were measured in the
serum of patients by means of enzyme-linked immunosorbent assays. Analysis of H.
pylori genomic DNA, by use of the polymerase chain reaction, was performed to
determine the cagA and vacA genotypes. Acute and chronic inflammation,
epithelial cell degeneration, mucin depletion, intestinal metaplasia, glandular
atrophy, and vacuolation were each scored in a blinded manner. Reported P values
are two-sided.
RESULTS: Persons harboring cagA+ strains (n = 20) had significantly higher
gastric epithelial proliferation scores than persons infected with cagA-strains
(n = 9) or uninfected persons (n = 21) (P = .025 and P<.001, respectively), but
the difference in cell proliferation between the latter two groups was not
statistically significant. The number of apoptotic cells per 100 epithelial
cells (apoptotic index) in persons infected with cagA+ strains was lower than in
persons infected with cagA-strains (P = .05). Apoptotic indices in the cagA+
group were similar to those in the uninfected group (P = .2). Epithelial cell
proliferation was significantly correlated with acute gastric inflammation, but
only in the cagA+ group (r = .44; P = .006). The cagA+ and vacA s1a genotypes
were found to be concordant, confirming the close relationship between these
virulence-related genotypes.
CONCLUSIONS: Gastric mucosal proliferation was significantly correlated with the
severity of acute gastritis in persons infected with cagA+ vacA s1a strains of
H. pylori. This increased proliferation was not accompanied by a parallel
increase in apoptosis.
IMPLICATIONS: Increased cell proliferation in the absence of a corresponding
increase in apoptosis may explain the heightened risk for gastric carcinoma that
is associated with infection by cagA+ vacA s1a strains of H. pylori. Apoptosis, or programmed cell death, is a physiological form of cell death that
plays a critical role in the development and maintece of multicellular
organisms. Apoptosis is characterized based on morphological and biochemical
criteria. Morphological characteristics include cell shrinkage, cytoplasmic
condensation, chromatin segregation and condensation, membrane blebbing, and the
formation of membrane-bound apoptotic bodies, whereas the biochemical hallmark
of apoptosis is internucleosomal DNA cleavage into oligonucleosome-length
fragments. A great deal of research is aimed at defining the molecular
mechanisms that play a role in apoptosis. As one of the common end points of
experiments related to apoptosis is in fact the death of the cell, it has become
important to develop reliable assays to measure cell death that may be compared
among the various systems being investigated. This chapter reviews many of the
current methods used to measure apoptotic cell death and points out strengths
and weaknesses of each approach with respect to the system being examined and
the questions being asked. Traditional cell-based methods, including light and
electron microscopy, vital dyes, and nuclear stains, are described. Biochemical
methods such as DNA laddering, lactate dehydrogenase enzyme release, and MTT/XTT
enzyme activity are described as well. Additionally, terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end labeling of DNA fragments (TUNEL) and
in situ end labeling (ISEL) techniques are reviewed, which when used in
conjunction with standard flow cytometric staining methods may yield informative
data relating cell death to various cellular parameters, including cell cycle
and cell phenotype. The use of one or more of the methods described in this
chapter for measuring cell death should enable investigators to accurately
assess apoptosis in the context of the various models being examined and help
define causal relationships between the mechanisms that regulate apoptosis and
the cell death event itself. The embryonic outflow tract is a simple tubular structure that connects the
single primitive ventricle with the aortic sac and aortic arch arteries. This
structure undergoes a complex sequence of morphogenetic processes to become the
portion of the heart that aligns the right and left ventricles with the
pulmonary artery and aorta. Abnormalities of the outflow tract are involved in
many clinically significant congenital cardiac defects; however, the cellular
and molecular processes governing the development of this important structure
are incompletely understood. Histologic and tissue-tagging studies indicate that
the outflow tract tissues compact and are incorporated predomitly into a
region of the right ventricle. The hypothesis tested in the current study was
that cell death or apoptosis in the muscular portion of the outflow tract is an
important cellular mechanism for outflow tract shortening. The tubular outflow
tract myocardium was specifically marked by infecting myocytes of the chicken
embryo heart with a recombit replication-defective adenovirus expressing
beta-galactosidase (beta-gal) under the control of the cytomegalovirus promoter.
Histochemical detection of the beta -gal-labeled outflow tract myocytes revealed
that the tubular structure shortened to become a compact ring at the level of
the pulmonic infundibulum over several days of development (stages 25-32,
embryonic days 4-8). The appearance of apoptotic cardiomyocytes was correlated
with OFT shortening by two histologic assays, TUNEL labeling of DNA fragments
and AnnexinV binding. The rise and fall in the number of apoptotic myocytes
detected by histologic analyses paralleled the change in activity levels of
Caspase-3, a protease in the apoptotic cascade, measured in outflow tract
homogenates. These results suggest that the elimination of myocytes by
programmed cell death is one mechanism by which the outflow tract myocardium
remodels to form the proper connection between the ventricular chambers and the
appropriate arterial trunks. Apoptosis is an organized, energy dependent process, which leads to cell death.
Its definition is based on distinct morphological features [10] and
demonstration of internucleosomal DNA degradation [27], executed by selectively
activated DNAses [4, 22]. The morphologic hallmarks of apoptosis include
chromatic margination, nuclear condensation and fragmentation, and condensation
of the cell with preservation of organelles. The process is followed by
fragmentation of the cell into membrane-bound apoptotic bodies, which undergo
phagocytosis by nearby cells without associated inflammation [10, 11]. Apoptosis
characteristically occurs in insolated single cells. The duration of apoptosis
is estimated to be from 12 to 24 hours, but in cell culture visible morphologic
changes are accomplished in less than two hours [10, 16]. Non-apoptotic cell
death, a prototype of which is cell death due to ischemia (oncosis), is
characterized by depletion of intracellular ATP stores, swelling of the cell
with disruption of organelles and rupture of the plasma membrane [15]. Groups of
necrotic cells and inflammation are found in tissues [10, 15]. The significance
of apoptosis has mostly been studied using the TUNEL assay that detects DNA
strand breaks in tissue sections and allows quantification of apoptotic cells by
light microscopy [6]. Common experience seems to be that the TUNEL assay is
prone to false positive or negative findings. This has been explained by the
dependence of the staining kinetics on the reagent concentration [17], fixation
of the tissue [2] and the extent of proteolysis [17]. Active RNA synthesis [12]
and DNA damage in necrotic cells [17, 19] may cause non-specific staining. To
obtain reliable and reproducible results, TUNEL assay should be carefully
standardized by using tissue sections treated with DNAse (positive control of
apoptosis). Quantification of apoptosis should include enough microscopic fields
and identification of the cell type undergoing apoptosis. The specificity of the
results can be substantiated by combining other methods with TUNEL, such as
assessment of the pattern of DNA fragmentation or evaluation of the
morphological features. Even though there is high variation in the results
obtained in consecutive studies under the same circumstances, increasing
evidence shows that TUNEL-positive cardiomyocytes and internucleosomal DNA
fragmentation are associated with various cardiac diseases, including acute
myocardial infarction and heart failure [reviewed in 5, 9]. Some morphological
features of apoptosis have been observed in TUNEL-positive cardiomyocytes using
light microscopy (Figure 1) or confocal microscopy [20]. Electron microscopic
evidence of apoptosis has been found in the degenerating conduction system [7],
in experimental heart failure [23], and in human hibernating myocardium [3]. In
acutely ischemic myocardium the interpretation of the findings remains
controversial, since only non-apoptotic cell morphology has been found in
electron microscopy [8, 19]. One explanation might be abortion of the apoptotic
program due to the lack of ATP before the morphologic features are fully evident
[14]. Another explanation is the possibility that non-apoptotic cell death and
apoptosis share common mechanisms in the early phases of the processes [14, 19].
The exact mechanisms of ischemic cell death remain to be clarified and the
classification between apoptosis and non-apoptosis cell death to be specified.
Recently, caspase activation has emerged as the central molecular event leading
to apoptosis, preceding DNA degradation and the development of apoptotic
morphology [22, 25]. New methods have been developed to demonstrate caspase
activation [1, 13]. Inhibition of caspase may be an efficient way to prevent
apoptotic cardiomyocyte death as well as to define and specifically probe the
significance of apoptotic cell death in cardiac diseases. The purpose of this experimental work was to investigate whether apoptosis
contributes to tissue remodelling during distraction bone healing. In a rabbit
model of mandibular distraction osteogenesis, we quantitatively analysed the
extent of apoptotic cell death in relation to differently applied mechanical
loadings. Apoptotic cells were identified by means of an in situ detection assay
for nuclear DNA fragmentation using a modified TUNEL procedure and by electron
microscopical examination for typical morphological features of programmed cell
death. TUNEL-positive cells were frequently detected in samples distracted at
higher strain magnitudes. Ultrastructurally, these apoptotic cells displayed a
condensed chromatin and fragmented nuclei, while the continuity of their plasma
membranes remained intact. Our results clearly indicated that the discontinuous
traction of osteotomized mandibles induced enhanced apoptosis. In contrast to
non-distracted samples and mandibles distracted at low strain magnitudes, in
which only minimal evidence of apoptotic cell death was detected, the
application of hyperphysiological strain magnitudes resulted in an increased
apoptosis rate. Thus, mechanical loading seems to be a triggering factor for
apoptotic changes in osteoblastic cells. These findings suggest a
pathophysiological role of apoptotic cell death in the control of tissue
integrity during distraction osteogenesis. Apoptotic cells possess specific morphological and biochemical markers. Various
methods have been developed to detect apoptosis based on these markers. One of
the most common is the fragmentation of genomic DNA. In addition to
electrophoresis for the identification of the characteristic 200 base pair
ladders and terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick
end-labeling, DNA content analysis is often employed. This technique is based on
the fact that permeablized apoptotic cells release fragmented DNA, resulting in
DNA content that is less than that in live diploid cells. Although widely used,
we have found that the number of apoptotic cells detected by DNA content
analysis is often lower than that detected by other methods. We have developed a
simplified version of the flow cytometry-based protocol that detects a number of
apoptotic cells closer to that detected by other methods, and which requires a
dramatically reduced number of cells. In addition, this simplified protocol
allows preparation of a large number of samples at the same time. Apoptosis or programmed cell death is a genetically controlled response of cells
to commit suicide and is associated with DNA fragmentation or laddering. The
common inducers of apoptosis include Ca2+i and oxygen free radicals/oxidative
stress, which are also implicated in the pathogenesis of exercise-induced
myopathies. To examine training-induced apoptosis, Thoroughbred horses were
subjected to 3 months training programme on a treadmill. At the end of the
training programme venous blood samples were taken for a creatine kinase (CK)
assay. In addition, muscle biopsy samples were obtained for a membrane lipid
peroxidation measurement by malondialdehyde (MDA) assay and for apoptosis
detection. Apoptosis was studied by visualising the apoptotic myocytes on the
paraffin sections by the modified TUNEL method. DNA laddering was evaluated by
subjecting the DNA obtained from the biopsies to 1.5% agarose gel
electrophoresis. There was a significant increase (P<0.05) of protein-bound MDA,
and a nonsignificant trend (P = 0.14) for the control group to have higher
levels of CK compared to the trained group. Under light microscopy, percentage
of the TUNEL positive cells was higher (P<0.001) in the training group. This
result was corroborated with the findings of DNA fragmentation by gel
electrophoresis, which showed higher ladders of DNA band at the same group. In
conclusion, these results clearly demonstrate that there is training-induced
apoptosis in skeletal muscle. It is probable that apoptosis allows the
work/recovery/rebound/supercompensation cycle, when unaccustomed muscle cells
activate programmed cell death and are replaced by new and stronger cells, which
is the mechanism for training-induced increases in fitness. The ability of frog virus 3 (FV3), the type species of the family Iridoviridae,
to induce apoptosis was examined by monitoring DNA cleavage, chromatin
condensation, and cell-surface expression of phosphotidylserine (PS) in fathead
minnow (FHM) and baby hamster kidney (BHK) cells. In productively infected FHM
cells, DNA fragmentation was first noted at 6-7 h postinfection and was clearly
seen by 17 h postinfection, while chromatin condensation was detected at 8.5 h
postinfection. As with some other viruses, FV3-induced apoptosis did not require
de novo viral gene expression as both heat-inactivated and UV-inactivated virus
readily triggered DNA fragmentation in FHM cells. Moreover, FV3-induced
apoptosis was blocked in FHM cells by the pan-caspase inhibitor Z-VAD-FMK,
suggesting that virus infection triggers programmed cell death through
activation of the caspase cascade. FV3 infection also triggered apoptosis in BHK
cells as monitored by TUNEL and annexin V binding assays. To determine whether
FV3, similar to other large DNA viruses, encoded proteins that block or delay
apoptosis, mock- and FV3-infected FHM cells were osmotically shocked and assayed
for DNA fragmentation 3 hours later. DNA fragmentation was clearly seen whether
or not shocked cells were previously infected with FV3, indicating that
infection with FV3 did not block apoptosis induced by osmotic shock in FHM
cells. The above results demonstrate that iridoviruses triggered apoptosis and
that the induction of programmed cell death did not require viral gene
expression. However, it remains to be determined if virion attachment to target
cells is sufficient to induce cell death, or if apoptosis is triggered directly
or indirectly by one or more virion-associated proteins. BACKGROUND: Single-cell gel electrophoresis, or the comet assay, a technique
widely used for DNA damage analysis, has been used recently for detecting DNA
fragmentation in cells undergoing apoptosis. However, the number of variants of
this assay used thus far primarily detected the late stages of DNA
fragmentation. Therefore, monitoring the progression of DNA fragmentation, which
could greatly improve the analysis of cell death induction and progression at
the single-cell level, has not been possible with this assay.
METHODS: In the present study, a modification of the original neutral comet
assay developed by Ostling and Johanson (Biochem Biophys Res Commun 123:291-298,
1984) was used to detect various stages of DNA fragmentation. This assay
involves cell lysis with anionic detergents at nearly neutral pH (9.5) and does
not include high salt concentration, unlike most other published methods. BMG-1
human glioma cells were induced to undergo programmed cell death by treating
with a large dose (100 microM) of etoposide, and comets were prepared after
different durations (1-24 h) of treatment.
RESULTS: In contrast to results of previously published studies, comets with
different shapes reflecting progressive stages of DNA fragmentation were
observed. Of these, six distinct shapes were identified and divided into three
different categories based on the extent of fragmentation. Type A comets had a
large head separated by a narrow "neck" region from an oval bulging tail that
indicated initiation of fragmentation. Type B and C comets had a constantly
diminishing head associated with a corresponding expansion of the tail and
reflected intermediate and late stages of fragmentation, respectively. Type A
and B comets appeared at a high frequency during early time points (1-6 h),
whereas type C comets that indicated late stages of fragmentation were observed
only after extended treatment (24 h). As a result, an elaborate kinetics of the
progression of DNA fragmentation could be obtained.
CONCLUSION: The present single-cell gel electrophoresis assay offers a
significant improvement in monitoring the kinetics of DNA fragmentation induced
during programmed cell death. Coupled with its simplicity and the ability to
detect responses of small cell subpopulations, this method can be used for a
reliable and sensitive analysis of the progression of cell death in different
cell types and treatment conditions. Programmed cell death or apoptosis is a physiological process by which
genetically damaged cells or undesired cells can be eliminated. Various
morphological and molecular changes undergoing during the process of apoptosis
are the formation of apoptotic blebs of the cell membrane, cell shrinkage,
condensation of chromatin and the disruption of deoxyribonucleic acid (DNA) into
typical fragments of multiples of 180 base pairs. These changes can be detected
in a number of ways. DNA ladder formation, which is observed following gel
electrophoresis technique although is widely accepted but does not reflect the
DNA breakdown in individual cell and also may miss contributions from small
sub-populations in a heterogeneous cell population. Alkaline comet assay as
measured by single cell gel electrophoresis, on the other hand, accurately
measures DNA fragmentation on a single cell level and allows analysis of
subpopulation of cells. The assay was originally developed for measuring DNA
damage of cells exposed to any genotoxic agent. However, the comet image
generated by an apoptotic cell is different from that obtained with a cell
treated for a short time with a genotoxic agent. Correlation of comet formation
with various other established parameters of apoptosis is very important. The
present study aims to correlate different features of apoptosis with the
formation of comet tail in human leukemia K-562 cells using tea extracts.
Apoptosis as measured by formation of apoptotic bodies, flow cytometric
analysis, activation of caspase 3 and 8, and expressions of apoptosis related
genes such as bcl-2 and bax showed high degree of correlation with comet tail
moment. This indicates that comet assay can accurately reflect measure of DNA
fragmentation and hence can be used to detect a cell undergoing apoptosis. As programmed cell death (PCD), or apoptosis, has emerged as an important
regulator of development and homeostasis in multicellular organisms, methods to
quantify apoptosis and to distinguish it from necrosis have been developed.
Necrosis refers to the morphology usually associated with accidental cell death,
while apoptosis is seen when cell death is programmed or physiologically
regulated. This unit presents a set of assays for these purposes, many of which
are technically very simple. Featured in this unit is the TUNEL method of
detecting cells that exhibit DNA fragmentation, which can also be performed on
tissue sections to locate apoptotic cells in situ. As programmed cell death (PCD) or apoptosis has emerged as an important
regulator of development and homeostasis in multicellular organisms, methods to
quantify apoptosis and to distinguish it from necrosis have been developed. This
unit presents a set of assays for these purposes, many of which are technically
very simple and ideally suited to the study of hematopoietic cells. The first
basic protocol allows the qualitative and quantitative assessment of apoptosis
in lymphocyte cell cultures using light or fluorescent microscopy. Three
protocols follow that are designed to detect nuclear DNA fragmentation and
support protocols describe methods to radiolabel the DNA and cytoplasm of the
cells to be tested. Techniques that quantitate apoptotic cells using flow
cytometry are then described and support protocols provide methods for priming T
cell clones and freshly isolated lymph node cells, respectively, for T cell
receptor (TCR)-induced apoptosis. Quantitative detection of DNA fragmentation in
apoptotic cells is also described. TdT-mediated dUTP-biotin nick end-labeling
(TUNEL) methods are provided for the detection of apoptotic cells, along with
procedures for the flow cytometric quantitation of apoptotic cells using TUNEL,
and TUNEL, staining of tissue sections to identify apoptotic cells. Since much
remains incompletely understood about the molecular pathways of programmed
death, and it is probably best to perform more than one of the basic protocols
to confirm an observation of apoptotic cell death. Apoptosis plays a crucial role in many biological processes and pathogenesis of
various maligcies and diseases of the immune system. In this paper, we
described a novel method for sensitive detection of drug-induced apoptosis by
using fluorescence correlation spectroscopy (FCS). The principle of this method
is based on the assay of DNA fragmentation in the process of the drug-induced
apoptosis. FCS is a single molecule method, and it can be used for sensitive and
selective assay of DNA fragmentation without separation. We first developed a
highly sensitive method for characterization of DNA fragments using a home-built
FCS system and SYBR Green I as fluorescent DNA-intercalating dye, and then
established a model of drug-induced apoptosis using human pancreatic cancer
cells and a drug lidamycin. Furthermore, FCS method established was used to
directly detect the fragmentation of DNA extracted from apoptotic cells or in
the apoptotic cell lysate. In FCS assay, the single-component model and the
multiple-components model were used to fit raw FCS data. The characteristic
diffusion time of DNA fragments was used as an important parameter to
distinguish the apoptotic status of cells. The obtained data documented that the
characteristic diffusion time of DNA fragments from apoptotic cells
significantly decreased with an increase of lidamycin concentration, which
implied that DNA fragmentation occurred in lidamycin-induced apoptosis. The FCS
results are well in line with the data obtained from flow cytometer and gel
electrophoresis. Compared to current methods, the method described here is
sensitive and simple, and more importantly, our detection volume is less than 1
fL, and the sample requirement can easily be reduced to nL level using a
droplets array technology. Therefore, our method probably becomes a high
throughput detection platform for early detection of cell apoptosis and
screening of apoptosis-based anticancer drugs. BACKGROUND AND PURPOSE: The electric field and the concomitant heat
(electrohyperthermia) can synergistically induce cell death in tumor tissue, due
to elevated glycolysis, ion concentration, and permittivity in maligt
compared with nonmaligt tissues. Here we studied the mechanism and time
course of tumor destruction caused by electrohyperthermia.
MATERIAL AND METHODS: Bilateral implants of HT29 colorectal cancer in the
femoral regions of Balb/c (nu/nu) mice were treated with a single 30-min shot of
modulated, 13.56-MHz, radiofrequency-generated electrohyperthermia (mEHT).
Tumors at 0, 1, 4, 8, 14, 24, 48, and 72 h posttreatment were studied for
morphology, DNA fragmentation, and cell death response-related protein
expression using tissue microarrays, immunohistochemistry, Western immunoblots,
and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays.
RESULTS: Modulated EHT treatment induced significant tumor destruction in HT29
xenografts with a peak of a sevenfold increase compared with the untreated
controls. The significant treatment-related elevation of DNA
fragmentation--detected with TUNEL assay--and apoptotic bodies between 24 and 72
h posttreatment was proof of a programmed cell death response. This was
associated with significant mitochondrial accumulation of bax and
mitochondrial-to-cytoplasmic release of cytochrome c proteins between 8 and 14
h. Cleaved caspase-3 levels were low and mainly localized to inflammatory cells.
The substantial cytoplasmic-to-nuclear translocation of apoptosis-inducing
factor (AIF) and its 57-kDa activated fragment detected between 14 and 24 h
after treatment indicated AIF as an effector for DNA fragmentation.
CONCLUSION: Modulated EHT treatment can induce programmed cell death-related
tumor destruction in HT29 colorectal adenocarcinoma xenografts, which domitly
follows a caspase-independent subroutine. |
Does triiodothyronine stimulate red blood cell sodium potassium pump? | An inverse correlation between this enzymatic action and free triiodothyronine (FT3) levels.
The effect of triiodothyronine (T3) on Na+,K(+)-ATPase activity in red blood cells may be different in vivo and in vitro. | The effect of triiodothyronine (T3) on Na+,K(+)-ATPase activity of K562 human
erythroleukemic cell was studied to understand why the erythrocyte sodium pump
activity is decreased in hyperthyroidism. Na+,K(+)-ATPase activity of K562 cell
lysates was assayed by measuring the release of inorganic phosphate (Pi) from
ATP. Na+,K(+)-ATPase activity of K562 cell grown in the presence of T3 for 48
hours was significantly higher than that of control (0.98 +/- 0.05 mumol Pi h-1
mg protein-1 vs 0.82 +/- 0.10 mumol Pi h-1 mg protein-1, p < 0.05). The
Na+,K(+)-ATPase activity could be stimulated in a time- and
concentration-dependent manner; maximum stimulatory effect of T3 was seen at a
concentration of 10(-7) mol/L. When an inducer
[cytosine-beta-D-arabino-furanoside (ARA-C)] was added to the culture medium,
the K562 cells showed signs of differentiation and synthesised haemoglobin. At
the same time, the Na+,K(+)-ATPase activity remained high. We conclude that T3
stimulates Na+,K(+)-ATPase activity of K562 cells and in the presence of T3
during differentiation, the enzyme activity remains high. In patients suffering from hyperthyroidism dependent on Graves' disease, a
reduction in Na+,K+ATPase activity has been demonstrated in red blood cells
(RBCs), as well as an inverse correlation between this enzymatic action and free
triiodothyronine (FT3) levels. The restoration of normal FT3 values also brings
about a normalization of Na+,K+ATPase activity in erythrocytes. These results
have made it possible to hypothesize that the thyroid hormones control
Na+,K+ATPase activity and that this control is manifested by means of variations
in the number of ouabain-binding sites. For this reason, the measurement of the
activity of the Na/K pump can be considered as a further indicator of the
peripheral effects of thyroid hormones. With a view to assess the relation
between the course of treated hyperthyroidism and Na+,K+ATPase activity during
antithyroid therapy and after surgical thyroidectomy followed by replacement
therapy, we studied 24 patients affected by Graves' disease (group Graves [GG]).
They were compared with 24 female Graves' patients who underwent total
thyroidectomy for nontoxic and diffuse nodular goiter (NDNG) (group control
[GC]) and with 24 normal healthy women (group normal [GN]). When Graves'
hyperthyroidism was diagnosed, the Na+,K+ATPase activity in RBCs was impaired in
all GG patients. Thionamide treatment restored the normal activity of the Na/K
pump, accompanied by normalization of the number of ouabain-binding sites. One
hundred eighty days after thyroidectomy, in conditions of clinical and
biochemical euthyroidism due to replacement therapy with levothyroxine, the
activity of Na+,K+ATPase in RBCs was once again reduced in GG, while appearing
normal in GC and GN (1.77 +/- 0.16 mmol Pi h(-1) L(-1) RBCs v 2.09 +/- 0.26 v
2.09 +/- 0.24, P < .05). Different instrumental or biochemical parameters, such
as glycemia, serum lipids, ions, serum alkaline phosphatase (AIPh), serum
creatine phosphokinase (CPK), blood pressure, and heart rate, were evaluated and
appeared normalized in GG and GC 180 days after surgery. We conclude that (1) in
patients suffering from Graves' disease, subjected to total thyroidectomy
followed by levothyroxine replacement therapy, there is a reduction in the
activity of the Na+,K+ATPase on erythrocytes 6 months after the surgical
approach; and (2) a similar alteration is not observed in patients subjected to
thyroidectomy for NDNG. These findings allow the formulation of the hypothesis
that (1) treatment with levothyroxine for 180 days after thyroidectomy in GG is
not long enough to restore the normality of all the peripheral indicators of
action of the thyroid hormones; and (2) levothyroxine replacement therapy is
unable to guarantee euthyroidism in all the tissues in GG (eg, during
hematopoiesis in the bone marrow). |
Which hormone concentrations are altered in patients with the Allan–Herndon–Dudley syndrome? | Thyroid hormone concentrations are altered in patients with the Allan-Herndon-Dudley syndrome. In particular, high serum T3 levels and low-normal to low T4 serum levels are common in the Allan-Herndon-Dudley syndrome. It is, an X linked condition, is characterized by severe intellectual disability, dysarthria, athetoid movements, muscle hypoplasia and spastic paraplegia in combination. | Allan-Herndon-Dudley syndrome was among the first of the X-linked mental
retardation syndromes to be described (in 1944) and among the first to be
regionally mapped on the X chromosome (in 1990). Six large families with the
syndrome have been identified, and linkage studies have placed the gene locus in
Xq13.2. Mutations in the monocarboxylate transporter 8 gene (MCT8) have been
found in each of the six families. One essential function of the protein encoded
by this gene appears to be the transport of triiodothyronine into neurons.
Abnormal transporter function is reflected in elevated free triiodothyronine and
lowered free thyroxine levels in the blood. Infancy and childhood in the
Allan-Herndon-Dudley syndrome are marked by hypotonia, weakness, reduced muscle
mass, and delay of developmental milestones. Facial manifestations are not
distinctive, but the face tends to be elongated with bifrontal narrowing, and
the ears are often simply formed or cupped. Some patients have myopathic facies.
Generalized weakness is manifested by excessive drooling, forward positioning of
the head and neck, failure to ambulate independently, or ataxia in those who do
ambulate. Speech is dysarthric or absent altogether. Hypotonia gives way in
adult life to spasticity. The hands exhibit dystonic and athetoid posturing and
fisting. Cognitive development is severely impaired. No major malformations
occur, intrauterine growth is not impaired, and head circumference and genital
development are usually normal. Behavior tends to be passive, with little
evidence of aggressive or disruptive behavior. Although clinical signs of
thyroid dysfunction are usually absent in affected males, the disturbances in
blood levels of thyroid hormones suggest the possibility of systematic detection
through screening of high-risk populations. Thyroid hormone is essential for the proper development and function of the
brain. The active form of thyroid hormone is T(3), which binds to nuclear
receptors. Recently, a transporter specific for T(3), MCT8 (monocarboxylate
transporter 8) was identified. MCT8 is highly expressed in liver and brain. The
gene is located in Xq13 and mutations in MCT8 are responsible for an X-linked
condition, Allan-Herndon-Dudley syndrome (AHDS). This syndrome is characterized
by congenital hypotonia that progresses to spasticity with severe psychomotor
delays. Affected males also present with muscle hypoplasia, generalized muscle
weakness, and limited speech. Importantly, these patients have elevated serum
levels of free T(3), low to below normal serum levels of free T(4), and levels
of thyroid stimulating hormone that are within the normal range. This
constellation of measurements of thyroid function enables quick screening for
AHDS in males presenting with cognitive impairment, congenital hypotonia, and
generalized muscle weakness. Thyroid hormones are known to be essential for growth, development, and
metabolism. Recently, the monocarboxylate transporter 8 (MCT8) was identified as
a thyroid hormone transporter, and MCT8 mutations have been associated with
Allan-Herndon-Dudley syndrome, an X linked condition characterized by severe
mental retardation, dysarthria, athetoid movements, muscle hypoplasia, and
spastic paraplegia. Here we describe in detail the clinical and biochemical
features and the response to thyroid hormone (L-thyroxine (LT4)) administration
in a boy with an MCT8 mutation (c.1649delA) that truncates the protein in the
twelfth transmembrane domain. It is of note that brain magnetic resoce
imaging (MRI) revealed delayed myelination from infancy. Endocrine functions
other than thyroid hormone regulation and metabolism were intact, resulting in
normal hypothalamic/pituitary function tests. While LT4 administration
suppressed thyrotropin (TSH) secretion, no significant changes in thyroid
hormone values or clinical symptoms were observed.
CONCLUSION: the characteristic thyroid hormone function tests and brain MRI
findings may allow screening of high-risk populations for a better understanding
of MCT8 pathophysiology. Mutations in the thyroid monocarboxylate transporter 8 gene (MCT8/SLC16A2) have
been reported to result in X-linked mental retardation (XLMR) in patients with
clinical features of the Allan-Herndon-Dudley syndrome (AHDS). We performed MCT8
mutation analysis including 13 XLMR families with LOD scores >2.0, 401 male MR
sibships and 47 sporadic male patients with AHDS-like clinical features. One
nonsense mutation (c.629insA) and two missense changes (c.1A>T and c.1673G>A)
were identified. Consistent with previous reports on MCT8 missense changes, the
patient with c.1673G>A showed elevated serum T3 level. The c.1A>T change in
another patient affects a putative translation start codon, but the same change
was present in his healthy brother. In addition normal serum T3 levels were
present, suggesting that the c.1A>T (NM_006517) variation is not responsible for
the MR phenotype but indicates that MCT8 translation likely starts with a
methionine at position p.75. Moreover, we characterized a de novo translocation
t(X;9)(q13.2;p24) in a female patient with full blown AHDS clinical features
including elevated serum T3 levels. The MCT8 gene was disrupted at the
X-breakpoint. A complete loss of MCT8 expression was observed in a fibroblast
cell-line derived from this patient because of unfavorable nonrandom
X-inactivation. Taken together, these data indicate that MCT8 mutations are not
common in non-AHDS MR patients yet they support that elevated serum T3 levels
can be indicative for AHDS and that AHDS clinical features can be present in
female MCT8 mutation carriers whenever there is unfavorable nonrandom
X-inactivation. Monocarboxylate transporter 8 (MCT8 or SLC16A2) is important for the neuronal
uptake of triiodothyronine (T3) in its function as a specific and active
transporter of thyroid hormones across the cell membrane, thus being essential
for human brain development. We report on a German male with
Allan-Herndon-Dudley syndrome presenting with severe intellectual and motor
disability, paroxysmal dyskinesia combined with truncal muscular hypotonia, and
peripheral muscular hypertonia at his current age of 9 years. Additionally, the
patient has a lesion in the left putamen region revealed by magnetic resoce
imaging and elevated serum T3 levels. The male appeared to have a hemizygous
mutation (R271H) in the MCT8 gene that was sequenced directly from genomic DNA
and occurred de novo in the maternal germline, as both his mother and his sister
were not carriers of the mutation. Ruling out a common polymorphism, 50 normal
individuals of the same ethnic background did not harbour the mutation. The
identified MCT8 gene mutation (R271H) is very likely to be the genetic cause for
neuronal hypothyroidism despite elevated serum T3 levels. Thyroid hormone is a pleiotropic hormone with widespread biological actions. For
instance, adequate levels of thyroid hormone are critical for the development of
different tissues such as the central nervous system, but are also essential for
the regulation of metabolic processes throughout life. The biological activity
of thyroid hormone depends not only on serum thyroid hormone levels, but is also
regulated at the tissue level by the expression and activity of deiodinases,
which activate thyroid hormone or mediate its degradation. In addition, thyroid
hormone transporters are necessary for the uptake of thyroid hormone into target
tissues. With the discovery of monocarboxylate transporter 8 (MCT8) as a
specific thyroid hormone transporter and the finding that mutations in this
transporter lead to a syndrome of severe psychomotor retardation and elevated
serum 3,3',5-tri-iodothyronine levels known as the Allan-Herndon-Dudley
syndrome, the interest in this area of research has greatly increased. In this
review, we will focus on the molecular aspects of thyroid hormone transporters,
including MCT8, MCT10, organic anion transporting polypeptides, and the effects
of genetic variation in these transporters. AIM: Mutations in the SLC16A2 gene have been implicated in Allan-Herndon-Dudley
syndrome (AHDS), an X-linked learning disability* syndrome associated with
thyroid function test (TFT) abnormalities. Delayed myelination is a non-specific
finding in individuals with learning disability whose genetic basis is often
uncertain. The aim of this study was to describe neuroimaging findings and
neurological features in males with SLC16A2 gene mutations.
METHOD: We reviewed brain magnetic resoce imaging (MRI) findings and
neurological features in a cohort of five males aged between 1 year 6 months and
6 years (median 4y) from four families harbouring SLC16A2 gene mutations.
RESULTS: The participants presented aged between 4 and 9 months with initial
hypotonia and subsequent spastic paraparesis with dystonic posturing and
superimposed paroxysmal dyskinesias. Dystonic cerebral palsy was the most common
initial clinical diagnosis, and AHDS was suspected only retrospectively,
considering the characteristically abnormal thyroid function tests, with high
serum tri-iodothyronine (T(3)), as the most consistent finding. Brain MRI showed
absent or markedly delayed myelination in all five participants, prompting the
suspicion of Pelizaeus-Merzbacher disease in one patient.
INTERPRETATION: Our findings indicate a consistent association between defective
neuronal T(3) uptake and delayed myelination. SLC16A2 involvement should be
considered in males with learning disability, an associated motor or movement
disorder, and evidence of delayed myelination on brain MRI. Although dysmorphic
features suggestive of AHDS are not always present, T(3) measurement is a
reliable screening test. The Allan-Herndon-Dudley syndrome (AHDS;MIM 300523) of X-linked mental
retardation and hypotonia is caused by mutations in a thyroid hormone
transporter gene--the monocarboxylate transporter 8 (MCT8 also known as SLC16A2)
gene. A 23-month-old boy with severe developmental delay, hypotonia, recurrent
emesis, and irritability is described. He was diagnosed with hypothyroidism at
the age of 4 months. However, T3 level was elevated. Molecular analysis of the
MCT8 gene detected a single base duplication in exon 5 c.1614dupC (p.Ile539fs),
consistent with a diagnosis of AHDS. While T3 is the best marker for this
disorder, elevations in TSH should alert to the diagnosis. Thyroid hormone (TH) is crucial for the development of different organs, in
particular the brain, as disturbances in TH supply cause severe neurological
abnormalities. TH transporters are necessary for the intracellular availability
of TH to have access to the deiodinases and nuclear receptors inside the cell.
The clinical importance of TH transporters is dramatically shown in patients
with mutations in MCT8, suffering from severe X-linked psychomotor retardation
in combination with disturbed TH levels, especially high serum T(3) levels, now
referred as Allan-Herndon-Dudley Syndrome (AHDS). Worldwide >45 families have
now been identified with MCT8 mutations. Most MCT8 mutations result in a
complete loss of TH transport function when tested in vitro, but some mutations
show significant residual activity and are associated with a somewhat milder
clinical phenotype. It is difficult to identify MCT8 patients only on the basis
of the clinical characteristics of X-linked mental retardation. Therefore, the
criterion for MCT8 mutation screening in these patients is the profile of
increased T(3) and low-normal to low FT(4) serum levels. Monocarboxylate transporter 8 (MCT8, SLC16A2) is a thyroid hormone (TH)
transmembrane transport protein mutated in Allan-Herndon-Dudley syndrome, a
severe X-linked psychomotor retardation. The neurological and endocrine
phenotypes of patients deficient in MCT8 function underscore the physiological
significance of carrier-mediated TH transmembrane transport. MCT8 belongs to the
major facilitator superfamily of 12 transmembrane-spanning proteins and mediates
energy-independent bidirectional transport of iodothyronines across the plasma
membrane. Structural information is lacking for all TH transmembrane
transporters. To gain insight into structure-function relations in TH transport,
we chose human MCT8 as a paradigm. We systematically performed conventional and
liquid chromatography-tandem mass spectrometry-based uptake measurements into
MCT8-transfected cells using a large number of compounds structurally related to
iodothyronines. We found that human MCT8 is specific for L-iodothyronines and
requires at least one iodine atom per aromatic ring. Neither thyronamines,
decarboxylated metabolites of iodothyronines, nor triiodothyroacetic acid and
tetraiodothyroacetic acid, TH derivatives lacking both chiral center and amino
group, are substrates for MCT8. The polyphenolic flavonoids naringenin and
F21388, potent competitors for TH binding at transthyretin, did not inhibit T(3)
transport, suggesting that MCT8 can discriminate its ligand better than
transthyretin. Bioinformatic studies and a first molecular homology model of
MCT8 suggested amino acids potentially involved in substrate interaction.
Indeed, alanine mutation of either Arg(445) (helix 8) or Asp(498) (helix 10)
abrogated T(3) transport activity of MCT8, supporting their predicted role in
substrate recognition. The MCT8 model allows us to rationalize potential
interactions of amino acids including those mutated in patients with
Allan-Herndon-Dudley syndrome. Thyroid hormones are known to be essential for growth, development and
metabolism. Recently mutations in the SLC16A2 gene coding for the
monocarboxylate thyroid hormone transporter 8, MCT8, have been associated with
Allan-Herndon-Dudley syndrome (AHDS), an X-linked condition characterized by
severe mental retardation, dysarthria, athetoid movements, muscle hypoplasia and
spastic paraplegia. Here we describe in detail the clinical and biochemical
features in a boy affected by AHDS with severe neurological abnormalities and a
novel de novo SLC16A2 gene insertion, 1343-1344insGCCC, resulting in a truncated
protein lacking the last four transmembrane domains (TMDs) as well as the
carboxyl cytoplasmic end. He presents mental retardation, axial hypotonia,
hypertonia of arms and legs, paroxysmal dyskinesias, seizures. The endocrine
phenotype showed low serum total and free thyroxine (T4), very elevated total
and free triiodothyronine (T3) and normal thyrotropin (TSH) with blunted
response to thyrotropin-releasing hormone (TRH). The latter finding was
unexpected and suggested that the lack of functional MCT8 was counterbalanced at
the thyrotrope cell level by high serum T3 concentration and/or by increased
intrapituitary type 2 deiodinase (D2) activity. Our case constitutes a relevant
contribution to better characterize this disorder and to elucidate the
functional consequences of SLC16A2 gene mutations. Thyroid hormones are essential for brain development. The active thyroid
hormone, T3, binds to several products of two genes, the nuclear thyroid hormone
receptors alpha and beta, and thus regulates gene expression. Mutations in a
thyroid hormone transmembrane transport protein, monocarboxylate transporter 8
(MCT8), underlie one of the first described X-linked mental retardation
syndromes, the Allan-Herndon-Dudley syndrome. This discovery sparked great
interest in the process of thyroid hormone transmembrane transport.
Iodothyronines are charged amino acid derivatives and require protein
facilitators to cross cellular membranes. Thyroid hormones are translocated
across lipid bilayers by several members of the major facilitator superfamily,
including monocarboxylate transporters, amino acid transporters, and organic
anion transporting polypeptides. Although until recently few researchers
considered thyroid hormone transporters an important object of study, there is
now a large number of candidate transporters to be reckoned with in the brain.
Moreover, to finally cross the neuronal plasma membrane, any iodothyronine
molecule on its way toward a neuronal nucleus has to cross consecutively the
lumenal and ablumenal membranes of the capillary endothelium, enter astrocytic
foot processes, and leave the astrocyte through the plasma membrane. Moreover,
microglia, oligodendrocytes, and precursor and stem cells are thyroid hormone
responsive and likely express thyroid hormone transporters. Hence, the many
roles played by thyroid hormones in the development, function, and regeneration
of the nervous system are dependent on the spatiotemporal expression of several
transmembrane transport proteins. Cellular thyroid hormone uptake and efflux are mediated by transmembrane
transport proteins. One of these, monocarboxylate transporter 8 (MCT8) is
mutated in Allan-Herndon-Dudley syndrome, a severe mental retardation associated
with abnormal thyroid hormone constellations. Since mice deficient in Mct8
exhibit a milder neurological phenotype than patients, we hypothesized that
alternative thyroid hormone transporters may compensate in murine brain cells
for the lack of Mct8. Using qPCR, Western Blot, and immunocytochemistry, we
investigated the expression of three different thyroid hormone transporters,
i.e., Mct8 and L-type amino acid transporters Lat1 and Lat2, in mouse brain. All
three thyroid hormone transporters are expressed from corticogenesis and peak
around birth. Primary cultures of neurons and astrocytes express Mct8, Lat1, and
Lat2. Microglia specifically expresses Mct10 and Slco4a1 in addition to high
levels of Lat2 mRNA and protein. As in vivo, a brain microvascular endothelial
cell line expressed Mct8 and Lat1. 158N, an oligodendroglial cell line expressed
Mct8 protein, consistent with delayed myelination in MCT8-deficient patients.
Functional T(3)- and T(4)-transport assays into primary astrocytes showed K(M)
values of 4.2 and 3.7 μM for T(3) and T(4). Pharmacological inhibition of L-type
amino acid transporters by BCH and genetic inactivation of Lat2 reduced
astrocytic T(3) uptake to the same extent. BSP, a broad spectrum inhibitor,
including Mct8, reduced T(3) uptake further suggesting the cooperative activity
of several T(3) transporters in astrocytes. Mutations of the monocarboxylate transporter 8 gene (MCT8, SLC16A2) cause the
Allan-Herndon-Dudley syndrome, an X-linked syndrome of severe intellectual
deficit and neurological impairment. Mct8 transports thyroid hormones (T4 and
T3), and the Allan-Herndon-Dudley syndrome is likely caused by lack of T3
transport to neurons during critical periods of fetal brain development. To
evaluate the role of Mct8 in thyroid hormone action in the fetal brain we
administered T4 or T3 to thyroidectomized pregt dams treated with
methyl-mercapto-imidazol to produce maternal and fetal hypothyroidism. Gene
expression was then measured in the fetal cerebral cortex. T4 increased Camk4,
Sema3c, and Slc7a3 expression, but T3 was without effect. To investigate the
cause for the lack of T3 action we analyzed the expression of organic anion
transport polypeptide (Oatp14, Slco1c1), a T4 transporter, and Mct8 (Slc16a2), a
T4 and T3 transporter, by confocal microscopy. Both proteins were present in the
brain capillaries forming the blood-brain barrier and in the epithelial cells of
the choroid plexus forming the blood-cerebrospinal fluid barrier. It is
concluded that T4 from the maternal compartment influences gene expression in
the fetal cerebral cortex, possibly after transport via organic anion
transporter polypeptide and/or Mct8, and conversion to T3 in the astrocytes. On
the other hand, T3 does not reach the target neurons despite the presence of
Mct8. The data indicate that T4, through local deiodination, provides most T3 in
the fetal rat brain. The role of Mct8 as a T3 transporter in the fetal rat brain
is therefore uncertain. PURPOSE OF REVIEW: To discuss the recent advances on thyroid hormone transport
in the brain. A special attention is paid to the X-linked thyroid hormone cell
transport (THCT) defect (also known as the Allan-Herndon-Dudley syndrome),
caused by mutations of the specific thyroid hormone transporter MCT8 gene.
RECENT FINDINGS: MCT8 is involved in thyroid hormone transport in the brain. MRI
of patients with THCT defect showed myelination delays, probably related to
impaired thyroid hormone action on oligodendrocytes. MCT8 is also expressed in
the thyroid and has an important role in thyroid hormone secretion. The altered
circulating concentrations of thyroid hormone in the patients are partly because
of impaired secretion and altered peripheral metabolism. Increased deiodinase
activity is important in the pathophysiology of the syndrome. High D1 activity
in liver and kidney increases T4 and rT3 deiodination, and contributes to the
increased serum T3. High D2 activity in the brain contributes to compensate the
deficient T3 transport by increasing local T3 production.
SUMMARY: Patients with suspected X-linked leukoencephalopathy should be screened
for MCT8 gene mutations. Research on the brain pathophysiology of the THCT
defect should focus on the specific role of Mct8 on oligodendrocytes and
myelination. BACKGROUND: The monocarboxylate transporter 8 (MCT8) is a member of the major
facilitator superfamily (MFS) and transports specificly iodothyronines. MCT8
mutations are the underlying cause of a syndrome of severe X-linked psychomotor
retardation known as the Allan-Herndon-Dudley syndrome. This syndrome is
characterized by abnormally high T3, low/normal T4 serum levels and slightly
elevated serum TSH. To date, more than 25 pathogenic mutations in hMCT8 are
known and they are valuable indicators of important regions for structural and
functional MCT8 properties.
METHODS: We designed a structural human MCT8 model and studied reported
pathogenic missense mutations with focus on the estimation of those amino acid
positions which are probably sensitive for substrate transport. Furthermore,
assuming similarities between determits of T3 binding observed in the
published crystal structure of the thyroid hormone receptor beta occupied by its
ligand T3 and the structural MCT8 model, we explore potential T3 binding sites
in the MCT8 substrate channel cavity.
RESULTS: We found that all known pathogenic missense mutations are located
exclusively in the transmembrane helices and to a high degree at conserved
residues among the MCT family. Furthermore, mutations either of or to
prolines/glycines are located mainly at helices 9-12 and are expected to cause
steric clashes or structural misfolding. In contrast, several other mutations
are close to the potential substrate channel and affected amino acids are likely
involved in the switching mechanism between different transporter conformations.
Finally, three potential substrate binding sites are predicted for MCT8.
CONCLUSIONS: Naturally occurring mutations of MCT8 provide molecular insights
into protein regions important for protein folding, substrate binding and the
switching mechanism during substrate transport. Future studies guided by this
information should help to clarify structure-function relationships at MCT8
which may bear broader relevance for other members of the MCT family. This
includes decoding of the complete set of transport-sensitive residue positions
and description of structural re-arrangements during transport. OBJECTIVE: The monocarboxylate transporter 8 (MCT8; SLC16A2) has a pivotal role
in neuronal triiodothyronine (T(3)) uptake. Mutations of this transporter
determine a distinct X-linked psychomotor retardation syndrome
(Allan-Herndon-Dudley syndrome (AHDS)) that is attributed to disturbed thyroid
hormone levels, especially elevated T(3) levels. We describe the genetic
analysis of the MCT8 gene in a patient suspected for AHDS and the clinical and
endocrine effects of L-thyroxine (LT(4)) or liothyronine (LT(3)) treatment
intending to overcome the T(3) uptake resistance through alternative
transporters.
METHODS: The six exons of the MCT8 gene were amplified individually by PCR. As
multiple exons were missing, the length of the X-chromosomal deletion was
determined by a dense SNP array, followed by PCR-based fine mapping to define
the exact borders of the deleted segment. The clinical and endocrine data of the
patient during 6.5 years of LT(4) treatment and two periods (3 months each) of
low- and high-dose LT(3) were evaluated.
RESULTS: A partial deletion of the MCT8 gene (comprising five of six exons) was
detected, confirming the suspected AHDS. MCT8 dysfunction was associated with
partial resistance to T(3) at the hypothalamus and pituitary level, with normal
responsiveness at the peripheral organs (liver and cardiovascular system).
Thyroid hormone administration had no beneficial effect on the neurological
status of the patient.
CONCLUSION: We identified a 70 kb deletion encompassing exons 2-6 of the MCT8
gene in our AHDS patient. Both LT(4) and LT(3) administration had no therapeutic
effect. Alternatively, treatment of AHDS patients with thyroid hormone analogs
should be considered. Thyroid hormone is essential for normal proliferation and differentiation of
chondrocytes. Thus, untreated congenital hypothyroidism is marked by severe
short stature. The monocarboxylate transporter 8 (MCT8) is a highly specific
transporter for thyroid hormone. The hallmarks of Allan-Herndon-Dudley syndrome,
caused by MCT8 mutations, are severe psychomotor retardation and elevated T(3)
levels. However, growth is mostly normal. We therefore hypothesized that growth
plate chondrocytes use transporters other than MCT8 for thyroid hormone uptake.
Extensive analysis of thyroid hormone transporter mRNA expression in mouse
chondrogenic ATDC5 cells revealed that monocarboxylate transporter 10 (Mct10)
was most abundantly expressed among the transporters known to be highly specific
for thyroid hormone, namely Mct8, Mct10, and organic anion transporter 1c1.
Expression levels of Mct10 mRNA diminished with chondrocyte differentiation in
these cells. Accordingly, Mct10 mRNA was expressed most abundantly in the growth
plate resting zone chondrocytes in vivo. Small interfering RNA-mediated
knockdown of Mct10 mRNA in ATDC5 cells decreased [(125)I]T(3) uptake up to 44%
compared with negative control (P < 0.05). Moreover, silencing Mct10 mRNA
expression abolished the known effects of T(3), i.e. suppression of
proliferation and enhancement of differentiation, in ATDC5 cells. These results
suggest that Mct10 functions as a thyroid hormone transporter in chondrocytes
and can explain at least in part why Allan-Herndon-Dudley syndrome patients do
not exhibit significant growth impairment. BACKGROUND: Iodothyronines are charged amino acid derivatives that cannot
passively cross a phospholipid bilayer. Transport of thyroid hormones across
plasma membranes is mediated by integral membrane proteins belonging to several
gene families. These transporters therefore allow or limit access of thyroid
hormones into brain. Since thyroid hormones are essential for brain development
and cell differentiation, it is expected that genetic deficiency of such
transporters would result in neurodevelopmental derangements.
SCOPE OF REVIEW: We introduce concepts of thyroid hormone transport into the
brain and into brain cells. Important thyroid hormone transmembrane transporters
are presented along with their expression patterns in different brain cell
types. A focus is placed on monocarboxylate transporter 8 (MCT8) which has been
identified as an essential thyroid hormone transporter in humans. Mutations in
MCT8 underlie one of the first described X-linked mental retardation syndromes,
the Allan-Herndon-Dudley syndrome.
MAJOR CONCLUSIONS: Thyroid hormone transporter molecules are expressed in a
developmental and cell type-specific pattern. Any thyroid hormone molecule has
to cross consecutively the luminal and abluminal membranes of the capillary
endothelium, enter astrocytic foot processes, and leave the astrocyte through
the plasma membrane to finally cross another plasma membrane on its way towards
its target nucleus.
GENERAL SIGNIFICANCE: We can expect more transporters being involved in or
contributing to in neurodevelopmental or neuropsychiatric disease. Due to their
expression in cellular components regulating the hypothalamus-pituitary-thyroid
axis, mutations and polymorphisms are expected to impact on negative feedback
regulation and hormonal setpoints. This article is part of a Special Issue
entitled Thyroid hormone signalling. Two siblings with psychomotor retardation, congenital hypotonia, spasticity, and
no speech acquisition underwent MRI and Tc ethyl cysteinate dimer SPECT imaging.
The SPECT images showed a reduction in regional cerebral blood flow in the
bilateral frontal cortex and cerebellum in both cases. T2-weighted and fluid
attenuated inversion recovery images obtained using MRI showed delayed
myelination and cortical atrophy in mainly the frontal lobes. Based on the MRI
findings, the abnormal serum levels of thyroid hormone, and the gene mutation,
the siblings were diagnosed as having monocarboxylate transporter 8 deficiency.
A reduction in regional cerebral blood flow, as observed using SPECT, may be a
common feature of monocarboxylate transporter 8 deficiency. Cellular entry is an important step preceding intracellular metabolism and
action of thyroid hormone (TH). Transport of TH across the plasma membrane does
not take place by simple diffusion but requires transporter proteins. One of the
most effective and specific TH transporters identified to date is
monocarboxylate transporter 8 (MCT8), the gene of which is located on the X
chromosome. Although MCT8 is expressed in many tissues, its function appears to
be most critical in the brain. Hemizygous MCT8 mutations in males cause severe
psychomotor retardation, known as the Allan-Herndon-Dudley syndrome (AHDS), and
abnormal serum TH levels. AHDS thus represents a type of TH resistance caused by
a defect in cellular TH transport. Allan-Herndon-Dudley syndrome (AHDS), an X linked condition, is characterized by
severe intellectual disability, dysarthria, athetoid movements, muscle
hypoplasia and spastic paraplegia in combination with altered TH levels, in
particular, high serum T3 levels. Mutations in the MCT8 gene coding for the
monocarboxylate thyroid hormone transporter 8 have been associated with AHDS.
Here we describe a family with the presence of a MCT8 gene mutation, p.A224T, in
three consecutive generations. In two generations its presence was detected in
the hemizygous state in two males with neurological abnormalities including
mental retardation, axial hypotonia, hypertonia of arms and legs and athetoid
movements. One of them presented normal thyroid hormone levels. Mutation was
also detected, although in the heterozygous state, in three females showing
thyroid hormone levels in the normal range. Our results show the difficulty of
distinguishing AHDS from patients with X-linked intellectual disability solely
on the basis of clinical features and biochemical tests, and we advise screening
for MCT8 mutations in either young or older patients with severe intellectual
disability, axial hypotonia/dystonia, poor head control, spastic paraplegia, and
athetoid movements even when they have normal thyroid hormone profiles. Monocarboxylate transporter 8 (MCT8) is a thyroid hormone (TH)-specific
transporter. Mutations in the MCT8 gene are associated with Allan-Herndon-Dudley
Syndrome (AHDS), consisting of severe psychomotor retardation and disturbed TH
parameters. To study the functional consequences of different MCT8 mutations in
detail, we combined functional analysis in different cell types with live-cell
imaging of the cellular distribution of seven mutations that we identified in
patients with AHDS. We used two cell models to study the mutations in vitro: 1)
transiently transfected COS1 and JEG3 cells, and 2) stably transfected Flp-in
293 cells expressing a MCT8-cyan fluorescent protein construct. All seven
mutants were expressed at the protein level and showed a defect in T3 and T4
transport in uptake and metabolism studies. Three mutants (G282C, P537L, and
G558D) had residual uptake activity in Flp-in 293 and COS1 cells, but not in
JEG3 cells. Four mutants (G221R, P321L, D453V, P537L) were expressed at the
plasma membrane. The mobility in the plasma membrane of P537L was similar to WT,
but the mobility of P321L was altered. The other mutants studied (insV236,
G282C, G558D) were predomitly localized in the endoplasmic reticulum. In
essence, loss of function by MCT8 mutations can be divided in two groups:
mutations that result in partial or complete loss of transport activity (G221R,
P321L, D453V, P537L) and mutations that mainly disturb protein expression and
trafficking (insV236, G282C, G558D). The cell type-dependent results suggest
that MCT8 mutations in AHDS patients may have tissue-specific effects on TH
transport probably caused by tissue-specific expression of yet unknown
MCT8-interacting proteins. The major product secreted by the thyroid is thyroxine (T4), whereas most of the
biologically active triiodothyronine (T3) derives from the peripheral conversion
of T4 into T3. The deiodinase enzymes are involved in activation and
inactivation of thyroid hormones (THs). Type 1 and type 2 deiodinase (D1 and D2)
convert T4 into T3 whereas D3 degrades T4 and T3 into inactive metabolites and
is thus the major physiological TH inactivator. The
hypothalamic-pituitary-thyroid axis maintains circulating TH levels constant,
while the deiodinases tissue-specifically regulate intracellular thyroid status
by controlling TH action in a precise spatio-temporal fashion. Here we review
the data related to the recent identification of a paraneoplastic syndrome
called "consumptive hypothyroidism," which exemplifies how deiodinases alter
substantially the concentration of TH in blood. This syndrome results from the
aberrant uncontrolled expression of D3 that can induce a severe form of
hypothyroidism by inactivating T4 and T3 in defined tumor tissue. This rare TH
insufficiency generally affects patients in the first years of life, and has
distinct features in terms of diagnosis, treatment, and prognosis with respect
to other forms of hypothyroidism. Allan-Herndon-Dudley Syndrome (AHDS), an X linked condition, is characterized by
congenital hypotonia that progresses to spasticity with severe psychomotor
delays, in combination with altered thyroid hormone levels, in particular, high
serum T3 levels. Recently, this disease was proved to be caused by mutations in
SLC16A2 coding for the monocarboxylate thyroid hormone transporter 8 (MCT8).
Here we describe a 26-year -old Japanese patient with AHDS who had deletion of
exon 3 of SLC16A2. INTRODUCTION: Allan-Herndon-Dudley syndrome is an X-linked condition caused by
mutations of the monocarboxylate transporter 8 gene. This syndrome is
characterized by axial hypotonia, severe mental retardation, dysarthria,
athetoid movements, spastic paraplegia, and a typical thyroid hormone profile.
In most of the cases reported so far, brain magnetic resoce imaging showed
delayed myelination of the central white matter and this finding greatly affects
the diagnosis of the syndrome.
CASE REPORT: We present a new case studied with magnetic resoce imaging and
spectroscopy and we reviewed all the articles published between 2004 and 2012
containing information on brain neuroimaging in this syndrome. An Italian boy,
showing a classical phenotype of the syndrome, was diagnosed at 17months of age.
Genetic analysis revealed a new frameshift mutation of the monocarboxylate
transporter 8 gene. His brain magnetic resoce imaging and spectroscopy,
performed at the age of 14months, were normal.
DISCUSSION: Among the 33 cases reported in the literature, 3 cases had normal
neuroimaging and in 7 of 14 cases, having a longitudinal follow-up, the initial
finding of delayed myelination gradually improved. Our case and the review of
the pertinent literature suggest that Allan-Herndon-Dudley syndrome should be
suspected in males with the typical neurological and thyroid profile, even in
cases with normal brain myelination. Thyroid function tests (TFTs) are amongst the most commonly requested laboratory
investigations in both primary and secondary care. Fortunately, most TFTs are
straightforward to interpret and confirm the clinical impression of
euthyroidism, hypothyroidism or hyperthyroidism. However, in an important
subgroup of patients the results of TFTs can seem confusing, either by virtue of
being discordant with the clinical picture or because they appear incongruent
with each other [e.g. raised thyroid hormones (TH), but with non-suppressed
thyrotropin (TSH); raised TSH, but with normal TH]. In such cases, it is
important first to revisit the clinical context, and to consider potential
confounding factors, including alterations in normal physiology (e.g.
pregcy), intercurrent (non-thyroidal) illness, and medication usage (e.g.
thyroxine, amiodarone, heparin). Once these have been excluded, laboratory
artefacts in commonly used TSH or TH immunoassays should be screened for, thus
avoiding unnecessary further investigation and/or treatment in cases where there
is assay interference. In the remainder, consideration should be given to
screening for rare genetic and acquired disorders of the
hypothalamic-pituitary-thyroid (HPT) axis [e.g. resistance to thyroid hormone
(RTH), thyrotropinoma (TSHoma)]. Here, we discuss the main pitfalls in the
measurement and interpretation of TFTs, and propose a structured algorithm for
the investigation and management of patients with anomalous/discordant TFTs. |
Is selumetinib effective in thyroid cancer? | Yes, selumetinib was shown to be effective treatment for thyroid cancer. Selumetinib may reverse radioiodine uptake in patients with radioiodine-refractory differentiated thyroid cancer. Clinical efficacy of selumetinib was also investigated in other solid tumors. | Over the past 5 years, patients with progressive radioactive iodine-refractory
thyroid cancer have responded to "targeted" multikinase inhibitors, which
inhibit angiogenesis and not the tumor cell. Here, selumetinib targets the
mitogen-activated protein kinase pathway in papillary thyroid carcinoma and
shows limited single-agent activity in the patients with tumors that harbor the
(V600E)BRAF mutation. BACKGROUND: Metastatic thyroid cancers that are refractory to radioiodine
(iodine-131) are associated with a poor prognosis. In mouse models of thyroid
cancer, selective mitogen-activated protein kinase (MAPK) pathway antagonists
increase the expression of the sodium-iodide symporter and uptake of iodine.
Their effects in humans are not known.
METHODS: We conducted a study to determine whether the MAPK kinase (MEK) 1 and
MEK2 inhibitor selumetinib (AZD6244, ARRY-142886) could reverse refractoriness
to radioiodine in patients with metastatic thyroid cancer. After stimulation
with thyrotropin alfa, dosimetry with iodine-124 positron-emission tomography
(PET) was performed before and 4 weeks after treatment with selumetinib (75 mg
twice daily). If the second iodine-124 PET study indicated that a dose of
iodine-131 of 2000 cGy or more could be delivered to the metastatic lesion or
lesions, therapeutic radioiodine was administered while the patient was
receiving selumetinib.
RESULTS: Of 24 patients screened for the study, 20 could be evaluated. The
median age was 61 years (range, 44 to 77), and 11 patients were men. Nine
patients had tumors with BRAF mutations, and 5 patients had tumors with
mutations of NRAS. Selumetinib increased the uptake of iodine-124 in 12 of the
20 patients (4 of 9 patients with BRAF mutations and 5 of 5 patients with NRAS
mutations). Eight of these 12 patients reached the dosimetry threshold for
radioiodine therapy, including all 5 patients with NRAS mutations. Of the 8
patients treated with radioiodine, 5 had confirmed partial responses and 3 had
stable disease; all patients had decreases in serum thyroglobulin levels (mean
reduction, 89%). No toxic effects of grade 3 or higher attributable by the
investigators to selumetinib were observed. One patient received a diagnosis of
myelodysplastic syndrome more than 51 weeks after radioiodine treatment, with
progression to acute leukemia.
CONCLUSIONS: Selumetinib produces clinically meaningful increases in iodine
uptake and retention in a subgroup of patients with thyroid cancer that is
refractory to radioiodine; the effectiveness may be greater in patients with
RAS-mutant disease. (Funded by the American Thyroid Association and others;
ClinicalTrials.gov number, NCT00970359.). BACKGROUND AND AIM: Selumetinib is a promising and interesting targeted therapy
agent as it may reverse radioiodine uptake in patients with
radioiodine-refractory differentiated thyroid cancer. We conduct this meta-
analysis to compare the efficacy and safety of selumetinib with current
therapies in patients with advanced cancer.
METHODS: An electronic search was conducted using PubMed/ Medicine, EMBASE and
Cochrane library databases. Statistical analyses were carried out using either
random-effects or fixed-effects models according to the heterogeneity of
eligible studies.
RESULTS: Six eligible trials involved 601 patients were identified. Compared
with current therapies, treatment schedules with selumetinib did not improve
progression free survival (hazard ratio, 0.91; 95%CI 0.70-1.17, P= 0.448), but
did identify better clinical benefits (odds ratio, 1.24; 95%CI 0.69- 2.24, P =
0.472) and less disease progression (hazard ratio, 0.72; 95%CI 0.51-1.00, P =
0.052) though its impact was not statistically significant. Sub-group analysis
resulted in significantly improved progression free survival (hazard ratio,
0.61; 95%CI 0.49-0.57, P = 0.00), clinical benefits (odds ratio, 3.04; 95%CI
1.60-5.77, P = 0.001) and reduced disease progression (hazard ratio, 0.35; 95%CI
0.18-0.67, P = 0.001) in patients administrated selumetinib. Dermatitis
acneiform (risk ratio, 9.775; 95%CI 3.143-30.395, P = 0.00) and peripheral edema
(risk ratio, 2.371; 95%CI 1.690-3.327, P = 0.00) are the most frequently
observed adverse effects associated with selumetinib.
CONCLUSIONS: Compared with current chemotherapy, selumetinib has modest clinical
activity as monotherapy in patients with advanced cancer, but combinations of
selumetinib with cytotoxic agents in patients with BRAF or KRAS mutations hold
great promise for cancer treatment. Dermatitis acneiform and peripheral edema
are the most frequently observed adverse effects in patients with selumetinib. Most of the genetic events implicated in the pathogenesis of thyroid cancer (TC)
involve genes with kinase activity. Thus, kinase inhibitors (KIs) are very
relevant in this field. KIs are considered the most suitable treatment for
patients with iodine-refractory differentiated TC; these patients comprise the
subgroup with the poorer prognosis. To date, only sorafenib has been approved
for this indication, but promising results have been reported with several other
KIs. In particular, lenvatinib has demonstrated excellent efficacy, with both
progression-free survival and objective tumour response being better than with
sorafenib. Despite being considered to be well tolerated, both sorafenib and
lenvatinib have shown a remarkable toxicity, which has led to dose reductions in
the majority of patients and to treatment discontinuation in a significant
proportion of cases. The role of KIs in differentiated TC may be revolutionised
by the finding that selumetinib may restore a clinical response to radioactive
iodine (RAI). Vandetanib and cabozantinib have been approved for the treatment
of advanced, progressive medullary TC (MTC). Nevertheless, the toxicity of both
compounds suggests their selective use in those patients with strong disease
progression. Treatment with the mTOR-inhibitor everolimus, alone or in
combination with somatostatin analogues, should be studied in metastatic MTC
patients with slow progression of disease, these representing the vast majority
of patients. KIs did not significantly impact on the clinical features of
anaplastic TC (ATC). Thyroid cancer is increasing all over the world. The exact cause of this
increase is still debated and there are conflicting reports. Sophisticated
molecular studies suggest that environmental chemicals may have effects of
thyroid carcinogenesis. The development of powerful molecular biology techniques
has enabled targeted next-generation sequencing for detection of mutations in
thyroid cancer, and this technique can make a specific diagnosis of thyroid
cancer in cytologically indeterminate cases. The initial treatment of
well-differentiated thyroid cancer (DTC) is surgery followed by radioiodine
remt ablation. However, further studies are needed to determine the optimal
dosage of radioactive iodine for DTC patients with lateral neck metastasis. DTC
is an indolent tumor and may cause death even decades later. Thus, long-term
follow-up is mandatory. Recently, dynamic risk stratification (DRS) has begun to
use stimulated thyroglobulin level at 1 year after the initial treatment and
restratified the risk in accordance with the response to the initial treatment.
This DRS strategy accurately predicts disease free survival and can be widely
used in daily clinical settings. For the iodine refractory metastatic disease,
redifferentiation therapy and targeted therapy are two promising alternative
treatments. Sorafenib is the first approved agent for the treatment of
progressive iodine refractory advanced thyroid cancer in Korea and may be very
helpful for radioactive-refractory locally advanced or metastatic DTC.
Selumetinib may be an effective redifferentiating agent and could be used within
several years. |
Where in the cell does the proteins S100A4 and p53 interact ? | S100A4 interacts with p53 in the cell nucleus. | A physical and functional interaction between the Ca(2+)-binding protein Mts1
(S100A4) and the tumor suppressor p53 protein is shown here for the first time.
We demonstrate that Mts1 binds to the extreme end of the C-terminal regulatory
domain of p53 by several in vitro and in vivo approaches:
co-immunoprecipitation, affinity chromatography, and far Western blot analysis.
The Mts1 protein in vitro inhibits phosphorylation of the full-length p53 and
its C-terminal peptide by protein kinase C but not by casein kinase II. The Mts1
binding to p53 interferes with the DNA binding activity of p53 in vitro and
reporter gene transactivation in vivo, and this has a regulatory function. A
differential modulation of the p53 target gene (p21/WAF, bax, thrombospondin-1,
and mdm-2) transcription was observed upon Mts1 induction in tet-inducible cell
lines expressing wild type p53. Mts1 cooperates with wild type p53 in apoptosis
induction. Our data imply that the ability of Mts1 to enhance p53-dependent
apoptosis might accelerate the loss of wild type p53 function in tumors. In this
way, Mts1 can contribute to the development of a more aggressive phenotype
during tumor progression. Metastasis-associated protein S100A4 (Mts1) induces invasiveness of primary
tumors and promotes metastasis. S100A4 belongs to the family of small
calcium-binding S100 proteins that are involved in different cellular processes
as transducers of calcium signal. S100A4 modulates properties of tumor cells via
interaction with its intracellular targets, heavy chain of non-muscle myosin and
p53. Here we report identification of a new molecular target of the S100A4
protein, liprin beta1. Liprin beta1 belongs to the family of leukocyte common
antigen-related (LAR) transmembrane tyrosine phosphatase-interacting proteins
that may regulate LAR protein properties via interaction with another member of
the family, liprin alpha1. We showed by the immunoprecipitation analysis that
S100A4 interacts specifically with liprin beta1 in vivo. Immunofluorescence
staining demonstrated the co-localization of S100A4 and liprin beta1 in the
cytoplasm and particularly at the protrusion sites of the plasma membrane. We
mapped the S100A4 binding site at the C terminus of the liprin beta1 molecule
between amino acid residues 938 and 1005. The S100A4-binding region contains two
putative phosphorylation sites by protein kinase C and protein kinase CK2.
S100A4-liprin beta1 interaction resulted in the inhibition of liprin beta1
phosphorylation by both kinases in vitro. Metastasis-promoting Mts1(S100A4) protein belongs to the S100 family of
Ca(2+)-binding proteins. A mouse strain with a germ-line inactivation of the
S100A4 gene was generated. The mice were viable and did not display
developmental abnormalities in the postnatal period. However, an abnormal sex
ratio was observed in the litters with the S100A4-/- genotype, raising the
possibility of a certain level of embryonic lethality in this strain. In all,
10% of 10-14-month-old S100A4-null animals developed tumors. This is a
characteristic feature of mouse strains with inactivated tumor suppressor genes.
Spontaneous tumors of S100A4-/- mice were p53 positive. Recently, we have shown
that S100A4 interacts with p53 tumor suppressor protein and induces apoptosis.
We proposed that impairment of this interaction could affect the
apoptosis-promoting function of p53 that is involved in its tumor suppressor
activity. The frequency of apoptosis in the spleen of S100A4-/- animals after
whole-body gamma-irradiation was reduced compared to the wild-type animals. The
same was true for the transcriptional activation of the p53 target genes -
waf/p21/cip1 and bax. Taken together, these observations indicate that
spontaneous tumors in S100A4-/- mice are a result of functional destabilization
of p53 tumor suppressor gene. The effects of hyperthermia on the expression of p53, the apoptosis-associated
genes Bax and Bcl-2, Notch and S100A4 have been studied in the HepG2 cell line
and the HUT cell line derived from HepG2, adapted for growth in hyperthermic
conditions. Hyperthermia inhibits cell proliferation and induces apoptosis.
HepG2 and HUT cells differed in respect of anchorage to growth surface, degree
of proliferation and apoptosis and expression of p53, Bax, Bcl-2, Notch, and
S100A4 genes. The induction of apoptosis and the inhibition of cell
proliferation occurred independently of p53, and independently also of
involvement of the apoptosis family genes Bax and Bcl-2. We demonstrate novel
and marked differences between transient heat shock and heat adaptation in
respect of pathways of signaling and generation of phenotypic effects in vitro.
Different signaling patterns have been identified here. Pathways of signaling by
S100A4, by its interaction with and sequestration of p53, and by Notch also seem
differentially operational in the induction of apoptosis, and both appear to be
activated as alternative pathways in the context of hyperthermia signaling
independently of p53. S100A4 (also known as Mts1, metastasin, p9Ka, pEL98, CAPL, calvasculin, Fsp-1,
placental calcium-binding protein) belongs to the family of EF-hand
calcium-binding proteins, whose expression is elevated in a number of
pathological conditions. Although it is well documented that S100A4 is expressed
in cancer cells and contributes to tumor cell motility and metastatic
progression, the exact underlying mechanisms remain elusive. An important
characteristic feature of S100 proteins is their dual function, inside and
outside the cell. In this review, we focus on the intracellular function of
S100A4. The review contains structural analysis of S1004 in comparison with
other members of S100 proteins. Possible modes of the interaction of S100
proteins with targets are described. Several examples of best-studied molecular
interactions involving S100A4 with heavy chain of nonmuscle myosin IIA,
LAR-interacting protein liprin beta1 and tumor suppressor protein p53 are
provided. We suggest that the binding of S100A4 to these molecules is critical
for the S100A4 function. Further studies of the implications of these
interactions in different molecular pathways may shed additional light on the
role of S100A4 protein in the control of tumor cell motility and migration. We
discuss the approaches for down-regulation of S100A4 expression and their
potential for application in the clinics. Proteins of the S100 family bind to the intrinsically disordered transactivation
domain (TAD; residues 1-57) and C-terminus (residues 293-393) of the tumor
suppressor p53. Both regions provide sites that are subject to posttranslational
modifications, such as phosphorylation and acetylation, that can alter the
affinity for interacting proteins such as p300 and MDM2. Here, we found that
S100A1, S100A2, S100A4, S100A6, and S100B bound to two subdomains of the TAD
(TAD1 and TAD2). Both subdomains were mandatory for high-affinity binding to
S100 proteins. Phosphorylation of Ser and Thr residues increased the affinity
for the p53 TAD. Conversely, acetylation and phosphorylation of the C-terminus
of p53 decreased the affinity for S100A2 and S100B. In contrast, we found that
nitrosylation of S100B caused a minor increase in binding to the p53 C-terminus,
whereas binding to the TAD remained unaffected. As activation of p53 is usually
accompanied by phosphorylation and acetylation at several sites, our results
suggest that a shift in binding from the C-terminus in favor of the N-terminus
occurs upon the modification of p53. We propose that binding to the p53 TAD
might be involved in the stimulation of p53 activity by S100 proteins. S100 proteins modulate p53 activity by interacting with its tetramerization
(p53TET, residues 325-355) and transactivation (residues 1-57) domains. In this
study, we characterized biophysically the binding of S100A1, S100A2, S100A4,
S100A6 and S100B to homologous domains of p63 and p73 in vitro by fluorescence
anisotropy, analytical ultracentrifugation and analytical gel filtration. We
found that S100A1, S100A2, S100A4, S100A6 and S100B proteins bound different p63
and p73 tetramerization domain variants and naturally occurring isoforms with
varying affinities in a calcium-dependent manner. Additional interactions were
observed with peptides derived from the p63 and p73 N-terminal transactivation
domains. Importantly, S100 proteins bound p63 and p73 with different affinities
in their different oligomeric states, similarly to the differential modes of
binding to p53. On the basis of our data, we hypothesize that S100 proteins
regulate the oligomerization state of all three p53 family members and their
isoforms, with a potential physiological relevance in developmental and
disease-related processes. The regulation of the p53 family by S100 is
complicated and depends on the target preference of each individual S100
protein, the concentration of the proteins and calcium, as well as the splicing
variation of p63 or p73. Our results outlining the complexity of the interaction
should be considered when studying the functional effects of S100 proteins in
their biological context. Nuclear localization of the metastasis-associated protein S100A4 has been shown
to correlate with advanced disease stage in primary colorectal carcinomas (CRC),
but nuclear function and its relevance for the metastatic capacity of tumor
cells is still unclear. Among several nuclear interacting protein partners
suggested for S100A4, the tumor suppressor protein p53 has attracted particular
interest, and previous studies suggest direct and indirect modes of interaction
between the two proteins. The present study was undertaken to assess
coexpression and potential interaction in CRC. TP53 mutational status and S100A4
expression were investigated in a selected series of primary CRC specimens (n =
40) and cell lines (n = 17) using DNA sequencing, western blot, and double
immunostaining. Additionally, S100A4 and p53 were experimentally up- and
down-regulated in vitro to assess reciprocal effects. For the first time, S100A4
and p53 coexpression was demonstrated in individual CRC cells, with nuclear
colocalization as a particularly interesting feature. In contrast to previous
studies, no correlation was observed between TP53 mutational status and S100A4
expression, and no evidence was obtained to support reciprocal regulation
between the two molecules in the HCT116 isogenic cell line model. In conclusion,
S100A4 and p53 were shown to be colocalized in individual nuclei of CRC cells,
and it might be speculated whether the proteins interact in this subcellular
compartment. S100 proteins interact with the transactivation domain and the C-terminus of
p53. Further, S100B has been shown to interact with MDM2, a central negative
regulator of p53. Here, we show that S100B bound directly to the folded
N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and
surface plasmon resoce experiments. This interaction with MDM2 (2-125) is a
general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also
interact with MDM2 (2-125). These interactions with S100 proteins do not result
in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability
of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53
ubiquitylation in vitro. S100A4 is a small calcium-binding protein that is commonly overexpressed in a
range of different tumor types, and it is widely accepted that S100A4 has an
important role in the process of cancer metastasis. In vitro binding assays has
shown that S100A4 interacts with the tumor suppressor protein p53, indicating
that S100A4 may have additional roles in tumor development. In the present
study, we show that endogenous S100A4 and p53 interact in complex samples, and
that the interaction increases after inhibition of MDM2-dependent p53
degradation using Nutlin-3A. Further, using proximity ligation assay, we show
that the interaction takes place in the cell nucleus. S100A4 knockdown
experiments in two p53 wild-type cell lines, A549 and HeLa, resulted in
stabilization of p53 protein, indicating that S100A4 is promoting p53
degradation. Finally, we demonstrate that S100A4 knockdown leads to
p53-dependent cell cycle arrest and increased cisplatin-induced apoptosis. Thus,
our data add a new layer to the oncogenic properties of S100A4 through its
inhibition of p53-dependent processes. |
What are the applications of a Dermaroller ? | Microneedling with dermaroller is a new treatment modality for the treatment of scars, especially acne scars, stretch marks, wrinkles, and for facial rejuvenation. It is a simple and relatively cheap modality that also can be used for transdermal drug delivery.
Microneedling is a safe and a promising tool in hair stimulation and also is useful to treat hair loss refractory to Minoxidil therapy. | |
Are immune cells affected in Amyotrophic Lateral Sclerosis? | In ALS T-cell deficiency increases neuronal loss, while boosting T cell levels reduces it. | The intrathymic injection of donor spleen cells into antilymphocyte serum
(ALS)-treated mice induces significant prolongation of donor skin grafts. The
intrathymic route of antigen presentation in this model is superior to the
intravenous route in achieving unresponsiveness. To elucidate possible
mechanisms involved in the induction of unresponsiveness in ALS-treated mice
after intrathymic injection of donor spleen cells, we have analysed the
reactivity of lymphoid cells from unresponsive mice in various ways. Deletion of
donor reactive cells has been studied using the Mls antigen system. Functional
inactivation was analysed by a sequential study of the frequency of donor
reactive cells. Suppressor cell activity was studied using an adoptive transfer
assay. Deletion of donor reactive cells was partial and occurred largely in the
spleen in both early and late stages of unresponsiveness. The frequency of donor
reactive cytotoxic cells was suppressed in both spleen and lymph nodes from day
+/- 13 until grafts were rejected, with the exception of a rebound period at day
+/- 22. In contrast, donor reactive cells were not deleted or inactivated in the
thymus. Suppressor cell activity could only be detected in mice bearing
long-term grafts. These results suggest that donor reactive cytotoxic cells are
functionally inactivated in the spleen and nodes in the early and late stages of
unresponsiveness after intrathymic injection of antigen. In contrast, donor
reactive cells in the thymus do not appear to be affected. Immunological disturbances have been implicated in the pathogenesis of
amyotrophic lateral sclerosis (ALS). Chemokines are involved in the recruitment
of immune cells. Regulated upon activation, normal T-cell expressed and secreted
(RANTES) is a C-C beta-chemokine with strong chemo-attractant activity for
T-lymphocytes and monocytes. We examined serum levels of RANTES in 20 patients
with amyotrophic lateral sclerosis (ALS), 14 patients with non-inflammatory
neurological disorders (NIND) and 13 control subjects (CTRL) and cerebrospinal
fluid (CSF) levels of RANTES in ALS and NIND group patients in order to
investigate whether RANTES as index of immune activation is present in ALS
patients. Patients with ALS had higher RANTES levels compared with the NIND
patients and CTRL subjects (p = 0.005 and p = 0.02, respectively). CSF RANTES
levels were also higher compared with the NIND patients (p = 0.007). No
correlation of serum and CSF RANTES levels with disease duration was found.
These results may suggest an activated microglia induced recruitment of
peripheral inflammatory cells to sites of inflammation in ALS patients. The immune system has been found to be involved with positive and negative
effects in the nervous system of amyotrophic lateral sclerosis (ALS) patients.
In general, T cells, B cells, NK cells, mast cells, macrophages, dendritic
cells, microglia, antibodies, complement and cytokines participate in limiting
damage. Several mechanisms of action, such as production of neurotrophic growth
factors and interaction with neurons and glial cells, have been shown to
preserve these latter from injury and stimulate growth and repair. The immune
system also participates in proliferation of neural progenitor stem cells and
their migration to sites of injury and this activity has been documented in
various neurologic disorders including traumatic injury, ischemic and
hemorrhagic stroke, multiple sclerosis, infection, and neurodegenerative
diseases (Alzheimer's disease, Parkinson's disease and ALS). Many therapies have
been shown to stimulate the protective and regenerative aspects of the immune
system in humans, such as intravenous immunoglobulins, and other experimental
interventions such as vaccination, minocycline, antibodies and neural stem
cells, have shown promise in animal models of ALS. Consequently, several
immunosuppressive and immunomodulatory therapies have been tried in ALS,
generally with no success, in particular intravenous immunoglobulins. The
multiple aspects of the immune response in ALS are beginning to be appreciated,
and their potential as pharmacologic targets in neurologic disease is being
explored. Amyotrophic lateral sclerosis (ALS) is a rapidly progressing fatal
neurodegenerative disorder characterized by the selective death of motor neurons
(MN) in the spinal cord, and is associated with local neuroinflammation.
Circulating CD4(+) T cells are required for controlling the local detrimental
inflammation in neurodegenerative diseases, and for supporting neuronal
survival, including that of MN. T-cell deficiency increases neuronal loss, while
boosting T cell levels reduces it. Here, we show that in the mutant superoxide
dismutase 1 G93A (mSOD1) mouse model of ALS, the levels of natural killer T
(NKT) cells increased dramatically, and T-cell distribution was altered both in
lymphoid organs and in the spinal cord relative to wild-type mice. The most
significant elevation of NKT cells was observed in the liver, concomitant with
organ atrophy. Hepatic expression levels of insulin-like growth factor (IGF)-1
decreased, while the expression of IGF binding protein (IGFBP)-1 was augmented
by more than 20-fold in mSOD1 mice relative to wild-type animals. Moreover,
hepatic lymphocytes of pre-symptomatic mSOD1 mice were found to secrete
significantly higher levels of cytokines when stimulated with an NKT ligand,
ex-vivo. Immunomodulation of NKT cells using an analogue of α-galactosyl
ceramide (α-GalCer), in a specific regimen, diminished the number of these cells
in the periphery, and induced recruitment of T cells into the affected spinal
cord, leading to a modest but significant prolongation of life span of mSOD1
mice. These results identify NKT cells as potential players in ALS, and the
liver as an additional site of major pathology in this disease, thereby
emphasizing that ALS is not only a non-cell autonomous, but a non-tissue
autonomous disease, as well. Moreover, the results suggest potential new
therapeutic targets such as the liver for immunomodulatory intervention for
modifying the disease, in addition to MN-based neuroprotection and systemic
treatments aimed at reducing oxidative stress. Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease
with selective loss of upper and lower motor neurons. At sites of motor neuron
injury, neuroinflammation is a prominent pathological finding and is
characterized by microglial activation, astrogliosis, and infiltration of
monocytes and T-cells. Both innate and adaptive immune responses actively
influence disease progression in animal models and in ALS patients, and promote
neuroprotection or neurotoxicity at different stages of disease. The early
immune reaction to signals from injured motor neurons is to rescue and repair
damaged tissue. As disease accelerates, a shift occurs from beneficial immune
responses (involving M2 microglia and regulatory T-cells) to deleterious immune
responses (involving M1 microglia and Th1 cells). In this review, we underscore
the importance of immune-mediated mechanisms in the pathogenesis of ALS and
discuss the alterations and distinct phenotypes of immune cells at the different
stages of disease. The better we understand the dynamic changes that occur
within the immune system over the course of disease, the better we will be able
to develop effective therapeutic regimens in ALS. Immune cell infiltration to the brain's territory was considered for decades to
reflect a pathological process in which immune cells attack the central nervous
system (CNS); such a process is observed in the inflammatory autoimmune disease,
multiple sclerosis (MS). As neuroinflammatory processes within the CNS
parenchyma are also common to other CNS pathologies, regardless of their
etiology, including neurodegenerative disorders such as Alzheimer's disease (AD)
and Amyotrophic lateral sclerosis (ALS), these pathologies have often been
compared to MS, a disease that benefits from immunosuppressive therapy. Yet,
over the last decade, it became clear that autoimmunity has a bright side, and
that it plays a pivotal role in CNS repair following damage. Specifically,
autoimmune T cells were found to facilitate CNS healing processes, such as in
the case of sterile mechanical injuries to the brain or the spinal cord, mental
stress, or biochemical insults. Even more intriguingly, autoimmune T cells were
found to be involved in supporting fundamental processes of brain functional
integrity, such as in the maintece of life-long brain plasticity, including
spatial learning and memory, and neurogenesis. Importantly, autoimmune T cells
are part of a cellular network which, to operate efficiently and safely,
requires tight regulation by other immune cell populations, such as regulatory T
cells, which are indispensable for maintece of immunological self-tolerance
and homeostasis. Here, we suggest that dysregulation of the balance between
peripheral immune suppression, on one hand, and protective autoimmunity, on the
other, is an underlying mechanism in the emergence and progression of the
neuroinflammatory response associated with chronic neurodegenerative diseases
and brain aging. Mitigating chronic neuroinflammation under these conditions
necessitates activation, rather than suppression, of the peripheral immune
response directed against self. Accordingly, we propose that fighting off acute
and chronic neurodegenerative conditions requires breaking peripheral immune
tolerance to CNS self-antigens, in order to boost protective autoimmunity.
Nevertheless, the optimal approach to fine tune such immune response must be
individually explored for each condition. Amyotrophic lateral sclerosis (ALS) is a devastating fatal motor neuron disease,
for which there is currently no cure or effective treatment. In this disease,
local neuroinflammation develops along the disease course and contributes to its
rapid progression. In several models of CNS pathologies, circulating immune
cells were shown to display an indispensable role in the resolution of the
neuroinflammatory response. The recruitment of such cells to the CNS involves
activation of the choroid plexus (CP) of the brain for leukocyte trafficking,
through a mechanism that requires IFN-γ signaling. Here, we found that in the
mutant SOD1(G93A) (mSOD1) mouse model of ALS, the CP does not support leukocyte
trafficking during disease progression, due to a local reduction in IFN-γ
levels. Therapeutic immunization of mSOD1 mice with a myelin-derived peptide led
to CP activation, and was followed by the accumulation of immunoregulatory
cells, including IL-10-producing monocyte-derived macrophages and Foxp3(+)
regulatory T cells, and elevation of the neurotrophic factors IGF-1 and GDNF in
the diseased spinal cord parenchyma. The immunization resulted in the
attenuation of disease progression and an increased life expectancy of the mSOD1
mice. Collectively, our results demonstrate that recruitment of immunoregulatory
cells to the diseased spinal cord in ALS, needed for fighting off the pathology,
can be enhanced by transiently boosting peripheral immunity to myelin antigens. |
Are OATP1B1 and OATP1B3 associated with bilirubin transport? | Yes, OATP1B1 and OATP1B3 are involved in the transport of bilirubin. | OATP1B1 (a.k.a. OATP-C, OATP2, LST-1, or SLC21A6) is a liver-specific organic
anion uptake transporter and has been shown to be a higher affinity bilirubin
uptake transporter than OATP1B3. Using human embryonic kidney (HEK 293) cells
stably transfected with OATP1B1, we have studied the effects of indinavir,
saquinavir, cyclosporin A, and rifamycin SV on human OATP1B1 transport function.
These drugs are potent inhibitors of OATP1B1 transport activity in vitro. We
further provide evidence that the calculated fraction of OATP1B1 inhibited at
the clinical exposure level correlated very well with the observed
hyperbilirubinemia outcome for these drugs in humans. Our data support the
hypothesis that inhibition of OATP1B1 is an important mechanism for drug-induced
unconjugated hyperbilirubinemia. Inhibition of OATPs may be an important
mechanism in drug-drug and drug-endogenous substance interactions. 1. Elevated serum bilirubin levels are caused mainly by liver diseases,
haematolysis, genetic defects and drug intake. Unconjugated bilirubin (UCB) is
taken up into hepatocytes by human organic anion transporting polypeptide 1B1
(OATP1B1; encoded for by the SLCO1B1 gene). The present study was performed to
determine the association between SLCO1B1 gene polymorphisms and serum bilirubin
levels in vivo. Moreover, the effects of administration of low-dose rifampicin
on serum bilirubin levels in different SLCO1B1 genotypes was examined. 2. Serum
bilirubin levels were examined in 42 healthy volunteers who had been analysed
for SLCO1B1 genotype (seven, 13, 14 and eight with SLCO1B1 genotypes *1a/*1a,
*1b/*1b, *1b/*15 and *15/*15, respectively). Among them, 24 subjects (seven,
seven, eight and two with SLCO1B1 genotypes *1a/*1a, *1b/*1b, *1b/*15 and
*15/*15, respectively) were selected to participate in an open-label, two-phase
clinical trial. Each was given 450 mg rifampicin orally once daily at 2000 hours
for 5 consecutive days. Serum bilirubin concentrations at 0800 hours on the 1st
and 6th days were compared between the different SLCO1B1 genotypes. 3. In the 42
volunteers, the mean (+/-SD) serum UCB in both SLCO1B1*1b/*15 and *15/*15 groups
was significantly higher than that in the SLCO1B1*1b/*1b group (11.07 +/- 2.31,
13.01 +/- 3.87 and 8.21 +/- 2.68 micromol/L, respectively; P = 0.009 and P <
0.001). Total bilirum (T.BIL) in both the SLCO1B1*1b/*15 and *15/*15 groups was
significantly higher than that in the SLCO1B1*1b/*1b group (16.69 +/- 4.09,
20.71 +/- 5.12 and 13.06 +/- 5.12 micromol/L, respectively; P = 0.029 and P <
0.001). The direct bilirubin (D.BIL) in the SLCO1B1*15/*15 group was
significantly higher than that in the SLCO1B1*1b/*1b group (7.69 +/- 1.81 vs
4.85 +/- 1.81 micromol/L, respectively; P = 0.001). Rifampicin significantly
increased UCB, T.BIL and D.BIL concentrations in 24 healthy volunteers (17.68
+/- 5.96 vs 13.95 +/- 4.44 micromol/L (P = 0.040), 5.72 +/- 2.01 vs 4.35 +/-
1.50 micromol/L (P = 0.028) and 12.00 +/- 4.26 vs 9.61 +/- 3.15 micromol/L (P =
0.035), respectively). However, the extent of the increase in serum bilirubin
caused by 450 mg rifampicin for 5 days was not affected by SLCO1B1 genotype. 4.
Genetic polymorphism in SLCO1B1 is a major determit of interindividual
variability in the serum bilirubin level. SLCO1B1*15 carriers had higher
baseline serum UCB, T.BIL and D.BIL levels compared with subjects with the
SLCO1B1*1a/*1a and SLCO1B1*1b/*1b genotypes. SLCO1B1*15/*15 homozygotes are more
susceptible to hyperbilirubinaemia. Serum bilirubin levels could be increased by
low-dose rifampicin administration, but the extent of the increase was not
associated with SLCO1B1 genotype. PURPOSE: Transport of the hepatobiliary scintigraphy agent Tc-99m mebrofenin
(MEB) was characterized and simulation studies were conducted to examine the
effects of altered hepatic transport on MEB pharmacokinetics in humans.
METHODS: MEB transport was investigated in Xenopus laevis oocytes expressing
OATP1B1 or OATP1B3, and in membrane vesicles prepared from HEK293 cells
transfected with MRP2 or MRP3. A pharmacokinetic model was developed based on
blood, urine and bile concentration-time profiles obtained in healthy humans,
and the effect of changes in hepatic uptake and/or excretion associated with
disease states (hyperbilirubinemia and cholestasis) on MEB disposition was
simulated.
RESULTS: MEB (80 pM) transport by OATP1B1 and OATP1B3 was inhibited by
rifampicin (50 microM) to 10% and 4% of control, respectively. MEB (0.4 nM)
transport by MRP2 was inhibited to 12% of control by MK571 (50 microM);
MRP3-mediated transport was inhibited to 5% of control by
estradiol-17-beta-glucuronide (100 microM). A two-compartment model described
MEB (2.5 mCi) systemic disposition in humans (systemic clearance = 16.2 +/- 2.7
ml min(-1) kg(-1)); biliary excretion was the predomit route of hepatic
elimination (efflux rate constants ratio canalicular/sinusoidal = 3.4 +/- 0.8).
Based on simulations, altered hepatic transport markedly influenced MEB systemic
and hepatic exposure.
CONCLUSIONS: MEB may be a useful probe to assess how altered hepatic function at
the transport level modulates hepatobiliary drug disposition. Eleven members of the human organic anion transporter (OATP) family (grouped
into six families) facilitate the Na(+)- independent transmembrane transport of
various endo- and xenobiotics (bile acids, bilirubin, steroid hormone
conjugates, thyroid hormones, prostaglandins, clinically used drugs, and
toxins). OATPs are 12-transmembrane glycoproteins (643-722 amino acids) and
contain many conserved structural features, for example, eleven cysteines in the
large extracellular loop 5. They are important for proper transport, for which
translocation of substrates through a central, positively-charged pore in a
rocker-switch-type mechanism has been proposed. Although OATPs are expressed in
various cells and tissues, some members show a more restricted pattern
(well-studied OATP1B1/OATP1B3 in liver, OATP4C1 in kidney, and OATP6A1 in
testis). In cancer, the distribution pattern is no longer maintained, and OATPs,
like OATP1B3, become upregulated in maligt tissues (colon, breast, prostate).
Studies in cell lines and animal models further revealed that the expression of
OATPs is regulated in a cell- and tissue-specific way by cytokines and
activation of nuclear receptors (LXR, FXR, PXR, CAR, HNF4). Also epigenetic
mechanisms and postranslational modifications influence their expression and
function. Therefore, changes in the expression of OATPs under pathological
conditions will influence transport processes causing an altered accumulation of
OATP substrates in cells of excretory organs (intestine, liver, kidney) and on
various blood/organ barriers (such as brain, testis, placenta). For drugs, this
may result in increased toxicity and adverse drug reactions. Therefore, it is
important to improve the knowledge on the regulation and function of individual
OATPs, and to apply it for therapeutic considerations. Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted
by the liver into bile. Failure of this system can lead to a buildup of
conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis
of bilirubin excretion and hyperbilirubinemia syndromes is largely understood,
but that of Rotor syndrome, an autosomal recessive disorder characterized by
conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic
uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8
Rotor-syndrome families and found that Rotor syndrome was linked to mutations
predicted to cause complete and simultaneous deficiencies of the organic anion
transporting polypeptides OATP1B1 and OATP1B3. These important
detoxification-limiting proteins mediate uptake and clearance of countless drugs
and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1
polymorphisms have previously been linked to drug hypersensitivities. Using mice
deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we
found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b
transporters mediate their hepatic reuptake. Transgenic expression of human
OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing
liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this
shuttle may allow flexible transfer of bilirubin conjugates (and probably also
drug conjugates) formed in upstream hepatocytes to downstream hepatocytes,
thereby preventing local saturation of further detoxification processes and
hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin
glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains
Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may
confer substantial drug toxicity risks. Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted
by the liver into bile. Failure of this system can lead to a buildup of
conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis
of bilirubin excretion and hyperbilirubinemia syndromes is largely understood,
but that of Rotor syndrome, an autosomal recessive disorder characterized by
conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic
uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8
Rotor-syndrome families and found that Rotor syndrome was linked to mutations
predicted to cause complete and simultaneous deficiencies of the organic anion
transporting polypeptides OATP1B1 and OATP1B3. These important
detoxification-limiting proteins mediate uptake and clearance of countless drugs
and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1
polymorphisms have previously been linked to drug hypersensitivities. Using mice
deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we
found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b
transporters mediate their hepatic re uptake. Transgenic expression of human
OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing
liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this
shuttle may allow flexible transfer of bilirubin conjugates (and probably also
drug conjugates) formed in upstream hepatocytes to downstream hepatocytes,
thereby preventing local saturation of further detoxification processes and
hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin
glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains
Rotor-type hyperbilirubinemia.Moreover, OATP1B1 and OATP1B3 null mutations may
confer substantial drug toxicity risks. Hyperbilirubinemia may arise due to inadequate clearance of bilirubin from the
body. Bilirubin elimination is a multifaceted process consisting of uptake of
bilirubin into the hepatocytes facilitated by OATP1B1 and OATP1B3. Once in the
hepatocytes, it is extensively glucuronidated by UGT1A1. Eventually, the
glucuronide metabolite is excreted into the bile via MRP2. UGT1A1 inhibition has
been previously shown to be linked with hyperbilirubinemia. However, because
drug transporters also contribute to bilirubin elimination, the purpose of this
work was to investigate the in vitro inhibition of OATP1B1, OATP1B3, MRP2, and
BSEP of select test drugs known to elicit hyperbilirubinemia. Test drugs
investigated in this study were atazanavir and indinavir, which are associated
with hyperbilirubinemia and elevations in serum transaminase; ritonavir and
nelfinavir, which are not associated with hyperbilirubinemia; and bromfenac,
troglitazone, and trovafloxacin, which are associated with severe idiosyncratic
hepatotoxicity exhibiting elevations in serum bilirubin and transaminase. Due to
limited solubility and poor ionization of bilirubin and its glucuronide, the
formation of estradiol 3-glucuronide was used as a surrogate to assess UGT1A1
activity, while the transport of pitavastatin, CDCF, and taurocholate were used
as surrogate probe substrates to monitor the function of OATP1B1/OATP1B3, MRP2,
and BSEP, respectively. It was assumed that any inhibition of the surrogate
probe substrates by test drugs is indicative of the potential impact of test
drugs to modulate the function of proteins involved in bilirubin disposition. In
vitro inhibition was determined by calculating IC50. Moreover, Cmax and
Cmax,free were integrated with IC50 values to calculate R and Rfree,
respectively, which represents the ratio of probe drug glucuronidation/transport
in the absence and presence of test drugs. Analysis of the data showed that
Rfree demonstrated the best correlation to hyperbilirubinemia. Specifically,
Rfree was above the 1.1 target threshold against UGT1A1, OATP1B1, and BSEP for
atazanavir and indinavir. In contrast, Rfree was below this threshold for
ritonavir and nelfinavir as well as for bromfenac, troglitazone, and
trovafloxacin. For all test drugs examined, only minor inhibition against
OATP1B3 and MRP2 were observed. These data suggest that the proposed surrogate
probe substrates to evaluate the in vitro inhibition of UGT1A1, OATP1B1, and
BSEP may be suitable to assess bilirubin disposition. For protease inhibitors,
inclusion of OATP1B1 and BSEP inhibition may improve the predictability of
hyperbilirubinemia. 1. Transient benign unconjugated hyperbilirubinemia has been observed
clinically with several drugs including indinavir, cyclosporine, and rifamycin
SV. Genome-wide association studies have shown significant association of
OATP1B1 and UGT1A1 with elevations of unconjugated bilirubin, and OATP1B1
inhibition data correlated with clinical unconjugated hyperbilirubinemia for
several compounds. 2. In this study, inhibition of OATP1B3 and UGT1A1, in
addition to OATP1B1, was explored to determine whether one measure offers value
over the other as a potential prospective tool to predict unconjugated
hyperbilirubinemia. OATP1B1 and OATP1B3-mediated transport of bilirubin was
confirmed and inhibition was determined for atazanavir, rifampicin, indinavir,
amprenavir, cyclosporine, rifamycin SV and saquinavir. To investigate the
intrinsic inhibition by the drugs, both in vivo Fi (fraction of intrinsic
inhibition) and R-value (estimated maximum in vivo inhibition) for OATP1B1,
OATP1B3 and UGT1A1 were calculated. 3. The results indicated that in vivo Fi
values >0.2 or R-values >1.5 for OATP1B1 or OATP1B3, but not UGT1A1, are
associated with previously reported clinical cases of drug-induced unconjugated
hyperbilirubinemia. 4. In conclusion, inhibition of OATP1B1 and/or OATP1B3
along with predicted human pharmacokinetic data could be used pre-clinically to
predict potential drug-induced benign unconjugated hyperbilirubinemia in the
clinic. Bilirubin, a major end product of heme breakdown, is an important constituent of
bile, responsible for its characteristic colour. Over recent decades, our
understanding of bilirubin metabolism has expanded along with the processes of
elimination of other endogenous and exogenous anionic substrates, mediated by
the action of multiple transport systems at the sinusoidal and canalicular
membrane of hepatocytes. Several inherited disorders characterised by impaired
bilirubin conjugation (Crigler-Najjar syndrome type I and type II, Gilbert
syndrome) or transport (Dubin-Johnson and Rotor syndrome) result in various
degrees of hyperbilirubinemia of either the predomitly unconjugated or
predomitly conjugated type. Moreover, disrupted regulation of hepatobiliary
transport systems can explain jaundice in many acquired liver disorders. In this
review, we discuss the recent data on liver bilirubin handling based on the
discovery of the molecular basis of Rotor syndrome. The data show that a
substantial fraction of bilirubin conjugates is primarily secreted by MRP3 at
the sinusoidal membrane into the blood, from where they are subsequently
reuptaken by sinusoidal membrane-bound organic anion transporting polypeptides
OATP1B1 and OATP1B3. OATP1B proteins are also responsible for liver clearance of
bilirubin conjugated in splanchnic organs, such as the intestine and kidney, and
for a number of endogenous compounds, xenobiotics and drugs. Absence of one or
both OATP1B proteins thus may have serious impact on toxicity of commonly used
drugs cleared by this system such as statins, sartans, methotrexate or
rifampicin. The liver-blood cycling of conjugated bilirubin is impaired in
cholestatic and parenchymal liver diseases and this impairment most likely
contributes to jaundice accompanying these disorders. Increased concentrations of bilirubin glucuronides in blood plasma indicate
hepatocellular dysfunction. Elucidation of the transport processes of bilirubin
conjugates across the basolateral (sinusoidal) and the canalicular plasma
membrane domains of hepatocytes has decisively contributed to our current
understanding of the molecular basis of conjugated hyperbilirubinemia in human
liver diseases. Under normal conditions, unconjugated bilirubin is taken up into
hepatocytes by transporters of the organic anion-transporting polypeptide (OATP)
family, followed by conjugation with glucuronic acid, and ATP-dependent
transport into bile. This efflux across the canalicular membrane is mediated by
multidrug resistance protein 2 (MRP2 or ABCC2), which is a 190-kDa glycoprotein
transporting with high affinity and efficiency monoglucuronosyl bilirubin and
bisglucuronosyl bilirubin into bile. MRP2 is hereditarily deficient in human
Dubin-Johnson syndrome. Under pathophysiological conditions such as cholestatic
liver injury and MRP2 inhibition, the basolateral efflux pump multidrug
resistance protein 3 (MRP3 or ABCC3) is responsible for the occurrence of
conjugated hyperbilirubinemia. MRP3 is a glycoprotein with a similar molecular
mass as MRP2, with 48% amino acid identity, and with overlapping substrate
specificity. Human MRP3 is the only basolateral efflux pump shown to transport
bilirubin glucuronides. In human and rat hepatocytes, MRP3/Mrp3 is strongly
upregulated under conditions of cholestasis and MRP2 deficiency. This is in line
with the concept that basolateral efflux pumps of the hepatocyte compensate for
impaired canalicular efflux of compounds into bile and contribute to balance the
rate of uptake or synthesis of compounds in hepatocytes with the capacity for
efflux into bile. New molecular entities (NMEs) are evaluated using a rigorous set of in vitro and
in vivo studies to assess their safety and suitability for testing in humans.
Regulatory health authorities require that therapeutic and supratherapeutic
doses be administered, by the intended route of administration, to two
nonclinical species prior to human testing (ICH Expert Working Group. The
international conference on harmonization of technical requirements for
registration of pharmaceuticals for human use (ICH); Multidisciplinary
guidelines; Nonclinical safety studies (M3).
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Multidisciplinary/M3_R2/Step4/M3_R2__Guideline.pdf
, 2009). The purpose of these studies is to identify potential target organ
toxicity and to determine if the effects are reversible. Liver is a potential
site for toxicity caused by orally administered NMEs due to high exposure during
first pass after oral administration. A range of clinical chemistry analytes are
routinely measured in both nonclinical and clinical studies to evaluate and
monitor for hepatotoxicity. While bilirubin itself circulates within a wide
range of concentrations in many animal species and humans, without causing
adverse effects and possibly providing benefits (Sedlak and Snyder. Pediatrics
113(6):1776-1782, 2004), bilirubin is one of the few readily monitored
circulating biomarkers that can provide insight into liver function. Therefore,
any changes in plasma or urine bilirubin levels must be carefully evaluated.
Changes in bilirubin may occur as a result of adaptive nontoxic changes or
severe toxicity. Examples of adaptive nontoxic changes in liver function, which
may elevate direct (conjugated) and/or indirect (unconjugated) bilirubin above
baseline levels, include reversible inhibition of UGT1A1-mediated bilirubin
metabolism and OATP1B1-, OATP1B3-, or MRP2-mediated transport (Keogh. Adv
Pharmacol 63:1-42, 2012). Alternatively, hepatocellular necrosis,
hypoalbuminuria, or cholestasis may also lead to elevation of bilirubin; in some
cases, these effects may be irreversible (FDA/CDER. Guidance for industry
drug-induced liver injury: premarketing clinical evaluation.
http://www.fda.gov/downloads/Drugs/…/Guidances/UCM174090.pdf , 2012).This
chapter aims to demonstrate application of enzyme kinetic principles in
understanding the risk of bilirubin elevation through inhibition of multiple
processes-involving both enzymes and transporters. In the sections that follow,
we first provide a brief summary of bilirubin formation and disposition. Two
case examples are then provided to illustrate the enzyme kinetic studies needed
for risk assessment and for identifying the mechanisms of bilirubin elevation.
Caveats of methods and data interpretation are discussed in these case studies.
The data presented in this chapter is unpublished at the time of compilation of
this book. It has been incorporated in this chapter to provide a sense of
complexities in enzyme kinetics to the reader. Faldaprevir, an investigational agent for hepatitis C virus treatment, is well
tolerated but associated with rapidly reversible, dose-dependent, clinically
benign, unconjugated hyperbilirubinemia. Multidisciplinary preclinical and
clinical studies were used to characterize mechanisms underlying this
hyperbilirubinemia. In vitro, faldaprevir inhibited key processes involved in
bilirubin clearance: UDP glucuronosyltransferase (UGT) 1A1 (UGT1A1) (IC50 0.45
µM), which conjugates bilirubin, and hepatic uptake and efflux transporters,
organic anion-transporting polypeptide (OATP) 1B1 (IC50 0.57 µM), OATP1B3 (IC50
0.18 µM), and multidrug resistance-associated protein (MRP) 2 (IC50 6.2 µM),
which transport bilirubin and its conjugates. In rat and human hepatocytes,
uptake and biliary excretion of [(3)H]bilirubin and/or its glucuronides
decreased on coincubation with faldaprevir. In monkeys, faldaprevir (≥20 mg/kg
per day) caused reversible unconjugated hyperbilirubinemia, without hemolysis or
hepatotoxicity. In clinical studies, faldaprevir-mediated hyperbilirubinemia was
predomitly unconjugated, and levels of unconjugated bilirubin correlated with
the UGT1A1*28 genotype. The reversible and dose-dependent nature of the clinical
hyperbilirubinemia was consistent with competitive inhibition of bilirubin
clearance by faldaprevir, and was not associated with liver toxicity or other
adverse events. Overall, the reversible, unconjugated hyperbilirubinemia
associated with faldaprevir may predomitly result from inhibition of
bilirubin conjugation by UGT1A1, with inhibition of hepatic uptake of bilirubin
also potentially playing a role. Since OATP1B1/1B3 are known to be involved in
hepatic uptake of circulating bilirubin glucuronides, inhibition of OATP1B1/1B3
and MRP2 may underlie isolated increases in conjugated bilirubin. As such,
faldaprevir-mediated hyperbilirubinemia is not associated with any liver injury
or toxicity, and is considered to result from decreased bilirubin elimination
due to a drug-bilirubin interaction. |
What is the mechanism of action of eprotirome? | Eprotirome belongs to thyromimetics and has selective TRβ1 activity. It has shown to be effective in dyslipidemia by the lipid-lowering action of TH in the liver and also in obesity. | BACKGROUND: Dyslipidemia increases the risk of atherosclerotic cardiovascular
disease and is incompletely reversed by statin therapy alone in many patients.
Thyroid hormone lowers levels of serum low-density lipoprotein (LDL) cholesterol
and has other potentially favorable actions on lipoprotein metabolism.
Consequently, thyromimetic drugs hold promise as lipid-lowering agents if
adverse effects can be avoided.
METHODS: We performed a randomized, placebo-controlled, double-blind,
multicenter trial to assess the safety and efficacy of the thyromimetic compound
eprotirome (KB2115) in lowering the level of serum LDL cholesterol in patients
with hypercholesterolemia who were already receiving simvastatin or
atorvastatin. In addition to statin treatment, patients received either
eprotirome (at a dose of 25, 50, or 100 microg per day) or placebo. Secondary
outcomes were changes in levels of serum apolipoprotein B, triglycerides, and
Lp(a) lipoprotein. Patients were monitored for potential adverse thyromimetic
effects on the heart, bone, and pituitary.
RESULTS: The addition of placebo or eprotirome at a dose of 25, 50, or 100
microg daily to statin treatment for 12 weeks reduced the mean level of serum
LDL cholesterol from 141 mg per deciliter (3.6 mmol per liter) to 127, 113, 99,
and 94 mg per deciliter (3.3, 2.9, 2.6, and 2.4 mmol per liter), respectively,
(mean reduction from baseline, 7%, 22%, 28%, and 32%). Similar reductions were
seen in levels of serum apolipoprotein B, triglycerides, and Lp(a) lipoprotein.
Eprotirome therapy was not associated with adverse effects on the heart or bone.
No change in levels of serum thyrotropin or triiodothyronine was detected,
although the thyroxine level decreased in patients receiving eprotirome.
CONCLUSIONS: In this 12-week trial, the thyroid hormone analogue eprotirome was
associated with decreases in levels of atherogenic lipoproteins in patients
receiving treatment with statins. (ClinicalTrials.gov number, NCT00593047.) PURPOSE OF REVIEW: The article is principally intended to describe the recent
evolutions in the field of research concerned with the metabolic actions of
thyroid hormones and those of some of their metabolites or derivatives.
Mitochondria, as a result of their functions, represent the principal objective
of scientists investigating the mechanisms underlying the effects of thyroid
hormones or their metabolites/derivatives.
RECENT FINDINGS: Indeed, some important recent findings concern these
organelles, and in particular mitochondrial uncoupling and its modulation by
effectors. Traditionally, thyroxine (T4) and tri-iodo-L-thyronine (T3) were the
only thyroid hormones considered to have metabolic effects, and they alone were
considered for potential as agents that might counteract some important
abnormalities such as dyslipidaemias and obesity. Several observations, however,
led to a reconsideration of this idea. In recent years, studies dealing with the
biological activities of some natural metabolites or structural analogues of
thyroid hormones have revealed abilities to ameliorate some major worldwide
medical problems, such as artherosclerosis, obesity and cardiovascular diseases.
Among natural metabolites, 3,5-diiodothyronine (T2) has been shown to powerfully
reduce adiposity and dyslipidaemia and to reverse hepatic steatosis without
unfavourable side-effects usually observed when T3 or T4 is used. Examples of
synthetic analogues are GC-1 (or sobetirome) and KB2115 (or eprotirome) which
show ipolipidaemic and antiaterogenic capacities. Clinical trials are in
progress for these last agents.
SUMMARY: In view of the above-mentioned actions, some of these compounds are now
undergoing clinical trials and may have important implications for clinical
practice or researches in the field of both endocrinology and metabolic-related
abnormalities such as diabetes and dyslipidaemias. The concept of thyroid hormone (TH) analogs that retain the beneficial effects
of TH excess on lipid lowering and fat metabolism, while avoiding any harmful
effects on the heart, muscle, bone and other tissues, has interested scientists
and physicians for several decades. While there have been many attempts to
develop selective TH receptor (TR) modulators (STRMs) for safe lipid lowering,
these approaches have failed consistently. This review details recent advances
in the development of TRβ subtype- and liver-selective STRM analogs, and
presents the results of preliminary clinical trials with one of these compounds,
eprotirome (KB-2115; Karo Bio AB). Thyroid hormone (TH) is known to have many beneficial effects on vital organs,
but its extrapolation to be used therapeutically has been restricted by the fact
that it does have concurrent adverse effects. Recent finding of various thyroid
hormone receptors (TR) isoforms and their differential pattern of tissue
distribution has regained interest in possible use of TH analogues in
therapeutics. These findings were followed by search of compounds with
isoform-specific or tissue-specific action on TR. Studying the
structure-activity relationship of TR led to the development of compounds like
GC1 and KB141, which preferentially act on the β1 isoform of TR. More recently,
eprotirome was developed and has been studied in humans. It has shown to be
effective in dyslipidemia by the lipid-lowering action of TH in the liver and
also in obesity. Another compound, 3,5-diiodothyropropionic acid (DITPA), binds
to both α- and β-type TRs with relatively low affinity and has been shown to be
effective in heart failure (HF). In postinfarction models of HF and in a pilot
clinical study, DITPA increased cardiac performance without affecting the heart
rate. TR antagonists like NH3 can be used in thyrotoxicosis and cardiac
arrhythmias. However, further larger clinical trials on some of these promising
compounds and development of newer compounds with increased selectivity is
required to achieve higher precision of action and avoid adverse effects seen
with TH. Thyroid hormone (T3) stimulates various metabolic pathways and the hepatic
actions of T3 are mediated primarily through the thyroid hormone receptor beta
(TRβ). Hypothyroidism has been linked with low grade inflammation, elevated risk
of hepatic steatosis and atherosclerosis. Secretory phospholipases (sPLA2) are
associated with inflammation, hyperlipidemia and atherosclerosis. Due to
potential linkage between thyroid hormone and sPLA2, we investigated the effect
of thyroid hormone status on the regulation of secretory phospholipases in mice,
rats and human liver. T3 suppressed the expression of the sPLA2 group IIa
(PLA2g2a) gene in the liver of BALB/c mice and C57BL/6 transgenic mice
expressing the human PLA2g2a. PLA2g2a was elevated with hypothyroidism and high
fat diets which may contribute to the low grade inflammation associated with
hypothyroidism and diet induced obesity. We also examined the effects of the TRβ
agonist eprotirome on hepatic gene regulation. We observed that eprotirome
inhibited the expression of selected sPLA2 genes and furthermore the cytokine
mediated induction PLA2g2a was suppressed. In addition, eprotirome induced genes
involved in fatty acid oxidation and cholesterol clearance while inhibiting
lipogenic genes. Our results indicate that in vivo thyroid hormone status
regulates the abundance of sPLA2 and the inhibition of PLA2g2a by T3 is
conserved across species. By regulating sPLA2 genes, T3 may impact processes
associated with atherosclerosis and inflammation and TRβ agonists may ameliorate
inflammation and hyperlipidemia. BACKGROUND: Eprotirome is a liver-selective thyroid hormone receptor agonist
that has been shown to lower plasma LDL cholesterol concentrations in previous
phase 1 and 2 studies of patients with dyslipidaemia. We aimed to assess the
long-term safety and efficacy of 50 μg and 100 μg eprotirome in patients with
familial hypercholesterolaemia.
METHODS: For this randomised, double-blind, placebo-controlled, parallel-group,
phase 3 clinical trial, we enrolled patients between Oct 3, 2011, and Feb 14,
2012, at 53 sites in 11 countries in Europe, Africa, and south Asia. Patients
were eligible for enrolment if they were aged 18 years or older, diagnosed with
heterozygous familial hypercholesterolaemia, and had not reached target LDL
cholesterol concentrations after at least 8 weeks of statin therapy with or
without ezetimibe. We used a computer-generated randomisation sequence to
allocate patients to one of three groups: 50 μg eprotirome, 100 μg eprotirome,
or placebo. This trial was planned for 52-76 weeks, with primary efficacy
analysis at 12 weeks, but it was prematurely terminated when another study found
that eprotirome causes cartilage damage in dogs. Although it was impossible to
meet the predefined study outcomes, we analysed changes in the concentrations of
LDL cholesterol and other lipids, liver parameters, thyroid hormone
concentrations, and adverse effects of treatment with eprotirome versus placebo
at 6 weeks of treatment. Analysis was done in all patients who received 6 weeks
of treatment. This study is registered with ClinicalTrials.gov, number
NCT01410383.
FINDINGS: We enrolled 236 patients, randomly allocating 80 to receive placebo,
79 to receive 50 μg eprotirome, and 77 to receive 100 μg eprotirome. 69 patients
reached the 6 week timepoint (23 given placebo, 24 given 50 μg eprotirome, and
22 given 100 μg eprotirome). Mean LDL cholesterol concentrations increased by 9%
(95% CI -2 to 20) in the placebo group, decreased by 12% (-28 to 4%; p=0.0677 vs
placebo) in the 50 μg eprotirome group, and decreased by 22% (-32 to -13%;
p=0.0045 vs placebo) in the 100 μg eprotirome group. We noted statistically
significant increases between both eprotirome groups and placebo in aspartate
aminotransferase (AST; p<0.0001), alanine aminotransferase (ALT; p<0.0001),
conjugated bilirubin (p=0.0006), and gamma-glutamyltranspeptidase (p<0.0001).
Four patients had to discontinue or interrupt study treatment before trial
termination due to AST increases between the upper limit of normal (ULN) and six
times ULN, and ALT concentrations between three and seven times ULN. Although we
detected no changes in serum concentrations of thyroid-stimulating hormone or
free tri-iodothyronine, free tetra-iodothyronine decreased by 19% (23 to 16) in
the 50 μg eprotirome group and 27% (30 to 23) in the 100 μg eprotirome group
(p<0.0001 vs placebo for both groups).
INTERPRETATION: Our findings show that eprotirome can lower LDL cholesterol
concentrations in patients with familial hypercholesterolaemia when added to
conventional statin treatment with or without ezetimibe, but that it has the
potential to induce liver injury. These findings, along with findings of
cartilage damage in dogs, raise serious doubts about selective thyroid hormone
mimetics as a therapeutic approach to lower LDL cholesterol concentrations.
FUNDING: Karo Bio AB. Reduced plasma LDL-cholesterol is a hallmark of hyperthyroidism and is caused by
transcriptional stimulation of LDL receptors in the liver. Here, we investigated
whether thyroid hormone (TH) actions involve other mechanisms that may also
account for the reduction in LDL-cholesterol, including effects on proprotein
convertase subtilisin/kexin type 9 (PCSK9) and bile acid synthesis. Twenty
hyperthyroid patients were studied before and after clinical normalization, and
the responses to hyperthyroidism were compared with those in 14 healthy
individuals after 14 days of treatment with the liver-selective TH analog
eprotirome. Both hyperthyroidism and eprotirome treatment reduced circulating
PCSK9, lipoprotein cholesterol, apoB and AI, and lipoprotein(a), while
cholesterol synthesis was stable. Hyperthyroidism, but not eprotirome treatment,
markedly increased bile acid synthesis and reduced fibroblast growth factor
(FGF) 19 and dietary cholesterol absorption. Eprotirome treatment, but not
hyperthyroidism, reduced plasma triglycerides. Neither hyperthyroidism nor
eprotirome treatment altered insulin, glucose, or FGF21 levels. TH reduces
circulating PSCK9, thereby likely contributing to lower plasma LDL-cholesterol
in hyperthyroidism. TH also stimulates bile acid synthesis, although this
response is not critical for its LDL-lowering effect. |
What is the role of music therapy in coma patients. | Several studies have shown that music can boost cognitive functions in patients with a disorder of consciousness but it is difficult to conclude since they did not use quantified measures and a control condition/group. Active improvised music therapy may offer an adjuvant form of treatment in the early rehabilitation of severe brain-injured patients. | Both from a theoretical perspective and by means of several case examples, the
article focuses on the issue of overcoming the disturbed pre-verbal
communication behaviour presented by patients in the early stage following
severe craniocerebral trauma. In patients with brain lesion, a pre-verbal,
emotionally-focussed tonal language almost invariably is capable of reaching the
still healthy sections of the person. Hence, it is possible for music therapy to
both establish contact with the seemingly non-responsive patient and
re-stimulate the person's fundamental communication competencies and experience
at the emotional, social and cognitive levels. This paper presents an overview of the studies directed at helping post-coma
persons with minimally conscious state improve their adaptive behavior.
Twenty-one studies were identified for the 2000-2010 period (i.e., a period in
which an intense debate has occurred about diagnostic, rehabilitative,
prognostic, and ethical issues concerning people with severe acquired brain
injury). Three of the 21 studies involved transcortical magnetic or deep brain
stimulation. Six studies focused on the provision of multisensory stimulation or
music therapy. The remaining 12 studies involved the use of response-related
(contingent) stimulation and assistive technology. The outcomes of the studies,
which were generally reported as positive, were discussed in terms of (a) the
size (quantitative relevance) of the changes obtained, (b) the
credibility/reliability of the changes, in light of the methodological
conditions of the studies, and (c) the level of engagement and interaction
involvement of the participants. Relevant issues for future research were also
examined. This review presents an overview of the use of music therapy in neurological
early rehabilitation of patients with coma and other disorders of consciousness
(DOC) such as unresponsive wakefulness syndrome (UWS) or minimally conscious
state (MCS). There is evidence that patients suffering from UWS show emotional
processing of auditory information, such as listening to speech. Thus, it seems
reasonable to believe that music listening-as part of an enriched environment
setting-may be of therapeutic value in these patients. There is, however, a
considerable lack of evidence. The authors strongly encourage further studies to
evaluate the efficacy of music listening in patients with DOC in neurological
early rehabilitation. These studies should consider a precise clinical
definition and homogeneity of the patient cohort with respect to the quality
(coma vs. UWS vs. MCS), duration (rather weeks to months than days) and cause
(traumatic vs. non-traumatic) of DOC, a standardized intervention protocol,
valid clinical outcome parameters over a longer observation period (weeks to
months), monitoring of neurophysiological and vegetative parameters and, if
available, neuroimaging to confirm diagnosis and to demonstrate responses and
functional changes in the patients' brains. |
Which is the causative agent of malaria? | Four Plasmodium species commonly infect humans (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale). Plasmodium falciparum infects about 5-10% of the world human population per year and it is the causative agent of the most severe and lethal form of malaria. P. falciparum causes fatal cerebral malaria and is responsible for most deaths, particularly in pregnant women and children under the age of five. P. falciparum is transmitted to the human host by Anopheles mosquitoes and is the most tremendous malaria vector in sub-Saharan Africa. Plasmodium vivax is the causative agent of benign malaria in more temperate climates of the world. Plasmodium gallinaceum is the main bird malaria causative agent and Plasmodium yoelli is the principle rodent malaria agent. | Several species of Plasmodium have been shown to contain a circular
extrachromosomal DNA molecule which is widely supposed to be mitochondrial DNA.
However, it has recently been shown to have a number of features in common with
chloroplast DNA. Here, a phylogenetic analysis of RNA polymerase coding
sequences from the Plasmodium molecule has been carried out using distance
matrix, maximum likelihood, parsimony and operator invariant methods. The
analysis indicates that the molecule is in fact derived from an oxygenic
photosynthetic organism and should be regarded as plastid DNA. This suggests
that Plasmodium originated from a phototroph that has lost the capacity to
photosynthesize. On a model pair Aedes aegypti--Plasmodium gallinaceum in has been shown that
changes in the conditions of larvae development caused by the addition into the
water medium of the live culture of Synochocystis sp. cyanobacteria or green
seaweeds Chlorella vulgaris, acetone extracts from the live culture precipitate
or Chlorella powder, as well as nitrogen-containing fertilizer--ammonium
chloride did not lower the sensitivity of the imago flying to malaria parasites.
The results of the experiments assessing the effect of biologically active
compounds introduced into the larvae habitation medium on the ability to change
sensitivity of the survived mosquito females to malaria agent have been summed
up. The data obtained are indicative of the high level of mutual adaptation
between mosquito-carriers and malaria parasites. The lack of repeated bloodsucking does not affect essentially the infection of
Ae. aegypti mosquitoes with malaria agent, P. gallinaceum. High resolution 31P-NMR has been used for the non-invasive observation of
metabolites and metabolic rates in blood of normal mice and of mice infected
with Plasmodium berghei, the causative agent of malaria. 31P-NMR was used to
quantitate levels of 2,3-diphosphoglycerate in whole cells as a function of the
degree of parasitemia and yielded good agreement with the results of enzymatic
assays. The time-dependence of 31P metabolites was monitored in both normal and
infected erythrocytes, greater rates of decay of 2,3-diphosphoglycerate being
observed in malarial blood which correlate with the level of parasitemia. Very
high metabolic rates of infected cells render measurement of intracellular pH
unreliable on freshly drawn whole blood. When appropriate measures are taken to
avoid this complication, no difference is observed in the intracellular pH of
parasitized and non-parasitized erythrocytes from infected animals. In both
normal and parasitized mice the intraerythrocytic pH is more acidic than that of
the suspending medium by 0.15 pH unit at 25 degrees C. Unlike free-living
protozoa, the parasitic protozoan Plasmodium does not contain detectable levels
of phosphonates or polyphosphates, in either whole cells or perchloric acid
extracts thereof. Plasmodium falciparum is the causative agent of malaria tropica in man.
Biochemical studies were focused on the asexual, intraerythrocytic stages of P.
falciparum, because of their role in the clinical phase of the disease and the
possibility of propagation in a cell culture system. In this report, we describe
the in-culture labeling of malarial glycolipids and the analysis of their
hydrophilic moieties. They were identified as glycosylphosphatidylinositols
(GPIs) by: 1) labeling with [3H]mannose, [3H]glucosamine, and [3H]ethanolamine
and 2) sensitivity toward glycosylphosphatidylinositol-specific phospholipase D,
phospholipase A2, and nitrous acid. Malarial GPIs are shown to be unaffected by
treatment with phosphatidylinositol-specific phospholipase C, regardless of
prior treatment with mild base commonly used for inositol deacylation. Two
candidates for putative GPI-anchor precursors to malarial membrane proteins with
the structures ethanolamine-phosphate-6(Man alpha 1-2)Man alpha 1-2Man alpha
1-6Man alpha 1-4 GlcN-PI (Pfg1 alpha) and ethanolamine-phosphate-6Man alpha
1-2Man alpha 1-6Man-alpha 1-4-GlcN-PI (Pfg1 beta) were identified. Plasmodium falciparum is the major causative agent of malaria, a disease of
worldwide importance. Resistance to current drugs such as chloroquine and
mefloquine is spreading at an alarming rate, and our antimalarial armamentarium
is almost depleted. The malarial parasite encodes two homologous aspartic
proteases, plasmepsins I and II, which are essential components of its
hemoglobin-degradation pathway and are novel targets for antimalarial drug
development. We have determined the crystal structure of recombit plasmepsin
II complexed with pepstatin A. This represents the first reported crystal
structure of a protein from P. falciparum. The crystals contain molecules in two
different conformations, revealing a remarkable degree of interdomain
flexibility of the enzyme. The structure was used to design a series of
selective low molecular weight compounds that inhibit both plasmepsin II and the
growth of P. falciparum in culture. This study describes the synergistic interaction of two calcium channel
blockers, verapamil (VR) and SR33557 or fantofarone (SR), in reversing
chloroquine resistance in Plasmodium falciparum, the causative agent of human
malaria. The two calcium channel blockers exhibited an intrinsic antimalarial
activity at 10 and 1 microM for verapamil and fantofarone, respectively.
Isobolograms revealed that chloroquine and verapamil, and chloroquine and
fantofarone, acted synergistically against chloroquine-resistant strains of P.
falciparum. When used at subinhibitory concentrations, verapamil appeared 2 to 3
times more potent than fantofarone in reversing chloroquine resistance. Indeed,
verapamil completely reversed the chloroquine resistance in P. falciparum, while
fantofarone did so only partially. In the highly chloroquine-resistant strain
FcB1, VR and SR acted synergistically to reverse CQ resistance, and the
concentrations of VR used in these combinations could be reduced 10- or 100-fold
(e.g. 100 nM and 10 nM) those required when this drug was used alone. In the
moderately chloroquine-resistant strain K1, a combination of VR and SR for CQ
resistance reversal allowed us to reduce the concentration of these
chemosensitizers 1000- and 100-fold, respectively. The maximum tolerable plasma
level beyond which side-effects occurred when using verapamil is 2.5 microM.
Thus, the approach described, which allowed us to lower the doses of
chemosensitizers, could well prevent toxic effects in humans and enlighten the
advantages of polychemotherapy. The gene of an NADP+-specific glutamate dehydrogenase was cloned from Plasmodium
falciparum, the causative agent of tropical malaria. Southern-blot analysis
indicates a single-copy gene. The gene encodes a protein with 470 residues which
has 50% of all residues identical with those of the glutamate dehydrogenases
from other low eukaryotes and eubacteria. In contrast, the sequence identity
with the human enzyme is marginal, which underlines the long evolutionary
distance between parasite and host. The gene was overexpressed in Escherichia
coli. The kinetic properties of the recombit enzyme are in good agreement
with those of the authentic enzyme. The parasite enzyme is inhibited by
D-glutamate and glutarate, but not by chloroquine. Like other coenzyme-specific
glutamate dehydrogenases, but in contrast to the dual-specific mammalian
enzymes, the P. falciparum enzyme is not affected by GTP and ADP. The physical
and chemical properties of the protein are in accordance with the cytosol being
the major localization. The gene does not encode a cleavable mitochondrial
presequence and the Mr of the recombit protein and the protein isolated from
the parasite are indistinguishable on SDS/PAGE. Western-blot analysis of
stage-specific parasites shows that glutamate dehydrogenase is present in all
intraerythrocytic stages. The signal increased continuously from rings, early
trophozoites to late trophozoites and decreased slightly in the segmenter stage.
Glutamate dehydrogenase, suggested to be the major source of NADPH in the
parasite, is an attractive target molecule for the rational development of new
antimalarial drugs. A laboratory model of circulation of the malaria causative agent P. gallinaceum
has been used to show that the effect of precocene (antijuvenoid) leads to a
statistically significant reduction in the proportion of infected females
developing eggs after blood suction. The females failing to develop eggs are not
infected. Trichopol (antiexdisone) inhibits vitellogenesis The females
undeveloping eggs become susceptible to the causative agent though to a lesser
degree than those developing them. The findings suggest that there is an
association of the mosquito susceptibility to the malaria causative agent with
the balance of hormones in the body of disease the carrier. The polyamines putrescine, spermidine and spermine play an essential role in
cell differentiation and proliferation. Inhibition of the rate-limiting enzymes
of polyamine biosynthesis, ornithine decarboxylase (ODC) and
S-adenosylmethionine decarboxylase (AdoMetDC), has been proposed as a
therapeutic strategy against cancer and parasitic infections. In the case of
Plasmodium falciparum, the causative agent of malaria tropica, this approach is
especially interesting, because here both key enzymes, ODC and AdoMetDC, are
combined in a bifunctional protein, ODC/AdoMetDC. This arrangement has not been
found in any other organism investigated so far. We report the cloning and
recombit expression of the ODC domain of P. falciparum in Escherichia coli.
First, we expressed the mere recombit ODC domain (rPfODC). Secondly, we
expressed the recombit ODC domain in conjunction with the preceding part of
the hinge region of the bifunctional ODC/AdoMetDC (rPfHinge-ODC). K(m) values
for L-ornithine were 47.3 microM for the rPfHinge-ODC and 161. 5 microM for the
rPfODC. Both recombit enzymes were inhibited by putrescine, but the K(i)
value for the rPfHinge-ODC was 50.4 microM (IC(50)=157 microM), whereas the
IC(50) for the rPfODC was 500 microM. Spermidine was a weak inhibitor in both
cases. alpha-Difluoromethylornithine inhibited the rPfHinge-ODC with a K(i)
value of 87.6 microM. For two novel ODC inhibitors, CGP52622A and CGP54619A, the
K(i) values of the rPfHinge-ODC were in the omolar range. A putative glutathione peroxidase gene (Swiss-Prot accession number Z 68200) of
Plasmodium falciparum, the causative agent of tropical malaria, was expressed in
Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid
hydroperoxide glutathione peroxidase of mammals, it proved to be monomeric. It
was active with H(2)O(2) and organic hydroperoxides but, unlike phospholipid
hydroperoxide glutathione peroxidase, not with phosphatidylcholine
hydroperoxide. With glutathione peroxidases it shares the ping-pong mechanism
with infinite V(max) and K(m) when analyzed with GSH as substrate. As a
homologue with selenocysteine replaced by cysteine, its reactions with
hydroperoxides and GSH are 3 orders of magnitude slower than those of the
selenoperoxidases. Unexpectedly, the plasmodial enzyme proved to react faster
with thioredoxins than with GSH and most efficiently with thioredoxin of P.
falciparum (Swiss-Prot accession number 202664). It is therefore reclassified as
thioredoxin peroxidase. With plasmodial thioredoxin, the enzyme also displays
ping-pong kinetics, yet with a limiting K(m) of 10 microm and a k(1)' of 0.55
s(-)1. The apparent k(1)' for oxidation with cumene, t-butyl, and hydrogen
peroxides are 2.0 x 10(4) m(-1) s(-1), 3.3 x 10(3) m(-1) s(-1), and 2.5 x 10(3)
m (-1) s(-1), respectively. k(2)' for reduction by autologous thioredoxin is 5.4
x 10(4) m(-1) s(-1) (21.2 m(-1) s(-1) for GSH). The newly discovered enzymatic
function of the plasmodial gene product suggests a reconsideration of its
presumed role in parasitic antioxidant defense. In Plasmodium falciparum, the causative agent of human malaria, the catalytic
subunit gene of cAMP-dependent protein kinase (Pfpka-c) exists as a single copy.
Interestingly, its expression appears developmentally regulated, being at higher
levels in the pathogenic asexual stages than in the sexual forms of parasite
that are responsible for transmission to the mosquito vector. Within asexual
parasites, PfPKA activity can be readily detected in schizonts. Similar to
endogenous PKA activity of noninfected red blood cells, the parasite enzyme can
be stimulated by cAMP and inhibited by protein kinase inhibitor.Importantly, ex
vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89
leads to a block in parasite growth. This suggests that the PKA activities of
infected red blood cells are essential for parasite multiplication. Finally,
structural considerations suggest that drugs targeting the parasite, rather than
the erythrocyte enzyme, might be developed that could help in the fight against
malaria. Despite vast efforts and expenditures in the past few decades, malaria continues
to kill millions of persons every year, and new approaches for disease control
are urgently needed. To complete its life cycle in the mosquito, Plasmodium, the
causative agent of malaria, has to traverse the epithelia of the midgut and
salivary glands. Although strong circumstantial evidence indicates that parasite
interactions with the two organs are specific, hardly any information is
available about the interacting molecules. By use of a phage display library, we
identified a 12-aa peptide--salivary gland and midgut peptide 1 (SM1)--that
binds to the distal lobes of the salivary gland and to the luminal side of the
midgut epithelium, but not to the midgut surface facing the hemolymph or to
ovaries. The coincidence of the tissues with which parasites and the SM1 peptide
interact suggested that the parasite and peptide recognize the same surface
ligand. In support of this hypothesis, the SM1 peptide strongly inhibited
Plasmodium invasion of salivary gland and midgut epithelia. These experiments
suggest a new strategy for the genetic manipulation of mosquito vectorial
capacity. Malaria is a major human health problem and is responsible for over 2 million
deaths per year. It is caused by a number of species of the genus Plasmodium,
and Plasmodium falciparum is the causative agent of the most lethal form.
Consequently, the development of a vaccine against this parasite is a priority.
There are a number of stages of the parasite life cycle that are being targeted
for the development of vaccines. Important candidate antigens include proteins
on the surface of the asexual merozoite stage, the form that invades the host
erythrocyte. The development of methods to manipulate the genome of Plasmodium
species has enabled the construction of gain-of-function and loss-of-function
mutants and provided new strategies to analyse the role of parasite proteins.
This has provided new information on the role of merozoite antigens in
erythrocyte invasion and also allows new approaches to address their potential
as vaccine candidates. Trypanosomes do not inhabit or grow in anopheles mosquitoes, the vector for the
transmission of Plasmodium parasites the causative agent for malaria. The
possession of lytic factors by the anopheline mosquito was thus considered. Head
and midgut sections prepared in phosphate buffered saline were tested for
trypanocidal action against T. congolense. While the head section was inactive
towards the trypanosomes, the midgut extract at 0.2 mg ml(-1) diminished the
motility of the parasites within 2 min of incubation; killing 50% of the
population after 5 min. At 0.5 mg ml(-1) of the extract, about 90% of the
parasites were killed within 2 min of incubation. The midgut fraction was
subjected to a purification protocol involving successive chromatography on:
octyl-sepharose, reactive brown agarose and fetuin-agarose columns. A final
trypanocidal active fraction (gp45), which moved homogeneously during
electrophoresis as a 45-kDa protein, was recovered from the fetuin-agarose
column. The protein reacted positively with thiobarbituric acid, which suggests
it is a sialoglycoprotein. Desialylation of the glycoprotein nullified its
trypanocidal activity on T. congolense. Similarly, when the saccharides,
lactose, methyl-beta-galactoside, lactulose,
methyl-umbelliferyl-beta-galactoside (MU-Gal), were included in the culture
medium, they inhibited the gp45 trypanocidal activity. Asialo-fetuin and
asialo-RBC also inhibited the gp45-induced killing of T. congolense cells. The
potential use of anopheline 45 kDa protein in the production of transgenic
tsetse flies (Glossina spp.) in the control of trypanosomiasis is discussed. A novel method for the in vitro detection of the protozoan Plasmodium, the
causative agent of malaria, has been developed. It comprises a protocol for
cleanup of whole blood samples, followed by direct ultraviolet laser desorption
(LD) time-of-flight mass spectrometry. Intense ion signals are observed from
intact ferriprotoporphyrin IX (heme), sequestered by malaria parasites during
their growth in human red blood cells. The LD mass spectrum of the heme is
structure-specific, and the signal intensities are correlated with the sample
parasitemia (number of parasites per unit volume of blood). Parasitemia levels
on the order of 10 parasites/microL blood can be unambiguously detected by this
method. Consideration of laser beam parameters (spot size, rastering across the
sample surface) and actual sample consumption suggests that the detection limits
can be further improved by at least an order of magnitude. The influence of
experimental factors, such as desorbed ion polarity, laser exposure and fluence,
sample size, and parasite growth stage, on the threshold for parasite detection
is also addressed. Cell-mediated immunity plays a crucial role in the control of many infectious
diseases, necessitating the need for adjuvants that can augment cellular immune
responses elicited by vaccines. It is well established that protection against
one such disease, malaria, requires strong CD8(+) T cell responses targeted
against the liver stages of the causative agent, Plasmodium spp. In this report
we show that the dendritic cell-specific chemokine, dendritic cell-derived CC
chemokine 1 (DC-CK1), which is produced in humans and acts on naive lymphocytes,
can enhance Ag-specific CD8(+) T cell responses when coadministered with either
irradiated Plasmodium yoelii sporozoites or a recombit adenovirus expressing
the P. yoelii circumsporozoite protein in mice. We further show that these
enhanced T cell responses result in increased protection to malaria in immunized
mice challenged with live P. yoelii sporozoites, revealing an adjuvant activity
for DC-CK1. DC-CK1 appears to act preferentially on naive mouse lymphocytes, and
its adjuvant effect requires IL-12, but not IFN-gamma or CD40. Overall, our
results show for the first time an in vivo role for DC-CK1 in the establishment
of primary T cell responses and indicate the potential of this chemokine as an
adjuvant for vaccines against malaria as well as other diseases in which
cellular immune responses are important. Plasmodium falciparum is the causative agent of the most burdensome form of
human malaria, affecting 200-300 million individuals per year worldwide. The
recently sequenced genome of P. falciparum revealed over 5,400 genes, of which
60% encode proteins of unknown function. Insights into the biochemical function
and regulation of these genes will provide the foundation for future drug and
vaccine development efforts toward eradication of this disease. By analyzing the
complete asexual intraerythrocytic developmental cycle (IDC) transcriptome of
the HB3 strain of P. falciparum, we demonstrate that at least 60% of the genome
is transcriptionally active during this stage. Our data demonstrate that this
parasite has evolved an extremely specialized mode of transcriptional regulation
that produces a continuous cascade of gene expression, beginning with genes
corresponding to general cellular processes, such as protein synthesis, and
ending with Plasmodium-specific functionalities, such as genes involved in
erythrocyte invasion. The data reveal that genes contiguous along the
chromosomes are rarely coregulated, while transcription from the plastid genome
is highly coregulated and likely polycistronic. Comparative genomic
hybridization between HB3 and the reference genome strain (3D7) was used to
distinguish between genes not expressed during the IDC and genes not detected
because of possible sequence variations. Genomic differences between these
strains were found almost exclusively in the highly antigenic subtelomeric
regions of chromosomes. The simple cascade of gene regulation that directs the
asexual development of P. falciparum is unprecedented in eukaryotic biology. The
transcriptome of the IDC resembles a "just-in-time" manufacturing process
whereby induction of any given gene occurs once per cycle and only at a time
when it is required. These data provide to our knowledge the first comprehensive
view of the timing of transcription throughout the intraerythrocytic development
of P. falciparum and provide a resource for the identification of new
chemotherapeutic and vaccine candidates. Cyclin dependent protein kinases (CDKs) have become attractive drug targets in
an effort to identify effective inhibitors of the parasite Plasmodium
falciparum, the causative agent of the most severe form of human malaria. We
tested known CDK inhibitors for their ability to inhibit two malarial CDKs:
Pfmrk and PfPK5. Many broad spectrum CDK inhibitors failed to inhibit Pfmrk
suggesting that the active site differs from other CDKs in important ways. By
screening compounds in the Walter Reed chemical database, we identified
oxindole-based compounds as effective inhibitors of Pfmrk (IC(50) = 1.5 microM).
These compounds have low cross-reactivity against PfPK5 and human CDK1
demonstrating selectivity for Pfmrk. Amino acid comparison of the active sites
of Pfmrk and PfPK5 identified unique amino acid differences that may explain
this selectivity and be exploited for further drug development efforts. Plasmodium, the causative agent of malaria, must first infect hepatocytes to
initiate a mammalian infection. Sporozoites migrate through several hepatocytes,
by breaching their plasma membranes, before infection is finally established in
one of them. Here we show that wounding of hepatocytes by sporozoite migration
induces the secretion of hepatocyte growth factor (HGF), which renders
hepatocytes susceptible to infection. Infection depends on activation of the HGF
receptor, MET, by secreted HGF. The malaria parasite exploits MET not as a
primary binding site, but as a mediator of signals that make the host cell
susceptible to infection. HGF/MET signaling induces rearrangements of the
host-cell actin cytoskeleton that are required for the early development of the
parasites within hepatocytes. Our findings identify HGF and MET as potential
targets for new approaches to malaria prevention. Thioredoxin reductase (TrxR) is the homodimeric flavoenzyme that catalyzes
reduction of thioredoxin disulfide (Trx). For Plasmodium falciparum, a causative
agent of tropical malaria, TrxR is an essential protein which has been validated
as a drug target. The high-throughput screening of 350000 compounds has
identified Mannich bases as a new class of TrxR mechanism-based inhibitors.
During catalysis, TrxR conducts reducing equivalents from the NADPH-reduced
flavin to Trx via the two redox-active cysteine pairs, Cys88-Cys93 and
Cys535'-Cys540', referred to as N-terminal and C-terminal cysteine pairs. The
structures of unsaturated Mannich bases suggested that they could act as
bisalkylating agents leading to a macrocycle that involves both C-terminal
cysteines of TrxR. To confirm this hypothesis, different Mannich bases
possessing one or two electrophilic centers were synthesized and first studied
in detail using glutathione as a model thiol. Michael addition of glutathione to
the double bond of an unsaturated Mannich base (3a) occurs readily at
physiological pH. Elimination of the amino group, promoted by base-catalyzed
enolization of the ketone, is followed by addition of a second nucleophile. The
intermediate formed in this reaction is an alpha,beta-unsaturated ketone that
can react rapidly with a second thiol. When studying TrxR as a target of Mannich
bases, we took advantage of the fact that the charge-transfer complex formed
between the thiolate of Cys88 and the flavin in the reduced enzyme can be
observed spectroscopically. The data show that it is the C-terminal Cys
535'-Cys540' pair rather than the N-terminal Cys88-Cys93 pair that is modified
by the inhibitor. Although alkylated TrxR is unable to turn over its natural
substrate Trx, it can reduce low M(r) electron acceptors such as methyl
methanethiolsulfonate by using its unmodified N-terminal thiols. On the basis of
results with chemically distinct Mannich bases, a detailed mechanism for the
inactivation of TrxR is proposed. Plasmodium, the causative agent of malaria, has to undergo sexual
differentiation and development in anopheline mosquitoes for transmission to
occur. To isolate genes specifically induced in both organisms during the early
stages of Plasmodium differentiation in the mosquito, two cDNA libraries were
constructed, one enriched for sequences expressed in differentiating Plasmodium
berghei ookinetes and another enriched for sequences expressed in Anopheles
stephensi guts containing invading ookinetes and early oocysts. Sequencing of
457 ookinete library clones and 652 early oocyst clones represented 175 and 346
unique expressed sequence tags, respectively. Nine of 13 Plasmodium and four of
the five Anopheles novel expressed sequence tags analyzed on Northern blots were
induced during ookinete differentiation and mosquito gut invasion. Ancaspase-7,
an Anopheles effector caspase, is proteolytically activated during Plasmodium
invasion of the midgut. WARP, a gene encoding a Plasmodium surface protein with
a von Willebrand factor A-like adhesive domain, is expressed only in ookinetes
and early oocysts. An anti-WARP polyclonal antibody strongly inhibits (70-92%)
Plasmodium development in the mosquito, making it a candidate antigen for
transmission blocking vaccines. The present results and those of an accompanying
report (Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro,
J. M. C., Dimopoulos G., Kafatos, F. C., Adams, J. H., and Jacobs-Lorena, M.
(2004) J. Biol. Chem. 279, 5581-5587) provide the foundation for further
analysis of Plasmodium differentiation in the mosquito and of mosquito responses
to the parasite. In spite of research efforts to develop vaccines against the causative agent of
human malaria, Plasmodium falciparum, effective control remains elusive. The
predomit vaccine strategy focuses on targeting parasite blood stages in the
vertebrate host. An alternative approach has been the development of
transmission-blocking vaccines (TBVs). TBVs target antigens on parasite sexual
stages that persist within the insect vector, anopheline mosquitoes, or target
mosquito midgut proteins that are presumed to mediate parasite development. By
blocking parasite development within the insect vector, TBVs effectively disrupt
transmission and the resultant cascade of secondary infections. Using a mosquito
midgut-specific mouse monoclonal antibody (MG96), we have partially
characterized membrane-bound midgut glycoproteins in Anopheles gambiae and
Anopheles stephensi. These proteins are present on the microvilli of midgut
epithelial cells in both blood-fed and unfed mosquitoes, suggesting that the
expression of the protein is not induced as a result of blood feeding. MG96
exhibits a dose-dependent blocking effect against Plasmodium yoelii development
in An. stephensi. We achieved 100% blocking of parasite development in the
mosquito midgut. Preliminary deglycosylation assays indicate that the epitope
recognized by MG96 is a complex oligosaccharide. Future investigation of the
carbohydrate epitope as well as gene identification should provide valuable
insight into the possible mechanisms of ookinete attachment and invasion of
mosquito midgut epithelial cells. Plasmodium falciparum, the causative agent of the most lethal form of human
malaria, relies on de novo pyrimidine biosynthesis. A gene encoding orotate
phosphoribosyltransferase (OPRT), the fifth enzyme of the de novo pathway
catalyzing formation of orotidine 5'-monophosphate (OMP) and pyrophosphate
(PP(i)) from 5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate, was identified
from P. falciparum (pfOPRT). The deduced amino acid sequence for pfOPRT was
compared with OPRTs from other organisms and found to be most similar to that of
Escherichia coli. The catalytic residues and consensus sequences for substrate
binding in the enzyme were conserved among other organisms. The pfOPRT was
exceptional in that it contained a unique insertion of 20 amino acids and an
amino-terminal extension of 66 amino acids, making the longest amino acid
sequence (281 amino acids with a predicted molecular mass of 33kDa). The cDNA of
the pfOPRT gene was cloned, sequenced and functionally expressed in soluble
form. The recombit pfOPRT was purified from the E. coli lysate by two steps,
nickel metal-affinity and gel-filtration chromatography. From 1l E. coli
culture, 1.2-1.5mg of pure pfOPRT was obtained. SDS-PAGE revealed that the
pfOPRT had a molecular mass of 33kDa and analytical gel-filtration
chromatography showed that the enzyme activity eluted at approximately 67kDa.
Using dimethyl suberimidate to cross-link neighboring subunits of the pfOPRT, it
was confirmed that the native enzyme exists in a dimeric form. The steady state
kinetics of initial velocity and product inhibition studies indicate that the
enzyme pfOPRT follows a random sequential kinetic mechanism. Compounds aimed at
the pfOPRT nexus may act against the parasite through at least two mechanisms:
by directly inhibiting the enzyme activity, or be processed to an inhibitor of
thymidylate synthase. This study provides a working system with which to
investigate new antimalarial agents targeted against P. falciparum OPRT. Antibacterial, antiparasitidal and antiviral properties have recently been
attributed to members of the secreted phospholipases A(2) (sPLA(2)s)
superfamily. Seven sPLA(2)s from groups IA, IB, IIA and III, were tested here in
different culture conditions for inhibition of the in vitro intraerythrocytic
development of Plasmodium falciparum, the causative agent of the most severe
form of human malaria. In the presence of human serum, all sPLA(2)s were
inhibitory, with three out of seven exhibiting IC(50)<0.1 nM. In all cases,
inhibition could be induced by enzymatic pre-treatment of the serum. By
contrast, no effect was observed when parasites were grown in a semi-defined
medium (AlbuMAX II) devoid of lipoproteins and containing 10 times less
phospholipids than the medium with human serum, strongly suggesting that
hydrolysis of serum generating toxic lipid by-products, rather than a direct
interaction of the sPLA(2) with the infected erythrocyte, is a general feature
of the anti-Plasmodium properties of sPLA(2)s. Furthermore, in serum, six out of
the seven sPLA(2)s were toxic against both trophozoite and schizont stages of
the parasite development, contrasting with the trophozoite-selective bee venom
enzyme's toxicity. Deciphering the molecular mechanisms at play in the
phenotypic singularity of the bee venom enzyme toxicity might offer new
prospects in antimalarial fight. Plasmodium falciparum is the causative agent of the most severe form of human
malaria. The rapid multiplication of the parasite within human erythrocytes
requires an active production of new membranes. Phosphatidylcholine is the most
abundant phospholipid in Plasmodium membranes, and the pathways leading to its
synthesis are attractive targets for chemotherapy. In addition to its synthesis
from choline, phosphatidylcholine is synthesized from serine via an unknown
pathway. Serine, which is actively transported by Plasmodium from human serum
and readily available in the parasite, is subsequently converted into
phosphoethanolamine. Here, we describe in P. falciparum a plant-like
S-adenosyl-l-methionine-dependent three-step methylation reaction that converts
phosphoethanolamine into phosphocholine, a precursor for the synthesis of
phosphatidylcholine. We have identified the gene, PfPMT, encoding this activity
and shown that its product is an unusual phosphoethanolamine methyltransferase
with no human homologs. P. falciparum phosphoethanolamine methyltransferase
(Pfpmt) is a monopartite enzyme with a single catalytic domain that is
responsible for the three-step methylation reaction. Interestingly, Pfpmt
activity is inhibited by its product phosphocholine and by the phosphocholine
analog, miltefosine. We show that miltefosine can also inhibit parasite
proliferation within human erythrocytes. The importance of this enzyme in P.
falciparum membrane biogenesis makes it a potential target for malaria
chemotherapy. Plasmodium falciparum, the causative agent of malaria, relies extensively on
glycolysis coupled with homolactic fermentation during its blood-borne stages
for energy production. Selective inhibitors of the parasite lactate
dehydrogenase (LDH), central to NAD(+) regeneration, therefore potentially
provide a route to new antimalarial drugs directed against a novel molecular
target. A series of heterocyclic, azole-based compounds are described that
preferentially inhibit P. falciparum LDH at sub-micromolar concentrations,
typically at concentrations about 100-fold lower than required for human lactate
dehydrogenase inhibition. Crystal structures show these competitive inhibitors
form a network of interactions with amino acids within the active site of the
enzyme, stacking alongside the nicotinamide ring of the NAD(+) cofactor. These
compounds display modest activity against parasitized erythrocytes, including
parasite strains with known resistance to existing anti-malarials and against
Plasmodium berghei in BALB/c mice. Initial toxicity data suggest the azole
derivatives have generally low cytotoxicity, and preliminary pharmoco-kinetic
data show favorable bioavailability and circulation times. These encouraging
results suggest that further enhancement of these structures may yield
candidates suitable for consideration as new therapeutics for the treatment of
malaria. In combination these studies also provide strong support for the
validity of targeting the Plasmodium glycolytic pathway and, in particular, LDH
in the search for novel anti-malarials. Plasmodium falciparum, the causative agent of the most lethal form of human
malaria, totally depends on de novo pyrimidine biosynthetic pathway. Orotate
phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase
(OMPDC), the fifth and sixth enzymes in the pathway catalyzing formation of
uridine 5'-monophosphate (UMP), remain largely uncharacterized in the protozoan
parasite. In this study, we achieved purification of OPRT and OMPDC to near
homogeneity from P. falciparum cultivated in vitro. The OPRT and OMPDC
activities were co-eluted in all chromatographic columns during purification,
suggesting the purified proteins exist as a multienzyme complex with a molecular
mass of 140+/-8 kDa and contain two subunits each of OPRT and OMPDC. Monomeric
forms of OPRT and OMPDC had molecular masses of 32+/-3 and 38+/-3 kDa,
respectively, in agreement with those of proteins predicted from P. falciparum
genome database. Interestingly, kinetic parameters and inhibitory constants of
both OPRT and OMPDC activities were found to be different to those of the
bifunctional human red cell UMP synthase. Our evidence provides the first
example of OPRT and OMPDC existing as a multienzyme complex. Comparative genomic analysis of the malaria causative agent, Plasmodium
falciparum, with other eukaryotes for which the complete genome is available,
revealed that the genome from P. falciparum was more similar to the genome of a
plant, Arabidopsis thaliana, than to other non-apicomplexan taxa. Plant-like
sequences are thought to result from horizontal gene transfers after a secondary
endosymbiosis involving an algal ancestor. The use of the A. thaliana genome and
proteome as a reference gives an opportunity to refine our understanding of the
extreme compositional bias in the P. falciparum genome that leads to a
proteome-wide amino acid bias. A set of pairs of non-redundant protein
homologues was selected owing to rigorous genome-wide sequence comparison
methods. The introduction of A. thaliana as a reference was a mean to weight the
magnitude of the protein evolutionary divergence in P. falciparum. The
correlation of the amino acid proportions with evolutionary time supports the
hypothesis that amino acids encoded by GC-rich codons are directionally
substituted into amino acids encoded by AT-rich codons in the P. falciparum
proteome. The long-term deviation of codons in malarial sequences appears as a
possible consequence of a genome-wide tri-nucleotidic signature imprinting.
Additionally, this study suggests possible working guidelines to improve the
accuracy of P. falciparum sequence comparisons, for homology searches and
phylogenetic studies. Plasmodium falciparum, the causative agent of the most lethal form of human
malaria, uses multiple ligand-receptor interactions to invade host red blood
cells (RBCs). We studied the invasion of P falciparum into abnormal RBCs from
humans carrying the Southeast Asian ovalocytosis (SAO) trait. One particular
parasite line, 3D7-A, invaded these cells efficiently, whereas all other lines
studied invaded SAO RBCs to only about 20% of the extent of normal (non-SAO)
cells. This result is consistent with the clinical observation that SAO
individuals can experience high-density P falciparum infections and provides an
explanation for previous discrepant results on invasion of SAO RBCs.
Characterization of the invasion phenotype of 3D7-A revealed that efficient
invasion of SAO RBCs was paralleled by relatively efficient invasion of normal
RBCs treated with either neuraminidase, trypsin, or chymotrypsin and a novel
capacity to invade normal RBCs treated sequentially with both neuraminidase and
trypsin. Our results suggest that only parasites able to use some particular
invasion pathways can invade SAO RBCs efficiently in culture. A similar
situation might occur in the field. In this study, the epidemiological characteristics of malaria cases in Edirne
province were investigated. Between the years of 1994-2002, a total of 317,087
blood samples were collected from soldiers in the province with selective active
surveillance and from the resident population with active or passive
surveillance methods, by the medical staff of Malaria Control Department and
Health Centers, to search the presence of Plasmodium. In 281 of them Plasmodium
spp. were detected, and the characteristics of malaria cases were investigated.
Of the cases, 238 (84.7%) were detected in the first three years and mostly in
September. While the indigenous cases were detected in the districts where rice
planted intensely, the imported cases were detected in the districts heavily
populated by military staff. Of the imported cases, 62% originated from
Diyarbakir, Batman and Sanliurfa provinces (Southeast part of Turkey). P. vivax
was detected as the causative agent in all blood samples except one P. ovale.
This latter case has been the only one in Turkey so far and he was a student
from Afghanistan. Attaching importance to fight off mosquitoes in intensely rice
planted districts and strictly surveying the military staff, particularly from
the region of Southern-East Anatolia, have led to successful control of the
malaria cases in Edirne region. In the Republic of Yemen, Plasmodium falciparum is the predomit causative
agent of malaria and is associated with adverse consequences for pregt women
and their babies. The prevalence and clinical manifestations of malaria among
500 pregt (260) and non-pregt (240) women were compared. Clinical
examinations, laboratory investigations and a structured questionnaire were used
to collect data. The prevalence of malaria was higher among pregt women (55%)
than non-pregt women (20%). Anaemia was significantly more prevalent among
pregt woman than non-pregt women and also more prevalent in pregt women
with malaria than non-pregt women with malaria. Plasmodium falciparum is the main causative agent of tropical malaria, the most
severe parasitic disease in the world. Growing resistance of Plasmodia towards
available drugs is an increasing problem in countries where malaria is endemic.
As Plasmodia are sensitive to oxidative stress, augmenting this in the parasite
represents a promising principle for the development of novel antimalarial
drugs. The NADP-dependent glutamate dehydrogenase (GDH) of P.falciparum is
largely responsible for the production of NADPH in the parasite, which in turn
serves as electron source for the antioxidative enzymes glutathione reductase
and thioredoxin reductase. As GDH does not occur in the host erythrocyte, GDH is
a particularly attractive target for drug therapy. The three-dimensional
structure of P.falciparum GDH in the unligated state has been determined by
X-ray crystallography to a resolution of 2.7A. Compared to the mammalian
enzymes, two amino acid residues are exchanged in the putative active site of
the parasite GDH. The most obvious differences between parasite and human GDH
are the subunit interfaces of the hexameric proteins. In the parasite protein,
several salt-bridges mediate contacts between the subunits whereas in the human
enzyme these interactions are mainly of hydrophobic nature. Furthermore,
P.falciparum GDH possesses a unique N-terminal extension that does not occur in
any other GDH sequence so far studied. These findings might be exploited for the
design of peptidomimetics capable of disrupting the oligomeric organisation of
the parasite enzyme. BACKGROUND: Malaria affects 200-300 million individuals per year worldwide.
Plasmodium falciparum is the causative agent of the most severe and mortal type
of malaria. The need for new antimalarials comes from the widespread resistance
to those in current use. New antimalarial targets are required to increase
chemical diversity and effectiveness of the drugs. The research for such new
targets and drug chemotypes is aided by structure-based drug design. We present
a model of the TBP-TFIIB complex from P. falciparum (pfTBP-pfTFIIB) and a
detailed study of the interactions at the TBP-TFIIB interface.
METHODS: The model was built using standard methodology, optimized energetically
and evaluated structurally. We carried out an analysis of the interface
considering its evolution, available experimental data on TBP and TFIIB mutants,
and the main conserved and non-conserved interactions. To support the
perspective of using this complex as a new target for rational antimalarial
design, we present the comparison of the pfTBP-pfTFIIB interface with its human
homolog.
RESULTS: Despite the high residue conservation at the interface, we identified a
potential region, composed of species-specific residues that can be used for
rational antimalarial design.
CONCLUSIONS: Currently there are no antimalarial drugs targeted to stop the
nuclear transcription process, a vital event for all replication stages of P.
falciparum. Due to its absolute requirement in transcription initiation, we
consider the pfTBP-pfTFIIB interface as a new potential target for novel
antimalarial chemotypes. BACKGROUND: To date, only a few transcription factors have been identified in
the genome of the parasite Plasmodium falciparum, the causative agent of
malaria. Moreover, no detailed molecular analysis of its basal transcription
machinery, which is otherwise well-conserved in the crown group of eukaryotes,
has yet been reported. In this study, we have used a combination of sensitive
sequence analysis methods to predict the existence of several parasite encoded
general transcription factors associated with RNA polymerase II.
RESULTS: Several orthologs of general transcription factors associated with RNA
polymerase II can be predicted among the hypothetical proteins of the P.
falciparum genome using the two-dimensional Hydrophobic Cluster Analysis (HCA)
together with profile-based search methods (PSI-BLAST). These predicted
orthologous genes encoding putative transcription factors include the large
subunit of TFIIA and two candidates for its small subunit, the TFIIE
beta-subunit, which would associate with the previously known TFIIE
alpha-subunit, the TFIIF beta-subunit, as well as the p62/TFB1 subunit of the
TFIIH core. Within TFIID, the putative orthologs of TAF1, TAF2, TAF7 and TAF10
were also predicted. However, no candidates for TAFs with classical histone fold
domain (HFD) were found, suggesting an unusual architecture of TFIID complex of
RNA polymerase II in the parasite.
CONCLUSION: Taken together, these results suggest that more general
transcription factors may be present in the P. falciparum proteome than
initially thought. The prediction of these orthologous general transcription
factors opens the way for further studies dealing with transcriptional
regulation in P. falciparum. These alternative and sensitive sequence analysis
methods can help to identify candidates for other transcriptional regulatory
factors in P. falciparum. They will also facilitate the prediction of biological
functions for several orphan proteins from other apicomplexan parasites such as
Toxoplasma gondii, Cryptosporidium parvum and Eimeria. IFN-gamma secretion by natural killer (NK) cells is pivotal to several tumor and
viral immune responses, during which NK and dendritic cells cooperation is
required. We show here that macrophages are mandatory for NK cell IFN-gamma
secretion in response to erythrocytes infected with Plasmodium falciparum (Pf),
a causative agent of human malaria. In addition, direct sensing of Pf infection
by NK cells induces their production of the proinflammatory chemokine CXCL8,
without triggering their granule-mediated cytolytic programs. Despite their
reported role in Pf recognition, Toll-like receptor (TLR) 2, TLR9, and TLR11 are
individually dispensable for NK cell activation induced by Pf-infected
erythrocytes. However, IL-18R expression on NK cells, IL-18 production by
macrophages, and MyD88 on both cell types are essential components of this
previously undescribed pathway of NK cell activation in response to a parasite
infection. Plasmodium vivax is an important human pathogen causing malaria in more
temperate climates of the world. Similar to Plasmodium falciparum, the causative
agent for malaria tropica, drug resistance is beginning to emerge for this
parasite species and this hampers adequate treatment of infection. We have used
a short-term ex vivo drug assay to monitor activity of OZ277 (RBx-11160), a
fully synthetic anti-malarial peroxide, and the diamidine DB75 against P. vivax.
For both compounds as well as the anti-malarial reference compounds artesunate,
artemether, and chloroquine, the in vitro IC(50) values were determined in
one-cycle hypoxanthine incorporation assays. Results from such assays were found
to be very similar compared to IC(50) values obtained from one-cycle P.
falciparum hypoxanthine assays. We demonstrate the anti-parasite activity of
OZ277 and the reference compounds to be faster than that of DB75. These data
warrant clinical testing of OZ277 against P. vivax malaria and support recent
data on clinical activity against P. vivax for DB75. Plasmodium falciparum, the causative agent of the fatal form of malaria,
synthesizes GMP primarily from IMP and, hence, needs active GMPS (GMP
synthetase) for its survival. GMPS, a G-type amidotransferase, catalyses the
amination of XMP to GMP with the reaction occurring in two domains, the GAT
(glutamine amidotransferase) and ATPPase (ATP pyrophosphatase). The GAT domain
hydrolyses glutamine to glutamate and ammonia, while the ATPPase domain
catalyses the formation of the intermediate AMP-XMP from ATP and XMP.
Co-ordination of activity across the two domains, achieved through channelling
of ammonia from GAT to the effector domain, is the hallmark of
amidotransferases. Our studies aimed at understanding the kinetic mechanism of
PfGMPS (Plasmodium falciparum GMPS) indicated steady-state ordered binding of
ATP followed by XMP to the ATPPase domain with glutamine binding in a random
manner to the GAT domain. We attribute the irreversible, Ping Pong step seen in
initial velocity kinetics to the release of glutamate before the attack of the
adenyl-XMP intermediate by ammonia. Specific aspects of the overall kinetic
mechanism of PfGMPS are different from that reported for the human and
Escherichia coli enzymes. Unlike human GMPS, absence of tight co-ordination of
activity across the two domains was evident in the parasite enzyme. Variations
seen in the inhibition by nucleosides and nucleotide analogues between human
GMPS and PfGMPS highlighted differences in ligand specificity that could serve
as a basis for the design of specific inhibitors. The present study represents
the first report on recombit His-tagged GMPS from parasitic protozoa. We have evaluated a technology called transcriptionally active PCR (TAP) for
high throughput identification and prioritization of novel target antigens from
genomic sequence data using the Plasmodium parasite, the causative agent of
malaria, as a model. First, we adapted the TAP technology for the highly AT-rich
Plasmodium genome, using well-characterized P. falciparum and P. yoelii antigens
and a small panel of uncharacterized open reading frames from the P. falciparum
genome sequence database. We demonstrated that TAP fragments encoding six
well-characterized P. falciparum antigens and five well-characterized P. yoelii
antigens could be amplified in an equivalent manner from both plasmid DNA and
genomic DNA templates, and that uncharacterized open reading frames could also
be amplified from genomic DNA template. Second, we showed that the in vitro
expression of the TAP fragments was equivalent or superior to that of
supercoiled plasmid DNA encoding the same antigen. Third, we evaluated the in
vivo immunogenicity of TAP fragments encoding a subset of the model P.
falciparum and P. yoelii antigens. We found that antigen-specific antibody and
cellular immune responses induced by the TAP fragments in mice were equivalent
or superior to those induced by the corresponding plasmid DNA vaccines. Finally,
we developed and demonstrated proof-of-principle for an in vitro humoral
immunoscreening assay for down-selection of novel target antigens. These data
support the potential of a TAP approach for rapid high throughput functional
screening and identification of potential candidate vaccine antigens from
genomic sequence data. In Plasmodium falciparum, the causative agent of cerebral malaria, silent
information regulator 2 (Sir2) has been implicated in pathogenesis through its
role in var gene silencing. P. falciparum Sir2 (PfSir2) in addition to the
catalytic core, has a 13 residue N-terminal and 4 residue C-terminal extension
over the shorter Archaeoglobus fulgidus Sir2. In this paper, we highlight our
studies aimed at understanding the kinetic mechanism of PfSir2 and the role of
N- and C-terminal extensions in protein function and oligomerization.
Bisubstrate kinetic analysis showed that PfSir2 exhibits a rapid equilibrium
ordered sequential mechanism, with peptide binding preceding NAD(+). This study
also reports on surfactin as a novel Sir2 inhibitor exhibiting competitive
inhibition with respect to NAD(+) and uncompetitive inhibition with acetylated
peptide. This inhibition pattern with surfactin provides further support for
ordered binding of substrates. Surfactin was also found to be a potent inhibitor
of intra-erythrocytic growth of P. falciparum with 50% inhibitory concentration
in the low micromolar range. PfSir2, like the yeast homologs (yHst2 and Sir2p),
is a trimer in solution. However, dissociation of trimer to monomers in the
presence of NAD(+) is characteristic of the parasite enzyme. Oligomerization
studies on N- and/or C-terminal deletion constructs of PfSir2 highlight the role
of C-terminus of the protein in mediating homotrimerization. N-terminal deletion
resulted in reduced catalytic efficiency although substrate affinity was not
altered in the constructs. Interestingly, deletion of both the ends relaxed
NAD(+) specificity. An essential requisite for transmission of Plasmodium, the causative agent of
malaria, is the successful completion of a complex developmental cycle in its
mosquito vector. Of hundreds of ookinetes that form in the mosquito midgut, only
few transform into oocysts, a loss attributed to the action of the mosquito
immune system. However, once oocysts form, they appear to be resistant to
mosquito defences. During oocyst development, a thick capsule forms around the
parasite and appears to function as a protective cover. Little information is
available about the composition of this capsule. Here we report on the
identification and partial characterization of the first Plasmodium oocyst
capsule protein (PbCap380). Genetic analysis indicates that the gene is
essential and that PbCap380(-) mutant parasites form oocysts in normal numbers
but are gradually eliminated. As a result, mosquitoes infected with PbCap380(-)
parasites do not transmit malaria. Targeting of the oocyst capsule may provide a
new strategy for malaria control. The causative agent of malaria, Plasmodium falciparum posses a single
aquaglyceroporin (PfAQP) which represents a potential drug target for treatment
of the disease. PfAQP is localized to the parasite membrane to transport water,
glycerol, ammonia and possibly glycolytic intermediates. In order to enable
design of inhibitors we set out to determine the 3D structure of PfAQP, where
the first bottleneck to overcome is achieving high enough yield of recombit
protein. The wild type PfAQP gene was expressed to low or undetectable levels in
the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to
be due to different genomic A+T content and different codon usage. Thus, two
codon-optimized PfAQP genes were generated. The Opt-PfAQP for E. coli still did
not result in high production yields, possibly due to folding problems. However,
PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for
production and in Saccharomyces cerevisiae for functional studies. In S.
cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport
could not be confirmed. Following high-level membrane-localized expression in P.
pastoris (estimated to 64mg PfAQP per liter cell culture) PfAQP was purified to
homogeneity (18mg/L) and initial attempts at crystallization of the protein
yielded several different forms. Plasmodium falciparum, the causative agent of malaria, relies on a complex
protein-secretion system for protein targeting into numerous subcellular
destinations. Recently, a homologue of the Golgi re-assembly stacking protein
(GRASP) was identified and used to characterise the Golgi organisation in this
parasite. Here, we report on the presence of a splice variant that leads to the
expression of a GRASP isoform. Although the first GRASP protein (GRASP1) relies
on a well-conserved myristoylation motif, the variant (GRASP2) displays a
different N-terminus, similar to GRASPs found in fungi. Phylogenetic analyses
between GRASP proteins of numerous taxa point to an independent evolution of the
unusual N-terminus that could reflect unique requirements for Golgi-dependent
protein sorting and organelle biogenesis in P. falciparum. Golgi association of
GRASP2 depends on the hydrophobic N-terminus that resembles a signal anchor,
leading to a unique mode of Golgi targeting and membrane attachment. Ferroquine (FQ or SR97193) is a unique ferrocene antimalarial drug candidate
which just entered phase IIb clinical trials in autumn 2007. FQ is able to
overcome the chloroquine (CQ) resistance problem, an important limit to the
control of Plasmodium falciparum, the principal causative agent of malaria.
However, as for other therapeutic agents such as chloroquine (CQ) and artemisin,
its mechanism of action remains partially unknown. Most investigations have so
far focused on comparing the activity of FQ to that of CQ in order to understand
how the ferrocene core contributes to a stronger antiplasmodial activity.
Studies have already shown that the ferrocene altered the shape, volume,
lipophilicity, basicity and also electronic profile of the parent molecule and,
hence, its pharmacodynamic behavior. However, few investigations have been
undertaken to probe the real contribution of redox properties of the ferrocene
(iron(II))/ferricinium (iron(III)) system in FQ as reported in this article. In
our experimental and theoretical approach, we considered the redox profile of
the ferrocene core of FQ in the specific conditions (acidic and oxidizing) of
the parasitic digestive vacuole as a possible discriminating property from CQ in
the antimalarial activity. In tropical regions millions of people still live at risk of malaria infection.
Indeed the emergence of resistance to chloroquine and other drugs in use in
these areas reinforces the need to implement alternative prophylactic
strategies. Genistein is a naturally occurring compound that is widely used as a
food supplement and is thought to be effective in countering several
pathologies. Results presented here show that genistein inhibits liver infection
by the Plasmodium parasite, the causative agent of malaria. In vitro, genistein
decreased the infection rates of both mouse and human hepatoma cells by
inhibiting the early stages of the parasite's intracellular development. Oral or
intraperitoneal administration of genistein decreased the liver parasite load of
P. berghei-infected mice. Moreover, mice fed on a genistein-supplemented diet
showed a significant reduction in Plasmodium liver infection as well as a
reduced blood parasitemia and partial protection from severe disease. Since
genistein is a safe, low-cost, natural compound that can be used permanently in
a diet, we propose its use as a prophylactic agent against malaria for endemic
populations and long-time travelers. A three-dimensional structure of histo-aspartic protease (HAP), a pepsin-like
enzyme from the causative agent of malaria Plasmodium falciparum, is suggested
on the basis of homologous modeling followed by equilibration by the method of
molecular dynamics. The presence of a His residue in the catalytic site instead
of an Asp residue, which is characteristic of pepsin-like enzymes, and
replacement of some other conserved residues in the active site make it possible
for the enzyme to function by the covalent mechanism inherent in serine
proteases. The detailed structures of HAP complexes with pepstatin, a
noncovalent inhibitor of aspartic proteases, and phenylmethylsulfonyl fluoride,
a covalent inhibitor of serine proteases, as well as with a pentapeptide
substrate are discussed. BACKGROUND: Of the 5,484 predicted proteins of Plasmodium falciparum, the main
causative agent of malaria, about 60% do not have sufficient sequence similarity
with proteins in other organisms to warrant provision of functional assignments.
Non-homology methods are thus needed to obtain functional clues for these
uncharacterized genes.
RESULTS: We present PlasmoDraft http://atgc.lirmm.fr/PlasmoDraft/, a database of
Gene Ontology (GO) annotation predictions for P. falciparum genes based on
postgenomic data. Predictions of PlasmoDraft are achieved with a Guilt By
Association method named Gonna. This involves (1) a predictor that proposes GO
annotations for a gene based on the similarity of its profile (measured with
transcriptome, proteome or interactome data) with genes already annotated by
GeneDB; (2) a procedure that estimates the confidence of the predictions
achieved with each data source; (3) a procedure that combines all data sources
to provide a global summary and confidence estimate of the predictions. Gonna
has been applied to all P. falciparum genes using most publicly available
transcriptome, proteome and interactome data sources. Gonna provides predictions
for numerous genes without any annotations. For example, 2,434 genes without any
annotations in the Biological Process ontology are associated with specific GO
terms (e.g. Rosetting, Antigenic variation), and among these, 841 have
confidence values above 50%. In the Cellular Component and Molecular Function
ontologies, 1,905 and 1,540 uncharacterized genes are associated with specific
GO terms, respectively (740 and 329 with confidence value above 50%).
CONCLUSION: All predictions along with their confidence values have been
compiled in PlasmoDraft, which thus provides an extensive database of GO
annotation predictions that can be achieved with these data sources. The
database can be accessed in different ways. A global view allows for a quick
inspection of the GO terms that are predicted with high confidence, depending on
the various data sources. A gene view and a GO term view allow for the search of
potential GO terms attached to a given gene, and genes that potentially belong
to a given GO term. A new lycorine derivative LT1 (4) was isolated from the aerial part and bulbs of
Lycoris traubii Hayward (Amaryllidaceae). Its structure including absolute
configuration was established by spectroscopic analysis and semi-synthesis to be
1-O-(3'S)-hydroxybutanoyllycorine. Some lycorine ester derivatives including LT1
were examined for their inhibitory activity against Trypanosoma brucei brucei,
the parasite associated with sleeping sickness, and against Plasmodium
falciparum, the causative agent of malaria. Among them, 2-O-acetyllycorine (6)
showed the most potent activity against parasitic T. b. brucei, and LT1 (4),
1-O-(3'R)-hydroxybutanoyllycorine (8), 1,2-di-O-butanoyllycorine (11), and
1-O-propanoyllycorine (12) showed significant activity against P. falciparum in
an in vitro experiment. Ferroquine (FQ or SR97193) is a novel antimalarial drug candidate, currently in
development at Sanofi-Aventis. In contrast to conventional drugs, FQ is the
first organometallic drug: a ferrocenyl group covalently flanked by a
4-aminoquinoline and a basic alkylamine. FQ is able to overcome the CQ
resistance problem, an important limit to the control of Plasmodium falciparum,
the principal causative agent of malaria. After fifteen years of effort, it is
now possible to propose a multifactorial mechanism of action of FQ by its
capacity to target lipids, to inhibit the formation of hemozoin and to generate
reactive oxygen species. Malaria is a devastating disease. For transmission to occur, Plasmodium, the
causative agent of malaria, must complete a complex developmental cycle in its
mosquito vector. Thus, the mosquito is a potential target for disease control.
Plasmodium ookinetes, which develop within the mosquito midgut, must first cross
the midgut's peritrophic matrix (PM), a thick extracellular sheath that
completely surrounds the blood meal. The PM poses a partial, natural barrier
against parasite invasion of the midgut and it is speculated that modifications
to the PM may lead to a complete barrier to infection. However, such strategies
require thorough characterization of the structure of the PM. Here, we describe
for the first time, the complete PM proteome of the main malaria vector,
Anopheles gambiae. Altogether, 209 proteins were identified by mass
spectrometry. Among them were nine new chitin-binding peritrophic matrix
proteins, expanding the list from three to twelve peritrophins. Lastly, we
provide a model for the putative interactions among the proteins identified in
this study. L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent
for the most severe form of malaria, has shown remarkable similarities to L:
-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs
characterized in archae and other prokaryotes. Initial sequence analysis and
identification of critical amino acid residues involved in inter-subunit
salt-bridge interactions predict tetrameric structure for PfMDH. The
catalytically active recombit PfMDH was characterized as a tetramer. The
enzyme is localized primarily in the parasites cytosol. To gain molecular
insights into PfMDH/PfLDH relationships and to understand the quaternary
structure of PfMDH, dimers were generated by mutation to the potential
salt-bridge interacting sites. The R183A and R214G mutations, which snapped the
salt bridges between the dimers and resulted in lower dimeric state, did not
affect catalytic properties of the enzyme. The mutant dimers of PfMDH were
active equally as the wild-type PfMDH. The studies reveal structure of PfMDH as
a dimer of dimers. The tetrameric state of PfMDH was not essential for catalytic
functions of the enzyme but may be an evolutionary adaptation for cytosolic
localization to support its role in NAD/NADH coupling, an important metabolic
function for survival of the malaria parasite. BACKGROUND: Malaria is a global health emergency, and yet our understanding of
the energy metabolism of the principle causative agent of this devastating
disease, Plasmodium falciparum, remains rather basic. Glucose was shown to be an
essential nutritional requirement nearly 100 years ago and since this original
observation, much of the current knowledge of Plasmodium energy metabolism is
based on early biochemical work, performed using basic analytical techniques
(e.g. paper chromatography), carried out almost exclusively on avian and rodent
malaria. Data derived from malaria parasite genome and transcriptome studies
suggest that the energy metabolism of the parasite may be more complex than
hitherto anticipated. This study was undertaken in order to further characterize
the fate of glucose catabolism in the human malaria parasite, P. falciparum.
METHODS: Products of glucose catabolism were determined by incubating
erythrocyte-freed parasites with D-[1-13C] glucose under controlled conditions
and metabolites were identified using 13C-NMR spectroscopy.
RESULTS: Following a 2 h incubation of freed-P. falciparum parasites with 25 mM
D-[1-13C] glucose (n = 4), the major metabolites identified included; [3-13C]
lactate, [1,3-13C] glycerol, [3-13C] pyruvate, [3-13C] alanine and [3-13C]
glycerol-3-phosphate. Control experiments performed with uninfected erythrocytes
incubated under identical conditions did not show any metabolism of D-[1-13C]
glucose to glycerol or glycerol-3-phosphate.
DISCUSSION: The identification of glycerol as a major glucose metabolite
confirms the view that energy metabolism in this parasite is more complex than
previously proposed. It is hypothesized here that glycerol production by the
malaria parasite is the result of a metabolic adaptation to growth in O2-limited
(and CO2 elevated) conditions by the operation of a glycerol-3-phosphate shuttle
for the re-oxidation of assimilatory NADH. Similar metabolic adaptations have
been reported previously for other microaerobic/anaerobic organisms, such as
yeast, rumen protozoa and human parasitic protozoa.
CONCLUSION: These data highlight the need to re-evaluate the carbon and redox
balance of this important human pathogen, ultimately leading to a better
understanding of how the parasite is able to adapt to the variable environments
encountered during parasite development and disease progression. Plasmodium falciparum, the most important causative agent of human malaria,
undergoes antigenic variation as a means of prolonging infection and ensuring
transmission between hosts. Clonal variation is observed in the surface adhesins
expressed on infected erythrocytes: primarily in the PfEMP1 adhesin encoded by
the large var gene family. The sirtuin PfSIR2A was the first protein discovered
to have a major influence on antigenic variation in P. falciparum. In the
absence of PfSIR2A, normal silencing of the variantly-expressed var gene family
is partially deregulated. To thoroughly investigate the role of PfSIR2A in
controlling antigenic variation, multiple independent clones of wildtype and
PfSIR2A-knockout (DeltaSir2a) parasites were generated. var gene expression was
then measured qualitatively, quantitatively and longitudinally over extended
periods in culture. DeltaSir2a parasites were found to activate about 10
specific var genes in every independent clone analyzed. The activated genes were
biased towards the upsA, upsBA and upsEvar gene subclasses. The total var
transcript level was two to three-fold higher in DeltaSir2a parasites than in
wildtype parasites and at least one transcript - encoding the pregcy malaria
adhesin VAR2CSA - was successfully translated and expressed on the infected cell
surface. In the absence of PfSIR2A, antigenic switching over time was also
diminished, although not abolished. This work expands our understanding of
clonal antigenic variation in this important human pathogen and demonstrates a
central role for PfSIR2A in regulating both the variant expression of specific
var gene subsets and the overall quantity of var gene expression. Plasmodium falciparum, the causative agent of maligt malaria, is among the
most severe human infectious diseases. The closest known relative of P.
falciparum is a chimpanzee parasite, Plasmodium reichenowi, of which one single
isolate was previously known. The co-speciation hypothesis suggests that both
parasites evolved separately from a common ancestor over the last 5-7 million
years, in parallel with the divergence of their hosts, the hominin and
chimpanzee lineages. Genetic analysis of eight new isolates of P. reichenowi,
from wild and wild-born captive chimpanzees in Cameroon and Côte d'Ivoire, shows
that P. reichenowi is a geographically widespread and genetically diverse
chimpanzee parasite. The genetic lineage comprising the totality of global P.
falciparum is fully included within the much broader genetic diversity of P.
reichenowi. This finding is inconsistent with the co-speciation hypothesis.
Phylogenetic analysis indicates that all extant P. falciparum populations
originated from P. reichenowi, likely by a single host transfer, which may have
occurred as early as 2-3 million years ago, or as recently as 10,000 years ago.
The evolutionary history of this relationship may be explained by two critical
genetic mutations. First, inactivation of the CMAH gene in the human lineage
rendered human ancestors unable to generate the sialic acid Neu5Gc from its
precursor Neu5Ac, and likely made humans resistant to P. reichenowi. More
recently, mutations in the domit invasion receptor EBA 175 in the P.
falciparum lineage provided the parasite with preference for the overabundant
Neu5Ac precursor, accounting for its extreme human pathogenicity. Infection by Plasmodium, the causative agent of malaria, is associated with
hemolysis and therefore with release of hemoglobin from RBC. Under inflammatory
conditions, cell-free hemoglobin can be oxidized, releasing its heme prosthetic
groups and producing deleterious free heme. Here we demonstrate that survival of
a Plasmodium-infected host relies strictly on its ability to prevent the
cytotoxic effects of free heme via the expression of the heme-catabolyzing
enzyme heme oxygenase-1 (HO-1; encoded by the Hmox1 gene). When infected with
Plasmodium chabaudi chabaudi (Pcc), wild-type (Hmox1(+/+)) BALB/c mice resolved
infection and restored homeostasis thereafter (0% lethality). In contrast, HO-1
deficient (Hmox1(-/-)) BALB/c mice developed a lethal form of hepatic failure
(100% lethality), similar to the one occurring in Pcc-infected DBA/2 mice (75%
lethality). Expression of HO-1 suppresses the pro-oxidant effects of free heme,
preventing it from sensitizing hepatocytes to undergo TNF-mediated programmed
cell death by apoptosis. This cytoprotective effect, which inhibits the
development of hepatic failure in Pcc-infected mice without interfering with
pathogen burden, is mimicked by pharmacological antioxidants such as
N-acetylcysteine (NAC). When administered therapeutically, i.e., after Pcc
infection, NAC suppressed the development of hepatic failure in Pcc-infected
DBA/2 mice (0% lethality), without interfering with pathogen burden. In
conclusion, we describe a mechanism of host defense against Plasmodium
infection, based on tissue cytoprotection against free heme and limiting disease
severity irrespectively of parasite burden. Malaria parasites are known to invade and develop in erythrocytes and
reticulocytes, but little is known about their infection of nucleated erythroid
precursors. We used an in vitro cell system that progressed through basophilic,
polychromatic, orthochromatic, and reticulocyte stages to mature erythrocytes.
We show that orthochromatic cells are the earliest stages that may be invaded by
Plasmodium falciparum, the causative agent of fatal human malaria.
Susceptibility to invasion is distinct from intracellular survival and occurs at
a time of extensive erythroid remodeling. Together these data suggest that the
potential for complexity of host interactions involved in infection may be
vastly greater than hitherto realized. Few studies have investigated the pathophysiologic mechanisms responsible for
what seems to be a possible interaction between Plasmodium falciparum, the
causative agent of malaria, and HIV-1 in dually infected patients. It has been
shown that Plasmodium parasites detoxify heme molecules into a pigment called
hemozoin (HZ), which can significantly modulate the immune system. The primary
objective of this study was to determine whether exposure of human primary
monocyte-derived macrophages (MDMs) to the malaria pigment influences the
process of HIV-1 infection. We report here that HIV-1 replication is
significantly diminished in HZ-loaded MDMs. The HZ-mediated reduction in virus
replication is due to a block at a step in the virus life cycle occurring
between the completion of full-length reverse transcripts and integration of
viral DNA within the host chromosome. Understanding the pathological mechanisms
involved in P. falciparum and HIV-1 co-infection is of high importance because
of possible therapeutic ramifications. Plasmodium falciparum, the causative agent of human malaria, invades host
erythrocytes using several proteins on the surface of the invasive merozoite,
which have been proposed as potential vaccine candidates. Members of the
multi-gene PfRh family are surface antigens that have been shown to play a
central role in directing merozoites to alternative erythrocyte receptors for
invasion. Recently, we identified a large structural polymorphism, a 0.58Kb
deletion, in the C-terminal region of the PfRh2b gene, present at a high
frequency in parasite populations from Senegal. We hypothesize that this region
is a target of humoral immunity. Here, by analyzing 371 P. falciparum isolates
we show that this major allele is present at varying frequencies in different
populations within Senegal, Africa, and throughout the world. For allelic
dimorphisms in the asexual stage antigens, Msp-2 and EBA-175, we find minimal
geographic differentiation among parasite populations from Senegal and other
African localities, suggesting extensive gene flow among these populations
and/or immune-mediated frequency-dependent balancing selection. In contrast, we
observe a higher level of inter-population divergence (as measured by F(st)) for
the PfRh2b deletion, similar to that observed for SNPs from the sexual stage
Pfs45/48 loci, which is postulated to be under directional selection. We confirm
that the region containing the PfRh2b polymorphism is a target of humoral immune
responses by demonstrating antibody reactivity of endemic sera. Our analysis of
inter-population divergence suggests that in contrast to the large allelic
dimorphisms in EBA-175 and Msp-2, the presence or absence of the large PfRh2b
deletion may not elicit frequency-dependent immune selection, but may be under
positive immune selection, having important implications for the development of
these proteins as vaccine candidates. BACKGROUND: Plasmodium falciparum is the main causative agent of malaria. Of the
5 484 predicted genes of P. falciparum, about 57% do not have sufficient
sequence similarity to characterized genes in other species to warrant
functional assignments. Non-homology methods are thus needed to obtain
functional clues for these uncharacterized genes. Gene expression data have been
widely used in the recent years to help functional annotation in an
intra-species way via the so-called Guilt By Association (GBA) principle.
RESULTS: We propose a new method that uses gene expression data to assess
inter-species annotation transfers. Our approach starts from a set of likely
orthologs between a reference species (here S. cerevisiae and D. melanogaster)
and a query species (P. falciparum). It aims at identifying clusters of
coexpressed genes in the query species whose coexpression has been conserved in
the reference species. These conserved clusters of coexpressed genes are then
used to assess annotation transfers between genes with low sequence similarity,
enabling reliable transfers of annotations from the reference to the query
species. The approach was used with transcriptomic data sets of P. falciparum,
S. cerevisiae and D. melanogaster, and enabled us to propose with high
confidence new/refined annotations for several dozens hypothetical/putative P.
falciparum genes. Notably, we revised the annotation of genes involved in
ribosomal proteins and ribosome biogenesis and assembly, thus highlighting
several potential drug targets.
CONCLUSIONS: Our approach uses both sequence similarity and gene expression data
to help inter-species gene annotation transfers. Experiments show that this
strategy improves the accuracy achieved when using solely sequence similarity
and outperforms the accuracy of the GBA approach. In addition, our experiments
with P. falciparum show that it can infer a function for numerous hypothetical
genes. Plasmodium falciparum is the causative agent of malaria, a disease where new
drug targets are required due to increasing resistance to current
anti-malarials. TMPK (thymidylate kinase) is a good candidate as it is essential
for the synthesis of dTTP, a critical precursor of DNA and has been much studied
due to its role in prodrug activation and as a drug target. Type I TMPKs, such
as the human enzyme, phosphorylate the substrate AZT
(3'-azido-3'-deoxythymidine)-MP (monophosphate) inefficiently compared with type
II TMPKs (e.g. Escherichia coli TMPK). In the present paper we report that
eukaryotic PfTMPK (P. falciparum TMPK) presents sequence features of a type I
enzyme yet the kinetic parameters for AZT-MP phosphorylation are similar to
those of the highly efficient E. coli enzyme. Structural information shows that
this is explained by a different juxtaposition of the P-loop and the azide of
AZT-MP. Subsequent formation of the transition state requires no further
movement of the PfTMPK P-loop, with no steric conflicts for the azide moiety,
allowing efficient phosphate transfer. Likewise, we present results that confirm
the ability of the enzyme to uniquely accept dGMP as a substrate and shed light
on the basis for its wider substrate specificity. Information resulting from two
ternary complexes (dTMP-ADP and AZT-MP-ADP) and a binary complex with the
transition state analogue AP5dT [P1-(5'-adenosyl)-P5-(5'-thymidyl)
pentaphosphate] all reveal significant differences with the human enzyme,
notably in the lid region and in the P-loop which may be exploited in the
rational design of Plasmodium-specific TMPK inhibitors with therapeutic
potential. Dendritic cells (DC) and macrophages phagocytose pathogens and degrade them in
their phagosomes to allow for proper presentation of foreign antigens to other
cells of the immune system. The Plasmodium parasite, causative agent of malaria,
infects RBC that are phagocytosed by DC and macrophages during the course of
infection. Under specific conditions, the functionality of these cells can be
affected by phagocytosis of Plasmodium-infected RBC. We investigated whether
phagosomal maturation and degradation of Plasmodium yoelii-infected RBC in
phagosomes is affected in DC and macrophages. We show that recruitment of the
phagolysosomal marker Lamp-1 and of MHC-II, as well as acidification of
phagosomes, was achieved in a timely manner. Using P. yoelii-infected RBC
labelled with a fluorescent dye or transgenic green fluorescent protein
(GFP)-expressing parasites, we found a gradual, rapid decrease in the phagosome
fluorescence signal, indicating that P. yoelii-infected RBC are efficiently
degraded in macrophages and DC. We also observed that pre-incubation of DC with
infected RBC did not affect phagosomal maturation of newly internalized P.
yoelii-infected RBC. In conclusion, after phagocytosis, Plasmodium-infected RBC
are degraded by DC and macrophages, suggesting that the process of phagosomal
maturation is effectively completed in malaria. A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified
with potent activity against the erythrocyte-stage of Plasmodium falciparum
(Pf), the most common causative agent of malaria. A systematic SAR study
resulted in the identification of compound 40 which exhibits good potency
against both wild-type and drug resistant parasites and exhibits good in vivo
pharmacokinetic properties. Hsp90 is an important cellular chaperone and attractive target for therapeutics
against both cancer and infectious organisms. The Hsp90 protein from the
parasite Plasmodium falciparum, the causative agent of malaria, is critical for
this organism's survival; the anti-Hsp90 drug geldanamycin is toxic to P.
falciparum growth. We have solved the structure of the N-terminal ATP-binding
domain of P. falciparum Hsp90, which contains a principal drug-binding pocket,
in both apo and ADP-bound states at 2.3 A resolution. The structure shows that
P. falciparum Hsp90 is highly similar to human Hsp90, and likely binds agents
such as geldanamycin in an identical manner. Our results should aid in the
structural understanding of Hsp90-drug interactions in P. falciparum, and
provide a scaffold for future drug-discovery efforts. The culminating step of the intraerythrocytic development of Plasmodium
falciparum, the causative agent of malaria, is the spectacular release of
multiple invasive merozoites on rupture of the infected erythrocyte membrane.
This work reports for the first time that the whole process, taking place in
time scales as short as 400 milliseconds, is the result of an elastic
instability of the infected erythrocyte membrane. Using high-speed differential
interference contrast (DIC) video microscopy and epifluorescence, we demonstrate
that the release occurs in 3 main steps after osmotic swelling of the infected
erythrocyte: a pore opens in ~ 100 milliseconds, ejecting 1-2 merozoites, an
outward curling of the erythrocyte membrane is then observed, ending with a fast
eversion of the infected erythrocyte membrane, pushing the parasites forward. It
is noteworthy that this last step shows slight differences when infected
erythrocytes are adhering. We rationalize our observations by considering that
during the parasite development, the infected erythrocyte membrane acquires a
spontaneous curvature and we present a subsequent model describing the dynamics
of the curling rim. Our results show that sequential erythrocyte membrane
curling and eversion is necessary for the parasite efficient angular dispersion
and might be biologically essential for fast and numerous invasions of new
erythrocytes. Plasmodium falciparum, the major causative agent of human malaria, contains
three separate genomes. The apicoplast (an intracellular organelle) contains an
∼35-kb circular DNA genome of unusually high A/T content (>86%) that is
replicated by the nuclear-encoded replication complex Pfprex. Herein, we have
expressed and purified the DNA polymerase domain of Pfprex [KPom1 (Klenow-like
polymerase of malaria 1)] and measured its fidelity using a LacZ-based forward
mutation assay. In addition, we analyzed the kinetic parameters for the
incorporation of both complementary and noncomplementary nucleotides using Kpom1
lacking 3'→5' exonucleolytic activity. KPom1 exhibits a strongly biased
mutational spectrum in which T→C is the most frequent single-base substitution
and differs significantly from the closely related Escherichia coli DNA
polymerase I. Using E. coli harboring a temperature-sensitive polymerase I
allele, we established that KPom1 can complement the growth-defective phenotype
at an elevated temperature. We propose that the error bias of KPom1 may be
exploited in the complementation assay to identify nucleoside analogs that mimic
this base-mispairing and preferentially inhibit apicoplast DNA replication. Toxoplasma gondii is a member of the phylum Apicomplexa that includes several
important human pathogens, such as Cryptosporidium and Plasmodium falciparum,
the causative agent of human malaria. It is an obligate intracellular parasite
that can cause severe disease in congenitally infected neonates and
immunocompromised individuals. Despite the importance of attachment and invasion
to the success of the parasite, little is known about the underlying mechanisms
that drive these processes. Here we describe a screen to identify small
molecules that block the process of host cell invasion by the T. gondii
parasite. We identified a small molecule that specifically and irreversibly
blocks parasite attachment and subsequent invasion of host cells. Using tandem
orthogonal proteolysis-activity-based protein profiling, we determined that this
compound covalently modifies a single cysteine residue in a poorly characterized
protein homologous to the human protein DJ-1. Mutation of this key cysteine
residue in the native gene sequence resulted in parasites that were resistant to
inhibition of host cell attachment and invasion by the compound. Further
analysis of the invasion phenotype confirmed that modification of Cys127 on
TgDJ-1 resulted in a block of microneme secretion and motility, even in the
presence of direct stimulators of calcium release. Together, our results suggest
that TgDJ-1 plays an important role that is likely downstream of the calcium
flux required for microneme secretion, parasite motility, and subsequent
invasion of host cells. Plasmodium falciparum, the causative agent of the most severe form of malaria in
humans invades erythrocytes using multiple ligand-receptor interactions. The P.
falciparum reticulocyte binding-like homologue proteins (PfRh or PfRBL) are
important for entry of the invasive merozoite form of the parasite into red
blood cells. We have analysed two members of this protein family, PfRh2a and
PfRh2b, and show they undergo a complex series of proteolytic cleavage events
before and during merozoite invasion. We show that PfRh2a undergoes a cleavage
event in the transmembrane region during invasion consistent with activity of
the membrane associated PfROM4 protease that would result in release of the
ectodomain into the supernatant. We also show that PfRh2a and PfRh2b bind to red
blood cells and have defined the erythrocyte-binding domain to a 15 kDa region
at the N-terminus of each protein. Antibodies to this receptor-binding region
block merozoite invasion demonstrating the important function of this domain.
This region of PfRh2a and PfRh2b has potential in a combination vaccine with
other erythrocyte binding ligands for induction of antibodies that would block a
broad range of invasion pathways for P. falciparum into human erythrocytes. OBJECTIVE: To demonstrate malaria situation analysis, stratification and
planning for an endemic area in southern Iran.
METHODS: Data on health system, population, meteorological parameters, malaria
cases, anopheline vectors, and control activities during 2005-2007 was obtained
from Minab Health Center, Minab Meteorological Station and published documents
about malaria elements in the study area. A datasheet was created in excel 2003
for analysis.
RESULTS: There were 644 health staff working in Minab District including 99
health staff in malaria control program. The health facilities are distributed
as follow: 1 hospital with 96 beds, 23 health centers including private centers
(10 in Minab city and 13 in rural area of Minab District) and 119 health houses
in rural areas of Minab District. A nopheles stephensi was the domit species
in Minab District, however, Anopheles dthali, Anopheles superpictus, Anopheles
fluviatilis, Anopheles multicolor, Anopheles pulcherrimus and Anopheles turkhudi
can also be found in the area. Anopheles stephensi was reported susceptible to
malathion, propoxur, primphos-methyl, lambda-cyhalothrin permethrin and
deltamethrin, and resistant to DDT and dieldrin in the area. During the study
period a total of 10 665 positive cases were reported, mainly due to local
transmission (99.6%). Plasmodium vivax was the main causative agent followed by
Plasmodium falciparum. There were reports about drug resistance of Plasmodium
falciparum in the area.
CONCLUSIONS: Using different parameters, Minab was classified into 3 strata. A
plan was designed based on described goal, objectives and targets. The
approaches of this plan were categorized into: health education, early detection
and correct treatment, and vector control. Main constraints of these approaches
are population movement between Iran, Pakistan and Afghanistan; vector control
challenges at district, inadequate skilled medical staff in malaria case
management and weak inter-sectorial coordination for malaria control, especially
in urban areas. Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which
are morphologically similar to Plasmodium falciparum, the causative agent of
malaria in humans. Like Plasmodium, different species of Babesia are tuned to
infect different mammalian hosts, including rats, dogs, horses and cattle. Most
species of Plasmodium and Babesia possess an essential bifunctional enzyme for
nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate
synthase. Although thymidylate synthase is highly conserved across organisms,
the bifunctional form of this enzyme is relatively uncommon in nature. The
structural characterization of dihydrofolate reductase-thymidylate synthase in
Babesia bovis, the causative agent of babesiosis in livestock cattle, is
reported here. The apo state is compared with structures that contain dUMP, NADP
and two different antifolate inhibitors: pemetrexed and raltitrexed. The
complexes reveal modes of binding similar to that seen in drug-resistant malaria
strains and point to the utility of applying structural studies with proven
cancer chemotherapies towards infectious disease research. Recent reports highlight the severity and the morbidity of disease caused by the
long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties
in the laboratory-propagation of P. vivax, the biology of this parasite has not
been adequately explored. While the proteome of P. falciparum, the causative
agent of cerebral malaria, has been extensively explored from several sources,
there is limited information on the proteome of P. vivax. We have, for the first
time, examined the proteome of P. vivax isolated directly from patients without
adaptation to laboratory conditions. We have identified 153 proteins from
clinical P. vivax, majority of which do not show homology to any previously
known gene products. We also report 29 new proteins that were found to be
expressed in P. vivax for the first time. In addition, several proteins
previously implicated as anti-malarial targets, were also found in our analysis.
Most importantly, we found several unique proteins expressed by P. vivax.This
study is an important step in providing insight into physiology of the parasite
under clinical settings. The 23-megabase genome of Plasmodium falciparum, the causative agent of severe
human malaria, contains ∼5300 genes, most of unknown function or lacking
homologs in other organisms. Identification of these gene functions will help in
the discovery of novel targets for the development of antimalarial drugs and
vaccines. The P. falciparum genome is unusually A+T-rich, which hampers cloning
and expressing these genes in heterologous systems for functional analysis. The
large repertoire of genetic tools available for Saccharomyces cerevisiae makes
this yeast an ideal system for large scale functional complementation analyses
of parasite genes. Here, we report the construction of a cDNA library from P.
knowlesi, which has a lower A+T content compared with P. falciparum. This
library was applied in a yeast complementation assay to identify malaria genes
involved in the decarboxylation of phosphatidylserine. Transformation of a
psd1Δpsd2Δdpl1Δ yeast strain, defective in phosphatidylethanolamine synthesis,
with the P. knowlesi library led to identification of a new parasite
phosphatidylserine decarboxylase (PkPSD). Unlike phosphatidylserine
decarboxylase enzymes from other eukaryotes that are tightly associated with
membranes, the PkPSD enzyme expressed in yeast was equally distributed between
membrane and soluble fractions. In vitro studies reveal that truncated forms of
PkPSD are soluble and undergo auto-endoproteolytic maturation in a
phosphatidylserine-dependent reaction that is inhibited by other anionic
phospholipids. This study defines a new system for probing the function of
Plasmodium genes by library-based genetic complementation and its usefulness in
revealing new biochemical properties of encoded proteins. BACKGROUND: The merozoite surface protein (MSP)-1 of Plasmodium falciparum, the
causative agent of malaria tropica, is considered to be a promising vaccine
candidate. Although its stable cloning and expression has been difficult in the
past, adenoviral vectors expressing the complex protein are described in the
present study.
METHODS: Codon-optimized msp-1 was used to construct a set of first generation
(ΔE1Ad) and high-capacity adenovirus (HC-Ad) vectors, and cellular and humoral
immune responses induced by the vectors were characterized in detail in mice.
RESULTS: Generation of stable ΔE1Ad and HC-Ad vectors expressing full-length
MSP-1 and their production to high vector titers was found to be feasible.
Epitope identification and analysis of frequencies of specific CD8 T-cells
revealed that MSP-1 expressing HC-Ad vectors induced higher frequencies of
interferon-γ + CD8 T-cells than ΔE1 vectors. Irrespective of the vector format,
higher titers of MSP-1 specific antibodies were generated by Ad vectors
expressing MSP-1 from a chicken β-actin (CAG) promoter comprising the
cytomegalovirus early enhancer element and the chicken β-actin promoter.
CONCLUSIONS: The findings of the present study suggest that Ad vectors
expressing full-length codon-optimized MSP-1 are promising candidate vaccines
against P. falciparum infections. Use of the HC-Ad vector type for delivery, as
well as the CAG promoter to control MSP-1 expression, may further increase the
efficacy of this vaccine candidate. A causative agent of human malaria, Plasmodium falciparum, is transmitted by
Anopheles mosquitoes. The malaria parasite is under intensive attack from the
mosquito's innate immune system during its sporogonic development. We have used
genetic engineering to create immune-enhanced Anopheles stephensi mosquitoes
through blood meal-inducible expression of a transgene encoding the IMD
pathway-controlled NF-kB Rel2 transcription factor in the midgut and fat-body
tissue. Transgenic mosquitoes showed greater resistance to Plasmodium and
microbial infection as a result of timely concerted tissue-specific immune
attacks involving multiple effectors. The relatively weak impact of this genetic
modification on mosquito fitness under laboratory conditions encourages further
investigation of this approach for malaria control. The causative agent of malaria, Plasmodium, possesses three translationally
active compartments: the cytosol, the mitochondrion and a relic plastid called
the apicoplast. Aminoacyl-tRNA synthetases to charge tRNA are thus required for
all three compartments. However, the Plasmodiumfalciparum genome encodes too few
tRNA synthetases to supply a unique enzyme for each amino acid in all three
compartments. We have investigated the subcellular localisation of three tRNA
synthetases (AlaRS, GlyRS and ThrRS), which occur only once in the nuclear
genome, and we show that each of these enzymes is dually localised to the P.
falciparum cytosol and the apicoplast. No mitochondrial fraction is apparent for
these three enzymes, which suggests that the Plasmodium mitochondrion lacks at
least these three tRNA synthetases. The unique Plasmodium ThrRS is the presumed
target of the antimalarial compound borrelidin. Borrelidin kills P. falciparum
parasites quickly without the delayed death effect typical of apicoplast
translation inhibitors and without an observable effect on apicoplast
morphology. By contrast, mupirocin, an inhibitor of the apicoplast IleRS, kills
with a delayed death effect that inhibits apicoplast growth and division.
Because inhibition of dual targeted tRNA synthetases should arrest translation
in all compartments of the parasite, these enzymes deserve further investigation
as potential targets for antimalarial drug development. Life cell imaging is a tool for cell biology that has provided invaluable
insights into many dynamic processes such as cell division, morphogenesis, or
endo- and exocytosis. While observing cells by time-lapse imaging is a standard
procedure in many systems, this technique was until recently not available for
blood stages of Plasmodium falciparum, the causative agent of the most severe
form of human malaria. Here, we provide a detailed description of the procedure
for time-lapse-based four-dimensional microscopy in blood stages of this
important pathogen. With the widespread use of P. falciparum transfection to
fluorescently tag proteins of interest, this technique provides a new tool to
study the biology of malaria blood stages that is hoped to lead to a better
appreciation of the dynamic processes in this life cycle phase. BACKGROUND: Plasmodium falciparum, the causative agent of human malaria,
expresses two aminopeptidases, PfM1AAP and PfM17LAP, critical to generating a
free amino acid pool used by the intraerythrocytic stage of the parasite for
proteins synthesis, growth and development. These exopeptidases are potential
targets for the development of a new class of anti-malaria drugs.
METHODOLOGY/PRINCIPAL FINDINGS: To define the substrate specificity of
recombit forms of these two malaria aminopeptidases we used a new library
consisting of 61 fluorogenic substrates derived both from natural and unnatural
amino acids. We obtained a detailed substrate fingerprint for recombit forms
of the enzymes revealing that PfM1AAP exhibits a very broad substrate tolerance,
capable of efficiently hydrolyzing neutral and basic amino acids, while PfM17LAP
has narrower substrate specificity and preferentially cleaves bulky, hydrophobic
amino acids. The substrate library was also exploited to profile the activity of
the native aminopeptidases in soluble cell lysates of P. falciparum malaria.
CONCLUSIONS/SIGNIFICANCE: This data showed that PfM1AAP and PfM17LAP are
responsible for majority of the aminopeptidase activity in these extracts. These
studies provide specific substrate and mechanistic information important for
understanding the function of these aminopeptidases and could be exploited in
the design of new inhibitors to specifically target these for anti-malaria
treatment. The synthesis of the recently characterized depsipeptide szentiamide (1), which
is produced by the entomopathogenic bacterium Xenorhabdus szentirmaii, is
described. Whereas no biological activity was previously identified for 1, the
material derived from the efficient synthesis enabled additional bioactivity
tests leading to the identification of a notable activity against insect cells
and Plasmodium falciparum, the causative agent of malaria. Malaria is a widespread and infectious disease that is a leading cause of death
in many parts of the world. Eradication of malaria has been a major world health
goal for decades, but one that still remains elusive. Other diseases have been
eradicated using vaccination, but traditional vaccination methods have thus far
been unsuccessful for malaria. Infection by Plasmodium species, the causative
agent of malaria, is currently treated with drug-based therapies, but an
increase in drug resistance has led to the need for new methods of treatment. A
promising strategy for malaria treatment is to combine transmission blocking
vaccines (TBVs) that prevent spread of disease with drug-based therapies to
treat infected individuals. TBVs can be developed against surface protein
antigens that are expressed during parasite reproduction in the mosquito. When
the mosquito ingests blood from a vaccinated individual harboring the Plasmodium
parasite, the antibodies generated by vaccination prevent completion of the
parasites life-cycle. Animal studies have shown that immunization with Pfs48/45
results in the production of malaria transmission blocking antibodies; however,
the development of this vaccine candidate has been hindered by poor expression
in both prokaryotic and eukaryotic hosts. Recently, the chloroplast of
Chlamydomonas reinhardtii has been used to express complex recombit proteins.
In this study, we show that the C-terminal antigenic region of the Pfs48/45
antigen can be expressed in the chloroplast of the green algae C. reinhardtii
and that this recombit protein has a conformation recognized by known
transmission blocking antibodies. Production of this protein in algae has the
potential to scale to the very large volumes required to meet the needs of
millions at risk for contracting malaria. It is generally accepted that the mitochondria play central roles in energy
production of most eukaryotes. In contrast, it has been thought that Plasmodium
spp., the causative agent of malaria, rely mainly on cytosolic glycolysis but
not mitochondrial oxidative phosphorylation for energy production during blood
stages. However, Plasmodium spp. possesses all genes necessary for the
tricarboxylic acid (TCA) cycle and most of the genes for electron transport
chain (ETC) enzymes. Therefore, it remains elusive whether oxidative
phosphorylation is essential for the parasite survival. To elucidate the role of
TCA metabolism and ETC in malaria parasites, we deleted the gene for
flavoprotein (Fp) subunit, Pbsdha, one of four components of complex II, a
catalytic subunit for succinate dehydrogenase activity. The Pbsdha(-) parasite
grew normally at blood stages in mouse. In contrast, ookinete formation of
Pbsdha(-) parasites in the mosquito stage was severely impaired. Finally,
Pbsdha(-) ookinetes failed in oocyst formation, leading to complete malaria
transmission blockade. These results suggest that malaria parasite may switch
the energy metabolism from glycolysis to oxidative phosphorylation to adapt to
the insect vector where glucose is not readily available for ATP production. Plasmodium falciparum is the causative agent of malaria, a deadly infectious
disease for which treatments are scarce and drug-resistant parasites are now
increasingly found. A comprehensive method of identifying and quantifying
metabolites of this intracellular parasite could expand the arsenal of tools to
understand its biology, and be used to develop new treatments against the
disease. Here, we present two methods based on liquid chromatography tandem mass
spectrometry for reliable measurement of water-soluble metabolites involved in
phospholipid biosynthesis, as well as several other metabolites that reflect the
metabolic status of the parasite including amino acids, carboxylic acids,
energy-related carbohydrates, and nucleotides. A total of 35 compounds was
quantified. In the first method, polar compounds were retained by hydrophilic
interaction chromatography (amino column) and detected in negative mode using
succinic acid-(13)C(4) and fluorovaline as internal standards. In the second
method, separations were carried out using reverse phase (C18) ion-pair liquid
chromatography, with heptafluorobutyric acid as a volatile ion pairing reagent
in positive detection mode, using d(9)-choline and 4-aminobutanol as internal
standards. Standard curves were performed in P. falciparum-infected and
uninfected red blood cells using standard addition method (r(2)>0.99). The
intra- and inter-day accuracy and precision as well as the extraction recovery
of each compound were determined. The lower limit of quantitation varied from
50pmol to 100fmol/3×10(7)cells. These methods were validated and successfully
applied to determine intracellular concentrations of metabolites from uninfected
host RBCs and isolated Plasmodium parasites. The effectiveness of methylene blue (MB) combined with pyrimethamine (PYR),
chloroquine (CQ) or quinine (Q) was examined in a classical four-day suppressive
test against a causative agent of rodent malaria, Plasmodium berghei. A marked
potentiation was observed when MB was administered at a non-curative dose of 15
mg/kg/day in combination with PYR (0.19 mg/kg/day) or Q (25 mg/kg/day). No
synergy was found between MB (15 mg/Kg) and CQ (0.75 mg/Kg). Our results suggest
that the combination of MB with PYR or Q may improve the efficacy of these
currently used antimalarial drugs. The global area for Plasmodium ovale is small as compared with that for other
species of human malaria pathogens. It has expanded in Asian areas and remained
as before in the African ones. In the past 20 years, there have been 2129
malaria cases imported from far abroad to Russia, including 84 (4%) cases of
vivax malaria (P. ovale). The patients were most foreign citizens: 70 from 20
African countries and 7 from two countries of Oceania, such as Papua New Guinea
and Indonesia. The other 7 patients were Russia's people who had returned from
different countries of Africa. For this period there have been a total of 5
cases of mixed infection: tropical P. falciparum malaria + vivax P. ovale
malaria. The mission of detected new sympatric subspecies (P. ovale curtisi and
P. ovale wallikeri) inhabiting the tropical countries with continuous local
transmission remains unclear. Only a thorough study of these subspecies will be
able to effectively apply preventive measures and to carry out their elimination
in future. Sphingolipids are essential components of eukaryotic cell membranes,
particularly the plasma membrane, and are involved in a diverse array of signal
transduction pathways. Mammals produce sphingomyelin (SM) as the primary complex
sphingolipid via the well characterised SM synthase. In contrast yeast, plants
and some protozoa utilise an evolutionarily related inositol phosphorylceramide
(IPC) synthase to synthesise IPC. This activity has no mammalian equivalent and
IPC synthase has been proposed as a target for anti-fungals and anti-protozoals.
However, detailed knowledge of the sphingolipid biosynthetic pathway of the
apicomplexan protozoan parasites was lacking. In this study bioinformatic
analyses indicated a single copy orthologue of the putative SM synthase from the
apicomplexan Plasmodium falciparum (the causative agent of malaria) was a bona
fide sphingolipid synthase in the related model parasite, Toxoplasma gondii
(TgSLS). Subsequently, TgSLS was indicated, by complementation of a mutant cell
line, to be a functional orthologue of the yeast IPC synthase (AUR1p),
demonstrating resistance to the well characterised AUR1p inhibitor aureobasidin
A. In vitro, recombit TgSLS exhibited IPC synthase activity and, for the
first time, the presence of IPC was demonstrated in T. gondii lipid extracts by
mass spectrometry. Furthermore, host sphingolipid biosynthesis was indicated to
influence, but be non-essential for, T. gondii proliferation, suggesting that
whilst scavenging does take place de novo sphingolipid synthesis may be
important for parasitism. A model betalainic dye was semisynthesized from betanin, the magenta pigment of
the red beet, and was effective for live-cell imaging of Plasmodium-infected red
blood cells. This water-soluble fluorescent probe is photostable, excitable in
the visible region and cell membrane-permeable, and its photophysical properties
are not notably pH-sensitive. Fluorescence imaging microscopy of erythrocytes
infected with Plasmodium falciparum, a causative agent of malaria in humans,
showed that only the parasite was stained. Z-stacking analysis suggested that
the probe accumulates proximal to the nucleus of the parasite. Indicaxanthin,
one of the natural fluorescent betalains found in the petals of certain flowers,
did not stain the parasite or the red blood cell. Plasmodium falciparum is the causative agent of malaria, a disease that kills
almost one million persons each year, mainly in sub-Saharan Africa. P.
falciparum is transmitted to the human host by the bite of an Anopheles female
mosquito, and Anopheles gambiae sensus stricto is the most tremendous malaria
vector in Africa, widespread throughout the afro-tropical belt. An. gambiae s.s.
is subdivided into two distinct molecular forms, namely M and S forms. The two
molecular forms are morphologically identical but they are distinct genetically,
and differ by their distribution and their ecological preferences. The
epidemiological importance of the two molecular forms in malaria transmission
has been poorly investigated so far and gave distinct results in different
areas. We have developed a real-time quantitative PCR (qPCR) assay, and used it
to detect P. falciparum at the oocyst stage in wild An. gambiae s.s. mosquitoes
experimentally infected with natural isolates of parasites. Mosquitoes were
collected at immature stages in sympatric and allopatric breeding sites and
further infected at the adult stage. We next measured the infection prevalence
and intensity in female mosquitoes using the qPCR assay and correlated the
infection success with the mosquito molecular forms. Our results revealed
different prevalence of infection between the M and S molecular forms of An.
gambiae s.s. in Cameroon, for both sympatric and allopatric populations of
mosquitoes. However, no difference in the infection intensity was observed.
Thus, the distribution of the molecular forms of An. gambiae s.s. may impact on
the malaria epidemiology, and it will be important to monitor the efficiency of
malaria control interventions on the two M and S forms. BACKGROUND: Plasmodium falciparum the main causative agent of malaria is an
important public health vector. With the use of PCR, its genetic diversity has
been extensively studied with dearth information from Nigeria.
METHODS: In this study, 100 P. falciparum strains merozoite surface protein
1(msp-1), merozoite surface protein 2 (msp-2) and Glutamate rich protein (Glurp)
from Ogun State General Hospitals were characterized. The genetic diversity of
P. falciparum isolates was analyzed by restriction fragment length polymorphism
following gel electrophoresis of DNA products from nested polymerase chain
reactions (PCR) of their respective allelic families KI, MAD 20, RO33
(MSP-1);FC27, 3D7 (MSP-2) and Glutamate rich protein respectively.
RESULTS: Majority of the patients showed monoclonal infections while
multiplicity of the infection for msp-1 and msp-2 were 1.1 and 1.2 respectively.
The estimated number of genotypes was 8 msp-1 (4 KI; 3 MAD; 1 RO33) and 6 msp-2
(3 FC27; 3 3D7). 80% of the isolates coded for Glurp with allelic size ranged
between 700 and 900 bp.
CONCLUSION: The allelic distributions however were similar to those previously
reported in other endemic malaria countries. Future studies will be designed to
include other malaria endemic regions of Nigeria such as the oil exploration
regions. Malaria is characterized by cyclical fevers and high levels of inflammation, and
while an early inflammatory response contributes to parasite clearance,
excessive and persistent inflammation can lead to severe forms of the disease.
Here, we show that Plasmodium falciparum-infected erythrocytes contain uric acid
precipitates in the cytoplasm of the parasitophorous vacuole, which are released
when erythrocytes rupture. Uric acid precipitates are highly inflammatory
molecules that are considered a danger signal for innate immunity and are the
causative agent in gout. We determined that P. falciparum-derived uric acid
precipitates induce maturation of human dendritic cells, increasing the
expression of cell surface co-stimulatory molecules such as CD80 and CD86, while
decreasing human leukocyte antigen-DR expression. In accordance with this, uric
acid accounts for a significant proportion of the total stimulatory activity
induced by parasite-infected erythrocytes. Moreover, the identification of uric
acid precipitates in P. falciparum- and P. vivax-infected erythrocytes obtained
directly from malaria patients underscores the in vivo and clinical relevance of
our findings. Altogether, our data implicate uric acid precipitates as a
potentially important contributor to the innate immune response to Plasmodium
infection and may provide a novel target for adjunct therapies. Vaccines that interrupt malaria transmission are of increasing interest and a
robust functional assay to measure this activity would promote their development
by providing a biologically relevant means of evaluating potential vaccine
candidates. Therefore, we aimed to qualify the standard membrane-feeding assay
(SMFA). The assay measures the transmission-blocking activity of antibodies by
feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the
presence of the test antibodies and measuring subsequent mosquito infection. The
International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline
Q2(R1) details characteristics considered in assay validation. Of these
characteristics, we decided to qualify the SMFA for Precision, Linearity, Range
and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested
over 6 feeding experiments at several concentrations to determine four suitable
concentrations that were tested in triplicate in the qualification experiments
(3 additional feeds) to evaluate Precision, Linearity and Range. For
Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined
intra- and inter-assay variability of % inhibition of mean oocyst intensity at
each concentration of 4B7 (lower concentrations showed higher variability). We
also showed that % inhibition was dependent on 4B7 concentration and the
activity is specific to 4B7. Since obtaining empirical data is time-consuming,
we generated a model using data from all 9 feeds and simulated the effects of
different parameters on final readouts to improve the assay procedure and
analytical methods for future studies. For example, we estimated the effect of
number of mosquitoes dissected on variability of % inhibition, and simulated the
relationship between % inhibition in oocyst intensity and % inhibition of
prevalence of infected mosquitos at different mean oocysts in the control. SMFA
is one of the few biological assays used in preclinical and early clinical
development of transmission-blocking vaccines, and this study strongly supports
its further development and application. Insecticide-resistance threatens the control of mosquito-borne diseases like
malaria or dengue fever. To ensure sustainable vector control we need a full
understanding of the factors driving the evolution of resistance. We test the
hypothesis that the expression of insecticide-resistance depends on the
available resources by rearing genetically DDT-resistant and sensitive larvae of
Anopheles mosquitoes at three diet regimes, which correspond to 40%, 70% and
100% of the normal diet and exposing the adult females to DDT 5, 10 and 15 days
after emergence. In both colonies post-exposure survival decreased with age at
exposure. Additionally, the food levels and DDT-resistance were positively
correlated in both colonies, although only in the DDT-resistant one was this
relationship statistically significant. The impact of larval diet was smaller
than the effect of age at exposure. We discuss our results and explain the
implication of this study to resistance monitoring for public health and vector
management. |
What is the major adverse effect of adriamycin(doxorubicin)? | Cardiac toxicity is a major adverse effect caused by doxorubicin (DOX) therapy | A phase II multicenter clinical study of epirubicin, a new anthracycline
anticancer agent, was carried out in 46 patients with advanced breast cancer.
The treatment schedule consisted of either 60 mg/m2 every three weeks or 40 to
50 mg/m2 on day 1 and day 8 every four weeks. Objective responses were observed
in 23.7% of 38 evaluable patients (1 CR and 8 PR). Response rates according to
previous chemotherapy were 50.0% (4/8) in previously non-treated patients and
36.4% (4/11) in patients previously treated with non-anthracyclines. The major
adverse effect was bone-marrow suppression; leukopenia was observed in 82.1% of
patients, anemia in 53.8% and thrombocytopenia in 20.0%. Other toxicities
frequently observed were anorexia (55.0%), nausea-vomiting (55.0%) and alopecia
(66.7%), but these seemed to be milder than those produced by doxorubicin. A new anthracycline analog, epirubicin (4'-epi-Adriamycin) was evaluated at
eleven institutes in a phase II clinical study in patients with maligt
lymphoma. Epirubicin was administered intravenously mainly with using the
following two regimens; 50 to 60 mg/m2 every three weeks and 40 mg/m2 weekly. A
total of 46 cases were entered into the study and 41 cases were evaluable.
Clinical responses, complete plus partial remissions, were observed in 27 cases
(65.9%) with 8 of these showing complete remission. There was no significant
difference of response between the two regimens. Response rates taking into
account previous chemotherapy were 90.9% (10/11) in previously nontreated cases,
61.9% (8/13) in cases previously treated with non-anthracyclines and 52.9%
(9/17) in cases treated with anthracyclines. The major adverse effect was bone
marrow suppression; leukopenia was observed in 83.8%, anemia in 60.5% and
thrombocytopenia in 15.4%. Other adverse effects frequently observed were
anorexia (59.0%), nausea-vomiting (48.8%) and alopecia (55.6%). These adverse
effects seemed milder than those produced by doxorubicin. The results indicated
that epirubicin seemed to be a markedly useful drug against maligt lymphoma. A Phase II clinical trial of a new anthracycline,
(2''R)-4'-0-tetrahydropyranyladriamycin (THP), was performed in 137 patients
with urological maligcies. Out of them, 111 patients were evaluated for tumor
responses and 125 patients were evaluated for adverse effects. In cases of
intravenous administration, overall response rate was 18.5% (22.2% for bladder
cancer, 30.0% for tumors of the renal pelvis and ureter, and 6.7% for prostatic
cancer). In the case of intra-arterial administration, overall response rate was
42.9% (50.0% for bladder cancer). For 50 patients with superficial bladder
cancer intravesical chemotherapy with THP was performed. Sixteen patients showed
complete disappearance of the tumor, 2 patients showed more than 90% tumor
regression and 12 patients showed more than 50% tumor regression, respectively.
Overall response rate was 60%. Cardiotoxicity was minimal. Alopecia was noted in
a total of 16 patients, but this was minimal. Leukocytopenia was the major
adverse effect among patients undergoing systemic THP administration. In
conclusion, THP was most effective against transitional cell carcinoma of the
urinary tract. The anthracycline glycoside antibiotics represent a group of potent anticancer
agents with a wide spectrum of activity against solid tumours and haematological
maligcies, and are the mainstay of a large number of clinical protocols for
the treatment of adult and childhood neoplastic diseases. Their clinical
activity is limited, however, by acute and chronic adverse effects.
Myelosuppression, predomitly neutropenia and leucopenia, is the dose-limiting
toxicity; in addition to this, mucositis, nausea, vomiting and alopecia are
frequent, whereas hepatopathy, characterised by elevated bilirubin
concentrations, occurs less frequently. Cardiotoxicity is a major adverse effect
of the anthracycline antibiotics and can be acute or chronic; in the acute
setting, electrocardiographic abnormalities may be seen, including ST-T
elevations and arrhythmias, but chronic cardiotoxicity represents a serious
adverse effect that may be lethal due to the development of irreversible,
cumulative dose-dependent, congestive cardiomyopathy. The occurrence of toxicity
displays a marked interindividual variation, and for this reason the
pharmacokinetics and pharmacodynamics of anthracyclines have been extensively
investigated in order to identify integrated models that can be used in the
clinical setting to prevent the development of serious toxicity, mainly
leucopenia, and maximise tumour exposure. Pharmacokinetics has been recognised
to influence both the toxicity and the activity of anthracyclines; in
particular, there is increasing evidence that the mode of administration plays
an important role for cumulative cardiotoxicity and data indicate that bolus
administration, rather than continuous infusion, appears to be an important risk
factor for anthracycline-induced cardiomyopathy, thus implying that this type of
toxicity is maximum concentration-dependent. On the contrary, exposure to the
drug, as measured by area under the curve, seems best related to the occurrence
of leucopenia. Finally, the development of pharmacokinetic-pharmacodynamic
models allows the simulation of drug effects and ultimately dose optimisation in
order to anticipate important toxicities and prevent their occurrence by the
administration of prophylactic treatments. A pilot trial of combined chemotherapy with paclitaxel, doxorubicin and
cisplatin was conducted in patients with advanced endometrial cancer. Between
June 2000 and March 2002 8 patients were treated with combined chemotherapy,
consisting of paclitaxel, 135 mg/m2; doxorubicin, 30 mg/m2; and cisplatin, 50
mg/m2 (TAP therapy). Patients received 3 to 5 courses of TAP therapy every 4
weeks. The major adverse effect was myelosuppression. All patients had grade 3
or 4 neutropenia, but did not have any severe infection with uncontrollable
fever. Only 1 patient discontinued additional therapy due to grade 3
thrombocytopenia after 3 cycles. Grade 2 neurotoxicity occurred in 5 patients,
but grade 3 was not observed. Among 5 patients with measurable tumors, 4
achieved partial response and 1 had no change of tumor size, indicating a
response rate of 80.0%. We found that TAP therapy was feasible with G-CSF
support and shows potential for high efficacy in advanced endometrial cancer. Cardiac toxicity is a major adverse effect caused by doxorubicin (DOX) therapy.
Many recent studies have shown that DOX toxicity involves generation of reactive
oxygen species (ROS). Although protection or alleviation of DOX toxicity can be
achieved by administration of antioxidant vitamins such as ascorbic acid and
vitamin E, their cardioprotective effect remains controversial. Thus alternative
naturally occurring antioxidants may potentially be candidates for antioxidant
therapy. In this study, we investigated the antioxidative and cytoprotective
effects of Phyllanthus urinaria (PU) against DOX toxicity using H9c2 cardiac
myoblasts. The total antioxidant capacity of PU (1 mg/ml) was 5306.75+/-461.62
FRAP value (microM). DOX IC50 values were used to evaluate the cytoprotective
effects of PU ethanolic extract (1 or 10 microg/ml) in comparison with those of
ascorbic acid (VIT C, 100 microM) and N-acetylcysteine (NAC, 100 microM). PU
treatments (1 or 10 microg/ml) dose dependently caused rightward DOX IC50 shifts
of 2.8- and 8.5-fold, respectively while treatments with VIT C and NAC increased
DOX IC50 by 3.3- and 4.2-fold, respectively. Additionally, lipid peroxidation
and caspase-3 activity were parameters used to evaluate cytoprotective effect.
All antioxidants completely inhibited cellular lipid peroxidation and caspase-3
activation induced by DOX (1 microM). Endogenous antioxidant defense such as
total glutathione (tGSH), catalase and superoxide dismutase (SOD) activity was
also modulated by the antioxidants. PU treatment alone dose dependently
increased tGSH, and this effect was retained in the presence of DOX. Similar
effect was observed in the assessment of catalase and SOD enzyme activity. The
nuclear factor kappaB (NFkappaB) transcription factor assay demonstrated that
all antioxidants significantly inhibited DOX-induced NFkappaB activation. Our
results suggest that PU protection against DOX cardiotoxicity was mediated
through multiple pathways and this plant may serve as an alternative source of
antioxidants for prevention of DOX cardiotoxicity. Doxorubicin (DOX) is a widely used chemotherapy agent. The major adverse effect
of DOX treatment in cancer patients is the onset of cardiomyopathy and heart
failure. Reactive oxygen species (ROS) are proposed to be responsible for DOX
cardiotoxicity. Curcumin, a natural compound extracted from Curcuma Longa L., is
known for its anti-oxidant properties. It has been identified as increased
apoptosis in several cancer cell lines in combination with doxorubicin, but
there are few studies about the effect of curcumin and doxorubicin on normal
cardiac cells. Therefore, we evaluated the effects of curcumin on apoptosis
induced by DOX in cardiac muscle cells. Pretreatment with curcumin significantly
increased DOX-induced apoptosis of cardiac muscle cells through down regulation
of Bcl-2, up-regulation of caspase-8 and caspase-9. The Bax/Bcl-2 ratio
increased significantly after 1h pretreatment with curcumin. As well, curcumin
increases ROS generation by DOX. In response to DOX, NF-κB was activated.
However, curcumin was able to inhibit NF-κB activation. In conclusion, our
results indicated that pretreatment with nontoxic concentrations of curcumin
sensitized H9c2 cells to DOX-mediated apoptosis by generation of ROS. |
What is GDF10? | The growth/differentiation factor-10 (GDF-10) is a new member of the transforming growth factor-beta (TGF-beta) superfamily. It is highly related to bone morphogenetic protein-3 (BMP-3) and often referred to as BMP3b. The nucleotide sequence of GDF-10 encodes a predicted protein of 476 amino acids with a molecular weight of approximately 52,000. The GDF-10 polypeptide contains a potential signal sequence for secretion, a putative RXXR proteolytic processing site, and a carboxy-terminal domain with considerable homology to other known members of the TGF-beta superfamily. GDF10 is found primarily in murine uterus, adipose tissue, and brain and to a lesser extent in liver and spleen. In addition, GDF-10 mRNA was present in both neonatal and adult bone samples, with higher levels being detected in calvaria than in long bone. These results suggest that GDF10 may play multiple roles in regulating cell differentiation events, including those involved in skeletal morphogenesis. Gdf10 was mapped to the proximal region of mouse chromosome 14 close to a region known to contain a spontaneous recessive mutation that is associated with a craniofacial defect. | BMP-3b (also termed GDF-10) is a novel BMP-3 related protein recently discovered
in rat femur tissue by molecular cloning. In this paper, we have isolated cDNA
and the gene for human BMP-3b and determined their structure. Cloned human
BMP-3b cDNA with a size of 2632 base pairs encodes a 478 amino acid precursor
protein sharing 83% identity with rat BMP-3b. The human BMP-3b gene is composed
of 3 exons and spans approximately 13 kilobases of DNA. The 5' flanking region
carries no typical TATA box but G+C rich sequences. Southern blot analysis
revealed that the BMP-3b gene is situated in a single locus of chromosome 10. By
Northern analysis, human BMP-3b transcripts were detected primarily in femur,
brain, lung, skeletal muscle, pancreas and testis. We have identified a new member of the transforming growth factor-beta
(TGF-beta) superfamily, growth/differentiation factor-10 (GDF-10), which is
highly related to bone morphogenetic protein-3 (BMP-3). The nucleotide sequence
of GDF-10 encodes a predicted protein of 476 amino acids with a molecular weight
of approximately 52,000. The GDF-10 polypeptide contains a potential signal
sequence for secretion, a putative RXXR proteolytic processing site, and a
carboxy-terminal domain with considerable homology to other known members of the
TGF-beta superfamily. In the mature carboxy-terminal domain GDF-10 is more
homologous to BMP-3 (83% amino acid sequence identity) than to any other
previously identified TGF-beta family member. GDF-10 also shows significant
homology to BMP-3 (approximately 30% amino acid sequence identity) in the pro-
region of the molecule. Based on these sequence comparisons, GDF-10 and BMP-3
define a new subgroup within the larger TGF-beta superfamily. By Northern
analysis, GDF-10 mRNA was detected primarily in murine uterus, adipose tissue,
and brain and to a lesser extent in liver and spleen. In addition, GDF-10 mRNA
was present in both neonatal and adult bone samples, with higher levels being
detected in calvaria than in long bone. These results suggest that GDF10 may
play multiple roles in regulating cell differentiation events, including those
involved in skeletal morphogenesis. Gdf10 was mapped to the proximal region of
mouse chromosome 14 close to a region known to contain a spontaneous recessive
mutation that is associated with a craniofacial defect. Growth differentiation factor 10 (Gdf10), also known as Bmp3b, is a member of
the transforming growth factor (TGF)-ß superfamily. Gdf10 is expressed in
Bergmann glial cells, which was investigated by single-cell transcriptional
profiling (Koirala and Corfas, (2010) PLoS ONE 5: e9198). Here we provide a
detailed characterization of Gdf10 expression from E14, the stage at which Gdf10
is expressed for the first time in the cerebellum, until P28. We detected Gdf10
expression in both germinal zones: in the ventricular zone (VZ) of the 4th
ventricle as well as in the rhombic lip (RL). The VZ has been postulated to give
rise to GABAergic neurons and glial cells, whereas the RL gives rise to
glutamatergic neurons. Thus, it was very surprising to discover a gene that is
expressed exclusively in glial cells and is not restricted to an expression in
the VZ, but is also present in the RL. At postnatal stages Gdf10 was distributed
equally in Bergmann glial cells of the cerebellum. Furthermore, we found Gdf10
to be regulated by Sonic hedgehog (Shh), which is secreted by Purkinje cells of
the cerebellum. In the conditional Shh mutants, glial cells showed a reduced
expression of Gdf10, whereas the expression of Nestin and Vimentin was
unchanged. Thus, we show for the first time, that Gdf10, expressed in Bergmann
glial cells, is affected by the loss of Shh as early as E18.5, suggesting a
regulation of glial development by Shh. |
Is thyroid hormone therapy indicated in patients with heart failure? | There are several experimental and clinical evidences of the potential benefits of Thyroid hormone replacement therapy in heart failure. Initial clinical data showed also a good safety profile and tolerance of TH replacement therapy in patients withheart failure.
However currently there is no indication to treat patients with heart failure withTHreplacementtherapy. | The effects of spontaneous and experimentally induced congestive heart failure
on serum thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3'5'-triiodothyronine
(reverse T3), free T4, free T3 concentrations, and the serum T4 and T3
concentrations in response to administration of thyrotropin were studied. Serum
thyroid hormone concentrations were not different between eight dogs with
spontaneous congestive heart failure and normal age matched control dogs. Seven
dogs with experimental heart failure were tested before and after induction of
congestive heart failure by rapid ventricular pacing. Mean serum T4 and free T3
concentrations were decreased and mean serum reverse T3 concentration was
increased following induction of heart failure. The serum T4 and T3 responses to
thyrotropin were not altered. Thyroid gland morphology appeared normal in dogs
with experimental heart failure. Experimental congestive heart failure, similar
to some other nonthyroidal illnesses, alters thyroid hormone secretion and
metabolism in dogs. In heart failure, cardiac output is insufficient to meet the needs of the body
for oxygen delivery. Available data suggest that alterations in thyroid hormone
metabolism may contribute to defective myocardial performance. Accordingly,
thyroid hormone or a thyroid hormone analogue that improves cardiac performance
might be useful in the treatment of heart failure and has been studied.
Experimental and theoretical results of these studies are reviewed and indicate
that thyroid hormone increases cardiac output by a combination of effects on the
heart and peripheral circulation, specifically by increasing myocardial
contractile performance and decreasing venous compliance. In the rat
postinfarction model of heart failure, treatment with low doses of thyroxine
(1.5 micrograms/100 g) for 3 days produced a positive inotropic response,
including an increase in rate of change of left ventricular pressure and a
decrease in left ventricular end-diastolic pressure. These changes could be
attributed to conversion to triiodothyronine, the active intracellular form of
thyroid hormone. When treatment with thyroxine was continued at the same or
higher doses (3 to 15 micrograms/100 g) for 10 to 12 days, heart rate increased
and improvement in left ventricular end-diastolic pressure was not sustained.
More favorable results were obtained with 3,5-diiodothyropropionic acid, a
cardiotonic thyroid hormone analogue administered at doses of 375 microgram/100
g, given in combination with captopril. Thus, triiodothyronine or a thyroid
hormone analogue may be a useful adjunct to other measures in the treatment of
heart failure. The possibility that thyroid hormone or a thyroid hormone analogue that improves
cardiac performance might be useful in the treatment of heart failure has-been
examined. In the rat postinfarction model of heart failure, treatment with low
doses (1.5 micrograms/100 g) of thyroxine (T4) for 3 days produced a positive
inotropic response, including an increase in left ventricular (LV) dP/dt and a
decrease in LV end-diastolic pressure (LVEDP). When treatment with T4 was
continued at the same or higher doses (3 to 15 micrograms/100 g) for 10-12 days,
heart rate was increased and improvement in LVEDP was not sustained. To identify
an analogue with a more favorable hemodynamic profile, single- and double-ring
compounds related to T4 were screened for thyromimetic activity in heart cell
cultures and for their ability to bind thyroid hormone receptors. One of the
analogues selected, 3,5-diiodothyropropionic acid (DITPA), was found to have
inotropic selectivity in hypothyroid rats. When administered (375 micrograms/100
g) to rats with ventricular dysfunction after myocardial infarction in
combination with captopril, there was improvement of the resting and stressed
cardiac index and LV filling pressure. Similar improvement in cardiac
performance was obtained when DITPA was administered to rabbits after
infarction. Thus a thyroid hormone analogue with inotropic selectivity may be a
useful adjunct to other measures in the treatment of heart failure. Though thyroid hormone abnormalities have been identified in many cardiac
conditions, the role of thyroid hormones in congestive heart failure has not
been well defined. In a population of patients with advanced heart failure, a
reduction in triiodothyronine (T3) with an increase in reverse T3 was identified
in many patients, with an abnormally low ratio of T3/reverse T3 being the
strongest predictor of mortality. Normalization of thyroid indices appeared to
be necessary for prolonged survival to occur. To address the concern of T3
administration possibility exacerbating a hypermetabolic state, basal metabolic
rate was measured in a group of advanced heart failure patients and was found to
be generally within the normal range. A preliminary safety study of short-term
intravenous T3 administration (bolus +/- 6 h infusion, total dose 0.15-2.7
micrograms/kg) was then performed in 23 patients under hemodynamic and
electrocardiographic monitoring. There were neither adverse events nor
substantial hemodynamic changes, but some patients had an increase in cardiac
output, consistent with a peripheral vasodilatory effect. With this foundation,
further investigation into the possible role of T3 and its analogs in congestive
heart failure therapy may be pursued. We asked whether thyroid hormone (T4) would improve heart function in left
ventricular hypertrophy (LVH) induced by pressure overload (aortic banding).
After banding for 10-22 wk, rats were treated with T4 or saline for 10-14 d.
Isovolumic LV pressure and cytosolic [Ca2+] (indo-1) were assessed in perfused
hearts. Sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban, and alpha-
and beta-myosin heavy chain (MHC) proteins were assayed in homogenates of
myocytes isolated from the same hearts. Of 14 banded hearts treated with saline,
8 had compensated LVH with normal function (LVHcomp), whereas 6 had abnormal
contraction, relaxation, and calcium handling (LVHdecomp). In contrast, banded
animals treated with T4 had no myocardial dysfunction; these hearts had
increased contractility, and faster relaxation and cytosolic [Ca2+] decline
compared with LVHcomp and LVHdecomp. Myocytes from banded hearts treated with T4
were hypertrophied but had increased concentrations of alpha-MHC and SERCA
proteins, similar to physiological hypertrophy induced by exercise. Thus thyroid
hormone improves LV function and calcium handling in pressure overload
hypertrophy, and these beneficial effects are related to changes in myocyte gene
expression. Induction of physiological hypertrophy by thyroid hormone-like
signaling might be a therapeutic strategy for treating cardiac dysfunction in
pathological hypertrophy and heart failure. Most patients with advanced congestive heart failure have altered thyroid
hormone metabolism. A low triiodothyronine level is associated with impaired
hemodynamics and is an independent predictor of poor survival. This study sought
to evaluate safety and hemodynamic effects of short-term intravenous
administration of triiodothyronine in patients with advanced heart failure. An
intravenous bolus dose of triiodothyronine, with or without a 6- to 12-hour
infusion (cumulative dose 0. 1 5 to 2.7 microg/kg), was administered to 23
patients with advanced heart failure (mean left ventricular ejection fraction
0.22 +/- 0.01). Cardiac rhythm and hemodynamic status were monitored for 12
hours, and basal metabolic rate by indirect calorimetry, echocardiographic
parameters of systolic function and valvular regurgitation, thyroid hormone, and
catecholamine levels were measured at baseline and at 4 to 6 hours.
Triiodothyronine was well tolerated without episodes of ischemia or clinical
arrhythmia. There was no significant change in heart rate or metabolic rate and
there was minimal increase in core temperature. Cardiac output increased with a
reduction in systemic vascular resistance in patients receiving the largest
dose, consistent with a peripheral vasodilatory effect. Acute intravenous
administration of triiodothyronine is well tolerated in patients with advanced
heart failure, establishing the basis for further investigation into the safety
and potential hemodynamic benefits of longer infusions, combined infusion with
inotropic agents, oral triiodothyronine replacement therapy, and new
triiodothyronine analogs. After an initial safety study in 7 normal volunteers, a randomized double-blind
comparison was made between 3,5-diiodothyropropionic acid (DITPA) and placebo in
19 patients with moderately severe congestive failure. In heart failure patients
receiving the drug for 4 weeks, cardiac index was increased (p = 0.04) and
systemic vascular resistance index was decreased (p = 0.02). Systolic cardiac
function was unchanged but isovolumetric relaxation time was decreased
significantly, suggesting improvement in diastolic function. Total serum
cholesterol (p = 0.005) and triglycerides (p = 0.01) also were decreased
significantly. DITPA could represent a useful new agent for treatment of
congestive heart failure. We examined the effects of thyroid hormones (THs) on left ventricular (LV)
function and myocyte remodeling in rats with spontaneously hypertensive heart
failure (SHHF). SHHF rats were treated with three different TH doses from 20-21
mo of age. In terminal experiments, LV function (as determined by
echocardiography and catheterization) and isolated myocyte shape were examined
in SHHF rat groups and age-matched Wistar-Furth control animals. Compared with
Wistar-Furth rats, the ratio of alpha- to beta-myosin was reduced in untreated
SHHF rats. The alpha-to-beta-myosin ratio increased in all TH groups, which
suggests a reversal of the fetal gene program. Low-dose TH produced no changes
in LV myocyte size or function, but high-dose TH produced signs of
hyperthyroidism (e.g., increased heart weight, tachycardia). The chamber
diameter-to-wall thickness ratio declined with increasing dose due to reduced
chamber diameter and increased wall thickness. This resulted in a 38% reduction
in LV systolic wall stress in the middle- and high-dose groups despite sustained
hypertension. Isolated myocyte data indicated that chamber remodeling and
reduced wall stress were due to a unique alteration in myocyte transverse shape
(e.g., reduced major diameter and increased minor diameter). Based on our
present understanding of ventricular remodeling and wall stress, we believe
these changes are likely beneficial. Results suggest that TH may be an important
regulator of myocyte transverse shape in heart disease. Thyroid hormone (T3 and T4) has many beneficial effects including enhancing
cardiac function, promoting weight loss and reducing serum cholesterol. Excess
thyroid hormone is, however, associated with unwanted effects on the heart, bone
and skeletal muscle. We therefore need analogs that harness the beneficial
effects of thyroid hormone without the untoward effects. Such work is largely
based on understanding the cellular mechanisms of thyroid hormone action,
specifically the crystal structure of the nuclear receptor proteins. In clinical
studies, use of naturally occurring thyroid hormone analogs can suppress TSH
levels in patients with thyroid cancer without producing tachycardia. Many
thyromimetic compounds have been tested in animal models and shown to increase
total body oxygen consumption, and to lower weight and serum cholesterol and
triglyceride levels while having minor effects on heart rate. Alternatively,
analogs that specifically enhance both systolic and diastolic function are
potentially useful in the treatment of chronic congestive heart failure. In
addition to analogs that are thyroid hormone receptor agonists, several
compounds that are thyroid hormone receptor antagonists have been identified and
tested. This Review discusses the potential application of thyroid hormone
analogs (both agonists and antagonists) in a variety of human disease states. OBJECTIVE: Previous experimental studies have provided evidence showing that
changes in thyroid hormone signaling correspond to alterations in myocardial
function in animal models of heart failure. The present study further explores
whether thyroid hormone alterations are correlated with the functional status of
the myocardium in patients with heart failure.
METHODS: In this study, 37 patients with mean ejection fraction (EF%) of 26.2
(8.2) were included. Myocardial performance was assessed by echocardiography and
cardiopulmonary exercise testing. Total tri-iodothyronine (T3), thyroxine, and
TSH levels were measured in plasma.
RESULTS: Total T3 was strongly correlated with VO2max (r = 0.78, P = 2 x
10(-8)). Furthermore, multivariate analysis revealed that total T3 was an
independent predictor of VO2max (P = 0.000 005). A weaker but significant
correlation was also found between total T3 and EF% (r = 0.56, P = 0.0004),
systolic (r = 0.43, P = 0.009) and diastolic (r = 0.46, P = 0.004) blood
pressure.
CONCLUSIONS: changes in thyroid hormone were closely correlated to myocardial
functional status in patients with heart failure. These data probably indicate a
possible role of thyroid hormone in the pathophysiology of heart failure and
confirm previous experimental reports. CONTEXT: Low-T(3) syndrome is a predictor of poor outcome in patients with
cardiac dysfunction. The study aimed to assess the short-term effects of
synthetic L-T(3) replacement therapy in patients with low-T(3) syndrome and
ischemic or nonischemic dilated cardiomyopathy (DC).
DESIGN: A total of 20 clinically stable patients with ischemic (n = 12) or
nonischemic (n = 8) DC were enrolled. There were 10 patients (average age 72 yr,
range 66-77; median, 25-75th percentile) who underwent 3-d synthetic L-T(3)
infusion (study group); the other 10 patients (average age 68 yr, range 64-71)
underwent placebo infusion (control group). Clinical examination,
electrocardiography, cardiac magnetic resoce, and bio-humoral profile (free
thyroid hormones, TSH, plasma renin activity, aldosterone, noradrenaline,
N-terminal-pro-B-Type natriuretic peptide, and IL-6) were assessed at baseline
and after 3-d synthetic L-T(3) (initial dose: 20 microg/m(2) body surface.d) or
placebo infusion.
RESULTS: After T(3) administration, free T(3) concentrations increased until
reaching a plateau at 24-48 h (3.43, 3.20-3.84 vs. 1.74, 1.62-1.93 pg/ml; P =
0.03) without side effects. Heart rate decreased significantly after T(3)
infusion (63, 60-66 vs. 69, 60-76 beats per minute; P = 0.008). Plasma
noradrenaline (347; 270-740 vs. 717, 413-808 pg/ml; P = 0.009), N-terminal
pro-B-Type natriuretic peptide (3000, 438-4005 vs. 3940, 528-5628 pg/ml; P =
0.02), and aldosterone (175, 152-229 vs. 231, 154-324 pg/ml; P = 0.047)
significantly decreased after T(3) administration. Neurohormonal profile did not
change after placebo infusion in the control group. After synthetic L-T(3)
administration, left-ventricular end-diastolic volume (142, 132-161 vs. 133,
114-158 ml/m(2) body surface; P = 0.02) and stroke volume (40, 34-44 vs. 35,
28-39 ml/m(2) body surface; P = 0.01) increased, whereas external and
intracardiac workload did not change.
CONCLUSIONS: In DC patients, short-term synthetic L-T(3) replacement therapy
significantly improved neuroendocrine profile and ventricular performance. These
data encourage further controlled trials with more patients and longer periods
of synthetic L-T(3) administration. Interest in the role of thyroid hormones (TH) in heart failure is steadily
increasing due to evidence for a physiological, homeostatic role of TH and the
effects of altered TH metabolism on the cardiovascular system, particularly in
presence of heart failure. Experimental studies have shown that altered TH
metabolism modifies cardiovascular homeostasis by inducing alterations of
cardiac histology, cardiomyocyte morphology and gene expression and
consequently, of diastolic and systolic myocardial function. Clinical studies
have shown that mild forms of thyroid dysfunction, both primary (subclinical
hypothyroidism and subclinical hyperthyroidism) and secondary (low T(3)
syndrome) have negative prognostic impact in patients with heart failure. In
these patients, the administration of synthetic triiodothyronine (T(3)) was well
tolerated and induced significant improvement in cardiac function without
increased heart rate and metabolic demand. Large multicenter, placebo-controlled
prospective studies are necessary to evaluate the safety and prognostic effects
of chronic treatment with TH replacement therapy in patients with heart failure.
The article also discusses recent patents in this field. BACKGROUND: Patients with congestive heart failure (CHF) often have low serum
triiodothyronine (T(3)) concentrations. In a rodent model of myocardial
infarction-induced CHF and low serum T(3), we hypothesized that replacing T(3)
to euthyroid levels would improve left ventricular function without producing
untoward signs of thyrotoxicosis.
METHODS AND RESULTS: Adult male Sprague-Dawley rats were subjected to left
anterior descending coronary artery ligation (myocardial infarction). One week
post-myocardial infarction, left ventricular fractional shortening was
significantly reduced to 22+/-1% in CHF animals versus 38+/-1% for sham-operated
controls (P<0.001). Serum T(3) concentration was also significantly reduced
(80+/-3 versus 103+/-6 ng/dL; P<0.001), in CHF animals versus Shams. At 9 weeks
post-myocardial infarction, systolic function (+dP/dt max) was significantly
attenuated in CHF animals (4773+/-259 versus 6310+/-267 mmHg/s; P<0.001) as well
as diastolic function measured by half time to relaxation (15.9+/-1.2 versus
11.1+/-0.3 ms; P<0.001). alpha-myosin heavy chain expression was also
significantly reduced by 77% (P<0.001), and beta-myosin heavy chain expression
was increased by 21%. Continuous T(3) replacement was initiated 1 week
post-myocardial infarction with osmotic mini-pumps (6 microg/kg/d), which
returned serum T(3) concentrations to levels similar to Sham controls while
resting conscious heart rate, arterial blood pressure and the incidence of
arrhythmias were not different. At 9 weeks, systolic function was significantly
improved by T(3) replacement (6279+/-347 mmHg/s; P<0.05) and a trend toward
improved diastolic function (12.3+/-0.6 ms) was noted. T(3) replacement in CHF
animals also significantly increased alpha- and reduced beta-MHC expression,
(P<0.05).
CONCLUSIONS: These data indicate that T(3) replacement to euthyroid levels
improves systolic function and tends to improve diastolic function, potentially
through changes in myocardial gene expression. 3,5,3'-Levo-triiodothyronine (L-T3) is essential for DNA transcription,
mitochondrial biogenesis and respiration, but its circulating levels rapidly
decrease after myocardial infarction (MI). The main aim of our study was to test
whether an early and sustained normalization of L-T3 serum levels after MI
exerts myocardial protective effects through a mitochondrial preservation.
Seventy-two hours after MI induced by anterior interventricular artery ligation,
rats were infused with synthetic L-T3 (1.2 μg/kg/day) or saline over 4 weeks.
Compared to saline, L-T3 infusion restored FT3 serum levels at euthyroid state
(3.0 ± 0.2 versus 4.2 ± 0.3 pg/ml), improved left ventricular (LV) ejection
fraction (39.5 ± 2.5 versus 65.5 ± 6.9%), preserved LV end-systolic wall
thickening in the peri-infarct zone (6.34 ± 3.1 versus 33.7 ± 6.21%) and reduced
LV infarct-scar size by approximately 50% (all P < 0.05). Moreover, L-T3
significantly increased angiogenesis and cell survival and enhanced the
expression of nuclear-encoded transcription factors involved in these processes.
Finally, L-T3 significantly increased the expression of factors involved in
mitochondrial DNA transcription and biogenesis, such as hypoxic inducible
factor-1α, mitochondrial transcription factor A and peroxisome proliferator
activated receptor γ coactivator-1α, in the LV peri-infarct zone. To further
explore mechanisms of L-T3 protective effects, we exposed isolated neonatal
cardiomyocytes to H(2)O(2) and found that L-T3 rescued mitochondrial biogenesis
and function and protected against cell death via a mitoKATP dependent pathway.
Early and sustained physiological restoration of circulating L-T3 levels after
MI halves infarct scar size and prevents the progression towards heart failure.
This beneficial effect is likely due to enhanced capillary formation and
mitochondrial protection. Thyroid hormones have relevant activity at cardiac and vascular level, by
influencing heart rate, myocardial excitability as well as inotropic and
lusitropic status, systemic vascular resistance and blood pressure. Moreover,
they interact with neuro-hormonal systems such as sympathetic nervous system and
renin-angiotensin-aldosterone system thus also indirectly influencing
cardiovascular function. Due to these effects, both hypothyroidism and
hyperthyroidism, either in their overt or subclinical forms, can have an
unfavourable impact in the setting of cardiovascular diseases. The aim of this
review is to focus on the prognostic consequences of thyroid disorders in heart
failure patients. Moreover, the therapeutical approach and the possible
beneficial effects of restoring euthyroidism are reviewed. AIM: To assess whether levothyroxine treatment improves functional capacity in
patients with chronic heart failure (New York Heart Association class i-iii) and
subclinical hypothyroidism.
METHODS: One hundred and sixty-three outpatients with stable chronic heart
failure followed up for at least 6 months were enrolled. A physical examination
was performed, and laboratory tests including thyroid hormone levels, Doppler
echocardiogram, radionuclide ventriculography, and Holter monitoring were
requested. Functional capacity was assessed by of the 6-min walk test. Patients
with subclinical hypothyroidism were detected and, after undergoing the s6-min
walk test, were given replacement therapy. When they reached normal thyrotropin
(TSH) levels, the 6-min walk test was performed again. The distance walked in
both tests was recorded, and the difference in meters covered by each patient
was analyzed.
RESULTS: Prevalence of subclinical hypothyroidism in patients with heart failure
was 13%. These patients walked 292±63m while they were hypothyroid and 350±76m
when TSH levels returned to normal, a difference of 58±11m (P<.011). Patients
with normal baseline TSH levels showed no significant difference between the 2
6-min walk tests.
CONCLUSIONS: Patients with chronic heart failure and subclinical hypothyroidism
significantly improved their physical performance when normal TSH levels were
reached. |
Is there a mouse model for Fanconi anemia? | A number of mouse models have already been generated with a targeted disruption of several Fanconi anemia genes, such as FANCA, FANCF, FANCM, FANCD1, etc. | Despite the cloning of four disease-associated genes for Fanconi anemia (FA),
the molecular pathogenesis of FA remains largely unknown. To study FA
complementation group A using the mouse as a model system, we cloned and
characterized the mouse homolog of the human FANCA cDNA. The mouse cDNA (Fanca)
encodes a 161-kDa protein that shares 65% amino acid sequence identity with
human FANCA. Fanca is located at the distal region of mouse chromosome 8 and has
a ubiquitous pattern of expression in embryonic and adult tissues. Expression of
the mouse cDNA in human FA-A cells restores the cellular drug sensitivity to
normal levels. Thus, the expression pattern, protein structure, chromosomal
location, and function of FANCA are conserved in the mouse. We also isolated a
novel zinc finger protein, Zfp276, which has five C(2)H(2) domains.
Interestingly, Zfp276 is situated in the Fanca locus, and the 3'UTR of its cDNA
overlaps with the last four exons of Fanca in a tail-to-tail manner. Zfp276 is
expressed in the same tissues as Fanca, but does not complement the mitomycin C
(MMC)-sensitive phenotype of FA-A cells. The overlapping genomic organization
between Zfp276 and Fanca may have relevance to the disease phenotype of FA. Fanconi anemia (FA) is a chromosomal instability syndrome characterized by the
presence of pancytopenia, congenital malformations and cancer predisposition.
Six genes associated with this disorder have been cloned, and mice with targeted
disruptions of several of the FA genes have been generated. These mouse models
display the characteristic FA feature of cellular hypersensitivity to DNA
cross-linking agents. Although they do not develop hematological or
developmental abnormalities spontaneously, they mimic FA patients in their
reduced fertility. Studies using these animal models provide valuable insights
into the involvement of apoptotic pathways in FA, and help characterize the
defects in FA hematopoietic cells. In addition, mouse models are also useful for
testing treatments for FA. OBJECTIVE: Fanconi anemia (FA) is a genetically heterogeneous disorder
associated with defects in at least eight genes. The biochemical function(s) of
the FA proteins are unknown, but together they define the FA pathway, which is
involved in cellular responses to DNA damage and in other cellular processes. It
is currently unknown whether all FA proteins are involved in controlling a
single function or whether some of the FA proteins have additional roles. The
aim of this study was 1) to determine whether the FA group A and group C genes
have identical or partially distinct functions, and 2) to have a better model
for human FA.
MATERIALS AND METHODS: We generated mice with a targeted mutation in fanca and
crossed them with fancc disrupted animals. Several phenotypes including
sensitivity to DNA cross linkers and ionizing radiation, hematopoietic colony
growth, and germ cell loss were analyzed in fanca-/-, fancc-/-, fanca/fancc
double -/-, and controls.
RESULTS: Fibroblast cells and hematopoietic precursors from fanca/fancc
double-mutant mice were not more sensitive to MMC than those of either single
mutant. fanca/fancc double mutants had no evidence for an additive phenotype at
the cellular or organismal level.
CONCLUSIONS: These results support a model where both FANCA and FANCC are part
of a multi-protein nuclear FA complex with identical function in cellular
responses to DNA damage and germ cell survival. Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone
marrow failure and cancer predisposition. So far, 8 complementation groups have
been identified, although mutations in FANCA account for the disease in the
majority of FA patients. In this study we characterized the hematopoietic
phenotype of a Fanca knockout mouse model and corrected the main phenotypic
characteristics of the bone marrow (BM) progenitors using retroviral vectors.
The hematopoiesis of these animals was characterized by a modest though
significant thrombocytopenia, consistent with reduced numbers of BM
megakaryocyte progenitors. As observed in other FA models, the hematopoietic
progenitors from Fanca(-/-) mice were highly sensitive to mitomycin C (MMC). In
addition, we observed for the first time in a FA mouse model a marked in vitro
growth defect of Fanca(-/-) progenitors, either when total BM or when purified
Lin(-)Sca-1(+) cells were subjected to in vitro stimulation. Liquid cultures of
Fanca(-/-) BM that were stimulated with stem cell factor plus interleukin-11
produced low numbers of granulocyte macrophage colony-forming units, contained a
high proportion of apoptotic cells, and generated a decreased proportion of
granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to
correct the phenotype of Fanca(-/-) progenitors, purified Lin(-)Sca-1(+) cells
were transduced with retroviral vectors encoding the enhanced green fluorescent
protein (EGFP) gene and human FANCA genes. Lin(-)Sca-1(+) cells from Fanca(-/-)
mice were transduced with an efficiency similar to that of samples from
wild-type mice. More significantly, transductions with FANCA vectors corrected
both the MMC hypersensitivity as well as the impaired ex vivo expansion ability
that characterized the BM progenitors of Fanca(-/-) mice. We have investigated the hematopoietic phenotype of mice with a hypomorphic
mutation in the Brca2/Fancd1 gene (Brca2(Delta27/Delta27) mutation). In contrast
to observations made in other Fanconi anemia (FA) mouse models, low numbers of
hematopoietic colony-forming cells (CFCs) were noted in Brca2(Delta27/Delta27)
mice, either young or adult. Additionally, a high incidence of spontaneous
chromosomal instability was observed in Brca2(Delta27/Delta27) bone marrow (BM)
cells, but not in Brca2(+/Delta27) or Fanca(-/-) BM cells. Although
Brca2(Delta27/Delta27) CFCs were not hypersensitive to ionizing radiation, a
very severe hematopoietic syndrome was observed in irradiated
Brca2(Delta27/Delta27) mice. Conventional BM competition experiments showed a
marked repopulation defect in Brca2(Delta27/Delta27) hematopoietic stem cells
(HSCs), compared to wild-type HSCs. Moreover, we have observed for the first
time in a DNA repair disease model a very significant proliferation defect in
Brca2(Delta27/Delta27) HSCs maintained in their natural physiological
environment. The progressive repopulation of wild-type HSCs transplanted into
unconditioned Brca2(Delta27/Delta27) recipients is reminiscent of the somatic
mosaicism phenomenon observed in a number of genetic diseases, including FA. The
hematopoietic phenotype associated with the Brca2(Delta27/Delta27) mutation
suggests that this FA-D1 mouse model will constitute an important tool for the
development of new therapies for FA, including gene therapy. Fanconi anemia (FA) is an inherited recessive DNA repair disorder mainly
characterized by bone marrow failure and cancer predisposition. Studies in
mosaic FA patients have shown that reversion of one inherited germ-line mutation
resulting in a functional allele in one or a few hematopoietic stem cells (HSCs)
can lead to the proliferation advantage of corrected cells, thus over time
normalizing the hematologic status of the patient. In contrast to these
observations, it is still unclear whether ex vivo genetic correction of FA HSCs
also provides a similar proliferation advantage to FA HSCs. Using an FA mouse
model with a marked hematopoietic phenotype, the FA-D1 (Brca2(Delta27/Delta27))
mice, we demonstrate that the lentivirus-mediated gene therapy of FA HSCs
results in the progressive expansion of genetically corrected clones in
mild-conditioned FA-D1 recipients. Consistent with these data, hematopoietic
progenitors from FA recipients progressively became mitomycin C resistant and
their chromosomal instability was reverted. No evidence of myelodysplasia,
leukemias, or abnormal clonal repopulation was observed at multiple time points
in primary or secondary recipients. Our results demonstrate that ectopic
expression of BRCA2 confers a beneficial in vivo proliferation advantage to
FA-D1 HSCs that enables the full hematopoietic repopulation of FA recipients
with genetically corrected cells. Fanconi anemia is a rare inherited disease characterized by congenital
anomalies, growth retardation, aplastic anemia and an increased risk of acute
myeloid leukemia and squamous cell carcinomas. The disease is caused by mutation
in genes encoding proteins required for the Fanconi anemia pathway, a response
mechanism to replicative stress, including that caused by genotoxins that cause
DNA interstrand crosslinks. Defects in the Fanconi anemia pathway lead to
genomic instability and apoptosis of proliferating cells. To date, 13
complementation groups of Fanconi anemia were identified. Five of these genes
have been deleted or mutated in the mouse, as well as a sixth key regulatory
gene, to create mouse models of Fanconi anemia. This review summarizes the
phenotype of each of the Fanconi anemia mouse models and highlights how genetic
and interventional studies using the strains have yielded novel insight into
therapeutic strategies for Fanconi anemia and into how the Fanconi anemia
pathway protects against genomic instability. The Fanconi anemia (FA) core complex member FANCM remodels synthetic replication
forks and recombination intermediates. Thus far, only one FA patient with FANCM
mutations has been described, but the relevance of these mutations for the FA
phenotype is uncertain. To provide further experimental access to the FA-M
complementation group we have generated Fancm-deficient mice by deleting exon 2.
FANCM deficiency caused hypogonadism in mice and hypersensitivity to
cross-linking agents in mouse embryonic fibroblasts (MEFs), thus phenocopying
other FA mouse models. However, Fancm(Delta2/Delta2) mice also showed unique
features atypical for FA mice, including underrepresentation of female
Fancm(Delta2/Delta2) mice and decreased overall and tumor-free survival. This
increased cancer incidence may be correlated to the role of FANCM in the
suppression of spontaneous sister chromatid exchanges as observed in MEFs. In
addition, FANCM appeared to have a stimulatory rather than essential role in
FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that
FANCM functions both inside and outside the FA core complex to maintain genome
stability and to prevent tumorigenesis. The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is
critical for DNA damage response. SLX4 nuclease complexes mediate repair during
replication and can also resolve Holliday junctions formed during homologous
recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the
mouse ortholog of SLX4, which recapitulates many key features of the human
genetic illness Fanconi anemia. Btbd12-deficient animals are born at
sub-Mendelian ratios, have greatly reduced fertility, are developmentally
compromised and are prone to blood cytopenias. Btbd12(-/-) cells prematurely
senesce, spontaneously accumulate damaged chromosomes and are particularly
sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial
requirement for Btbd12 (also known as Slx4) to interact with the
structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The
Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia
and genetically links a regulator of nuclease incision complexes to the Fanconi
anemia DNA crosslink repair pathway. Fanconi anaemia (FA) is a rare recessive disorder marked by developmental
abnormalities, bone marrow failure, and a high risk for the development of
leukaemia and solid tumours. The inactivation of FA genes, in particular FANCF,
has also been documented in sporadic tumours in non-FA patients. To study
whether there is a causal relationship between FA pathway defects and tumour
development, we have generated a mouse model with a targeted disruption of the
FA core complex gene Fancf. Fancf-deficient mouse embryonic fibroblasts
displayed a phenotype typical for FA cells: they showed an aberrant response to
DNA cross-linking agents as manifested by G(2) arrest, chromosomal aberrations,
reduced survival, and an inability to monoubiquitinate FANCD2. Fancf homozygous
mice were viable, born following a normal Mendelian distribution, and showed no
growth retardation or developmental abnormalities. The gonads of Fancf mutant
mice functioned abnormally, showing compromised follicle development and
spermatogenesis as has been observed in other FA mouse models and in FA
patients. In a cohort of Fancf-deficient mice, we observed decreased overall
survival and increased tumour incidence. Notably, in seven female mice, six
ovarian tumours developed: five granulosa cell tumours and one luteoma. One
mouse had developed tumours in both ovaries. High-resolution array comparative
genomic hybridization (aCGH) on these tumours suggests that the increased
incidence of ovarian tumours correlates with the infertility in Fancf-deficient
mice and the genomic instability characteristic of FA pathway deficiency. |
Could Hyperthermic intraperitoneal chemotherapy (HIPEC) be effective for the treatment of recurrent ovarian cancer? | There is level-one evidence suggesting the benefit of postoperative adjuvant intraperitoneal chemotherapy for patients with advanced ovarian cancer after cytoreductive surgery, albeit catheter-related complications resulted after treatment discontinuation. Studies report the use of HIPEC predominantly in the setting of recurrent disease and have demonstrated encouraging results, which merits further investigation in future clinical trials | OBJECTIVES: To assess feasibility, complications and efficacy of secondary
surgical cytoreduction (CRS) and hyperthermic intraperitoneal chemotherapy
(HIPEC) in a selected group of platinum-sensitive recurrent ovarian cancer
patients.
METHODS: Recurrent ovarian cancer patients with a platinum-free interval of at
least 6 months were prospectively enrolled. After complete CRS they were
submitted to intraperitoneal perfusion of oxaplatinum (460 mg/m(2)) heated to
41.5 degrees C for 30 min. Then they received systemic chemotherapy with
taxotere 75 mg/m(2) and oxaliplatin 100 mg/m(2) for 6 cycles. Patients were
followed up routinely until recurrence or death.
RESULTS: Twenty-five recurrent ovarian cancer patients were valuable for the
study. The median Platinum Free Interval (PFI) was 25 months (range 7-67). The
majority of the patients (76%) had diffuse carcinosis. Nobody had ascites. An
optimal residual disease was obtained in all patients. The median duration of
CRS+HIPEC was 312 min (range 138-619). Median intensive care unit (ICU) stay was
2 days (1-6), median hospital stay was 13 days (7-30). Post-operative major
complications were observed in 7 patients (28%). Post-operative mortality was
0%. With a median follow-up time of 18 months (range 3-38), 24 patients (96%)
are alive, but seven women (28%) have relapsed.
CONCLUSIONS: Adequate pre-operative selection can improve feasibility of CRS and
HIPEC. Morbidity rate is comparable to aggressive cytoreduction without HIPEC.
Although associated with some post-operative morbidity, long-term results are
encouraging, waiting for larger series and longer follow-up data. BACKGROUND: The present study reviews our 12-year results with cytoreductive
surgery and HIPEC in patients with advanced primary and recurrent ovarian
cancer.
METHODS: During the period from January 1995 to December 2007, 56 patients (31
with primary and 25 with recurrent epithelial ovarian cancer) underwent
cytoreductive surgery and HIPEC (Doxorubicin intra-operatively, and cisplatin
next 1-5 postoperative days) at our department.
RESULTS: 52 (92.8%) patients had no gross residual disease after the complete
surgical procedure (Sugarbaker completeness of cytoreduction CC, score 0-1), and
4 patients had macroscopic residual disease (CC-2 or CC-3) Average PCI
(peritoneal cancer index) was 13.4 (4-28). Mean follow-up was 56 months (range,
1-135). The median operation time was 279min (range 190 + or - 500min). Median
total blood loss was 850mL (range 250 + or - 1550mL). The median survival time
was 34.1 months for primary, 40.1 for recurrent ovarian cancer without
statistically significance difference (p>0.05) and median disease-free survival
was 26.2 months. The PCI was equal or less than 12 in 31 patients and their
median survival time was statistically significant longer than median survival
time of months for the 25 patients with PCI greater then 12 (p<0.01). Morbidity
and mortality rate were 17.8% (10/56) and 1.8% (1/56).
CONCLUSION: This series indicates that in the majority of patients with primary
and recurrent advanced ovarian cancer, cytoreductive surgery combined with HIPEC
can lead to a substantial increase in subsequent rates of disease-free and
overall survival. BACKGROUND: Advanced and recurrent ovarian cancer results in extensive spread of
tumor on the peritoneal surfaces of the abdomen and pelvis. We collectively
review studies in the literature that report the efficacy of cytoreductive
surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) for ovarian
cancer peritoneal carcinomatosis.
METHODS: An electronic search of all relevant studies published in peer-reviewed
journals before May 2009 was performed on three databases. The quality of each
study was independently assessed and classified according to the time point of
HIPEC use in various setting of ovarian cancer from the consensus statement of
the Peritoneal Surface Oncology Group. Clinical efficacy was synthesized through
a narrative review with full tabulation of the results of each included study.
RESULTS: Nineteen studies each of more than ten patients reporting treatment
results of HIPEC of patients with both advanced and recurrent ovarian cancer
were included and data were extracted. All studies were observational case
series. The overall rate of severe perioperative morbidity ranged from 0 to 40%
and mortality rate varied from 0 to 10%. The overall median survival following
treatment with HIPEC ranged from 22 to 64 months with a median disease-free
survival ranging from 10 to 57 months. In patients with optimal cytoreduction, a
5-year survival rate ranging from 12 to 66% could be achieved.
CONCLUSION: Despite the heterogeneity of the studies reviewed, current evidence
suggest that complete CRS and HIPEC may be a feasible option with potential
benefits that are comparable with the current standard of care. A randomized
trial is required to establish the role of HIPEC in ovarian cancer. Hyperthermic intraperitoneal chemotherapy (HIPEC) represents a new treatment
strategy aimed to improve outcome of patients with advanced ovarian cancer.
Based on theoretical and experimental basis, HIPEC should stand as an effective
treatment for ovarian cancer. Literature review reveals a number of different
experiences on feasibility of this technique, but scientific evidence of current
studies remains poor. Much more research is still required to elucidate
uswered questions. Before this technique can be routinely used, some
controversial aspects have to be defined: which drug is the best to deliver and
at what temperature, is it necessary to use mono- or poly-chemotherapy regimens,
which is the time-point for HIPEC in the natural history of ovarian cancer: at
front-line therapy, at interval debulking following initial neo-adjuvant
chemotherapy, at consolidation following front-line therapy, or at the time of
recurrence. Nevertheless, we must not forget that the most important issue in
ovarian cancer prognosis is maximal cytoreductive surgery, with no residual
disease at completion of initial surgery. OBJECTIVES: To analyze the outcomes of cytoreductive surgery and HIPEC in
patients with peritoneal carcinomatosis from ovarian cancer.
METHODS: Fifty-three patients with peritoneal carcinomatosis from primary (45
cases) and recurrent (8 cases) ovarian cancer were previously treated by
systemic chemotherapy with platinum and taxanes and then submitted to surgical
cytoreduction and HIPEC (cisplatin and mitomycin-C) with a closed abdomen
technique. The median follow-up period was 27 months (range: 3-107).
RESULTS: At the end of operation a complete cytoreduction (CCR-0) was obtained
in 37 patients (70%). Major morbidity occurred in 12 patients (23%); reoperation
was necessary in 2 patients (4%), and no postoperative mortality was observed.
Overall 5-year survival probability was 55%; it was 71% in CCR-0, 44% in CCR-1,
and none in patients with CCR-2 or CCR-3 residual tumor (log-rank test: P =
0.017). The cumulative risk of recurrence in 37 CCR-0 cases was 54% at 5 years
from operation.
CONCLUSIONS: The results of our study indicate the feasibility and the potential
benefit of a protocol including systemic chemotherapy, surgical cytoreduction
and HIPEC in patients with peritoneal carcinomatosis from ovarian cancer. A
phase III trial to compare this approach with conventional treatment is needed. Favorable oncological outcomes have been reported in several trials with the
introduction of Cytoreductive Surgery (CRS) and Hyperthermic Intraperitoneal
Chemotherapy (HIPEC) in the treatment of Advanced Epithelial Ovarian Cancer
(EOC). However most of the studies testing the combined approach are
observational and have been conducted in inhomogeneous series so that the
evidence supporting the performance of this combined treatment is still poor.
Median Overall and Disease Free Survivals of up to 64 months and 57 months,
respectively have been reported. Although a rate of morbidity of up to 40% has
been observed in some series the CRS + HIPEC continues to gain an increased
popularity. Several prospective randomized trials are ongoing using the
procedure in various time points of the disease. In this review several issues
such as the impact of cytoreduction and residual disease (RD) on outcomes as
well as the role of HIPEC will be updated from the literature evidence. Some
controversial points HIPEC related will also be discussed. Recent experiences
regarding the introduction of a more aggressive surgical approach to upper
abdomen to resect peritoneal carcinomatosis (PC) allowed increased rates of
optimal cytoreduction and has demonstrated an apparent better outcome. This
evidence associated with the positive results phase III trial testing
normothermic intraperitoneal as first-line chemotherapy is guiding some
investigators to propose the CRS + HIPEC in the primary setting. Several
prospective phase II and III trials have recently been launched to validate the
role of the combined treatment in various time points of disease natural
evolution. OBJECTIVE: To evaluate morbidity and mortality rates associated with the use of
hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC) after optimal
cytoreduction (CRS) in a large single-institutional series of platinum-sensitive
recurrent ovarian cancer patients. Moreover, disease free (DFS) and overall
survival (OS) of previously studied patients have been assessed after a longer
follow-up period.
METHOD: From May 2005 to October 2010, recurrent ovarian cancer patients with a
platinum-free interval of at least 6 months have been prospectively enrolled in
a protocol of CRS plus HIPEC with oxaplatinum (460 mg/m(2)) heated to 41.5 °C
for 30 min, followed by 6 cycles of systemic chemotherapy with taxotere 75
mg/m(2) and oxaliplatin 100 mg/m(2).
RESULTS: Forty-one patients experienced 43 procedures (CRS+HIPEC). An optimal
cytoreduction was achieved in all cases (CC-0 95.3%; CC-1 4.7%). A complication
rate of 34.8% was registered, with no case of intraoperative death or within 30
days after surgery. Survival curves have been calculated in a group of 25
patients with a minimum follow-up of 18 months, obtaining a median DFS and OS of
24 (range 6-60) and 38 months (range 18-60), respectively.
CONCLUSION: In recurrent platinum-sensitive ovarian cancer patients, the use of
CRS plus HIPEC represents a safe treatment, able to significantly influence the
survival rates compared to chemotherapy alone or surgery plus standard
chemotherapy. PURPOSE: Peritoneal recurrence of ovarian cancer is frequent after primary
surgery and chemotherapy and has poor long-term survival. De novo cytoreductive
surgery is crucial with the potential to improve prognosis, especially when
combined with hyperthermic intraperitoneal chemotherapy (HIPEC).
METHODS: The sampled data of 40 consecutive patients were retrospectively
analyzed. Thirty-one patients were treated with cytoreductive surgery combined
with hyperthermic intraperitoneal chemotherapy.
RESULTS: No patient was lost in the perioperative period, and the combined
procedure was performed with acceptable morbidity. Colon-preserving
cytoreductive surgery was associated with reduced morbidity.
CONCLUSIONS: Patients suffering from peritoneal recurrence of ovarian cancer
should be considered for radical reoperation with HIPEC in a center with
expertise in multimodal therapeutic options. Organ-preserving cytoreductive
surgery allows complete cytoreduction with the goal of decreasing morbidity. OBJECTIVE: Although standard treatment for advanced epithelial ovarian cancer
(EOC) consists of surgical debulking and intravenous platinum- and taxane-based
chemotherapy, favorable oncological outcomes have been recently reported with
the use of cytoreductive surgery (CRS) and hyperthermic intraperitoneal
chemotherapy (HIPEC). The aim of the study was to analyze feasibility and
results of CRS and HIPEC in patients with advanced EOC.
MATERIALS/METHODS: This is an open, prospective phase 2 study including patients
with primary or recurrent peritoneal carcinomatosis due to EOC. Thirty-nine
patients with a mean (SD) age of 57.3 (9.7) years (range, 34-74 years) were
included between September 2005 and December 2009. Thirty patients (77%) had
recurrent EOC and 9 (23%) had primary EOC.
RESULTS: For HIPEC, cisplatin and paclitaxel were used for 11 patients (28%),
cisplatin and doxorubicin for 26 patients (66%), paclitaxel and doxorubicin for
1 patient (3%), and doxorubicin alone for 1 patient (3%). The median
intra-abdominal outflow temperature was 41.5°C. The mean peritoneal cancer index
(PCI) was 11.1 (range, 1-28); and according to the intraoperative tumor extent,
the tumor volume was classified as low (PCI <15) or high (PCI ≥15) in 27
patients (69%) and 12 patients (31%), respectively. Microscopically complete
cytoreduction was achieved for 35 patients (90%), macroscopic cytoreduction was
achieved for 3 patients (7%), and a gross tumor debulking was performed for 1
patient (3%). Mean hospital stay was 23.8 days. Postoperative complications
occurred in 7 patients (18%), and reoperations in 3 patients (8%). There was one
postoperative death. Recurrence was seen in 23 patients (59%) with a mean
recurrence time of 14.4 months (range, 1-49 months).
CONCLUSIONS: Hyperthermic intraperitoneal chemotherapy after extensive CRS for
advanced EOC is feasible with acceptable morbidity and mortality. Complete
cytoreduction may improve survival in highly selected patients. Additional
follow-up and further studies are needed to determine the effects of HIPEC on
survival. The standard treatment for advanced ovarian cancer consists in complete
cytoreductive surgery (CRS) and intravenous combination chemotherapy with a
platinum compound and a taxane. Although response rates to initial therapy are
high, many patients will recur and die of peritoneal carcinomatosis. The
addition of Hyperthermic IntraPEritoneal Chemotherapy (HIPEC) to the standard
therapy aims at increasing survival by reducing peritoneal recurrence. This
review describes the survival results of HIPEC at the different time-points of
the treatment of ovarian cancer: at upfront CRS, at interval CRS, at
consolidation CRS after complete response to initial therapy, at secondary CRS
after incomplete response, at salvage CRS for recurrence and as palliative
treatment without CRS for unresectable ovarian cancer with chemotherapy
resistant ascites. The available evidence suggests that a potential survival
benefit of adding HIPEC may be largest in the settings of secondary CRS for
stage III ovarian cancer and salvage CRS for recurrent ovarian cancer, two
time-points representing failure of initial standard therapy. There is much less
evidence for a potential benefit of HIPEC for less advanced stages (I-II) and
for earlier time-points in the treatment of ovarian cancer (upfront, interval
and consolidation). Postoperative mortality is not higher after CRS and HIPEC
(0.7%) than after CRS only (1.4%). Four randomised trials are ongoing and their
results are eagerly awaited. Palliative HIPEC without CRS might be used more in
patients with incapacitating ascites due to recurrent ovarian cancer which has
become resistant to systemic chemotherapy. Advanced epithelial ovarian carcinoma has a dismal prognosis. Notwithstanding
the good initial response to primary therapy, 75% of the patients with advanced
disease will develop recurrent disease, causing approximately 60-80% of patients
to die within 5 years of initial diagnosis. The chemotherapy regimens of the
past century are summarized and the focus is on current systemic therapies and
future developments in advanced epithelial ovarian cancer. Recently various
promising treatments emerged. Attempts to optimize chemotherapy have included
weekly scheduling of paclitaxel and intraperitoneal chemotherapy. Trials using
angiogenesis inhibition and poly-ADP ribose polymerase (PARP) inhibitors show an
advantage in progression-free survival while further overall survival data are
awaited. Trabectedin, Hyperthermic intraperitoneal chemotherapy (HIPEC) and
chemo-immunotherapy may be become a promising therapy for the treatment of
ovarian cancer. This Editorial encapsulates these new developments in ovarian
cancer that are described extensively in the current theme issue Advances in
Epithelial Ovarian Cancer Therapy. The outcome of ovarian cancer remains poor with conventional therapy.
Intraperitoneal chemotherapy has some advantages over systemic chemotherapy,
including favorable pharmacokinetics and optimal treatment timing.
Intraoperative hyperthermic intraperitoneal chemotherapy (HIPEC) provides
improved exposure of the entire seroperitoneal surface to the agent and utilizes
the direct cytoxic and drug-enhancing effect of hyperthermia. While standard
normothermic, nonintraoperative, intraperitoneal chemotherapy has been
demonstrated to be beneficial in randomized trials and meta-analyses, there are
no data from randomized HIPEC trials available yet. Cautious extrapolation of
data from standard normothermic, nonintraoperative, intraperitoneal chemotherapy
and data from Phase II and nonrandomized comparative studies suggest that HIPEC
delivered at the time of surgery for ovarian cancer has definite potential. Data
from ongoing randomized HIPEC trials to adequately answer the question of
whether the addition of HIPEC actually prolongs survival in patients with
peritoneal dissemination of primary and recurrent ovarian cancer are awaited in
the near future. OBJECTIVES: To compare survival data in platinum-sensitive recurrent ovarian
cancer patients submitted to secondary cytoreduction (SCR) plus hyperthermic
intraperitoneal intraoperative chemotherapy (HIPEC) (Cases) and a similar group
of women not experiencing HIPEC (Controls).
METHODS: Case-control study, matching 30 Cases with 37 Controls, with at least
24 months of follow-up.
RESULTS: Groups were comparable for all characteristics, except for a higher
proportion of patients with single-nodule relapses is the Controls (19 vs. 6;
p=0.011). Median follow-up time was 46 months in the Cases and 36 months in the
Controls. Twenty patients (66.6%) experienced secondary recurrence in the Cases
and 37 women (100%) in the Controls (p=0.001). Moreover, 7 (23.3%) and 23
(62.2%) patients died of disease in the Cases and Controls respectively
(p=0.003). The duration of secondary response was 26 months in the Cases and 15
months in the Controls (p=0.004).
CONCLUSIONS: The combination of SCR and HIPEC seems to improve survival rate in
patients suffering from platinum-sensitive EOC recurrence with respect to
no-HIPEC treatments. This result further supports the need of a randomized
trial. PURPOSE: TO REVIEW THE TWO MAIN APPROACHES OF INTRAPERITONEAL (IP) CHEMOTHERAPY
DELIVERY IN OVARIAN CANCER: postoperative adjuvant IP chemotherapy after
cytoreductive surgery (CRS) and intraoperative hyperthermic intraperitoneal
chemotherapy (HIPEC).
METHODS: A literature search was conducted to identify studies that employed
postoperative adjuvant IP chemotherapy after CRS or combined CRS and
intraoperative HIPEC in patients with ovarian cancer. Data of interest included
chemotherapy protocol, morbidity and mortality, and survival data.
RESULTS: Three large randomized controlled trials comprising 707 patients with
advanced ovarian cancer who received postoperative adjuvant IP chemotherapy were
reviewed. Morbidity rate ranged from 56% to 94% in IP chemotherapy, and
mortality rate ranged from 1% to 2%. Median disease-free survival ranged from 24
to 28 months, and overall survival ranged from 49 to 66 months. Planned
chemotherapy completion rates ranged from 42% to 71%. Twenty-four nonrandomized
studies that reported HIPEC comprised 1167 patients with both advanced and
recurrent ovarian cancer. In patients with advanced ovarian cancer, mortality
ranged from 0% to 5%, minor morbidity ranged from 16% to 90%, and major
morbidity ranged from 0% to 40%. Median disease-free survival ranged from 13 to
56 months, and overall survival ranged from 14 to 64 months. Survival at 5 years
ranged from 35% to 70%. In patients with recurrent ovarian cancer, the mortality
rate ranged from 0% to 10%, minor morbidity ranged from 7% to 90%, and major
morbidity ranged from 0% to 49%. Median disease-free survival ranged from 13 to
24 months and overall survival from 23 to 49 months. Survival at 5 years ranged
from 12% to 54%.
CONCLUSION: There is level-one evidence suggesting the benefit of postoperative
adjuvant intraperitoneal chemotherapy for patients with advanced ovarian cancer
after cytoreductive surgery, albeit catheter-related complications resulted
after treatment discontinuation. Studies report the use of HIPEC predomitly
in the setting of recurrent disease and have demonstrated encouraging results,
which merits further investigation in future clinical trials. BACKGROUND: The aim of this study is to report the perioperative outcomes of CRS
and HIPEC from a single institution and review those factors that are associated
with a poor perioperative outcome in patients with peritoneal dissemination from
primary or recurrent ovarian cancer.
PATIENTS AND METHOD: A retrospective cohort study setting was conducted in a
third level hospital peritoneal surface maligcy program. Ninety one patients
diagnosed with ovarian peritoneal carcinomatosis, primary and recurrent without
extraperitoneal metastasis were included for cytoreductive surgery and HIPEC
with paclitaxel. We analyzed the postoperative morbidity rates and a univariate
and multivariate analysis of factors associated with overall (grade I-IV) and
major (grade III-IV) postoperative morbidity were performed.
RESULTS: Peritoneal Cancer Index (PCI) upper than 12 (OR = 2.942 95%:
1.892-9.594 p = 0.044) was an independent factor associated with the occurrence
of I-IV postoperative morbidity. Regarding major complications (grade III-IV),
on multivariate analysis, in addition to PCI >12 (OR = 6.692, 95% CI: 1974-45,
674, p = 0.032), the need to carry out intestinal resection (OR = 4.987, 95% CI:
1350-27, 620, p = 0.046) was an independent factor related with major morbidity
(grade III-IV).
CONCLUSIONS: The use of HIPEC after aggressive cytoreductive surgery in patients
with ovarian cancer with peritoneal dissemination can be performed with
acceptable postoperative morbidity rates. Knowledge of the factors associated
with the onset of these postoperative adverse events allows better management of
the same and offers the patient a safe procedure. AIM: Ovarian cancer may be considered as an "intraperitoneal disease" by itself.
When surgical removal associated with systemic chemotherapy fails, usually, the
history of the patients is characterized by poor prognosis. Some encouraging
results have been reported by the treatment of peritoneal carcinomatosis (PC)
from ovarian cancer by complete surgical cytoreduction, peritonectomy and
hyperthermic intraperitoneal chemotherapy (HIPEC). The purpose of this article
was to evaluate the survival benefit and the morbidity of patients with ovarian
cancer treated at our institution by cytoreductive surgery associated with
hyperthermic intraperitoneal perioperative chemotherapy (HIPEC).
METHODS: Between October 1995 and December 2012 more than 600 operations for PC
were performed; in 308 cases surgical cytoreduction associated with HIPEC was
carried out. Eighty-five patients treated by cytoreduction associated with HIPEC
were affected by recurrent epithelial ovarian cancer (EOC). Statistical analysis
was performed on 70 patients (last 15 patients were too recent for evaluation).
Two trials were applied: 1) patients presenting first peritoneal relapse after
surgery and systemic chemotherapy (CT), 6 months later from last CT
administration; 2) multiple relapse patients.
RESULTS: On 70 patients, morbidity and mortality rates were 35.7% and 7.1%,
respectively. Overall median survival was 42.0 months, but in primary EOC was
48.0 months and in recurrent EOC was 28 months (P=0.12). Statistical analysis
revealed that the completeness of cytoreduction was the most statistically
significant factor related to survival: in completely citoreduced patients,
overall survival was 48 months.
CONCLUSION: Citoreductive surgery associated to platinum compounds HIPEC is
feasible and relatively safe in recurrent and primary PC from ovarian cancer.
Better selection of patients and second-look surgery in high risk-patients have
to be investigated to improve those encouraging results. BACKGROUND: Approximately 70% of women diagnosed with advanced-stage ovarian
cancer experience disease recurrence. Survival data were compared between a
group of recurrent epithelial ovarian cancer (rEOC) patients treated by
cytoreduction and hyperthermic intraperitoneal chemotherapy (HIPEC) and a
matched group of rEOC patients treated by systemic chemotherapy only (without
surgery). Treatment outcome in relation to the patients' BRCA status was
compared.
METHODS: Twenty-seven rEOC patients treated by cytoreduction and HIPEC were
selected from our database and matched (1:3) with 84 rEOC patients treated with
chemotherapy only. Progression-free survival (PFS) and overall survival (OS) in
the two groups were analyzed and compared.
RESULTS: The estimated median PFS was 15 months in the HIPEC group and 6 months
in the systemic chemotherapy group (P = 0.001). The median OS following HIPEC
treatment has not been reached, since more than 70% of the women were alive at
the time of analysis. The 5-year survival rate was significantly higher in the
HIPEC treated patients compared to that of the controls (79% vs. 45%,
P = 0.016). BRCA status did not affect PFS.
CONCLUSIONS: HIPEC after surgical cytoreduction in patients with rEOC appears
beneficial compared to systemic chemotherapy treatment alone. The benefit is
even greater in BRCA mutation carriers. |
Which is the most common editing modification in eukaryotic mRNA? | One of the most common forms of pre-mRNA editing is A-to-I editing, in which adenosine is deaminated to inosine, which is read as guanosine during translation. | RNA editing is a post-transcriptional modification of pre-mRNA that results in
increased diversity in transcriptomes and proteomes. It occurs in a wide variety
of eukaryotic organisms and in some viruses. One of the most common forms of
pre-mRNA editing is A-to-I editing, in which adenosine is deaminated to inosine,
which is read as guanosine during translation. This phenomenon has been observed
in numerous transcripts, including the mammalian 5-HT(2C) receptor, which can be
edited at five distinct sites. Methods used to date to quantify 5-HT(2C)
receptor editing are labor-intensive, expensive and provide limited information
regarding the relative abundance of 5-HT(2C) receptor editing variants. Here, we
present a novel, ultra high-throughput method to quantify 5-HT(2C) receptor
editing, compare it to a more conventional method, and use it to assess the
effect of a range of genetic and pharmacologic manipulations on 5-HT(2C)
editing. We conclude that this new method is powerful and economical, and we
provide evidence that alterations in 5-HT(2C) editing appear to be a result of
regional changes in brain activity, rather than a mechanism to normalize
5-HT(2C) signaling. The present review surveys the available data on the involvement of adenine
deamination in RNA molecules in the formation of structurally and functionally
diverse RNA and protein subforms in eukaryotic cells. Deamination of adenine by
adenosine deaminases that act on RNA (ADARs) leads to the conversion of adenine
into inosine (A-I editing) recognized by the splicing and translation systems as
guanine. This may modify splicing sites in pre-mRNA and codons in translated
regions ofmRNA and also affect the RNA secondary structure. Apart from mRNA,
editing also involves microRNAs whose regulatory functions in multicellular
animals are associated with the inhibition of transcription of target genes or
with the degradation of certain RNA transcripts. ADARs can inhibit the
production of mature microRNAs or modify microRNAs so that their specificity to
target genes is altered. Adenosine deaminases editing adenines in transport RNAs
(ADATs) convert adenine into inosine in tRNAs of all eukaryotes; as a result,
the diversity of tRNA forms in the cell increases. RNA editing is an alteration in the primary nucleotide sequences resulting from
a chemical change in the base. RNA editing is observed in eukaryotic mRNA,
transfer RNA, ribosomal RNA, and non-coding RNAs (ncRNA). The most common RNA
editing in the mammalian central nervous system is a base modification, where
the adenosine residue is base-modified to inosine (A to I). Studies from ADAR
(adenosine deaminase that act on RNA) mutants in Caenorhabditis elegans,
Drosophila, and mice clearly show that the RNA editing process is an absolute
requirement for nervous system homeostasis and normal physiology of the animal.
Understanding the mechanisms of editing and findings of edited substrates has
provided a better knowledge of the phenotype due to defective and hyperactive
RNA editing. A to I RNA editing is catalyzed by a family of enzymes knows as
ADARs. ADARs modify duplex RNAs and editing of duplex RNAs formed by ncRNAs can
impact RNA functions, leading to an altered regulatory gene network. Such
altered functions by A to I editing is observed in mRNAs, microRNAs (miRNA) but
other editing of small and long ncRNAs (lncRNAs) has yet to be identified. Thus,
ncRNA and RNA editing may provide key links between neural development, nervous
system function, and neurological diseases. This review includes a summary of
seminal findings regarding the impact of ncRNAs on biological and pathological
processes, which may be further modified by RNA editing. NcRNAs are
non-translated RNAs classified by size and function. Known ncRNAs like miRNAs,
smallRNAs (smRNAs), PIWI-interacting RNAs (piRNAs), and lncRNAs play important
roles in splicing, DNA methylation, imprinting, and RNA interference. Of note,
miRNAs are involved in development and function of the nervous system that is
heavily dependent on both RNA editing and the intricate spatiotemporal
expression of ncRNAs. This review focuses on the impact of dysregulated A to I
editing and ncRNAs in neurodegeneration. Non-melanoma skin cancers (NMSC) are the most common maligcies in caucasians
worldwide. Insulin-like growth factor-binding protein-7 (IGFBP7) was suggested
to function as a tumor suppressor gene in several cancers, and to play a role in
the proliferation of keratinocytes. A-to-I RNA editing is a post-transcriptional
mechanism frequently used to expand and diversify transcriptome and proteome
repertoire in eukaryotic cells. A-to-I RNA editing can alter codons, substitute
amino acids and affect protein sequence, structure, and function. Two editing
sites were identified within the IGFBP7 transcript. To evaluate the expression
and editing of IGFBP7 mRNA in NMSC compared to normal epidermis. We examined the
expression and mRNA editing level of IGFBP7 in 22 basal cell carcinoma (BCC), 15
squamous cell carcinoma (SCC), and 18 normal epidermis samples that were
surgically removed from patients by the Mohs Micrographic Surgery procedure. We
studied the effect of IGFBP7 editing on an immortalized HaCaT keratinocyte cell
model. IGFBP7 mRNA is over expressed in BCC and SCC compared to normal
epidermis. Moreover, the IGFBP7 transcript is highly edited in normal epidermis,
but its editing is significantly reduced in BCC and SCC. The edited form of
IGFBP7 can inhibit proliferation and induce senescence in cultured
keratinocytes. This study describes for the first time A-to-I editing in the
coding sequence of a tumor suppressor gene in humans, and suggests that IGFBP7
editing serves as a fine-tuning mechanism to maintain the equilibrium between
proliferation and senescence in normal skin. RNA editing increases transcriptome diversity through post-transcriptional
modifications of RNA. Adenosine deaminases that act on RNA (ADARs) catalyze the
adenosine-to-inosine (A-to-I) conversion, the most common type of RNA editing in
higher eukaryotes. Caenorhabditis elegans has two ADARs, ADR-1 and ADR-2, but
their functions remain unclear. Here, we profiled the RNA editomes of C. elegans
at different developmental stages of wild-type and ADAR mutants. We developed a
new computational pipeline with a "bisulfite-seq-mapping-like" step and achieved
a threefold increase in identification sensitivity. A total of 99.5% of the
47,660 A-to-I editing sites were found in clusters. Of the 3080 editing
clusters, 65.7% overlapped with DNA transposons in noncoding regions and 73.7%
could form hairpin structures. The numbers of editing sites and clusters were
highest at the L1 and embryonic stages. The editing frequency of a cluster
positively correlated with the number of editing sites within it. Intriguingly,
for 80% of the clusters with 10 or more editing sites, almost all expressed
transcripts were edited. Deletion of adr-1 reduced the editing frequency but not
the number of editing clusters, whereas deletion of adr-2 nearly abolished RNA
editing, indicating a modulating role of ADR-1 and an essential role of ADR-2 in
A-to-I editing. Quantitative proteomics analysis showed that adr-2 mutant worms
altered the abundance of proteins involved in aging and lifespan regulation.
Consistent with this finding, we observed that worms lacking RNA editing were
short-lived. Taken together, our results reveal a sophisticated landscape of RNA
editing and distinct modes of action of different ADARs. The advent of deep sequencing technologies has greatly improved the study of
complex eukaryotic genomes and transcriptomes, providing the unique opportunity
to investigate posttranscriptional molecular mechanisms as alternative splicing
and RNA editing at single base-pair resolution. RNA editing by adenosine
deamination (A-to-I) is widespread in humans and can lead to a variety of
biological effects depending on the RNA type or the RNA region involved in the
editing modification. Hereafter, we describe an easy and reproducible
computational protocol for the identification of candidate RNA editing sites in
human using deep transcriptome (RNA-Seq) and genome (DNA-Seq) sequencing data. |
Is Rac1 involved in cancer cell invasion? | A number of signalling pathways have been found to converge to and activate Rac1, which, in turn, activates a number of downstream targets to control actin-cytoskeleton rearrangements at membrane ruffles, as well as formation and activity of lamellipodia, to regulate the migratory processes leading to cell invasion. | INTRODUCTION: The metastatic progression of cancer is a direct result of the
disregulation of numerous cellular signaling pathways, including those
associated with adhesion, migration, and invasion. Members of the Rac family of
small GTPases are known to act as regulators of actin cytoskeletal structures
and strongly influence the cellular processes of integrin-mediated adhesion and
migration. Even though hyperactivated Rac proteins have been shown to influence
metastatic processes, these proteins have never been directly linked to
metastatic progression.
METHODS: To investigate a role for Rac and Cdc42 in metastatic breast cancer
cell invasion and migration, relative endogenous Rac or Cdc42 activity was
determined in a panel of metastatic variants of the MDA-MB-435 metastatic human
breast cancer cell line using a p21-binding domain-PAK pull down assay. To
investigate the migratory and invasive potential of the Rac isoforms in human
breast cancer, namely Rac1 and the subsequently cloned Rac3, we stably expressed
either domit active Rac1 or domit active Rac3 into the least metastatic
cell variant. Domit negative Rac1 or domit negative Rac3 were stably
expressed in the most metastatic cell variant. Cell lines expressing mutant Rac1
or Rac3 were analyzed using in vitro adhesion, migration and invasion assays.
RESULTS: We show that increased activation of Rac proteins directly correlates
with increasing metastatic potential in a panel of cell variants derived from a
single metastatic breast cancer cell line (MDA-MB-435). The same correlation
could not be found with activated Cdc42. Expression of a domit active Rac1 or
a domit active Rac3 resulted in a more invasive and motile phenotype.
Moreover, expression of either domit negative Rac1 or domit negative Rac3
into the most metastatic cell variant resulted in decreased invasive and motile
properties.
CONCLUSION: This study correlates endogenous Rac activity with high metastatic
potential and implicates Rac in the regulation of cell migration and invasion in
metastatic breast cancer cells. Taken together, these results suggest a role for
both the Rac1 and Rac3 GTPases in human breast cancer progression. Glioblastoma multiforme (GBM) is the most maligt glioma type with diffuse
borders due to extensive tumor cell infiltration. Therefore, understanding the
mechanism of GBM cell dispersal is critical for developing effective therapies
to limit infiltration. We identified neuropilin-1 as a mediator of cancer cell
invasion by a functional proteomic screen and showed its role in GBM cells.
Neuropilin-1 is a receptor for semaphorin3A (Sema3A), a secreted chemorepellent
that facilitates axon guidance during neural development. Although neuropilin-1
expression in GBMs was previously shown, its role as a Sema3A receptor remained
elusive. Using fluorophore-assisted light inactivation and RNA interference , we
showed that neuropilin-1 is required for GBM cell migration. We also showed that
GBM cells secrete Sema3A endogenously, and RNA interference-mediated
downregulation of Sema3A inhibits migration and alters cell morphology that is
dependent on Rac1 activity. Sema3A depletion also reduces dispersal, which is
recovered by supplying Sema3A exogenously. Extracellular application of Sema3A
decreases cell-substrate adhesion in a neuropilin-1-dependent manner. Using
immunohistochemistry, we showed that Sema3A is overexpressed in a subset of
human GBMs compared with the non-neoplastic brain. Together, these findings
implicate Sema3A as an autocrine signal for neuropilin-1 to promote GBM
dispersal by modulating substrate adhesion and suggest that targeting
Sema3A-neuropilin-1 signaling may limit GBM infiltration. IMPORTANCE OF THE FIELD: Rho GDP dissociation inhibitor 2 (RhoGDI2) has been
identified as a regulator of Rho GTPases that play important roles in the
development of numerous aspects of the maligt phenotype, including cell cycle
progression, resistance to apoptotic stimuli, neovascularization, tumor cell
motility, invasiveness, and metastasis. Although RhoGDI2 has been known to be
expressed only in hematopoietic tissues, recent studies suggest that this
protein is also aberrantly expressed in several human cancers and contributes to
aggressive phenotypes, such as invasion and metastasis. Hence, RhoGDI2 appears
to be a target of interest for therapeutic manipulation.
AREAS COVERED IN THIS REVIEW: Here, we summarize the role of RhoGDI2 in human
cancers, specifically metastasis-related processes, and discuss its potential as
a therapeutic target.
WHAT THE READER WILL GAIN: RhoGDI2 modulates the invasiveness and metastatic
ability of cancer cells through regulation of Rac1 activity.
TAKE HOME MESSAGE: RhoGDI2 may be a useful marker for tumor progression in human
cancers, and interruption of the RhoGDI2-mediated cancer cell invasion and
metastasis by an interfacial inhibitor may be a powerful therapeutic approach to
cancer. There is considerable experimental evidence that hyperactive Ras proteins
promote breast cancer growth and development including invasiveness, despite the
low frequency of mutated forms of Ras in breast cancer. We have previously shown
that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small
GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor
(EGF) plays an important role in aberrant growth and metastasis formation of
many tumor types including breast cancer. The present study aims to investigate
the correlation between EGF-induced invasiveness and Ras activation in four
widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities
and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell
lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA
(siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive
phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover,
siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in
these cells. Taken together, this study characterized human breast cancer cell
lines with regard to the relationship between H-Ras activation and the invasive
phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and
the downstream molecule Rac1 correlates with EGF-induced breast cancer cell
invasion, providing important information on the regulation of maligt
progression in mammary carcinoma cells. Cudratricusxanthone G (CTXG), a natural bioactive cudratricusxanthone extracted
from C. tricuspidata, has shown anti-cancer properties. However, the function
and mechanism of CTXG in tumor invasion have not been elucidated to date. In
this study, we investigated the inhibitory effect of CTXG on the proliferation,
migration and invasion of SW620 cells. We found that MMP-2, a pivotal factor in
tumor invasion, was suppressed in both expression and activation by CTXG in a
dose-dependent manner. The suppression of MMP-2 expression by CTXG led to an
inhibition of SW620 cells invasion and migration by inactivating Rac1 and Cdc42
but not RhoA GTPase. Furthermore, CTXG also inhibited the transcriptional
activity of AP-1 (activator protein-1). In conclusion, our data demonstrate that
CTXG exerted anti-invasion action in SW620 cells by targeting MMP-2 though
regulating the activities of Rac1, Cdc42 and their downstream transcriptional
factor AP-1. These results are the first to reveal the novel functions of CTXG
in cancer cell invasion and its molecular basis for the anti-cancer action. BACKGROUND: Small GTPase proteins, including RhoA, RhoB, RhoC, Rac1, and cdc42,
are important molecules for linking cell shape and cell-cycle progression
because of their role in both cytoskeletal arrangements and mitogenic signaling.
Over-expression of wild-type or constitutively active forms of RhoA has been
shown to induce invasive behavior in non-invasive rat hepatoma cells in vitro.
In addition, over-expression of RhoC has been found in melanoma cells with
increasing metastatic activity as well as inflammatory breast cancer. These
results indicate that overexpression of Rho proteins contributes to cancer cell
invasion and metastasis. Rho GDP dissociation inhibitor 2 (RhoGDI2) was recently
shown to act as a metastasis suppressor gene in bladder cancer. The purpose of
this study was to clarify the clinical significance of this gene expression in
patients with colorectal carcinoma.
METHODS: Fifty pairs of normal mucosa and cancer specimens obtained at the time
of surgery from patients with colorectal cancer (CRC) were subjected to reverse
transcription-polymerase chain reaction for RhoGDI2.
RESULTS: No patients with RhoGDI2-higher expression tumors had liver metastasis
(0 in 8 cases); however, 33.3% (14 in 42 cases) of patients with RhoGDI2-lower
expression tumors had liver metastasis. With regard to outcome in relation to
RhoGDI2-positivity, RhoGDI2-higher expression tumors had a significant
correlation with superior relapse-free survival (RFS) time as compared to
RhoGDI2-lower expression tumors in stage III CRC (log-rank test, P < 0.05).
Moreover, multivariate analysis indicated that RhoGDI2 was an independent
prognostic factor for RFS.
CONCLUSION: RhoGDI2 is a novel predictor of RFS in patients with colorectal
carcinoma. The Rho GTPases organize the actin cytoskeleton and are involved in cancer
metastasis. Previously, we demonstrated that RhoC GTPase was required for PC-3
prostate cancer cell invasion. Targeted down-regulation of RhoC led to sustained
activation of Rac1 GTPase and morphological, molecular and phenotypic changes
reminiscent of epithelial to mesenchymal transition. We also reported that Rac1
is required for PC-3 cell diapedesis across a bone marrow endothelial cell
layer. In the current study, we queried whether Rac3 and RhoG GTPases also have
a role in prostate tumor cell diapedesis. Using specific siRNAs we demonstrate
roles for each protein in PC-3 and C4-2 cell adhesion and diapedesis. We have
shown that the chemokine CCL2 induces tumor cell diapedesis via Rac1 activation.
Here we find that RhoG partially contributes to CCL2-induced tumor cell
diapedesis. We also find that Rac1 GTPase mediates tight binding of prostate
cancer cells to bone marrow endothelial cells and promotes retraction of
endothelial cells required for tumor cell diapedesis. Finally, Rac1 leads to β1
integrin activation, suggesting a mechanism that Rac1 can mediate tight binding
with endothelial cells. Together, our data suggest that Rac1 GTPase is key
mediator of prostate cancer cell-bone marrow endothelial cell interactions. P-cadherin is a cell-cell adhesion molecule, whose expression is highly
associated with undifferentiated cells in normal adult epithelial tissues, as
well as with poorly differentiated carcinomas. Its expression has been already
reported in human embryonic stem cells and it is presumed to be a marker of stem
or progenitor cells of some epithelial tissues. In normal breast, P-cadherin has
an essential role during ductal mammary branching, being expressed by the
monolayer of epithelial cap cells at the end buds. In mature mammary tissue, its
expression is restricted to the myoepithelium; it has been postulated that it
may also be present in early luminal progenitor cells. In breast cancer,
P-cadherin is frequently overexpressed in high-grade tumours, being a
well-established indicator of poor patient prognosis. It has been reported as an
important inducer of cancer cell migration and invasion, with underlying
molecular mechanisms involving the signalling mediated by its juxtamembrane
domain, the secretion of matrix metalloproteases to the extracellular media, and
the cleavage of a P-cadherin soluble form with pro-invasive activity.
Intracellularly, this protein interferes with the endogenous cadherin/catenin
complex, inducing p120-catenin delocalization to the cytoplasm, and the
consequent activation of Rac1/Cdc42 and associated alterations in the actin
cytoskeleton. Considering P-cadherin's role in cancer cell invasion and
metastasis formation, a humanized monoclonal antibody was recently produced to
antagonize P-cadherin-associated signalling pathways, which is currently under
Phase I clinical trials. In this review, the most important findings about the
role of P-cadherin in normal breast development and cancer will be illustrated
and discussed, with emphasis on the most recent data. We report that Binder of Arl Two (BART) plays a role in inhibiting cell invasion
by regulating the activity of the Rho small guanosine triphosphatase protein
Rac1 in pancreatic cancer cells. BART was originally identified as a binding
partner of ADP-ribosylation factor-like 2, a small G protein implicated as a
regulator of microtubule dynamics and folding. BART interacts with active forms
of Rac1, and the BART-Rac1 complex localizes at the leading edges of migrating
cancer cells. Suppression of BART increases active Rac1, thereby increasing cell
invasion. Treatment of pancreatic cancer cells in which BART is stably knocked
down with a Rac1 inhibitor decreases invasiveness. Thus, BART-dependent
inhibition of cell invasion is likely associated with decreased active Rac1.
Suppression of BART induces membrane ruffling and lamellipodial protrusion and
increases peripheral actin structures in membrane ruffles at the edges of
lamellipodia. The Rac1 inhibitor inhibits the lamellipodia formation that is
stimulated by suppression of BART. Our results imply that BART regulates
actin-cytoskeleton rearrangements at membrane ruffles through modulation of the
activity of Rac1, which, in turn, inhibits pancreatic cancer cell invasion. Dysregulation of cell adhesion and motility is known to be an important factor
in the development of tumor maligcy. Actopaxin (α-parvin) is a paxillin,
integrin-linked kinase, and F-actin binding focal adhesion protein with several
serine phosphorylation sites in the amino terminus that contribute to the
regulation of cell spreading and migration. Here, phosphorylation of actopaxin
is shown to contribute to the regulation of matrix degradation and cell
invasion. Osteosarcoma cells stably expressing wild type (WT),
nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate
that actopaxin phosphorylation is necessary for efficient Src and matrix
metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to
be required for actopaxin-induced matrix degradation whereas inhibition of
myosin contractility promoted degradation in the phosphomutant-expressing Quint
cells, indicating that a balance of Rho GTPase signaling and regulation of
cellular tension are important for the process. Furthermore, actopaxin forms a
complex with the Rac1/Cdc42 GEF β-PIX and Rac1/Cdc42 effector PAK1, to regulate
actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in
the invasive breast cancer cell line MDA-MB-231 compared with normal breast
epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin
in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT
actopaxin promotes invasion in MCF10A cells. Taken together, this study
demonstrates a new role for actopaxin phosphorylation in matrix degradation and
cell invasion via regulation of Rho GTPase signaling. Cell motility proceeds by cycles of edge protrusion, adhesion and retraction.
Whether these functions are coordinated by biochemical or biomechanical
processes is unknown. Tumor invasion and metastasis is directly related to cell
motility. We showed that stimulation of proteinase-activated receptor-1 (PAR1)
can trigger an array of responses that would promote tumor cell growth and
invasion. Thus, we examined aspects of PAR1 activation related to cell
morphological change that might contribute to cell motility. We established a
PAR1 stably transfected MKN45 gastric cancer cell line (MKN45/PAR1). We examined
morphological changes, Rho family activation and overexpression of cytoskeletal
protein in cells exposed to PAR1 agonists (α-thrombin and TFLLR-NH2). MKN45/PAR1
grows with an elongated and polarized morphology, extending pseudopodia at the
leading edge. However, in the presence of PAR1 antagonist, MKN45/PAR1 did not
show any changes in cell shape upon addition of either α-thrombin or TFLLR-NH2.
Activated PAR1 induced RhoA and Rac1 phosphorylation, and subsequent
overexpression of myosin IIA and filamin B which are stress fiber components
that were identified by PMF analysis of peptide mass data obtained by
MALDI-TOF/MS measurement. Upon stimulation of MKN45/PAR1 for 24 h with either
α-thrombin or TFLLR-NH2, the distribution of both myosin IIA and filamin B
proteins shifted to being distributed throughout the cytoplasm to the membrane,
with more intense luminescence signals than in the absence of stimulation. These
results demonstrate that PAR1 activation induces cell morphological change
associated with cell motility via Rho family activation and cytoskeletal protein
overexpression, and has a critical role in gastric cancer cell invasion and
metastasis. Expression of the Rac-guanine nucleotide exchange factor (RacGEF), P-Rex1 is a
key determit of progression to metastasis in a number of human cancers. In
accordance with this proposed role in cancer cell invasion and metastasis, we
find that ectopic expression of P-Rex1 in an immortalised human fibroblast cell
line is sufficient to drive multiple migratory and invasive phenotypes. The
invasive phenotype is greatly enhanced by the presence of a gradient of serum or
platelet-derived growth factor, and is dependent upon the expression of
functional PDGF receptor β. Consistently, the invasiveness of WM852 melanoma
cells, which endogenously express P-Rex1 and PDGFRβ, is opposed by siRNA of
either of these proteins. Furthermore, the current model of P-Rex1 activation is
advanced through demonstration of P-Rex1 and PDGFRβ as components of the same
macromolecular complex. These data suggest that P-Rex1 has an influence on
physiological migratory processes, such as invasion of cancer cells, both
through effects upon classical Rac1-driven motility and a novel association with
RTK signalling complexes. Castration-refractory prostate cancer (CRPC) is treated with taxane-based
chemotherapy, but eventually becomes drug resistant. It is thus essential to
identify novel therapeutic targets for taxane resistance in CRPC patients. We
investigated the role of the chemokine (C-C motif) receptor 1 (CCR1) and its
ligand, chemokine (C-C motif) ligand 5 (CCL5), in taxane-resistant CRPC using
paclitaxel-resistant prostate cancer cells (PC3PR) established from PC3 cells.
We found that the expression levels of CCR1 mRNA and protein were up-regulated
in PC3PR cells compared to PC3 cells. In order to investigate the role of
increased CCR1 in PC3PR cells, we stimulated cells with CCL5, one of the
chemokine ligands of CCR1. In CCL5-stimulated PC3PR cells, siRNA-mediated
knockdown of CCR1 expression reduced phosphorylation of ERK1/2 and Rac1/cdc42.
Furthermore, CCR1 knockdown and MEK1/2 inhibition decreased CCL5-stimulated
secretion of MMPs 2 and 9, which play important roles in cancer cell invasion
and metastasis. In the Matrigel invasion assay, knockdown of CCR1 and inhibition
of the ERK and Rac signaling pathways significantly decreased the number of
invading cells. Finally, the serum CCL5 protein level as measured by ELISA was
not different among the three groups of patients: those with negative prostate
biopsy, those at initial diagnosis of prostate cancer, and those with
taxane-resistant prostate cancer. These results demonstrated for the first time
that the interaction of CCR1 with CCL5 caused by increased expression of CCR1
promotes invasion of PC3PR cells by increasing secretion of MMPs 2 and 9 and by
activating ERK and Rac signaling. Our findings suggest that CCR1 could be a
novel therapeutic target for taxane-resistant CRPC. |
Which are the APOBEC3 protein family members able to inhibit Vif-deficient HIV-1 replication? | APOBEC3G, APOBEC3F, APOBEC3DE, APOBEC3A, and APOBEC3H haplotypes II, V, and VII, provide protection against Vif-deficient HIV-1, through hypermutation of the viral genome, inhibition of reverse transcription, and inhibition of viral DNA integration into the host genome. | In the human genome the apolipoprotein B mRNA-editing enzyme catalytic
polypeptide (APOBEC)3 gene has expanded into a tandem array of genes termed
APOBEC3A-G. Two members of this family, APOBEC3G and APOBEC3F, have been found
to have potent activity against virion infectivity factor deficient (Deltavif)
human immunodeficiency virus 1 (HIV-1). These enzymes become encapsidated in
Deltavif HIV-1 virions and in the next round of infection deaminate the newly
synthesized reverse transcripts. The lentiviral Vif protein prevents the
deamination by inducing the degradation of APOBEC3G and APOBEC3F. We report here
that two additional APOBEC3 family members, APOBEC3B and APOBEC3C, have potent
antiviral activity against simian immuno-deficiency virus (SIV), but not HIV-1.
Both enzymes were encapsidated in HIV-1 and SIV virions and were active against
Deltavif SIV(mac) and SIV(agm). SIV Vif neutralized the antiviral activity of
APOBEC3C, but not that of APOBEC3B. APOBEC3B induced abundant G --> A mutations
in both wild-type and Deltavif SIV reverse transcripts. APOBEC3C induced
substantially fewer mutations. APOBEC3F was found to be active against SIV and
sensitive to SIV(mac) Vif. These findings raise the possibility that the
different APOBEC3 family members function to neutralize specific lentiviruses. Human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) inhibit replication
of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif
overcomes these host restriction factors by binding to them and inducing their
proteasomal degradation. The Vif-A3G and Vif-A3F interactions are attractive
targets for antiviral drug development because inhibiting the interactions could
allow the host defense mechanism to control HIV-1 replication. It was recently
reported that the Vif amino acids D(14)RMR(17) are important for functional
interaction and degradation of the previously identified Vif-resistant mutant of
A3G (D128K-A3G). However, the Vif determits important for functional
interaction with A3G and A3F have not been fully characterized. To identify
these determits, we performed an extensive mutational analysis of HIV-1 Vif.
Our analysis revealed two distinct Vif determits, amino acids Y(40)RHHY(44)
and D(14)RMR(17), which are essential for binding to A3G and A3F, respectively.
Interestingly, mutation of the A3G-binding region increased Vif's ability to
suppress A3F. Vif binding to D128K-A3G was also dependent on the Y(40)RHHY(44)
region but not the D(14)RMR(17) region. Consistent with previous observations,
subsequent neutralization of the D128K-A3G antiviral activity required
substitution of Vif determit D(14)RMR(17) with SEMQ, similar to the SERQ
amino acids in simian immunodeficiency virus SIV(AGM) Vif, which is capable of
neutralizing D128K-A3G. These studies are the first to clearly identify two
distinct regions of Vif that are critical for independent interactions with A3G
and A3F. Pharmacological interference with the Vif-A3G or Vif-A3F interactions
could result in potent inhibition of HIV-1 replication by the APOBEC3 proteins. Human APOBEC3G and other APOBEC3 cytidine deaminases inhibit a variety of
retroviruses, including Vif-deficient HIV-1. These host proteins are packaged
into viral particles and inhibit the replication of virus in new target cells.
A3G and A3F are known to be efficiently packaged into HIV-1 virions by binding
to 7SL RNA through the Gag NC domain; however, the packaging mechanisms of other
APOBEC3 proteins are poorly defined. We have now demonstrated that APOBEC3C
(A3C) can be efficiently packaged into HIV-1 virions that are deficient for
viral genomic RNA. Inhibition of the encapsidation of 7SL RNA into HIV-1 virions
blocked the packaging of A3G, but not A3C. While the NC domain is required for
efficient packaging of A3G, deletion of this domain had little effect on A3C
packaging into HIV-1 Gag particles. A3C interacted with HIV-1 Gag which was MA
domain-dependent and RNA-dependent. Deletion of the MA domain of HIV-1 Gag
inhibited A3C but not A3G packaging into HIV-1 Gag particles. Thus, A3G and A3C
have evolved to use distinct mechanisms for targeting retroviruses. Human APOBEC3G (A3G) and APOBEC3F (A3F) inhibit the replication of Vif-deficient
human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host
restriction factors by binding to them and inducing their degradation. Thus, the
Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug
development, as inhibiting these interactions could allow the host defense
mechanism to control HIV-1 replication. Recently, it has been reported that
amino acids 105 to 156 of A3G are involved in the interaction with Vif; however,
to date, the region of A3F involved in Vif binding has not been identified.
Using our previously reported Vif mutants that are capable of binding to only
A3G (3G binder) or only A3F (3F binder), in conjunction with a series of A3G-A3F
chimeras, we have now mapped the APOBEC3-Vif interaction domains. We found that
the A3G domain that interacts with the Vif YRHHY region is located between amino
acids 126 and 132 of A3G, which is consistent with the conclusions reported in
previous studies. The A3F domain that interacts with the Vif DRMR region did not
occur in the homologous domain but instead was located between amino acids 283
and 300 of A3F. These studies are the first to identify the A3F domain that
interacts with the Vif DRMR region and show that distinct domains of A3G and A3F
interact with different Vif regions. Pharmacological inhibition of either or
both of these Vif-A3 interactions should prevent the degradation of the APOBEC3
proteins and could be used as a therapy against HIV-1. The human APOBEC3 (A3) cytidine deaminases, such as APOBEC3G (A3G) and APOBEC3F
(A3F), are potent inhibitors of Vif-deficient human immunodeficiency virus type
1 (HIV-1). HIV-1 Vif (viral infectivity factor) binds A3 proteins and targets
these proteins for ubiquitination and proteasomal degradation. As such, the
therapeutic blockage of Vif-A3 interaction is predicted to stimulate natural
antiviral activity by rescuing APOBEC expression and virion packaging. In this
study, we describe a successful application of the Protein Fragment
Complementation Assay (PCA) based on the enzyme TEM-1 β-lactamase to study
Vif-A3 interactions. PCA is based on the interaction between two protein binding
partners (e.g., Vif and A3G), which are fused to the two halves of a dissected
marker protein (β-lactamase). Binding of the two partners reassembles
β-lactamase and hence reconstitutes its activity. To validate our assay, we
studied the effect of well-described Vif (DRMR, YRHHY) and A3G (D128K) mutations
on the interaction between the two proteins. Additionally, we studied the
interaction of human Vif with other members of the A3 family: A3F and APOBEC3C
(A3C). Our results demonstrate the applicability of PCA as a simple and reliable
technique for the assessment of Vif-A3 interactions. Furthermore, when compared
with co-immunoprecipitation assays, PCA appeared to be a more sensitive
technique for the quantitative assessment of Vif-A3 interactions. Thus, with our
results, we conclude that PCA could be used to quantitatively study specific
domains that may be involved in the interaction between Vif and APOBEC proteins. Successful intracellular pathogens must evade or neutralize the innate immune
defenses of their host cells and render the cellular environment permissive for
replication. For example, to replicate efficiently in CD4(+) T lymphocytes,
human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral
infectivity factor (Vif) that promotes pathogenesis by triggering the
degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3
proteins have been implicated in HIV-1 restriction, but the relevant repertoire
remains ambiguous. Here we present the first comprehensive analysis of the
complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction.
In addition to APOBEC3G, we find that three other human APOBEC3 proteins,
APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors.
These four proteins are expressed in CD4(+) T lymphocytes, are packaged into and
restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral
DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F,
APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict
Vif-deficient HIV-1 when stably expressed in T cells, and they are all
neutralized by the simian immunodeficiency virus Vif protein. On the other hand,
neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant
impact on HIV-1 replication. These data strongly implicate a combination of four
APOBEC3 proteins--APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H--in HIV-1
restriction. Several members of the APOBEC3 family of cellular restriction factors provide
intrinsic immunity to the host against viral infection. Specifically, APOBEC3DE,
APOBEC3F, APOBEC3G, and APOBEC3H haplotypes II, V, and VII provide protection
against HIV-1Δvif through hypermutation of the viral genome, inhibition of
reverse transcription, and inhibition of viral DNA integration into the host
genome. HIV-1 counteracts APOBEC3 proteins by encoding the viral protein Vif,
which contains distinct domains that specifically interact with these APOBEC3
proteins to ensure their proteasomal degradation, allowing virus replication to
proceed. Here, we review our current understanding of APOBEC3 structure, editing
and non-editing mechanisms of APOBEC3-mediated restriction, Vif-APOBEC3
interactions that trigger APOBEC3 degradation, and the contribution of APOBEC3
proteins to restriction and control of HIV-1 replication in infected patients. |
Is the gene SLC6A2 associated with orthostatic intolerance? | Yes, variants of the SLC6A2 (or NET) gene are associated with orthostatic intolerance. | Orthostatic intolerance (OI) or postural tachycardia syndrome (POTS) is a
syndrome primarily affecting young females, and is characterized by
lightheadedness, palpitations, fatigue, altered mentation, and syncope primarily
occurring with upright posture and being relieved by lying down. There is
typically tachycardia and raised plasma norepinephrine levels on upright
posture, but little or no orthostatic hypotension. The pathophysiology of OI is
believed to be very heterogeneous. Most studies of the syndrome have focused on
abnormalities in norepinephrine release. Here the hypothesis that abnormal
norepinephrine transporter (NET) function might contribute to the
pathophysiology in some patients with OI was tested. In a proband with
significant orthostatic symptoms and tachycardia, disproportionately elevated
plasma norepinephrine with standing, impaired systemic, and local clearance of
infused tritiated norepinephrine, impaired tyramine responsiveness, and a
dissociation between stimulated plasma norepinephrine and DHPG elevation were
found. Studies of NET gene structure in the proband revealed a coding mutation
that converts a highly conserved transmembrane domain Ala residue to Pro.
Analysis of the protein produced by the mutant cDNA in transfected cells
demonstrated greater than 98% reduction in activity relative to normal. NE,
DHPG/NE, and heart rate correlated with the mutant allele in this family.
CONCLUSION: These results represent the first identification of a specific
genetic defect in OI and the first disease linked to a coding alteration in a
Na+/Cl(-)-dependent neurotransmitter transporter. Identification of this
mechanism may facilitate our understanding of genetic causes of OI and lead to
the development of more effective therapeutic modalities. Orthostatic intolerance is a debilitating syndrome characterized by tachycardia
on assumption of upright posture. The norepinephrine (NE) transporter (NET) has
been implicated in a genetic form of the disorder. We assessed the combined
central and peripheral effects of pharmacological NET blockade on cardiovascular
regulation and baroreflex sensitivity in rats. NE reuptake was blocked
chronically in female Sprague-Dawley rats by the NET antagonist desipramine
(DMI). Treated animals demonstrated an elevated supine heart rate, reduced
tyramine responsiveness, and a reduced plasma ratio of the intraneuronal NE
metabolite dihydroxyphenylglycol relative to NE, all of which are consistent
with observations in human NET deficiency. Spectral analysis revealed a dramatic
decrease in low-frequency spectral power after DMI that was consistent with
decreased sympathetic outflow. Stimulation of the baroreflex with the
vasodilator nitroprusside revealed an attenuated tachycardia in DMI-treated
animals. This indicated that the DMI-induced sympathoinhibitory effects of
increased NE in the brain stem predominates over the functional elevation of NE
stimulation of peripheral targets. Thus attenuated baroreflex function and
reduced sympathetic outflow may contribute to the orthostatic intolerance of
severe NET deficiency. BACKGROUND: Orthostatic intolerance (OI) is a syndrome characterized by
lightheadedness, palpitations, fatigue, blurred vision, dizziness, chest
discomfort, cognitive impairment, and occasionally syncope. These symptoms
usually occur after upright posture and are associated with tachycardia and high
plasma concentrations of norepinephrine. It has been proposed that a mutation in
exon 9 of the norepinephrine transporter gene (Ala457Pro), resulting in more
than 98% loss of function compared with the wild type, might provide a
pathogenetic mechanism to explain the clinical symptoms of patients with OI.
METHODS: We studied 46 young men from military service who had sought medical
advice because of dizziness while standing. Every patient underwent a tilt-table
test, with monitoring of blood pressure, heart rate, and plasma catecholamines
in supine position and during 30 minutes of standing. Fourteen patients showing
the full-blown OI syndrome (30 bpm increase in heart rate and 600 pg/mL plasma
norepinephrine levels while standing) underwent direct DNA sequencing of exon 9
of the norepinephrine-transporter gene.
RESULTS AND CONCLUSIONS: The specific mutation (Ala457Pro) was not detected in
any of the 14 OI patients. Based on these findings, we doubt that this specific
genetic transport defect is a frequent cause of the impaired uptake of
norepinephrine in OI patients. Its routine determination will therefore not be
helpful to establish the clinical diagnosis of OI. The norepinephrine transporter (NET) mediates reuptake of norepinephrine
released from neurons, and, as such, it is an important regulator of
noradrenergic neurotransmission. Recently, our laboratory reported a
polymorphism in the human NET (hNET) gene A457P in an individual with the
autonomic disorder orthostatic intolerance (OI). The presence of the hNET-A457P
allele tracked with elevated heart rates and plasma NE levels in family members.
hNET-A457P lacks >98% transport activity in several heterologous expression
systems. In the present work, Western blot and biotinylation analyses performed
in transiently transfected COS-7 cells revealed impairment in processing of
hNET-A457P to the fully glycosylated form and a decrease in surface expression
to approximately 30% of hNET-wild type (hNET-wt). Because the hNET-A457P
mutation is carried on a single allele in OI subjects, we examined the influence
of cotransfection of hNET-wt and hNET-A457P and found that hNET-A457P exerts a
domit-negative effect on hNET-wt uptake activity. Experiments to determine
oligomerization as a potential mechanism of the domit-negative effect
demonstrated that hNET-A457P coimmunoprecipitates with, and diminishes surface
expression of, hNET-wt. These results reveal that hNET-A457P causes a
conformational disruption that interferes with transporter biosynthetic
progression and trafficking of both the mutant transporter and hNET-wt. These
results elucidate a molecular mechanism for the disrupted NE homeostasis and
cardiovascular function evident in OI patients with the hNET-A457P mutation. The role of norepinephrine (NE) in attention, memory, affect, stress, heart
rate, and blood pressure implicates NE in psychiatric and cardiovascular
disease. The norepinephrine transporter (NET) mediates reuptake of released
catecholamines, thus playing a role in the limitation of signaling strength in
the central and peripheral nervous systems. Nonsynonymous single nucleotide
polymorphisms (SNPs) in the human NET (hNET) gene that influence transporter
function can contribute to disease, such as the nonfunctional transporter,
A457P, identified in orthostatic intolerance. Here, we examine additional amino
acid variants that have been identified but not characterized in populations
that include cardiovascular phenotypes. Variant hNETs were expressed in COS-7
cells and were assayed for protein expression and trafficking using cell-surface
biotinylation and Western blot analysis, transport of radiolabeled substrate,
antagonist interaction, and regulation through protein kinase C (PKC)-linked
pathways by the phorbol ester beta-phorbol-12-myristate-13-acetate. We observed
functional perturbations in 6 of the 10 mutants studied. Several variants were
defective in trafficking and transport, with the most dramatic effect observed
for A369P, which was completely devoid of the fully glycosylated form of
transporter protein, was retained intracellularly, and lacked any transport
activity. Furthermore, A369P and another trafficking variant, N292T, impeded
surface expression of hNET when coexpressed. F528C demonstrated increased
transport and, remarkably, exhibited both insensitivity to down-regulation by
PKC and a decrease in potency for the tricyclic antidepressant desipramine.
These findings reveal functional deficits that are likely to compromise NE
signaling in SNP carriers in the population and identify key regions of NET
contributing to transporter biosynthesis, activity, and regulation. OBJECTIVE: The postural tachycardia syndrome (POTS) has multiple symptoms, chief
among which are tachycardia, weakness, and recurrent blackouts while standing.
Previous research has implicated dysfunction of the norepinephrine transporter.
A coding mutation in the norepinephrine transporter gene (SLC6A2) sequence has
been reported in 1 family kindred only. The goal of the present study was to
further characterize the role and regulation of the SLC6A2 gene in POTS.
METHODS AND RESULTS: Sympathetic nervous system responses to head-up tilt were
examined by combining norepinephrine plasma kinetics measurements and muscle
sympathetic nerve activity recordings in patients with POTS compared with that
in controls. The SLC6A2 gene sequence was investigated in leukocytes from POTS
patients and healthy controls using single nucleotide polymorphisms genotyping,
bisulphite sequencing, and chromatin immunoprecipitation assays for histone
modifications and binding of the transcriptional regulatory complex, methyl-CpG
binding protein 2. The expression of norepinephrine transporter was lower in
POTS patients compared with healthy volunteers. In the absence of altered SLC6A2
gene sequence or promoter methylation, this reduced expression was directly
correlated with chromatin modifications.
CONCLUSIONS: We propose that chromatin-modifying events associated with SLC6A2
gene suppression may constitute a mechanism of POTS. |
Other than protein coding potential, what features set apart long non-coding RNAs from protein coding genes? | Compared to protein coding genes, long non-coding RNAs (lncRNAs) display a bias towards two-exon transcripts. They are predominantly localized in the chromatin and nucleous. They are lower expressed and display a more tissue-specific expression pattern. LncRNAs are overall more weakly conserved than protein coding genes. | Long transcripts that do not encode protein have only rarely been the subject of
experimental scrutiny. Presumably, this is owing to the current lack of evidence
of their functionality, thereby leaving an impression that, instead, they
represent "transcriptional noise." Here, we describe an analysis of 3122 long
and full-length, noncoding RNAs ("macroRNAs") from the mouse, and compare their
sequences and their promoters with orthologous sequence from human and from rat.
We considered three independent signatures of purifying selection related to
substitutions, sequence insertions and deletions, and splicing. We find that the
evolution of the set of noncoding RNAs is not consistent with neutralist
explanations. Rather, our results indicate that purifying selection has acted on
the macroRNAs' promoters, primary sequence, and consensus splice site motifs.
Promoters have experienced the greatest elimination of nucleotide substitutions,
insertions, and deletions. The proportion of conserved sequence (4.1%-5.5%) in
these macroRNAs is comparable to the density of exons within protein-coding
transcripts (5.2%). These macroRNAs, taken together, thus possess the imprint of
purifying selection, thereby indicating their functionality. Our findings should
now provide an incentive for the experimental investigation of these macroRNAs'
functions. Experimental evidence suggests that half or more of the mammalian transcriptome
consists of noncoding RNA. Noncoding RNAs are divided into short noncoding RNAs
(including microRNAs) and long noncoding RNAs (lncRNAs). We defined
complementary DNAs (cDNAs) lacking any positive-strand open reading frames
(ORFs) longer than 30 amino acids, as well as cDNAs lacking any evidence of
interspecies conservation of their longer-than-30-amino acid ORFs, as noncoding.
We have identified 5446 lncRNA genes in the human genome from approximately
24,000 full-length cDNAs, using our new ORF-prediction pipeline. We combined
them nonredundantly with lncRNAs from four published sources to derive 6736
lncRNA genes. In an effort to distinguish standalone and antisense lncRNA genes
from database artifacts, we stratified our catalog of lncRNAs according to the
distance between each lncRNA gene candidate and its nearest known protein-coding
gene. We concurrently examined the protein-coding capacity of known genes
overlapping with lncRNAs. Remarkably, 62% of known genes with "hypothetical
protein" names actually lacked protein-coding capacity. This study has greatly
expanded the known human lncRNA catalog, increased its accuracy through manual
annotation of cDNA-to-genome alignments, and revealed that a large set of
hypothetical-protein genes in GenBank lacks protein-coding capacity. In
addition, we have developed, independently of existing NCBI tools, command-line
programs with high-throughput ORF-finding and BLASTP-parsing functionality,
suitable for future automated assessments of protein-coding capacity of novel
transcripts. Large numbers of long RNAs with little or no protein-coding potential [long
noncoding RNAs (lncRNAs)] are being identified in eukaryotes. In parallel,
increasing data describing the expression profiles, molecular features and
functions of individual lncRNAs in a variety of systems are accumulating. To
enable the systematic compilation and updating of this information, we have
developed a database (lncRNAdb) containing a comprehensive list of lncRNAs that
have been shown to have, or to be associated with, biological functions in
eukaryotes, as well as messenger RNAs that have regulatory roles. Each entry
contains referenced information about the RNA, including sequences, structural
information, genomic context, expression, subcellular localization,
conservation, functional evidence and other relevant information. lncRNAdb can
be searched by querying published RNA names and aliases, sequences, species and
associated protein-coding genes, as well as terms contained in the annotations,
such as the tissues in which the transcripts are expressed and associated
diseases. In addition, lncRNAdb is linked to the UCSC Genome Browser for
visualization and Noncoding RNA Expression Database (NRED) for expression
information from a variety of sources. lncRNAdb provides a platform for the
ongoing collation of the literature pertaining to lncRNAs and their association
with other genomic elements. lncRNAdb can be accessed at:
http://www.lncrnadb.org/. The human genome contains thousands of long noncoding RNAs (ncRNAs) transcribed
from diverse genomic locations. A large set of long ncRNAs is transcribed
independent of protein-coding genes. We have used the GENCODE annotation of the
human genome to identify 3019 long ncRNAs expressed in various human cell lines
and tissue. This set of long ncRNAs responds to differentiation signals in
primary human keratinocytes and is coexpressed with important regulators of
keratinocyte development. Depletion of a number of these long ncRNAs leads to
the repression of specific genes in their surrounding locus, supportive of an
activating function for ncRNAs. Using reporter assays, we confirmed such
activating function and show that such transcriptional enhancement is mediated
through the long ncRNA transcripts. Our studies show that long ncRNAs exhibit
functions similar to classically defined enhancers, through an RNA-dependent
mechanism. Accumulating evidence over the last decade has presented us with the intriguing
observation that the majority of eukaryotic genomes are pervasively transcribed
to encode a complex network of small and long noncoding RNAs. Long noncoding
RNAs are of particular interest, as they were once thought to be restricted to
housekeeping functions and are now linked to a wide variety of biological
functions related to physiology, embryology and development. Emerging evidence
indicates that a subset of long noncoding RNAs mediate their biological
functions by using chromatin as a substrate, to index the genetic information
encoded in the genome. This chapter will discuss how noncoding RNAs and the
processes underlying their transcription mediate transcriptional regulation, by
epigenetically regulating the structure of chromatin in various biological
contexts. Mammalian genomes contain numerous genes for long noncoding RNAs (lncRNAs). The
functions of the lncRNAs remain largely unknown but their evolution appears to
be constrained by purifying selection, albeit relatively weakly. To gain
insights into the mode of evolution and the functional range of the lncRNA, they
can be compared with much better characterized protein-coding genes. The
evolutionary rate of the protein-coding genes shows a universal negative
correlation with expression: highly expressed genes are on average more
conserved during evolution than the genes with lower expression levels. This
correlation was conceptualized in the misfolding-driven protein evolution
hypothesis according to which misfolding is the principal cost incurred by
protein expression. We sought to determine whether long intergenic ncRNAs
(lincRNAs) follow the same evolutionary trend and indeed detected a moderate but
statistically significant negative correlation between the evolutionary rate and
expression level of human and mouse lincRNA genes. The magnitude of the
correlation for the lincRNAs is similar to that for equal-sized sets of
protein-coding genes with similar levels of sequence conservation. Additionally,
the expression level of the lincRNAs is significantly and positively correlated
with the predicted extent of lincRNA molecule folding (base-pairing), however,
the contributions of evolutionary rates and folding to the expression level are
independent. Thus, the anticorrelation between evolutionary rate and expression
level appears to be a general feature of gene evolution that might be caused by
similar deleterious effects of protein and RNA misfolding and/or other factors,
for example, the number of interacting partners of the gene product. The transcriptome of a cell is represented by a myriad of different RNA
molecules with and without protein-coding capacities. In recent years, advances
in sequencing technologies have allowed researchers to more fully appreciate the
complexity of whole transcriptomes, showing that the vast majority of the genome
is transcribed, producing a diverse population of non-protein coding RNAs
(ncRNAs). Thus, the biological significance of non-coding RNAs (ncRNAs) have
been largely underestimated. Amongst these multiple classes of ncRNAs, the long
non-coding RNAs (lncRNAs) are apparently the most numerous and functionally
diverse. A small but growing number of lncRNAs have been experimentally studied,
and a view is emerging that these are key regulators of epigenetic gene
regulation in mammalian cells. LncRNAs have already been implicated in human
diseases such as cancer and neurodegeneration, highlighting the importance of
this emergent field. In this article, we review the catalogs of annotated
lncRNAs and the latest advances in our understanding of lncRNAs. The human genome encodes thousands of long noncoding RNAs (lncRNAs). Although
most remain functionally uncharacterized biological "dark matter," lncRNAs have
garnered considerable attention for their diverse roles in human biology,
including developmental programs and tumor suppressor gene networks. As the
number of lncRNAs associated with human disease grows, ongoing research efforts
are focusing on their regulatory mechanisms. New technologies that enable
enumeration of lncRNA interaction partners and determination of lncRNA structure
are well positioned to drive deeper understanding of their functions and
involvement in pathogenesis. In turn, lncRNAs may become targets for therapeutic
intervention or new tools for biotechnology. The human genome contains many thousands of long noncoding RNAs (lncRNAs). While
several studies have demonstrated compelling biological and disease roles for
individual examples, analytical and experimental approaches to investigate these
genes have been hampered by the lack of comprehensive lncRNA annotation. Here,
we present and analyze the most complete human lncRNA annotation to date,
produced by the GENCODE consortium within the framework of the ENCODE project
and comprising 9277 manually annotated genes producing 14,880 transcripts. Our
analyses indicate that lncRNAs are generated through pathways similar to that of
protein-coding genes, with similar histone-modification profiles, splicing
signals, and exon/intron lengths. In contrast to protein-coding genes, however,
lncRNAs display a striking bias toward two-exon transcripts, they are
predomitly localized in the chromatin and nucleus, and a fraction appear to
be preferentially processed into small RNAs. They are under stronger selective
pressure than neutrally evolving sequences-particularly in their promoter
regions, which display levels of selection comparable to protein-coding genes.
Importantly, about one-third seem to have arisen within the primate lineage.
Comprehensive analysis of their expression in multiple human organs and brain
regions shows that lncRNAs are generally lower expressed than protein-coding
genes, and display more tissue-specific expression patterns, with a large
fraction of tissue-specific lncRNAs expressed in the brain. Expression
correlation analysis indicates that lncRNAs show particularly striking positive
correlation with the expression of antisense coding genes. This GENCODE
annotation represents a valuable resource for future studies of lncRNAs. Dorsal root ganglia (DRG) neurons spontaneously undergo robust neurite growth
after axotomy. Long noncoding RNAs (lncRNAs) are an important class of pervasive
genes involved in a variety of biological functions. However, the functions of
lncRNAs in the regulation of responses of DRG neurons to injury stimuli remain
untested. Here, lncRNA microarray analysis was performed to profile the lncRNAs
in L4-L6 DRGs following rat sciatic nerve resection. The 105 lncRNAs were
identified to be differentially expressed at 0, 1, 4, 7 d post injury. A
coexpression network of 24 down-regulated lncRNAs and coding genes was
constructed, and 115 targets of these 24 lncRNAs were found to be mainly
involved in cell phenotype modulation, including glial cell migration,
purinergic nucleotide receptor signaling pathway, vasodilation, regulation of
multi-organism process, and neuropeptide signaling pathway, and also to be
potentially associated with several key regeneration signaling pathways,
including MAPK signaling pathway, and neuroactive ligand-receptor interaction.
LncRNA BC089918 was selected from 24 down-regulated lncRNAs for validation by
quantitative real-time polymerase chain reaction and in situ hybridization. And
silencing of BC089918 with small interfering RNAs indicted that the lncRNA had a
particular promoting effect on neurite outgrowth. Our data demonstrated a
distinct involvement of lncRNAs in DRGs after nerve injury, thus contributing to
illustration of molecular mechanisms responsible for nerve regeneration. Long noncoding RNAs (lncRNAs) have gained widespread attention in recent years
as a potentially new and crucial layer of biological regulation. lncRNAs of all
kinds have been implicated in a range of developmental processes and diseases,
but knowledge of the mechanisms by which they act is still surprisingly limited,
and claims that almost the entirety of the mammalian genome is transcribed into
functional noncoding transcripts remain controversial. At the same time, a small
number of well-studied lncRNAs have given us important clues about the biology
of these molecules, and a few key functional and mechanistic themes have begun
to emerge, although the robustness of these models and classification schemes
remains to be seen. Here, we review the current state of knowledge of the lncRNA
field, discussing what is known about the genomic contexts, biological
functions, and mechanisms of action of lncRNAs. We also reflect on how the
recent interest in lncRNAs is deeply rooted in biology's longstanding concern
with the evolution and function of genomes. |
Is macitentan an ET agonist? | No, macitentan is anendothelin receptor antagonist. | Macitentan, also called Actelion-1 or ACT-064992
[N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4-yl]-N'-propylaminosulfonamide],
is a new dual ET(A)/ET(B) endothelin (ET) receptor antagonist designed for
tissue targeting. Selection of macitentan was based on inhibitory potency on
both ET receptors and optimization of physicochemical properties to achieve high
affinity for lipophilic milieu. In vivo, macitentan is metabolized into a major
and pharmacologically active metabolite, ACT-132577. Macitentan and its
metabolite antagonized the specific binding of ET-1 on membranes of cells
overexpressing ET(A) and ET(B) receptors and blunted ET-1-induced calcium
mobilization in various natural cell lines, with inhibitory constants within the
omolar range. In functional assays, macitentan and ACT-132577 inhibited
ET-1-induced contractions in isolated endothelium-denuded rat aorta (ET(A)
receptors) and sarafotoxin S6c-induced contractions in isolated rat trachea
(ET(B) receptors). In rats with pulmonary hypertension, macitentan prevented
both the increase of pulmonary pressure and the right ventricle hypertrophy, and
it markedly improved survival. In diabetic rats, chronic administration of
macitentan decreased blood pressure and proteinuria and prevented end-organ
damage (renal vascular hypertrophy and structural injury). In conclusion,
macitentan, by its tissue-targeting properties and dual antagonism of ET
receptors, protects against end-organ damage in diabetes and improves survival
in pulmonary hypertensive rats. This profile makes macitentan a new agent to
treat cardiovascular disorders associated with chronic tissue ET system
activation. Macitentan (ACT-064992), under development by Actelion Ltd in collaboration with
Japanese licensee Nippon Shinyaku Co Ltd, is an orally active, non-peptide dual
endothelin (ET)(A) and ET(B) receptor antagonist for the potential treatment of
idiopathic pulmonary fibrosis (IPF) and pulmonary arterial hypertension (PAH).
Scientific evidence suggests that the ET system may play an important role in
the pathobiology of several cardiovascular diseases. A major therapeutic advance
for the treatment of patients with PAH and IPF has been the pharmacological
control of the activated ET system with ET receptor antagonists. Macitentan,
because of its ability to target the tissues and to block both ET(A) and ET(B)
receptors, is emerging as a new agent to treat cardiovascular disorders
associated with chronic tissue ET system activation. The phase I and II clinical
trials conducted to date have demonstrated that macitentan increases plasma
levels of ET-1, displays dose-dependent pharmacokinetics, and was well tolerated
in healthy volunteers and patients. At the time of publication, a phase II trial
in patients with IPF and a phase III trial in patients with PAH was ongoing. It
is expected that the results of these trials will validate the safety and
efficacy of macitentan. Potential treatments for ovarian cancers that have become resistant to standard
chemotherapies include modulators of tumor cell survival, such as endothelin
receptor (ETR) antagonist. We investigated the therapeutic efficacy of the dual
ETR antagonist, macitentan, on human ovarian cancer cells, SKOV3ip1 and IGROV1,
growing orthotopically in nude mice. Mice with established disease were treated
with vehicle (control), paclitaxel (weekly, intraperitoneal injections),
macitentan (daily oral administrations), or a combination of paclitaxel and
macitentan. Treatment with paclitaxel decreased tumor weight and volume of
ascites. Combination therapy with macitentan and paclitaxel reduced tumor
incidence and further reduced tumor weight and volume of ascites when compared
with paclitaxel alone. Macitentan alone occasionally reduced tumor weight but
alone had no effect on tumor incidence or ascites. Immunohistochemical analyses
revealed that treatment with macitentan and macitentan plus paclitaxel inhibited
the phosphorylation of ETRs and suppressed the survival pathways of tumor cells
by decreasing the levels of pVEGFR2, pAkt, and pMAPK. The dose of macitentan
necessary for inhibition of phosphorylation correlated with the dose required to
increase antitumor efficacy of paclitaxel. Treatment with macitentan enhanced
the cytotoxicity mediated by paclitaxel as measured by the degree of apoptosis
in tumor cells and tumor-associated endothelial cells. Collectively, these
results show that administration of macitentan in combination with paclitaxel
prevents the progression of ovarian cancer in the peritoneal cavity of nude mice
in part by inhibiting survival pathways of both tumor cells and tumor-associated
endothelial cells. Macitentan is a dual endothelin receptor antagonist under phase 3 investigation
in pulmonary arterial hypertension. We investigated the effect of cyclosporine
(Cs) and rifampin on the pharmacokinetics of macitentan and its metabolites
ACT-132577 and ACT-373898 in healthy male subjects. In addition, in vitro
studies were performed to investigate interactions between macitentan and its
active metabolite ACT-132577 with human organic anion-transporting polypeptides
(OATPs). The clinical study (AC-055-111) was conducted as a two-part,
one-sequence, crossover study. Ten subjects in each part received multiple-dose
macitentan followed by multiple-dose co-administration of Cs (part A) or
rifampin (part B). In the presence of Cs, steady-state area under the plasma
concentration-time profiles during a dose interval (AUC(τ)) for macitentan and
ACT-373898 increased 10% and 7%, respectively, and decreased 3% for ACT-132577.
Steady-state AUC(τ) of macitentan and ACT-373898 in the presence of rifampin
decreased 79% and 64%, respectively. For ACT-132577, no relevant difference in
AUC(τ) between the two treatments was observed. Macitentan co-administered with
Cs or rifampin was well tolerated. The complementary in vitro studies
demonstrated no marked differences in uptake rates of macitentan and ACT-132577
between the wild-type and OATP over-expressing cells over the concentration
range tested. Concomitant treatment with Cs did not have any clinically relevant
effect on the exposure to macitentan or its metabolites, at steady-state.
Concomitant treatment with rifampin reduced significantly the exposure to
macitentan and its metabolite ACT-373898 at steady-state but did not affect the
exposure to the active metabolite ACT-132577 to a clinically relevant extent. Endothelin receptors (ETRs) are often overexpressed in ovarian tumors, which can
be resistant to conventional therapies. Thus, we investigated whether blockage
of the ETR pathways using the dual ETR antagonist macitentan combined with taxol
or cisplatinum can produce therapy for orthotopically growing
multidrug-resistant (MDR) human ovarian carcinoma. In several studies, nude mice
were injected in the peritoneal cavity with HeyA8-MDR human ovarian cancer
cells. Ten days later, mice were randomized to receive vehicle (saline),
macitentan (oral, daily), taxol (intraperitoneal, weekly), cisplatinum
(intraperitoneal, weekly), macitentan plus taxol, or macitentan plus
cisplatinum. Moribund mice were killed, and tumors were collected, weighed, and
prepared for immunohistochemical analysis. The HeyA8-MDR tumors did not respond
to taxol, cisplatinum, or macitentan administered as single agents. In contrast,
combination therapy with macitentan and taxol or macitentan and cisplatinum
significantly decreased the tumor incidence and weight and significantly
increased the survival of mice and their general condition. Multiple
immunohistochemical analyses revealed that treatment with macitentan and
macitentan plus taxol or cisplatinum inhibited the phosphorylation of ETRs,
decreased the levels of pVEGFR2, pAkt, and pMAPK in tumor cells after 2 weeks of
treatment and induced a first wave of apoptosis in tumor-associated endothelial
cells followed by apoptosis in surrounding tumor cells. Our study shows that
ovarian cancer cells, which express the endothelin axis and are multidrug
resistant, are exquisitely sensitive to treatment with a dual ET antagonist and
can be resensitized to both taxol and cisplatinum. This combined therapy led to
a significant reduction in tumor weight. Starting from the structure of bosentan (1), we embarked on a medicinal
chemistry program aiming at the identification of novel potent dual endothelin
receptor antagonists with high oral efficacy. This led to the discovery of a
novel series of alkyl sulfamide substituted pyrimidines. Among these, compound
17 (macitentan, ACT-064992) emerged as particularly interesting as it is a
potent inhibitor of ET(A) with significant affinity for the ET(B) receptor and
shows excellent pharmacokinetic properties and high in vivo efficacy in
hypertensive Dahl salt-sensitive rats. Compound 17 successfully completed a
long-term phase III clinical trial for pulmonary arterial hypertension. Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are
currently approved for the treatment of pulmonary arterial hypertension (PAH), a
devastating disease involving an activated endothelin system and aberrant
contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC).
The novel ERA macitentan has recently concluded testing in a Phase III
morbidity/mortality clinical trial in PAH patients. Since the association and
dissociation rates of G protein-coupled receptor antagonists can influence their
pharmacological activity in vivo, we used human PASMC to characterize inhibitory
potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan
using calcium release and inositol-1-phosphate (IP(1)) assays. In calcium
release assays macitentan, ambrisentan and bosentan were highly potent ERAs with
K(b) values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not
ambrisentan and bosentan, displayed slow apparent receptor association kinetics
as evidenced by increased antagonistic potency upon prolongation of antagonist
pre-incubation times. In compound washout experiments, macitentan displayed a
significantly lower receptor dissociation rate and longer receptor occupancy
half-life (ROt(1/2)) compared to bosentan and ambrisentan (ROt(1/2):17 minutes
versus 70 seconds and 40 seconds, respectively). Because of its lower
dissociation rate macitentan behaved as an insurmountable antagonist in calcium
release and IP(1) assays, and unlike bosentan and ambrisentan it blocked
endothelin receptor activation across a wide range of endothelin-1 (ET-1)
concentrations. However, prolongation of the ET-1 stimulation time beyond
ROt(1/2) rendered macitentan a surmountable antagonist, revealing its
competitive binding mode. Bosentan and ambrisentan behaved as surmountable
antagonists irrespective of the assay duration and they lacked inhibitory
activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with
significantly slower receptor dissociation kinetics than the currently approved
ERAs. Slow dissociation caused insurmountable antagonism in functional
PASMC-based assays and this could contribute to an enhanced pharmacological
activity of macitentan in ET-1-dependent pathologies. Macitentan is a new non-selective endothelin-1 receptor antagonist under
development for the treatment of pulmonary arterial hypertension. Information on
the potential for macitentan to influence the pharmacokinetics of concomitantly
administered drugs by inhibition or induction of drug metabolising enzymes or
drug transporters is sparse. We therefore studied the potential of macitentan to
inhibit and induce critical targets of drug metabolism and drug distribution
(transporters) in vitro. Induction was quantified at the mRNA level by real-time
RT-PCR in LS180 cells and revealed that macitentan significantly induced mRNA
expression of cytochrome P450 3A4 (CYP3A4), P-glycoprotein (P-gp, ABCB1), solute
carrier of organic anions 1B1 (SLCO1B1), and
uridinediphosphate-glucuronosyltransferase 1A3 (UGT1A9). By means of a reporter
gene assay our study establishes macitentan as a potent activator of prege X
receptor (PXR). Inhibition of drug transporters was evaluated by using
transporter over-expressing cell lines and fluorescent specific substrates of
the respective transporters and revealed that macitentan is an inhibitor of
P-gp, breast cancer resistance protein (BCRP), SLCO1B1, and SLCO1B3. Using
commercial kits macitentan was demonstrated to be a moderate inhibitor of CYP3A4
and CYP2C19. In conclusion our data provide a comprehensive analysis of the
interaction profile of macitentan with drug metabolising and transporting
enzymes in vitro. Although macitentan has a similar or higher potency for
induction and inhibition of drug metabolising enzymes and transporters than
bosentan, its low plasma concentrations and minimal accumulation in the liver
suggest that it will be markedly less prone to drug-drug interactions than
bosentan. This multiple-ascending-dose study investigated safety, tolerability,
pharmacokinetics, and pharmacodynamics, of macitentan, a new endothelin receptor
antagonist (ERA) with sustained receptor binding and enhanced tissue penetration
properties compared to other ERAs. Healthy male subjects (n = 32) received once
daily oral doses of macitentan (1 - 30 mg) or placebo for 10 days.
Administration of macitentan was safe and well tolerated. Macitentan had no
effect on bile salts, suggesting an improved liver safety profile. The
multiple-dose pharmacokinetics of macitentan were dose-proportional and were
characterized by a median tmax and apparent elimination half-life varying from
6.0 to 8.5 and 14.3 to 18.5 hours, respectively, for the different doses and
minimal accumulation. ACT-132577, a metabolite with lower potency than
macitentan, had a half-life of about 48 hours and accumulated approximately
8.5-fold. Compared to placebo, administration of macitentan caused a
dose-dependent increase in plasma ET-1 with maximum effects attained at 10 mg. A
small dose-dependent increase in the 6β-hydroxycortisol/cortisol urinary
excretion ratio was observed, although there were no statistically significant
differences between treatments including placebo. Effects of macitentan on
cytochrome P450 enzyme 3A4 should be further evaluated in dedicated studies. The
present results support investigation of macitentan in the management of
pulmonary arterial hypertension and ET-1-dependent pathologies. Pulmonary arterial hypertension (PAH) is a progressive disease characterised by
remodelling of small pulmonary arteries leading to an increased pulmonary
vascular resistance, right ventricular failure and death. Available treatments
try to re-establish the equilibrium on three signalling pathways: the
prostacyclin, the endothelin (ET)-1 and the nitric oxide. Prostanoids, such as
epoprostenol or treprostinil have a vasodilator, antiproliferative and
immunomodulatory effect and, despite the administration inconveniences,
represent established therapies for severe cases of PAH. Recently oral
prostacyclin receptor agonists have shown encouraging results. Many clinical
studies targeting the vasoconstrictor ET-1 pathway with receptor antagonists
like bosentan and ambrisentan have shown strong results, even more optimism
coming from macitentan, the newest drug. Sildenafil and tadalafil, two
phosphodiesterase type-5 inhibitors, have shown improved exercise capacity by
increasing the nitric oxide level. Riociguat, acting on the same nitric oxide
pathway, as a guanylatecyclase activator, has shown promising results in
clinical trials and will be available soon. Long-awaited results for
tyrosin-kinase inhibitor, imatinib, as an antiproliferative therapy in PAH have
been disappointing, due to severe adverse events. In conclusion, although it
remains a disease with severe prognosis, the past 20 years have represented a
huge progress in terms of treatments for PAH with interesting opportunities for
the future. Macitentan (Opsumit®) is a novel dual endothelin receptor antagonist (ERA) with
sustained receptor binding properties developed by Actelion Pharmaceuticals Ltd.
In October 2013, oral macitentan 10 mg once daily received its first global
approval in the US, followed closely by Canada, for the treatment of pulmonary
arterial hypertension (PAH). The drug has also received a positive opinion in
the EU from the Committee for Medicinal Products for Human Use for the treatment
of PAH, and is under regulatory review in several other countries for the same
indication. Endothelin (ET)-1 influences pathological changes via two ET
receptor subtypes (ETA and ETB), to which it binds with high affinity. ET-1 is
implicated in several forms of vascular disease making it a valid target for the
treatment of pulmonary vascular diseases such as PAH. Clinical development is
underway for other indications, including Eisenmenger syndrome, ischaemic
digital ulcers secondary to systemic sclerosis, and glioblastoma. Macitentan was
also evaluated in idiopathic pulmonary fibrosis; however, a phase 2 trial did
not meet its primary endpoint and further investigation in this indication was
discontinued. Macitentan was developed by modifying the structure of bosentan in
the search for an optimal dual ERA with improved efficacy and tolerability
compared with other ERAs. This article summarizes the milestones in the
development of macitentan leading to this first approval for PAH. AIMS: The endothelin (ET) system is a tissular system, as the production of ET
isoforms is mostly autocrine or paracrine. Macitentan is a novel dual ETA/ETB
receptor antagonist with enhanced tissue distribution and sustained receptor
binding properties designed to achieve a more efficacious ET receptor blockade.
To determine if these features translate into improved efficacy in vivo, a study
was designed in which rats with either systemic or pulmonary hypertension and
equipped with telemetry were given macitentan on top of maximally effective
doses of another dual ETA/ETB receptor antagonist, bosentan, which does not
display sustained receptor occupancy and shows less tissue distribution.
MAIN METHODS: After establishing dose-response curves of both compounds in
conscious, hypertensive Dahl salt-sensitive and pulmonary hypertensive
bleomycin-treated rats, macitentan was administered on top of the maximal
effective dose of bosentan.
KEY FINDINGS: In hypertensive rats, macitentan 30 mg/kg further decreased mean
arterial blood pressure (MAP) by 19 mm Hg when given on top of bosentan 100
mg/kg (n=9, p<0.01 vs. vehicle). Conversely, bosentan given on top of macitentan
failed to induce an additional MAP decrease. In pulmonary hypertensive rats,
macitentan 30 mg/kg further decreased mean pulmonary artery pressure (MPAP) by 4
mm Hg on top of bosentan (n=8, p<0.01 vs. vehicle), whereas a maximal effective
dose of bosentan given on top of macitentan did not cause any additional MPAP
decrease.
SIGNIFICANCE: The add-on effect of macitentan on top of bosentan in two
pathological models confirms that this novel compound can achieve a superior
blockade of ET receptors and provides evidence for greater maximal efficacy. OBJECTIVES: Endothelin (ET) is a major therapeutic target in cardiopulmonary
diseases. The purpose of this review is to present the main concepts concerning
ET biology, its pathophysiological roles and the major pharmacological and
medical advances recently developed around the concept of ET receptor blockade.
METHODS: Analysis of PubMed database (keywords: endothelin, endothelin receptor
antagonists, pulmonary hypertension, etc.), and of abstract originating from
recent international meetings.
RESULTS: ET is a peptide produced by vascular endothelial cells as well as by
many other tissues. Both its production and its effects are activated in
pathological situations associated with endothelial dysfunction. ET is
characterized by a strong tropism toward tissues because of its polarized
release, the strong tissue receptor density and high affinity of the receptors
for the peptide. ET exerts several vascular effects, including vasoconstriction,
proliferation and hypertrophy, as well as non-vascular effects, notably
stimulation of cardiac hypertrophy, tissue fibrosis and inflammation. Both
vascular and non-vascular effects depend on the stimulation of two receptor
subtypes, ETA and ETB. ET receptor antagonists (ERA) demonstrated beneficial
effects in many different pre-clinical models of cardiovascular and pulmonary
diseases, and constitute a first-line treatment of patients with pulmonary
arterial hypertension (PAH). Recently, the targeted search for a novel ERA led
to the development of macitentan which, compared to existing ERA, show optimized
tissue penetration, increased receptor affinity and in vivo pharmacological
efficacy in pre-clinical models, associated with a favorable profile, in terms
of hepatic safety and drug interactions. The clinical efficacy of macitentan in
the treatment of PAH was recently demonstrated in the SERAPHIN trial, which
contrasts with previous PAH trials because of its long duration, the high number
of patients enrolled, and its primary endpoint evaluating morbidity/mortality.
Results show a significant reduction of the primary composite
morbidity/mortality endpoint (taking into account both progression of PAH and
death) by 30 and 45% with macitentan 3 and 10mg, respectively, compared to
placebo, and confirm on the large scale the favorable tolerance profile,
especially at the hepatic level.
CONCLUSION: The extensive knowledge on the complexity of the ET system allowed
the synthesis of a new antagonist optimized, in terms of pharmacological
efficacy and safety, which also show promising therapeutic effects in PAH
patients, with demonstrated results in a prospective study using a composite
primary endpoint of morbidity-mortality. BACKGROUND AND OBJECTIVES: Macitentan is a novel dual endothelin (ET)-1 receptor
antagonist to be used in patients with pulmonary arterial hypertension. This
study aimed to assess the pharmacokinetics (PK) and pharmacodynamics (PD) of
macitentan after administration of multiple doses to healthy Korean male
subjects.
METHODS: A randomized, double-blind, placebo-controlled, multiple-ascending dose
study was performed in 30 healthy male subjects receiving oral macitentan (3,
10, or 30 mg) or placebo once daily for 10 days. Plasma concentrations of
macitentan, its active metabolite ACT-13277, and ET-1 were evaluated. Safety and
tolerability measurements were conducted throughout the study.
RESULTS: The concentration-time profile of macitentan was characterized by slow
absorption (median time to maximum plasma concentration [t(max)] 9-10 h) and
slow elimination (mean elimination half-life [t ½] 11-15 h). After repeated
doses of 3, 10, and 30 mg of macitentan over the course of 10 days, the peak
concentration (C(max)) increased as the dose increased and the area under the
plasma concentration-time curve during the dosing interval (AUC(τ)) increased in
a dose-proportional manner. Plasma concentrations showed approximately 1.5- to
1.9-fold accumulation on day 10 compared with day 1. ACT-132577 showed higher
levels of exposure than macitentan, its mean half-life was 46-48 h, and it
accumulated 7- to 12-fold. Macitentan increased plasma ET-1 concentrations at
all doses tested and was well tolerated and elicited no serious adverse events.
CONCLUSION: Multiple oral doses of 3, 10, and 30 mg of macitentan were well
tolerated in healthy Korean subjects, and its pharmacokinetics correlated
positively with ET-1 concentrations. Macitentan (Opsumit(®)) is an orally active, dual endothelin receptor antagonist
(ERA) with tissue targeting properties. Macitentan was approved recently in the
EU (as monotherapy or combination therapy) for the long-term treatment of
pulmonary arterial hypertension (PAH) in adults of WHO functional class II or
III, and in the USA for the treatment of PAH (WHO group I) to delay disease
progression and reduce hospitalization for PAH. This article reviews the
pharmacological properties, efficacy and tolerability data relevant to the use
of macitentan in this indication. Treatment with macitentan 10 mg once daily
significantly reduced the risk for the primary composite endpoint of morbidity
and mortality in patients with PAH (mostly WHO functional class II or III) in
the large, randomized, placebo-controlled SERAPHIN study. Other efficacy
outcomes, including exercise capacity, haemodynamic parameters and
health-related quality of life also improved significantly with macitentan
relative to placebo. Macitentan was generally well tolerated in this study. As
with other ERAs, haemoglobin levels decreased with macitentan therapy; however,
these were not progressive and stabilized following longer-term treatment.
Although comparative studies are needed to definitively position macitentan with
respect to other approved agents, current evidence suggests that macitentan is a
useful treatment option for initial therapy in patients with WHO functional
class II or III PAH, which has the potential advantage of once-daily
administration. The efficacy of endothelin (ET) receptor antagonist bosentan in patients with
severe pulmonary arterial hypertension (PAH) remains limited, partly because its
higher doses for potential blockade of ET receptors have never been tested due
to liver dysfunction. We hypothesized that rigorous blockade of ET receptors
using the novel dual ET receptor antagonist macitentan would be effective in
treating severe PAH without major side effects in a preclinical model
appropriately representing the human disorder. In normal rats, 30 mg·kg·d of
macitentan completely abolished big ET-1-induced increases in right ventricle
(RV) systolic pressure. Adult male rats were injected with SU5416, a vascular
endothelial growth factor blocker, and exposed to hypoxia for 3 weeks and then
to normoxia for an additional 5 weeks (total 8 weeks). In intrapulmonary
arterial rings isolated from rats with severe PAH, macitentan concentration
dependently inhibited ET-1-induced contraction. Long-term treatment with
macitentan (30 mg·kg·d, from week 3 to 8) reversed the high RV systolic pressure
with preserved cardiac output. Development of RV hypertrophy, luminal occlusive
lesions and medial wall thickening were also significantly improved without
increasing serum levels of liver enzymes by macitentan. In conclusion,
efficacious blockade of ET receptors with macitentan would reverse severe PAH
without major adverse effects. The competitive endothelin receptor antagonists (ERA) bosentan and ambrisentan,
which have long been approved for the treatment of pulmonary arterial
hypertension, are characterized by very short (1 min) occupancy half-lives at
the ET(A) receptor. The novel ERA macitentan, displays a 20-fold increased
receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced
signaling in pulmonary arterial smooth muscle cells. We show here that the slow
ET(A) receptor dissociation rate of macitentan was shared with a set of
structural analogs, whereas compounds structurally related to bosentan displayed
fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact
structure in aqueous solution and molecular modeling suggests that this
conformation tightly fits into a well-defined ET(A) receptor binding pocket. In
contrast the structurally different and negatively charged bosentan-type
molecules only partially filled this pocket and expanded into an extended
endothelin binding site. To further investigate these different ET(A)
receptor-antagonist interaction modes, we performed functional studies using
ET(A) receptor variants harboring amino acid point mutations in the presumed ERA
interaction site. Three ET(A) receptor residues significantly and differentially
affected ERA activity: Mutation R326Q did not affect the antagonist activity of
macitentan, however the potencies of bosentan and ambrisentan were significantly
reduced; mutation L322A rendered macitentan less potent, whereas bosentan and
ambrisentan were unaffected; mutation I355A significantly reduced bosentan
potency, but not ambrisentan and macitentan potencies. This suggests that--in
contrast to bosentan and ambrisentan--macitentan-ET(A) receptor binding is not
dependent on strong charge-charge interactions, but depends predomitly on
hydrophobic interactions. This different binding mode could be the reason for
macitentan's sustained target occupancy and insurmountable antagonism. The high mortality of epithelial ovarian cancer (EOC) is mainly caused by
resistance to the available therapies. In EOC, the endothelin-1 (ET-1,
EDN1)-endothelin A receptor (ETAR, EDNRA) signaling axis regulates the
epithelial-mesenchymal transition (EMT) and a chemoresistant phenotype. However,
there is a paucity of knowledge about how ET-1 mediates drug resistance. Here,
we define a novel bypass mechanism through which ETAR/β-arrestin-1 (β-arr1,
ARRB1) links Wnt signaling to acquire chemoresistant and EMT phenotype. We found
that ETAR/β-arr1 activity promoted nuclear complex with β-catenin and p300,
resulting in histone acetylation, chromatin reorganization, and enhanced
transcription of genes, such as ET-1, enhancing the network that sustains
chemoresistance. Silencing of β-arr1 or pharmacologic treatment with the dual
ETAR/ETBR antagonist macitentan prevented core complex formation and restored
drug sensitivity, impairing the signaling pathways involved in cell survival,
EMT, and invasion. In vivo macitentan treatment reduced tumor growth,
vascularization, intravasation, and metastatic progression. The combination of
macitentan and cisplatinum resulted in the potentiation of the cytotoxic effect,
indicating that macitentan can enhance sensitivity to chemotherapy.
Investigations in clinical specimens of chemoresistant EOC tissues confirmed
increased recruitment of β-arr1 and β-catenin to ET-1 gene promoter. In these
tissues, high expression of ETAR significantly associated with poor clinical
outcome and chemoresistance. Collectively, our findings reveal the existence of
a novel mechanism by which ETAR/β-arr1 signaling is integrated with the
Wnt/β-catenin pathway to sustain chemoresistance in EOC, and they offer a solid
rationale for clinical evaluation of macitentan in combination with chemotherapy
to overcome chemoresistance in this setting. OBJECTIVES: This study sought to evaluate the effect of macitentan on
hospitalization of patients with symptomatic pulmonary arterial hypertension
(PAH).
BACKGROUND: PAH is a progressive, life-threatening disease often requiring
hospitalization.
METHODS: In the multicenter, double-blind, randomized, event-driven, phase III
SERAPHIN (Study with an Endothelin Receptor Antagonist in Pulmonary arterial
Hypertension to Improve cliNical outcome) trial, patients with symptomatic PAH
were randomized (1:1:1) to receive placebo or 3 mg or 10 mg of macitentan.
Effects of macitentan on the risk, rate, and number of hospital days for
all-cause and PAH-related hospitalizations were compared with those for placebo.
Risk and causes of hospitalizations unrelated to PAH were investigated.
RESULTS: Of 742 randomized patients, 250 received placebo, 250 received 3 mg of
macitentan, and 242 received 10 mg of macitentan; the overall median duration of
treatment was 115 weeks. Risk of all-cause hospitalization was reduced by 18.9%
(p = 0.1208) and 32.3% (p = 0.0051) in the macitentan 3-mg and 10-mg arm,
respectively. Rates of all-cause hospitalizations and numbers of hospital days
were reduced by 20.5% (p = 0.0378) and 30.6% (p = 0.0278), respectively, with 3
mg of macitentan and by 33.1% (p = 0.0005) and 31.0% (p = 0.0336), respectively,
with 10 mg of macitentan. Risk of PAH-related hospitalizations were reduced by
42.7% (p = 0.0015) and 51.6% (p < 0.0001) in the macitentan 3-mg and 10-mg arms,
respectively. Rate of PAH-related hospitalizations and numbers of hospital days
were reduced by 44.5% (p = 0.0004) and 53.3% (p = 0.0001), respectively, with 3
mg of macitentan, and reduced by 49.8% (p < 0.0001) and 52.3% (p = 0.0003),
respectively, with 10 mg of macitentan. Risk of non-PAH-related hospitalization
was similar between treatment arms.
CONCLUSIONS: Macitentan 10 mg significantly reduced the risk and rate of
all-cause hospitalization, which was driven by reductions in the risk and rate
of PAH-related hospitalization. (Study of Macitentan [ACT-064992] on Morbidity
and Mortality in Patients With Symptomatic Pulmonary Arterial Hypertension;
NCT00660179). INTRODUCTION: Pulmonary arterial hypertension (PAH) is a chronic disorder of the
pulmonary vasculature characterized by elevated mean pulmonary arterial pressure
eventually leading to right-sided heart failure and premature death. Macitentan
is an oral, once-daily, dual endothelin (ET)A and ETB receptor antagonist with
high affinity and sustained receptor binding that was approved in the USA,
Europe, Canada, and Switzerland for the treatment of PAH.
AREAS COVERED: This review discusses the pharmacokinetics (PK) and
pharmacodynamics (PD) of macitentan and its drug interaction potential based on
preclinical and clinical data.
EXPERT OPINION: Up to date, macitentan is the only registered treatment for PAH
that significantly reduced morbidity and mortality as a combined endpoint in a
long-term event-driven study. The safety profile of macitentan is favorable with
respect to hepatic safety and edema/fluid retention and may be better than that
of other ET receptor antagonists such as bosentan and ambrisentan. The PK
profile supports a once-a-day dosing regimen. Macitentan has limited
interactions with other drugs. Based on these characteristics macitentan is an
important new addition to the treatment of PAH. |
Can we use platelet biomarkers to study Alzheimer's disease? | Yes, platelet biomarkers can be used to study Alzheimer's disease. | Platelets play a fundamental role in hemostasis. Because they do not have a
nucleus, proteomics is an ideal way to approach their biochemistry. Platelet
proteomics is still a young field that emerged a decade ago. Initial platelet
proteomic research focused on general proteome mapping followed by the
exploration of sub-cellular compartments, the membrane proteome, and signaling
pathways. The initial studies were later completed with the analysis of the
platelet releasate and microparticle proteome. The success of these studies led
to the application of platelet proteomics to the study of several pathologies
where platelets play a fundamental role. Those include platelet-related
disorders, such as storage pool disease, gray platelet syndrome, and Quebec
platelet disorder; diseases where unwanted platelet activation is highly
relevant, such as thrombosis and cardiovascular disease; and other diseases,
such as cystic fibrosis, uremia, or Alzheimer's disease. In the present review
article, we revise the most relevant proteomic studies on platelet-related
diseases carried out to date, paying special attention to sample preparation
requirements for platelet clinical proteomic studies. This article is part of a
Special Issue entitled: Integrated omics. The search for diagnostic and prognostic markers in Alzheimer's disease (AD) has
been an area of active research in the last decades. Biochemical markers are
correlates of intracerebral changes that can be identified in biological fluids,
namely: peripheral blood (total blood, red and white blood cells, platelets,
plasma and serum), saliva, urine and cerebrospinal fluid. An important feature
of a biomarker is that it can be measured objectively and evaluated as (1) an
indicator of disease mechanisms (markers of core pathogenic processes or the
expression of downstream effects of these processes), or (2) biochemical
responses to pharmacological or therapeutic intervention, which can be
indicative of disease modification. Platelets have been used in
neuropharmacological models since the mid-fifties, as they share several
homeostatic functions with neurons, such as accumulation and release of
neurotransmitters, responsiveness to variations in calcium concentration, and
expression of membrane-bound compounds. Recent studies have shown that platelets
also express several components related to the pathogenesis of AD, in particular
to the amyloid cascade and the regulation of oxidative stress: thus they can be
used in the search for biomarkers of the disease process. For instance,
platelets are the most important source of circulating forms of the amyloid
precursor protein and other important proteins such as Tau and glycogen synthase
kinase-3B. Moreover, platelets express enzymes involved in membrane homeostasis
(e.g., phospholipase A2), and markers of the inflammatory process and oxidative
stress. In this review we summarize the available literature and discuss
evidence concerning the potential use of platelet markers in AD. |
Which genetic defects are observed in Prader-Willi syndrome? | The predominant genetic defects in Prader-Willi syndrome are 15q11-13 deletions of paternal origin and maternal chromosome 15 uniparental disomy, or rare imprinting mutations, combined with monoallelic expression of the paternal alleles. | The genetic defects in Prader-Willi syndrome (PWS) and Angelman syndrome (AS)
map to 15q11-13. Using microdissection, we have recently isolated several DNA
probes for the critical region. Here we report that microclone MN7 detects
multiple loci in 15q11-13 and 16p11.2. Eight yeast artificial chromosome (YAC)
clones, two genomic phage clones, and two placenta cDNA clones were isolated to
analyze these loci in detail. Two of the YAC clones map to 16p. Six YAC clones
and two genomic phage clones contain a total of four or five different MN7
copies, which are spread over a large distance within 15q11-13. One cDNA clone
is from chromosome 15 and one is from chromosome 16. The chromosome 15 cDNA
detects transcripts of 14 and 8 kilobases in various human tissues. The presence
of multiple copies of the MN7 gene family in proximal 15q may conceivably be
related to the instability of this region and thus to the etiology of associated
disorders. Prader-Willi syndrome (PWS) is caused by absence of a paternal contribution of
the chromosome region 15q11-q13, resulting from paternal deletions, maternal
uniparental disomy, or rare imprinting mutations. Laboratory diagnosis is
currently performed using fluorescence in situ hybridization (FISH), DNA
polymorphism (microsatellite) analysis, or DNA methylation analysis at locus
PW71 (D15S63). We examined another parent-of-origin-specific DNA methylation
assay at exon alpha of the small nuclear ribonucleoprotein-associated
polypeptide N gene (SNRPN) in patients referred with clinical suspicion of PWS
or Angelman syndrome (AS). These included 30 PWS and 17 AS patients with known
deletion or uniparental disomy status, and a larger cohort of patients (n = 512)
suspected of PWS who had been analyzed previously for their methylation status
at the PW71 locus. Results of SNRPN methylation were consistent with known
deletion or uniparental disomy (UPD) status as determined by other molecular
methods in all 47 cases of PWS and AS. In the larger cohort of possible PWS
patients, SNRPN results were consistent with clinical diagnosis by examination
and with PW71 methylation results in all cases. These data provide support for
the use of SNRPN methylation as a diagnostic method. Because methylation
analysis can detect all three major classes of genetic defects associated with
PWS (deletion, UPD, or imprinting mutations), methylation analysis with either
PW71 or SNRPN is an efficient primary screening test to rule out a diagnosis of
PWS. Only patients with an abnormal methylation result require further
diagnostic investigation by FISH or DNA polymorphism analysis to distinguish
among the three classes for accurate genetic counseling and recurrence-risk
assessment. Imprinted genes are marked in the germline and retain molecular memory of their
parental origin, resulting in allelic expression differences during development.
Abnormalities in imprinted inheritance occur in several genetic diseases and
cancer, and are exemplified by the diverse genetic defects involving chromosome
15q11-q13 in Prader-Willi (PWS) and Angelman (AS) syndromes. PWS involves loss
of function of multiple paternally expressed genes, while mutations in a single
gene, UBE3A, which is subject to spatially restricted imprinting, occur in some
AS patients. Identification of mutations in the imprinting process in PWS and AS
has led to a definition of an imprinting center (IC), involving the promoter (in
PWS) or an alternative transcript of the SNRPN gene (in AS). The IC regulates
initiation of imprint switching for all genes in a 2 Mb imprinted domain during
gametogenesis. Imprinting mutations define a novel mechanism of genetic disease
because they have no direct effect in the affected patient but, rather, it is
the parental germline effect of an IC mutation that leads to disease in the
offspring. The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are human
neurogenetic disorders involving the imprinting mechanism, at the 15q11-13
chromosome region. The predomit genetic defects in PW are 15q11-13 deletions
of paternal origin and maternal chromosome 15 uniparental disomy. In contrast,
maternal deletions and paternal chromosome 15 uniparental disomy are associated
with a different neurogenetic disorder, the AS. In both disorders, these
mutations are associated with parent-of-origin specific methylation at several
15q11-13 loci. We studied 5 patients suspect of PWS and 4 patients suspect of AS
who were referred to the Medical Genetics Unit at the University Hospital of
Medical School from Ribeirão Preto. Our objective was to establish the correct
clinical and etiological diagnosis in these cases. We used conventional
cytogenetics, methylation analysis with the probe KB17 (CpG island of the SNRPN
gene) by Southern blotting after digestion with the Xba I and Not I restriction
enzymes. We studied in patients and their parents the segregation of the (CA)***
repeats polymorphisms by PCR, using the primers 196 and IR4-3R. All the patients
had normal conventional cytogenetical analysis. We confirmed 3 cases of PWS: one
by de novo deletion, one by maternal chromosome 15 uniparental disomy and one
case with no defined cause determined by the used primers. We confirmed 2 cases
of AS, caused by de novo deletion at the 15q11-13 region, and one case with
normal molecular analysis but with strong clinical characteristics. OBJECTIVE: Prader-Willi syndrome (PWS) is an example of a human genetic disorder
that involves imprinting genes on the proximal long arm of chromosome 15 and
SNRPN gene as a candidate gene for this syndrome. The purpose of this study was
to show the molecular genetic defects and genomic imprinting basis in Chinese
PWS patients and to evaluate the clinical applications of a differential
diagnostic test for PWS.
METHODS: Fluorescence in situ hybridization (FISH) and methylation-specific PCR
(MSPCR) techniques were applied for 4 clinically suspected PWS patients. Using
three probes, including SNRPN probe for identification of the critical locus in
PWS region, D15Z1 and PML control probes for identification of the 15p arm and
15q arm, the authors detected the deletions 15q in PWS. MSPCR was based on
sodium bisulfite treatment of DNA and PCR primers specific for the maternal and
paternal allele.
RESULTS: When hybridized with mixed probes, it was found in 2 patients that the
central specific signal was absent, but both the flanking control signals were
retained, indicating SNRPN gene deletion of chromosome 15q11-13.
Bisulfite-modified DNA from all PWS children amplified with methylated
allele-specific primer pair showed only maternal 131bp PCR product, indicating
the maternal uniparental disomy (UPD15).
CONCLUSION: Genomic imprinting plays an important role in the molecular
pathogenesis of PWS that caused by paternal microdeletions of 15q11-q13 or
maternal UPD of chromosome 15. The basic defect seemed to be an absence of
function of PWS genes that are normally expressed only from the paternal
chromosome 15. MSPCR is a rapid and simple PCR-based assay compared with other
cyto-molecular tests and its results were consistent with the clinical diagnosis
of PWS, so it seems to be a reliable diagnostic method for PWS patients who show
abnormal methylation at SNRPN. The genetic differential tests for PWS are
important in determining familial recurrence risk. Prader-Willi syndrome (PWS) is caused by the disturbed expression of genes from
the imprinted region of 15q11-q13, but the specific contributions of individual
genes remain unknown. Most paternal PWS deletions are bracketed by recurrent
breakpoints BP1 or BP2 and BP3. Atypical deletions are very rare. In the present
work, we describe the molecular analysis of two patients with atypical deletions
using microsatellite analysis, methylation-specific MLPA, and microarray CGH. A
deletion of about 2 Mb in Patient 1 started at BP2 and ended in the middle of
the typically deleted region within the UBE3A gene. The deletion in Patient 2
started 1.3 Mb distal from BP2 within the C15ORF2 gene, extended over 9.5 Mb,
and ended within the AVEN gene in proximal 15q14. In Patient 1 both deletion
breakpoints involved repetitive regions, which precluded cloning of the junction
and pointed to non-allelic homologous recombination as a possible mechanism of
this rearrangement. The breakpoints in Patient 2 were sequenced, and their
structure suggested non-homologous end joining as the most likely cause of this
deletion. The phenotype of both patients did not depart significantly from the
typical clinical picture of PWS, although some symptoms in Patient 2 were also
reminiscent of the phenotype of individuals with the recently described 15q13.3
microdeletion syndrome. Our findings support previous observations of relatively
mild phenotypic effects resulting from deletions that extend distally from the
PWS region and observations of the modest effects of different types of genetic
defects on the spectrum and severity of symptoms in PWS. INTRODUCTION: Prader-Willi syndrome is a complex genetic disease caused by lack
of expression of paternally inherited genes on chromosome 15q11-q13. The
prevalence of Prader-Willi syndrome is estimated to be one in 10,000 to 25,000.
However, descriptions of the oral and dental phenotype are rare.
CASE PRESENTATION: We describe the clinical presentation and periodontal
findings in a 20-year-old Japanese man with previously diagnosed Prader-Willi
syndrome. Clinical and radiographic findings confirmed the diagnosis of
periodontitis. The most striking oral findings were anterior open bite, and
crowding and attrition of the lower first molars. Periodontal treatment
consisted of tooth-brushing instruction and scaling. Home care involved
recommended use of adjunctive chlorhexidine gel for tooth brushing twice a week
and chlorhexidine mouthwash twice daily. Gingival swelling improved, but further
treatment will be required and our patient's oral hygiene remains poor. The
present treatment of tooth-brushing instruction and scaling every three weeks
therefore only represents a temporary solution.
CONCLUSIONS: Rather than being a direct result of genetic defects, periodontal
diseases in Prader-Willi syndrome may largely result from a loss of cuspid
guidance leading to traumatic occlusion, which in turn leads to the development
of periodontal diseases and dental plaque because of poor oral hygiene. These
could be avoided by early interventions to improve occlusion and regular
follow-up to monitor oral hygiene. This report emphasizes the importance of
long-term follow-up of oral health care by dental practitioners, especially
pediatric dentists, to prevent periodontal disease and dental caries in patients
with Prader-Willi syndrome, who appear to have problems maintaining their own
oral health. |
What is the most likely age of diagnosis of Crohn's disease (CD)? | Crohn's disease has a bimodal age distribution of disease onset diagnosis. The peaks (20 and 50 years) may represent different phenotypes or different genetic and/or environmental influences between younger- and older-onset individuals. When the age-related incidence of Crohn's disease was plotted for all countries from which such data were available, the peaks of greatest case frequency occurred at ages 15 to 25 years and paralleled a similar peak representing the number of Peyer's patches as a function of age. For those with biologic use, average age at time of diagnosis of Crohn's disease was 32.3 ± 12.2 years, compared with 43.7 ± 16.3 years for those who had not received biologics (P = 0.005). | BACKGROUND: Our objectives were to assess the differences in perforating disease
behavior, disease severity, and extraintestinal manifestations (EIM) in patients
with Crohn's disease (CD) by race.
MATERIALS AND METHODS: We identified outpatients with CD evaluated at the
University of Maryland Gastroenterology Faculty Practice office or the Baltimore
Veterans Affairs Maryland Health Care System, from 1997 to 2005. We assessed age
at diagnosis, disease behavior, disease location, need for surgery and EIM.
RESULTS: Race was not associated with perforating disease behavior (relative
risk [RR] 0.79, 95% confidence interval [CI] 0.46-1.35), need for surgery (RR
0.89, 95% CI 0.56-1.12), and EIM of CD (RR 0.77, 95% CI 0.46-1.27). White
patients were significantly more likely to have ileal disease, whereas African
American patients were significantly more likely to have ileocolonic and colonic
disease. Age at diagnosis younger than 40 years (odds ratio [OR] 4.41, 95% CI
1.84-10.56) and ileocolonic disease (OR 2.39, 95% CI 1.24-4.63) were independent
risk factors for perforating disease behavior. Similarly, age at diagnosis
younger than 40 (OR 2.79, 95% CI 1.45-5.33), ileal disease (OR 3.76, 95% CI
1.66-8.48), and ileocolonic disease (OR 2.57, 95% CI1.21-5.46) were associated
with the need for surgery. Female gender (OR 4.23, 95% CI 1.87-9.58) and a
positive family history of CD (OR 3.45, 95% CI 1.49-8.0) were associated with
joint manifestations of CD.
DISCUSSION: We did not detect differences in disease behavior, severity, or
joint EIM by race. Although African American patients were more likely to have
ileocolonic or colonic disease, these factors did not affect disease behavior or
severity. The aim of the study was to examine the influence of age at diagnosis of Crohn's
disease on disease site and course in Tunisian patients.
METHODS: All hospital patients for Crohn's disease between 1993 and 2002 were
included. They were segregated by age at diagnosis as follows: younger than 20
years, 20-39 years, and 40 years or older. And all patients were classified at
the time of the latest visit into one of three subtypes of disease (non
complicating, stricturing, and fistulizing) according Vienna's classification.
Crohn's disease was devised also by site (ileum, ileocecal, colon and higher
site).
RESULTS: Sixty one patients (50.4%) were 20-39 years old and 43 patients (35.5%)
were 40 years and older. Colonic involvement was significantly more common
(46,5%) in the 40 years and older group compared with 20-39 years group (24.6%)
(p = 0.01). The subtype without complication was significantly more common
(58.1%) in the 40 years and older group compared with 20-39 years group (39.3%)
(p = 0.05). The frequency of the need for surgery for any indication for Crohn's
disease didn't differ significantly according to age.
CONCLUSION: In this study, Crohn's disease diagnosed in tunisian patients that
were 40 years and older had often a colonic site and a less severe phenotype
supporting the concept of genetic heterogeneity. BACKGROUND: For patients with Crohn's disease, age at onset is known to
influence the clinical course of the illness.
AIMS: The aim of this study is to seek an association between age at onset of
Crohn's disease and use of biologic agents for its treatment.
METHODS: We reviewed the medical records of 127 veteran patients with Crohn's
disease treated at our hospital, and compared differences in age at disease
onset between patients who had received biologics and those who had not.
RESULTS: The mean age of our patients was 54.9 ± 15.4 years, and 34% were
currently receiving or had previously received treatment with a biologic agent.
For those with biologic use, average age at time of diagnosis of Crohn's disease
was 32.3 ± 12.2 years, compared with 43.7 ± 16.3 years for those who had not
received biologics (P = 0.005). This relationship remained significant even
after controlling for disease severity. The frequency of use of biologic agents
varied inversely with age at diagnosis. For patients diagnosed before age 21
years, 55.5% had used biologics, whereas no patient >70 years of age at time of
diagnosis had used biologics. We found no significant correlation between
biologic use and duration of disease, smoking or ethnicity. Perianal disease and
concomitant arthritis were both significantly associated with use of biologics.
CONCLUSIONS: In our veteran patients with Crohn's disease, frequency of
treatment with a biologic agent varied inversely with age at disease onset. INTRODUCTION: It is unclear whether recent therapeutic advances have improved
the growth of children with Crohn's disease (CD).
AIM: To assess the frequency of short stature and poor growth and their
relationship to disease course and therapy in children with CD. WHAT IS ALREADY
KNOWN ON THIS TOPIC: Growth retardation may occur in children with Crohn's
disease (CD). Current therapy for CD in the UK is less likely than previously to
involve the use of long-term glucocorticoids.
WHAT THIS STUDY ADDS: Despite advances in therapy, short stature and slow growth
continue to be encountered in children with CD. There is a need for simple and
consistent definitions of growth that can identify poor growth in children with
chronic disease.
METHODS: The anthropometric and treatment details of 116 children (68 male) with
a mean (range) age at diagnosis of 10.8 years (4.9-15.5) and a mean age at
maximum follow-up (MF) of 15.4 years (9.4-19.3) were studied retrospectively at
diagnosis (T0), at 1 (T1), 2 (T2) and 3 years (T3) after diagnosis and at MF.
RESULTS: At T0, mean height SD score (HtSDS) was -0.5 (-3.3 to 2.6) compared to
a mid-parental HtSDS of 0.2 (-2.0 to 01.4) (p=0.002). At T1, T2, T3 and MF, mean
HtSDS was -0.6 (-4.8 to 7.8), -0.6 (-2.9 to 2.2), -0.7 (-3.6 to 2.5) and -0.5
(-3.5 to 2.9), respectively. Mean Ht velocity (HV) SDS at T1, T2, T3 and MF was
-1.4 (-7.4 to 7.4), -0.6 (-7.5 to 6.1), -0.1 (-6.6 to 7.6) and 0.6 (-4.8 to
7.8), respectively (p<0.05). In final models, HtSDS was associated negatively
with the use of prednisolone (p=0.0001), azathioprine (p=0.0001), methotrexate
(p=0.0001) and weight SDS (WtSDS) (p=0.0001). HVSDS was associated positively
with age (p=0.0001) and WtSDS (p=0.01). ΔHtSDS was associated negatively with
use of prednisolone (p<0.02).
CONCLUSION: Although current therapy for CD is associated with improved rate of
growth for the first few years, a substantial proportion of children remain
short. This study also highlights the need for consistency in describing growth
in children with chronic diseases. BACKGROUND: We analyzed the characteristics associated with increased risk of
osteoporosis in patients with Crohn's disease in Malta.
METHOD: Eighty-three patients with histologically and endoscopically confirmed
Crohn's disease underwent a DEXA bone density scan and their phenotypic
characteristics were analyzed.
RESULTS: There was a significant association between body mass index and bone
mineral density (P = 0.004) and a significant difference in the T scores of
patients according to age at diagnosis (Montreal Classification: P = 0.0006)
with patients diagnosed <17 years (n = 13) having lower T scores than those
diagnosed at older age groups (n = 70). There was a significant difference
between the T scores of patients on infliximab (n = 33) and those not on
biological therapy (n = 50, P = 0.0058). Patients with high cumulative
corticosteroid doses (>10 mg/d for >3 mo, n = 18) had lower bone mineral
densities than patients who received smaller corticosteroid doses (P = 0.013).
There was however no significant difference in the T scores of patients
according to disease location (P = 0.18), disease type (P = 0.64), gender (P =
0.30), and history of ileal resection (P = 0.68). There was also no significant
correlation between disease duration and T scores (hip) (P = 0.61).
CONCLUSIONS: Low body mass index, early disease onset, high corticosteroid doses
and, anti-tumor necrosis factor α therapy are associated with increased risk of
osteoporosis. Lower T scores in patients on infliximab occur as patients
receiving this therapy have more severe inflammation, which is associated with
elevated osteoclastogenic factors, rather than as a side-effect of the
anti-tumor necrosis factor-α therapy. BACKGROUND & AIMS: Crohn's disease (CD) diagnosed in pediatric patients has been
reported to have a more aggressive phenotype and course, with a greater
prevalence of upper gastrointestinal involvement, than in adults. However,
studies have not accounted for differences in diagnostic tests. We aimed to
discern whether, in fact, CD diagnosed in childhood has a different outcome than
CD diagnosed in adults.
METHODS: We performed comprehensive medical chart reviews of 571 patients with
CD (451 with complete data) who were followed in a single referral inflammatory
bowel disease clinic in Winnipeg, Canada, from 1993-2012. For specific time
intervals, we determined types and numbers of imaging studies performed and
parameters of disease phenotype, including age at diagnosis according to the
Montreal classification (A1 diagnosed <17 years of age, A2 diagnosed 17-40
years, and A3 diagnosed >40 years).
RESULTS: Within 1 year of diagnosis, a higher proportion of A1 patients had
upper gastrointestinal involvement and ileocolonic (L3) disease than A2 or A3
patients. These differences could be partly accounted for by the diagnostic
tests performed during this time period. Although A1 patients underwent more
extensive imaging studies, they had a lower prevalence of complicated disease,
particularly compared with A3 patients. After a median follow-up period of 11.1
years, complicated disease behavior (B2 [structuring] or B3 [penetrating]) was
similar among the 3 groups. Nonetheless, at the end of the study period, rates
of inflammatory bowel disease-related abdominal surgery were significantly lower
for A1 than A2 patients (odds ratio, 0.63; 95% confidence interval, 0.41-0.98)
but not for A3 patients (odds ratio, 0.71; 95% confidence interval, 0.40-1.27).
CONCLUSIONS: On the basis of a database analysis of different age groups of
patients with CD, studies of disease phenotypes among different cohorts should
account for different patterns of diagnostic imaging evaluation. Our data show
that although children are at increased risk of panenteric disease, they are not
more likely to have more complicated disease or undergo surgery than adults. PURPOSE: Fifteen percent of incident Crohn's disease (CD) cases are diagnosed at
older ages and demonstrate colonic location and inflammatory behavior. Serologic
responses to gut microbial antigens are associated with specific phenotypes, and
may differ by age at diagnosis. Our aim was to identify an association between
age at diagnosis of CD and responses to gut microbial antigens.
PATIENTS AND METHODS: Levels of anti-Saccharomyces cerevisiae antibodies (ASCA)
immunoglobulins A and G (IgA and IgG), antibodies to Escherichia coli outer
membrane porin-C (anti-Omp-C), antibodies to clostridial flagellin
(anti-CBir-1), and perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCA)
were compared in patients by age in three diagnosis groups: patients diagnosed
at ages of <40, ≥40-59, and ≥60 years. For each antigen, patients with antibody
levels in the first, second, third, and fourth quartile were assigned a score of
1, 2, 3, or 4, respectively. Individual scores were added to create a quartile
sum score representing cumulative quantitative immune response.
RESULTS: Eighteen, 17, and 12 patients were diagnosed at ages <40, 40-59, and
≥60 years, respectively. The majority (71%) had ileocolonic disease in the
youngest group, compared to 36% in the oldest group (P=0.001). Mean ASCA IgA and
IgG titers were increased in the youngest age group compared to the older groups
(P=0.19 and P=0.13, respectively). Mean quartile sum scores for antibody levels
were 7.2±2.8 in those patients diagnosed at ages <40 years, 4.9±2.9 in the
40-59-year-old age group, and 5.6±2.6 in the ≥60-year-old age group (P=0.06).
CONCLUSION: A trend toward decreased cumulative immune responses to
CD-associated gut antigens was observed in CD patients diagnosed at older ages
compared to younger patients. Host responses to microbial antigens may be less
important in older onset IBD and may contribute to the distinct phenotype in
this group. |
How does thyroid hormone affect insulin resistance in the heart? | T3 potentiates insulin signaling and improves insulin sensitivity. In addition, T3 lowers blood glucose in a model of type 2 diabetes. TRalpha P398H mutation is associated with insulin resistance. Circulating T(1)AM is produced from thyroid hormones and is found to be increased in diabetic patients. | Thyroid hormone has profound effects on metabolic homeostasis, regulating both
lipogenesis and lipolysis, primarily by modulating adrenergic activity. We
generated mice with a point mutation in the thyroid hormone receptor alpha
(TRalpha) gene producing a domit-negative TRalpha mutant receptor with a
proline to histidine substitution (P398H). The heterozygous P398H mutant mice
had a 3.4-fold (p < 0.02) increase in serum thyrotropin (TSH) levels. Serum
triiodothyronine (T3) and thyroxine (T4) concentrations were slightly elevated
compared with wild-type mice. The P398H mice had a 4.4-fold increase in body fat
(as a fraction of total body weight) (p < 0.001) and a 5-fold increase in serum
leptin levels (p < 0.005) compared with wild-type mice. A 3-fold increase in
serum fasting insulin levels (p < 0.002) and a 55% increase in fasting glucose
levels (p < 0.01) were observed in P398H compared with wild-type mice. There was
a marked reduction in norepinephrine-induced lipolysis, as reflected in reduced
glycerol release from white adipose tissue isolated from P398H mice. Heart rate
and cold-induced adaptive thermogenesis, mediated by thyroid
hormone-catecholamine interaction, were also reduced in P398H mice. In
conclusion, the TRalpha P398H mutation is associated with visceral adiposity and
insulin resistance primarily due to a marked reduction in
catecholamine-stimulated lipolysis. The observed phenotype in the TRalpha P398H
mouse is likely due to interference with TRalpha action as well as influence on
other metabolic signaling pathways. The physiologic significance of these
findings will ultimately depend on understanding the full range of actions of
this mutation. BACKGROUND AND PURPOSE: The thyroid hormone, triiodothyronine (T3) has many
metabolic functions. Unexpectedly, exogenous T3 lowered blood glucose in db/db
mice, a model of type 2 diabetes. Here, we have explored this finding and its
possible mechanisms further.
EXPERIMENTAL APPROACH: db/db and lean mice were treated with T3, the
phosphoinositide 3- kinase (PI3-kinase) inhibitor, LY294002, plus T3, or
vehicles. Blood glucose, insulin sensitivity, levels and synthesis were
measured. Effects of T3 on intracellular insulin signaling were analyzed in
3T3-L1 pre-adipocytes with Western blotting. Knock-down of the thyroid hormone
receptor α1 (TRα1) in 3T3-L1 cells was achieved with an appropriate silencing
RNA (siRNA).
KEY RESULTS: Single injections of T3 (7 ng·g⁻¹ i.p.) rapidly and markedly
attenuated hyperglycemia. Treatment with T3 (14 ng·g⁻¹·day⁻¹, 18 days)
dose-dependently attenuated blood glucose and increased insulin sensitivity in
db/db mice. Higher doses of T3 (28 ng·g⁻¹·day⁻¹) reversed insulin resistance in
db/db mice. T3 also increased insulin levels in plasma and the neurogenic
differentiation factor (an insulin synthesis transcription factor) and insulin
storage in pancreatic islets in db/db mice. These anti-diabetic effects of T3
were abolished by the PI3-kinase inhibitor (LY294002). In 3T3-L1 preadipocytes,
T3 enhanced insulin-induced tyrosine phosphorylation of insulin receptor
substrate (IRS)-1 and activation of PI3-kinase, effects blocked by siRNA for
TRα1.
CONCLUSIONS AND IMPLICATIONS: T3 potentiated insulin signaling, improved insulin
sensitivity, and increased insulin synthesis, which may contribute to its
anti-diabetic effects. These findings may provide new approaches to the
treatment of type 2 diabetes. CONTEXT AND OBJECTIVE: The primary purpose of this study was to detect and
quantify 3-iodothyronamine (T(1)AM), an endogenous biogenic amine related to
thyroid hormone, in human blood.
DESIGN: T(1)AM, total T(3), and total T(4) were assayed in serum by a novel HPLC
tandem mass spectrometry assay, which has already been validated in animal
investigations, and the results were related to standard clinical and laboratory
variables.
SETTING AND PATIENTS: The series included one healthy volunteer, 24 patients
admitted to a cardiological ward, and 17 ambulatory patients suspected of
thyroid disease, who underwent blood sampling at admission for routine
diagnostic purposes. Seven patients were affected by type 2 diabetes, and six
patients showed echocardiographic evidence of impaired left ventricular
function.
INTERVENTIONS: No intervention or any patient selection was performed.
MAIN OUTCOME MEASURES: serum T(1)AM, total and free T(3) and T(4), routine
chemistry, routine hematology, and echocardiographic parameters were measured.
RESULTS: T(1)AM was detected in all samples, and its concentration averaged
0.219 ± 0.012 pmol/ml. The T(1)AM concentration was significantly correlated to
total T(4) (r = 0.654, P < 0.001), total T(3) (r = 0.705, P < 0.001), glycated
hemoglobin (r = 0.508, P = 0.013), brain natriuretic peptide (r = 0.543, P =
0.016), and γ-glutamyl transpeptidase (r = 0.675, P < 0.001). In diabetic vs.
nondiabetic patients T(1)AM concentration was significantly increased (0.232 ±
0.014 vs. 0.203 ± 0.006 pmol/ml, P = 0.044), whereas no significant difference
was observed in patients with cardiac dysfunction.
CONCLUSIONS: T(1)AM is an endogenous messenger that can be assayed in human
blood. Our results are consistent with the hypothesis that circulating T(1)AM is
produced from thyroid hormones and encourage further investigations on the
potential role of T(1)AM in insulin resistance and heart failure. |
Which are the state-of-the-art computational tools for the prediction of gene fusion events? | Gene fusion detection - also known as the 'Rosetta Stone' method - involves the identification of fused composite genes in a set of reference genomes, which indicates potential interactions between its un-fused counterpart genes in query genomes. A few methods/tools and computational pipelines for the detection of gene fusion events have been introduced. The basic steps followed in these approaches consist of (i) all-against-all sequence comparison, (ii) detection of non-overlapping similarities of two genes/proteins (components) to a single gene/protein (composite), and optionally (iii) elimination of putative spurious hits (e.g. due to promiscuous domains) achieves via clustering based on sequence similarity and examining dense regions of the resulting graph or by querying the PFAM database. An advantage of gene fusion analysis is that functional associations can be predicted even in cases of genes of unknown function. Due to the computationally intense nature of these approaches, precompiled data of this type are often organized in specialized databases. Tools and databases developed for this purpose include (in alphabetical order): fdfBLAST, FusionDB, InPrePPI, (Integrated method for Prediction of Protein-Protein Interactions), MosaicFinder, Phydbac2, PLEX, Predictome, Rosetta Stone method, STRING. | A large-scale effort to measure, detect and analyse protein-protein interactions
using experimental methods is under way. These include biochemistry such as
co-immunoprecipitation or crosslinking, molecular biology such as the two-hybrid
system or phage display, and genetics such as unlinked noncomplementing mutant
detection. Using the two-hybrid system, an international effort to analyse the
complete yeast genome is in progress. Evidently, all these approaches are
tedious, labour intensive and inaccurate. From a computational perspective, the
question is how can we predict that two proteins interact from structure or
sequence alone. Here we present a method that identifies gene-fusion events in
complete genomes, solely based on sequence comparison. Because there must be
selective pressure for certain genes to be fused over the course of evolution,
we are able to predict functional associations of proteins. We show that 215
genes or proteins in the complete genomes of Escherichia coli, Haemophilus
influenzae and Methanococcus jannaschii are involved in 64 unique fusion events.
The approach is general, and can be applied even to genes of unknown function. The current deluge of genomic sequences has spawned the creation of tools
capable of making sense of the data. Computational and high-throughput
experimental methods for generating links between proteins have recently been
emerging. These methods effectively act as hypothesis machines, allowing
researchers to screen large sets of data to detect interesting patterns that can
then be studied in greater detail. Although the potential use of these putative
links in predicting gene function has been demonstrated, a central repository
for all such links for many genomes would maximize their usefulness. Here we
present Predictome, a database of predicted links between the proteins of 44
genomes based on the implementation of three computational methods--chromosomal
proximity, phylogenetic profiling and domain fusion--and large-scale
experimental screenings of protein-protein interaction data. The combination of
data from various predictive methods in one database allows for their comparison
with each other, as well as visualization of their correlation with known
pathway information. As a repository for such data, Predictome is an ongoing
resource for the community, providing functional relationships among proteins as
new genomic data emerges. Predictome is available at http://predictome.bu.edu. BACKGROUND: It has recently been shown that the detection of gene fusion events
across genomes can be used for predicting functional associations of proteins,
including physical interaction or complex formation. To obtain such predictions
we have made an exhaustive search for gene fusion events within 24 available
completely sequenced genomes.
RESULTS: Each genome was used as a query against the remaining 23 complete
genomes to detect gene fusion events. Using an improved, fully automatic
protocol, a total of 7,224 single-domain proteins that are components of gene
fusions in other genomes were detected, many of which were identified for the
first time. The total number of predicted pairwise functional associations is
39,730 for all genomes. Component pairs were identified by virtue of their
similarity to 2,365 multidomain composite proteins. We also show for the first
time that gene fusion is a complex evolutionary process with a number of
contributory factors, including paralogy, genome size and phylogenetic distance.
On average, 9% of genes in a given genome appear to code for single-domain,
component proteins predicted to be functionally associated. These proteins are
detected by an additional 4% of genes that code for fused, composite proteins.
CONCLUSIONS: These results provide an exhaustive set of functionally associated
genes and also delineate the power of fusion analysis for the prediction of
protein interactions. Functional links between proteins can often be inferred from genomic
associations between the genes that encode them: groups of genes that are
required for the same function tend to show similar species coverage, are often
located in close proximity on the genome (in prokaryotes), and tend to be
involved in gene-fusion events. The database STRING is a precomputed global
resource for the exploration and analysis of these associations. Since the three
types of evidence differ conceptually, and the number of predicted interactions
is very large, it is essential to be able to assess and compare the significance
of individual predictions. Thus, STRING contains a unique scoring-framework
based on benchmarks of the different types of associations against a common
reference set, integrated in a single confidence score per prediction. The
graphical representation of the network of inferred, weighted protein
interactions provides a high-level view of functional linkage, facilitating the
analysis of modularity in biological processes. STRING is updated continuously,
and currently contains 261 033 orthologs in 89 fully sequenced genomes. The
database predicts functional interactions at an expected level of accuracy of at
least 80% for more than half of the genes; it is online at
http://www.bork.embl-heidelberg.de/STRING/. Pairs of genes that function together in a pathway or cellular system can
sometimes be found fused together in another organism as a Rosetta Stone
protein--a fusion protein whose separate domains are homologous to the two
functionally-related proteins. The finding of such a Rosetta Stone protein
allows the prediction of a functional linkage between the component proteins.
The significance of these deduced functional linkages, however, varies depending
on the prevalence of each of the two domains. Here, we develop a statistical
measure for the significance of predicted functional linkages, and test this
measure for proteins of E. coli on a functional benchmark based on the KEGG
database. By applying this statistical measure, proteins can be linked with over
70% accuracy. Using the Rosetta Stone method and this scoring scheme, we find
all significant functional linkages for proteins of E. coli, P. horikshii and S.
cerevisiae, and measure the extent of the resulting protein networks. Phydbac (phylogenomic display of bacterial genes) implemented a method of
phylogenomic profiling using a distance measure based on normalized BLAST
scores. This method was able to increase the predictive power of phylogenomic
profiling by about 25% when compared to the classical approach based on Hamming
distances. Here we present a major extension of Phydbac (named here Phydbac2),
that extends both the concept and the functionality of the original web-service.
While phylogenomic profiles remain the central focus of Phydbac2, it now
integrates chromosomal proximity and gene fusion analyses as two additional
non-similarity-based indicators for inferring pairwise gene functional
relationships. Moreover, all presently available (January 2004) fully sequenced
bacterial genomes and those of three lower eukaryotes are now included in the
profiling process, thus increasing the initial number of reference genomes (71
in Phydbac) to 150 in Phydbac2. Using the KEGG metabolic pathway database as a
benchmark, we show that the predictive power of Phydbac2 is improved by 27% over
the previous version. This gain is accounted for on one hand, by the increased
number of reference genomes (11%) and on the other hand, as a result of
including chromosomal proximity into the distance measure (16%). The expanded
functionality of Phydbac2 now allows the user to query more than 50 different
genomes, including at least one member of each major bacterial group, most major
pathogens and potential bio-terrorism agents. The search for co-evolving genes
based on consensus profiles from multiple organisms, the display of Phydbac2
profiles side by side with COG information, the inclusion of KEGG metabolic
pathway maps the production of chromosomal proximity maps, and the possibility
of collecting and processing results from different Phydbac queries in a common
shopping cart are the main new features of Phydbac2. The Phydbac2 web server is
available at http://igs-server.cnrs-mrs.fr/phydbac/. BACKGROUND: Although many genomic features have been used in the prediction of
protein-protein interactions (PPIs), frequently only one is used in a
computational method. After realizing the limited power in the prediction using
only one genomic feature, investigators are now moving toward integration. So
far, there have been few integration studies for PPI prediction; one failed to
yield appreciable improvement of prediction and the others did not conduct
performance comparison. It remains unclear whether an integration of multiple
genomic features can improve the PPI prediction and, if it can, how to integrate
these features.
RESULTS: In this study, we first performed a systematic evaluation on the PPI
prediction in Escherichia coli (E. coli) by four genomic context based methods:
the phylogenetic profile method, the gene cluster method, the gene fusion
method, and the gene neighbor method. The number of predicted PPIs and the
average degree in the predicted PPI networks varied greatly among the four
methods. Further, no method outperformed the others when we tested using three
well-defined positive datasets from the KEGG, EcoCyc, and DIP databases. Based
on these comparisons, we developed a novel integrated method, named InPrePPI.
InPrePPI first normalizes the AC value (an integrated value of the accuracy and
coverage) of each method using three positive datasets, then calculates a weight
for each method, and finally uses the weight to calculate an integrated score
for each protein pair predicted by the four genomic context based methods. We
demonstrate that InPrePPI outperforms each of the four individual methods and,
in general, the other two existing integrated methods: the joint observation
method and the integrated prediction method in STRING. These four methods and
InPrePPI are implemented in a user-friendly web interface.
CONCLUSION: This study evaluated the PPI prediction by four genomic context
based methods, and presents an integrated evaluation method that shows better
performance in E. coli. The method described in this chapter can be used to infer putative functional
links between two proteins. The basic idea is based on the principle of "guilt
by association." It is assumed that two proteins, which are found to be
transcribed by a single transcript in one (or several) genomes are likely to be
functionally linked, for example by acting in a same metabolic pathway or by
forming a multiprotein complex. This method is of particular interest for
studying genes that exhibit no, or only remote, homologies with already
well-characterized proteins. Combined with other non-homology based methods,
gene fusion events may yield valuable information for hypothesis building on
protein function, and may guide experimental characterization of the target
protein, for example by suggesting potential ligands or binding partners. This
chapter uses the FusionDB database (http://www.igs.cnrs-mrs.fr/FusionDB/) as
source of information. FusionDB provides a characterization of a large number of
gene fusion events at hand of multiple sequence alignments. Orthologous genes
are included to yield a comprehensive view of the structure of a gene fusion
event. Phylogenetic tree reconstruction is provided to evaluate the history of a
gene fusion event, and three-dimensional protein structure information is used,
where available, to further characterize the nature of the gene fusion. For
genes that are not comprised in FusionDB, some instructions are given as how to
generate a similar type of information, based solely on publicly available web
tools that are listed here. Gene fusion and fission events are key mechanisms in the evolution of gene
architecture, whose effects are visible in protein architecture when they occur
in coding sequences. Until now, the detection of fusion and fission events has
been performed at the level of protein sequences with a post facto removal of
supernumerary links due to paralogy, and often did not include looking for
events defined only in single genomes. We propose a method for the detection of
these events, defined on groups of paralogs to compensate for the gene
redundancy of eukaryotic genomes, and apply it to the proteomes of 12 fungal
species. We collected an inventory of 1,680 elementary fusion and fission
events. In half the cases, both composite and element genes are found in the
same species. Per-species counts of events correlate with the species genome
size, suggesting a random mechanism of occurrence. Some biological functions of
the genes involved in fusion and fission events are slightly over- or
under-represented. As already noted in previous studies, the genes involved in
an event tend to belong to the same functional category. We inferred the
position of each event in the evolution tree of the 12 fungal species. The event
localization counts for all the segments of the tree provide a metric that
depicts the "recombinational" phylogeny among fungi. A possible interpretation
of this metric as distance in adaptation space is proposed. During the course of evolution genes undergo both fusion and fission by which
ORFs are joined or separated. These processes can amend gene function and
represent an important factor in the evolution of protein interaction networks.
Gene fusions have been suggested to be useful characters for identifying
evolutionary relationships because they constitute synapomorphies or cladistic
characters. To investigate the fidelity of gene-fusion characters, we developed
an approach for identifying differentially distributed gene fusions among
whole-genome datasets: fdfBLAST. Applying this tool to the Fungi, we identified
63 gene fusions present in two or more genomes. Using a combination of
phylogenetic and comparative genomic analyses, we then investigated the
evolution of these genes across 115 fungal genomes, testing each gene fusion for
evidence of homoplasy, including gene fission, convergence, and horizontal gene
transfer. These analyses demonstrated 110 gene-fission events. We then
identified a minimum of three mechanisms that drive gene fission: separation,
degeneration, and duplication. These data suggest that gene fission plays an
important and hitherto underestimated role in gene evolution. Gene fusions
therefore are highly labile characters, and their use for polarizing
evolutionary relationships, without reference to gene and species phylogenies,
is limited. Accounting for these considerable sources of homoplasy, we
identified fusion characters that provide support for multiple nodes in the
phylogeny of the Fungi, including relationships within the deeply derived
flagellum-forming fungi (i.e., the chytrids). MOTIVATION: Gene fusion is an important evolutionary process. It can yield
valuable information to infer the interactions and functions of proteins. Fused
genes have been identified as non-transitive patterns of similarity in triplets
of genes. To be computationally tractable, this approach usually imposes an a
priori distinction between a dataset in which fused genes are searched for, and
a dataset that may have provided genetic material for fusion. This reduces the
'genetic space' in which fusion can be discovered, as only a subset of triplets
of genes is investigated. Moreover, this approach may have a high-false-positive
rate, and it does not identify gene families descending from a common fusion
event.
RESULTS: We represent similarities between sequences as a network. This leads to
an efficient formulation of previous methods of fused gene identification, which
we implemented in the Python program FusedTriplets. Furthermore, we propose a
new characterization of families of fused genes, as clique minimal separators of
the sequence similarity network. This well-studied graph topology provides a
robust and fast method of detection, well suited for automatic analyses of big
datasets. We implemented this method in the C++ program MosaicFinder, which
additionally uses local alignments to discard false-positive candidates and
indicates potential fusion points. The grouping into families will help
distinguish sequencing or prediction errors from real biological fusions, and it
will yield additional insight into the function and history of fused genes.
AVAILABILITY: FusedTriplets and MosaicFinder are published under the GPL license
and are freely available with their source code at this address:
http://sourceforge.net/projects/mosaicfinder.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. |
Is CHEK2 involved in cell cycle control? | CHEK2 is a key cell cycle control gene encoding a pluripotent kinase that can cause arrest or apoptosis in response to unrepaired DNA damage. | Checkpoint kinase 2 (hCHK2/hCds1) is a tumor suppressor gene involved in
cell-cycle control. A hCHK2/hCds1 polymorphism in codon 84 (A-->G at nucleotide
252) was recently identified in Li-Fraumeni syndrome patients. Because cell
cycle regulates DNA repair that is associated with cancer risk, we hypothesized
that this new polymorphism exists in the general population and is associated
with cancer risk. To test this hypothesis, we evaluated the role of this
polymorphism in a case-control study of 215 non-Hispanic white patients with
newly diagnosed squamous cell carcinoma of the head and neck (SCCHN) and 229
frequency-matched cancer-free controls. We found that the hCHK2/hCds1 codon 84
variant was rare and less frequent in non-Hispanic white cases (0.0186) than in
controls (0.0437; P = 0.033). Although no variant homozygotes were detected in
these cases and controls, heterozygosity protected against SCCHN, representing a
60% reduction of risk (adjusted odds ratio = 0.40; 95% confidence intervals,
0.17-0.93) compared with wild-type homozygotes. The variant allele was also rare
in other ethnic groups (0.0487, 0.0095 and 0.0541 in 115 African Americans, 105
Hispanic Americans and 111 native Chinese, respectively), and only one variant
homozygous individual (a Chinese subject) was identified. These results suggest
that this hCHK2/hCds1 codon 84 polymorphism is rare and may have a protective
role in the aetiology of SCCHN in non-Hispanic whites. Larger studies are
warranted to confirm this finding and further mechanistic studies are needed to
understand biological relevance of this polymorphism. High-fidelity maintece of genomic integrity in eukaryotes is ensured by cell
cycle checkpoints and DNA repair. The checkpoint kinase, Chk2, has been
implicated in both of these responses. In response to DNA damage, Chk2 is
initially phosphorylated at Thr-68, which leads to its full activation. The
fully activated Chk2 then phosphorylates downstream substrates of cell cycle
control. However, the mechanism of inactivation of Chk2 is still unknown.
Protein phosphatase type 2A (PP2A) plays an essential role in cell cycle
regulation and induction of G2 arrest by a mechanism of
phosphorylation/dephosphorylation with a variety of protein kinases. Data from
our investigation provide evidence that, in response to cisplatin exposure, PP2A
associates with Chk2 as a complex in cells and functions as a negative regulator
of Chk2 activation by dephosphorylating p-Chk2. Results from immunostaining and
coimmunoprecipitation demonstrate that Chk2 and PP2A can colocalize in cells,
and the holoenzyme of PP2A (subunits A, B and C) coimmunoprecipitates with
p-Chk2. Further, inhibition of PP2A by okadaic acid, an inhibitor of PP2A, and
by small interfering RNA (siRNA) to PP2A results in enhanced Chk2
phosphorylation, implicating a direct enzyme-substrate relationship. An in vitro
PP2A dephosphorylation assay shows that PP2A dephosphorylates p-Chk2 in a
cell-free system. These findings suggest that the protein serine/threonine
kinase, Chk2, is activated after cisplatin exposure and negatively regulated by
a tightly associated protein serine/threonine phosphatase, PP2A. CHEK2 is a key cell cycle control gene encoding a pluripotent kinase that can
cause arrest or apoptosis in response to unrepaired DNA damage. We report a
large case-control study of a non-functional variant that had previously been
expected to increase cancer rates. Four thousand and fifteen cancer patients
(2250 lung, 811 squamous upper aero-digestive and 954 kidney) and 3052 controls
in central Europe were genotyped for the mis-sense variant rs17879961
(replacement of T by C), which changes an amino acid (I157T) in an active site
of the gene product. The heterozygous (T/C) genotype was associated with a
highly significantly lower incidence of lung cancer than the common T/T genotype
[relative risk (RR), T/C versus T/T, 0.44, with 95% confidence interval (CI)
0.31-0.63, P < 0.00001] and with a significantly lower incidence of upper
aero-digestive cancer (RR 0.44, CI 0.26-0.73, P = 0.001; P = 0.000001 for lung
or upper aero-digestive cancer). Protection was significantly greater for
squamous than adenomatous lung cancer (P = 0.001). There was an increase of
borderline significance in kidney cancer (RR 1.44, CI 0.99-2.00, P = 0.06). This
unexpected halving of tobacco-related cancer (since replicated independently)
implies much greater absolute risk reduction in smokers than in non-smokers. The
mechanism is unknown: perhaps squamous stem cell apoptosis following smoke
exposure causes net harm (e.g. by forcing nearby stem cells to divide before
they have repaired their own DNA damage from tobacco smoke). If so, reducing the
rate of apoptosis by reducing CHEK2 activity could be protective-although not
smoking would be far more so. Deregulated epigenetic mechanisms are likely involved in the pathogenesis of
myelodysplastic syndromes (MDSs). Which genes are silenced by aberrant promotor
methylation during MDS hematopoiesis has not been equivalently investigated.
Using an in vitro differentiation model of human hematopoiesis, we generated
defined differentiation stages (day 0, day 4, day 7, day 11) of erythro-,
thrombo- and granulopoiesis from 13 MDS patients and seven healthy donors.
Promotor methylation analysis of key regulatory genes involved in cell cycle
control (p14, p15, p16, CHK2), DNA repair (hMLH1), apoptosis (p73, survivin,
DAPK), and differentiation (RARb, WT1) was performed by methylation-specific
polymerase chain reaction. Corresponding gene expression was analyzed by
microarray (Affymetrix, HG-U133A). We provide evidence that p16, survivin, CHK2,
and WT1 are affected by promotor hypermethylation in MDSs displaying a selective
International Prognostic Scoring System risk association. A
methylation-associated mRNA downregulation for specific hematopoietic lineages
and differentiation stages is demonstrated for survivin, CHK2, and WT1. We
identified a suppressed survivin mRNA expression in methylated samples during
erythropoiesis, whereas WT1 and CHK2 methylation-related reduction of mRNA
expression was found during granulopoiesis in all MDS risk types. Our data
suggest that lineage-specific methylation-associated gene silencing of survivin,
CHK2, and WT1 in MDS hematopoietic precursor cells may contribute to the
MDS-specific phenotype In the current study, we evaluated the possible associations of seven common
variants of the DNA repair and cell cycle control genes BRCA2 and CHEK2 with
maligt melanoma (MM). We genotyped 630 unselected MM patients and over 3700
controls (newborns, age- and sex-matched healthy adults with negative cancer
family histories, and the adults selected at random by family doctors) for the
prevalence of three common variants of the BRCA2 (T1915M, N991D and N372H) and
four common variants of the CHEK2 (1100delC, VS2+1G --> A, I157T and del5395).
Our study strongly suggests that the common variant of the BRCA2 gene -- the
N991D variant is associated with maligt melanoma risk (OR=1.8, p=0.002 after
Bonferroni correction). Patients homozygote for the N991D variant were present
in 0.32% of cases and only 0.13% of controls. The other variants studied were
not over-represented among MM patients when compared to the general population.
In conclusion, we report an increased melanoma risk among carriers of the N991D
change of the BRCA2 and no association of the CHEK2 changes with maligt
melanoma. It is possible that reduced function of DNA repair and cell-cycle control genes
increases the individual susceptibility to maligt melanoma. As CHEK2 is a
cell-cycle master controller, we tested the hypothesis that heterozygosity for
the frameshift alteration CHEK2*1100delC is associated with increased risk of
maligt melanoma. First, we performed case-control studies of 1,152 Danish and
752 German individuals with maligt melanoma compared with 9,142 Danish and
3,718 German controls. Second, we performed a meta-analysis of CHEK2*1100delC
and maligt melanoma, involving 2,619 cases and 17,481 controls. Third, we
examined the risk of maligt melanoma associated with CHEK2*1100delC
heterozygosity in an analysis stratified for sun exposure, as well as for
subtype and location on the body. The odds ratios for maligt melanoma for
CHEK2(*)1100del heterozygotes compared with those for noncarriers were 2.01 (95%
confidence interval (CI), 1.03-3.91) in Danes, 1.42 (95% CI, 0.46-4.31) in
Germans, and 1.79 (95% CI, 1.02-3.17) in Danes and Germans combined. In a
meta-analysis, the odds ratio of maligt melanoma for CHEK2*1100delC
heterozygotes compared with that for noncarriers was 1.81 (95% CI, 1.07-3.05).
Stratifications did not alter these results. CHEK2*1100delC heterozygotes have a
twofold risk of maligt melanoma compared with noncarriers. |
Describe mechanism of action of PLX3397 drug. | PLX3397 works by inhibiting colony-stimulating-factor-1 receptor (CSF1R). | PURPOSE: Gastrointestinal stromal tumor (GIST) is the most common human sarcoma
and a model of targeted molecular therapy. GIST depends on oncogenic KIT
signaling and responds to the tyrosine kinase inhibitor imatinib. However,
imatinib is rarely curative. We hypothesized that PLX3397, which inhibits KIT
and colony-stimulating-factor-1 receptor (CSF1R), would be more efficacious than
imatinib in GIST by also depleting tumor-associated macrophages, which are
generally thought to support tumor growth.
EXPERIMENTAL DESIGN: We treated Kit(V558del/+) mice that develop GIST or mice
with subcutaneous human GIST xenografts with imatinib or PLX3397 and analyzed
tumor weight, cellular composition, histology, molecular signaling, and
fibrosis. In vitro assays on human GIST cell lines were also performed.
RESULTS: PLX3397 was more effective than imatinib in reducing tumor weight and
cellularity in both Kit(V558del)(/+) murine GIST and human GIST xenografts. The
superiority of PLX3397 did not depend on depletion of tumor-associated
macrophages, because adding CSF1R inhibition did not improve the effects of
imatinib. Instead, PLX3397 was a more potent KIT inhibitor than imatinib in
vitro. PLX3397 therapy also induced substantial intratumoral fibrosis, which
impaired the subsequent delivery of small molecules.
CONCLUSIONS: PLX3397 therapy has greater efficacy than imatinib in preclinical
GIST models and warrants study in patients with GIST. The resultant intratumoral
fibrosis may represent one of the barriers to achieving complete tumor
eradication. PURPOSE: Maligt peripheral nerve sheath tumor (MPNST) is a highly aggressive
tumor type that is resistant to chemotherapy and there are no effective
therapies. MPNSTs have been shown to have gene amplification for receptor
tyrosine kinases (RTK), PDGFR and c-Kit. We tested the c-Kit inhibitor,
imatinib, and PLX3397, a selective c-Fms and c-Kit inhibitor, to evaluate their
efficacy against MPNST cells in vitro and in vivo.
EXPERIMENTAL DESIGN: We tested the efficacy of imatinib or PLX3397 either alone
or in combination with TORC1 inhibitor rapamycin in a cell proliferation assay
in vitro and by immunoblotting to determine target inhibition. Immunoblotting
and immunohistochemical analysis was further carried out using xenograft samples
in vivo.
RESULTS: Our in vitro studies show that imatinib and PLX3397 similarly inhibit
cell growth and this can be enhanced with rapamycin with comparable target
specificity. However, in vivo studies clearly demonstrate that compared with
imatinib, PLX3397 results in sustained blockade of c-Kit, c-Fms, and PDGFRβ,
resulting in significant suppression of tumor growth. Moreover, staining for
Iba-1, a marker for macrophages, indicates that PLX3397 results in significant
depletion of macrophages in the growing tumors. The combination of PLX3397 and
rapamycin results in even greater macrophage depletion with continued growth
suppression, even when the drug treatment is discontinued.
CONCLUSIONS: Taken together, our data strongly suggest that PLX3397 is superior
to imatinib in the treatment of MPNSTs, and the combination of PLX3397 with a
TORC1 inhibitor could provide a new therapeutic approach for the treatment of
this disease. Macrophage colony stimulating factor (CSF1) is a cytokine that is upregulated in
several diseases of the central nervous system (CNS). To examine the effects of
CSF1 overexpression on microglia, transgenic mice that overexpress CSF1 in the
glial fibrillary acidic protein (GFAP) compartment were generated. CSF1
overexpressing mice have increased microglial proliferation and increased
microglial numbers compared with controls. Treatment with PLX3397, a small
molecule inhibitor of the CSF1 receptor CSF1R and related kinases, decreases
microglial numbers by promoting microglial apoptosis in both CSF1 overexpressing
and control mice. Microglia in CSF1 overexpressing mice exhibit gene expression
profiles indicating that they are not basally M1 or M2 polarized, but they do
have defects in inducing expression of certain genes in response to the
inflammatory stimulus lipopolysaccharide. These results indicate that the CSF1
overexpression observed in CNS pathologies likely has pleiotropic influences on
microglia. Furthermore, small molecule inhibition of CSF1R has the potential to
reverse CSF1-driven microglial accumulation that is frequently observed in CNS
pathologies, but can also promote apoptosis of normal microglia. Tumor associated macrophages (TAM) can promote angiogenesis, invasiveness and
immunosuppression. The cytokine CSF-1 (or M-CSF) is an important factor of TAM
recruitment and differentiation and several pharmacological agents targeting the
CSF-1 receptor (CSF-1R) have been developed to regulate TAM in solid cancers. We
show that the kinase inhibitor PLX3397 strongly dampened the systemic and local
accumulation of macrophages driven by B16F10 melanomas, without affecting
Gr-1(+) myeloid derived suppressor cells. Removal of intratumoral macrophages
was remarkably efficient and a modest, but statistically significant, delay in
melanoma outgrowth was observed. Importantly, CSF-1R inhibition strongly
enhanced tumor control by immunotherapy using tumor-specific CD8 T cells.
Elevated IFNγ production by T cells was observed in mice treated with the
combination of PLX3397 and immunotherapy. These results support the combined use
of CSF-1R inhibition with CD8 T cell immunotherapy, especially for
macrophage-stimulating tumors. With severe injury or disease, microglia become chronically activated and damage
the local brain environment, likely contributing to cognitive decline. We
previously discovered that microglia are dependent on colony-stimulating factor
1 receptor (CSF1R) signaling for survival in the healthy adult brain, and we
have exploited this dependence to determine whether such activated microglia
contribute deleteriously to functional recovery following a neuronal lesion.
Here, we induced a hippocampal lesion in mice for 25 d via neuronal expression
of diphtheria toxin A-chain, producing both a neuroinflammatory reaction and
behavioral alterations. Following the 25 d lesion, we administered PLX3397, a
CSF1R inhibitor, for 30 d to eliminate microglia. This post-lesion treatment
paradigm improved functional recovery on elevated plus maze and Morris water
maze, concomitant with reductions in elevated proinflammatory molecules, as well
as normalization of lesion-induced alterations in synaptophysin and PSD-95.
Further exploration of the effects of microglia on synapses in a second cohort
of mice revealed that dendritic spine densities are increased with long-term
microglial elimination, providing evidence that microglia shape the synaptic
landscape in the adult mouse brain. Furthermore, in these same animals, we
determined that microglia play a protective role during lesioning, whereby
neuronal loss was potentiated in the absence of these cells. Collectively, we
demonstrate that microglia exert beneficial effects during a diphtheria
toxin-induced neuronal lesion, but impede recovery following insult.
SIGNIFICANCE STATEMENT: It remains unknown to what degree, and by what
mechanisms, chronically activated microglia contribute to cognitive deficits
associated with brain insults. We induced a genetic neuronal lesion in mice for
25 d and found activated microglia to increase inflammation, alter synaptic
surrogates, and impede behavioral recovery. These lesion-associated deficits
were ameliorated with subsequent microglial elimination, underscoring the
importance of developing therapeutics aimed at eliminating/modulating chronic
microglial activation. Additionally, we found long-term microglial depletion
globally increases dendritic spines by ∼35% in the adult brain, indicating that
microglia continue to sculpt the synaptic landscape in the postdevelopmental
brain under homeostatic conditions. Microglial manipulation can therefore be
used to investigate the utility of increasing dendritic spine numbers in
postnatal conditions displaying synaptic aberrations. BACKGROUND: Expression of the colony-stimulating factor 1 (CSF1) gene is
elevated in most tenosynovial giant-cell tumors. This observation has led to the
discovery and clinical development of therapy targeting the CSF1 receptor
(CSF1R).
METHODS: Using x-ray co-crystallography to guide our drug-discovery research, we
generated a potent, selective CSF1R inhibitor, PLX3397, that traps the kinase in
the autoinhibited conformation. We then conducted a multicenter, phase 1 trial
in two parts to analyze this compound. In the first part, we evaluated
escalations in the dose of PLX3397 that was administered orally in patients with
solid tumors (dose-escalation study). In the second part, we evaluated PLX3397
at the chosen phase 2 dose in an extension cohort of patients with tenosynovial
giant-cell tumors (extension study). Pharmacokinetic and tumor responses in the
enrolled patients were assessed, and CSF1 in situ hybridization was performed to
confirm the mechanism of action of PLX3397 and that the pattern of CSF1
expression was consistent with the pathological features of tenosynovial
giant-cell tumor.
RESULTS: A total of 41 patients were enrolled in the dose-escalation study, and
an additional 23 patients were enrolled in the extension study. The chosen phase
2 dose of PLX3397 was 1000 mg per day. In the extension study, 12 patients with
tenosynovial giant-cell tumors had a partial response and 7 patients had stable
disease. Responses usually occurred within the first 4 months of treatment, and
the median duration of response exceeded 8 months. The most common adverse
events included fatigue, change in hair color, nausea, dysgeusia, and
periorbital edema; adverse events rarely led to discontinuation of treatment.
CONCLUSIONS: Treatment of tenosynovial giant-cell tumors with PLX3397 resulted
in a prolonged regression in tumor volume in most patients. (Funded by
Plexxikon; ClinicalTrials.gov number, NCT01004861.). |
What disease is small bowel lymphoma commonly associated with | Small bowel lymphoma is commonly associated with celiac disease. | An increased incidence of small bowel lymphoma in patients with long-standing
celiac sprue is well documented in the literature. Less common is the
association of adenocarcinoma of the small intestine. We report a patient with
celiac sprue who initially responded to a gluten-free diet. Eighteen months
later, diarrhea, abdominal cramps, and bloating was found to have its origin in
partial small bowel obstruction. At laparotomy, two distinct adenocarcinomas of
the jejunum were resected. Celiac patients who initially respond to gluten
withdrawal and subsequently suffer exacerbation while adhering to strict dietary
therapy should be carefully evaluated for evidence of a small bowel maligcy. 'Flat and flexible truths are beat out by every hammer' (Sir Thomas Browne,
writer and physician) The prevalence of gluten-sensitive enteropathy (GSE) or
coeliac disease is likely to be as high as 1:200 to 1:400 in the developed
world. Current medical practice leaves a significant proportion of these cases
undiagnosed. An association between untreated coeliac disease and intestinal
maligcy is well described so it is possible that patients with undiagnosed
coeliac disease constitute a significant reservoir of preventable
gastrointestinal maligcy. However, it is not clear whether all patients with
coeliac disease are equally at risk of maligcy nor are all cases of
intestinal maligcy necessarily associated with wheat protein sensitivity.
Thus the precise links between GSE, villous atrophy and maligcy have not yet
been established. However, there is evidence that products of activated T-cell
clones, be they antigen specific or maligt, influence epithelial cell
proliferation, differentiation and function thus contributing to the
histological lesion characteristic of GSE or small bowel lymphoma. Celiac disease is an autoimmune disorder triggered by ingestion of
gluten-containing foods. Epidemiologic studies dating from the 1950s established
its association with gastrointestinal maligcies, particularly small bowel
lymphoma. Corrao et al. recently demonstrated that patients with celiac disease
are at increased risk of mortality. Further, this risk is directly related to
compliance with a gluten-free diet. Continued research is needed regarding the
development of maligt complications related to celiac disease. A 62-yr-old man presented with a 5-yr history of intermittent abdominal
distention and pain. These symptoms persisted for several months and subsided
without treatment. A diagnosis of suspected small bowel lymphoma was made based
on plain radiograph and computerized tomogram findings, and he was referred to
our institution for further evaluation. Segmental resection of the small
intestine was performed and the diagnosis of marginal zone B-cell lymphoma
associated with amyloidosis was made. This is the first case of marginal zone
B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) in the small
intestine associated with amyloidosis in Korea. |
Are the proteins Erbin (LAP2) and Merlin cooperating? | Yes, Erbin and Merlin are cooperating. | Biallelic mutations in the neurofibromatosis 2 (NF2) gene are linked to
schwannoma and meningioma tumorigenesis. Cells with NF2 mutations exhibit
elevated levels of phosphorylated extracellular signal-regulated kinase (ERK)
and aberrant cell-cell and cell-matrix contacts. The NF2 gene product, merlin,
associates with adherens junction protein complexes, suggesting that part of its
function as a tumor suppressor involves regulating cell junctions. Here, we find
that a novel PDZ protein, called erbin, binds directly to the merlin-binding
partner, EBP0, and regulates adherens junction dissociation through a MAP
kinase-dependent mechanism. Reducing erbin expression using a targeted siRNA in
primary cultures of Schwann cells results in altered cell-cell interactions,
disruption of E-cadherin adherens junctions, increased cell proliferation, and
elevated levels of phosphorylated ERK, all phenotypes observed in cells that
lack merlin. Reduction of erbin expression also results in the dissociation of
merlin from adherens junction proteins and an increase in the levels of
phosphorylated merlin. These phenotypes can be rescued if cells with reduced
levels of erbin are treated with a pharmacological inhibitor of ERK kinase.
Collectively, these data indicate that erbin regulates MAP kinase activation in
Schwann cells and suggest that erbin links merlin to both adherens junction
protein complexes and the MAP kinase signaling pathway. Transforming growth factor beta (TGF-beta) family ligands are pleotropic
proteins with diverse cell-type-specific effects on growth and differentiation.
For example, PAK2 activation is critical for the proliferative/profibrotic
action of TGF-beta on mesenchymal cells, and yet it is not responsive to
TGF-beta in epithelial cells. We therefore investigated the regulatory
constraints that prevent inappropriate PAK2 activation in epithelial cultures.
The results show that the epithelial-enriched protein Erbin controls the
function of the NF2 tumor suppressor Merlin by determining the output of
Merlin's physical interactions with active PAK2. Whereas mesenchymal TGF-beta
signaling induces PAK2-mediated inhibition of Merlin function in the absence of
Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in
epithelia. These results not only identify Erbin as a key determit of
epithelial resistance to TGF-beta signaling, they also show that Erbin controls
Merlin tumor suppressor function by switching the functional valence of PAK2
binding. |
Which molecule is targeted by a monoclonal antibody Secukinumab? | Secukinumab (AIN457) is a fully human anti-interleukin-17A monoclonal antibody that neutralizes interleukin-17A. | Conflict of interest statement: Competing interests Dr Wolfgang Hueber is an
employee of Novartis Pharma and owns shares; Dr Bruce E Sands received
consulting fees for service on a scientific advisory board for Abbott
Immunology, Avaxia Biologics, Bristol-Myers Squibb, Elan Pharmaceuticals, Glaxo
SmithKline Welcome, Novartis Pharmaceuticals, Pfizer and Prometheus
Laboratories; consulting fees from Emmi Solutions; and holds common stock in
Avaxia Biologics (a company that is not publicly traded); Steve Lewitzky is an
employee of Novartis Pharma; Dr Marc Vandemeulebroecke is an employee of
Novartis Pharma and owns Novartis shares; Dr Walter Reinisch is a medical
advisor to Novartis; Dr Peter D R Higgins consults for Amgen, Genentech and JBR
Pharma, and receives honoraria from Abbott; Dr Jan Wehkamp has no conflict of
interest; Dr Brian G Feagan has been a scientific advisor for Protein Design
Labs, Astra Zeneca, Elan/Biogen, Celltech, Synta, Merck, Celgene, Novartis,
Given Imaging Inc., UCB Pharma, Salix Pharmaceuticals, Abbott Laboratories,
Centocor Inc. Pfizer, Axcan, Tillotts Pharma AG, Prometheus Laboratories, a
consultant for Synta, Millennium, Merck, Centocor, Elan/Biogen, Janssen-Ortho,
Protein Design Labs, ISIS, Teva Pharmaceuticals, Santarus, Bristol-Myers Squibb,
Celgene, UCB Pharma, Abbott, Proctor and Gamble, Genentech, Tillotts, Given
Imaging Inc., Salix Pharm., Ore Pharm. (previously GeneLogic), Novo Nordisk,
GSK, Actogenix, Prometheus Therapeutics and Diagnostics, Athersys, Alba
Therapeutics, Axcan, Pfizer, Shire, Wyeth, Zealand Pharm and has received a
research grant from Merck, Milllennium, Tillotts, Abbott, Engelheim, Novartis,
Centocor, Synta, Elan/Biogen, UCB Pharma, BMS, Proctor and Gamble, Genentech,
CombinatoRx, ActoGeniX; Dr Michael D Yao has nothing to disclose; Dr Marek
Karczewski was an external consultant for Novartis Pharma AG; Dr Jacek
Karczewski was an external consultant for Novartis Pharma AG; Nicole Pezous is
an employee of Novartis Pharma; Dr Stephan Bek is an employee of Novartis
Pharma; Dr Gerard Bruin is an employee of Novartis Pharma and owns Novartis
shares; Dr Bjoern Mellgard is an employee of Novartis Pharma; Claudia Berger is
an employee of Novartis Pharma and owns Novartis shares; Dr Marco Londei is an
employee of Novartis Pharma; Dr Arthur P Bertolino is an employee of Novartis
Pharma; Dr Gervais Tougas is an employee of Novartis Pharma and owns Novartis
shares; Dr Simon P L Travis has received honoraria for advisory boards or
consultancy from Abbott; Asahi; Aspreva; BMS; Centocor; Cosmo; Elan; Ferring;
Genentech; Genzyme; Giuliani; GSK; Glenmark; MSD; Novartis; Ocera; Procter &
Gamble Pharmaceuticals; PDLBiopharma; Santarus; Schering-Plough; Shire; Takeda;
Tillotts; UCB Pharma; Vertex; Vifor and Warner Chilcott. He has given expert
testimony on behalf of Elan, Cosmo, Ocera, Procter & Gamble, Santarus and
Tillotts, and received unrestricted educational grants from Abbott, Ferring,
MSD, Procter & Gamble, Schering Plough and Warner Chilcott. Our objective was to evaluate the efficacy of influenza and meningococcal
vaccinations in healthy subjects exposed to the anti-interleukin-17A (IL-17A)
monoclonal antibody (MAb) secukinumab. We used an open-label, parallel-group,
randomized single-center study of 50 healthy subjects. Subjects received a
single 150-mg dose of secukinumab or no treatment, followed by vaccination with
inactivated trivalent subunit influenza virus and conjugate group C
meningococcal vaccine (Agrippal and Menjugate, respectively) 2 weeks later.
Primary efficacy variables were responses of ≥4-fold increases in antibody titer
(hemagglutination inhibition [HI; for influenza virus] and serum bactericidal
assay [SBA; for Neisseria meningitides]) for meningococcus and influenza (at
least two out of three serotypes), both at 4 weeks postvaccination. All subjects
randomized to secukinumab (n = 25) or the control (n = 25) completed the study.
Antibody responses to vaccinations measured at 4 weeks were comparable in both
groups, with ≥4-fold increased responses following influenza virus vaccination
of 20/25 (80%) for both groups and following meningococcal vaccination of 19/25
(76%) for the secukinumab group and 18/25 (72%) for the control group.
Differences between groups were 0% (90% confidence intervals [CI], 19 and 19%)
and 4% (90% CI, 16 and 24%) for influenza virus and meningococcal vaccines,
respectively. Antibody responses were comparable between the 2 groups at
different time points. Headache was the most frequently reported adverse effect.
No deaths or serious adverse events were reported. Blockade of IL-17A by
secukinumab does not appear to interfere with efficacy of influenza and
meningococcal vaccinations, as assessed by the achievement of protective
antibody levels. A protective (≥4-fold) immune response to both vaccinations at
4 weeks was achieved in 80 and 76% of subjects exposed to secukinumab and the
control, respectively. BACKGROUND: Conventional systemic therapies for plaque psoriasis have not fully
met the needs of patients, and although current biologic treatments are
generally well tolerated, concerns exist with respect to long-term safety.
Interleukin (IL)-17A is believed to be an important effector cytokine in the
pathogenesis of psoriasis and is produced by Th17 cells, a class of helper T
cells that act outside the established Th1/Th2 paradigm for regulation of innate
and adaptive immunity.
OBJECTIVES: To assess the efficacy and safety of different doses of secukinumab,
a fully human anti-IL-17A IgG1κ monoclonal antibody, in patients with
moderate-to-severe plaque psoriasis.
METHODS: Patients (n = 125) were randomized 1 : 1 : 1 : 1 : 1 to receive
subcutaneous doses of placebo (n = 22) or secukinumab [1 × 25 mg (n = 29), 3 ×
25 mg (n = 26), 3 × 75 mg (n = 21) or 3 × 150 mg (n = 27)] at weeks 0, 4 and 8.
After the 12-week treatment period, patients entered a follow-up period of 24
weeks. The primary efficacy outcome was at least 75% improvement from baseline
in the Psoriasis Area and Severity Index score (PASI 75); secondary outcomes
included the Investigator's Global Assessment (IGA) and PASI 90 and 50 response
rates.
RESULTS: After 12 weeks of treatment, secukinumab 3 × 150 mg and 3 × 75 mg
resulted in significantly higher PASI 75 response rates vs. placebo (82% and 57%
vs. 9%; P < 0·001 and P = 0·002, respectively). Higher PASI 75 response rates
compared with placebo were maintained throughout the follow-up period with these
dosages [week 36, 26% (n = 7) and 19% (n = 4) vs. 4% (n = 1), respectively],
with a gradual decline of PASI 75 response over time after the dosing period.
IGA response rates were significantly higher in the 3 × 150 mg group vs. placebo
at week 12 (48% vs. 9%; P = 0·005) and were consistently higher for the 3 × 150
mg and 3 × 75 mg groups vs. placebo at all time points from week 4 onward. The
PASI 90 response rate was significantly higher in the 3 × 150 mg group vs.
placebo (52% vs. 5%) at week 12 and remained higher during the follow-up period.
Secukinumab was well tolerated. Two cases of neutropenia (≤ grade 2) were
reported in the 3 × 150 mg cohort.
CONCLUSIONS: Treatment with subcutaneous secukinumab 3 × 75 mg and 3 × 150 mg
met the primary outcome of PASI 75 response achievement after 12 weeks,
demonstrating efficacy in moderate-to-severe psoriasis. Genetic studies and correlative expression data in diseased tissues have pointed
to the role of interleukin (IL)-17 and Th17 cells in the pathogenesis of
autoimmune disorders such as psoriasis, inflammatory bowel disease and
seronegative spondyloarthropathies. Th17 cells are known to produce the
proinflammatory cytokine IL-17A as well as other effector cytokines, including
IL-17F and IL-22. Recent research has demonstrated that IL-17A is also expressed
by multiple lineages of the innate immune system, including mast cells,
neutrophils, dendritic cells, γδ-T cells, macrophages and natural killer cells.
It can thus be expected that the inhibition of IL-17A as a therapeutic target in
autoimmune disease would exert different physiological effects than the
suppression of Th17 cell activity. Early clinical data are now available on
secukinumab (AIN457), a recombit, highly selective, fully human monoclonal
anti-IL-17A antibody of the IgG1/κ isotype, enabling a preliminary assessment of
the effects of IL-17A inhibition in multiple autoimmune diseases. Rapid and
sustained symptom reductions in psoriasis, rheumatoid arthritis, psoriatic
arthritis and ankylosing spondylitis have been observed in secukinumab-treated
patients, with no overt safety signals. In conjunction with studies using the
humanised anti-IL-17A monoclonal antibody (mAb) ixekizumab (LY2439821) and the
fully human anti-IL-17RA mAb brodalumab (AMG 827), the findings on secukinumab
provide evidence for the role of IL-17A in the pathophysiology of autoimmune
disease and suggest the potential value of targeting this cytokine. PURPOSE: To determine the efficacy and safety of different doses of secukinumab,
a fully human monoclonal antibody for targeted interleukin-17A blockade, in
patients with noninfectious uveitis.
DESIGN: Three multicenter, randomized, double-masked, placebo-controlled,
dose-ranging phase III studies: SHIELD, INSURE, and ENDURE.
PARTICIPANTS: A total of 118 patients with Behçet's uveitis (SHIELD study); 31
patients with active, noninfectious, non-Behçet's uveitis (INSURE study); and
125 patients with quiescent, noninfectious, non-Behçet's uveitis (ENDURE study)
were enrolled.
METHODS: After an initial subcutaneous (s.c.) loading phase in each treatment
arm, patients received s.c. maintece therapy with secukinumab 300 mg every 2
weeks (q2w), secukinumab 300 mg monthly (q4w), or placebo in the SHIELD study;
secukinumab 300 mg q2w, secukinumab 300 mg q4w, secukinumab 150 mg q4w, or
placebo in the INSURE study; or secukinumab 300 mg q2w, secukinumab 300 mg q4w,
secukinumab 150 mg q4w, or placebo in the ENDURE study.
MAIN OUTCOME MEASURES: Reduction of uveitis recurrence or vitreous haze score
during withdrawal of concomitant immunosuppressive medication (ISM). Other end
points included best-corrected visual acuity, ISM use (expressed as a
standardized ISM score), and safety outcomes.
RESULTS: After completion or early termination of each trial, there were no
statistically significant differences in uveitis recurrence between the
secukinumab treatment groups and placebo groups in any study. Secukinumab was
associated with a significant reduction in mean total post-baseline ISM score (P
= 0.019; 300 mg q4w vs. placebo) in the SHIELD study. Likewise, secukinumab was
associated with a greater median reduction in ISM score versus placebo in the
INSURE study, although no statistical analysis of the difference was conducted
because of the small sample size. Overall, there was no loss in visual acuity
reported in any treatment group during follow-up in all 3 studies. According to
descriptive safety statistics, the frequencies of ocular and nonocular adverse
events seemed to be slightly higher among secukinumab groups versus placebo
across the 3 studies.
CONCLUSIONS: The primary efficacy end points of the 3 studies were not met. The
secondary efficacy data from these studies suggest a beneficial effect of
secukinumab in reducing the use of concomitant ISM. OBJECTIVE: To evaluate the efficacy and safety of secukinumab, a fully human,
anti-interleukin (IL)-17A monoclonal antibody, in patients with psoriatic
arthritis (PsA).
METHODS: 42 patients with active PsA fulfilling ClASsification for Psoriatic
ARthritis (CASPAR) criteria were randomly assigned (2:1) to receive two
intravenous secukinumab doses (10 mg/kg; n=28) or placebo (n=14) 3 weeks apart.
The primary endpoint was the proportion of American College of Rheumatology
(ACR) 20 responses at week 6 for secukinumab versus placebo (one-sided p<0.1).
RESULTS: Primary endpoint: ACR20 responses at week 6 were 39% (9/23) for
secukinumab versus 23% (3/13) for placebo (p=0.27). ACR20 responses were greater
with secukinumab versus placebo at week 12 (39% (9/23) vs 15% (2/13), p=0.13)
and week 24 (43% (10/23) vs 18% (2/11), p= 0.14). At week 6, 'good' European
League Against Rheumatism response was seen in 21.7% (5/23) secukinumab versus
9.1% (1/11) placebo patients. Compared with placebo at week 6, significant
reductions were observed among secukinumab recipients for C reactive protein
(p=0.039), erythrocyte sedimentation rate (p=0.038), Health Assessment
Questionnaire Disability Index (p=0.002) and Short Form Health Survey (SF-36;
p=0.030) scores. The overall adverse event (AE) frequency was comparable between
secukinumab (26 (93%)) and placebo (11 (79%)) recipients. Six serious AEs (SAEs)
were reported in four secukinumab patients and one SAE in one placebo patient.
CONCLUSIONS: Although the primary endpoint was not met, clinical responses,
acute-phase reactant and quality of life improvements were greater with
secukinumab versus placebo, suggesting some clinical benefit. Secukinumab
exhibited satisfactory safety. Larger clinical trials of secukinumab in PsA are
warranted. The IL-23/IL-17 pathway may be a novel therapeutic target for the treatment of
psoriatic arthritis (PsA). The potential beneficial effect of Th-17A antagonism
has been investigated by a randomized controlled trial in PsA patients with
secukinumab, a fully human, high-affinity, monoclonal antibody in a cohort of
patients with active PsA. Although this Phase II study presents bias that limits
the ability of this drug to meet the primary and some secondary end points, the
authors suggest that secukinumab may have biological effects and some clinical
benefits in PsA patients. Further studies are required to demonstrate if the
rationale to use drugs acting on the IL-23/IL-17 pathway is associated with
relevant efficacy and safety in the treatment of PsA. Clinical and experimental evidence suggest that interleukin-17A (IL-17A; also
known as IL-17) is an attractive therapeutic target in rheumatoid arthritis
(RA). Rheumatoid synovial tissue produces IL-17A, which causes cartilage and
bone degradation in synovial and bone explants. Overexpression of IL-17A induces
synovial inflammation and joint destruction in animal RA models. These effects
are attenuated in IL-17A-deficient animals and by agents that block IL-17A.
Serum IL-17A levels and, to a greater extent, synovial fluid IL-17A levels are
elevated in many patients with RA. In some RA cohorts, higher IL-17A levels have
been associated with a more severe clinical course. Several IL-17A blockers,
including the anti-IL-17A monoclonal antibodies secukinumab and ixekizumab, and
the anti-IL-17 receptor subunit A monoclonal antibody brodalumab have been
evaluated in phase II clinical trials. Of these, secukinumab is the most
advanced with respect to clinical evaluation in RA, with phase III trials
ongoing in patients on background methotrexate who had inadequate responses to
previous tumor necrosis factor blocker therapy. The contribution of Th17 cells to the development of colitis is well described.
The effector cytokines IL-17A and IL-17F have been proposed as potential
therapeutic targets for the treatment of patients with inflammatory bowel
disease. In a proof-of-concept study for the treatment of patients with Crohn's
disease, secukinumab, a monoclonal antibody directed against IL-17A, was
ineffective and associated with more adverse events than placebo. Wedebye
Schmidt et al. propose that blockade of both IL-17A and IL-17F, rather than
either cytokine alone, attenuates the development of colitis in a T-cell
transfer model of experimental colitis. These findings suggest that combined
blockade of IL-17A and IL-17F may be an effective strategy for the treatment of
patients with inflammatory bowel disease. BACKGROUND: Ankylosing spondylitis is a chronic immune-mediated inflammatory
disease characterised by spinal inflammation, progressive spinal rigidity, and
peripheral arthritis. Interleukin 17 (IL-17) is thought to be a key inflammatory
cytokine in the development of ankylosing spondylitis, the prototypical form of
spondyloarthritis. We assessed the efficacy and safety of the anti-IL-17A
monoclonal antibody secukinumab in treating patients with active ankylosing
spondylitis.
METHODS: We did a randomised double-blind proof-of-concept study at eight
centres in Europe (four in Germany, two in the Netherlands, and two in the UK).
Patients aged 18-65 years were randomly assigned (in a 4:1 ratio) to either
intravenous secukinumab (2×10 mg/kg) or placebo, given 3 weeks apart.
Randomisation was done with a computer-generated block randomisation list
without a stratification process. The primary efficacy endpoint was the
percentage of patients with a 20% response according to the Assessment of
SpondyloArthritis international Society criteria for improvement (ASAS20) at
week 6 (Bayesian analysis). Safety was assessed up to week 28. This study is
registered with ClinicalTrials.gov, number NCT00809159.
FINDINGS: 37 patients with moderate-to-severe ankylosing spondylitis were
screened, and 30 were randomly assigned to receive either intravenous
secukinumab (n=24) or placebo (n=6). The final efficacy analysis included 23
patients receiving secukinumab and six patients receiving placebo, and the
safety analysis included all 30 patients. At week 6, ASAS20 response estimates
were 59% on secukinumab versus 24% on placebo (99·8% probability that
secukinumab is superior to placebo). One serious adverse event (subcutaneous
abscess caused by Staphylococcus aureus) occurred in the secukinumab-treated
group.
INTERPRETATION: Secukinumab rapidly reduced clinical or biological signs of
active ankylosing spondylitis and was well tolerated. It is the first targeted
therapy that we know of that is an alternative to tumour necrosis factor
inhibition to reach its primary endpoint in a phase 2 trial.
FUNDING: Novartis. IMPORTANCE: Accumulating evidence suggests that IL-17 A has broad pathogenic
roles in multiple autoimmune and immune-mediated inflammatory diseases,
including psoriasis and rheumatoid arthritis (RA). The development of new
therapies that inhibit IL-17 pathway signaling is of clinical significance.
OBJECTIVES: This review aims to summarize the current preclinical evidence on
the role of Th17 cells and IL-17 and related cytokines in immune-mediated
disease pathophysiology, with a focus on psoriasis and rheumatoid arthritis, as
well as to summarize recent clinical trials in these indications with newly
developed IL-17 pathway inhibitors.
METHODS: A systematic literature search was conducted of PubMed using relevant
keywords. Studies were assessed according to recent relevance to IL-17-mediated
pathophysiology and clinical IL-17 inhibition. Experimental animal models of
autoimmune disease and clinical studies that focused on IL-17 pathway inhibitors
were included.
RESULTS: Preclinical studies suggest that IL-17A is an attractive therapeutic
target. Several IL-17A inhibitors have advanced into clinical trials, including
the anti-IL-17A monoclonal antibodies, secukinumab and ixekizumab, and the
anti-17RA monoclonal antibody brodalumab. Each has shown variable and sometimes
favorable results in proof-of-concept and phase II clinical trials and is
currently undergoing further clinical evaluation in a range of immune-mediated
diseases.
CONCLUSION: Targeting the IL-17 pathway shows promise as strategy to treat
immune-mediated diseases ranging from skin to joints. Psoriasis is a chronic inflammatory skin disease affecting about 1-3% of the
general population. Moderate-to-severe psoriasis is commonly associated with
various comorbidities, including psoriatic arthritis (PsA) and cardio-metabolic
disorders such as obesity, hypertension, diabetes, and metabolic syndrome. There
is increasing recognition that systemic inflammation accompanies severe skin
disease. Abnormal innate and adaptive immune responses in the skin are involved
in pathogenesis. The cytokine interleukin (IL)-17A is produced by T helper 17
(Th17) cells, neutrophils, mast cells, and T cytotoxic 17 cells. IL-17 plays a
key role in host defense against extracellular bacteria and fungi. IL-17A acts
on keratinocytes to increase expression of chemokines involved in recruiting
myeloid dendritic cells, Th17 cells, and neutrophils to the lesion site. IL-17A
also induces the production of antimicrobial peptides and pro-inflammatory
cytokines that, in turn, may amplify and sustain immune responses in the skin.
Blocking IL-17A improved psoriasis-like pathology in experimental models, and
reduction in IL-17 signaling is part of the mechanism of action of tumor
necrosis factor-α blockers. Three agents neutralizing IL-17 (i.e., secukinumab
and ixekizumab) or antagonizing its receptor (i.e., brodalumab) are currently
being tested for efficacy and safety in the treatment of plaque psoriasis and
PsA. Secukinumab is a fully human IgG1 monoclonal antibody that selectively
binds and neutralizes IL-17A whose efficacy in the therapy of chronic plaque
psoriasis has been demonstrated in different phase II clinical trial. No new
safety signals have emerged so far. The family of interleukin 17 receptors (IL17Rs), subtypes IL17RA-IL17RE, is
targeted by the group of pro-inflammatory IL17 cytokines (IL17A-F) and moreover
the newly developed anti-IL17A antibody secukinumab (AIN457) has shown promise
in Phase II trials in multiple sclerosis. Here, we show that human astrocytes,
isolated from a fetal cerebral cortex, express IL17RA and IL17RC and in vitro
treatment with IL17A increases protein levels of IL6 in human astrocytes, which
is enhanced in the presence of TNFα, as determined by homogeneous time resolved
fluorescence. Studies on acutely isolated mouse astrocytes are comparable to
human astrocytes although the protein levels of IL6 are lower in mouse
astrocytes, which also show a lower response to IL17F and IL1β in promoting IL6
levels. In human astrocytes, IL17A and TNFα also induce mRNA expression of IL6,
IL8 and the Th17 cytokines CXCL1, CXCL2, and CCL20, with little effect on Th1
cytokines CXCL9, CXCL10, and CXCL11. The effects of IL17A are associated with
nuclear translocation of the NF-κB transcription factor, as determined by
immunocytochemistry, where treatment of human astrocytes with the inhibitors of
the NF-κB pathway and with secukinumab inhibits the IL17A and IL17A/TNFα-induced
increase in nuclear translocation of NF-κB and levels of IL6. Taken together the
data shows that IL17A signaling plays a key role in regulating the levels of
cytokines, such as IL6, in human astrocytes via a mechanism that involves NF-κB
signaling and that selective inhibition of IL17A signaling attenuates levels of
pro-inflammatory molecules in astrocytes. PURPOSE OF REVIEW: Various novel therapies for spondyloarthritis (SpA) are
currently under development. In this review, we discuss the scientific rational
to target the interleukin (IL)-23/IL-17 axis in SpA and give an overview of the
proof-of-concept trials with drugs directed towards this axis.
RECENT FINDINGS: Cumulative evidence from genetics (e.g. the strong genetic
association with the IL-23 receptor gene), in-vitro models (e.g. the increased
IL-23 production upon HLA-B27 misfolding), human expression studies (e.g. the
expansion of IL-17 producing innate cells in SpA) and animal models (e.g. the
increased IL-17 production in HLA-B27 transgenic rats) strongly supports the
involvement of the IL-23/IL-17 axis in the pathogenesis of SpA. Ustekinumab (a
monoclonal antibody directed against the common p40 subunit of IL-23 and IL-12),
secukinumab, ixekizumab (both monoclonal antibodies directed against IL-17A),
and brodalumab a monoclonal antibody against the IL-17RA receptor) have been
recently used in proof-of-concept and randomized trials in the ankylosing
spondylitis and/or psoriatic arthritis subforms of SpA, with overall very
promising clinical efficacy.
SUMMARY: The first results for novel drugs blocking key cytokines in the
IL-23/IL-17 axis are promising in SpA and more novel compounds are upcoming.
This will teach us more on the role of the IL-23/IL-17 axis in the
pathophysiology of SpA. This article discusses the scientific rationale for the use of cytokine
inhibitors, including ustekinumab, an inhibitor of the interleukin (IL)-12 and
IL-23 pathways in psoriasis. Also addressed are the efficacy and safety data for
this agent, as well as for several emerging therapies that target other cytokine
pathways in psoriasis: the IL-17 inhibitors secukinumab, ixekizumab, and
brodalumab, the IL-23 blocker tildrakizumab, and the small-molecule kinase
inhibitors apremilast (a phosphodiesterase-4 blocker) and tofacitinib (a Janus
kinase inhibitor). BACKGROUND: Secukinumab is a fully human anti-interleukin-17A monoclonal
antibody.
OBJECTIVE: Determine the efficacy, safety and usability of secukinumab
administered via autoinjector/pen.
METHODS: This phase III trial randomized subjects with moderate to severe plaque
psoriasis to secukinumab 300 mg, 150 mg or placebo self-injection once weekly to
Week 4, then every 4 weeks. Co-primary end points at Week 12 were ≥75%
improvement in Psoriasis Area and Severity Index (PASI 75) and clear/almost
clear skin by investigator's global assessment 2011 modified version (IGA mod
2011 0/1). Secondary end points included autoinjector usability, assessed by
successful, hazard-free self-injection and subject-reported acceptability on
Self-Injection Assessment Questionnaire.
RESULTS: Week 12 PASI 75 and IGA mod 2011 0/1 responses were superior with
secukinumab 300 mg (86.7% and 73.3%, respectively) and 150 mg (71.7% and 53.3%,
respectively) vs. placebo (3.3% and 0%, respectively) (P < 0.0001 for all). All
subjects successfully self-administered treatment at Week 1, without critical
use-related hazards. Subject acceptability of autoinjector was high throughout
12 weeks. Adverse events were higher with secukinumab (300 mg, 70.0%; 150 mg,
63.9%) vs. placebo (54.1%), with differences largely driven by mild/moderate
nasopharyngitis.
CONCLUSION: Secukinumab delivered by autoinjector/pen is efficacious,
well-tolerated and associated with high usability in moderate to severe plaque
psoriasis. Secukinumab, a fully human anti-IL-17A monoclonal antibody, neutralizes IL-17A,
a key cytokine in the pathogenesis of psoriasis. Efficacy and safety of
secukinumab was evaluated in Japanese patients with moderate-to-severe plaque
psoriasis as part of a large Phase 3 global study (ERASURE). In this 52-week,
double-blind study (ClinicalTrials.gov Identifier: NCT01365455,
JapicCTI-111529), 87 patients from Japan (11.8% of 738 patients randomized in
the overall study population) were equally randomized to receive secukinumab
300 mg or 150 mg, or placebo once weekly at baseline and at Weeks 1, 2, 3 and 4,
then every 4 weeks. Co-primary endpoints (Week 12) were ≥75% improvement in
psoriasis area-and-severity index (PASI 75) from baseline and a score of 0
(clear) or 1 (almost clear) on a 5-point Investigator's Global Assessment scale
(IGA mod 2011 0/1) versus placebo. PASI 75 and IGA mod 2011 0/1 responses at
Week 12 were superior with secukinumab 300 mg (82.8% and 55.2%, respectively) or
150 mg (86.2% and 55.2%, respectively) versus placebo (6.9% and 3.4%,
respectively; P < 0.0001 for all). Greater than 90% improvement in PASI (PASI
90) was also superior with secukinumab 300 mg (62.1%) or 150 mg (55.2%) versus
placebo (0.0%) at Week 12 (P < 0.0001 for both). Clinical responses were
sustained up to Week 52 in the majority of patients. During a 12-week induction
period, adverse event incidences were 48.3% with secukinumab 300 mg, 55.2% with
150 mg, and 41.4% with placebo. Secukinumab showed robust and sustainable
efficacy in symptom reduction for moderate-to-severe plaque psoriasis in the
Japanese patients. Anti-cytokine autoantibodies have been widely reported to be present in human
plasma, both in healthy subjects and in patients with underlying autoimmune
conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal
dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic,
they can cause or facilitate a wide range of diseases including opportunistic
infections. The potential therapeutic value of specific neutralizing
anti-cytokine autoantibodies has not been thoroughly investigated. Here we used
mammalian cell display to isolate IL17A-specific antibodies from a thymoma
patient with proven high-titer autoantibodies against the same. We identified 3
distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro
assay. Their potencies were comparable to those of known neutralizing
antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently
in clinical development for the treatment of various inflammatory disorders.
These data clearly demonstrate that the human autoantibody repertoire can be
mined for antibodies with high therapeutic potential for clinical development. |
Which calcium/calmodulin dependent protein phosphatase is involved in the activation of the family of NFAT transcription factors (Nuclear Factors of Activated T cells)? | The activity of NFAT proteins is tightly regulated by the Ca(2+)/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN).Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization. | The nuclear factor of activated T cells (NFAT) group of transcription factors is
retained in the cytoplasm of quiescent cells. NFAT activation is mediated in
part by induced nuclear import. This process requires calcium-dependent
dephosphorylation of NFAT caused by the phosphatase calcineurin. The c-Jun
amino-terminal kinase (JNK) phosphorylates NFAT4 on two sites. Mutational
removal of the JNK phosphorylation sites caused constitutive nuclear
localization of NFAT4. In contrast, JNK activation in calcineurin-stimulated
cells caused nuclear exclusion of NFAT4. These findings show that the nuclear
accumulation of NFAT4 promoted by calcineurin is opposed by the JNK signal
transduction pathway. Transcription factors belonging to the nuclear factor of activated T cells
(NFAT) family regulate the expression of cytokine genes and other inducible
genes during the immune response. The functions of NFAT proteins are directly
controlled by the calcium- and calmodulin-dependent phosphatase calcineurin.
Here we show that the binding of calcineurin to NFAT is substantially increased
when calcineurin is activated with calmodulin and calcium. FK506.FKBP12
drug-immunophilin complexes inhibited the interaction of NFAT with activated
calcineurin much more effectively than they inhibited the interaction with
inactive calcineurin, suggesting that part of the interaction with activated
calcineurin involved the enzyme active site. We have previously shown that NFAT
is targeted to inactive calcineurin at a region distinct from the calcineurin
active site (Aramburu, J., Garcia-Cozar, F. J., Raghavan, A., Okamura, H., Rao,
A., and Hogan, P. G. (1998) Mol. Cell 1, 627-637); this region is also involved
in NFAT binding to activated calcineurin, since binding is inhibited by an NFAT
peptide spanning the calcineurin docking site on NFAT. The interacting surfaces
are located on the catalytic domain of the calcineurin A chain and on an
86-amino acid fragment of the NFAT regulatory domain. NFAT binding to the
calcineurin catalytic domain was inhibited by the calcineurin autoinhibitory
domain and the RII substrate peptide, which bind in the calcineurin active site,
as well as by the NFAT docking site peptide, which binds to a region of
calcineurin distinct from the active site. We propose that, in resting cells,
NFAT is targeted to a region of the calcineurin catalytic domain that does not
overlap the calcineurin active site. Upon cell activation, displacement of the
autoinhibitory domain by calmodulin binding allows NFAT to bind additionally to
the calcineurin active site, thus positioning NFAT for immediate
dephosphorylation at functional phosphoserine residues. Transcription factors of the NFAT (nuclear factor of activated T cells) family
are expressed in most immune system cells and in a range of other cell types.
Signaling through NFAT is implicated in the regulation of transcription for the
immune response and other processes, including differentiation and apoptosis.
NFAT normally resides in the cytoplasm, and a key aspect of the NFAT activation
pathway is the regulation of its nuclear import by the
Ca(2+)/calmodulin-dependent phosphatase calcineurin. In a cell line stably
expressing green fluorescent protein (GFP)-NFAT, this import can be triggered by
elevation of intracellular calcium and visualized in live cells. Here we show
that the inducible nuclear import of GFP-NFAT is efficiently blocked at early
stages of herpes simplex virus (HSV) infection. This is a specific effect, since
we observed abundant nuclear accumulation of a test viral protein and no
impediment to general nuclear localization signal-dependent nuclear import and
retention in infected cells. We show that virus binding at the cell surface is
not itself sufficient to inhibit the signaling that induces NFAT nuclear
translocation. Since the block occurs following infection in the presence of
phosphonoacetic acid but not cycloheximide, we infer that the entry of the
virion and early gene transcription are required but the effect is independent
of DNA replication or late virus gene expression. A consequence of the block to
GFP-NFAT import is a reduction in NFAT-dependent transcriptional activation from
the interleukin-2 promoter in infected cells. This HSV-mediated repression of
the NFAT pathway may constitute an immune evasion strategy or subversion of
other NFAT-dependent cellular processes to promote viral replication. NFAT (nuclear factor of activated T cell) proteins are expressed in most immune
system cells and regulate the transcription of cytokine genes critical for the
immune response. The activity of NFAT proteins is tightly regulated by the
Ca(2+)/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN).
Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization.
Current immunosuppressive drugs such as cyclosporin A and FK506 block CaN
activity thus inhibiting nuclear translocation of NFAT and consequent cytokine
gene transcription. The inhibition of CaN in cells outside of the immune system
may contribute to the toxicities associated with cyclosporin A therapy. In a
search for safer immunosuppressive drugs, we identified a series of
3,5-bistrifluoromethyl pyrazole (BTP) derivatives that block Th1 and Th2
cytokine gene transcription. The BTP compounds block the activation-dependent
nuclear localization of NFAT as determined by electrophoretic mobility shift
assays. Confocal microscopy of cells expressing fluorescent-tagged NFAT
confirmed that the BTP compounds block calcium-induced movement of NFAT from the
cytosol to the nucleus. Inhibition of NFAT was selective because the BTP
compounds did not affect the activation of NF-kappaB and AP-1 transcription
factors. Treatment of intact T cells with the BTP compounds prior to calcium
ionophore-induced activation of CaN caused NFAT to remain in a highly
phosphorylated state. However, the BTP compounds did not directly inhibit the
dephosphorylation of NFAT by CaN in vitro, nor did the drugs block the
dephosphorylation of other CaN substrates including the type II regulatory
subunit of protein kinase A and the transcription factor Elk-1. The data suggest
that the BTP compounds cause NFAT to be maintained in the cytosol in a
phosphorylated state and block the nuclear import of NFAT and, hence,
NFAT-dependent cytokine gene transcription by a mechanism other than direct
inhibition of CaN phosphatase activity. The novel inhibitors described herein
will be useful in better defining the cellular regulation of NFAT activation and
may lead to identification of new therapeutic targets for the treatment of
autoimmune disease and transplant rejection. Calcineurin signaling has been implicated in a broad spectrum of developmental
processes in a variety of organ systems. Calcineurin is a calmodulin-dependent,
calcium-activated protein phosphatase composed of catalytic and regulatory
subunits. The serine/threonine-specific phosphatase functions within a signal
transduction pathway that regulates gene expression and biological responses in
many developmentally important cell types. Calcineurin signaling was first
defined in T lymphocytes as a regulator of nuclear factor of activated T cells
(NFAT) transcription factor nuclear translocation and activation. Recent studies
have demonstrated the vital nature of calcium/calcineurin/NFAT signaling in
cardiovascular and skeletal muscle development in vertebrates. Inhibition,
mutation, or forced expression of calcineurin pathway genes result in defects or
alterations in cardiomyocyte maturation, heart valve formation, vascular
development, skeletal muscle differentiation and fiber-type switching, and
cardiac and skeletal muscle hypertrophy. Conserved calcineurin genes are found
in invertebrates such as Drosophila and Caenorhabditis elegans, and genetic
studies have demonstrated specific myogenic functions for the phosphatase in
their development. The ability to investigate calcineurin signaling pathways in
vertebrates and model genetic organisms provides a great potential to more fully
comprehend the functions of calcineurin and its interacting genes in heart,
blood vessel, and muscle development. The calcium-regulated protein phosphatase calcineurin (PP2B) functions as a
regulator of gene expression in diverse tissues through the dephosphorylation
and activation of a family of transcription factors known as nuclear factor of
activated T cells (NFAT). Here we show that NFATc3, in addition to being calcium
responsive, is regulated through an indirect recruitment of class II histone
deacetylases (HDACs). Specifically, yeast two-hybrid screening with the rel
homology domain of NFATc3 identified the chaperone mammalian relative of DnaJ
(Mrj) as a specific interacting factor. Mrj and NFATc3 were shown to directly
associate with one another in mammalian cells and in vitro. Mrj served as a
potent inhibitor of NFAT transcriptional activity within the nucleus through a
mechanism involving histone deacetylase recruitment in conjunction with heat
shock stimulation. Indeed, Mrj was determined to interact with class II histone
deacetylases, each of which translocated to the nucleus following heat shock
stimulation. Mrj also decreased NFATc3 occupancy of the tumor necrosis
factor-alpha promoter in cardiomyocytes in an HDAC-dependent manner, and Mrj
blocked calcineurin-induced cardiomyocyte hypertrophic growth. Conversely,
small-interfering-RNA-mediated reduction of Mrj augmented NFAT transcriptional
activity and spontaneously induced cardiac myocyte growth. Collectively, our
results define a novel response pathway whereby NFATc3 is negatively regulated
by class II histone deacetylases through the DnaJ (heat shock protein-40)
superfamily member Mrj. The heart responds to injury and chronic pressure overload by pathologic growth
and remodeling, which frequently result in heart failure and sudden death.
Calcium-dependent signaling pathways promote cardiac growth and associated
changes in gene expression in response to stress. The
calcium/calmodulin-dependent phosphatase calcineurin, which signals to nuclear
factor of activated T cells (NFAT) transcription factors, serves as a transducer
of calcium signals and is sufficient and necessary for pathologic cardiac
hypertrophy and remodeling. Transient receptor potential (TRP) proteins regulate
cation entry into cells in response to a variety of signals, and in skeletal
muscle, expression of TRP cation channel, subfamily C, member 3 (TRPC3) is
increased in response to neurostimulation and calcineurin signaling. Here we
show that TRPC6 was upregulated in mouse hearts in response to activated
calcineurin and pressure overload, as well as in failing human hearts. Two
conserved NFAT consensus sites in the promoter of the TRPC6 gene conferred
responsiveness to cardiac stress. Cardiac-specific overexpression of TRPC6 in
transgenic mice resulted in heightened sensitivity to stress, a propensity for
lethal cardiac growth and heart failure, and an increase in NFAT-dependent
expression of beta-myosin heavy chain, a sensitive marker for pathologic
hypertrophy. These findings implicate TRPC6 as a positive regulator of
calcineurin-NFAT signaling and a key component of a calcium-dependent regulatory
loop that drives pathologic cardiac remodeling. In cells of the immune system that are stimulated by antigen or antigen-antibody
complexes, Ca(2+) entry from the extracellular medium is driven by depletion of
endoplasmic reticulum Ca(2+) stores and occurs through specialized
store-operated Ca(2+) channels known as Ca(2+)-release-activated Ca(2+) (CRAC)
channels. The process of store-operated Ca(2+) influx is essential for
short-term as well as long-term responses by immune-system cells. Short-term
responses include mast cell degranulation and killing of target cells by
effector cytolytic T cells, whereas long-term responses typically involve
changes in gene transcription and include T and B cell proliferation and
differentiation. Transcription downstream of Ca(2+) influx is in large part
funneled through the transcription factor nuclear factor of activated T cells
(NFAT), a heavily phosphorylated protein that is cytoplasmic in resting cells,
but that enters the nucleus when dephosphorylated by the calmodulin-dependent
serine/threonine phosphatase calcineurin. The importance of the
Ca(2+)/calcineurin/NFAT signalling pathway for lymphocyte activation is
underscored by the finding that the underlying defect in a family with a
hereditary severe combined immune deficiency (SCID) syndrome is a defect in CRAC
channel function, store-operated Ca(2+) entry, NFAT activation and transcription
of cytokines, chemokines and many other NFAT target genes whose transcription is
essential for productive immune defence. We recently used a two-pronged genetic
approach to identify Orai1 as the pore subunit of the CRAC channel. On the one
hand, we initiated a positional cloning approach in which we utilised
genome-wide single nucleotide polymorphism (SNP) mapping to identify the genomic
region linked to the mutant gene in the SCID family described above. In
parallel, we used a genome-wide RNAi screen in Drosophila to identify critical
regulators of NFAT nuclear translocation and store-operated Ca(2+) entry. These
approaches, together with subsequent mutational and electrophysiological
analyses, converged to identify human Orai1 as a pore subunit of the CRAC
channel and as the gene product mutated in the SCID patients. Calcium activated gene transcription through Nuclear Factor of Activated
T-cells, (NFAT) proteins, is emerging as a ubiquitous mechanism for the control
of important physiological processes. Of the five mammalian NFAT isoforms,
transcriptional activities of NFATs 1-4 are stimulated by a calcium driven
association between the ubiquitous phosphatase calcineurin and the
calcium-sensing protein calmodulin. Published in vitro evidence has suggested
that other members of the calmodulin super-family, in particular the neuronal
calcium sensor (NCS) proteins, can similarly modulate calcineurin activity. In
this study we have assessed the ability of NCS proteins to interact directly
with calcineurin in vitro and report a specific if weak association between
various NCS proteins and the phosphatase. In an extension to these analyses we
have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on
calcineurin signalling in HeLa cells in experiments examining the
dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin
activity. Results from these experiments indicate that NCS-1 was not able to
detectably modulate calcineurin/NFAT signalling in a live mammalian cell system,
findings that are consistent with the idea that calmodulin and not NCS-1 or
other NCS family proteins is the physiologically relevant modulator of
calcineurin activity. |
Is abdominal pain a common symptom in autism? | Yes, although there are no precise data. There is data that Lactase deficiency, not associated with intestinal inflammation or injury, is common in autistic children and may contribute to abdominal discomfort, pain and observed aberrant behavior. | The purpose of this study was to estimate the prevalence of chronic
gastrointestinal symptoms in a general population of children with autism or
autistic spectrum disorder (ASD). The study site was a clinic specializing in
ASD in a large pediatric medical center serving a 10 county area in the
midwestern USA. In a sample of 137 children, age 24-96 months, classified as
having autism or ASD by the Autism Diagnostic Observation Schedule-Generic, 24
percent had a history of at least one chronic gastrointestinal symptom. The most
common symptom was diarrhea, which occurred in 17 percent. There was no
association between chronic gastrointestinal symptoms and a history of
developmental regression. The potential phenotypic association between autism
and gastrointestinal symptoms is discussed. AIM: To investigate whether children with autistic spectrum disorder (ASD) have
bowel symptoms consistent with underlying enterocolitis.
METHODS: Information on children's stool patterns and gut symptoms collected by
questionnaire at 4 weeks and at 6, 18, 30 and 42 months of age were available
for 12,984 children from the Avon Longitudinal Study of Parents and Children
(ALSPAC). Data on the 78 children identified by local health and/or education
systems to have special educational provision for ASD were compared with the
12,906 remaining children in the cohort.
RESULTS: Comparison of the ASD and control group during the first 3.5 years of
life showed no major differences in stool colour or consistency, or in frequency
of diarrhoea, constipation, bloody stools or abdominal pain. The ASD children
had similar stool frequency up to 18 months, but there was a trend for ASD
children to pass more stools at 30 months (OR 3.73, 95% CI 1.11 to 12.6; p =
0.004) and at 42 months (OR 6.46, 95% CI 1.83 to 22.7; p<0.001), although only
three children passed more than 4 stools/day. Repeating the analysis on only
those cases diagnosed as having classical childhood autism resulted in very
similar findings.
CONCLUSIONS: During the first 42 months of life, ASD children had a stool
pattern that was very similar to that of other children, apart from a slight
increase in stool frequency at 30 and 42 months. There were no symptoms to
support the hypothesis that ASD children had enterocolitis. Autistic behavior is often accompanied by numerous disturbing symptoms on the
part of gastrointestinal system, such as abdominal pain, constipation or
diarrhea. These problems are often connected with deregulation of physiological
microflora in intestine. The aim of this study was to determine differences in
intestinal microflora of autistic and healthy children. Strains of Clostridium
spp. and enterococci were isolated more frequently from stool samples of
autistic children and rarely lactobacilli. Quantitative differences were
observed maliny among staphylococci, Candida spp. and Clostridium perfringens.
Monitoring and stabilization of intestinal microflora and knowledge about role
of particular strains in etiology of autistic disorders can increase the chances
for appropriate therapy. Intestinal disaccharidase activities were measured in 199 individuals with
autism to determine the frequency of enzyme deficiency. All patients had
duodenal biopsies that were evaluated morphologically and assayed for lactase,
sucrase, and maltase activity. Frequency of lactase deficiency was 58% in
autistic children ≤ 5 years old and 65% in older patients. As would be expected,
patients with autism at age 5 > years demonstrated significant decline in
lactase activity (24%, p = .02) in comparison with ≤ 5 years old autistic
patients. Boys ≤ 5 years old with autism had 1.7 fold lower lactase activity
than girls with autism (p = .02). Only 6% of autistic patients had intestinal
inflammation. Lactase deficiency not associated with intestinal inflammation or
injury is common in autistic children and may contribute to abdominal
discomfort, pain and observed aberrant behavior. Most autistic children with
lactose intolerance are not identified by clinical history. Children with autism spectrum disorders (ASD) experience high rates of anxiety,
sensory processing problems, and gastrointestinal (GI) problems; however, the
associations among these symptoms in children with ASD have not been previously
examined. The current study examined bivariate and multivariate relations among
anxiety, sensory over-responsivity, and chronic GI problems in a sample of 2,973
children with ASD enrolled in the Autism Treatment Network (ages 2-17 years,
81.6 % male). Twenty-four percent of the sample experienced at least one type of
chronic GI problem (constipation, abdominal pain, bloating, diarrhea, and/or
nausea lasting three or more months). Children with each type of GI problem had
significantly higher rates of both anxiety and sensory over-responsivity.
Sensory over-responsivity and anxiety were highly associated, and each provided
unique contributions to the prediction of chronic GI problems in logistic
regression analyses. The results indicate that anxiety, sensory
over-responsivity and GI problems are possibly interrelated phenomenon for
children with ASD, and may have common underlying mechanisms. Many children with autism spectrum disorders (ASDs) suffer from gastrointestinal
problems such as diarrhoea, constipation and abdominal pain. This has stimulated
investigations into possible abnormalities of intestinal microbiota in autistic
patients. Therefore, we designed this study to identify differences (and/or
similarities) in the microbiota of children with autism (without
gastrointestinal dysfunction: n = 23; with gastrointestinal dysfunction: n = 28)
and their neurotypical siblings (n = 53) who share a similar environment using
bacterial tag-encoded FLX amplicon pyrosequencing. Regardless of the diagnosis
and sociodemographic characteristics, overall, Firmicutes (70%), Bacteroidetes
(20%) and Proteobacteria (4%) were the most domit phyla in samples. Results
did not indicate clinically meaningful differences between groups. The data do
not support the hypothesis that the gastrointestinal microbiota of children with
ASD plays a role in the symptomatology of ASD. Other explanations for the
gastrointestinal dysfunction in this population should be considered including
elevated anxiety and self-restricted diets. The objective of this study is to investigate whether parentally-reported
gastro-intestinal (GI) symptoms are increased in a population-derived sample of
children with autism spectrum disorders (ASD) compared to controls. Participants
included 132 children with ASD and 81 with special educational needs (SEN) but
no ASD, aged 10-14 years plus 82 typically developing (TD) children. Data were
collected on GI symptoms, diet, cognitive abilities, and developmental
histories. Nearly half (weighted rate 46.5 %) of children with ASD had at least
one individual lifetime GI symptom compared with 21.8 % of TD children and
29.2 % of those with SEN. Children with ASD had more past and current GI
symptoms than TD or SEN groups although fewer current symptoms were reported in
all groups compared with the past. The ASD group had significantly increased
past vomiting and diarrhoea compared with the TD group and more abdominal pain
than the SEN group. The ASD group had more current constipation (when defined as
bowel movement less than three times per week) and soiling than either the TD or
SEN groups. No association was found between GI symptoms and intellectual
ability, ASD severity, ASD regression or limited or faddy diet. Parents report
more GI symptoms in children with ASD than children with either SEN or TD
children but the frequency of reported symptoms is greater in the past than
currently in all groups. |
Are cyclophilins ubiquitously expressed? | Yes, cyps (cyclophilins) are ubiquitous proteins of the immunophilin superfamily. | Cyclophilins belong to the family of peptidyl-prolyl cis/trans isomerases
(PPIases), which are ubiquitous and highly conserved enzymes capable of
cis/trans isomerizing Xaa-Pro peptide bonds. Members of the CyP40-type
cyclophilins have originally been described as components of hormone receptor
complexes. Here, we describe NcCyP41, a CyP40 ortholog from Neurospora crassa,
its expression in Escherichia coli and subsequent purification. Characterization
of NcCyP41 reveals that it is a heat shock protein, which is active as a
cyclosporin A-sensitive PPIase. Affinity chromatography using immobilized
recombit NcCyP41 yielded two major NcCyP41-binding proteins: Hsp80 (a Hsp90
ortholog from N.crassa) and CyPBP37. CyPBP37 has not been described. In
addition, this is the first record describing an interaction between a member of
Cyp40-type cyclophilins and of CyPBP37-type proteins, respectively. CyPBP37
expression is repressed by thiamine and in the stationary phase in N.crassa.
CyPBP37 is present in different isoforms. The expression of a CyPBP37 ortholog
in yeast, Thi4p, is diminished in a mutant lacking one of the two CyP40
orthologs (Cpr7p). In addition, the DeltaCpr7p deletion mutant shows a
thiamine-dependent growth defect. We conclude that, in yeast, Cpr7p and Thi4p
interact functionally. Cyclophilins (CyPs) are a large class of highly conserved ubiquitous
peptidyl-prolyl cis-trans isomerases. CyPs have also been identified as being a
specific receptor for the immunosuppressive drug cyclosporin A and are involved
in a variety of biological functions. CyPJ is a novel member of the CyP family
and human CyPJ (hCyPJ) is the protein encoded by a cyclophilin-like gene from
human foetal brain, which shows 50% sequence identity to human cyclophilin A
(hCyPA). Recombit hCyPJ was expressed in Escherichia coli and purified. The
three-dimensional structure of hCyPJ has been determined by molecular
replacement using the hCyPA structure as the search model and has been refined
at 2.6 angstroms resolution. The hCyPJ molecule contains four helices and one
beta-barrel composed of eight antiparallel beta-strands. The overall secondary
and tertiary structures of hCyPJ are similar to those of hCyPA, but hCyPJ
contains an additional disulfide bridge and four segments with conformations
that are strikingly different from those of hCyPA. His43 and Gln52 of hCyPJ are
expected to be the active sites based on sequence alignment with hCyPA. The
hCyPJ structure shows a conserved water molecule close to His43 and Gln52 which
appears to support the solvent-assisted mechanism. Originally identified as the cellular targets of immunosuppressant drugs, the
immunophilins encompass two ubiquitous protein families: the FK-506 binding
proteins or FKBPs, and the cyclosporin-binding proteins or cyclophilins. Present
in organisms ranging from bacteria to animals and plants, these proteins are
characterized by their enzymatic activity; the peptidyl-prolyl cis-trans
isomerization of polypeptides. Whilst this function is important for protein
folding, it has formed the functional basis for more complex interactions
between immunophilins and their target proteins. Beginning with a brief
historical overview of the immunophilin family, and a representative
illustration of the current state of knowledge that has accumulated for these
proteins in diverse organisms, a detailed description is presented of the recent
advances in the elucidation of the role of this ubiquitous protein family in
plant biology. Though still in its infancy, investigation into the function of
plant immunophilins has so far yielded interesting results--as a significant
component of the chloroplast proteome, the abundance of immunophilins located in
the thylakoid lumen suggests that these proteins may play important roles in
this relatively uncharacterized subcellular compartment. Moreover, the
importance of the complex multidomain immunophilins in functions pertaining to
development is underscored by the strong phenotypes displayed by their
corresponding mutants. Immunophilins are ubiquitous enzymes responsible for proline isomerisation
during protein synthesis and for the chaperoning of several membrane proteins.
These activities can be blocked by the immunosuppressants cyclosporin A, FK506
and rapamycin. It has been shown that all three immunosuppressants have
neurotrophic activity and can modulate neurotransmitter release, but the
molecular basis of these effects is currently unknown. Here, we show that
synapsin I, a synaptic vesicle-associated protein, can be purified from Torpedo
cholinergic synaptosomes through its affinity to cyclophilin B, an immunophilin
that is particularly abundant in brain. The interaction is direct and conserved
in mammals, and shows a dissociation constant of about 0.5 microM in vitro. The
binding between the two proteins can be disrupted by cyclosporin A and inhibited
by physiological concentrations of ATP. Furthermore, cyclophilin B co-localizes
with synapsin I in rat synaptic vesicle fractions and its levels in synaptic
vesicle-containing fractions are decreased in synapsin knockout mice. These
results suggest that immunophilins are involved in the complex protein networks
operating at the presynaptic level and implicate the interaction between
cyclophilin B and synapsins in presynaptic function. Cyps (cyclophilins) are ubiquitous proteins of the immunophilin superfamily with
proposed functions in protein folding, protein degradation, stress response and
signal transduction. Conserved cysteine residues further suggest a role in redox
regulation. In order to get insight into the conformational change mechanism and
functional properties of the chloroplast-located CYP20-3, site-directed
mutagenized cysteine-->serine variants were generated and analysed for enzymatic
and conformational properties under reducing and oxidizing conditions. Compared
with the wild-type form, elimination of three out of the four cysteine residues
decreased the catalytic efficiency of PPI (peptidyl-prolyl cis-trans isomerase)
activity of the reduced CYP20-3, indicating a regulatory role of
dithiol-disulfide transitions in protein function. Oxidation was accompanied by
conformational changes with a predomit role in the structural rearrangement
of the disulfide bridge formed between Cys(54) and Cys(171). The rather negative
E(m) (midpoint redox potential) of -319 mV places CYP20-3 into the redox
hierarchy of the chloroplast, suggesting the activation of CYP20-3 in the light
under conditions of limited acceptor availability for photosynthesis as realized
under environmental stress. Chloroplast Prx (peroxiredoxins) were identified as
interacting partners of CYP20-3 in a DNA-protection assay. A catalytic role in
the reduction of 2-Cys PrxA and 2-Cys PrxB was assigned to Cys(129) and
Cys(171). In addition, it was shown that the isomerization and
disulfide-reduction activities are two independent functions of CYP20-3 that
both are regulated by the redox state of its active centre. Cyclophilins are folding helper enzymes belonging to the class of
peptidyl-prolyl cis-trans isomerases (PPIases; EC 5.2.1.8) that catalyze the
cis-trans isomerization of peptidyl-prolyl bonds in proteins. They are
ubiquitous proteins present in almost all living organisms analyzed to date,
with extremely rare exceptions. Few cyclophilins have been described in
Actinobacteria, except for three reported in the genus Streptomyces and another
one in Mycobacterium tuberculosis. In this study, we performed a complete
phylogenetic analysis of all Actinobacteria cyclophilins available in sequence
databases and new Streptomyces cyclophilin genes sequenced in our laboratory.
Phylogenetic analyses of cyclophilins recovered six highly supported groups of
paralogy. Streptomyces appears as the bacteria having the highest cyclophilin
diversity, harboring proteins from four groups. The first group was named "A"
and is made up of highly conserved cytosolic proteins of approximately 18 kDa
present in all Actinobacteria. The second group, "B," includes cytosolic
proteins widely distributed throughout the genus Streptomyces and closely
related to eukaryotic cyclophilins. The third group, "M" cyclophilins, consists
of high molecular mass cyclophilins ( approximately 30 kDa) that contain
putative membrane binding domains and would constitute the only membrane
cyclophilins described to date in bacteria. The fourth group, named "C"
cyclophilins, is made up of proteins of approximately 18 kDa that are
orthologous to Gram-negative proteobacteria cyclophilins. Ancestral character
reconstruction under parsimony was used to identify shared-derived (and likely
functionally important) amino acid residues of each paralogue. Southern and
Western blot experiments were performed to determine the taxonomic distribution
of the different cyclophilins in Actinobacteria. Cyclophilin is a ubiquitous peptidyl prolyl cis/trans isomerase that plays
critical roles in many biological processes. A number of cyclophilin inhibitors
have been designed based on the structure of the immunosuppressant cyclosporin
A. To discover inhibitors that have other structures, the authors established
the high-throughput screening (HTS) method using FDSS6000 real-time fluorescence
detector. The inhibitors identified with this HTS showed significant correlation
with direct interaction as measured by surface plasmon resoce. This
high-throughput assay system is a powerful tool for the discovery of
peptidylprolyl isomerase inhibitors. Drug development against Leishmania donovani, the pathogen that causes visceral
leishmaniasis in humans, is currently an active area of research given the
widespread prevalence of the disease and the emergence of resistant strains. The
immunosuppressive drug cyclosporin is known to have antiparasitic activity
against a variety of pathogens. The receptor for cyclosporin is the protein
cyclophilin, which is a ubiquitous peptidylprolyl isomerase. The crystal
structure of cyclophilin from L. donovani complexed with cyclosporin has been
solved at 2.6 A resolution. The thermodynamic parameters of the interaction have
been determined using spectroscopic and calorimetric techniques. A detailed
effort has been made to predict the thermodynamic parameters of binding from
computations based on the three-dimensional crystal structure. These results
were in good agreement with the corresponding experimental values. Furthermore,
the structural and biophysical results have been discussed in the context of
leishmanial drug resistance and could also set the stage for the design of
potent non-immunosuppressive antileishmanials. Cyclophilins constitute a subgroup of large family of proteins called
immunophilins, which also include FKBPs and Parvulins. They are remarkably
conserved in all genera, highlighting their pivotal role in important cellular
processes. Most cyclophilins display PPIase enzymatic activity, multiplicity,
diverse cellular locations and active role in protein folding which render them
to be included in the class of diverse set of proteins called molecular
chaperones. Due to their distinct PPIase function, besides protein disulfide
isomerases and protein foldases, cyclophilins have been deemed necessary for in
vivo chaperoning activity. Unlike other cellular chaperones, these proteins are
specific in their respective targets. Not all cyclophilin proteins possess
PPIase activity, indicating a loss of their PPIase activity during the course of
evolution and gain of function independent of their PPIase activity. The PPIase
function of cyclophilins is also compensated by their functional homologs, like
FKBPs. Multiple cyclophilin members in plants like Arabidopsis and rice have
been reported to be associated with diverse functions and regulatory pathways
through their foldase, scaffolding, chaperoning or other unknown activities.
Although many functions of plant cyclophilins were reported or suggested, the
physiological relevance and molecular basis of stress-responsive expression of
plant cyclophilins is still largely unknown. However, their wide distribution
and ubiquitous nature signifies their fundamental importance in plant survival.
Several of these members have also been directly linked to multiple stresses.
This review attempts to deal with plant cyclophilins with respect to their role
in stress response. |
Are adenylyl cyclases always transmembrane proteins? | Adenylyl cyclases exists both as transmembrane and soluble proteins. | Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling
molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from
the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs).
Mammalian sAC is distributed throughout the cytoplasm and it may be present in
the nucleus and inside mitochondria. sAC activity is directly stimulated by
HCO3(-), and sAC has been confirmed to be a HCO3(-) sensor in a variety of
mammalian cell types. In addition, sAC can functionally associate with carbonic
anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains
of sAC are related to HCO3(-)-regulated adenylyl cyclases from cyanobacteria,
suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing
CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are
still limited but are rapidly accumulating. In shark gills, sAC senses blood
alkalosis and triggers compensatory H(+) absorption. In the intestine of bony
fishes, sAC modulates NaCl and water absorption. And in sea urchin sperm, sAC
may participate in the initiation of flagellar movement and in the acrosome
reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are
present in most animal phyla. This review summarizes the current knowledge on
the physiological roles of sAC in aquatic animals and suggests additional
functions in which sAC may be involved. Recently published findings indicate that a knockout (KO) of soluble adenylyl
cyclase (sAC, also known as AC-10) gene expression in mice leads to defective
glucoregulation that is characterized by reduced pancreatic insulin secretion
and reduced intraperitoneal glucose tolerance. Summarized here are current
concepts regarding the molecular basis for this phenotype, with special emphasis
on the potential role of sAC as a determit of glucose-stimulated insulin
secretion. Highlighted is new evidence that in pancreatic beta cells, oxidative
glucose metabolism stimulates mitochondrial CO₂production that in turn generates
bicarbonate ion (HCO(3)(-)). Since HCO(3)(-) binds to and directly stimulates
the activity of sAC, we propose that glucose-stimulated cAMP production in beta
cells is mediated not simply by transmembrane adenylyl cyclases (TMACs), but
also by sAC. Based on evidence that sAC is expressed in mitochondria, there
exists the possibility that beta-cell glucose metabolism is linked to
mitochondrial cAMP production with consequent facilitation of oxidative
phosphorylation. Since sAC is also expressed in the cytoplasm, sAC catalyzed
cAMP production may activate cAMP sensors such as PKA and Epac2 to control ion
channel function, intracellular Ca²⁺ handling, and Ca²⁺-dependent exocytosis.
Thus, we propose that the existence of sAC in beta cells provides a new and
unexpected explanation for previously reported actions of glucose metabolism to
stimulate cAMP production. It seems possible that alterations of sAC activity
might be of importance when evaluating new strategies for the treatment of type
2 diabetes (T2DM), or when evaluating why glucose metabolism fails to stimulate
insulin secretion in patients diagnosed with T2DM. This article is part of a
Special Issue entitled: The role of soluble adenylyl cyclase in health and
disease. Axon regeneration in the mature central nervous system is limited by extrinsic
inhibitory signals and a postnatal decline in neurons' intrinsic growth
capacity. Neuronal levels of the second messenger cAMP are important in
regulating both intrinsic growth capacity and neurons' responses to extrinsic
factors. Approaches which increase intracellular cAMP in neurons enhance neurite
outgrowth and facilitate regeneration after injury. Thus, understanding the
factors which affect cAMP in neurons is of potential therapeutic importance.
Recently, soluble adenylyl cyclase (sAC, ADCY10), the ubiquitous,
non-transmembrane adenylyl cyclase, was found to play a key role in neuronal
survival and axon growth. sAC is activated by bicarbonate and cations and may
translate physiologic signals from metabolism and electrical activity into a
neuron's decision to survive or regenerate. Here we critically review the
literature surrounding sAC and cAMP signaling in neurons to further elucidate
the potential role of sAC signaling in neurite outgrowth and regeneration. This
article is part of a Special Issue entitled: The role of soluble adenylyl
cyclase in health and disease. Cyclic adenosine 3',5'-monophosphate (cAMP), the first second messenger to be
described, plays a central role in cell signaling in a wide variety of cell
types. Over the last decades, a wide body of literature addressed the different
roles of cAMP in cell physiology, mainly in response to neurotransmitters and
hormones. cAMP is synthesized by a wide variety of adenylyl cyclases that can
generally be grouped in two types: transmembrane adenylyl cyclase and soluble
adenylyl cyclases. In particular, several aspects of sperm physiology are
regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka
sAC, SACY). The signature that identifies sAC among other ACs, is their direct
stimulation by bicarbonate. The essential nature of cAMP in sperm function has
been demonstrated using gain of function as well as loss of function approaches.
This review unifies state of the art knowledge of the role of cAMP and those
enzymes involved in cAMP signaling pathways required for the acquisition of
fertilizing capacity of mammalian sperm. This article is part of a Special Issue
entitled: The role of soluble adenylyl cyclase in health and disease. Adenylyl cyclase (AC) is a key enzyme that synthesizes cyclic AMP (cAMP) at the
onset of the signaling pathway to activate sperm motility. Here, we showed that
both transmembrane AC (tmAC) and soluble AC (sAC) are distinctly involved in the
regulation of sperm motility in the ascidian Ciona intestinalis. A tmAC
inhibitor blocked both cAMP synthesis and the activation of sperm motility
induced by the egg factor sperm activating and attracting factor (SAAF), as well
as those induced by theophylline, an inhibitor of phoshodiesterase. It also
significantly inhibited cAMP-dependent phosphorylation of a set of proteins at
motility activation. On the other hand, a sAC inhibitor does not affect on
SAAF-induced transient increase of cAMP, motility activation or protein
phosphorylation, but it reduced swimming velocity to half in
theophylline-induced sperm. A sAC inhibitor KH-7 induced circular swimming
trajectory with smaller diameter and significantly suppressed chemotaxis of
sperm to SAAF. These results suggest that tmAC is involved in the basic
mechanism for motility activation through cAMP-dependent protein
phosphorylation, whereas sAC plays distinct roles in increase of flagellar beat
frequency and in the Ca2+-dependent chemotactic movement of sperm. BACKGROUND AND PURPOSE: H2 O2 is widely understood to regulate intracellular
signalling. In airway epithelia, H2 O2 stimulates anion secretion primarily by
activating an autocrine PGE2 signalling pathway via EP4 and EP1 receptors to
initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion.
This study investigated signalling downstream of the receptors activated by H2
O2 .
EXPERIMENTAL APPROACH: Anion secretion by differentiated bronchial epithelial
cells was measured in Ussing chambers during stimulation with H2 O2 , an EP4
receptor agonist or β2 -adrenoceptor agonist in the presence and absence of
inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca(2+) ]I )
changes were followed by microscopy using fura-2-loaded cells and PKA activation
followed by FRET microscopy.
KEY RESULTS: Transmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were
both necessary for H2 O2 and EP4 receptor-mediated CFTR activation in bronchial
epithelia. H2 O2 and EP4 receptor agonist stimulated tmAC to increase exchange
protein activated by cAMP (Epac) activity that drives PLC activation to raise
[Ca(2+) ]i via Ca(2+) store release (and not entry). Increased [Ca(2+) ]i led to
sAC activation and further increases in CFTR activity. Stimulation of sAC did
not depend on changes in [HCO3 (-) ]. Ca(2+) -activated apical KCa 1.1 channels
and cAMP-activated basolateral KV 7.1 channels contributed to H2 O2 -stimulated
anion currents. A similar Epac-mediated pathway was seen following β2
-adrenoceptor or forskolin stimulation.
CONCLUSIONS AND IMPLICATIONS: H2 O2 initiated a complex signalling cascade that
used direct stimulation of tmACs by Gαs followed by Epac-mediated Ca(2+)
crosstalk to activate sAC. The Epac-mediated Ca(2+) signal constituted a
positive feedback loop that amplified CFTR anion secretion following stimulation
of tmAC by a variety of stimuli. Forward motility stimulating factor (FMSF), a glycoprotein isolated from buffalo
serum, binds to the surface of the mature sperm cells to promote their
progressive motility. This article reports the mode of signal transduction of
this extracellular factor in goat sperm. The mechanism was investigated by
assaying intracellular second messenger level and forward motility in presence
of different pharmacological modulators. Mg++-dependent Forskolin responsive
form of transmembrane adenylyl cyclase (tmAC) of goat spermatozoa was probed for
its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of
tmACs, was used to identify the role of this enzyme in the scheme of
FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has
been inspected using GTPγS. Participation of protein kinase A (PKA) and tyrosine
kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC
activity in a dose-dependent manner through receptor/G-protein activation to
enhance intracellular cAMP and forward motility. Motility boosting effects of
this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF
displayed substantial motility promoting activity when movement of spermatozoa
was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase
indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in
mediating FMSF action was confirmed by the application of dibutyryl cAMP.
Observed motility regulatory effects with IP20 and genistein indicate
contribution of PKA and tyrosine kinase in FMSF activity; enhanced
phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this
regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that
augments intracellular cAMP, which through downstream crosstalk of
phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus,
this article for the first time describes conventional tmAC-dependent profound
activation of progressive motility by a physiologic extracellular factor in a
mammalian species. |
What histone trimethylation has been associated to RNA splicing? | Mostly H3K36me3 but there is some evidence that H3K4me3 may also play a role in splicing | Trimethylation of histone H3 on lysine 4 (H3K4me3) localizes near the 5' region
of genes and is tightly associated with active loci. Several proteins, such as
CHD1, BPTF, JMJD2A, and the ING tumor suppressor family, directly recognize this
lysine methyl mark. However, how H3K4me3 recognition participates in active
transcription remains poorly characterized. Here we identify specific
CHD1-interacting proteins via H3K4me3 affinity purification, including numerous
factors mediating postinitiation events. Conventional biochemical purification
revealed a stable complex between CHD1 and components of the spliceosome.
Depletion of CHD1 in extracts dramatically reduced splicing efficiency in vitro,
indicating a functional link between CHD1 and the spliceosome. Knockdown of CHD1
and H3K4me3 levels by siRNA reduced association of U2 snRNP components with
chromatin and, more importantly, altered the efficiency of pre-mRNA splicing on
active genes in vivo. These findings suggest that methylated H3K4 serves to
facilitate the competency of pre-mRNA maturation through the bridging of
spliceosomal components to H3K4me3 via CHD1. Nucleosome positioning is constrained at eukaryotic transcription start sites
and implicated in transcriptional regulation. Moreover, recent observations
indicate that chromatin structure, transcription and splicing are functionally
intertwined, and that modified nucleosomes with trimethylation of lysine 36 in
histone subunit 3 (H3K36me3) are enriched at internal exons and the downstream
flanking intronic regions of highly expressed genes. However, the position of
nucleosomes in the interior of genes has been thought to be largely random. Here
we show, by analysis of data sets from human sperm and T cells and medaka
(Japanese killifish, Oryzias latipes) blastulae, that internal exons of genes
are characterized by sharply elevated average nucleosome occupancy in comparison
to flanking intronic sequences. We also show that the preferential positioning
of nucleosomes at internal exons is independent of their modification status,
and of the GC content, conservation or the expression level of the exon. These
findings show that the location of exons is recorded in the chromatin structure
and may be inherited across generations. Such embedded information may underpin
transcriptionally coupled exon recognition and splice site selection. Several lines of recent evidence support a role for chromatin in splicing
regulation. Here, we show that splicing can also contribute to histone
modification, which implies bidirectional communication between epigenetic
mechanisms and RNA processing. Genome-wide analysis of histone methylation in
human cell lines and mouse primary T cells reveals that intron-containing genes
are preferentially marked with histone H3 Lys36 trimethylation (H3K36me3)
relative to intronless genes. In intron-containing genes, H3K36me3 marking is
proportional to transcriptional activity, whereas in intronless genes, H3K36me3
is always detected at much lower levels. Furthermore, splicing inhibition
impairs recruitment of H3K36 methyltransferase HYPB (also known as Setd2) and
reduces H3K36me3, whereas splicing activation has the opposite effect. Moreover,
the increase of H3K36me3 correlates with the length of the first intron,
consistent with the view that splicing enhances H3 methylation. We propose that
splicing is mechanistically coupled to recruitment of HYPB/Setd2 to elongating
RNA polymerase II. A chromatin code appears to mark introns and exons with distinct patterns of
nucleosome enrichment and histone methylation. We investigated whether a causal
relationship exists between splicing and chromatin modification by asking
whether splice-site mutations affect the methylation of histone H3K36. Deletions
of the 3' splice site in intron 2 or in both introns 1 and 2 of an integrated
β-globin reporter gene caused a shift in relative distribution of H3K36
trimethylation away from 5' ends and toward 3' ends. The effects of splice-site
mutations correlated with enhanced retention of a U5 snRNP subunit on
transcription complexes downstream of the gene. In contrast, a poly(A) site
mutation did not affect H3K36 methylation. Similarly, global inhibition of
splicing by spliceostatin A caused a rapid repositioning of H3K36me3 away from
5' ends in favor of 3' ends. These results suggest that the cotranscriptional
splicing apparatus influences establishment of normal patterns of histone
modification. BACKGROUND: The packaging of DNA into chromatin regulates transcription from
initiation through 3' end processing. One aspect of transcription in which
chromatin plays a poorly understood role is the co-transcriptional splicing of
pre-mRNA.
RESULTS: Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks
introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this
organism reveals that this modification is enriched in coding regions and that
its levels peak at the transcribed regions of two characteristic subgroups of
genes. First, long genes are more likely to have higher levels of H2BK123ub1,
correlating with the postulated role of this modification in preventing cryptic
transcription initiation in ORFs. Second, genes that are highly transcribed also
have high levels of H2BK123ub1, including the ribosomal protein genes, which
comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a
feature of introns in the yeast genome, and the disruption of this modification
alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3),
which functionally correlates with alternative RNA splicing in humans. In
addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in
combination with an htb-K123R mutation, leads to synthetic lethality.
CONCLUSION: These data suggest that H2BK123ub1 facilitates cross talk between
chromatin and pre-mRNA splicing by modulating the distribution of intronic and
exonic histone modifications. The protein Vezf1 plays multiple roles important for embryonic development. In
Vezf1(-/-) mouse embryonic stem (mES) cells, our earlier data showed widespread
changes in gene-expression profiles, including decreased expression of the
full-length active isoform of Dnmt3b methyltransferase and concomitant
genome-wide reduction in DNA methylation. Here we show that in HeLaS3 cells
there is a strong genome-wide correlation between Vezf1 binding and peaks of
elongating Ser2-P RNA polymerase (Pol) ll, reflecting Vezf1-dependent slowing of
elongation. In WT mES cells, the elongating form of RNA pol II accumulates near
Vezf1 binding sites within the dnmt3b gene and at several other Vezf1 sites, and
this accumulation is significantly reduced at these sites in Vezf1(-/-) mES
cells. Depending upon genomic location, Vezf1-mediated Pol II pausing can have
different regulatory roles in transcription and splicing. We find examples of
genes in which Vezf1 binding sites are located near cassette exons, and in which
loss of Vezf1 leads to a change in the relative abundance of alternatively
spliced messages. We further show that Vezf1 interacts with Mrg15/Mrgbp, a
protein that recognizes H3K36 trimethylation, consistent with the role of
histone modifications at alternatively spliced sites. BACKGROUND: Zebrafish embryos are transcriptionally silent until activation of
the zygotic genome during the 10th cell cycle. Onset of transcription is
followed by cellular and morphological changes involving cell speciation and
gastrulation. Previous genome-wide surveys of transcriptional changes only
assessed gene expression levels; however, recent studies have shown the
necessity to map isoform-specific transcriptional changes. Here, we perform
isoform discovery and quantification on transcriptome sequences from before and
after zebrafish zygotic genome activation (ZGA).
RESULTS: We identify novel isoforms and isoform switches during ZGA for genes
related to cell adhesion, pluripotency and DNA methylation. Isoform switching
events include alternative splicing and changes in transcriptional start sites
and in 3' untranslated regions. New isoforms are identified even for
well-characterized genes such as pou5f1, sall4 and dnmt1. Genes involved in
cell-cell interactions such as f11r and magi1 display isoform switches with
alterations of coding sequences. We also detect over 1000 transcripts that
acquire a longer 3' terminal exon when transcribed by the zygote compared to
their maternal transcript counterparts. ChIP-sequencing data mapped onto skipped
exon events reveal a correlation between histone H3K36 trimethylation peaks and
skipped exons, suggesting epigenetic marks being part of alternative splicing
regulation.
CONCLUSIONS: The novel isoforms and isoform switches reported here include
regulators of transcriptional, cellular and morphological changes taking place
around ZGA. Our data display an array of isoform-related functional changes and
represent a valuable resource complementary to existing early embryo
transcriptomes. MOTIVATION: In addition to alternative splicing, alternative polyadenylation has
also been identified as a critical and prevalent regulatory mechanism in human
gene expression. However, the mechanism of alternative polyadenylation selection
and the involved factors is still largely unknown.
RESULTS: We use the ENCODE data to scan DNA functional elements, including
chromatin accessibility and histone modification, around transcript cleavage
sites. Our results demonstrate that polyadenylation sites tend to be less
sensitive to DNase I. However, these polyadenylation sites have preference in
nucleosome-depleted regions, indicating the involvement of chromatin
higher-order structure rather than nucleosomes in the resultant lower chromatin
accessibility. More interestingly, for genes using two polyadenylation sites,
the distal sites show even lower chromatin accessibility compared with the
proximal sites or the unique sites of genes using only one polyadenylation site.
We also observe that the histone modification mark, histone H3 lysine 36
tri-methylation (H3K36Me3), exhibits different patterns around the cleavage
sites of genes using multiple polyadenylation sites from those of genes using a
single polyadenylation site. Surprisingly, the H3K36Me3 levels are comparable
among the alternative polyadenylation sites themselves. In summary,
polyadenylation and alternative polyadenylation are closely related to
functional elements on the DNA level.
CONTACT: [email protected]. |
What was the purpose of the FANTOM4 project? | The international Functional Annotation Of the Mammalian Genomes 4 (FANTOM4) research collaboration set out to better understand the transcriptional network that regulates macrophage differentiation and to uncover novel components of the transcriptome employing a series of high-throughput experiments. | |
Does low T3 negatively affect prognosis of patients after cardiac surgery? | Low cardiac output syndrome after cardiac surgery for congenital heart diseases is associated with decreased T3
Low T3 concentrations are associated with occurrence of post operative atrial fibrillation
Low T3 concentrations are inversely correlated with the days of post operative hospitalization | A significant reduction in plasma free triiodothyronine (T3) (P less than
0.0001) has been observed in patients undergoing open heart surgery. The
beneficial effect of T3 would appear to be associated with increased synthesis
and utilization of myocardial high energy stores. We have therefore administered
T3 (4-10 micrograms iv) to 10 patients either when difficulty was being
experienced in weaning from cardiopulmonary bypass (CPB) support (n = 5), or
when myocardial function remained extremely poor (n = 5), despite inotropic and
intraaortic balloon pump support. Mean preoperative NYHA functional class of the
10 patients was 3.2, left ventricular enddiastolic pressure (LVEDP) 20 mm Hg and
ejection fraction (EF) 40%. The mean myocardial ischaemia time was 72 min (range
40-120 min). Within 1 h of T3 administration the mean plasma free T3 level had
risen from 1.03 to 3.56 micrograms/ml and CPB was discontinued in all 5 cases.
Balloon pump support (n = 2) was no longer essential within 3 h. At 1 h, the
mean arterial pressure (MAP) had risen from 42 to 78 mm Hg, and heart rate (HR)
from 90 to 104 beats/min; the left atrial pressure (LAP) had fallen from 30 to
14 mm Hg, and the central venous pressure (CVP) from 20 to 11 cm H2O. (All
changes significant at P less than 0.0001.) Inotropic support had been
significantly reduced or discontinued. To our knowledge, T3 has not been
administered previously as an inotropic agent to patients who have undergone
cardiac surgery. We believe that T3 may have an important role in the rescue of
failing hearts following a period of myocardial ischaemia in patients who have
undergone open heart surgery. Thyroid hormone status was assessed in 132 children with congenital heart
defects undergoing cardiac surgery (median age 3.1 y; range 2 d to 16.2 y).
Plasma TSH, thyroxine (T4), free thyroxine (fT4), triiodothyronine (T3), reverse
triiodothyronine (rT3), thyroglobulin (Tg), and urinary iodine excretion were
measured before and every other day after cardiac surgery (d 1-21). After
surgery we observed strikingly low plasma concentrations of TSH (0.4 mU/L;
0.2-1.3), T3 (0.6 nmol/L; 0.3-1.2), T4 (48.9 nmol/L; 12.9-82.4), IT4 (12.9
pmol/L; 5.1-19.3), and Tg (9.4 micrograms/L; 1.5-20.6), whereas rT3 plasma
concentrations increased (0.13 pmol/L; 0.05-0.3; n = 40). The maximal
post-operative changes of TSH and rT3 preceded changes of T3, T4, fT4, and Tg.
Postoperative urinary iodine excretion increased significantly (n = 109).
Thyroid hormone plasma concentrations were lowest after cardiopulmonary bypass
operations and in patients treated with dopamine. In patients with postoperative
T3 plasma concentrations less than 0.6 nmol/L (n =52) the period of mechanical
ventilation and intensive care treatment was significantly prolonged.
Furthermore, the cumulative doses of inotropic and vasoactive catecholamines and
furosemide were significantly higher in this patient group. Our results
demonstrate transient secondary hypothyroidism in children after cardiac surgery
that may contribute to postoperative cardiac and respiratory dysfunction and may
delay recovery. Possible benefits of thyroid hormone replacement therapy need to
be thoroughly examined. The cardiovascular system is an important target for thyroid hormones. The
present study evaluates the changes affecting thyroid hormone metabolism during
and 6 days after coronary artery bypass and their relationship with the
post-operative outcome of the patients. Thirty-three patients were enrolled in
the study; their thyroid hormone profiles were determined at 13 sampling points
during surgery and for 6 days afterwards. Serum total tri-iodothyronine (T3) and
free T3 (FT3) concentrations decreased significantly after surgery (P<0.001) and
they remained significantly low until the end of the study. Free thyroxine (FT4)
and T4 declined significantly immediately after surgery (P<0.05 for FT4, P<0.001
for T4) but they returned to baseline values (24 h and 96 h post-surgery
respectively). Serum reverse T3 increased remarkably 36 h after surgery
(P<0.001) and remained significantly higher than the baseline value throughout
the study. A relevant finding was that the days of post-operative
hospitalization (10+/-3 days, means+/-S.D.) was inversely correlated with the
slope of the recovery of T3 concentration (P<0.001) or with the area under the
plasma curves of T3 (P=0.024, time range 72-144 h) and the FT3/FT4 ratio
(P=0.037, time range 72-144 h) during the post-operative period. Our data
suggest a prolonged reduction of T4 to T3 conversion in patients undergoing
cardiac surgery and indicate that the recovery period is the most critical in
the evaluation of a possibly successful approach for T3 substitutive therapy. OBJECTIVE: Despite improved perioperative management, atrial fibrillation (AF)
after coronary artery bypass grafting (CABG) remains a relevant clinical
problem, whose pathogenetic mechanisms remain incompletely explained. A reduced
incidence of postoperative AF has been described in CABG patients receiving IV
tri-iodothyronine (T3). This study was designed to define the role of thyroid
metabolism on the genesis of postoperative AF.
METHODS AND RESULTS: Free T3 (fT3), free thyroxine (fT4), and thyroid
stimulating hormone were assayed at admission in 107 consecutive patients
undergoing isolated CABG surgery. Patients with thyroid disease or taking drugs
known to interfere with thyroid function were excluded. A preoperative rhythm
other than sinus rhythm was considered an exclusion criterion. Thirty-three
patients (30.8%) had postoperative AF. An older age (P=0.03), no therapy with
beta-blockers (P=0.08), chronic obstructive pulmonary disease (P=0.08), lower
left ventricle ejection fraction (P=0.09) and lower fT3 concentration (P=0.001),
were univariate predictors of postoperative AF. On multivariate analysis, low
fT3 concentration and lack of beta-blocking therapy were independently related
with the development of postoperative AF (odds ratio, OR, 4.425; 95% confidence
interval, CI, 1.745-11.235; P=0.001 and OR 3.107; 95% CI 1.087-8.875; P=0.03,
respectively). Postoperative AF significantly prolonged postoperative hospital
stay (P=0.002).
CONCLUSIONS: Low basal fT3 concentration can reliably predict the occurrence of
postoperative AF in CABG patients. OBJECTIVE: The purpose of this study was to evaluate serum triiodothyronine
levels as a trigger of postoperative atrial fibrillation (AF) in elderly
patients undergoing cardiac surgery and to study the possible association of
serum triiodothyronine levels with preoperative and postoperative hemodynamics.
DESIGN: Prospective study.
SETTING: University hospital.
PARTICIPANTS: Forty-six consecutive nonemergency patients 65 years or older
undergoing cardiac surgery during 1999 to 2000 in Tampere University Hospital,
Tampere, Finland.
INTERVENTIONS: Free serum T3 concentration was used as a measure of serum
triiodothyronine levels. Samples were taken preoperatively, on the fourth
postoperative day, and at the 3-month follow-up. The hemodynamic state of the
patients was estimated by whole-body impedance cardiography preoperatively,
during the intensive care unit period, daily until the fourth postoperative day,
and at the 3-month follow-up.
MEASUREMENTS AND MAIN RESULTS: AF occurred in 43% of the patients. The patients
in the AF group had significantly more grafts (3.9 v 3.1, p = 0.02), and there
was a small difference in age between the AF and non-AF groups (73 years v 69
years, p = 0.06). The free T3 concentration on the fourth postoperative day was
significantly lower in the AF group (3.5 nmol/L v 4.6 nmol/L, p = 0.04). In
logistic regression analysis, the independent predictors of AF were age, number
of grafts, and serum free T3 concentration on the fourth postoperative day. In
the group with low T3 concentration, the cardiac index was lower (1.4 v 1.8, p =
0.05) and the systemic vascular resistance index was higher (4,064 v 2,969, p =
0.04) but only immediately after the operation. Although the AF mostly appeared
during the second to fourth postoperative days, there were no longer any
differences in the hemodynamic state at that time.
CONCLUSIONS: In a group of elderly patients undergoing cardiac surgery, there
was a strong association between a postoperative decrease of serum
triiodothyronine levels and atrial fibrillation. The decrease of serum
triiodothyronine levels was related to the changes of hemodynamic parameters
only in the immediate postoperative period. Cardiac surgery using cardiopulmonary bypass produces a generalized systemic
inflammatory response, resulting in increased postoperative morbidity and
mortality. Under these circumstances, a typical pattern of thyroid abnormalities
is seen in the absence of primary disease, defined as sick euthyroid syndrome
(SES). The presence of postoperative SES mainly in small children and neonates
exposed to long bypass times and the pharmacological profile of thyroid hormones
and their effects on the cardiovascular physiology make supplementation therapy
an attractive treatment option to improve postoperative morbidity and mortality.
Many studies have been performed with conflicting results. In this article, we
review the important literature on the development of SES in paediatric
postoperative cardiac patients, analyse the existing information on thyroid
hormone replacement therapy in this patient group and try to summarize the
findings for a recommendation. PURPOSE OF REVIEW: Here we review typical thyroid function alterations observed
in the critically ill pediatric patient.
RECENT FINDINGS: Abnormalities in the hypothalamic-pituitary-thyroid axis have
recently been confirmed to be prevalent in similar proportions in pediatric and
adult patients. Significant benefits of therapy have yet to be demonstrated.
SUMMARY: At present, there is no evidence of benefit in giving thyroid hormone
to patients with nonthyroidal illness who have low serum T3 or T4
concentrations, including preterm infants and postcardiac surgery patients. OBJECTIVES: We evaluated the effects of thyroid hormone levels and interleukin-8
levels on prognosis in patients undergoing congenital heart surgery under
cardiopulmonary bypass (CPB).
STUDY DESIGN: The study included 41 consecutive children (19 boys, 22 girls;
mean age 3.4 ± 3.1 years; range 0.3 to 12 years). The patients were divided into
two groups based on the presence or absence of postoperative low cardiac output
state (LCOS). The definition of LCOS included oliguria, tachycardia, metabolic
acidosis, and increased plasma lactate level. Plasma total (tT4) and free (fT4)
thyroxine, total (tT3) and free (fT3) triiodothyronine, thyroid stimulating
hormone (TSH), and interleukin-8 (IL-8) levels were measured preoperatively and
at 48 hours postoperatively.
RESULTS: Postoperatively, nine patients (22%) developed LCOS. While the two
groups were similar with respect to preoperative levels of thyroid hormones,
lactate, and IL-8, postoperative tT3 and fT3 levels were significantly lower,
and lactate and IL-8 levels were significantly higher in the LCOS group
(p<0.05). In correlation analysis, postoperative IL-8 level showed significant
correlations with CPB time (r=0.66), duration of mechanical ventilation
(r=0.68), and inotropic requirement (r=0.59) (for all p<0.001). On the other
hand, LCOS was significantly correlated with the following: preoperative tT4
(r=-0.32, p=0.043) and postoperative fT3 (r=-0.44, p=0.004) levels, duration of
mechanical ventilation (r=0.56, p<0.001), intensive care unit stay (r=0.45,
p=0.003), and cross-clamp time (r=0.43, p=0.005). Regression analysis showed
preoperative level of tT4 as the independent predictor of LCOS (t=-2.092,
p=0.045). Mortality occurred in four patients (9.8%) in the early postoperative
period, all of whom were in the LCOS group.
CONCLUSION: Our findings suggest that the development of LCOS after congenital
heart surgery is associated with decreased total and free T3, and increased IL-8
levels at 48 hours, and preoperative tT4 level is an independent predictor of
LCOS. |
Which deficiency is the cause of restless leg syndrome | Iron deficiency (low serum ferritin) is a recognized cause for RLS. Further, in the striatum of subjects with restless legs syndrome, the dopamine transporter is decreased, which leads to impaired dopaminergic neurotransmission. There is also a report of magnesium deficiency underlying RLS. | INTRODUCTION: The restless legs syndrome is characterized by an unpleasant
sensation in the legs which causes an imperative need to move the legs and is
therefore considered to be a disorder of movement. When it appears before going
to sleep, it may interfere with falling asleep and lead to a sleep-deficit.
DEVELOPMENT AND CONCLUSIONS: It is a clinical condition with a satisfactory
treatment, and improvement of the associated sleep disorder. The etiology is
unknown, sometimes it is familial. The syndrome is increasingly often diagnosed,
particularly in association with iron deficiency, during pregcy, in chronic
renal failure and in patients with peripheral neuropathy. Polysomnography is not
necessary, unless one suspects an associated disorder of periodic leg movements.
Treatment is by dopaminergic, opiate, benzodiazepine, anticonvulsant drugs or
clonidine. Iron is the most important transitional metal in the body, as it is implicated
in many metabolic processes, mostly related to its capacity as an electron
donor/acceptor. Iron deficiency has been long been known to cause anaemia, iron
excess to cause haemochromatosis. As excess free iron can cause oxidative
damage, it is important that the levels of iron in the body are tightly
regulated which appears to be done only by digestive absorption, as there is no
known regulating mechanism for elimination of iron. The amount of free iron is
also kept to a minimum thanks to binding to transferrin for transport, and to
ferritin for storage. Recent research has put emphasis on the possible role of
excess iron in the brain in several degenerative diseases. Iron deficiency in
the central nervous system is known to cause motor impairment and cognitive
deficits; more recently, it has been suggested that it may play a role in the
pathophysiology of the restless leg syndrome. 2001 Harcourt Publishers Ltd Restless leg syndrome (RLS) and periodic limb movement disorder (PLMD) are
considered to be a continuum of a neurological sleep disorder associated with
abnormal iron metabolism or deficiency. I describe a case of RLS and PLMD in a
cystic fibrosis patient with iron deficiency from chronic hemoptysis. This is
the first case that reports RLS and PLMD manifesting from iron deficiency caused
by chronic hemoptysis in advanced cystic fibrosis lung disease. Restless legs syndrome (RLS) is a common condition that is frequently
unrecognized, misdiagnosed and poorly managed. It is characterized by
uncomfortable sensations deep in the legs developing at rest that compel the
person to move; symptoms are worst at night and sleep disturbance is common. RLS
occurs in 7%-11% of the population in Western countries, and many such people
experience troublesome symptoms. Primary RLS is familial in up to two thirds of
patients. RLS may also be secondary to a number of conditions including iron
deficiency, pregcy and end-stage renal failure and, perhaps, neuropathy.
Secondary RLS is most common in those presenting for the first time in later
life. The pathogenesis of RLS probably involves the interplay of systemic or
brain iron deficiency and impaired dopaminergic neurotransmission in the
subcortex of the brain. RLS is very responsive to dopaminergic therapies.
Rebound of RLS symptoms during the early morning and development of severe
symptoms earlier in the day (augmentation) are problematic in those treated for
a prolonged period with levodopa. Consequently, dopamine agonists have become
first line treatment. Anti-convulsant medications and opioids are helpful in
some patients. Correction of underlying problem wherever possible is important
in the management of secondary RLS. Restless legs syndrome (RLS) has gradually been recognized as a cause for
insomnia in adults, but there have been few reports about children with RLS in
Japan. Here we described seven pediatric RLS patients. All of the parents of our
patients had difficult times to make their children sleep due to irritability,
restlessness, and demanding bedtime routines. All patients had asked their
parents to rub their feet in bed, and it took more than half an hour to soothe
them until they fell asleep. Their mothers had been exhausted from this
night-time routine. However, they did not consider the routine abnormal, as it
had been their habitual behavior since infancy. Some parents were too distressed
or embarrassed to describe the symptoms of their child properly. Five patients
had clear family history and none had obvious periodic leg movements during
sleep. All patients showed low levels of ferritin and iron supplementation was
effective in five cases. In the severest two cases, pramipexole, but not iron,
was dramatically effective. Both patients started to show RLS symptoms in the
early days of infancy, which may suggest more severe hereditary dopaminergic
dysfunction. RLS does occur in childhood and pediatricians should bear it in
mind as one of the differential diagnoses when seeing children who are irritated
and/or having difficulty in initiating their sleep. OBJECTIVE: Iron deficiency is associated with paediatric sleep disturbances; in
particular, restless leg syndrome (RLS) and periodic limb movement disorder
(PLMD). Correction of iron deficiency has been shown to improve sleep disordered
breathing (SDB) in certain adult populations. We evaluated the iron status of
children diagnosed with SDB undergoing adenotonsillectomy.
METHODS: Consecutive children undergoing adenotonsillectomy for SDB between
January 2007 and January 2008 were analysed. Routine blood tests were performed
including full blood count and iron studies. Children were grouped according to
age; 0-2 years (group A), 2-6 years (group B) and above 6 years (group C).
Results were compared to local normal values and published data regarding normal
values for paediatric populations.
RESULTS: 94 children were included (60 male, 34 female). Mean age was 3.9 years
(range 1.2-13.4 years). Iron deficiency was most marked in group A (n=8), with
levels of ferritin (12.4), Mean Cell Haemoglobin (MCH) (25.0), iron saturation
(16%, normal 20-40%) all below normal and Hb at the bottom of the normal range
(11.0, normal 11-14.5). In group B (n=76), ferritin (19.4) and MCH (26.5) were
again below normal but were higher than for group A. Average Hb for group B was
11.9.
CONCLUSION: The association between low iron and SDB in children has not
previously been described. The results of this study highlighted low iron status
in the children undergoing adenotonsillectomy for SDB. This was most severe in
children under 6 years old. It is unclear whether low iron levels represent a
cause or effect of SDB. Restless leg syndrome (RLS) is a sensorimotor disorder. Clinical studies have
implicated the dopaminergic system in RLS, while others have suggested that it
is associated with insufficient levels of brain iron. To date, alterations in
brain iron status have been demonstrated but, despite suggestions from the
clinical literature, there have been no consistent findings documenting a
dopaminergic abnormality in RLS brain tissue. In this study, the substantia
nigra and putamen were obtained at autopsy from individuals with primary RLS and
a neurologically normal control group. A quantitative profile of the
dopaminergic system was obtained. Additional assays were performed on a
catecholaminergic cell line and animal models of iron deficiency. RLS tissue,
compared with controls, showed a significant decrease in D2R in the putamen that
correlated with severity of the RLS. RLS also showed significant increases in
tyrosine hydroxylase (TH) in the substantia nigra, compared with the controls,
but not in the putamen. Both TH and phosphorylated (active) TH were
significantly increased in both the substantia nigra and putamen. There were no
significant differences in either the putamen or nigra for dopamine receptor 1,
dopamine transporters or for VMAT. Significant increases in TH and
phosphorylated TH were also seen in both the animal and cell models of iron
insufficiency similar to that from the RLS autopsy data. For the first time, a
clear indication of dopamine pathology in RLS is revealed in this autopsy study.
The results suggest cellular regulation of dopamine production that closely
matches the data from cellular and animal iron insufficiency models. The results
are consistent with the hypothesis that a primary iron insufficiency produces a
dopaminergic abnormality characterized as an overly activated dopaminergic
system as part of the RLS pathology. There may be a relationship between proton pump inhibitors (PPIs) and iron
absorption. PPIs may decrease the amount of iron absorbed gastrointestinally
specifically due to alteration of the pH in the duodenum. Restless legs syndrome
(RLS) is a sensorimotor disorder that includes an urge to move legs, accompanied
or caused by uncomfortable and unpleasant sensations in the legs; the urge to
move begins or worsens during periods of rest or inactivity, the urge to move is
partially or totally relieved by movement, and the urge is worse or only occurs
at night. In the majority of the restless leg syndrome population, the sensation
is deep seated, often described as being in the shin bones, and most commonly
felt between the knee and ankle. It may be described as a creepy, shock-like,
tense, electric, buzzing, itchy, or even numb sensation. A subpopulation of this
restless leg syndrome patient population experiences restless leg syndrome
associated pain (RLSAP) that has been described as a deep "achy pain." This pain
has not been found to be relieved by many of the typical over the counter
analgesics. Often, constant movement of the legs appears to be the only remedy,
as these sensations usually appear during periods of rest. Furthermore, there
appears to be an association between iron deficiency and those suffering from
Restless Leg Syndrome (RLS). The authors theorize that there may be a possible
correlation between PPIs and the symptoms (e.g. pain) associated with RLS. The
authors propose that PPIs, such as omeprazole, may interfere with iron
absorption in certain patients and that a subpopulation of patients who develop
significant iron deficiency characterized by low serum ferritin levels while on
PPIs may also develop RLS-like symptoms (including RLSAP). While there is no
robust direct evidence to support any associations of PPIs and iron deficiency
or PPIs associated with RLS-like symptoms (including RLSAP), it is hoped that
this manuscript may spark research efforts on this issue. BACKGROUND AND OBJECTIVES: Iron depletion is common in regular blood donors. The
objective of the study was to investigate the frequency and severity of iron
depletion in regular blood donors and whether IV iron is more effective than
oral to avoid iron depletion and symptoms thereof, especially restless legs
syndrome (RLS).
METHOD: One hundred and twenty blood donors with at least five previous whole
blood donations were randomized to receive either IV iron sucrose (Venofer(®),
RenaPharma/Vifor, Uppsala, Sweden), 200 mg, or to 20×100 mg of oral iron
sulphate (Duroferon(®), GlaxoSmithKline, Stockholm, Sweden), after each blood
donation during 1 year. Iron status and RLS incidence and severity were
investigated.
RESULTS: Iron status was generally poor among regular blood donors, especially
in women, with a high incidence of iron depletion (>20%) and RLS (18%). The IV
iron group increased storage iron to a greater extent than the oral iron group
after 12 months (P=0·0043). Female donors were more responsive to IV iron
sucrose compared to oral iron sulphate, particularly female donors below 50
years of age. RLS severity scores were significantly lower in the IV iron group.
The two treatments were safe.
CONCLUSION: Iron status is poor in regular blood donors, restless legs syndrome
is common, and the routine iron supplementation is insufficient. IV iron sucrose
substitutes iron loss in blood donors more efficiently compared with oral iron
sulphate, especially in women. Iron substitution to blood donors should be
individualized and based on P-ferritin monitoring. BACKGROUND: Iron deficiency is a frequent side effect of blood donation. In
recent years, several studies have described genetic variants associated with
iron concentrations. However, the impact of these variants on iron levels is
unknown in blood donors. Knowledge of genetic variants that predispose donors to
iron deficiency would allow bleeding frequency and iron supplementation to be
tailored to the individual donor.
STUDY DESIGN AND METHODS: The genotypes of five specific single-nucleotide
polymorphisms (SNPs) in three genes that have been previously associated with
iron status and/or restless leg syndrome (RLS) were investigated in two groups
of female blood donors. The first group had low iron stores (serum ferritin ≤
12 µg/L, n = 657), and the second group had normal to high iron stores (serum
ferritin > 30 µg/L, n = 645). Genotype distribution for each of the SNPs was
compared between the two groups.
RESULTS: Homozygosity for the T-allele of BTBD9 rs9296249 was associated with
lower serum ferritin. The odds ratio for low serum ferritin was 1.35 (95%
confidence interval, 1.02-1.77; p = 0.03) when comparing donors with the TT
genotype with donors with the CT genotype.
CONCLUSION: A frequent polymorphism in BTBD9 was significantly associated with
serum ferritin. This polymorphism has previously been associated with RLS, but
not low iron stores in blood donors. The ubiquitously expressed iron storage protein ferritin plays a central role in
maintaining cellular iron homeostasis. Cytosolic ferritins are composed of heavy
(H) and light (L) subunits that co-assemble into a hollow spherical shell with
an internal cavity where iron is stored. The ferroxidase activity of the
ferritin H chain is critical to store iron in its Fe3+ oxidation state, while
the L chain shows iron nucleation properties. We describe a unique case of a
23-yr-old female patient affected by a homozygous loss of function mutation in
the L-ferritin gene, idiopathic generalized seizures, and atypical restless leg
syndrome (RLS). We show that L chain ferritin is undetectable in primary
fibroblasts from the patient, and thus ferritin consists only of H chains.
Increased iron incorporation into the FtH homopolymer leads to reduced cellular
iron availability, diminished levels of cytosolic catalase, SOD1 protein levels,
enhanced ROS production and higher levels of oxidized proteins. Importantly, key
phenotypic features observed in fibroblasts are also mirrored in reprogrammed
neurons from the patient's fibroblasts. Our results demonstrate for the first
time the pathophysiological consequences of L-ferritin deficiency in a human and
help to define the concept for a new disease entity hallmarked by idiopathic
generalized seizure and atypical RLS. |
List the results of mutated casein kinase 1 epsilon. | Mutation in casein kinase 1 epsilon results in a short circadian period, abnormal entrainment to light cycles, and potentiated resetting responses to light.
Mutations of CK1epsilon found in breast cancer can suppress Wnt/beta-catenin as well as promote the Wnt/Rac-1/JNK and Wnt/NFAT pathways, thus contributing to breast cancer development via effects on cell adhesion and migration.
Csnk1e is regulating not only the timing of sleep, but also the REM sleep amount and NREM sleep architecture. | The vertebrate circadian clock was thought to be highly localized to specific
anatomical structures: the mammalian suprachiasmatic nucleus (SCN), and the
retina and pineal gland in lower vertebrates. However, recent findings in the
zebrafish, rat and in cultured cells have suggested that the vertebrate
circadian timing system may in fact be highly distributed, with most if not all
cells containing a clock. Our understanding of the clock mechanism has
progressed extensively through the use of mutant screening and forward genetic
approaches. The first vertebrate clock gene was identified only a few years ago
in the mouse by such an approach. More recently, using a syntenic comparative
genetic approach, the molecular basis of the the tau mutation in the hamster was
determined. The tau gene in the hamster appears to encode casein kinase 1
epsilon, a protein previously shown to be important for PER protein turnover in
the Drosophila circadian system. A number of additional clock genes have now
been described. These proteins appear to play central roles in the
transcription-translation negative feedback loop responsible for clock function.
Post-translational modification, protein dimerization and nuclear transport all
appear to be essential features of how clocks are thought to tick. The tau mutation in the Syrian hamster resides in the enzyme casein kinase 1
epsilon (CK1epsilon), resulting in a dramatic acceleration of wheel-running
activity cycles to about 20 hours. tau also impacts growth, energy, metabolism,
feeding behavior, and circadian mechanisms underpinning seasonal timing, causing
accelerated reproductive and neuroendocrine responses to photoperiodic changes.
Modeling and experimental studies suggest that tau acts as a gain of function on
specific residues of PER, consistent with hamster studies showing accelerated
degradation of PER in the suprachiasmatic nucleus in the early circadian night.
We have created null and tau mutants of Ck1epsilon in mice. Circadian period
lengthens in CK1epsilon(/), whereas CK1epsilon(tau/tau) shortens circadian
period of behavior in vivo in a manner nearly identical to that of the Syrian
hamster. CK1epsilon(tau/tau) also accelerates molecular oscillations in
peripheral tissues, demonstrating its global circadian role. CK1epsilon(tau)
acts by promoting degradation of both nuclear and cytoplasmic PERIOD, but not
CRYPTOCHROME, proteins. Our studies reveal that tau acts as a gain-of-function
mutation, to accelerate degradation of PERIOD proteins. tau has consistent
effects in both hamsters and mice on the circadian organization of behavior and
metabolism, highlighting the global impact of this mutation on mammalian
clockwork in brain and periphery. Circadian rhythms rely on the interaction of highly conserved
transcription-translation loops. Casein kinase I epsilon (CK1epsilon)
post-transcriptionally regulates circadian rhythms by phosphorylating clock
genes, and the tau mutation, an arginine to cysteine substitution at residue
128, results in a short circadian period, abnormal entrainment to light cycles,
and potentiated resetting responses to light. Each of these effects could be
attributed to changes in the regulation of the core molecular circadian loops.
We now demonstrate that the mutation results in a heightened sensitivity to
light, suggesting that CK1epsilon also regulates the photic entrainment pathway. INTRODUCTION: Breast cancer is one of the most common types of cancer in women.
One of the genes that were found mutated in breast cancer is casein kinase 1
epsilon (CK1epsilon). Because CK1epsilon is a crucial regulator of the Wnt
signaling cascades, we determined how these CK1epsilon mutations interfere with
the Wnt pathway and affect the behavior of epithelial breast cancer cell lines.
METHODS: We performed in silico modeling of various mutations and analyzed the
kinase activity of the CK1epsilon mutants both in vitro and in vivo.
Furthermore, we used reporter and small GTPase assays to identify how mutation
of CK1epsilon affects different branches of the Wnt signaling pathway. Based on
these results, we employed cell adhesion and cell migration assays in MCF7 cells
to demonstrate a crucial role for CK1epsilon in these processes.
RESULTS: In silico modeling and in vivo data showed that autophosphorylation at
Thr 44, a site adjacent to the breast cancer point mutations in the N-terminal
lobe of human CK1epsilon, is involved in positive regulation of the CK1epsilon
activity. Our data further demonstrate that, in mammalian cells, mutated forms
of CK1epsilon failed to affect the intracellular localization and
phosphorylation of Dvl2; we were able to demonstrate that CK1epsilon mutants
were unable to enhance Dvl-induced TCF/LEF-mediated transcription, that
CK1epsilon mutants acted as loss-of-function in the Wnt/beta-catenin pathway,
and that CK1epsilon mutants activated the noncanonical Wnt/Rac-1 and NFAT
pathways, similar to pharmacological inhibitors of CK1. In line with these
findings, inhibition of CK1 promoted cell migration as well as decreased cell
adhesion and E-cadherin expression in the breast cancer-derived cell line MCF7.
CONCLUSIONS: In summary, these data suggest that the mutations of CK1epsilon
found in breast cancer can suppress Wnt/beta-catenin as well as promote the
Wnt/Rac-1/JNK and Wnt/NFAT pathways, thus contributing to breast cancer
development via effects on cell adhesion and migration. In terms of molecular
mechanism, our data indicate that the breast cancer point mutations in the
N-terminal lobe of CK1epsilon, which are correlated with decreased
phosphorylation activities of mutated forms of CK1epsilon both in vitro and in
vivo, interfere with positive autophosphorylation at Thr 44. |
Does neuroglobin has neuroprotective properties in the setting of traumatic brain injury? | Yes, neuroglobin has neuroprotective properties in the setting of traumatic brain injury. | Neuroglobin has shown rich neuroprotective effects against cerebral hypoxia, and
therefore has the potential to impact outcomes after traumatic brain injury
(TBI). However, to date an association between genetic variation within the
human neuroglobin (NGB) gene and recovery post-TBI has not been reported. The
purpose of this study was to explore the relationship between NGB genotypes and
outcomes (as assessed by the Glasgow Outcome Scale [GOS], the Disability Rating
Scale [DRS], and the Neurobehavioral Rating Scale-Revised [NRS-R]) after severe
TBI. Genotyping using TaqMan allele discrimination for two tagging single
nucleotide polymorphisms (tSNPs) that represent the two haplotype blocks for NGB
(rs3783988 and rs10133981) was completed on DNA obtained from 196 Caucasian
patients recovering from severe TBI. Patients were dichotomized based on the
presence of the variant allele for each tSNP. Chi-square and Fisher's exact
tests were used to compare characteristics between groups. Multivariate linear
regression was used to examine NGB tSNPs and recovery from severe TBI. Subjects
with the TT genotype (wild-type) for rs3783988 were more likely to have better
GOS and DRS scores at 3, 6, 12, and 24 months, while rs10133981 genotype was not
significantly related to functional outcome. After controlling for age, gender,
and Glasgow Coma Scale (GCS) score, those subjects with the rs3783988 TT
genotype had more than a 2.65-times greater likelihood of better functional
outcomes than individuals with genotypes harboring a variant allele. Data
suggest that the haplotype block represented by rs3783988 in NGB appears to
influence recovery after severe TBI. Represented within this haplotype block of
NGB is the region that codes for the oxygen-binding portion of NGB. OBJECTIVE: To study the expression changes of neuroglobin in rats with the model
of diffuse traumatic brain injury and explore the relationship between the
neuroglobin and neuron apoptosis in traumatic brain injury.
METHODS: The diffuse traumatic brain injury of rats was induced by the
Marmarou's 'weight-drop' device. And the immunohistochemical technique was used
to detect the expression changes of neuroglobin and neuron apoptosis in rat
brain at different time points post-injury.
RESULTS: The expression of neuroglobin increased twice and reached peaks at 2
hours and 72 hours post-injury respectively. And the increased expression of
neuroglobin from 30 minutes to 1 hour post-injury and from 48 hours to 72 hours
post-injury accompanied with the decreased expression ratio of Bax to Bcl-2.
CONCLUSION: The increased expression of neuroglobin in traumatic brain injury
informed us that neuroglobin had anti-apoptosis action in post-injury neuron. It
could protect the neuron from traumatic stress and secondary ischemia and
hypoxia insults during ultra-early and acute stages. Neuroglobin (NGB) is a recently discovered globin, which is widely expressed in
vertebrates central and peripheral nervous systems. Previous studies have shown
that NGB is important in protecting neurons from hypoxic/ischemic brain
injuries. However, there are no reports on the neuroprotective effects of NGB
after mechanical injury. Currently, we showed that the NGB expression level in
neurons increased continuously from 2 h after injury, and reached a peak at 16 h
(p<0.01), after which it decreased sharply. NGB that was overexpressed in
mechanically injured B104 cells showed significant neuroprotective effects.
Lactate dehydrogenase (LDH) activity decreased and cell survival rates increased
(p<0.01, n=5). In the rat model of focal brain trauma, the NGB expression
increased sharply at 1 h, after which it increased continuously until it reached
a peak at 6 h, and then gradually decreased (p<0.01, n=5). Furthermore, moderate
and severe injury resulted in significantly higher NGB levels than did mild
injury (p<0.01, n=5). Our results indicate that NGB exerts significant
neuroprotective effects after mechanical injury, and thus has important
implications for the prognosis and cure of traumatic brain injury. BACKGROUND: Accumulating evidence has demonstrated that over-expression of
Neuroglobin (Ngb) is neuroprotective against hypoxic/ischemic brain injuries. In
this study we tested the neuroprotective effects of Ngb over-expression against
traumatic brain injury (TBI) in mice.
RESULTS: Both Ngb over-expression transgenic (Ngb-Tg) and wild-type (WT) control
mice were subjected to TBI induced by a controlled cortical impact (CCI) device.
TBI significantly increased Ngb expression in the brains of both WT and Ngb-Tg
mice, but Ngb-Tg mice had significantly higher Ngb protein levels at the
pre-injury baseline and post-TBI. Production of oxidative tissue damage
biomarker 3NT in the brain was significantly reduced in Ngb-Tg mice compared to
WT controls at 6 hours after TBI. The traumatic brain lesion volume was
significantly reduced in Ngb Tg mice compared to WT mice at 3 weeks after TBI;
however, there were no significant differences in the recovery of sensorimotor
and spatial memory functional deficits between Ngb-Tg and WT control mice for up
to 3 weeks after TBI.
CONCLUSION: Ngb over-expression reduced traumatic lesion volume, which might
partially be achieved by decreasing oxidative stress. OBJECTIVE: This study aims to investigate the potentially protective effect of
neuroglobin (Ngb) gene-modified bone marrow mesenchymal stem cells (BMSCs) on
traumatic spinal cord injury (SCI) in rabbits.
METHODS: A lentiviral vector containing an Ngb gene was constructed and used to
deliver Ngb to BMSCs. Ngb gene-modified BMSCs were then injected at the SCI
sites 24 hours after SCI. The motor functions of the rabbits were evaluated by
the Basso-Beattie-Bresnahan rating scale. Fluorescence microscopy, quantitative
real-time PCRs, Western blots, malondialdehyde (MDA) tests, and terminal
deoxynucleotidyltransferase-mediated UTP end labeling assays were also
performed.
RESULTS: Ngb expression in the Ngb-BMSC group increased significantly. A more
significant functional improvement was observed in the Ngb-BMSC group compared
with those in the other groups. Traumatic SCI seemingly led to an increase in
MDA level and number of apoptotic cells, which can be prevented by Ngb-BMSC
treatment.
CONCLUSION: This study demonstrates that Ngb gene-modified BMSCs can strengthen
the therapeutic benefits of BMSCs in reducing secondary damage and improving the
neurological outcome after traumatic SCI. Therefore, the combined strategy of
BMSC transplantation and Ngb gene therapy can be used to treat traumatic SCI. There is a significant need for novel treatments that will improve traumatic
brain injury (TBI) outcomes. One potential neuroprotective mechanism is to
increase oxygen binding proteins such as neuroglobin. Neuroglobin has a high
affinity for oxygen, is an effective free radical scavenger, and is
neuroprotective within the brain following hypoxia and ischemia. The purpose of
this study was to determine whether neuroglobin overexpression improves
sensorimotor outcomes following TBI in transgenic neuroglobin overexpressing
(NGB) mice. Additional study aims were to determine if and when an endogenous
neuroglobin response occurred following TBI in wild-type (WT) mice, and in what
brain regions and cell types the response occurred. Controlled cortical impact
(CCI) was performed in adult (5 month) C57/BL6 WT mice, and NGB mice
constitutively overexpressing neuroglobin via the chicken beta actin promoter
coupled with the cytomegalovirus distal enhancer. The gridwalk task was used for
sensorimotor testing of both WT and NGB mice, prior to injury, and at 2, 3, and
7 days post-TBI. NGB mice displayed significant reductions in the average number
of foot faults per minute walking at 2, 3, and 7 days post-TBI when compared to
WT mice at each time point. Neuroglobin mRNA expression was assessed in the
injured cortex of WT mice prior to injury, and at 1, 3, 7, and 14 days post-TBI
using quantitative real time polymerase chain reaction (qRT-PCR). Neuroglobin
mRNA was significantly increased at 7 days post-TBI. Immunostaining showed
neuroglobin primarily localized to neurons and glial cells in the injured cortex
and ipsilateral hippocampus of WT mice, while neuroglobin was present in all
brain regions of NGB mice at 7 days post-TBI. These results showed that
overexpression of neuroglobin reduced sensorimotor deficits following TBI, and
that an endogenous increase in neuroglobin expression occurs during the subacute
period. Increasing neuroglobin expression through novel therapeutic
interventions during the acute period after TBI may improve recovery. In this study, we used a rat model of severe closed traumatic brain injury to
explore the relationship between neuroglobin, brain injury and neuronal
apoptosis. Real-time PCR showed that neuroglobin mRNA expression rapidly
increased in the rat cerebral cortex, and peaked at 30 minutes and 48 hours
following traumatic brain injury. Immunohistochemical staining demonstrated that
neuroglobin expression increased and remained high 2 hours to 5 days following
injury. The rate of increase in the apoptosis-related Bax/Bcl-2 ratio greatly
decreased between 30 minutes and 1 hour as well as between 48 and 72 hours post
injury. Expression of neuroglobin and the anti-apoptotic factor Bcl-2 greatly
increased, while that of the proapoptotic factor decreased, in the cerebral
cortex post severe closed traumatic brain injury. It suggests that neuroglobin
might protect neurons from apoptosis after traumatic injury by regulating
Bax/Bcl-2 pathway. |
What gene test is recommended for clopidogrel? | The genetic test recommended for clopidogrel is CYP2C19 genotyping. | OBJECTIVES: The aim of this study was to evaluate the effect of polymorphisms
affecting the clopidogrel metabolism (CYP3A4 IVS10+12G/A and CYP2C19*2) and the
P2Y12 receptor (P2Y12 T744C) on modulating platelet function in acute coronary
syndrome patients on dual antiplatelet treatment.
BACKGROUND: Residual platelet reactivity (RPR) phenomenon on antiplatelet
therapy requires clarification. P2Y12 T744C, CYP3A4 IVS10+12G/A and, in healthy
individuals only, CYP2C19*2 polymorphisms have been investigated; however, the
influence on platelet reactivity in a large population of high-risk vascular
patients on dual antiplatelet treatment has not yet been elucidated.
METHODS: A total of 1419 acute coronary syndrome patients on dual antiplatelet
treatment were studied. Platelet function was evaluated by platelet-rich plasma
aggregation. Electronic ochips and restriction-fragment length polymorphism
were used for analysis of polymorphisms.
RESULTS: Only CYP2C19*2, out of the three investigated polymorphisms, is
associated with higher platelet reactivity. Carriers of the *2 allele had
significantly higher platelet aggregation values after arachidonic acid (AA;
P=0.043), 2 micromol/l adenosine 5' diphosphate (ADP; P<0.0001) and 10
micromol/l ADP (P=0.001) stimuli. The genotype distribution of CYP2C19*2
polymorphism significantly differed between patients with and without RPR, as
evaluated by 10-micromol/l ADP-induced platelet aggregation (P=0.002) and by
AA-induced platelet aggregation (P=0.045). At the multivariate linear regression
analysis, the CYP2C19*2 polymorphism remained a significant and independent risk
factor for dual antiplatelet treatment variability.
CONCLUSIONS: This study demonstrates, for the first time, that the *2 CYP2C19
allele is associated with higher platelet aggregability and RPR in high-risk
vascular patients on dual antiplatelet treatment. These findings can have a
significant impact on the future design of pharmacogenetic antiaggregant
strategies for high-risk vascular patients on dual antiplatelet treatment. CONTEXT: Clopidogrel therapy improves cardiovascular outcomes in patients with
acute coronary syndromes and following percutaneous coronary intervention by
inhibiting adenosine diphosphate (ADP)-dependent platelet activation. However,
nonresponsiveness is widely recognized and is related to recurrent ischemic
events.
OBJECTIVE: To identify gene variants that influence clopidogrel response.
DESIGN, SETTING, AND PARTICIPANTS: In the Pharmacogenomics of Antiplatelet
Intervention (PAPI) Study (2006-2008), we administered clopidogrel for 7 days to
429 healthy Amish persons and measured response by ex vivo platelet
aggregometry. A genome-wide association study was performed followed by
genotyping the loss-of-function cytochrome P450 (CYP) 2C19*2 variant
(rs4244285). Findings in the PAPI Study were extended by examining the relation
of CYP2C19*2 genotype to platelet function and cardiovascular outcomes in an
independent sample of 227 patients undergoing percutaneous coronary
intervention.
MAIN OUTCOME MEASURE: ADP-stimulated platelet aggregation in response to
clopidogrel treatment and cardiovascular events.
RESULTS: Platelet response to clopidogrel was highly heritable (h(2) = 0.73; P <
.001). Thirteen single-nucleotide polymorphisms on chromosome 10q24 within the
CYP2C18-CYP2C19-CYP2C9-CYP2C8 cluster were associated with diminished
clopidogrel response, with a high degree of statistical significance (P = 1.5 x
10(-13) for rs12777823, additive model). The rs12777823 polymorphism was in
strong linkage disequilibrium with the CYP2C19*2 variant, and was associated
with diminished clopidogrel response, accounting for 12% of the variation in
platelet aggregation to ADP (P = 4.3 x 10(-11)). The relation between CYP2C19*2
genotype and platelet aggregation was replicated in clopidogrel-treated patients
undergoing coronary intervention (P = .02). Furthermore, patients with the
CYP2C19*2 variant were more likely (20.9% vs 10.0%) to have a cardiovascular
ischemic event or death during 1 year of follow-up (hazard ratio, 2.42; 95%
confidence interval, 1.18-4.99; P = .02).
CONCLUSION: CYP2C19*2 genotype was associated with diminished platelet response
to clopidogrel treatment and poorer cardiovascular outcomes. BACKGROUND: The cytochrome P450 (CYP) 2C19 isoenzyme plays an important role in
clopidogrel metabolization. A recently explored CYP2C19*17 allelic variant has
been linked to increased transcriptional activity, resulting in extensive
metabolization of CYP2C19 substrates, which may lead to an enhanced platelet
response to clopidogrel treatment. The aim of this study was to assess the
impact of CYP2C19*17 on ADP-induced platelet aggregation, the risk of bleeding,
and stent thrombosis in clopidogrel-treated patients undergoing percutaneous
coronary intervention.
METHODS AND RESULTS: The study population included 1524 patients undergoing
percutaneous coronary intervention after pretreatment with 600 mg clopidogrel.
Genotypes were determined with a TaqMan assay. ADP-induced platelet aggregation
was assessed on a Multiplate analyzer. The primary clinical safety end point was
the 30-day incidence of bleeding defined according to Thrombolysis in Myocardial
Infarction criteria, and the primary clinical efficacy end point was the 30-day
incidence of stent thrombosis. For both heterozygous (*wt/*17; n=546) and
homozygous (*17/*17; n=76) allele carriers, significantly lower ADP-induced
platelet aggregation values were found compared with wild-type homozygotes
(*wt/*wt; n=902; P=0.039 and P=0.008, respectively). CYP2C19*17 allele carriage
was significantly associated with an increased risk of bleeding; the highest
risk was observed for CYP2C19*17 homozygous patients (P=0.01, chi(2) test for
trend). Multivariate analysis confirmed the independent association of
CYP2C19*17 allele carriage with platelet aggregation values (P<0.001) and the
occurrence of bleeding (P=0.006). No significant influence of CYP2C19*17 on the
occurrence of stent thrombosis was found (P=0.79).
CONCLUSIONS: CYP2C19*17 carrier status is significantly associated with enhanced
response to clopidogrel and an increased risk of bleeding. The cytochrome P450 (CYP) 2C19*2 loss-of-function allele has been associated
with impaired clopidogrel response and worse prognosis in clopidogrel-treated
patients. The benefit of tailored therapy according to platelet function test
results remains unclear, and the potential effect of genotypes on this benefit
has not been addressed in unstable patients. The present study was designed to
evaluate the benefit of tailored therapy with a higher maintece dose
according to CYP2C19 genotypes in patients identified as nonresponders who
underwent percutaneous coronary intervention for non-ST-segment elevation acute
coronary syndromes. Three hundred forty-six consecutive patients were enrolled
and received a loading dose of 600 mg, including 86 *2 carriers (13 homozygotes
and 73 heterozygotes) and 260 *2 noncarriers. Clopidogrel response, assessed
with platelet reactivity index vasoactive-stimulated phosphoprotein, was
significantly affected by genotype, with lower clopidogrel response in CYP2C19*2
allele carriers (p = 0.01). Accordingly, the rate of clopidogrel nonresponse was
higher in CYP2C19*2 allele carriers (53% vs 41%, p = 0.04). All clopidogrel
nonresponders (n = 151), including 105 *2 noncarriers and 46 *2 carriers,
received high 150-mg clopidogrel maintece doses at discharge to overcome
initial low response. After 1 month, high maintece doses overcame clopidogrel
low response in only 44% of the whole population and significantly less
frequently in *2 carriers than in noncarriers (28% vs 50%, p = 0.01). In
conclusion, higher clopidogrel maintece doses were able to overcome
clopidogrel low response in fewer than half of clopidogrel low responders who
underwent percutaneous coronary intervention for non-ST-segment elevation acute
coronary syndromes. The benefit of this tailored therapy was significantly
reduced in CYP2C19*2 carriers. Therefore, these patients might require
alternative strategies with new P2Y₁₂ blockers. CONTEXT: Variants in the CYP2C19 gene influence the pharmacologic and clinical
response to the standard 75-mg daily maintece dose of the antiplatelet drug
clopidogrel.
OBJECTIVE: To test whether higher doses (up to 300 mg daily) improve the
response to clopidogrel in the setting of loss-of-function CYP2C19 genotypes.
DESIGN, SETTING, AND PATIENTS: ELEVATE-TIMI 56 was a multicenter, randomized,
double-blind trial that enrolled and genotyped 333 patients with cardiovascular
disease across 32 sites from October 2010 until September 2011.
INTERVENTIONS: Maintece doses of clopidogrel for 4 treatment periods, each
lasting approximately 14 days, based on genotype. In total, 247 noncarriers of a
CYP2C19*2 loss-of-function allele were to receive 75 and 150 mg daily of
clopidogrel (2 periods each), whereas 86 carriers (80 heterozygotes, 6
homozygotes) were to receive 75, 150, 225, and 300 mg daily.
MAIN OUTCOME MEASURES: Platelet function test results (vasodilator-stimulated
phosphoprotein [VASP] phosphorylation and VerifyNow P2Y(12) assays) and adverse
events.
RESULTS: With 75 mg daily, CYP2C19*2 heterozygotes had significantly higher
on-treatment platelet reactivity than did noncarriers (VASP platelet reactivity
index [PRI]: mean, 70.0%; 95% CI, 66.0%-74.0%, vs 57.5%; 95% CI, 55.1%-59.9%,
and VerifyNow P2Y(12) reaction units [PRU]: mean, 225.6; 95% CI, 207.7-243.4, vs
163.6; 95% CI, 154.4-173.9; P < .001 for both comparisons). Among CYP2C19*2
heterozygotes, doses up to 300 mg daily significantly reduced platelet
reactivity, with VASP PRI decreasing to 48.9% (95% CI, 44.6%-53.2%) and PRU to
127.5 (95% CI, 109.9-145.2) (P < .001 for trend across doses for both). Whereas
52% of CYP2C19*2 heterozygotes were nonresponders (≥230 PRU) with 75 mg of
clopidogrel, only 10% were nonresponders with 225 or 300 mg (P < .001 for both).
Clopidogrel, 225 mg daily, reduced platelet reactivity in CYP2C19*2
heterozygotes to levels achieved with standard clopidogrel, 75 mg, in
noncarriers (mean ratios of platelet reactivity, VASP PRI, 0.92; 90% CI,
0.85-0.99, and PRU, 0.94; 90% CI, 0.84-1.04). In CYP2C19*2 homozygotes, even
with 300 mg daily of clopidogrel, mean VASP PRI was 68.3% (95% CI, 44.9%-91.6%)
and mean PRU, 287.0 (95% CI, 170.2-403.8).
CONCLUSION: Among patients with stable cardiovascular disease, tripling the
maintece dose of clopidogrel to 225 mg daily in CYP2C19*2 heterozygotes
achieved levels of platelet reactivity similar to that seen with the standard
75-mg dose in noncarriers; in contrast, for CYP2C19*2 homozygotes, doses as high
as 300 mg daily did not result in comparable degrees of platelet inhibition.
TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01235351. BACKGROUND: The antiplatelet effect of clopidogrel has been linked to cytochrome
P450 2C19 (CYP2C19) carrier status. The presence of loss of function and gain of
function variants were found to have a gene-dose effect on clopidogrel
metabolism. However, genotyping is only one aspect of predicting response to
clopidogrel and several platelet function tests are available to measure
platelet response. Patients and methods We studied the influence of CYP2C19
allelic variants on on-treatment platelet reactivity as assessed by light
transmission aggregometry (LTA), the VerifyNow P2Y12 assay, the VASP assay,
multiple electrode aggregometry (MEA), and the Impact-R in 288 patients after
stenting for cardiovascular disease. Allelic variants of CYP2C19 were determined
with the Infiniti® CYP450 2C19+ assay and categorized into four metabolizer
states (ultrarapid, extensive, intermediate, poor).
RESULTS: Platelet reactivity increased linearly from ultrarapid to poor
metabolizers using the VerifyNow P2Y12 assay (P = 0.04), the VASP assay (P =
0.02) and the Impact-R (P = 0.04). The proportion of patients with high
on-treatment residual platelet reactivity (HRPR) identified by LTA, the
VerifyNow P2Y12 assay and the VASP assay increased when the metabolizer status
decreased, while no such relationship could be identified for results of MEA and
Impact-R. The presence of loss of function variants (*2/*2, *2-8*/wt, *2/*17)
was an independent predictor of HRPR in LTA and the VASP assay while it did not
reach statistical significance in the VerifyNow P2Y12 assay, MEA, and the
Impact-R.
CONCLUSION: Depending on the type of platelet function test differences in the
association of on-treatment platelet reactivity with CYP2C19 carrier status are
observed. BACKGROUND: Prospective assessment of pharmacogenetic strategies has been
limited by an inability to undertake bedside genetic testing. The CYP2C19*2
allele is a common genetic variant associated with increased rates of major
adverse events in individuals given clopidogrel after percutaneous coronary
intervention (PCI). We used a novel point-of-care genetic test to identify
carriers of the CYP2C19*2 allele and aimed to assess a pharmacogenetic approach
to dual antiplatelet treatment after PCI.
METHODS: Between Aug 26, 2010, and July 7, 2011, 200 patients were enrolled into
our prospective, randomised, proof-of-concept study. Patients undergoing PCI for
acute coronary syndrome or stable angina were randomly assigned to rapid
point-of-care genotyping or to standard treatment. Individuals in the rapid
genotyping group were screened for the CYP2C19*2 allele. Carriers were given 10
mg prasugrel daily, and non-carriers and patients in the standard treatment
group were given 75 mg clopidogrel daily. The primary endpoint was the
proportion of CYP2C19*2 carriers with high on-treatment platelet reactivity
(P2Y12 reactivity unit [PRU] value of more than 234) after 1 week of dual
antiplatelet treatment, which is a marker associated with increased adverse
cardiovascular events. Interventional cardiologists and data analysts were
masked to genetic status and treatment. Patients were not masked to treatment
allocation. All analyses were by intention to treat. This study is registered
with ClinicalTrials.gov, NCT01184300.
FINDINGS: After randomisation, 187 patients completed follow-up (91 rapid
genotyping group, 96 standard treatment). 23 individuals in each group carried
at least one CYP2C19*2 allele. None of the 23 carriers in the rapid genotyping
group had a PRU value of more than 234 at day 7, compared with seven (30%) given
standard treatment (p=0·0092). The point-of-care genetic test had a sensitivity
of 100% (95% CI 92·3-100) and a specificity of 99·3% (96·3-100).
INTERPRETATION: Point-of-care genetic testing after PCI can be done effectively
at the bedside and treatment of identified CYP2C19*2 carriers with prasugrel can
reduce high on-treatment platelet reactivity.
FUNDING: Spartan Biosciences. OBJECTIVES: This study sought to evaluate the influence of single nucleotide
polymorphisms (SNPs) on the pharmacodynamic effect of high- or standard-dose
clopidogrel after percutaneous coronary intervention (PCI).
BACKGROUND: There is a lack of prospective, multicenter data regarding the
effect of different genetic variants on clopidogrel pharmacodynamics over time
in patients undergoing PCI.
METHODS: The GRAVITAS (Gauging Responsiveness with A VerifyNow assay-Impact on
Thrombosis And Safety) trial screened patients with platelet function testing
after PCI and randomly assigned those with high on-treatment reactivity (OTR) to
either high- or standard-dose clopidogrel; a cohort of patients without high OTR
were also followed. DNA samples obtained from 1,028 patients were genotyped for
41 SNPs in 17 genes related to platelet reactivity. After adjusting for clinical
characteristics, the associations between the SNPs and OTR using linear
regression were evaluated.
RESULTS: CYP2C19*2 was significantly associated with OTR at 12 to 24 h (R(2) =
0.07, p = 2.2 × 10(-15)), 30 days (R(2) = 0.10, p = 1.3 × 10(-7)), and 6 months
after PCI (R(2) = 0.07, p = 1.9 × 10(-11)), whereas PON1, ABCB1 3435 C→T, and
other candidate SNPs were not. Carriers of 1 and 2 reduced-function CYP2C19
alleles were significantly more likely to display persistently high OTR at 30
days and 6 months, irrespective of treatment assignment. The portion of the risk
of persistently high OTR at 30 days attributable to reduced-function CYP2C19
allele carriage was 5.2% in the patients randomly assigned to high-dose
clopidogrel.
CONCLUSIONS: CYP2C19, but not PON1 or ABCB1, is a significant determit of the
pharmacodynamic effects of clopidogrel, both early and late after PCI. In
patients with high OTR identified by platelet function testing, the CYP2C19
genotype provides limited incremental information regarding the risk of
persistently high reactivity with clopidogrel 150-mg maintece dosing.
(Genotype Information and Functional Testing Study [GIFT]; NCT00992420). BACKGROUND: A recent clinical trial has demonstrated that patients with acute
coronary syndromes (ACS) and the reduced function allele CYP2C19*2 (*2 allele),
who are treated with thienopyridines, have an increased risk of adverse cardiac
events with clopidogrel, but not with prasugrel. The frequency of the *2 allele
varies by ethnicity and the Maoris, Asians and Pacific Islanders of New Zealand
have a relatively high incidence.
OBJECTIVE: Our objective was to evaluate, from a New Zealand health system
perspective, the cost effectiveness of treating all ACS patients with generic
clopidogrel compared with prasugrel, and also compared with the genetically
guided strategy that *2 allele carriers receive prasugrel and non-carriers
receive clopidogrel.
METHODS: A decision-tree model consisting of five health states (myocardial
infarction, stroke, bleeding, stent thrombosis and cardiovascular death) was
developed. Clinical outcome data (two TRITON-TIMI 38 genetic sub-studies)
comparing clopidogrel and prasugrel for both *2 allele carriers and non-carriers
were combined with the prevalence of the heterozygosity for the *2 allele in New
Zealand Europeans (15%), Maoris (24%), Asians (29%) and Pacific Islanders (45%)
to determine the predicted adverse event rate for the New Zealand population.
National hospital diagnosis-related group (DRG) discharge codes were used to
determine alternative adverse event rates, along with the costs of
hospitalizations during the 15 months after patients presented with an ACS. The
primary outcome measure was the incremental cost per QALY (calculated using
literature-reported weights). Monte Carlo simulations and alternative scenario
analysis based on both clinical trial and national hospital incidence were used.
Additional analysis considered the overall TRITON-TIMI 38 rates. Costs (in New
Zealand dollars [$NZ], year 2009 values) and benefits were discounted at 3% per
annum.
RESULTS: Actual hospital-based adverse event rates were higher than those
reported in the TRITON-TIMI 38 randomized controlled trial and the genetic
sub-studies, especially for myocardial infarction and cardiovascular death, and
for Maoris and Pacific Islanders. For both sources of adverse event rates,
treating the population with prasugrel was associated with worse outcomes
(QALYs) than clopidogrel. However, prasugrel became cost effective
($NZ31 751/QALY) when the overall TRITON-TIMI 38 rates were used. A genetic test
to guide the selected use of prasugrel was cost effective ($NZ8702/QALY versus
$NZ24 617/QALY) for hospital and clinical trial incidence, respectively. Based
on the hospital rates, the genetically guided strategy was especially cost
effective for Maoris ($NZ7312/QALY) and Pacific Islanders ($NZ7041/QALY). These
results were robust to the sensitivity analysis, except the genetically guided
strategy under the 15-month clinical trial event rate scenario ($NZ168 748/QALY)
did not remain cost effective under a $NZ50 000 threshold.
CONCLUSIONS: Use of a genetic test to guide thienopyridine treatment in patients
with ACS is a potentially cost-effective treatment strategy, especially for
Maoris and Pacific Islanders. This treatment strategy also has the potential to
reduce ethnic health disparities that exist in New Zealand. However, the results
comparing clopidogrel and prasugrel are sensitive to whether the genetic
sub-studies or the overall TRITON-TIMI 38 rates are used. While the national
hospital event rates may be more appropriate for the New Zealand population,
many assumptions are required when they are used to adjust the genetic
sub-studies rates. |
What is the role of eteplirsen in DMD patients? | AVI-4658(eteplirsen) induces skipping of dystrophin exon 51 in patients with relevant deletions, restores the open reading frame and induces dystrophin protein expression after intramuscular (i.m.) injection. | We previously conducted a proof of principle; dose escalation study in Duchenne
muscular dystrophy (DMD) patients using the morpholino splice-switching
oligonucleotide AVI-4658 (eteplirsen) that induces skipping of dystrophin exon
51 in patients with relevant deletions, restores the open reading frame and
induces dystrophin protein expression after intramuscular (i.m.) injection. We
now show that this dystrophin expression was accompanied by an elevated
expression of α-sarcoglycan, β-dystroglycan (BDG) and--in relevant
cases--neuronal nitric oxide synthase (nNOS) at the sarcolemma, each of which is
a component of a different subcomplex of the dystrophin-associated glycoprotein
complex (DAPC). As expected, nNOS expression was relocalized to the sarcolemma
in Duchenne patients in whom the dystrophin deletion left the nNOS-binding
domain (exons 42-45) intact, whereas this did not occur in patients with
deletions that involved this domain. Our results indicate that the novel
internally deleted and shorter dystrophin induced by skipping exon 51 in
patients with amenable deletions, can also restore the dystrophin-associated
complex, further suggesting preserved functionality of the newly translated
dystrophin. Restoration of the open reading frame of the DMD gene and dystrophin protein
production in Duchenne muscular dystrophy (DMD) can be achieved by exon skipping
using antisense oligomers (AOs) targeted to splicing elements. Several such
RNA-based gene therapy approaches are in clinical development in which all
studies to date have assessed AO efficacy by semiquantitative nested
reverse-transcription polymerase chain reaction (RT-PCR). Precise evaluation of
dystrophin protein levels is complex and hindered by the large size and low
abundance of dystrophin; thus an accurate and standardized measurement of DMD
exon skipping at the RNA level remains important to assess and compare patient
responses in DMD exon skipping clinical trials. Here we describe the development
of a Taqman quantitative (q)RT-PCR assay to quantify exon skipping and highlight
its use to determine the levels of exon skipping in DMD patients treated
intramuscularly with a morpholino AO to skip exon 51, eteplirsen (AVI-4658). The
muscle biopsies of these patients were previously thoroughly characterized,
providing a valuable benchmark for the evaluation of novel methodology. We
demonstrate that levels of dystrophin protein restoration, and thus patient
response, correlate accurately with the RNA level. Furthermore, this sensitive
assay detects revertant exon 51 skipped fibers in untreated biopsies, providing
an important baseline to precisely quantify treatment success. This study
represents the first quantitative assessment of exon skipping in a clinical
trial setting. We present a standardized and reproducible method to assess
patient response that will complement protein studies in future preclinical and
clinical exon skipping-based gene therapy studies for DMD. OBJECTIVE: In prior open-label studies, eteplirsen, a phosphorodiamidate
morpholino oligomer, enabled dystrophin production in Duchenne muscular
dystrophy (DMD) with genetic mutations amenable to skipping exon 51. The present
study used a double-blind placebo-controlled protocol to test eteplirsen's
ability to induce dystrophin production and improve distance walked on the
6-minute walk test (6MWT).
METHODS: DMD boys aged 7 to 13 years, with confirmed deletions correctable by
skipping exon 51 and ability to walk 200 to 400 m on 6 MWT, were randomized to
weekly intravenous infusions of 30 or 50 mg/kg/wk eteplirsen or placebo for 24
weeks (n = 4/group). Placebo patients switched to 30 or 50 mg/kg eteplirsen
(n=2/group) at week 25; treatment was open label thereafter. All patients had
muscle biopsies at baseline and week 48. Efficacy included dystrophin-positive
fibers and distance walked on the 6MWT.
RESULTS: At week 24, the 30 mg/kg eteplirsen patients were biopsied, and
percentage of dystrophin-positive fibers was increased to 23% of normal; no
increases were detected in placebo-treated patients (p≤0.002). Even greater
increases occurred at week 48 (52% and 43% in the 30 and 50 mg/kg cohorts,
respectively), suggesting that dystrophin increases with longer treatment.
Restoration of functional dystrophin was confirmed by detection of sarcoglycans
and neuronal nitric oxide synthase at the sarcolemma. Ambulation-evaluable
eteplirsen-treated patients experienced a 67.3 m benefit compared to
placebo/delayed patients (p≤0.001).
INTERPRETATION: Eteplirsen restored dystrophin in the 30 and 50 mg/kg/wk
cohorts, and in subsequently treated, placebo-controlled subjects. Duration,
more than dose, accounted for dystrophin production, also resulting in
ambulation stability. No severe adverse events were encountered. PURPOSE OF REVIEW: The most encouraging recent advances regarding
pharmacological agents for treating Duchenne muscular dystrophy (DMD) are
summarized. Emphasis is given to compounds acting downstream of dystrophin, the
protein lacking in DMD, on cellular pathways leading to pathological
consequences. The author highlights the progress that may have the greatest
potential for clinical use in DMD.
RECENT FINDINGS: Modifying the transcripts of the mutated gene by exon skipping
has led to expression of shortened dystrophins in DMD patients. Currently, the
most promising potential drugs are the exon-skipping agents eteplirsen and
drisapersen. Biglycan and SMTC1100 upregulate utrophin. The steroid receptor
modulating compounds VBP15 and tamoxifen, and specific antioxidants appear
promising agents for symptomatic therapy.
SUMMARY: The past 18 months have seen a strong increase in the number of
exciting reports on novel therapeutic agents for DMD. Exon-skipping agents have
been fine-tuned to improve tissue delivery and stability. Impressive discoveries
regarding pathogenic events in cellular signalling have revealed targets that
were unknown in the context of DMD, thus enabling approaches that limit
inflammation, fibrosis and necrosis. The targets are nuclear hormone receptors,
NADPH-oxidases and Ca channels. Inhibition of NF-KB, transforming growth
factor-alpha (TGF-α) and transforming growth factor-beta (TGF-β)/myostatin
production or action are also promising routes in counteracting the complex
pathogenesis of DMD. Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in
the dystrophin gene that disrupt the open reading frame, while in frame
mutations result in Becker muscular dystrophy (BMD). Ullrich congenital muscular
dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific
muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring
the outcome of therapeutic interventions and disease progression. We quantified
miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD
and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but
unchanged in UCMD compared to controls. Milder DMD patients had higher levels of
dystromirs than more severely affected patients. Patients with low forced vital
capacity (FVC) values, indicating respiratory muscle weakness, had low levels of
serum miR-1 and miR-133b. There was no significant difference in the level of
the dystromirs in BMD compared to controls. We also assessed the effect of
dystrophin restoration on the expression of the five dystromirs in serum of DMD
patients treated systemically for 12 weeks with antisense oligomer eteplirsen
that induces skipping of exon 51 in the dystrophin gene. The dystromirs were
also analysed in muscle biopsies of DMD patients included in a single dose
intramuscular eteplirsen clinical trial. Our analysis detected a trend towards
normalization of these miRNA between the pre- and post-treatment samples of the
systemic trial, which however failed to reach statistical significance. This
could possibly be due to the small number of patients and the short duration of
these clinical trials. Although longer term studies are needed to clarify the
relationship between dystrophin restoration following therapeutic intervention
and the level of circulating miRNAs, our results indicate that miR-1 and miR-133
can be considered as exploratory biomarkers for monitoring the progression of
muscle weakness and indirectly the remaining muscle mass in DMD. |
Describe clinical presentation of Parkinsonism with dementia of Guadeloupe syndrome. | Parkinsonism with dementia of Guadeloupe is a unique combination of levodopa-resistant parkinsonism, tremor, myoclonus, hallucinations, REM sleep behavior disorder and fronto-subcortical dementia. Based on the presence or the absence of supranuclear gaze palsy, two subgroups of patients can be distinguished. | Steele, Richardson and Olszweski in 1964 described a distinctive clinical and
pathological entity they called progressive supranuclear palsy (PSP). Now on
Guadeloupe in the Carribbean French West Indies, Caparros-Lefebvre is
identifying many patients with similar clinical and histological features.
Others have a clinical syndrome of atypical parkinsonism that resembles the
parkinsonism-dementia complex (PDC) of Guam and the Kii peninsula of Japan
(PDC). But in those Pacific foci the histology is different and the abnormal tau
is of Alzheimer's type rather than the PSP type of Guadeloupe. In both locales,
neurotoxins of local foods are implicated in etiology. Future studies will
confirm if Guadeloupean parkinsonism is truly a geographic focus of PSP, and if
dietary factors account for both. In Guadeloupe, there is an abnormally high frequency of atypical parkinsonism.
Only one-third of the patients that develop parkinsonian symptoms were reported
to present the classical features of idiopathic Parkinson disease and one-third
a syndrome resembling progressive supranuclear palsy (PSP). The others were
unclassifiable, according to established criteria. We carried out a
cross-sectional study of 160 parkinsonian patients to: (i) define more precisely
the clinical phenotypes of the PSP-like syndrome and the parkinsonism that was
considered unclassifiable in comparison with previously known disorders; (ii)
define the neuropsychological and brain imaging features of these patients;
(iii) evaluate to what extent a candidate aetiological factor, the mitochondrial
complex I inhibitor annonacin contained in the fruit and leaves of the tropical
plant Annona muricata (soursop) plays a role in the neurological syndrome.
Neuropsychological tests and MRI were used to classify the patients into those
with Parkinson's disease (31%), Guadeloupean PSP-like syndrome (32%),
Guadeloupean parkinsonism-dementia complex (PDC, 31%) and other
parkinsonism-related disorders (6%). Patients with a PSP-like syndrome developed
levodopa-resistant parkinsonism, associated with early postural instability and
supranuclear oculomotor dysfunction. They differed, however, from classical PSP
patients by the frequency of tremor (>50%), dysautonomia (50%) and the
occurrence of hallucinations (59%). PDC patients had levodopa-resistant
parkinsonism associated with frontosubcortical dementia, 52% of these patients
had hallucinations, but, importantly, none had oculomotor dysfunction. The
pattern of neuropsychological deficits was similar in both subgroups. Cerebral
atrophy was seen in the majority of the PSP-like and PDC patients, with
enlargement of the third ventricle and marked T2-hypointensity in the basal
ganglia, particularly the substantia nigra. Consumption of soursop was
significantly greater in both PSP-like and PDC patients than in controls and
Parkinson's disease patients. In conclusion, atypical Guadeloupean parkinsonism
comprises two forms of parkinsonism and dementia that differ clinically by the
presence of oculomotor signs, but have similar cognitive profiles and
neuroimaging features, suggesting that they may constitute a single disease
entity, and both were similarly exposed to annonaceous neurotoxins, notably
annonacin. STUDY OBJECTIVE: To describe sleep characteristics and rapid eye movement (REM)
sleep behavior disorder in patients with Guadeloupean atypical parkinsonism
(Gd-PSP), a tauopathy resembling progressive supranuclear palsy that mainly
affects the midbrain. It is possibly caused by the ingestion of sour sop
(corossol), a tropical fruit containing acetogenins, which are mitochondrial
poisons.
DESIGN: Sleep interview, motor and cognitive tests, and overnight
videopolysomnography.
PATIENTS: Thirty-six age-, sex-, disease-duration- and disability-matched
patients with Gd-PSP (n = 9), progressive supranuclear palsy (a tauopathy, n =
9), Parkinson disease (a synucleinopathy, n = 9) and controls (n = 9).
SETTINGS: Tertiary-care academic hospital.
RESULTS: REM sleep behavior disorder was found in 78% patients with Gd-PSP (43%
of patients reported having this disorder several years before the onset of
parkinsonism), 44% of patients with idiopathic Parkinson disease, 33% of
patients with progressive supranuclear palsy, and no controls. The percentage of
muscle activity during REM sleep was greater in patients with Gd-PSP than in
controls (limb muscle activity, 8.3%+/-8.7% vs 0.1%+/- 0.2%; chin muscle
activity, 24.3%+/- 23.7% vs 0.7%+/-2.0%) but similar to that of other patient
groups. The latency and percentage of REM sleep were similar in patients with
Gd-PSP, patients with Parkinson disease, and controls, whereas patients with
progressive supranuclear palsy had delayed and shortened REM sleep.
CONCLUSION: Although Gd-PSP is a tauopathy, most patients experience REM sleep
behavior disorder. This suggests that the location of neuronal loss or
dysfunction in the midbrain, rather than the protein comprising the histologic
lesions (synuclein versus tau aggregation), is responsible for suppressing
muscle atonia during REM sleep. Subjects with idiopathic REM sleep behavior
disorder should avoid eating sour sop. On the French West Indian island of Guadeloupe, atypical parkinsonian patients
represent two-thirds of all cases of parkinsonism, which is exceptionally
frequent compared to epidemiological data from European countries where atypical
parkinsonism accounts for only approximately 5% of all cases. The clinical
entity was a unique combination of levodopa-resistant parkinsonism, tremor,
myoclonus, hallucinations, REM sleep behavior disorder and fronto-subcortical
dementia. Based on the presence or the absence of supranuclear gaze palsy, two
subgroups of patients were distinguished. In patients with oculomotor signs that
came to autopsy, neuronal loss was found to predominate in the substantia nigra
and the striatum but other brain areas were also affected, including the frontal
cortex. In addition, tau-containing lesions were detected throughout the brain.
Epidemiological data suggested a close association of the disease with the
regular consumption of soursop, a tropical annonaceous plant. Experimental
studies performed in midbrain cell cultures identified annonacin, a selective
mitochondrial complex I inhibitor contained in the fruit and leaves of soursop,
as a probable etiological factor. Consistent with this view, chronic
administration of annonacin to rats through Alzet osmotic minipumps showed that
annonacin was able to reproduce the brain lesions characteristic of the human
disease. Atypical parkinsonism is extremely frequent in Guadeloupe and may have an
environmental cause. One-half of the patients with this tauopathy have
dopa-resistant parkinsonism, tremor, subcortical dementia and abnormal eye
movements suggestive of progressive supranuclear palsy (PSP). They also have
hallucinations, dysautonomia, which are not characteristic of PSP. Furthermore,
the oculomotor abnormalities and the tremor, which is jerky, differ from what is
observed in classical PSP patients. We therefore undertook an
electrophysiological study to characterize these features in greater detail.
Nine representative Guadeloupean PSP-like (Gd-PSP) patients were selected for
electro-oculographic recordings of horizontal eye movements [visually guided
saccades (VGS), antisaccades (AS) and smooth pursuit], clinical evaluation of
vertical saccade velocity and electrophysiological analysis of abnormal limb
movements [electromyographic polygraphy, EEG jerk-locked-back-averaging (JLBA)
and long-loop C-reflex]. Vertical saccade velocity was reduced in five patients.
The velocity of horizontal VGS was normal, although the latencies were increased
and horizontal smooth pursuit (HSP) was mostly saccadic. The AS error rate was
above 70% in most patients. Myoclonus was detected in 89% of the Gd-PSP
patients. It was mainly small amplitude rest and action myoclonus in the upper
limbs, characterized by short arrhythmic 24-76 ms bursts and was of cortical
origin, as confirmed by JLBA in five patients. In conclusion, Gd-PSP patients
have cortical myoclonus and cortical oculomotor impairments, but only minor
signs of brainstem oculomotor dysfunction, suggesting that cortical dysfunction
predominates over brainstem impairments. This electrophysiological study, added
to previous clinical, neuropsychological and neuroradiological studies, has
enriched the characterization of Guadeloupean atypical parkinsonism, which thus
appears to be a new clinical entity. INTRODUCTION: : On Guadeloupe, atypical parkinsonism is abnormally frequent, and
represents 75% of progressive parkinsonism while Parkinson's disease (PD)
accounts for only 25%, which is an inversed percentage in comparison with
Europe. Herbal tea made with Annonaceae leaves (containing
benzyltetrahydroisoquinolines (Be-TIQ), tetrahydroprotoberberines (THPB) and
acetogenins (potent mitochondrial complex I inhibitors) are commonly used on
Guadeloupe.
CLINICAL STUDY: : Of 265 patients studied on Guadeloupe, 66 (25%) had PD, and
199 (75%) had atypical parkinsonism. This latter group includes 58 patients
(29%) with progressive supranuclear palsy (PSP), and 100 patients (50%) with
unclassifiable parkinsonism (UP). This focus resembles the parkinsonism-dementia
complex (PDC) described on Guam, where a very high prevalence of atypical
parkinsonism has been reported since the second World War, including one-third
of PSP. A preliminary case-control study on Guadeloupe showed a significant
higher consumption of fruits and herbal tea of Annonaceae in atypical
parkinsonian cases (PSP and unclassifiable parkinsonism, UP), compared to
hospital controls and to idiopathic PD.
DISCUSSION: : The overrepresentation of atypical parkinsonism on Guadeloupe and
Guam could be related to the consumption of plants containing (simultaneously)
isoquinoline derivates which are toxic for dopaminergic neurons and inhibitors
of the mitochondrial respiratory chain such as acetogenins. This hypothesis is
in keeping with epidemiologic data and experimental studies showing neuronal
loss after exposure to isoquinolines or acetogenins. |
Does the concentration of protein HIF-1α increase after the administration of the cytoprotective prodrug"amifostine" (ethyol) ? | The key-protein that when associated with HREs leads to the activation of all of these genes, is identified as“Hypoxia Inducible Factor-1” (HIF1). It is a heterodimer composed of two subunits (IIF1a 120kDa and HIF-1b 91-94kDa), both of which belong to the group of "basic helix-loop-helix" (bHLH)-Pas proteins. The heterodimer HIF1 and IIF2 increase in the cytoplasm of cells exposed to hypoxia. | PURPOSE: The cytoprotective mechanism of amifostine (WR-2721) implies free
radical scavenging and DNA repair activities. We investigated additional
cytoprotective pathways involving intracellular hypoxia and the activation of
the hypoxia-inducible factor (HIF) pathway, a key transcription factor
regulating glycolysis, angiogenesis and apoptosis, which is also linked with
radioresistance.
MATERIALS AND METHODS: The glucose and oxygen levels in the peripheral blood of
patients receiving 1000 mg amifostine were determined at various time-points in
order to investigate the metabolic changes induced by amifostine. MDA468 breast
tumor cell lines were incubated with a high amifostine concentration (10 m M) to
overcome the natural resistance of cancer cells to influx of the non-hydrolyzed
WR-2721, and the HIF1 alpha protein levels were determined by Western blot
analysis. In vivo experiments with Wistar rats were performed in order to assess
immunohistochemically changes in the intracellular accumulation of HIF1 alpha
induced by amifostine (200 mg/kg).
RESULTS: By 30 min following amifostine administration, the hemoglobin oxygen
saturation and pO(2) levels had increased in the peripheral blood while glucose
levels had reduced, providing evidence that normal tissue metabolism switches to
glycolytic pathways. Incubation of cell lines with amifostine resulted in HIF1
alpha induction. In Wistar rats administration of amifostine resulted in
increased HIF1 alpha accumulation in normal tissues.
CONCLUSIONS: Since it is doubtful whether dephosphorylation of amifostine to the
active metabolite WR-1065 occurs within tumoral tissues (an acidic environment
that lacks vascular alkaline phosphatase activity), intracellular hypoxia and
upregulation of HIF1 alpha represents an additional, normal tissue-specific,
amifostine cytoprotective pathway. PURPOSE: Tumor hypoxia and low intrinsic radiosensitivity may counteract the
efficacy of standard radiotherapy for locally advanced head and neck cancer
(HNC). We investigated the involvement of hypoxia-regulated proteins (Hypoxia
inducible factors HIF1alpha, HIF2alpha and carbonic anhydrase CA9) in HNC
resistance to accelerated and hypofractionated radiotherapy.
MATERIALS AND METHODS: Thirty-nine patients with locally advanced HNC received
15 daily fractions of 3.4 Gy amounting to a total tumor dose of 51 Gy
(equivalent to 63 Gy in four weeks--one week split); this was combined with
platinum chemotherapy and amifostine cytoprotection administered subcutaneously.
Immunohistochemical analysis of hypoxia-regulated proteins, namely HIF1alpha,
HIF2alpha and CA9, was performed in formalin-fixed paraffin-embedded tissues
obtained prior to radio-chemotherapy.
RESULTS: HIF1alpha and HIF2alpha were expressed in the nuclei and cytoplasm of
cancer cells, while CA9 had a membrane reactivity. A high expression of
HIF1alpha, HIF2alpha and CA9 was noted in 21/39 (53.8%), 20/39 (51.3%) and 23/39
(58.9%) cases, respectively. Complete response was obtained in 85.2% of patients
and HIF1alpha was marginally related with persistent disease after RT (p =
0.05). HIF1alpha was significantly associated with poor local relapse free
survival (LRFS) (p = 0.006) and overall survival (p = 0.008), whilst HIF2alpha
was not. A significant association of CA9 expression with poor LRFS was noted (p
= 0.01).
CONCLUSION: In accord with previously reported studies, high levels of the
hypoxia regulated proteins HIF1alpha and CA9 in HNC predict resistance to
platinum based radio-chemotherapy. Whether HIF2alpha expressing tumors are more
sensitive to larger radiotherapy fractions, compared to standard radiotherapy
fractionation, is an issue that deserves further investigation. Doxorubicin (DOX) is widely used in combination cocktails for treatment of
childhood hematological cancers and solid tumors. A major factor limiting DOX
usage is DOX-induced cardiotoxicity. However, it is not known whether
protectants like dexrazoxane (DXR) and amifostine (AMF) can prevent DOX-mediated
bone damage. The present study investigated whether administration of AMF alone
or in combination with DXR would prevent any DOX-mediated bone damage. Male rat
pups were treated with DOX, DXR, AMF, and their combinations. On neonate day 38,
the bone mineral density (BMD), bone mineral content (BMC) and the
micro-architecture of the lumbar vertebrae were analyzed. We have shown that
when male rats are treated with DOX, DXR, DOX+DXR, AMF, DOX+AMF or DOX+DXR+AMF,
there is a decrease in lumbar vertebral BMD (p<0.05). Furthermore, the relative
bone volume (BV/TV) was decreased by DXR, DOX+DXR, and DOX+AMF treatments.
Interestingly, DOX+AMF significantly increased BV/TV when compared to DXR
treatment (p<0.04). The trabecular number (Tb.N) decreased with DXR and DOX+DXR
and increased with DOX+AMF treatments. This information will be useful in
designing better cancer combination therapies that do not lead to vertebrae
deterioration. BACKGROUND: Amifostine (WR-2721, delivered as Ethyol) is a phosphorylated
aminothiol compound clinically used in addition to cis-platinum to reduce the
toxic side effects of therapeutic treatment on normal cells without reducing
their efficacy on tumour cells. Its mechanism of action is attributed to the
free radical scavenging properties of its active dephosphorylated metabolite
WR-1065. However, amifostine has also been described as a potent hypoxia-mimetic
compound and as a strong p53 inducer; both effects are known to potently
modulate vascular endothelial growth factor (VEGF-A) expression. The angiogenic
properties of this drug have not been clearly defined.
METHODS: Cancer cell lines and endothelial cells were used in culture and
treated with Amifostine in order to study (i) the expression of angiogenesis
related genes and proteins and (ii) the effects of the drug on VEGF-A induced in
vitro angiogenesis.
RESULTS: We demonstrated that the treatment of several human cancer cell lines
with therapeutical doses of WR-1065 led to a strong induction of different
VEGF-A mRNA isoforms independently of HIF-1alpha. VEGF-A induction by WR-1065
depends on the activation of the eIF2alpha/ATF4 pathway. This up-regulation of
VEGF-A mRNA was accompanied by an increased secretion of VEGF-A proteins fully
active in stimulating vascular endothelial cells (EC). Nevertheless, direct
treatment of EC with amifostine impaired their ability to respond to exogenous
VEGF-A, an effect that correlated to the down-regulation of VEGFR-2 expression,
to the reduction in cell surface binding of VEGF-A and to the decreased
phosphorylation of the downstream p42/44 kinases.
CONCLUSIONS: Taken together, our results indicate that amifostine treatment
modulates tumour angiogenesis by two apparently opposite mechanisms - the
increased VEGF-A expression by tumour cells and the inhibition of EC capacity to
respond to VEGF-A stimulation. Topoisomerase I inhibitors down-regulate HIF-1α leading to tumor growth
inhibition, but only while maintaining sustained levels of drug exposure.
EZN-2208, a multi-arm 40 kDa pegylated, releasable SN38-drug conjugate, provides
higher, longer lasting exposure of tumors to SN38 in contrast to SN38 that is
released from CPT-11. EZN-2208 also consistently has greater antitumor activity
than CPT-11 in a variety of solid and hematological tumor models. In this
report, the ability of PEG-SN38 to down-regulate HIF-1α and its downstream
targets, in a more potent, sustained manner compared with CPT-11 was examined.
To do so, U251 glioma xenografts that stably expressed a hypoxia response
element-dependent luciferase reporter gene were implanted in mice. After
treatment it was found that EZN-2208 induced potent, sustained HIF-1α
down-regulation (37% at 48 h and 83% at 120 h) in the tumors, whereas CPT-11
caused only minor, transient HIF-1α down-regulation. In addition, EZN-2208
down-regulated mRNA levels of HIF-1α targeted genes (MMP2, VEGF1, Glut1, Glut3
and TGFβ1). Further, western blot analyses of xenograft tumors demonstrated that
EZN-2208 had significantly more effect than CPT-11 in down-regulating HIF-1α,
VEGF, Glut1 and MMP2 protein levels. Significant down-regulation of HIF-1α and
VEGF proteins translated to EZN-2208's superior anti-angiogenic activity
compared with CPT-11, confirmed by microvessel density reduction in a
chorioallantoic membrane assay and in CD-31 immunohistochemistry studies.
Additional studies done with matrigel implants devoid of tumor cells show that
EZN-2208 significantly inhibits angiogenesis while CPT-11 has little or no
effect. It is concluded that the superior antitumor activity of EZN-2208
compared with CPT-11 is attributed, in part, to an anti-angiogenic effect.
Ongoing clinical Phase I and Phase II studies will assess safety and efficacy of
EZN-2208. OBJECTIVE: Burn-induced gut dysfunction plays an important role in the
development of sepsis and multiple organ dysfunction. Emerging evidence suggests
that hypoxia-inducible factor-1α (HIF-1α) is critical in paracellular barrier
functions via regulating vascular endothelial growth factor (VEGF) and myosin
light chain kinase (MLCK) expression. Previous studies have also demonstrated
that histone deacetylase inhibitors (HDACIs) can repress HIF-1α. This study aims
to examine whether valproic acid (VPA), a HDACI, protects against burn-induced
gut barrier dysfunction via repressing HIF-1α-dependent upregulation of VEGF and
MLCK expression.
METHODS: Rats were subjected to third degree 55% TBSA burns and treated with/
without VPA (300 mg/kg). Intestinal barrier dysfunction was evaluated by
permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran
and histologic evaluation. Histone acetylation, tight junction protein zonula
occludens 1 (ZO-1), VEGF, MLCK and HIF-1α were measured. In addition, CaCO2
cells were transfected with siRNA directed against HIF-1α and were stimulated
with CoCl2 (1mM) for 24 hours with/without VPA (2mM) followed by analysis of
HIF-1α, MLCK, VEGF and ZO-1.
RESULTS: Burn insults resulted in a significant increase in intestinal
permeability and mucosal damage, accompanied by a significant reduction in
histone acetylation, ZO-1, upregulation of VEGF, MLCK expression, and an
increase in HIF-1α accumulation. VPA significantly attenuated the increase in
intestinal permeability, mucosa damage, histone deacetylation and changes in
ZO-1 expression. VPA also attenuated the increased VEGF, MLCK and HIF-1α protein
levels. VPA reduced HIF-1α, MLCK and VEGF production and prevented ZO-1 loss in
CoCl2-stimulated Caco-2 cells. Moreover, transfection of siRNA directed against
HIF-1α led to inhibition of MLCK and VEGF production, accompanied by
upregulation of ZO-1.
CONCLUSIONS: These results indicate that VPA can protect against burn-induced
gut barrier dysfunction. These protective effects may be due to its inhibitory
action on HIF-1α, leading to a reduction in intestinal VEGF and MLCK expression
and minimizing ZO-1 degradation. |
Which diseases can Oncotype DX be used for? | Oncotype can be used for predicting breast cancer and colon cancer recurrence. | Advances in molecular genetics aimed at individualizing breast cancer treatment
have been validated. We examined the use of gene assays predictive of distant
recurrence in breast cancer and their impact on adjuvant treatment. A
retrospective chart review of 58 T1/T2, node-negative, estrogen-receptor
positive breast cancer patients that underwent Oncotype DX gene assay testing
between January and December 2006 was performed. We compared treatment received
after gene assay evaluation to treatment based on National Comprehensive Cancer
Network guidelines. Patients were grouped using these recommendations: Low-risk
group (T1a/T1b), no chemotherapy; High-risk group (T1c/T2), chemotherapy.
Oncotype DX recommendations are as follows: Low recurrence risk, no
chemotherapy; high recurrence risk, chemotherapy. A change in management was
defined as chemotherapy for T1a/T1b disease and no chemotherapy for T1c/T2
disease. Two T1a/T1b patients had high risk of recurrence per gene assay scores
and were treated with chemotherapy (P < 0.05). Eighteen T1c/T2 patients had low
recurrence risk scores; 13 (72%) were spared chemotherapy. The recurrence score
increased the number of patients classified as low risk of recurrence by 12 per
cent and downstaged 63 per cent of high-risk patients (P < 0.003). Gene assay
results changed management in 15 of 58 (26%) patients (P < 0.05). The use of
gene assays allowed us to better tailor treatment in a significant number of our
patients. Recently, recommendations for the use of the Oncotype DX assay in estrogen
receptor-positive node-negative breast cancer patients were incorporated into
guidelines from both the American Society of Clinical Oncology and the National
Comprehensive Cancer Network. The Oncotype DX assay is a diagnostic test which
measures changes in a set of 21 genes in order to predict the likelihood of
disease recurrence and also to predict which patients are most likely to respond
to chemotherapy. Oncotype DX has been available commercially since January 2004
and has been used for more than 85,000 patients. Drs. William J. Gradishar, Nora
M. Hansen, and Barbara Susnik answered questions regarding the incorporation of
the Oncotype DX breast cancer assay into routine clinical practice. This expert
dialog offers an update and clinical insights into when, how, and why clinicians
might incorporate the Oncotype DX assay into the management of their breast
cancer patients. Also, the latest research into the benefit of the Oncotype DX
assay in node-positive patients is discussed. Finally, sample case studies offer
clinically relevant examples of the practical application of the Oncotype DX
assay. The Oncotype DX assay is one of the molecular tests that provide predictive and
prognostic information to breast cancer patients with estrogen receptor
(ER)-positive and node-negative disease. This study evaluates the association of
Forkhead-box protein A1 (FOXA1) and GATA-binding protein 3 (GATA3) expressions
with Oncotype DX recurrences scores in 77 cases of patients with ER-positive
node-negative breast carcinomas diagnosed at Indiana University. The data were
correlated with patient age, tumor size, histologic type,
Scarff-Bloom-Richardson score, histologic grade, and progesterone receptor
status. The median FOXA1 and GATA3 scores were 240 and 200, respectively. The
Oncotype DX recurrence scores were low in 57%, intermediate in 30%, and high in
13% of cases. FOXA1 expression correlated negatively with Oncotype DX recurrence
scores (P=0.004), and histologic type (P=0.0004). Oncotype DX recurrences score
also correlated negatively with progesterone receptor (P=0.035) with 100% of
progesterone receptor-negative cases having high or intermediate Oncotype DX
scores. FOXA1 and GATA3 expressions correlated positively (P=0.014). The
correlation between FOXA1 expression and Oncotype DX recurrence scores remained
significant after adjusting for multiple comparisons and controlling for
confounders such as histological type, grade, and progesterone receptor. A
statistically significant correlation between the Oncotype DX recurrence scores
and FOXA1 expression in our diverse cohort of ER-positive breast cancer patients
was observed. We propose that this may represent a more cost-effective strategy
to further risk stratify patients with good prognosis in whom chemotherapy may
be omitted. To confirm these findings, further studies in a larger cohort of
patients are warranted. BACKGROUND: Genomic recurrence risk test results now inform clinical decisions
about adjuvant treatment for women with early-stage breast cancer. We sought to
understand patients' knowledge of these tests and correlates of their knowledge.
METHODS: Participants in this cross-sectional study were 78 women, treated for
early-stage, estrogen receptor-positive breast cancer with 0-3 positive lymph
nodes, whose medical records indicated they received Oncotype DX testing
earlier. We mailed a questionnaire that assessed knowledge of genomic recurrence
risk testing (13 item scale, alpha=0.83) and reviewed medical charts of
consenting patients.
RESULTS: Knowledge about genomic recurrence risk testing was low (mean knowledge
score=67%, SD=0.23). Low knowledge scores were more commonly due to responses of
'don't know' than incorrect answers. Most women (91%) clearly understood that
test results can aid decisions about chemotherapy, and few (22%) understood that
the test's estimate of the chance of metastasis assumes the patient is receiving
hormone therapy. Higher knowledge about genomic recurrence risk testing was
associated with higher education, reading ability, and numeracy. Knowledge was
higher among women who recalled receiving both verbal and printed information
about the test and among women who had active roles in deciding about their
treatments. Higher knowledge was also associated with having fewer concerns
about genomic testing.
DISCUSSION: Among early-stage breast cancer patients who received Oncotype DX,
we found low knowledge about many aspects of genomic recurrence risk testing.
Research is needed to understand testing information provided to patients and
best practices for patient education. Overall five-year survival for patients with stage-II colon cancer averages 75%
after surgery alone. However, some of these patients have poorer outcomes,
similar to patients with stage-III disease. The proposed use of the Oncotype DX
assay is to improve risk stratification for recurrence in stage-II colon cancer. BACKGROUND: The Oncotype DX Colon Cancer Assay is a new diagnostic test for
determining the likelihood of recurrence in stage II colon cancer patients after
surgical resection using fixed paraffin embedded (FPE) primary colon tumor
tissue. Like the Oncotype DX Breast Cancer Assay, this is a high complexity,
multi-analyte, reverse transcription (RT) polymerase chain reaction (PCR) assay
that measures the expression levels of specific cancer-related genes. By
capturing the biology underlying each patient's tumor, the Oncotype DX Colon
Cancer Assay provides a Recurrence Score (RS) that reflects an individualized
risk of disease recurrence. Here we describe its analytical performance using
pre-determined performance criteria, which is a critical component of molecular
diagnostic test validation.
RESULTS: All analytical measurements met pre-specified performance criteria. PCR
amplification efficiency for all 12 assays was high, ranging from 96% to 107%,
while linearity was demonstrated over an 11 log2 concentration range for all
assays. Based on estimated components of variance for FPE RNA pools, analytical
reproducibility and precision demonstrated low SDs for individual genes (0.16 to
0.32 CTs), gene groups (≤ 0.05 normalized/aggregate CTs) and RS (≤ 1.38 RS
units).
CONCLUSIONS: Analytical performance characteristics shown here for both
individual genes and gene groups in the Oncotype DX Colon Cancer Assay
demonstrate consistent translation of specific biology of individual tumors into
clinically useful diagnostic information. The results of these studies
illustrate how the analytical capability of the Oncotype DX Colon Cancer Assay
has enabled clinical validation of a test to determine individualized recurrence
risk after colon cancer surgery. Node-negative breast cancer is a prevalent form of the disease worldwide,
particularly in regions with rigorous screening and disease awareness efforts.
Although there is a common biology between node-negative and node-positive
breast tumors, it is still important to specifically address risk assessment and
predictive factors in node-negative breast cancer. The relative risks and
benefits are more pronounced in these patients, but there is no single
prognostic factor available for deciding whether to administer chemotherapy and
selecting the best adjuvant chemotherapy regimen. In the absence of universal
predictive factors, the trend is to give chemotherapy to all patients to ensure
the highest possibility for cure. Tumor grade is important in that it is
predictive of risk over time, but lacks standardization. Adjuvant! Online, a
web-based algorithm, is also used to guide treatment decisions. Recently, the
urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor
type 1 (PAI-1) biomarkers have been used to determine disease risk and,
consequently, determine whether or not chemotherapy is needed. However, the
assay requires a fresh-frozen tissue sample, which is not always feasible. The
oncotype DX® genomic assay has also been used to help prognosis estimation and
treatment decisions. It is currently under evaluation in conjunction with the
uPA/PAI-1 assay in the Plan B trial. The question that remains in adjuvant
chemotherapy today for patients with node-negative disease is proper patient
selection. Node-negative breast cancer does not automatically suggest a good
prognosis, or the preclusion of chemotherapy benefits, and additional biomarkers
are needed to help identify patients who do benefit from chemotherapy. A molecular test providing clear identification of individuals at highest risk
for developing metastatic disease from among early stage breast cancer patients
has proven to be of great benefit in breast cancer treatment planning and
therapeutic management. Patients with high risk of disease recurrence can also
get an estimate of the magnitude of benefit to be gained by adding chemotherapy
to surgery and hormonal therapy. Developing this clinical test was made possible
by the availability of technologies capable of identifying molecular biomarkers
from the gene expression profiles of preserved surgical specimens. Molecular
tests such as the Oncotype DX(®) breast cancer test are proving to be more
effective tools for individualized patient stratification and treatment planning
than traditional methods such as patient demographic variables and
histopathology indicators.Molecular biomarkers must be clinically validated
before they can be effectively applied toward patient management in clinical
practice. The most effective and efficient means of clinical validation is to
use archived surgical specimens annotated with well-characterized clinical
outcomes. However, carrying out this type of clinical study requires
optimization of traditional molecular expression profiling techniques to analyze
RNA from fixed, paraffin-embedded (FPE) tissues. In order to develop our
clinically validated breast cancer assay, we modified molecular methods for RNA
extraction, RNA quantitation, reverse transcription, and quantitative PCR to
work optimally in archived clinical samples. Here, we present an updated
description of current best practices for isolating both mRNA and microRNA from
FPE tissues for RT-PCR-based expression profiling. BACKGROUND: Half of all breast cancers are early stage, lymph node negative, and
hormone receptor positive. A 21-gene (Oncotype DX®; Genomic Health, Inc.,
Redwood City, CA) recurrence score (RS) is prognostic for recurrence and
predictive of chemotherapy benefit. We explored the ability of oncologists to
predict the RS using standard prognostic criteria.
METHODS: Standard demographic and tumor prognostic criteria were obtained from
patients with an available RS. Two academic pathologists provided tumor grade,
histologic type, and hormone receptor status. Six academic oncologists predicted
the RS category (low, intermediate, or high) and provided a recommendation for
therapy. The oncologists were then given the actual RS and provided
recommendations for therapy. Analysis for agreement was performed.
RESULTS: Thirty-one cases, including nine additional cases with variant
pathology reads, were presented. There was substantial agreement in oncologists'
ability to discriminate between true low or true intermediate and true high (κ =
0.75; p < .0001). Predictions between low and intermediate were not consistent.
The most common discrepancies were predictions of a low RS risk when cases were
true intermediate and predictions of an intermediate RS risk when cases were
true low. The actual RS resulted in a change in the treatment recommendations in
19% of cases. Of the 186 scenarios and six oncologists in aggregate, five fewer
chemotherapy recommendations resulted with the actual RS.
CONCLUSIONS: Oncologists are able to differentiate between a low or intermediate
RS and a high RS using standard prognostic criteria. However, provision of the
actual RS changed the treatment recommendations in nearly 20% of cases,
suggesting that the RS may reduce chemotherapy use. This effect was observed in
particular in intermediate-risk cases. Prospective clinical trials are necessary
to determine whether decisions based on the RS change outcomes. The 21-gene recurrence score (Oncotype DX: RS) appears to augment
clinico-pathologic prognostication and is predictive of adjuvant chemotherapy
benefit in node-negative (N-) and node-positive (N+), endocrine-sensitive breast
cancer. RS is a costly assay that is associated with good 'value for money' in
N- disease, while economic evaluations in N+ disease based on most recent data
have not been conducted. We examined the cost-utility (CU) of a RS-guided
adjuvant strategy, compared to current practice without RS in N- and N+,
endocrine-sensitive, breast cancer from a Canadian health care system
perspective. A generic state-transition model was developed to compute
cumulative costs and quality-adjusted life years (QALYs) over a 25-year horizon.
Patient outcomes with and without chemotherapy in RS-untested cohorts and in
those with low, intermediate and high RS were examined based on the reported
prognostic and predictive impact of RS in N- and N+ disease. Chemotherapy
utilization (current vs. RS-guided), unit costs and utilities were derived from
a Nova Scotia Canadian population-based cohort, local unit costs and the
literature. Costs and outcomes were discounted at 3% annually, and costs were
reported in 2011 Canadian dollars ($). Probabilistic and one-way sensitivity
analyses were conducted for key model parameters. Compared to a non-RS-guided
strategy, RS-guided adjuvant therapy was associated with $2,585 and $864
incremental costs, 0.27 and 0.06 QALY gains, and resultant CUs of $9,591 and
$14,844 per QALY gained for N- and N+ disease, respectively. CU estimates were
robust to key model parameters, and were most sensitive to chemo utilization
proportions. RS-guided adjuvant therapy appears to be a cost-effective strategy
in both N- and N+, endocrine-sensitive breast cancer with resultant CU ratios
well below commonly quoted thresholds. Use of chemotherapy for patients with estrogen receptor (ER)-positive breast
cancer has been a conflicting issue. Recent studies have identified predictive
markers allowing identification of poor-prognosis ER-positive breast cancers in
need of more aggressive therapy. In general, tumours belonging to the so-called
luminal B class, tumours expressing a high Ki67, human epidermal growth factor
receptor 2 (HER-2) overexpression or a high score on the Oncotype DX gene
expression profile reveal a poor prognosis compared with ER-rich tumours of the
luminal A class. In contrast, recent studies have shown these tumours,
contrasting tumours of the luminal A class, to benefit from more aggressive
anthracycline-containing chemotherapy including a taxane. In the case of
metastatic disease, patients with HER-2-positive, ER-positive tumours may
benefit from having endocrine therapy and an anti-HER-2 agent administered in
combination. In this paper, we attempt to quantify the prognostic information embedded in
multi-parametric histologic biopsy images to predict disease aggressiveness in
estrogen receptor-positive (ER+) breast cancers (BCa). The novel methodological
contribution is in the use of a multi-field-of-view (multi-FOV) framework for
integrating image-based information from differently stained histopathology
slides. The multi-FOV approach involves a fixed image resolution while
simultaneously integrating image descriptors from many FOVs corresponding to
different sizes. For each study, the corresponding risk score (high scores
reflecting aggressive disease and vice versa), predicted by a molecular assay
(Oncotype DX), is available and serves as the surrogate ground truth for
long-term patient outcome. Using the risk scores, a trained classifier is used
to identify disease aggressiveness for each FOV size. The predictions for each
FOV are then combined to yield the final prediction of disease aggressiveness
(good, intermediate, or poor outcome). Independent multi-FOV classifiers are
constructed for (1) 50 image features describing the spatial arrangement of
cancer nuclei (via Voronoi diagram, Delaunay triangulation, and minimum spanning
tree graphs) in H and E stained histopathology and (2) one image feature
describing the vascular density in CD34 IHC stained histopathology. In a cohort
of 29 patients, the multi-FOV classifiers obtained by combining information from
the H and E and CD34 IHC stained channels were able to distinguish low- and
high-risk patients with an accuracy of 0.91 ± 0.02 and a positive predictive
value of 0.94 ± 0.10, suggesting that a purely image-based assay could
potentially replace more expensive molecular assays for making disease
prognostic predictions. In February 2010, the Medical Advisory Secretariat (MAS) began work on
evidence-based reviews of published literature surrounding three pharmacogenomic
tests. This project came about when Cancer Care Ontario (CCO) asked MAS to
provide evidence-based analyses on the effectiveness and cost-effectiveness of
three oncology pharmacogenomic tests currently in use in Ontario.Evidence-based
analyses have been prepared for each of these technologies. These have been
completed in conjunction with internal and external stakeholders, including a
Provincial Expert Panel on Pharmacogenomics (PEPP). Within the PEPP, subgroup
committees were developed for each disease area. For each technology, an
economic analysis was also completed by the Toronto Health Economics and
Technology Assessment Collaborative (THETA) and is summarized within the
reports.THE FOLLOWING REPORTS CAN BE PUBLICLY ACCESSED AT THE MAS WEBSITE AT:
www.health.gov.on.ca/mas or at
www.health.gov.on.ca/english/providers/program/mas/mas_about.htmlGENE EXPRESSION
PROFILING FOR GUIDING ADJUVANT CHEMOTHERAPY DECISIONS IN WOMEN WITH EARLY BREAST
CANCER: An Evidence-Based and Economic AnalysisEpidermal Growth Factor Receptor
Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine
Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung
Cancer: An Evidence-Based and Ecopnomic AnalysisK-RAS testing in Treatment
Decisions for Advanced Colorectal Cancer: an Evidence-Based and Economic
Analysis
OBJECTIVE: To review and synthesize the available evidence regarding the
laboratory performance, prognostic value, and predictive value of Oncotype-DX
for the target population.
CLINICAL NEED: CONDITION AND TARGET POPULATION The target population of this
review is women with newly diagnosed early stage (stage I-IIIa) invasive breast
cancer that is estrogen-receptor (ER) positive and/or progesterone-receptor (PR)
positive. Much of this review, however, is relevant for women with early stage
(I and II) invasive breast cancer that is specifically ER positive, lymph node
(LN) negative and human epidermal growth factor receptor 2 (HER-2/neu) negative.
This refined population represents an estimated incident population of 3,315 new
breast cancers in Ontario (according to 2007 data). Currently it is estimated
that only 15% of these women will develop a distant metastasis at 10 years;
however, a far great proportion currently receive adjuvant chemotherapy,
suggesting that more women are being treated with chemotherapy than can benefit.
There is therefore a need to develop better prognostic and predictive tools to
improve the selection of women that may benefit from adjuvant chemotherapy.
TECHNOLOGY OF CONCERN: The Oncotype-DX Breast Cancer Assay (Genomic Health,
Redwood City, CA) quantifies gene expression for 21 genes in breast cancer
tissue by performing reverse transcription polymerase chain reaction (RT-PCR) on
formalin-fixed paraffin-embedded (FFPE) tumour blocks that are obtained during
initial surgery (lumpectomy, mastectomy, or core biopsy) of women with early
breast cancer that is newly diagnosed. The panel of 21 genes include genes
associated with tumour proliferation and invasion, as well as other genes
related to HER-2/neu expression, ER expression, and progesterone receptor (PR)
expression.
RESEARCH QUESTIONS: What is the laboratory performance of Oncotype-DX?How
reliable is Oncotype-DX (i.e., how repeatable and reproducible is
Oncotype-DX)?How often does Oncotype-DX fail to give a useable result?What is
the prognostic value of Oncotype-DX?Is Oncotype-DX recurrence score associated
with the risk of distant recurrence or death due to any cause in women with
early breast cancer receiving tamoxifen?What is the predictive value of
Oncotype-DX?Does Oncoytpe-DX recurrence score predict significant benefit in
terms of improvements in 10-year distant recurrence or death due to any cause
for women receiving tamoxifen plus chemotherapy in comparison to women receiving
tamoxifen alone?How does Oncotype-DX compare to other known predictors of risk
such as Adjuvant! Online?How does Oncotype-DX impact patient quality of life and
clinical/patient decision-making?
SEARCH STRATEGY: A literature search was performed on March 19(th), 2010 using
OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, the
Cumulative Index to Nursing & Allied Health Literature (CINAHL), the Cochrane
Library, and the International Agency for Health Technology Assessment (INAHTA)
for studies published from January 1(st), 2006 to March 19(th), 2010. A starting
search date of January 1(st), 2006 was because a comprehensive systematic review
of Oncotype-DX was identified in preliminary literature searching. This
systematic review, by Marchionni et al. (2008), included literature up to
January 1(st), 2007. All studies identified in the review by Marchionni et al.
as well as those identified in updated literature searching were used to form
the evidentiary base of this review. The quality of the overall body of evidence
was identified as high, moderate, low or very low according to GRADE
methodology.
INCLUSION CRITERIA: Any observational trial, controlled clinical trial,
randomized controlled trial (RCT), meta-analysis or systematic review that
reported on the laboratory performance, prognostic value and/or predictive value
of Oncotype-DX testing, or other outcome relevant to the Key Questions, specific
to the target population was included.
EXCLUSION CRITERIA: Studies that did not report original data or original data
analysis,Studies published in a language other than English,Studies reported
only in abstract or as poster presentations (such publications were not sought
nor included in this review since the MAS does not generally consider evidence
that is not subject to peer review nor does the MAS consider evidence that lacks
detailed description of methodology).
OUTCOMES OF INTEREST: Outcomes of interest varied depending on the Key Question.
For the Key Questions of prognostic and predictive value (Key Questions #2 and
#3), the prospectively defined primary outcome was risk of 10-year distant
recurrence. The prospectively defined secondary outcome was 10-year death due to
any cause (i.e., overall survival). All additional outcomes such as risk of
locoregional recurrence or disease-free survival (DFS) were not prospectively
determined for this review but were reported as presented in included trials;
these outcomes are referenced as tertiary outcomes in this review. Outcomes for
other Key Questions (i.e., Key Questions #1, #4 and #5) were not prospectively
defined due to the variability in endpoints relevant for these questions.
SUMMARY OF FINDINGS: A total of 26 studies were included. Of these 26 studies,
only five studies were relevant to the primary questions of this review (Key
Questions #2 and #3). The following conclusions were drawn from the entire body
of evidence: There is a lack of external validation to support the reliability
of Oncotype-DX; however, the current available evidence derived from internal
industry validation studies suggests that Oncotype-DX is reliable (i.e.,
Oncotype-DX is repeatable and reproducible).Current available evidence suggests
a moderate failure rate of Oncotype-DX testing; however, the failure rate
observed across clinical trials included in this review is likely inflated; the
current Ontario experience suggests an acceptably lower rate of test failure.In
women with newly diagnosed early breast cancer (stage I-II) that is
estrogen-receptor positive and/or progesterone-receptor positive and lymph-node
negative:There is low quality evidence that Oncotype-DX has prognostic value in
women who are being treated with adjuvant tamoxifen or anastrozole (the latter
for postmenopausal women only),There is very low quality evidence that
Oncotype-DX can predict which women will benefit from adjuvant CMF/MF
chemotherapy in women being treated with adjuvant tamoxifen.In postmenopausal
women with newly diagnosed early breast cancer that is estrogen-receptor
positive and/or progesterone-receptor positive and lymph-node positive:There is
low quality evidence that Oncotype-DX has limited prognostic value in women who
are being treated with adjuvant tamoxifen or anastrozole,There is very low
quality evidence that Oncotype-DX has limited predictive value for predicting
which women will benefit from adjuvant CAF chemotherapy in women who are being
treated with adjuvant tamoxifen.There are methodological and statistical
limitations that affect both the generalizability of the current available
evidence, as well as the magnitude and statistical strength of the observed
effect sizes; in particular:Of the major predictive trials, Oncotype-DX scores
were only produced for a small subset of women (<40% of the original randomized
population) potentially disabling the effects of treatment randomization and
opening the possibility of selection bias;Data is not specific to
HER-2/neu-negative women;There were limitations with multivariate statistical
analyses.Additional trials of observational design may provide further
validation of the prognostic and predictive value of Oncotype-DX; however, it is
unlikely that prospective or randomized data will become available in the near
future due to ethical, time and resource considerations.There is currently
insufficient evidence investigating how Oncoytpe-DX compares to other known
prognostic estimators of risk, such as Adjuvant! Online, and there is
insufficient evidence investigating how Oncotype-DX would impact
clinician/patient decision-making in a setting generalizable to Ontario. Personalized medicine in the sense of individualized therapy concepts plays an
important role in breast cancer. In early breast cancer the molecular subtypes
luminal A and B and basal-like are important for planning adjuvant systemic
therapy. Prognostic and predictive markers, such as hormone receptor status,
HER2, Ki-67, uPA/PAI-1 or multiple gene tests, such as Oncotype DX® currently
allow avoidance of an over therapy or under therapy. In early and also advanced
breast cancer there are an increasing number of new targeted therapies which
represent an augmentation of standard endocrine and chemotherapy and in the
future could at least partially replace them. As a whole the therapy regimens
for breast cancer have become more complex due to the inclusion of molecular
information, new therapies and the withdrawal of conventional treatment
concepts. Decisive for the future will be the confirmation of this development
by modern study concepts contemporarily with adequate evidence. It could then be
expected that a personalized therapy for early breast cancer and in particular
adjuvant chemotherapy would only be used for those patients for whom it is
really necessary. In advanced stage disease there is justified hope that the
survival time in the sense of a chronic disease can be improved by the use of
targeted therapy. |
Which are the DNA (cytosine-5-)-methyltransferases inhibitors? | DNA (Cytosine-5-)-methyltransferases are a family of enzymes that methylate DNA at the C5 position of cytosine residues. Given that methylation of tumour suppressor gene promoters leads to carcinogenesis, inhibition of DNA (Cytosine-5-)-methyltransferases is a promising strategy for the treatment of cancer. There are several inhibitors of DNA (Cytosine-5-)-methyltransferases that uses different modes of action: 5-azacytidine (5-aza-CR, Vidaza®), 5-azadeoxycytidine (5-aza-CdR, decitabine, Dacogen®), 5-azacytosine (ZCyt), 5-fluorodeoxycytidine (FdC), 5,6-dihydro-5-azacytosine (DZCyt), 4'-thio-2'-deoxycytidine, hydralazine, 2-(1H)-pyrimidinone riboside (zebularine), 2-(1H)-pyrimidinone (zebularine aglycon), procaine, procainamide, psammaplin A, and RSC133, a new synthetic derivative of indoleacrylic acid/indolepropionic acid. | The mechanism of inhibition of DNA (cytosine-5-)-methyltransferases by the
mechanism-based inhibitor 5-azacytosine has remained unclear, mainly because of
the unavailability of a substrate in which the inhibitor, but not normal
cytosine, is present at the target site. We synthesized an oligonucleotide
duplex containing a single target site for the EcoRII methyltransferase, in
which the target base is 5-azacytosine. This substrate formed a stable covalent
complex with EcoRII methyltransferase in the absence and in the presence of the
cofactor S-adenosylmethionine. The complex formed in the presence of the
cofactor was resistant to SDS and moderate heat treatment, and a methyl group
was incorporated into the complex. Enzyme titration and kinetic studies of
inhibition suggest that methyl transfer to the complex occurred only during the
first turnover of the reaction. These results suggest that, when the enzyme
binds to 5-azacytosine in the presence of the cofactor, a methyl group is
transferred to the N-5 position of the base, resulting in the inactivation of
the enzyme. 4'-Thio-2'-deoxycytidine was synthesized as a 5'- protected phosphoramidite
compatible with solid phase DNA synthesis. When incorporated as the target
cytosine (C*) in the GC*GC recognition sequence for the DNA methyltransferase M.
HhaI, methyl transfer was strongly inhibited. In contrast, these same
oligonucleotides were normal substrates for the cognate restriction endonuclease
R. HhaI and its isoschizomer R. Hin P1I. M. HhaI was able to bind both
4'-thio-modified DNA and unmodified DNA to equivalent extents under equilibrium
conditions. However, the presence of 4'-thio-2'-deoxycytidine decreased the
half-life of the complex by >10-fold. The crystal structure of a ternary complex
of M. HhaI, AdoMet and DNA containing 4'-thio-2'-deoxycytidine was solved at
2.05 A resolution with a crystallographic R-factor of 0.186 and R-free of 0.231.
The structure is not grossly different from previously solved ternary complexes
containing M. HhaI, DNA and AdoHcy. The difference electron density suggests
partial methylation at C5 of the flipped target 4'-thio-2'-deoxycytidine. The
inhibitory effect of the 4'sulfur atom on enzymatic activity may be traced to
perturbation of a step in the methylation reaction after DNA binding but prior
to methyl transfer. This inhibitory effect can be partially overcome after a
considerably long time in the crystal environment where the packing prevents
complex dissociation and the target is accurately positioned within the active
site. 5-Azacytidine inhibits DNA synthesis and to a lesser proportion RNA synthesis in
S. antibioticus. The biosynthesis of proteins is not affected. The main
inhibitory effect of 5-azacytidine on DNA and RNA synthesis is probably caused
by its incorporation into newly synthesized DNA or RNA and the formation of
covalent complexes between cytosine-specific methyltransferases and the modified
DNA or RNA templates. To analyze whether such effects could occur at the oriC
region of S. antibioticus we analyzed the methylation status of this region
using the bisulphite assisted genomic sequencing method. One of the cytosine
residues found to be partially methylated was contained within an unique NaeI
sequence (GCCGGC) in oriC. Subsequent analysis shows chromosomal DNA from S.
antibioticus to be resistant to R.NaeI restriction indicating that this strain
contains a NaeI-specific cytosine C5-methyltransferase activity. Following
5-azacytidine treatment the NaeI site within the oriC region becomes partially
demethylated. Our results suggest that some of the 5-azacytidine effects on DNA
and RNA synthesis might indeed be related to the complex formation and
inhibition of a cytosine-specific DNA methyltransferase. The incorporation of 5-azacytosine residues into DNA causes potent inhibition of
DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides
incorporating single or multiple 5-aza-2'-deoxycytidine residues at precise
sites was undertaken to generate an array of sequences containing the reactive
5-azacytosine base as specific target sites for enzymatic methylation.
Preparation of these modified oligonucleotides requires the use of
2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic
amino functions. These groups are removed under mild conditions, thus avoiding
conventional protocols that are detrimental to the integrity of the
5-azacytosine ring. Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the
reaction pathway, have been deployed extensively in the analysis of metabolic
pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA
methyltransferases (C5 MTases) by oligodeoxynucleotides containing
5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a
well-documented example of mechanism-based inhibition of enzymes central to
nucleic acid metabolism. Here, we describe the interaction between the C5 MTase
from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex
containing 2-H pyrimidinone, an analogue often referred to as zebularine and
known to give rise to high-affinity complexes with MTases. X-ray crystallography
has demonstrated the formation of a covalent bond between M.HhaI and the 2-H
pyrimidinone-containing oligodeoxynucleotide. This observation enables a
comparison between the mechanisms of action of 2-H pyrimidinone with other
mechanism-based inhibitors such as FdC. This novel complex provides a molecular
explanation for the mechanism of action of the anti-cancer drug zebularine. The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition
of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro.
Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification
that can alter gene activity and chromosomal stability, influencing both
differentiation and tumorigenesis. Thus, it is important to understand the
critical mechanistic determits of ZCyt's inhibitory action. Although several
DNA C5-MTases have been reported to undergo essentially irreversible binding to
ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl
transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate
that formation of stable complexes between HhaI methyltransferase (M.HhaI) and
oligodeoxyribonucleotides containing ZCyt at the target position for methylation
(ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and
AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing
conditions and take on a compact conformation that increases their
electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can
occur only in the presence of AdoMet, these results suggest (1) that the
inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable,
tightly closed conformation of M.HhaI that prevents DNA and co-factor release
and (2) that methylation of ZCyt in DNA is not required for inhibition of
M.HhaI. DNA methyltransferases (DNMTs) are a family of enzymes that methylate DNA at the
C5 position of cytosine residues, and their inhibition is a promising strategy
for the treatment of various developmental and proliferative diseases,
particularly cancers. In the present study, a binding model for hydralazine,
with a validated homology model of human DNMT, was developed by the use of
automated molecular docking and molecular dynamics simulations. The docking
protocol was validated by predicting the binding mode of 2'-deoxycytidine,
5-azacytidine, and 5-aza-2'-deoxycytidine. The inhibitory activity of
hydralazine toward DNMT may be rationalized at the molecular level by similar
interactions within the binding pocket (e.g., by a similar pharmacophore) as
established by substrate-like deoxycytidine analogues. These interactions
involve a complex network of hydrogen bonds with arginine and glutamic acid
residues that also play a major role in the mechanism of DNA methylation.
Despite the different scaffolds of other non-nucleoside DNMT inhibitors such as
procaine and procainamide, the current modeling work reveals that these drugs
exhibit similar interactions within the DNMT1 binding site. These findings are
valuable in guiding the rational design and virtual screening of novel DNMT
inhibitors. Aberrant cytosine methylation in promoter regions leads to gene silencing
associated with cancer progression. A number of DNA methyltransferase inhibitors
are known to reactivate silenced genes; including 5-azacytidine and
2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less
cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic
basis for this difference, we carried out a detailed comparisons of the
interaction between purified DNA methyltransferases and
oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or
2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When
incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by
both nucleosides is essentially identical. However, the stability and
reversibility of the enzyme complex in the absence and presence of cofactor
differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that
are irreversible when the 5-azacytosine ring is intact. ODNs containing
2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of
both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the
ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the
cofactor S-adenosyl methionine are maintained through the formation of a
reversible covalent interaction. The differing stability and reversibility of
the covalent bonds may partially account for the observed differences in
cytotoxicity between zebularine and 5-azacytidine inhibitors. 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. Histone deacetylase inhibitors (HDACi) are promising antitumor drugs acting
through reactivation of silenced tumor suppressor genes. Several HDACi are
currently in clinical trials both for hematological and solid tissue
maligcies. Cooperative action of HDACi and DNA methylation inhibitors (DNMTi)
has been reported, making combined treatment an attractive choice for cancer
therapy. There is some evidence that synergistic effects of HDACi and DNMTi are
achieved by their action on common targets, including DNA methyltransferase 1
(DNMT1). To further analyze this interaction, we investigated the effect of the
HDACi trichostatin A on global and gene-specific DNA methylation and applied
methods with single molecule sensitivity, confocal laser scanning microscopy
with avalanche photodiode detectors (APD imaging) and fluorescence correlation
spectroscopy (FCS), to study its effect on the nuclear dynamics of DNMT1 in live
cells. Our data show that trichostatin A treatment reduces global DNA
methylation and the DNMT1 protein level and alters DNMT1 nuclear dynamics and
interactions with chromatin. The mechanisms underlying these effects are
apparently distinct from the mechanisms of action of the DNMT inhibitor
5-azacytidine. Our study sheds light on the molecular mechanisms underlying the
synergistic action of HDACi and DNMTi and may also help to define improved
policies for cancer treatment. Apoptosis genes, such as TP53 and p16/CDKN2A, that mediate responses to
cytotoxic chemotherapy, are frequently nonfunctional in melanoma.
Differentiation may be an alternative to apoptosis for inducing melanoma cell
cycle exit. Epigenetic mechanisms regulate differentiation, and DNA methylation
alterations are associated with the abnormal differentiation of melanoma cells.
The effects of the deoxycytidine analogue decitabine (5-aza-2'-deoxycytidine),
which depletes DNA methyl transferase 1 (DNMT1), on melanoma differentiation
were examined. Treatment of human and murine melanoma cells in vitro with
concentrations of decitabine that did not cause apoptosis inhibited
proliferation accompanied by cellular differentiation. A decrease in promoter
methylation, and increase in expression of the melanocyte late-differentiation
driver SOX9, was followed by increases in cyclin-dependent kinase inhibitors
(CDKN) p27/CDKN1B and p21/CDKN1A that mediate cell cycle exit with
differentiation. Effects were independent of the TP53, p16/CDKN2A and also the
BRAF status of the melanoma cells. Resistance, when observed, was pharmacologic,
characterized by diminished ability of decitabine to deplete DNMT1. Treatment of
murine melanoma models in vivo with intermittent, low-dose decitabine,
administered sub-cutaneously to limit high peak drug levels that cause
cytotoxicity and increase exposure time for DNMT1 depletion, and with
tetrahydrouridine to decrease decitabine metabolism and further increase
exposure time, inhibited tumor growth and increased molecular and tumor stromal
factors implicated in melanocyte differentiation. Modification of decitabine
dose, schedule and formulation for differentiation rather than cytotoxic
objectives inhibits the growth of melanoma cells in vitro and in vivo. DNA methyltransferase 1 (DNMT1) is an emerging target for the treatment of
cancer, brain disorders, and other diseases. Currently, there are only a few
DNMT1 inhibitors with potential application as therapeutic agents or research
tools. 5,5-Methylenedisalicylic acid is a novel scaffold previously identified
by virtual screening with detectable although weak inhibitory activity of DNMT1
in biochemical assays. Herein, we report enzyme inhibition of a structurally
related compound, trimethylaurintricarboxylic acid (NSC97317) that showed a low
micromolar inhibition of DNMT1 (IC(50) = 4.79 μM). Docking studies of the new
inhibitor with the catalytic domain of DNMT1 suggest that NSC97317 can bind into
the catalytic site. Interactions with amino acid residues that participate in
the mechanism of DNA methylation contribute to the binding recognition. In
addition, NSC97317 had a good match with a structure-based pharmacophore model
recently developed for inhibitors of DNMT1. Trimethylaurintricarboxylic acid can
be a valuable biochemical tool to study DNMT1 inhibition in cancer and other
diseases related to DNA methylation. Aberrant DNA methylation is a critical epigenetic process involved in gene
expression of tumor cells. Diverse DNA methyltransferase inhibitors are being
studied as potential anticancer drugs, and there is interest in developing novel
and more effective DNMTIs. We evaluated zebularine, a stable and low-toxic
cytidine analog, effects on human promyelocytic leukemia cell lines, NB4 and
KG1. Zebularine caused a dose- and time-dependent NB4 and KG1 cell growth
inhibition, did not induce myeloid differentiation but triggered
concentration-dependent apoptosis as manifested by procaspase-3 and PAR-1
cleavage and the occurrence of early apoptosis detected by Annexin-V-propidium
iodide. Zebularine co-treatment with all-trans retinoic acid (RA) at
pharmacological dose (1 μM for NB4 cells) and higher (3 μM for KG1 cells)
increased granulocytic differentiation in both cell lines. Pretreatment for 24
or 48 h with zebularine before the treatment with different doses of RA alone or
RA with histone deacetylase inhibitors, phenyl butyrate, and BML-210, resulted
in significant acceleration and enhancement of differentiation and cell cycle
arrest at G0/1. Zebularine alone or in sequential combination with RA decreased
expression of DNMT1, caused fast and time-dependent expression of pan-cadherin
and partial demethylation of E-cadherin but not tumor suppressor p15. When used
in combination with RA, zebularine increased expression of both genes transcript
and protein. Zebularine induced regional chromatin remodeling by local histone
H4 acetylation and histone H3-K4 methylation in promoter sites of methylated
E-cadherin and also in the promoter of unmethylated p21 as evidenced by
chromatin immunoprecipitation assay. Our results extend the spectrum of
zebularine effects and the evaluation its utility in acute myeloid leukemia
therapy based on epigenetics. DNA methyltransferases (DNMTs) are responsible for DNA methylation, an
epigenetic modification involved in gene regulation. Families of conjugates of
procainamide, an inhibitor of DNMT1, were conceived and produced by rapid
synthetic pathways. Six compounds resulted in potent inhibitors of the murine
catalytic Dnmt3A/3L complex and of human DNMT1, at least 50 times greater than
that of the parent compounds. The inhibitors showed selectivity for C5 DNA
methyltransferases. The cytotoxicity of the inhibitors was validated on two
tumour cell lines (DU145 and HCT116) and correlated with the DNMT inhibitory
potency. The inhibition potency of procainamide conjugated to phthalimide
through alkyl linkers depended on the length of the linker; the dodecane linker
was the best. Zebularine is a novel potent inhibitor of both cytidine deaminase and DNA
methylation. We examined the effect of zebularine on mammary tumor growth in
genetically engineered MMTV-PyMT transgenic mice that develop mammary tumors at
60 days of age with 100% penetrance. The MMTV-PyMT transgenic mice were
randomized at 46 days of age into control (n = 25) and zebularine (n = 25)
treatment groups and monitored for parameters of tumor growth. Zebularine was
administered at 5 mg/mL in drinking water. We observed a significant delay in
the growth of mammary tumors in zebularine-treated mice with a statistically
significant reduction (P = 0.0135) in total tumor burden at 94 days of age when
the mice were sacrificed. After 48 days of zebularine treatment, the tumors were
predomitly necrotic compared with untreated animals. In addition, a high
apoptotic index by terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling assay was observed as early as 13 days following treatment. Immunoblot
analysis showed depletion of DNMT1 and partial depletion of DNMT3b after
zebularine treatment. Microarray analyses of global gene expression identified
upregulation of twelve methylation-regulated genes as well as a set of candidate
cancer genes that participate in cell growth and apoptosis. In summary,
zebularine inhibits the growth of spontaneous mammary tumors and causes early
onset of tumor cell necrosis and apoptosis in a genetically engineered mouse
model of breast cancer. Defining the parameters of zebularine-mediated tumor
inhibition may advance the future development of DNA methyltransferase
inhibitors as an effective cancer treatment. BACKGROUND: DNA methylation of CpG islands within the promoters of specific
genes may play roles in tumor initiation and progression. It has been suggested
such events may serve as critical check points.
METHODS: The present study analyzed the methylation status of CpG islands within
the promoters of secreted frizzled-related proteins (SFRPs) in 87 acute leukemia
(AL) patients, 20 normal controls, and four AL cell lines. 5-aza-2'-
deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, was employed to
determine its effect on SFRP expression.
RESULT: Methylation of at least one SFRP promoter was observed in 69% of the AL
patients analyzed. In addition, methylation of all four SFRP promoters was
observed in Molt-4, Jurkat, HL60 and NB4 cells. In Jurkat cells, methylation
levels of four SFRP promoters decreased in a dose-dependent manner upon
treatment with 5-Aza-CdR, which coincided with increased mRNA expression. With
increasing 5-Aza-CdR concentrations, the expression of DNA methyltransferases,
DNMT3A and DNMT3B, significantly decreased in a dose-dependent manner.
CONCLUSION: The present study demonstrated that SFRP gene methylation may be
involved in AL progression, with a possible epigenetic mechanism influencing Wnt
signaling. Gemcitabine is indicated in combination with cisplatin as first-line therapy for
solid tumours including non-small cell lung cancer (NSCLC), bladder cancer and
mesothelioma. Gemcitabine is an analogue of pyrimidine cytosine and functions as
an anti-metabolite. Structurally, however, gemcitabine has similarities to
5-aza-2-deoxycytidine (decitabine/Dacogen®), a DNA methyltransferase inhibitor
(DNMTi). NSCLC, mesothelioma and prostate cancer cell lines were treated with
decitabine and gemcitabine. Reactivation of epigenetically silenced genes was
examined by RT-PCR/qPCR. DNA methyltransferase activity in nuclear extracts and
recombit proteins was measured using a DNA methyl-transferase assay, and
alterations in DNA methylation status were examined using methylation-specific
PCR (MS-PCR) and pyrosequencing. We observe a reactivation of several
epigenetically silenced genes including GSTP1, IGFBP3 and RASSF1A. Gemcitabine
functionally inhibited DNA methyltransferase activity in both nuclear extracts
and recombit proteins. Gemcitabine dramatically destabilised DNMT1 protein.
However, DNA CpG methylation was for the most part unaffected by gemcitabine. In
conclusion, gemcitabine both inhibits and destabilises DNA methyltransferases
and reactivates epigenetically silenced genes having activity equivalent to
decitabine at concentrations significantly lower than those achieved in the
treatment of patients with solid tumours. This property may contribute to the
anticancer activity of gemcitabine. DNA methyltransferase inhibitors (DNMTIs), including decitabine (DAC) and
azacitidine (AZA), have recently been highlighted for the treatment of high-risk
myelodysplastic syndrome (MDS); however, their action mechanisms have not been
clearly defined. Therefore, we investigated the effects of DNMTIs on MDS-derived
cell lines in vitro. An MDS-derived cell line MDS92 and its blastic subline
MDS-L and HL-60 were used. All three cell lines were sensitive to DNMTIs, but
MDS-L was the most susceptible. DAC-induced cell death in MDS-L was preceded by
DNA damage-induced G2 arrest via a p53-independent pathway. AZA did not
influence the pattern of cell cycle, although it induced DNA damage response.
The IC(50) of DAC or AZA on MDS-L cells was associated with the dose inducing
the maximal hypomethylation in long interspersed nuclear elements-1 (LINE-1)
methylation assay. AZA suppressed the level of methylation in a time-dependent
manner (days 4, 7, and 10), whereas DAC maintained the level of methylation from
day 4 to 11. The protein expression of DNMT1 and DNMT3a decreased with the
suppression of growth and methylation. We conclude that this study provides
in vitro models for understanding the effects of DNMTIs on cell growth and gene
regulation, including differences in the possible action mechanism of DAC and
AZA. Booster of pluripotency: RSC133, a new synthetic derivative of indoleacrylic
acid/indolepropionic acid, exhibits dual activity by inhibiting histone
deacetylase and DNA methyltransferase. Furthermore it potently improves the
reprogramming of human somatic cells into a pluripotent state and aids the
growth and maintece of human pluripotent stem cells (hPSCs). |
Describe armoured brain syndrome. | Armoured brain syndrome is defined by calcified chronic subdural haematoma. | Calcification of chronic subdural haematoma is called "armoured brain" when it
covers most of the cortical surface. We report high-field magnetic resoce
images of the armoured brain, and discuss the relationship between operative
findings, computer assisted tomographic (CT) findings and the change in
relaxation time on magnetic resoce images. In our case, low, iso, and high
density layers were detected on computer assisted tomography. The change in
relaxation time of a liquefied haematoma showed good agreement with
chronological change in intracerebral haematoma, and the material was easily
detected on magnetic resoce images. But with a grainy and mud-like haematoma,
the change in relaxation time did not coincide with the state of the
intracerebral haematoma. It is generally said that in the detection of a
calcified mass, computer assisted tomography is superior to magnetic resoce
images and this was also true in the present case. While there are a few reports
on computer assisted tomographic findings for the armoured brain, this is
probably the first report on high-field (1.5T) magnetic resoce imaging of the
armoured brain. The optimal surgical procedure for patients with calcified and organized chronic
subdural haematoma (CSDH), or "armoured brain", has not been established because
it is difficult to obtain good re-expansion of the brain after surgery. We
present herein the case of a 32-year-old woman with huge calcified CSDH
manifesting as refractory headache, periods of unconsciousness, and unsteady
gait who obtained favourable results after craniotomy. Thinning of the thick
calcified inner membrane using high-speed air drilling was performed after
removal of the organized CSDH. The patient obtained good re-expansion after
surgery. This observation illustrates that it is possible to perform such a
surgery even on a huge calcified CSDH. Calcified chronic subdural collection (armoured brain) is a known long-standing
complication of shunt overdrainage. We report a young male who became
symptomatic eleven years after a shunt surgery. Radiology showed bilateral
calcified subdural collections. Drainage of these collections did not help, but
shunt revision did. Patients with armoured brain syndrome who suddenly become
symptomatic should possibly undergo shunt revision before the more extensive and
morbid procedure of drilling the membranes. Armoured brain is a rare condition where dense calcification occurs over the
brain. It can result in mass effect and raised intracranial pressure. Most
often, it happens due to trauma, subdural effusion, infection, or after VP
shunt. There is controversy in its treatment. Most published literature does not
support removing the calcification. We describe a rare case of idiopathic
chronic calcified subdural hematoma with relatively short history which was
successfully treated by microsurgical removal of calcification over the brain.
This resulted in complete expansion of the brain with relief in symptoms. |
Which are the main histone modifications associated with enhancers? | Histone 3 lysine 4 mono- (H3K4me1) and di-methylation (H3K4me2) are the main post-transcriptional histone modifications related to enhancer activity. | Enhancers cause a high level of transcription and activation of chromatin
structure at target genes. Hyperacetylation of histones H3 and H4, a mark of
active chromatin, is established broadly across target loci by enhancers that
function over long distances. In the present study, we studied the role of an
enhancer in methylation of various lysine residues on H3 by comparing a model
gene locus having an active enhancer with one in which the enhancer has been
inactivated within the context of minichromosomes. The intact enhancer affected
histone methylation at K4, K9 and K36 in distinct ways depending on the
methylation level and the location in the locus. All three lysine residues were
highly tri-methylated in the coding region of the gene linked to the active
enhancer but not the inactive enhancer. However di-methylation of K9 and K36 was
not affected by the enhancer. The enhancer region itself was marked by
mono-methylation at K4 and K9, distinguishing it from the methyl marks in the
gene coding region. These results indicate that an enhancer has roles in
establishing active histone methylation patterns linked with gene transcription
rather than removing methylation linked with gene inactivation. Modifications to the core histones are thought to contribute to ESC pluripotency
by priming tissue-specific promoters and enhancers for later activation.
However, it is unclear how these marks are targeted in ESCs and maintained
during differentiation. Here, we show that the ESC factor Sox2 targets H3K4
methylation to monovalent and bivalent domains. In ESCs, Sox2 contributes to the
formation of a monovalent mark at an enhancer in the pro/pre-B cell-specific
lambda5-VpreB1 locus. Binding of Foxd3 suppresses intergenic transcription of
the enhancer and surrounding sequences. In pro-B cells, enhancer activity is
dependent on the Sox and Fox binding sites, and the enhancer is bound by Sox4,
which is required for efficient expression of lambda5. Our results lead us to
propose a factor relay model whereby ESC factors establish active epigenetic
marks at tissue specific elements before being replaced by cell type-specific
factors as cells differentiate. |
What is the role of Thyrotropin Releasing Hormone in the treatment of comatose patients? | Thyrotropin Releasing Hormone and its analogs are used for treatment of comatose patients. In animal models, Thyrotropin Releasing Hormone and its analogs have been shown to improve the disturbance of consciousness caused by head concussion and pentobarbital. This analeptic action is attributable to stimulation of cholinergic neurons in the septo-hippocampal area and to the presence of terminals containing TRH in the lateral septum and TRH receptors concentrated especially in the medial septum and diagonal band of Broca. It has also been suggested that TRH localized in the pineal gland has a part in activating the neuronal mechanisms of arousal. Associated with the arousal effect and especially evident in variously originated shock conditions are the activating effects of TRH on vegetative functions (body temperature, circulation, the gastrointestinal tract). These stimulatory activities on the CNS were the rationale for therapeutic use of TRH in the initial treatment of coma due to brain trauma. Thyrotropin Releasing Hormone has been shown to induce recovery in comatose patients with extrapontine and pontine myelinosis syndromes. | Hypothalamic hormones as well as anterior pituitary hormones were detected in
the peripheral plasma after the diagnosis of brain death. It is possible that
residual hypothalamic tissue was functioning after satisfying the usual criteria
of total brain death. To examine this possibility, endocrinological and
morphological alterations of the hypothalamic-pituitary system was evaluated in
28 brain dead patients. Intrinsic ADH was depleted in the plasma shortly after
the diagnosis of brain death. Anterior pituitary hormones were initially
detected in all patients, but gradually disappeared. The direct TRH (thyrotropin
releasing hormone) stimulation to the anterior lobe was responded to well.
Morphological studies showed a partial necrosis of the anterior lobe and the
preservation of the posterior lobe for as long as a week. These data prove that
the pituitary is partially preserved after brain death. LH-RH (luteinizing
hormone releasing hormone) was detected in the peripheral plasma of all patients
and GRF (growth hormone releasing factor) was detected in half of the patients
for as long as 15 days, but autopsy revealed the fact that the brain tissue
including the hypothalamus became extensively necrotic after the sixth day of
brain death. In order to solve this controversy it is proposed that these
hormones originate from extracranial tissues such as pancreas. The detection of
hypothalamic hormones after the diagnosis of brain death therefore is not
contradictory to the concept of total brain death. A 46-year-old female motorcyclist, who suffered injuries to the brain stem in a
traffic accident, showed hypotensive and bradycardiac responses to
thyrotropin-releasing hormone (TRH) given to counter consciousness disturbance.
The cardiodepressive responses to TRH were reduced with i.v. pretreatment with
atropine sulfate, suggesting an involvement of the vagal nervous system in the
development of the responses. Furthermore, this patient had complicated
impairments in the sympathetic nervous system, which were revealed by the
results of testing baroreceptor reflex sensitivity to pharmacological
alterations in blood pressure. We thus speculate that the hypotensive and
bradycardiac effects of TRH observed in this patient may result from
derangements of the sympathetic nervous system caused by the injuries. This case
report is believed to be a novel description of the cardiodepressive effects of
TRH. Patients suffering from severe cranio-cerebral trauma show alterations of the
secretory patterns of thyroid stimulating hormone (TSH) and human growth hormone
(HGH) which may be of prognostic significance. We studied 10 patients following
severe brain injury and prospectively compared a new synthetic human growth
hormone releasing factor (HGHRF) test with the thyrotropin releasing hormone
(TRH) test. On admission, all patients had a Glasgow Coma Scale score of 3 or 4.
All patients had a low T3 syndrome. In the patients who died the TSH response
after stimulation with TRH was also absent. In the patients who survived a
significant TSH increase was observed (p less than 0.05). In comparison to the
patients who died those who survived showed a significant (p less than 0.001)
HGH increase after HGHRF stimulation. This test might be useful as an additional
tool in establishing early prognosis in patients with severe brain injury. Pharmacological interest in the tripeptide thyrotropin-releasing hormone (TRH)
is due to the multiple effects it produces. In fact, apart from taking part in
regulating the activity of the hypothalamo-pituitary-thyroid axis, TRH produces
various neuropharmacological effects which indicate a biological role that is
probably more important than that of a releasing hormone. Trials performed in
animals have shown, for example, the dose-dependent capacity of TRH to induce
analgesia, probably by interacting with the opioid peptide system. Motor
activity is affected by TRH. In fact this tripeptide elicits an increase in
spontaneous motor and explorative activities by interacting with the
dopaminergic neurotransmitter system at the nucleus accumbens level. The
neuropharmacological activities of TRH include an interesting arousal effect and
an analeptic action on generalized depression of the CNS whether this depression
is of natural origin, such as hibernation, or induced pharmacologically
(barbiturates, ethanol) or of a traumatic origin (coma). This analeptic action
is attributable to stimulation of cholinergic neurons in the septo-hippocampal
area and to the presence of terminals containing TRH in the lateral septum and
TRH receptors concentrated especially in the medial septum and diagonal band of
Broca. It has also been suggested that TRH localized in the pineal gland has a
part in activating the neuronal mechanisms of arousal. Associated with the
arousal effect and especially evident in variously originated shock conditions
are the activating effects of TRH on vegetative functions (body temperature,
circulation, the gastrointestinal tract). These stimulatory activities on the
CNS were the rationale for therapeutic use of TRH in the initial treatment of
coma due to brain trauma and for the treatment of endogenous depression. A most
interesting property of TRH is that of counteracting the neurological deficit
due to experimental lesion of the spinal cord particularly with regard to
spasticity and ataxia. Electrophysiological trials have shown that TRH
depolarizes the motoneurons in frog spinal cord thereby increasing the
monosynaptic reflex. Furthermore, TRH has recently been shown to have a trophic
effect on cultures of rat fetus spinal cord. On this basis TRH has been used
successfully for the treatment of amyotropic lateral sclerosis (Charcot's
syndrome) and spinocerebellar degeneration. Further support for this therapeutic
strategy is given by the demonstration that deafferentiation of rat spinal cord
produces an increased density of TRH spinal receptors. Recent studies have also
given encouraging results on the possible therapeutic use of TRH for the
treatment of Alzheimer's disease.(ABSTRACT TRUNCATED AT 400 WORDS) The typical patient with post-traumatic hypopituitarism is a young adult male
presenting months to years after an automobile accident, following which he was
unconscious for several days. He will probably have sustained a fracture of the
base of the skull and on recovery is likely to have permanent visual or other
neurological sequelae. Temporary or permanent diabetes insipidus may have
occurred. The features of panhypopituitarism such as weight loss, fatigue,
faintness, loss of libido, and impotence may have been ascribed to depression or
the "postconcussion syndrome" and often inappropriate treatment and
rehabilitation advised. The striking feature on review of the literature is that
the pathological consequences of head injury to the pituitary and hypothalamus
have been well described, while only 47 cases of traumatic hypopituitarism have
been reported. The most likely reason for this disparity is that head injury of
sufficient severity to cause hypothalamic and pituitary damage commonly led to
death. More patients now survive, owing to the availability of intensive care;
accordingly, most cases have been reported in the last 15 years. However,
several patients are described in whom the initiating head injury was not
associated with a skull fracture or followed by coma. We recommend that patients
with major head injury (defined by post-traumatic amnesia greater than 24
hours), and in particular those with fractures of the base of the skull or
diabetes insipidus should be closely monitored for symptoms and signs of
endocrine dysfunction and appropriate dynamic pituitary-function tests
performed. A 65-year-old man was admitted with a sudden onset of dyspnea. Severe mitral
regurgitation due to torn chordae tendinae was revealed on UCG. Mitral valve
replacement was undergone. The postoperative course was complicated with a low
cardiac output syndrome which was successfully treated with IABP, catecholamines
and vasodilators. However, stupor developed on the 4 th postoperative day,
following by tetrapregia on the 8 th day, and deep coma on the 15 th day
respectively. Laboratory studies of the 4 th day disclosed the following values,
serum sodium 150 mEq/l, blood urea nitrogen 84.7 mg/dl, blood sugar 184 mg/dl
and calculated serum osmolality 354 mOsm/l. Cranial CT of the 15 th day showed
an obscure low density area in the central pons which was strongly suggestive of
central pontine myelinolysis (CPM). His CNS symptoms improved dramatically after
administration of thyrotropin-releasing hormone tartrate (TRH-T). A diagnosis of
CPM was made on MRI of the 41 st day. He discharged without any neurological
deficit on the 62 nd day. Effects of a novel TRH analog, montirelin hydrate (NS-3), on the coma caused by
head concussion and narcosis induced by pentobarbital were compared with those
of TRH in mice. Head concussion caused a behavioral comatose state with loss of
the righting reflex and spontaneous motor activity. NS-3 shortened the latent
periods to the recovery of the righting reflex (0.03-0.1 mg/kg, i.v.) and
spontaneous motor activity (0.1 mg/kg, i.v.) following the head concussion. In
the case of TRH, higher doses were needed to induce such effects. NS-3 (0.1-0.3
mg/kg, i.v.) reversed the pentobarbital-induced narcosis in a dose-dependent
manner. A similar effect was elicited by 30- to 100-fold higher doses of TRH
than NS-3. The analeptic effect of NS-3 in the pentobarbital-narcotized mice was
antagonized by SCH23390, a dopamine D1 antagonist or by the combined treatment
with prazosin and scopolamine, while neither prazosin nor scopolamine alone
antagonized the analeptic effect of NS-3. Taken together with the finding that
NS-3 did not bind to dopamine, adrenaline or muscarine receptors, it is
suggested that NS-3 may restore the disturbance of consciousness by activating
the brain dopamine, noradrenaline and acetylcholine neurons without stimulating
these receptors directly. Central pontine and extra-pontine myelinolysis are a well known complication of
hyponatremia. Other causes may be present. We report a case of head injury in a
13 year-old girl, who recovered well after surgery for extra-dural hematoma, but
presented endocrinological disorders with hyperglycemia followed by a severe
hyponatremia. Despite the correction of these metabolic disorders, the patient
became comatose, and MRI, on T2 weighted image, showed hyperintense signals in
the basal ganglia consistent with extra-pontine myelinolysis. The patient's
state remained unchanged for six weeks. Since S. Konno and H. Wakui published
cases of myelinolysis who dramatically improved after TRH treatment, the patient
was given 0.6 mg i.v daily of TRH for six weeks. Improvement began within a few
days, and continued until complete recovery. |
Do Conserved noncoding elements act as enhancers? | An important percentage of noncoding elements conserved across distant species shows enhancer activity and other forms of regulatory functionality. | 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. Determining how transcriptional regulatory signals are encoded in vertebrate
genomes is essential for understanding the origins of multicellular complexity;
yet the genetic code of vertebrate gene regulation remains poorly understood. In
an attempt to elucidate this code, we synergistically combined genome-wide
gene-expression profiling, vertebrate genome comparisons, and transcription
factor binding-site analysis to define sequence signatures characteristic of
candidate tissue-specific enhancers in the human genome. We applied this
strategy to microarray-based gene expression profiles from 79 human tissues and
identified 7187 candidate enhancers that defined their flanking gene expression,
the majority of which were located outside of known promoters. We
cross-validated this method for its ability to de novo predict tissue-specific
gene expression and confirmed its reliability in 57 of the 79 available human
tissues, with an average precision in enhancer recognition ranging from 32% to
63% and a sensitivity of 47%. We used the sequence signatures identified by this
approach to successfully assign tissue-specific predictions to approximately
328,000 human-mouse conserved noncoding elements in the human genome. By
overlapping these genome-wide predictions with a data set of enhancers validated
in vivo, in transgenic mice, we were able to confirm our results with a 28%
sensitivity and 50% precision. These results indicate the power of combining
complementary genomic data sets as an initial computational foray into a global
view of tissue-specific gene regulation in vertebrates. 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. A large-scale enhancer detection screen was performed in the zebrafish using a
retroviral vector carrying a basal promoter and a fluorescent protein reporter
cassette. Analysis of insertional hotspots uncovered areas around developmental
regulatory genes in which an insertion results in the same global expression
pattern, irrespective of exact position. These areas coincide with vertebrate
chromosomal segments containing identical gene order; a phenomenon known as
conserved synteny and thought to be a vestige of evolution. Genomic comparative
studies have found large numbers of highly conserved noncoding elements (HCNEs)
spanning these and other loci. HCNEs are thought to act as transcriptional
enhancers based on the finding that many of those that have been tested direct
tissue specific expression in transient or transgenic assays. Although gene
order in hox and other gene clusters has long been known to be conserved because
of shared regulatory sequences or overlapping transcriptional units, the
chromosomal areas found through insertional hotspots contain only one or a few
developmental regulatory genes as well as phylogenetically unrelated genes. We
have termed these regions genomic regulatory blocks (GRBs), and show that they
underlie the phenomenon of conserved synteny through all sequenced vertebrate
genomes. After teleost whole genome duplication, a subset of GRBs were retained
in two copies, underwent degenerative changes compared with tetrapod loci that
exist as single copy, and that therefore can be viewed as representing the
ancestral form. We discuss these findings in light of evolution of vertebrate
chromosomal architecture and the identification of human disease mutations. Cephalochordates, urochordates, and vertebrates evolved from a common ancestor
over 520 million years ago. To improve our understanding of chordate evolution
and the origin of vertebrates, we intensively searched for particular genes,
gene families, and conserved noncoding elements in the sequenced genome of the
cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets.
Special attention was given to homeobox genes, opsin genes, genes involved in
neural crest development, nuclear receptor genes, genes encoding components of
the endocrine and immune systems, and conserved cis-regulatory enhancers. The
amphioxus genome contains a basic set of chordate genes involved in development
and cell signaling, including a fifteenth Hox gene. This set includes many genes
that were co-opted in vertebrates for new roles in neural crest development and
adaptive immunity. However, where amphioxus has a single gene, vertebrates often
have two, three, or four paralogs derived from two whole-genome duplication
events. In addition, several transcriptional enhancers are conserved between
amphioxus and vertebrates--a very wide phylogenetic distance. In contrast,
urochordate genomes have lost many genes, including a diversity of homeobox
families and genes involved in steroid hormone function. The amphioxus genome
also exhibits derived features, including duplications of opsins and genes
proposed to function in innate immunity and endocrine systems. Our results
indicate that the amphioxus genome is elemental to an understanding of the
biology and evolution of nonchordate deuterostomes, invertebrate chordates, and
vertebrates. 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. The Lmo2 gene encodes a transcriptional cofactor critical for the development of
hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell
acute lymphoblastic leukemia (T-ALL) patients and severe combined
immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled
Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2
regulation has been published so far. By comparative genomics, we identified 17
highly conserved noncoding elements, 9 of which revealed specific acetylation
marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays
performed across 250 kb of the Lmo2 locus in 11 cell types covering different
stages of hematopoietic differentiation. All candidate regulatory regions were
tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed
strong endothelial activity, while the distal promoter showed weak forebrain
activity. Eight of the 15 distal candidate elements functioned as enhancers,
which together recapitulated the full expression pattern of Lmo2, directing
expression to endothelium, hematopoietic cells, tail, and forebrain.
Interestingly, distinct combinations of specific distal regulatory elements were
required to extend endothelial activity of the LMO2 promoter to yolk sac or
fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and
Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus
identifying key upstream regulators and positioning Lmo2 within hematopoietic
regulatory networks. Animal genomes possess highly conserved cis-regulatory sequences that are often
found near genes that regulate transcription and development. Researchers have
proposed that the strong conservation of these sequences may affect the
evolution of the surrounding genome, both by repressing rearrangement, and
possibly by promoting duplicate gene retention. Conflicting data, however, have
made the validity of these propositions unclear. Here, we use a new
computational method to identify phylogenetically conserved noncoding elements
(PCNEs) in a manner that is not biased by rearrangement and duplication. This
method is powerful enough to identify more than a thousand PCNEs that have been
conserved between vertebrates and the basal chordate amphioxus. We test 42 of
our PCNEs in transgenic zebrafish assays--including examples from vertebrates
and amphioxus--and find that the majority are functional enhancers. We find that
PCNEs are enriched around genes with ancient synteny conservation, and that this
association is strongest for extragenic PCNEs, suggesting that cis-regulatory
interdigitation plays a key role in repressing genome rearrangement. Next, we
classify mouse and zebrafish genes according to association with PCNEs, synteny
conservation, duplication history, and presence in bidirectional promoter pairs,
and use these data to cluster gene functions into a series of distinct
evolutionary patterns. These results demonstrate that subfunctionalization of
conserved cis-regulation has not been the primary determinate of gene duplicate
retention in vertebrates. Instead, the data support the gene balance hypothesis,
which proposes that duplicate retention has been driven by selection against
dosage imbalances in genes with many protein connections. We have sequenced and analyzed Hox gene clusters from elephant shark, a
holocephalian cartilaginous fish. Elephant shark possesses 4 Hox clusters with
45 Hox genes that include orthologs for a higher number of ancient gnathostome
Hox genes than the 4 clusters in tetrapods and the supernumerary clusters in
teleost fishes. Phylogenetic analysis of elephant shark Hox genes from 7
paralogous groups that contain all of the 4 members indicated an ((AB)(CD))
topology for the order of Hox cluster duplication, providing support for the 2R
hypothesis (i.e., 2 rounds of whole-genome duplication during the early
evolution of vertebrates). Comparisons of noncoding sequences of the elephant
shark and human Hox clusters have identified a large number of conserved
noncoding elements (CNEs), which represent putative cis-regulatory elements that
may be involved in the regulation of Hox genes. Interestingly, in fugu more than
50% of these ancient CNEs have diverged beyond recognition in the duplicated
(HoxA, HoxB, and HoxD) as well as the singleton (HoxC) Hox clusters.
Furthermore, the b-paralogs of the duplicated fugu Hox clusters are virtually
devoid of unique ancient CNEs. In contrast to fugu Hox clusters, elephant shark
and human Hox clusters have lost fewer ancient CNEs. If these ancient CNEs are
indeed enhancers directing tissue-specific expression of Hox genes, divergence
of their sequences in vertebrate lineages might have led to altered expression
patterns and presumably the functions of their associated Hox genes. Conserved noncoding elements (CNEs) in vertebrate genomes often act as
developmental enhancers, but a critical issue is how well orthologous CNE
sequences retain the same activity in their respective species, a characteristic
important for generalization of model organism studies. To quantify how well CNE
enhancer activity has been preserved, we compared the anatomy-specific
activities of 41 zebra fish CNEs in zebra fish embryos with the activities of
orthologous human CNEs in mouse embryos. We found that 13/41 (∼30%) of the
orthologous CNE pairs exhibit conserved positive activity in zebra fish and
mouse. Conserved positive activity is only weakly associated with either
sequence conservation or the absence of bases undergoing accelerated evolution.
A stronger effect is that disparate activity is associated with transcription
factor binding site divergence. To distinguish the contributions of cis- versus
trans-regulatory changes, we analyzed 13 CNEs in a three-way experimental
comparison: human CNE tested in zebra fish, human CNE tested in mouse, and
orthologous zebra fish CNE tested in zebra fish. Both cis- and trans-changes
affect a significant fraction of CNEs, although human and zebra fish sequences
exhibit disparate activity in zebra fish (indicating cis regulatory changes)
twice as often as human sequences show disparate activity when tested in mouse
and zebra fish (indicating trans regulatory changes). In all four cases where
the zebra fish and human CNE display a similar expression pattern in zebra fish,
the human CNE also displays a similar expression pattern in mouse. This suggests
that the endogenous enhancer activity of ∼30% of human CNEs can be determined
from experiments in zebra fish alone, and to identify these CNEs, both the zebra
fish and the human sequences should be tested. BACKGROUND: The modern coelacanth (Latimeria) is the extant taxon of a basal
sarcopterygian lineage and sister group to tetrapods. Apart from certain
apomorphic traits, its morphology is characterized by a high degree of retention
of ancestral vertebrate structures and little morphological change. An insight
into the molecular evolution that may explain the unchanged character of
Latimeria morphology requires the analysis of the expression patterns of
developmental regulator genes and their cis-regulatory modules (CRMs).
RESULTS: We describe the comparative and functional analysis of the sonic
hedgehog (shh) genomic region of Latimeria menadoensis. Several putative
enhancers in the Latimeria shh locus have been identified by comparisons to
sarcopterygian and actinopterygian extant species. Specific sequence
conservation with all known actinopterygian enhancer elements has been detected.
However, these elements are selectively missing in more recently diverged
actinopterygian and sarcopterygian species. The functionality of the putative
Latimeria enhancers was confirmed by reporter gene expression analysis in
transient transgenic zebrafish and chick embryos.
CONCLUSIONS: Latimeria shh CRMs represent the ancestral set of enhancers that
have emerged before the split of lobe-finned and ray-finned fishes. In contrast
to lineage-specific losses and differentiations in more derived lineages,
Latimeria shh enhancers reveal low levels of sequence diversification. High
overall sequence conservation of shh conserved noncoding elements (CNE) is
consistent with the general trend of high levels of conservation of noncoding
DNA in the slowly evolving Latimeria genome. The vertebrate Lhx2 is a member of the LIM homeobox family of transcription
factors. It is essential for the normal development of the forebrain, eye,
olfactory system and liver as well for the differentiation of lymphoid cells.
However, despite the highly restricted spatio-temporal expression pattern of
Lhx2, nothing is known about its transcriptional regulation. In mammals and
chicken, Crb2, Dennd1a and Lhx2 constitute a conserved linkage block, while the
intervening Dennd1a is lost in the fugu Lhx2 locus. To identify functional
enhancers of Lhx2, we predicted conserved noncoding elements (CNEs) in the
human, mouse and fugu Crb2-Lhx2 loci and assayed their function in transgenic
mouse at E11.5. Four of the eight CNE constructs tested functioned as
tissue-specific enhancers in specific regions of the central nervous system and
the dorsal root ganglia (DRG), recapitulating partial and overlapping expression
patterns of Lhx2 and Crb2 genes. There was considerable overlap in the
expression domains of the CNEs, which suggests that the CNEs are either
redundant enhancers or regulating different genes in the locus. Using a large
set of CNEs (810 CNEs) associated with transcription factor-encoding genes that
express predomitly in the central nervous system, we predicted four
over-represented 8-mer motifs that are likely to be associated with expression
in the central nervous system. Mutation of one of them in a CNE that drove
reporter expression in the neural tube and DRG abolished expression in both
domains indicating that this motif is essential for expression in these domains.
The failure of the four functional enhancers to recapitulate the complete
expression pattern of Lhx2 at E11.5 indicates that there must be other Lhx2
enhancers that are either located outside the region investigated or divergent
in mammals and fishes. Other approaches such as sequence comparison between
multiple mammals are required to identify and characterize such enhancers. |
Can the iPS cell technology be used in Fanconi anemia therapy? | iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications in Fanconi anemia. | The generation of induced pluripotent stem (iPS) cells has enabled the
derivation of patient-specific pluripotent cells and provided valuable
experimental platforms to model human disease. Patient-specific iPS cells are
also thought to hold great therapeutic potential, although direct evidence for
this is still lacking. Here we show that, on correction of the genetic defect,
somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency
to generate patient-specific iPS cells. These cell lines appear
indistinguishable from human embryonic stem cells and iPS cells from healthy
individuals. Most importantly, we show that corrected Fanconi-anaemia-specific
iPS cells can give rise to haematopoietic progenitors of the myeloid and
erythroid lineages that are phenotypically normal, that is, disease-free. These
data offer proof-of-concept that iPS cell technology can be used for the
generation of disease-corrected, patient-specific cells with potential value for
cell therapy applications. The generation of patient-specific induced pluripotent stem cells (iPSCs) offers
unprecedented opportunities for modeling and treating human disease. In
combination with gene therapy, the iPSC technology can be used to generate
disease-free progenitor cells of potential interest for autologous cell therapy.
We explain a protocol for the reproducible generation of genetically corrected
iPSCs starting from the skin biopsies of Fanconi anemia patients using
retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the
fibroblasts and/or keratinocytes of the patients are genetically corrected with
lentiviruses expressing FANCA. The same approach may be used for other diseases
susceptible to gene therapy correction. Genetically corrected, characterized
lines of patient-specific iPSCs can be obtained in 4-5 months. |
Which drug is considered as the first line treatment of fibromyalgia? | Pregabalin is, therefore, a valuable option in the first-line treatment of patients with fibromyalgia. | PURPOSE: Fibromyalgia is a syndrome of unknown origin with a high prevalence.
Multimodal approaches seem to be the treatment of choice in fibromyalgia. A
multidisciplinary program was developed and implemented for patients with
fibromyalgia in the primary care setting. The program included education (seven
sessions) and physical therapy (25 sessions).
METHOD: Patients were referred to the program by their general practitioner or
by a medical specialist. A prospective non-controlled treatment study was
performed, patients were evaluated before, after and three months after the
program (single group time series design). The following measurements were
performed: The Fibromyalgia Impact Questionnaire, RAND 36, the Pain Coping and
Cognition List, the Tampa scale for kinesiophobia, two physical tests and a
qualitative evaluation. Data of 65 patients with fibromyalgia were analysed, of
whom 97% were female. The mean age was 44 and the mean duration of pain was nine
years.
RESULTS: Data of 65 patients with fibromyalgia were analysed, patients
significantly improved on the domains feeling good, pain, fatigue, stiffness,
quality of life, catastrophizing and on the physical tests.
CONCLUSION: The multidisciplinary program fibromyalgia implemented in primary
care seems feasible and the results are promising. BACKGROUND: Valproic acid and its sodium salt (sodium valproate) are
antiepileptic drugs that are sometimes used to treat chronic neuropathic pain
and fibromyalgia, although they are not licensed for this use.
OBJECTIVES: To evaluate the analgesic efficacy and adverse effects of valproic
acid and sodium valproate in the management of chronic neuropathic pain and
fibromyalgia.
SEARCH STRATEGY: We identified randomised controlled trials (RCTs) of valproic
acid and sodium valproate in acute, and chronic pain by searching MEDLINE,
EMBASE and Cochrane CENTRAL to June 2011, together with reference lists of
retrieved papers and reviews.
SELECTION CRITERIA: RCTs that were double blind and of eight-weeks duration or
longer, reporting on analgesic effects and adverse events with valproic acid and
sodium valproate in the treatment of chronic neuropathic pain and fibromyalgia.
DATA COLLECTION AND ANALYSIS: Two review authors independently extracted results
and scored for quality. We extracted efficacy and adverse event data, and
examined issues of study quality.
MAIN RESULTS: We included three studies, two in diabetic neuropathy (42
participants treated with valproate, 42 with placebo), and one in post-herpetic
neuralgia (23 treated with divalproex sodium, 22 with placebo). Study duration
was eight or 12 weeks. No studies were found in fibromyalgia.Only one study
reported one of our primary outcomes (≥ 50% pain relief), while all three
reported group means for pain reduction from baseline to endpoint. In all three
studies; efficacy results were given only for participants who completed the
study. One study in diabetic neuropathy and the study in post-herpetic neuralgia
reported significant differences between active and placebo groups, but there
were insufficient data for reliable pooled analysis.More adverse events were
reported with active treatment than placebo, and included nausea, drowsiness and
abnormal liver function tests. One participant taking sodium valproate withdrew
due to serious derangement of liver enzymes.
AUTHORS' CONCLUSIONS: These three studies no more than hint that sodium
valproate may reduce pain in diabetic neuropathy, and divalproex sodium in
post-herpetic neuralgia, but the use of 'completer' analysis may overestimate
efficacy, and there were too few data for pooled analysis of efficacy or harm,
or to have confidence in the results of the individual studies. There is
insufficient evidence to support the use of valproic acid or sodium valproate as
a first-line treatment for neuropathic pain. There is more robust evidence of
greater efficacy for a small number of other drugs. BACKGROUND: Amitriptyline is a tricyclic antidepressant that is widely used to
treat chronic neuropathic pain (pain due to nerve damage) and fibromyalgia, and
is recommended in many guidelines. These types of pain can be treated with
antidepressant drugs in doses below those at which the drugs act as
antidepressants.
OBJECTIVES: To assess the analgesic efficacy of amitriptyline for chronic
neuropathic pain and fibromyalgia.To assess the adverse events associated with
the clinical use of amitriptyline for chronic neuropathic pain and fibromyalgia.
SEARCH METHODS: We searched CENTRAL, MEDLINE, and EMBASE to September 2012,
together with reference lists of retrieved papers, previous systematic reviews,
and other reviews; we also used our own handsearched database for older studies.
SELECTION CRITERIA: We included randomised, double-blind studies of at least
four weeks' duration comparing amitriptyline with placebo or another active
treatment in chronic neuropathic pain or fibromyalgia.
DATA COLLECTION AND ANALYSIS: We extracted efficacy and adverse event data, and
two study authors examined issues of study quality independently. We performed
analysis using two tiers of evidence. The first tier used data meeting current
best standards, where studies reported the outcome of at least 50% pain
intensity reduction over baseline (or its equivalent), without the use of last
observation carried forward (LOCF) or other imputation method for dropouts,
reported an intention-to-treat (ITT) analysis, lasted 8 to 12 weeks or longer,
had a parallel-group design, and where there were at least 200 participants in
the comparison. The second tier used data that failed to meet this standard and
were therefore subject to potential bias.
MAIN RESULTS: Twenty-one studies (1437 participants) were included; they
individually involved between 15 and 235 participants, only four involved over
100 participants, and the median study size was 44 participants. The median
duration was six weeks. Ten studies had a cross-over design. Doses of
amitriptyline were generally between 25 mg and 125 mg, and dose escalation was
common.There was no top-tier evidence for amitriptyline in treating neuropathic
pain or fibromyalgia.Second-tier evidence indicated no evidence of effect in
cancer-related neuropathic pain or HIV-related neuropathic pain, but some
evidence of effect in painful diabetic neuropathy (PDN), mixed neuropathic pain,
and fibromyalgia. Combining the classic neuropathic pain conditions of PDN,
postherpetic neuralgia (PHN) and post-stroke pain with fibromyalgia for
second-tier evidence, in eight studies and 687 participants, there was a
statistically significant benefit (risk ratio (RR) 2.3, 95% confidence interval
(CI) 1.8 to 3.1) with a number needed to treat (NNT) of 4.6 (3.6 to 6.6). The
analysis showed that even using this potentially biased data, only about 38% of
participants benefited with amitriptyline and 16% with placebo; most
participants did not get adequate pain relief. Potential benefits of
amitriptyline were supported by a lower rate of lack of efficacy withdrawals;
8/153 (5%) withdrew because of lack of efficacy with amitriptyline and 14/119
(12%) with placebo.More participants experienced at least one adverse event; 64%
of participants taking amitriptyline and 40% taking placebo. The RR was 1.5 (95%
CI 1.4 to 1.7) and the number needed to treat to harm was 4.1 (95% CI 3.2 to
5.7). Adverse event and all-cause withdrawals were not different.
AUTHORS' CONCLUSIONS: Amitriptyline has been a first-line treatment for
neuropathic pain for many years. The fact that there is no supportive unbiased
evidence for a beneficial effect is disappointing, but has to be balanced
against decades of successful treatment in many patients with neuropathic pain
or fibromyalgia. There is no good evidence of a lack of effect; rather our
concern should be of overestimation of treatment effect. Amitriptyline should
continue to be used as part of the treatment of neuropathic pain or
fibromyalgia, but only a minority of patients will achieve satisfactory pain
relief. Limited information suggests that failure with one antidepressant does
not mean failure with all.It is unlikely that any large randomised trials of
amitriptyline will be conducted in specific neuropathic pain conditions or in
fibromyalgia to prove efficacy. |
What are the signatures of aggressive periodontitis? | Aggressive periodontitis does not differ from chronic periodontitis from a microbial profile point of view but there are distinctive immunological signatures, including a higher expression in IgG against most periodontal pathogens and a more intense regulatory mechanism of metabolic processes. | This pilot study examined gene expression signatures in pathological gingival
tissues of subjects with chronic or aggressive periodontitis, and explored
whether new subclasses of periodontitis can be identified based on gene
expression profiles. A total of 14 patients, seven with chronic and seven with
aggressive periodontitis, were examined with respect to clinical periodontal
status, composition of subgingival bacterial plaque assessed by checkerboard
hybridizations, and levels of serum IgG antibodies to periodontal bacteria
assayed by checkerboard immunoblotting. In addition, at least two pathological
pockets/patient were biopsied, processed for RNA extraction, amplification and
labeling, and used to study gene expression using Affymetrix U-133 A arrays.
Based on a total of 35 microarrays, no significantly different gene expression
profiles appeared to emerge between chronic and aggressive periodontitis.
However, a de novo grouping of the 14 subjects into two fairly robust clusters
was possible based on similarities in gene expression. These two groups had
similar clinical periodontal status and subgingival bacterial profiles, but
differed significantly with respect to serum IgG levels against the important
periodontal pathogens Porphyromonas gingivalis, Tannerella forsythensis and
Campylobacter rectus. These early data point to the usefulness of gene
expression profiling techniques in the identification of subclasses of
periodontitis with common pathobiology. |
With which complexes is the protein SUS1 associated? | Sus1/ENY2 is a component of the SAGA and TREX-2 complexes | Sus1 is a central component of the yeast gene gating machinery, the process by
which actively transcribing genes such as GAL1 become associated with nuclear
pore complexes. Sus1 is a component of both the SAGA transcriptional
co-activator complex and the TREX-2 complex that binds to nuclear pore
complexes. TREX-2 contains two Sus1 chains that have an articulated helical
hairpin fold, enabling them to wrap around an extended alpha-helix in Sac3,
following a helical hydrophobic stripe. In SAGA, Sus1 binds to Sgf11 and has
been proposed to provide a link between SAGA and TREX-2. We present here the
crystal structure of the complex between Sus1 and the N-terminal region of Sgf11
that forms an extended alpha-helix around which Sus1 wraps in a manner that
shares some similarities with the Sus1-Sac3 interface in TREX-2. However, the
Sus1-binding site on Sgf11 is somewhat shorter than on Sac3 and is based on a
narrower hydrophobic stripe. Engineered mutants that disrupt the Sgf11-Sus1
interaction in vitro confirm the importance of the hydrophobic helical stripe in
molecular recognition. Helix alpha1 of the Sus1-articulated hairpin does not
bind directly to Sgf11 and adopts a wide range of conformations within and
between crystal forms, consistent with the presence of a flexible hinge and also
with results from previous extensive mutagenesis studies (Klöckner, C.,
Schneider, M., Lutz, S., Jani, D., Kressler, D., Stewart, M., Hurt, E., and
Köhler, A. (2009) J. Biol. Chem. 284, 12049-12056). A single Sus1 molecule
cannot bind Sgf11 and Sac3 simultaneously and this, combined with the structure
of the Sus1-Sgf11 complex, indicates that Sus1 forms separate subcomplexes
within SAGA and TREX-2. BACKGROUND: Gene expression is achieved by the coordinated action of multiple
factors to ensure a perfect synchrony from chromatin epigenetic regulation
through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and
is a key player in coupling transcription initiation, elongation and mRNA
export. In the nucleus, Sus1 is associated to the transcriptional co-activator
SAGA and to the NPC associated complex termed TREX2/THSC. Through these
associations, Sus1 mediates the nuclear dynamics of different gene loci and
facilitate the export of the new transcripts.
RESULTS: In this study, we have investigated whether the yeast Sus1 protein is
linked to factors involved in mRNA degradation pathways. We provide evidence for
genetic interactions between SUS1 and genes coding for components of P-bodies
such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is
synthetic lethal with 5'-->3' decay machinery components LSM1 and PAT1 and has a
strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1
overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can
co-localise with components of P-bodies and stress granules. In addition, we
have identified novel physical interactions between Sus1 and factors associated
to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes
the Sus1-TREX2 association.
CONCLUSIONS: In this study, we found genetic and biochemical association between
Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1
accumulates in discrete cytoplasmic granules, which partially co-localise with
P-bodies and stress granules under specific conditions. These interactions
suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in
addition to its nuclear function. SAGA is a transcriptional coactivator complex that is conserved across
eukaryotes and performs multiple functions during transcriptional activation and
elongation. One role is deubiquitination of histone H2B, and this activity
resides in a distinct subcomplex called the deubiquitinating module (DUBm),
which contains the ubiquitin-specific protease Ubp8, bound to Sgf11, Sus1, and
Sgf73. The deubiquitinating activity depends on the presence of all four DUBm
proteins. We report here the 1.90 angstrom resolution crystal structure of the
DUBm bound to ubiquitin aldehyde, as well as the 2.45 angstrom resolution
structure of the uncomplexed DUBm. The structure reveals an arrangement of
protein domains that gives rise to a highly interconnected complex, which is
stabilized by eight structural zinc atoms that are critical for enzymatic
activity. The structure suggests a model for how interactions with the other
DUBm proteins activate Ubp8 and allows us to speculate about how the DUBm binds
to monoubiquitinated histone H2B in nucleosomes. BACKGROUND AND AIMS: Differential responses of closely related species to
submergence can provide insight into the evolution and mechanisms of submergence
tolerance. Several traits of two wetland species from habitats with contrasting
flooding regimes, Rorippa amphibia and Rorippa sylvestris, as well as F(1)
hybrid Rorippa × anceps were analysed to unravel mechanisms underlying
submergence tolerance.
METHODS: In the first submergence experiment (lasting 20 d) we analysed biomass,
stem elongation and carbohydrate content. In the second submergence experiment
(lasting 3 months) we analysed survival and the effect of re-establishment of
air contact on biomass and carbohydrate content. In a separate experiment we
analysed expression of two carbohydrate catabolism genes, ADH1 and SUS1, upon
re-establishment of air contact following submergence.
KEY RESULTS: All plants had low mortality even after 3 months of submergence.
Rorippa sylvestris was characterized by 100 % survival and higher carbohydrate
levels coupled with lower ADH1 gene expression as well as reduced growth
compared with R. amphibia. Rorippa amphibia and the hybrid elongated their stems
but this did not pay-off in higher survival when plants remained submerged. Only
R. amphibia and the hybrid benefited in terms of increased biomass and
carbohydrate accumulation upon re-establishing air contact.
CONCLUSIONS: Results demonstrate contrasting 'escape' and 'quiescence'
strategies between Rorippa species. Being a close relative of arabidopsis,
Rorippa is an excellent model for future studies on the molecular mechanism(s)
controlling these strategies. Transcription and mRNA export are linked processes. However, the molecular
mechanisms of this coordination are not clear. Sus1 (hENY2) participates in this
coordination as part of two protein complexes: SAGA, a transcriptional
co-activator; TREX-2, which functions in mRNA biogenesis and export. Here, we
investigate the coordinated action of SAGA and TREX-2 required for gene
expression. We demonstrate that TREX-2 subunit Sem1 also participates in
transcription activation. Like Sus1, Sem1 is required for the induction of ARG1
and GAL1, these being SAGA-regulated genes. Chromatin immunoprecipitations show
that proper recruitment of certain SAGA subunits to the GAL1 promoter depends on
Sem1. Notably, both in vivo and in vitro analyses reveal that Sem1 influences
SAGA-dependent histone H2B deubiquitylation. Most of these phenotypes are also
found to depend on another TREX-2 subunit, Thp1. These results unveil a new role
for Sem1 in the activation of the SAGA-dependent gene GAL1 and influencing H2B
deubiquitylation. Our work provides insights into a novel functional
relationship between Sem1 and the SAGA complex. The conserved Sac3:Thp1:Sem1:Sus1:Cdc31 (TREX2) complex binds to nuclear pore
complexes (NPCs) and, in addition to integrating mRNA nuclear export with
preceding steps in the gene expression pathway, facilitates re-positioning of
highly regulated actively transcribing genes (such as GAL1) to NPCs. Although
TREX2 is thought to bind NPC protein Nup1, defining the precise role of this
interaction has been frustrated by the complex pleiotropic phenotype exhibited
by nup1Δ strains. To provide a structural framework for understanding the
binding of TREX2 to NPCs and its function in the gene expression pathway, we
have determined the structure of the Nup1:TREX2 interaction interface and used
this information to engineer a Sac3 variant that impairs NPC binding while not
compromising TREX2 assembly. This variant inhibited the NPC association of both
de-repressed and activated GAL1 and also produced mRNA export and growth
defects. These results indicate that the TREX2:Nup1 interaction facilitates the
efficient nuclear export of bulk mRNA together with the re-positioning of GAL1
to NPCs that is required for transcriptional control that is mediated by removal
of SUMO from repressors by NPC-bound Ulp1. |
Can Preimplantation Genetic Diagnosis (PGD) be used for gender selection? | Preimplantation Genetic Diagnosis can be used for gender selection. | PGD has been successfully used for several years. Over 40 babies have been born
worldwide by use of these techniques. Unfortunately, a number of misdiagnoses
have been made, a distressing consequence of a new frontier. Significant
advances have been made to improve the efficiency and accuracy of PCR and FISH.
The widespread use of this technology awaits further documentation of safety and
accuracy. Other issues must also be addressed. First, the cost-effectiveness of
the techniques relative to the traditional alternatives must be evaluated. A
number of ethical issues regarding embryo screening must be addressed including
what diseases are serious enough to warrant the procedure. Another concern is
the use of this technology for nongenetic disorders such as gender selection.
Finally, the experimental nature of these procedures must continually be
discussed with patients, and long-term follow-up studies must be undertaken.
Development of more accurate and less expensive assays coupled with improved IVF
success rates may make PGD a more widely used clinical tool. The future awaits
these developments. The use of PGD for sex selection arouses considerable debate, especially in
countries like India that have a marked cultural preference for boys. It is
argued that using PGD for sex selection is a treatment option that can be
ethically offered to couples who desire to use this technology to plan their
families. The use of preimplantation genetic diagnosis (PGD) to screen embryos for
aneuploidy and genetic disease is growing. New uses of PGD have been reported in
the past year for screening embryos for susceptibility to cancer, for late-onset
diseases, for HLA-matching for existing children, and for gender. These
extensions have raised questions about their ethical acceptability and the
adequacy of regulatory structures to review new uses. This article describes
current and likely future uses of PGD, and then analyses the ethical issues
posed by new uses of PGD to screen embryos for susceptibility and late-onset
conditions, for HLA-matching for tissue donation to an existing child, and for
gender selection. It also addresses ethical issues that would arise in more
speculative scenarios of selecting embryos for hearing ability or sexual
orientation. The article concludes that except for sex selection of the first
child, most current extensions of PGD are ethically acceptable, and provides a
framework for evaluating future extensions for nonmedical purposes that are
still speculative. Preimplantation genetic diagnosis (PGD) was originally developed for couples
whose potential offspring were at risk of severe Mendelian disorders, but has
since been extended to other indications. One possible use of PGD is to perform
gender selection for couples whose offspring are at increased risk of disorders
that do not follow Mendelian inheritance, but which are substantially more
common in one sex than another (unequal sex incidence). Here, we examine the
clinical and ethical issues to be considered prior to offering PGD gender
selection to reduce the risk of a child being affected by a non-Mendelian
condition with unequal sex incidence. Factors to be considered include: the risk
that a child of either sex will be affected by the condition; the overall
reduction in risk provided by gender selection and the potential harms of the
procedure. Consideration should also be given to the interests of the family and
the child to be born, the seriousness of the condition and the couple's
procreative autonomy. To illustrate these issues we use the example of autism, a
non-Mendelian disorder that is considerably more common in males than in
females. The purpose of this article is to ascertain and appraise the ethical issues
inherent to the utilisation of preimplantation genetic diagnosis for gender
selection in infertile patients anticipating undergoing a medically indicated
assisted reproductive technique procedure. Performance of preimplantation
genetic diagnosis per request specifically for gender selection by an infertile
couple undergoing medically indicated assisted reproductive technique may not
breach the principles of ethics, and is unlikely to alter the population balance
of sexes. OBJECTIVE: To examine information on preimplantation genetic diagnosis (PGD)
presented on IVF clinic websites.
DESIGN: We systematically sampled every third IVF clinic on the 2004 Centers for
Disease Control provider list.
SETTING: The Internet.
PATIENT(S): None.
INTERVENTION(S): None.
MAIN OUTCOME MEASURE(S): Benefits, risks, and other types of information
mentioned regarding PGD.
RESULT(S): Of 135 sites examined, 88.1% had websites, and 70% mentioned PGD, of
which 27% were university- or hospital-based and 63% were private clinics. Sites
mentioning PGD listed uses and benefits of PGD far more than the risks involved.
Of these sites, 76% described testing for single-gene diseases, but fewer
mentioned risks of missing target diagnoses (35%) or risks for loss of embryo
(18%), and 14% described PGD as new or controversial. Private clinics were more
likely than other programs to be on either the East or West Coast, list certain
PGD risks (e.g., diagnostic error), note that PGD was new or controversial,
reference source of PGD information, provide accuracy rates of genetic testing
of embryos, and offer gender selection for social reasons.
CONCLUSION(S): Most IVF clinics advertise PGD online, but the scope and quality
of information about it varies widely, emphasizing benefits while minimizing
risks. Clinics and patients may benefit from more thorough and consistent
presentation of PGD, drawing on available evidence to best provide a realistic
portrayal of PGD. OBJECTIVES: To investigate Hong Kong couples' attitudes and concerns about using
preimplantation genetic diagnosis (PGD) and human leukocyte antigens (HLA)
typing to conceive a disease-free and tissue-compatible 'Saviour Child (SC)' to
save an afflicted sibling.
METHODS: Two cohorts of Chinese couples, one with natural pregcies and the
other receiving in vitro fertilization (IVF) services, were studied using a
structured questionnaire.
RESULTS: Although most couples believed that embryos possess moral rights, they
considered it acceptable to reproduce a donor child if it was safe for the
child, and tissue transplantation was the only available treatment for the sick
sibling. Most couples believed that the donor child would not suffer adverse
physical or psychological effects but rather would gain positive psychological
benefits, and opined that parents using PGD/HLA-typing suffer sacrificially for
their children. In response to one specific question, one-third of the couples
agreed to use the donor child as a lifetime organ donor and supported the use of
PGD for non-medical gender selection. One-quarter were willing to reject
PGD/HLA-typing because of its potential for non-medical genetic enhancement.
CONCLUSION: Probably influenced by the Chinese tradition of strong familism,
couples in Hong Kong generally show positive attitudes towards using
PGD/HLA-typing to reproduce a 'SC' to save a sibling affected with
life-threatening diseases amenable to treatment with genetically compatible
tissue. BACKGROUND: Preimplantation genetic diagnosis (PGD) is an appealing option for
couples at risk of having a child with hemophilia A (HA). Although many clinics
offer PGD for HA by gender selection, an approach that detects the presence of
the underlying F8 mutation has several advantages.
OBJECTIVES: To develop and validate analysis protocols combining indirect and
direct methods for identifying F8 mutations in single cells, and to apply these
protocols clinically for PGD.
METHODS: A panel of microsatellite markers in linkage disequilibrium with F8
were validated for single-cell multiplex polymerase chain reaction. For point
mutations, a primer extension genotyping assay was included in the multiplex.
Amplification efficiency was evaluated using buccal cells and blastomeres. Four
clinical PGD analyses were performed, for two families.
RESULTS: Across all validation experiments and the clinical PGD cases,
approximately 80% of cells were successfully genotyped. Following one of the PGD
cycles, healthy twins were born to a woman who carries the F8 intron 22
inversion. The PGD analysis for the other family was complicated by possible
germline mosaicism associated with a de novo F8 mutation, and no pregcy was
achieved.
CONCLUSIONS: PGD for the F8 intron 22 inversion using microsatellite linkage
analysis was validated by the birth of healthy twins to one of the couples. The
other family's situation highlighted the complexities associated with de novo
mutations, and possible germline mosaicism. As many cases of HA result from de
novo mutations, these factors must be considered when assessing the reproductive
options for such families. In the last two decades, the use of preimplantation genetic testing has
increased dramatically. This testing is used for identifying singlegene
disorders, chromosomal abnormalities, mitochondrial disorders, gender selection
in non-mendelian disorders with unequal gender distribution, aneuploidy
screening, and other preconceptually identified genetic abnormalities in
prospective parents. Genetic testing strategies and diagnostic accuracy
continues to improve, but not without risks or controversies. In this review the
authors discuss the techniques and clinical application of preimplantation
genetic diagnosis, and the debate surrounding its associated uncertainty and
expanded use. |
What is the principle of ATAC (Assay for Transposase-Accessible Chromatin) technique? | ATAC-seq (Assay for Transposase-Accessible Chromatin) is an assay for transposase-accessible chromatin using sequencing, based on direct in vitro transposition of sequencing adaptors into native chromatin. ATAC is a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500-50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. | |
Which are the main NMD factors in Saccharomyces cerevisiae? | Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that accelerates the degradation of mRNAs containing premature translation termination codons. This quality control pathway depends on the NMD-specific factors, Upf1p, Upf2p/Nmd2p, and Upf3p, as well as the two release factors, eRF1 and eRF3 (respectively designated Sup45p and Sup35p in yeast). NMD activation is also enabled by the absence of the poly(A)-binding protein, Pab1p, downstream of a termination event | Transcripts harboring premature signals for translation termination are
recognized and rapidly degraded by eukaryotic cells through a pathway known as
nonsense-mediated mRNA decay (NMD). In addition to protecting cells by
preventing the translation of potentially deleterious truncated peptides,
studies have suggested that NMD plays a broader role in the regulation of the
steady-state levels of physiologic transcripts. In Saccharomyces cerevisiae,
three trans-acting factors (Upf1p to Upf3p) are required for NMD. Orthologues of
Upf1p have been identified in numerous species, showing that the NMD machinery,
at least in part, is conserved through evolution. In this study, we demonstrate
additional functional conservation of the NMD pathway through the identification
of Upf2p homologues in Schizosaccharomyces pombe and humans (rent2). Disruption
of S. pombe UPF2 established that this gene is required for NMD in fission
yeast. rent2 was demonstrated to interact directly with rent1, a known
trans-effector of NMD in mammalian cells. Additionally, fragments of rent2 were
shown to possess nuclear targeting activity, although the native protein
localizes to the cytoplasmic compartment. Finally, novel functional domains of
Upf2p and rent2 with homology to eukaryotic initiation factor 4G (eIF4G) and
other translational regulatory proteins were identified. Directed mutations
within these so-called eIF4G homology (4GH) domains were sufficient to abolish
the function of S. pombe Upf2p. Furthermore, using the two-hybrid system, we
obtained evidence for direct interaction between rent2 and human eIF4AI and
Sui1, both components of the translation initiation complex. Based on these
findings, a novel model in which Upf2p and rent2 effects decreased translation
and accelerated decay of nonsense transcripts through competitive interactions
with eIF4G-binding partners is proposed. In Saccharomyces cerevisiae, nonsense-mediated mRNA decay (NMD) requires Upf1p,
Upf2p, and Upf3p to accelerate the decay rate of two unique classes of
transcripts: (1) nonsense mRNAs that arise through errors in gene expression,
and (2) naturally occurring transcripts that lack coding errors but have
built-in features that target them for accelerated decay (error-free mRNAs). NMD
can trigger decay during any round of translation and can target Cbc-bound or
eIF-4E-bound transcripts. Extremely low concentrations of the Upf proteins
relative to the total pool of transcripts make it difficult to understand how
nonsense transcripts are selectively recruited. To stimulate debate, we propose
two alternative mechanisms for selecting nonsense transcripts for NMD and for
assembling components of the surveillance complex, one for the first (pioneer)
round of translation, called "nuclear marking," and the other for subsequent
rounds, called "reverse assembly." The model is designed to accommodate (1) the
low abundance of NMD factors, (2) the role of nucleocytoplasmic shuttling
proteins in NMD, (3) the independent and nonobligate order of assembly of two
different subcomplexes of NMD factors, and (4) the ability of NMD to
simultaneously reduce or eliminate the synthesis of truncated proteins produced
by nonsense transcripts while down-regulating but not completely eliminating
functional proteins produced from error-free NMD-sensitive transcripts Among a large collection of nonsense (termination) suppressors of Saccharomyces
cerevisiae, a few remained obscure for their molecular nature. Of those, a group
of weak and recessive suppressors, sup111, sup112 and sup113, is of particular
interest because of their dependency on [PSI+], a yeast prion. From the facts
that these suppressors map at positions quite similar to the UPF2, UPF3 and UPF1
genes, respectively, and that some mutations in the UPF genes confer termination
suppressor activity, we suspected that sup111, sup112 and sup113 would very well
be mutant alleles of the UPF genes. We tested our speculation and found that
sup113, sup111 and sup112 were in fact complemented with the wild-type alleles
of UPF1, UPF2 and UPF3, respectively. We further obtained evidence that the
UPF1, UPF2 and UPF3 loci of the strains carrying sup113, sup111 and sup112,
respectively, had point mutations. From these results, we conclude that sup111,
sup112 and sup113 are mutant alleles of UPF2, UPF3 and UPF1, respectively, and
thus attribute suppressor activity of these mutations to defects in the NMD
(nonsense-mediated mRNA decay) machinery. BACKGROUND: The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid
degradation of mRNAs containing premature termination codons (PTCs). In yeast
Saccharomyces cerevisiae, the activity of the NMD pathway depends on the
recognition of the PTC by the translational machinery. Translation termination
factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of
protein synthesis but also in mRNA degradation and translation initiation via
interaction with such proteins as Pab1, Upf1, Upf2 and Upf3.
RESULTS: In this work we have used previously isolated sup45 mutants of S.
cerevisiae to characterize degradation of aberrant mRNA in conditions when
translation termination is impaired. We have sequenced his7-1, lys9-A21 and
trp1-289 alleles which are frequently used for analysis of nonsense suppression.
We have established that sup45 nonsense and missense mutations lead to
accumulation of his7-1 mRNA and CYH2 pre-mRNA. Remarkably, deletion of the UPF1
gene suppresses some sup45 phenotypes. In particular, sup45-n upf1Delta double
mutants were less temperature sensitive, and more resistant to paromomycin than
sup45 single mutants. In addition, deletion of either UPF2 or UPF3 restored
viability of sup45-n double mutants.
CONCLUSION: This is the first demonstration that sup45 mutations do not only
change translation fidelity but also acts by causing a change in mRNA stability. In Saccharomyces cerevisiae, mRNA transcripts with premature termination codons
are targeted for deadenylation independent decapping and 5' to 3' decay in a
quality control pathway termed nonsense-mediated decay (NMD). Critical factors
in NMD include Upf1, Upf2, and Upf3, as well as the decapping enzyme, Dcp2/Dcp1.
Loss of Upf2 or Upf3 leads to the accumulation of not only Upf1 and Dcp2 in
P-bodies, but also of the decapping-activators Pat1, Dhh1, and Lsm1. An
interaction between Upf1 and Dcp2 has been identified, which might recruit Dcp2
to the NMD decapping complex. To determine the nature and significance of the
Dcp2-Upf1 interaction, we utilized the yeast two-hybrid assay to assess Upf1
interactions with various mRNA decapping factors. We find that although Dcp2 can
interact with Upf1, this interaction is indirect and is largely dependent on the
Edc3 protein, which interacts with the N-terminal domain of Upf1 at an
overlapping, but not identical, site as Upf2. We also found that Pat1 has an
independent two-hybrid interaction with the N-terminus of Upf1. Assessment of
both reporter and endogenous NMD transcripts suggest that the decapping
stimulators, including Edc3 and Pat1, as well as Edc1 and Edc2, are not
essential for NMD under normal conditions. This work defines a larger decapping
complex involved in NMD, but indicates that components of that complex are not
required for general NMD and might either regulate a subset of NMD transcripts
or be essential for proper NMD under different environmental conditions. Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that accelerates
the degradation of mRNAs containing premature translation termination codons.
This quality control pathway depends on the NMD-specific factors, Upf1p,
Upf2p/Nmd2p, and Upf3p, as well as the two release factors, eRF1 and eRF3
(respectively designated Sup45p and Sup35p in yeast). NMD activation is also
enabled by the absence of the poly(A)-binding protein, Pab1p, downstream of a
termination event. Since Sup35p interacts with both Upf1p and Pab1p we
considered the possibility that differential binding of the latter factors to
Sup35p may be a critical determit of NMD sensitivity for an mRNA. Here we
describe three approaches to assess this hypothesis. First, we tethered
fragments or mutant forms of Sup35p downstream of a premature termination codon
in a mini-pgk1 nonsense-containing mRNA and showed that the inhibition of NMD by
tethered Sup35p does not depend on the domain necessary for the recruitment of
Pab1p. Second, we examined the Sup35p interaction properties of Upf1p and Pab1p
in vitro and showed that these two proteins bind differentially to Sup35p.
Finally, we examined competitive binding between the three proteins and observed
that Upf1p inhibits Pab1p binding to Sup35p whereas the interaction between
Upf1p and Sup35p is relatively unaffected by Pab1p. These data indicate that the
binding of Upf1p and Pab1p to Sup35p may be more complex than anticipated and
that NMD activation could involve more than just simple competition between
these factors. We conclude that activation of NMD at a premature termination
codon is not solely based on the absence of Pab1p and suggest that a specific
recruitment step must commit Upf1p to the process and Upf1p-associated mRNAs to
NMD. |
Which histone marks are deposited by Set7? | Set7 is H4K20 monomethyltransferase. Upregulation of PR-Set7 expression upon loss of HCF-1 leads to improper mitotic H4-K20 methylation. Set7 (or some variant) has also been reported to perform mono-methylation on lysine-4 of H3. | We have purified a human histone H4 lysine 20 methyltransferase and cloned the
encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second
instar larval death coincides with the loss of H4 lysine 20 methylation,
indicating a fundamental role for PR-Set7 in development. Transcriptionally
competent regions lack H4 lysine 20 methylation, but the modification coincided
with condensed chromosomal regions on polytene chromosomes, including
chromocenter and euchromatic arms. The Drosophila male X chromosome, which is
hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20
methylation compared to that of females. In vitro, methylation of lysine 20 and
acetylation of lysine 16 on the H4 tail are competitive. Taken together, these
results support the hypothesis that methylation of H4 lysine 20 maintains silent
chromatin, in part, by precluding neighboring acetylation on the H4 tail. We describe distinct patterns of histone methylation during human cell cycle
progression. Histone H4 methyltransferase activity was found to be cell
cycle-regulated, consistent with increased H4 Lys 20 methylation at mitosis.
This increase closely followed the cell cycle-regulated expression of the H4 Lys
20 methyltransferase, PR-Set7. Localization of PR-Set7 to mitotic chromosomes
and subsequent increase in H4 Lys 20 methylation were inversely correlated to
transient H4 Lys 16 acetylation in early S-phase. These data suggest that H4 Lys
20 methylation by PR-Set7 during mitosis acts to antagonize H4 Lys 16
acetylation and to establish a mechanism by which this mark is epigenetically
transmitted. Expression of viral proteins causes important epigenetic changes leading to
abnormal cell growth. Whether viral proteins directly target histone
methyltransferases (HMTs), a key family enzyme for epigenetic regulation, and
modulate their enzymatic activities remains elusive. Here we show that the E6
proteins of both low-risk and high-risk human papillomavirus (HPV) interact with
three coactivator HMTs, CARM1, PRMT1 and SET7, and downregulate their enzymatic
activities in vitro and in HPV-transformed HeLa cells. Furthermore, these three
HMTs are required for E6 to attenuate p53 transactivation function.
Mechanistically, E6 hampers CARM1- and PRMT1-catalyzed histone methylation at
p53-responsive promoters, and suppresses the binding of p53 to chromatinized DNA
independently of E6-mediated p53 degradation. p53 pre-methylated at lysine-372
(p53K372 mono-methylation) by SET7 protects p53 from E6-induced degradation.
Consistently, E6 downregulates p53K372 mono-methylation and thus reduces p53
protein stability. As a result of the E6-mediated inhibition of HMT activity,
expression of p53 downstream genes is suppressed. Together, our results not only
reveal a clever approach for the virus to interfere with p53 function, but also
demonstrate the modulation of HMT activity as a novel mechanism of epigenetic
regulation by a viral oncoprotein. |
Has proteomics been used in the study of the dry eye syndrome? | Yes, tears obtained from patients with the dry eye syndrome have been analyzed using different proteomic technologies. | The proteins found in tears have an important role in the maintece of the
ocular surface and changes in the quality and quantity of tear components
reflect changes in the health of the ocular surface. In this study, we have used
quantitative proteomics, iTRAQ technology coupled with 2D-oLC-o-ESI-MS/MS
and with a statistical model to uncover proteins that are significantly and
reliably changed in the tears of dry eye patients in an effort to reveal
potential biomarker candidates. Fifty-six patients with dry eye and 40 healthy
subjects were recruited for this study. In total, 93 tear proteins were
identified with a ProtScore >or=2 (>or=99% confidence). Associated with dry eye
were 6 up-regulated proteins, alpha-enolase, alpha-1-acid glycoprotein 1, S100
A8 (calgranulin A), S100 A9 (calgranulin B), S100 A4 and S100 A11 (calgizzarin)
and 4 down-regulated proteins, prolactin-inducible protein (PIP), lipocalin-1,
lactoferrin and lysozyme. Receiver operating curves (ROC) were evaluated for
individual biomarker candidates and a biomarker panel. With the use of a
4-protein biomarker panel, the diagnostic accuracy for dry eye was 96%
(sensitivity, 91.0%; specificity, 90.0%). Two biomarker candidates
(alpha-enolase and S100 A4) generated from iTRAQ experiments were successfully
verified using an ELISA assay. The levels of these 10 tear proteins reflect
aqueous secretion deficiency by lacrimal gland, inflammatory status of the
ocular surface. The clinical classification of the severity of the dry eye
condition was successfully correlated to the proteomics by using three proteins
that are associated with inflammation, alpha1-acid glycoprotein 1, S100 A8 and
S100 A9. The nine tear protein biomarker candidates (except alpha1-acid
glycoprotein 1) were also verified using an independent age-matched patient
sample set. This study demonstrated that iTRAQ technology combined with
2D-oLC-oESI-MS/MS quantitative proteomics is a powerful tool for biomarker
discovery. Tear proteomics, by 2-DE, can give a fingerprint of the protein profile, which
is well suited in clinical proteomics for biomarker identification and in
diagnostics. The mode of tear collection can influence the representation of the
proteins in the tear and therefore it is important to use the appropriate
method. In this study, capillary and Schirmer mode of tear collection was done
in the healthy controls and the Schirmer method was validated in dry eye
syndrome conditions. 2-D PAGE of normal and dry eye tear was performed using pH
3-10 linear IPG strips followed by 13% SDS-PAGE. The spot intensity was analyzed
by the PD quest software. The two methods were compared using Bland-Altman
statistical tool. The 2-D map of capillary and Schirmer tear showed 147 ± 8
spots and 145 ± 7 spots respectively. Both the collection methods were in
agreement with each other and were comparable. Dry eye tear protein showed
differential expression of proteins as observed in 25-35 kDa region. One of the
significantly reduced protein was identified as proline-rich 4 protein. Schirmer
method of tear collection is reliable in patients with dry eye, which can
display the differential protein expression and help in biomarker
identification. Submandibular gland autotransplantation is effective for treating severe dry eye
syndrome. However, more than 40% of patients show epiphora within 3-6 months
after treatment. The mechanism underlying the hypersecretion in epiphora remains
to be elucidated for developing novel interventions. Since salivary gland
secretion is dependent on a variety of proteins, we analyzed the changes in
protein expression in transplanted glands of epiphora patients with 2-D gel
electrophoresis and electrospray ionization quadrupole/time-of-flight mass
spectrometry and evaluated their possible roles in epiphora. There were 23
proteins that showed altered expression in the glands of epiphora patients, 15
being up-expressed and 8 being down-expressed. The expression of secretory
proteins was decreased in these glands, including alpha-amylase, cystatin S, SA,
and SN. In contrast, cytoskeletal proteins were all up-regulated, including
actin and vimentin. Immunofluorescence revealed that the intensity ratio of
F-actin in apical and lateral cytoplasm to total F-actin in acini was decreased
in the glands of epiphora patients. Carbachol stimulation induced a similar
redistribution of F-actin in the control glands. Phosphorylation of
extracellular signal-regulated kinase 1/2 (ERK1/2) was increased in both
carbachol-stimulated and epiphora glands. Preincubation of submandibular glands
with ERK1/2 inhibitors PD98059 or U0126 inhibited carbachol-induced F-actin
redistribution. These results indicated that differentially expressed proteins
participated in the hypersecretion of transplanted submandibular glands and the
redistribution of F-actin might be involved in this hypersecretion in an
ERK1/2-dependent manner. Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder
of the tear and ocular surface. DES with a high prevalence world over needs
identification of potential biomarkers so as to understand not only the disease
mechanism but also to identify drug targets. In this study we looked for
differentially expressed proteins in tear samples of DES to arrive at
characteristic biomarkers. As part of a prospective case-control study, tear
specimen were collected using Schirmer strips from 129 dry eye cases and 73 age
matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis
(DIGE) was done to identify differentially expressed proteins. One of the
differentially expressed protein in DES is lacrimal proline rich 4 protein
(LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay
(ELISA). LPRR4 was down regulated significantly in all types of dry eye cases,
correlating with the disease severity as measured by clinical investigations.
Further characterization of the protein is required to assess its therapeutic
potential in DES. PURPOSE: Diabetes mellitus has been shown to be associated with and complicated
by dry eye syndrome. We sought to examine and compare the tear film proteome of
type 2 diabetic patients with or without dry eye syndrome and normal subjects
using two-dimensional o-liquid chromatography coupled with tandem mass
spectrometry (MS)-based proteomics.
METHODS: Tears were collected from eight type 2 diabetes patients with dry eye
syndrome, eight type 2 diabetes patients without dry eye syndrome, and eight
normal subjects. Tear breakup time (BUT) was determined, and tear proteins were
prepared and analyzed using two-dimensional strong
cation-exchange/reversed-phase o-scale liquid chromatography MS. All MS/MS
spectra were identified by using SEQUEST against the human International Protein
Index (IPI) database and the relative abundance of individual proteins was
assessed by spectral counting.
RESULTS: Tear BUT was significantly lower in patients with diabetes and dry eye
syndrome than in patients with diabetes only and normal subjects. Analysis of
spectral counts of tear proteins showed that, compared to healthy controls,
patients with diabetes and dry eye syndrome had increased expression of
apoptosis-related proteins, like annexin A1, and immunity- and
inflammation-related proteins, including neutrophil elastase 2 and clusterin,
and glycometabolism-related proteins, like apolipoprotein A-II.
CONCLUSIONS: Dry eye syndrome in diabetic patients is associated with aberrant
expression of tear proteins, and the findings could lead to identification of
novel pathways for therapeutic targeting and new diagnostic markers. |
What is the indication for isradipine? | Isradipine is safe and effective when administered long-term in the treatment of hypertensive patients | One-year open Multicentric Isradipine Study (MIS) performed in 7 centres in
Czechoslovakia included 144 patients with mild and moderate hypertension.
Isradipine was given at a dose of 2.5 mg daily. If normalization of diastolic
blood pressure (BP) had not been reached, the dosage was increased to 5 mg.
Monotherapy with isradipine normalized diastolic BP in 44% of patients.
Isradipine (5 mg daily) was combined with bopindolol in patients in whom
isradipine alone failed to normalize diastolic BP. These had higher mean
systolic and diastolic BP, body weight, erythrocyte and platelet counts at the
beginning of the study. The combination of isradipine with bopindolol normalized
diastolic BP in 87% of the group at the end of 48 weeks' treatment. Tolerance
was excellent in 82% of patients. Treatment was discontinued in 8% patients,
undesirable effects being the reason in 2%, ineffective therapy in 2% and poor
adherence to therapy in 4%. Isradipine in monotherapy or in combination with
bopindolol did not exert an adverse effect on the metabolic risk factors of
ischaemic heart disease (cholesterol, glycaemia). Treatment with the new calcium antagonist isradipine significantly reduced
diastolic blood pressure to less than 90 mm Hg in 64% of fourteen blacks with
mild or moderately severe essential hypertension. There was a significant
reduction in the echocardiographic measures of left ventricular wall thickness
and mass in these patients. There was also an increase in fractional left
ventricular mass index, shortening and ejection fraction per 100 g left
ventricular mass and no indication in mean circumferential shortening. There was
another indication of improved left ventricular performance. With the reduced
left ventricular mass and diastolic blood pressure, there was a reduction in the
ratio of peak systolic wall stress to fractional shortening per 100 g left
ventricular mass. There was a significant relationship between peak systolic
wall stress and fractional shortening per 100 g left ventricular mass index. The
directional change after left ventricular mass reduction with isradipine
indicated improved left ventricular function. There was an increase in left
ventricular wall thickness and mass both in those patients not controlled on
isradipine combined with those treated with placebo (n = 10), and in those
treated with placebo (n = 5) there was an increase in wall thickness. These
changes occurred in five weeks. There was no regression to a lower mean of left
ventricular mass or wall thickness during placebo. There was reduction in
electrocardiogram (ECG) ST-T changes of ischemia in those patients with
diastolic blood pressure reduced to less than 90 mm Hg. Isradipine monotherapy
was an effective antihypertensive drug in blacks with essential hypertension,
resulting in regression of left ventricular wall thickness and mass and
augmentation of fractional shortening per 100 g left ventricular mass. The effects of isradipine in a rat model of embolic stroke [permanent occlusion
of the left middle cerebral artery (MCA)] are reviewed. Isradipine, when present
or given up to 4 hours after the onset of stroke, reduces the infarct size,
determined by magnetic resoce imaging (MRI) 24 hours, and by histology 5
days, after MCA occlusion. These cytoprotective effects seem to be permanent and
are paralleled by an improvement in the neurological deficit. Isradipine has
proved to be the most potent and effective calcium antagonist for reducing the
infarct size compared with other representatives of this class of drugs such as
nimodipine, nicardipine and flunarizine. Isradipine is cytoprotective after a
stroke when used as an antihypertensive: at doses which normalise high blood
pressure in spontaneously hypertensive rats, isradipine reduces by more than 60%
the infarct size caused by a subsequent stroke. Since the lowering of blood
pressure, e.g. by a calcium antagonist that does not cross the blood-brain
barrier, is ineffective in reducing the infarct size, normalisation of blood
pressure alone cannot account for the reductions in infarct size observed with
isradipine. The antihypertensive drug isradipine seems rather to offer the
additional benefit of attenuating the consequences of an eventual stroke. If
clinically confirmed, this will be of considerable therapeutic importance.
Evidence is presented that isradipine has at least 2 mechanisms within the brain
that might be responsible for cytoprotection in stroke.(ABSTRACT TRUNCATED AT
250 WORDS) One hundred eighteen patients, aged between 65 and 87 years with
mild-to-moderate hypertension were given placebo or isradipine (1.25 to 5 mg
twice daily, or 2.5 to 10 mg once daily) following a three-week placebo period
to evaluate its safety and efficacy in elderly patients. Dosage step-up was at
three-week intervals. Blood pressure measurements were taken immediately before
the morning dose (12 to 24 hours after the previous dose). At the end of the
study, both supine and standing diastolic blood pressures were significantly
lower with isradipine than with placebo. A 10-mm Hg reduction in supine
diastolic blood pressure was achieved with once-daily isradipine in 65 percent
and with twice-daily isradipine in 52 percent of patients, indicating sustained
blood pressure control over the dose interval (12 to 24 hours). Adverse events
were usually mild, transient, and typical for vasodilators. Isradipine did not
produce more laboratory, clinical, or electrocardiographic abnormalities than
did placebo. In conclusion, isradipine either once or twice daily at doses
between 2.5 and 10 mg is an effective and well-tolerated treatment for
mild-to-moderate hypertension in elderly patients. A total of 152 patients with essential hypertension (World Health Organization
classification I/II) entered a multicenter randomized study to assess the safety
and efficacy of isradipine compared with, and in combination with, the
beta-blocker atenolol. After a three-week placebo treatment period, patients
received either isradipine (2.5, 5, 7.5, or 10 mg twice daily) or atenolol (50
or 100 mg once daily) for seven weeks. Patients who did not have an adequate
response (diastolic blood pressure less than 90 mm Hg) with the maximal doses of
single-drug therapy were given a combination of both drugs for a further seven
weeks. Both drugs resulted in significant and clinically relevant reductions in
blood pressures. Although the reduction in diastolic blood pressure was the same
for patients receiving the two drugs, there was a statistically significant
greater reduction in systolic blood pressure in the isradipine group, suggesting
that this drug has a more favorable effect on vascular impedance. Of the 14
patients who did not attain normal blood pressure levels (sitting diastolic
blood pressure less than 90 mm Hg) receiving monotherapy, 12 patients reached
this goal with a combination of the two drugs. The safety and antihypertensive efficacy of PN 200-110 (isradipine), a novel
calcium antagonist, are discussed in a preliminary report of double-blind,
multicenter, controlled, phase III clinical trials for essential hypertension.
Patients who qualified for entry after a 3 week placebo-washout period were
enrolled in 1 of 5 studies; 2 studies were placebo controlled; 3 studies
evaluated PN 200-110 against 1 of 3 active controls: hydrochlorothiazide (HCTZ)
propranolol or prazosin. A separate study assessing the effects of PN 200-110 in
combination with HCTZ versus propranolol plus HCTZ is also discussed. Compared
with placebo, PN 200-110 decreased mean supine systolic and diastolic blood
pressures by 20/16 mm Hg versus 4/6 mm Hg. Compared with placebo and active
control drugs, PN 200-110 normalized supine diastolic blood pressure to less
than or equal to 90 mm Hg in 72% of patients, versus 13% in the placebo group,
74% in the HCTZ group, 45% in the propranolol group and 69% in the prazosin
group. In the ability to decrease diastolic blood pressure by greater than or
equal to 10 mm Hg, PN 200-110 compared favorably to prazosin (81% vs 81%), and
was superior to HCTZ (81% vs 61%) and propranolol (81% vs 36%). In combination
with HCTZ, PN 200-110 exerted as great an antihypertensive effect as propranolol
plus HCTZ. Long-term therapy with PN 200-110 was also effective. Supine systolic
and diastolic blood pressures decreased a mean of 15/13 mm Hg at 3 months, and
18/20 mm Hg at 12 months. PN 200-110 is well tolerated with relatively few
adverse effects reported. The incidence of vasodilation-related events (flushing, headache, dizziness and
oedema) was determined in a total of 37,670 patients treated with diltiazem,
nicardipine, isradipine or amlodipine and studied by Prescription-Event
Monitoring between 1984 and 1991. Event rates are expressed as the percentage of
patients who experienced these events during the six months after the first
prescription. The rates for all these events with the newer vasoselective
dihydropyridines (nicardipine, isradipine and amlodipine) were higher than those
with diltiazem. Among the three dihydropyridines, there were large individual
differences in the rates. With nicardipine, the frequency of each of the four
vasodilation-related events were similar to one another (approximately 3%). With
isradipine, the rates were also similar to one another but all were
approximately twice those measured in the nicardipine study (approximately 6%).
These differences may have been due to confounding factors such as the publicity
about adverse drug reactions, the indication for use by individual patients or
the doses actually being used at the time the event occurred. With amlodipine,
in contrast, the rate for oedema was two to four times larger than the rates for
flushing, headache or dizziness. In the treatment of hypertension in renally impaired patients, normalization of
blood pressure alone may not be sufficient to prevent significant morbidity to
the kidneys. Treatment must reduce pressure in the renal vasculature, otherwise
glomerular filtration rate and renal plasma flow will continue to deteriorate.
Isradipine a dihydropyridine calcium-channel blocker, has been investigated as a
suitable treatment in this setting. Isradipine maintains glomerular filtration
rate, preserves or enhances renal plasma flow, decreases renal vascular
resistance, maintains or reduces filtration fraction, and exerts a sustained
natriuretic effect, all of which may enable isradipine to slow the rate of
progression of renal deterioration. In addition, isradipine may decrease
proteinuria and may decrease glomerular capillary pressure by dilating both the
efferent and afferent arterioles. Unlike older calcium-channel blockers,
isradipine exhibits minimal cardiodepressant activity and is not associated with
any negative inotropic effects. It is metabolized in the liver and dosage
adjustments may not be necessary when administered to patients with renal
insufficiency. Isradipine has a favorable renal effect profile and also has
several properties that meet the requirements of other patient populations where
an extra measure of antihypertensive safety is required, such as diabetics,
dialysis patients, and transplant recipients. Side effects with isradipine are
usually mild and transient, occurring in a dose-dependent manner. Since the earlier review in Drugs substantial additional data have accumulated
regarding the antihypertensive efficacy of isradipine in various clinical
situations, as well as data on its clinical effects in atherosclerosis. Recent
therapeutic trials confirm that the efficacy of isradipine in the treatment of
patients with mainly mild to moderate hypertension, when administered orally as
a conventional or modified release preparation, is similar to that of titrated
dosages of amlodipine, felodipine, nifedipine, diltiazem, captopril, methyldopa,
metoprolol, prazosin and hydrochlorothiazide. A further decrease in blood
pressure can be expected when isradipine is combined with another
antihypertensive drug in patients who have not responded adequately to
monotherapy. Initial studies have shown that intravenous isradipine is effective
in controlling hypertension following coronary artery bypass graft surgery and
that it appears useful in the treatment of intraoperative hypertension and
hypertensive crisis, and in hypertensive disorders in pregcy, when
administered orally or intravenously. A large study, the Multicentre Isradipine
Diuretic Atherosclerosis Study (MIDAS), was designed to compare the efficacy of
isradipine and hydrochlorothiazide in reducing the rate of progression of
carotid artery wall thickness, measured by B-mode ultrasound, as a surrogate for
early atherosclerosis. Results indicated that wall thickness increased
significantly less with isradipine than hydrochlorothiazide after 6 months of
therapy. Thereafter the rate of progression remained parallel for the remainder
of the 3-year trial. The confirmation of its antihypertensive efficacy, along
with its favourable haemodynamic profile and reversal of left ventricular
hypertrophy, minimal effect on glucose and lipid metabolism, preservation of
quality of life and good tolerability, makes isradipine a suitable drug for the
treatment of most patients with mild to moderate hypertension. Isradipine, a 1,4 dihydropyridine calcium channel antagonist, is a potent
coronary artery dilator that increases coronary blood flow with little effect on
cardiac contractility. Isradipine is an approved antihypertensive agent, but its
antianginal effects have not been well documented. In this placebo-controlled,
double-blind, parallel-group design study we evaluated the duration of effects
and safety of isradipine 10 mg bid in male patients with chronic stable angina
pectoris. Seventy-two patients experiencing moderately severe angina between 3
and 7.5 minutes during a standard Bruce exercise test received placebo in a
single-blind manner for 8-14 days. Sixty-one of these patients had reproducible
treadmill exercise test results on three consecutive occasions and underwent
further exercise tests at 3, 8, and 12 hours after a placebo period. Patients
were then randomized (double blind) to either placebo or isradipine 10 mg bid
for 2 weeks. Symptom-limited exercise tests were repeated predose and at 3, 8,
and 12 hours after the 0800 hour dose dosing. Exercise duration increased
significantly from baseline (last qualifying test during the single-blind
placebo therapy, i.e., 0800 hours predose at visit 4) in the isradipine group
compared to the placebo group prior to the administration of the 0800 hour dose
(i.e., 12 hours after the 2000 hour dose) by 51 vs. 18 seconds, p = 0.04; and
after the administration of the 0800 hour dose at 3 hours by 78 vs. 29 seconds,
p = 0.005; and at 8 hours by 54 vs. 18 seconds, p = 0.04. Similarly, statistical
significance was achieved when exercise data were analyzed using visit 4
(single-blind placebo therapy) corresponding time points as baseline. At 12
hours after the 0800 hour dose, exercise tolerance did not increase
significantly after isradipine compared to placebo. Time to 1-mm ST-segment
depression increased significantly after isradipine at 3 hours post 0800 hour
dose compared to placebo (87 vs. 7 seconds, p < 0.01) but not at the 0, 8, or
12-hour postdose time points, regardless of which baseline was used. Isradipine
therapy did not affect the rate-pressure double product. A significant
correlation between the mean increase in total exercise time and mean plasma
isradipine concentration was also present (p = 0.0295). During double-blind
treatment, drug-related adverse events were experienced by four patients in the
isradipine group and two patients in the placebo group. None of the patients
experienced ischemic complications during the study.(ABSTRACT TRUNCATED AT 400
WORDS) The aim of the present study was to evaluate the effect of dihydropyridine
calcium antagonist isradipine on left ventricular (LV) structure and function in
patients with essential hypertension. Cuff blood pressure and Doppler
echocardiographic variables were assessed in 26 patients with mild to moderate
hypertension (diastolic blood pressure range 95-110 mmHg) before and after 12
weeks of therapy with either isradipine 5 mg daily or enalapril 20 mg daily. The
study was of double-blind, parallel design, with a placebo run-in period of 15
days. Three subjects withdrew from isradipine treatment because of flushing and
2 from enalapril treatment due to cough before completing the study. Both drugs
significantly reduced cuff systolic and diastolic blood pressure (p < 0.001)
without affecting heart rate. By virtue of the decrease in both septal wall (p <
0.01) and posterior wall thicknesses (p < 0.05), isradipine treatment produced a
significant reduction in LV mass adjusted for height (p < 0.001) in comparison
with placebo; also LV end-systolic dimension showed a slight decrease (p <
0.05). Enalapril induced a similar reduction in LV end-systolic dimension (p <
0.05) but the changes of wall thickness and LV mass did not reach statistical
significance. In conclusion, our results indicate that isradipine treatment
improves LV systolic function and causes a significant reduction in LV mass.
This reduction is observed early in the course of antihypertensive treatment and
is effective in both patients with and without LV hypertrophy. These are the preliminary data of an open multicenter trial of antihypertensive
treatment with isradipine as monotherapy (dose, 4.55 +/- 0.56 mg twice daily; n
= 11) or isradipine (7.5 +/- 0.63 mg twice daily) in combination with bopindolol
(1.16 +/- 0.12 mg once daily; n = 30) administered for 3 years to patients with
essential hypertension (WHO classification I or II). Blood pressure was
significantly decreased in both treatment groups and there was no indication of
resistance to therapy. Plasma levels of total cholesterol and triglycerides were
decreased by the end of the second year of treatment, and there was a tendency
toward increase in plasma levels of high-density lipoprotein cholesterol (HDL2
or HDL3). The atherogenic index (ratio between total cholesterol and HDL2 plus
HDL3) was also decreased. Blood glucose levels remained unchanged in both
normoglycemic patients and those with non-insulin-dependent diabetes mellitus
(NIDDM) during 3 years of therapy. It is concluded that isradipine is safe and
effective when administered long-term in the treatment of hypertensive patients
with either hyperlipidemia or NIDDM. BACKGROUND: Hypertension commonly occurs after cardiac surgery and requires
therapy to prevent the potentially deleterious effects.
METHODS AND RESULTS: After coronary artery bypass graft surgery (CABG), 177
patients with elevated blood pressure > or = 90 mm Hg during the initial 6-hour
postsurgical period were selected for this random blinded, parallel study to
receive intravenous infusions of either isradipine (n = 90) or sodium
nitroprusside (n = 87). Isradipine produced a statistically significant decrease
in mean arterial pressure (MAP, delta-23 mmHg) during a 90-minute treatment
period. Target MAP (< or = 85 mmHg or a decrease of 10 mmHg, if baseline MAP was
between 90 and 95 mmHg) was achieved in 94% of patients 30 minutes after
initiation of isradipine infusion (total mean dose, 411 micrograms); target MAP
was achieved in 75% of nitroprusside-treated patients (total mean dose, 1708
micrograms). The mean time to control MAP was 18 minutes for isradipine compared
with 24 minutes for nitroprusside. Global smoothness in MAP control was graded
on a scale of 0 (not controlled) to 5 (excellent). Approximately 76% of
isradipine-treated patients received a rating of > or = 3 (mean score, 3.5); 40%
of the sodium nitroprusside-treated patients achieved a score of > or = 3 (mean
score, 2.0). Both isradipine and nitroprusside produced statistically
significant reductions in systolic and diastolic blood pressures, a decrease in
systemic vascular resistance, and increases in heart rate, cardiac index, and
stroke volume index. Isradipine produced no significant decreases in pulmonary
artery occlusion wedge pressure compared with nitroprusside.
CONCLUSIONS: Intravenous isradipine was effective and well tolerated in patients
with hypertension after CABG and offers an additional therapeutic option to
treat patients after cardiac surgery. The antihypertensive effect of isradipine was studied in 45 patients with
mild-to-moderate hypertension (mean age 59 years) using casual and ambulatory
24-h blood pressure measurement. Patients were included into the study according
to their casual blood pressure. Isradipine was started at a dose of 1.25 mg
twice daily for 4 weeks, and increased to 2.5 mg twice daily if casual blood
pressure was not normalized. If necessary, 3 mg of spirapril, a new
angiotensin-converting enzyme (ACE) inhibitor, (n = 1) or 5 mg of pindolol (n =
1) was added. The active-treatment period lasted 24 weeks. At the end of the
therapy, casual blood pressure was significantly decreased (p < 0.001) from
173/103 to 150/86 mmHg, and mean ambulatory blood pressure, from 146/87 to
140/83 mmHg (p < 0.05). When patients were divided into three groups according
to initial whole-day ambulatory blood pressure values (group I: < 140/90 mmHg;
group II: > or = 140/90 mmHg; group III: > or = 140/<90 mmHg), no effect of
treatment was detected in group I. However, whole-day blood pressure fell
significantly (p < 0.001) in group II (155/96 vs 143/88 mmHg) as did systolic
blood pressure (p < 0.01) in group III (150/83 vs 142/81 mmHg), whereas
diastolic blood pressure remained unchanged. Thus, ambulatory blood pressure
measurement may be superior to casual measurement in the decision-making process
to treat hypertension, avoiding not only the phenomenon of 'white-coat
hypertension', but also ineffective treatment. This conclusion, however, should
be confirmed by prospective studies. Until recently, only three calcium channel antagonists--verapamil, diltiazem and
nifedipine--were available for managing cardiovascular disorders such as
hypertension and ischemic heart disease. In the past few years, however, several
dihydropyridine calcium channel antagonists, including nicardipine, isradipine,
felodipine, nimodipine, and amlodipine, have been marketed. Others are currently
awaiting FDA approval. In addition, bepridil, which belongs to a new class of
calcium channel antagonists, has recently been marketed for refractory angina
pectoris. Clinical uses of calcium channel antagonists have been expanded since
the 1970s to include management of cardiovascular disorders such as
supraventricular arrhythmias, CHF secondary to diastolic dysfunction, and
myocardial reinfarction in selected patients. Calcium channel antagonists are
also being investigated for prevention of atherosclerosis. Calcium channel
antagonists are a heterogeneous group of pharmacologic agents. Differences in
tissue selectivity are largely responsible for the variations in hemodynamic and
electrophysiologic properties of these agents. Thus, their clinical uses and
side effect profiles differ. These differences must be taken into consideration
in the selection of the most appropriate agent for a specific indication.
Potential advantages of some of the newer dihydropyridine calcium channel
antagonists include less frequent dosing (amlodipine and isradipine) and little
or no negative inotropic effect (nicardipine, felodipine, amlodipine,
isradipine) compared with the prototype calcium channel antagonists. Additional
clinical experience with these newer agents is required, however, before their
role in the management of cardiovascular disorders can be fully delineated. The
availability of sustained-release formulations of verapamil, diltiazem,
nifedipine, felodipine, and nicardipine, as well as the recent marketing of
calcium channel antagonists with relatively long half-lives (amlodipine and
isradipine), makes once- or twice-daily dosing possible with most calcium
channel blockers. However, selection of a particular agent will depend on
several factors, including clinical efficacy, side effect profile, cost, and
patient characteristics such as concomitant disease states and baseline
hemodynamic status. To assess left ventricular (LV) structural and functional changes, 45
hypertensive patients were studied by echocardiography after 2 weeks of placebo
and 6 months of isradipine monotherapy. Although LV cavity size did not change,
LV wall thickness decreased dramatically (P < .0001), producing a significant
decrease in LV mass index (from 158 g/m2 to 136 g/m2; P < .0001). In addition,
LV fractional shortening (FS) did not change (1.2%; P = NS) whereas the cardiac
index increased (6.4%; P = .0007) due to a modest tachycardia accompanied by a
reduction in total peripheral resistance (-22.1%; P < .0001). The magnitude of
the reduction of LV mass was related to the degree of FS increase (r = -0.70; P
< .0001), an indication of beneficial LV remodeling. It can be concluded that
isradipine antihypertensive therapy leads to regression of LV hypertrophy
without depression of LV pump function. The study covered influence of Isradipine (2.5 mg administered twice daily
during 6 weeks) on blood circulation in the heart and occupationally important
functions and traits among 30 drivers having mild and moderate arterial
hypertension (AH). Systolic and diastolic pressure demonstrate reliable decrease
in all the examinees with mild AH and in 93.8% of the examinees with moderate
AH. Isradipine proved to influence positively decrease of hypertensive reactions
and subclinical myocardial ischemia. Isradipine presented statistically
significant improvement of the studied psychophysiologic parameters (quickness
of latent and motor visual reaction, number of errors in color choice, exactness
in following the mobile object). Thus, all above enables to recommend Isradipine
(Lomir) as effective and safe method correcting arterial hypertension in
drivers. The peroxidation step in lipid transformation is considered to be essential in
the pathogenesis of atherosclerosis. Calcium antagonists (CA) appear to have
antioxidant effects in addition to their potent vasorelaxant properties. In the
present study, we compared the antioxidative efficacy of CA (amlodipine,
lacidipine, nifedipine, isradipine, diltiazem, and semotiadil) in the
copper-catalysed oxidation of low-density lipoprotein (LDL) with that of
glycated(g)/glycoxidated(go) LDL. This issue is of great importance when
considering the potential therapeutic use of antioxidant drugs in
diabetes-associated vasculopathy. Oxidation of native LDL was inhibited most
efficiently (>90%) by lacidipine and semotiadil in the concentration range
10(-4)-10(-3) M. We found, however, a dramatic decrease in antioxidant activity
towards g/goLDL as compared to native LDL in all the CA tested. Only lacidipine
significantly inhibited copper-mediated oxidation of g/goLDL in the whole
concentration range tested (10(-5) M-10(-3) M). This probably resulted from the
increased auto-oxidative potential introduced by early and advanced glycation
end products (AGE) into the g/goLDL. We noted that coincubation of LDL with
10(-3) M CA and 0.5 M glucose under oxidative/non-oxidative conditions partially
or fully restored the antioxidant capacity of the different CA to inhibit the
subsequent copper-catalysed oxidation of the modified LDL. This is a clear
indication that CA inhibit glycative or glycoxidative LDL changes during the
preceding long-term glycation period. The notion that both oxidative changes and
long-term glycation effects were reduced by CA was corroborated by fluorescence
analysis, AGE-ELISA, quantitation of lipid peroxidation, and thiobarbituric acid
reactive substance (TBARS) measurement of long-term g/goLDL. The strongest
antioxidative effects during long-term glycation of LDL were seen with
isradipine, lacidipine, nifedipine, and semotiadil. Diltiazem was the only CA
that could not prevent TBARS formation in LDL during the long-term glycation
period. In contrast, Amadori product formation, as measured by the generation of
fructosamines, was not significantly reduced by any CA tested. Thus CA, like
other antioxidants, significantly retard AGE formation, while initial glycation
reactions, such as Amadori product formation, are only weakly inhibited. Many children with hypertension, particularly those with new-onset hypertension
related to glomerulonephritis, organ transplantation, or other forms of
secondary hypertension, require treatment with a short-acting antihypertensive
in order to quickly achieve blood pressure (BP) control. We administered
isradipine, a short-acting, second-generation calcium antagonist, to 72 such
children. Retrospective data collection was undertaken to determine the effects
of isradipine treatment. The mean age of children treated with isradipine was
74+/-55 months (mean+/-SD). Nearly all of these children had secondary
hypertension and were initially treated as hospital inpatients for newly
diagnosed hypertension. Mean isradipine dose was 0.36+/-0.17 mg/kg per day, with
no significant variation in dose according to patient age. Isradipine was
administered three times per day in most instances, but 21% of the time it was
administered four times per day. An extemporaneous isradipine suspension was
used in 62% of treatment courses. BP control was achieved with isradipine alone
in 38 children; the remainder received isradipine in combination with additional
antihypertensives. Comparison of pre-treatment BP with BP obtained 8+/-9 days
later demonstrated a significant BP reduction with isradipine treatment, with a
mean reduction of 14+/-13 mmHg for systolic BP and 13+/-15 mmHg for diastolic
BP. There was no effect of isradipine treatment on heart rate. Adverse effects
occurred in 9.5% of treatment courses, and included headache, flushing,
dizziness, and tachycardia. We conclude that isradipine successfully lowers BP
in hypertensive children with secondary forms of hypertension. Use of isradipine
suspension allows infants and young children to be treated as readily as older
children. Calcium channel antagonists (CCAs) may either be divided into the
dihydropyridines (e.g. amlodipine, felodipine, isradipine, lacidipine,
nilvadipine, nifedipine, nicardipine etc.), the phenylalkylamines (e.g.
verapamil) and the benzothiazepines (e.g. diltiazem) according to their chemical
structure, or into first generation agents (nifedipine, verapamil and diltiazem)
and second generation agents (subsequently developed
dihydropyridine-derivatives). Second generation CCAs are characterized by
greater selectivity for calcium channels in vascular smooth muscle cells than
the myocardium, a longer duration of action and a small trough-to-peak variation
in plasma concentrations. Heart failure is characterized by decreased cardiac
output resulting in inadequate oxygen delivery to peripheral tissues. Although
the accompanying neurohormonal activation, leading to vasoconstriction and
increased blood pressure, is initially beneficial in increasing tissue
perfusion, prolonged activation is detrimental because it increases afterload
and further reduces cardiac output. At the level of the myocyte, heart failure
is associated with increased intracellular calcium levels which are thought to
impair diastolic function. These changes indicate that the CCAs would be
beneficial in patients with heart failure. There has been a strong interest and
increasing experience in the use of CCAs in patients with heart failure. Despite
potential beneficial effects in initial small trials, findings from larger
trials suggest that CCA may have detrimental effects upon survival and
cardiovascular events. However, this may not necessarily be a 'class b' effect
of the CCAs as there is considerable heterogeneity in the chemical structure of
individual agents. Clinical experience with different CCAs in patients with
heart failure includes trials that evaluated their effects on hemodynamic
parameters, exercise tolerance and on symptomatology. However, the most relevant
results are those from randomized clinical trials that assessed mortality as the
primary endpoint. First generation CCAs have direct negative inotropic effects
and even sustained release formulations have not proved any beneficial effect
upon survival. With second generation CCAs, some benefit on hemodynamic
parameters has been observed but none on survival, alone or in combination with
ACE inhibitors. It is noteworthy that although amlodipine had a neutral effect
on morbidity and mortality in large, randomized, placebo-controlled trials in
patients with heart failure, the drug was well tolerated. There is no specific
indication for CCAs (first or second generation) in patients with systolic heart
failure, alone or in combination with ACE inhibitors, but amlodipine may be a
considered in the management of hypertension or coronary artery disease in
patients with heart failure. As it was reported earlier that isradipine, a Ca superset 2+ antagonist of
dihydropyridine derivative class, caused regression of nifedipine-induced
hyperplasia of human gingiva, experiments were performed to examine whether or
not isradipine would solely inhibit the proliferation of cultured gingival
fibroblasts. Normal human gingival fibroblast Gin-1 cells were used to test the
impact of this medication. Fibroblast proliferation in the presence of
isradipine (10 microM) was examined by using the reagent water-soluble
tetrazolium-1 (WST-1). The level of basic fibroblast growth factor (bFGF) in the
cell-free supernatant of each well was determined by using an enzyme-linked
immunosorvent assay (ELISA) kit. The production of type I collagen was assayed
by ELISA. Isradipine significantly enhanced the cell proliferation from the
second day of the culture period. Also, isradipine raised the level of bFGF in
the culture medium. The same concentration, also significantly enhanced the
production of type I collagen. In conclusion, we were able to prove that
isradipine causes the proliferation of cultured gingival fibroblasts as well as
other dihydropyridine-derivative Ca superset 2+ antagonists do. In order to
prevent the gingival overgrowth, it is advisable to be very careful in the use
of isradipine as a therapy for hypertension and other indications. |
List non-surgical treatment modalities that are included in the Stupp protocol. | Radiotherapy and chemotherapy are non-surgical treatment modalities that are included in the Stupp protocol. This protocol is widely used for treatment of glioblastoma. | The coincidence of multiple sclerosis (MS) and glioblastoma has been reported in
several anecdotal reports. Little is known concerning the effects of radio-
and/or chemotherapy on demyelinating brain lesions in MS patients. Moreover,
there are no data concerning the effect of concomitant radiochemotherapy
according to the STUPP protocol on the course ofMS in patients with coexisting
glioblastoma. A 43-year-old male patient was diagnosed for relapsing-remitting
MS in 1997. He received interferon and glatiramer acetate for immunomodulatory
treatment and was stable until 2006 (EDSS < 1.5), when neurological
deterioration occurred. He developed a left-sided hemiparesis, and an MRI showed
right temporal contrast-enhancing mass lesion. A subsequent tumor resection was
performed and histology revealed a glioblastoma. At the beginning of
radiochemotherapy, treatment for multiple sclerosis (glatiramer acetate) was
stopped. The tumor responded well to treatment and was clinically as well as
radiologically stable until 9 months after diagnosis of glioblastoma. The
typical radiological MS lesions remained unchanged. The patient died 12 months
after diagnosis of glioblastoma due to tumor progression. This report
demonstrates that concomitant radiochemotherapy according to the STUPP protocol,
was safe in our patient with respect to the radiological as well as the clinical
course of multiple sclerosis. The present work is a retrospective study on glioblastomas treated in the Angers
and Nice Hospital Departments of Neurosurgery between 2006 and 2007. This study
was conducted 2 years after the audit on incident glioblastoma in France in
2004. New events that may modify the care or survival of glioblastoma have
occurred since 2004, justifying the present study. The results show that the
Karnowsky Index is more often included in the clinical files and that the rate
of complete resection has increased, indicating that neurosurgeons are becoming
aware of neuro-oncology. Patients with total resection still have the longest
survival (14 months). Surprisingly, less than half the patients having surgery
received concomitant radiochemotherapy according to the Stupp protocol. Median
overall survival remains at 9 months with intention to treat. For patients
treated with concomitant chemoradiotherapy with temozolomide, the median
survival is 12 months. For patients having a total resection, the median
survival is 14 months, whatever adjuvant treatment is used. Median survival for
patients having total resection and chemoradiotherapy with temozolomide is 18
months, with a 23.3% 2-year survival rate, less than the ORTC trial rate. BACKGROUND: Changes in promoter DNA methylation pattern of genes involved in key
biological pathways have been reported in glioblastoma. Genome-wide assessments
of DNA methylation levels are now required to decipher the epigenetic events
involved in the aggressive phenotype of glioblastoma, and to guide new treatment
strategies.
RESULTS: We performed a whole-genome integrative analysis of methylation and
gene expression profiles in 40 newly diagnosed glioblastoma patients. We also
screened for associations between the level of methylation of CpG sites and
overall survival in a cohort of 50 patients uniformly treated by surgery,
radiotherapy and chemotherapy with concomitant and adjuvant temozolomide (STUPP
protocol). The methylation analysis identified 616 CpG sites differentially
methylated between glioblastoma and control brain, a quarter of which was
differentially expressed in a concordant way. Thirteen of the genes with
concordant CpG sites displayed an inverse correlation between promoter
methylation and expression level in glioblastomas: B3GNT5, FABP7, ZNF217, BST2,
OAS1, SLC13A5, GSTM5, ME1, UBXD3, TSPYL5, FAAH, C7orf13, and C3orf14. Survival
analysis identified six CpG sites associated with overall survival. SOX10
promoter methylation status (two CpG sites) stratified patients similarly to
MGMT status, but with a higher Area Under the Curve (0.78 vs. 0.71, p-value <
5e-04). The methylation status of the FNDC3B, TBX3, DGKI, and FSD1 promoters
identified patients with MGMT-methylated tumors that did not respond to STUPP
treatment (p-value < 1e-04).
CONCLUSIONS: This study provides the first genome-wide integrative analysis of
DNA methylation and gene expression profiles obtained from the same GBM cohort.
We also present a methylome-based survival analysis for one of the largest
uniformly treated GBM cohort ever studied, for more than 27,000 CpG sites. We
have identified genes whose expression may be tightly regulated by epigenetic
mechanisms and markers that may guide treatment decisions. BACKGROUND: O(6) -methylguanine-DNA methyltransferase (MGMT) promoter
methylation status was proposed as a prognostic biomarker for patients with
glioblastoma. However, the prognostic impact of MGMT in patients with newly
diagnosed glioblastoma who receive carmustine-releasing wafers (Gliadel) along
with temozolomide (TMZ) is still unknown.
METHODS: MGMT promoter methylation status and protein expression were analyzed
in formalin-fixed, paraffin-embedded tumor specimens obtained from 111 French
patients with newly diagnosed glioblastoma. Patients received the Gliadel wafers
followed by radiotherapy plus concomitant and adjuvant TMZ chemotherapy while
they were enrolled in a French multicenter prospective study.
RESULTS: For the whole cohort, the median overall survival (OS) was 17.5 months,
and the progression-free survival was 10.3 months. Patients with tumors that
harbored MGMT methylation had a significantly longer OS compared with patients
who had wild-type MGMT (21.7 months vs 15.1 months; P = .025). Similarly,
patients who had low MGMT protein expression (≤15%) had a significantly improved
OS compared with patients who had high MGMT expression (27.0 months vs 15.1
months; P = .021). The extent of resection was the strongest clinical predictor
of outcome. In multivariate Cox models that were adjusted for sex, performance
status, and extent of surgery, both MGMT methylation and protein expression were
identified as independent prognosticators, and the finding was validated
internally using a bootstrap resampling technique. Discrepancies were identified
between protein expression and MGMT methylation status, thus suggesting that the
2 assays probably assess different biologic features.
CONCLUSIONS: MGMT promoter methylation status and low MGMT expression both were
identified as positive prognosticators in patients with newly diagnosed
glioblastoma who underwent surgical resection and received Gliadel wafer
implants followed by adjuvant radiotherapy and concomitant oral TMZ chemotherapy
(the Stupp protocol). PURPOSE: For the last few years wafers of Gliadel have been inserted into the
operation cavity in patients with glioblastoma multiforme. This is followed by
concurrent radio-chemotherapy with temozolomide (TMZ) according to the Stupp
protocol. Only a few studies have investigated this kind of treatment regimen
and the impact in terms of survival and toxicity of the combination of Gliadel
with TMZ and radiotherapy.
METHODS AND MATERIALS: From November 2006 to January 2010, 24 patients with a
newly diagnosed glioblastoma have undergone a tumour resection which was
considered to be macroscopically complete in 12 cases and with tumour residue in
another 12 cases. The mean age at the moment of diagnosis was 60.25years and the
median age 63. Twenty-three patients underwent subsequently concurrent
radio-chemotherapy with TMZ followed by cycles of elevated doses of TMZ as an
adjuvant treatment. One patient had adjuvant radiotherapy alone followed by
adjuvant chemotherapy. Thirteen were able to receive 6 or more cycles of
adjuvant TMZ. Seven patients had received less than 6 cycles of TMZ as an
adjuvant therapy. Two patients did not receive adjuvant TMZ at all.
RESULTS: The median overall survival of our group was 19.2months and the median
progression free survival was 12.3months. Overall survival for the
macroscopically complete-resection patients was 14months, and 12.85months in
subtotal-resection patients. The median OS was 14.25months for patients PS 0 - 1
at the moment of diagnosis and 12.65 for PS 2 patients. Chemotherapy with TMZ
had to be stopped prematurely in 10 cases due to haematotoxicity, digestive
toxicity or early relapse.
CONCLUSIONS: The concomitant use of surgery with implantation of BCNU wafers and
radio-chemotherapy seems to be well tolerated. Despite the small number of
patients treated in our group, particular attention should be paid to the
potential haematological consequences of this multimodal treatment regimen. BACKGROUND: To compare survival and hematological toxicity rates between two
postoperative therapy regimens in patients with primary glioblastoma (GBM),
namely temozolomide (TMZ) concomitant to radiation, followed by adjuvant TMZ,
versus adjuvant TMZ after radiation only.
PATIENTS AND METHODS: A total of 191 patients with primary GBM were
postoperatively treated with either radiation and concomitant TMZ, followed by
adjuvant TMZ (Stupp protocol) (n = 154), or radiation followed by adjuvant TMZ
(n = 37). The incidence of hematological adverse effects (AE) was recorded for
all patients. From both treatment groups, 26 patients were matched according to
age, Karnofsky performance scale (KPS) score, and
O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation.
RESULTS: Hematological AEs were mild in both unmatched groups, but were
significantly more frequent in the concomitant plus adjuvant TMZ group (p <
0.001). Matched-pair analysis confirmed significantly more frequent
hematological AEs in the concomitant and adjuvant group compared to the
sequential (adjuvant) TMZ group (p = 0,012). Patients treated with concomitant
plus adjuvant TMZ showed significantly longer progression-free survival (PFS)
(10.6 versus 6.6 months; p = 0.014), but no prolonged overall survival (OS)
(16.9 vs. 15.6 months; p = 0.717) compared to patients who received the
sequential treatment regimen.
CONCLUSION: In this retrospective study, the OS in patients with primary GBM
treated with sequential TMZ following radiation appeared to be similar to that
in patients treated with concomitant plus adjuvant TMZ. Given the significantly
higher risk of hematological AE for concomitant treatment, the role of
concomitant plus adjuvant TMZ use compared to sequential administration of TMZ,
especially for patients with MGMT-unmethylated tumors, should be further
evaluated. 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. BACKGROUND: Patients with glioblastoma treated with BCNU wafer implantation for
recurrence frequently receive frontline chemoradiotherapy with temozolomide as
part of the Stupp protocol. A retrospective investigation was conducted of
surgical complications in a cohort of these patients treated at a single
institution.
METHODS: We searched our institutional database for patients treated between
January 2006 and October 2012 who had recurrent glioblastoma previously treated
with open surgery followed by the Stupp protocol and then underwent repeat
resection with or without BCNU wafers for recurrent disease. Rates of select
post-operative complications within 3 months of surgery were estimated.
RESULTS: We identified 95 patients with glioblastoma who underwent resection
followed by the Stupp protocol as frontline treatment. At disease recurrence
(first and second recurrence), 63 patients underwent repeat resection with BCNU
wafer implantation and 32 without implantation. Generally, BCNU wafer use was
associated with minor to moderate increases in rates of select complications
versus non-implantation-wound healing abnormalities (14.2 vs. 6.2 %),
cerebrospinal fluid leak (7.9 vs. 3.1 %), hydrocephalus requiring
ventriculoperitoneal shunt (6.3 vs. 9.3 %), chemical meningitis (3.1 vs. 0 %),
cerebral infections (3.1 vs. 0 %), cyst formation (3.1 vs. 3.1 %), cerebral
edema (4.7 vs. 0 %), and empyema formations (1.5 vs. 0 %). Performance status
was well maintained post-operatively in both groups. Median progression-free
survival from the time of first recurrence was 6.0 and 5.0 months, respectively.
CONCLUSIONS: The use of the Stupp protocol as frontline therapy in patients with
glioblastoma does not preclude the use of BCNU wafers at the time of
progression. Primary spinal glioblastoma (GBM) is a rare spinal tumour and is considered to
have poor prognosis. We describe a case of a 17-year-old adolescent boy with a
cervical spine GBM presenting with neck pain and right upper limb weakness.
Initial spinal MRI demonstrated a 4.5 cm lesion extending from C2 to C5
suspicious for demyelination. Despite high-dose corticosteroids, his weakness
progressed resulting in quadriparesis. Subsequent laminectomy and biopsy
confirmed spinal GBM. Shortly after surgery the patient continued to deteriorate
and was essentially bedbound. Standard chemoradiotherapy as per the Stupp
protocol, together with multimodal rehabilitation, resulted in substantial
functional improvement within 6 weeks of initiation. Continued functional
improvement was observed for a period of 11 months. Although an Eastern
Cooperative Oncology Group (ECOG) performance score of 4 would normally preclude
chemoradiotherapy, a prolonged response to treatment and return to independent
function were observed. Maligt glioma, ie, anaplastic astrocytoma and glioblastoma, is the most
common type of primary maligt brain tumor in the People's Republic of China,
and is particularly aggressive. The median survival of patients with newly
diagnosed glioblastoma is only 12-14 months despite advanced therapeutic
strategies. Treatment of maligt glioma consists mainly of surgical resection
followed by adjuvant radiation and chemotherapy. Temozolomide (TMZ), a
second-generation oral alkylating agent, is playing an increasingly important
role in the treatment of maligt glioma in Chinese patients. Since the
publication of a study by Stupp et al in 2005, which used a protocol of
conventional fractionated irradiation with concomitant TMZ followed by standard
TMZ for six cycles, many clinical studies in the People's Republic of China have
demonstrated that such a treatment strategy has significantly improved efficacy
with limited side effects for newly diagnosed glioblastoma after surgery as
compared with strategies that do not contain TMZ. However, as a relatively new
agent, the history and development of TMZ for maligt glioma is not well
documented in Chinese patients. Multicenter, randomized controlled trials
including appropriately sized patient populations investigating multiple aspects
of TMZ therapy and related combination therapies are warranted in patients with
maligt glioma. This review provides an update on the efficacy, mechanism of
action, adverse reactions, and clinical role of TMZ in the treatment of
maligt glioma in Chinese patients. Gliomas are the most frequent primary brain tumors. Their care is difficult
because of the proximity of organs at risk. The treatment of glioblastoma
includes surgery followed by chemoradiation with the protocol of Stupp et al.
The addition of bevacizumab allows an increase in progression-free survival by 4
months but it does not improve overall survival. This treatment is reserved for
clinical trials. Intensity modulation radiotherapy may be useful to reduce the
neurocognitive late effects in different types of gliomas. In elderly patients
an accelerated radiotherapy 40 Gy in 15 fractions allows a similar survival to
standard radiotherapy. O(6)-methylguanine-DNA methyltransferase (MGMT) status
may help to choose between chemotherapy and radiotherapy. There is no standard
for the treatment of recurrent gliomas. Re-irradiation in stereotactic
conditions allows a median survival of 8 to 12.4 months. Anaplastic gliomas with
1p19q mutation have a greater sensibility to chemotherapy by procarbazine,
lomustine and vincristine. Chemoradiotherapy in these patients has become the
standard treatment. Many studies are underway testing targeted therapies, their
place in the therapeutic management and new radiotherapy techniques. OBJECT: The objective of this study was to report the authors' experience with
the long-term administration of temozolomide (TMZ; > 6 cycles, up to 101) in
patients with newly diagnosed glioblastoma and to analyze its feasibility and
safety as well as its impact on survival. The authors also compared data
obtained from the group of patients undergoing long-term TMZ treatment with data
from patients treated with a standard TMZ protocol.
METHODS: A retrospective analysis was conducted of 37 patients who underwent
operations for glioblastoma between 2004 and 2012. Volumetric analysis of
postoperative Gd-enhanced MR images, obtained within 48 hours, confirmed tumor
gross-total resection (GTR) in all but 2 patients. All patients received the
first cycle of TMZ at a dosage of 150 mg/m(2) starting on the second or third
postsurgical day. Afterward, patients received concomitant radiochemotherapy
according to the Stupp protocol. With regard to adjuvant TMZ therapy, the 19
patients in Group A, aged 30-72 years (mean 56.1 years), received 150 mg/m(2)
for 5 days every 28 days for more than 6 cycles (range 7-101 cycles). The 18
patients in Group B, aged 46-82 years (mean 64.8 years), received the same dose,
but for no more than 6 cycles. O(6)-methylguanine-DNA methyltransferase (MGMT)
promoter methylation status was analyzed for both groups and correlated with
overall survival (OS) and progression-free survival (PFS). The impact of age,
sex, Karnofsky Performance Scale score, and Ki 67 staining were also considered.
RESULTS: All patients but 1 in Group A survived at least 18 months (range 18-101
months), and patients in Group B survived no more than 17 months (range 2-17
months). The long-term survivors (Group A), defined as patients who survived at
least 12 months after diagnosis, were 51.3% of the total (19/37). Kaplan-Meier
curve analysis showed that patients treated with more than 6 TMZ cycles had OS
and PFS that was significantly longer than patients receiving standard treatment
(median OS 28 months vs 8 months, respectively; p = 0.0001; median PFS 20 months
vs 4 months, respectively; p = 0.0002). By univariate and multivariate Cox
proportional hazard regression analysis, MGMT methylation status and number of
TMZ cycles appeared to be survival prognostic factors in patients with
glioblastoma. After controlling for MGMT status, highly significant differences
related to OS and PFS between patients with standard and long-term TMZ treatment
were still detected. Furthermore, in Group A and B, the statistical correlation
of MGMT status to the number of TMZ cycles showed a significant difference only
in Group A patients, suggesting that MGMT promoter methylation was predictive of
response for long-term TMZ treatment. Prolonged therapy did not confer
hematological toxicity or opportunistic infections in either patient group.
CONCLUSIONS: This study describes the longest experience so far reported with
TMZ in patients with newly diagnosed glioblastomas, with as many as 101 cycles,
who were treated using GTR. Statistically significant data confirm that median
survival correlates with MGMT promoter methylation status as well as with the
number of TMZ cycles administered. Long-term TMZ therapy appears feasible and
safe. |
Which genes are associated with Ehlers-Danlos syndrome type I/II? | It is currently estimated that approximately 50% of patients with a clinical diagnosis of classic Ehlers-Danlos syndrome harbor mutations in the COL5A1 and the COL5A2 gene, encoding the α1 and the α2-chain of type V collagen, respectively | Ehlers-Danlos syndrome (EDS) is a group of heritable disorders of connective
tissue with skin, ligaments and blood vessels being the main sites affected. The
commonest variant (EDS II) exhibits an autosomal domit mode of inheritance
and is characterized by joint hypermobility, cigarette paper scars, lax skin and
excessive bruising. As yet no gene has been linked to EDS II, nor has linkage
been established to a specific region of the genome. However, several candidate
genes encoding proteins of the extracellular matrix have been excluded. Using an
intragenic simple sequence repeat polymorphism, we report linkage of the COL5A1
gene, which encodes the alpha 1(V) chain of type V collagen, to EDS II. A
maximum LOD score (Zmax) for linkage of 8.3 at theta = 0.00 was generated for a
single large pedigree. Type V collagen is a constituent of type I collagen-rich fibrils in many
connective tissues and is a regulator of fibril diameter. In tissues, type V
collagen is a heterotrimer with the molecular structure: alpha 1(V)2 alpha 2(V)
or alpha 1(V) alpha 2(V) alpha 3(V). We report that genomic polymorphisms at the
pro alpha 1(V) gene (COL5A1) locus cosegregated with the gravis form of
Ehlers-Danlos syndrome (EDS) (type I) in a three generation family. Affected
family members, who had classical features including joint hyperextensibility,
fragile skin, and widened, atrophic scars, were heterozygous for a 4 bp deletion
at positions from +3 to +6 of intron 65, which resulted in removal of exon 65
sequences from processed mRNAs. Since exon 65 encodes 78 residues of the
carboxyl propeptide, the expected result of this mutation is reduced efficiency
in incorporating mutant pro alpha 1(V) chains into type V collagen molecules and
reduced type V collagen synthesis. These studies indicate that heterozygous
mutations in COL5A1 can result in EDS type I. However, linkage studies in other
EDS I families indicate the disorder is heterogeneous; linkage to both COL5A1
and COL5A2 was excluded in two other families with EDS I while a fourth family
was concordant for linkage to COL5A1 (Z = 2.11; theta = 0.00). To investigate the role of COL5A1 as a candidate gene for Ehlers-Danlos syndrome
(EDS), we have carried out linkage studies in two large British families with
EDS type I/II and type II, respectively. Fourteen living, affected individuals
were identified by family history, clinical examination and ultrastructural
analysis. A polymorphic intragenic simple sequence repeat at the COL5A1 locus
showed linkage to EDS without recombination to give a combined lod score of 5.7.
We have previously reported linkage to COL5A1 in an EDS type I/II family which
brings the total lod score to 9.8 at zero recombination. Taken together, these
data implicate COL5A1 as an important cause of EDS and confirm that types I and
II are allelic. Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue
disorders. Recently mutations have been found in the genes for type V collagen
in a small number of people with the most common forms of EDS, types I and II.
Here we characterise a COL5A2 mutation in an EDS II family. Cultured dermal
fibroblasts obtained from an affected subject synthesised abnormal type V
collagen. Haplotype analysis excluded COL5A1 but was concordant with COL5A2 as
the disease locus. The entire open reading frame of the COL5A2 cDNA was directly
sequenced and a single base mutation detected. It substituted a glycine residue
within the triple helical domain (G934R) of alpha2(V) collagen, typical of the
domit negative changes in other collagens, which cause various other
inherited connective tissue disorders. All three affected family members
possessed the single base change, which was absent in 50 normal chromosomes. Ehlers-Danlos syndrome (EDS) types I and II, which comprise the classical
variety, are well characterized from the clinical perspective, but it has been
difficult to identify the molecular basis of the disorder in the majority of
affected individuals. Several explanations for this failure to detect mutations
have been proposed, including genetic heterogeneity, failure of allele
expression, and technical difficulties. Genetic heterogeneity has been confirmed
as an explanation for such failure, since causative mutations have been
identified in the COL5A1, COL5A2, and tenascin X genes and since they have been
inferred in the COL1A2 gene. Nonetheless, in the majority of families with
autosomal domit inheritance of EDS, there appears to be linkage to loci that
contain the COL5A1 or COL5A2 genes. To determine whether allele-product
instability could explain failure to identify some mutations, we analyzed
polymorphic variants in the COL5A1 gene in 16 individuals, and we examined mRNA
for the expression of both alleles and for alterations in splicing. We found a
splice-site mutation in a single individual, and we determined that, in six
individuals, the mRNA from one COL5A1 allele either was not expressed or was
very unstable. We identified small insertions or deletions in five of these cell
strains, but we could not identify the mutation in the sixth individual. Thus,
although as many as one-half of the mutations that give rise to EDS types I and
II are likely to lie in the COL5A1 gene, a significant portion of them result in
very low levels of mRNA from the mutant allele, as a consequence of
nonsense-mediated mRNA decay. Ehlers-Danlos syndrome (EDS) type I (the classical variety) is a domitly
inherited, genetically heterogeneous connective-tissue disorder. Mutations in
the COL5A1 and COL5A2 genes, which encode type V collagen, have been identified
in several individuals. Most mutations affect either the triple-helical domain
of the protein or the expression of one COL5A1 allele. We identified a novel
splice-acceptor mutation (IVS4-2A-->G) in the N-propeptide-encoding region of
COL5A1, in one patient with EDS type I. The outcome of this mutation was
complex: In the major product, both exons 5 and 6 were skipped; other products
included a small amount in which only exon 5 was skipped and an even smaller
amount in which cryptic acceptor sites within exon 5 were used. All products
were in frame. Pro-alpha1(V) chains with abnormal N-propeptides were secreted
and were incorporated into extracellular matrix, and the mutation resulted in
dramatic alterations in collagen fibril structure. The two-exon skip occurred in
transcripts in which intron 5 was removed rapidly relative to introns 4 and 6,
leaving a large (270 nt) composite exon that can be skipped in its entirety. The
transcripts in which only exon 5 was skipped were derived from those in which
intron 6 was removed prior to intron 5. The use of cryptic acceptor sites in
exon 5 occurred in transcripts in which intron 4 was removed subsequent to
introns 5 and 6. These findings suggest that the order of intron removal plays
an important role in the outcome of splice-site mutations and provide a model
that explains why multiple products derive from a mutation at a single splice
site. Classic Ehlers-Danlos syndrome is a heritable connective tissue disorder
characterized by skin hyperextensibility, fragile and soft skin, delayed wound
healing with formation of atrophic scars, easy bruising, and generalized joint
hypermobility. It comprises Ehlers-Danlos syndrome type I and Ehlers-Danlos
syndrome type II, but it is now apparent that these form a continuum of clinical
findings and differ only in phenotypic severity. It is currently estimated that
approximately 50% of patients with a clinical diagnosis of classic Ehlers-Danlos
syndrome harbor mutations in the COL5A1 and the COL5A2 gene, encoding the α1 and
the α2-chain of type V collagen, respectively. However, because no prospective
molecular studies of COL5A1 and COL5A2 have been performed in a clinically
well-defined patient group, this number may underestimate the real proportion of
patients with classic Ehlers-Danlos syndrome harboring a mutation in one of
these genes. In the majority of patients with molecularly characterized classic
Ehlers-Danlos syndrome, the disease is caused by a mutation leading to a
nonfunctional COL5A1 allele and resulting in haploinsufficiency of type V
collagen. A smaller proportion of patients harbor a structural mutation in
COL5A1 or COL5A2, causing the production of a functionally defective type V
collagen protein. Most mutations identified so far result in a reduced amount of
type V collagen in the connective tissues available for collagen
fibrillogenesis. Inter- and intrafamilial phenotypic variability is observed,
but no genotype-phenotype correlations have been observed. No treatment for the
underlying defect is presently available for Ehlers-Danlos syndrome. However, a
series of preventive guidelines are applicable. |
Which factors are considered in the ABCD2 score? | Age, Blood pressure, Clinical features, Duration of symptoms and Diabetes are included in the ABCD2 score, which is used to identify patients having a transient ischemic attack who are at high risk for imminent stroke. | STUDY OBJECTIVE: We evaluate, in admitted patients with transient ischemic
attack, the accuracy of the ABCD(2) (age [A], blood pressure [B], clinical
features [weakness/speech disturbance] [C], transient ischemic attack duration
[D], and diabetes history [D]) score in predicting ischemic stroke within 7
days.
METHODS: At 16 North Carolina hospitals, we enrolled a prospective,
nonconsecutive sample of admitted patients with transient ischemic attack and
with no stroke history, presenting within 24 hours of transient ischemic attack
symptom onset. We conducted a medical record review to determine ischemic stroke
outcomes within 7 days. According to a modified Rankin Scale Score, strokes were
classified as disabling (>2) or nondisabling (< or =2).
RESULTS: During a 35-month period, we enrolled 1,667 patients, of whom 373 (23%)
received a diagnosis of an ischemic stroke within 7 days. Eighteen percent
(69/373) of all strokes were disabling. We were unable to calculate an ABCD(2)
score in 613 patients (37%); however, our imputed analysis indicated this did
not significantly alter results. The discriminatory power of the ABCD(2) score
was modest for ischemic stroke in 7 days (c statistic 0.59), and fair for
disabling ischemic stroke within 7 days (c statistic 0.71). Patients
characterized as low risk according to ABCD(2) score (< or =3) were at low risk
for experiencing a disabling stroke within 7 days, with a negative likelihood
ratio of 0.16 (95% confidence interval [CI] 0.04 to 0.64) with missing values
excluded and 0.34 (95% CI 0.15 to 0.76) when missing values were imputed.
CONCLUSION: Our analysis suggests the best application of the ABCD(2) score may
be to identify patients at low risk for an early disabling ischemic stroke.
Further study of the ability to determine an ABCD(2) score in all patients is
needed, along with validation in a large, consecutive population of patients
with transient ischemic attack. Transient ischemic attack (TIA) is a medical emergency, which has been newly
termed as "acute cerebrovascular syndrome" (ACVS). TIA is often ignored or
unrecognized by patients or their families since its symptoms are naturally
subsided without any treatment. TIA is also usually underestimated or
nonprioritized by physicians because it is regarded merely as a minor stroke.
However, stroke risk is very high in patients early after TIA. Therefore, rapid
evaluation followed by immediate treatment is essential in TIA patients. TIA
patients should be directly referred to stroke specialists in TIA clinics to
consider hospitalization for specific emergent treatments. Early stroke risk is
especially high in TIA patients with a high ABCD2 score of 4 or more (A age over
60 years [1 point]: B blood pressure > 140/90 mmHg [1 point]: C Clinical
features, including unilateral weakness [2 points] and speech disturbance
without weakness [1 point] D2: Diabetes [1 point] and Duration of symptoms [1
point for < 60 min and 2 points for > 60 min]), acute ischemic lesions on
diffusion weighted image, > 50% carotid stenosis, severe intracranial artery
stenosis, microembolic signals on transcranial Doppler, atrial fibrillation, or
hypercoagulable states. It has been reported that immediate starting treatment
with statins, antiplatelet agents, and antihypertensives substantially reduces
the risk of stroke within 90 days after TIA. US National Stroke Association
guidelines recommend assessments using computed tomography (CT)/ CT angiography
(CTA), magnetic resoce imaging (MRI)/MR angiography (MRA), and carotid
ultrasonography as well as immediate starting antiplatelet therapy in patients
with non-cardioembolic TIA or oral anticoagulant therapy in patients with
cardioembolic TIA within 24 hours during the first week after TIA. A large
international, multicenter cooperative, observational study (TIA Registry. Org.)
on 5,000 patients with TIA or minor stroke within 7 days of onset is being
initiated. Now, we should say "Time is TIA". OBJECTIVE: Urgent evaluation and treatment of transient ischemic attack (TIA)
patients in a dedicated TIA clinic may reduce the 90-day stroke risk by 80%.
ABCD2 (Age, Blood pressure, Clinical features, Duration, Diabetes) score and
magnetic resoce imaging abnormalities help to identify patients at high risk
of stroke. Our aim was to determine whether the use of transcranial Doppler
(TCD) examination on arrival at the TIA clinic yields additional information
that facilitates the identification of patients at high risk of stroke
recurrence.
METHODS: Between January 2003 and December 2007, 1,881 patients were admitted to
SOS-TIA clinic (a TIA clinic with around-the-clock access). Clinical and
vascular assessment included TCD performed by a neurologist immediately after
admission. Stroke prevention measures were initiated on arrival, in accordance
with guidelines. All patients were followed for 1 year after presentation to the
SOS-TIA clinic.
RESULTS: A total of 1,823 TCD examinations were performed within 4 hours of
admission. Intracranial narrowing or occlusion was found in 8.8% of patients,
and was independently associated with age, hypertension, and diabetes. After
1-year follow-up on best preventive therapy, the incidence of recurrent vascular
events (intracranial revascularization for TIA recurrence, stroke, myocardial
infarction, and vascular death combined) was 7.0% in patients with intracranial
narrowing or occlusion and 2.4% in those without (log-rank, p = 0.007). The
hazard ratio of combined outcome for the presence of intracranial narrowing or
occlusion was 2.29 (95% confidence interval [CI], 1.15-4.56; p = 0.02) in
multivariate analysis including age, gender, hypertension, and diabetes, and was
2.50 (95%CI, 1.24-5.05; p = 0.01) in multivariate analysis including ABCD2 score
> or =4.
INTERPRETATION: Immediate TCD examination on arrival at the TIA clinic is
feasible and could help to identify patients at high risk of vascular events
recurrence. This study supports a systematic intracranial vascular examination
in the initial management of TIA. BACKGROUND: Modifications to the age, blood pressure, clinical symptoms,
duration of symptoms, and diabetes (ABCD2) score, which incorporate history of
hypertension and acute hyperglycemia in addition to acute blood pressure (BP)
elevation and history of diabetes, have been proposed to increase the predictive
value of the score. In addition, the timing of acute BP measurement may be
important in the emergency department (ED) setting, given the phenomenon of "ED
triage hypertension".
METHODS: The standard ABCD2 score was compared to modified scores incorporating
various combinations of acute BP elevation or hyperglycemia, history of
hypertension or diabetes, and subsequent versus initial ED BP measurements. The
number of patients reclassified into an alternate risk category
(low/moderate/high) with different schemes was determined. Predictive value
using the composite outcome of stroke, death, or high-risk transient ischemic
attack mechanism was assessed using c statistics.
RESULTS: Modified ABCD2 scores resulted in few patients shifting risk categories
(between 2% and 10% for six alternate schemes), and did not improve the
performance of the ABCD2 score (c-statistics, 0.61-0.65, compared to 0.63 for
the standard score). ED triage hypertension was frequent (mean systolic blood
pressure [SBP]/diastolic blood pressure [DBP] decrease of 8/9 mm Hg on
subsequent measurement; P < .001), but the use of second BP did not reclassify
many patients (10%) nor did it improve score performance (c-statistic, 0.61).
CONCLUSIONS: Modifications of the ABCD2 score changed the risk category for few
patients and did not improve the overall predictive value of the score. BACKGROUND AND PURPOSE: The risk of stroke after a transient ischaemic attack
(TIA) can be predicted by scores incorporating age, blood pressure, clinical
features, duration (ABCD-score), and diabetes (ABCD2-score). However, some
patients have strokes despite a low predicted risk according to these scores. We
designed the ABCDE+ score by adding the variables 'etiology' and ischaemic
lesion visible on diffusion-weighted imaging (DWI) -'DWI-positivity'- to the
ABCD-score. We hypothesized that this refinement increases the predictability of
recurrent ischaemic events.
METHODS: We performed a prospective cohort study amongst all consecutive TIA
patients in a university hospital emergency department. Area under the computed
receiver-operating curves (AUCs) were used to compare the predictive values of
the scores with regard to the outcome stroke or recurrent TIA within 90 days.
RESULTS: Amongst 248 patients, 33 (13.3%, 95%-CI 9.3-18.2%) had a stroke (n =
13) or a recurrent TIA (n = 20). Patients with recurrent ischaemic events more
often had large-artery atherosclerosis as the cause for TIA (46% vs. 14%, P <
0.001) and positive DWI (61% vs. 35%; P = 0.01) compared with patients without
recurrent events. Patients with and those without events did not differ with
regard to age, clinical symptoms, duration, blood pressure, risk factors, and
stroke preventive treatment. The comparison of AUCs [95%CI] showed superiority
of the ABCDE+ score (0.67[0.55-0.75]) compared to the ABCD(2) -score
(0.48[0.37-0.58]; P = 0.04) and a trend toward superiority compared to the
ABCD-score (0.50[0.40-0.61]; P = 0.07).
CONCLUSION: In TIA patients, the addition of the variables 'etiology' and
'DWI-positivity' to the ABCD-score seems to enhance the predictability of
subsequent cerebral ischaemic events. BACKGROUND: The ABCD2 score (Age, Blood pressure, Clinical features, Duration of
symptoms and Diabetes) is used to identify patients having a transient ischemic
attack who are at high risk for imminent stroke. However, despite its widespread
implementation, the ABCD2 score has not yet been prospectively validated. We
assessed the accuracy of the ABCD2 score for predicting stroke at 7 (primary
outcome) and 90 days.
METHODS: This prospective cohort study enrolled adults from eight Canadian
emergency departments who had received a diagnosis of transient ischemic attack.
Physicians completed data forms with the ABCD2 score before disposition. The
outcome criterion, stroke, was established by a treating neurologist or by an
Adjudication Committee. We calculated the sensitivity and specificity for
predicting stroke 7 and 90 days after visiting the emergency department using
the original "high-risk" cutpoint of an ABCD2 score of more than 5, and the
American Heart Association recommendation of a score of more than 2.
RESULTS: We enrolled 2056 patients (mean age 68.0 yr, 1046 (50.9%) women) who
had a rate of stroke of 1.8% at 7 days and 3.2% at 90 days. An ABCD2 score of
more than 5 had a sensitivity of 31.6% (95% confidence interval [CI] 19.1-47.5)
for stroke at 7 days and 29.2% (95% CI 19.6-41.2) for stroke at 90 days. An
ABCD2 score of more than 2 resulted in sensitivity of 94.7% (95% CI 82.7-98.5)
for stroke at 7 days with a specificity of 12.5% (95% CI 11.2-14.1). The
accuracy of the ABCD2 score as calculated by either the enrolling physician
(area under the curve 0.56; 95% CI 0.47-0.65) or the coordinating centre (area
under the curve 0.65; 95% CI 0.57-0.73) was poor.
INTERPRETATION: This multicentre prospective study involving patients in
emergency departments with transient ischemic attack found the ABCD2 score to be
inaccurate, at any cut-point, as a predictor of imminent stroke. Furthermore,
the ABCD2 score of more than 2 that is recommended by the American Heart
Association is nonspecific. BACKGROUND: Several clinical scales have been developed for predicting stroke
recurrence. These clinical scores could be extremely useful to guide triage
decisions. Our goal was to compare the very early predictive accuracy of the
most relevant clinical scores [age, blood pressure, clinical features and
duration of symptoms (ABCD) score, ABCD and diabetes (ABCD2) score, ABCD and
brain infarction on imaging score, ABCD2 and brain infarction on imaging score,
ABCD and prior TIA within 1 week of the index event (ABCD3) score, California
Risk Score, Essen Stroke Risk Score and Stroke Prognosis Instrument II] in
consecutive transient ischemic attack (TIA) patients.
METHODS: Between April 2008 and December 2009, we included 1,255 consecutive TIA
patients from 30 Spanish stroke centers (PROMAPA study). A neurologist treated
all patients within the first 48 h after symptom onset. The duration and
typology of clinical symptoms, vascular risk factors and etiological work-ups
were prospectively recorded in a case report form in order to calculate
established prognostic scores. We determined the early short-term risk of stroke
(at 7 and 90 days). To evaluate the performance of each model, we calculated the
area under the receiver operating characteristic curve. Cox proportional hazards
multivariate analyses determining independent predictors of stroke recurrence
using the different components of all clinical scores were calculated.
RESULTS: We calculated clinical scales for 1,137 patients (90.6%). Seven-day and
90-day stroke risks were 2.6 and 3.8%, respectively. Large-artery
atherosclerosis (LAA) was observed in 190 patients (16.7%). We could confirm the
predictive value of the ABCD3 score for stroke recurrence at the 7-day follow-up
[0.66, 95% confidence interval (CI) 0.54-0.77] and 90-day follow-up (0.61, 95%
CI 0.52-0.70), which improved when we added vascular imaging information and
derived ABCD3V scores by assigning 2 points for at least 50% symptomatic
stenosis on carotid or intracranial imaging (0.69, 95% CI 0.57-0.81, and 0.63,
95% CI 0.51-0.69, respectively). When we evaluated each component of all
clinical scores using Cox regression analyses, we observed that prior TIA and
LAA were independent predictors of stroke recurrence at the 7-day follow-up
[hazard ratio (HR) 3.97, 95% CI 1.91-8.26, p < 0.001, and HR 3.11, 95% CI
1.47-6.58, p = 0.003, respectively] and 90-day follow-up (HR 2.35, 95% CI
1.28-4.31, p = 0.006, and HR 2.20, 95% CI 1.15-4.21, p = 0.018, respectively).
CONCLUSION: All published scores that do not take into account vascular imaging
or prior TIA when identifying stroke risk after TIA failed to predict risk when
applied by neurologists. Clinical scores were not able to replace extensive
emergent diagnostic evaluations such as vascular imaging, and they should take
into account unstable patients with recent prior transient episodes. This study aimed to examine outcome in low risk transient ischaemic attack (TIA)
patients presenting to emergency departments (ED) in a regional Australian
setting discharged on antiplatelet therapy with expedited neurology review. All
patients presenting to Gosford or Wyong Hospital ED with TIA, for whom faxed
referrals to the neurology department were received between October 2008 and
July 2010, were included in this prospective cohort study. Classification of low
risk was based on an age, blood pressure, clinical features, duration of
symptoms and diabetes (ABCD2) score <4 and the absence of high risk features,
including known carotid disease, crescendo TIA, or atrial fibrillation. Patients
with ABCD2 scores > or =4 or with high risk features were discussed with the
neurologist on call (a decision regarding discharge or admission was then made
at the neurologist's discretion). Patients were investigated with a brain CT
scan and/or CT angiography, routine pathology, and an electrocardiogram. All
discharged patients were commenced on antiplatelet therapy and asked to follow
up with their local medical officer within 7 days. The patients were contacted
by the neurology department to arrange follow-up. Our primary outcome was the
number of subsequent strokes occurring within 90 days. Of 200 discharged
patients for whom referrals were received, three patients had a stroke within 90
days. None of these would have been prevented through hospitalisation. In
conclusion, medical assessment, expedited investigation with immediate
commencement of secondary prevention and outpatient neurology review may be a
reasonable alternative to admission for low risk patients presenting to the ED
with TIA. The 'accuracy' of age, blood pressure, clinical features, duration and diabetes
(ABCD(2)) scoring by non-stroke specialists referring patients to a daily Rapid
Access Stroke Prevention (RASP) service is unclear, as is the accuracy of
ABCD(2) scoring by trainee residents. In this prospective study, referrals were
classified as 'confirmed TIAs' if the stroke specialist confirmed a clinical
diagnosis of possible, probable or definite TIA, and 'non-TIAs' if patients had
a TIA mimic or completed stroke. ABCD(2) scores from referring physicians were
compared with scores by experienced stroke specialists and neurology/geriatric
medicine residents at a daily RASP clinic; inter-observer agreement was
examined. Data from 101 referrals were analysed (mean age=60.0years, 58% male).
The median interval between referral and clinic assessment was 1day. Of 101
referrals, 52 (52%) were 'non-TIAs': 45 (86%) of 52 were 'TIA mimics' and 7
(14%) of 52 were completed strokes. There was only 'fair' agreement in total
ABCD(2) scoring between referring physicians and stroke specialists (κ=0.37).
Agreement was 'excellent' between residents and stroke specialists (κ=0.91).
Twenty of 29 patients scored as 'moderate to high risk' (score 4-6) by stroke
specialists were scored 'low risk' (score 0-3) by referring physicians. ABCD(2)
scoring by referring doctors is frequently inaccurate, with a tendency to
underestimate stroke risk. These findings emphasise the importance of urgent
specialist assessment of suspected TIA patients, and that ABCD(2) scores by
non-stroke specialists cannot be relied upon in isolation to risk-stratify
patients. Inter-observer agreement in ABCD(2) scoring was 'excellent' between
residents and stroke specialists, indicating short-term training may improve
accuracy. |
Is Tuberous Sclerosis a genetic disease? | Tuberous sclerosis is a genetic disorder with an autosomal dominant pattern of inheritance, variable expressivity, and incomplete penetrance. Two thirds of TSC cases result from sporadic genetic mutations, not inheritance, but their offspring may inherit it from them. Current genetic tests have difficulty locating the mutation in approximately 20% of individuals diagnosed with the disease. So far it has been mapped to two genetic loci, TSC1 and TSC2.
TSC1 encodes for the protein hamartin, is located on chromosome 9 q34 and was discovered in 1997. TSC2 encodes for the protein Tuberin, is located on chromosome 16 p13.3 and was discovered in 1993. TSC2 is contiguous with PKD1, the gene involved in one form of polycystic kidney disease (PKD). Gross deletions affecting both genes may account for the 2% of individuals with TSC who also develop PKD in childhood. TSC2 has been associated with a more severe form of TSC. However, the difference is subtle and cannot be used to identify the mutation clinically. Estimates of the proportion of TSC caused by TSC2 range from 55% to 80-90%. | The existence of locus heterogeneity for a genetic disease may complicate
linkage studies considerably, especially when very few large families with the
disease are available. In this situation a modest collection of families is
unlikely to be sufficient for successful localisation of one or more disease
genes. Recently, eight research groups working on tuberous sclerosis (TSC)
brought together linkage data pertaining to the candidate chromosomes 9, 11, and
12 for a large group of families. In a series of simulation studies we
determined the probability of detecting linkage and linkage heterogeneity in
this set of families. On average TSC families are very small; in most cases
there are fewer than two informative meioses. The size distribution of
chromosome 9 linked families was similar to that of non-linked families. This
indicates that a dramatic difference in the clinical severity of major genetic
forms of TSC is unlikely. The results of our simulation studies show that this
set of families can generate highly significant evidence for linkage and
heterogeneity. When two TSC genes are equally common, the strongest evidence for
linkage and heterogeneity could be obtained using a method based on the
incorporation of multiple candidate regions in a single analysis, with an
average lod score of 24.27. Renal angiomyolipomas were present in 23 out of a series of 38 patients with
proven tuberous sclerosis (60.5%). Multiplicity and bilateral localization of
combined renal angiomyolipomas were important differences between this category
and the isolated, usually solitary, angiomyolipomas. One of the parents of a
patient with tuberous sclerosis had small renal angiomyolipomas without signs of
tuberous sclerosis. This indicates that renal angiomyolipomas might be a forme
fruste of tuberous sclerosis. Two patients with suspected isolated renal
angiomyolipomas proved to have tuberous sclerosis. From this study we can
conclude that multiple angiomyolipomas, or a combination of a single renal
hamartoma with one of the signs suggestive of tuberous sclerosis, warrant a
thorough examination to exclude tuberous sclerosis. BACKGROUND AND DESIGN: Tuberous sclerosis (TS) is a genetic disease with
prominent cutaneous and brain involvement whose clinical and molecular genetics
are reviewed.
OBSERVATIONS: Tuberous sclerosis is a systemic disorder (incidence one in
10,000) characterized by benign growths (hamartias and hamartomas) in multiple
organ systems. Involvement of the brain can result in persistent seizures and
mental retardation; skin involvement includes facial angiofibromas, subungual
fibromas, hypomelanotic macules, forehead fibrous plaques, and Shagreen's
patches. Approximately 60% of TS occurs as apparent sporadic cases. In families,
it has autosomal domit inheritance with high penetrance (approximately 95%),
with careful clinical and radiologic evaluation. Genetic linkage analysis
indicates that about half of all TS families show linkage to chromosome 9q34,
and about half to chromosome 16p13. There are no distinguishing features in the
two groups of families showing linkage to the two genomic regions, nor strong
evidence for a third causative gene. Positional cloning efforts for both
chromosomal regions have limited the region containing the gene to about 1 to 2
million bases.
CONCLUSIONS: Identification of the two TS genes should illuminate the
pathogenesis of TS and provide opportunities for genetic counseling, prenatal
diagnosis, and therapeutic intervention. Tuberous sclerosis complex (TSC) is an autosomal domitly inherited disease
with a high mutation rate. It is clinically a very variable disorder and
hamartomas can occur in many different organs. TSC shows genetic heterogeneity;
one gene, TSC1, is on chromosome 9q34, and the second gene, TSC2, on chromosome
16p13.3. Clinical criteria for diagnosis have been established, but diagnosis of
patients with minimal expression of the disease can be very difficult. In
children the phenotype is often incomplete or not fully assessable. Hence mildly
affected subjects, at risk for severely affected offspring, may remain
undiagnosed. The detection of (small) mutations in the tuberous sclerosis gene
located on chromosome 16 (TSC2) has recently become possible and may be helpful
in the diagnosis of ambiguous cases. To our knowledge, this is the first report
of a point mutation in the TSC2 gene in a familial case of tuberous sclerosis. A
nonsense mutation was detected in a family in which the father had only minor
signs hinting at tuberous sclerosis. The son had multiple cardiac tumours and
white patches, but full clinical investigation was impossible in this child.
This case illustrates that mutation analysis can contribute to a diagnosis of
tuberous sclerosis in families with an incomplete phenotype. Tuberous sclerosis is a rare genetic disease with protean clinical
manifestations. The lesion most commonly described in the lung is
lymphangiomyomatosis. There have been recent reports of multifocal micronodular
pneumocyte hyperplasia, as well as a single case documentation of a clear cell
tumor of the lung, in patients affected by the disease. We detail a case of a
female Chinese patient with tuberous sclerosis who was incidentally discovered
to have bilateral pneumothoraces. The open lung biopsy revealed combined
histological features of multifocal micronodular pneumocyte hyperplasia,
lymphangiomyomatosis and clear cell micronodules. Tuberous sclerosis (TSC) is a frequent autosomal-domit condition (affecting 1
in 6000 individuals) caused by various mutations in either the hamartin (TSC1)
or the tuberin gene (TSC2). This allelic and non-allelic heterogeneity makes
genetic counseling and prenatal diagnosis difficult, especially as a significant
proportion of TSC cases are due to de novo mutations. For this reason the
identification of the disease causing mutation is mandatory for accurate
counseling, yet current mutation detection methods such as single-strand
conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis
(DGGE) are labor intensive with limited detection efficiency. Denaturing
high-performance liquid chromatography (DHPLC) is a high-throughput,
semi-automated mutation detection system with a reported mutation detection rate
close to 100% for PCR fragments of up to 800 bp. We used a recently described
DHPLC assay allowing the efficient detection of mutations in TSC1 to analyze the
DNA extracted from a chorion villus sample in order to perform a prenatal
diagnosis for TSC. The fetus was found not to have inherited the deleterious
mutation and the DHPLC diagnosis was confirmed by haplotype analysis. This
represents the first DHPLC-based prenatal diagnosis of a genetic disease. Mutation in either the TSC1 or TSC2 tumor suppressor gene is responsible for the
inherited genetic disease of tuberous sclerosis complex. TSC1 and TSC2 form a
physical and functional complex to regulate cell growth. Recently, it has been
demonstrated that TSC1.TSC2 functions to inhibit ribosomal S6 kinase and
negatively regulate cell size. TSC2 is negatively regulated by Akt
phosphorylation. Here, we report that TSC2, but not TSC1, associates with 14-3-3
in vivo. Phosphorylation of Ser(1210) in TSC2 is required for its association
with 14-3-3. Our data indicate that 14-3-3 association may inhibit the function
of TSC2 and represents a possible mechanism of TSC2 regulation. Tuberous sclerosis complex (TSC) is a genetic disease caused by mutation in
either TSC1 or TSC2. The TSC1 and TSC2 gene products form a functional complex
and inhibit phosphorylation of S6K and 4EBP1. These functions of TSC1/TSC2 are
likely mediated by mTOR. Here we report that TSC2 is a GTPase-activating protein
(GAP) toward Rheb, a Ras family GTPase. Rheb stimulates phosphorylation of S6K
and 4EBP1. This function of Rheb is blocked by rapamycin and domit-negative
mTOR. Rheb stimulates the phosphorylation of mTOR and plays an essential role in
regulation of S6K and 4EBP1 in response to nutrients and cellular energy status.
Our data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR
to regulate cell growth. Tuberous sclerosis complex (TSC) is a genetic disease caused by a mutation in
either the tsc1 or tsc2 tumor suppressor gene. Recent studies have demonstrated
that TSC2 displays GAP (GTPase-activating protein) activity specifically towards
the small G protein Rheb and inhibits its ability to stimulate the mTOR
signaling pathway. Rheb and TSC2 comprise a unique pair of GTPase and GAP,
because Rheb has high basal GTP levels and TSC2 does not have the catalytic
arginine finger found in Ras-GAP. To investigate the function of TSC2 and Rheb
in mTOR signaling, we analyzed the TSC2-stimulated Rheb GTPase activity. We
found that Arg15, a residue equivalent to Gly12 in Ras, is important for Rheb to
function as a substrate for TSC2 GAP. In addition, we identified asparagine
residues essential for TSC2 GAP activity. We demonstrated a novel catalytic
mechanism of the TSC2 GAP and Rheb that TSC2 uses a catalytic "asparagine thumb"
instead of the arginine finger found in Ras-GAP. Furthermore, we discovered that
farnesylation and membrane localization of Rheb is not essential for Rheb to
stimulate S6 kinase (S6K) phosphorylation. Analysis of TSC1 binding defective
mutants of TSC2 shows that TSC1 is not required for the TSC2 GAP activity but
may function as a regulatory component in the TSC1/TSC2 complex. Our data
further demonstrate that GAP activity is essential for the cellular function of
TSC2 to inhibit S6K phosphorylation. Tuberous sclerosis complex (TSC) is a multiorgan genetic disease caused by
inactivation of either the TSC1 or TSC2 genes. The disorder typically has
profound neurologic involvement and often presents early in life with epilepsy,
developmental delay, mental retardation, and autism. These features are
generally accepted to result from structural brain abnormalities that are found
in patients with TSC. Although much progress has recently been made in
discerning the function(s) of the TSC genes, many questions remain as to the
role of these genes in brain development and homeostasis. This review will
summarize recent progress and suggest future avenues of basic science research. Tuberous sclerosis is a genetic disease with autosomal domit inheritance,
associated with hamartomas in several organs and various skin findings. A case
of a ten year old boy is presented here to highlight the multisystem involvement
in tuberous sclerosis. The child had seizures, facial papular naevi and
peri-ungual fibromas. MRI revealed cortical tubers, white matter lesions and
subependymal nodules. Orbital ultrasound showed retinal hamartoma on the left
side. Ultrasound of the abdomen revealed a soft tissue mass at the upper pole of
left kidney with a small cyst in right kidney. Tuberous sclerosis complex (TSC) is a relatively rare autosomal domit
disorder characterized by widespread benign tumor formation in a variety of
organs. Mutations in either TSC1 or TSC2 tumor suppressor gene are responsible
for TSC. The gene products of TSC1 and TSC2, also known as hamartin and tuberin,
respectively, form a physical and functional complex and inhibit the mammalian
target of rapamycin complex 1 (mTORC1) signaling. The mTORC1 pathway is an
evolutionarily conserved growth promoting pathway. mTORC1 plays an essential
role in a wide array of cellular processes including translation, transcription,
trafficking and autophagy. In this review, we will discuss recent progresses in
the TSC-mTOR field and their physiological functions and alterations of this
pathway in pathophysiology. The tuberous sclerosis gene 2 product tuberin is an important regulator of the
mammalian target of rapamycin (mTOR). In addition, tuberin is known to bind to
the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) and to regulate its
stability and localization via mTOR-independent mechanisms. Recently, evidence
has been provided that tuberin also affects p27 localization via regulating
mTOR's potential to activate the serum- and glucocorticoid-inducible kinase
(SGK1) to phosphorylate p27. Taken together, these findings strengthen the
argument that besides mTOR-inhibitors, such as rapamycin analogues, p27 and CDKs
could also be considered targets for hamartoma therapeutics in tuberous
sclerosis. CONTEXT: Lymphangioleiomyomatosis (LAM) is a cystic lung disease that can be
included in the wide group of proliferative lesions named PEComas (perivascular
epithelioid cell tumors). These proliferative tumors are characterized by the
coexpression of myogenic and melanogenesis-related markers. In all these
lesions, genetic alterations related to the tuberous sclerosis complex (TSC)
have been demonstrated. Striking improvements in the understanding of the
genetic basis of this autosomal domit genetic disease are coupled to the
understanding of the mechanisms that link the loss of TSC1 (9q34) or TSC2
(16p13.3) genes with the regulation of the Rheb/m-TOR/p70S6K pathway. These data
have opened a new era in the comprehension of the pathogenesis of LAM and have
also suggested new therapeutic strategies for this potentially lethal disease.
OBJECTIVE: To present and discuss the pathologic and molecular features of LAM
within the spectrum of PEComas, providing a rational approach to their
diagnosis.
DATA SOURCES: The published literature and personal experience.
CONCLUSIONS: The inclusion of LAM within the PEComa category is supported by a
variety of biologic data and can significantly help in providing a comprehensive
view of this interesting and clinically relevant group of lesions. The
demonstration of molecular alterations of the mTOR pathway in LAM and other
PEComas represents a rational basis for innovative therapeutic approaches with
inhibitors of mTOR signaling. PURPOSE OF REVIEW: Tuberous sclerosis complex (TSC) is a multiorgan genetic
disease caused by mutations in the TSC1 or TSC2 genes. TSC has been recognized
for many years as an important cause of severe neurological disease with
patients suffering from epilepsy, developmental delay, autism, and psychiatric
problems. During the last year, there have been enormous advances in basic and
translational research pertaining to TSC.
RECENT FINDINGS: In this review, I discuss the basic science findings that
position the TSC1 and TSC2 genes as critical regulators of the mammalian target
of rapamycin kinase within mammalian target of rapamycin complex 1. In addition,
I will discuss the development of new animal models, translational data, and
recent clinical trials using mammalian target of rapamycin complex 1 inhibitors
such as rapamycin.
SUMMARY: The past few years have seen spectacular advances that have energized
TSC-related research and challenged existing symptomatic treatments. Although it
remains to be seen whether use of mammalian target of rapamycin complex 1
inhibitors will revolutionize the care of patients with TSC, the application of
basic and translational research towards a specific clinical disorder emphasizes
the potential and promise of molecular medicine. PURPOSE OF REVIEW: Mendelian disorders that affect cognition provide a unique
opportunity to study the mechanisms of neurodevelopmental disorders through the
examination of genetic defects in animals and development of hypotheses that can
be tested in human beings. Tuberous sclerosis complex (TSC) is a genetic disease
that presents with epilepsy, autism, and intellectual disability. Here we review
recent advances in our understanding of TSC pathogenesis and signaling pathways
that may be modulated to treat the neurological symptoms.
RECENT FINDINGS: Accumulating evidence suggests that TSC patients have nontuber
abnormalities that contribute to the development of the neurological phenotype-
in particular, disorganization of axon tracts and deficient myelination. TSC
mouse models have failed to replicate the human neuropathology entirely, but
have shed light on the cellular abnormalities and the neurobehavioral
phenotypes. Most importantly, cell culture and animal models have identified the
mTORC1 pathway as a therapeutic target in this disease.
SUMMARY: Preclinical data strongly suggest that TSC is a disease of abnormal
neuronal connectivity. The high incidence of neurodevelopmental deficits, early
detection of the disease in very young ages, and availability of mTORC1
inhibitors make TSC a model for other Mendelian disorders of neurocognition and
an avenue for the mechanism-based treatment trials of neurodevelopmental
disorders. The effects of missense changes and small in-frame deletions and insertions on
protein function are not easy to predict, and the identification of such
variants in individuals at risk of a genetic disease can complicate genetic
counselling. One option is to perform functional tests to assess whether the
variants affect protein function. We have used this strategy to characterize
variants identified in the TSC1 and TSC2 genes in individuals with, or suspected
of having, Tuberous Sclerosis Complex (TSC). Here we present an overview of our
functional studies on 45 TSC1 and 107 TSC2 variants. Using a standardized
protocol we classified 16 TSC1 variants and 70 TSC2 variants as pathogenic. In
addition we identified eight putative splice site mutations (five TSC1 and three
TSC2). The remaining 24 TSC1 and 34 TSC2 variants were classified as probably
neutral. Tuberous Sclerosis Complex (TSC) is a multiorgan genetic disease caused by loss
of function of either the TSC1 (encodes hamartin) or TSC2 (encodes tuberin)
genes. Patients with TSC have benign tumors (hamartomas) in multiple organs
though brain involvement is typically the most disabling aspect of the disease
as very high rates of neurodevelopmental disorders are seen. While first
described well over 120 years ago, recent advances have transformed TSC into a
prototypical disorder that exemplifies the methods and potential of molecular
medicine. This review will detail historical aspects of TSC and its strong
associations with neurodevelopmental disorders focusing on epilepsy and autism.
Finally, promising new approaches for the treatment of epilepsy and autism in
patients with TSC as well as those in the general population will be discussed. Tuberous Sclerosis Complex (TSC) is a multiorgan genetic disease that
prominently features brain malformations (tubers) with many patients suffering
from epilepsy and autism. These malformations typically exhibit neuronal as well
as glial cell abnormalities and likely underlie much of the neurological
morbidity seen in TSC. Tuber pathogenesis remains poorly understood though
upregulation of the mTORC1 signaling pathway in TSC has been consistently
demonstrated. Here we address abnormal brain development in TSC by inactivating
the mouse Tsc1 gene in embryonic neural progenitor cells. This strategy permits
evaluation of the role of the Tsc1 gene in both neuronal as well as glial cell
lineages. Tsc1(Emx1-Cre) conditional knockout (CKO) animals die by 25 days of
life. Their brains have increased size and contain prominent large cells within
the cerebral cortex that have greatly increased mTORC1 signaling and decreased
mTORC2 signaling. Severe defects of cortical lamination, enlarged dysmorphic
astrocytes and decreased myelination were also found. Tsc1(Emx1-Cre) CKO mice
were then treated with rapamycin to see if the premature death and brain
abnormalities can be rescued. Postnatal rapamycin treatment completely prevented
premature death and largely reversed the glia pathology but not abnormal
neuronal lamination. These findings support a model that loss of function of the
TSC genes in embryonic neural progenitor cells causes cortical malformations in
patients with TSC. The dramatic effect of rapamycin suggests that even with
extensive multi-lineage abnormalities, a postnatal therapeutic window may exist
for patients with TSC. Tuberous sclerosis complex (TSC) is a genetic disease with severe neurologic and
psychiatric manifestations including epilepsy, developmental delay, and autism.
Despite much progress in defining abnormal signaling pathways including the
contribution of increased mTORC1 signaling, specific abnormalities that underlie
the severe neurologic features in TSC remain poorly understood. We hypothesized
that epilepsy and autism in TSC result from abnormalities of γ-aminobutyric
acidergic (GABAergic) interneurons. To test this hypothesis, we generated
conditional knockout mice with selective deletion of the Tsc1 gene in GABAergic
interneuron progenitor cells. These interneuron-specific Tsc1 conditional
knockout (CKO) mice have impaired growth and decreased survival. Cortical and
hippocampal GABAergic interneurons of CKO mice are enlarged and show increased
mTORC1 signaling. Total numbers of GABAergic cells are reduced in the cortex
with differential reduction of specific GABAergic subtypes. Ectopic clusters of
cells with increased mTORC1 signaling are also seen suggesting impaired
interneuron migration. The functional consequences of these cellular changes are
evident in the decreased seizure threshold on exposure to the proconvulsant
flurothyl. These findings support an important role for the Tsc1 gene during
GABAergic interneuron development, function, and possibly migration. Tuberous sclerosis complex (TSC) is a multiorgan genetic disease in which brain
involvement causes epilepsy, intellectual disability, and autism. The hallmark
pathological finding in TSC is the cerebral cortical tuber and its unique
constituent, giant cells. However, an animal model that replicates giant cells
has not yet been described. Here, we report that mosaic induction of Tsc1 loss
in neural progenitor cells in Tsc1(cc) Nestin-rtTA(+) TetOp-cre(+) embryos by
doxycycline leads to multiple neurological symptoms, including severe epilepsy
and premature death. Strikingly, Tsc1-null neural progenitor cells develop into
highly enlarged giant cells with enlarged vacuoles. We found that the vacuolated
giant cells had multiple signs of organelle dysfunction, including markedly
increased mitochondria, aberrant lysosomes, and elevated cellular stress. We
found similar vacuolated giant cells in human tuber specimens. Postnatal
rapamycin treatment completely reversed these phenotypes and rescued the mutants
from epilepsy and premature death, despite prenatal onset of Tsc1 loss and mTOR
complex 1 activation in the developing brain. This TSC brain model provides
insights into the pathogenesis and organelle dysfunction of giant cells, as well
as epilepsy control in patients with TSC. Tuberous sclerosis is a rare genetic disease with autosomal domit
inheritance, associated with multiple hamartomas in several organs, such as the
brain, skin, lung, kidney, heart and eyes. The authors of this study report a
case of a 30 years old female patient with tuberous sclerosis, presenting
multiple angiofibromas on face treated with high frequency equipment
(radiofrequency), and discuss the therapeutic options for treatment of
individuals with extensive cutaneous involvement in tuberous sclerosis. Tuberous sclerosis (TSC) is an autosomal-domit genetic disease characterized
by a spectrum of pathologic manifestations involving skin, brain, kidney, and
heart. These manifestations include neuroectodermal, mesodermal, and skin
lesions as well as a variety of associated tumors and hamartomas. We report an
11-year-old male with previously diagnosed TSC who presented with a laryngeal
mass shown on histology to be fetal cellular rhabdomyoma. Cardiac rhabdomyomas
are common in TSC patients, but to our knowledge, the association between TSC
and extracardiac rhabdomyomas has not been previously reported. New epilepsy treatments are needed that not only inhibit seizures
symptomatically (antiseizure) but also prevent the development of epilepsy
(antiepileptogenic). The mammalian target of rapamycin (mTOR) pathway may
mediate mechanisms of epileptogenesis and serve as a rational therapeutic
target. mTOR inhibitors have antiepileptogenic and antiseizure effects in animal
models of the genetic disease, tuberous sclerosis complex. The mTOR pathway is
also implicated in epileptogenesis in animal models of acquired epilepsy and
infantile spasms, although the effects of mTOR inhibitors are variable depending
on the specific conditions and model. Furthermore, beneficial effects on
seizures are lost when treatment is withdrawn, suggesting that mTOR inhibitors
are "epileptostatic" in only stalling epilepsy progression during treatment.
Clinical studies of rapamycin in human epilepsy are limited, but suggest that
mTOR inhibitors at least have antiseizure effects in tuberous sclerosis
patients. Further studies are needed to assess the full potential of mTOR
inhibitors for epilepsy treatment. Renal cysts are a common radiological finding in both adults and children. They
occur in a variety of conditions, and the clinical presentation, management, and
prognosis varies widely. In this article, we discuss the major causes of renal
cysts in children and adults with a particular focus on the most common genetic
forms. Many cystoproteins have been localized to the cilia centrosome complex
(CCC). We consider the evidence for a universal 'cilia hypothesis' for cyst
formation and the evidence for non-ciliary proteins in cyst formation. Tuberous sclerosis complex (TSC) is a genetic disease characterized by
multiorgan benign tumors as well as neurological manifestations. Epilepsy and
autism are two of the more prevalent neurological complications and are usually
severe. TSC is caused by mutations in either the TSC1 (encodes hamartin) or the
TSC2 (encodes tuberin) genes with TSC2 mutations being associated with worse
outcomes. Tuberin contains a highly conserved GTPase-activating protein (GAP)
domain that indirectly inhibits mammalian target of rapamycin complex 1
(mTORC1). mTORC1 dysregulation is currently thought to cause much of the
pathogenesis in TSC but mTORC1-independent mechanisms may also contribute. We
generated a novel conditional allele of Tsc2 by flanking exons 36 and 37 with
loxP sites. Mice homozygous for this knock-in Tsc2 allele are viable and fertile
with normal appearing growth and development. Exposure to Cre recombinase then
creates an in-frame deletion involving critical residues of the GAP domain.
Homozygous conditional mutant mice generated using Emx1(Cre) have increased
cortical mTORC1 signaling, severe developmental brain anomalies, seizures, and
die within 3 weeks. We found that the normal levels of the mutant Tsc2 mRNA,
though GAP-deficient tuberin protein, appear unstable and rapidly degraded. This
novel animal model will allow further study of tuberin function including the
requirement of the GAP domain for protein stability. Tuberous sclerosis complex (TSC) is a genetic multisystem disorder characterized
by widespread hamartomas in several organs, including the brain, heart, skin,
eyes, kidney, lung, and liver. The affected genes are TSC1 and TSC2, encoding
hamartin and tuberin respectively. The hamartin-tuberin complex inhibits the
mammalian-target-of-Rapamycin (mTOR) pathway, which controls cell growth and
proliferation. Variations in the distribution, number, size, and location of
lesions cause the clinical syndrome to vary even between relatives. About 85% of
children and adolescents with TSC have CNS complications, including epilepsy,
cognitive impairment, challenging behavioral problems, and autism-like symptoms.
Epilepsy generally begins during the first year of life, with focal seizures and
spasms. The discovery of the mTOR pathway upregulation in TSC-associated lesions
presents new possibilities for treatment strategy. Increasing understanding of
the molecular abnormalities caused by TSC may enable improved management of the
disease. Tuberous sclerosis complex (TSC) is an autosomal domit neurogenetic disorder
characterized by hamartomas in multiple organs and is caused by a wide spectrum
of mutations in 1 of 2 causative genes (TSC1 or TSC2). Here, we present
mutational analyses of the TSC1 and TSC2 genes in 4 cases of TSC in Chinese Han
children, including 2 familial and 2 sporadic cases, using PCR and DNA
sequencing of the entire coding region as well as exon-intron boundaries of
these genes. Three mutations were identified in the TSC2 gene. Of these
mutations, 2 mutations (c.3312-3313delGA and c.45delT) were novel, and the 3rd
mutation (c.5238-5255del) was previously reported in Chinese Han and other
populations. These mutations were not present in healthy family members or in
100 unrelated normal controls. The identification of these mutations in this
study further expands the spectrum of known TSC2 gene mutations and contributes
to prenatal molecular diagnosis and preimplantation genetic testing of TSC. Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with variable
expressivity. Heterozygous mutations in either of two genes, TSC1 (hamartin) or
TSC2 (tuberin), are responsible for most cases. Hamartin and tuberin form a
heterodimer that functions as a major cellular inhibitor of the mammalian target
of rapamycin complex 1 (mTORC1) kinase. Genotype-phenotype studies suggest that
TSC2 mutations are associated with a more severe neurologic phenotype, although
the biologic basis for the difference between TSC1- and TSC2-based disease is
unclear. Here we performed a study to compare and contrast the brain phenotypes
of Tsc1 and Tsc2 single and double mutants. Using Tsc1 and Tsc2 floxed alleles
and a radial glial transgenic Cre driver (FVB-Tg(GFAP-cre)25Mes/J), we deleted
Tsc1 and/or Tsc2 in radial glial progenitor cells. Single and double mutants had
remarkably similar phenotypes: early postnatal mortality, brain overgrowth,
laminar disruption, astrogliosis, a paucity of oligodendroglia, and myelination
defects. Double Tsc1/Tsc2 mutants died earlier than single mutants, and single
mutants showed differences in the location of heterotopias and the organization
of the hippocampal stratum pyramidale. The differences were not due to
differential mTORC1 activation or feedback inhibition on Akt. These data provide
further genetic evidence for individual hamartin and tuberin functions that may
explain some of the genotype-phenotype differences seen in the human disease. Tuberous sclerosis is a polymorphic, domitly inherited syndrome caused by an
inactivating mutation in a tumor suppressor gene. The disease involves benign
tumors in several distinct organs such as the skin, kidneys, heart and central
nervous system. The tumors interfere with organ function, but only some exhibit
a significant tendency to grow. The clinical picture of tuberous sclerosis
varies from nearly symptomless to a severe disease. Treatment of growing tumors
associated with tuberous sclerosis is changing significantly, since their growth
can be suppressed with rapamycin and its derivatives. CONTEXT: Tuberous sclerosis complex (TSC) is a genetic disease in the group
known as neurocutaneous syndromes, with domit autosomal inheritance. It is
characterized by skin and adnexal lesions and central and peripheral nervous
system tumors, with neurological and psychiatric findings. It may affect the
heart, kidneys, eyes, face, bones, lungs, stomach and dentition.
CASE REPORT: We present the case of a 66-year-old man with dermatological signs
that included hypopigmented maculae, confetti-like lesions, shagreen plaque,
angiofibromas on nasolabial folds, neck and back, nail dystrophy and periungual
fibromas on fingers and toes. An electroencephalogram produced normal results,
but magnetic resoce imaging showed a nodular image measuring 1.2 x 1.0 cm
close to the Monro foramen, which was similar to cerebral parenchyma and
compatible with a subependymal giant-cell astrocytoma. A conservative approach
was taken, through control imaging examinations on the lesion for seven years,
with absence of any expansive process or neurological symptoms. Abdominal
ultrasonography revealed a solid, heterogenic and echogenic mass with a
calcified focus, measuring 4.6 x 3.4 cm, in the rightkidney, compatible with
angiomyolipoma. The patient was treated by means of complete nephrectomy because
of maligt areas seen on histopathological examination and died one month
after the procedure. This case report illustrates the importance of oral
clinical findings such as dental enamel pits and angiofibromas in making an
early diagnosis of TSC, with subsequent screening examinations, treatment and
genetic counseling. |
Does TRIM37 gene mutation causes Mulibrey nanism? | Yes, Mulibrey nanism is caused by recessive mutations in the TRIM37 gene encoding for the peroxisomal TRIM37 protein with ubiquitin-ligase activity. | Mulibrey ism is a rare growth disorder of prenatal onset caused by mutations
in the TRIM37 gene, which encodes a RING-B-box-coiled-coil protein. The
pathogenetic mechanisms of mulibrey ism are unknown. We have used transiently
transfected cells and antibodies raised against the predicted TRIM37 protein to
characterize the TRIM37 gene product and to determine its intracellular
localization. We show that the human TRIM37 cDNA encodes a peroxisomal protein
with an apparent molecular weight of 130 kD. Peroxisomal localization is
compromised in mutant protein representing the major Finnish TRIM37 mutation but
is retained in the protein representing the minor Finnish mutation.
Colocalization of endogenous TRIM37 with peroxisomal markers was observed by
double immunofluorescence staining in HepG2 and human intestinal smooth muscle
cell lines. In human tissue sections, TRIM37 shows a granular cytoplasmic
pattern. Endogenous TRIM37 is not imported into peroxisomes in peroxin 1
(PEX1(-/-)) and peroxin 5 (PEX5(-/-)) mutant fibroblasts but is imported
normally in peroxin 7 (PEX7(-/-)) deficient fibroblasts, giving further evidence
for a peroxisomal localization of TRIM37. Fibroblasts derived from patients with
mulibrey ism lack C-terminal TRIM37 immunoreactivity but stain normally for
both peroxisomal matrix and membrane markers, suggesting apparently normal
peroxisome biogenesis in patient fibroblasts. Taken together, this molecular
evidence unequivocally indicates that TRIM37 is located in the peroxisomes, and
Mulibrey ism thus can be classified as a new peroxisomal disorder. Mulibrey ism (muscle-liver-brain-eye ism; MUL) is an autosomal recessively
transmitted disease characterized by severe growth delays of prenatal onset
caused by mutations in the TRIM37 gene. Recent studies on the subcellular
localization revealed that the TRIM37 (KIAA0898) protein is located in
peroxisomes. Therefore, MUL has been classified as a new peroxisomal disorder.
Up to now, four mutations have been reported, all of which lead to frameshifts
and truncated proteins. In this study, mutation screening was performed for the
coding region of the TRIM37 gene in a Turkish family by means of RT-PCR and
direct cDNA sequencing. We have identified a novel mutation resulting in a
frameshift cosegregating within the family. Finally, we report on the presence
of novel splice variants observed in lymphoblastoid cells and muscle tissue of
normal subjects and patients. Mulibrey ism (MUL) is an autosomal recessive disease caused by mutations in
the TRIM37 gene encoding the peroxisomal TRIM37 protein of unknown function. In
this work, we analysed the clinical characteristics of 85 Finnish patients with
MUL, most of whom were homozygous for the Finn major mutation of TRIM37. The
patients' hospital records from birth to the time of the diagnosis at age
0.02-52 years (median 2.1 years) were retrospectively analysed. All except four
of the patients (95%) had a prenatal onset growth failure without postnatal
catch up growth. The mean length standard deviation score (SDS) was -3.1 and
-4.0 at birth and at diagnosis, respectively. In infancy, feeding difficulties,
and respiratory tract infections were the most common problems. Congestive heart
failure and pericardial constriction were diagnosed during infancy in 12% and 6%
of the patients, respectively. At the time of the diagnosis, characteristic
craniofacial features of scaphocephaly, facial triangularity, high and broad
forehead, and low nasal bridge were evident in over 90% of the patients. In
addition, practically all patients were gracile and had thin extremities. Other
findings included a peculiar high-pitched voice (96%), yellowish dots in ocular
fundi (79%), cutaneous naevi flammei (65%), hepatomegaly (45%), and fibrous
dysplasia of long bones (25%). Mild muscular hypotonicity (68%) was the only
neurological abnormality. The clinical features of the Finnish patients with MUL
formed a distinct entity. The most consistent findings were growth failure and
characteristic craniofacial features. However, organ manifestations varied
considerably in early childhood. Based on these findings, we propose new
diagnostic criteria for MUL. Mulibrey ism is an autosomal recessive prenatal-onset growth disorder of
unknown pathogenesis. The main clinical features are pre- and postnatal growth
failure, characteristic dysmorphic craniofacial features, heart disease, and
hepatomegaly. Five truncating mutations in the TRIM37 gene have previously been
reported in Mulibrey ism patients. The TRIM37 protein encodes a novel protein
of unknown function. It contains a tripartite motif (TRIM, also denoted the
RING-B-box-Coiled-coil or RBCC domain) and a TRAF (tumor necrosis
factor-receptor associated factor) domain. TRIM37 localizes to peroxisomes
classifying Mulibrey ism as a peroxisomal disorder. Here we have
characterized the genomic structure of the TRIM37 gene, which has 24 exons
spanning approximately 109 kb of genomic DNA. Further, we report six novel
disease-associated mutations, five of which predict a truncated protein:
c.745C>T (p.Gln249X), c.1411C>T (p.Arg471X), c.2056C>T (p.Arg686X), and an 8.6
kb genomic deletion (c.1314+507_1668-207del resulting in p.Arg439fsX4). The
sixth mutation (c.965G>T) is the first missense mutation (p.Gly322Val)
associated with Mulibrey ism. It affects the TRAF domain of TRIM37 and
results in altered subcellular localization of the mutant TRIM37 protein,
further suggesting that it is pathogenic. The TRIM37 gene encodes a peroxisomal protein of unknown function. Mutations in
TRIM37 underlie mulibrey ism, a rare autosomal recessively inherited disorder
with severe growth failure of prenatal onset, constrictive pericardium,
hepatomegaly and characteristic dysmorphic features. Eleven mulibrey
ism-associated mutations have been identified. We here characterised TRIM37
further by mapping the transcription initiation site and promoter region as well
as by analysing splice variants. By primer extension analysis, several
transcription initiation sites were localised to a region between -246 and -373
relative to the ATG codon for translation initiation. Basal promoter activity
was mapped within 600 nucleotides upstream from the translation initiation site
using promoter-luciferase reporter constructs. Several alternative splice
variants of TRIM37 exist in databases. Most of these predict non-functional
protein products, are expressed at low levels and are thus likely to be targets
for nonsense-mediated mRNA decay. A novel splice variant, TRIM37b, with an
alternative termination codon and 3'untranslated region (UTR) transcribed from
an exon 16 kb downstream from exon 24, predicts an identical protein product
with the previously identified transcript, TRIM37a. As seen by Northern blot
analysis and quantitative real-time PCR, both transcripts are highly expressed
in testis, whereas in other tissues TRIM37a is prominent. The 3'UTR of the PPM1E
gene in the opposite strand overlaps TRIM37b. These data suggest that TRIM37
expression is regulated by several mechanisms: through nonsense surveillance of
non-functional transcripts, as well as through 3'UTR regulatory sequences and/or
naturally occurring antisense RNAs especially in testis. Mulibrey ism is an autosomal recessive growth disorder caused by mutations in
the TRIM37 gene encoding a protein of unknown function. More than half of female
patients with Mulibrey ism develop benign mesenchymal tumors of ovarian sex
cord-stromal origin. In this work, we characterize the gynecological tumors of
female patients with Mulibrey ism in detail. In addition to tumors of the
fibrothecoma group, 18% (4/22) of the patients were observed with epithelial
neoplasias, including 2 ovarian adenofibromas, 1 ovarian poorly differentiated
adenocarcinoma and 1 endometrial adenocarcinoma. To investigate the possible
involvement of TRIM37 alterations in the pathogenesis of sporadic fibrothecomas,
we analyzed the TRIM37 cDNA for mutations and alternatively spliced transcripts
and TRIM37 expression in fibrothecomas of women without Mulibrey ism. No
mutations in the open-reading frame of TRIM37 were detected. Two alternatively
spliced variants were found, one lacking exon 23 and one exon 2. TRIM37del2 was
also found in normal ovary but in a proportion of sporadic fibrothecomas, the
TRIM37del2:TRIM37 ratio was increased. In normal ovary, TRIM37 was localized in
the cytoplasm of stromal cells, especially theca cells surrounding developing
follicles. TRIM37 transcript was found in all sporadic fibrothecomas examined,
but 80% (20/25) of the tumors showed reduced or absent expression of TRIM37
protein. Allelic loss at the TRIM37 locus (17q22-23) was observed in 6% of
sporadic fibrothecomas. Nearly half of the sporadic fibrothecomas showed
evidence of CpG promoter methylation, suggesting promoter downregulation as one
mechanism of reduced TRIM37 expression. In conclusion, inherited biallelic
inactivation of TRIM37 (Mulibrey ism) predisposes to both mesenchymal and
epithelial ovarian tumors and dysregulation of TRIM37 may also be involved in
the pathogenesis of sporadic fibrothecomas. Mulibrey ism (MUL) is a monogenic disorder with prenatal-onset growth
failure, typical clinical characteristics, cardiopathy and tendency for a
metabolic syndrome. It is caused by recessive mutations in the TRIM37 gene
encoding for the peroxisomal TRIM37 protein with ubiquitin-ligase activity. In
this work, the frequency and pathology of maligt and benign tumours were
analysed in a national cohort of 89 Finnish MUL patients aged 0.7-76 years. The
subjects had a clinical and radiological evaluation, and histological and
immunohistocemical analyses on specimens obtained from biopsy, surgery or
autopsy, were performed. The results show that the MUL patients have disturbed
architecture with ectopic tissues and a high frequency of both benign and
maligt tumours detectable in several internal organs. A total of 210 tumorous
lesions were detected in 66/89 patients (74%). Fifteen maligcies occurred in
13 patients (15%), seven of them in the kidney (five Wilms' tumours), three in
the thyroid gland, two gynaecological cancers, one gastrointestinal carcinoid
tumour, one neuropituitary Langerhans cell histiocytosis and one case of acute
lymphoblastic leukaemia (ALL). Tumours detected by radiology in the liver and
other organs mainly comprised strongly dilated blood vessels (peliosis),
vascularized cysts and nodular lesions. The lesions showed strong expression of
the endothelial cell markers CD34 and CD31 as well as the myocyte marker
alpha-smooth muscle actin (alpha-SMA). Our findings show that MUL is associated
with frequent maligt tumours and benign adenomatous and vascular lesions, as
well as disturbed organ development. Mulibrey ism (MUL) is a rare autosomal recessive disorder with severe
primordial growth retardation and multiorgan involvement, caused by mutations in
TRIM37. Early clinical detection is important since more than 50 % of the
patients develop congestive heart failure. We report a 12-year-old patient who
presented in infancy with severe growth retardation, dysmorphic features, and
cleft palate. Clinical diagnosis of MUL was established at the age of 5 years.
Postmortem, molecular diagnostic confirmed MUL as a novel 1-bp deletion
(c.1233delA) in exon 14 of the TRIM37 coding region. Cardiac examination at the
age of 6 years revealed constrictive pericarditis with significant elevation of
atrial filling pressures, consecutive hepatomegaly, and protein loosing
enteropathy. Since the parents refused pericardectomy, surgery was delayed until
the age of 12 years, when congestive heart failure deteriorated. Despite
pericardectomy, the boy died from persistent right heart failure.
CONCLUSION: Our report underlines the necessity of early clinical diagnosis of
Mulibrey ism. Careful cardiologic examination is required to detect
constrictive pericarditis, which is a major factor of mortality in these
patients. Pericardectomy should be performed early, to avoid sequelae of
persisting congestive heart failure. |
Have thyronamines effects on fat tissue? | thyronamines cause reduction of fat mass | Thyronamines (TAMs) are a newly identified class of endogenous signaling
compounds. Their structure is identical to that of thyroid hormone and
deiodinated thyroid hormone derivatives, except that TAMs do not possess a
carboxylate group. Despite some initial publications dating back to the 1950s,
TAMs did not develop into an independent area of research until 2004, when they
were rediscovered as potential ligands to a class of G protein-coupled receptors
called trace-amine associated receptors. Since this discovery, two
representatives of TAMs, namely 3-iodothyronamine (3-T(1)AM) and thyronamine
(T(0)AM), have been detected in vivo. Intraperitoneal or central injection of
3-T(1)AM or T(0)AM into mice, rats, or Djungarian hamsters caused various prompt
effects, such as metabolic depression, hypothermia, negative chronotropy,
negative inotropy, hyperglycemia, reduction of the respiratory quotient,
ketonuria, and reduction of fat mass. Although their physiological function
remains elusive, 3-T(1)AM and T(0)AM have already revealed promising therapeutic
potential because they represent the only endogenous compounds inducing
hypothermia as a prophylactic or acute treatment of stroke and might thus be
expected to cause fewer side effects than synthetic compounds. This review
article summarizes the still somewhat scattered data on TAMs obtained both
recently and more than 20 yr ago to yield a complete and updated picture of the
current state of TAM research. |
What is the genetic basis of progeria | Mutations in the LMNA gene cause Hutchinson-Gilford progeria syndrome in around 90% of patients | Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder
characterized by features reminiscent of marked premature ageing. Here, we
present evidence of mutations in lamin A (LMNA) as the cause of this disorder.
The HGPS gene was initially localized to chromosome 1q by observing two cases of
uniparental isodisomy of 1q-the inheritance of both copies of this material from
one parent-and one case with a 6-megabase paternal interstitial deletion.
Sequencing of LMNA, located in this interval and previously implicated in
several other heritable disorders, revealed that 18 out of 20 classical cases of
HGPS harboured an identical de novo (that is, newly arisen and not inherited)
single-base substitution, G608G(GGC > GGT), within exon 11. One additional case
was identified with a different substitution within the same codon. Both of
these mutations result in activation of a cryptic splice site within exon 11,
resulting in production of a protein product that deletes 50 amino acids near
the carboxy terminus. Immunofluorescence of HGPS fibroblasts with antibodies
directed against lamin A revealed that many cells show visible abnormalities of
the nuclear membrane. The discovery of the molecular basis of this disease may
shed light on the general phenomenon of human ageing. Accelerated aging or progeria has been a puzzling disease for many years. The
recent findings involving the lamin A/FACE-1 (substrate/protease) system in the
etiology of Hutchinson-Gilford progeria syndrome and related pathologies have
shed some light on the mechanisms underlying the development of these
devastating conditions. Thus, genetic defects in the nuclear envelope protein
prelamin A or in the FACE-1 metalloprotease (also called Zmspte24) involved in
prelamin A proteolytic maturation, cause the accumulation of an abnormal form of
this protein and the subsequent disruption of nuclear envelope integrity.
Recently, we and others have observed how this disruption leads to alterations
in chromatin organization, genomic instability, transcriptional changes, and
activation of a p53-linked signaling pathway. By using genetic manipulation
approaches in mouse, we have shown that lowering prelamin A levels results in a
total recovery of Zmpste24-deficient mice from the accelerated aging process.
Moreover, p53 nullizygosity allows a modest but significant improvement in the
premature aging phenotype, and contributes to delaying the onset of the
progeroid condition. On the basis of these results, we propose different
potential therapeutic approaches that could be tested in Zmpste24-deficient
mice. These strategies, some of which are based on existing drugs, might
contribute to the development of effective treatments for these dramatic
pathologies. CONTEXT: Mandibuloacral dysplasia (MAD) is a rare autosomal recessive progeroid
syndrome due to mutations in genes encoding nuclear lamina proteins, lamins A/C
(LMNA) or prelamin A processing enzyme, and zinc metalloproteinase (ZMPSTE24).
OBJECTIVE: The aim of the study was to investigate the underlying genetic and
molecular basis of the phenotype of a 7-yr-old girl with MAD belonging to a
consanguineous pedigree and with severe progeroid features and lipodystrophy.
DESIGN AND PATIENT: The patient developed mandibular hypoplasia during infancy
and joint stiffness, skin thinning, and mottled hyperpigmentation at 15 months.
Progressive clavicular hypoplasia, acroosteolysis, and severe loss of hair from
the temporal and occipital areas were noticed at 3 yr. At 5 yr, cranial sutures
were still open and lipodystrophy of the limbs was prominent. GH therapy from
the ages of 3-7 yr did not improve the short stature. Severe joint contractures
resulted in abnormal posture and decreased mobility. We studied her skin
fibroblasts for nuclear morphology and immunoblotting and determined the in
vitro effects of various pharmacological interventions on fibroblasts.
RESULTS: LMNA gene sequencing revealed a homozygous missense mutation,
c.1579C>T, p.Arg527Cys. Immunoblotting of skin fibroblast lysate with lamin A/C
antibody revealed no prelamin A accumulation. Immunofluorescence staining of the
nuclei for lamin A/C in fibroblasts revealed marked nuclear morphological
abnormalities. This abnormal phenotype could not be rescued with inhibitors of
farnesyl transferase, geranylgeranyl transferase, or histone deacetylase.
CONCLUSION: Severe progeroid features in MAD could result from LMNA mutation,
which does not lead to accumulation of prenylated lamin A or prelamin A. Hutchinson-Gilford progeria is a rare genetic disorder resulting from mutations
in the LMNA gene encoding lamin A/C. In addition to the classical phenotype
usually caused by the 1824C>T mutation of LMNA, a number of atypical progeroid
syndromes have been described. They have some distinct features, such as
skeletal deformities or scleroderma-like skin changes. The underlying defect is
usually a homozygous mutation of LMNA, or a combined defect of LMNA and another
gene, for example, ZMPSTE-24. We present a 2-year-old girl born to
consanguineous parents affected by progeroid syndrome with scleroderma-like skin
changes. Genetic analysis revealed the homozygous LMNA mutation 1303C>T (R435C).
The same heterozygous mutation was found in the patient's parents and 11 other
family members. The progeroid syndrome in our patient shares the signs of two
laminopathies: progeria and restrictive dermatopathy. Two other children in the
family died at the age of 2 due to a disease similar to that in the proposita.
On the basis of the family pedigree we presume that these children probably had
the same homozygous LMNA mutation. Scleroderma-like skin changes in infants,
associated with growth retardation and dysmorphic features, suggest premature
aging syndrome, requiring genetic testing and counseling of asymptomatic
carriers of LMNA mutations. CONTEXT: Hutchinson-Gilford progeria syndrome (HGPS) and mandibuloacral
dysplasia are well-recognized allelic autosomal domit and recessive progeroid
disorders, respectively, due to mutations in lamin A/C (LMNA) gene. Heterozygous
LMNA mutations have also been reported in a small number of patients with a less
well-characterized atypical progeroid syndrome (APS).
OBJECTIVE: The objective of the study was to investigate the underlying genetic
and molecular basis of the phenotype of patients presenting with APS.
RESULTS: We report 11 patients with APS from nine families, many with novel
heterozygous missense LMNA mutations, such as, P4R, E111K, D136H, E159K, and
C588R. These and previously reported patients now reveal a spectrum of clinical
features including progeroid manifestations such as short stature, beaked nose,
premature graying, partial alopecia, high-pitched voice, skin atrophy over the
hands and feet, partial and generalized lipodystrophy with metabolic
complications, and skeletal anomalies such as mandibular hypoplasia and mild
acroosteolysis. Skin fibroblasts from these patients when assessed for lamin A/C
expression using epifluorescence microscopy revealed variable nuclear
morphological abnormalities similar to those observed in patients with HGPS.
However, these nuclear abnormalities in APS patients could not be rescued with
48 h treatment with farnesyl transferase inhibitors, geranylgeranyl transferase
inhibitors or trichostatin-A, a histone deacetylase inhibitor. Immunoblots of
cell lysates from fibroblasts did not reveal prelamin A accumulation in any of
these patients.
CONCLUSIONS: APS patients have a few overlapping but some distinct clinical
features as compared with HGPS and mandibuloacral dysplasia. The pathogenesis of
clinical manifestations in APS patients seems not to be related to accumulation
of mutant farnesylated prelamin A. |
What type of DNA repair pathways is initiated by AlkA glycosylase? | The AlkA protein (3-methyladenine DNA glycosylase II protein) is a monofunctional DNA glycosylase that recognizes a broad range of oxidized and alkylated base lesions and catalyzes the hydrolysis of the N-glycosidic bond to initiate the base excision repair (BER) pathway. | BACKGROUND: Reactive oxygen species, ionizing radiation, and other free radical
generators initiate the conversion of guanine (G) residues in DNA to
8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with
adenine (A) during replication. Bacteria counter this threat with a
multicomponent system that excises the lesion, corrects OG:A mispairs and
cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence
has suggested the existence of base-excision DNA repair proteins specific for OG
in eukaryotes, little is known about these proteins.
RESULTS: Using substrate-mimetic affinity chromatography followed by a
mechanism-based covalent trapping procedure, we have isolated a base-excision
DNA repair protein from Saccharomyces cerevisiae that processes OG opposite
cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome
database using peptide sequences from the protein identified a gene, OGG1,
encoding a predicted 43 kDa (376 amino acid) protein, identical to one
identified independently by complementation cloning. Ogg1 has OG:C-specific
base-excision DNA repair activity and also intrinsic beta-lyase activity, which
proceeds through a Schiff base intermediate. Targeted disruption of the OGG1
gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named
Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G.
CONCLUSIONS: S. cerevisiae has two OG-specific glycosylase/lyases, which differ
significantly in their preference for the base opposite the lesion. We suggest
that one of these, Ogg1, is closely related in overall three-dimensional
structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase
that acts on fragmented and oxidatively damaged pyrimidines. We have recently
shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases,
is structurally homologous to endo III. We have now identified a shared active
site motif amongst these three proteins. Using this motif as a protein database
searching tool, we find that it is present in a number of other base-excision
DNA repair proteins that process diverse lesions. Thus, we propose the existence
of a DNA glycosylase superfamily, members of which possess a common fold yet act
upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to
alkylated or oxidized bases. Methylating and ethylating agents are used in the chemical industry and produced
during tobacco smoking. They generate DNA base damage whose role in cancer
induction has been documented. Alkylated bases are repaired by the base excision
repair pathway. We have established the repair efficiency of methylated and
ethylated bases by various Escherichia coli repair proteins, namely
3-methyladenine-DNA-glycosylase I (TagA protein), which excises 3-methyladenine
and 3-methylguanine, 3-methyladenine-DNA-glycosylase II (AlkA protein), which
has a broad substrate specificity including 3- and 7-alkylated purines and the
formamidopyrimidine(Fapy)-DNA-glycosylase (Fpg protein) repairing imidazole
ring-opened 7-methylguanine. The comparison of the Km values of these various
enzymes showed that methylated bases were excised more efficiently than
ethylated bases. Several 3-alkyladenine derivatives have been synthesized and
examined for their ability to inhibit the activity of the various repair
proteins. We have shown that 3-ethyl-, 3-propyl-, 3-butyl- and 3-benzyladenine
were much more efficient inhibitors of TagA protein than 3-methyladenine. The
inhibitory effect was increased with the increase of the size of alkyl-group and
IC50 for 3-benzyladenine was 0.4 +/- 0.1 microM as compared to 1.5 +/- 0.3 mM
for 3-methyladenine. These compounds inhibited neither the AlkA protein nor
human 3-methyladenine-DNA-glycosylase (ANPG protein). Moreover,
3-hydroxyethyladenine did not affect the activity of any of these enzymes. Taken
together, these results suggest that hydrophobic interactions are involved in
the mechanism of inhibition and/or recognition and excision of alkylated purines
by TagA protein. A variety of alkylated base adducts are repaired by 3-methyladenine DNA
glycosylases, one of the base excision repair enzymes. In this study, we
examined the DNA adducts induced by hepsulfam and determined whether alkylated
base adducts can be substrates for bacterial and mammalian 3-methyladenine DNA
glycosylases by electrophoresis methods. Hepsulfam, a synthetic analogue of
busulfan, is known to alkylate DNA and form interstrand cross-links. The extent
of DNA interstrand cross-links induced by hepsulfam and busulfan was found to be
similar but significantly lower than that induced by chlorambucil, as measured
by an agarose gel assay. The major monofunctional alkylation site of hepsulfam
was observed at the N7 position of guanine, and not at the N3 position of
adenine. Both compounds did not exhibit any sequence selective DNA alkylation
patterns. The excision of hepsulfam-induced DNA adducts has been determined by
treatment with homogeneous recombit bacterial, rat and human 3-methyladenine
DNA glycosylases and successive treatments by formamidopyrimidine-DNA
glycosylase. The Escherichia coli alkA protein was shown to completely excise N7
guanine adducts, whereas mammalian 3-methyladenine DNA glycosylase failed to
excise them. In addition, the cytotoxicity assay showed that E. coli mutant
strains defective in the alkA gene or the uvrA gene were more sensitive to
killing by hepsulfam than the wild type. The role of base excision repair in the repair of alkylation damage produced by
a series of sequence specific oligopyrrole-containing analogues of distamycin A
that tether benzoic acid mustard (BAM) has been examined. Whereas BAM alkylates
and cross-links in the major groove of DNA, attachment to pyrrole units produces
monoalkylations in the minor groove of DNA at AT tracts. Both sequence
specificity of alkylation and cytotoxicity increase from one to three attached
pyrrole units (compounds 1-3), and with 3 alkylation is selective for purine-N3
in the sequence 5'-TTTTGPu (where Pu = guanine or adenine). In a model bacterial
(Escherichia coli) system repair of the sequence specific minor groove
alkylations produced by 2 and 3 does not appear to involve BER, since neither a
formamidopyrimidine-DNA glycosylase repair deficient E. coli mutant (BH 20, fpg-
mutant) nor a 3-methyladenine-DNA glycosylase repair deficient mutant (GC 4803,
tag-alkA- mutant) showed increased cytotoxicity to 2 or 3 compared with the wild
type, AB 1157. The monopyrrole compound 1 was, however, approximately 4-fold
more cytotoxic to the GC 4803 mutant compared with wild type and BH 20,
suggesting a role for the 3-methyladenine-DNA glycosylase in the recognition and
excision of the adducts formed by 1. In contrast, increased sensitivity (>
10-fold) was observed for the conventional nitrogen mustard BAM in the BH 20
strain, suggesting a role for the formamidopyrimidine-DNA glycosylase in the
repair of the lesions produced by the agent. In a cell-free system the E. coli
3-methyladenine-DNA glycosylase (AlkA) was shown to remove alkylations at
5'-TTTTGPu sequences. However, the efficiency in removing the adducts formed by
the oligopyrrole compounds decreased dramatically from compound 1 to compound 3.
Increasing the size of the DNA adduct formed in the minor groove therefore
decreased the efficiency of recognition and removal of the adduct by the DNA
glycosylase. DNA is constantly exposed to endogenous andexogenous alkylating agents that can
modify its bases,resulting in mutagenesis in the absence of DNA repair [1,2].
Alkylation damage is removed by the action of DNA glycosylases, which initiate
the base excision repair pathway and protect the sequence information of the
genome [3-5]. We have identified a new class of methylpurine DNA glycosylase,
designated MpgII, that is a member of the endonuclease III family of DNA repair
enzymes. We expressed and purified MpgII from Thermotoga maritima and found that
the enzyme releases both 7-methylguanine and 3-methyladenine from DNA. We cloned
the MpgII genes from T. maritima and from Aquifex aeolicus and found that both
genes could restore methylmethanesulfonate (MMS) resistance to Escherichia coli
alkA tagA double mutants, which are deficient in the repair of alkylated bases.
Analogous genes are found in other Bacteria and Archaea and appear to be the
only genes coding for methylpurine DNA glycosylase activity in these organisms.
MpgII is the fifth member of the endonuclease III family of DNA repair enzymes,
suggesting that the endonuclease III protein scaffold has been modified during
evolution to recognize and repair a variety of DNA damage. The Escherichia coli AlkA protein is a base excision repair glycosylase that
removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of
AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme
flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend
in the DNA with a marked widening of the minor groove. The position of the
1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the
glycosylase reaction, in which the essential catalytic Asp238 provides direct
assistance for base removal. Catalytic selectivity might result from the
enhanced stacking of positively charged, alkylated bases against the aromatic
side chain of Trp272 in conjunction with the relative ease of cleaving the
weakened glycosylic bond of these modified nucleotides. The structure of the
AlkA-DNA complex offers the first glimpse of a helix-hairpin-helix (HhH)
glycosylase complexed to DNA. Modeling studies suggest that other HhH
glycosylases can bind to DNA in a similar manner. Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine
glycosylase (AlkA) are base excision repair glycosylases that recognize and
excise a variety of alkylated bases from DNA. The crystal structures of these
enzymes have provided insight into their substrate specificity and mechanisms of
catalysis. Both enzymes utilize DNA bending and base-flipping mechanisms to
expose and bind substrate bases. Crystal structures of AAG complexed to DNA
suggest that the enzyme selects substrate bases through a combination of
hydrogen bonding and the steric constraints of the active site, and that the
enzyme activates a water molecule for an in-line backside attack of the
N-glycosylic bond. In contrast to AAG, the structure of the AlkA-DNA complex
suggests that AlkA substrate recognition and catalytic specificity are
intimately integrated in a S(N)1 type mechanism in which the catalytic Asp238
directly promotes the release of modified bases. Base excision repair of DNA alkylation damage is initiated by a methylpurine DNA
glycosylase (MPG) function. Such enzymes have previously been characterized from
bacteria and eukarya, but not from archaea. We identified activity for the
release of methylated bases from DNA in cell-free extracts of Archaeoglobus
fulgidus, an archaeon growing optimally at 83 degrees C. An open reading frame
homologous to the alkA gene of Escherichia coli was overexpressed and identified
as a gene encoding an MPG enzyme (M(r) = 34 251), hereafter designated afalkA.
The purified AfalkA protein differs from E. coli AlkA by excising alkylated
bases only, from DNA, in the following order of efficiency: 3-methyladenine
(m(3)A) >> 3-methylguanine approximately 7-methyladenine >> 7-methylguanine.
Although the rate of enzymatic release of m(3)A is highest in the temperature
range of 65-75 degrees C, it is only reduced by 50% at 45 degrees C, a
temperature that does not support growth of A. fulgidus. At temperatures above
75 degrees C, nonenzymatic release of methylpurines predominates. The results
suggest that the biological function of AfalkA is to excise m(3)A from DNA at
suboptimal and maybe even mesophilic temperatures. This hypothesis is further
supported by the observation that the afalkA gene function suppresses the
alkylation sensitivity of the E. coli tag alkA double mutant. The amino acid
sequence similarity and evolutionary relationship of AfalkA with other MPG
enzymes from the three domains of life are described and discussed. Deamination of DNA bases can occur spontaneously, generating highly mutagenic
lesions such as uracil and hypoxanthine. In Escherichia coli two enzymes
initiate repair at hypoxanthine residues in DNA. The alkylbase DNA glycosylase,
AlkA, initiates repair by removal of the damaged base, whereas endonuclease V,
Endo V, hydrolyses the second phosphodiester bond 3' to the lesion. We have
identified and characterised a mouse cDNA with striking homology to the E.coli
nfi gene, which also has significant similarities to motifs required for
catalytic activity of the UvrC endonuclease. The 37-kDa mouse enzyme (mEndo V)
incises the DNA strand at the second phosphodiester bond 3' to hypoxanthine- and
uracil-containing nucleotides. The activity of mEndo V is elevated on
single-stranded DNA substrate in vitro. Expression of the mouse protein in a DNA
repair-deficient E.coli alkA nfi strain suppresses its spontaneous mutator
phenotype. We suggest that mEndo V initiates an alternative excision repair
pathway for hypoxanthine removal. It thus appears that mEndo V has properties
overlapping the function of alkylbase DNA glycosylase (Aag) in repair of
deaminated adenine, which to some extent could explain the absence of phenotypic
abnormalities associated with Aag knockout in mice. Schizosaccharomyces pombe has two paralogues of 3-methyladenine DNA glycosylase,
Mag1p and Mag2p, which share homology with Escherichia coli AlkA. To clarify the
function of these redundant enzymes in base excision repair (BER) of alkylation
damage, we performed several genetic analyses. The mag1 and mag2 single mutants
as well as the double mutant showed no obvious methyl methanesulfonate (MMS)
sensitivity. Deletion of mag1 or mag2 from an nth1 mutant resulted in tolerance
to MMS damage, indicating that both enzymes generate AP sites in vivo by removal
of methylated bases. A rad16 mutant that is deficient in nucleotide excision
repair (NER) exhibited moderate MMS sensitivity. Deletion of mag1 from the rad16
mutant greatly enhanced MMS sensitivity, and the mag2 deletion also weakened the
resistance to MMS of the rad16 mutant. A mag1/mag2/rad16 triple mutant was most
sensitive to MMS. These results suggest that the NER pathway obscures the mag1
and mag2 functions in MMS resistance and that both paralogues initiate the BER
pathway of MMS-induced DNA damage at the same level in NER-deficient cells or
that Mag2p tends to make a little lower contribution than Mag1p. Mag1p and Mag2p
functioned additively in vivo. Expression of mag1 and mag2 in the triple mutant
confirmed the contribution of Mag1p and Mag2p to BER of MMS resistance. 3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes
alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme
belongs to the helix-hairpin-helix (HhH) superfamily of DNA glycosylases and
possesses broad substrate specificity. In the genome of Deinococcus radiodurans,
two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584).
Dr_2074 is a homologue of human AlkA (MPG or AAG) and Dr_2584 is a homologue of
bacterial AlkAs. Here, the three-dimensional structure of Dr_2584 (DrAlkA2) is
presented and compared with the previously determined structure of Escherichia
coli AlkA (EcAlkA). The results show that the enzyme consists of two
helical-bundle domains separated by a wide DNA-binding cleft and contains an HhH
motif. Overall, the protein fold is similar to the two helical-bundle domains of
EcAlkA, while the third N-terminal mixed α/β domain observed in EcAlkA is
absent. Substrate-specificity analyses show that DrAlkA2, like EcAlkA, is able
to remove both 3-methyladenine (3meA) and 7-methylguanine (7meG) from DNA;
however, the enzyme possesses no activity towards 1,N(6)-ethenoadenine (ℇA) and
hypoxanthine (Hx). In addition, it shows activity towards the AlkB dioxygenase
substrates 3-methylcytosine (3meC) and 1-methyladenine (1meA). Thus, the enzyme
seems to preferentially repair methylated bases with weakened N-glycosidic
bonds; this is an unusual specificity for a bacterial AlkA protein and is
probably dictated by a combination of the wide DNA-binding cleft and a highly
accessible specificity pocket. Alkylating agents are widespread in the environment and also occur endogenously.
They can be cytotoxic or mutagenic to the cells introducing alkylated bases to
DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to
counteract the effects of DNA alkylation: the most cytotoxic lesion,
N(3)-methyladenine (3meA), is excised by AlkA glycosylase initiating base
excision repair (BER); toxic N(1)-methyladenine (1meA) and N(3)-methylcytosine
(3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic
and cytotoxic O(6)-methylguanine (O(6) meG) is repaired by Ada
methyltransferase. In Escherichia coli, Ada response involves the expression of
four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA,
AlkB, and AidB. The Ada response is conserved among many bacterial species;
however, it can be organized differently, with diverse substrate specificity of
the particular proteins. Here, an overview of the organization of the Ada
regulon and function of individual proteins is presented. We put special effort
into the characterization of AlkB dioxygenases, their substrate specificity, and
function in the repair of alkylation lesions in DNA/RNA. |
Is there any protein that undergoes both mono-ubiquitination and poly-ubiquitination? | Yes, there are some rare cases where a protein can be both mono-ubiquitinated and poly-ubiquitinated. | Recently, we demonstrated that hydrogen peroxide (H2O2) inhibits the
internalization of the epidermal growth factor (EGF) receptor and the
EGF-induced mono-ubiquitination of EGF receptor pathway substrate clone #15
(Eps15) in fibroblasts. In addition, it was suggested that EGF receptor
internalization might be inhibited by H2O2 by inhibition of ubiquitination of
proteins involved in endocytosis. Here, we show that H2O2 also inhibits the
poly-ubiquitination of the EGF receptor in fibroblasts. Furthermore, recovery of
the cells resulted in re-establishment of ubiquitination of both the EGF
receptor and Eps15 and coincided with restoration of internalization of those
receptors that had bound EGF in the presence of H2O2. In addition, EGF receptor
internalization was inhibited by the sulphydryl reagent N-ethylmaleimide (NEM),
indicating that intact SH groups might be required for receptor-mediated
endocytosis. Furthermore, H2O2 rapidly induced an increase in the cellular ratio
of GSSG:GSH (oxidized glutathione:reduced glutathione) and removal of H2O2
resulted in a fast restoration of the ratio of GSSG:GSH. Therefore, these
results suggest a relation between the inhibition of internalization
ubiquitination and an increase in GSSG:GSH ratio, which strengthens the
hypothesis that H2O2 inhibits EGF receptor internalization by an inhibition of
ubiquitination of proteins involved in EGF receptor-mediated endocytosis. Anti-Ro/SSA antibodies are antinuclear antibodies most commonly found in
patients with Sjögren's syndrome, a chronic autoimmune disease characterized by
dryness of the eyes and mouth. The autoantibodies recognize a RING-finger
protein, Ro52/SSA (52 kDa), whose function is still unknown. In this study, the
ubiquitination of Ro52 was investigated. We found that Ro52 was strongly
conjugated by a single molecule of ubiquitin in cells. Although the biological
relevance of this mono-ubiquitination was not defined, the function of Ro52
might be modified by the mono-ubiquitination. We also found that Ro52 was
conjugated with poly-ubiquitin chain in cells (poly-ubiquitination), suggesting
that Ro52 may be downregulated by the ubiquitin-proteasome pathway in vivo.
Interestingly, sera from patients with Sjögren's syndrome showed heterogeneity
in their reactivity to poly-ubiquitinated Ro52, probably because of their
differing antigenic determits. This heterogeneity of the reactivity might be
associated with the varying clinical features found in patients with Sjögren's
syndrome. Smad4 is an essential signal transducer of all transforming growth factor-beta
(TGF-beta) superfamily pathways that regulate cell growth and differentiation,
and it becomes inactivated in human cancers. Receptor-activated (R-) Smads can
be poly-ubiquitinated in the cytoplasm or the nucleus, and this regulates their
steady state levels or shutdown of the signaling pathway. Oncogenic mutations in
Smad4 and other Smads have been linked to protein destabilization and
proteasomal degradation. We analyzed a panel of missense mutants derived from
human cancers that map in the N-terminal Mad homology (MH) 1 domain of Smad4 and
result in protein instability. We demonstrate that all mutants exhibit enhanced
poly-ubiquitination and proteasomal degradation. In contrast, wild type Smad4 is
a relatively stable protein that undergoes mono- or oligo-ubiquitination, a
modification not linked to protein degradation. Analysis of Smad4 deletion
mutants indicated efficient mono- or oligo-ubiquitination of the C-terminal MH2
domain. Mass spectrometric analysis of mono-ubiquitinated Smad4 MH2 domain
identified lysine 507 as a major target for ubiquitination. Lysine 507 resides
in the conserved L3 loop of Smad4 and participates in R-Smad C-terminal
phosphoserine recognition. Mono- or oligo-ubiquitinated Smad4 exhibited enhanced
ability to oligomerize with R-Smads, whereas mutagenesis of lysine 507 led to
inefficient Smad4/R-Smad hetero-oligomerization and defective transcriptional
activity. Finally, overexpression of a mutant ubiquitin that only leads to
mono-ubiquitination of Smad4 enhanced Smad transcriptional activity. These data
suggest that oligo-ubiquitination positively regulates Smad4 function, whereas
poly-ubiquitination primarily occurs in unstable cancer mutants and leads to
protein degradation. We previously reported that oxidative stress is associated with
unloading-mediated ubiquitination of muscle proteins. To further elucidate the
involvement of oxidative stress in ubiquitination, we examined the
ubiquitination profile in rat myoblastic L6 cells after treatment with hydrogen
peroxide. Hydrogen peroxide induced many ubiquitinated proteins with low
molecular masses (less than 60 kDa) as well as high molecular masses (more than
160 kDa). Among them, a 42-kDa-ubiquitinated protein was abundantly accumulated
and immediately disappeared after the treatment. Microsequencing revealed that
the 42-kDa-protein was identical to the mono-ubiquitinated form of rat lactate
dehydrogenase A (LDH-A), and we confirmed that hydrogen peroxide induced the
mono-ubiquitination of LDH-A in COS7 cells overexpressing LDH-A and ubiquitin.
Under unloading conditions, such as tail-suspension and spaceflight,
mono-ubiquitinated LDH was accumulated in gastrocnemius muscle. Interestingly,
E-64-d plus pepstatin, lysosomal protease inhibitors, further accumulated
mono-ubiquitinated LDH-A in the cells after treatment with hydrogen peroxide,
while they did not affect the amount of poly-ubiquitinated LDH. In contrast,
epoxomicin, a potent proteasome inhibitor, did not change the amount of
mono-ubiquitinated LDH-A in L6 cells treated with hydrogen peroxide, although it
significantly increased the amount of poly-ubiquitinated LDH. Our results
suggest that oxidative stress induces not only poly-ubiquitination but also
mono-ubiquitination of LDH-A, which may be involved in its lysosomal degradation
during unloading. Whereas poly-ubiquitination targets protein substrates for proteasomal
degradation, mono-ubiquitination is known to regulate protein trafficking in the
endosomal system and to target cargo proteins for lysosomal degradation. The
role of the de-ubiquitinating enzymes AMSH and UBPY in endosomal trafficking of
cargo proteins such as the epidermal growth factor receptor (EGFR) has only very
recently been the subject of study and is already a matter of debate. Although
one report (Mizuno, E., Iura, T., Mukai, A., Yoshimori, T., Kitamura, N., and
Komada, M. (2005) Mol. Biol. Cell 16, 5163-5174) concludes that UBPY negatively
regulates EGFR degradation by de-ubiquitinating the EGFR on endosomes, another
report (Row, P. E., Prior, I. A., McCullough, J., Clague, M. J., and Urbe, S.
(2006) J. Biol. Chem. 281, 12618-12624) concludes that UBPY-mediated EGFR
de-ubiquitination is essential for EGFR degradation. Here, we demonstrate that
Usp8/UBPY, the mammalian ortholog of budding yeast Ubp4/Doa4, constitutively
co-precipitates in a bivalent manner with the EGFR. Moreover, UBPY is a
substrate for Src-family tyrosine kinases that are activated after
ligand-induced EGFR activation. Using overexpression of three different
recombit domit negative UBPY mutants (UBPY C748A mutant, UBPY 1-505, and
UBPY 640-1080) in NIH3T3 and HEK293 cells, we demonstrate that UBPY affects both
constitutive and ligand-induced (i) EGFR ubiquitination, (ii) EGFR expression
levels, and (iii) the appearance of intermediate EGFR degradation products as
well as (iv) downstream mitogen-activated protein kinase signal transduction.
Our findings provide further evidence in favor of the model that UBPY-mediated
EGFR de-ubiquitination promotes EGFR degradation. Smad1, a downstream regulator of the bone morphogenetic protein (BMP) receptors,
is tightly regulated by the ubiquitin-proteasomal degradation system. To dissect
the mechanisms that underlie the regulation of Smad1, it is important to
investigate the specific ubiquitination site(s) in Smad1. Here we report that
the alpha-NH(2) group of the N terminus and the epsilon-NH(2) groups of internal
lysine residues 116, 118 and 269 (K116, K118 and K269) of Smad1 are ubiquitin
acceptor sites mediated by the carboxyl terminus of Hsc70-interacting protein
(CHIP). The in vitro degradation assay indicates that ubiquitination at the N
terminus partially contributes to the degradation of Smad1. Furthermore, we
demonstrate that the ubiquitination level of pseudo-phosphorylated Smad1 by CHIP
is stronger than that of wild-type Smad1 and can be strongly inhibited by a
phosphorylated tail of Smad1, PIS(pS)V(pS). Third, our results indicate that
Hsp70 facilitates CHIP-mediated poly-ubiquitination of Smad1 whereas it
attenuates CHIP-meditated mono-ubiquitination of Smad1. Finally, consistent with
the in vitro observation, we show that CHIP preferentially mediates the
degradation of phospho-Smad1/5 in vivo. Taken together, these results provide us
a hint that CHIP might preferentially regulate phosphorylated Smad1 and thus the
BMP signaling. Mutations in the parkin gene cause autosomal recessive, juvenile-onset
parkinsonism. Parkin is an E3 ubiquitin ligase that mediates the ubiquitination
of protein substrates. Disease-associated mutations cause a loss-of-function of
parkin which may compromise the poly-ubiquitination and proteasomal degradation
of specific protein substrates, potentially leading to their deleterious
accumulation. Here, we identify the molecular chaperones, Hsp70 and Hsc70, as
substrates for parkin. Parkin mediates the ubiquitination of Hsp70 both in vitro
and in cultured cells. Parkin interacts with Hsp70 via its second RING finger
domain and mutations in/near this domain compromise Hsp70 ubiquitination.
Ubiquitination of Hsp70 fails to alter its steady-state levels or turnover, nor
does it promote its proteasomal degradation. Consistent with this observation,
Hsp70 levels remain unaltered in brains from parkin-deficient autosomal
recessive, juvenile-onset parkinsonism subjects, whereas alternatively, Hsp70
levels are elevated in the detergent-insoluble fraction of sporadic Parkinson's
disease/dementia with Lewy bodies brains. Parkin mediates the multiple
mono-ubiquitination of Hsp70/Hsc70 consistent with a degradation-independent
role for this ubiquitin modification. Our observations support a novel
functional relationship between parkin and Hsc/Hsp70 and support the notion that
parkin is a multi-purpose E3 ubiquitin ligase capable of modifying proteins
either via attachment of alternatively linked poly-ubiquitin chains or through
multiple mono-ubiquitination to achieve alternate biological outcomes. Emerging evidence suggests that ubiquitination serves as a protein trafficking
signal in addition to its well characterized role in promoting protein
degradation. The yeast G protein alpha subunit Gpa1 represents a rare example of
a protein that undergoes both mono- and poly-ubiquitination. Whereas
mono-ubiquitinated Gpa1 is targeted to the vacuole, poly-ubiquitinated Gpa1 is
directed instead to the proteasome. Here we investigate the structural
requirements for mono- and poly-ubiquitination of Gpa1. We find that variants of
Gpa1 engineered to be unstable are more likely to be poly-ubiquitinated and less
likely to be mono-ubiquitinated. In addition, mutants that cannot be
myristoylated are no longer mono-ubiquitinated but are still polyubiquitinated.
Finally, we show that the ubiquitin ligase Rsp5 is necessary for Gpa1
mono-ubiquitination in vivo and that the purified enzyme is sufficient to
catalyze Gpa1 mono-ubiquitination in vitro. Taken together, these data indicate
that mono- and poly-ubiquitination have distinct enzyme and substrate
recognition requirements; whereas poly-ubiquitination targets misfolded protein
for degradation, a distinct ubiquitination apparatus targets the fully mature,
fully myristoylated G protein for mono-ubiquitination and delivery to the
vacuole. gamma-Secretase is an enzymatic complex, composed of presenilin 1 (PS1),
nicastrin, pen-2, and aph-1, and is responsible for the intramembranous cleavage
of various type-I membrane proteins. The level of each component is tightly
regulated in a cell via proteasomal degradation. On the other hand, it has
previously been reported that PS1/gamma-secretase is involved in the activation
of phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway. PI3K is inhibited in
Alzheimer's disease (AD) brain, whereas the effects of PI3K inhibition on the
metabolism of PS1/gamma-secretase have not been elucidated. Here, we demonstrate
that the treatment of neurons with PI3K inhibitors leads to increased levels of
PS1/gamma-secretase components through an inhibitory effect on their
degradation. Moreover, PI3K inhibition accelerated ubiquitination of PS1. We
further show the evidence that the PS1 ubiquitination after PI3K inhibition is
represented by the multiple mono-ubiquitination, instead of poly-ubiquitination.
Accordingly, treatment of cells with PI3K inhibitor led to a differential
intracellular redistribution of PS1 from the one observed after the proteasomal
inhibition. These results suggest that PI3K inhibition may trigger the multiple
mono-ubiquitination of PS1, which precludes the degradation of
PS1/gamma-secretase through the proteasomal pathway. Since PS1/gamma-secretase
is deeply involved in the production of Abeta protein, a deeper knowledge into
its metabolism could contribute to a better elucidation of AD pathogenesis. Major histocompatibility class II (MHC class II) molecules are glycoproteins
that present extracellular antigens to CD4(+) T cells and are essential for
initiation of adaptive immune responses. MHC class II expression requires
recruitment of a master regulator, the class II transactivator (CIITA), to the
MHC class II promoter. Others and we have earlier linked CIITA to the
ubiquitin-proteasome system by showing that mono-ubiquitination of CIITA
increases its transactivity, whereas poly-ubiquitination of CIITA leads to its
degradation. We have further shown that the 26S proteasome also has
non-proteolytic functions in MHC class II transcription, as 19S ATPase subunits
of the 26S proteasome positively regulate MHC class II transcription and are
necessary for stable promoter binding of CIITA. Although these basic
requirements of the proteasome to initiate MHC class II transcription are known,
how CIITA is recruited, stabilized, and degraded remains unclear. Here, we
identify a novel N-terminal 19S ATPase-binding domain of CIITA. The
ATPase-binding domain lies within the proline/serine/threonine-rich region of
CIITA and encompasses a majority of the CIITA degron sequence. Absence of the
ATPase-binding domain increases the half-life of CIITA, but blocks MHC class II
surface expression, indicating that CIITA requires interaction with the 19S
ATPases for both appropriate deployment and destruction. Many DNA lesions cause pausing of replication forks at lesion sites; thus,
generating gaps in the daughter strands that are filled-in by post-replication
repair (PRR) pathways. In Saccharomyces cerevisiae, PRR involves translesion
synthesis (TLS) mediated by Poleta or Polzeta, or Rad5-dependent gap filling
through a poorly characterized error-free mechanism. We have developed an assay
to monitor error-free and mutagenic TLS across single DNA lesions in
Schizosaccharomyces pombe. For both main UV photolesions, we have delineated a
major error-free pathway mediated by a distinct combination of TLS polymerases.
Surprisingly, these TLS pathways require enzymes needed for poly-ubiquitination
of proliferating cell nuclear antigen (PCNA) as well as those required for
mono-ubiquitination. For pathways that require several TLS polymerases the
poly-ubiquitin chains of PCNA may facilitate their recruitment through specific
interactions with their multiple ubiquitin-binding motifs. These error-free TLS
pathways may at least partially account for the previously described
poly-ubiquitination-dependent error-free branch of PRR. This work highlights
major differences in the control of lesion tolerance pathways between S. pombe
and S. cerevisiae despite the homologous sets of PRR genes these organisms
share. Major histocompatibility (MHC) class II molecules are cell surface glycoproteins
that present extracellular antigens to CD4(+) T cells and are essential for
initiation of the adaptive immune response. MHC class II expression requires
recruitment of a master regulator, the class II transactivator (CIITA), to the
MHC class II promoter. Post-translational modifications to CIITA play important
roles in modulating CIITA mediated transcription of various genes in different
cell types. We have previously linked regulation of CIITA to the Ubiquitin
Proteasome System (UPS), and we and others have demonstrated that
mono-ubiquitination of CIITA dramatically increases its transactivity whereas
poly-ubiquitination leads to CIITA degradation. Here we identify three degron
proximal lysine residues, Lys-315, Lys-330, and Lys-333, and a phosphorylation
site, Ser-280, located within the CIITA degron, that regulate CIITA
ubiquitination, stability, and MHC class II expression. Together, these findings
contribute to the developing post-translational modification code for CIITA. TGF-β signalling is regulated by post-translational modifications of Smad
proteins to translate quantitative difference in ligand concentration into
proportional transcriptional output. Previous studies in cell culture systems
suggested that Smad ubiquitination regulatory factors (Smurfs) act in this
regulation by targeting Smads for proteasomal degradation, but whether this
mechanism operates under physiological conditions is not clear. Here, we
generated mice harbouring a target-disrupted Smurf2 allele. Using primary mouse
embryonic fibroblasts and dermal fibroblasts, we show that TGF-β-mediated,
Smad-dependent transcriptional responses are elevated in the absence of Smurf2.
Instead of promoting poly-ubiquitination and degradation, we show that Smurf2
actually induces multiple mono-ubiquitination of Smad3 in vivo. Phosphorylation
of T179, immediately upstream of the Smad3 PY motif, enhances Smurf2 and Smad3
interaction and Smad3 ubiquitination. We have mapped Smurf2-induced Smad3
ubiquitination sites to lysine residues at the MH2 domain, and demonstrate that
Smad3 ubiquitination inhibits the formation of Smad3 complexes. Thus, our data
support a model in which Smurf2 negatively regulates TGF-β signalling by
attenuating the activity of Smad3 rather than promoting its degradation. PTEN is one of the most frequently mutated or deleted tumor suppressors in human
cancers. NEDD4-1 was recently identified as the E3 ubiquitin ligase for PTEN;
however, a number of important questions remain regarding the role of
ubiquitination in regulating PTEN function and the mechanisms by which PTEN
ubiquitination is regulated. In the present study, we demonstrated that p34,
which was identified as a binding partner of NEDD4-1, controls PTEN
ubiquitination by regulating NEDD4-1 protein stability. p34 interacts with the
WW1 domain of NEDD4-1, an interaction that enhances NEDD4-1 stability.
Expression of p34 promotes PTEN poly-ubiquitination, leading to PTEN protein
degradation, whereas p34 knockdown results in PTEN mono-ubiquitination. Notably,
an inverse correlation between PTEN and p34/NEDD4-1 levels was confirmed in
tumor samples from colon cancer patients. Thus, p34 acts as a key regulator of
the oncogenic behavior of NEDD4-1 and PTEN. |
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