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Does Vitamin D induce autophagy? | Yes, vitamin D induces autophagy. | We evaluated the effects of administration of 1,25-dihydroxyvitamin D
(1,25(OH)2D) during pregcy on relieving adverse outcomes of preeclampsia and
the pathologic and biochemical changes in reduction in uteroplacental perfusion
(RUPP) model of rats. On day 1, 7, and 14 of pregcy, rats in pregt RUPP
plus 1,25(OH)2D (RUPP+VD) group (n = 15) received 120 ng/100 g body weight/week
of 1,25(OH)2D by subcutaneous injection, while rats in normal pregt (n = 12)
and the RUPP group (n = 14) received 1,25(OH)2D vehicle (saline solution). On
day 19 of pregcy, after measure of blood pressure and cardiac function and
urine collection, rats were euthanized, and fetal and maternal serum, placenta,
and heart and kidney were collected. Fetal mortality, urinary protein, glucose,
and parameters for kidney function in serum were measured. We evaluated vitamin
D receptor expression and pathological and ultrastructural changes in rat heart,
kidney, and placenta. Levels of oxidative stress, endoplasmic reticulum (ER)
stress, apoptosis, and autophagy were measured in placenta. Compared to RUPP
rats, 1,25(OH)2D decreased fetal mortality, mean blood pressure, 24-h urinary
protein, urine microalbumin, and hyperglycemia in RUPP+VD rats. These were
consistent with the improvements of structure impairment in heart, kidney, and
placenta of RUPP rat by 1,25(OH)2D. In placenta of RUPP rat, the decrease in
oxidative stress and ER stress by 1,25(OH)2D treatment was accompanied by
autophagy activation and apoptosis attenuation. 1,25(OH)2D plays a beneficial
effect on preeclampsia at the early gestation and might be used as a potential
protective agent for preeclampsia. However, the RUPP model only recapitulated
the hypoxic origin of preeclampsia; further randomized controlled trial is
expected to be performed for validation and evaluation. Vitamin D had an anti-infection effect and benefited to the intestinal health.
Autophagy signaling pathway was regulated by vitamin D3 to inhibit the infection
of human immunodeficiency virus type-1. Rotavirus (RV) was a major cause of the
severe diarrheal disease in young children and young animals. Although evidence
suggested that vitamin D3 attenuates the negative effects of RV infection via
the retinoic acid-inducible gene I signaling pathway, little is known of its
antiviral effect whether through the regulation of autophagy. The present study
was performed to investigate whether vitamin D3 alleviates RV infection in pig
and porcine small intestinal epithelial cell line (IPEC-J2) models via
regulating the autophagy signaling pathway. RV administration increased the
Beclin 1 mRNA abundance in porcine jejunum and ileum. 5000 IU/kg dietary vitamin
D3 supplementation greatly up-regulated LC3-II/LC3-I ratios and PR-39 mRNA
expression under the condition of RV challenged. The viability of IPEC-J2 was
significantly inhibited by RV infection. Incubation with 25-hydroxyvitamin D3
significantly decreased the concentrations of RV antigen and non-structural
protein 4 (NSP4), and up-regulated the mRNA expression of Beclin 1 and PR-39 in
the RV-infected IPEC-J2 cells. And then, based on the 25-hydroxyvitamin D3
treatment and RV infection, LC3-II mRNA expression in cells was inhibited by an
autophagy inhibitor 3-methyladenine (3-MA). Bafilomycin A1 (Baf A1, a class of
inhibitors of membrane ATPases, inhibits maturation of autophagic vacuoles)
treatment numerically enhanced the LC3-II mRNA abundance, but had no effect on
NSP4 concentration. Furthermore, 25-hydroxyvitamin D3 decreased the p62 mRNA
expression and increased porcine cathelicidins (PMAP23, PG1-5 and PR-39) mRNA
expression in the RV-infected cells. Taken together, these results indicated
that vitamin D3 attenuates RV infection through regulating autophagic maturation
and porcine cathelicidin genes expression. Epidemiological evidence indicates that vitamin D is involved in defense against
diabetes; however, the precise underlying mechanism remains to be elucidated. In
the present study, the effect of vitamin D on the pathogenesis of diabetes was
investigated, with an emphasis on its direct effect on pancreatic β‑cells. A
streptozotocin (STZ)‑induced type 1 diabetes mellitus (T1DM) mouse model and
MIN6 mouse insulinoma β‑cells were subjected to vitamin D treatment.
Histopathological analysis of pancreatic islets was performed to investigate
insulitis, and reverse transcription-quantitative polymerase chain reaction and
western blotting were used to determine the mRNA and protein expression levels
of markers of autophagy [microtubule-associated protein 1A/1B‑light chain 3
(LC3) and Beclin 1] and regulation of apoptosis [B-cell lymphoma 2 (Bcl-2)].
Apoptosis of MIN6 cells was examined by flow cytometry following annexin
V/propidium iodide labeling. The secretion of insulin was measured by ELISA. The
results revealed that vitamin D reduced the incidence of T1DM, enhanced insulin
secretion and relieved pancreatic inflammation in STZ‑treated mice. Furthermore,
vitamin D increased the mRNA expression levels of LC3 and Beclin 1, and
increased Bcl‑2 protein expression levels in STZ‑treated MIN6 cells, while
decreasing the apoptosis rate. The results of the present study demonstrated,
for the first time to the best of our knowledge, that vitamin D induces
autophagy and suppresses apoptosis of pancreatic β‑cells, as well as preventing
insulitis. These findings regarding vitamin D provide insights into its
involvement in diabetes, and suggest a potential novel strategy for the
treatment of diabetes via agents enhancing autophagy in pancreatic β-cells. |
What is sQTLseekeR? | sQTLseekeR is an R package for the identification of genetic variants associated with alternative splicing. It is based on a statistical framework that uses a distance-based approach to compute the variability of splicing ratios across observations, and a non-parametric analogue to multivariate analysis of variance. | |
What is Creutzfeldt-Jakob Disease (CJD)? | Creutzfeldt-Jakob disease (CJD) is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein (PrP(Sc)). The origin of CJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous prion protein (PrP(C)) into infectious PrP(Sc). | Creutzfeldt-Jakob disease (CJD) is presumably caused by a slow infectious
pathogen or prion. The principal clinical features of Creutzfeldt-Jakob disease
are dementia, pyramidal and extrapyramidal symptoms and signs, cerebellar
dysfunction, and myoclonus. The patient rapidly deteriorates, declines to a
vegetative state, becomes comatous, and is ultimately dead within several
months. The authors present a case of Creutzfeldt-Jakob disease, proved by
clinical findings, typical serial EEG, and pathologic features. Creutzfeldt-Jakob disease (CJD) is a rare neurodegenerative disorder. Since the
emergence of variant CJD (vCJD) vigilance concerning the disease's incidence has
increased and the interest in accurate in vivo diagnosis has augmented. So far,
a large number of biomarkers has been investigated as aid in the differential
diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) and vCJD. These include,
among others, neuron-specific enolase (NSE), microtubuli associated protein Tau,
S-100beta, amyloid-beta (Abeta(1-42)) and the 14-3-3 protein. Multiple studies
have confirmed that CSF detection of 14-3-3 protein by Western blot was the best
single biomarker for sCJD with an average sensitivity and specificity of 92%.
Also, in genetic and iatrogenic CJD (iCJD) patients with an average disease
duration of less than 1 year, 14-3-3 is the best differential biomarker.
Unfortunately, the 14-3-3 protein has a lower sensitivity if the disease
duration exceeds beyond 1 year in both sporadic CJD and other CJD types (vCJD,
and specific genetic or iatrogenic CJD types). Creutzfeldt-Jakob disease (CJD) is a rare, degenerative and fatal brain disease
that appears to be caused by an abnormal form of a protein called a prion. Due
to the lack of an effective treatment for CJD, support for patients and family
members is crucial. Appropriate education of the healthcare community on this
rare disease and provision of palliative care are critically needed. The CJD
Foundation and CJD Insight were formed to provide services to patients and
families affected by prion diseases. OBJECTIVES: To develop a scale sensitive for the neurological manifestations of
Creutzfeldt-Jakob disease (CJD).
METHODS: A 26-item CJD neurological status scale (CJD-NS) was created based on
characteristic disease manifestations. Each sign was assigned to one of eight
neurological systems to calculate a total scale score (TSS) and a system
involvement score (SIS). The scale was administered to 37 CJD patients, 101
healthy first-degree relatives of the patients and 14 elderly patients with
Parkinson's disease (PD).
RESULTS: The mean TSS (±SD) was significantly higher in patients with CJD (13.19
± 5.63) compared with normal controls (0.41 ± 0.78) and PD patients (9.71 ±
3.05). The mean SIS was also significantly different between the CJD (5.19 ±
1.22) and PD (2.78 ± 1.18 P ≤ 0.01) groups reflecting the disseminated nature of
neurological involvement in CJD. Using a cutoff of TSS > 4 yielded a sensitivity
of 97% for CJD, and specificity of 100% against healthy controls. All individual
items showed excellent specificity against healthy subjects, but sensitivity was
highly variable. Repeat assessments of CJD patients over 3-9 months revealed a
time-dependent increase in both the TSS and the SIS reflecting the scale's
ability to track disease progression.
CONCLUSIONS: The CJD-NS scale is sensitive to neurological signs and their
progression in CJD patients. Creutzfeldt-Jakob disease (CJD) is a rare neurodegenerative condition with a
rapid disease course and a mortality rate of 100%. Several forms of the disease
have been described, and the most common is the sporadic type. The most
challenging aspect of this disease is its diagnosis-the gold standard for
definitive diagnosis is considered to be histopathological confirmation-but
newer tests are providing means for an antemortem diagnosis in ways less
invasive than brain biopsy. Imaging studies, electroencephalography, and
biomarkers are used in conjunction with the clinical picture to try to make the
diagnosis of CJD without brain tissue samples, and all of these are reviewed in
this article. The current diagnostic criteria are limited; test sensitivity and
specificity varies with the genetics of the disease as well as the clinical
stage. Physicians may be unsure of all diagnostic testing available, and may
order outdated tests or prematurely request a brain biopsy when the diagnostic
workup is incomplete. The authors review CJD, discuss the role of brain biopsy
in this patient population, provide a diagnostic pathway for the patient
presenting with rapidly progressive dementia, and propose newer diagnostic
criteria. Creutzfeldt-Jakob disease is a rare, but rapidly progressive, up to now
untreatable and fatal neurodegenerative disorder. Clinical diagnosis of
Creutzfeldt-Jakob disease (CJD) is difficult; however, it can be facilitated by
suitable biomarkers. Aim of the present study is to compare levels of
cerebrospinal fluid biomarkers (total tau protein, phosphorylated-tau protein,
protein 14-3-3 and amyloid beta) in Slovak population of CJD suspect cases,
retrospectively in over a 10-year period. One thousand three hundred sixty-four
CSF samples from patients with suspect CJD, forming a homogenous group in terms
of geographical as well as of equal transport conditions, storage and laboratory
processing, were analysed. Definite diagnosis of Creutzfeldt-Jakob disease was
confirmed in 101 patients with genetic form, and 60 patients with its sporadic
form of the disease. Specificity of protein 14-3-3 and total tau in both forms
CJD was similar (87 % for P14-3-3/85 % for total tau), sensitivity to P 14-3-3
and total tau was higher in sporadic Creutzfeldt-Jakob disease (sCJD) (90/95 %)
than in genetic Creutzfeldt-Jakob disease (gCJD) (89/74 %). As expected, the
total tau levels were significantly higher in CJD patients than in controls, but
there was also significant difference between gCJD and sCJD (levels in gCJD were
lower; p = 0.003). There was no significant difference in p-tau and Aβ 1-42
levels neither between both CJD forms nor between CJD patients and control
group. |
Describe what is athelia syndrome? | Athelia is a very rare entity that is defined by the absence of the nipple-areola complex. | The absence of nipple-areola complex is a rare entity and is always associated
with other anomalies. This paper described a case of bilateral athelia without
other alterations. The atrophy of the dense mesenchyme due to absence of
parathyroid hormone-related protein produced in epithelium may lead to nipple
involution. Further cases should be studied to corroborate this theory. Athelia is a very rare entity that is defined by the absence of the
nipple-areola complex. It can affect either sex and is mostly part of syndromes
including other congenital or ectodermal anomalies, such as limb-mammary
syndrome, scalp-ear-nipple syndrome, or ectodermal dysplasias. Here, we report
on three children from two branches of an extended consanguineous Israeli Arab
family, a girl and two boys, who presented with a spectrum of nipple anomalies
ranging from unilateral hypothelia to bilateral athelia but no other
consistently associated anomalies except a characteristic eyebrow shape. Using
homozygosity mapping after single nucleotide polymorphism (SNP) array genotyping
and candidate gene sequencing we identified a homozygous frameshift mutation in
PTPRF as the likely cause of nipple anomalies in this family. PTPRF encodes a
receptor-type protein phosphatase that localizes to adherens junctions and may
be involved in the regulation of epithelial cell-cell contacts, peptide growth
factor signaling, and the canonical Wnt pathway. Together with previous reports
on female mutant Ptprf mice, which have a lactation defect, and disruption of
one allele of PTPRF by a balanced translocation in a woman with amastia, our
results indicate a key role for PTPRF in the development of the nipple-areola
region. |
Is RASA2 involved in melanoma? | Yes. Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer. | Author information:
(1)Molecular Cell Biology Department, Weizmann Institute of Science, Rehovot,
Israel.
(2)Melanoma Institute Australia, Sydney, New South Wales, Australia.
(3)Discipline of Pathology, Sydney Medical School, University of Sydney, Sydney,
New South Wales, Australia.
(4)National Human Genome Research Institute, US National Institutes of Health,
Bethesda, Maryland, USA.
(5)National Cancer Institute, US National Institutes of Health, Bethesda,
Maryland, USA.
(6)Institute of Biochemistry, Food Science and Nutrition, Hebrew University,
Rehovot, Israel.
(7)Department of Biological Services, Weizmann Institute of Science, Rehovot,
Israel.
(8)QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
(9)Department of Biological Regulation, Weizmann Institute of Science, Rehovot,
Israel.
(10)Centre for Cancer Research, Westmead Millennium Institute for Medical
Research, University of Sydney, Sydney, New South Wales, Australia.
(11)Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital,
Sydney, New South Wales, Australia. |
What is BioCreative? | A community wide effort to evaluate biomedical information extraction and text mining. | We present a maximum entropy-based system for identifying named entities (NEs)
in biomedical abstracts and present its performance in the only two biomedical
named entity recognition (NER) comparative evaluations that have been held to
date, namely BioCreative and Coling BioNLP. Our system obtained an exact match
F-score of 83.2% in the BioCreative evaluation and 70.1% in the BioNLP
evaluation. We discuss our system in detail, including its rich use of local
features, attention to correct boundary identification, innovative use of
external knowledge resources, including parsing and web searches, and rapid
adaptation to new NE sets. We also discuss in depth problems with data
annotation in the evaluations which caused the final performance to be lower
than optimal. BACKGROUND: Genome sciences have experienced an increasing demand for efficient
text-processing tools that can extract biologically relevant information from
the growing amount of published literature. In response, a range of text-mining
and information-extraction tools have recently been developed specifically for
the biological domain. Such tools are only useful if they are designed to meet
real-life tasks and if their performance can be estimated and compared. The
BioCreative challenge (Critical Assessment of Information Extraction in Biology)
consists of a collaborative initiative to provide a common evaluation framework
for monitoring and assessing the state-of-the-art of text-mining systems applied
to biologically relevant problems.
RESULTS: The Second BioCreative assessment (2006 to 2007) attracted 44 teams
from 13 countries worldwide, with the aim of evaluating current
information-extraction/text-mining technologies developed for one or more of the
three tasks defined for this challenge evaluation. These tasks included the
recognition of gene mentions in abstracts (gene mention task); the extraction of
a list of unique identifiers for human genes mentioned in abstracts (gene
normalization task); and finally the extraction of physical protein-protein
interaction annotation-relevant information (protein-protein interaction task).
The 'gold standard' data used for evaluating submissions for the third task was
provided by the interaction databases MINT (Molecular Interaction Database) and
IntAct.
CONCLUSION: The Second BioCreative assessment almost doubled the number of
participants for each individual task when compared with the first BioCreative
assessment. An overall improvement in terms of balanced precision and recall was
observed for the best submissions for the gene mention (F score 0.87); for the
gene normalization task, the best results were comparable (F score 0.81)
compared with results obtained for similar tasks posed at the first BioCreative
challenge. In case of the protein-protein interaction task, the importance and
difficulties of experimentally confirmed annotation extraction from full-text
articles were explored, yielding different results depending on the step of the
annotation extraction workflow. A common characteristic observed in all three
tasks was that the combination of system outputs could yield better results than
any single system. Finally, the development of the first text-mining meta-server
was promoted within the context of this community challenge. BACKGROUND: Research scientists and companies working in the domains of
biomedicine and genomics are increasingly faced with the problem of efficiently
locating, within the vast body of published scientific findings, the critical
pieces of information that are needed to direct current and future research
investment.
RESULTS: In this report we describe approaches taken within the scope of the
second BioCreative competition in order to solve two aspects of this problem:
detection of novel protein interactions reported in scientific articles, and
detection of the experimental method that was used to confirm the interaction.
Our approach to the former problem is based on a high-recall protein annotation
step, followed by two strict disambiguation steps. The remaining proteins are
then combined according to a number of lexico-syntactic filters, which deliver
high-precision results while maintaining reasonable recall. The detection of the
experimental methods is tackled by a pattern matching approach, which has
delivered the best results in the official BioCreative evaluation.
CONCLUSION: Although the results of BioCreative clearly show that no tool is
sufficiently reliable for fully automated annotations, a few of the proposed
approaches (including our own) already perform at a competitive level. This
makes them interesting either as standalone tools for preliminary document
inspection, or as modules within an environment aimed at supporting the process
of curation of biomedical literature. BACKGROUND: In the absence of consolidated pipelines to archive biological data
electronically, information dispersed in the literature must be captured by
manual annotation. Unfortunately, manual annotation is time consuming and the
coverage of published interaction data is therefore far from complete. The use
of text-mining tools to identify relevant publications and to assist in the
initial information extraction could help to improve the efficiency of the
curation process and, as a consequence, the database coverage of data available
in the literature. The 2006 BioCreative competition was aimed at evaluating
text-mining procedures in comparison with manual annotation of protein-protein
interactions.
RESULTS: To aid the BioCreative protein-protein interaction task, IntAct and
MINT (Molecular INTeraction) provided both the training and the test datasets.
Data from both databases are comparable because they were curated according to
the same standards. During the manual curation process, the major cause of data
loss in mining the articles for information was ambiguity in the mapping of the
gene names to stable UniProtKB database identifiers. It was also observed that
most of the information about interactions was contained only within the
full-text of the publication; hence, text mining of protein-protein interaction
data will require the analysis of the full-text of the articles and cannot be
restricted to the abstract.
CONCLUSION: The development of text-mining tools to extract protein-protein
interaction information may increase the literature coverage achieved by manual
curation. To support the text-mining community, databases will highlight those
sentences within the articles that describe the interactions. These will supply
data-miners with a high quality dataset for algorithm development. Furthermore,
the dictionary of terms created by the BioCreative competitors could enrich the
synonym list of the PSI-MI (Proteomics Standards Initiative-Molecular
Interactions) controlled vocabulary, which is used by both databases to annotate
their data content. Proteins and their interactions govern virtually all cellular processes, such as
regulation, signaling, metabolism, and structure. Most experimental findings
pertaining to such interactions are discussed in research papers, which, in
turn, get curated by protein interaction databases. Authors, editors, and
publishers benefit from efforts to alleviate the tasks of searching for relevant
papers, evidence for physical interactions, and proper identifiers for each
protein involved. The BioCreative II.5 community challenge addressed these tasks
in a competition-style assessment to evaluate and compare different
methodologies, to make aware of the increasing accuracy of automated methods,
and to guide future implementations. In this paper, we present our approaches
for protein-named entity recognition, including normalization, and for
extraction of protein-protein interactions from full text. Our overall goal is
to identify efficient individual components, and we compare various compositions
to handle a single full-text article in between 10 seconds and 2 minutes. We
propose strategies to transfer document-level annotations to the sentence-level,
which allows for the creation of a more fine-grained training corpus; we use
this corpus to automatically derive around 5,000 patterns. We rank sentences by
relevance to the task of finding novel interactions with physical evidence,
using a sentence classifier built from this training corpus. Heuristics for
paraphrasing sentences help to further remove unnecessary information that might
interfere with patterns, such as additional adjectives, clauses, or bracketed
expressions. In BioCreative II.5, we achieved an f-score of 22 percent for
finding protein interactions, and 43 percent for mapping proteins to UniProt
IDs; disregarding species, f-scores are 30 percent and 55 percent, respectively.
On average, our best-performing setup required around 2 minutes per full text.
All data and pattern sets as well as Java classes that extend- - third-party
software are available as supplementary information (see Appendix). BACKGROUND: The vast amount of data published in the primary biomedical
literature represents a challenge for the automated extraction and codification
of individual data elements. Biological databases that rely solely on manual
extraction by expert curators are unable to comprehensively annotate the
information dispersed across the entire biomedical literature. The development
of efficient tools based on natural language processing (NLP) systems is
essential for the selection of relevant publications, identification of data
attributes and partially automated annotation. One of the tasks of the
Biocreative 2010 Challenge III was devoted to the evaluation of NLP systems
developed to identify articles for curation and extraction of protein-protein
interaction (PPI) data.
RESULTS: The Biocreative 2010 competition addressed three tasks: gene
normalization, article classification and interaction method identification. The
BioGRID and MINT protein interaction databases both participated in the
generation of the test publication set for gene normalization, annotated the
development and test sets for article classification, and curated the test set
for interaction method classification. These test datasets served as a gold
standard for the evaluation of data extraction algorithms.
CONCLUSION: The development of efficient tools for extraction of PPI data is a
necessary step to achieve full curation of the biomedical literature. NLP
systems can in the first instance facilitate expert curation by refining the
list of candidate publications that contain PPI data; more ambitiously, NLP
approaches may be able to directly extract relevant information from full-text
articles for rapid inspection by expert curators. Close collaboration between
biological databases and NLP systems developers will continue to facilitate the
long-term objectives of both disciplines. BACKGROUND: The overall goal of the BioCreative Workshops is to promote the
development of text mining and text processing tools which are useful to the
communities of researchers and database curators in the biological sciences. To
this end BioCreative I was held in 2004, BioCreative II in 2007, and BioCreative
II.5 in 2009. Each of these workshops involved humanly annotated test data for
several basic tasks in text mining applied to the biomedical literature.
Participants in the workshops were invited to compete in the tasks by
constructing software systems to perform the tasks automatically and were given
scores based on their performance. The results of these workshops have benefited
the community in several ways. They have 1) provided evidence for the most
effective methods currently available to solve specific problems; 2) revealed
the current state of the art for performance on those problems; 3) and provided
gold standard data and results on that data by which future advances can be
gauged. This special issue contains overview papers for the three tasks of
BioCreative III.
RESULTS: The BioCreative III Workshop was held in September of 2010 and
continued the tradition of a challenge evaluation on several tasks judged basic
to effective text mining in biology, including a gene normalization (GN) task
and two protein-protein interaction (PPI) tasks. In total the Workshop involved
the work of twenty-three teams. Thirteen teams participated in the GN task which
required the assignment of EntrezGene IDs to all named genes in full text papers
without any species information being provided to a system. Ten teams
participated in the PPI article classification task (ACT) requiring a system to
classify and rank a PubMed® record as belonging to an article either having or
not having "PPI relevant" information. Eight teams participated in the PPI
interaction method task (IMT) where systems were given full text documents and
were required to extract the experimental methods used to establish PPIs and a
text segment supporting each such method. Gold standard data was compiled for
each of these tasks and participants competed in developing systems to perform
the tasks automatically.BioCreative III also introduced a new interactive task
(IAT), run as a demonstration task. The goal was to develop an interactive
system to facilitate a user's annotation of the unique database identifiers for
all the genes appearing in an article. This task included ranking genes by
importance (based preferably on the amount of described experimental information
regarding genes). There was also an optional task to assist the user in finding
the most relevant articles about a given gene. For BioCreative III, a user
advisory group (UAG) was assembled and played an important role 1) in producing
some of the gold standard annotations for the GN task, 2) in critiquing IAT
systems, and 3) in providing guidance for a future more rigorous evaluation of
IAT systems. Six teams participated in the IAT demonstration task and received
feedback on their systems from the UAG group. Besides innovations in the GN and
PPI tasks making them more realistic and practical and the introduction of the
IAT task, discussions were begun on community data standards to promote
interoperability and on user requirements and evaluation metrics to address
utility and usability of systems.
CONCLUSIONS: In this paper we give a brief history of the BioCreative Workshops
and how they relate to other text mining competitions in biology. This is
followed by a synopsis of the three tasks GN, PPI, and IAT in BioCreative III
with figures for best participant performance on the GN and PPI tasks. These
results are discussed and compared with results from previous BioCreative
Workshops and we conclude that the best performing systems for GN, PPI-ACT and
PPI-IMT in realistic settings are not sufficient for fully automatic use. This
provides evidence for the importance of interactive systems and we present our
vision of how best to construct an interactive system for a GN or PPI like task
in the remainder of the paper. BACKGROUND: The BioCreative challenge evaluation is a community-wide effort for
evaluating text mining and information extraction systems applied to the
biological domain. The biocurator community, as an active user of biomedical
literature, provides a diverse and engaged end user group for text mining tools.
Earlier BioCreative challenges involved many text mining teams in developing
basic capabilities relevant to biological curation, but they did not address the
issues of system usage, insertion into the workflow and adoption by curators.
Thus in BioCreative III (BC-III), the InterActive Task (IAT) was introduced to
address the utility and usability of text mining tools for real-life biocuration
tasks. To support the aims of the IAT in BC-III, involvement of both developers
and end users was solicited, and the development of a user interface to address
the tasks interactively was requested.
RESULTS: A User Advisory Group (UAG) actively participated in the IAT design and
assessment. The task focused on gene normalization (identifying gene mentions in
the article and linking these genes to standard database identifiers), gene
ranking based on the overall importance of each gene mentioned in the article,
and gene-oriented document retrieval (identifying full text papers relevant to a
selected gene). Six systems participated and all processed and displayed the
same set of articles. The articles were selected based on content known to be
problematic for curation, such as ambiguity of gene names, coverage of multiple
genes and species, or introduction of a new gene name. Members of the UAG
curated three articles for training and assessment purposes, and each member was
assigned a system to review. A questionnaire related to the interface usability
and task performance (as measured by precision and recall) was answered after
systems were used to curate articles. Although the limited number of articles
analyzed and users involved in the IAT experiment precluded rigorous
quantitative analysis of the results, a qualitative analysis provided valuable
insight into some of the problems encountered by users when using the systems.
The overall assessment indicates that the system usability features appealed to
most users, but the system performance was suboptimal (mainly due to low
accuracy in gene normalization). Some of the issues included failure of species
identification and gene name ambiguity in the gene normalization task leading to
an extensive list of gene identifiers to review, which, in some cases, did not
contain the relevant genes. The document retrieval suffered from the same
shortfalls. The UAG favored achieving high performance (measured by precision
and recall), but strongly recommended the addition of features that facilitate
the identification of correct gene and its identifier, such as contextual
information to assist in disambiguation.
DISCUSSION: The IAT was an informative exercise that advanced the dialog between
curators and developers and increased the appreciation of challenges faced by
each group. A major conclusion was that the intended users should be actively
involved in every phase of software development, and this will be strongly
encouraged in future tasks. The IAT Task provides the first steps toward the
definition of metrics and functional requirements that are necessary for
designing a formal evaluation of interactive curation systems in the BioCreative
IV challenge. There is an increasing interest in developing ontologies and controlled
vocabularies to improve the efficiency and consistency of manual literature
curation, to enable more formal biocuration workflow results and ultimately to
improve analysis of biological data. Two ontologies that have been successfully
used for this purpose are the Gene Ontology (GO) for annotating aspects of gene
products and the Molecular Interaction ontology (PSI-MI) used by databases that
archive protein-protein interactions. The examination of protein interactions
has proven to be extremely promising for the understanding of cellular
processes. Manual mapping of information from the biomedical literature to
bio-ontology terms is one of the most challenging components in the curation
pipeline. It requires that expert curators interpret the natural language
descriptions contained in articles and infer their semantic equivalents in the
ontology (controlled vocabulary). Since manual curation is a time-consuming
process, there is strong motivation to implement text-mining techniques to
automatically extract annotations from free text. A range of text mining
strategies has been devised to assist in the automated extraction of biological
data. These strategies either recognize technical terms used recurrently in the
literature and propose them as candidates for inclusion in ontologies, or
retrieve passages that serve as evidential support for annotating an ontology
term, e.g. from the PSI-MI or GO controlled vocabularies. Here, we provide a
general overview of current text-mining methods to automatically extract
annotations of GO and PSI-MI ontology terms in the context of the BioCreative
(Critical Assessment of Information Extraction Systems in Biology) challenge.
Special emphasis is given to protein-protein interaction data and PSI-MI terms
referring to interaction detection methods. Author information:
(1)National Center for Biotechnology Information, National Library of Medicine,
National Institutes of Health, Bethesda, MD 20894, USA.
(2)Institute for Research in Immunology and Cancer, Université de Montréal,
Montréal, QC H3C 3J7, Canada.
(3)Lewis-Sigler Institute for Integrative Genomics, Princeton University,
Princeton, NJ 08544, USA.
(4)National Centre for Text Mining, School of Computer Science, University of
Manchester, Manchester, UK.
(5)DETI/IEETA, University of Aveiro, Campus Universitário de Santiago, 3810-193
Aveiro, Portugal.
(6)BMD Software, Lda, Rua Calouste Gulbenkian 1, 3810-074 Aveiro, Portugal.
(7)Graduate Institute of Biomedical Informatics, College of Medical Science and
Technology, Taipei Medical University, Taipei, Taiwan.
(8)School of Public Health and Community Medicine, University of New South
Wales, Kensington NSW 2033, Australia Prince of Wales Clinical School,
University of New South Wales, Kensington NSW 2033, Australia.
(9)Department of Computer Science and Information Engineering, National Taitung
University, Taitung, Taiwan.
(10)Institute of Information Science, Academia Sinica, Taipei, Taiwan Department
of Information Management, National Taiwan University, Taipei, Taiwan.
(11)Department of Computer Science, National Tsing Hua University, Hsinchu,
Taiwan.
(12)Institute of Information Science, Academia Sinica, Taipei, Taiwan.
(13)Department of Information Management, National Taiwan University, Taipei,
Taiwan.
(14)Computer & Information Sciences, University of Delaware, Newark, DE 19716,
USA.
(15)Computer & Information Sciences, University of Delaware, Newark, DE 19716,
USA Center for Bioinformatics & Computational Biology, University of Delaware,
Newark, DE 19716, USA.
(16)Department of Computer Engineering, Boğaziçi University, Bebek, 34342
Istanbul, Turkey.
(17)Department of Biomedical Informatics, Asan Medical Center, 138-736 Seoul,
South Korea.
(18)Department of Computer Engineering, Myongji University, 449-728 Yongin,
South Korea.
(19)Institute for Research in Immunology and Cancer, Université de Montréal,
Montréal, QC H3C 3J7, Canada The Lunenfeld-Tanenbaum Research Institute, Mount
Sinai Hospital, Toronto, Ontario, Canada.
(20)National Center for Biotechnology Information, National Library of Medicine,
National Institutes of Health, Bethesda, MD 20894, USA [email protected]. |
What is Contrave prescribed for? | Contrave(?) is a combination of naltrexone hydrochloride extended release and bupropion hydrochloride extended release for the treatment of obesity | In March 2010, Orexigen(R) Therapeutics submitted a new drug application (NDA)
for approval of naltrexone sustained release (SR)/bupropion SR (Contrave(R)) for
the treatment of obesity in the US. The tablet contains naltrexone SR 32 mg and
bupropion SR 360 mg. The drug has been tested in four randomized, double-blind,
placebo-controlled, phase III trials and the co-primary endpoints were met in
each case. This review discusses the key development milestones and clinical
trial program to date. INTRODUCTION: Central neurochemical systems including the monoamine, opioid, and
cannabinoid systems have been promising targets for antiobesity drugs that
modify behavioral components of obesity. In addition to modulating eating
behavior, centrally acting antiobesity drugs are also likely to alter emotional
behavior and cognitive function due to the high expression of receptors for the
neurochemical systems targeted by these drugs within the fronto-striatal and
limbic circuitry.
METHODS: This paper reviewed the neuropsychiatric adverse effects of past and
current antiobesity drugs, with a central mechanism of action, linking the
adverse effects to their underlying neural substrates and neurochemistry.
RESULTS: Antiobesity drugs were found to have varying neuropsychiatric adverse
event profiles. Insomnia was the most common adverse effect with drugs targeting
monoamine systems (sibutramine, bupropion and tesofensine). These drugs had some
positive effects on mood and anxiety and may have added therapeutic benefits in
obese patients with comorbid depression and anxiety symptoms. Sedation and
tiredness were the most common adverse effects reported with drugs targeting the
m-opioid receptors (i.e., naltrexone) and combination therapies targeting the
opioid and monoamine systems (i.e., Contrave™). Cognitive impairments were most
frequently associated with the antiepileptic drugs, topiramate and zonisamide,
consistent with their sedative properties. Drugs targeting the cannabinoid
system (rimonabant and taranabant) were consistently associated with symptoms of
anxiety and depression, including reports of suicidal ideation. Similar adverse
events have also been noted for the D₁/D₅ antagonist ecopipam.
CONCLUSION: These findings highlight the need to assess neuropsychiatric adverse
events comprehensively using sensitive and validated methods early in the
clinical development of candidate antiobesity drugs with a central mechanism of
action. Obesity is a global problem that is predomitly caused by the increasing
adoption of a low-cost, Westernised diet that is rich in fat and sugar and a
more sedentary lifestyle. The costs of this epidemic are substantial increases
in Type 2 diabetes, cardiovascular disease and some types of cancer that are
certain to place a huge burden on individuals, healthcare providers and society.
In this review, we provide an overview of the chequered history of
pharmacotherapy for the treatment of obesity and an analysis of the regulatory
and commercial challenges for developing new centrally-acting drugs in this
metabolic indication. The efficacy and safety of the drug candidates that are
currently at the pre-registration phase, i.e., lorcaserin, Qnexa and Contrave,
are critically assessed. The main focus, however, is to provide a comprehensive
review of the wide range of novel CNS compounds that are in the discovery phase
or early clinical development. The profiles of various clinical candidates in
animal models of obesity predict that several new CNS approaches in the clinic
have the potential to deliver greater weight-loss than existing agents. This
article is part of a Special Issue entitled 'Central Control of Food Intake'. OBJECTIVE: To examine the effects of naltrexone/bupropion (NB) combination
therapy on weight and weight-related risk factors in overweight and obese
participants.
DESIGN AND METHODS: CONTRAVE Obesity Research-II (COR-II) was a double-blind,
placebo-controlled study of 1,496 obese (BMI 30-45 kg/m(2) ) or overweight
(27-45 kg/m(2) with dyslipidemia and/or hypertension) participants randomized
2:1 to combined naltrexone sustained-release (SR) (32 mg/day) plus bupropion SR
(360 mg/day) (NB32) or placebo for up to 56 weeks. The co-primary endpoints were
percent weight change and proportion achieving ≥ 5% weight loss at week 28.
RESULTS: Significantly (P < 0.001) greater weight loss was observed with NB32
versus placebo at week 28 (-6.5% vs. -1.9%) and week 56 (-6.4% vs. -1.2%). More
NB32-treated participants (P < 0.001) experienced ≥ 5% weight loss versus
placebo at week 28 (55.6% vs. 17.5%) and week 56 (50.5% vs. 17.1%). NB32
produced greater improvements in various cardiometabolic risk markers,
participant-reported weight-related quality of life, and control of eating. The
most common adverse event with NB was nausea, which was generally mild to
moderate and transient. NB was not associated with increased events of
depression or suicidality versus placebo.
CONCLUSION: NB represents a novel pharmacological approach to the treatment of
obesity, and may become a valuable new therapeutic option. Oral naltrexone extended-release/bupropion extended-release (naltrexone
ER/bupropion ER; Contrave(®), Mysimba(™)) is available as an adjunct to a
reduced-calorie diet and increased physical activity in adults with an initial
body mass index (BMI) of ≥ 30 kg/m(2) (i.e. obese) or a BMI of ≥ 27 kg/m(2)
(i.e. overweight) in the presence of at least one bodyweight-related
comorbidity, such as type 2 diabetes mellitus, hypertension or dyslipidaemia. In
56-week phase III trials in these patient populations, oral naltrexone
ER/bupropion ER 32/360 mg/day was significantly more effective than placebo with
regard to percentage bodyweight reductions from baseline and the proportion of
patients who achieved bodyweight reductions of ≥ 5 and ≥ 10%. Significantly
greater improvements in several cardiometabolic risk factors were also observed
with naltrexone ER/bupropion ER versus placebo, as well as greater improvements
in glycated haemoglobin levels in obese or overweight adults with type 2
diabetes. Naltrexone ER/bupropion ER was generally well tolerated in phase III
trials, with nausea being the most common adverse event. Thus, naltrexone
ER/bupropion ER 32/360 mg/day as an adjunct to a reduced-calorie diet and
increased physical activity, is an effective and well tolerated option for
chronic bodyweight management in obese adults or overweight adults with at least
one bodyweight-related comorbidity. Weight loss is associated with improved quality of life in some, but not all,
weight loss trials. We evaluated changes at 56 weeks in quality of life,
measured by the Impact of Weight on Quality of Life-Lite (IWQOL-Lite)
questionnaire, in a pooled analysis of patient-level data from four randomized
controlled Phase 3 studies of naltrexone/bupropion (NB32 or Contrave®). The
total number of subjects was 3362 (NB32 = 2043; placebo = 1319; mean body mass
index = 36.3 kg m(2); mean age = 46). Improvements in IWQOL-Lite Total Score
were greater in subjects treated with NB32 (11.9 points [SE 0.3]) vs. placebo
(8.2 points [SE 0.3]; P < 0.001), corresponding to weight reductions of 7.0% (SE
0.2) and 2.3% (SE 0.2), respectively. Greater improvements were also observed
for NB32 vs. placebo on all five subscale scores of the IWQOL-Lite. Fifty per
cent of NB32-treated subjects achieved clinically meaningful improvements in
IWQOL-Lite Total Score vs. 32.3% of placebo-treated subjects (odds ratio, 95%
confidence interval; 2.09, 1.79-2.44). Subjects losing the most weight (≥ 15% of
baseline weight) experienced the greatest improvement in IWQOL-Lite Total Score
(19.3 points [SE 0.7] for NB32 and 18.7 points [SE 1.3] for placebo; P = 0.624).
Improved quality of life was associated with weight reduction and was achieved
in more subjects treated with NB32 than placebo. Obesity and metabolic syndrome (MS) are risk factors for diabetes, cancer, some
cardiovascular and musculoskeletal diseases. Pharmacotherapy should be used when
the body mass index (BMI) exceeds 30 kg/m² or 27 kg/m² with comorbidity.
Efficacy and safety of pharmacotherapy depend on the mechanism of action of
drugs. In this context, drugs affecting the central and peripheral mediator
systems such as cannabinoid receptor antagonists (Rimonabant), neuronal reuptake
inhibitor of NE and 5 HT (Sibutramine), neuronal reuptake inhibitor of NE 5-HT
DA (Tesofensine), agonist of 5 HT 2C receptors (Lorcaserin) have a high risk of
side effects on the central nervous and cardiovascular systems when used for a
long period. Apparently, the drugs design targeting obesity should screen safer
drugs that affect fat absorption (Orlistat), activate energy metabolism
(Adipokines), inhibit MetAP2 (Beloranib) and other peripheral metabolic
processes. The use of synergies of anti-obesity drugs with different mechanisms
of action is an effective approach for developing new combined pharmaceutical
compositions (Contrave®, EmpaticTM, Qsymia et al). The purpose of this article
is to review the currently available anti-obesity drugs and some new promising
trends in development of anti-obesity therapy. Contrave(®) is a combination of naltrexone hydrochloride extended release and
bupropion hydrochloride extended release for the treatment of obesity, and is
used with lifestyle modification. Its safety and efficacy were assessed in four
randomized, double-blind, placebo-controlled, 56-week Phase III clinical trials
in 4536 adult subjects: COR-1, COR-II, COR-BMOD and COR-DM. All four studies
demonstrated statistically significant and clinically meaningful weight loss
following up to 52 weeks of treatment with naltrexone/bupropion compared with
placebo. The average weight loss from baseline across the four studies was
approximately 11-22 lbs (5-9 kg). Results show the efficacy of Contrave for
weight loss, as well as significant improvements in cardiometabolic markers.
This review focuses on the four studies, their outcomes and the mechanism of
action of Contrave. Naltrexone/Bupropion ER (Contrave): Newly Approved Treatment Option for Chronic
Weight Management in Obese Adults. |
Which is the chromosome area that the human gene coding for the dopamine transporter (DAT1) is located to? | The gene encoding DAT1 consists of 15 exons spanning 60 kb and is located on chromosome 5p15.3. | The human dopamine transporter (DAT1) gene is localized to chromosome 5p15.3 by
in situ hybridization and PCR amplification of rodent somatic cell hybrid DNA.
Analysis of a 40-bp repeat in the 3' untranslated region of the message revealed
variable numbers of the repeat ranging from 3 to 11 copies. These results will
aid in the investigation of a role for this gene in genetic disorders of the
dopaminergic system in humans. In 29 adults with attention deficit hyperactivity disorder (ADHD) striatal
dopamine transporter (DAT) availability was assessed by [(99m)Tc]TRODAT-1 SPECT
and correlated with 3' VNTR polymorphism of the DAT gene on chromosome 5p15.3.
Seventeen patients showed homozygosity for the 10-repeat allele, two
homozygosity of the 9 allele and 10 were heterozygous (9-10). No statistically
significant difference in DAT availability was found between patients with 10-10
carriers (DAT 1.28 +/- 0.34) and with at least one 9 allele (DAT 1.31 +/- 0.27);
when smokers were excluded, DAT availability was 1.38 +/- 0.28 in the 10-10
carriers (n = 12) and 1.42 +/- 0.19 in the 9-10 and 9-9 carriers (n = 7). In
conclusion, no higher striatal DAT was found in patients with homozygosity of
the 10 allele of the DAT gene in this study. These results differ from a study
in 11 Korean children with ADHD, in which 10-10 carriers showed higher DAT
availability in [(123)I]IPT SPECT. Discrepancies may be explained by differences
in patient's age, ethnical differences, different imaging techniques or the
limited number of patients included in both studies. Previously, we had reported a genome-wide scan for
attention-deficit/hyperactivity disorder (ADHD) in 102 families with affected
sibs of German ancestry; the highest multipoint LOD score of 4.75 was obtained
on chromosome 5p13 (parametric HLOD analysis under a domit model) near the
dopamine transporter gene (DAT1). We genotyped 30 single nucleotide
polymorphisms (SNPs) in this candidate gene and its 5' region in 329 families
(including the 102 initial families) with 523 affected offspring. We found that
(1) SNP rs463379 was significantly associated with ADHD upon correction for
multiple testing (P=0.0046); (2) the global P-value for association of
haplotypes was significant for block two upon correction for all (n=3) tested
blocks (P=0.0048); (3) within block two we detected a nominal P=0.000034 for one
specific marker combination. This CGC haplotype showed relative risks of 1.95
and 2.43 for heterozygous and homozygous carriers, respectively; and (4)
finally, our linkage data and the genotype-IBD sharing test (GIST) suggest that
genetic variation at the DAT1 locus explains our linkage peak and that rs463379
(P<0.05) is the only SNP of the above haplotype that contributed to the linkage
signal. In sum, we have accumulated evidence that genetic variation at the DAT1
locus underlies our ADHD linkage peak on chromosome 5; additionally solid
association for a single SNP and a haplotype were shown. Future studies are
required to assess if variation at this locus also explains other positive
linkage results obtained for chromosome 5p. |
What type of mutation is causing the industrial melanism phenotype in peppered moths? | The mutation event giving rise to industrial melanism in Britain was the insertion of a large, tandemly repeated, transposable element into the first intron of the gene cortex. | Industrial melanism in peppered moths has been studied most intensively in
Britain. The first melanic phenotype (effectively solid black) was recorded near
Manchester in 1848. By 1895 about 98% of the specimens near Manchester were
melanic, and this once rare phenotype had spread across regions of the country
blackened by industrial soot. In rural, unpolluted regions, well away from
industrial centers, the pale phenotype (peppered with white and black scales)
remained the predomit form. During the latter half of the 20th century,
following legislation designed to improve air quality, melanics began to decline
in frequency and are now rare where once they had been common. Similar
evolutionary changes have occurred elsewhere, but records from outside Britain
are fragmentary. We have extended previous surveys of American peppered moth
populations and present a composite picture of the recent decline in melanism in
northern industrial states-Michigan and Pennsylvania-where melanic phenotypes
decreased from more than 90% in 1959 to 6% by 2001. We contrast these changes to
the near absence of melanism in a southern state-Virginia-during that same
period. As in Britain, the decline in melanism in American peppered moths
followed clean air legislation. |
What gene is mutated in Huntington's disease? | Huntington disease (HD; OMIM 143100), a progressive neurodegenerative disorder, is caused by an expanded trinucleotide CAG (polyQ) motif in the HTT gene. Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington's disease (HD). | Huntingtons disease (HD) is a hereditary disorder involving the central nervous
system. Its effects are devastating, to the affected person as well as his
family. The Department of Medical and Molecular Genetics at Indiana University
(IU) plays an integral part in Huntingtons research by providing computerized
repositories of HD family information for researchers and families. The National
Huntingtons Disease Research Roster, founded in 1979 at IU, and the Huntingtons
Disease in Venezuela Project database contain information that has proven to be
invaluable in the worldwide field of HD research. This paper addresses the types
of information stored in each database, the pedigree database program (MEGADATS)
used to manage the data, and significant findings that have resulted from access
to the data. The deletion of 3bp at codon positions 2642-2645 (delta 2642) of the gene
mutated in Huntington's disease (HD) was analysed on the normal (N) and HD
chromosomes of 79 French families affected with HD, and previously typed for the
(CAG)n repeats. delta 2642 Polymorphism has been found over-represented on HD
chromosomes, the relative risk of HD with the deletion being at a value of 8.26.
In this study, the presence of the deleted allele on HD chromosomes increases
the (CAG)n number (47.93 +/- 1.80 versus 43.50 +/- 2.78) and decreases the age
of onset (41.34 +/- 2.09 versus 36.90 +/- 2.41) in the patients with versus
without delta 2642; so the deletion may add to the severity of the disease. Our
studies of delta 2642 on N chromosomes confirm that the deletion event occurs on
N chromosomes with a (CAG)n allele length at the upper end of the normal size
range. Huntington's disease is an inherited disorder caused by expansion of a CAG
trinucleotide repeat in the IT15 gene, which leads to expansion of a
polyglutamine tract within the protein called huntingtin. Despite the
characterization of the IT15 gene and the mutation involved in the disease, the
normal function of huntingtin and the effects of the mutation on its function
and on its neuronal location remain unknown. To study whether mutated huntingtin
has the same neuronal distribution and intracellular location as normal
huntingtin, we analyzed immunohistochemically both forms of this protein in the
brain of 5 controls and 5 patients with Huntington's disease. We show that the
distribution of mutated huntingtin is, like that of the normal form,
heterogeneous throughout the brain, but is not limited to vulnerable neurons in
Huntington's disease, supporting the hypothesis that the presence of the mutated
huntingtin in a neuron is not in itself sufficient to lead to neuronal death.
Moreover, whereas normal huntingtin is detected in some neuronal perikarya,
nerve fibers, and nerve endings, the mutated form is observed in some neuronal
perikarya and proximal nerve processes but is not detectable in nerve endings.
Our results suggest that the expression or processing of the mutated huntingtin
in perikarya and nerve endings differs quantitatively or qualitatively from the
expression of the normal form in the same neuronal compartments. Huntington's disease (HD) is a neurodegenerative disorder characterized by the
expansion of CAG repeats in exon 1 of the HD gene. To clarify the instability of
expanded CAG repeats in HD patients, an HD model mouse has been generated by
gene replacement with human exon 1 of the HD gene with expansion to 77 CAG
repeats. Chimeric proteins composed of human mutated exon 1 and mouse huntingtin
are expressed ubiquitously in brain and peripheral tissues. One or two CAG
repeat expansion was found in litters from paternal transmission, whereas
contraction of CAG repeat in litters was observed through maternal transmission.
Elderly mice show greater CAG repeat instability than younger mice, and a unique
case was observed of an expanded 97 CAG repeat mouse. Somatic CAG repeat
instability is particularly pronounced in the liver, kidney, stomach, and brain
but not in the cerebellum of 100-week-old mice. The same results of expanded CAG
repeat instability as observed in this HD model mouse were confirmed in the
human brain of HD patients. Glial fibrillary acidic protein (GFAP)-positive
cells have been found to be increased in the substantia nigra (SN), globus
pallidus (GP), and striatum (St) in the brains of 40-week-old affected mice,
although without neuronal cell death. The CAG repeat instability and increase in
GFAP-positive cells in this mouse model appear to mirror the abnormalities in HD
patients. The HD model mouse may therefore have advantages for investigations of
molecular mechanisms underlying instability of CAG repeats. Huntington's disease (HD) is a late onset, incurable, autosomal
domitly-inherited, progressive neuropsychiatric disease, characterised by
chorea, changes in personality, mood and behaviour, and dementia. Huntington's
disease is a clinical diagnosis. The advent of DNA diagnosis has made
predictive, prenatal and preimplantation testing possible for at-risk persons or
asymptomatic carriers. The prevalence is estimated to be 3-10/100,000 among
individuals of European descent; HD is less common in other ethnic groups.
Huntington's disease is caused by an expanded trinucleotide CAG repeat in the HD
gene on chromosome 4. The gene encodes for the protein huntingtin, with an as
yet unknown function. The mutated huntingtin has an elongated stretch of
glutamines which leads to a gain of function such as overactivity,
excitotoxicity, or to interactions with other proteins. Huntington's disease (HD) is a neurological condition of progressive course that
results from abnormally increased number of CAG repeats within IT-15 gene,
coding for huntington. The main symptoms consist of choraetic movements,
dementia, and characteropathy. The aim of the present study was to search for
possible correlation between the age of the onset of HD, time from the onset,
clinical status of the patients, and CAG repeats number. Ten patients were
studied altogether. Modified UHDRS (MUHDRS) was applied for the estimation of
patients' clinical status. The number of CAG repeats in examination of the IT-15
gene was determined by polymerase chain reaction (PCR) and separation of
radioisotope labelled PCR product against DNA size marker in polyacrylamide gel.
A negative significant correlation was found between the CAG repeats number and
the disease onset age (r = -0.67; p < 0.05) and MUHDRS score (r = 0.75; p <
0.05), as well. Negative significant correlation between time from the onset and
MUHDRS score (r = -0.95; p < 0.05) and negative correlation between summarised:
time from the onset and CAG number on the one site and MUHDRS on the other (p =
-0.91) were found, as well. Our findings indicate an interdependence between CAG
repeats number within the IT-15 gene, the course of the disease and the clinical
status of HD patients. We report a group of 252 patients with a Huntington's disease-like (HDL)
phenotype, including 60 with typical Huntington's disease, who had tested
negative for pathological expansions in the IT15 gene, the major mutation in
Huntington's disease. They were screened for repeat expansions in two other
genes involved in HDL phenotypes: those encoding the junctophilin-3 (JPH3/HDL2)
and prion (PRNP/HDL1) proteins. In addition, because of the clinical overlap
between patients with HDL disease and autosomal domit cerebellar ataxia or
dentatorubral and pallidoluysian atrophy (DRPLA), we investigated trinucleotide
repeat expansions in genes encoding the TATA-binding protein (TBP/SCA17) and
atrophin-1 (DRPLA). Two patients carried 43 and 50 uninterrupted CTG repeats in
the JPH3 gene. Two other patients had 44 and 46 CAA/CAG repeats in the TBP gene.
Patients with expansions in the TBP or JPH3 genes had HDL phenotypes
indistinguishable from Huntington's disease. Taking into account patients with
typical Huntington's disease, their frequencies were evaluated as 3% each in our
series of typical HDL patients. Interestingly, incomplete penetrance of the 46
CAA/CAG repeat in the TBP gene was observed in a 59-year-old transmitting, but
healthy, parent. Furthermore, we report a new configuration of the expanded TBP
allele, with 11 repeats on the first polymorphic stretch of CAGs. Expansions in
the DRPLA gene and insertions in the PRNP gene were not found in our group of
patients. Further genetic heterogeneity of the HDL phenotype therefore exists. Understanding molecular genetical background of hereditary chorea has recently
been progressed so far. Triplet repeat expansion diseases including, Huntington
disease, in which CAG expansion has been identified in the IT-15 or Huntingtin
gene, and Huntington disease like-2, in which CTG expansion in junctophilin-3
(JPH3) gene occurs, causes selective degeneration of striatum in the brain.
Octapeptide repeat expansion in the prion gene in Huntington disease like-1 has
been also identified. Neuroacanthocytosis syndromes including McLeod syndrome
and chorea-acanthocytosis cause acanthocytosis in the red blood cells and chorea
due to the degeneration of caudate nucleus in the brain. The XK gene on the X
chromosome is mutated to lose its function in McLeod syndrome. CHAC gene coding
chorein is mutated to lead loss of function in chorea-acanthocytosis. Selective
degeneration in the striatum, especially in the caudate nucleus might be
associated with the molecular cascade of expanded polyglutamine or polyleucine
or octapeptide and the loss of function of the XK protein and of chorein
protein. The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has
been identified as the major cause of Huntington's disease (HD) and determines
42-73% of the variance in the age-at-onset of the disease. Polymorphisms in
huntingtin interacting or associated genes are thought to modify the course of
the disease. To identify genetic modifiers influencing the age at disease onset,
we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17
genes and analysed seven of them by association studies in 980 independent
European HD patients. Screening for unknown sequence variations we found besides
several silent variations three polymorphisms in the ZDHHC17 gene. These and
polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to
their association with the HD age-at-onset. Although some of the factors have
been defined as genetic modifier factors in previous studies, none of the genes
encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier
for HD. BACKGROUND AND PURPOSE: The aim of this study was to perform DNA analysis in
patients with clinical diagnosis of Huntington's disease (HD) after molecular
exclusion of HD and further molecular examinations for other neurodegenerative
diseases such as Huntington's disease-like 2 (HDL-2; gene JPH3), dentatorubral
pallidoluysian atrophy (DRPLA; gene ATN1) and spinocerebellar ataxia type 17
(SCA17; gene TBP).
MATERIAL AND METHODS: The material comprised 224 DNA samples isolated from
peripheral blood from patients suspected of HD and 100 DNA samples from
unaffected controls. The control group was used to determine the normal range of
the number of CAG/CTG repeats in genes JPH3, ATN1 and TBP in the Polish
population. Molecular analysis was carried out by PCR reaction, embracing
microsatellite repeats in genes JPH3, ATN1 and TBP with specific, fluorescently
labelled primers. PCR products were separated in polyacrylamide gels. The normal
ranges of the number of repeats established for the control group in genes JPH3,
ATN1 and TBP were 7-19, 9-27 and 29-45, respectively.
RESULTS: Molecular analysis of DNA from 224 individuals suspected of HD (117
women and 107 men) revealed one case of dynamic mutation - 55 CAG repeats - in
the TBP locus (SCA17). No cases of DRPLA or HDL-2 were detected. The range of
CAG/CTG repeats for the JPH3 gene in the patient group was 11-19, with the most
common alleles containing 14 and 16 repeats. For the ATN1 gene in patients the
range of 8-27 repeats was established and the most frequent allele with 16
triplets was present.
CONCLUSIONS: The study on 244 patients referred with the clinical diagnosis of
HD and without mutation of the IT15 gene revealed one case of SCA17 but did not
disclose the presence of two other diseases with a similar clinical
manifestation: DRPLA and HDL2. We report a cluster of patients from a Karaite Jew community with a movement
disorder suggestive of Huntington disease (HD), in some cases associated with
repeat lengths below the edge of 36 CAG repeats. The study describes the
clinical and genetic features of four patients who were followed over several
years. Patients belonged to an inbred family in whom progressive chorea,
manifesting predomitly with dystonia and cerebellar features, developed
during middle age. Although severe psychiatric symptoms ultimately developed in
two of the four patients, cognitive function remained reasonably well preserved
in all of them even after several disease years. Moderate cognitive deficits
were limited to the visuomotor organization and abstract thinking subtests in
three of the four patients. Qualitative brain imaging showed atrophy of brain
predomitly involving cortex and cerebellum. Genetic testing revealed a
variable mutation penetrance among family members, some affected members showing
an upper allele size ranging from 34 to 49, whereas others remained unaffected
despite the presence of the full mutation beyond 40 CAG repeats. Co-morbidity
with recessive hereditary inclusion body myopathy was found in two subjects from
one family. Although the main diagnosis of HD remains to be confirmed by further
neuropathological studies, these cases may suggest that HD could manifest with
as few as 34 CAG repeats, in some geographic areas, the disease phenotype most
probably being influenced by additional, as yet unidentified, genes. INTRODUCTION: Huntington's disease is a neurodegenerative, autosomal domit
disease that mainly affects the basal ganglia. The disorder is caused by
mutation in the gene encoding the huntingtin protein (Htt), producing
intracellular aggregates. The adult form (chorea with dementia) is the most
frequent. It is characterized by unpredictable, spasmodic, involuntary and
constant movements, mostly associated with psychiatric and cognitive
alterations.
DEVELOPMENT: The main characteristics of Huntington's disease are: neuronal
loss, glyosis, and accumulations of mutated Htt, all associated with different
underlying channels in the pathogenesis of the disease, such as: excitotoxicity,
energy deficit (ATP depletion), reduction in the synthesis and release of
neurotrophic factors (BDNF and GDNF), and oxidative stress. Oxidative stress is
involved in the pathogenesis of various neurodegenerative diseases, including
Huntington's disease, and numerous studies have shown the existence of oxidative
damage in the plasma and tissue of patients suffering from this disease.
CONCLUSIONS: Oxidative stress in patients with Huntington's disease may be used
as an evolutionary-prognostic marker of both the disease and therapeutic
effectiveness, as well as an interesting field of research for the development
of new therapeutic strategies. Huntington disease is a monogenic, autosomal domit, progressive
neurodegenerative disorder caused by a trinucleotide CAG repeat expansion in
exon 1 of the huntingtin (HTT) gene; age of onset of clinical symptoms inversely
correlates with expanded CAG repeat length. HD leads to extensive degeneration
of the basal ganglia, hypothalamic nuclei, and selected cortical areas, and a
wide range of molecular mechanisms have been implicated in disease pathology in
animal or cellular models expressing mutated HTT (mHTT) proteins, either
full-length or amino-terminal fragments. However, HD cellular models that
recapitulate the slow progression of the disease have not been available due to
the toxicity of overexpressed exogenous mHTT or to limitations with using
primary cells for long-term studies. Most investigations of the effects of mHTT
relied on cytotoxicity or aggregation end points in heterologous systems or in
primary embryonic neuroglial cultures derived from HD mouse models. More
innovative approaches are currently under active investigation, including
screening using electrophysiological endpoints, as well as the recent use of
primary blood mononuclear cells and of human embryonic stem cells derived from a
variety of HD research participants. Here we describe how these cellular systems
are being used to investigate HD biology as well as to identify mechanisms with
therapeutic potential. Huntington's disease (HD) is an autosomal domit neurodegenerative disorder.
The causative mutation is an expansion of more than 36 CAG repeats in the first
exon of IT15 gene. Many studies have shown that the IT15 interacts with several
modifier genes to regulate the age at onset (AO) of HD. Our study aims to
investigate the implication of CAG expansion and 9 modifiers in the age at onset
variance of 15 HD Tunisian patients and to establish the correlation between
these modifiers genes and the AO of this disease. Despite the small number of
studied patients, this report consists of the first North African study in
Huntington disease patients. Our results approve a specific effect of modifiers
genes in each population. Huntington disease (HD; OMIM 143100), a progressive neurodegenerative disorder,
is caused by an expanded trinucleotide CAG (polyQ) motif in the HTT gene.
Cardiovascular symptoms, often present in early stage HD patients, are, in
general, ascribed to dysautonomia. However, cardio-specific expression of polyQ
peptides caused pathological response in murine models, suggesting the presence
of a nervous system-independent heart phenotype in HD patients. A positive
correlation between the CAG repeat size and severity of symptoms observed in HD
patients has also been observed in in vitro HD cellular models. Here, we test
the suitability of human embryonic stem cell (hESC) lines carrying HD-specific
mutation as in vitro models for understanding molecular mechanisms of cardiac
pathology seen in HD patients. We have differentiated three HD-hESC lines into
cardiomyocytes and investigated CAG stability up to 60 days after starting
differentiation. To assess CAG stability in other tissues, the lines were also
subjected to in vivo differentiation into teratomas for 10 weeks. Neither
directed differentiation into cardiomyocytes in vitro nor in vivo
differentiation into teratomas, rich in immature neuronal tissue, led to an
increase in the number of CAG repeats. Although the CAG stability might be cell
line-dependent, induced pluripotent stem cells generated from patients with
larger numbers of CAG repeats could have an advantage as a research tool for
understanding cardiac symptoms of HD patients. Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by
a single-gene mutation: a CAG expansion in the huntingtin (HTT) gene that
results in production of a mutated protein, mutant HTT, with a polyglutamine
tail (polyQ-HTT). Although the molecular pathways of polyQ-HTT toxicity are not
fully understood, because protein misfolding and aggregation are central
features of HD, it has long been suspected that cellular housekeeping processes
such as autophagy might be important to disease pathology. Indeed, multiple
lines of research have identified abnormal autophagy in HD, characterized
generally by increased autophagic induction and inefficient clearance of
substrates. To date, the origin of autophagic dysfunction in HD remains unclear
and the search for actors involved continues. To that end, recent studies have
suggested a bidirectional relationship between autophagy and primary cilia,
signaling organelles of most mammalian cells. Interestingly, primary cilia
structure is defective in HD, suggesting a potential link between autophagic
dysfunction, primary cilia and HD pathogenesis. In addition, because polyQ-HTT
also accumulates in primary cilia, the possibility exists that primary cilia
might play additional roles in HD: perhaps by disrupting signaling pathways or
acting as a reservoir for secretion and propagation of toxic, misfolded
polyQ-HTT fragments. Here, we review recent research suggesting potential links
between autophagy, primary cilia and HD and speculate on possible pathogenic
mechanisms and future directions for the field. Author information:
(1)Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière University Hospital,
Department of Genetics, Paris, France2Assistance Publique-Hôpitaux de Paris,
Pitié-Salpêtrière University Hospital, Department of Neurology, Paris, France.
(2)Institut du Cerveau et de la Moelle Epinière, Paris, France4Institut National
de la Santé et de la Récherche Médicale Unité 1127, Centre National de la
Recherche Scientifique Unité Mixte de Recherche 7225, Sorbonne Universités,
Université Pierre et Marie Curie University Paris 06 Unité Mixte de Recherche
S1127, Paris, France5Ecole Pratique des Hautes Etudes, Paris, France.
(3)Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière University Hospital,
Department of Genetics, Paris, France.
(4)Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière University Hospital,
Department of Neurology, Paris, France6Assistance Publique-Hôpitaux de Paris,
Unité de Neurologie de la Mémoire et du Langage, Centre Hospitalier Saint-Anne,
Paris, France.
(5)Assistance Publique-Hôpitaux de Paris, Reference Centre for Huntington's
Disease, Unité Fonctionnelle de Neurologie Cognitive, Henri Mondor University
Hospital, Créteil, France8Institut National de la Santé et de la Récherche
Médicale Unité 955 Team 1, Institut Mondor de Recherche Biomédicale, Faculté de
Médecine de Créteil, Créteil, France9Institut d'Etude de la Cognition, Ecole
Normale Supérieure, Paris, France.
(6)Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière University Hospital,
Department of Neurology, Paris, France10Department of Neurology, McGovern
Medical School, UTHealth, Houston, Texas.
(7)Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière University Hospital,
Department of Neurology, Paris, France3Institut du Cerveau et de la Moelle
Epinière, Paris, France.
(8)Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière University Hospital,
Department of Neurology, Paris, France3Institut du Cerveau et de la Moelle
Epinière, Paris, France4Institut National de la Santé et de la Récherche
Médicale Unité 1127, Centre National de la Recherche Scientifique Unité Mixte de
Recherche 7225, Sorbonne Universités, Université Pierre et Marie Curie
University Paris 06 Unité Mixte de Recherche S1127, Paris, France.
(9)Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière University Hospital,
Department of Genetics, Paris, France3Institut du Cerveau et de la Moelle
Epinière, Paris, France4Institut National de la Santé et de la Récherche
Médicale Unité 1127, Centre National de la Recherche Scientifique Unité Mixte de
Recherche 7225, Sorbonne Universités, Université Pierre et Marie Curie
University Paris 06 Unité Mixte de Recherche S1127, Paris, France.
(10)Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière University
Hospital, Department of Genetics, Paris, France3Institut du Cerveau et de la
Moelle Epinière, Paris, France4Institut National de la Santé et de la Récherche
Médicale Unité 1127, Centre National de la Recherche Scientifique Unité Mixte de
Recherche 7225, Sorbonne Universités, Université Pierre et Marie Curie
University Paris 06 Unité Mixte de Recherche S1127, Paris, France5Ecole Pratique
des Hautes Etudes, Paris, France. |
Which software package is available for the analysis of conserved genomic loci? | PHYLUCE is a software package for the analysis of conserved genomic loci. It identifies targeted, enriched loci from the off-target background data; aligns enriched contigs representing conserved loci to one another; and prepare and manipulate these alignments for subsequent phylogenomic inference. PHYLUCE is an efficient and easy-to-install software package that accomplishes these tasks across hundreds of taxa and thousands of enriched loci | Targeted enrichment of conserved and ultraconserved genomic elements allows
universal collection of phylogenomic data from hundreds of species at multiple
time scales (<5 Ma to > 300 Ma). Prior to downstream inference, data from these
types of targeted enrichment studies must undergo preprocessing to assemble
contigs from sequence data; identify targeted, enriched loci from the off-target
background data; align enriched contigs representing conserved loci to one
another; and prepare and manipulate these alignments for subsequent phylogenomic
inference. PHYLUCE is an efficient and easy-to-install software package that
accomplishes these tasks across hundreds of taxa and thousands of enriched loci.
AVAILABILITY AND IMPLEMENTATION: PHYLUCE is written for Python 2.7. PHYLUCE is
supported on OSX and Linux (RedHat/CentOS) operating systems. PHYLUCE source
code is distributed under a BSD-style license from
https://www.github.com/faircloth-lab/phyluce/ PHYLUCE is also available as a
package (https://binstar.org/faircloth-lab/phyluce) for the Anaconda Python
distribution that installs all dependencies, and users can request a PHYLUCE
instance on iPlant Atmosphere (tag: phyluce). The software manual and a tutorial
are available from http://phyluce.readthedocs.org/en/latest/ and test data are
available from doi: 10.6084/m9.figshare.1284521.
CONTACT: [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. |
Mutation of which gene is implicated in the Christianson syndrome? | Christianson syndrome is caused by mutations in SLC9A6 and is characterized by severe intellectual disability, absent speech, microcephaly, ataxia, seizures, and behavioral abnormalities. | Interstitial deletions of chromosome band Xq26.3 are rare. We report on a
2-year-old boy in whom array comparative genomic hybridization analysis revealed
an interstitial 314 kb deletion in Xq26.3 affecting SLC9A6 and FHL1. Mutations
in SLC9A6 are associated with Christianson syndrome (OMIM 300243), a syndromic
form of X-linked mental retardation (XLMR) characterized by microcephaly, severe
global developmental delay, ataxia and seizures. FHL1 mutations cause
Emery-Dreifuss muscular dystrophy (OMIM 310300), X-linked myopathy with postural
muscle atrophy (XMPMA, OMIM 300696), scapuloperoneal myopathy (OMIM 300695), or
reducing body myopathy (OMIM 300717, 300718). The clinical problems of the
patient reported here comprised severe intellectual disability, absent speech,
ataxia, epilepsy, and gastroesophageal reflux, and could mostly be attributed to
SLC9A6 insufficiency. In contrast to the majority of reported Christianson
syndrome patients who were microcephalic, this patient was normocephalic, but
his head circumference had decelerated from the 50th centile at birth to the
25th centile at the age of 2 ²/¹² years. Muscle problems due to the FHL1
deletion are not to be expected before late childhood, which is the earliest age
of onset for FHL1 associated Emery-Dreifuss muscular dystrophy. This patient
broadens the spectrum of SLC9A6 mutations and contributes to the clinical
delineation of Christianson syndrome. This is also the first patient with a
deletion affecting both SLC9A6 and the complete FHL1 gene. Mutations in solute carrier family 9 isoform 6 on chromosome Xq26.3 encoding
sodium-hydrogen exchanger 6, a protein mainly expressed in early and recycling
endosomes are known to cause a complex and slowly progressive degenerative human
neurological disease. Three resulting phenotypes have so far been reported: an
X-linked Angelman syndrome-like condition, Christianson syndrome and
corticobasal degeneration with tau deposition, with each characterized by severe
intellectual disability, epilepsy, autistic behaviour and ataxia. Hypothesizing
that a sodium-hydrogen exchanger 6 deficiency would most likely disrupt the
endosomal-lysosomal system of neurons, we examined Slc9a6 knockout mice with
tissue staining and related techniques commonly used to study lysosomal storage
disorders. As a result, we found that sodium-hydrogen exchanger 6 depletion
leads to abnormal accumulation of GM2 ganglioside and unesterified cholesterol
within late endosomes and lysosomes of neurons in selective brain regions, most
notably the basolateral nuclei of the amygdala, the CA3 and CA4 regions and
dentate gyrus of the hippocampus and some areas of cerebral cortex. In these
select neuronal populations, histochemical staining for β-hexosaminidase
activity, a lysosomal enzyme involved in the degradation of GM2 ganglioside, was
undetectable. Neuroaxonal dystrophy similar to that observed in lysosomal
disease was observed in the cerebellum and was accompanied by a marked and
progressive loss of Purkinje cells, particularly in those lacking the expression
of Zebrin II. On behavioural testing, Slc9a6 knockout mice displayed a discrete
clinical phenotype attributable to motor hyperactivity and cerebellar
dysfunction. Importantly, these findings show that sodium-hydrogen exchanger 6
loss of function in the Slc9a6-targeted mouse model leads to compromise of
endosomal-lysosomal function similar to lysosomal disease and to conspicuous
neuronal abnormalities in specific brain regions, which in concert could provide
a unified explanation for the cellular and clinical phenotypes in humans with
SLC9A6 mutations. Christianson syndrome (CS) is caused by mutations in SLC9A6 and is characterized
by severe intellectual disability, absent speech, microcephaly, ataxia,
seizures, and behavioral abnormalities. The clinical phenotypes of CS and
Angelman syndrome (AS) are similar. Differentiation between CS and AS is
important in terms of genetic counseling. We report on two children with CS and
confirmed mutations in SLC9A6 focusing on neuroimaging findings and review the
available literature. Cerebellar atrophy (CA) occurs in approximately 60% of the
patients with CS and develops after the age of 12 months. Hyperintense signal of
the cerebellar cortex (CbC) is less common, and may be diffuse, patchy, or
involve only the inferior part of the cerebellum and is best seen on coronal
fluid attenuation inversion recovery images. CA and CbC-hyperintensity are not
neuroimaging features of AS. In a child with the phenotype of AS, CA and/or
CbC-hyperintensity are rather specific for CS and should prioritize sequencing
of SLC9A6. Angelman syndrome (AS) is caused by a lack of expression of the maternally
inherited UBE3A gene in the brain. However, about 10% of individuals with a
clinical diagnosis of AS do not have an identifiable molecular defect. It is
likely that most of those individuals have an AS-like syndrome that is
clinically and molecularly distinct from AS. These AS-like syndromes can be
broadly classified into chromosomal microdeletion and microduplication
syndromes, and single-gene disorders. The microdeletion/microduplication
syndromes are now easily identified by chromosomal microarray analysis and
include Phelan–McDermid syndrome (chromosome 22q13.3 deletion), MBD5
haploinsufficiency syndrome (chromosome 2q23.1 deletion), and KANSL1
haploinsufficiency syndrome (chromosome 17q21.31 deletion). The single-gene
disorders include Pitt–Hopkins syndrome (TCF4), Christianson syndrome (SLC9A6),
Mowat–Wilson syndrome (ZEB2), Kleefstra syndrome (EHMT1), and Rett (MECP2)
syndrome. They also include disorders due to mutations in HERC2,
adenylosuccinase lyase (ADSL), CDKL5, FOXG1, MECP2 (duplications), MEF2C, and
ATRX. Although many of these single-gene disorders can be caused by chromosomal
microdeletions resulting in haploinsufficiency of the critical gene, the
individual disorders are often caused by intragenic mutations that cannot be
detected by chromosomal microarray analysis. We provide an overview of the
clinical features of these syndromes, comparing and contrasting them with AS, in
the hope that it will help guide clinicians in the diagnostic work-up of
individuals with AS-like syndromes. Several genetic disorders are characterized by normal head size at birth,
followed by deceleration in head growth resulting in postnatal microcephaly.
Among these are classic disorders such as Angelman syndrome and MECP2-related
disorder (formerly Rett syndrome), as well as more recently described clinical
entities associated with mutations in CASK, CDKL5, CREBBP, and EP300
(Rubinstein-Taybi syndrome), FOXG1, SLC9A6 (Christianson syndrome), and TCF4
(Pitt-Hopkins syndrome). These disorders can be identified clinically by
phenotyping across multiple neurodevelopmental and neurobehavioral realms, and
enough data are available to recognize these postnatal microcephaly disorders as
separate diagnostic entities in their own right. A second diagnostic grouping,
comprised of Warburg MICRO syndrome, Cockayne syndrome, and
Cerebral-oculo-facial skeletal syndrome, share similar features of somatic
growth failure, ophthalmologic, and dysmorphologic features. Many postnatal
microcephaly syndromes are caused by mutations in genes important in the
regulation of gene expression in the developing forebrain and hindbrain,
although important synaptic structural genes also play a role. This is an
emerging group of disorders with a fascinating combination of brain
malformations, specific epilepsies, movement disorders, and other complex
neurobehavioral abnormalities. OBJECTIVE: Recently, Christianson syndrome (CS) has been determined to be caused
by mutations in the X-linked Na(+) /H(+) exchanger 6 (NHE6). We aimed to
determine the diagnostic criteria and mutational spectrum for CS.
METHODS: Twelve independent pedigrees (14 boys, age = 4-19 years) with mutations
in NHE6 were administered standardized research assessments, and mutations were
characterized.
RESULTS: The mutational spectrum was composed of 9 single nucleotide variants, 2
indels, and 1 copy number variation deletion. All mutations were
protein-truncating or splicing mutations. We identified 2 recurrent mutations
(c.1498 c>t, p.R500X; and c.1710 g>a, p.W570X). Otherwise, all mutations were
unique. In our study, 7 of 12 mutations (58%) were de novo, in contrast to prior
literature wherein mutations were largely inherited. We also report prominent
neurological, medical, and behavioral symptoms. All CS participants were
nonverbal and had intellectual disability, epilepsy, and ataxia. Many had prior
diagnoses of autism and/or Angelman syndrome. Other neurologic symptoms included
eye movement abnormalities (79%), postnatal microcephaly (92%), and magnetic
resoce imaging evidence of cerebellar atrophy (33%). Regression was noted in
50%, with recurrent presentations involving loss of words and/or the ability to
walk. Medical symptoms, particularly gastrointestinal symptoms, were common.
Height and body mass index measures were below normal ranges in most
participants. Behavioral symptoms included hyperkinetic behavior (100%), and a
majority exhibited high pain threshold.
INTERPRETATION: This is the largest cohort of independent CS pedigrees reported.
We propose diagnostic criteria for CS. CS represents a novel neurogenetic
disorder with general relevance to autism, intellectual disability, Angelman
syndrome, epilepsy, and regression. BACKGROUND: Mutations of SLC9A6 may cause an X-linked clinical syndrome first
described by Christianson in 1999 in which affected males exhibited profound
intellectual disability, autism, drug-resistant epilepsy, ophthalmoplegia, mild
craniofacial dysmorphism, microcephaly, and ataxia.
METHODS: We describe a child with an SLC9A6 mutation and an
electroencephalographic pattern consistent with electrographic status
epilepticus of sleep.
RESULTS: Our patient's electrographic status epilepticus of sleep resolved after
treatment with felbamate. Following treatment, he remained seizure-free but did
not make significant or lasting gains in language.
CONCLUSION: Our report extends the clinical epilepsy phenotype in children with
SLC9A6 mutations to include electrographic status epilepticus of sleep. In
addition, felbamate was an effective treatment for electrographic status
epilepticus of sleep in our patient. Christianson syndrome (CS) is an X-linked neurodevelopmental and neurological
disorder characterized in males by core symptoms that include non-verbal status,
intellectual disability, epilepsy, truncal ataxia, postnatal microcephaly and
hyperkinesis. CS is caused by mutations in the SLC9A6 gene, which encodes a
multipass transmembrane sodium (potassium)-hydrogen exchanger 6 (NHE6) protein,
functional in early recycling endosomes. The extent and variability of the CS
phenotype in female heterozygotes, who presumably express the wild-type and
mutant SLC9A6 alleles mosaically as a result of X-chromosome inactivation (XCI),
have not yet been systematically characterized. Slc9a6 knockout mice (Slc9a6 KO)
were generated by insertion of the bacterial lacZ/β-galactosidase (β-Gal)
reporter into exon 6 of the X-linked gene. Mutant Slc9a6 KO male mice have been
shown to develop late endosomal/lysosomal dysfunction associated with glycolipid
accumulation in selected neuronal populations and patterned degeneration of
Purkinje cells (PCs). In heterozygous female Slc9a6 KO mice, β-Gal serves as a
transcriptional/XCI reporter and thus facilitates testing of effects of mosaic
expression of the mutant allele on penetrance of the abnormal phenotype. Using
β-Gal, we demonstrated mosaic expression of the mutant Slc9a6 allele and
mosaically distributed lysosomal glycolipid accumulation and PC pathology in the
brains of heterozygous Slc9a6 KO female mice. At the behavioral level, we showed
that heterozygous female mice suffer from visuospatial memory and motor
coordination deficits similar to but less severe than those observed in
X-chromosome hemizygous mutant males. Our studies in heterozygous Slc9a6 KO
female mice provide important clues for understanding the likely phenotypic
range of Christianson syndrome among females heterozygous for SLC9A6 mutations
and might improve diagnostic practice and genetic counseling by helping to
characterize this presumably underappreciated patient/carrier group. Christianson syndrome (OMIM 300243), caused by mutations in the X-linked SLC9A6
gene, is characterized by severe global developmental delay and intellectual
disability, developmental regression, epilepsy, microcephaly and impaired ocular
movements. It shares many common features with Angelman syndrome. Carrier
females have been described as having learning difficulties with mild to
moderate intellectual disability, behavioural issues and psychiatric illnesses.
There is little literature on the carrier female phenotype of Christianson
syndrome. We describe a large extended family with three affected males, four
carrier females, one presumed carrier female and one obligate carrier female
with a c.190G>T, p.E64X mutation known to cause a premature stop codon in
SLC9A6. We characterize and expand the clinical phenotype of female SLC9A6
mutation carriers by comparing our described family with female carriers
previously discussed in the literature. In particular, we highlight the
neurodevelopmental and psychiatric phenotypes observed in our family and
previous reports. BACKGROUND: Christianson Syndrome, a recently identified X-linked
neurodevelopmental disorder, is caused by mutations in the human gene SLC9A6
encoding the recycling endosomal alkali cation/proton exchanger NHE6. The
patients have pronounced limitations in cognitive ability, motor skills and
adaptive behaviour. However, the mechanistic basis for this disorder is poorly
understood as few of the more than 20 mutations identified thus far have been
studied in detail.
METHODS: Here, we examined the molecular and cellular consequences of a 6
base-pair deletion of amino acids Glu(287) and Ser(288) (∆ES) in the predicted
seventh transmembrane helix of human NHE6 expressed in established cell lines
(CHO/AP-1, HeLa and neuroblastoma SH-SY5Y) and primary cultures of mouse
hippocampal neurons by measuring levels of protein expression, stability,
membrane trafficking, endosomal function and cell viability.
RESULTS: In the cell lines, immunoblot analyses showed that the nascent mutant
protein was properly synthesized and assembled as a homodimer, but its
oligosaccharide maturation and half-life were markedly reduced compared to
wild-type (WT) and correlated with enhanced ubiquitination leading to both
proteasomal and lysosomal degradation. Despite this instability, a measurable
fraction of the transporter was correctly sorted to the plasma membrane.
However, the rates of clathrin-mediated endocytosis of the ∆ES mutant as well as
uptake of companion vesicular cargo, such as the ligand-bound transferrin
receptor, were significantly reduced and correlated with excessive endosomal
acidification. Notably, ectopic expression of ∆ES but not WT induced apoptosis
when examined in AP-1 cells. Similarly, in transfected primary cultures of mouse
hippocampal neurons, membrane trafficking of the ∆ES mutant was impaired and
elicited marked reductions in total dendritic length, area and arborization, and
triggered apoptotic cell death.
CONCLUSIONS: These results suggest that loss-of-function mutations in NHE6
disrupt recycling endosomal function and trafficking of cargo which ultimately
leads to neuronal degeneration and cell death in Christianson Syndrome. |
What is Eteplirsen (Exondys 51)? | Eteplirsen (Exondys 51) is an antisense oligonucleotide designed to induce exon 51 skipping that is developed by Sarepta Therapeutics. Intravenous eteplirsen has received accelerated approval from the US FDA for the treatment of Duchenne muscular dystrophy (DMD) in patients with a confirmed mutation of the DMD gene amenable to exon 51 skipping.
By the method of exon skipping in dystrophin pre-mRNA the reading frame is restored and the internally deleted but functional dystrophin is produced. | AVI-4658 is a phosphorodiamidate morpholino oligomer (PMO) designed to induce
skipping of dystrophin exon 51 and restore its expression in patients with
Duchenne muscular dystrophy (DMD). Preclinically, restoration of dystrophin in
the dystrophic mdx mouse model requires skipping of exon 23, achieved with the
mouse-specific PMO, AVI-4225. Herein, we report the potential toxicological
consequences of exon skipping and dystrophin restoration in mdx mice using
AVI-4225. We also evaluated the toxicological effects of AVI-4658 in both mdx
and wild-type mice. In both studies, animals were dosed once weekly for 12 weeks
up to the maximum feasible dose of 960 mg/kg per injection. Both AVI-4658 and
AVI-4225 were well-tolerated at all doses. Findings in AVI-4225-treated animals
were generally limited to mild renal tubular basophilia/vacuolation, without any
significant changes in renal function and with evidence of reversing. No
toxicity associated with the mechanism of action of AVI-4225 in a dystrophic
animal was observed. 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 caused mostly by internal deletions in the
gene for dystrophin, a protein essential for maintaining muscle cell membrane
integrity. These deletions abrogate the reading frame and the lack of dystrophin
results in progressive muscle deterioration. DMD patients experience progressive
loss of ambulation, followed by a need for assisted ventilation, and eventual
death in mid-twenties. By the method of exon skipping in dystrophin pre-mRNA the
reading frame is restored and the internally deleted but functional dystrophin
is produced. Two oligonucleotide drugs that induce desired exon skipping are
currently in advanced clinical trials. Eteplirsen (Exondys 51) is an antisense oligonucleotide designed to induce exon
51 skipping that is developed by Sarepta Therapeutics. Intravenous eteplirsen
has received accelerated approval from the US FDA for the treatment of Duchenne
muscular dystrophy (DMD) in patients with a confirmed mutation of the DMD gene
amenable to exon 51 skipping. Eteplirsen has orphan drug designation in the USA
and EU, and rare paediatric disease designation in the USA for use in DMD. In
the phase III PROMOVI trial, eteplirsen significantly increased dystrophin
levels from baseline in muscle tissues of 12 evaluable patients with DMD after
48 weeks of treatment. This finding is supported by data from phase II trials.
Long-term treatment with eteplirsen was associated with a decrease in the rate
of decline in ambulation and pulmonary function in an open-label extension of a
phase II trial. Eteplirsen was generally well tolerated in clinical trials. This
article summarizes the milestones in the development of eteplirsen leading to
this first approval for DMD. Exon skipping is a therapeutic approach for Duchenne muscular dystrophy (DMD)
that has been in development for close to two decades. This approach uses
antisense oligonucleotides (AONs) to modulate pre-mRNA splicing of dystrophin
transcripts to restore the disrupted DMD reading frame. The approach has moved
from in vitro proof of concept studies to the clinical trial phase and marketing
authorization applications with regulators. The first AON (eteplirsen) has
recently received accelerated approval by the Food and Drug Administration in
the US. Areas covered: In this review the authors explain the antisense-mediated
exon skipping approach, outline how it needs be tailored for different DMD
mutation types and describe the challenges and opportunities for each mutation
type. The authors summarize the clinical development of antisense-mediated exon
51 skipping, and discuss methods to improve efficiency. Finally, the authors
provide their opinion on current developments and identify topics for future
prioritization. Expert opinion: Exon skipping development has been a learning
experience for all those involved. Aside from an approved therapy, its
development has yielded side benefits including the development of tools for
clinical trials and has increased collaboration between academics, patients,
industry and regulators. |
What are the SINEUPs? | SINEUPs represent a new class of natural and synthetic antisense long non-coding RNAs that activate translation. These molecules have been named SINEUPs since their function requires the activity of an embedded inverted SINEB2 sequence to UP-regulate translation. Natural SINEUPs suggest that embedded Transposable Elements may represent functional domains in long non-coding RNAs. Synthetic SINEUPs may be designed by targeting the antisense sequence to the mRNA of choice representing the first scalable tool to increase protein synthesis of potentially any gene of interest. | Antisense (AS) transcripts are RNA molecules that are transcribed from the
opposite strand to sense (S) genes forming S/AS pairs. The most prominent
configuration is when a lncRNA is antisense to a protein coding gene. Increasing
evidences prove that antisense transcription may control sense gene expression
acting at distinct regulatory levels. However, its contribution to brain
function and neurodegenerative diseases remains unclear. We have recently
identified AS Uchl1 as an antisense to the mouse Ubiquitin carboxy-terminal
hydrolase L1 (Uchl1) gene (AS Uchl1), the synthenic locus of UCHL1/PARK5. This
is mutated in rare cases of early-onset familial Parkinson's Disease (PD) and
loss of UCHL1 activity has been reported in many neurodegenerative diseases.
Importantly, manipulation of UchL1 expression has been proposed as tool for
therapeutic intervention. AS Uchl1 induces UchL1 expression by increasing its
translation. It is the representative member of SINEUPs (SINEB2 sequence to
UP-regulate translation), a new functional class of natural antisense lncRNAs
that activate translation of their sense genes. Here we take advantage of
FANTOM5 dataset to identify the transcription start sites associated to S/AS
pair at Uchl1 locus. We show that AS Uchl1 expression is under the regulation of
Nurr1, a major transcription factor involved in dopaminergic cells'
differentiation and maintece. Furthermore, AS Uch1 RNA levels are strongly
down-regulated in neurochemical models of PD in vitro and in vivo. This work
positions AS Uchl1 RNA as a component of Nurr1-dependent gene network and target
of cellular stress extending our understanding on the role of antisense
transcription in the brain. Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and
unveiling their functions in a wide range of biological processes their
applications as biotechnological or therapeutic tools are still at their
infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the
Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1
(Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional
level. Its activity requires two RNA elements: an embedded inverted SINEB2
sequence to increase translation and the overlapping region to target its sense
mRNA. This functional organization is shared with several mouse lncRNAs
antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs
as enhancers of target mRNA translation remains unexplored. Here we define AS
Uchl1 as the representative member of a new functional class of natural and
synthetic antisense lncRNAs that activate translation. We named this class of
RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to
UP-regulate translation in a gene-specific manner. The overlapping region is
indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element
is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed
targeting mRNAs of interest. SINEUPs function in an array of cell lines and can
be efficiently directed toward N-terminally tagged proteins. Their biological
activity is retained in a miniaturized version within the range of small RNAs
length. Its modular structure was exploited to successfully design synthetic
SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to
be active in different neuronal cell lines. In summary, SINEUPs represent the
first scalable tool to increase synthesis of proteins of interest. We propose
SINEUPs as reagents for molecular biology experiments, in protein manufacturing
as well as in therapy of haploinsufficiencies. Over the past 10 years, it has emerged that pervasive transcription in mammalian
genomes has a tremendous impact on several biological functions. Most of
transcribed RNAs are lncRNAs and repetitive elements. In this review, we will
detail the discovery of a new functional class of natural and synthetic
antisense lncRNAs that stimulate translation of sense mRNAs. These molecules
have been named SINEUPs since their function requires the activity of an
embedded inverted SINEB2 sequence to UP-regulate translation. Natural SINEUPs
suggest that embedded Transposable Elements may represent functional domains in
long non-coding RNAs. Synthetic SINEUPs may be designed by targeting the
antisense sequence to the mRNA of choice representing the first scalable tool to
increase protein synthesis of potentially any gene of interest. We will discuss
potential applications of SINEUP technology in the field of molecular biology
experiments, in protein manufacturing as well as in therapy of
haploinsufficiencies. Non-coding RNAs provide additional regulatory layers to gene expression as well
as the potential to being exploited as therapeutic tools. Non-coding RNA-based
therapeutic approaches have been attempted in domit diseases, however their
use for treatment of genetic diseases caused by insufficient gene dosage is
currently more challenging. SINEUPs are long antisense non-coding RNAs that
up-regulate translation in mammalian cells in a gene-specific manner, although,
so far evidence of SINEUP efficacy has only been demonstrated in in vitro
systems. We now show that synthetic SINEUPs effectively and specifically
increase protein levels of a gene of interest in vivo. We demonstrated that
SINEUPs rescue haploinsufficient gene dosage in a medakafish model of a human
disorder leading to amelioration of the disease phenotype. Our results
demonstrate that SINEUPs act through mechanisms conserved among vertebrates and
that SINEUP technology can be successfully applied in vivo as a new research and
therapeutic tool for gene-specific up-regulation of endogenous functional
proteins. |
What is trichotillomania? | Trichotillomania is a hair pulling disorder. | Trichotillomania (hair pulling disorder, HPD) is characterized by significant
psychological distress, childhood-onset, and, in adults, certain cognitive
deficits such as inhibitory control. A total absence of such literature exists
within pediatric HPD samples, including research investigating neurocognitive
aspects of disparate pulling-styles. The present study aims to address these
gaps in the literature. Youth with HPD and healthy controls (N = 45) were
compared on an automated neurocognitive task--stop-signal task (SST)--assessing
inhibitory control. Youth with HPD (n = 17), controlling for age and attention
issues, were found to perform better on the stop-signal reaction time compared
to controls (n = 28). No significant relationships between performance on the
SST and HPD severity, distress/impairment, or pulling-styles were noted.
Findings from the current study suggest that children with HPD may not exhibit
deficits in motor inhibition as compared to controls when the effects of age and
attentional problems are controlled. Author information:
(1)Texas A&M University, Department of Psychology, 4235 TAMU, College Station,
TX 77843-4235, United States. Electronic address: [email protected].
(2)Radboud University-Nijmegen, Department of Clinical Psychology, P.O. Box
9104, NL-6500 HE Nijmegen, The Netherlands. Electronic address:
[email protected].
(3)Utah State University, Department of Psychology, 2810 Old Main Hill, Logan,
UT 84322-2810, United States. Electronic address: [email protected].
(4)Marquette University, Department of Psychology, 328E Cramer Hall, Milwaukee,
WI 53233, United States. Electronic address: [email protected].
(5)Duke University School of Medicine, Department of Psychiatry and Behavioral
Sciences, DUMC 3527, Durham, NC 27710, United States. Electronic address:
[email protected].
(6)Kent State University, Department of Psychological Sciences, 203 Kent Hall
Addition, Kent, OH 44242, United States. Electronic address: [email protected].
(7)University of Pennsylvania School of Medicine, Department of Psychiatry, 3535
Market Street, 6th Floor, Philadelphia, PA 19104, United States. Electronic
address: [email protected].
(8)Texas A&M University, Department of Psychology, 4235 TAMU, College Station,
TX 77843-4235, United States. Electronic address: [email protected]. |
What is the mechanism of action of raxibacumab? | Raxibacumab is a recombinant human IgG1 monoclonal antibody that binds the protective antigen of Bacillus anthracis, thus blocking toxin effects.
It is approved to treat inhalational anthrax. | BACKGROUND: Inhalational anthrax caused by Bacillus anthracis is associated with
high mortality primarily due to toxin-mediated injury. Raxibacumab is a human
IgG1lambda monoclonal antibody directed against protective antigen, a component
of the anthrax toxin.
METHODS: We evaluated the efficacy of raxibacumab as a prophylactic agent and
after disease onset in a total of four randomized, placebo-controlled studies
conducted in rabbits and monkeys. Animals were exposed to an aerosolized target
exposure of B. anthracis spores that was approximately 100 times (in the
prophylactic studies) and 200 times (in the therapeutic-intervention studies)
the median lethal dose. In the therapeutic-intervention studies, animals were
monitored for the onset of symptoms. Animals with detectable protective antigen
in serum, a significant increase in temperature, or both received a single
intravenous bolus of placebo or raxibacumab at a dose of either 20 mg per
kilogram of body weight or 40 mg per kilogram. The primary end point was
survival at day 14 (in rabbits) or at day 28 (in monkeys). Safety studies were
conducted with intravenous raxibacumab (40 mg per kilogram) in 333 healthy human
volunteers.
RESULTS: In both rabbits and monkeys, the time to detection of protective
antigen correlated with the time to bacteremia (r=0.9, P<0.001). In the
therapeutic-intervention studies, the survival rate was significantly higher
among rabbits that received raxibacumab at a dose of 40 mg per kilogram (44% [8
of 18]) than among rabbits that received placebo (0% [0 of 18]; P=0.003).
Raxibacumab treatment also significantly increased survival in monkeys (64% [9
of 14], vs. 0% [0 of 12] with placebo; P<0.001). In human subjects, intravenous
raxibacumab at a dose of 40 mg per kilogram had a half-life of 20 to 22 days and
provided a maximum concentration of the drug in excess of levels that are
protective in animals. Concentrations of raxibacumab provide a surrogate end
point that should be predictive of clinical benefit.
CONCLUSIONS: A single dose of raxibacumab improved survival in rabbits and
monkeys with symptomatic inhalational anthrax. (ClinicalTrials.gov number,
NCT00639678.) Raxibacumab (ABthrax) is a human IgG1 monoclonal antibody against Bacillus
anthracis protective antigen. HGS is currently providing stockpiles of the agent
to the US government for use in the prevention and treatment of inhalation
anthrax. As of May 2009, the candidate was undergoing review by the US Food and
Drug Administration. The availability of bioterrorism countermeasures has become
more important since the September 2001 anthrax attacks, and development of
raxibacumab is a significant advance in this area. IMPORTANCE OF THE FIELD: Inhalational anthrax is a disease with a high lethality
potential and current therapeutic interventions with antibiotics to manage the
bacteraemia might not always be fully effective. Blocking the activity of the
toxins with raxibacumab, a fully-human mAb directed against the protective
antigen of Bacillus anthracis, may serve as an adjunct treatment. The existing
ones are aimed at preventing post-exposure bacteraemia but might not be always
fully protective. Therefore, more specific 'therapies' are needed and the
protective antigen of B. anthracis might be targeted effectively with
raxibacumab, which is a blocking human mAb. AREAS COVERED IN THIS PAPER: To
discuss the results of experimental and human studies evaluating raxibacumab for
inhalational anthrax.
WHAT THE READER WILL GAIN: Raxibacumab was efficacious prophylactically after
exposure and therapeutically before exposure in rabbit and monkey animal models
of inhalational anthrax and exhibited good safety and pharmacokinetic profiles
in healthy humans.
TAKE HOME MESSAGE: Raxibacumab is a promising prophylactic and therapeutic for
inhalational anthrax. Inhaled Bacillus anthracis spores germinate and the subsequent vegetative growth
results in bacteremia and toxin production. Anthrax toxin is tripartite: the
lethal factor and edema factor are enzymatic moieties, while the protective
antigen (PA) binds to cell receptors and the enzymatic moieties. Antibiotics can
control B. anthracis bacteremia, whereas raxibacumab binds PA and blocks lethal
toxin effects. This study assessed plasma PA kinetics in rabbits following an
inhaled B. anthracis spore challenge. Additionally, at 84 h post-challenge, 42%
of challenged rabbits that had survived were treated with either
levofloxacin/placebo or levofloxacin/raxibacumab. The profiles were modeled
using a modified Gompertz/second exponential growth phase model in untreated
rabbits, with added monoexponential PA elimination in treated rabbits. Shorter
survival times were related to a higher plateau and a faster increase in PA
levels. PA elimination half-lives were 10 and 19 h for the levofloxacin/placebo
and levofloxacin/raxibacumab groups, respectively, with the difference
attributable to persistent circulating PA-raxibacumab complex. PA kinetics were
similar between untreated and treated rabbits, with one exception: treated
rabbits had a plateau phase nearly twice as long as that for untreated rabbits.
Treated rabbits that succumbed to disease had higher plateau PA levels and
shorter plateau duration than surviving treated rabbits. INTRODUCTION: Present-day rational drug design approaches are based on
exploiting unique features of the target biomolecules, small- or macromolecule
drug candidates and physical forces that govern their interactions. The 2013
Nobel Prize in chemistry awarded 'for the development of multiscale models for
complex chemical systems' once again demonstrated the importance of the tailored
drug discovery that reduces the role of the trial-and-error approach to a
minimum. The intentional dissemination of Bacillus anthracis spores in 2001 via
the so-called anthrax letters has led to increased efforts, politically and
scientifically, to develop medical countermeasures that will protect people from
the threat of anthrax bioterrorism.
AREAS COVERED: This article provides an overview of the recent rational drug
design approaches for discovering inhibitors of anthrax toxin. The review also
directs the readers to the vast literature on the recognized advances and future
possibilities in the field.
EXPERT OPINION: Existing options to combat anthrax toxin lethality are limited.
With the only anthrax toxin inhibiting therapy (protective antigen-targeting
with a monoclonal antibody, raxibacumab) approved to treat inhalational anthrax,
the situation, in our view, is still insecure. Further, the FDA's animal rule
for drug approval, which clears compounds without validated efficacy studies on
humans, creates a high level of uncertainty, especially when a
well-characterized animal model does not exist. Better identification and
validation of anthrax toxin therapeutic targets at the molecular level as well
as elucidation of the parameters determining the corresponding therapeutic
windows are still necessary for more effective therapeutic options. Anthrax is a highly contagious and potentially fatal human disease caused by
Bacillus anthracis, an aerobic, Gram-positive, spore-forming rod-shaped
bacterium with worldwide distribution as a zoonotic infection in herbivore
animals. Bioterrorist attacks with inhalational anthrax have prompted the
development of more effective treatments. Antibodies against anthrax toxin have
been shown to decrease mortality in animal studies. Raxibacumab is a recombit
human monoclonal antibody developed against inhalational anthrax. The drug
received approval after human studies showed its safety and animal studies
demonstrated its efficacy for treatment as well as prophylaxis against
inhalational anthrax. It works by preventing binding of the protective antigen
component of the anthrax toxin to its receptors in host cells, thereby blocking
the toxin's deleterious effects. Recently updated therapy guidelines for
Bacillus anthracis recommend the use of antitoxin treatment. Raxibacumab is the
first monoclonal antitoxin antibody made available that can be used with the
antibiotics recommended for treatment of the disease. When exposure is
suspected, raxibacumab should be given with anthrax vaccination to augment
immunity. Raxibacumab provides additional protection against inhalational
anthrax via a mechanism different from that of either antibiotics or active
immunization. In combination with currently available and recommended therapies,
raxibacumab should reduce the morbidity and mortality of inhalational anthrax. Although antibiotics treat bacteremia in inhalational anthrax, pathogenesis is
mainly driven by bacterial exotoxins. Raxibacumab, an IgG1 monoclonal antibody,
binds the protective antigen (PA) of Bacillus anthracis, thus blocking toxin
effects and leading to improved survival in the rabbit and monkey models of
inhalational anthrax. To assess raxibacumab's added benefit over levofloxacin
(LVX) alone, rabbits surviving to 84 h after a challenge with 200 times the
median (50%) lethal dose of B. anthracis spores were randomized to receive 3
daily intragastric LVX doses of 50 mg/kg of body weight, with the first LVX dose
administered just prior to administration of a single intravenous dose of
placebo or 40 mg/kg raxibacumab. The percentages of animals alive at 28 days
following the last LVX dose were compared between the 2 treatment groups using a
two-sided likelihood-ratio chi-square test. The 82% survival rate for the
LVX-raxibacumab combination was higher than the 65% survival rate for LVX alone
(P=0.0874). There were nearly 2-fold fewer deaths for the combination (7 deaths;
n=39) than for LVX alone (13 deaths; n=37), and the survival time was prolonged
for the combination (P=0.1016). Toxin-neutralizing-activity titers were similar
for both treatment groups, suggesting that survivors in both groups were able to
mount a toxin-neutralizing immune response. Microscopic findings considered
consistent with anthrax were present in animals that died or became moribund on
study in both treatment groups, and there were no anthrax-related findings in
animals that survived. Overall, raxibacumab provided a meaningful benefit over
antibiotic alone when administered late in the disease course. INTRODUCTION AND OBJECTIVE: Bacillus anthracis is one of biological agents which
may be used in bioterrorism attacks. The aim of this study a review of the new
treatment possibilities of anthrax, with particular emphasis on the treatment of
pulmonary anthrax. Abbreviated description of the state of knowledge. Pulmonary
anthrax, as the most dangerous clinical form of the disease, is also extremely
difficult to treat. Recently, considerable progress in finding new drugs and
suitable therapy for anthrax has been achieved, for example, new antibiotics
worth to mentioning, levofloxacin, daptomycin, gatifloxacin and dalbavancin.
However, alternative therapeutic options should also be considered, among them
the antimicrobial peptides, characterized by lack of inducible mechanisms of
pathogen resistance. Very promising research considers bacteriophages lytic
enzymes against selected bacteria species, including antibiotic-resistant
strains.
RESULTS: Interesting results were obtained using monoclonal antibodies:
raxibacumab, cAb29 or cocktails of antibodies. The application of CpG
oligodeoxynucleotides to boost the immune response elicited by Anthrax Vaccine
Adsorbed and CMG2 protein complexes, also produced satisfying therapy results.
Furthermore, the IFN-α and IFN-β, PA-domit negative mutant, human inter-alpha
inhibitor proteins and LF inhibitors in combination with ciprofloxacin, also
showed very promising results.
CONCLUSIONS: Recently, progress has been achieved in inhalation anthrax
treatment. The most promising new possibilities include: new antibiotics,
peptides and bacteriophages enzymes, monoclonal antibodies, antigen PA mutants,
and inter alpha inhibitors applications. In the case of the possibility of
bioterrorist attacks, the examination of inhalation anthrax treatment should be
intensively continued. Author information:
(1)Critical Care Medicine Department, Clinical Center, National Institutes of
Health, Bldg 10, Rm 2C145, Bethesda, MD, 20892, USA. [email protected].
(2)Critical Care Medicine Department, Clinical Center, National Institutes of
Health, Bldg 10, Rm 2C145, Bethesda, MD, 20892, USA. [email protected].
(3)Critical Care Medicine Department, Clinical Center, National Institutes of
Health, Bldg 10, Rm 2C145, Bethesda, MD, 20892, USA. [email protected].
(4)Critical Care Medicine Department, Clinical Center, National Institutes of
Health, Bldg 10, Rm 2C145, Bethesda, MD, 20892, USA. [email protected].
(5)Critical Care Medicine Department, Clinical Center, National Institutes of
Health, Bldg 10, Rm 2C145, Bethesda, MD, 20892, USA. [email protected].
(6)Critical Care Medicine Department, Clinical Center, National Institutes of
Health, Bldg 10, Rm 2C145, Bethesda, MD, 20892, USA. [email protected].
(7)National Heart, Lung and Blood Institute, National Institutes of Health,
Bethesda, MD, USA. [email protected].
(8)Critical Care Medicine Department, Clinical Center, National Institutes of
Health, Bldg 10, Rm 2C145, Bethesda, MD, 20892, USA. [email protected].
(9)National Institute of Allergy and Infectious Disease, National Institutes of
Health, Bethesda, MD, USA. [email protected].
(10)National Institute of Allergy and Infectious Disease, National Institutes of
Health, Bethesda, MD, USA. [email protected].
(11)Critical Care Medicine Department, Clinical Center, National Institutes of
Health, Bldg 10, Rm 2C145, Bethesda, MD, 20892, USA. [email protected]. On December 14, 2012, the FDA approved Raxibacumab, the first monoclonal
antibody product developed under Project BioShield to achieve this milestone,
and the first biologic product to be approved through the FDA animal efficacy
rule (or "Animal Rule"). Raxibacumab is approved for the treatment of adult and
pediatric patients with inhalational anthrax due to Bacillus anthracis in
combination with appropriate antibiotic drugs and for prophylaxis of
inhalational anthrax when alternative therapies are not available or not
appropriate. The developmental process required for approval of Raxibacumab
illustrates many of the challenges that product developers may encounter when
pursuing approval under the Animal Rule and highlights a number of important
regulatory and policy issues. |
What is the effect of CPEB3 binding to the CPE domain? | The cytoplasmic polyadenylation element (CPE) is the binding platform for CPE-binding protein (CPEB), which promotes polyadenylation-induced translation. | Nearly two decades ago, Xenopus oocytes were found to contain mRNAs harboring a
small sequence in their 3' untranslated regions that control cytoplasmic
polyadenylation and translational activation during development. This
cytoplasmic polyadenylation element (CPE) is the binding platform for
CPE-binding protein (CPEB), which promotes polyadenylation-induced translation.
Since then, the biochemistry and biology of CPEB has grown rather substantially:
mechanistically, CPEB nucleates a complex of factors that regulates poly(A)
elongation through, of all things, a deadenylating enzyme; biologically, CPEB
mediates many processes including germ-cell development, cell division and
cellular senescence, and synaptic plasticity and learning and memory. These
observations underscore the growing complexities of CPEB involvement in cell
function. Poly(A) tail elongation after export of an messenger RNA (mRNA) to the cytoplasm
is called cytoplasmic polyadenylation. It was first discovered in oocytes and
embryos, where it has roles in meiosis and development. In recent years,
however, has been implicated in many other processes, including synaptic
plasticity and mitosis. This review aims to introduce cytoplasmic
polyadenylation with an emphasis on the factors and elements mediating this
process for different mRNAs and in different animal species. We will discuss the
RNA sequence elements mediating cytoplasmic polyadenylation in the 3'
untranslated regions of mRNAs, including the CPE, MBE, TCS, eCPE, and C-CPE. In
addition to describing the role of general polyadenylation factors, we discuss
the specific RNA binding protein families associated with cytoplasmic
polyadenylation elements, including CPEB (CPEB1, CPEB2, CPEB3, and CPEB4),
Pumilio (PUM2), Musashi (MSI1, MSI2), zygote arrest (ZAR2), ELAV like proteins
(ELAVL1, HuR), poly(C) binding proteins (PCBP2, αCP2, hnRNP-E2), and Bicaudal C
(BICC1). Some emerging themes in cytoplasmic polyadenylation will be
highlighted. To facilitate understanding for those working in different
organisms and fields, particularly those who are analyzing high throughput data,
HUGO gene nomenclature for the human orthologs is used throughout. Where human
orthologs have not been clearly identified, reference is made to protein
families identified in man. The family of cytoplasmic polyadenylation element binding proteins CPEB1, CPEB2,
CPEB3, and CPEB4 binds to the 3'-untranslated region (3'-UTR) of mRNA, and plays
significant roles in mRNA metabolism and translation regulation. They have a
common domain organization, involving two consecutive RNA recognition motif
(RRM) domains followed by a zinc finger domain in the C-terminal region. We
solved the solution structure of the first RRM domain (RRM1) of human CPEB3,
which revealed that CPEB3 RRM1 exhibits structural features distinct from those
of the canonical RRM domain. Our structural data provide important information
about the RNA binding ability of CPEB3 RRM1. CPEB (Cytoplasmic Polyadenylation Element Binding) proteins are a family of four
RNA-binding proteins that regulate the translation of maternal mRNAs controlling
meiotic cell cycle progression. But CPEBs are not limited to the
transcriptionally silent germline; they are also expressed, in various
combinations, in somatic cells, yet their role in regulation of mitosis-related
gene expression is largely unknown. Deregulation of CPEB1 and CPEB4 have been
linked to tumor development. However, a systematic analysis addressing their
requirements for the temporal regulation of mitotic gene expression has yet to
be performed. This study addresses the requirements of each of the four CPEBs
for mitotic phase transitions, with a particular focus on cytoplasmic
polyadenylation and translational regulation. We demonstrate that CPEB3 is the
only member dispensable for mitotic cell division, whereas the other three
members, CPEB1, 2, and 4, are essential to successful mitotic cell division.
Thus, CPEB1 is required for prophase entry, CPEB2 for metaphase and CPEB4 for
cytokinesis. These three CPEBs have sequential non-redundant functions that
promote the phase-specific polyadenylation and translational activation of
CPE-regulated transcripts in the mitotic cell cycle. |
Is SUMOylation a post-translational modification in eukaryotes? | Yes, SUMOylation that is the conjugation of target proteins with SUMO (small ubiquitin-related modifier), is a type of post-translational modification in eukaryotes. | Post-translational modification is a major mechanism by which protein function
is regulated in eukaryotes. Instead of single-site action, many proteins such as
histones, p53, RNA polymerase II, tubulin, Cdc25C and tyrosine kinases are
modified at multiple sites by modifications like phosphorylation, acetylation,
methylation, ubiquitination, sumoylation and citrullination. Multisite
modification on a protein constitutes a complex regulatory program that
resembles a dynamic 'molecular barcode' and transduces molecular information to
and from signaling pathways. This program imparts effects through
'loss-of-function' and 'gain-of-function' mechanisms. Among the latter, covalent
modifications specifically recruit a diverse array of modules, including the SH2
domain, 14-3-3, WW domain, Polo box, BRCT repeat, bromodomain, chromodomain,
Tudor domain and motifs binding to ubiquitin and other protein modifiers. Such
recruitments are often modulated by modifications occurred at neighboring and
distant sites. Multisite modification thus coordinates intermolecular and
intramolecular signaling for the qualitative and quantitative control of protein
function in vivo. SUMO (Small Ubiquitin-like MOdifier) conjugation is a post-translational
modification implicated in a variety of cellular functions including
transcriptional regulation, nuclear location and signal transduction.
Sumoylation, although conserved and vital in eukaryotes, has not been studied in
malaria parasites. Here, we identify SUMO conjugation of blood stage parasites
of Plasmodium falciparum. Antibodies raised against synthetic peptides of the
plasmodial SUMO orthologue PfSUMO, a 100-amino-acid protein, reacted with
distinctive subcellular compartments of the parasitized erythrocyte during blood
stage development. Anti-PfSUMO stains the nucleus and parasite cytoplasm. We
also found antibody reactivity in the host cell cytoplasm with the
parasite-derived structures called Maurer's clefts. Anti-PfSUMO reacts in
Western blot with a number of blood stage proteins ranging from approximately
40-250 kDa. Parasites expressing FLAG-tagged PfSUMO gave similar results in
Immunofluorescence assay and Western blots. In addition, we show that
anti-PfSUMO identified PfSir2, a telomere-associated nuclear protein involved in
var gene silencing, as a target for sumoylation. Furthermore, LC-MS/MS analysis
of a two-step immunoprecipitation (IP) with anti-FLAG and anti-PfSUMO antibodies
reveals a number of putative P. falciparum sumoylated proteins. Our results
imply that SUMO conjugation has an essential function in a number of different
biological processes in P. falciparum. PML is a potent tumor suppressor and proapoptotic factor and is functionally
regulated by post-translational modifications such as phosphorylation,
sumoylation, and ubiquitination. Histone deacetylase (HDAC) inhibitors are a
promising class of targeted anticancer agents and induce apoptosis in cancer
cells by largely unknown mechanisms. We report here a novel post-transcriptional
modification, acetylation, of PML. PML exists as an acetylated protein in HeLa
cells, and its acetylation is enhanced by coexpression of p300 or treatment with
a HDAC inhibitor, trichostatin A. Increased PML acetylation is associated with
increased sumoylation of PML in vitro and in vivo. PML is involved in
trichostatin A-induced apoptosis and PML with an acetylation-defective mutation
shows an inability to mediate apoptosis, suggesting the importance of PML
acetylation. Our work provides new insights into PML regulation by
post-translational modification and new information about the therapeutic
mechanism of HDAC inhibitors. In the last decade, SUMOylation has emerged as an essential post-translational
modification in eukaryotes. In plants, the biological role of SUMO (small
ubiquitin-related modifier) has been studied through genetic approaches that
together with recent biochemical studies suggest that the plant SUMOylation
system has a high degree of complexity. The present review summarizes our
current knowledge on the SUMOylation system in Arabidopsis, focusing on the
mechanistic properties of the machinery components identified. During infection, bacterial pathogens interfere with many different
post-translational modifications of the host cell to promote their own survival
and replication. By stimulating or counteracting host post-translational
modifications, these pathogens may control locally and specifically the fate and
function of host factors critical for the infection process. Besides
phosphorylation or ubiquitylation, for which many examples of modulation by
pathogens exist, a post-translational modification called SUMOylation was
recently shown to be targeted by pathogenic bacteria. Group III metabotropic glutamate receptors (mGluRs) undergo post-translational
modification by SUMO in in vitro assays but the SUMOylation of full-length
mGluRs in mammalian cells has not been reported. Here we investigated
SUMOylation of mGluR7 in HEK293 cells and primary cortical neurons in an attempt
to confirm SUMOylation and define physiological effects on mGluR7 function.
Using a recombit bacterial expression assay we validated in vitro SUMOylation
of the C-terminal domain of mGluR7 by both SUMO-1 and SUMO-2 and show that a
single lysine residue (K889) in mGluR7 is required for SUMOylation. However,
using a range of approaches, we were unable to detect SUMOylation of full-length
mGluR7 in either heterologous cells or neurons. Further, we observed no
differences in receptor stability or surface expression between wild-type and a
non-SUMOylatable point mutant mGluR7. Thus, our results question whether mGluR7,
and by implication other group III mGluRs, are physiologically relevant neuronal
SUMO substrates. SUMOylation is a relevant protein post-translational modification in eukaryotes.
The C terminus of proteolytically activated small ubiquitin-like modifier (SUMO)
is covalently linked to a lysine residue of the target protein by an isopeptide
bond, through a mechanism that includes an E1-activating enzyme, an
E2-conjugating enzyme, and transfer to the target, sometimes with the assistance
of a ligase. The modification is reversed by a protease, also responsible for
SUMO maturation. A number of proteins have been identified as SUMO targets,
participating in the regulation of cell cycle progression, transcription,
translation, ubiquitination, and DNA repair. In this study, we report that
orthologous genes corresponding to the SUMOylation pathway are present in the
etiological agent of Chagas disease, Trypanosoma cruzi. Furthermore, the
SUMOylation system is functionally active in this protozoan parasite, having the
requirements for SUMO maturation and conjugation. Immunofluorescence analysis
showed that T. cruzi SUMO (TcSUMO) is predomitly found in the nucleus. To
identify SUMOylation targets and get an insight into their physiological roles
we generated transfectant T. cruzi epimastigote lines expressing a double-tagged
T. cruzi SUMO, and SUMOylated proteins were enriched by tandem affinity
chromatography. By two-dimensional liquid chromatography-mass spectrometry a
total of 236 proteins with diverse biological functions were identified as
potential T. cruzi SUMO targets. Of these, metacaspase-3 was biochemically
validated as a bona fide SUMOylation substrate. Proteomic studies in other
organisms have reported that orthologs of putative T. cruzi SUMOylated proteins
are similarly modified, indicating conserved functions for protein SUMOylation
in this early divergent eukaryote. Förster resoce energy transfer (FRET) is a powerful tool in biological
research and has been widely used in the study of biomolecular interactions.
SUMOylation is an important post-translational modification that is involved in
many key biological processes. As a multi-step cascade reaction, SUMOylation
involves multiple enzymes and protein-protein interactions. Here, we report the
development of an in vitro FRET-based high-throughput screening (HTS) assay in
SUMOylation. This assay is based on steady state and high efficiency of the
fluorescent energy transfer between CyPet and YPet fused to SUMO1 and Ubc9,
respectively. We optimized the assay and performed a small-scale pilot study to
validate the screening platform. Carried out in 384-well plate format, our
FRET-based HTS provides a powerful tool for large-scale and high-throughput
applications. BACKGROUND: Small Ubiquitin-like MOdifier protein (SUMO) is a key regulator of
nuclear functions but little is known regarding the role of the
post-translational modification sumoylation outside of the nucleus, particularly
in the Central Nervous System (CNS).
METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that the expression levels of
SUMO-modified substrates as well as the components of the sumoylation machinery
are temporally and spatially regulated in the developing rat brain.
Interestingly, while the overall sumoylation is decreasing during brain
development, there are progressively more SUMO substrates localized at synapses.
This increase is correlated with a differential redistribution of the
sumoylation machinery into dendritic spines during neuronal maturation.
CONCLUSIONS/SIGNIFICANCE: Overall, our data clearly demonstrate that the
sumoylation process is developmentally regulated in the brain with high levels
of nuclear sumoylation early in the development suggesting a role for this
post-translational modification during the synaptogenesis period and a
redistribution of the SUMO system towards dendritic spines at a later
developmental stage to modulate synaptic protein function. Bicoid (Bcd) is a Drosophila morphogenetic protein and a transcriptional
activator. Genetic studies have suggested a role of sumoylation in Bcd function,
but it is unknown how Bcd activity is affected specifically by its own
sumoylation status. Here we show that Bcd is sumoylated in Drosophila cells. We
identify a lysine residue of Bcd as the primary sumoylation site. Using a Bcd
mutant defective in being sumoylated, we show that sumoylation of Bcd is
inhibitory to its ability to activate transcription. We provide evidence
suggesting that the SUMO moiety has an intrinsic inhibitory activity for the
activator function of Bcd. Small ubiquitin-like modifier (SUMO) is a small (∼12kDa) protein that occurs in
all eukaryotes and participates in the reversible posttranslational modification
of target cellular proteins. The three-dimensional structure of SUMO and
ubiquitin (Ub) are superimposable although there is very little similarity in
their primary amino acid sequences. In all organisms, conjugation and
deconjugation of Ub and SUMO proceed by the same reactions while using
pathway-specific enzymes. SUMO conjugation in plants is a part of the controls
governing important biological processes such as growth, development, flowering,
environmental (abiotic) stress responses, and response to pathogen infection.
Most of the evidence for this comes from genetic analyses. Recent efforts to
dissect the function of sumoylation have focused on uncovering targets of SUMO
conjugation by using either a yeast two-hybrid screen employing components of
the SUMO cycle as bait or by using affinity purification of SUMO-conjugated
proteins followed by identification of these proteins by mass spectrometry. This
chapter reviews the current knowledge regarding sumoylation in plants, with
special focus on the model plant Arabidopsis thaliana. Using in silico analysis a number of potential sites for post-translational
modifications has been revealed within the human O6-methylguanine-DNA
methyltransferase (MGMT) protein. In particular these were the acetylation of
Gly3 residue in the N-terminus of protein and internal residues Lys132 and
Lys135; Arg166 residue methylation; Lys63 SUMOylation and ubiquitination of
Lys31, Lys39, Lys49, Lys63, Lys67, Lys135, Lys156, Lys196, Lys209. Also it has
been predicted 16 novel potential phosphorylation sites of serine residues
(positions 13, 124, 144, 182, 183, 190, 215, 216 and 230), tyrosine residues
(positions 100 and 189) and threonine residues (positions 23, 69, 94, 126 and
229), as well as five binding sites for kinases and other proteins (Serl3 with
14-3-3, Val21 and Ile172 with D-domain, Pro78 and Pro111 with SH3-domain, Pro111
with MAPK3). Some kinases predicted by the authors are known as partners of the
MGMT protein, that confirms the probability of modification of the given sites.
Potential sites require further experimental confirmation of modifications and
investigation of their influence on stability and DNA-repair activity of this
protein. The ubiquitin-proteasome system (UPS) is the major intracellular degradation
system, and its proper function is critical to the health and function of
cardiac cells. Alterations in cardiac proteasomes have been linked to several
pathological phenotypes, including cardiomyopathies, ischemia-reperfusion
injury, heart failure, and hypertrophy. Defects in proteasome-dependent cellular
protein homeostasis can be causal for the initiation and progression of certain
cardiovascular diseases. Emerging evidence suggests that the UPS can
specifically target proteins that govern pathological signaling pathways for
degradation, thus altering downstream effectors and disease outcomes.
Alterations in UPS-substrate interactions in disease occur, in part, due to
direct modifications of 19S, 11S or 20S proteasome subunits. Post-translational
modifications (PTMs) are one facet of this proteasomal regulation, with over 400
known phosphorylation sites, over 500 ubiquitination sites and 83 internal
lysine acetylation sites, as well as multiple sites for caspase cleavage,
glycosylation (such as O-GlcNAc modification), methylation, nitrosylation,
oxidation, and SUMOylation. Changes in cardiac proteasome PTMs, which occur in
ischemia and cardiomyopathies, are associated with changes in proteasome
activity and proteasome assembly; however several features of this regulation
remain to be explored. In this review, we focus on how some of the less common
PTMs affect proteasome function and alter cellular protein homeostasis. This
article is part of a Special Issue entitled "Protein Quality Control, the
Ubiquitin Proteasome System, and Autophagy". Post-translational modification by SUMO is a highly conserved pathway in
eukaryotes that plays very important regulatory roles in many cellular
processes. Deregulation of the SUMO pathway contributes to the development and
progression of many diseases including cancer. Therefore, identifying additional
SUMO substrates and studying how their cellular and biological functions are
regulated by sumoylation should provide new insights. Our studies showed that
sumoylation activity was significant in Xenopus egg extracts, and that a high
level of sumoylation was associated with sperm chromatin when SUMO was incubated
with Xenopus egg extracts. By isolating SUMO-conjugated substrates using
His-tagged SUMO1 or SUMO2 proteins under denaturing conditions, we identified
346 proteins by mass spectrometry analysis that were not present in control
pull-downs. Among them, 167 proteins were identified from interphase egg
extracts, 86 proteins from mitotic phase egg extracts, and 93 proteins from
both. Thirty-three proteins were pulled down by SUMO1, 85 proteins by SUMO2, and
228 proteins by both. We validated the sumoylation of five candidates, CKB,
ATXN10, BTF3, HABP4, and BZW1, by co-transfecting them along with SUMO in
HEK293T cells. Gene ontology analysis showed that SUMO substrates identified in
this study were involved in diverse biological processes. Additionally, SUMO
substrates identified from different cell cycle stages or pulled down by
different SUMO homologs were enriched for distinct cellular components and
functional categories. Our results comprehensively profile the sumoylation
occurring in the Xenopus egg extract system. Post-translational modifications are able to regulate protein function and
cellular processes in a rapid and reversible way. SUMOylation, the
post-translational modification of proteins by the addition of SUMO, is a highly
conserved process that seems to be present in modern cells. However, the
mechanism of protein SUMOylation in earlier divergent eukaryotes, such as
Giardia lamblia, is only starting to become apparent. In this work, we report
the presence of a single SUMO gene encoding to SUMO protein in Giardia.
Monoclonal antibodies against recombit Giardia SUMO protein revealed the
cytoplasmic localization of native SUMO in wild-type trophozoites. Moreover, the
over-expression of SUMO protein showed a mainly cytoplasmic localization, though
also neighboring the plasma membrane, flagella, and around and even inside the
nuclei. Western blot assays revealed a number of SUMOylated proteins in a range
between 20 and 120 kDa. The genes corresponding to putative enzymes involved in
the SUMOylation pathway were also explored. Our results as a whole suggest that
SUMOylation is a process conserved in the eukaryotic lineage, and that its study
is significant for understanding the biology of this interesting parasite and
the role of post-translational modification in its evolution. SUMOylation is a reversible post-translational modification essential for genome
stability. Using high-resolution MS, we have studied global SUMOylation in human
cells in a site-specific manner, identifying a total of >4,300 SUMOylation sites
in >1,600 proteins. To our knowledge, this is the first time that >1,000
SUMOylation sites have been identified under standard growth conditions. We
quantitatively studied SUMOylation dynamics in response to SUMO protease
inhibition, proteasome inhibition and heat shock. Many SUMOylated lysines have
previously been reported to be ubiquitinated, acetylated or methylated, thus
indicating cross-talk between SUMO and other post-translational modifications.
We identified 70 phosphorylation and four acetylation events in proximity to
SUMOylation sites, and we provide evidence for acetylation-dependent SUMOylation
of endogenous histone H3. SUMOylation regulates target proteins involved in all
nuclear processes including transcription, DNA repair, chromatin remodeling,
precursor-mRNA splicing and ribosome assembly. SUMOylation, the covalent attachment of a member of the small ubiquitin-like
modifier (SUMO) family of proteins to lysines in target substrates, is an
essential post-translational modification in eukaryotes. Microbial manipulation
of SUMOylation recently emerged as a key virulence strategy for viruses and
facultative intracellular bacteria, the latter of which have only been shown to
deploy effectors that negatively regulate SUMOylation. Here, we demonstrate that
the obligate intracellular bacterium, Anaplasma phagocytophilum, utilizes an
effector, AmpA (A. phagocytophilum post-translationally modified protein A) that
becomes SUMOylated in host cells and this is important for the pathogen's
survival. We previously discovered that AmpA (formerly APH1387) localizes to the
A. phagocytophilum-occupied vacuolar membrane (AVM). Algorithmic prediction
analyses denoted AmpA as a candidate for SUMOylation. We verified this
phenomenon using a SUMO affinity matrix to precipitate both native AmpA and
ectopically expressed green fluorescent protein (GFP)-tagged AmpA. SUMOylation
of AmpA was lysine dependent, as SUMO affinity beads failed to precipitate a
GFP-AmpA protein when its lysine residues were substituted with arginine.
Ectopically expressed and endogenous AmpA were poly-SUMOylated, which was
consistent with the observation that AmpA colocalizes with SUMO2/3 at the AVM.
Only late during the infection cycle did AmpA colocalize with SUMO1, which
terminally caps poly-SUMO2/3 chains. AmpA was also detected in the cytosol of
infected host cells, further supporting its secretion and likely participation
in interactions that aid pathogen survival. Indeed, whereas siRNA-mediated
knockdown of Ubc9 - a necessary enzyme for SUMOylation - slightly bolstered
A. phagocytophilum infection, pharmacologically inhibiting SUMOylation in
infected cells significantly reduced the bacterial load. Ectopically expressed
GFP-AmpA served as a competitive agonist against native AmpA in infected cells,
while lysine-deficient GFP-AmpA was less effective, implying that modification
of AmpA lysines is important for infection. Collectively, these data show that
AmpA becomes directly SUMOylated during infection, representing a novel tactic
for A. phagocytophilum survival. Nucleolin is a ubiquitously expressed protein and participates in many important
biological processes, such as cell cycle regulation and ribosomal biogenesis.
The activity of nucleolin is regulated by intracellular localization and
post-translational modifications, including phosphorylation, methylation, and
ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of recently
verified forms of post-translational modifications and exerts various effects on
the target proteins. In the studies reported here, we discovered SUMOylational
modification of human nucleolin protein at Lys-294, which facilitated the mRNA
binding property of nucleolin by maintaining its nuclear localization. In
response to arsenic exposure, nucleolin-SUMO was induced and promoted its
binding with gadd45α mRNA, which increased gadd45α mRNA stability and protein
expression, subsequently causing GADD45α-mediated cell death. On the other hand,
ectopic expression of Mn-SOD attenuated the arsenite-generated superoxide
radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced
apoptosis by reducing GADD45α expression. Collectively, our results for the
first time demonstrate that nucleolin-SUMO at K294R plays a critical role in its
nucleus sequestration and gadd45α mRNA binding activity. This novel biological
function of nucleolin is distinct from its conventional role as a
proto-oncogene. Therefore, our findings here not only reveal a new modification
of nucleolin protein and its novel functional paradigm in mRNA metabolism but
also expand our understanding of the dichotomous roles of nucleolin in terms of
cancer development, which are dependent on multiple intracellular conditions and
consequently the appropriate regulations of its modifications, including
SUMOylation. Sumoylation is a post-translational modification essential in most eukaryotes
that regulates stability, localization, activity, or interaction of a multitude
of proteins. It is a reversible process wherein counteracting ligases and
proteases, respectively, mediate the conjugation and deconjugation of SUMO
molecules to/from target proteins. Apart from attachment of single SUMO moieties
to targets, formation of poly-SUMO chains occurs by the attachment of additional
SUMO molecules to lysine residues in the N-terminal extensions of SUMO. In
Saccharomyces cerevisiae there are apparently only two SUMO(Smt3)-specific
proteases: Ulp1 and Ulp2. Ulp2 has been shown to be important for the control of
poly-SUMO conjugates in cells and to dismantle SUMO chains in vitro, but the
mechanism by which it acts remains to be elucidated. Applying an in vitro
approach, we found that Ulp2 acts sequentially rather than stochastically,
processing substrate-linked poly-SUMO chains from their distal ends down to two
linked SUMO moieties. Furthermore, three linked SUMO units turned out to be the
minimum length of a substrate-linked chain required for efficient binding to and
processing by Ulp2. Our data suggest that Ulp2 disassembles SUMO chains by
removing one SUMO moiety at a time from their ends (exo mechanism). Apparently,
Ulp2 recognizes surfaces at or near the N terminus of the distal SUMO moiety, as
attachments to this end significantly reduce cleavage efficiency. Our studies
suggest that Ulp2 controls the dynamic range of SUMO chain lengths by trimming
them from the distal ends. SUMOylation is an important post-translational modification, and Akt SUMOylation
was found to regulate cell proliferation, tumorigenesis and cell cycle, but the
molecular mechanism of Akt SUMOylation is less well known. Here, we show both
endogenous and ectopic Akt SUMOylation and Lys276 is the major SUMO acceptor on
Akt. Further, Akt SUMOylation is Akt phosphorylation dependent and Akt
SUMOylation increases Akt kinase activity without affecting the phosphorylation
level of Akt. Moreover, endogenous Akt SUMOylation is enhanced by insulin
treatment and this is Akt activity dependent. Heat-shock stimulus also increases
Akt SUMOylation and it is also Akt activity dependent. Endogenous Akt
SUMOylation is also found in the rat brain and it is enhanced by insulin-like
growth factor-1 stimulation. In addition, Akt directly phosphorylates Ubc9 at
Thr35 and phosphorylates SUMO1 at Thr76. Ubc9 phosphorylation at Thr35 promotes
Ubc9 thioester bond formation and SUMO1 phosphorylation at Thr76 stabilizes the
SUMO1 protein. Through these distinct mechanisms, Akt SUMOylation regulates
global SUMOylation, including Akt and Ubc9 SUMOylation, and substrate
SUMOylation specificity, including STAT1 and CREB SUMOylation, in different
manners. Akt SUMOylation also enhances phosphatase and tensin homolog (PTEN)
SUMOylation through Akt phosphorylation of Ubc9 and SUMO1, which serves as an
endogenous mechanism to stop the positive feedback loop resulted from Akt
activation. Further, Akt SUMOylation increases cyclin D1 expression and cell
proliferation, and these effects are also mediated through Ubc9 phosphorylation
at Thr35 and SUMO1 phosphorylation at Thr76. Here, we have identified a novel
mechanism for SUMOylation regulation. Because of the important role Akt plays in
tumorigenesis, this mechanism may also be involved in Akt-regulated
tumorigenesis. Small ubiquitin-like modifier (SUMO), a reversible post-translational protein
modifier, plays important roles in diverse cellular mechanisms. Three enzymes,
E1 (activating enzyme), E2 (conjugating enzyme) and E3 (ligase), are involved in
SUMO modification. SUMOylation system and process in higher eukaryotes have been
well studied. However, in protozoa, such as Trypanosoma brucei (T. brucei),
these remain poorly understood. Herein, we identified the E1 (TbAos1/TbUba2) and
E2 (TbUbc9) enzymes of SUMOylation pathway in T. brucei by sequence analysis and
GST pull-down assay. Furthermore, we successfully reconstructed the SUMOylation
system in vitro with recombit enzymes. Using this system, the active site of
TbUba2 and TbUbc9 was revealed to be located at Cys343 and Cys132, respectively,
and a centrin homologue (TbCentrin3) was identified to be a target of
SUMOylation in T. brucei. Altogether, our results demonstrate that TbAos1/TbUba2
and TbUbc9 are the bona fide E1 and E2 enzymes of the SUMOylation system in T.
brucei. Akt/PKB, a serine/threonine kinase member of the AGC family of proteins, is
involved in the regulation of a plethora of cellular processes triggered by a
wide diversity of extracellular signals and is thus considered a key signalling
molecule in higher eukaryotes. Deregulation of Akt signalling is associated with
a variety of human diseases, revealing Akt-dependent pathways as an attractive
target for therapeutic intervention. Since its discovery in the early 1990s, a
large body of work has focused on Akt phosphorylation of two residues, Thr308
and Ser473, and modification of these two sites has been established as being
equivalent to Akt activation. More recently, Akt has been identified as a
substrate for many different post-translational modifications, including not
only phosphorylation of other residues, but also acetylation, glycosylation,
oxidation, ubiquitination and SUMOylation. These modifications could provide
additional regulatory steps for fine-tuning Akt function, Akt trafficking within
the cell and/or for determining the substrate specificity of this signalling
molecule. In the present review, we provide an overview of these different
post-translational modifications identified for Akt, focusing on their
consequences for this kinase activity. MOTIVATION: Post-translational modification by the Small Ubiquitin-like Modifier
(SUMO) proteins, a process termed SUMOylation, is involved in many fundamental
cellular processes. SUMO proteins are conjugated to a protein substrate,
creating an interface for the recruitment of cofactors harboring
SUMO-interacting motifs (SIMs). Mapping both SUMO-conjugation sites and SIMs is
required to study the functional consequence of SUMOylation. To define the best
candidate sites for experimental validation we designed JASSA, a Joint Analyzer
of SUMOylation site and SIMs.
RESULTS: JASSA is a predictor that uses a scoring system based on a Position
Frequency Matrix derived from the alignment of experimental SUMOylation sites or
SIMs. Compared with existing web-tools, JASSA displays on par or better
performances. Novel features were implemented towards a better evaluation of the
prediction, including identification of database hits matching the query
sequence and representation of candidate sites within the secondary structural
elements and/or the 3D fold of the protein of interest, retrievable from
deposited PDB files.
AVAILABILITY AND IMPLEMENTATION: JASSA is freely accessible at
http://www.jassa.fr/. Website is implemented in PHP and MySQL, with all major
browsers supported.
CONTACT: [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. BACKGROUND: Cells have developed many ways to cope with external stress. One
distinctive feature in acute proteotoxic stresses, such as heat shock (HS), is
rapid post-translational modification of proteins by SUMOs (small ubiquitin-like
modifier proteins; SUMOylation). While many of the SUMO targets are chromatin
proteins, there is scarce information on chromatin binding of SUMOylated
proteins in HS and the role of chromatin SUMOylation in the regulation of
transcription.
RESULTS: We mapped HS-induced genome-wide changes in chromatin occupancy of
SUMO-2/3-modified proteins in K562 and VCaP cells using ChIP-seq. Chromatin
SUMOylation was further correlated with HS-induced global changes in
transcription using GRO-seq and RNA polymerase II (Pol2) ChIP-seq along with
ENCODE data for K562 cells. HS induced a rapid and massive rearrangement of
chromatin SUMOylation pattern: SUMOylation was gained at active promoters and
enhancers associated with multiple transcription factors, including heat shock
factor 1. Concomitant loss of SUMOylation occurred at inactive intergenic
chromatin regions that were associated with CTCF-cohesin complex and SETDB1
methyltransferase complex. In addition, HS triggered a dynamic chromatin binding
of SUMO ligase PIAS1, especially onto promoters. The HS-induced SUMOylation on
chromatin was most notable at promoters of transcribed genes where it positively
correlated with active transcription and Pol2 promoter-proximal pausing.
Furthermore, silencing of SUMOylation machinery either by depletion of UBC9 or
PIAS1 enhanced expression of HS-induced genes.
CONCLUSIONS: HS-triggered SUMOylation targets promoters and enhancers of
actively transcribed genes where it restricts the transcriptional activity of
the HS-induced genes. PIAS1-mediated promoter SUMOylation is likely to regulate
Pol2-associated factors in HS. Plants have evolved to cope with changing environmental conditions. One way
plants achieve this is through post-translational modification of target
proteins by ubiquitination and SUMOylation. These post-translational modifiers
(PMs) can alter stability, protein-protein interactions, and the overall fate of
the protein. Both of these systems have remarkable similarities in terms of the
process leading to attachment of the PM to its substrate : having to undertake
activation, conjugation, and finally ligation to the target. In the ubiquitin
system, there are a vast number of ubiquitin ligase enzymes (E3s) that provide
specificity for the attachment of ubiquitin. With the SUMO system, only a small
number of SUMO E3 ligases have so far been identified in the fully sequenced
plant genomes. In Arabidopsis thaliana, there are only two SUMO E3s, compared to
over 1400 ubiquitin E3s, a trend also observed in crop species such as Oryza
sativa and Zea mays Recent research indicates that removing SUMO from its
substrate by the enzymatically active SUMO proteases is a vital part of this
system. A class of SUMO proteases called ubiquitin-like proteases (ULPs) are
widespread in all eukaryotes; within plants, both monocot and dicot kingdoms
have conserved and divergent ULPs and ULP-like proteases. This paper examines
the roles ULPs have in stress responses and highlights the 'fine-tuning' of SUMO
attachment/removal in balancing growth versus stress. Author information:
(1)State Key Laboratory of Crop Biology, Shandong Agricultural University,
Tai-An, Shandong, China; MOA Key Laboratory of Horticultural Crop Biology and
Germplasm Innovation, Shandong Agricultural University, Tai-An, Shandong, China;
College of Horticulture Science and Engineering, Shandong Agricultural
University, Tai-An, Shandong, China.
(2)State Key Laboratory of Crop Biology, Shandong Agricultural University,
Tai-An, Shandong, China; MOA Key Laboratory of Horticultural Crop Biology and
Germplasm Innovation, Shandong Agricultural University, Tai-An, Shandong, China;
College of Horticulture Science and Engineering, Shandong Agricultural
University, Tai-An, Shandong, China. Electronic address: [email protected].
(3)State Key Laboratory of Crop Biology, Shandong Agricultural University,
Tai-An, Shandong, China; MOA Key Laboratory of Horticultural Crop Biology and
Germplasm Innovation, Shandong Agricultural University, Tai-An, Shandong, China;
College of Horticulture Science and Engineering, Shandong Agricultural
University, Tai-An, Shandong, China. Electronic address: [email protected]. SUMOylation constitutes a major post-translational modification (PTM) used by
the eukaryote cellular machinery to modulate protein interactions of the
targeted proteins. The small ubiquitin-like modifier-1 (SUMO-1) features a
central and conserved cysteine residue (Cys52) that is located in the
hydrophobic core of the protein and in tight contact with Phe65, suggesting the
occurrence of an S/π interaction. To investigate the importance of Cys52 on
SUMO-1 thermal stability and biochemical properties, we produced by total
chemical synthesis SUMO-1 or SUMO-1 Cys52Ala peptide-protein conjugates
featuring a native isopeptidic bond between SUMO-1 and a peptide derived from
p53 tumor suppressor protein. The Cys52Ala modification perturbed SUMO-1
secondary structure and resulted in a dramatic loss of protein thermal
stability. Moreover, the cleavage of the isopeptidic bond by the deconjugating
enzyme Upl1 was significantly less efficient than for the wild-type conjugate.
Similarly, the in vitro SUMOylation of RanGap1 by E1/E2 conjugating enzymes was
significantly less efficient with the SUMO-1 C52A analog compared to wild-type
SUMO-1. These data demonstrate the critical role of Cys52 in maintaining SUMO-1
conformation and function and the importance of keeping this cysteine intact for
the study of SUMO-1 protein conjugates. |
What is the Shelterin complex? | Human telomeres are associated with the shelterin complex which consists of six telomere-associated proteins that specifically bind to telomeric DNA. Alterations or removal of individual shelterin components would lead to telomere uncapping and telomere dysfunction, resulting in cellular senescence and transformation to a malignant state. | Telomeres interact with numerous proteins, including components of the shelterin
complex, whose alteration, similarly to proliferation-induced telomere
shortening, initiates cellular senescence. In tumors, telomere length is
maintained by Telomerase activity or by the Alternative Lengthening of Telomeres
mechanism, whose hallmark is the telomeric localization of the promyelocytic
leukemia (PML) protein. Whether PML contributes to telomeres maintece in
normal cells is unknown. We show that in normal human fibroblasts the PML
protein associates with few telomeres, preferentially when they are damaged.
Proliferation-induced telomere attrition or their damage due to alteration of
the shelterin complex enhances the telomeric localization of PML, which is
increased in human T-lymphocytes derived from patients genetically deficient in
telomerase. In normal fibroblasts, PML depletion induces telomere damage,
nuclear and chromosomal abnormalities, and senescence. Expression of the
leukemia protein PML/RARα in hematopoietic progenitors displaces PML from
telomeres and induces telomere shortening in the bone marrow of pre-leukemic
mice. Our work provides a novel view of the physiologic function of PML, which
participates in telomeres surveillance in normal cells. Our data further imply
that a diminished PML function may contribute to cell senescence, genomic
instability, and tumorigenesis. Author information:
(1)Department of Biochemistry, Biophysics and Structural Biology, Institute for
Integrative Biology of the Cell (I2BC), CEA, UMR 9198 CNRS, Université
Paris-Sud, Batiment 144, CEA Saclay, Gif-sur-Yvette, F-91191, France.
(2)Institute for Research on Cancer and Aging, Nice (IRCAN); CNRS UMR7284/INSERM
U1081; Faculty of Medicine; Nice, 06107, France.
(3)Synchrotron SOLEIL, L'Orme des Merisiers, Saint-Aubin BP 48, 91192
GIF-SUR-YVETTE Cedex, France Institut National de la Recherche Agronomique,
Unité Biopolymères, Interactions, Assemblages, 44316 Nantes, France.
(4)Synchrotron SOLEIL, L'Orme des Merisiers, Saint-Aubin BP 48, 91192
GIF-SUR-YVETTE Cedex, France.
(5)CEA, iBiTecS, F-91191 Gif-sur-Yvette, France.
(6)Institute for Research on Cancer and Aging, Nice (IRCAN); CNRS UMR7284/INSERM
U1081; Faculty of Medicine; Nice, 06107, France Department of Genetics, CHU;
Nice, 06107, France.
(7)Department of Biochemistry, Biophysics and Structural Biology, Institute for
Integrative Biology of the Cell (I2BC), CEA, UMR 9198 CNRS, Université
Paris-Sud, Batiment 144, CEA Saclay, Gif-sur-Yvette, F-91191, France
[email protected]. Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the
DNA damage response (DDR) by the shelterin complex. To understand how shelterin
protects telomere ends, we investigated the structural organization of telomeric
chromatin in human cells using super-resolution microscopy. We found that
telomeres form compact globular structures through a complex network of
interactions between shelterin subunits and telomeric DNA, but not by DNA
methylation, histone deacetylation, or histone trimethylation at telomeres and
subtelomeric regions. Mutations that abrogate shelterin assembly or removal of
individual subunits from telomeres cause up to a 10-fold increase in telomere
volume. Decompacted telomeres accumulate DDR signals and become more accessible
to telomere-associated proteins. Recompaction of telomeric chromatin using an
orthogonal method displaces DDR signals from telomeres. These results reveal the
chromatin remodeling activity of shelterin and demonstrate that
shelterin-mediated compaction of telomeric chromatin provides robust protection
of chromosome ends against the DDR machinery. DNA ends get exposed in cells upon either normal or dysfunctional cellular
processes or molecular events. Telomeres need to be protected by the shelterin
complex to avoid junctions occurring between chromosomes while failing
topoisomerases or clustered DNA damage processing may produce double-strand
breaks, thus requiring swift repair to avoid cell death. The rigorous study of
the great many proteins involved in the maintece of DNA integrity is a
challenging task because of the innumerous unspecific electrostatic and/or
hydrophobic DNA-protein interactions that arise due to the chemical nature of
DNA. We devised a technique that discriminates the proteins recruited
specifically at DNA ends from those that bind to DNA because of a generic
affinity for the double helix. Our study shows that the DNA ends proteome
comprises proteins of an unexpectedly wide functional spectrum, ranging from DNA
repair to ribosome biogenesis and cytoskeleton, including novel proteins of
undocumented function. A global mapping of the identified proteome on published
DNA repair protein networks demonstrated the excellent specificity and
functional coverage of our purification technique. Finally, the native
nucleoproteic complexes that assembled specifically onto DNA ends were shown to
be endowed with a highly efficient DNA repair activity. Mammalian chromosomes terminate in stretches of repetitive telomeric DNA that
act as buffers to avoid loss of essential genetic information during
end-replication. A multiprotein complex known as shelterin prevents recognition
of telomeric sequences as sites of DNA damage. Telomere erosion contributes to
human diseases ranging from BM failure to premature aging syndromes and cancer.
The role of shelterin telomere protection is less understood. Mutations in genes
encoding the shelterin proteins TRF1-interacting nuclear factor 2 (TIN2) and
adrenocortical dysplasia homolog (ACD) were identified in dyskeratosis
congenita, a syndrome characterized by somatic stem cell dysfunction in multiple
organs leading to BM failure and other pleiotropic manifestations. Here, we
introduce the biochemical features and in vivo effects of individual shelterin
proteins, discuss shelterin functions in hematopoiesis, and review emerging
knowledge implicating the shelterin complex in hematological disorders. Telomeres are highly regulated and dynamic complexes that protect the genomic
DNA and prevent the end of linear chromosomes from being misrecognized as a
broken DNA. Due to the end replication problem, telomeres of somatic cells
shorten with each cell division, inducing cell senescence. Telomerase is a
reverse transcriptase capable of compensating telomere attrition by adding
telomere repeats to the ends of chromosomes. Human telomeres are associated with
the shelterin complex which consists of six telomere-associated proteins that
specifically bind to telomeric DNA. Alterations or removal of individual
shelterin components would lead to telomere uncapping and telomere dysfunction,
resulting in cellular senescence and transformation to a maligt state.
Another complex of multifunctional proteins, named non-shelterin complex, is
thought to prevent telomere degradation and facilitate telomerase-based telomere
elongation. As telomerase is highly expressed in most human tumor cells, it is
considered an attractive target for new therapeutic strategies. In this review,
we will summarize the characteristics of telomeres and telomerase in lymphoid
maligcies and discuss the role of telomere-associated proteins in these
entities. |
What disease is the drug aducanumab targeting? | Aducanumab is an anti-Aβ antibody being developed for the treatment of Alzheimer's disease (AD). | Despite continuing debate about the amyloid β-protein (or Aβ hypothesis, new
lines of evidence from laboratories and clinics worldwide support the concept
that an imbalance between production and clearance of Aβ42 and related Aβ
peptides is a very early, often initiating factor in Alzheimer's disease (AD).
Confirmation that presenilin is the catalytic site of γ-secretase has provided a
linchpin: all domit mutations causing early-onset AD occur either in the
substrate (amyloid precursor protein, APP) or the protease (presenilin) of the
reaction that generates Aβ. Duplication of the wild-type APP gene in Down's
syndrome leads to Aβ deposits in the teens, followed by microgliosis,
astrocytosis, and neurofibrillary tangles typical of AD Apolipoprotein E4, which
predisposes to AD in > 40% of cases, has been found to impair Aβ clearance from
the brain. Soluble oligomers of Aβ42 isolated from AD patients' brains can
decrease synapse number, inhibit long-term potentiation, and enhance long-term
synaptic depression in rodent hippocampus, and injecting them into healthy rats
impairs memory. The human oligomers also induce hyperphosphorylation of tau at
AD-relevant epitopes and cause neuritic dystrophy in cultured neurons. Crossing
human APP with human tau transgenic mice enhances tau-positive neurotoxicity. In
humans, new studies show that low cerebrospinal fluid (CSF) Aβ42 and amyloid-PET
positivity precede other AD manifestations by many years. Most importantly,
recent trials of three different Aβ antibodies (solanezumab, crenezumab, and
aducanumab) have suggested a slowing of cognitive decline in post hoc analyses
of mild AD subjects. Although many factors contribute to AD pathogenesis, Aβ
dyshomeostasis has emerged as the most extensively validated and compelling
therapeutic target. Alzheimer's disease (AD) is characterized by deposition of amyloid-β (Aβ)
plaques and neurofibrillary tangles in the brain, accompanied by synaptic
dysfunction and neurodegeneration. Antibody-based immunotherapy against Aβ to
trigger its clearance or mitigate its neurotoxicity has so far been
unsuccessful. Here we report the generation of aducanumab, a human monoclonal
antibody that selectively targets aggregated Aβ. In a transgenic mouse model of
AD, aducanumab is shown to enter the brain, bind parenchymal Aβ, and reduce
soluble and insoluble Aβ in a dose-dependent manner. In patients with prodromal
or mild AD, one year of monthly intravenous infusions of aducanumab reduces
brain Aβ in a dose- and time-dependent manner. This is accompanied by a slowing
of clinical decline measured by Clinical Dementia Rating-Sum of Boxes and Mini
Mental State Examination scores. The main safety and tolerability findings are
amyloid-related imaging abnormalities. These results justify further development
of aducanumab for the treatment of AD. Should the slowing of clinical decline be
confirmed in ongoing phase 3 clinical trials, it would provide compelling
support for the amyloid hypothesis. Calcium homeostasis plays a major role in maintaining neuronal function under
physiological conditions. Amyloid-β (Aβ) initiates pathological processes that
include disruption in intracellular calcium levels, so amelioration of the
calcium alteration could serve as an indirect functional indicator of treatment
efficacy. Therefore, calcium dynamics were used as a measure of functional
outcome. We evaluated the effects of the anti-Aβ antibody aducanumab on calcium
homeostasis and plaque clearance in aged Tg2576 mice with in vivo multiphoton
imaging. Acute topical application of aducanumab to the brain resulted in
clearance of amyloid plaques. Although chronic systemic administration of
aducanumab in 22-month-old mice did not clear existing plaques, calcium overload
was ameliorated over time. Therefore, this antibody likely restores neuronal
network function that possibly underlies cognitive deficits, indicating promise
as a clinical treatment. In addition, functional readouts such as calcium
overload may be a more useful outcome measure to monitor treatment efficacy in
models of Alzheimer's disease compared with amyloid burden alone.
SIGNIFICANCE STATEMENT: Alzheimer's disease (AD) is a progressive
neurodegenerative disorder that is currently without a cure. Aducanumab is an
anti-amyloid-β antibody being developed for the treatment of AD. Interim
analyses of a phase 1b clinical trial have suggested potential beneficial
effects on amyloid pathology and cognitive status in patients treated with
aducanumab (Sevigny et al., 2016). Here, we show that a murine analog of
aducanumab clears amyloid plaques in an acute setting and restores calcium
homeostasis disrupted in a mouse model of AD upon chronic treatment. Therefore,
we demonstrate that aducanumab reverses a functional outcome measure reflective
of neural network activity. |
Which human syndromes have been detected with Fluorescence in situ hybridization (FISH)? | To explore the clonal evolution of monosomy 7 in patients with aplastic anemia (AA).Monosomy 7 (-7) in 81 AA patients with normal karyotype at diagnosis and 46 AA treated with immunosuppressive therapy (IST) and more than 6 months of recombinant human granulocyte colony-stimulating factor (rhuG-CSF) were detected by interphase- fluorescence in situ hybridization (FISH) retrospectively.There were 5.4% - 7.6% of -7 cells in 11 (13.6%) of 81 patients at diagnosis, the survival and response rate to IST in -7 positive patients did not differ s Former molecular cytogenetic studies showed that such aberrations as an equivalent of intrinsic radiosensitivity can be detected by fluorescence in-situ hybridization (FISH) techniques using whole chromosome painting (wcp) probes. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. Using this technique, followed by cell-by-cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. | A 15-year-old male with myelodysplastic syndrome (MDS) characterized by monosomy
7 was cytogenetically evaluated by metaphase karyotyping and fluorescence in
situ hybridization (FISH) of interphase cells at six different points during the
course of his disease. At diagnosis, there was complete agreement between
metaphase and interphase findings. Interphase analysis alone provided important
cytogenetic information on the first specimens received following intensive
combination chemotherapy and bone marrow transplantation where metaphase
analyses were uninformative. The detection of a minor post-treatment monosomy 7
population by interphase but not metaphase studies may have identified minimal
residual disease prior to recurrence of MDS. From this longitudinal study, it is
concluded that metaphase and interphase cytogenetic analyses form complementary
approaches and that use of both provides greater analytical power when
appropriate chromosome markers are available. Fluorescence in situ hybridization (FISH) using two cosmid probes (41A and P13)
from the Miller-Dieker syndrome (MDS) critical region in 17p13.3 was performed
in a blinded comparison of three MDS patients with submicroscopic deletions and
in four normal relatives used as controls. The controls showed both chromosome
17 homologues labeled in 85%-95% of cells, while each patient showed only one
homologue labeled in 75%-80% of cells. Two MDS patients with cryptic
translocations were also studied. In one case, a patient and her mother had the
same der(17) (p+), but the reciprocal product of the translocation could not be
identified in the mother by G-banding (i.e., it was a "half-cryptic"
translocation). FISH revealed a 3q;17p translocation. The other case involved a
patient with apparently normal karyotype. Because a large molecular deletion was
found, a translocation involving two G-negative telomeres (i.e., a
"full-cryptic" translocation) was postulated. FISH studies on her father and
normal brother showed an 8q;17p translocation. These studies demonstrate that in
situ hybridization is an efficient method for deletion detection in
Miller-Dieker syndrome. More important, parental studies by FISH on patients
demonstrating molecular deletions and a normal karyotype may identify cryptic
translocation events, which cannot be detected by other molecular genetic
strategies. Similar in situ strategies for deletion detection can be developed
for other microdeletion syndromes, such as Prader-Willi/Angelman syndrome or
DiGeorge syndrome. Structural anomalies of the X chromosome, especially translocations, are rare
events in myelodysplastic syndromes (MDS). In a series of 2270 MDS patients
analyzed between 1983 and 1994 (Center for Human Genetics, Leuven), 9 cases were
found with translocations involving the X chromosome. These aberrations were not
restricted to specific FAB subtypes and were the sole anomalies in 3 cases. In
the remaining 6 patients, they were associated with other abnormalities,
including 5q-, observed in three cases. Fluorescence in situ hybridization
(FISH) was retrospectively performed on 8 patients and was shown to be a useful
complement for the characterization of the translocations involving the X
chromosome. In 3 cases, we could identify translocation partners and breakpoint
regions only by using chromosome painting. No recurrent chromosome partners were
observed. The breakpoints could be localized along the whole X chromosome. There
was, however, a cluster in the Xq13 region involved in 4 of the 9 patients. The
previously reported association of Xq13 anomalies with refractory anemia with
ringed sideroblasts (RARS) was found in only one case. Despite the lack of
characteristic translocations involving the X chromosome, the occurrence of such
changes as the sole karyotypic anomaly suggests that they could play a role in
the pathogenesis of some myelodysplastic syndromes. Two patients with classical features of Angel-man syndrome (AS) and one with
Prader-Willi syndrome (PWS) had unbalanced reciprocal translocations involving
the chromosome 15 proximal long arm and the telomeric region of chromosomes 7, 8
and 10. Fluorescence in situ hybridization (FISH) was used for the detection of
chromosome 15(q11-13) deletions (with probes from the PWS/AS region) and to
define the involvement of the telomere in the derivative chromosomes (with
library probes and telomere-specific probes). The 15(q11-13) region was not
deleted in one patient but was deleted in the other two. The telomere on the
derivative chromosomes 7, 8 and 10 was deleted in all three cases. Thus, these
are true reciprocal translocations in which there has been loss of the small
satellited reciprocal chromosome (15) fragment. Chromosomal changes through pericentric inversions play an important role in the
origin of species. Certain pericentric inversions are too minute to be detected
cytogenetically, thus hindering the complete reconstruction of hominoid
phylogeny. The advent of the fluorescence in situ hybridization (FISH) technique
has facilitated the identification of many chromosomal segments, even at the
single gene level. Therefore the cosmid probe for Prader-Willi (PWS)/Angelman
syndrome to the loci on human chromosome 15 [q11-13] is being used as a marker
to highlight the complementary sequence in higher primates. We hybridized
metaphase chromosomes of chimpanzee (PTR), gorilla (GGO), and orangutan (PPY)
with this probe (Oncor) to characterize the chromosomal segments because the
nature of these pericentric inversions remains relatively unknown. Our
observations suggest that a pericentric inversion has occurred in chimpanzee
chromosome (PTR 16) which corresponds to human chromosome 15 at PTR 16 band
p11-12, while in gorilla (GGO 15) and orangutan (PPY 16) the bands q11-13
complemented to human chromosome 15 band q11-13. This approach has proven to be
a better avenue to characterize the pericentric inversions which have apparently
occurred during human evolution. "Genetic" divergence in the speciation process
which occurs through "chromosomal" rearrangement needs to be reevaluated and
further explored using newer techniques. Mouse Tec is a non-receptor type protein-tyrosine kinase and is highly expressed
in many hematopoietic cell lines. To investigate the roles of the Tec kinase in
the human hematopoietic system, we isolated cDNAs encoding the human Tec kinase.
The human tec cDNAs can encode a peptide of 631 amino acid residues with a
calculated molecular mass of 73,624. The predicted human Tec protein is highly
homologous to those of the members of the Tec family including mouse Tec type IV
(94% homology), mouse Tsk/Itk (60%), and human Btk (57%). The homology between
human Tec and other members of the Tec family can be observed not only in the
Src homology 3 (SH3), SH2, and kinase domains, but also in the N-terminal unique
domain. Northern blot analysis demonstrated that the major transcripts of tec
could be detected at 2.6 kb and 3.6 kb in a wide range of human hematopoietic
cell lines including myeloid, B-, and T-cell lineages. Interestingly, high
expression of the tec gene could be detected in all of the three patients
examined with myelodysplastic syndrome. The human tec gene was mapped by
fluorescence in situ hybridization (FISH) to chromosome 4p12. Fluorescence in situ hybridization (FISH) with a chromosome-region-specific DNA
probe was used prospectively on uncultured amniocyte interphase cells to detect
an unbalanced chromosome abnormality that resulted in cri du chat or 5p-
syndrome. Confirmation was performed by routine cytogenetics. We used fluorescence DNA in situ hybridization (FISH) to detect chromosomal
abnormalities as an indicator of minimal residual disease in follow-up samples
from the bone marrow (BM), or peripheral blood, of 25 patients with leukemia,
lymphoma and myelodysplastic syndromes. Trisomies were detected by interphase
FISH with repeat-sequence probes (RSP) or by using metaphase FISH with
whole-chromosome paint probes (WCP). Specific translocations were detected using
WCP probes. Translocations were observed using metaphase FISH in two patients in
uncertain or complete remission (CR), who both later suffered relapse. Five
patients with no abnormal cells remained in CR. Four patients with trisomies
detected during CR suffered relapse; metaphase FISH detected the trisomy in
0.17-16% of metaphase cells. Five patients for whom the trisomy occurred in
0.034% of cells remained in CR. Trisomic nuclei were observed in 0.27-2.3% of
interphase cells, by means of RSPs, in four patients who later suffered relapse.
Five patients with trisomic nuclei in 0.061% remained in CR. When two probes
were used simultaneously in a sample from one patient, 1% of the residual cells
were abnormal. The patient later suffered relapse. In one patient with
anaplastic large cell lymphoma, CD30-positive interphase cells were shown to
have trisomic chromosome 7 by immunophenotyping and FISH. Our results suggest
that metaphase FISH using WCP probes is a sensitive and specific method for
detecting minimal residual disease especially in patients with translocations. We performed conventional cytogenetic (CC) and interphase fluorescence in situ
hybridization (FISH) analysis with an alpha satellite chromosome 7 specific DNA
centromeric probe (p alpha 7t1) on bone marrow material prepared for CC in 11
controls and 80 cases of myelodysplastic syndromes (MDS). In controls, a mean of
4.3 +/- 1% of the 700 cells examined showed only one FISH signal for chromosome
7, and the finding of > 6.3% (mean +2 standard deviations) of cells with one
FISH signal was considered to indicate the presence of a clone with -7. By CC,
clonal -7 was found in 11 patients, whereas two patients had -7 in only one
mitose (non-clonal -7). In eight of the 11 cases of clonal -7 by CC, interphase
FISH confirmed -7. In the remaining three patients, 5.1%, 6.3% and 18.4%
respectively of the cells had one signal. Those three patients had, in addition
to -7 by CC, a marker chromosome which was shown to be constituted of chromosome
7 pericentromeric material by FISH analysis on metaphase spreads (metaphase
FISH). Of the two patients with non-clonal -7 by CC, one had a -7 clone by
interphase FISH whereas the other patient had normal FISH results. Five of the
67 patients with no -7 mitose by CC had clonal -7 by interphase FISH, with one
chromosome 7 signal in 14.4 to 39% of the cells examined. At least three mitoses
with -7 were found in two of them by metaphase FISH. Three of the five patients
were reexamined 12 to 17 months later: CC and metaphase FISH found no -7,
whereas interphase FISH still showed a -7 clone. Three of the patients with
clonal -7 by CC and by FISH were reexamined in complete hematological remission
after intensive therapy. CC found no -7 and interphase FISH was normal in all
three patients. Our findings suggest that interphase FISH may improve the
detection of -7 in MDS. Conventional cytogenetics should still be performed in
parallel to FISH, however, because of possible false negative FISH results when
a pericentromeric chromosome 7 marker is present in patients with -7. Larger
numbers of cases with minor -7 clones, detectable by FISH only, and longer
follow-up in those cases will be necessary to determine the significance of this
finding, the evolution of this minor clone, and the outcome of the patients. Fluorescence in situ hybridization (FISH) with a chromosome 7 specific alpha
satellite DNA probe was used to detect monosomy 7 in interphase and metaphase
cells obtained from patients with myelodysplastic syndrome (MDS) and acute
nonlymphocytic leukemia (ANLL). Chromosome analysis revealed monosomy 7, either
alone or as part of a complex chromosome abnormality, in all cell samples. FISH
analyses of 12 marrow samples and a blood sample using a chromosome 7 specific
alpha satellite DNA probe revealed a single fluorescence spot in 80.5-97.5% of
interphase cells indicating monosomy 7. In contrast, 83.5-92.0% of the same
cells had two copies of chromosome 17 as two fluorescent spots were detected
using a chromosome 17 specific alpha satellite DNA probe used as a positive
control. The proportion of interphase cells with monosomy 7 did not correlated
with the percentage of metaphase cells with monosomy 7 detected by conventional
karyotyping or with the percentage of blast cells in the bone marrow. The small nuclear ribonucleoprotein polypeptide N (SNRPN) gene is regarded as
one of the candidates for Prader-Willi syndrome (PWS). We describe two sibs with
typical PWS presenting deletion of SNRPN detected by fluorescence in situ
hybridization (FISH). Neither a cytogenetically detectable 15q12 deletion nor a
deletion for the D15S11, D15S10, and GABRB3 cosmid probes were found in either
patient. This implies a smaller deletion limited to the PWS critical region.
FISH with a SNRPN probe will permit analysis of PWS patients with limited
deletions not detectable with other probes. The lineage involvement in myelodysplastic syndromes (MDS) is still unclear. To
determine the clonality and the evolution of the disorder, a retrospective study
on bone marrow smears from seven MDS patients with trisomy 8 was performed using
fluorescence in situ hybridization (FISH). We observed that the trisomy of
chromosome 8 was selectively expressed in the myeloid-derived cells. No mature
lymphocytes or plasma cells expressed three signals. Our studies demonstrate
here the value of FISH for identifying the affected cell lineage. Furthermore,
the easy quantification of the abnormal cells can help in assessing the
progression of the disease. The majority of patients with acute myelogenous leukaemia (AML) and
myelodysplastic syndromes (MDS) relapse, especially those with unfavourable
cytogenetics. This study was designed to investigate the presence and frequency
of minimal residual disease (MRD) in patients with AML or MDS (n=35) and
numerical abnormalities of chromosomes 6, 7, 8, 9, 10, 17 and 18 in clinical
remission by using a combination of fluorescence activated cell sorting (FACS),
fluorescence in-situ hybridization (FISH) and labelling with bromodeoxyuridine
(BUdR). The technique enables the detection of as few as three leukaemic cells
in 10(5) normal cells. MRD was detected in 33/35 patients in complete remission
(CR). 16 patients relapsed (8/11 with monosomy 7, 4/17 with trisomy 8, and 4/7
with other cytogenetic abnormalities) after a median of 4.8 months (range 3-13).
Levels of MRD (P=0.007) and proliferation index (P=0.011) were significantly
higher in patients with monosomy 7 than in patients with trisomy 8 or other
cytogenetic abnormalities. The percentage of cells in S-phase, the number of
abnormal cells and cytogenetic class were related to time to relapse (P=0.001)
with S-phase being the single most important prognostic factor (P=0.0001). We
conclude that the combination of FACS/FISH/BUdR, which determines the number,
phenotype and proliferation rate of very rare leukaemic cells in patients with
AML or MDS in clinical remission, provides information that is useful in the
identification of patients with high and low likelihood of relapse. Myelodysplastic syndrome (MDS) is believed to be a stem-cell disorder involving
cytopenia and dysplastic changes in three hematopoietic lineages. However, the
involvement of pluripotent stem cells and progenitor cells has not been
clarified conclusively. To address this issue, we used fluorescence in situ
hybridization (FISH) of blood and bone marrow (BM) smears for mature cells and
FISH of cells sorted by fluorescence-activated cell sorting for progenitor
cells. Seven patients with MDS associated with trisomy 8 were studied. FISH
showed +8 in granulocytes, monocytes, and erythroblasts, but not in lymphocytes.
Sorted cells of T (CD3(+)), B (CD19(+)), and NK cells (CD3(-)CD56(+)) from
peripheral blood did not contain +8, nor did CD34(+) subpopulations from BM
including B (CD34(+)CD19(+)), T/NK (CD34(+)CD7(+)) progenitors, and pluripotent
stem cells (CD34(+)Thy1(+)). The +8 chromosome abnormality was identified in
stem cells only at the level of colony-forming unit of
granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM; CD34(+)CD33(+)). It
may thus be concluded that cells affected by trisomy 8 in the context of MDS are
at the CFU-GEMM level and that cells of lymphoid lineage are not involved. These
results provide new insights into the biology of MDS and suggest that intensive
chemotherapy and autologous BM transplantation may become important therapeutic
strategies. Pallister Killian syndrome (PKS) is the most frequent form of partial autosomal
tetrasomy 12p in humans. Sufferers have a mosaic of isochromosome 12p [i(12p)].
We report the first pre-natal diagnosis on fetal blood cells after cordocentesis
during the second trimester. The extra chromosome was first diagnosed by in situ
hybridization. Fluorescence in situ hybridization (FISH) was used to count the
interphase and/or metaphase cells containing the isochromosome. A review of the
literature identified 27 other reports of PKS diagnosed pre-natally. We showed
that the most consistent pre-natal ultrasound findings include hypertelorism,
broad neck, shorts limbs, abnormal hands or feet, diaphragmatic hernia and
hydramnios. Recognition of this congenital malformation pattern pre-natally may
allow utilization of FISH. Conventional cytogenetics (CC) is proven as a diagnostic and prognostic factor
in myelodysplastic syndrome (MDS). However, CC may be hampered by insufficient
metaphase preparation and cannot analyze interphase nuclei. These problems are
solved by using comparative genomic hybridization (CGH). The CGH was applied to
samples from 45 patients with MDS, and the results were compared with CC and
fluorescence in situ hybridization (FISH). The CC detected aberrations in 12 of
45 samples, including chromosomes 3 (n = 1), 5 (n = 9), 7 (n = 2),8(n = 1), 18(n
= 1),21 (n = 1), X (n = 1), and Y(n = 2). In one patient, loss of B and C group
chromosomes and a marker chromosome were seen. The CGH revealed chromosomal
imbalances in 18 of 45 samples, including chromosomes 5 (n = 11), 7 (n = 2), 8
(n = 1), 18(n = 1), 20(n = 1), 21 (n = 1), X (n = 1), and Y (n = 2). All
unbalanced aberrations found by CC were detected by CGH, too. In two patients,
the CGH found additional aberrations and redefined the aberrations of the
chromosomes of the B and C group in one sample. The FISH confirmed these
aberrations. Additionally performed FISH for chromosomes 5, 7, and 8 gave normal
findings in all patients found to be normal in CC and CGH. The CGH and FISH
confirmed the results obtained by CC. All three techniques showed changes of
chromosomes 5 and 7 as the most frequent aberrations, emphasizing the importance
of these chromosomes in the development of MDS. Furthermore, the CC is proven as
the basic technique for cytogenetic evaluation of MDS. Klinefelter syndrome is the first human sex chromosomal abnormality to be
reported. The majority of Klinefelter syndrome patients have the XXY karyotype.
Approximately 15% of Klinefelter patients, however, are mosaics with variable
phenotypes. Among the variant Klinefelter genotypes are such karyotypes as
XY/XXY and XX/XXY. The variation in phenotypes most likely depends on the number
of abnormal cells and their location in body tissues. In this paper we report
the case of a 42-year-old patient with Klinefelter syndrome and a rare variant
mosaic XXY/XX karyotype initially identified by GTG-banding. This was confirmed
by fluorescence in situ hybridization (FISH) using a dual-color X/Y probe. The
patient presented with erectile dysfunction and few other physical findings.
Thus, this case illustrates a rare variant of Klinefelter syndrome with a
relatively mild phenotype. It also illustrates the utility of FISH as an adjunct
to conventional cytogenetics in assessing the chromosome copy number in each
cell line of a mosaic. In our case, FISH also detected the presence of a small
population of cells with the XY karyotype not previously detected in the initial
30-cell GTG-banding analysis. Thus, through a combination of GTG-banding and
FISH, the patient was determined to be an XXY/XX/XY mosaic. Given that most
individuals with Klinefelter syndrome are infertile, and that these individuals
may wish to reproduce with the aid of modern reproductive technology, such as
testicular fine needle aspiration and intracytoplasmic sperm injection, it is
important that accurate estimation of the frequency of abnormal cells be
obtained for accurate risk estimation and genetic counseling, as recent studies
in patients with mosaic Klinefelter syndrome revealed that germ cells with sex
chromosomal abnormalities were nevertheless capable of completing meiosis. The 22q11.2 deletion syndrome, which includes DiGeorge and velocardiofacial
syndromes (DGS/VCFS), is the most common microdeletion syndrome. The majority of
deleted patients share a common 3 Mb hemizygous deletion of 22q11.2. The
remaining patients include those who have smaller deletions that are nested
within the 3 Mb typically deleted region (TDR) and a few with rare deletions
that have no overlap with the TDR. The identification of chromosome 22-specific
duplicated sequences or low copy repeats (LCRs) near the end-points of the 3 Mb
TDR has led to the hypothesis that they mediate deletions of 22q11.2. The entire
3 Mb TDR has been sequenced, permitting detailed investigation of the LCRs and
their involvement in the 22q11.2 deletions. Sequence analysis has identified
four LCRs within the 3 Mb TDR. Although the LCRs differ in content and
organization of shared modules, those modules that are common between them share
97-98% sequence identity with one another. By fluorescence in situ hybridization
(FISH) analysis, the end-points of four variant 22q11.2 deletions appear to
localize to the LCRs. Pulsed-field gel electrophoresis and Southern
hybridization have been used to identify rearranged junction fragments from
three variant deletions. Analysis of junction fragments by PCR and sequencing of
the PCR products implicate the LCRs directly in the formation of 22q11.2
deletions. The evolutionary origin of the duplications on chromosome 22 has been
assessed by FISH analysis of non-human primates. Multiple signals in Old World
monkeys suggest that the duplication events may have occurred at least 20-25
million years ago. The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood
may represent a new approach to noninvasive prenatal diagnosis. Although
promising, these techniques require highly accurate separation of NFCs from
nucleated cells of maternal origin; the two major problems limiting these
techniques are the relative rarity of fetal cells in maternal blood and the need
to establish their fetal origin. We now report a novel procedure that has
allowed accurate separation of NFCs from maternal cells. The technique reported
involves direct micromanipulator isolation of histochemically identified
hemoglobin F-positive nucleated cells to obtain fetal nucleated red blood cells
(FNRBCs) of high yield and purity. Using this technique, followed by
cell-by-cell multicolor fluorescence in situ hybridization (FISH) analysis of
purified FNRBCs, we were able to detect some of the most common human
aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in
33 pregt women referred for amniocentesis. The procedure used, which can be
completed in <72 hrs, produced complete concordance with the results of
amniocentesis. We also confirm findings of prior studies suggesting that the
number of FNRBCs in maternal circulation is remarkably higher in abnormal
pregcies than in normal pregcies, especially in women carrying a fetus
with trisomy 21. OBJECTIVE: To investigate the value of interphase fluorescence in situ
hybridization(FISH) in the detection of monosomy 7 (-7)or deletion of long arm
of chromosome 7(7q-) in the cases of myelodysplastic syndrome (MDS).
METHODS: Forty-six cases of MDS and 10 normal controls were studied
simultaneously by conventional karyotype analysis and interphase FISH technique
using SpectrumRed directly labeled DNA specific probe for 7q32. Two hundred
interphase cells were analyzed for each case and the cells with one red
hybridization spot<7% were regarded as positive.
RESULTS: Three cases displayed -7/7q- by conventional cytogenetics(CC) and were
confirmed by interphase FISH. Six cases in 43 cases who did not show -7/7q- by
CC displayed -7/7q- by interphase FISH.
CONCLUSION: Interphase FISH is very useful for the detection of -7 or 7q- in MDS
and it is more sensitive than CC. OBJECTIVE: To explore the value of interphase fluorescence in situ hybridization
(FISH) in the detection of trisomy 8 in myelodysplastic syndromes (MDS).
METHODS: Conventional cytogenetics (CC) and interphase FISH using SpectrumGreen
labelled chromosome 8 centromere specific probe were simultaneously carried out
to detect trisomy 8 in 69 MDS and 6 normal individuals.
RESULTS: Two hundred interphase cells were counted and cells with three green
hybridization spots > 3% was assigned. Eleven cases displayed trisomy 8 by CC
and were confirmed in 10 by FISH. In 7 cases, the percentage of trisomy 8 cells
was significantly lower by FISH than by CC. Seven cases displayed trisomy 8 by
FISH in 58 cases who did not show trisomy 8 by CC. Of the 7 cases, two had 2 and
3 marker chromosomes respectively, 4 had normal karyotypes.
CONCLUSIONS: Interphase FISH was a useful method for the detection of trisomy 8
in MDS, especially in patients with normal karyotype or marker chromosome. It
was a important complement to CC. Saethre-Chotzen syndrome is a common craniosynostosis syndrome characterized by
craniofacial and limb anomalies. Intragenic mutations of the TWIST gene within
7p21 have been identified as a cause of this disorder. There is phenotypic
overlap with other craniosynostosis syndromes, and intragenic mutations in FGFR2
(fibroblast growth factor receptor 2) and FGFR3 (fibroblast growth factor
receptor 3) have been demonstrated in the other conditions. Furthermore,
complete gene deletions of TWIST have also been found in a significant
proportion of patients with Saethre-Chotzen syndrome. We investigated 11
patients clinically identified as having the Saethre-Chotzen phenotype and 4
patients with craniosynostosis but without a clear diagnosis. Of the patients
with the Saethre-Chotzen phenotype, four were found to carry the FGFR3 P250R
mutation, three were found to be heterozygous for three different novel
mutations in the coding region of TWIST, and two were found to have a deletion
of one copy of the entire TWIST gene. Developmental delay was a distinguishing
feature of the patients with deletions, compared to patients with intragenic
mutations of TWIST, in agreement with the results of Johnson et al. [1998: Am J
Hum Genet 63:1282-1293]. No mutations were found for the four patients with
craniosynostosis without a clear diagnosis. Therefore, 9 of our 11 patients
(82%) with the Saethre-Chotzen phenotype had detectable genetic changes in FGFR3
or TWIST. We propose that initial screening for the FGFR3 P250R mutation,
followed by sequencing of TWIST and then fluorescence in situ hybridization
(FISH) for deletion detection of TWIST, is sufficient to detect mutations in >
80% of patients with the Saethre-Chotzen phenotype. In this study, we used spectral karyotyping (SKY) and fluorescence in situ
hybridization (FISH) as complementary techniques for the analysis of two
therapy-related secondary myelodysplastic syndrome (t-MDS) cases with complex
karyotypes, previously analyzed by G-banding. Different types of SKY's
cytogenetic contributions include confirmation of G-banding results,
identification of partially characterized rearrangements, identification of
marker chromosomes unidentified by G-banding, and detection of cryptic
reciprocal translocations. In particular, the ability of SKY to clarify a number
of markers led to the comprehension of clonal evolution. The common aberration
found in these two t-MDS cases was the fragility of chromosome 5 and monosomy of
chromosome 18. We clearly present that the use of SKY combined with conventional
G-banding analysis and FISH has assisted in the identification of important
chromosomal events that may play a key role in the development of t-MDS. The purpose of this study was to compare the detection of trisomy 8 in
myelodysplastic syndrome (MDS) patients with interphase fluorescence in situ
hybridization (FISH) and cytogenetic karyotype analysis. Using Spectrum Green
labeled chromosome 8 centromere probe, interphase FISH was established. The
trisomy 8 clones were simultaneously detected in 48 MDS cases with FISH and
conventional cytogenetic analysis (CCA). Results showed that the CCA revealed no
significant difference of constitutional proportion between MDS-RA and MDS-RAEB
with karyotypes of whole +8, partial +8 and one +8. With FISH, detectable rates
were 66.1% for whole +8. Partial +8 and sole +8 were significantly higher than
one +8 and complex +8, respectively. The percentages of trisomy 8 were similar
in MDS-RA and MDS-RAEB. Trisomy 8 was detected in 1 of 15 specimens with normal
or abnormal karyotype without trisomy 8 by FISH. There was linear correlation
between the percentages of partial +8 detected by FISH and CCA. Two patients
received CCA and FISH examination at diagnosis and during treatment, the
percentage of trisomy 8 was increased with progress of disease. In conclusion,
our results showed that FISH is a sensitive and accurate technique to detect
trisomy 8 in MDS patients. It can provide contribute to diagnosis, assessment of
curative effect and predicting progress of disease in MDS. Clone size of trisomy
8 does not related to classification of MDS, but sole +8 is seems to see in
MDS-RA frequently. The year 2001 witnessed the sequencing of 90% of the euchromatic region in the
human genome but the ultimate goal to delineate the positions of all genes is
yet to be achieved. Fluorescence In Situ Hybridization (FISH) is one of the
methods for localizing genes on chromosomes. In the present study, diagnostic
utility of single-, dual-, and multicolor FISH was evaluated for prenatal
diagnosis, cancer genetics, and screening of various congenital anomalies (sex
chromosomal and autosomal). Centromeric probes for chromosomes X and Y were used
for screening minor aneuploid cell lines (XXY, XO, and XXX) in the cases of
primary amenorrhea and suspected Klinefelter syndrome. The cases with ambiguous
genitalia were analyzed using a probe specific for the sex-determining region
(SRY). Suspected cases of Down syndrome were subjected to FISH using probe
specific for chromosome 21. FISH was also used to study gene alterations in
retinoblastoma and myeloid leukemias. Prenatal diagnosis was done to screen for
aneuploidies of chromosomes 13, 18, 21, X, and Y using FISH on uncultured cells
from amniotic fluid and chorionic villi sampling. The screening for common
aneuploidies was extended to abortuses from spontaneous abortions. Using FISH,
low-level mosaicism could be identified in some cases of primary amenorrhea and
suspected Klinefelter syndrome. Submicroscopic gene rearrangements could be
detected using FISH in cases of ambiguous genitalia and cancers. Further
interphase FISH could provide results within 24 hours. To conclude, FISH adds to
the diagnostic utility of routine cytogenetics and its use on interphase nuclei
overcomes the difficulty of conventional cytogenetics, thereby reducing the time
between sampling and diagnosis to 24 hr. OBJECTIVE: To evaluate the value of a panel fluorescence in situ hybridization
(FISH) in the detection of common chromosome abnormalities in myelodysplastic
syndrome (MDS).
METHODS: Twenty cases of MDS patients, whose karyotypes were unknown by the FISH
examiner beforehand, were analyzed with a panel FISH using YAC248F5 (5q31),
YAC938G5 (7q32), CEP8 and YAC 912C3 (20q12) probes to detect the frequently
occurring chromosome abnormalities (-5/5q, -/7q-, +8, 20q-) in MDS. Then the
results were compared to those of conventional cytogenetics (CC).
RESULTS: Among 20 cases, 13 cases were found to carry common chromosome
abnormalities by panel FISH (trisomy 8, five cases; -5/5q-, one case; 20q-, five
cases; 5q- accompanying 20q-, one case; complex abnormalities, one case).
However, on CC examination, only five cases were found to have common
chromosomal abnormalities (20q-, four cases; 5q- accompanying 20q-, one case).
In addition, trisomy 21, marker chromosome and complex abnormalities comprising
-5, -7 and marker chromosomes were seen in one case each, the rest were normal.
CONCLUSION: Panel FISH is a useful tool of molecular cytogenetics in the
detection of common chromosome abnormalities in MDS. In order to explore the value of interphase fluorescence in situ hybridization
(FISH) in the detection of partial deletion of the long arm of chromosome 20
(20q(-)) in patients with myelodysplastic syndrome (MDS), spectrum Green
fluorescein directly labeled yeast artificial chromosome (YAC) clone 912C3 which
spans the breakpoint cluster region in band 20q12 was used as probes to perform
interphase FISH on the marrow cells from 52 cases of MDS and 5 normal controls.
200 to 300 cells were scored for each case and cases which had cells with a
green hybridization signal>7.16% were defined as 20q(-) positive. The results of
FISH were compared with those of conventional cytogenetics (CC) assay. The
results showed that among 52 cases of MDS, 7 (13.5%) cases were positive by
FISH, however, of which, 4 cases were positive and the other 3 cases were
negative by CC assay. It is concluded that YAC912C3 and interphase FISH
providing a powerful technique in the detection of 20q(-) in MDS is an important
complement to CC assay. 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. The in vivo effect of recombit human erythropoietin (rHuEpo) and granulocyte
colony-stimulating factor (G-CSF) combined treatment on CD34(+) cells was
evaluated by fluorescence in situ hybridization (FISH) in 13 myelodysplastic
syndrome (MDS) patients with known cytogenetic abnormalities. After treatment,
responsive patients presented a significantly lower proportion of FISH abnormal
CD34(+) cells than before treatment (P = 0.003), and in comparison with
unresponsive cases (P = 0.007). Response to treatment was associated with a
reduced degree of apoptosis in CD34(+) cells (P = 0.021): however, no difference
in telomere length was observed in responsive patients after growth factor
administration. Although the number of patients analysed was relatively small,
the present data suggest that, in MDS patients, response to rHuEpo and G-CSF may
be related to the proliferation of karyotypically normal but potentially
defective CD34(+) progenitor cells. Duplication of the long arm of chromosome 1 (1q) is widely reported in human
neoplasia, including the myelodysplastic syndromes (MDS). So far, it has not
been described as a single aberration in the chronic myelomonocytic leukemia
(CMML), a subtype of MDS. Rather, trisomy 1q was always a part of complex
chromosome changes affecting the subtypes of MDS other than CMML. We report on a
patient with CMML with an unbalanced translocation of the entire 1q onto the
short arm of chromosome 14 as a sole cytogenetic abnormality. Fluorescence in
situ hybridization (FISH) analysis with an alpha-satellite probe for the
paracentric region of the long arm of chromosome 1 confirmed the presence of
trisomy 1q in a derivative chromosome, der(14)t(1;14)(q12;p11). The discrepant
results between the metaphase cytogenetics (100% abnormal) and interphase
cytogenetic (71% nuclei with 3 signals) suggest that trisomy 1q, even in the
absence of additional cytogenetic changes, has a sufficient leukemogenic
potential to confer a proliferative advantage on hematopoietic cells committed
to monocyte stemline both in vitro and in vivo. The literature data on partial
and complete trisomy 1q in CMML is reviewed. OBJECTIVE: To explore the value of multiplex fluorescence in situ hybridization
(M-FISH) technique in the detection of the complex chromosomal aberrations
(CCAs) in myelodysplastic syndromes (MDS).
METHODS: M-FISH was used in ten MDS patients with R-banding CCAs to refine the
complex chromosomal rearrangements, the constitute of marker chromosomes, and to
identify the cryptic translocations.
RESULTS: Thirty-seven kinds of structural rearrangements were detected by M-FISH
including insertion, deletion, translocation and derivative chromosomes, among
which 34 kinds were unbalanced rearrangements, and 3 were balanced
rearrangements including t(6;22) (q21; q12), t(9; 19) (q13; p13) and t(3;5) (?;
?). Seven abnormalities in the present paper were first reported in the
literature. In addition, chromosome 17 aberrations (7/10) and -5/5q - (7/10)
were the two most frequent abnormalities.
CONCLUSIONS: M-FISH could refine CCAs in MDS patients, find or correct the
missed or misidentified abnormalities analysed by conventional cytogenetics. Abnormalities in DNA copy number are frequently found in patients with multiple
anomaly syndromes and mental retardation. Array-based comparative genomic
hybridization (array-CGH) is a high-resolution, whole-genome technology that
improves detection of submicroscopic aberrations underlying these syndromes.
Eight patients with mental disability, multiple congenital anomalies, and
dysmorphic features were screened for submicroscopic chromosomal imbalances
using the GenoSensor Array 300 Chip. Subtelomeric aberrations previously
detected by fluorescence in situ hybridization (FISH) analysis were confirmed in
two patients, and accurate diagnosis was provided in two previously undiagnosed
complex cases. Microdeletions at 15q11.2-q13 in a newborn with hypotonia,
cryptorchidism, and hypopigmentation were detected with few discrepancies
between the array results and FISH analysis. Contiguous microdeletion of GSCL,
HIRA and TBX1 genes at 22q11.2 was identified in a previously undiagnosed boy
with an unusual presentation of the VCF/DiGeorge spectrum. In a newborn with
aniridia, a borderline false-negative WT1 deletion was observed, most probably
because of differences between the size of the genomic deletion and the
microarray probe. A false-positive rate of 0.2% was calculated for
clone-by-clone analysis, whereas the per patient false-positive rate was 20%.
Array-CGH is a powerful tool for the rapid and accurate detection of genetic
disorders associated with copy number abnormalities and can significantly
improve clinical genetic diagnosis and care. INTRODUCTION: In the course of the Down-screening protocol there are
possibilities today for rapid diagnosis of aneuploidies among high-risk
pregcies identified by non-invasive screening tests, however, the diagnostic
value of these molecular genetic tests are debated.
AIM OF THE STUDY: In this prospective study, data about the reliability of one
of the rapid tests, namely; interphase fluorescence in situ hybridization
(int-FISH) was to be gathered by the authors.
METHODS: For the period between May 2002 and September 2006 all of the 1279
fetal sample were examined both with int-FISH and full karyotyping.
RESULTS: Extra or absent signal was detected in 47 cases (3.7%) (trisomy 21 in
32, various other numerical abnormalities in 15 cases). All of these numerical
aberrations were confirmed by metaphase analysis without false positivity or
negativity. In 19 cases the finding of int-FISH was negative, however, full
karyotyping disclosed abnormalities (in 12 of these 19 cases, the abnormality
was balanced). Only 4 of the 1279 fetuses (0.3%) (3 small extra marker
chromosomes, 1 de novo unbalanced translocation) were to be found, who would
have been born with phenotypical abnormalities without metaphase analysis (2 of
them had suspect ultrasound signs).
CONCLUSION: Although more analysis are needed, based on the results of this
study it is to be concluded that rapid molecular genetic methods like int-FISH
might be accepted as a diagnostic tests of fetal aneuploidy, if its use were
restricted to high risk pregcies identified by advanced maternal age and
non-invasive maternal screening only. However, full karyotyping is needed in
cases with familial translocation and abnormal 2nd trimester ultrasound signs. OBJECTIVE: To identify the abnormal karyotypes by fluorescence in situ
hybridization (FISH) and explore prognostic implications in patients with
myelodysplastic syndromes (MDS).
METHODS: FISH was used to detect the frequently occurring chromosome
abnormalities (-5/5q, +8, -7/7q-) in 37 MDS cases. SPSS 11.5 software and
correlation analysis were used to analyze the relativity among the abnormal
chromosomes, the prognosis and the disease conversion in 37 MDS patients.
RESULTS: Karyotype abnormalities were found in 21 (56.8%) of 37 cases, among
which 6 (16.2%) were complex karyotypes, 9 (24.3%) +8, 2(5.4%) -5/5q-, 2(5.4%)
-7/7q-. In the median time of follow-up of 12 months, 12 cases transformed into
acute leukemia. Complex karyotypes were significantly associated with the poor
prognosis and leukemia transformation. + 8 and -7/7q- abnormalities were
correlated with the death.
CONCLUSIONS: FISH was more sensitive than conventional cytogenetics for
detecting mini-clonal abnormality. There are some differences in abnormal
karyotypes between patients in China and the western countries. Multi-probes
used in cytogenetic detections may predict the patient' s prognosis more
accurately. The higher proportion of abnormal karyotypes the poorer prognosis. As chromosomal instability may contribute to leukemogenesis in patients with
congenital bone marrow failure (CBMF) disorders, it was the aim of this study to
characterize chromosomally aberrant clones that arise during the clinical course
of disease by means of R-banding and fluorescence in situ hybridization (FISH)
analyses. In addition, multicolor-FISH and array-comparative genomic
hybridization (CGH) were applied to characterize clonal chromosome aberrations
in more detail. Between January 2004 and December 2005, we prospectively
analyzed 90 samples of 73 patients with proven or suspected CBMF disorders
enrolled in a German Study Network of CBMF diseases. Clonal aberrations could be
identified in four of 73 patients examined. In one child with congenital
thrombocytopenia, Jacobsen syndrome [del(11)(q24)c] was diagnosed, and thus a
CBMF could be excluded. In a girl with Shwachman-Diamond syndrome, two
independent clones, one with an isochromosome i(7)(q10), another with a complex
aberrant karyotype, were identified. Simultaneously, transition into a
myelodysplastic syndrome (MDS) occurred. The brother, who was also afflicted
with Shwachman-Diamond syndrome, showed an isochromosome i(7q) as a single
aberration. In the fourth patient with severe congenital neutropenia, an
add(21)(q22) marker containing a low-level amplification of the AML1 gene was
identified at the time point of transition into acute myelogenous leukemia
(AML). In summary, we suggest that follow-up of patients with CBMF using
chromosome and FISH analyses will be helpful for the early detection of
transition into MDS or AML and thus should be an integral part of the clinical
management of these patients. BACKGROUND: Rarely, patients who present with pancytopenia and are diagnosed
initially with aplastic anemia (AA) subsequently develop a myelodysplastic
syndrome (MDS). There has been controversy regarding whether the initial
diagnosis of AA is correct or whether these patients have hypocellular MDS at
the onset of pancytopenia.
METHODS: The authors studied bone marrow (BM) specimens from patients who were
diagnosed initially with AA and subsequently with MDS from a cohort of 128
consecutive patients who had AA during the period from 1993 to 2004. Cytogenetic
and fluorescence in situ hybridization (FISH) analyses were performed to assess
for monosomy 7 retrospectively in a subset of patients.
RESULTS: Twelve patients were identified (age range, 26-79 years). At the time
they were diagnosed with AA, there was no evidence of dysplasia, the median BM
cellularity was 5% (range, from <1% to 15%), and all patients had a normal
karyotype. Therapy for 11 patients included immunomodulating agents, which were
accompanied by growth factors in 4 patients and 1 patient underwent BM
transplantation. One patient received growth factors only. The median interval
to the diagnosis of MDS was 9 months (range, 2-43 months). The median BM
cellularity was 30% (range, 5-90%), and dysplastic changes were observed in all
patients. Nine patients had an abnormal karyotype, and monosomy 7 was the most
common abnormality (n = 5 patients). FISH detected monosomy 7 in 6 samples at
the time MDS was diagnosed and in 2 samples at the time AA was diagnosed.
CONCLUSIONS: The detection of monosomy 7 in specimens that were considered AA
and the short time interval to a subsequent diagnosis of MDS suggests that these
patients had hypoplastic MDS at the onset of pancytopenia. Therapy may allow the
detection of MDS by enhancing cell growth. BACKGROUND: Prader-Willi Syndrome (PWS) is a complex human genetic disease
arising from a loss of paternal allele expression of imprinting genes on
chromosome 15q11-q13. Normally the CpG islands at this site are heavily
methylated in the maternal allele, but unmethylated in the paternal allele and
therefore activated in gene expression. only the methylated allele should
present in pws patients when methylation-specific pcr (msp) is analyzed.
METHODS: This paper reports an analysis of PWS in Thai patients using consensus
diagnostic criteria based on a combination of clinical data, basic G-banding and
fluorescence in situ hybridization (FISH) cytogenetics, PCR-based methylation
assay, and bisulfite sequencing of the CpG islands of SNRPN to confirm 15q
deletion or the methylation pattern of the SNRPN promoter and exon 1. Lack of
complete clinical reports or inadequacy of the minimum laboratory support
required had made it difficult to diagnose PWS, Angelman syndrome and other
microdeletion disorders.
RESULTS: Accuracy of 100% was obtained for diagnosis of the PWS study patients
using the minimum requirements necessary. A total of 20 patients were diagnosed
as PWS based on clinical criteria and the scoring tool for PWS, and the same
approach was applied to four separate patients with some unmatched criteria but
phenotypic similarity to PWS. Findings showed that 70% of those clinically
diagnosed as PWS patients (14/20) had a deletion at 15q11-q13 according to FISH,
while all 20 patients showed MSP positive of SNRPN gene. Six cases (30%) without
a paternal deletion were confirmed to have maternal uniparental disomy (mUPD) of
PWS by MSP and methylation sequencing approaches. Noteworthy, two of the six
cases with mUPD were 3.5 year-old twins. None of the five cases with scores
lower than the reported consensus criteria showed positive G-band, FISH or MSP
results.
CONCLUSIONS: We demonstrate here the high power of combining clinical findings,
FISH and MSP in definitive diagnosis of PWS and in distinguishing between the
two major different types of molecular mechanisms. No false positives or false
negatives were observed in our analysis. 22q11 Deletion syndrome (22q11DS) is the most common microdeletion syndrome in
humans, occurring with an incidence of 1 in 4,000. In most cases the
submicroscopic deletion spans 3 Mb, but there are a number of other overlapping
and non-overlapping deletions that generate a similar phenotype. The majority of
the 22q11.2 microdeletions can be ascertained using a standard fluorescence in
situ hybridization (FISH) assay probing for TUPLE1 or N25 on 22q11.2. However,
this test fails to detect deletions that are either proximal or distal to the
FISH probes, and does not provide any information about the length of the
deletion. In order to increase the detection rate of 22q11.2 deletion and to
better characterize the size and position of such deletions we undertook a study
of 22q11.2 cases using multiplex ligation dependent probe amplification (MLPA).
We used MLPA to estimate the size of the 22q11.2 deletions in 51 patients
positive for TUPLE1 or N25 (FISH) testing, and to investigate 12 patients with
clinical features suggestive of 22q11DS and negative FISH results. MLPA analysis
confirmed a microdeletion in all 51 FISH-positive samples as well as
microduplications in three samples. Further, it allowed us to delineate
deletions not previously detected using standard clinical FISH probes in 2 of 12
subjects with clinical features suggestive of 22q11DS. We conclude that MLPA is
a cost-effective and accurate diagnostic tool for 22q11DS with a higher
sensitivity than FISH alone. Additional advantages of MLPA testing in our study
included determination of deletion length and detection of 22q11.2 duplications.
(c) 2007 Wiley-Liss, Inc. Fluorescent in situ hybridisation (FISH) is a complementary cytogenetic method
which has an important role in discovering unsolved cases of mental retardation
and multiple anomalies. The ability of this method to detect complex and cryptic
chromosomal rearrangements exceeds the resolution of the usual cytogenetic
banding techniques; therefore it has a wide implementation in modern cytogenetic
laboratories - in routine work, as well as for research purposes. We analysed 19
patients with microdeletion syndromes - 9 patients with Williams syndrome, 4
patients with Prader-Willi syndrome, and 6 patients with DiGeorge syndrome. On
the basis of evaluation of facial dysmorphism and the presence of specific major
anomalies, all the patients met the criteria for the diagnosis of the syndrome.
FISH studies were performed, confirming the suspected syndrome in patients. This unit opens with an overview of microdeletions and methods for their
detection. It goes on to describe a vast array of autosomal microdeletion
syndromes, X-linked microdeletion syndromes, and microduplication syndromes. The
final portion of the unit offers guidance for detecting such syndromes with
Fluorescence in situ Hybridization (FISH). OBJECTIVE: To evaluate the conventional cytogenetic methods in genetic diagnosis
and prenatal diagnosis in the family with a proband of Angelman syndrome (AS).
METHODS: High-resolution G-banding karyotyping and fluorescence in situ
hybridization (FISH) on metaphase chromosomes were performed.
RESULTS: Two AS patients and 1 normal fetus in the family were successfully
detected by FISH.
CONCLUSION: Our result demonstrated that patient with type I AS could be
detected by combining the techniques of high-resolution G-banding and FISH with
clinical observation, which would offer accurate genetic counseling information
to the geneticists and provide the prenatal diagnosis for the AS family. A proportion of cytogenetic abnormalities in myelodysplastic syndromes (MDS) and
acute myeloid leukaemia (AML) may escape detection by high-resolution genomic
technologies, but can be identified by conventional cytogenetic and molecular
analysis. Here, we report the detection of a reciprocal translocation
t(7;21)(p22;q22) in the marrow of two adults with MDS and AML, using
conventional cytogenetic analysis and fluorescence-in situ-hybridization (FISH).
Reverse-transcription polymerase chain reaction (RT-PCR) and sequence analysis
identified a fusion between RUNX1 and the gene encoding ubiquitin specific
peptidase-42 (USP42), with splice-variants and variable break-points within
RUNX1. Combined cytomorphology and FISH studies in MDS marrow revealed abnormal
RUNX1 signals within megakaryocytes, suggesting that the acquisition of
t(7;21)(p22;q22) does not confer complete differentiation arrest and may
represent an early genetic event in leukaemogenesis. Single nucleotide
polymorphism-arrays failed to detect additional sub-microscopic genomic changes
predisposing to or associated with t(7;21). Molecular analysis of 100 MDS and
AML marrow specimens by RT-PCR did not reveal new cases with the RUNX1-USP42
fusion. Thus, our studies have identified t(7;21)(p22;q22) as a rare but
recurrent abnormality in MDS/AML, with the existence of alternative spliced
forms of the RUNX1-USP42 transcript in different patients. Further studies are
required to identify the potential contribution of these splice-variants to
disease heterogeneity. Although conventional cytogenetics is considered the gold standard to detect
chromosomal abnormalities in myelodysplastic syndromes (MDS), fluorescence in
situ hybridization (FISH) is being increasingly used additionally. However, the
real contribution of FISH analysis in the cytogenetic diagnosis of MDS has not
been well defined. The aim of this study was to evaluate whether FISH studies
are able to reveal chromosomal abnormalities in MDS patients undetected by
conventional cytogenetics. One hundred seventy-four FISH studies were performed
on bone marrow samples of 60 patients with MDS. The number of FISH studies in
each patient was variable (1-5). FISH studies confirmed the G-banding
cytogenetic findings in 99.4% (153/154) of samples and detected cytogenetic
abnormalities in 25% (5/20) of cases in which the conventional cytognetic study
failed. These results indicate that FISH studies provide relevant information in
MSD in which the conventional cytogenetic analysis was unsuccessful but add
little value to a normal katyotype in conventional cytogenetic analysis. TET2 haplo-insufficiency occurs through different molecular mechanisms and is
promptly revealed by array comparative genomic hybridization, single nucleotide
polymorphism (SNP) array, and next-generation sequencing (NGS). Fluorescence in
situ hybridization (FISH) can effectively demonstrate TET2 deletions and is
often used to validate molecular results. In the present study 41 MDS patients
with and without 4q abnormalities were analyzed with a series of bacterial
artificial chromosome (BAC) probes spanning the 4q22.3-q25 region. On
conventional cytogenetic (CC) studies, a structural defect of the long arm of
chromosome 4 (4q) was observed in seven patients. In three, one each with a
t(1;4)(p21;q24), an ins(5;4)(q23;q24qter), and a t(4;17)(q31;p13) as the sole
chromosomal abnormality, FISH with the RP11-356L5 and RP11-16G16 probes, which
cover the TET2 locus, produced one signal only. Unexpectedly, this same result
was achieved in 3 of the remaining 34 patients. Thus, a TET2 deletion was
observed in a total of six patients (14.6%). TET2 deletion was not correlated
with any particular clinical findings or outcome. These findings demonstrate
that 1) FISH is an effective and economical method to reveal cryptic
abnormalities of band 4q22-q24 resulting in TET2 deletions; 2) in these
patients, TET2 deletion is the unifying genetic event; and 3) the different
breakpoints within the 4q22-q25 region suggest that deletions are not mediated
by repetitive sequences. BACKGROUND & OBJECTIVES: Microdeletion syndromes are characterized by small (<5
Mb) chromosomal deletions in which one or more genes are involved. These are
frequently associated with multiple congenital anomalies. The phenotype is the
result of haploinsufficiency of genes in the critical interval. Fluorescence in
situ hybridization (FISH) technique is commonly used for precise genetic
diagnosis of microdeletion syndromes. This study was conducted to assess the
role of FISH in the diagnosis of suspected microdeletion syndrome.
METHODS: FISH was carried out on 301 clinically suspected microdeletion syndrome
cases for the confirmation of clinical diagnosis using non-commercial probes. Of
these, 177 cases were referred for 22q11.2 microdeletion, 42 cases were referred
for William syndrome, 38 cases were referred for Prader Willi/Angelman and 44
cases were referred for other suspected microdeletion syndromes.
RESULTS: FISH was confirmatory in 23 cases only (7.6%). There were 17 cases of
22q11.2 microdeletion, four cases of Prader Willi syndrome and two cases of
William syndrome.
INTERPRETATION & CONCLUSION: We conclude that FISH should not be the method of
choice for clinically suspected microdeletion syndromes. We propose to follow
strict clinical criteria for FISH testing or preferably to follow better methods
(genotype first approach). Whole genome screening may be used as first line of
test and FISH may be used for confirmation of screening result, screening of
family members and prenatal diagnosis. Somatic chromosomal mosaicism arising from post-zygotic errors is known to cause
several well-defined genetic syndromes as well as contribute to phenotypic
variation in diseases. However, somatic mosaicism is often under-diagnosed due
to challenges in detection. We evaluated 10,362 patients with a custom-designed,
exon-targeted whole-genome oligonucleotide array and detected somatic mosaicism
in a total of 57 cases (0.55%). The mosaicism was characterized and confirmed by
fluorescence in situ hybridization (FISH) and/or chromosome analysis. Different
categories of abnormal cell lines were detected: (1) aneuploidy, including sex
chromosome abnormalities and isochromosomes (22 cases), (2) ring or marker
chromosomes (12 cases), (3) single deletion/duplication copy number variations
(CNVs) (11 cases), (4) multiple deletion/duplication CNVs (5 cases), (5) exonic
CNVs (4 cases), and (6) unbalanced translocations (3 cases). Levels of mosaicism
calculated based on the array data were in good concordance with those observed
by FISH (10-93%). Of the 14 cases evaluated concurrently by chromosome analysis,
mosaicism was detected solely by the array in 4 cases (29%). In summary, our
exon-targeted array further expands the diagnostic capability of high-resolution
array comparative genomic hybridization in detecting mosaicism for cytogenetic
abnormalities as well as small CNVs in disease-causing genes. OBJECTIVE: To compare the results of fluorescence in situ hybridization (FISH)
versus conventional cytogenetics (CC) in the detection of common chromosomal
abnormalities and evaluate the significance of FISH in myelodysplastic syndrome
(MDS) .
METHODS: A total of 344 patients with de novo MDS from June 2008 to October 2012
were detected by 6 pairs of probes, including CSF1R/D5S23-D5S721 (5q33) , EGR1/
D5S23-D5S721 (5q31) , D7S486 (7q31) /CSP7, D7S522 (7q31) /CSP7, D20S108/CSP8
(20q12/CSP8) and CSPX/CSPY. The results were compared with those of CC.
RESULTS: CC revealed cytogenetic abnormalities in 168/344 cases (48.8%) and the
frequency of common aberrations such as +8, 20q-, -7/7q-, -5/5q- and -Y were
18.9% (65/344) , 9.3% (32/344), 8.4% (29/344), 8.4% (29/344) and 2.4% (5/206)
respectively. While FISH revealed chromosome abnormalities in 147/344 patients
(42.7%) and the frequency of +8, 20q-, -7/7q-, -5/5q- and -Y were 20.9%
(72/344), 11.6% (40/344), 11.6% (40/344), 10.2% (35/344) and 2.9% (6/206)
respectively. Overall 187/344 patients (54.4%) carried clonal aberrations by a
combination of CC and FISH. Among 158 patients with normal karyotype by CC, 14
cases (8.9%) were detected to have clonal aberrations by FISH. FISH also
confirmed 4 carriers of clonal aberrations out of 9 patients with non clonal
abnormalities by CC.
CONCLUSIONS: FISH is effective for improving the probability of detecting
chromosome abnormalities in MDS cases with normal karyotypes and karyotype
failure. FISH may provide rationales for clonal abnormalities in patients with
non clonal aberrations by CC. A combination of FISH and CC shows complementary
advantages. Author information:
(1)a Hospital Arnau de Vilanova , Lleida , Spain.
(2)b Universitat de Lleida , Lleida , Spain.
(3)c Institut de Recerca contra la Leucèmia Josep Carreras (IJC) , Badalona,
Barcelona , Spain.
(4)d Hospital Clínico Universitario de Valencia , Valencia , Spain.
(5)e Hospital Universitario Central de Asturias , Oviedo , Spain.
(6)f Hospital Vall d'Hebron , Barcelona , Spain.
(7)g Hospital del Mar , Barcelona , Spain.
(8)h Hospital General de Castellón , Castellón , Spain.
(9)i Hospital Universitario Marqués de Valdecilla , Santander , Spain.
(10)j Hospital General Universitario Gregorio Marañón , Madrid , Spain.
(11)k Hospital Universitario 12 de Octubre , Madrid , Spain.
(12)l Hospital de Basurto , Bilbao , Spain.
(13)m Hospital General Universitario de València , Valencia , Spain.
(14)n Hospital Universitario Virgen Macarena , Sevilla , Spain.
(15)o Fundación Pública Galega Medicina Xenómica, Hospital Clínico Universitario
, Santiago , Spain.
(16)p Hospital Universitari i Politècnic La Fe , Valencia , Spain. Author information:
(1)Department of Hematology and Medical Oncology, University Medicine of
Goettingen, Germany.
(2)Department of Hematology and Oncology, Technical University of Munich,
Germany.
(3)Department of Hematology and Oncology, University of Dresden, Germany.
(4)Department of Hematology and Oncology, University of Duesseldorf, Germany.
(5)Department of Hematology and Oncology, University Hospital of Mannheim,
Germany.
(6)Department of Hematology and Oncology, Marienhospital, Duesseldorf, Germany.
(7)Department of Hematology and Oncology, University of Freiburg Medical Center,
Freiburg, Germany.
(8)Department of Hematology, Stanford University Cancer Center, Stanford, CA.
(9)University of Rochester Medical Center, Rochester, NY.
(10)Institut De Recerca Contra La Leukemia Josep Carreras, Badalona, Spain.
(11)Sonora Quest Laboratories, Phoenix, AZ.
(12)Tokyo Medical University, Tokyo, Japan.
(13)Section of Hematology and Oncology, University of Chicago, Chicago, IL.
(14)Hanusch Hospital Boltzmann Institute for Leukemia Research, Vienna, Austria.
(15)Third Medical Department for Hematology and Oncology and L. Boltzmann
Cluster Oncology, Hanusch Hospital, Vienna, Austria.
(16)Department of Human Genetics, University of Düsseldorf, Düsseldorf, Germany.
(17)Department of Hematology, Oncology and Clinical Immunology, St. Johannes
Hospital, Duisburg, Germany.
(18)Department of Internal Medicine, Innsbruck Medical University, Innsbruck,
Austria.
(19)Department of Internal Medicine I, Division of Hematology and Hemostaseology
and Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna,
Austria.
(20)Department of Medical Genetics, Medical University of Vienna, Vienna,
Austria. The most common chromosomal abnormalities in myelodysplastic syndrome (MDS) and
acute myeloid leukemia (AML) are -5/del(5q) and -7/del(7q). When -5/del(5q) and
-7/del(7q) coexist in patients, a poor prognosis is typically associated. Given
that -5/del(5q) and/or -7/del(7q) often are accompanied with additional
recurrent chromosomal alterations, genetic change(s) on the accompanying
chromosome(s) other than chromosomes 5 and 7 may be important factor(s)
affecting leukemogenesis and disease prognosis. Using an integrated analysis of
karyotype, FISH and array CGH results in this study, we evaluated the smallest
region of overlap (SRO) of chromosomes 5 and 7 as well as copy number
alterations (CNAs) on the other chromosomes. Moreover, the relationship between
the CNAs and del(5q) and -7/del(7q) was investigated by categorizing the cases
into three groups based on the abnormalities of chromosomes 5 and 7 [group I:
cases only with del(5q), group II: cases only with -7/del(7q) and group III:
concurrent del(5q) and del(7q) cases]. The overlapping SRO of chromosome 5 from
groups I and III was 5q31.1-33.1 and of chromosome 7 from groups II and III was
7q31.31-q36.1. A total of 318 CNAs were observed; ~ 78.3% of them were
identified on chromosomes other than chromosomes 5 and 7, which were defined as
'other CNAs'. Group III was a distinctive group carrying the most high number
(HN) CNAs, cryptic CNAs and 'other CNAs'. The loss of TP53 was highly associated
with del(5q). The loss of ETV6 was specifically associated with group III. These
CNAs or genes may play a secondary role in disease progression and should be
further evaluated for their clinical significance and influence on therapeutic
approaches in patients with MDS/AML carrying del(5q) and/or -7/del(7q) in
large-scale, patient population study. Recurrent translocations are uncommon in myelodysplastic syndromes (MDS). Three
new recurrent translocations, namely der(12)t(3;12)(q13;p13),
t(11;13;22)(q13;q14;q12) and der(17)t(13;17)(q21;p13), identified by
conventional cytogenetics (CC) in 4 MDS patients, were further characterized
using a panel of commercial and homemade fluorescence in situ hybridization
(FISH) probes. The goal of this study was to determine the precise breakpoints
and to identify genes that could be related with the neoplastic process. Half of
the breakpoints (4/8) were precisely identified and in the remaining half they
were narrowed to a region ranging from 14 to 926 kb. All the studied breakpoints
had interstitial or terminal deletions ranging from 536 kb to 89 Mb, and only
those 7 Mb were detected by CC. The genes located in or around the breakpoints
described in our study have not been previously related to MDS. The deleted
regions include the ETV6 and RB1 genes, among others, and exclude the TP53 gene.
FISH studies were useful to refine the breakpoints of the translocations, but
further studies are needed to determine the role of the involved genes in the
neoplastic process. Chromosomal rearrangements involving 3q26 are recurrent findings in myeloid
maligcies leading to MECOM overexpression, which has been associated with a
very poor prognosis. Other 3q abnormalities have been reported and cryptic MECOM
rearrangements have been identified in some cases. By fluorescence in situ
hybridization (FISH) analysis, we investigated 97 acute myeloid
leukemia/myelodysplastic syndrome patients with various 3q abnormalities to
determine the role and the frequency of the involvement of MECOM. We identified
MECOM rearrangements in 51 patients, most of them showed 3q26 involvement by
chromosome banding analysis (CBA): inv(3)/t(3;3) (n = 26) and other balanced
3q26 translocations (t(3q26)) (n = 15); the remaining cases (n = 10) showed
various 3q abnormalities: five with balanced translocations involving 3q21 or
3q25; two with homogenously staining region (hsr) on 3q; and three with other
various 3q abnormalities. Complex rearrangements with multiple breakpoints on
3q, masking 3q26 involvement, were identified in cases with 3q21/3q25
translocations. Furthermore, multiple breaks were observed in two cases with
t(3q26), suggesting that complex rearrangement may also occur in apparently
simple t(3q26). Intrachromosomal gene amplification was another mechanism
leading to MECOM overexpression in two cases with hsr on 3q. In the last three
cases, FISH analysis revealed 3q26 involvement that was missed by CBA because of
metaphases' suboptimal quality. All cases with MECOM rearrangements showed
overexpression by real-time quantitative PCR. Finally, MECOM rearrangements can
occur in patients with 3q abnormalities even in the absence of specific 3q26
involvement, underlining that their frequency is underestimated. As MECOM
rearrangement has been associated with very poor prognosis, its screening should
be performed in patients with any 3q abnormalities. OBJECTIVE: To investigate the clinical application of fluorescent in situ
hybridization (FISH) for the differential diagnosis of myelodysplastic syndromes
(MDS) and aplastic anemia (AA).
METHODS: A FISH kit capable of detecting the chromosomal abnormalities related
to MDS was used to analyze 94 patients who were suspected to have AA by bone
marrow morphology.
RESULTS: Cytogenetic abnormalities were detected in 11 of the 94 patients, which
included trisomy 8 (5 cases), 20q- (1 case) and -Y (1 case). There were 4 cases
related to MDS, which included 3 cases of 5q-, in which 1 case carry 20q- at the
same time, and 7q- (1 case). No significant difference was found between the MDS
and AA groups in terms of age, sex or routine blood examination including
absolute neutrophil count, hemoglobin content and platelet count.
CONCLUSION: FISH can detect certain cytogenetic abnormalities related to MDS in
patients morphologically diagnosed as AA. The heterogeneous phenotype of known syndromes is a clinical challenge, and
harmonized description using globally accepted ontology is desirable. This
report attempts phenotypic analysis in a patient of constitutional mosaic
trisomy 13 in mesoderm and ectoderm to make globally comparable clinical
description. Phenotypic features (minor/major abnormalities) were recorded and
matched with the Human Phenotype Ontology terms that were used to query
web-based tool Phenomizer. We report here a case of 24-year-old girl born to non
consanguineous parents with history of one abortion. Her phenotypic evaluation
included short columella, low-set ears, seizures, enlarged naris, bifid tongue,
infra-orbital fold, smooth philtrum, microtia, microcephaly, carious teeth,
downslanted palpebral fissures, proportionate short stature, high palate, thin
upper lip vermilion, small for gestational age, broad fingertip, broad hallux,
mandibular prognathia and dental malocclusion. Karyotype and interphase FISH
(Fluorescence in situ hybridization) was done in blood cells. Interphase FISH
was also performed on buccal epithelial cells. Cytogenetic analysis demonstrated
trisomy 13 mosaicism in 25% cells i.e. 47, XX,+13(9)/46,XX(27). The interphase
FISH in blood cells showed trisomy 13 in 15%, whereas in buccal mucosa cells
showed nearly 6%. Mosaic aneuploidy in constitutional karyotype can be
responsible for variation in clinical and morphological presentation of patient
with genetic disorder. Karyotype, bone marrow blast percentage and cytopenia influence the prognosis of
myelodysplastic syndrome. We studied the abnormalities detected by fluorescence
in situ hybridization (FISH) in myelodysplastic syndrome and associated
haematological profile with abnormalities detected by FISH. Complete blood
counts, peripheral blood and bone marrow of patients were evaluated for
cytopenia, dysplasia and blasts. FISH probes were used to detect del(5q), gain
of chromosome 8, de (7q/-7) and del(20 q). Multiple regression analysis was used
to study the association of FISH abnormalities, age and sex with haematological
profile. Mc Nemar's test studied the relationship between FISH abnormalities and
dysplastic features in bone marrow. Cytogenetic abnormalities were detected by
FISH in 25.7% of patients. Del(20 q) was seen in 14.2% of patients. FISH was
able to predict changes in peripheral blood blast count by 80% (p ˂ 0.0001).
Cytogenetic abnormalities were not seen in 74.2% of patients. Groups with FISH
abnormalities have a different haematological profile, and these abnormalities
have a significant effect on blast percentage. |
List symptoms of EAST syndrome. | Epilepsy, ataxia, sensorineural deafness, and tubulopathy comprise EAST syndrome that is associated with recessive mutations in the KCNJ10 gene. | Mutations of the KCNJ10 (Kir4.1) K(+) channel underlie autosomal recessive
epilepsy, ataxia, sensorineural deafness, and (a salt-wasting) renal tubulopathy
(EAST) syndrome. We investigated the localization of KCNJ10 and the homologous
KCNJ16 in kidney and the functional consequences of KCNJ10 mutations found in
our patients with EAST syndrome. Kcnj10 and Kcnj16 were found in the basolateral
membrane of mouse distal convoluted tubules, connecting tubules, and cortical
collecting ducts. In the human kidney, KCNJ10 staining was additionally observed
in the basolateral membrane of the cortical thick ascending limb of Henle's
loop. EM of distal tubular cells of a patient with EAST syndrome showed reduced
basal infoldings in this nephron segment, which likely reflects the
morphological consequences of the impaired salt reabsorption capacity. When
expressed in CHO and HEK293 cells, the KCNJ10 mutations R65P, G77R, and R175Q
caused a marked impairment of channel function. R199X showed complete loss of
function. Single-channel analysis revealed a strongly reduced mean open time.
Qualitatively similar results were obtained with coexpression of KCNJ10/KCNJ16,
suggesting a domice of KCNJ10 function in native renal KCNJ10/KCNJ16
heteromers. The decrease in the current of R65P and R175Q was mainly caused by a
remarkable shift of pH sensitivity to the alkaline range. In summary, EAST
mutations of KCNJ10 lead to impaired channel function and structural changes in
distal convoluted tubules. Intriguingly, the metabolic alkalosis present in
patients carrying the R65P mutation possibly improves residual function of
KCNJ10, which shows higher activity at alkaline pH. The K+ channel expressed by the KCNJ10 gene (Kir4.1) has previously demonstrated
importance in retinal function in animal experiments. Recently, mutations in
KCNJ10 were recognised as pathogenic in man, causing a constellation of
symptoms, including epilepsy, ataxia, sensorineural deafness and a renal
tubulopathy designated as EAST syndrome. We have studied the impact of KCNJ10
mutations on the human electroretinogram (ERG) in four unrelated patients with
EAST syndrome. Corneal ganzfeld ERGs were elicited in response to flash stimuli
of strengths of 0.001–10 phot cd s/m2 presented scotopically, and 0.3–10 phot cd
s/m2 presented photopically. ERG waveforms from light-adapted retinae of all
patients showed reduced amplitudes of the photopic negative response (PhNR) (P <
0.001). The photopic ERGs showed a delay in b-wave time to peak, but the
photopic hill, i.e. the relative variation of time to peak and amplitude with
lumice flash strength, was preserved. Scotopic ERGs to flash strengths 0.01
to 0.1 phot cd s/m2 showed a delay of up to 20 ms before the onset of the b-wave
in two patients compared to controls. Stimulus–response functions were fitted by
Michaelis–Menten equations and showed significantly lower retinal sensitivity in
two patients than in controls (P < 0.001). Our study for the first time in the
human ERG shows changes in association with KCNJ10 mutations affecting a Muller
cell K+ channel. These data illustrate the role of KCNJ10 function in the
physiology of proximal and possibly also the distal human retina. The heteromeric inwardly rectifying Kir4.1/Kir5.1 K(+) channel underlies the
basolateral K(+) conductance in the distal nephron and is extremely sensitive to
inhibition by intracellular pH. The functional importance of Kir4.1/Kir5.1 in
renal ion transport has recently been highlighted by mutations in the human
Kir4.1 gene (KCNJ10) that result in seizures, sensorineural deafness, ataxia,
mental retardation, and electrolyte imbalance (SeSAME)/epilepsy, ataxia,
sensorineural deafness, and renal tubulopathy (EAST) syndrome, a complex
disorder that includes salt wasting and hypokalemic alkalosis. Here, we
investigated the role of the Kir5.1 subunit in mice with a targeted disruption
of the Kir5.1 gene (Kcnj16). The Kir5.1(-/-) mice displayed hypokalemic,
hyperchloremic metabolic acidosis with hypercalciuria. The short-term responses
to hydrochlorothiazide, an inhibitor of ion transport in the distal convoluted
tubule (DCT), were also exaggerated, indicating excessive renal Na(+) absorption
in this segment. Furthermore, chronic treatment with hydrochlorothiazide
normalized urinary excretion of Na(+) and Ca(2+), and abolished acidosis in
Kir5.1(-/-) mice. Finally, in contrast to WT mice, electrophysiological
recording of K(+) channels in the DCT basolateral membrane of Kir5.1(-/-) mice
revealed that, even though Kir5.1 is absent, there is an increased K(+)
conductance caused by the decreased pH sensitivity of the remaining homomeric
Kir4.1 channels. In conclusion, disruption of Kcnj16 induces a severe renal
phenotype that, apart from hypokalemia, is the opposite of the phenotype seen in
SeSAME/EAST syndrome. These results highlight the important role that Kir5.1
plays as a pH-sensitive regulator of salt transport in the DCT, and the
implication of these results for the correct genetic diagnosis of renal
tubulopathies is discussed. Recessive mutations in KCNJ10, which encodes an inwardly rectifying potassium
channel, were recently identified as the cause of EAST syndrome, a severe and
disabling multi-organ disorder consisting of epilepsy, ataxia, sensorineural
deafness and tubulopathy that becomes clinically apparent with seizures in
infancy. A Kcnj10 knockout mouse shows postnatal mortality and is therefore not
suitable for drug discovery. Because zebrafish are ideal for in vivo screening
for potential therapeutics, we tested whether kcnj10 knockdown in zebrafish
would fill this need. We cloned zebrafish kcnj10 and demonstrated that its
function is equivalent to that of human KCNJ10. We next injected splice- and
translation-blocking kcnj10 antisense morpholino oligonucleotides and reproduced
the cardinal symptoms of EAST syndrome - ataxia, epilepsy and renal tubular
defects. Several of these phenotypes could be assayed in an automated manner. We
could rescue the morphant phenotype with complementary RNA (cRNA) encoding human
wild-type KCNJ10, but not with cRNA encoding a KCNJ10 mutation identified in
individuals with EAST syndrome. Our results suggest that zebrafish will be a
valuable tool to screen for compounds that are potentially therapeutic for EAST
syndrome or its individual symptoms. Knockdown of kcnj10 represents the first
zebrafish model of a salt-losing tubulopathy, which has relevance for blood
pressure control. AIM: Recently, we reported a previously unrecognized symptom constellation
comprising epilepsy, ataxia, sensorineural deafness, and tubulopathy (EAST
syndrome) associated with recessive mutations in the KCNJ10 gene. Here, we
provide a detailed characterization of the clinical features of the syndrome to
aid patient management with respect to diagnosis, prognostic counselling, and
identification of best treatment modalities.
METHOD: We conducted a retrospective review of the detailed neurological and
neuroradiological features of nine children (four females, five males; age range
at last examination 6-20y) with genetically proven EAST syndrome.
RESULTS: All children presented with tonic-clonic seizures in infancy. Later,
non-progressive, cerebellar ataxia and hearing loss were noted. Whilst seizures
mostly responded well to treatment, ataxia proved to be the most debilitating
feature, with three patients non-ambulant. All available magnetic resoce
imaging (MRI) revealed subtle symmetrical signal changes in the cerebellar
dentate nuclei. Moreover, four patients had a small corpus callosum and
brainstem hypoplasia, and three had a small spinal cord. Regional quantitative
volumetric analysis of the images confirmed the corpus callosum and brainstem
hypoplasia and showed further patterns of variation from the norm.
INTERPRETATION: The neurological features of EAST syndrome appear to be
non-progressive, which is important for prognostic counselling. The spectrum of
EAST syndrome includes consistent abnormalities on brain MRI, which may aid
diagnosis. Further longitudinal documentation is required to determine the true
natural history of the disorder. |
When did the polio vaccine becomes available? | Inactivated poliovirus vaccine (IPV), developed in the USA by Jonas Salk in the early 1950s, was field tested in 1954, | It has been suggested that the human immunodeficiency virus (HIV), and thus the
acquired immunodeficiency syndrome (AIDS) it causes, was inadvertently
introduced to humans by the use of an oral polio vaccine (OPV) during a
vaccination campaign launched by the Wistar Institute, Philadelphia, PA, USA, in
the Belgian Congo in 1958 and 1959. The "OPV/AIDS hypothesis" suggests that the
OPV used in this campaign was produced in chimpanzee kidney epithelial cell
cultures rather than in monkey kidney cell cultures, as stated by H. Koprowski
and co-workers, who produced the OPV. If chimpanzee cells were indeed used, this
would lend support to the OPV/AIDS hypothesis, since chimpanzees harbor a simian
immunodeficiency virus, widely accepted to be the origin of HIV-1. We analyzed
several early OPV pools and found no evidence for the presence of chimpanzee
DNA; by contrast, monkey DNA is present. Routine and mass administration of oral polio vaccine (OPV) since 1961 has
prevented many millions of cases of paralytic poliomyelitis. The public health
value of this inexpensive and easily administered product has been
extraordinary. Progress of the Global Polio Eradication Initiative has further
defined the value of OPV as well as its risk through vaccine-associated
paralytic poliomyelitis (VAPP) and vaccine-derived polioviruses (VDPV). Although
both are rare, once wild poliovirus transmission has been interrupted by OPV,
the only poliomyelitis due to poliovirus will be caused by OPV. Poliovirus will
be eradicated only when OPV use is discontinued. This paradox provides a major
incentive for eventually stopping polio immunization or replacing OPV, but it
also introduces complexity into the process of identifying safe and
scientifically sound strategies for doing so. The core post eradication
immunization issues include the risk/benefits of continued OPV use, the extent
of OPV replacement with IPV, possible strategies for discontinuing OPV, and the
potential for development and licensure of a safe and effective replacement for
OPV. Formulation of an informed post eradication immunization policy requires
careful evaluation of polio epidemiology, surveillance capability, vaccine
availability, laboratory containment, and the risks posed by the very tool
responsible for successful interruption of wild poliovirus transmission. Inactivated poliovirus vaccine (IPV), developed in the USA by Jonas Salk in the
early 1950s, was field tested in 1954, and found to be safe and effective. The
year 2004 marks the golden jubilee of this breakthrough. From 1955 IPV was used
extensively in the US and polio incidence declined by more than 95 per cent.
However, in 1962, when oral poliovirus vaccine (OPV) became available, the
national policy was shifted to its exclusive use, for reasons other than science
and economics. The World Health Organisation (WHO) also adopted the policy of
the exclusive use of OPV in developing countries. Thus IPV fell into disrepute
in much of the world, while Northern European countries continued to use it. New
research led to improving its potency, reducing its manufacturing costs and
combining it with the diphtheria-tetanus-pertussis (DTP) vaccine to simplify its
administration and reduce programmatic costs. All countries that chose to
persist with IPV eliminated poliovirus circulation without OPV-induced polio or
the risk of live vaccine viruses reverting to wild-like nature. IPV is highly
immunogenic, confers mucosal immunity and exerts herd protective effect, all
qualities of a good vaccine. It can be used in harmony with the extendend
programme on immunization (EPI) schedule of infant immunisation with DTP, thus
reducing programmatic costs. During the last ten years IPV has once again
regained its popularity and some 25 industrialised countries use it exclusively.
The demand is increasing from other countries and the supply has not caught up,
leaving market forces to dictate the sale price of IPV. Anticipating such a turn
of events India had launched its own IPV manufacturing programme in 1987, but
the project was closed in 1992. Today it is not clear if we can complete the job
of global polio eradication without IPV, on account of the genetic instability
of OPV and the consequent tendency of vaccine viruses to revert to wild-like
properties. The option to use IPV is complicated since it is not yet licensed in
India, we do not manufacture it and imported vaccine would be prohibitively
costly. However, in this golden jubilee year we have much to celebrate as the
global eradication of wild polioviruses is within sight. Had we strictly
followed the principles of science and health economics, perhaps we could have
achieved success earlier and cheaper, with the absence of vaccine-induced polio
as the bonus. |
Is Annexin V an apoptotic marker? | Yes, annexin V is an early apoptotic marker. | In the early stages of apoptosis changes occur at the cell surface, which until
now have remained difficult to recognize. One of these plasma membrane
alterations is the translocation of phosphatidylserine (PS) from the inner side
of the plasma membrane to the outer layer, by which PS becomes exposed at the
external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding
protein with high affinity for PS. Hence this protein can be used as a sensitive
probe for PS exposure upon the cell membrane. Translocation of PS to the
external cell surface is not unique to apoptosis, but occurs also during cell
necrosis. The difference between these two forms of cell death is that during
the initial stages of apoptosis the cell membrane remains intact, while at the
very moment that necrosis occurs the cell membrane looses its integrity and
becomes leaky. Therefore the measurement of Annexin V binding to the cell
surface as indicative for apoptosis has to be performed in conjunction with a
dye exclusion test to establish integrity of the cell membrane. This paper
describes the results of such an assay, as obtained in cultured HSB-2 cells,
rendered apoptotic by irradiation and in human lymphocytes, following
dexamethasone treatment. Untreated and treated cells were evaluated for
apoptosis by light microscopy, by measuring the amount of hypo-diploid cells
using of DNA flow cytometry (FCM) and by DNA electrophoresis to establish
whether or not DNA fragmentation had occurred. Annexin V binding was assessed
using bivariate FCM, and cell staining was evaluated with fluorescein
isothiocyanate (FITC)-labelled Annexin V (green fluorescence), simultaneously
with dye exclusion of propidium iodide (PI) (negative for red fluorescence). The
test described, discriminates intact cells (FITC-/PI-), apoptotic cells
(FITC+/PI-) and necrotic cells (FITC+/PI+). In comparison with existing
traditional tests the Annexin V assay is sensitive and easy to perform. The
Annexin V assay offers the possibility of detecting early phases of apoptosis
before the loss of cell membrane integrity and permits measurements of the
kinetics of apoptotic death in relation to the cell cycle. More extensive FCM
will allow discrimination between different cell subpopulations, that may or may
not be involved in the apoptotic process. We have previously reported that neutrophilic granulocytes rapidly release part
of their Fc gamma RIII from the plasma membrane upon in vitro activation,
probably by proteolytic cleavage. In plasma and other body fluids, released or
soluble Fc gamma RIII has been found in considerable amounts. In the present
study, neutrophils were kept in maintece culture for 18 to 24 hours. Forty
percent of the neutrophils completely lost Fc gamma RIII, and the remainder of
the cells showed a 60% decrease in Fc gamma RIII expression on their surface.
Released Fc gamma RIII was detected in the culture supernatant. Nevertheless,
more than 90% of the cells was viable as judged by hydrolysis of fluorescein
diacetate. The presence of interferon gamma, granulocyte colony-stimulating
factor, or granulocyte-macrophage colony-stimulating factor, but not
interleukin-3 (IL-3), IL-6, or IL-8, in the culture medium increased the number
of cells that still expressed Fc gamma RIII. We found that this loss of Fc gamma
RIII was not the result of cell activation but correlated strongly with
apoptosis. The Fc gamma RIII-negative subpopulation exhibited typical
morphologic changes, such as nuclear condensation and DNA fragmentation.
Furthermore, this subpopulation appeared to have acquired the property of
binding Annexin V, a calcium-dependent, phospholipid-binding protein with high
affinity for phosphatidylserine. The external exposure of this phospholipid by
cells has been reported to occur during apoptosis. The property of Annexin V
binding was not shared by the nonapoptotic, Fc gamma RIII-positive
subpopulation. In this respect, we identified binding of Annexin V as an
convenient marker for apoptotic cells. Our results indicate that soluble Fc
gamma RIII in body fluids might be derived for a large part from neutrophils
undergoing apoptosis in the tissues. Annexin V is a Ca(++)-binding protein which is widely used as a marker for
apoptotic cells, as it binds to the phosphatidylserine residues exposed at the
surface of apoptotic cells. In this paper, we describe a method for the
immunogold-labeling of biotin-conjugated Annexin V, to detect apoptotic
thymocytes at electron microscopy. Etoposide-treated thymocytes were reacted in
tissue culture medium with biotin-conjugated Annexin V, fixed with
glutaraldehyde, and processed for resin embedding; thin sections were incubated
with antibiotin antibodies coupled with colloidal gold. Cytometric estimates of
the apoptotic index were also performed by evaluating either the DNA content
after propidium iodide staining or the light-scattering values, as well as the
positivity for fluorescein isothiocyanate-conjugated anti-biotin antibodies. At
electron microscopy, gold-labeling of Annexin V was located on the plasma
membrane only of apoptotic thymocytes and on cytoplasmic debris, likely
resulting from the typical apoptotic blebbing. Unlabeled thymocytes always
showed normal, non-apoptotic nuclear morphology. The application of Annexin V
labeling at electron microscopy will allow a more refined description of the
morphological events occurring during apoptosis. The present article compares the reliability of four previously described
cytofluorometric methods of apoptosis quantification for phenotyping apoptotic
human lymphocytes. Each of these assays detects distinct cellular alterations of
the apoptotic process. Alteration in plasma membrane integrity can be evaluated
following 7-AAD incorporation and the translocation of phosphatidylserine from
the inner to the outer layer of the plasma membrane can be detected through the
FITC annexin V staining. DNA strand breaks in apoptotic nuclei can be evidenced
by the ISNT assay and finally morphological modifications can be followed with
FSC/SSC criteria. Comparative analysis of apoptosis in cultured PBMC from
HIV-infected patients considering the FSC/SSC parameters, 7-AAD stainability and
annexin V fixation revealed that the latter identifies early apoptotic cells,
also characterized as 7-AAD(low) with a reduced FSC. Moreover these three
methods proved to be reliable and gave statistically similar results when
combined with cell surface detection of antigens such as CD4, CD8 and CD19 by
specific mAbs. Importantly, the 7-AAD assay easily allowed the identification of
debris/apoptotic bodies, which were still stained by anti-cell surface mAbs and
might therefore significantly distort the apoptosis percentage in a given
lymphocyte subset. In the present report we also point out that the ISNT assay
is not appropriate for phenotyping apoptotic lymphocytes in PBMC. Indeed it can
particularly underestimate the rate of apoptosis in the B-cell subset. This was
found to be related to the apoptosis-associated decrease in cell surface antigen
expression, which is dramatically exacerbated in the ISNT assay because of the
stripper effect of ethanol used for cell permeabilization. Finally, we propose a
three step analytical strategy to accurately phenotype apoptotic peripheral
human lymphocytes. It includes two gating steps performed on FSC/SSC criteria
and 7-AAD/FSC parameters to eliminate monocytes, granulocytes and
debris-apoptotic bodies, the third step being the phenotyping step itself,
performed in dual or triple staining experiments. Altogether these observations
emphasize that it is essential to assess critically the ability of a
cytofluorometric method to phenotype apoptotic cells in complex lymphoid
populations and that inaccurate identification of cell subsets undergoing
apoptosis can be readily overcome by gating properly the lymphoid population,
and using assays which preserve cell surface structure. The translocation of phosphatidylserine from the cytosol to the external surface
of the plasma membrane has been documented as a characteristic feature of
apoptosis in a number of cell types. Annexin V is a calcium-dependent
phospholipid binding protein that has high affinity for phosphatidylserine. To
investigate whether Annexin V provides a marker of apoptosis in the central
nervous system we carried out histochemical analysis of its binding in the
21-day-old rat brain at various time-points following a moderate unilateral
hypoxic-ischemic (HI) insult. The CA1 pyramidal neurons, which are selectively
vulnerable to HI injury and that die by an apoptotic mechanism showed an
increase in Annexin V binding 48-168 hours post-insult. Eosinophils, prominent cells in asthmatic inflammation, undergo apoptosis or
programmed cell death following deprivation of contact with survival-promoting
cytokines such as IL-5 and GM-CSF. The aim of this study was to assess a number
of techniques for the quantification of apoptosis in human eosinophils cultured
with or without IL-5 or GM-CSF and following staurosporine treatment. The
relationship between apoptosis and necrosis in eosinophils was also determined.
Eosinophils 'aged' in vitro for 48 h exhibited endonuclease DNA degradation,
apoptotic morphology, increased red autofluorescence and externalisation of
phosphatidylserine (PS) as assessed by binding of FITC-labelled annexin V.
Annexin V-FITC binding was first detectable in eosinophils maintained at 37
degrees C for 5 h post-purification. This method proved to be the most sensitive
marker of apoptosis. Morphological assessment of wet preparations of eosinophils
by Kimura staining was found to be the next most-sensitive marker followed by
increased red autofluorescence. The latter was a relatively insensitive method
for the detection of apoptosis. At 5, 20 and 24 h of culture trypan blue
exclusion indicated that eosinophil viability was high (85-90% viable cells).
However, propidium iodide (PI) staining and flow cytometry revealed that, by 24
h, approximately 75% of cells had compromised membrane integrity. Eosinophils
maintained in IL-5 or GM-CSF exhibited a non-apoptotic morphology and levels of
annexin V-FITC binding and PI uptake similar to that of freshly isolated cells.
Staurosporine (10(-5) M) treatment of eosinophils maintained in IL-5 or GM-CSF
resulted in significant levels of apoptotic morphology at 2 h (23.8% +/- 6.9, p
< 0.025) which was associated with negligible annexin binding. At 6 h
post-staurosporine treatment significant annexin-FITC binding (38% +/- 1.5, p <
0.025) was observed compared with 93% +/- 1.2 of eosinophils displaying
apoptotic morphology. Exclusion of PI demonstrated membrane integrity at all
time points up to 6 h. Thus, eosinophils aged in vitro in the absence of
viability-promoting cytokines exhibit evidence of both apoptosis and necrosis
simultaneously. In contrast, staurosporine-treated eosinophils exhibited both
membrane integrity and rapid apoptosis-associated morphological changes detected
by single step Kimura staining which preceded externalisation of PS. Either inadequate or excessive apoptosis (programmed cell death) is associated
with many diseases. A method to image apoptosis in vivo, rather than requiring
histologic evaluation of tissue, could assist with therapeutic decision making
in these disorders. Programmed cell death is associated with a
well-choreographed series of events resulting in the cessation of normal cell
function, and the ultimate disappearance of the cell. One component of apoptosis
is signaling adjacent cells that this cell is committing suicide by
externalizing phosphatidylserine to the outer leaflet of the cell membrane.
Annexin V, a 32-kDa endogenous human protein, has a high affinity for
membrane-bound phosphatidylserine. We have coupled annexin V with the
bifunctional hydrazinonicotinamide reagent (HYNIC) to prepare technetium-99m
HYNIC-annexin V and demonstrated localization of radioactivity in tissues
undergoing apoptosis in vivo. In this report we describe the results of a series
of experiments in mice and rats to characterize the biologic behavior of
(99m)Tc-HYNIC- annexin V. Biodistribution studies were performed in groups of
rats at 10-180 min after intravenous injection of (99m)Tc-HYNIC-annexin V. In
order to estimate the degree of apoptosis required for localization of
(99m)Tc-annexin V in vivo, mice were treated with dexamethasone at doses ranging
from 1 to 20 mg/kg, 5 h prior to (99m)Tc-HYNIC-annexin V administration, to
induce thymic apoptosis. Thymus was excised 1 h after radiolabeled HYNIC-annexin
V injection; thymocytes were isolated, incubated with Hoechst 33342 followed by
propidium iodide, and analyzed on a fluorescence-activated cell sorter. Each
sorted cell population was counted in a scintillation counter. To test
(99m)Tc-HYNIC-annexin V as a tracer for external radionuclide imaging of
apoptotic cell death, radionuclide imaging of Fas-defective mice (lpr/lpr mice)
and wild-type mice treated with the antibody to Fas (anti-Fas) was carried out 1
h post injection. Rat biodistribution studies demonstrated a blood clearance
half-time of less than 10 min for (99m)Tc-HYNIC-annexin V. The kidneys had the
highest concentration of radioactivity at all time points. Studies in the mouse
thymus demonstrated a 40-fold increase in (99m)Tc-HYNIC-annexin V concentration
in apoptotic thymocytes compared with the viable cell population. A correlation
of r=0.78 was found between radioactivity and flow cytometric and histologic
evidence of apoptosis. Imaging studies in the lpr/lpr and wild-type mice showed
a substantial increase of activity in the liver of wild-type mice treated with
anti-Fas, while there was no significant change, irrespective of anti-Fas
administration, in lpr/lpr mice. Excellent images of hepatic apoptosis were
obtained in wild-type mice 30 min after injection of (99m)Tc-HYNIC-annexin V.
The imaging results were consistent with histologic analysis in these animals.
In conlusion, these studies confirm the value of (99m)Tc-HYNIC-annexin V uptake
as a marker for the detection and quantification of apoptotic cells in vivo. Monocytes/macrophages (Mphis), the predomit cell types in subacute and
chronic inflammation, are attracted to and activated by monocyte chemotactic
peptide-1 (MCP-1). Mphis promote the resolution of inflammation through the
induction of apoptosis and phagocytosis of senescent (spent) and bystander
(superfluous) granulocytes. We wished to determine whether MCP-1, which
selectively binds to Mphis, could be used to image subacute and chronic
inflammation. We also sought to image granulocyte apoptosis within these lesions
with technetium-99m labeled annexin V, a marker of apoptotic cells. Sterile
inflammation was induced in 45 12-week-old male Sprague-Dawley rats by deep
intramuscular injection of turpentine into the right thigh. Groups of four to
six animals were then imaged 1 h after tail vein injection of 37-148 MBq (1-4
mCi) of 99mTc-labeled MCP-1 or annexin V 1-14 days after turpentine treatment.
Image analysis showed significantly greater activity of both MCP-1 and annexin V
in inflamed thighs than in control thighs (165%-290% and 188%-313%,
respectively; P<0.01) on days 1-5 after turpentine injection. Dual
autoradiography in animals co-injected with iodine-125 labeled bovine serum
albumin on days 1 and 4 showed specific location of MCP-1 to infiltrating Mphis
while annexin V localized to focal zones of apoptosis within granulocytic
infiltrates adjacent to abscess cavities. Scintillation well counting on day 5
demonstrated significantly higher (P<0.005) ratios of abscess to control thigh
specific activities for MCP-1 (5.83+/-2.17) and annexin V (9.24 +/- 2.8) as
compared to 125I-labeled bovine serum albumin (3.11 +/- 0.65). No significant
increases in uptake were noted at imaging or ex vivo analyses on days 13 and 14,
when lesions were predominately fibrotic. It is concluded that 99mTc-labeled
MCP-1 and 99mTc-labeled annexin V both localize in zones of subacute
inflammation, reflecting the density of Mphis and the incidence of apoptotic
granulocytes, respectively. These agents may be useful in the characterization
of subacute inflammation. In a previous study, we demonstrated that apoptosis increased according to
gestational age, accounting partly for the presence of free fetal DNA in
maternal plasma and serum. Using simultaneous TUNEL assay and FISH analysis, we
identified the fetal origin of part of the apoptotic cell population, but very
few TUNEL-positive cells showed hybridization signals since they were in a late
apoptosis stage and nuclei were destroyed. In the present study, the apoptotic
cell population was identified immunocytochemically using Annexin V, a marker of
cells in an early stage of apoptosis. The mean apoptosis rate in mononuclear
cells isolated from the peripheral blood of 20 pregt women in the 16th to
19th week of pregcy with Annexin V was 6.8 +/- 0.5% (range: 4.2-8.1%)
compared to 6.14 +/- 0.5% (range: 3.7-6.9%) obtained with ethidium bromide
staining. FISH using X and Y chromosome-specific probes was applied in 11 cases
known to be carrying male fetuses. Eighty percent of Annexin V+ cells showed
hybridization signals, while the proportion of apoptotic cells showing X/Y
signals was 7.8% (range: 5-12%). Although our results are still preliminary, it
seems that use of Annexin V antibody to detect the apoptotic cell population
improves FISH analysis and allows a more accurate estimate of the proportion of
fetal cells among the apoptotic cell population. 99mTc-Labeled annexin V has been used for the imaging of tumor apoptosis induced
by chemotherapy. However, owing to the short half-life of annexin V, multiple
injections of the radiotracer are necessary to capture the peak apoptotic
activity. In this study, we evaluated the imaging properties of an
(111)In-labeled, long-circulating annexin V.
METHODS: Both polyethylene glycol (PEG) and the metal chelator
diethylenetriaminepentaacetic acid (DTPA) were simultaneously introduced to
annexin V or ovalbumin through the use of a heterofunctional PEG precursor.
Imaging studies were performed in mice bearing subcutaneously inoculated human
mammary MDA-MB-468 tumors. The mice were treated with poly(L-glutamic
acid)-paclitaxel, monoclonal antibody C225, or a combination of poly(L-glutamic
acid)-paclitaxel and C225, followed by intravenous injection of
(111)In-DTPA-PEG-annexin V. Images were acquired 48 h after the injection of the
radiotracer. Autoradiography and TUNEL (terminal
deoxynucleotidyltransferase-mediated dUTP nick-end labeling) staining were
performed on adjacent tumor slices for the localization of apoptotic cells. The
imaging properties of unPEGylated annexin V and PEGylated ovalbumin were also
determined to permit assessment of the specificity of (111)In-DTPA-PEG-annexin
V.
RESULTS: Tumor apoptotic index increased from 1.67% +/- 0.31% at baseline to
7.60% +/- 0.72% and 11.07% +/- 1.81%, respectively, 4 d after treatment with
poly(L-glutamic acid)-paclitaxel or combined poly(L-glutamic acid)-paclitaxel
and C225. Tumor uptake (percentage of injected dose per gram of tumor [%ID/g])
of PEGylated (111)In-DTPA-PEG-annexin 4 d after treatment was significantly
higher in tumors treated with poly(L-glutamic acid)-paclitaxel (10.76 +/- 1.38
%ID/g; P = 0.001) and with combined poly(L-glutamic acid)-paclitaxel and C225
(9.84 +/- 2.51 %ID/g; P = 0.029) than in nontreated tumors (6.14 +/- 0.67
%ID/g), resulting in enhanced visualization of treated tumors.
(111)In-DTPA-PEG-annexin V distributed into the central zone of tumors, whereas
(111)In-DTPA-annexin V was largely confined to the tumor periphery. Furthermore,
uptake of (111)In-DTPA-PEG-annexin V by tumors correlated with apoptotic index
(r = 0.87, P = 0.02). Increase in tumor uptake of the nonspecific PEGylated
protein (111)In-DTPA-PEG-ovalbumin was also observed after poly(L-glutamic
acid)-paclitaxel treatment (55.6%), although this increase was less than that
observed for (111)In-DTPA-PEG-annexin V (96.7%).
CONCLUSION: Increased uptake of and improved visualization with
(111)In-DTPA-PEG-annexin V in solid tumors after chemotherapy are mediated
through both specific binding to apoptotic cells and nonspecific retention of
macromolecular contrast agents in the tumors. (111)In-Labeled, PEGylated annexin
V may be used to assess tumor response to chemotherapy. It has been proposed that direct and indirect mechanisms contribute to the
unresolved issue of CD4(+) T-cell depletion that results from HIV-1 infection.
We recently reported that plasma levels of tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL) are elevated in HIV-1-infected patients and
that they correlate with viral load. The present study investigates the
expression of TRAIL death receptor 5 (DR5) in the peripheral-blood mononuclear
cells (PBMCs) of HIV-1-infected patients and its role in CD4(+) T-cell death.
DR5 expression was elevated and associated with the apoptotic marker annexin V.
Apoptosis was reduced in CD4(+) T cells when cultured with anti-DR5 antibody.
CD4(+), but not CD8(+), T cells from uninfected donors expressed TRAIL, DR5, and
activated caspase-3 when cultured with infectious or noninfectious HIV-1,
resulting in preferential apoptosis of CD4(+) T cells. TRAIL, caspase-3
expression, and apoptosis were type 1 interferon (IFN) dependent. Induction of
apoptosis and DR5 expression required glycoprotein 120 (gp120)-CD4 interaction.
Finally, we analyzed DR5 expression by CD4(+) T cells in highly active
antiretroviral therapy (HAART)-treated patients. The decreased viral loads and
increased CD4 counts of HAART-responsive patients were associated with a
decrease in DR5 mRNA expression by CD4(+) T lymphocytes. We propose a novel
model in which a type 1 IFN-regulated TRAIL /DR5 mechanism induces apoptosis of
HIV-1-exposed CD4(+) T cells. BACKGROUND: The survival of endometriotic cells in the ectopic site has been
investigated from the aspect of susceptibility of endometriotic tissues to
apoptosis. In order to investigate the nature of abnormal survival of
endometriotic cells in ectopic locations, we compared drug-induced apoptosis in
endometrial and endometriotic cells.
METHODS: Endometrial stromal cells were obtained from normal endometrium in 11
patients who underwent hysterectomy for leiomyoma without endometriosis.
Endometriotic cells were isolated from the chocolate cyst linings of the ovary
in 13 patients who underwent laparoscopic surgery. Cells were cultured in the
presence or absence of staurosporine. Apoptotic cell death was evaluated by
staining nuclei with propidium iodide and phosphatidylserine (a marker of early
apoptotic events) with Annexin V as well as by DNA fragmentation assay. The
number of viable cells was estimated by modified MTT
[3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide WST-8] assay.
RESULTS: After 3 h of exposure to staurosporine, >50% of the endometrial stromal
cells became Annexin V positive. In contrast, >30% of the endometriotic cells
were Annexin V positive. DNA fragmentation was not clearly induced in the
endometriotic cells. Less than 20% of the endometrial cells survived after
staurosporine exposure, while >40% of the endometriotic cells survived. Cell
death induced by staurosporine was partially blocked by incubation with the
caspase inhibitor, N-benzyoxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl-ketone
(ZVAD-fmk), suggesting that a caspase cascade may play a role in the cell death
process.
CONCLUSIONS: Attenuated susceptibility to apoptosis in endometriotic stromal
cells may be associated with abnormal survival in ectopic sites in an
environment that is probably unfavourable. These results may be implicated in
the pathophysiology of endometriosis. Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates
apoptotic spermatozoa based on the externalization of phosphatidylserine
residues. The procedure delivers two sperm fractions: annexin V-negative
(nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine
whether the sperm fertilizing potential can be improved by selecting a
nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to
separation on a density gradient followed by MACS. Extent of apoptosis was
assessed by measuring levels of activated caspase 3 using fluorescein-labeled
inhibitors of caspase, alterations in mitochondrial membrane potential (MMP)
using a lipophilic cationic dye, and DNA fragmentation using terminal
deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay.
The sperm fertilization potential was assessed using hamster oocyte penetration
assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin
V-negative sperm displayed superior quality in terms of high motility, low
caspase 3 activation, MMP integrity, and small extent of DNA fragmentation.
Annexin V-negative sperm demonstrated higher oocyte penetration capacity but
comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the
annexin V-positive sperm presented with poor quality and fertilization
potential. The oocyte penetration rate was negatively correlated with apoptotic
marker expression, whereas SCD following ICSI was only associated with apoptosis
on sperm-damaged membranes. We conclude that apoptosis appears to impact
sperm-oocyte penetration rate; however, it does not seem to affect early stages
of fertilization such as SCD in spermatozoa of healthy donors. The selection of
nonapoptotic sperm by MACS may be used to enhance results of in vitro
fertilization by increasing sperm-oocyte penetration. PURPOSE: The purpose of this study was to assess the cytotoxic effects of the
fluoquinolone ofloxacin and the aminoglycoside netilmicin on stromal human
keratocytes in vitro.
METHODS: Cultured human keratocytes were exposed to various concentrations of
ofloxacin or netilmicin (0.16-5.0 mg/mL). Both cell proliferation (MTT assay)
and cell morphology (phase-contrast microscopy) were evaluated after 1, 4, 12,
and 24 hours of incubation. Measurement of annexin V binding performed in
association with the dye exclusion test using propidium iodide (PI) was also
performed by FACS analysis after 4 hours of exposure.
RESULTS: Both antimicrobials induced dose- and time-dependent morphologic
changes in keratocytes, yet the effects of netilmicin were minimal. After 24
hours of exposure, both drugs induced a dose-dependent inhibition of cell
proliferation; however, ofloxacin demonstrated significantly more toxic effects
than netilmicin (t test for ED50 values, P < 0.0001). Statistical differences
between 2 antibiotics start at concentrations above 1.25 mg/mL (ANOVA with
post-hoc test, P < 0.01). Expression of the apoptotic marker annexin V was
unaffected by antibiotic exposure, whereas the uptake of the necrotic marker PI
was increased by ofloxacin (5 mg/mL) but not by netilmicin (ofloxacin versus
netilmicin, ANOVA, P < 0.05).
CONCLUSIONS: Relative effects of aminoglycosides and fluoroquinolones on stromal
keratocytes appear to be different: netilmicin was shown to be significantly
less toxic than ofloxacin. This finding is particularly relevant in deciding the
optimal antibiotic to be applied in clinical situations in which the epithelium
is absent or compromised, as after photorefractive keratectomy, alkali burns, or
ulcerative keratitis. OBJECTIVE: To investigate the effects of quercetin on cell morphology and VEGF
expression of acute myeloblastic leukemia cells NB4 in vitro.
METHODS: The cytomorphology of NB4 cells was assessed by Wright-stain, apoptosis
rate by apoptotic marker Annexin V, and VEGF secretion level by ELISA.
RESULTS: Typical apoptosis was found in NB4 cells after treatment with
quercetin. Apoptotic marker Annexin V analysis showed that the apoptotic rate of
NB4 cells was increased after treatment with quercetin. The secretion of VEGF of
NB4 cells was significantly decreased after treatment with quercetin.
CONCLUSION: Quercetin can induce apoptosis and inhibit secretion of VEGF in NB4
leukemia cells. PURPOSE: It is well established that experimental testicular torsion induces
germ cell specific apoptosis. Annexin V (BD Pharmingentrade mark) binds
phosphatidylserine that becomes exposed on the cell membrane in apoptotic cells.
In vivo detection of apoptotic cells with fluorescently labeled annexin V is an
emerging technique that we evaluated for detecting apoptotic germ cells in a
mouse model of testicular torsion.
MATERIALS AND METHODS: Annexin V labeled with an indocyanine fluorophore
(bisfunctional succinimidyl ester of cyanine 5.5) (Amersham, Little Chalfont,
United Kingdom) was injected intravenously in mice 18 hours after the repair of
unilateral 720-degree testicular torsion for 2 hours. Serial fluorescence images
were obtained 21, 24, 28 and 42 hours after torsion repair. Relative fluorophore
localization was visualized in vivo using an optical small animal imaging system
mounted with a filter in near infrared light. Average fluorescence intensity in
torsed and sham testes was quantified in images of testes in situ exposed
through an abdominal incision and in ex vivo testes.
RESULTS: A significant increase in fluorescence intensity was found in images of
torsed vs sham operated testes. This was seen in ex vivo, exposed and in vivo
testes (215%, 250% and 161%, respectively, p <0.05). Bisfunctional succinimidyl
ester of cyanine 5.5 conjugated to dehydrogenase, a protein with a size similar
to that of annexin V, was used to assess for capillary leakage. It was also more
localized to the torsed testis relative to its contralateral sham control
whether exposed or ex vivo (174% and 176%, respectively).
CONCLUSIONS: To our knowledge this study demonstrates for the first time the
possibility of in vivo near infrared fluorescence imaging of apoptotic germ
cells after testicular torsion in mice. It shows important confounding factors
that must be considered as this new imaging technique is developed for detecting
apoptotic cells in vivo in testes or in any other organ. The mechanisms by which Ca(2+)-independent phospholipase A(2) (iPLA(2)) mediates
cell growth in p53-positive LNCaP and p53-negative PC-3 prostate cancer cell
lines were studied. Exposure of cells to the iPLA(2) selective inhibitor
bromoenol lactone (BEL; 0-20 microM) induced concentration- and time-dependent
decreases in cell growth based on 3-(4,
dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide staining and cell number.
Decreased cell growth was not caused by cell death as BEL exposure did not alter
nuclear morphology or increase annexin V (apoptotic cell marker) or propidium
iodide (necrotic cell marker) staining after 48 h. Decreased growth correlated
to a G(1)/G(0) arrest in LNCaP cells and aG(2)/M arrest in PC-3 cells. In LNCaP
cells, G(1) arrest was preceded by time- (0-48 h) and concentration-dependent
(0-10 microM) increases in the expression of the tumor suppressor protein p53
and the cyclin-dependent kinase inhibitor p21. Increases in p53 expression
preceded increases in p21 expression by 8 h. In LNCaP cells, BEL treatment
decreased the expression of the p53 antagonist Mdm2, while increasing Akt
phosphorylation. BEL treatment also increased Akt phosphorylation in PC-3 cells,
but Mdm2 was not detected. The ability of BEL to increase Akt phosphorylation
was inhibited by the phosphoinositide 3-kinase inhibitor LY294002
[2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]. BEL treatment also decreased
agonist-induced activation of the epidermal growth factor receptor. These data
suggest that inhibition of iPLA(2) decreases prostate cancer cell growth by
p53-dependent and independent mechanisms. Furthermore, alterations in Mdm2 and
epidermal growth factor receptor activation following BEL exposure suggest novel
roles for iPLA(2) in prostate cancer cell signaling. Recently, we developed a simple and potent therapeutic liposome cancer vaccine
consisting of a peptide antigen and a cationic lipid. The molecular mechanism of
the adjuvanticity of cationic liposome was studied and described in the current
report. First, cationic DOTAP liposome, but not the neutral liposome DOPC, was
shown to generate reactive oxygen species (ROS) in mouse bone marrow-derived
dendritic cells (BMDC). ROS generation by DOTAP was required for ERK and p38
activation and downstream chemokine/cytokine induction. Furthermore, ROS were
shown to be involved in the expression of the co-stimulatory molecules CD86/CD80
induced by DOTAP. However, as the DOTAP concentration increased from 50 to 800
microM, the apoptotic marker Annexin V and ROS double positive cells increased,
suggesting that high dose of DOTAP-generated ROS causes cell apoptosis. In vivo,
optimal amount of ROS in the draining lymph nodes (DLN) and anti-tumor (HPV
positive TC-1 tumor) activity induced by E7 peptide (antigen derived from E7
oncoprotein of human papillomavirus (HPV) type 16) formulated in 100 nmol DOTAP
were attenuated by incorporating DOPC in the formulation, suggesting that ROS
are essential for the vaccine induced anti-tumor activity. Moreover, 600 nmol
DOTAP/E7 generated huge amount of ROS in the DLN and showed no activity of tumor
regression. Interestingly, 600 nmol DOTAP/E7-induced ROS were tuned down to the
same level induced by 100 nmol DOTAP/E7 by adding DOPC in the formulation and
this formulation showed tumor regression activity. In conclusion, DOTAP is an
active DC stimulator resulting in the activation of ERK and p38 and induction of
chemokines, cytokines and co-stimulatory molecules mediated by appropriate
amount of ROS. Our data elucidated an important mechanism of adjuvant activity
of cationic liposome and could facilitate rational design of synthetic lipid
based adjuvants and vaccine formulation. Abscess formation causes systemic and localized up-regulation of neutrophil
[polymorphonuclear leukocytes (PMNs)] signaling pathways. In the abscess,
following bacterial ingestion or PMN activation by inflammatory mediators, PMN
apoptosis is elevated and leads to the externalization of phosphatidylserine.
Annexin-V (AnxV) has been shown to have high affinity to externalized
phosphatidylserine. We hypothesized that (99m)Tc-AnxV will target high densities
of apoptotic PMNs and image abscesses. AnxV, conjugated with
hydrazinenicaotinamide (HYNIC), was labeled with reduced (99m)TcO(4)(-) and its
purity was determined by instant thin-layer chromatography. Apoptosis was
induced in isolated human PMNs by incubation in 2% saline for 17 and 22 h at 37
degrees C. PMNs were then incubated with (99m)Tc-HYNIC-AnxV and associated
(99m)Tc was determined. Abscesses were induced in mice by intramuscular
injection of bacteria or turpentine. Following intravenous administration of
(99m)Tc-HYNIC-AnxV, mice were imaged and tissue distribution studied at 4 and 24
h. Radiochemical purity of (99m)Tc-HYNIC-AnxV was 84.9+/-8.11%. At 17 h,
(99m)Tc-HYNIC-AnxV bound to apoptotic PMNs was 71.6+/-0.01% and 48.6+/-0.01% for
experimental and control cells, respectively (P=.002). At 22 h, experimental
cells retained 74.9+/-0.02% and control cells retained 47.2+/-0.02% (P=.005).
(99m)Tc-HYNIC-AnxV associated with bacterial abscesses was 1.25+/-0.09 and
3.75+/-0.83 percent injected dose per gram (%ID/g) at 4 and 24 h compared to
turpentine abscesses which was 1.02+/-0.16 and 0.72+/-0.17 %ID/g at 4 (P<or=.05)
and 24 h (P<or=.01). (99m)Tc-HYNIC-AnxV represents a minimally invasive and
promising agent to image and potentially distinguish between infectious and
inflammatory abscesses. OBJECTIVE: Microalbuminuria is associated with an increased risk for all-cause
and cardiovascular mortality, but the pathophysiologic mechanism underlying the
association between urinary albumin excretion and cardiovascular disease remains
unclear. Here, we tested the hypothesis that enhanced endothelial apoptotic
microparticles and decreased endothelial progenitor cell (EPC) levels might
contribute to the pathophysiology of microalbuminuria or macroalbuminuria in
cardiovascular disease.
METHODS: Flow cytometry was used to assess endothelial cell apoptosis and
circulating EPC levels by quantification of circulating CD31/annexin V apoptotic
microparticles and EPC markers (defined as KDRCD133, CD34CD133, CD34KDR) in
peripheral blood.
RESULTS: In total, 125 patients with hypertension were enrolled in the study, of
whom 80 patients (64%) were with normoalbuminuria (albumin excretion rate of <20
microg/min, overnight urine samples), 35 patients (28%) with microalbuminuria
(an albumin excretion rate of 20-200 microg/min), and 10 patients (8%) with
macroalbuminuria (an albumin excretion rate >200 microg/min). Compared to
hypertensive patients with normoalbuminuria, patients with microalbuminuria or
macroalbuminuria had significantly more diabetes (P = 0.005), higher systolic
blood pressure (P = 0.018), and elevated serum creatinine levels (P < 0.001).
Among the three groups, patients with microalbuminuria or macroalbuminuria had
significantly increased CD31/annexin V apoptotic microparticles (1.8 +/- 2.2
versus 3.0 +/- 4.3 versus 5.2 +/- 6.2%, P = 0.044) and decreased circulating EPC
numbers (P < 0.05). By multivariate analysis, CD31/annexin V apoptotic
microparticle level was an independent predictor of urinary albumin excretion
rate in hypertensive patients (P < 0.001). Microparticles isolated from
hypertensive patients with microalbuminuria or macroalbuminuria attenuated EPC
proliferation, migration, and increased H2O2 production, cellular senescence and
apoptosis in comparison with those from hypertensive patients with
normoalbuminuria.
CONCLUSION: These findings suggest that hypertensive patients with
microalbuminuria or macroalbuminuria have increased endothelial apoptotic
microparticles and decreased circulating EPC levels, which may contribute to
atherosclerotic disease progression and enhanced cardiovascular risk in
hypertensive patients with nephropathy. To examine the effects of standardized (reference) tobacco preparations on human
oral cavity cells, two oral squamous cell carcinoma cell lines (101A, 101B) and
normal human gingival epithelial cells (HGEC) were treated with cigarette smoke
total particulate matter (TPM), smokeless tobacco extracted with complete
artificial saliva (ST/CAS), or whole-smoke conditioned media (WS-CM). EC-50
values, as determined by sulforhodamine B assays, varied among the cell types
and agents. When normalized to nicotine content, cytotoxicity for WS-CM and TPM
was higher compared to that observed with ST/CAS. Nicotine alone had no or only
minimal cytotoxicity for all cell types in the applied range. Activation of
pro-apoptotic caspase-3 was examined in all cell types at their respective EC-50
doses for the three agents. TPM, but not ST/CAS or WS-CM significantly activated
caspase-3 in all three cell types. Fluorescence-activated cell sorting (FACS)
for expression of the early apoptosis marker Annexin V and for nuclear staining
by 7-aminoactinomycin (7-AAD) revealed different extents of apoptosis versus
non-apoptotic cell death for the three agents. These data characterize
differential responses of normal and maligt oral cells after exposure to TPM,
ST/CAS, or WS-CM. They assist in understanding differential effects of
combustible versus non-combustible tobacco products, and in identifying novel
biomarkers for tobacco smoke exposure and effect in the oral cavity. The Ca(2+)-dependent binding of Annexin V to phosphatidylserine on cell surfaces
is a reliable marker for apoptosis that is widely used in flow cytometry based
apoptosis assays. In this paper, we report a new class of Annexin V-based probes
for apoptosis. Luciferase from Renilla reniformis (RLuc) was linked to Annexin V
and expressed successfully in a soluble form in Escherichia coli BL21 (DE3). The
new probe, Rluc/Annexin V, was purified and functionally assayed for detection
of apoptosis in actinomycin D-induced apoptotic Jurkat cells. Moreover, the
spontaneous apoptosis in neutrophils was shown using the new probe. The results
indicate that Rluc/Annexin V can bind to the apoptotic cells, and the signal of
Renilla luciferase can be detected by luminometric measurements. The
availability of Rluc/Annexin V may be of potential commercial interest for
improving current apoptosis assays. A new strategy for sensitive detection of early stage apoptosis was proposed
based on silver-enhanced gold oparticle (GNP) label method. Annexin-V
modified substrate was constructed via layer-by-layer (LBL) method for specific
capture of early stage apoptotic Jurkat cells. A new kind of aminophenylboronic
acid modified gold oparticle (APBA-GNP) was synthesized and utilized for
labeling cells, followed by silver enhancement. Anodic stripping voltammetry
(ASV) was applied to sensitive detection of Ag(+) dissolved from the deposited
silver particles, which reflected the number of cells. A good linear range from
1 × 10(2) to 3.5 × 10(3) cells was achieved, with a detection limit of 38
apoptotic cells. Moreover, the gray color of silver enhancement could be
observed by the naked eye, which could be used to tell apoptotic cells apart
from normal cells. Therefore, using the silver-enhanced GNP label method,
apoptotic cells could not only be sensitively detected via electrochemical
technique, but also can be discriminated from normal cells by the naked eye. We tested the effect of two different concentrations (150μg/l and 0.15μg/l) of
mycotoxin zearalenone (ZEA) on the reproductive parameters and expression of
testicular genes in male mice. In adult males, no reduction of body or
reproductive organ weight was observed, and the seminiferous tubules were
morphologically normal with ongoing spermatogenesis. However, we found decreased
sperm concentration, increase of morphologically abnormal spermatozoa and
increased binding of apoptotic marker annexin V. This study was also focused on
the evaluation of gene expression profiles of 28 genes playing important roles
during the processes occurring in the testicular tissue. We detected changes in
the expression of genes important for proper spermatogenesis. Surprisingly, we
observed a stronger effect after exposure to the lower dose of ZEA. Small cell lung cancer (SCLC) is an aggressive disease with a poor prognosis.
These cancers are deficient in glucocorticoid receptor (GR) expression, and
therefore, resistant to glucocorticoids. Overexpression of the GR both in vivo
and in vitro leads to apoptotic cell death suggesting that loss of GR is
favorable for cancer growth. Indeed, the GR promoter is silenced in SCLC cells
by methylation. We now show that treatment of the SCLC cell line (DMS79) cells
with the demethylating agent, 5-aza-2'-deoxycytidine (5-aza), results in
significant endogenous re-expression of both GRα and the ligand-independent
GR-P. The GR gene has a complex promoter region comprising nine alternative
promoters, the proximal seven of which lie within a CpG island. The endogenous
re-expression seen is attributed to the constitutive promoters 1B and 1C and 1J
but predomitly 1F, which we show to be heavily methylated in SCLC cells. Flow
cytometric analysis using the apoptotic marker, Annexin V, shows that this
endogenous re-expression is sufficient to drive the SCLC cells to apoptosis.
Apoptotic induction is specific to GR re-expression as cotreatment with 5-aza
and the GR antagonist, RU486 prevented apoptosis. Of the three functional GR
domains (the DNA binding domain, ligand binding domain, and transactivation
domain), we identified that the transactivation domain is essential for
apoptosis in SCLC. The discovery that endogenous re-expression of the GR in SCLC
cells is sufficient to induce apoptotic cell death by reversing a cancer-driven
DNA methylation effect may lead to the development of novel adjunct therapies. Author information:
(1)Department of Rheumatology and Clinical Immunology, University of Groningen,
University Medical Center Groningen, Groningen, The Netherlands, Division of
Rheumatology, Department of Internal Medicine, Seoul National University College
of Medicine, Seoul, Korea, Department of Pathology and Medical Biology and
Department of Laboratory Medicine, Section Medical Immunology, University of
Groningen, University Medical Center Groningen, Groningen, The Netherlands
Department of Rheumatology and Clinical Immunology, University of Groningen,
University Medical Center Groningen, Groningen, The Netherlands, Division of
Rheumatology, Department of Internal Medicine, Seoul National University College
of Medicine, Seoul, Korea, Department of Pathology and Medical Biology and
Department of Laboratory Medicine, Section Medical Immunology, University of
Groningen, University Medical Center Groningen, Groningen, The Netherlands
[email protected].
(2)Department of Rheumatology and Clinical Immunology, University of Groningen,
University Medical Center Groningen, Groningen, The Netherlands, Division of
Rheumatology, Department of Internal Medicine, Seoul National University College
of Medicine, Seoul, Korea, Department of Pathology and Medical Biology and
Department of Laboratory Medicine, Section Medical Immunology, University of
Groningen, University Medical Center Groningen, Groningen, The Netherlands. BACKGROUND/AIMS: Des-gamma-carboxy prothrombin (DCP), an aberrant prothrombin
produced by hepatocellular carcinoma (HCC) cells, is known as a marker for HCC.
Recent studies indicated that high levels of DCP are associated with the
maligt potential of HCC. In this study, we aimed to investigate the
association of DCP with gefitinib treatment failure in HCC and whether DCP
counteracts gefitinib-induced growth inhibition and apoptosis of HCC.
METHODS: The experiments were performed in HCC cell lines HepG2 and PLC/PRF/5.
The effects of gefitinib on HCC in the presence or absence of DCP were evaluated
by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)
assay. Apoptotic cells were identified by Annexin V-FITC/PI staining. Western
blotting was performed to analyze the expressions of molecules related to the
apoptotic caspase-dependent pathway and epidermal growth factor receptor (EGFR)
pathway.
RESULTS: Gefitinib inhibited HCC cell proliferation and induced apoptosis in HCC
cells. The effects of gefitinib on HCC cells were antagonized by DCP. In the
presence of DCP, HCC cells were resistant to the gefitinib-induced inhibition of
proliferation and stimulation of apoptosis. DCP prevented the activation of the
apoptotic caspase-dependent pathway induced by gefitinib. These antagonistic
effects of DCP also arose from its ability to up-regulate EGFR, c-Met and
hepatocyte growth factor (HGF) in HCC cells.
CONCLUSION: DCP antagonized gefitinib-induced HCC cell growth inhibition by
counteracting apoptosis and up-regulating the EGFR pathway. High levels of DCP
might thus lead to low response rates or possibly no response to gefitinib in
patients with HCC. Nonalcoholic steatohepatitis (NASH) is characterized by extensive hepatic
monocyte infiltration and monocyte-derived macrophages have an important role in
regulating the disease evolution. However, little is known about the functional
changes occurring in liver macrophages during NASH progression. In this study,
we investigated phenotypic and functional modifications of hepatic macrophages
in experimental NASH induced by feeding C57BL/6 mice with a methionine-choline
deficient (MCD) diet up to 8weeks. In mice with steatohepatitis liver
F4/80-positive macrophages increased in parallel with the disease progression
and formed small clusters of enlarged and vacuolated cells. At
immunofluorescence these cells contained lipid vesicles positive for the
apoptotic cell marker Annexin V suggesting the phagocytosis of apoptotic bodies
derived from dead fat-laden hepatocytes. Flow cytometry revealed that these
enlarged macrophages expressed inflammatory monocyte (CD11b, Ly6C, TNF-α)
markers. However, as compared to regular size macrophages the enlarged sub-set
was characterized by an enhanced production of arginase-1 and of the
anti-inflammatory mediators IL-10 and annexin A1. Similar vacuolated macrophages
producing annexin A1 were also evident in liver biopsies of NASH patients. In
mice with NASH, the accumulation of enlarged F4/80(+) cells paralleled with a
decline in the expression of the macrophage M1 activation markers iNOS, IL-12
and CXCL10, while the levels of M2 polarization markers arginase-1 and MGL-1
were unchanged. Interestingly, the lowering of IL-12 expression mainly involved
the macrophage sub-set with regular size. We conclude that during the
progression of NASH fat accumulation within liver macrophages promotes the
production of anti-inflammatory mediators that influence hepatic inflammatory
responses. Exendin-4 (ex-4) is a long-acting glucagon-like peptide-1 receptor (GLP-1R)
agonist which exerts beneficial effects on glycemic control and promotes cell
viability. In the present study, we investigated the anti-apoptotic effects of
ex-4, as well as the potential mechanisms responsible for these effects in rat
bone marrow-derived mesenchymal stem cells (BM-MSCs) under conditions of oxygen,
glucose and serum deprivation (OGD). The apoptosis of the MSCs was induced by
subjecting the cells to OGD conditions for 4 h and was detected by Annexin V/PI
and Hoechst 33258 staining. The MSCs were pre-conditioned with ex-4 for 12 h
prior to being subjected to OGD conditions, and the expression levels of an
apoptotic marker (cleaved caspase-3), endoplasmic reticulum (ER) stress markers
[phosphorylated (p-)protein kinase RNA-like endoplasmic reticulum kinase (PERK),
PERK, binding immunoglobulin protein (BIP), activating transcription factor 4
(ATF-4) and C/EBP homologous protein (CHOP)], as well as those of a survival
marker (Bcl-2) were measured by western blot analysis. Furthermore, the mRNA
levels of ATF-4 and CHOP were determined by RT-qPCR. ELISA was used to examine
the activity of intracellular cAMP. Moreover, the GLP-1R antagonist, exendin9-39
(ex9-39), the protein kinase A (PKA) inhibitor, H89, and small interfering RNA
(siRNA) targeting rat ATF-4 and CHOP were co-incubated with the MSCs. The
apoptotic rate was markedly diminished following pre-conditioning with ex-4 in a
dose‑dependent manner (P<0.05). The ER stress markers, p-PERK, BIP, ATF-4 and
CHOP, were upregulated in the cells subjected to OGD conditions. Ex-4
pre-conditioning significantly decreased the mRNA and protein levels of ATF-4
and CHOP (P<0.05), and increased the activity of intracellular cAMP (P<0.05).
Furthermore, the anti-apoptotic effects of ex-4 were almost reversed by
treatment with either H89 or ex9-39 (P<0.05); transfection with siRNA-CHOP
significantly reduced the apoptotic rate of the MSCs and did not impair the
cytoprotective effects of ex-4. Taken together, these findings suggest that ex-4
protects rat BM-MSCs from OGD-induced apoptosis through the activation of the
PKA/cAMP pathway and the attenuation of the ER stress signaling pathway. Ex-4
may thus prove to be a therapeutic agent with the potential to improve the
viability of MSCs in the ischemic milieu, and consequently, to optimize the
therapeutic effects of MSC therapy in acute myocardial infarction. |
Which are the lipid lowering drugs administered in patients with Coronary Artery Disease (CAD)? | The lipid lowering drugs administered in patients with Coronary Artery Disease (CAD) are:
1) Statins
2) Fibrates
3) Resins
4) Niacin
5) Cholesterol absorption-inhibiting drugs. | BACKGROUND: Recent studies have confirmed that treatment with lipid-lowering
drugs decreases cardiovascular morbidity and mortality in primary as well as
secondary prevention of cardiovascular diseases. In 1999, new Swiss
recommendations for treatment with lipid-lowering drugs have been published. We
therefore performed a study to estimate the proportion of patients with coronary
artery disease requiring lipid-lowering drugs.
METHODS: We included 637 patients with coronary heart disease who were referred
for coronary angiography during 1991-1993. We calculated the proportion of
patients requiring lipid-lowering drugs according to the new Swiss guidelines,
and compared them with European and US guidelines.
RESULTS: According to the 1999 Swiss recommendations, 79% of the study
population would have qualified for lipid-lowering treatment (males 80%, females
78%; patients aged up to 69 years 80%, patients aged 70 years and over 73%).
Agreement with both the Recommendations of the Second Joint Task Force of
European and other Societies on Coronary Prevention, and the ACC/AHA Guidelines
for Management of Patients with Acute Myocardial Infarction, was 96% (Kappa 0.79
and 0.83 respectively).
CONCLUSION: A large proportion of patients with coronary artery disease
qualifies for treatment with lipid-lowering drugs. The new Swiss recommendations
closely agree with European and US guidelines. High-resolution magnetic resoce imaging (MRI) with flow suppression not only
provides useful information on luminal and wall areas of the carotid artery but
also can identify the principal tissue components of the carotid atherosclerotic
plaque. The effects of intensive lipid-lowering therapy on these MRI tissue
characteristics were examined in patients with coronary disease (CAD). Eight CAD
patients who have been receiving intensive lipid-lowering treatment (niacin 2.5
g/d, lovastatin 40 mg/d, and colestipol 20 g/d) for 10 years in the Familial
Atherosclerosis Treatment Study (FATS) follow-up were randomly selected from
among 60 such treated patients. Eight CAD patients who were matched to the
treated patients for age (+/-3 years), baseline low density lipoprotein (+/-5
mg/dL), and triglycerides (+/-50 mg/dL) but who had never been treated with
lipid-lowering drugs were selected as controls. For each of these 32 carotid
arteries, luminal and plaque areas were measured by planimetry, in a blinded
protocol, from the magnetic resoce image that showed most plaque. Fibrous
tissue, calcium, and lipid deposits were identified on the basis of established
criteria. Plaque composition was estimated as a fraction of total planimetered
area. Patients treated with 10-year intensive lipid-lowering therapy, compared
with control subjects, had significantly lower low density lipoprotein
cholesterol levels (84 versus 158 mg/dL, respectively; P<0.001) and higher high
density lipoprotein cholesterol levels (51 versus 37 mg/dL, respectively;
P<0.001). As a group, treated patients, compared with untreated control
subjects, had a smaller core lipid area (0.7 versus 10.2 mm(2), respectively;
P=0.01) and lipid composition (1% versus 17%, respectively). Group differences
in luminal area (55 [treated] versus 44 [control] mm(2), P=NS) and plaque area
(58 [treated] versus 64 [control] mm(2), P=NS) tended to favor treatment. MRI
appears useful for estimating carotid plaque size and composition.
Hyperlipidemic CAD patients frequently (97%) have at least moderate (>/=40% area
stenosis) carotid plaque. In this case-control study, prolonged intensive
lipid-lowering therapy is associated with a markedly decreased lipid content, a
characteristic of clinically stable plaques. BACKGROUND: Lipid lowering with statins prevents adverse cardiac events. Both
lipid-lowering and antioxidant therapies may favorably affect vasomotor function
and thereby improve ischemia.
METHODS AND RESULTS: In a randomized, double-blind, placebo-controlled trial,
300 patients with stable coronary disease, a positive exercise treadmill test,
48-hour ambulatory ECG with > or =1 episode of ischemia, and fasting total
cholesterol of 180 to 250 mg/dL were assigned to 1-year treatment with intensive
atorvastatin to reduce LDL to <80 mg/dL (n=96), intensive atorvastatin to reduce
LDL to <80 mg/dL plus antioxidant vitamins C (1000 mg/d) and E (800 mg/d)
(n=101), or diet and low-dose lovastatin, if needed, to reduce LDL to <130 mg/dL
(n=103; control group). Ischemia end points, including ambulatory ECG monitoring
and exercise treadmill testing, and endothelial assessment using brachial artery
flow-mediated dilation were obtained at baseline and at 6 and 12 months.
Baseline characteristics were similar in all groups. LDL decreased from
approximately 153 mg/dL at baseline in the 2 atorvastatin groups to
approximately 83 mg/dL at 12 months (each P<0.0001) and from 147 to 120 mg/dL in
the control group (P<0.0001). During ambulatory ECG monitoring, mean number of
ischemic episodes per 48 hours decreased 31% to 61% in each group (each P<0.001;
P=0.15 across groups), without a change in daily heart rate activity. Mean
duration of ischemia for 48 hours decreased 26% to 62% in each group (each
P<0.001; P=0.06 across groups). Mean exercise duration to 1-mm ST-segment
depression significantly increased in each group, but total exercise duration
and mean sum of maximum ST depression were unchanged. Angina frequency decreased
in each group. There was no incremental effect of supplemental vitamins C and E
on any ischemia outcome. Flow-mediated dilation studies indicated no meaningful
changes.
CONCLUSIONS: Intensive lipid lowering with atorvastatin to an LDL level of 80
mg/dL, with or without antioxidant vitamins, does not provide any further
benefits in ambulatory ischemia, exercise time to onset of ischemia, and angina
frequency than moderate lipid lowering with diet and low-dose lovastatin to an
LDL level of <120 mg/dL. OBJECTIVE: To investigate the effect of intensive lipid lowering with high-dose
atorvastatin on the incidence of major cardiovascular events compared with
low-dose atorvastatin in patients with coronary artery disease and type 2
diabetes, with and without chronic kidney disease (CKD).
PATIENTS AND METHODS: Following 8 weeks' open-label therapy with atorvastatin
(10 mg/d), 10,001 patients with coronary artery disease were randomized to
receive double-blind therapy with either 80 mg/d or 10 mg/d of atorvastatin
between July 1, 1998, and December 31, 1999. Of 1501 patients with diabetes,
renal data were available for 1431. Patients with CKD were defined as having a
baseline estimated glomerular filtration rate (eGFR) below 60 mL/min per 1.73
m2, using the Modification of Diet in Renal Disease equation.
RESULTS: After a median follow-up of 4.8 years, 95 (17.4%) of 546 patients with
diabetes and CKD experienced a major cardiovascular event vs 119 (13.4%) of 885
patients with diabetes and normal eGFRs (hazard ratio [HR], 1.32; 95% confidence
interval [CI], 1.00-1.72; P<.05). Compared with 10 mg of atorvastatin, 80 mg of
atorvastatin reduced the relative risk of major cardiovascular events by 35% in
patients with diabetes and CKD (20.9% [57/273] vs 13.9% [38/273]; HR, 0.65; 95%
CI, 0.43-0.98; P=.04) and by 10% in patients with diabetes and normal eGFR
(14.1% [62/441] vs 12.8% [57/444]; HR, 0.90; 95% CI, 0.63-1.29; P=.56). The
absolute risk reduction in patients with diabetes and CKD was substantial,
yielding a number needed to treat of 14 to prevent 1 major cardiovascular event
over 4.8 years. Both treatments were well tolerated.
CONCLUSION: Patients with diabetes, stable coronary artery disease, and mild to
moderate CKD experience marked reduction in cardiovascular events with intensive
lipid lowering, in contrast to previous observations in patients with diabetes
and end-stage renal disease.
TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT00327691. Telomere erosion of EPCs (endothelial progenitor cells) may be a key factor in
endothelial cell senescence and is highly dependent on cellular oxidative
damage. The aim of the present study was to investigate whether LLT
(lipid-lowering therapy) with statins could attenuate EPC telomere erosion in
patients with CAD (coronary artery disease). The study included 100 patients
with stable CAD and 25 subjects without CAD as controls. CAD patients were
randomized to 12 months of intensive LLT with atorvastatin or moderate LLT with
pravastatin. EPCs were obtained from peripheral blood at baseline and after 12
months of statin therapy. Telomere length in EPCs was measured by FISH
(fluorescence in situ hybridization) and oxidative DNA damage by flow cytometry
of oxidized DNA bases. EPC telomere length was shorter in the CAD group than in
the controls, and oxidative DNA damage to EPCs was higher in the CAD group
compared with controls. After 12 months of therapy, changes in lipid profiles
were greater in the intensive LLT group than in the moderate LLT group.
Intensive LLT markedly increased EPC number and decreased oxidative DNA damage
in EPCs (both P<0.05), with no change in telomere length. In contrast, moderate
LLT did not change EPC counts or oxidative DNA damage, but showed telomere
shortening (P<0.05). There was a weak negative correlation between changes in
EPC number and LDL (low-density lipoprotein)-cholesterol levels after intensive
LLT, whereas there was no correlation between them after moderate LLT. With in
vitro culturing of EPCs subjected to oxidative stress, atorvastatin led to the
prevention of EPC telomere shortening compared with pravastatin. In conclusion,
the present study has demonstrated that intensive LLT may prevent EPC telomere
erosion in patients with CAD, possibly contributing to the beneficial effects of
intensive LLT in this disorder. OBJECTIVES: To investigate the impact of lipid lowering therapy by different
means on skin microvascular function in patients with dysglycaemia and coronary
artery disease (CAD).
DESIGN AND SETTING: Thirty-six patients were randomized to simvastatin 80 mg
daily (S80, n = 19) or ezetimibe 10 mg and simvastatin 10 mg daily (E10/S10, n =
17) for 6 weeks. Skin microvascular function was assessed by laser Doppler
fluxmetry (LDF) at rest, following arterial occlusion (peak postocclusive LDF)
and following local heating on the forearm (heat arm LDF) and foot (heat foot
LDF). LDF parameters and serum lipids were evaluated at baseline and follow-up.
RESULTS: At follow-up, LDL cholesterol had decreased from 3.1 (2.7-3.5) to 1.6
(1.5-1.8) (mmol L(-1)) and 3.0 (2.4-3.9) to 1.3 (1.1-1.8) (mmol L(-1)) in the
E10/S10 and S80 groups respectively. In the entire study group (n = 32), LDF
parameters increased significantly; postocclusive LDF from 22 (17-27) to 26
(21-32) perfusion units (PU) (P < 0.001), heat foot LDF from 61 (44-82) to 66
(45-83) PU (P < 0.001) and heat arm LDF from 60 (48-121) to 75 (54-125) PU (P <
0.01). The changes in LDF parameters did not differ between the E10/S10 and S80
groups.
CONCLUSIONS: Lipid lowering improves microvascular function in patients with
dysglycaemia and CAD. The data suggest that lipid lowering per se is more
important than pleiotropic effects of statins for this effect. BACKGROUND: It has been suggested that intensive lipid-lowering therapy using
statins significantly decreases atheromatous plaque volume. The effect of
rosuvastatin on plaque volume in patients with stable coronary artery disease
(CAD), including those receiving prior lipid-lowering therapy, was examined in
the present study.
METHODS AND RESULTS: A 76-week open-label trial was performed at 37 centers in
Japan. Eligible patients began treatment with rosuvastatin 2.5 mg/day, which
could be increased at 4-week intervals to <or=20 mg/day. A total of 214 patients
underwent intravascular ultrasound (IVUS) at baseline; 126 patients had
analyzable IVUS images at the end of the study. The change in the serum
low-density lipoprotein-cholesterol level from baseline to end of follow-up was
-38.6 +/-16.9%, whereas that of high-density lipoprotein-cholesterol was +19.8
+/-22.9% (both P<0.0001). Percent change of plaque volume, the primary endpoint,
was -5.1 +/-14.1% (P<0.0001).
CONCLUSIONS: Rosuvastatin exerted significant regression of coronary plaque
volume in Japanese patients with stable CAD, including those who had previously
used other lipid-lowering drugs. Rosuvastatin might be useful in the setting of
secondary prevention in patients with stable CAD. BACKGROUND: Recently we reported a decline of circulating myeloid (m) and
plasmacytoid (p) dendritic cells (DCs) in patients with coronary artery disease
(CAD). This study also determined the total blood DC numbers and focused on
effects of extent (one vs. three-vessel disease) and type (stable vs. unstable)
of CAD, and on endothelial cell function.
METHODS: Patients undergoing diagnostic coronarography were enrolled in four
groups: control patients (atypical chest pain, <50% narrowing, n=15), stable
one-vessel (n=15), stable three-vessel (n=15), and unstable one-vessel CAD
(n=16). Total blood DCs were identified as lineage (lin) and HLADR, and DC
subtypes with blood DC antigen (BDCA)-1 for mDCs and BDCA-2 for pDCs.
Flow-mediated dilatation (FMD) was measured in the brachial artery.
RESULTS: Numbers of total blood DCs, mDCs and pDCs declined in CAD patients
compared with control patients, but without differences between the CAD groups.
Interleukin-6 and high sensitivity C-reactive protein displayed inverse
associations with mDCs. A FMD below the median of the study population, use of
beta-blockers or of lipid-lowering drugs was associated with increased mDCs,
whereas pDCs were similar. Interestingly, the effects of drugs and FMD were
additive with that of CAD.
CONCLUSION: This study indicates that lower blood DCs do not result from
medication intake or endothelial dysfunction, and are an overall systemic effect
of atherosclerosis rather than CAD type (stable or unstable) or number of
stenotic coronary arteries. In view of discrete associations with cytokines,
FMD, beta-blockers and statins, mDCs and pDCs seem to behave differently and may
influence inflammation during atherosclerosis in different ways. OBJECTIVE: To assess the effectiveness and safety of low-density lipoprotein
(LDL) apheresis performed with the heparin-induced extracorporeal LDL
precipitation (HELP) system for the treatment of patients with refractory
homozygous (HMZ) and heterozygous (HTZ) familial hypercholesterolemia (FH).
BACKGROUND ON FAMILIAL HYPERCHOLESTEROLEMIA: Familial hypercholesterolemia is a
genetic autosomal domit disorder that is caused by several mutations in the
LDL-receptor gene. The reduced number or absence of functional LDL receptors
results in impaired hepatic clearance of circulating low-density lipoprotein
cholesterol (LDL-C) particles, which results in extremely high levels of LDL-C
in the bloodstream. Familial hypercholesterolemia is characterized by excess
LDL-C deposits in tendons and arterial walls, early onset of atherosclerotic
disease, and premature cardiac death. Familial hypercholesterolemia occurs in
both HTZ and HMZ forms. Heterozygous FH is one of the most common monogenic
metabolic disorders in the general population, occurring in approximately 1 in
500 individuals. Nevertheless, HTZ FH is largely undiagnosed and an accurate
diagnosis occurs in only about 15% of affected patients in Canada. Thus, it is
estimated that there are approximately 3,800 diagnosed and 21,680 undiagnosed
cases of HTZ FH in Ontario. In HTZ FH patients, half of the LDL receptors do not
work properly or are absent, resulting in plasma LDL-C levels 2- to 3-fold
higher than normal (range 7-15mmol/L or 300-500mg/dL). Most HTZ FH patients are
not diagnosed until middle age when either they or one of their siblings present
with symptomatic coronary artery disease (CAD). Without lipid-lowering
treatment, 50% of males die before the age of 50 and 25% of females die before
the age of 60, from myocardial infarction or sudden death. In contrast to the
HTZ form, HMZ FH is rare (occurring in 1 case per million persons) and more
severe, with a 6- to 8-fold elevation in plasma LDL-C levels (range 15-25mmol/L
or 500-1000mg/dL). Homozygous FH patients are typically diagnosed in infancy,
usually due to the presence of cholesterol deposits in the skin and tendons. The
main complication of HMZ FH is supravalvular aortic stenosis, which is caused by
cholesterol deposits on the aortic valve and in the ascending aorta. The average
life expectancy of affected individuals is 23 to 25 years. In Ontario, it is
estimated that there are 13 to 15 cases of HMZ FH. An Ontario clinical expert
confirmed that 9 HMZ FH patients have been identified to date.
DIAGNOSIS: There are 2 accepted clinical diagnostic criterion for the diagnosis
of FH: the Simon Broome FH Register criteria from the United Kingdom and the
Dutch Lipid Network criteria from the Netherlands. The criterion supplement
cholesterol levels with clinical history, physical signs and family history.
DNA-based-mutation-screening methods permit a definitive diagnosis of HTZ FH to
be made. However, given that there are over 1000 identified mutations in the LDL
receptor gene and that the detection rates of current techniques are low,
genetic testing becomes problematic in countries with high genetic
heterogeneity, such as Canada.
TREATMENT: The primary aim of treatment in both HTZ and HMZ FH is to reduce
plasma LDL-C levels in order to reduce the risk of developing atherosclerosis
and CAD. The first line of treatment is dietary intervention, however it alone
is rarely sufficient for the treatment of FH patients. Patients are frequently
treated with lipid-lowering drugs such as resins, fibrates, niacin, statins and
cholesterol absorption-inhibiting drugs (ezetimibe). Most HTZ FH patients
require a combination of drugs to achieve or approach target cholesterol levels.
A small number of HTZ FH patients are refractory to treatment or intolerant to
lipid-lowering medication. According to clinical experts, the prevalence of
refractory HTZ FH in Ontario is between 1 to 5%. Using the mean of 3%, it is
estimated that there are approximately 765 refractory HTZ FH patients in
Ontario, of which 115 are diagnosed and 650 are undiagnosed. Drug therapy is
less effective in HMZ FH patients since the effects of the majority of
cholesterol-lowering drugs are mediated by the upregulation of LDL receptors,
which are often absent or function poorly in HMZ FH patients. Some HMZ FH
patients may still benefit from drug therapy, however this rarely reduces LDL-C
levels to targeted levels. EXISTING TECHNOLOGY: PLASMA EXCHANGE An option
currently available in Ontario for FH patients who do not respond to standard
diet and drug therapy is plasma exchange (PE). Patients are treated with this
lifelong therapy on a weekly or biweekly basis with concomitant drug therapy.
Plasma exchange is nonspecific and eliminates virtually all plasma proteins such
as albumin, immunoglobulins, coagulation factors, fibrinolytic factors and
HDL-C, in addition to acutely lowering LDL-C by about 50%. Blood is removed from
the patient, plasma is isolated, discarded and replaced with a substitution
fluid. The substitution fluid and the remaining cellular components of the blood
are then returned to the patient. The major limitation of PE is its
nonspecificity. The removal of HDL-C prevents successful vascular remodeling of
the areas stenosed by atherosclerosis. In addition, there is an increased
susceptibility to infections, and costs are incurred by the need for replacement
fluid. Adverse events can be expected to occur in 12% of procedures. OTHER
ALTERNATIVES: Surgical alternatives for FH patients include portocaval shunt,
ileal bypass and liver transplantation. However, these are risky procedures and
are associated with a high morbidity rate. Results with gene therapy are not
convincing to date.
THE TECHNOLOGY BEING REVIEWED: LDL APHERESIS An alternative to PE is LDL
apheresis. Unlike PE, LDL apheresis is a selective treatment that removes LDL-C
and other atherogenic lipoproteins from the blood while minimally impacting
other plasma components such as HDL-C, total serum protein, albumin and
immunoglobulins. As with PE, FH patients require lifelong therapy with LDL
apheresis on a weekly/biweekly basis with concomitant drug therapy.
HEPARIN-INDUCED EXTRACORPOREAL LDL PRECIPITATION: Heparin-induced extracorporeal
LDL precipitation (HELP) is one of the most widely used methods of LDL
apheresis. It is a continuous closed-loop system that processes blood
extracorporeally. It operates on the principle that at a low pH, LDL and
lipoprotein (a) [Lp(a)] bind to heparin and fibrinogen to form a precipitate
which is then removed by filtration. In general, the total duration of treatment
is approximately 2 to 3 hours. Results from early trials indicate that LDL-C
concentration is reduced by 65% to 70% immediately following treatment in both
HMZ and HTZ FH and then rapidly begins to rise. Typically patients with HTZ FH
are treated every 2 weeks while patients with HMZ FH require weekly therapy.
Heparin-induced extracorporeal LDL precipitation also produces small transient
decreases in HDL-C, however levels generally return to baseline within 2 days.
After several months of therapy, long-term reductions in LDL-C and increases in
HDL-C have been reported. In addition to having an impact on plasma cholesterol
concentrations, HELP lowers plasma fibrinogen, a risk factor for
atherosclerosis, and reduces concentrations of cellular adhesion molecules,
which play a role in early atherogenesis. In comparison with PE, HELP LDL
apheresis does not have major effects on essential plasma proteins and does not
require replacement fluid, thus decreasing susceptibility to infections. One
study noted that adverse events were documented in 2.9% of LDL apheresis
treatments using the HELP system compared with 12% using PE. As per the
manufacturer, patients must weigh at least 30kgs to be eligible for treatment
with HELP.
REGULATORY STATUS: The H.E.L.P.® System (B.Braun Medizintechnologie GmbH,
Germany) has been licensed by Health Canada since December 2000 as a Class 3
medical device (Licence # 26023) for performing LDL apheresis to acutely remove
LDL from the plasma of 3 high-risk patient populations for whom diet has been
ineffective and maximum drug therapy has either been ineffective or not
tolerated. The 3 patient groups are as follows: Functional hypercholesterolemic
homozygotes with LDL-C >500 mg/dL (>13mmol/L);Functional hypercholesterolemic
heterozygotes with LDL-C >300 mg/dL (>7.8mmol/L);Functional hypercholesterolemic
heterozygotes with LDL-C >200 mg/dL (>5.2mmol/L) and documented CADNo other LDL
apheresis system is currently licensed in Canada.
REVIEW STRATEGY: The Medical Advisory Secretariat systematically reviewed the
literature to assess the effectiveness and safety of LDL apheresis performed
with the HELP system for the treatment of patients with refractory HMZ and HTZ
FH. A standard search methodology was used to retrieve international health
technology assessments and English-language journal articles from selected
databases. The GRADE approach was used to systematically and explicitly make
judgments about the quality of evidence and strength of recommendations.
SUMMARY OF FINDINGS: The search identified 398 articles published from January
1, 1998 to May 30, 2007. Eight studies met the inclusion criteria. Five case
series, 2 case series nested within comparative studies, and one retrospective
review, were included in the analysis. A health technology assessment conducted
by the Alberta Heritage Foundation for Medical Research, and a review by the
United States Food and Drug Administration were also included. Large
heterogeneity among the studies was observed. Studies varied in inclusion
criteria, baseline patient characteristics and methodology. Overall, the mean
acute relative decrease in LDL-C with HELP LDL apheresis ranged from 53 to 77%.
The mean acute relative reductions ranged as follows: total cholesterol (TC) 47
to 64%, HDL-C +0. (ABSTRACT TRUNCATED) BACKGROUND: Previous studies have demonstrated that intensive lipid lowering
using rosuvastatin results in regression of coronary plaques. However, few data
exist regarding lipid profiles over time, drug tolerability, and the effects of
prior use of lipid lowering agents in patients on rosuvastatin treatment.
Therefore, we studied these matters in a subanalysis of the Coronary
Atherosclerosis Study Measuring Effects of Rosuvastatin Using Intravascular
Ultrasound in Japanese Subjects (COSMOS).
METHODS: Rosuvastatin was titrated for 76 weeks to attain LDL-C < 80 mg/dL in
213 Japanese dyslipidemic patients with CAD. Clinic visits were scheduled for
every 4 weeks during the 76-week study period. Changes over time in lipid
parameters, changes in those according to prior lipid-lowering therapy, and
changes in those according to baseline lipid levels were evaluated in this
subanalysis.
RESULTS: Overall, 126 patients completed the study. The mean rosuvastatin dose
at the last observation carried forward was 16.9 mg (range, 2.5-20 mg).
Rosuvastatin significantly increased HDL-C, lowered LDL-C, and improved the
LDL-C/HDL-C ratio (all, P < 0.0001). Increases in serum HDL-C levels were
significantly greater in patients with HDL-C < 40 mg/dL than in those with
HDL-C ≥ 40 mg/dL at baseline (P = 0.0005). The estimated glomerular filtration
rate increased significantly by 2.84 ± 9.01 mL/min/1.73 m(2) (P < 0.0001). Of
166 adverse events in 74 patients, 113 events in 54 patients were laboratory
values beyond the normal range.
CONCLUSION: Rosuvastatin significantly improved lipid profiles, with an
acceptable safety profile, contributing to plaque regression in Japanese
patients with CAD. In the Clinical Outcomes Utilizing Revascularization and Aggressive Drug
Evaluation (COURAGE) study, a revascularization strategy trial with optimal
medical therapy in both arms, the low-density lipoprotein (LDL) cholesterol goal
was 60 to 85 mg/dl; this was revised to <70 mg/dl in 2004. COURAGE patients (n =
2,287) were titrated with increasing statin doses to achieve the initial LDL
cholesterol goal using a prespecified protocol. Ezetimibe was not available when
study enrollment began in 1999 but became available after approval in 2003.
After maximizing statin dose, ezetimibe was added to reach the LDL cholesterol
goal in 34% of patients (n = 734). Median baseline LDL cholesterol was higher in
patients who received ezetimibe than in those who did not (109 vs 96 mg/dl). At
baseline, 18% of patients who would later receive ezetimibe had LDL cholesterol
<85 mg/dl, and 8% had LDL cholesterol <70 mg/dl. On maximum tolerated statin
(with or without other lipid-lowering drugs), 40% had LDL cholesterol <85 mg/dl
and 23% had LDL cholesterol <70 mg/dl before starting ezetimibe. At the final
study visit, 68% of ezetimibe patients achieved LDL cholesterol <85 mg/dl, and
46% achieved LDL cholesterol <70 mg/dl. Using Cox regression analysis, the most
significant factors associated with achieving LDL cholesterol goals were lower
baseline LDL cholesterol, average statin dose, and ezetimibe use. In conclusion,
after maximizing statin dose, the addition of ezetimibe results in a substantial
increase in the percentage of patients who reach LDL cholesterol goal, a key
component of optimal medical therapy. Chronic kidney disease (CKD) is an independent risk factor for coronary artery
disease (CAD). Coronary artery disease is the leading cause of morbidity and
mortality in patients with CKD. The outcomes of CAD are poorer in patients with
CKD. In addition to traditional risk factors, several uremia-related risk
factors such as inflammation, oxidative stress, endothelial dysfunction,
coronary artery calcification, hyperhomocysteinemia, and immunosuppressants have
been associated with accelerated atherosclerosis. A number of uremia-related
biomarkers are identified as predictors of cardiac outcomes in CKD patients. The
symptoms of CAD may not be typical in patients with CKD. Both dobutamine stress
echocardiography and radionuclide myocardial perfusion imaging have moderate
sensitivity and specificity in detecting obstructive CAD in CKD patients.
Invasive coronary angiography carries a risk of contrast nephropathy in patients
with advanced CKD. It should be reserved for those patients with a high risk for
CAD and those who would benefit from revascularization. Guideline-recommended
therapies are, in general, underutilized in renal patients. Medical therapy
should be considered the initial strategy for clinically stable CAD. The effects
of statins in patients with advanced CKD have been neutral despite a
lipid-lowering effect. Compared to non-CKD population, percutaneous coronary
intervention (PCI) is associated with higher procedure complications,
restenosis, and future cardiac events even in the drug-eluting stent era in
patients with CKD. Compared with PCI, coronary artery bypass grafting (CABG)
reduces repeat revascularizations but is associated with significant
perioperative morbidity and mortality. Screening for CAD is an important part of
preoperative evaluation for kidney transplant candidates. OBJECTIVES: This study sought to investigate the effect of daily glucose
fluctuation on coronary plaque properties in patients with coronary artery
disease (CAD) pre-treated with lipid-lowering therapy.
BACKGROUND: There is growing evidence that glucose fluctuation, as a residual
risk apart from dyslipidemia, is an important factor contributing to the
development of CAD.
METHODS: This prospective study enrolled 70 consecutive CAD patients who were
referred for percutaneous coronary intervention and whose low-density
lipoprotein cholesterol level was <120 mg/dl under statin treatment or <100
mg/dl without statins. Daily glucose fluctuation was analyzed by measuring the
mean amplitude of glycemic excursion (MAGE). The plaque properties in the
culprit and nonculprit lesions were assessed by virtual histology intravascular
ultrasound, and the volume percentage of necrotic core within the plaque (%NC)
and the presence of thin-cap fibroatheroma were evaluated.
RESULTS: In total, 165 lesions were evaluated in 70 patients (40 diabetic and 30
nondiabetic patients). %NC was well correlated with MAGE (r = 0.490, p <0.001).
A linear mixed effect model showed that MAGE had the strongest effect on %NC
(coefficient β = 0.080 ± 0.020 [standard error], p < 0.001). The generalized
linear mixed effect model revealed that MAGE was the only independent predictor
of the presence of thin-cap fibroatheroma (odds ratio: 1.037; 95% confidence
interval: 1.010 to 1.065; p = 0.007).
CONCLUSIONS: Daily glucose fluctuation may have an effect on coronary plaque
vulnerability in patients with CAD pre-treated with lipid-lowering therapy.
Further investigations should address the rationale for the early detection and
control of glucose fluctuation in the era of universal statin use for CAD
patients. BACKGROUND: Many disease entities, including coronary artery disease (CAD),
demonstrate abnormalities in the activity of cholinesterases. As CAD is
characterized by an increase in cholesterol level, patients with this disease
are treated with lipid-lowering drugs. The present study attempts to determine
how statin or combined statin and ezetimibe therapy influences cholinesterase
activity.
METHODS: Plasma and erythrocytes were isolated from the peripheral blood of CAD
patients (n=61) and healthy subjects (n=63). The patients were randomized into
three groups: 20mg/day rosuvastatin, 40mg/day atorvastatin, and combined
10mg/day atorvastatin with 10mg/day ezetimibe. The following parameters were
studied: activity of acetylcholinesterase (AChE) and butyrylcholinoesterase
(BChE) and lipid levels.
RESULTS: Patients with CAD demonstrated significant increase in AChE and BChE
activity. We observed increase in the level of low-density lipoprotein
cholesterol (LDL) and triglycerides (TG) level, and decrease in high density
lipoprotein cholesterol (HDL) level. After atorvastatin monotherapy, the
following decrease in activity were observed: 17% LDL, 43% total cholesterol
(TC) level, 33% AChE and 17% BChE. The following decrease in activity were
observed following rosuvastatin monotherapy: 26% LDL level, 26% AChE and 18%
BChE. After combined atorvastatin+ezetimibe therapy, the following decrease in
activity occurred: 27% of LDL level, 15% TC, 33% of AChE and 20% BChE.
CONCLUSIONS: Our results suggest that intensive lipid-lowering therapy has a
beneficial effect on AChE and BChE activity and lipid levels. Combination
atorvastatin+ezetimibe therapy was found to have similar effects on the tested
parameters as statin monotherapy. |
What are PD-1 inhibitors? | The programmed death-1 (PD-1) pathway negatively regulates T-cell activation and has an important role in regulating antitumor host immunity. Monoclonal antibodies directed against PD-1 or the PD-1 ligand (PD-L1) are used to treat cancer. | The receptor programmed death 1 (PD-1) inhibits T cell proliferation and plays a
critical role in suppressing self-reactive T cells, and it also compromises
antiviral and antitumor responses. To determine how PD-1 signaling inhibits T
cell proliferation, we used human CD4(+) T cells to examine the effects of PD-1
signaling on the molecular control of the cell cycle. The ubiquitin ligase
SCF(Skp2) degrades p27(kip1), an inhibitor of cyclin-dependent kinases (Cdks),
and PD-1 blocked cell cycle progression through the G(1) phase by suppressing
transcription of SKP2, which encodes a component of this ubiquitin ligase. Thus,
in T cells stimulated through PD-1, Cdks were not activated, and two critical
Cdk substrates were not phosphorylated. Activation of PD-1 inhibited
phosphorylation of the retinoblastoma gene product, which suppressed expression
of E2F target genes. PD-1 also inhibited phosphorylation of the transcription
factor Smad3, which increased its activity. These events induced additional
inhibitory checkpoints in the cell cycle by increasing the abundance of the G(1)
phase inhibitor p15(INK4) and repressing the Cdk-activating phosphatase Cdc25A.
PD-1 suppressed SKP2 transcription by inhibiting phosphoinositide 3-kinase-Akt
and Ras-mitogen-activated and extracellular signal-regulated kinase kinase
(MEK)-extracellular signal-regulated kinase (ERK) signaling. Exposure of cells
to the proliferation-promoting cytokine interleukin-2 restored activation of
MEK-ERK signaling, but not Akt signaling, and only partially restored SKP2
expression. Thus, PD-1 blocks cell cycle progression and proliferation of T
lymphocytes by affecting multiple regulators of the cell cycle. Two PD-1 inhibitors, Bristol-Myers Squibb's nivolumab and Merck's MK-3475, both
demonstrated positive results in phase I trials of previously treated patients
with non-small cell lung cancer, reported at the World Conference on Lung Cancer
in Sydney, Australia. PURPOSE: Blocking the interaction between the programmed cell death (PD)-1
protein and one of its ligands, PD-L1, has been reported to have impressive
antitumor responses. Therapeutics targeting this pathway are currently in
clinical trials. Pembrolizumab and nivolumab are the first of this anti-PD-1
pathway family of checkpoint inhibitors to gain accelerated approval from the US
Food and Drug Administration (FDA) for the treatment of ipilimumab-refractory
melanoma. Nivolumab has been associated with improved overall survival compared
with dacarbazine in patients with previously untreated wild-type
serine/threonine-protein kinase B-raf proto-oncogene BRAF melanoma. Although the
most mature data are in the treatment of melanoma, the FDA has granted approval
of nivolumab for squamous cell lung cancer and the breakthrough therapy
designation to immune- checkpoint inhibitors for use in other cancers:
nivolumab, an anti-PD-1 monoclonal antibody, for Hodgkin lymphoma, and
MPDL-3280A, an anti-PD-L1 monoclonal antibody, for bladder cancer and non-small
cell lung cancer. Here we review the literature on PD-1 and PD-L1 blockade and
focus on the reported clinical studies that have included patients with
melanoma.
METHODS: PubMed was searched to identify relevant clinical studies of
PD-1/PD-L1-targeted therapies in melanoma. A review of data from the current
trials on clinicaltrial.gov was incorporated, as well as data presented in
abstracts at the 2014 annual meeting of the American Society of Clinical
Oncology, given the limited number of published clinical trials on this topic.
FINDINGS: The anti-PD-1 and anti-PD-L1 agents have been reported to have
impressive antitumor effects in several maligcies, including melanoma. The
greatest clinical activity in unselected patients has been seen in melanoma.
Tumor expression of PD-L1 is a suggestive, but inadequate, biomarker predictive
of response to immune-checkpoint blockade. However, tumors expressing little or
no PD-L1 are less likely to respond to PD-1 pathway blockade. Combination
checkpoint blockade with PD-1 plus cytotoxic T-lymphocyte antigen (CTLA)-4
blockade appears to improve response rates in patients who are less likely to
respond to single-checkpoint blockade. Toxicity with PD-1 blocking agents is
less than the toxicity with previous immunotherapies (eg, interleukin 2, CTLA-4
blockade). Certain adverse events can be severe and potentially life
threatening, but most can be prevented or reversed with close monitoring and
appropriate management.
IMPLICATIONS: This family of immune-checkpoint inhibitors benefits not only
patients with metastatic melanoma but also those with historically less
responsive tumor types. Although a subset of patients responds to single-agent
blockade, the initial trial of checkpoint-inhibitor combinations has reported a
potential to improve response rates. Combination therapies appear to be a means
of increasing response rates, albeit with increased immune-related adverse
events. As these treatments become available to patients, education regarding
the recognition and management of immune-related effects of immune-checkpoint
blockade will be essential for maximizing clinical benefit. DNA vaccines have demonstrated antitumor efficacy in multiple preclinical
models, but low immunogenicity has been observed in several human clinical
trials. This has led to many approaches seeking to improve the immunogenicity of
DNA vaccines. We previously reported that a DNA vaccine encoding the
cancer-testis antigen SSX2, modified to encode altered epitopes with increased
MHC class I affinity, elicited a greater frequency of cytolytic, multifunctional
CD8(+) T cells in non-tumor-bearing mice. We sought to test whether this
optimized vaccine resulted in increased antitumor activity in mice bearing an
HLA-A2-expressing tumor engineered to express SSX2. We found that immunization
of tumor-bearing mice with the optimized vaccine elicited a surprisingly
inferior antitumor effect relative to the native vaccine. Both native and
optimized vaccines led to increased expression of PD-L1 on tumor cells, but
antigen-specific CD8(+) T cells from mice immunized with the optimized construct
expressed higher PD-1. Splenocytes from immunized animals induced PD-L1
expression on tumor cells in vitro. Antitumor activity of the optimized vaccine
could be increased when combined with antibodies blocking PD-1 or PD-L1, or by
targeting a tumor line not expressing PD-L1. These findings suggest that
vaccines aimed at eliciting effector CD8(+) T cells, and DNA vaccines in
particular, might best be combined with PD-1 pathway inhibitors in clinical
trials. This strategy may be particularly advantageous for vaccines targeting
prostate cancer, a disease for which antitumor vaccines have demonstrated
clinical benefit and yet PD-1 pathway inhibitors alone have shown little
efficacy to date. BACKGROUND: Monoclonal antibodies that target the programmed death-1
(PD-1)/programmed death-ligand 1(PD-L1) pathway have shown antitumour activity
in metastatic renal cell carcinoma (mRCC) and are currently being developed in
first-line (in combination) and in previously treated patients. The efficacy
targeted therapy (TT) after PD-1/PD-L1 blockade is still unknown.
METHODS: Medical records of mRCC patients treated with investigational PD-1 or
PD-L1 inhibitors at 4 academic institutions were reviewed. Patients who received
subsequent treatment with TT were selected to collect outcome measures of
subsequent TT.
RESULTS: Of 99 patients who received PD-1/PD-L1 blockade as part of clinical
trials, 56 patients have received subsequent therapy: 44 patients received
vascular endothelial growth factor (VEGF)/vascular endothelial growth factor
receptor (VEGFR) inhibitors and 12 received mammalian target of rapamycin (mTOR)
inhibitors as first subsequent TT. Median follow up, from the start of
subsequent TT was 16.1 months (range: 0.2, 30.6 months). TT post PD-1/PD-L1
blockade was administered as second-line, third-line or beyond third-line in 9
(16%), 24 (43%) and 23 patients (41%) respectively. Median time to treatment
failure on subsequent TT was 6.6 months (range: 0.2+, 23.0). 1-year and 2 year
overall survival from the initiation of subsequent TT was 58% (95% confidence
interval (CI): 41-72%) and 36% (95% CI: 18-54%), respectively.
CONCLUSION: Both VEGF/VEGFR and mTOR inhibitors demonstrate antitumour activity
following PD-1/PD-L1 blockade. In non-small cell lung cancer (NSCLC), the first immune checkpoint inhibitor to
be approved by the US Food and Drug Administration was nivolumab, based on a
survival advantage over docetaxel in recurrent squamous NSCLC, a
difficult-to-treat histology. In addition, several other immune checkpoint
inhibitors are also in late-stage development. Most of these agents inhibit the
programmed cell death protein 1 (PD-1) pathway, targeting either the PD-1
receptor or its ligand, programmed cell death ligand 1 (PD-L1). In addition to
nivolumab, pembrolizumab is a PD-1 inhibitor under investigation in NSCLC, and
atezolizumab (MPDL3280A), durvalumab (MEDI4736), and avelumab (MSB0010718C) are
PD-L1 inhibitors under investigation. The cytotoxic T-lymphocyte-associated
antigen 4 (CTLA-4) immune checkpoint inhibitors ipilimumab and tremelimumab are
also under investigation in NSCLC, largely as part of combination approaches
rather than as monotherapy. PD-L1 expression as a potential biomarker to select
patients most likely to respond to inhibitors of the PD-1 pathway has been
widely studied. Manipulation of co-stimulatory or co-inhibitory checkpoint proteins allows for
the reversal of tumor-induced T-cell anergy observed in cancer. The field has
gained credence given success with CTLA-4 and PD-1 inhibitors. These molecules
include immunoglobulin family members and the B7 subfamily as well as the TNF
receptor family members. PD-L1 inhibitors and LAG-3 inhibitors have progressed
through clinical trials. Other B7 family members have shown promise in
preclinical models. TNFR superfamily members have shown variable success in
preclinical and clinical studies. As clinical investigation in tumor immunology
gains momentum, the next stage becomes learning how to combine checkpoint
inhibitors and agonists with each other as well as with traditional
chemotherapeutic agents. BACKGROUND: The programmed death-1 (PD-1) pathway negatively regulates T-cell
activation and has an important role in regulating antitumor host immunity.
Monoclonal antibodies directed against PD-1 or the PD-1 ligand (PD-L1) have
shown activity in several tumor types with preliminary data suggesting a
relationship between PD-L1 expression and response. The aim of this study was to
establish the frequency of PD-L1 expression in muscle-invasive bladder cancer
and associated lymph node metastasis using immunohistochemistry and to
investigate the feasibility of using PD-L1 expression as a biomarker to select
patients for PD-1-directed therapy.
PATIENTS AND METHODS: Cases of radical cystectomy for muscle-invasive bladder
cancer with no exposure to previous chemotherapy were identified and
representative slides from archived paraffin-embedded blocks stained with
anti-PD-L1 antibody (5H1 clone) were identified. PD-L1 positivity was defined by
a 5% expression threshold.
RESULTS: Fifty-two radical cystectomy specimens were reviewed. PD-L1 was
overexpressed in the tumor cells of 5/52 (9.6%) of cystectomy specimens in this
cohort with 17/52 (32.7%) of cases showing PD-L1 overexpression in
tumor-infiltrating immune cells. Discordance was observed between PD-L1
expression in lymph node metastasis and the primary tumor.
CONCLUSION: Standard assays for PD-L1 expression have yet to be established. The
observation of discordance between PD-L1 expression in metastatic sites and
primary tumors suggests that prospective biomarker studies should aim to acquire
material immediately before treatment initiation rather than archived tissue
from resected specimens that might not reflect the current immune-active
microenvironment. BACKGROUND: Emerging agents blocking the programmed cell death 1 (PD-1) pathway
show activity in metastatic clear cell renal cell carcinoma (mRCC). The aim of
this study was to evaluate the efficacy and safety of vascular endothelial
growth factor (VEGF)/VEGF receptor (VEGFR)-tyrosine kinase inhibitor (TKI)
therapy after PD-1 inhibition.
PATIENTS AND METHODS: Patients with mRCC treated with anti-PD-1 antibody (aPD-1)
monotherapy or in combination (with VEGFR-TKI or ipilimumab) that subsequently
received VEGFR-TKI were retrospectively reviewed. The efficacy end points were
objective response rate (ORR) and progression-free survival (PFS) stratified by
the type of prior PD-1 regimen. Safety by the type and PD-1 exposure was also
evaluated.
RESULTS: Seventy patients were included. Forty-nine patients received prior
therapy with immune checkpoint inhibitors (CPIs) alone and 21 had combination
therapy of aPD-1 and VEGFR-TKI. Overall, ORR to VEGFR-TKI after PD-1 inhibition
was 28% (19/68) and the median PFS was 6.4 months (mo) (4.3-9.5). ORR to
VEGFR-TKI after aPD-1 in combination with VEGFR-TKI was lower than that in
patients treated with VEGFR-TKI after CPI alone (ORR 10% versus 36%, P = 0.039).
In the multivariable analysis, patients treated with prior CPI alone were more
likely to achieve an objective response than those treated with aPD-1 in
combination with VEGFR-TKI (OR = 5.38; 95% CI 1.12-26.0, P = 0.03). There was a
trend toward numerically longer median PFS in the VEGFR-TKI after the CPI alone
group, 8.4 mo (3.2-12.4) compared with 5.5 mo (2.9-8.3) for those who had
VEGFR-TKI after aPD-1 in combination with VEGFR-TKI (P = 0.15). The most common
adverse events (AEs) were asthenia, hypertension, and diarrhea.
CONCLUSIONS: The efficacy and safety of VEGFR-TKIs after PD-1 inhibition were
demonstrated in this retrospective study. The response rate was lower and the
median progression-free survival was shorter in those patients who received
prior PD-1 in combination with VEGFR-TKI. PD-1 exposure does not seem to
significantly influence the safety of subsequent VEGFR-TKI treatment. Blockading the interaction of programmed death-1 (PD-1) protein with its ligands
(PD-Ls, such as PD-L1) was proved to be a pathway for suppressing the
development of tumors and other degradations of biological species. Thus,
finding PD-1 inhibitors situated at the convergence point of drug discovery. In
addition to some monoclonal antibodies applied to treat cancers clinically, the
screening of organic molecules for hindering the interaction of PD-1 with PD-L1
became an efficient strategy in the development of PD-1 inhibitors. We herein
applied resorcinol and 3-hydroxythiophenol as the core to link with
N,N-dimethylcarbamate and other alkyl-substituted amines to afford 13
amine-appended phenyl dimethylcarbamates (AAPDs). The test for blockading the
combination of PD-1 with PD-L1 revealed that abilities of 13 AAPDs were higher
than that of sulfamethizole, a successful PD-1 inhibitor. In particular, large
hydrophobic substituents at amine moiety or a nitro at resorcinol skeleton
enhanced the inhibitory effect of AAPD even higher than that of
sulfamethoxypyridazine, another successful PD-1 inhibitor. The present results
may provide valuable information for further investigation on synthetic PD-1
inhibitors. The use of antibodies against programmed death (PD)1, such as nivolumab and
pembrolizumab, has dramatically improved the prognosis of patients with advanced
melanoma. Nivolumab is also approved in advanced squamous cell nonsmall-cell
lung cancer. These immunotherapies are associated with a unique set of
toxicities termed immune-related adverse events, which are different from
toxicities observed with conventional cytotoxic chemotherapy. We report the case
of a 56-year-old man who was diagnosed with metastatic melanoma and who received
nivolumab. One week after the second infusion, he developed pulmonary symptoms,
dry eye syndrome and a bilateral swelling of the parotid glands. Investigations
were negative for infection. The bronchoalveolar lavage differential cell count
showed 32% lymphocytes with an increased CD4 : CD8 ratio, and bronchial biopsies
revealed noncaseating epithelioid granulomas, without maligt cells. The
clinical and radiological courses were rapidly favourable with oral
corticosteroid. This case illustrates that sarcoidosis can be induced by
nivolumab treatment. With the increasing use of anti-PD1 inhibitors in patients
with advanced melanoma and squamous cell nonsmall-cell lung cancer, clinicians
should be aware of this potential associated immune-related adverse event. IMPORTANCE: The development of programmed cell death 1 (PD-1)/PD-1 ligand 1
(PD-L1) checkpoint inhibitors has changed the landscape of non-small-cell lung
cancer (NSCLC) therapy, with 2 approvals from the US Food and Drug
Administration of PD-1 inhibitors for second-line therapy. However, the rational
use of these agents has been limited by the lack of a definitive predictive
biomarker.
OBSERVATIONS: Tumor PD-L1 expression is associated with an increased likelihood
of NSCLC response to these agents, although responses can still occur at a low
rate in PD-L1-negative tumors. The use of PD-L1 as a predictive biomarker for
use of PD-1/PD-L1 inhibitors is limited by the multitude of PD-L1 antibodies,
assays, scoring systems, and thresholds for positivity currently used.
Alternative biomarkers such as tumor neoantigens identified through whole-exome
sequencing and clinical parameters (eg, smoking or oncogene driver status) may
also have predictive value. Biomarkers that can direct the rational use of
PD-1/PD-L1 checkpoint inhibitors are crucial given the risk of life-threatening
immune-related complications associated with these therapies and the reality
that most patients still do not benefit from their use.
CONCLUSIONS AND RELEVANCE: The refinement of existing biomarkers and
identification of novel predictive biomarkers will be key to ensuring the
effective and safe use of these agents. Since most patients still do not benefit
from these agents, it is critical to continue to work to define the select
patient population who will derive durable benefit from PD-1/PD-L1 inhibition
and identify markers that could have predictive value for combination therapies
that could expand the population who benefit. Historically, lung cancer was long considered a poorly immunogenic maligcy.
In recent years, however, immune checkpoint inhibitors have emerged as promising
therapeutic agents in non-small cell lung cancer (NSCLC). To date, the best
characterized and most therapeutically relevant immune checkpoints have been
cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell
death protein-1 (PD-1) pathway. In early studies, PD-1/programmed cell death
ligand-1 (PD-L1) inhibitors demonstrated promising antitumor activity and
durable clinical responses in a subset of patients. Based on these encouraging
results, multiple different PD-1/PD-L1 inhibitors have entered clinical
development, and two agents (nivolumab and pembrolizumab) have gained regulatory
approval in the United States for the treatment of NSCLC. In several large,
randomized studies, PD-1/PD-L1 inhibitors have produced significant improvements
in overall survival compared with single-agent docetaxel delivered in the
second-line setting, effectively establishing a new standard of care in NSCLC.
In the present report, we provide an overview of the rationale for checkpoint
inhibitors in lung cancer, recent clinical trial data, and the need for
predictive biomarkers.
THE ONCOLOGIST: 2017;22:81-88 IMPLICATIONS FOR PRACTICE: Strategies targeting
negative regulators (i.e., checkpoints) of the immune system have demonstrated
significant antitumor activity across a range of solid tumors. In non-small cell
lung cancer (NSCLC), programmed cell death protein-1 (PD-1) pathway inhibitors
have entered routine clinical use because of the results from recent randomized
studies demonstrating superiority against single-agent chemotherapy in
previously treated patients. The present report provides an overview of immune
checkpoint inhibitors in lung cancer for the practicing clinician, focusing on
the rationale for immunotherapy, recent clinical trial data, and future
directions. Colorectal cancers (CRCs) have been identified as potential targets for
immunotherapy with programmed cell death (PD)-1 inhibitors. English-language
publications from MedLine and Embase that evaluated PD-1/PD ligand 1 (PD-L1) in
the CRC tumor microenvironment and clinical trials that assessed PD-1 inhibitors
were included. Sixteen abstracts were screened. Fifteen met the inclusion
criteria. After review of the full texts, this resulted in a final reference
list of 8 studies eligible for review. Five studies that assessed PD-1/PD-L1 in
CRC and 3 trials that assessed PD-1 inhibitors were included. PD-1-positive
(PD-1+) tumor-infiltrating lymphocytes and PD-L1+ cancer cells featured more
prominently in high-level microsatellite instability (MSI-H) CRCs compared with
microsatellite stable (MSS) CRCs, except in 1 study in which PD-L1 expression
was higher in MSS CRCs. In the 3 trials that assessed PD-1 inhibitor, all 3
studies recruited patients with metastatic CRC (mCRC). One study also included
patients with recurrent CRC. The objective response according to the Response
Evaluation Criteria in Solid Tumors criteria was 0% (19 CRC patients with
unknown microsatellite instability status) in the nivolumab study. In the
pembrolizumab study, the objective response to PD-1 inhibitor was 40% and 0% in
patients with MSI-H and MSS mCRC, respectively (10 patients in the MSI-H group,
18 patients in the MSS group). Seventy-eight percent of the patients in the
MSI-H mCRC group compared with 11% in the MSS mCRC group (P < .005) showed no
further disease progression at 12 weeks. In the nivolumab with or without
ipilimumab study, objective partial response at 12 weeks to PD-1 inhibitor with
or without cytotoxic T-lymphocyte-associated protein 4 inhibitor was 25.5% to
33.3% and 5% in the MSI-H and MSS groups, respectively (100 patients in the
MSI-H group, 20 patients in the MSS group). Clinical trials that assessed PD-1
inhibitor immunotherapy in patients with CRC have recruited only small cohorts
of patients with mCRC. Studies on the tumor microenvironment have been on the
basis of archival specimens with different antibody PD-1 and PD-L1 preparations
for immunohistochemistry, independent from immunotherapy trials. Immunotherapy
with PD-1 therapy has potential benefit for immunogenic MSI-H CRCs whereas there
is no evidence to date to suggest immunotherapy benefit in MSS CRCs. The
available data are limited, and there is no information on non-mCRCs. Future
trials are under way to determine its benefits. Metastatic melanoma is a fatal form of skin cancer that has a tendency to
proliferate more rapidly than any other solid tumor. Since 2010, treatment
options for metastatic melanoma have been developed including chemotherapies,
checkpoint inhibition immunotherapies, e.g., anti‑cytotoxic T‑lymphocyte
antigen‑4 (CTLA‑4) and anti‑programmed death‑1 (PD‑1), and molecular-targeted
therapies, e.g., BRAF and MEK inhibitors. These treatments have shown not only
high response rates yet also side‑effects and limitations. Notwithstanding its
limitations, stem cell therapy has emerged as a new auspicious therapy for
various tumor types. Since stem cells possess the ability to serve as a novel
vehicle for delivering therapeutic or suicide genes to primary or metastatic
cancer sites, these cells can function as part of gene‑directed enzyme prodrug
therapy (GDEPT). This review focuses on introducing engineered neural stem
cells (NSCs), which have tumor‑tropic behavior that allows NSCs to selectively
approach primary and invasive tumor foci, as a potential gene therapy for
melanoma. Therapy using engineered NSCs with cytotoxic agents resulted in
markedly reduced tumor volumes and significantly prolonged survival rates in
preclinical models of various tumor types. This review elucidates current
treatment options for metastatic melanoma and introduces a promising NSC
therapy. PURPOSE: Anti-programmed cell death receptor-1 (PD-1) antibodies have
demonstrated antitumor activity in many cancer entities. Hepatic adverse events
(AEs) are one of its major side effects, but the overall risks have not been
systematically evaluated. Thus, we conducted this meta-analysis to investigate
the overall incidence and risk of developing hepatic AEs in cancer patients
treated with PD-1 inhibitors.
METHODS: PubMed, Embase, and oncology conference proceedings were searched for
relevant studies. Eligible studies were randomized controlled trials of cancer
patients treated with PD-1 inhibitors with adequate data on hepatic AEs.
RESULTS: A total of nine randomized controlled trials with a variety of solid
tumors were eligible for the meta-analysis. The use of PD-1 inhibitors
significantly increased the risk of developing all-grade hepatic AEs but not for
high-grade hepatic AEs in comparison with chemotherapy or everolimus control.
Additionally, the risk of all-grade and high-grade hepatic AEs with a
nivolumab/ipilimumab combination was substantially higher than ipilimumab. No
significant differences in the risk of all-grade and high-grade hepatic AEs were
found between PD-1 inhibitors monotherapy and ipilimumab.
CONCLUSION: While the use of PD-1 inhibitors is associated with an increased
risk of developing hepatic AEs in cancer patients, this is primarily for lower
grade events. The field of immuno-oncology has witnessed unprecedented success in recent
years, with several PD=1 and PD-L1 inhibitors obtaining US FDA registration and
breakthrough drug therapy designation in multiple tumor types. Despite its clear
efficacy in certain cancers, treatment with these agents carries a risk of
immune-related toxicities and substantial ficial burden. It is, therefore,
critical to identify patients likely to benefit from such immunotherapies and
develop strategies to differentiate responders from nonresponders early during
treatment. Here we discuss the development of predictive and treatment response
biomarkers for immune checkpoint inhibitors. We first examine the role of PD-L1
expression, the most extensively studied predictive biomarker of response, and
further discuss emerging putative predictive biomarkers. We also detail
challenges faced in the development of response assessments for
immunotherapeutics and propose other biomarkers that may be useful as surrogate
intermediate end points of response. |
Is the protein lefty an inhibitor of nodal? | Yes, lefty is an inhibitor of nodal. | The nodal signaling pathway has been shown to play crucial roles in inducing and
patterning the mesoderm and endoderm, as well as in regulating neurogenesis and
left-right axis asymmetry. Here, we present the first complete cDNA and genomic
sequences as well as the promoter predication of the Dnah9 gene in the Japanese
flounder. The 15,558-bp-long cDNA is divided into 96 exons and spread over 138
kb of genomic DNA. Protein sequence comparison showed that it shares higher
identity with other vertebrate orthologs, with an ATP binding dynein motor, AAA
domain and microtubule binding stalk of dynein motor. Dnah9 exhibited maternal
and ubiquitous expression in all cells of the early development stages, but
became concentrated in the head at 1 DAH, as identified by qRT-PCR and in situ
hybridization methods. Furthermore, after nodal signaling was inhibited, the
level of Southpaw did not change significantly at early development stage (50 %
epiboly) but increased significantly at late stages (27-somite stages and 1
DAH), as well as the expression of Lefty, an inhibitor of nodal signaling,
increased continuously. On the other hand, the expression level of Dnah9
decreased. The transcription factor binding site of FAST-1 (SMAD interacting
protein) was identified in the transcription region of Dnah9 by the promoter
analysis, which might format the complexes of SMADs, FAST-1 and the
transcription region of Dnah9 served as a bridge of Dnah9 and nodal signaling.
All evidences indicated that Dnah9 might be downstream of nodal during the early
development stages, and an indirect function through SMADs for nodal signaling
pathway. Morphogen signaling is critical for the growth and patterning of tissues in
embryos and adults, but how morphogen signaling gradients are generated in
tissues remains controversial. The morphogen Nodal was proposed to form a
long-range signaling gradient via a reaction-diffusion system, on the basis of
differential diffusion rates of Nodal and its antagonist Lefty. Here we use a
specific zebrafish Nodal biosensor combined with immunofluorescence for
phosphorylated Smad2 to demonstrate that endogenous Nodal is unlikely to diffuse
over a long range. Instead, short-range Nodal signaling activation in a temporal
window is sufficient to determine the dimensions of the Nodal signaling domain.
The size of this temporal window is set by the differentially timed production
of Nodal and Lefty, which arises mainly from repression of Lefty translation by
the microRNA miR-430. Thus, temporal information is transformed into spatial
information to define the dimensions of the Nodal signaling domain and,
consequently, to specify mesendoderm. |
List drugs withdrawn from the market for cardiovascular adverse events. | Over the years, a number of different drugs have been withdrawn for Cardiotoxicity. Drugs like Clobutinol, that induce cardiac arrhythmias by a blockade of the potassium channel coded by the hERG channel, sibutramine for weight loss and Cox2 inhibitors, Rofecoxib and Valdecoxib have been withdrawn from the market. | In a very short time, COX-2 enzyme inhibitors have gone from the darlings to the
pariahs of the pharmaceutical industry. These drugs were developed based on the
hypothesis whereby selective inhibition of the COX enzyme would lead to
reduction in pain and inflammation without associated gastrointestinal and
bleeding risks. However, in September 2004, rofecoxib was voluntarily removed
from the market for increased cardiovascular risk and in April 2005, valdecoxib
was also withdrawn, at least in part, due to excess cardiovascular risk.
Celecoxib was the first COX-2 inhibitor introduced and the only remaining one on
the US market. There is consequently a justified concern that cardiovascular
toxicity is a class effect of all COX-2 inhibitors. This article systematically
reviews the evidence surrounding COX-2 inhibitors and cardiovascular risk.
Although the evidence suggests a fairly consistent cardiovascular risk with
rofecoxib, the evidence for cardiovascular risk with celecoxib is more
equivocal. Although isolated studies have suggested some cardiovascular risk for
celecoxib, the totality of the evidence suggests that any risk is likely to be
small and comparable to traditional NSAIDs. The cardiovascular risks of COX-2
inhibitors appear heterogeneous, influenced not only by the drug class, but also
individual drug, dosage and patient characteristics. Specific modifying factors
of the cardiovascular risk of COX-2 inhibitors including dose, concomitant
drugs, individual cardiac and genetic risk profiles, will require further study. Sudden death as a side effect of action of non-antiarrhythmic drugs is a major
pharmacological safety concern facing the pharmaceutical industry and the health
regulatory authorities. A number of drugs have been withdrawn from the market in
recent years due to cardiovascular toxicity associated with undesirable blockade
of hERG potassium channel. Pharmaceuticals of widely varying structure have been
shown to interact with hERG. Defining the molecular features that confer hERG
inhibitory activity has therefore become a focus of considerable computational
and statistical modeling efforts. Some of the approaches are aimed primarily at
filtering out potential hERG blockers in the context of virtual libraries, while
others involve understanding structure-activity relationships governing
hERG-drug interactions. The ability of models to produce structural hypotheses
that can be tested by the project teams has become the key prerequisite driving
their organization-wide adoption. A common over-the-counter (OTC) non-opioid antitussive drug, clobutinol, was
recently withdrawn from the market due to its potential to induce cardiac
arrhythmias by a blockade of the potassium channel coded by the human
ether-à-go-go-related gene (hERG). In this study, we investigated the effects of
a number of antitussive compounds on the hERG ion channel current using
patch-clamp electrophysiology, and compared the effects to that of clobutinol.
The compounds clobutinol, pentoxyverine, dextromethorphan, and codeine inhibited
the outward current in hERG transfected cells with half-maximal inhibition
concentrations (IC50) of 1.9 microM, 3.0 microM, 5.1 microM, and 97 microM,
respectively. For theobromine, no significant effect on the hERG current at a
concentration up to 100 microM was detected. Safety margins between the effects
of the drugs on the hERG ion channel current and their calculated maximal free
therapeutic plasma concentration were calculated. These results were compared to
assess potential risks of the compounds to induce torsade de pointes-type
arrhythmias. BACKGROUND: In September 2004, rofecoxib was voluntarily withdrawn from the
worldwide market. Our objective was to determine whether and when analysis of
published and unpublished placebo-controlled trials could have revealed
cardiovascular risk associated with rofecoxib before its withdrawal as an
example to inform future postmarket pharmaceutical safety surveillance efforts.
METHODS: We conducted a cumulative subject-level pooled analysis of data from
all randomized, placebo-controlled trials of rofecoxib conducted by the
manufacturer before September 2004. Our main outcome measurement was incidence
of any investigator-reported death from any cause or cardiovascular
thromboembolic (CVT) adverse event.
RESULTS: We identified 30 randomized, placebo-controlled trials of rofecoxib
that enrolled a combined 20 152 subjects. Trial duration ranged from 4 weeks to
4 years; enrollment ranged from 17 to 2586 subjects prescribed either rofecoxib
or placebo; and rofecoxib dose ranged from 12.5 mg to 50 mg. As of December
2000, 21 of these trials had been completed (70%), and the risk of a CVT adverse
event or death was greater among subjects assigned to the rofecoxib group (rate
ratio [RR], 2.18; 95% confidence interval [CI], 0.93-5.81) (P = .07), raising
concerns from a safety standpoint. Subsequently collected data through June 2001
showed that rofecoxib was associated with a 35% increased risk of a CVT adverse
event or death (RR, 1.35; 95% CI, 1.00-1.96) (P = .05). Analyzing data available
as of April 2002, we found a 39% increased risk (RR, 1.39; 95% CI, 1.07-1.80) (P
= .02), and using data available as of September 2004, we found a 43% increased
risk (RR,1.43; 95% CI, 1.16-1.76) (P < .001).
CONCLUSION: Cumulative pooled analysis of all randomized, placebo-controlled
trials demonstrates a trend toward increased cardiovascular risk associated with
rofecoxib compared with placebo as early as December 2000, the comparison
reaching a P value of .05 by June 2001, nearly 3(1/2) years before the
manufacturer's voluntary market withdrawal. BACKGROUND: Concerns regarding the safety of transfused blood have led to the
development of a range of interventions to minimise blood loss during major
surgery. Anti-fibrinolytic drugs are widely used, particularly in cardiac
surgery, and previous reviews have found them to be effective in reducing blood
loss, the need for transfusion, and the need for re-operation due to continued
or recurrent bleeding. In the last few years questions have been raised
regarding the comparative performance of the drugs. The safety of the most
popular agent, aprotinin, has been challenged, and it was withdrawn from world
markets in May 2008 because of concerns that it increased the risk of
cardiovascular complications and death.
OBJECTIVES: To assess the comparative effects of the anti-fibrinolytic drugs
aprotinin, tranexamic acid (TXA), and epsilon aminocaproic acid (EACA) on blood
loss during surgery, the need for red blood cell (RBC) transfusion, and adverse
events, particularly vascular occlusion, renal dysfunction, and death.
SEARCH STRATEGY: We searched: the Cochrane Injuries Group's Specialised Register
(July 2010), Cochrane Central Register of Controlled Trials (The Cochrane
Library 2010, Issue 3), MEDLINE (Ovid SP) 1950 to July 2010, EMBASE (Ovid SP)
1980 to July 2010. References in identified trials and review articles were
checked and trial authors were contacted to identify any additional studies. The
searches were last updated in July 2010.
SELECTION CRITERIA: Randomised controlled trials (RCTs) of anti-fibrinolytic
drugs in adults scheduled for non-urgent surgery. Eligible trials compared
anti-fibrinolytic drugs with placebo (or no treatment), or with each other.
DATA COLLECTION AND ANALYSIS: Two authors independently assessed trial quality
and extracted data.
MAIN RESULTS: This review summarises data from 252 RCTs that recruited over
25,000 participants. Data from the head-to-head trials suggest an advantage of
aprotinin over the lysine analogues TXA and EACA in terms of reducing
perioperative blood loss, but the differences were small. Compared to control,
aprotinin reduced the probability of requiring RBC transfusion by a relative 34%
(relative risk [RR] 0.66, 95% confidence interval [CI] 0.60 to 0.72). The RR for
RBC transfusion with TXA was 0.61 (95% CI 0.53 to 0.70) and was 0.81 (95% CI
0.67 to 0.99) with EACA. When the pooled estimates from the head-to-head trials
of the two lysine analogues were combined and compared to aprotinin alone,
aprotinin appeared more effective in reducing the need for RBC transfusion (RR
0.90; 95% CI 0.81 to 0.99).Aprotinin reduced the need for re-operation due to
bleeding by a relative 54% (RR 0.46, 95% CI 0.34 to 0.62). This translates into
an absolute risk reduction of 2% and a number needed-to-treat (NNT) of 50 (95%
CI 33 to 100). A similar trend was seen with EACA (RR 0.32, 95% CI 0.11 to 0.99)
but not TXA (RR 0.80, 95% CI 0.55 to 1.17). The blood transfusion data were
heterogeneous and funnel plots indicate that trials of aprotinin and the lysine
analogues may be subject to publication bias.When compared with no treatment
aprotinin did not increase the risk of myocardial infarction (RR 0.87, 95% CI
0.69 to 1.11), stroke (RR 0.82, 95% CI 0.44 to 1.52), renal dysfunction (RR
1.10, 95% CI 0.79 to 1.54) or overall mortality (RR 0.81, 95% CI 0.63 to 1.06).
Similar trends were seen with the lysine analogues, but data were sparse. These
data conflict with the results of recently published non-randomised studies,
which found increased risk of cardiovascular complications and death with
aprotinin. There are concerns about the adequacy of reporting of uncommon events
in the small clinical trials included in this review.When aprotinin was compared
directly with either, or both, of the two lysine analogues it resulted in a
significant increase in the risk of death (RR 1.39, 95% CI 1.02, 1.89), and a
non-significant increase in the risk of myocardial infarction (RR 1.11 95% CI
0.82, 1.50). Most of the data contributing to this added risk came from a single
study - the BART trial (2008).
AUTHORS' CONCLUSIONS: Anti-fibrinolytic drugs provide worthwhile reductions in
blood loss and the receipt of allogeneic red cell transfusion. Aprotinin appears
to be slightly more effective than the lysine analogues in reducing blood loss
and the receipt of blood transfusion. However, head to head comparisons show a
lower risk of death with lysine analogues when compared with aprotinin. The
lysine analogues are effective in reducing blood loss during and after surgery,
and appear to be free of serious adverse effects. The global incidence of obesity continues to rise and is a major driver of
morbidity and mortality through cardiovascular and cerebrovascular diseases.
Animal models used in the discovery of novel treatments for obesity range from
straightforward measures of food intake in lean rodents to long-term studies in
animals exhibiting obesity due to the continuous access to diets high in fat.
The utility of these animal models can be extended to determine, for example,
that weight loss is due to fat loss and/or assess whether beneficial changes in
key plasma parameters (e.g. insulin) are evident. In addition, behavioural
models such as the behavioural satiety sequence can be used to confirm that a
drug treatment has a selective effect on food intake. Typically, animal models
have excellent predictive validity whereby drug-induced weight loss in rodents
subsequently translates to weight loss in man. However, despite this, at the
time of writing orlistat (Europe; USA) remains the only drug currently marketed
for the treatment of obesity, with sibutramine having recently been withdrawn
from sale globally due to the increased incidence of serious, non-fatal
cardiovascular events. While the utility of rodent models in predicting clinical
weight loss is detailed, the review also discusses whether animals can be used
to predict adverse events such as those seen with recent anti-obesity drugs in
the clinic. BACKGROUND: Concerns regarding the safety of transfused blood have led to the
development of a range of interventions to minimise blood loss during major
surgery. Anti-fibrinolytic drugs are widely used, particularly in cardiac
surgery, and previous reviews have found them to be effective in reducing blood
loss, the need for transfusion, and the need for re-operation due to continued
or recurrent bleeding. In the last few years questions have been raised
regarding the comparative performance of the drugs. The safety of the most
popular agent, aprotinin, has been challenged, and it was withdrawn from world
markets in May 2008 because of concerns that it increased the risk of
cardiovascular complications and death.
OBJECTIVES: To assess the comparative effects of the anti-fibrinolytic drugs
aprotinin, tranexamic acid (TXA), and epsilon aminocaproic acid (EACA) on blood
loss during surgery, the need for red blood cell (RBC) transfusion, and adverse
events, particularly vascular occlusion, renal dysfunction, and death.
SEARCH STRATEGY: We searched: the Cochrane Injuries Group's Specialised Register
(July 2010), Cochrane Central Register of Controlled Trials (The Cochrane
Library 2010, Issue 3), MEDLINE (Ovid SP) 1950 to July 2010, EMBASE (Ovid SP)
1980 to July 2010. References in identified trials and review articles were
checked and trial authors were contacted to identify any additional studies. The
searches were last updated in July 2010.
SELECTION CRITERIA: Randomised controlled trials (RCTs) of anti-fibrinolytic
drugs in adults scheduled for non-urgent surgery. Eligible trials compared
anti-fibrinolytic drugs with placebo (or no treatment), or with each other.
DATA COLLECTION AND ANALYSIS: Two authors independently assessed trial quality
and extracted data. This version of the review includes a sensitivity analysis
excluding trials authored by Prof. Joachim Boldt.
MAIN RESULTS: This review summarises data from 252 RCTs that recruited over
25,000 participants. Data from the head-to-head trials suggest an advantage of
aprotinin over the lysine analogues TXA and EACA in terms of reducing
perioperative blood loss, but the differences were small. Compared to control,
aprotinin reduced the probability of requiring RBC transfusion by a relative 34%
(relative risk [RR] 0.66, 95% confidence interval [CI] 0.60 to 0.72). The RR for
RBC transfusion with TXA was 0.61 (95% CI 0.53 to 0.70) and was 0.81 (95% CI
0.67 to 0.99) with EACA. When the pooled estimates from the head-to-head trials
of the two lysine analogues were combined and compared to aprotinin alone,
aprotinin appeared more effective in reducing the need for RBC transfusion (RR
0.90; 95% CI 0.81 to 0.99).Aprotinin reduced the need for re-operation due to
bleeding by a relative 54% (RR 0.46, 95% CI 0.34 to 0.62). This translates into
an absolute risk reduction of 2% and a number needed-to-treat (NNT) of 50 (95%
CI 33 to 100). A similar trend was seen with EACA (RR 0.32, 95% CI 0.11 to 0.99)
but not TXA (RR 0.80, 95% CI 0.55 to 1.17). The blood transfusion data were
heterogeneous and funnel plots indicate that trials of aprotinin and the lysine
analogues may be subject to publication bias.When compared with no treatment
aprotinin did not increase the risk of myocardial infarction (RR 0.87, 95% CI
0.69 to 1.11), stroke (RR 0.82, 95% CI 0.44 to 1.52), renal dysfunction (RR
1.10, 95% CI 0.79 to 1.54) or overall mortality (RR 0.81, 95% CI 0.63 to 1.06).
Similar trends were seen with the lysine analogues, but data were sparse. These
data conflict with the results of recently published non-randomised studies,
which found increased risk of cardiovascular complications and death with
aprotinin. There are concerns about the adequacy of reporting of uncommon events
in the small clinical trials included in this review.When aprotinin was compared
directly with either, or both, of the two lysine analogues it resulted in a
significant increase in the risk of death (RR 1.39, 95% CI 1.02, 1.89), and a
non-significant increase in the risk of myocardial infarction (RR 1.11 95% CI
0.82, 1.50). Most of the data contributing to this added risk came from a single
study - the BART trial (2008).
AUTHORS' CONCLUSIONS: Anti-fibrinolytic drugs provide worthwhile reductions in
blood loss and the receipt of allogeneic red cell transfusion. Aprotinin appears
to be slightly more effective than the lysine analogues in reducing blood loss
and the receipt of blood transfusion. However, head to head comparisons show a
lower risk of death with lysine analogues when compared with aprotinin. The
lysine analogues are effective in reducing blood loss during and after surgery,
and appear to be free of serious adverse effects. INTRODUCTION: Despite substantial progress in the pharmacological treatment of
schizophrenia, antipsychotic drugs are associated with insufficient effects on
negative and cognitive symptoms, adverse events, poor compliance and drug
discontinuation. Sertindole , first withdrawn from the market for cardiovascular
safety concerns, is currently available in many countries. It has high affinity
for dopamine D2, serotonin 5-HT2A, 5-HT2C and a1-adrenergic receptors, and a low
potential for extrapyramidal symptoms.
AREAS COVERED: The pharmacodynamics, pharmacokinetics, clinical efficacy, safety
and tolerability, and cost-effectiveness of sertindole are covered in this
article, based on a literature review (PubMed) from 1990 to 2014.
EXPERT OPINION: The reviewed studies suggest a beneficial effect of sertindole
on positive, negative and cognitive symptoms, and on relapse prevention.
Although generally well tolerated, sertindole is associated with a dose-related
QT-interval prolongation; thus, its administration requires electrocardiogram
screening and monitoring. The presence of congenital long QT syndrome,
prolongated QTc at baseline, cardiac disease and drug interactions should be
carefully considered before prescribing sertindole. Further direct
'head-to-head' comparisons with other second-generation antipsychotics (SGAs),
trials on special populations and further clarification on cardiac safety are
needed to better define the role of sertindole in the treatment of
schizophrenia. Obesity is a major health problem worldwide. Although diet and physical activity
are crucial in the management of obesity, the long-term success rate is low.
Therefore antiobesity drugs are of great interest, especially when lifestyle
modification has failed. As obesity is not an immediate life-threatening
disease, these drugs are required to be safe. Antiobesity drugs that have been
developed so far have limited efficacies and considerable adverse effects
affecting tolerability and safety. Therefore, most antiobesity drugs have been
withdrawn. Fenfluramine and dexfenfluramine were withdrawn because of the
potential damage to heart valves. Sibutramine was associated with an increase in
major adverse cardiovascular events in the Sibutramine Cardiovascular Outcomes
(SCOUT) trial and it was withdrawn from the market in 2010. Rimonabant was
withdrawn because of significant psychiatric adverse effects. Orlistat was
approved in Europe and the United States for long-term treatment of obesity, but
many patients cannot tolerate its gastrointestinal side effects. Phentermine and
diethylpropion can only be used for less than 12 weeks because the long-term
safety of these drugs is unknown. Ephedrine and caffeine are natural substances
but the effects on weight reduction are modest. As a result there is a huge
unmet need for effective and safe antiobesity drugs. Recently lorcaserin and
topiramate plus phentermine have been approved for the treatment of obesity but
long-term safety data are lacking. BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to reduce
inflammatory pain and swelling in inflammatory bowel disease (IBD) patients with
rheumatological manifestations. While these drugs effectively reduce
musculoskeletal pain and stiffness, long-term use is limited by gastrointestinal
(GI) adverse effects (AEs) and disease exacerbation. As an alternative to
NSAIDs, selective cyclooxygenase 2 (COX-2) inhibitors were developed to improve
GI safety and tolerability. COX-2 inhibitors include drugs such as celecoxib,
rofecoxib, valdecoxib, etoricoxib, and lumiracoxib. Rofecoxib and valdecoxib
have been withdrawn from the market worldwide due to safety concerns (most
importantly for cardiovascular adverse events) and lumiracoxib has been
withdrawn in many countries due to liver toxicity. However, celecoxib and
etoricoxib continue to be available for use in many countries. Several studies
have examined whether COX-2 inhibitors can be safely used for the treatment of
rheumatological manifestations of IBD with inconsistent results. Some
investigators report acceptable safety profiles associated with these drugs
while others found that COX-2 inhibitors are associated with high rates of
disease exacerbation.
OBJECTIVES: The objective of this systematic review was to evaluate the
tolerability and safety of COX-2 inhibitors used for the treatment of
rheumatological manifestations of IBD.
SEARCH METHODS: We searched the following databases from inception to 19
September 2013: PubMed, EMBASE, MEDLINE and CENTRAL. The search was not limited
by language. Additional trials were identified by manually searching the
reference lists of relevant papers and conference proceedings and through
correspondence with experts and pharmaceutical companies.
SELECTION CRITERIA: Randomized controlled trials (RCTs) that compared COX-2
inhibitors to placebo were considered for inclusion. Participants were adult
patients with IBD presenting with rheumatological manifestations of at least two
weeks duration.
DATA COLLECTION AND ANALYSIS: Two authors independently assessed trial
eligibility and extracted data. Methodological quality was assessed using the
Cochrane risk of bias tool. The primary outcome measure was the proportion of
patients with disease exacerbation as defined by the included studies. Secondary
outcomes included GI adverse effects, renal toxicity, cardiovascular and
thrombotic events. Data were analysed on an intention-to-treat basis where
patients with missing final outcomes were assumed to have had an exacerbation of
IBD. We calculated the risk ratio (RR) and corresponding 95% confidence interval
(95% CI) for dichotomous outcomes. The overall quality of the evidence was
assessed using the GRADE criteria.
MAIN RESULTS: There were no RCTs that assessed the tolerability or safety of the
withdrawn COX-2 inhibitors rofecoxib, valdecoxib, or lumiracoxib. Two RCTs (n =
381 IBD patients with rheumatological manifestations) were included in the
review. One study (n = 159) compared etoricoxib (60 to 120 mg/day) to placebo in
IBD patients with quiescent or active ulcerative colitis or Crohn's disease. The
other study (n = 222) compared celecoxib (200 mg twice daily) to placebo in
patients with quiescent ulcerative colitis. Both studies were judged to be at
low risk of bias. The two included studies were not pooled for meta-analysis due
to differences in patient populations and treatment duration. There was no
statistically significant difference in exacerbation of IBD between etoricoxib
and placebo. After 12 weeks of treatment the IBD exacerbation rate was 17%
(14/82) in the etoricoxib group compared to 19% (15/77) in the placebo group (RR
0.88, 95% CI 0.45 to 1.69). A GRADE analysis indicated that the overall quality
of the evidence supporting this outcome was low due to very sparse data (29
events). There was no statistically significant difference in exacerbation of
ulcerative colitis between celecoxib and placebo. After two weeks of treatment
4% (5/112) of celecoxib patients experienced an exacerbation of ulcerative
colitis compared to 6% (7/110) of patients in the placebo group (RR 0.70, 95% CI
0.23 to 2.14). A GRADE analysis indicated that the overall quality of the
evidence supporting this outcome was low due to very sparse data (12 events).
The study comparing etoricoxib to placebo documented but did not report on AEs.
The proportion of patients who experienced AEs was similar in the celecoxib and
placebo groups (21% and 17%, respectively, P > 0.20). No patients in either
group died or experienced serious adverse events. Eleven percent of patients in
the celecoxib and placebo groups experienced GI AEs (RR 0.97, 95% CI 0.46 to
2.07). A GRADE analysis indicated that the overall quality of the evidence
supporting this outcome was low due to very sparse data (24 events). GI AEs led
to premature withdrawal from the study in 3% of patients in celecoxib and
placebo groups respectively. GI AEs included increased stool frequency, rectal
bleeding, and inflamed mucosa. No patients experienced any cardiovascular
adverse events. Renal toxicity or thrombotic AEs were not reported.
AUTHORS' CONCLUSIONS: The results for disease exacerbation and AEs between the
COX-2 inhibitors celecoxib and etoricoxib and placebo were uncertain. Thus no
definitive conclusions regarding the tolerability and safety of the short term
use of celecoxib and etoricoxib in patients with IBD can be drawn. The two
included studies suggest that celecoxib and etoricoxib do not exacerbate IBD
symptoms. However, it should be noted that both studies had relatively small
sample sizes and short follow-up durations. Clinicians need to continue to weigh
the risks and benefits of these drugs when treating patients IBD patients with
rheumatological manifestations in order to avoid disease exacerbation and other
adverse effects. Further RCTs are needed to determine the tolerability and
safety of celecoxib and etoricoxib in these patients. BACKGROUND: All major guidelines on antihypertensive therapy recommend weight
loss; anti-obesity drugs may be able to help in this respect.
PRIMARY OBJECTIVES: To assess the long-term effects of pharmacologically induced
reduction in body weight in adults with essential hypertension on all-cause
mortality, cardiovascular morbidity, and adverse events (including total serious
adverse events, withdrawal due to adverse events, and total non-serious adverse
events).
SECONDARY OBJECTIVES: To assess the long-term effects of pharmacologically
induced reduction in body weight in adults with essential hypertension on change
from baseline in systolic blood pressure, change from baseline in diastolic
blood pressure, and body weight reduction.
SEARCH METHODS: We obtained studies using computerised searches of the Cochrane
Hypertension Group Specialised Register, the Cochrane Central Register of
Controlled Trials (CENTRAL), Ovid MEDLINE, Ovid EMBASE, the clinical trials
registry ClinicalTrials.gov, and from handsearches in reference lists and
systematic reviews (status as of 13 April 2015).
SELECTION CRITERIA: Randomised controlled trials in hypertensive adults of at
least 24 weeks' duration that compared long-term pharmacologic interventions for
weight loss with placebo.
DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies,
assessed risk of bias, and extracted data. Where appropriate and in the absence
of significant heterogeneity between studies (P > 0.1), we pooled studies using
fixed-effect meta-analysis. When heterogeneity was present, we used the
random-effects method and investigated the cause of heterogeneity.
MAIN RESULTS: After updating the literature search, which was extended to
include four new weight-reducing drugs, we identified one additional study of
phentermine/topiramate, bringing the total number of studies to nine that
compare orlistat, sibutramine, or phentermine/topiramate to placebo and thus
fulfil our inclusion criteria. We identified no relevant studies investigating
rimonabant, liraglutide, lorcaserin, or naltrexone/bupropion. No study included
mortality and cardiovascular morbidity as predefined outcomes. Incidence of
gastrointestinal side effects was consistently higher in those participants
treated with orlistat versus those treated with placebo. The most frequent side
effects were dry mouth, constipation, and headache with sibutramine, and dry
mouth and paresthaesia with phentermine/topiramate. In participants assigned to
orlistat, sibutramine, or phentermine/topiramate body weight was reduced more
effectively than in participants in the usual-care/placebo groups. Orlistat
reduced systolic blood pressure as compared to placebo by -2.5 mm Hg (mean
difference (MD); 95% confidence interval (CI): -4.0 to -0.9 mm Hg) and diastolic
blood pressure by -1.9 mm Hg (MD; 95% CI: -3.0 to -0.9 mm Hg). Sibutramine
increased diastolic blood pressure compared to placebo by +3.2 mm Hg (MD; 95%
CI: +1.4 to +4.9 mm Hg). The one trial that investigated phentermine/topiramate
suggested it lowered blood pressure.
AUTHORS' CONCLUSIONS: In people with elevated blood pressure, orlistat and
sibutramine reduced body weight to a similar degree, while
phentermine/topiramate reduced body weight to a greater extent. In the same
trials, orlistat and phentermine/topiramate reduced blood pressure, while
sibutramine increased it. We could include no trials investigating rimonabant,
liraglutide, lorcaserin, or naltrexone/bupropion in people with elevated blood
pressure. Long-term trials assessing the effect of orlistat, liraglutide,
lorcaserin, phentermine/topiramate, or naltrexone/bupropion on mortality and
morbidity are unavailable and needed. Rimonabant and sibutramine have been
withdrawn from the market, after long-term trials on mortality and morbidity
have confirmed concerns about the potential severe side effects of these two
drugs. The European Medicines Agency refused marketing authorisation for
phentermine/topiramate due to safety concerns, while the application for
European marketing authorisation for lorcaserin was withdrawn by the
manufacturer after the Committee for Medicinal Products for Human Use judged the
overall benefit/risk balance to be negative. |
Is Migalastat used for treatment of Fabry Disease? | Yes, Migalastat is approved for treatment of Fabry disease. Migalastat is an oral pharmacological chaperone developed as an alternative to intravenous enzyme replacement therapy (ERT), stabilises specific mutant (amenable) forms of α-Gal to facilitate normal lysosomal trafficking. | Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA
gene. Fabry disease (FD) is an X-linked lysosomal storage disorder with a
heterogeneous spectrum of clinical manifestations that are caused by the
deficiency of α-galactosidase A (α-Gal-A) activity. Although useful for
diagnosis in males, enzyme activity is not a reliable biochemical marker in
heterozygous females due to random X-chromosome inactivation, thus rendering DNA
sequencing of the α-Gal-A gene, alpha-galactosidase gene (GLA), the most
reliable test for the confirmation of diagnosis in females. The spectrum of GLA
mutations is highly heterogeneous. Many polymorphic GLA variants have been
described, but it is unclear if haplotypes formed by combinations of such
variants correlate with FD, thus complicating molecular diagnosis in females
with normal α-Gal-A activity. We tested 67 female probands with clinical
manifestations that may be associated with FD and 110 control males with normal
α-Gal-A activity. Five different combinations of GLA polymorphic variants were
identified in 14 of the 67 females, whereas clearcut pathogenetic alterations,
p.Met51Ile and p.Met290Leu, were identified in two cases. The latter has not
been reported so far, and both mutant forms were found to be responsive to the
pharmacological chaperone deoxygalactonojirimycin (DGJ; migalastat
hydrochloride). Analysis of the male control population, as well as male
relatives of a suspected FD female proband, permitted the identification of
seven different GLA gene haplotypes in strong linkage disequilibrium. The
identification of haplotypes in control males provides evidence against their
involvement in the development of FD phenotypic manifestations. Conflict of interest statement: Competing Interests: Brandy Young-Gqamana is a
former employee of Amicus Therapeutics. Nastry Brignol, Hui-Hwa Chang, Richie
Khanna, Rebecca Soska, Sheela A. Sitaraman, Pol Boudes, David J. Lockhart,
Kenneth J. Valenzano, and Elfrida R. Benjamin are employed by Amicus
Therapeutics and are shareholders in the company. Maria Fuller was a
collaborator with Amicus Therapeutics. All other authors were investigators on
the Phase 2 clinical studies of migalastat HCl. Dominique P. Germain has
received research funding, consultancy fees, and travel expenses from Genzyme
and Shire HGT. Roberto Giugliani is a consultant and investigator for Actelion,
Amicus, BioMarin, Genzyme and Shire HGT. Derralynn A. Hughes is a consultant for
Amicus, Shire HGT, Genzyme, Actelion, has been on a Speaker's Bureau for Amicus,
Shire HGT, Genzyme, and Actelion, and has received grants from Amicus, Shire
HGT, and Genzyme. Atul Mehta has received honoraria, research funding,
consultancy fees, and travel expenses from Shire HGT, Genzyme, Actelion,
Protalix, and Amicus. Kathy Nicholls has received travel and research support
and speaker's honoraria from Amicus, Shire HGT and Genzyme. Amicus Therapeutics
funded the research and any publication fees. The α-Gal A (Fabrazyme) used in
this study is a product produced by Genzyme. Maria Fuller is employed by SA
Pathology, Adelaide. There are no further patents, products in development or
marketed products to declare. This does not alter the authors' adherence to all
of the PLOS ONE policies on sharing data and materials. BACKGROUND: Fabry disease (FD) is a genetic disorder resulting from deficiency
of the lysosomal enzyme α-galactosidase A (α-Gal A) which leads to
globotriaosylceramide (GL-3) accumulation in multiple tissues. We report on the
safety and pharmacodynamics of migalastat hydrochloride, an investigational
pharmacological chaperone given orally every other day (QOD) to females with FD.
METHODS: This was an open-label, uncontrolled, Phase 2 study of 12 weeks with
extension to 48 weeks in nine females with FD. Doses of 50mg, 150 mg and 250 mg
were given QOD. At multiple time points, α-Gal A activity and GL-3 levels were
quantified in blood cells, kidney and skin. GL-3 levels were also evaluated
through skin and renal histology. Each individual GLA mutation was
retrospectively categorized as being amenable or not to migalastat HCl based on
an in vitro α-Gal A transfection assay developed in human embryonic kidney
(HEK)-293 cells.
RESULTS: Migalastat HCl was generally well tolerated. Patients with amenable
mutations seem to demonstrate greater pharmacodynamic response to migalastat HCl
compared to patients with non-amenable mutations. The greatest declines in urine
GL-3 were observed in the three patients with amenable GLA mutations that were
treated with 150 or 250 mg migalastat HCl QOD. Additionally, these three
patients all demonstrated decreases in GL-3 inclusions in kidney peri-tubular
capillaries.
CONCLUSIONS: Migalastat HCl is a candidate oral pharmacological chaperone that
provides a potential novel genotype-specific treatment for FD. Treatment
resulted in GL-3 substrate decrease in female patients with amenable GLA
mutations. Phase 3 studies are ongoing. The prevalence rate for Fabry disease is conventionally considered to be 1 case
in 40,000; however, due to increased screening accuracy, reports now suggest
that prevalence is 1 case in 1,500 among male children, and it is likely that
the clinical importance of the condition will increase in the future. In
dialysis patients to date, prevalence rates are between 0.16 and 1.2 %.
Globotriaosylsphingosine (Lyso-GL-3), which is a substrate of α-galactosidase A
(α-Gal A), has surfaced as a new biomarker, and is also effective in the
determination and monitoring of the effects of enzyme replacement therapy. In
terms of genetic abnormalities, the E66Q mutation has recently become a topic of
discussion, and although doubts have been expressed over whether or not it is
the gene responsible for Fabry disease, there is still a strong possibility that
it is a functional genetic polymorphism. At present, the standard treatment for
Fabry disease is enzyme replacement therapy, and in order to overcome the
problems involved with this, a method of producing recombit human α-Gal A
using methanol-assimilating yeast, and chemical or medicinal chaperone treatment
are of current interest. Migalastat hydrochloride is known as a pharmacological
chaperone, but is currently in Phase III global clinical trials. Adding saposin
B to modified α-N-acetyl galactosaminidase is also under consideration as a
treatment method. Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational
pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A)
deficiency, which leads to Fabry disease, an X-linked, lysosomal storage
disorder. The currently approved, biologics-based therapy for Fabry disease is
enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or
agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in
combination with agalsidase is expected to result in the pharmacokinetic (PK)
enhancement of agalsidase in plasma by increasing the systemic exposure of
active agalsidase, thereby leading to increased cellular levels in
disease-relevant tissues. This Phase 2a study design consisted of an open-label,
fixed-treatment sequence that evaluated the effects of single oral doses of 150
mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused
agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected,
intravenous administration of agalsidase alone resulted in increased α-Gal A
activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs)
compared to baseline. Following co-administration of migalastat HCl and
agalsidase, α-Gal A activity in plasma was further significantly increased 1.2-
to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients
(95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were
seen following co-administration of migalastat HCl and agalsidase. The effects
were not related to the administered migalastat HCl dose, as the 150 mg dose of
migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose.
Conversely, agalsidase had no effect on the plasma PK of migalastat. No
migalastat HCl-related adverse events or drug-related tolerability issues were
identified.
TRIAL REGISTRATION: ClinicalTrials.gov NCT01196871. Migalastat (Galafold™)-a small molecule drug developed by Amicus Therapeutics
that restores the activity of specific mutant forms of α-galactosidase-has been
approved for the treatment of Fabry disease in the EU in patients with amenable
mutations. Fabry disease is a rare disorder that results in a deficiency or
absence of α-galactosidase, leading to accumulation of globotriaosylceramide in
the lysosomes of various cells. This article summarizes the milestones in the
development of migalastat leading to this first approval in the EU for the
long-term treatment of adults and adolescents aged ≥16 years with a confirmed
diagnosis of Fabry disease. PURPOSE: Fabry disease is an X-linked lysosomal storage disorder caused by
mutations in the α-galactosidase A gene. Migalastat, a pharmacological
chaperone, binds to specific mutant forms of α-galactosidase A to restore
lysosomal activity.
METHODS: A pharmacogenetic assay was used to identify the α-galactosidase A
mutant forms amenable to migalastat. Six hundred Fabry disease-causing mutations
were expressed in HEK-293 (HEK) cells; increases in α-galactosidase A activity
were measured by a good laboratory practice (GLP)-validated assay (GLP
HEK/Migalastat Amenability Assay). The predictive value of the assay was
assessed based on pharmacodynamic responses to migalastat in phase II and III
clinical studies.
RESULTS: Comparison of the GLP HEK assay results in in vivo white blood cell
α-galactosidase A responses to migalastat in male patients showed high
sensitivity, specificity, and positive and negative predictive values (≥0.875).
GLP HEK assay results were also predictive of decreases in kidney
globotriaosylceramide in males and plasma globotriaosylsphingosine in males and
females. The clinical study subset of amenable mutations (n = 51) was
representative of all 268 amenable mutations identified by the GLP HEK assay.
CONCLUSION: The GLP HEK assay is a clinically validated method of identifying
male and female Fabry patients for treatment with migalastat.Genet Med 19 4,
430-438. Author information:
(1)Department of Haematology, Royal Free London NHS Foundation Trust and
University College London, London, UK.
(2)Department of Nephrology, Royal Melbourne Hospital, Parkville, Victoria,
Australia.
(3)Section of Vitreoretinal Surgery & Diseases, Emory University, Atlanta,
Georgia, USA.
(4)Division of Nephrology and Dialysis, Department of Medicine III, Medical
University of Vienna, Vienna, Austria.
(5)Doernbecher Children's Hospital, Oregon Health & Science University,
Portland, Oregon, USA.
(6)Infusion Associates, Grand Rapids, Missouri, USA.
(7)Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USA.
(8)Department of Pediatrics, Osaka City University Hospital, Osaka-shi, Japan.
(9)Charles Dent Metabolic Unit, National Hospital for Neurology and
Neurosurgery, London, UK.
(10)Jikei University Hospital, Tokyo, Japan.
(11)Departmento Cuore e vasi, A.O.U. Careggi Firenze, Firenze, Italy.
(12)Osaka City University Hospital, Osaka-shi, Japan.
(13)Lysosmal Disorders Unit, Department of Medicine, Addenbrooke's Hospital,
Cambridge, UK.
(14)Genetics Center MS716, Children's Hospital of Wisconsin, Milwaukee,
Wisconsin, USA.
(15)Univeritair Ziekenhaus Anterpen, Edegem, Belgium.
(16)Division of Medical Genetics, University of Versailles, Paris-Saclay
University and Assistance Publique-Hôpitaux de Paris, Paris, France.
(17)O & O Alpan LLC, Springfield, Virginia, USA.
(18)Service de Médecine, Hôpital Claude Huriez-CHRU Lille, Lille, France.
(19)Department of Endocrinology and Metabolic Medicine, Salford Royal NHS
Foundation Trust, Salford, UK.
(20)Hospital das Clínicas FMUSP-Ribeirão Preto, São Paulo, Ribeirão Preto,
Brazil.
(21)Niigata University Graduate School of Medicine and Dental Sciences, Niigata,
Japan.
(22)Royal Perth Hospital, Perth, New South Wales, Australia.
(23)Department of Genetics, Emory University School of Medicine, Atlanta,
Georgia, USA.
(24)Clinical Research Division, Hôpital du Sacré-Coeur de Montreal, University
of Montreal, Montreal, Quebec, Canada.
(25)Institute of Metabolic Disease, Baylor Research Institute, Dallas, Texas,
USA.
(26)Agility Clinical Inc., Carlsbad, California, USA.
(27)Amicus Therapeutics Inc., Cranbury, New Jersey, USA.
(28)CymaBay Therapeutics, Inc., Newark, California, USA.
(29)TranscripTx, Inc., Sunnyvale, California, USA.
(30)Parkinson's Institute and Clinical Center, Sunnyvale, California, USA.
(31)Department of Medical Endocrinology, Rigshospital, Copenhagen University
Hospital, Copenhagen, Denmark. |
Where are Paneth cells located? | Paneth cells are located in the intestinal crypt base columnar cells (CBCCs). | In about 70% of patients Crohn's disease (CD) affects the small intestine. This
disease location is stable over time and associated with a genetic background
different from isolated colonic disease. A characteristic feature of small
intestinal host defense is the presence of Paneth cells at the bottom of the
crypts of Lieberkühn. These cells produce different broad spectrum antimicrobial
peptides (AMPs) most abundantly the α-defensins HD-5 and -6 (DEFA5 und DEFA6).
In small intestinal Crohn's disease both these PC products are specifically
reduced. As a functional consequence, ileal extracts from Crohn's disease
patients are compromised in clearing bacteria and enteroadherent E. coli
colonize the mucosa. Mechanisms for defective antimicrobial Paneth cell function
are complex and include an association with a NOD2 loss of function mutation, a
disturbance of the Wnt pathway transcription factor TCF7L2 (also known as TCF4),
the autophagy factor ATG16L1, the endosomal stress protein XBP1, the toll-like
receptor TLR9, the calcium mediated potassium channel KCNN4 as well as mutations
or inactivation of HD5. Thus we conclude that small intestinal Crohn's disease
is most likely a complex disease of the Paneth cell: Paneth's disease. Current models of necrotizing enterocolitis (NEC) propose that intraluminal
microbes destroy intestinal mucosa and activate an inflammatory cascade that
ends in necrosis. We suggest an alternate hypothesis wherein NEC is caused by
injury to Paneth cells (PCs) in the intestinal crypts. PCs are specialized
epithelia that protect intestinal stem cells from pathogens, stimulate stem cell
differentiation, shape the intestinal microbiota, and assist in repairing the
gut. Our novel model of NEC uses neonatal mice and ablates PCs followed by
enteral infection. We contrast this model with other animal examples of NEC and
the clinical disease. Selective destruction of PCs using dithizone likely
releases tumor necrosis factor-α and other inflammatory mediators. We propose
that this event produces inflammation in the submucosa, generates
platelet-activating factor, and induces a coagulopathy. The role of PCs in NEC
is consistent with the onset of disease in preterm infants after a period of
PC-related maturation, the central role of PCs in crypt-related homeostasis, the
anatomic location of pneumatosis intestinalis close to the crypts, and the
proximity of PCs to occluded blood vessels that cause coagulation necrosis of
the intestinal villi. We offer this hypothesis to promote new thoughts about how
NEC occurs and its potential prevention. Paneth cells have been reported in colorectal adenomas and adenocarcinomas;
however, the frequency of colonic Paneth cell-containing adenomas is unknown as
are their clinicopathologic features. A total of 152 consecutive colorectal
adenomas from 103 patients (57 males and 46 females) were reviewed. The
frequency of Paneth cells in this cohort of adenomas was determined and
correlated with patient demographics. Twenty-six adenomas (17.1%) from 22
(21.4%) patients harbored Paneth cells, which were not limited to the base of
the crypts but aberrantly located throughout the crypts. Patient age, adenoma
size, villous features, and grade of dysplasia were not different between these
2 groups. Not surprisingly, Paneth cell-containing adenomas were more likely to
occur in the proximal colon (84.6% vs. 55.6%; P=0.006). There was a strong
association between male sex and Paneth cell-containing adenomas, as 23 of 26
(88.5%) of these adenomas occurred in male individuals compared with 71 of 126
(56.3%) non-Paneth cell-containing adenomas (P=0.002). Upon review of an
additional 460 adenomas from 200 patients with varying numbers of adenomas (68
with 1 adenoma, 68 with 2 adenomas, and 64 with 3 or more adenomas), the risk of
harboring synchronous adenomas was associated with villous morphology, proximal
location, and the presence of a Paneth cell-containing adenoma. Thus, the
presence of a Paneth cell-containing adenoma may be a marker for increased risk
of developing colorectal neoplasia. The intestinal epithelium is a classic example of a rapidly self-renewing tissue
fueled by dedicated resident stem cells. These stem cells reside at the crypt
base, generating committed progeny that mature into the various functional
epithelial lineages while following a rapid migratory path toward the villi. Two
models of intestinal stem cell location were proposed half a century ago and
data have been presented in support of both models, dividing the scientific
community. Molecular markers have been identified and validated using new
techniques such as in vivo lineage tracing and ex vivo organoid culture. The
intestinal stem cell niche comprises both epithelial cells, in particular the
Paneth cell, and the stromal compartment, where cell-associated ligands and
soluble factors regulate stem cell behavior. This review highlights the recent
advances in identifying and characterizing the intestinal stem cells and their
defining niche. |
Which are the types of viral meningitis? | Aseptic meningitis is the most common type of meningitis and is characterized by meningeal inflammation that is not linked to identifiable bacterial pathogens in cerebrospinal fluid (CSF). It can be originated from infection from:
1) Varicella-zoster virus (VZV)
2) Herpes simplex types I and II (HSV-1, HSV-2)
3) Epstein-Barr virus (EBV)
4) Cytomegalovirus (CMV)
5) Enteroviruses (EV)
6) Parechoviruses (HPeV)
7) Human rhinoviruses (HRVs)
8) Echovirus types 6, 9, 11
9) West Nile Virus (WNV) | The majority of viral meningitis cases is known to be due to ECHO virus
infections on one hand, and mumps on the other. While the latter can be
diagnosed by IgM antibody detection from one serum sample in the acute stage,
diagnosis of enterovirus infections is by virus isolation and typing. An
IgM-antibody test for ECHO 9 and 11 viruses is presented to evaluate the
possibility of rapid serological diagnosis of ECHO virus meningitis cases. 36
cases from five local outbreaks due to ECHO 6, 9, 11, and 30 viruses were
characterized by virus isolation and serum neutralization tests. All sera (88
samples) were assayed by a MACRIA (M-antibody capturing radioimmunoassay) to
ECHO 9 and 11 viruses. While sera from all ECHO 9 and 11 cases, when taken at
appropriate times, had IgM antibodies to the infecting type, a varying degree of
cross-reactivity could be observed. Specificity problems are discussed in
comparison with isolated cases of enteroviral infections due to different types,
including Coxsackie B viruses. During the autumn of 1992, Western Australia experienced a large viral
meningitis outbreak of dual aetiology. Of the 161 cases, 64% were children under
15 years of age, with the highest notification rate being in children less than
5 years of age. Echovirus 9 caused 41% of cases and occurred mainly in the
metropolitan areas of Western Australia whereas echovirus 6, which caused 37% of
cases, was more widespread. An enterovirus was cultured from 70% of CSF
specimens, 88% of faecal specimens and 68% of upper respiratory tract specimens.
High CSF white cell counts and neutrophil predomice were common. Seven cases
had normal CSF white cell counts even though an enterovirus was isolated from
the CSF. Therefore, the CSF findings were of restricted value in excluding viral
meningitis, and did not reliably distinguish between bacterial and viral
meningitis. Aseptic meningitis is not an uncommon complication to primary genital herpes
infection caused by herpes simplex virus type 2 (HSV-2). Compared with other
types of viral meningitis, HSV-2-meningitis is associated with a significant
rate of neurological complications in the acute stage. In addition, some
patients will suffer from recurrent aseptic meningitis (Mollaret's meningitis)
later. We describe six patients, five women and one man, age 26-35 years, with
aseptic meningitis caused by HSV-2. All the patients showed serological evidence
of primary herpes infection (negative HSV-IgG and/or positive HSV-IgM in serum
samples). Polymerase chain reaction detected HSV-2 in cerebrospinal fluid in all
five of five cases, while virus cultures were positive in two of the six cases.
Only three patients showed clinical signs of simultaneous genital herpes
infection. One patient, a 28-year-old female, developed transient autonomic
nervous system dysfunction with urinary retention, constipation, and neuralgic
pain in the buttocks, perineum and lower limbs. 13 months later she was
hospitalised for a genital herpes infection with headache, parestesia and fever,
but spinal fluid examination showed no abnormality. The sporadic occurrence of Echo virus types 27 and 31 in association with
aseptic meningitis during the enterovirus seasons in Ontario in 1959 to 1962 is
reported.The etiological significance of both of these isolates was verified by
demonstration of type-specific serological response in meningitis patients. Echo
27, which up to now has not been encountered in association with human illness,
could be added to the list of viral agents capable of causing aseptic
meningitis. Isolation of Echo 31 from cerebrospinal fluid provides definite
proof of the etiological significance of this virus in cases of aseptic
meningitis. Adult cases of viral meningitis caused by echovirus type 13 (E13) were studied.
E13 was isolated from 8 of 11 adult patients (73%) with viral meningitis between
April and September 2002 in Fukui Prefecture. The mean age was 27.4 +/- 6.4
years (4 males and 4 females). The disease was prevalent among adults,
especially younger adults as well as children. The symptoms and signs were as
follows; headache (100%), fever (100%), nausea and/or vomiting (88%), Kernig's
sign (88%), and increased deep tendon reflexes (50%). The average cell counts in
cerebrospinal fluid (CSF) were 118 +/- 111/mm3. Of the 2 patients, polynuclear
cells were domit during the early phase of the disease. The prognosis was
good. Since May 2002, the number of patients with viral meningitis caused by E13
has rapidly increased. Most of the reported patients were children. We should
consider the possibility of E13 infection as a cause of adult viral meningitis. BACKGROUND: It is unclear whether or not the CSF cytokine profiles in viral
meningitis differ with the kind of causative virus.
METHODS: We measured the concentrations of interferon-gamma (IFN-gamma), tumor
necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, and IL-10
in CSF during the acute stage in 15 children with mumps meningitis (MM), and 34
with echovirus type 30 meningitis (EM).
RESULTS: The CSF IFN-gamma, IL-2, IL-6, and IL-10 levels were elevated in MM,
and the CSF IFN-gamma, IL-2, and IL-6 levels were elevated in EM. The CSF
IFN-gamma, IL-2, and IL-10 levels in MM were significantly higher than those in
EM (p<0.0001, p<0.0001, and p<0.0001, respectively). The CSF IL-6 levels in EM
were significantly higher than those in MM (p=0.0255). The CSF TNF-alpha and
IL-4 levels were not elevated in MM or EM. In MM, the IL-6 level was correlated
with the IL-2 and IL-10 levels in CSF (p=0.0347 and p=0.0120, respectively). In
EM, the IFN-gamma level was correlated with the IL-10 level in CSF (p=0.0002).
CONCLUSION: CSF cytokine profiles in MM were different from those in EM.
Therefore, it is likely that the pathogenesis of MM is different from that of
EM. Human enteroviruses are responsible for a wide spectrum of clinical diseases
affecting many different organ systems. Although infection is usually
asymptomatic, infections of the central nervous system manifested as meningitis
or encephalitis can pose a serious public health problem, especially during
outbreaks. In this study, samples from 218 patients diagnosed with enteroviral
meningitis between January 2000 and December 2002 were analysed in order to
assess the epidemiology of human enteroviruses as a cause of viral meningitis in
Cyprus. A new typing strategy, based on partial sequencing of the 5' non-coding
region (5'NCR), prediction of type, and selection of type-specific primers for
sensitive VP1 PCR amplification, was developed. As clustering in the 5'NCR was
concordant with clustering in the VP1 region, quick and reliable typing by VP1
sequencing was achieved without virus isolation in cell culture. The most
frequent enterovirus serotypes identified were Human echovirus 30 (55.5%), Human
echovirus 13 (15.1%), Human echovirus 6 (13.8%) and Human echovirus 9 (8.3%).
Human coxsackieviruses B2, B1 and B5, Human echovirus 4, Human enterovirus 71
and Human coxsackievirus A6 represented rather rare serotypes. This is the first
molecular epidemiological study of enterovirus meningitis in Cyprus. Serotype
distribution corresponded basically with observations in other European
countries, suggesting the spread of enteroviruses by tourism. OBJECTIVE: This study examined the burden of hospitalization of children with
viral meningitis in Department of Pediatric Infectious Diseases, Medical
University of Bialystok from 2003 to 2005.
METHODS: Data were extracted from Children Teaching Hospital database. A
hospitalization with viral meningitis was defined as any discharge with
diagnoses of mumps meningitis (B26.1), enteroviral meningitis (A85.0), tick-born
encephalitis (A84.1) and viral undifferentiated meningitis (A87.9) due to
ICD-10. Outcome measures included number of hospitalizations in total and due to
different etiology as well as their impact in the budget of the unit.
RESULTS: 316 hospitalizations with viral meningitis were identified. Of these
246 (77.6%) were diagnosed with mumps etiology, 10 (3.2%) with enteroviral, 1
(0.3%) with tick-born virus and 59 (18.7%) with unknown viral etiology. Most
cases of viral meningitis were hospitalized in 2003--132 (41.8%). Of these 93.9%
were diagnosed with mumps meningitis. In 2005, of 78 hospitalizations associated
with viral meningitis 10 (12.8%) were confirmed and further 38 (48.7%) were
suspected enteroviral meningitis. The percentages of costs associated with viral
meningitis in 2004 and 2005 (26% and 18.8%) were high in comparison to
percentage of hospitalizations with viral meningitis (10.7% and 7.3%,
respectively). BACKGROUND: Enteroviruses (EV) and parechoviruses (HPeV) are the most common
causes of aseptic meningitis, encephalitis and sepsis-like syndrome in neonates.
Detection by nucleic acid amplification methods improves patient management.
OBJECTIVE: Development of a real-time PCR assay on a LightCycler for
simultaneous detection of EV, HPeV and an internal control to monitor
inhibition.
STUDY DESIGN: We investigated the value of the new assay, prospectively, in a
variety of samples from patients suspected of having viral meningitis or
sepsis-like syndrome.
RESULTS: The assay detected 64 EV serotypes and HPeV types 1-4. Of 186 patients,
63 (33.9%) were EV positive and 18 (9.7%) HPeV positive in one or more samples.
In 43 of 159 feces and 6 of 57 throat samples viral culture and PCR were
positive. With real-time PCR 27 extra EV and 19 HPeV positives were found. Blood
and CSF were present from 33 patients. In 19 patients blood and CSF were
positive, one was only positive in CSF, two were only positive in blood, 11 were
negative. From 96 patients CSF and/or blood samples were tested and compared to
results in throat and/or feces samples. Forty patients were EV-PCR and 14
HPeV-PCR positive in blood and/or CSF. All of these were confirmed by a positive
PCR for the respective virus in feces and/or throat.
CONCLUSIONS: Simultaneous detection of EV and HPeV with this two-step real-time
PCR is specific, faster and more sensitive than viral culture. All systemic
infections (blood or CSF positive) were confirmed in feces. Culture is no longer
necessary for clinical diagnosis and should only be performed on PCR-positive
samples to obtain isolates for typing purposes. Application of this assay is an
important improvement for patient management since the outcome of the analysis
is available within the time frame of clinical decision-making. Matrix metalloproteinases are involved in leucocyte invasion into the central
nervous system (CNS) during meningitis. The aim of the study was to determine
whether there are differences in the expression patterns of matrix
metalloproteinase-9 (MMP-9) and the tissue inhibitor of metalloproteinase-1
(TIMP-1) in the cerebrospinal fluid (CSF) of patients with meningitis caused by
one of two known distinct viral agents. Concentrations were measured by using an
enzyme-linked immunosorbent assay (ELISA) in 16 children with mumps meningitis,
in 25 children with echovirus type 30 meningitis and in a control group of 23
children without any CNS infection. Increased levels of MMP-9 were found in
children with mumps (median 0.48 ng/ml; P < 0.001) and enteroviral meningitis
(median 2.76 ng/ml; P < 0.001) compared with that in controls (median: 0.01
ng/ml). Concentrations of TIMP-1 greatly exceeded concentrations of MMP-9 and
were elevated in children with mumps (median: 56 ng/ml) and echovirus type 30
meningitis (median: 55 ng/ml) compared to controls (median: 17 ng/ml). No
significant differences in MMP-9 or TIMP-1 levels were detected between the two
meningitis groups. The MMP-9/TIMP-1 ratio was greater in children with echovirus
type 30 than in those with mumps meningitis. There was no correlation between
MMP-9 levels and total CSF cell count. MMP-9 correlated with CSF absolute
neutrophil count in children with echovirus type 30 meningitis (r = 0.431; P <
0.05). The concentration of MMP-9 is higher in children with viral meningitis,
possibly because of infiltrating polymorphonuclear cells present in the initial
phase of the disease. A descriptive retrospective study was carried out to describe an epidemic
outbreak of enteroviral meningitis in Misiones. We reviewed records of 143
children from 1 month to 14 years of age who were hospitalized with aseptic
meningitis in the Pediatric Hospital of Posadas from August to December 2005.
Increased number of cases was observed between weeks 33 to 50 which reached a
maximum peak in weeks 47 and 48, confirming an outbreak. The median of age was 8
years old, 55.2% were males. Eighty percent of cases were in 5 to 14 years old
children. The average length of time spent in the hospital was 4.5+/-1.7 days,
no deaths were reported. We performed cell counts, chemical and bacterial
studies of CSF, and culture or RT-Nested/PCR for enteroviruses. Isolates were
serotyped by RT-PCR amplification and genetic sequencing. Cell counts were from
6 to 5040 cells/mm3. Ninety two percent had less than 500 cells/mm3 and 43.5%
had lymphocyte predomice. Glucose levels were normal with slightly elevated
protein counts in 56% of cases. Of the cultured samples, 28% (17/60) showed
cytopathic effect compatible with enterovirus. RT-n-PCR detected enterovirus in
73% (43/59) of the analyzed CSF. Echovirus type 4 was identified in 6 of them.
The positive indicator obtained by combining both techniques was 83% (58/70). BACKGROUND: In this retrospective study, our objective was to review the
epidemiology of viral meningitis and to compare clinical features associated
with enterovirus, herpes simplex virus (HSV), and varicella zoster virus (VZV)
infections in immunocompetent adults.
METHODS: Data on cerebrospinal fluid (CSF) samples submitted to the Trust
Virology Laboratory (Sheffield, UK) from April 2004 through April 2007 were
reviewed. Notes on immunocompetent adults who were polymerase chain reaction
(PCR) positive for enterovirus, HSV type 2, or VZV and who had presented to
local clinical departments were scrutinized (4 patients were positive for HSV
type 1 and did not meet the inclusion criteria).
RESULTS: A total of 2045 samples were analyzed for viral pathogens during the
3-year period. Of the 109 PCR-positive samples, 38 (35%) were from
immunocompetent adults, of whom 22 were infected with enterovirus, 8 were
infected with HSV type 2, and 8 were infected with VZV. The median ages were 32
years (range, 16-39 years), 39 years (range, 22-53 years), and 47.5 years
(range, 26-80 years), respectively. Rash occurred after the meningitis symptoms
in 5 patients infected with VZV (median time from meningitis symptoms to rash, 6
days). Protein levels were significantly higher in CSF samples from patients
infected with HSV type 2 (median, 1205 mg/L) and in samples from those infected
with VZV (median, 974 mg/L) than in samples from those infected with enterovirus
(median, 640 mg/L; P = .001 and P = .01, respectively). White blood cell counts
were significantly higher in CSF samples from patients infected with HSV type 2
(median, 240 x 10(6) cells/L) than in samples from those infected with
enterovirus (median, 51 x 10(6) cells/L; P = .01).
CONCLUSIONS: Enterovirus infection was the most common cause of viral meningitis
in immunocompetent adults in this study. White blood cell counts and protein
levels were significantly higher in CSF samples from patients infected with HSV
type 2 than in samples from patients with enterovirus infection. Zoster rash
often occurs after meningitis. PCR testing provides a rapid and specific
etiological diagnosis. Aseptic meningitis refers to a clinical syndrome of meningeal inflammation in
which bacteria cannot be identified in the cerebrospinal fluid (CSF). The viral
etiology and the epidemiological, clinical, and laboratory characteristics of
aseptic meningitis among children aged 2 months to 15 years in Shiraz, southern
Iran were determined. From May 2007 to April 2008, 65 patients were admitted to
the hospital with aseptic meningitis. Seven viruses, non-polio human
enteroviruses, mumps virus, herpes simplex virus (HSV), varicella-zoster virus
(VZV), human cytomegalovirus (HCMV), human herpes virus type 6 (HHV-6), and
Epstein-Barr virus (EBV) were investigated by polymerase chain reaction (PCR)
method. Viruses were detected in 30 (46.2%) patients in whom non-polio human
enterovirus and mumps virus were detected in 13 (43.3%) and 11 (36.7%),
respectively. The remaining 6 (20%) of the cases were caused by HSV, VZV, HCMV,
and HHV-6. Haemophilus influenzae and non-polio human enterovirus were detected
in one patient simultaneously. Viral meningitis was found to be more frequent
during spring and summer. The majority (66.6%) of the patients were treated in
the hospital for 10 days and had received antibiotics in the case of bacterial
meningitis. Rapid diagnosis of viral meningitis using PCR testing of CSF can
help shorten hospitalization, and avoid the unnecessary use of antibiotics. BACKGROUND: The mumps virus is frequently the causative agent in aseptic
meningitis and mumps has still prevailed in Japan. We compared data obtained
from patients with mumps meningitis and patients with aseptic meningitis caused
by other viruses in order to identify mumps meningitis-specific
cytokine/chemokine alterations in cerebrospinal fluid (CSF).
METHODS: We elucidated the cytokine/chemokine network based on the
cytokine/chemokine profiles in CSF from children with mumps meningitis and
meningitis due to other viral infections using multiplex cytokine measurement.
Seventeen cytokines/chemokines, namely interleukin (IL)-1β, IL-2, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17, interferon (IFN)-γ, tumor
necrosis factor (TNF)-α, granulocyte colony-stimulating factor (G-CSF),
granulocyte monocyte colony-stimulating factor (GM-CSF), monocyte
chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β
(MIP-1β), were measured simultaneously in CSF supernatants from eight children
with mumps meningitis, 11 children with other types of viral meningitis and
eight children with fever without neurological complications such as convulsion.
RESULTS: We found that IL-8, IL-10, IL-12, IL-13 and IFN-γ showed a
statistically significant increase in CSF from mumps meningitis when compared to
other types of viral meningitis and fever without neurological complications.
CONCLUSION: Mumps meningitis may induce a distinct immunological response when
compared with other types of viral meningitis. We measured levels of pro-inflammatory cytokines in the cerebrospinal fluid
(CSF) of patients with mumps meningitis, enteroviral echovirus 30 meningitis and
children without central nervous system infection to investigate whether these
molecules were involved in the pathogenesis of viral meningitis. The CSF was
obtained from 62 children suspected with meningitis. These patients were
classified to the mumps meningitis (n = 19), echovirus 30 meningitis (n = 22)
and non-meningitis (n = 21) groups. The concentrations of interleukin-1 (IL-1),
interleukin-1 soluble receptor type 2 (IL-1R2), interleukin-8 (IL-8), human
interferon gamma (IFN-γ) and human tumour necrosis factor alpha (TNF-α) were
determined by immunoassay. A significant increase was noted in the levels of
IL-8, TNF-α and IL-1R2 in the CSF of both meningitis groups as compared to
controls. The concentrations of IFN-γ and IL-1 differed significantly only
between the mumps group and control. The levels of IL-1, IFN-γ and TNF-α were
significantly higher in mumps meningitis when compared to the echovirus 30
group. Of all cytokines examined, only IFN-γ correlated with pleocytosis (r =
0.58) in the mumps meningitis group. The increased CSF cytokine levels are
markers of meningeal inflammation, and each virus may cause a specific profile
of the cytokine pattern. In previous reports on the viral causes of central nervous system (CNS)
infections, it has been generally recognized that HSV-1 is a major cause of
encephalitis, while HSV-2 is the predomit cause of aseptic meningitis in
adults. To examine this matter, the clinical characteristics in the two types of
HSV CNS infections were investigated. In a retrospective cohort study which
included all adult patients (≥16 years) between January 1999 and December 2013
in a 2,700-bed tertiary care hospital, all the patients in whom PCR of the CSF
for HSV was positive were identified. Ninety-five patients with positive CSF PCR
results for HSV were included, 21 with HSV-1 and 74 with HSV-2. Many patients
with HSV-1 had encephalitis (13/21, 61.9%), whereas most patients with HSV-2 had
meningitis (62/74, 83.8%). However, HSV-1 and HSV-2 accounted for similar
proportion of patients with HSV encephalitis (13/25, 52.0% vs. 12/25, 48.0%).
Neurological sequelae were more frequent among patients with HSV-1 (9/21, 42.9%
vs. 6/74, 8.1%; P = 0.001). The present study suggests that HSV-2 is not only a
major cause of aseptic meningitis, but also it may cause serious manifestation
as HSV-1 encephalitis in adults. Over 100 known enterovirus serotypes differ in their epidemiological and
pathogenic properties. Much less is known about variation of these features on a
sub-serotype level, such as genotypes. Echovirus 11 (E11) and E30 are amongst
the most frequent causative agents of aseptic meningitis. We studied the
molecular epidemiology of these pathogens to evaluate potential epidemiological
and pathogenic dissimilarities of their genotypes. The complete VP1 genome
region was sequenced for 97 E11 and 62 E30 isolates collected in Russia from
2008 to 2012, and they were studied in comparison with all 140 E11 and 432 E30
sequences available in GenBank. A geographic pattern of genotype prevalence was
observed for both types. Russian E11 isolates belonged mainly to A genotype,
which is common in Asia, and D5, which is predomit in Europe. For E30,
genotype III by classification of Ke et al. (2011), also termed genotype a by
Bailly et al. (2009), was endemic in Russia from 2003 to 2012, while it was not
detected in Europe and North America during this time. The E30 genotypes VI-B,
VI-G, and VI-H (e, f and h) were regularly introduced from different countries,
became predomit and vanished after no more than 4years. In addition to
geographic patterns, E11 genotypes also differed by isolation source. Genotype
A2 viruses were significantly more often found in sewage, compared to genotype
D5 that was isolated from both sewage and human samples. In addition, there was
evidence of a different capacity for international transfers among E11 GtA
subclusters. Human parechovirus (HPeV) belongs to the Picornaviridae family of RNA viruses.
HPeV infections can be asymptomatic, lead to mild respiratory and/or
gastrointestinal symptoms, or less frequently cause severe diseases such as
sepsis, meningitis, encephalitis, and myocarditis. Severe neurological HPeV
infections occur most commonly in infants and neonates. There are currently 16
recognized types of HPeV. HPeV type 3 (HPeV3) has been the predomit type
associated with severe central nervous system disease in neonates and newborns
since its discovery in 1999. Although HPeV-related infections have been reported
in adults, symptomatic HPeV3 infections in adolescents and adults are uncommon.
A case of severe HPeV3 myocarditis and encephalitis in an adolescent is
described. The aetiology of acute meningoencephalitis in Sri Lankan children and adults is
poorly understood. This study was carried out to determine pathogens responsible
for meningoencephalitis in Sri Lanka. A hospital-based cross-sectional study was
performed using cerebrospinal fluid samples (22 adult and 17 pediatric)
collected from August to December 2009 from patients clinically diagnosed with
acute meningoencephalitis at two tertiary care hospitals in Sri Lanka. Routine
microbiology for bacterial pathogens together with in-house RT-PCR and PCR
assays for the detection of dengue viruses, Japanese encephalitis virus, West
Nile virus, chikungunya virus, enteroviruses, mumps virus, measles virus, herpes
simplex viruses types 1 and 2, and varicella zoster virus were performed.
Bacterial pathogens were not isolated from any patient specimens. However, from
nine of the paediatric patients aged 1 month to 10 years (mean age 5.2 years)
echovirus 9 (E-9; family Picornaviridae, genus Enterovirus,species Enterovirus B
) was detected by RT-PCR. All nine patients presented with fever, six had
headache, and seven had vomiting. Neck stiffness indicating meningitis was
present in six of the patients. Phylogenetic analysis of partial VP1 and VP4-VP2
genes showed these E-9 strains to be most closely related to E-9 strains
detected in CSF from Korea and France in 2005 and 2006. The remaining patients
were negative for all other viruses tested. E-9 was the most common cause of
acute meningoencephalitis in the tested paediatric population from Sri Lanka in
2009, which likely reflects circulation of this E-9 strain between Europe and
Asia over several years. |
What are the different classes of orally administered drugs used to treat diabetes | There are a number of classes of medications tha are used to treat Type 2 diabetes. These include biguanides like metformin, which decreased hepatic glucose release; sulfonyureas like Glimepride, metglitinides like repaglin and d-phenylalanine derivatives, all of which stimulate pancreatic insulin release; Glitizones or thiazolidinediones which make the body more sensitive to insulin; DPP-4 inhibitors which lower the amount of glucose made in the body; alpha-glucosidase inhibitors which slow the absorbtion of carbohydrate in the blood; and bile acid sequestrants that lower blood glucose | Diabetes mellitus is a multifaceted disease which intervenes in the personal
lives of those afflicted in many different ways. In this study prescription drug
use among diabetics was analyzed in order to shed light on the characteristics
of diabetic morbidity. Prescription drug use among diabetics and non-diabetics
in a total population of 21,000 inhabitants in a defined geographic area were
studied. The diabetic population was categorized according to the type of
treatment received: insulin treatment, oral anti-diabetic treatment or dietary
treatment or dietary treatment only. The pattern of prescription drug use
differed between diabetics and non-diabetics and important differences were
observed also between diabetics according to type of treatment. Drug use among
those treated with insulin and those treated orally was substantially higher
than among non-diabetics while the difference between diabetics on dietary
regimen and non-diabetics was much smaller. All three treatment groups had
considerably higher consumption of cardiovascular drugs than non-diabetics.
Additional findings include more frequent antibiotic use among diabetics treated
orally and on diet only than among non-diabetics. The use of these drugs was
also common among insulin treated diabetics but did not differ significantly
from among non-diabetics. Use of psychotropics was more common among diabetics
treated with insulin and orally than among non-diabetics. Besides the considerable differences in the intestinal absorption of the
different antibiotic classes as described in part I of this paper galenic
preparations and the administration in form of a capsule -- tablet or sirup play
a major role too. The particle size of antimicrobial drugs in sirup form has to
be carefully selected. It should not be too small to prevent a rapid decay of
the substance by gastric acid; if it is too large the dissolution of the
substance is not fast enough for maximal absorption in the upper intestinal
tract. Enteric coating, soluble binders, the dissolution time of a tablet are
other important variables in the absorption of orally administered antibiotics.
The most important influence on the resorption of orally administered
antibiotics comes from the patient. Individual variations in the absorption are
exceptionally high with amoxycillin. Different age groups show considerable
variations in intestinal absorption which can amount to an extent of a two to
threefold increase or decrease over the mean. Simultaneous administration of
food or drugs have significant influences on the absorption of certain
antibiotics. The pharmacokinetic investigation and bioavailability studies are
done on healthy young adults. Diseases of the intestinal tract like acute
diarrhea or malabsorption syndromes influence the absorption of orally
administered antibiotics to a considerable extent which even renders some drugs
completely ineffective. In cystic fibrosis the enteral absorption is retarded
and pharmakokinetic parameters sizably altered. Rosiglitazone is a potent oral antidiabetic agent of the thiazolidinedione class
that works through activation of the peroxisome proliferator-activated nuclear
receptor. It improves insulin sensitivity in peripheral tissues and effectively
lowers blood glucose in patients with type 2 diabetes. Metformin is a
dimethyl-biguanide, also used in type 2 diabetes, that lowers fasting blood
glucose primarily by decreasing hepatic glucose output. Rosiglitazone and
metformin reduce plasma glucose concentrations via different mechanisms and thus
could potentially be used in combination to optimize glycemic control. This
study evaluated the effects of the coadministration of these two agents on the
pharmacokinetics of both rosiglitazone and metformin. Sixteen male volunteers
(22-55 years old) received oral metformin (500 mg every 12 hours), rosiglitazone
(2 mg every 12 hours), or the combination each for 4 days. Plasma collected on
day 4 of each regimen was assayed for rosiglitazone and metformin
concentrations. Oral doses of rosiglitazone and metformin were safe and well
tolerated when administered alone or in combination. There were no clinically
significant episodes of hypoglycemia or increased blood lactic acid levels
following treatment with any regimen. Coadministration of rosiglitazone and
metformin had no significant effects on the steady-state pharmacokinetics
(AUC(0-12 h), Cmax, tmax, or t1/2) of either drug. The authors conclude that
rosiglitazone can be safely administered with metformin and, due to the
different mechanisms of action of these agents, may offer a therapeutic
advantage in patients with type 2 diabetes mellitus. The drugs used to treat diabetes mellitus are diverse and include several
classes. One class is sulfonylureas which primarily cause serum glucose
reduction by stimulating the release of preformed insulin from the pancreatic
islets. Gliclazide, a second generation sulfonylurea, is used to control
glycemic levels in non-insulin-dependent diabetes mellitus. We report a 14
year-old non-diabetic girl who developed hepatitis, hemiplegia and dysphasia
after ingestion of an overdose of gliclazide (20 mg/kg/day) in a suicide
attempt. Our purpose is to draw attention to the severity of gliclazide-induced
neurological signs. To the best of our knowledge, gliclazide-induced hemiplegia
and dysphasia have not been previously reported in the literature. OBJECTIVE: To describe the use of oral antidiabetic drugs for management of type
2 diabetes in the U.S. from 1990 through 2001.
RESEARCH DESIGN AND METHODS: Data on oral antidiabetic drugs were derived from
two pharmaceutical marketing databases from IMS Health, the National
Prescription Audit Plus and the National Disease and Therapeutic Index.
RESULTS: In 1990, 23.4 million outpatient prescriptions of oral antidiabetic
agents were dispensed. By 2001, this number had increased 3.9-fold, to 91.8
million prescriptions. Glipizide and glyburide, two sulfonylurea medications,
accounted for approximately 77% of prescriptions of oral antidiabetic drugs in
1990 and 35.5% of prescriptions in 2001. By 2001, the biguanide metformin
(approved in 1995) had captured approximately 33% of prescriptions, and the
thiazolidinedione insulin sensitizers (rosiglitazone and pioglitazone marketed
beginning in 1999) accounted for approximately 17% of market share. Compared
with patients treated in 1990, those in 2001 were proportionately younger and
they more often used oral antidiabetic drugs and insulin in combination.
Internists and general and family practitioners were the primary prescribers of
this class of drugs.
CONCLUSIONS: Consistent with the reported increase in the prevalence of type 2
diabetes, the number of dispensed outpatient prescriptions of oral antidiabetic
drugs increased rapidly between 1990 and 2001. This period was marked by an
increase in the treatment of younger people and the use of oral antidiabetic
drugs in combination. With the approval in the last decade of several new types
of oral antidiabetic medications with different mechanisms of action, options
for management of type 2 diabetes have expanded. OBJECTIVE: To discuss a rational approach to improvement of glycemic control in
patients with type 2 diabetes mellitus with use of combination therapy.
METHODS: We review the mechanisms of action and clinical applications for the
various antidiabetic agents alone and in various combinations. Relevant studies
in the literature are reviewed.
RESULTS: Although diet and exercise remain the cornerstones of treatment, in
most patients with type 2 diabetes, pharmacologic agents are needed to achieve
optimal glycemic control and likely reduce the incidence of microvascular and
possibly macrovascular complications as well. Sulfonylureas have long been the
foundation of oral pharmacologic therapy and provide adequate glycemic control
for most patients for 5 to 10 years or longer. In the past, when treatment with
sulfonylureas was no longer effective, insulin therapy was inevitable. With the
approval of several new pharmacologic agents for the treatment of type 2
diabetes, however, the addition of one or more orally administered agents to
sulfonylurea therapy or use of other oral combination therapy is rapidly
evolving as a means of optimizing glycemic control. In many patients,
combination therapy can delay the need to add or switch to insulin, or it can
enhance glycemic control in patients already receiving insulin. In selected
patients treated solely with insulin, discontinuation of insulin treatment and
reinitiation of oral therapy may even be possible.
CONCLUSION: Currently, four classes of orally administered antidiabetic agents
are available for use in patients with type 2 diabetes: insulin secretagogues,
biguanides, a-glucosidase inhibitors, and thiazolidinediones. By taking
advantage of differing mechanisms of action, combination therapy is evolving as
a means of optimizing glycemic control in patients in whom a single agent or
insulin is inadequate. Combinations of orally administered agents can often
delay the need for insulin or in combination with insulin aid in achieving
glycemic goals. Continuing research will help optimize combination therapies
even further. The aims of the study were to evaluate differences in dietary, oral hygiene
habits and social class in children with Type I diabetes mellitus (DM), compared
to non-diabetics, and to investigate relationship between selected caries-risk
factors and caries experience in diabetics.
MATERIAL AND METHODS: 70 children with Type I DM and 70 age- and sex-matched
non-diabetic controls were included in the study. Metabolic control of diabetes
was categorized into well- to- moderately-controlled and poorly-controlled
groups based on glycosylated haemoglobin HbA1c. The study was based on the data
obtained from the questionnaire including information about dietary and oral
hygiene habits, pattern of dental visits and social class. Results showed that
the diabetic children had more frequent main meals and less snacking than their
controls: the mean number of main meals/day was 4.33 (SD = 0.93) in the
diabetics, and 2.53 (SD = 0.85) in the controls. Significantly less diabetics
(43%) used sweet drinks than their controls (79%). There were no differences
according to the frequency of toothbrushing as well as frequency of dental
visits between the diabetics and controls, however, significantly more diabetics
reported that they never used dental floss than non-diabetics. There were no
significant differences in the diet, toothbrushing frequency between the
diabetics with different metabolic control. Multiple logistic regression
analysis showed that among caries risk associated variables only age of children
(OR = 1.98; CI = 1.23-3.19) and level of metabolic control of diabetes (OR =
4.65; CI = 1.28-16.89) were statistically significantly associated with high
caries experience in the diabetics.
CONCLUSIONS: Frequent consumption of sweet drinks and snacks can influence
caries development in children. Amongst the diabetics, the differences in caries
prevalence can be explained by combination of biological and behavioral factors
rather than single dietary or oral hygiene elements. OBJECTIVE: To evaluate prescription drug use in the elderly and in particular,
to determine the number and types of medications taken, whether and to what
extent drugs that are contraindicated in this age group are being used, and what
type of prescription check may be performed by primary care physicians.
DESIGN: A survey was performed in a sample of non-institutionalised elderly
subjects (= 65 years). These were selected by cluster sampling in 11 of 20
Italian regions and were interviewed in the home by trained interviewers using a
standardised questionnaire.
RESULTS: Eighty-seven percent of interviewed subjects reported that they had
taken at least one medication in the previous year; higher frequencies were
found in age groups= 75 years. The most common therapeutic classes of drugs used
in all participating regions, in the previous week, were cardiovascular,
gastrointestinal, metabolic (including drugs to treat diabetes) and nervous
system. Among interviewed subjects, 45.3% reported using 4 or more different
drugs, though wide regional differences were observed (Campania 60.5%, Bolzano
35.6%); 7.2 % were taking potentially inappropriate drugs while 2.3% were taking
medications that may lead to potentially harmful interactions. In addition,
84.9% of subjects reported that their primary care physician regularly checked
their drug prescriptions.
CONCLUSIONS: The high frequency of prescription drug use observed in the elderly
is a diffuse phenomenon, related to the worsening health conditions that
inevitably accompany aging. Considering the extent of this phenomenon, care
should be taken to improve qualitative (i.e. contraindications in the elderly,
potential drug-interactions) and quantitative (high number of medications taken
by the elderly) appropriateness in physician prescribing. In addition, special
attention must be placed on regularly checking drug therapies in the elderly. OBJECTIVE: Significant race and ethnic disparities exist in diabetes-related
health care. Using a nationally representative database, we sought to determine
if use of thiazolidinediones (TZDs) differs by race and ethnicity. As a
secondary objective, we sought to determine if race and ethnicity is associated
with use of older oral antidiabetic agents, such as sulfonylureas and metformin.
RESEARCH DESIGN AND METHODS: Adult respondents to the 2003 Medical Expenditure
Panel Survey with diabetes, identified by diagnosis code or self-report, were
included. Race/ethnic groups were defined as: White/not-Hispanic;
Black/not-Hispanic; Hispanic; or Other/not-Hispanic. Associations between use of
oral antidiabetic agents (defined as > or = 1 prescription for a TZD,
sulfonylurea, or metformin) and race/ethnicity, sex, age, insurance status,
poverty status, and having a usual source of care were evaluated in univariate
analyses with chi(2) tests and in adjusted analyses using logistic regression
methods for survey data.
RESULTS: A total of 1873 US adults with diabetes were identified, with use of
oral antidiabetic agents varying by drug class: 23.1% received TZDs, 45.3%
received metformin, and 43.8% received sulfonylureas. Use of oral antidiabetic
agents, by drug class, did not differ significantly by race/ethnicity (p = 0.33
for TZDs, p = 0.43 for metformin, p = 0.38 for sulfonylureas). In univariate
analyses, only insurance status was significantly associated with use of TZDs (p
= 0.03), and no variables were associated with use of sulfonylureas or
metformin. In adjusted logistic regression analyses, there were no significant
predictors of the use of TZDs or metformin, and only age was significantly
associated with the use of sulfonylureas.
CONCLUSIONS: In a nationally representative database, fewer US adults with
diabetes received TZDs compared with sulfonylureas or metformin in 2003.
Although we were not able to differentiate between type 1 and type 2 diabetes,
nor did we assess oral agent monotherapy versus combination therapy, we found
that use of TZDs, sulfonylureas, and metformin did not differ based on
race/ethnicity or other demographic variables such as sex, insurance status,
poverty status, or having a usual source of health care. Type II diabetes is a heterogeneous disease where environment and genetics are
important factors for the expression of the disease. The high cost for treating
complications of diabetes is a burden for public health systems and governments
worldwide. Type II diabetes has been causing debilitation worldwide for many
decades, and a single drug that safely treats the disease has yet to be
discovered. Sulfonylureas, biguanides, alpha-glucosidase, meglitinides, DPP-4
inhibitors and thiazolidinediones are among the classes of oral hypoglycemic
drugs available to treat Type II diabetes, but concerns exist regarding safety
and efficacy of these drugs. In this article we present the pros and cons of the
six classes and discuss some of the latest advances towards the development of
new drugs for the treatment of Type II diabetes. OBJECTIVE: Although diabetes is conveniently assessed by self-report, few
validation studies have been performed. Therefore, we studied whether
self-report of prevalent and incident diabetes in Women's Health Initiative
(WHI) participants was concordant with other diagnostic evidence of diabetes.
STUDY DESIGN AND SETTING: A total of 161 808 postmenopausal women aged 50-79
were enrolled at 40 clinical centers in the U.S. in 1993-1998 and followed
prospectively. At baseline, prevalent medication treated diabetes was defined as
a self-report of physician diagnosis and treatment with insulin or oral
antidiabetic drugs. During followup, incident treated diabetes was defined as a
self-report of a new physician diagnosis of diabetes treated with insulin or
oral drugs. Diabetes self-reports were compared with medication inventories and
fasting glucose levels at baseline and during follow-up.
RESULTS: At baseline, self-reported treated diabetes was concordant with the
medication inventory in 79% of clinical trial, and 77% of observational study
participants. Self-reported incident treated diabetes was concordant with the
medication inventory in 78% between baseline and Year 1 in the clinical trials,
in 62% between Year 1 and Year 3 in the clinical trials, and in 72% between
baseline and Year 3 in the observational study. Over similar periods, 99.9% of
those who did not report treated diabetes had no oral antidiabetic drugs or
insulin in the medication inventory. At baseline, about 3% not reporting
diabetes had fasting glucose >126 mg/dl, and 88% of these subjects subsequently
reported treated diabetes during 6.9 years of follow-up.
LIMITATIONS: Incident self-reported diabetes treated by lifestyle alone was not
determined in WHI. Medication inventories may have been incomplete and fasting
glucose may have been lowered by treatment; therefore, concordance with
self-reported treatment or fasting glucose > or = 126 may have been
underestimated.
CONCLUSION: In the WHI, self-reported prevalent and incident diabetes was
consistent with medication inventories, and a high proportion of those with
undiagnosed diabetes subsequently reported diabetes treatment. Self-reports of
;treated diabetes' are sufficiently accurate to allow use in epidemiologic
studies. Vildagliptin is a potent and selective inhibitor of dipeptidyl peptidase-IV
(DPP-4), orally active, that improves glycemic control in patients with type 2
diabetes (T2DM) primarily by enhancing pancreatic (alpha and beta) islet
function. Thus vildagliptin has been shown both to improve insulin secretion and
to suppress the inappropriate glucagon secretion seen in patients with T2DM.
Vildagliptin reduces HbA(1c) when given as monotherapy, without weight gain and
with minimal hypoglycemia, or in combination with the most commonly prescribed
classes of oral hypoglycemic drugs: metformin, a sulfonylurea, a
thiazolidinedione, or insulin. Metformin, with a different mode of action not
addressing beta-cell dysfunction, has been used for about 50 years and still
represents the universal first line therapy of all guidelines. However, given
the multiple pathophysiological abnormalities in T2DM and the progressive nature
of the disease, intensification of therapy with combinations is typically
required over time. Recent guidelines imply that patients will require
pharmacologic combinations much earlier to attain and sustain the increasingly
stringent glycemic targets, with careful drug selection to avoid unwanted
adverse events, especially hypoglycemia. The combination of metformin and
vildagliptin offers advantages when compared to currently used combinations with
additive efficacy and complimentary mechanisms of action, since it does not
increase the risk of hypoglycemia and does not promote weight gain. Therefore,
by specifically combining these agents in a single tablet, there is considerable
potential to achieve better blood glucose control and to improve compliance to
therapy. BACKGROUND: Diabetes is associated with a higher risk for adverse cardiovascular
outcomes. To improve the health outcomes of patients with type 2 diabetes
(T2DM), the American Diabetes Association (ADA) recommended target goals for the
improvement of glycemic control and the reduction of cardiovascular risk factors
associated with the disease. This retrospective analysis calculated the absolute
benefit increase (ABI) of using exenatide once weekly (QW), a glucagon-like
peptide-1 (GLP-1) receptor agonist, vs an oral glucose-lowering medication or
insulin glargine to achieve ADA-recommended goals. The number needed to treat
(NNT) to achieve these goals was also calculated and provides a useful clinical
metric for comparing potential therapies from different drug classes.
METHODS: Patient data from three double-blind or open label, 26-week,
randomized, controlled trials were retrospectively analyzed separately. ABI and
NNT were calculated by comparing the percentage of patients treated with
exenatide QW (N = 641) vs metformin (N = 246), sitagliptin (N = 329),
pioglitazone (N = 328), or insulin glargine (N = 223), who achieved a single
glycemic, weight, blood pressure, or lipid goal or a composite of these
recommended goals, during the DURATION-2, -3, and -4 clinical trials.
RESULTS: Significant ABIs favoring exenatide QW over all four glucose-lowering
medications were observed for at least one HbA1c glycemic goal. NNTs of 4 and 5
were calculated when exenatide QW was compared to sitagliptin for attaining
HbA1c goals of <7.0% and ≤6.5%, respectively. Additionally, significantly more
patients using exenatide QW compared to sitagliptin, pioglitazone, or insulin
glargine attained the composite goal of HbA1c <7% or ≤6.5%, without weight gain
or hypoglycemia. Exenatide QW was also favored over sitagliptin and insulin
glargine for the achievement of the composite goals of HbA1c <7% (or ≤6.5%),
systolic blood pressure <130 mm Hg, and low-density lipoprotein <2.59 mmol/L.
For most goals, exenatide QW and metformin had similar effects in treatment
naïve patients.
CONCLUSIONS: This analysis assessed the between-therapy differences in achieving
therapeutic goals with therapies commonly used for glycemic control in patients
with T2DM. In clinical trials, exenatide QW assisted more patients in reaching
the majority of ADA-recommended therapeutic goals than treatment with
sitagliptin, pioglitazone, or insulin glargine.
TRIAL REGISTRATION: NCT00637273, NCT00641056, NCT00676338. Dipeptidyl peptidase-4 (DPP-4) inhibitors have recently emerged as a new class
of antidiabetic that show favorable results in improving glycemic control with a
minimal risk of hypoglycemia and weight gain. Teneligliptin, a novel DPP-4
inhibitor, exhibits a unique structure characterized by five consecutive rings,
which produce a potent and long-lasting effect. Teneligliptin is currently used
in cases showing insufficient improvement in glycemic control even after diet
control and exercise or a combination of diet control, exercise, and
sulfonylurea- or thiazolidine-class drugs. In adults, teneligliptin is orally
administered at a dosage of 20 mg once daily, which can be increased up to 40 mg
per day. Because the metabolites of this drug are eliminated via renal and
hepatic excretion, no dose adjustment is necessary in patients with renal
impairment. The safety profile of teneligliptin is similar to those of other
available DPP-4 inhibitors. However, caution needs to be exercised when
administering teneligliptin to patients who are prone to QT prolongation. One
study has reported that the postprandial blood glucose-lowering effects of
teneligliptin administered prior to breakfast were sustained throughout the day,
and the effects observed after dinner were similar to those observed after
breakfast or lunch. Thus, although clinical data for this new drug are limited,
this drug shows promise in stabilizing glycemic fluctuations throughout the day
and consequently suppressing the progression of diabetic complications. However,
continued evaluation in long-term studies and clinical trials is required to
evaluate the efficacy and safety of the drug as well as to identify additional
indications for its clinical use. INTRODUCTION: Dipeptidyl peptidase (DPP)-4 inhibitors belong to one class of
drugs that have been approved for treatment of type 2 diabetes (T2D) based on
the glucose-lowering actions of the gastrointestinal hormone glucagon-like
peptide (GLP)-1. Several different compounds are now available, and although
their mechanism of action (inhibition of the catalytic activity of DPP-4) is the
same, there are fundamental differences between them.
AREAS COVERED: The authors discuss the differences between different DPP-4
inhibitors and review their therapeutic efficacy and key safety data. The
literature covered includes original studies and meta-analyses identified in
PubMed, recent abstracts presented at major diabetes scientific conferences, and
clinical trials registered at ClinicalTrials.gov.
EXPERT OPINION: Although there are some differences in the pharmacokinetic and
pharmacodynamic profiles of the different DPP-4 inhibitors, all are small orally
active compounds with broadly similar HbA1c-lowering efficacy. They improve
glycaemic control in T2D, without increasing the risk of hypoglycaemia or
causing weight gain. They can be used as monotherapy or in combination with
other anti-diabetic therapies, including insulin, regardless of renal or hepatic
function, and are efficacious across the spectrum of patients with T2D,
including those with long-standing disease duration. DPP-4 inhibitors may also
have beneficial effects beyond glycaemic control, although this remains to be
demonstrated in purpose-designed clinical trials. Diabetes Mellitus is a chronic metabolic disease affecting wide range of people
across the globe. In India the rate of subjects being suffered from diabetes is
continuously increasing. So, the development of drugs for its effective
treatment is essential. Thereby, various attempts have been made to discover
newer drugs, to reduce the rate of anti diabetic occurrence. Anti-diabetic drugs
were found to treat diabetes mellitus by lowering glucose levels in the blood.
Both the use antidiabetic drugs as well as the changes in lifestyle and proper
diet can significantly affect the severity of diabetes mellitus and also reduces
the symptoms and occurrence of the disease. Researches in the past few years on
diabetes mellitus showed that this disease is spreading at a very faster rate,
thereby; various attempts have been made to treat it efficaciously. Development
and approval of antidiabetic drugs is quite necessary. There are different
classes of anti-diabetic drugs reported to treat diabetes. The objective of the
present review is to explore Invokana as a newly approved antidiabetic drug for
the effective treatment of type 2 diabetes. This review focuses mainly on the
various aspects of diabetes mellitus and its treatment perspectives. From the
various clinical studies done on Invokana, it was concluded that and Invokana
was found to be very effective for the efficacious therapy of diabetes mellitus. Patients with type 2 diabetes mellitus (T2DM) have a high or very high
cardiovascular risk. The clinical practice guidelines focus on the need to
achieve optimal glycemic control, and strategies for a multifactorial
therapeutic approach have shown significant cardiovascular benefits in these
patients. Inhibitors of sodium-glucose co-transporter 2 (SGLT-2) are a new class
of orally administered drugs in the treatment of T2DM, which act by inhibiting
reabsorption of glucose in the renal proximal tubule with consequent glycosuric
effect and lowering of blood glucose. Dapagliflozin, SGLT-2 inhibitor marketed
in Europe and Australia, has been shown to achieve glycosylated hemoglobin
reductions similar to other oral agents, as well as beneficial effects on major
comorbidities associated with T2DM. Therefore, it is considered of interest to
review the clinical efficacy of this new oral hypoglycemic on glycemic control,
risk of hypoglycemia, and its impact on body weight, blood pressure, lipid
profile and renal function. OBJECTIVE: Tofogliflozin is an oral hypoglycemic agent with a novel mechanism of
action that reduces blood glucose levels by promoting glucose excretion in
urine, achieved by selectively inhibiting sodium-glucose co-transporter 2
(SGLT2). We evaluated the effects of several selected anti-type 2 diabetes
mellitus (T2DM) drugs-glimepiride, metformin, sitagliptin, pioglitazone,
miglitol, nateglinide, and voglibose-on the pharmacokinetics and
pharmacodynamics of tofogliflozin, and the effects of tofogliflozin on the
pharmacokinetics of these anti-T2DM drugs in healthy male volunteers.
METHODS: A single dose of either tofogliflozin alone, one of the anti-T2DM drugs
alone, or co-administration of tofogliflozin and the anti-T2DM drug was
administered to 108 healthy men. Cmax, AUCinf, and cumulative urine glucose
excretion after co-administration of tofogliflozin and each of the anti-T2DM
drugs was evaluated relative to the values of those parameters after
administration of each drug alone.
RESULTS: None of the anti-T2DM drugs had any effect on tofogliflozin exposure.
Tofogliflozin had no or little effect on the exposure of any anti-T2DM drug. No
anti-T2DM drug had any major effect on the cumulative urine glucose excretion
induced by tofogliflozin. There were no safety concerns evident after
administration of any drug alone or in co-administration.
CONCLUSIONS: Neither the pharmacokinetics nor the pharmacodynamics of
tofogliflozin was affected by any of the anti-T2DM drugs evaluated in this
study, nor was the pharmacokinetics of any of the anti-T2DM drugs affected by
tofogliflozin in healthy male volunteers. Type 2 diabetes mellitus is a chronic metabolic disorder that has become the
fourth leading cause of death in the developed countries. The disorder is
characterized by pancreatic β-cells dysfunction, which causes hyperglycaemia
leading to several other complications. Treatment by far, which focuses on
insulin administration and glycaemic control, has not been satisfactory.
Glucagon-like peptide-1 (GLP1) is an endogenous peptide that stimulates
post-prandial insulin secretion. Despite being able to mimic the effect of
insulin, GLP1 has not been the target drug in diabetes treatment due to the
peptide's metabolic instability. After a decade-long effort to improve the
pharmacokinetics of GLP1, a number of GLP1 analogues are currently available on
the market. The current Minireview does not discuss these drugs but presents
strategies that were undertaken to address the weaknesses of the native GLP1,
particularly drug delivery techniques used in developing GLP1 oparticles and
modified GLP1 molecule. The article highlights how each of the selected
preparations has improved the efficacy of GLP1, and more importantly, through an
overview of these studies, it will provide an insight into strategies that may
be adopted in the future in the development of a more effective oral GLP1
formulation. A substantial proportion of patients with type 2 diabetes mellitus do not reach
glycemic targets, despite treatment with oral anti-diabetic drugs and basal
insulin therapy. Several options exist for treatment intensification beyond
basal insulin, and the treatment paradigm is complex. In this review, the
options for treatment intensification will be explored, focusing on drug classes
that act via the incretin system and paying particular attention to the
short-acting glucagon-like peptide-1 receptor agonists exenatide and
lixisenatide. Current treatment guidelines will be summarized and discussed. ©
2016 The Authors. Diabetes/Metabolism Research and Reviews Published by John
Wiley & Sons Ltd. BACKGROUND: Clinical guidelines form one of the cornerstones for providing
high-quality care for patients with diabetes. We compare the national guidelines
and the use of glucose lowering medication for type 2 diabetes (T2D) in Denmark,
Finland, Norway and Sweden.
METHODS: We compared how guidelines take comprehensive care into consideration,
what treatment targets and what antihyperglycemic medication was recommended.
The use of glucose-lowering medication was based on the sales of diabetes drugs
in these countries.
RESULTS: All guidelines stress the importance of comprehensive diabetes care.
Individualized glycemic targets are emphasized especially in the Danish and
Finnish guidelines. In 2013, sulfonylureas were the most common second-line
treatment after metformin in Denmark, Norway and Sweden; in Finland, this
position was taken by DPP-4 inhibitors. Recommended initial insulin type for
patients with T2D differs between the four countries. Danish, Norwegian and
Swedish guidelines also take economic aspects into account.
CONCLUSIONS: All guidelines stress regular and comprehensive diabetes care.
Danish and Finnish guidelines strongly underline the importance of
individualized glycemic targets. All guidelines recommend metformin as the
initial oral antihyperglycemic drug. In relation to recommended second line drug
therapy and initial insulin type for patients with T2D, the guidelines vary
largely between the four countries. INTRODUCTION: Treatment options for type 2 diabetes are becoming increasingly
complex with people often prescribed multiple medications, and may include both
oral and injectable therapies. There is ongoing debate about which drug classes
provide the optimum second-line and third-line treatment options. In the real
world, patient adherence and persistence determines medication effectiveness. A
better understanding of adherence may help inform the choice of second-line and
third-line drug classes.
METHODS AND ANALYSIS: This systematic review will compare adherence and
persistence rates across the different classes of medication available to people
with type 2 diabetes. It will include all identified studies comparing
medication adherence or persistence between two or more glucose-lowering
medications in people with type 2 diabetes. Research databases (MEDLINE, EMBASE,
The Cochrane Library, The Register of Controlled Trials, PsychINFO and CINAHL)
will be searched for relevant articles, using a comprehensive search strategy.
All identified medication trials and observational studies will be included
which compare adherence or persistence across classes of diabetes medication.
The characteristics and outcomes of all the included studies will be reported
along with a study quality grade, assessed using the Cochrane Risk Assessment
Tool. The quality of adjustment for confounders of adherence or persistence will
be reported for each study. Where multiple (n ≥ 3) studies provide compare
adherence or persistence across the same 2 medication classes, a meta-analysis
will be performed.
ETHICS AND DISSEMINATION: No ethics approval is required. This review and
meta-analysis (where possible) will provide important information on the
relative patient adherence and persistence, with the different classes of
diabetes therapies. Once complete, the results will be made available by
peer-reviewed publication.
TRIAL REGISTRATION NUMBER: CRD42015027865. BACKGROUND: Several antidiabetic drugs (i.e., sulfonylureas; SU, rosiglitazone)
have been reported to be associated with increased risks of cardiovascular
diseases (CVD) in patients with type 2 diabetes mellitus (T2DM). Dipeptidyl
peptidase-4 inhibitors (DPP4i) are newly available antidiabetic drugs. Most
studies only compared DPP4i with a placebo or SU, or targeted a specific CVD
event of interest (i.e., heart failure; HF). Comparative research of CVD risks
of DPP4i with other antidiabetic drugs (i.e., metformin, thiazolidinediones,
meglitinides, acarbose, and insulin) remains scarce. This study was aimed to
assess comparative risks of CVD, including ischemic stroke, myocardial
infarction (MI) and HF, and hypoglycemia of DPP4i with other antidiabetic drugs.
METHODS: We utilized Taiwan's National Health Insurance Research Database. A
total of 123,050 T2DM patients newly prescribed oral antidiabetic treatments
were identified in 2009-2010 and followed until 2013. Outcome endpoints included
a composite of CVD events: hospitalizations for ischemic stroke, MI and HF, and
hypoglycemia. Time-varying Cox proportional hazards regression was applied to
assess the time to event hazards of various antidiabetic drugs, adjusted for
patients' demographics, comorbidity, diabetic complications, and co-medications.
Additional analyses were performed for the patients with and without CVD
history, respectively.
RESULTS: DPP4i users had significantly lower CVD risks as compared to that of
non-DPP4i users (adjusted hazard ratio [aHR]: 0.83, 95 % confidence interval
[CI]: 0.76-0.91). Compared to DPP4i users, meglitinides (aHR 1.3, 95 % CI
1.20-1.43) and insulin users (aHR 3.73, 95 % CI 3.35, 4.14) had significantly
higher risks for composite CVD, as well as those for stroke, MI, HF, and
hypoglycemia. Additionally, metformin users had significantly lower risks for
composite CVD risk (aHR 0.87, 95 % CI 0.79-0.94), as well as those for MI, HF,
and hypoglycemia, as compared to those of DPP4i users. Although there was a
trend toward low CVD risks in pioglitazone users, the role of potential
confounding by indication cannot be excluded.
CONCLUSIONS: DPP4i-treated T2DM patients had lower risks for CVD as compared to
those for non-DPP4i users, except metformin users. In the last ten years, knowledge on pathophysiology of type 2 diabetes (T2DM)
has significantly increased, with multiple failures (decreased incretin effect,
increased lipolysis, increased glucagon secretion, neurotransmitters
dysfunction) recognized as important contributors, together with decreased
insulin secretion and reduced peripheral glucose uptake. As a consequence, the
pharmacologic therapy of T2DM has been progressively enriched by several novel
classes of drugs, trying to overcome these defects. The last, intriguing
compounds come into the market are SGLT2 inhibitors, framing the kidney in a
different scenario, not as site of a harmful disease complication, but rather as
the means to correct hyperglycemia and fight the disease. This review aims to
offer a short, updated overview of the role of these compounds in the treatment
of T2DM, focusing on efficacy, ancillary albeit relevant clinical effects,
safety, potential cardiovascular protection, positioning in common therapeutic
algorithms. |
Which pipelines are used for analyzing data from ChIP-nexus experiments? | PeakXus and Q-nexus enable comprehensive transcription factor binding site discovery from ChIP-nexus experiments. | MOTIVATION: Transcription factor (TF) binding can be studied accurately in vivo
with ChIP-exo and ChIP-Nexus experiments. Only fraction of TF binding mechanisms
are yet fully understood and accurate knowledge of binding locations and
patterns of TFs is key to understanding binding that is not explained by simple
positional weight matrix models. ChIP-exo/Nexus experiments can also offer
insight on the effect of single nucleotide polymorphism (SNP) at TF binding
sites on expression of the target genes. This is an important mechanism of
action for disease-causing SNPs at non-coding genomic regions.
RESULTS: We describe a peak caller PeakXus that is specifically designed to
leverage the increased resolution of ChIP-exo/Nexus and developed with the aim
of making as few assumptions of the data as possible to allow discoveries of
novel binding patterns. We apply PeakXus to ChIP-Nexus and ChIP-exo experiments
performed both in Homo sapiens and in Drosophila melanogaster cell lines. We
show that PeakXus consistently finds more peaks overlapping with a TF-specific
recognition sequence than published methods. As an application example we
demonstrate how PeakXus can be coupled with unique molecular identifiers (UMIs)
to measure the effect of a SNP overlapping with a TF binding site on the in vivo
binding of the TF.
AVAILABILITY AND IMPLEMENTATION: Source code of PeakXus is available at
https://github.com/hartonen/PeakXus
CONTACT: [email protected] or [email protected]. Author information:
(1)Institute for Medical and Human Genetics, Charité-Universitätsmedizin Berlin,
Augustenburger Platz 1, Berlin, 13353, Germany.
(2)Berlin Brandenburg Center for Regenerative Therapies (BCRT),
Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, Berlin, 13353,
Germany.
(3)Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and
Technology, Dr. Aiguader 88, Barcelona, 08003, Spain.
(4)Universitat Pompeu Fabra (UPF), Barcelona, Spain.
(5)Faculty of Biology, Johannes Gutenberg University Mainz, Ackermannweg 4,
Mainz, 55128, Germany.
(6)Institute of Molecular Biology, Ackermannweg 4, Mainz, 55128, Germany.
(7)Institute for Bioinformatics, Department of Mathematics and Computer Science,
Freie Universität Berlin, Arnimallee 14, Berlin, 14195, Germany.
(8)Labor für Pädiatrische Molekularbiologie, Charité-Universitätsmedizin Berlin,
Augustenburger Platz 1, Berlin, 13353, Germany.
(9)Institute for Medical and Human Genetics, Charité-Universitätsmedizin Berlin,
Augustenburger Platz 1, Berlin, 13353, Germany. [email protected].
(10)Berlin Brandenburg Center for Regenerative Therapies (BCRT),
Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, Berlin, 13353,
Germany. [email protected].
(11)Institute for Bioinformatics, Department of Mathematics and Computer
Science, Freie Universität Berlin, Arnimallee 14, Berlin, 14195, Germany.
[email protected].
(12)Max Planck Institute for Molecular Genetics, Inhestr. 63-73, Berlin, 14195,
Germany. [email protected].
(13)Current address: The Jackson Laboratory for Genomic Medicine, 10 Discovery
Drive, Farmington, 06032, CT, USA. [email protected]. |
What do statins do? | Statins lower high cholesterol | Recent large clinical trials have demonstrated that HMG-CoA reductase
inhibitors, or statins, markedly reduce morbidity and mortality when used in the
primary and secondary prevention of cardiovascular disease. It has been
established that the benefits of statin therapy in cardiovascular disease can be
explained not only by the lipid-lowering potential of statins but also by
nonlipid-related mechanisms (so-called "pleiotropic effects") that contribute to
the positive effect of statins on the incidence of cardiovascular events. The
coagulation and fibrinolytic systems are two separate but reciprocally linked
enzyme cascades that regulate the formation and breakdown of fibrin. Numerous
studies have demonstrated that disturbances of coagulation and fibrinolysis
contribute to the development and progression of atherosclerosis, and that they
affect the incidence of atherosclerosis-related clinical events. High plasma
levels or activities of fibrinogen, factor VII, factor VIII, von Willebrand
factor (vWF), soluble thrombomodulin, tissue plasminogen activator (tPA) and
plasminogen activator inhibitor-1 (PAI-1) are thought to be associated with
increased morbidity and mortality related to cardiovascular disease.
Experimental studies and many clinical studies have recently shown that statins
produce favourable effects on haemostatic parameters, including those that are
risk factors for cardiovascular disease. Statins diminish procoagulant activity,
which is observed at different stages of the coagulation cascade, including
tissue factor (TF) activity, conversion of prothrombin to thrombin and thrombin
activity. In some studies, statins also reduced fibrinogen levels. By altering
the levels and activities of tPA and PAI-1, statins seem to stimulate
fibrinolysis. The data on the effects of combined treatment with statins and
other drugs on haemostasis are rather limited. They suggest that statins
combined with fibric acid derivatives, omega-3 fatty acids and 17beta-estradiol
are superior to statins alone. The only two clinical studies performed in
patients with acute coronary syndromes showed a relatively weak effect of
statins on haemostasis in those patients. Although various statins may produce
different effects on individual variables, there are no convincing data showing
that differences in their physicochemical and pharmacokinetic properties
significantly alter their net effect on excessive procoagulant activity. Apart
from the lipid-lowering effect, statins suppress the synthesis of several
important nonsterol isoprenoids derived from the mevalonate pathway, especially
farnesyl and geranylgeranyl pyrophosphates, which via enhanced protein
prenylation, are involved in the regulation of many cellular processes. It is
presumed that the inhibitory effect of statins on the mevalonate pathway is
involved in the regulation of some key steps of coagulation and fibrinolysis
processes. In this way they probably regulate the synthesis of TF, tPA and
PAI-1, and perhaps they also control the generation and activity of thrombin.
The beneficial effects of statins on coagulation and fibrinolysis may be
responsible for their ability to decrease the number of cardiovascular events.
The lipid-independent effects of statins on haemostasis may contribute to the
marked decrease in the incidence rates of mortality, hospitalisation and
revascularisation in patients treated with these drugs. OBJECTIVE: HMG-CoA reductase inhibitors (statins) possess anti-inflammatory and
immunomodulatory properties that are independent of their lipid-lowering action.
As the CD40-CD40L signaling pathway is implicated in the modulation of
inflammatory responses between vascular cells, involving adhesion molecules,
pro-inflammatory cytokines, chemokines, we sought to investigate the potential
role of statins in regulating the expression of CD40.
METHODS AND RESULTS: Using Western blot, flow cytometry and immunohistochemistry
analyses, we observed that four different statins reduced IFN-gamma-induced CD40
expression in human vascular cells (endothelial cells, smooth muscle cells,
macrophages and fibroblasts). This effect was dose-dependent (from 5 microM to
80 nM) and reversed by addition of L-mevalonate. Activation of vascular cells by
human recombit CD40L, as measured by ELISA for IL-6, IL-8 and MCP-1, was
strongly reduced when cells were treated with statins. Immunostaining of human
carotid atherosclerotic lesions of patients subjected to statin treatment
revealed less CD40 expression on a 'per vascular cell' basis compared to control
patients. Although many pleiotropic effects of statins are mediated by nitric
oxide synthase (NOS)- or peroxisome proliferator-activated receptor
(PPAR)-dependent signaling pathways, we observed similar statin-induced
reduction of CD40 expression using NOS inhibitors or different PPAR ligands.
CONCLUSION: Statins decrease CD40 expression and CD40-related activation of
vascular cells. These effects are partially reversed by the HMG-CoA reductase
product L-mevalonate and are mediated by NOS- or PPAR-dependent pathways.
Altogether, these findings provide mechanistic insight into the beneficial
effects of statins on atherogenesis. They also provide a scientific rationale
for the use of statins as immunomodulators after organ transplantation. PURPOSE: Statins reduce cardiovascular events by more than can be explained by
their effects on lipids. We conducted a systematic review of how statins affect
vascular structure and function, differences among statins, and correlations
between the effects of statins on vascular outcomes and either lipid levels or
cardiovascular outcomes.
METHODS: We primarily searched MEDLINE (1980 to March 2004) to identify all
studies with at least 10 subjects that reported the effects of currently
available statins on coronary artery stenosis, carotid intima-media thickness,
and endothelial function (excluding studies of drug combinations and subjects
with organ transplants). Meta-analyses were performed when feasible.
RESULTS: Statins decrease the progression and increase the regression of
coronary artery lesions and luminal narrowing. Compared with placebo, statins
decrease the likelihood of coronary artery restenosis (summary risk ratio =
0.85; 95% confidence interval: 0.77 to 0.95). Statins appear to slow the
progression of carotid artery intima-media thickness. Although the effect of
statins on coronary endothelial function is uncertain, statins appear to improve
peripheral endothelial function. There is no conclusive evidence to suggest that
individual statins differ in their effects on these outcomes. Studies generally
found weak or no correlation between the effects of statins on vascular outcomes
and lipid levels. No study showed a correlation between vascular effect and
clinical outcome.
CONCLUSION: Statins slow the progression of, and may reverse, atherosclerosis.
The magnitude of these effects, however, is small compared with the effects of
statins on cardiovascular events. Statins also improve measures of vascular
function, which may contribute to their clinical benefits. There is insufficient
evidence to suggest that individual statins differ in their vascular effects. 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, statins, are
widely used cholesterol-lowering drugs and have been shown to have anticancer
effects in many models. We have investigated the effect of statins on Mdm2, a
p53-specific ubiquitin ligase. It was found that pravastatin induced Mdm2
phosphorylation at Ser166 and at 2A10 antibody-specific epitopes in HepG2 cells,
while mRNA levels were unchanged. Furthermore, pravastatin was found to induce
phosphorylation of mTOR at Ser2448. Ser166 phosphorylation of Mdm2 was abrogated
by an inhibitor of mTOR, rapamycin, but not by the PI3-kinase inhibitors
LY294002 and wortmannin. Ser166 phosphorylation of Mdm2 has been associated to
active Mdm2 and has been shown to increase its ubiquitin ligase activity and
lead to increased p53 degradation. Our data show that statins attenuated the p53
response to DNA damage. Thus, in HepG2 cells pravastatin and simvastatin
pretreatment attenuated the p53 response to DNA damage induced by 5-fluorouracil
and benzo(a)pyrene. Similar attenuation was induced when p53 stabilization was
induced by the inhibitor of nuclear export, leptomycin B. Furthermore, in the
DNA-damaged cells, half-lives of Mdm2 and p53 were decreased by statins,
indicating a more rapid formation of p53/Mdm2 complexes and facilitated p53
degradation. The induction of p53 responsive genes and apoptosis was attenuated.
Mdm2 and p53 were also studied in vivo in rat liver employing
immunohistochemistry, and it was found that constitutive Mdm2 expression was
changed in livers of pravastatin-treated rats. We also show that the p53
response to a challenging dose of diethylnitrosamine was attenuated in
hepatocytes in situ and in primary cultures of hepatocytes by pravastatin
pretreatment. Taken together, these data indicate that statins induce an
mTOR-dependent Ser166 phosphorylation of Mdm2, and this effect may attenuate the
duration and intensity of the p53 response to DNA damage in hepatocytes. Serious side effects of statins, including severe myopathy and rhabdomyolysis,
are rare but important in general practice. Hypothyroidism can cause secondary
hypercholesterolemia and myopathy. There have been few reports on the risk of
statins in patient with unnoticed hypothyroidism. We analyzed the
characteristics of 77 patients with primary hypothyroidism in our hospital. Nine
patients (11%) accidentally received statins in the treatment of
hypercholesterolemia without diagnosis of hypothyroidism. In such patients, free
T4 (FT4) levels were lower, and those of LDH, CK were higher than those in
patients not receiving statins. In patients accidentally receiving statins, an
inverse correlation between CK and FT4 could not be shown (which was recognized
in patients not receiving them). Even after FT4 levels were matched, levels of
CK were still higher in the patients accidentally receiving statins. Patients
with high CK levels over 1000 U/L were 5 times more frequent (56%) in patients
accidentally receiving statins than in those not receiving statins (11%). The
present study confirms that statins enhances levels of CK in patients with
hypothyroidism. We must not begin and continue to use these drugs without
checking the possibility of hypothyroidism. Statins can cause a necrotizing myopathy and hyperCKaemia which is reversible on
cessation of the drug. What is less well known is a phenomenon whereby statins
may induce a myopathy, which persists or may progress after stopping the drug.
We investigated the muscle pathology in 8 such cases. All had myofibre necrosis
but only 3 had an inflammatory infiltrate. In all cases there was diffuse or
multifocal up-regulation of MHC-I expression even in non-necrotic fibres.
Progressive improvement occurred in 7 cases after commencement of prednisolone
and methotrexate, and in one case spontaneously. These observations suggest that
statins may initiate an immune-mediated myopathy that persists after withdrawal
of the drug and responds to immunosuppressive therapy. The mechanism of this
myopathy is uncertain but may involve the induction by statins of an endoplasmic
reticulum stress response with associated up-regulation of MHC-I expression and
antigen presentation by muscle fibres. OBJECTIVES: The aims of this study were to determine if statins reduce adhesion
formation in vivo and to identify the mechanism of action in vitro.
BACKGROUND: : Intraperitoneal adhesions develop in up to 95% of patients
following laparotomy. Adhesions are reduced by mechanisms that up-regulate
fibrinolysis within the peritoneum. Statins promote fibrinolysis in the
cardiovascular system and may play a role in the prevention of adhesions.
METHODS: Adhesions were induced in rats (n = 102) using our previously described
ischemic button model. Rats received vehicle (controls), lovastatin (30 mg/kg),
or atorvastatin (30 mg/kg) as a single intraperitoneal dose at the time of
laparotomy. Animals were killed and adhesions were quantified at day 7.
Peritoneal fluid and tissue were collected at day 1 to measure tissue
plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) by
real-time PCR and ELISA. To assess the effects of statins on wound healing,
burst pressures were measured in anastomoses of the colon. The effects of
lovastatin on tPA and PAI-1 production were measured in vitro in human
mesothelial cells (HMC) in the presence or absence of mevalonate (MVA),
geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate (FPP), all
intermediates in the cholesterol pathway downstream of HMG-CoA. The effect of a
Rho protein inhibitor, exoenzyme C3 transferase, on tPA production was also
determined.
RESULTS: Lovastatin and atorvastatin reduced adhesion formation by 26% and 58%,
respectively (P < 0.05), without affecting anastomotic burst pressure. At 24
hours, tPA mRNA levels in peritoneal tissue and tPA activity in peritoneal fluid
from lovastatin-treated animals were increased by 57% and 379%, respectively (P
< 0.05), while PAI-1 levels were unchanged. HMC incubated with either lovastatin
or atorvastatin showed concentration-dependent increases in tPA production and
decreases in PAI-1 production (P < 0.05). These lovastatin-induced changes in
tPA and PAI-1 production were significantly reversed by the addition of MVA,
GGPP, and FPP. The Rho protein inhibitor increased tPA production and rescued
tPA production from the inhibitory effect of GGPP.
CONCLUSION: These data suggest that statins administered within the peritoneum
can up-regulate local fibrinolysis, while the in vitro studies show that this
effect may be mediated, in part, by intermediates of the cholesterol
biosynthetic pathway that regulate Rho protein signaling. Both statins and peroxisome proliferator-activated receptor (PPAR)gamma ligands
have been reported to protect against the progression of atherosclerosis. In the
present study, we investigated the effects of statins on PPARgamma activation in
macrophages. Statins increased PPARgamma activity, which was inhibited by
mevalonate, farnesylpyrophosphate, or geranylgeranylpyrophosphate. Furthermore,
a farnesyl transferase inhibitor and a geranylgeranyl transferase inhibitor
mimicked the effects of statins. Statins inhibited the membrane translocations
of Ras, RhoA, Rac, and Cdc42, and overexpression of domit-negative mutants of
RhoA (DN-RhoA) and Cdc42 (DN-Cdc42), but not of Ras or Rac, increased PPARgamma
activity. Statins induced extracellular signal-regulated kinase (ERK)1/2 and p38
mitogen-activated protein kinase (MAPK) activation. However, DN-RhoA and
DN-Cdc42 activated p38 MAPK, but not ERK1/2. ERK1/2- or p38 MAPK-specific
inhibitors abrogated statin-induced PPARgamma activation. Statins induced
cyclooxygenase (COX)-2 expression and increased intracellular
15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) levels through ERK1/2- and
p38 MAPK-dependent pathways, and inhibitors or small interfering RNA of COX-2
inhibited statin-induced PPARgamma activation. Statins also activate PPARalpha
via COX-2-dependent increases in 15d-PGJ(2) levels. We further demonstrated that
statins inhibited lipopolysaccharide-induced tumor necrosis factor alpha or
monocyte chemoattractant protein-1 mRNA expression, and these effects by statins
were abrogated by the PPARgamma antagonist T0070907 or by small interfering RNA
of PPARgamma or PPARalpha. Statins also induced ATP-binding cassette protein A1
or CD36 mRNA expression, and these effects were suppressed by small interfering
RNAs of PPARgamma or PPARalpha. In conclusion, statins induce COX-2-dependent
increase in 15d-PGJ(2) level through a RhoA- and Cdc42-dependent p38 MAPK
pathway and a RhoA- and Cdc42-independent ERK1/2 pathway, thereby activating
PPARgamma. Statins also activate PPARalpha via COX-2-dependent pathway. These
effects of statins may explain their antiatherogenic actions. Patients with high low-density lipoprotein cholesterol (LDLC) and asymptomatic
high creatine kinase (CK) (>or=250 but <2500 IU/L, 10x the laboratory upper
normal limit [UNL]) are often not started on statins or have statins stopped
because of concern about myositis-rhabdomyolysis. In the current report, we
prospectively examined the hypothesis that asymptomatic patients with high CK
(>or=250 but <2500 IU/L) tolerate statins well at doses reducing LDLC to target,
less than 100 mg/dL, without development of myalgia-myositis. We assessed
outcomes of 3 groups of patients referred to us because of asymptomatic high CK
(>or=250 but <2500 IU/L)--1 group (n = 29) on statins at referral and continued
on statins, 1 group (n = 20) not on statins and started on statins, and 1 group
(n = 19) not on statins and not given statins--all restudied 1 month after entry
and then every 3 months. Of the 68 patients, 59 (87%) had CK greater than 1 to 3
times the UNL, 7 (10%) had CK greater than 3 to 5 times the UNL, and 2 (3%) had
CK greater than 5 to 10 times the UNL. After 1.2 months of follow-up in 29
statin-->statin patients, median CK fell from 353 to 301 (P = .0018) and was 287
(P = .015) after 4 months. After 1.3 months of follow-up in 20 no
statin-->statin patients, median CK fell from 397 to 292 (P = .0094) and was 419
after 4.1 months. After 1.1 months of follow-up in 19 no statin-->no statin
patients, median CK fell from 392 to 323 (P = .14) and was 271 (P = .029) after
4.2 months. By repeated-measures analysis, there were no differences in entry CK
among the 3 treatment groups; CK fell (P = .04) in the no statin-->no statin
patients. Despite high baseline CK (48 patients with CK 1-5x the UNL, 1 with CK
5-10x UNL), no patients during follow-up on statins developed CK greater than 10
times the UNL (2500 IU/L), none discontinued statins or reduced statin dose
because of myalgia-myositis, and there was no rhabdomyolysis. High pretreatment
CK, particularly 1 to 5 times the UNL, should not be an impediment to start or
continue statins to lower LDLC. In gallbladder epithelial cells (GBEC), PPARalpha and PPARgamma ligands modulate
inflammation by suppression of TNFalpha production and prevent excessive
accumulation of cholesterol by ABCA1 activation. Recently, HMG-CoA reductase
inhibitors (statins) were shown to activate PPARalpha and PPARgamma in various
cells but no studies of their effects in GBEC have been conducted. The objective
of this study was, therefore, to determine the effects of statins on PPAR and
ABCA1 expression and the anti-inflammatory effect of statins in GBEC. Canine
GBEC were cultured on Petri dishes. Expression of the proteins PPARalpha,
PPARgamma, and ABCA1 was measured by western blotting analysis after treatment
with simvastatin, pravastatin, NO-pravastatin, PPARalpha ligand, or PPARgamma
ligand in the culture media. Expression of ABCA1 and LXRalpha mRNAs was
estimated by RT-PCR. Expression of TNFalpha mRNA was measured by RT-PCR after 24
h pre-treatment with the statins, preceding 1 h of lipopolysaccharide (LPS)
loading. Simvastatin, pravastatin, and NO-pravastatin increased expression of
the proteins PPARalpha, PPARgamma, and ABCA1, and expression of the mRNA of
ABCA1 and LXRalpha in GBEC. Pre-treatment with simvastatin, pravastatin, and
NO-pravastatin suppressed the production of TNFalpha mRNA induced by LPS. In
conclusion, statins probably contribute to the preservation of GBEC function by
activation of PPARalpha and PPARgamma, which have anti-inflammatory effects by
suppression of pro-inflammatory cytokines, and ABCA1 activation mediated by
LXRalpha, which prevents the accumulation of cholesterol in GBEC. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: * The increasing evidence of the
anti-inflammatory action of statins has stimulated interest in whether these
might be beneficial in disease management of rheumatoid arthritis (RA), a
chronic diseases characterized by high levels of inflammation. * The TARA trial
(McCarey 2004) suggested a significant reduction in disease activity outcomes in
RA patients randomized to atorvastatin compared with those assigned to the
placebo harm. * However, as the signal reported by the trial was small, more
evidence is needed.
WHAT THIS PAPER ADDS: * We investigated the possible anti-inflammatory effect of
statins in a cohort of RA patients using a large health insurance claims
database. * To our knowledge, this is the largest study ever conducted on the
anti-inflammatory effects of statins. * Our data do not show any beneficial
effect of statins in reducing disease inflammation in RA patients.
AIM: To investigate the possible anti-inflammatory effect of statins in a cohort
of rheumatoid arthritis (RA) patients.
METHODS: We conducted a cohort study consisting of all patients with at least
one claim for RA using LifeLink, a health insurance claims database. Initiation
and cessation of oral steroid (OS) therapy were treated as surrogate for
inflammatory flare-up and controlled inflammation, respectively. We split the RA
patients into two sub-cohorts based on whether they were using OS within a
specified time window of the RA index date (first recorded claim for RA in the
database). Cox proportional hazard models were used to evaluate the association
between time-varying exposure to any statins and (i) initiation of OS therapy in
the non-users of OS at RA index date and (ii) cessation of OS therapy in the
users of OS at RA index date controlling for potential confounders.
RESULTS: We found 31 451 non-users of OS at RA index date and 6026 users of OS
within the time window at RA index date. The results on both sub-cohorts were
both consistent with no association of statin exposure with the risk of
initiation/cessation of OS: the hazard ratio (HR) of initiating OS therapy was
0.96 (95% confidence interval 0.9, 1.01) in the sub-cohort of non-users and the
HR of cessation of OS therapy was 0.95 (0.87, 1.05) in the sub-cohort of users
of OS therapy at RA diagnosis.
CONCLUSIONS: These data do not show any beneficial effect of statins in reducing
disease inflammation in RA patients. OBJECTIVE: Statins, which are used as cholesterol-lowering agents, have
pleiotropic immunomodulatory properties. Although beneficial effects of statins
have been reported in autoimmune diseases, the mechanisms of these
immunomodulatory effects are still poorly understood. Type I interferons (IFNs)
and plasmacytoid dendritic cells (PDCs) represent key molecular and cellular
pathogenic components in autoimmune diseases such as systemic lupus
erythematosus (SLE). Therefore, PDCs may be a specific target of statins in
therapeutic strategies against SLE. This study was undertaken to investigate the
immunomodulatory mechanisms of statins that target the IFN response in PDCs.
METHODS: We isolated human blood PDCs by flow cytometry and examined the effects
of simvastatin and pitavastatin on PDC activation, IFNalpha production, and
intracellular signaling.
RESULTS: Statins inhibited IFNalpha production profoundly and tumor necrosis
factor alpha production modestly in human PDCs in response to Toll-like receptor
ligands. The inhibitory effect on IFNalpha production was reversed by
geranylgeranyl pyrophosphate and was mimicked by either geranylgeranyl
transferase inhibitor or Rho kinase inhibitor, suggesting that statins exert
their inhibitory actions through geranylgeranylated Rho inactivation. Statins
inhibited the expression of phosphorylated p38 MAPK and Akt, and the inhibitory
effect on the IFN response was through the prevention of nuclear translocation
of IFN regulatory factor 7. In addition, statins had an inhibitory effect on
both IFNalpha production by PDCs from SLE patients and SLE serum-induced
IFNalpha production.
CONCLUSION: Our findings suggest a specific role of statins in controlling type
I IFN production and a therapeutic potential in IFN-related autoimmune diseases
such as SLE. BACKGROUND: Muscle pain without elevation of serum creatine phosphokinase (CPK)
(myalgia) is the most common medication-related adverse effect of statin
therapy; it occurs in up to 10% of patients who are prescribed statin therapy.
Although much is known regarding risk factors for overt myositis, very few
studies have provided information on this common form of statin intolerance.
METHODS: We defined a detailed clinical and laboratory phenotype of a cohort of
patients referred to the lipid clinic of a governmental health maintece
organization for statin intolerance attributable to muscle pain without CPK
elevation (myalgia) and characterized their response to alternative
lipid-lowering therapy. Baseline and follow-up data were analyzed for 104
patients with statin intolerance attributable to myalgia and 211 statin-tolerant
control patients identified from the referral population.
RESULTS: Among patients with myalgia, more were white and had hypertension. The
prevalence of known risk factors for overt myositis, including renal disease,
type 2 diabetes mellitus, thyroid disease, and electrolyte abnormalities, did
not differ between statin intolerant and statin tolerant patients. Although
individual cases were identified in which the addition of interacting
medications was temporally associated with development of statin intolerance,
overall use of interacting medications was not more frequent among
statin-intolerant patients. The majority of patients were intolerant of two or
more statins; however, in more than one-half the cases, successful rechallenge
with an alternative statin was accomplished. Despite this and extensive use of
nonstatin lipid medications after lipid clinic referral, control of plasma
lipoproteins remained significantly worse in statin-intolerant patients.
CONCLUSIONS: Statin intolerance attributable to myalgia is a significant barrier
to effective treatment of hyperlipidemia. Conventional clinical risk factors for
myositis do not appear to predictive of statin-associated myalgia. These
findings underscore the need to better define the pathophysiology of
statin-induced myalgia and develop methodologies to guide treatment of
statin-intolerant patients. Myositis-myalgia is the most common cause of statin intolerance, leading to
cessation of statin use, with consequent failure to lower LDL cholesterol to
target levels for primary and secondary prevention of cardiovascular disease
(CVD). We hypothesize that symptomatic myositis-myalgia in hypercholesterolemic
statin-treated patients with concurrent 25 (OH) vitamin D deficiency and statin
intolerance may reflect a reversible interaction between vitamin D deficiency
and statins on skeletal muscle. In hypercholesterolemic, vitamin D deficient
patients, intolerant to statins because of myositis-myalgia, three non-blinded
clinical case series have uniformly demonstrated that after supplementation with
oral vitamin D2 which normalizes serum 25 (OH) vitamin D levels, statins can be
successfully re-instituted in >90% of patients, without recurrent
myositis-myalgia, with reduction of LDL cholesterol to target levels.
Empirically, in 68 hypercholesterolemic patients, unable to tolerate≥1 statin
because of myositis-myalgia, selected by low (<32 ng/ml) serum 25 (OH) vitamin
D, we have prospectively assessed whether resolution of vitamin D deficiency
would result in statin tolerance, free of myositis-myalgia. On no statins,
50,000 units of vitamin D2 was given twice/week for 3 weeks, and was then
continued once/week. After 3 weeks on vitamin D supplementation, statins were
restarted, and patients were re-assessed after 3 months on statins while
continuing vitamin D supplementation. At 3 months follow-up, on vitamin D
supplementation and re-instituted statins, 62 of 68 (91%) previously
statin-intolerant patients now tolerated statins well and were asymptomatic
without myositis-myalgia. In these 68 patients, on vitamin D supplementation and
statins, mean±SD vitamin D rose from 22±7 to 43±13 ng/ml (p<0.0001), and LDL
cholesterol fell from 162±55 to 101±35 mg/dl (p<0.0001). Despite published and
new empirical evidence, the medical establishment has refused to accept the
hypothesis, requiring placebo-controlled, double-blind studies, none having been
reported to date. A placebo-controlled, double-blind study is needed to document
that normalization of serum 25 (OH) vitamin D levels in vitamin D deficient,
statin intolerant patients would facilitate re-introduction of statins with
concurrent freedom from myositis-myalgia. The ability to reverse
myositis-myalgia in vitamin D deficient, statin intolerant, hypercholesterolemic
patients by vitamin D supplementation would be extraordinarily valuable,
facilitating reinstitution of statins to lower LDL cholesterol to reduce risk of
CVD events. We hypothesize that symptomatic myositis-myalgia in
hypercholesterolemic statin-treated patients with concurrent vitamin D
deficiency producing statin intolerance may reflect a reversible interaction
between vitamin D deficiency and statins on skeletal muscle. BACKGROUND: 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, also
known as statins, are the most commonly prescribed cholesterol-lowering
medications in the world. Statins have been shown to inhibit connective tissue
growth factor (CTGF) gene and protein expression in vitro, and previous studies
in the authors' laboratory have demonstrated that CTGF plays a significant role
in wound healing and scarring. The authors explore whether administration of
various statins to healing wounds has any effect on hypertrophic scar formation
using an established rabbit ear wounding model.
METHODS: Punch wounds were made on the ears of 31 rabbits (n = 372 total
wounds). Treatment wounds were injected with simvastatin, lovastatin, or
pravastatin at low, medium, or high doses on postwounding days 15, 20, and 25,
whereas control wounds were injected in a corresponding fashion. Wounds were
harvested after 35 days for histomorphometric analysis.
RESULTS: Low-dose (40 μM) simvastatin, lovastatin, and pravastatin each
demonstrated significant reductions in scar elevation index when compared with
control: 21.9 percent (p = 0.03), 25.8 percent (p = 0.02), and 22.8 percent (p =
0.01), respectively. There were no significant differences in scar elevation
index in the medium- (120 μM) and high-dose (400 μM) statin groups. Analysis of
mRNA in a subset of rabbits treated with low-dose simvastatin demonstrated a
significant reduction in CTGF expression (p = 0.009).
CONCLUSIONS: 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors reduce
hypertrophic scar formation by means of CTGF inhibition when administered at low
doses, and the novel application of these commonly prescribed medications may
lead to innovative and effective antiscarring therapies. INTRODUCTION: Statins reduce coronary events in patients with coronary artery
disease.
MATERIAL AND METHODS: Chart reviews were performed in 305 patients (217 men and
88 women, mean age 74 years) not treated with statins during the first year of
being seen in an outpatient cardiology practice but subsequently treated with
statins. Based on the starting date of statins use, the long-term outcomes of
myocardial infarction (MI), percutaneous coronary intervention (PCI), and
coronary artery bypass graft surgery (CABGs) before and after statin use were
compared.
RESULTS: Mean follow-up was 65 months before statins use and 66 months after
statins use. Myocardial infarction occurred in 31 of 305 patients (10%) before
statins, and in 13 of 305 patients (4%) after statins (p < 0.01). Percutaneous
coronary intervention had been performed in 66 of 305 patients (22%) before
statins and was performed in 41 of 305 patients (13%) after statins (p < 0.01).
Coronary artery bypass graft surgery had been performed in 56 of 305 patients
(18%) before statins and in 20 of 305 patients (7%) after statins (p < 0.001).
Stepwise logistic regression showed statins use was an independent risk factor
for MI (odds ratio = 0.0207, 95% CI, 0.0082-0.0522, p < 0.0001), PCI (odds ratio
= 0.0109, 95% CI, 0.0038-0.0315, p < 0.0001) and CABGs (odds ratio = 0.0177, 95%
CI = 0.0072-0.0431, p < 0.0001)
CONCLUSIONS: Statins use in an outpatient cardiology practice reduces the
incidence of MI, PCI, and CABGs. WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Statins have shown
broad spectrum anti-cancer properties in laboratory studies. In epidemiological
studies, use of statins has been associated with reduced risk of advanced
prostate cancer. However, the effects of statins on prostate cancer disease
progression following curative treatment have not been extensively studied, and
previous studies reported conflicting results. This study found no clear
association between overall statin use and risk of disease progression, as well
as lack of a monotone dose-response relationship between the use of statins,
whether it was use before or after prostatectomy, and prostate cancer disease
progression.
OBJECTIVE: To investigate whether use of HMG-CoA reductase inhibitors
('statins'), which have shown broad spectrum anti-cancer properties in
laboratory studies, is associated with a reduced risk of recurrence in patients
with prostate cancer who undergo radical prostatectomy.
PATIENTS AND METHODS: All men with incident prostate cancer diagnosed between
2004 and 2005 who subsequently underwent radical prostatectomy by the end of
2005 in the Kaiser Permanente Southern California (KPSC) health plan were
identified using KPSC's cancer registry. Subjects were followed for up to 5
years after prostatectomy for (i) biochemical recurrence, defined as a single
PSA measurement >0.2 ng/mL, and (ii) clinical disease progression, defined as
diagnosis of metastatic disease or prostate-cancer-related death. Information on
statin use, demographics, comorbidities, patho-clinical factors and outcomes
were ascertained from KPSC's electronic medical records. The effects of statin
use prior to and after prostatectomy were both examined using bivariate and
multivariate Cox models, adjusting for known prognostic factors. For
postoperative statin exposure, a time-dependent Cox model was used.
RESULTS: A total of 1200 men were included; 37% had preoperative and 56% had
postoperative statin use. Neither preoperative nor postoperative statin use was
associated with biochemical recurrence (hazard ratio [HR] = 1.00 [0.72-1.39] and
1.05 [0.76-1.46], respectively) or clinical disease progression (HR = 0.63
[0.31-1.27] and 1.20 [0.63-2.30], respectively). No clear dose-response
relationship was found for duration of use.
CONCLUSIONS: Statin use may not prevent prostate cancer progression following
radical prostatectomy. These findings do not provide support for the pursuit of
a prospective clinical trial of statin use as a secondary prevention among
surgically treated patients with prostate cancer. OBJECTIVES: The authors sought to evaluate the cost-effectiveness of statins for
primary prevention of myocardial infarction (MI) and stroke in patients with
chronic kidney disease (CKD).
BACKGROUND: Patients with CKD have an elevated risk of MI and stroke. Although
HMG Co-A reductase inhibitors (“statins”) may prevent cardiovascular events in
patients with non–dialysis-requiring CKD, adverse drug effects and competing
risks could materially influence net effects and clinical decision-making.
METHODS: We developed a decision-analytic model of CKD and cardiovascular
disease (CVD) to determine the cost-effectiveness of low-cost generic statins
for primary CVD prevention in men and women with hypertension and
mild-to-moderate CKD. Outcomes included MI and stroke rates, discounted
quality-adjusted life years (QALYs) and lifetime costs (2010 USD), and
incremental cost-effectiveness ratios.
RESULTS: For 65-year-old men with moderate hypertension and mild-to-moderate
CKD, statins reduced the combined rate of MI and stroke, yielded 0.10 QALYs, and
increased costs by $1,800 ($18,000 per QALY gained). For patients with lower
baseline cardiovascular risks, health and economic benefits were smaller; for
65-year-old women, statins yielded 0.06 QALYs and increased costs by $1,900
($33,400 per QALY gained). Results were sensitive to rates of rhabdomyolysis and
drug costs. Statins are less cost-effective when obtained at average retail
prices, particularly in patients at lower CVD risk.
CONCLUSIONS: Although statins reduce absolute CVD risk in patients with CKD, the
increased risk of rhabdomyolysis, and competing risks associated with
progressive CKD, partly offset these gains. Low-cost generic statins appear
cost-effective for primary prevention of CVD in patients with mild-to-moderate
CKD and hypertension. BACKGROUND: Statins are known to reduce cardiovascular morbidity and mortality
in primary and secondary prevention studies. Subsequently, a number of
nonrandomised studies have shown statins improve clinical outcomes in patients
with heart failure (HF). Small randomised controlled trials (RCT) also show
improved cardiac function, reduced inflammation and mortality with statins in
HF. However, the findings of two large RCTs do not support the evidence provided
by previous studies and suggest statins lack beneficial effects in HF. Two
meta-analyses have shown statins do not improve survival, whereas two others
showed improved cardiac function and reduced inflammation in HF. It appears
lipophilic statins produce better survival and other outcome benefits compared
to hydrophilic statins. But the two types have not been compared in direct
comparison trials in HF.
METHODS/DESIGN: We will conduct a systematic review and meta-analysis of
lipophilic and hydrophilic statin therapy in patients with HF. Our objectives
are:1. To determine the effects of lipophilic statins on (1) mortality, (2)
hospitalisation for worsening HF, (3) cardiac function and (4) inflammation.2.
To determine the effects of hydrophilic statins on (1) mortality, (2)
hospitalisation for worsening HF, (3) cardiac function and (4) inflammation.3.
To compare the efficacy of lipophilic and hydrophilic statins on HF outcomes
with an adjusted indirect comparison meta-analysis.We will conduct an electronic
search of databases for RCTs that evaluate statins in patients with HF. The
reference lists of all identified studies will be reviewed. Two independent
reviewers will conduct the search. The inclusion criteria include:1. RCTs
comparing statins with placebo or no statin in patients with symptomatic HF.2.
RCTs that employed the intention-to-treat (ITT) principle in data analysis.3.
Symptomatic HF patients of all aetiologies and on standard treatment.4. Statin
of any dose as intervention.5. Placebo or no statin arm as control.The exclusion
criteria include:1. RCTs involving cerivastatin in HF patients.2. RCTs with less
than 4 weeks of follow-up.
DISCUSSION: We will perform an adjusted indirect comparison meta-analysis of
lipophilic versus hydrophilic statins in patients with HF using placebo or no
statin arm as common comparator. Among the various hypolipidemic drugs, 3-hydroxy-3-methyl-glutaryl-coenzyme A
(HMG-CoA) reductase inhibitors (also known as "statins") belong to a
heterogeneous class of compounds, sharing an identical hypocholesterolemic
effect that develops through direct inhibition of a rate-limiting step in
endogenous cholesterol synthesis. Their mechanism of action entails competitive
inhibition of HMG-CoA reductase. Several lines of evidence suggest that the
pleiotropic effects of statins may also play a role in prevention of venous
thrombosis, wherein hypercholesterolemic patients are characterized by enhanced
thrombin generation, increased susceptibility to endothelial dysfunction and
platelet hyperreactivity, so that limiting or counteracting the burden of one or
more of these mechanisms would provide an effective means of prophylaxis.
Plausible biological links can also be found between statin therapy and
reduction of thrombotic risk, mainly targeting immune system, blood coagulation,
endothelium, lipid metabolism, and inflammation. The earlier JUPITER
(Justification for the Use of Statins in Primary Prevention) trial provided
appealing evidence that the risk of venous thrombosis may be lowered by statins.
The results of the following studies and those of recent meta-analyses have,
however, questioned this assumption. Currently, it seems thereby cautious to
conclude that the use of statins as part of the approach used for preventing
venous thromboembolism appears unwarranted. This is due to the existence of
controversial clinical evidence, to the large number of patients who would need
to be treated to prevent one case of venous thrombosis, as well as to the
tangible risk of side effects. More randomized and the larger studies are needed
before definitive conclusions can be drawn. Statins are the current basis of lipid-lowering therapy, despite which may have
limitations on efficacy and safety. In high risk patients who do not achieve
current lipid goals, in those intolerant to statins or those with atherogenic
dyslipidemia, it is possible combine two or more lipid lowering drugs, including
statins, ezetimibe, bile acid sequestrants, fibrates, niacin and prescription
omega-3 fatty acids. However, for most of these combination therapies pivotal
data on clinical outcomes are still lacking. SIGNIFICANCE: The 3-hydroxy-methylglutaryl coenzyme A reductase inhibitors or
statins are important therapeutic agents for lowering serum cholesterol levels.
However, recent studies suggest that statins may exert atheroprotective effects
beyond cholesterol lowering. These so-called "pleiotropic effects" include
effects of statins on vascular and inflammatory cells. Thus, it is important to
understand whether other signaling pathways that are involved in atherosclerosis
could be targets of statins, and if so, whether individuals with "overactivity"
of these pathways could benefit from statin therapy, regardless of serum
cholesterol level.
RECENT ADVANCES: Statins inhibit the synthesis of isoprenoids, which are
important for the function of the Rho/Rho-associated coiled-coil containing
kinase (ROCK) pathway. Indeed, recent studies suggest that inhibition of the
Rho/ROCK pathway by statins could lead to improved endothelial function and
decreased vascular inflammation and atherosclerosis. Thus, the Rho/ROCK pathway
has emerged as an important target of statin therapy for reducing
atherosclerosis and possibly cardiovascular disease.
CRITICAL ISSUES: Because atherosclerosis is both a lipid and an inflammatory
disease, it is important to understand how inhibition of Rho/ROCK pathway could
contribute to statins' antiatherosclerotic effects.
FUTURE DIRECTIONS: The role of ROCKs (ROCK1 and ROCK2) in endothelial, smooth
muscle, and inflammatory cells needs to be determined in the context of
atherogenesis. This could lead to the development of specific ROCK1 or ROCK2
inhibitors, which could have greater therapeutic benefits with less toxicity.
Also, clinical trials will need to be performed to determine whether inhibition
of ROCKs, with and without statins, could lead to further reduction in
atherosclerosis and cardiovascular disease. BACKGROUND: People with chronic kidney disease (CKD) have higher risks of
cardiovascular disease compared to the general population. Specifically,
cardiovascular deaths account most deaths in kidney transplant recipients.
Statins are a potentially beneficial intervention for kidney transplant patients
given their established benefits in patients at risk of cardiovascular disease
in the general population. This is an update of a review first published in
2009.
OBJECTIVES: We aimed to evaluate the benefits (reductions in all-cause and
cardiovascular mortality, major cardiovascular events, myocardial infarction and
stroke, and progression of CKD to requiring dialysis) and harms (muscle or liver
dysfunction, withdrawal, cancer) of statins compared to placebo, no treatment,
standard care, or another statin in adults with CKD who have a functioning
kidney transplant.
SEARCH METHODS: We searched the Cochrane Renal Group's Specialised Register to
29 February 2012 through contact with the Trials Search Co-ordinator using
search terms relevant to this review.
SELECTION CRITERIA: We included randomised controlled trials (RCTs) and
quasi-RCTs that compared the effects of statins with placebo, no treatment,
standard care, or statins on mortality, cardiovascular events, kidney function
and toxicity in kidney transplant recipients.
DATA COLLECTION AND ANALYSIS: Two authors independently extracted data and
assessed risk of bias. Treatment effects were expressed as mean difference (MD)
for continuous outcomes (lipids, glomerular filtration rate (GFR), proteinuria)
and relative risk (RR) for dichotomous outcomes (major cardiovascular events,
mortality, fatal or non-fatal myocardial infarction, fatal or non-fatal stroke,
elevated muscle or liver enzymes, withdrawal due to adverse events, cancer,
end-stage kidney disease (ESKD), acute allograft rejection) together with 95%
confidence intervals (CI).
MAIN RESULTS: We identified 22 studies (3465 participants); 17 studies (3282
participants) compared statin with placebo or no treatment, and five studies
(183 participants) compared two different statin regimens.From data generally
derived from a single high-quality study, it was found that statins may reduce
major cardiovascular events (1 study, 2102 participants: RR 0.84, CI 0.66 to
1.06), cardiovascular mortality (4 studies, 2322 participants: RR 0.68, CI 0.45
to 1.01), and fatal or non-fatal myocardial infarction (1 study, 2102
participants: RR 0.70, CI 0.48 to 1.01); although effect estimates lack
precision and include the possibility of no effect.Statins had uncertain effects
on all-cause mortality (6 studies, 2760 participants: RR 1.08, CI 0.63 to 1.83);
fatal or non-fatal stroke (1 study, 2102 participants: RR 1.18, CI 0.85 to
1.63); creatine kinase elevation (3 studies, 2233 participants: RR 0.86, CI 0.39
to 1.89); liver enzyme elevation (4 studies, 608 participants: RR 0.62, CI 0.33
to 1.19); withdrawal due to adverse events (9 studies, 2810 participants: RR
0.89, CI 0.74 to 1.06); and cancer (1 study, 2094 participants: RR 0.94, CI 0.82
to 1.07).Statins significantly reduced serum total cholesterol (12 studies, 3070
participants: MD -42.43 mg/dL, CI -51.22 to -33.65); low-density lipoprotein
cholesterol (11 studies, 3004 participants: MD -43.19 mg/dL, CI -52.59 to
-33.78); serum triglycerides (11 studies, 3012 participants: MD -27.28 mg/dL, CI
-34.29 to -20.27); and lowered high-density lipoprotein cholesterol (11 studies,
3005 participants: MD -5.69 mg/dL, CI -10.35 to -1.03).Statins had uncertain
effects on kidney function: ESKD (6 studies, 2740 participants: RR 1.14, CI 0.94
to 1.37); proteinuria (2 studies, 136 participants: MD -0.04 g/24 h, CI -0.17 to
0.25); acute allograft rejection (4 studies, 582 participants: RR 0.88, CI 0.61
to 1.28); and GFR (1 study, 62 participants: MD -1.00 mL/min, CI -9.96 to
7.96).Due to heterogeneity in comparisons, data directly comparing differing
statin regimens could not be meta-analysed. Evidence for statins in people who
have had a kidney transplant were sparse and lower quality due to imprecise
effect estimates and provided limited systematic evaluation of treatment harm.
AUTHORS' CONCLUSIONS: Statins may reduce cardiovascular events in kidney
transplant recipients, although treatment effects are imprecise. Statin
treatment has uncertain effects on overall mortality, stroke, kidney function,
and toxicity outcomes in kidney transplant recipients. Additional studies would
improve our confidence in the treatment benefits and harms of statins on
cardiovascular events in this clinical setting. BACKGROUND: Patients with ductal carcinoma-in-situ (DCIS) are currently not
prescribed adjuvant systemic treatment after surgery and radiotherapy.
Prediction of DCIS patients who would benefit from radiotherapy is warranted.
Statins have been suggested to exert radio-sensitizing effects. The target for
cholesterol-lowering statins is HMG-CoA reductase (HMGCR), the rate-limiting
enzyme in the mevalonate pathway. The aim of this study was to examine HMGCR
expression in DCIS and study its treatment predictive value.
METHODS: A population-based cohort including 458 women diagnosed with primary
DCIS between 1986 and 2004 were followed until November 2011 to study long-term
survival. Tumor tissue microarrays were constructed, and immunohistochemical
analyses were performed to detect cytoplasmic protein expression of HMGCR. The
association between DCIS HMGCR expression and invasive breast cancer
recurrence-free survival (RFSinv) and overall survival (OS) was analyzed by
Kaplan-Meier curves, log rank test, and Cox proportional hazard analysis.
RESULTS: HMGCR was strongly expressed in 24 % of the assessed DCIS samples,
moderately expressed in 46 %, and weakly expressed in 23 %; no expression was
detected in 7 % of the samples. During the follow-up time (median 13.8 years),
61 patients were diagnosed with an invasive breast cancer recurrence, and 80
patients died. A crude analysis showed no survival benefit from radiotherapy.
However, patients with strong HMGCR expression showed an improved RFSinv (log
rank, p = 0.03) and OS (log rank, p = 0.04) after radiotherapy. No statistically
significant interaction was observed for HMGCR and radiotherapy (RFSinv p = 0.69
and OS p = 0.29).
CONCLUSIONS: This study demonstrates HMGCR expression in DCIS and suggests HMGCR
as a predictive marker of response to postoperative radiotherapy in DCIS,
although the test for interaction was nonsignificant. Future DCIS studies
addressing the potential of statin treatment targeting HMGCR are warranted. AIMS: Statins are the main lipid-lowering treatment in both primary and
secondary prevention populations. Whether statins deteriorates glycemic control,
predisposing to the onset of diabetes mellitus has been a matter of recent
concern. Statins may accelerate progression to diabetes via molecular mechanisms
that impact insulin sensitivity and secretion. In this review, we debate the
relative effect of statins in driving insulin resistance and the impairment of
insulin secretion.
METHODS: Narrative overview of the literature synthesizing the findings of
literature was retrieved from searches of computerized databases, hand searches,
and authoritative texts employing the key words "Statins", "Randomized Clinical
Trial", "Insulin sensitivity", "Insulin resistance", "Insulin Secretion",
"Diabetes Mellitus" alone and/or in combination.
RESULTS: The weight of clinical evidence suggests a worsening effect of statins
on insulin resistance and secretion, anyway basic science studies did not find a
clear molecular explanation, providing conflicting evidence regarding both the
beneficial and the adverse effects of statin therapy on insulin sensitivity.
CONCLUSIONS: Although most of the clinical studies suggest a worsening of
insulin resistance and secretion, the cardiovascular benefits of statin therapy
outweigh the risk of developing insulin resistance, thus the data suggest the
need to treat dyslipidemia and to make patients aware of the possible risk of
developing type 2 diabetes or, if they already are diabetic, of worsening their
metabolic control. Statins, which specifically inhibit HMG Co-A reductase, the rate-limiting step
of cholesterol biosynthesis, are widely prescribed to reduce serum cholesterol
and cardiac risk, but many other effects are seen. We now show an effect of
these drugs to induce profound changes in the step-wise synthesis of
glycosphingolipids (GSLs) in the Golgi. Glucosylceramide (GlcCer) was increased
several-fold in all cell lines tested, demonstrating a widespread effect.
Additionally, de novo or elevated lactotriaosylceramide (Lc3Cer;
GlcNAcβ1-3Galβ1-4GlcCer) synthesis was observed in 70%. Western blot showed that
GlcCer synthase (GCS) was elevated by statins, and GCS and Lc3Cer synthase
(Lc3S) activities were increased; however, transcript was elevated for Lc3S
only. Supplementation with the isoprenoid precursor, geranylgeranyl
pyrophosphate (GGPP), a downstream product of HMG Co-A reductase, reversed
statin-induced glycosyltransferase and GSL elevation. The Rab geranylgeranyl
transferase inhibitor 3-PEHPC, but not specific inhibitors of farnesyl
transferase, or geranylgeranyl transferase I, was sufficient to replicate
statin-induced GlcCer and Lc3Cer synthesis, supporting a Rab
prenylation-dependent mechanism. While total cholesterol was unaffected, the
trans-Golgi network (TGN) cholesterol pool was dissipated and medial Golgi GCS
partially relocated by statins. GSL-dependent vesicular retrograde transport of
Verotoxin and cholera toxin to the Golgi/endoplasmic reticulum were blocked
after statin or 3-PEHPC treatment, suggesting aberrant, prenylation-dependent
vesicular traffic as a basis of glycosyltransferase increase and GSL remodeling.
These in vitro studies indicate a previously unreported link between Rab
prenylation and regulation of GCS activity and GlcCer metabolism. The mevalonate pathway synthesizes intermediates and products such as
cholesterol and nonsterol isoprenoids that are crucial for cell survival and
function. In the human placenta, the prenylation of proteins, rather than
cholesterol synthesis, represents the main "metabolic target" of mevalonate
metabolism. Major cellular functions depend on isoprenylation including
proliferation, migration, metabolism and protein glycosylation that are all
crucial for proper development of the embryo and the placenta. Statins are
inhibitors of HMG-CoA reductase, the enzyme that catalyzes the reduction of
HMG-CoA to mevalonic acid by NADPH. In vitro experiments using human placental
explants suggest that statins elicit a detrimental effect on placental growth.
However, animal and epidemiologic studies show no increase of fetal
malformations after exposure to statins during pregcy. Moreover, emerging
evidence from mouse studies suggest that statins may be useful in preventing
serious pregcy complications like preeclampsia. IMPORTANCE: High-intensity statin therapy is recommended for the secondary
prevention of atherosclerotic cardiovascular disease (ASCVD). Nevertheless,
statin therapy in general, and high-intensity statin therapy in particular, is
underused in patients with established ASCVD.
OBJECTIVE: To determine the association between all-cause mortality and
intensity of statin therapy in the Veterans Affairs health care system.
DESIGN, SETTING, AND PARTICIPANTS: A retrospective cohort analysis was conducted
of patients aged 21 to 84 years with ASCVD treated in the Veterans Affairs
health care system from April 1, 2013, to April 1, 2014. Patients who were
included had 1 or more International Classification of Diseases, Ninth Revision
codes for ASCVD on 2 or more different dates in the prior 2 years.
EXPOSURES: Intensity of statin therapy was defined by the 2013 American College
of Cardiology/American Heart Association guidelines, and use was defined as a
filled prescription in the prior 6 months. Patients were excluded if they were
taking a higher statin dose in the prior 5 years.
MAIN OUTCOMES AND MEASURES: The primary outcome was death from all causes
adjusted for the propensity to receive high-intensity statins.
RESULTS: The study sample included 509 766 eligible adults with ASCVD at
baseline (mean [SD] age, 68.5 [8.8] years; 499 598 men and 10 168 women),
including 150 928 (29.6%) receiving high-intensity statin therapy, 232 293
(45.6%) receiving moderate-intensity statin therapy, 33 920 (6.7%) receiving
low-intensity statin therapy, and 92 625 (18.2%) receiving no statins. During a
mean follow-up of 492 days, there was a graded association between intensity of
statin therapy and mortality, with 1-year mortality rates of 4.0% (5103 of
126 139) for those receiving high-intensity statin therapy, 4.8% (9703 of
200 709) for those receiving moderate-intensity statin therapy, 5.7% (1632 of
28 765) for those receiving low-intensity statin therapy, and 6.6% (4868 of
73 728) for those receiving no statin (P < .001). After adjusting for the
propensity to receive high-intensity statins, the hazard ratio for mortality was
0.91 (95% CI, 0.88-0.93) for those receiving high- vs moderate-intensity
statins. The magnitude of benefit of high- vs moderate-intensity statins was
similar, for an incident cohort hazard ratio of 0.93 (95% CI, 0.85-1.01). For
patients aged 76 to 84 years, the hazard ratio was 0.91 (95% CI, 0.87-0.95).
Patients treated with maximal doses of high-intensity statins had lower
mortality (hazard ratio, 0.90; 95% CI, 0.87-0.94) compared with those receiving
submaximal doses.
CONCLUSIONS AND RELEVANCE: We found a graded association between intensity of
statin therapy and mortality in a national sample of patients with ASCVD.
High-intensity statins were associated with a small but significant survival
advantage compared with moderate-intensity statins, even among older adults.
Maximal doses of high-intensity statins were associated with a further survival
benefit. |
Which cells secretes alpha defensin 5? | Human enteric α-defensins (HD5 and HD6), major antimicrobial peptides produced by Paneth cells in the intestine, play important roles in intestinal innate immunity. | Human enteric α-defensins (HD5 and HD6), major antimicrobial peptides produced
by Paneth cells in the intestine, play important roles in intestinal innate
immunity. Since their expression is decreased in Crohn's disease (CD), with
decreased expression being more pronounced in the presence of NOD2 mutations, it
would be extremely interesting to investigate the mechanism by which NOD2 may
regulate expression of human enteric α-defensins. Here we show that although
NOD2 by itself can slightly up-regulate expression of enteric α-defensins mainly
through activation of the NF-κB pathway, it can strongly down-regulates their
expression during differentiation of the Paneth cell lineage mainly by
inhibiting activation of the MAPK pathway. Since NOD2 is over-expressed in CD
and mutant NOD2 cannot result in NF-κB activity, our finding can provide an
explanation of the previous observation showing decreased expression of human
enteric α-defensin in CD and even more so in the presence of NOD2 mutations. In
addition, this finding provides a new view on the function of NOD2 in regulating
intestinal innate immunity. Author information:
(1)Centre for Neonatal Research and Education, University of Western Australia,
Perth, Western Australia, Australia; School of Paediatrics and Child Health,
University of Western Australia, Perth, Western Australia, Australia.
(2)Centre for Neonatal Research and Education, University of Western Australia,
Perth, Western Australia, Australia; School of Paediatrics and Child Health,
University of Western Australia, Perth, Western Australia, Australia; Neonatal
Clinical Care Unit, King Edward Memorial Hospital for Women, Perth, Western
Australia, Australia.
(3)School of Paediatrics and Child Health, University of Western Australia,
Perth, Western Australia, Australia.
(4)Centre for Neonatal Research and Education, University of Western Australia,
Perth, Western Australia, Australia; Neonatal Clinical Care Unit, King Edward
Memorial Hospital for Women, Perth, Western Australia, Australia.
(5)School of Public Health, Curtin University, Perth, Australia.
(6)School of Women's and Infants' Health, University of Western Australia,
Perth, Australia.
(7)Murdoch Childrens Research Institute, Parkville, Victoria, Australia;
University of Melbourne, Melbourne, Victoria, Australia.
(8)The University of Edinburgh/MRC Centre for Inflammation Research, Queen's
Medical Research Institute, Edinburgh, United Kingdom.
(9)Centre for Neonatal Research and Education, University of Western Australia,
Perth, Western Australia, Australia; School of Paediatrics and Child Health,
University of Western Australia, Perth, Western Australia, Australia; School of
Veterinary and Life Sciences, Murdoch University, Perth, Western Australia,
Australia. Human alpha defensins are a class of antimicrobial peptides with additional
antiviral activity. Such antimicrobial peptides constitute a major part of
mammalian innate immunity. Alpha defensins contain six cysteines, which form
three well defined disulfide bridges under oxidizing conditions. Residues
C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the
peptide. The major tissue in which HD5 is expressed is the crypt of the small
intestine, an anaerobic niche that should allow for substantial pools of both
oxidized and (partly) reduced HD5. We used ion mobility coupled to mass
spectrometry to track the structural changes in HD5 upon disulfide bond
reduction. We found evidence of stepwise unfolding of HD5 with sequential
reduction of the three disulfide bonds. Alkylation of free cysteines followed by
tandem mass spectrometry of the corresponding partially reduced states revealed
a domit pathway of reductive unfolding. The majority of HD5 unfolds by
initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional
evidence for a minor pathway that starts with reduction of C3-C31, followed by
C5-C20 and C10-C30. Our results provide insight into the pathway and
conformational landscape of disulfide bond reduction in HD5. |
Do statins cause diabetes? | The relationship between T2DM and statins is further complicated since these drugs can cause new onset diabetes (NOD) although there is an overall benefit in terms of preventing vascular events. | BACKGROUND: The survival benefit of statins in nontrial populations of persons
with diabetes is unknown.
OBJECTIVE: : We sought to determine all-cause mortality in fiscal year 2001
(FY01) after statin initiation in FY 99 and/or FY00 in individuals with diabetes
in the Veterans Healthcare Administration (VHA).
METHODS: Using a retrospective longitudinal cohort analysis, we analyzed 201,102
veterans with diabetes from 104 VHA facilities with medical, pharmacy, and
laboratory information from VHA and Medicare databases. Patients with statin
exposure, defined as having medication possession coverage >50% of eligible days
in FY99 and/or FY00, were characterized as initiators if no statin prescription
was found in FY98. Otherwise, they were characterized as continuing users. We
defined 4 statin exposure groups: FY99 only, FY00 only, both FY99 and 00, and
neither year. All-cause mortality was determined in FY01. Propensity score
matched comparisons were used to corroborate results from mixed effects logistic
models.
RESULTS: FY01 mortality was no different between FY99-only initiators and the
nonexposure group (odds ratio [OR] = 1.08, P = 0.429). In contrast, FY00-only
and FY99-00 initiator groups showed odds ratio of 0.75 (P < 0.0001) and 0.71 (P
< 0.0001), respectively. There was a similar benefit for continuing users.
Propensity analysis demonstrated consistent findings. Increased statin adherence
from >50% to >75% was associated with increased benefit (OR = 0.71, P < 0.0001
versus OR = 0.62, P = 0.0002).
CONCLUSIONS: One to 2-year statin exposure is associated with a 25% to 29% risk
reduction in all-cause mortality of the subsequent year in a high-risk diabetes
cohort. AIMS: (i) To examine the incidence of new onset treated diabetes in patients
treated with different types of statins and (ii) the relationship between the
duration and dose of statins and the subsequent development of new onset treated
diabetes.
METHODS: A retrospective cohort study was performed using the Irish Health
Services Executive Primary Care Reimbursement Services national pharmacy claims
database. Individuals who received any medicines were identified from January
2001 to January 2009 (n = 1 235 671). Patients newly treated with statins from 1
January 2002 to 31 December 2007 were identified (n = 239 628). Cases were
identified as individuals newly treated with antidiabetic medication (n =
38 503). Adjusted hazards ratios (HR) with 95% confidence intervals (CI) were
calculated to examine the association between statins (any vs. none) and time to
new onset treated diabetes using Cox proportional hazard regression. The dose
and duration response relationship between statins and new onset treated
diabetes was examined using restricted spline functions to assess the linearity
of the relationship.
RESULTS: Statin use was associated with an increased risk of new onset treated
diabetes (HR = 1.18, 95% CI 1.15, 1.22). Increased risk of new onset treated
diabetes was found with rosuvastatin (HR = 1.41, 95% CI 1.31, 1.52),
atorvastatin (HR = 1.23, 95% CI 1.19, 1.27) and simvastatin (HR = 1.15, 95% CI
1.05, 1.25). There were statistically significant overall dose and duration
effects for all statins, excepting fluvastatin, which only demonstrated a
duration effect.
CONCLUSION: An increased risk of new onset treated diabetes was found in those
treated with statins showing significant duration and dose effect. Further study
is required to confirm this association. BACKGROUND: Statins significantly reduce cardiovascular events in a broad
population of patients with hyperlipidemia. However, a small, but significant
risk of new-onset diabetes has been reported in patients treated with statins.
The mechanism by which statins cause diabetes has not been elucidated and
therefore preventive strategies have yet to be defined.
METHOD: Our goal was to study the differing effects of a lipophilic
(simvastatin) statin, hydrophilic (pravastatin) statin, and ezetimibe on glucose
transporter-4 (GLUT4) protein expression in 3T3-L1 adipocytes. We hypothesized
that the reductions in GLUT4 protein secondary to statin treatment would be
prevented when cells were co-incubated with coenzyme Q10 (CoQ10). GLUT4 protein
expression was determined using the In-Cell Western technique. Confluent
adipocytes were differentiated using a hormonal cocktail for 3 days; followed by
treatment with simvastatin, pravastatin, ezetimibe and CoQ10. Cell morphology
was observed after treatment using phase-contrast microscopy.
RESULTS: Treatment with simvastatin (P<0.001) and simvastatin plus ezetimibe
(P<0.001) significantly decreased GLUT4 protein expression in the adipocytes
compared to control conditions. GLUT4 protein levels were similar to control
after treatment with ezetimibe alone (P=0.52) or pravastatin (P=0.32). There was
no significant difference (P=0.098) in GLUT4 protein levels after co-treatment
with CoQ10 between any of the treatments and control conditions.
CONCLUSION: Our studies have shown that lipophilic statins (simvastatin) reduce
the GLUT4 protein levels in adipocytes, whereas hydrophilic statins
(pravastatin) or ezetimibe do not. Co-treatment with CoQ10 appears to prevent
the reduction in GLUT4 protein levels caused by simvastatin. Statins are widely prescribed cholesterol-lowering agents, which have been
demonstrated to significantly reduce cardiovascular morbidity and mortality.
However, recent trials have reported that statins cause worsening of
hyperglycemia and increase the risk of new-onset diabetes. The association
between the diabetogenic effect of statins with intensive dose and accompanying
major risk factors for diabetes has been demonstrated. However, statins do not
appear to have a class effect on insulin sensitivity in non-diabetic patients.
Numerous mechanisms have been suggested to explain how statins cause β-cell
insulin secretory dysfunction and peripheral insulin resistance leading to
incident diabetes. According to findings from an aggregate of large clinical
trials, the benefits of statin treatment appear to outweigh the risk of
new-onset diabetes. Therefore, it would be inappropriate to discontinue the use
of statins for prevention of cardiovascular events because of its potential risk
for development of incident diabetes. This review addresses the currently
available evidence related to statin use and new-onset diabetes from a clinical
perspective. AIMS: Statins are the main lipid-lowering treatment in both primary and
secondary prevention populations. Whether statins deteriorates glycemic control,
predisposing to the onset of diabetes mellitus has been a matter of recent
concern. Statins may accelerate progression to diabetes via molecular mechanisms
that impact insulin sensitivity and secretion. In this review, we debate the
relative effect of statins in driving insulin resistance and the impairment of
insulin secretion.
METHODS: Narrative overview of the literature synthesizing the findings of
literature was retrieved from searches of computerized databases, hand searches,
and authoritative texts employing the key words "Statins", "Randomized Clinical
Trial", "Insulin sensitivity", "Insulin resistance", "Insulin Secretion",
"Diabetes Mellitus" alone and/or in combination.
RESULTS: The weight of clinical evidence suggests a worsening effect of statins
on insulin resistance and secretion, anyway basic science studies did not find a
clear molecular explanation, providing conflicting evidence regarding both the
beneficial and the adverse effects of statin therapy on insulin sensitivity.
CONCLUSIONS: Although most of the clinical studies suggest a worsening of
insulin resistance and secretion, the cardiovascular benefits of statin therapy
outweigh the risk of developing insulin resistance, thus the data suggest the
need to treat dyslipidemia and to make patients aware of the possible risk of
developing type 2 diabetes or, if they already are diabetic, of worsening their
metabolic control. AIMS/HYPOTHESES: Current evidence indicates that statins increase the risk of
incident diabetes; however, the relationship between statins and glycaemic
control in people with established diabetes has not been well characterised. To
address this question, we conducted a meta-analysis of randomised controlled
trials (RCTs) of statins in patients with diabetes for whom there was available
data on glycaemic control.
METHODS: We identified studies published between January 1970 and November 2013
by searching electronic databases and reference lists. We included RCTs in which
the intervention group received statins and the control group received placebo
or standard treatment, with >200 participants enrolled, with the intervention
lasting >12 weeks and with pre- and post-intervention HbA1c reported. We
combined study-specific estimates using random-effects model meta-analysis.
RESULTS: In a pooled analysis of nine trials involving 9,696 participants (4,980
statin, 4,716 control) and an average follow-up of 3.6 years, the mean HbA1c of
participants randomised to statins was higher than those randomised to the
control group: mean difference (95% CI) was 0.12% (0.04, 0.20) or 1.3 mmol/mol
(0.4, 2.2); p = 0.003. There was moderate heterogeneity across the studies (I
(2) = 54%, p = 0.014) not explained by available study-level characteristics.
This review was limited by the small number of studies, available data on only
three statins and sparse reporting on changes in use of glucose-lowering
medications.
CONCLUSIONS/INTERPRETATION: Statin treatment is associated with a modest
increase in HbA1c in patients with diabetes. OBJECTIVE: 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins)
are used to control blood cholesterol levels and reduce cardiovascular disease.
It has been repeatedly reported that statins may cause new-onset diabetes
mellitus (DM). However, limited evidence exists from direct head to head
comparisons of statins on whether the risk of DM differs among statins. We
investigated the risk of development of new-onset diabetes in subjects treated
with different statins.
METHODS: We retrospectively enrolled consecutive 3680 patients without DM or
impaired fasting glucose who started receiving statin treatment for cholesterol
control. We evaluated the incidence of new-onset diabetes according to the type
of statin.
RESULTS: The mean duration of follow-up was 62.6±15.3 months. The incidence of
DM was significantly higher in the pitavastatin group (49 of 628; 7.8%) compared
to that in the other statin groups [atorvastatin (68 of 1327; 5.1%),
rosuvastatin (77 of 1191; 6.5%), simvastatin (11 of 326; 3.4%), and pravastatin
(12 of 298; 5.8%); p=0.041]. The risk of diabetes was the highest in the
pitavastatin group compared with that in the simvastatin group [hazard ratio
(HR)=2.68, p=0.011]. Other statins showed no significant risk differences
compared to that for simvastatin. Fasting blood glucose (FBG) level at baseline
and body-mass index (BMI) were associated with the development of diabetes [FBG,
HR=1.11, p<0.001; BMI, HR=1.02, p=0.005].
CONCLUSIONS: Among the five statins, pitavastatin showed the strongest effect on
the development of new-onset diabetes. In a review [1] published in this journal in 2014 we updated the role of statin
treatment in patients with type 2 diabetes mellitus (T2DM). This is an important
topic because the prevalence of T2DM is increasing and this disease is
associated with a high risk of cardiovascular disease (CVD) as well as
microvascular complications [1]. The relationship between T2DM and statins is
further complicated since these drugs can cause new onset diabetes (NOD)
although there is an overall benefit in terms of preventing vascular events [1,
2]. The cost implications of T2DM in terms of quality of life as well as
providing healthcare are obvious. This is a brief update of our earlier review
[1] based on recently published data. |
What is the effect of nocodazole cell treatment? | Nocodazole trigger mitotic arrest. | Zinc finger proteins are a massive, diverse family of proteins that serve a wide
variety of biological functions. However, the roles of them during meiosis are
not yet clearly defined. Here, we report that Zfp207 localizes at the
kinetochores during mouse oocyte meiotic maturation. Depletion of Zfp207 leads
to a significantly higher proportion of impaired spindle organization and
misaligned chromosomes in oocytes. This is coupled with the defective
kinetochore-microtubule attachments, and resultantly increasing incidence of
aneuploid metaphase II eggs. The precocious polar body extrusion and escape of
metaphase I arrest induced by nocodazole treatment in Zfp207-depleted oocytes
indicates that Zfp207 is essential for activation of SAC (Spindle Assembly
Checkpoint) activity. Notably, we find that Zfp207 binds to Bub3 to form a
complex and maintains its protein level in oocytes, and that overexpression of
Bub3 is able to partially rescue the occurrence of aneuploid eggs in
Zfp207-depleted oocytes. Collectively, we identify Zfp207 as a novel Bub3
binding protein in oocytes which plays an important role in controlling meiotic
chromosome alignment and SAC function. During mitosis, Promyelocytic leukemia nuclear bodies (PML NBs) change
dramatically in morphology and composition, but little is known about function
of PML in mitosis. Here, we show that PML is phosphorylated at T409 (PML p409)
in a mitosis-specific manner. More importantly, PML p409 contributes to maintain
the duration of pro-metaphase and regulates spindle checkpoint. Deficient PML
p409 caused a shortening of pro-metaphase and challenged the
nocodazole-triggered mitotic arrest. T409A mutation led to a higher frequency of
misaligned chromosomes on metaphase plate, and subsequently death in late
mitosis. In addition, inhibition of PML p409 repressed growth of tumor cells,
suggesting that PML p409 is a potential target for cancer therapy. Collectively,
our study demonstrated an important phosphorylated site of PML, which
contributed to explore the role of PML in mitosis. Cell cycle checkpoints are surveillance mechanisms that sequentially and
continuously monitor cell cycle progression thereby contributing to the
preservation of genetic stability. Among them, the spindle assembly checkpoint
(SAC) prevents the occurrence of abnormal divisions by halting the metaphase to
anaphase transition following the detection of erroneous
microtubules-kinetochore attachment(s). Most synchronization strategies are
based on the activation of cell cycle checkpoints to enrich the population of
cells in a specific phase of the cell cycle. Here, we develop a two-step
protocol of sequential cell synchronization and desynchronization employing
antimitotic SAC-inducing agents (i.e., nocodazole or paclitaxel) in combination
with the depletion of the SAC kinase MPS1. We describe cytofluorometric and
time-lapse videomicroscopy methods to detect cell cycle progression, including
the assessment of cell cycle distribution, quantification of mitotic cell
fraction, and analysis of single cell fate profile of living cells. We applied
these methods to validate the synchronization-desynchronization protocol and to
qualitatively and quantitatively determine the impact of SAC inactivation on the
activity of antimitotic agents. HeLa is one of the oldest and most commonly used cell lines in biomedical
research. Owing to the ease of which they can be effectively synchronized by
various methods, HeLa cells have been used extensively for studying the cell
cycle. Here, we describe several protocols for synchronizing HeLa cells from
different phases of the cell cycle, including G1 phase using the HMG-CoA
reductase inhibitor lovastatin, S phase with a double thymidine block procedure,
and G2 phase with the CDK1 inhibitor RO-3306. Cells can also be enriched in
mitosis using nocodazole and mechanical shake-off. Releasing the cells from
these blocks enables researchers to follow gene expression and other events
through the cell cycle. We also describe several protocols, including flow
cytometry, BrdU labeling, immunoblotting, and time-lapse microscopy, for
validating the synchrony of the cells and monitoring the progression of the cell
cycle. The initiation of T-cell signaling is critically dependent on the function of
the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor
(TCR) triggering, Lck kinase activity induces the nucleation of
signal-transducing hubs that regulate the formation of complex signaling network
and cytoskeletal rearrangement. In addition, the delivery of Lck function
requires rapid and targeted membrane redistribution, but the mechanism
underpinning this process is largely unknown. To gain insight into this process,
we considered previously described proteins that could assist in this process
via their capacity to interact with kinases and regulate their intracellular
translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1),
was chosen as a viable option, and its capacity to bind Lck and aid the process
of activation-induced redistribution of Lck was assessed. Our microscopic
observation showed that T-cell activation induces a rapid, concomitant, and
transient co-redistribution of Lck and RACK1 into the forming immunological
synapse. Consistent with this observation, the formation of transient RACK1-Lck
complexes were detectable in primary CD4+ T-cells with their maximum levels
peaking 10 s after TCR-CD4 co-aggregation. Moreover, RACK1 preferentially binds
to a pool of kinase active pY394Lck, which co-purifies with high molecular
weight cellular fractions. The formation of RACK1-Lck complexes depends on
functional SH2 and SH3 domains of Lck and includes several other signaling and
cytoskeletal elements that transiently bind the complex. Notably, the
F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence
of kinase active Lck suggesting that the formation of RACK1-pY394Lck-α-actinin-1
complex serves as a signal module coupling actin cytoskeleton bundling with
productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with
nocodazole, which disrupts the microtubular network, also blocked the formation
of RACK1-Lck complexes. Importantly, activation-induced Lck redistribution was
diminished in primary CD4+ T-cells by an adenoviral-mediated knockdown of RACK1.
These results demonstrate that in T cells, RACK1, as an essential component of
the multiprotein complex which upon TCR engagement, links the binding of kinase
active Lck to elements of the cytoskeletal network and affects the subcellular
redistribution of Lck. |
What is Path2PPI? | Path2PPI is an R package to identify protein-protein interaction (PPI) networks for fully sequenced organisms for which nearly none PPI are known. Path2PPI predicts PPI networks based on sets of proteins from well-established model organisms, providing an intuitive visualization and usability. It can be used to combine and transfer information of a certain pathway or biological process from several reference organisms to one target organism. | |
SPAG5 was implicated in which cancers? | SPAG5 was implicated in prostate cancer, lung cancer and cervical cancer. | OBJECTIVES: Despite the well-defined histological types of non-small cell lung
cancer (NSCLC), a given stage is often associated with wide-ranging survival
rates and treatment outcomes. This disparity has led to an increased demand for
the discovery and identification of new informative biomarkers.
METHODS: In the current study, we screened 81 NSCLC samples using Illumina
whole-genome gene expression microarrays in an effort to identify differentially
expressed genes and new NSCLC biomarkers.
RESULTS: We identified novel genes whose expression was upregulated in NSCLC,
including SPAG5, POLH, KIF23, and RAD54L, which are associated with mitotic
spindle formation, DNA repair, chromosome segregation, and dsDNA break repair,
respectively. We also identified several novel genes whose expression was
downregulated in NSCLC, including SGCG, NLRC4, MMRN1, and SFTPD, which are
involved in extracellular matrix formation, apoptosis, blood vessel leakage, and
inflammation, respectively. We found a significant correlation between RNA
degradation and survival in adenocarcinoma cases.
CONCLUSIONS: Even though the follow-up time was too limited to draw final
conclusions, we were able to show better prediction p values in a group
selection based on molecular profiles compared to histology. The current study
also uncovered new candidate biomarker genes that are likely to be involved in
diverse processes associated with NSCLC development. The rapidly growing collection of diverse genome-scale data from multiple tumor
types sheds light on various aspects of the underlying tumor biology. With the
objective to identify genes of importance for breast tumorigenesis in men and to
enable comparisons with genes important for breast cancer development in women,
we applied the computational framework COpy Number and EXpression In Cancer
(CONEXIC) to detect candidate driver genes among all altered passenger genes.
Unique to this approach is that each driver gene is associated with several gene
modules that are believed to be altered by the driver. Thirty candidate drivers
were found in the male breast cancers and 67 in the female breast cancers. We
identified many known drivers of breast cancer and other types of cancer, in the
female dataset (e.g. GATA3, CCNE1, GRB7, CDK4). In contrast, only three known
cancer genes were found among male breast cancers; MAP2K4, LHP, and ZNF217. Many
of the candidate drivers identified are known to be involved in processes
associated with tumorigenesis, including proliferation, invasion and
differentiation. One of the modules identified in male breast cancer was
regulated by THY1, a gene involved in invasion and related to
epithelial-mesenchymal transition. Furthermore, men with THY1 positive breast
cancers had significantly inferior survival. THY1 may thus be a promising novel
prognostic marker for male breast cancer. Another module identified among male
breast cancers, regulated by SPAG5, was closely associated with proliferation.
Our data indicate that male and female breast cancers display highly different
landscapes of candidate driver genes, as only a few genes were found in common
between the two. Consequently, the pathobiology of male breast cancer may differ
from that of female breast cancer and can be associated with differences in
prognosis; men diagnosed with breast cancer may consequently require different
management and treatment strategies than women. Previously, we found that sperm-associated antigen 5 (SPAG5) was upregulated in
pelvic lymph node metastasis-positive cervical cancer. The aim of this study is
to examine the role of SPAG5 in the proliferation and tumorigenicity of cervical
cancer and its clinical significance in tumor progression. In our study, SPAG5
expression in cervical cancer patients was detected using quantitative real-time
polymerase chain reaction, western blotting, and immunohistochemistry; cervical
cancer cell function with downregulated SPAG5 in vitro was explored using
tetrazolium assay, flow cytometry, and colony formation and Transwell assays.
SPAG5 was upregulated in tumor tissue compared with paired adjacent noncancerous
tissues; SPAG5 upregulation in tumor tissues indicated poor disease-free
survival, which was also an independent prognostic indicator for cervical cancer
patients. In vitro study demonstrated that SPAG5 downregulation inhibited cell
proliferation and growth significantly by G2/M arrest and induction of
apoptosis, and hindered cell migration and invasion. Under SPAG5 downregulation,
the sensitivity of cervical cancer cells differed according to taxol dose, which
correlated with mammalian target of rapamycin (mTOR) signaling pathway activity.
In general, SPAG5 upregulation relates to poor prognosis in cervical cancer
patients, and SPAG5 is a regulator of mTOR activity during taxol treatment in
cervical cancer. BACKGROUND: We conducted multiple microarray datasets analyses from clinical and
xenograft tumor tissues to search for disease progression-driving oncogenes in
prostate cancer (PCa). Sperm-associated antigen 5 (SPAG5) attracted our
attention. SPAG5 was recently identified as an oncogene participating in lung
cancer and cervical cancer progression. However, the roles of SPAG5 in PCa
progression remain unknown.
METHODS: SPAG5 expression level in clinical primary PCa, metastatic PCa,
castration resistant PCa, neuroendocrine PCa, and normal prostate tissues was
investigated. We established multiple in vivo xenografts models using
patient-derived tissues and investigated SPAG5 expression trend in these models.
We also investigated the functions of SPAG5 in vivo and in vitro studies.
Luciferase reporter assays were performed to investigate potential miRNAs that
can regulate SPAG5.
RESULTS: We identified that SPAG5 expression was gradually increased in PCa
progression and its level was significantly associated with lymph node
metastasis, clinical stage, Gleason score, and biochemical recurrence. Our
results indicated that SPAG5 knockdown can drastically inhibit PCa cell
proliferation, migration, and invasion in vitro and supress tumor growth and
metastasis in vivo. We identified that miR-539 can directly target SPAG5.
Ectopic overexpression of miR-539 can drastically inhibit SPAG5 expression and
the restoration of SPAG5 expression can reverse the inhibitory effects of
miR-539 on PCa cell proliferation and metastasis.
CONCLUSION: Our results collectively showed a progression-driving role of SPAG5
in PCa which can be regulated by miR-539, suggesting that miR-539/SPAG5 can
serve as a potential therapeutic target for PCa. BACKGROUND: Proliferation markers and profiles have been recommended for guiding
the choice of systemic treatments for breast cancer. However, the best molecular
marker or test to use has not yet been identified. We did this study to identify
factors that drive proliferation and its associated features in breast cancer
and assess their association with clinical outcomes and response to
chemotherapy.
METHODS: We applied an artificial neural network-based integrative data mining
approach to data from three cohorts of patients with breast cancer (the
Nottingham discovery cohort (n=171), Uppsala cohort (n=249), and Molecular
Taxonomy of Breast Cancer International Consortium [METABRIC] cohort; n=1980).
We then identified the genes with the most effect on other genes in the
resulting interactome map. Sperm-associated antigen 5 (SPAG5) featured
prominently in our interactome map of proliferation and we chose to take it
forward in our analysis on the basis of its fundamental role in the function and
dynamic regulation of mitotic spindles, mitotic progression, and chromosome
segregation fidelity. We investigated the clinicopathological relevance of SPAG5
gene copy number aberrations, mRNA transcript expression, and protein expression
and analysed the associations of SPAG5 copy number aberrations, transcript
expression, and protein expression with breast cancer-specific survival,
disease-free survival, distant relapse-free survival, pathological complete
response, and residual cancer burden in the Nottingham discovery cohort, Uppsala
cohort, METABRIC cohort, a pooled untreated lymph node-negative cohort (n=684),
a multicentre combined cohort (n=5439), the Nottingham historical early stage
breast cancer cohort (Nottingham-HES; n=1650), Nottingham early stage oestrogen
receptor-negative breast cancer adjuvant chemotherapy cohort
(Nottingham-oestrogen receptor-negative-ACT; n=697), the Nottingham
anthracycline neoadjuvant chemotherapy cohort (Nottingham-NeoACT; n=200), the MD
Anderson taxane plus anthracycline-based neoadjuvant chemotherapy cohort (MD
Anderson-NeoACT; n=508), and the multicentre phase 2 neoadjuvant clinical trial
cohort (phase 2 NeoACT; NCT00455533; n=253).
FINDINGS: In the METABRIC cohort, we detected SPAG5 gene gain or amplification
at the Ch17q11.2 locus in 206 (10%) of 1980 patients overall, 46 (19%) of 237
patients with a PAM50-HER2 phenotype, and 87 (18%) of 488 patients with
PAM50-LumB phenotype. Copy number aberration leading to SPAG5 gain or
amplification and high SPAG5 transcript and SPAG5 protein concentrations were
associated with shorter overall breast cancer-specific survival (METABRIC cohort
[copy number aberration]: hazard ratio [HR] 1·50, 95% CI 1·18-1·92, p=0·00010;
METABRIC cohort [transcript]: 1·68, 1·40-2·01, p<0·0001; and
Nottingham-HES-breast cancer cohort [protein]: 1·68, 1·32-2·12, p<0·0001). In
multivariable analysis, high SPAG5 transcript and SPAG5 protein expression were
associated with reduced breast cancer-specific survival at 10 years compared
with lower concentrations (Uppsala: HR 1·62, 95% CI 1·03-2·53, p=0·036;
METABRIC: 1·27, 1·02-1·58, p=0·034; untreated lymph node-negative cohort: 2·34,
1·24-4·42, p=0·0090; and Nottingham-HES: 1·73, 1·23-2·46, p=0·0020). In patients
with oestrogen receptor-negative breast cancer with high SPAG5 protein
expression, anthracycline-based adjuvant chemotherapy increased breast
cancer-specific survival overall compared with that for patients who did not
receive chemotherapy (Nottingham-oestrogen receptor-negative-ACT cohort: HR
0·37, 95% CI 0·20-0·60, p=0·0010). Multivariable analysis showed high SPAG5
transcript concentrations to be independently associated with longer distant
relapse-free survival after receiving taxane plus anthracycline neoadjuvant
chemotherapy (MD Anderson-NeoACT: HR 0·68, 95% CI 0·48-0·97, p=0·031). In
multivariable analysis, both high SPAG5 transcript and high SPAG5 protein
concentrations were independent predictors for a higher proportion of patients
achieving a pathological complete response after combination cytotoxic
chemotherapy (MD Anderson-NeoACT: OR 1·71, 95% CI, 1·07-2·74, p=0·024;
Nottingham-ACT: 8·75, 2·42-31·62, p=0·0010).
INTERPRETATION: SPAG5 is a novel amplified gene on Ch17q11.2 in breast cancer.
The transcript and protein products of SPAG5 are independent prognostic and
predictive biomarkers that might have clinical utility as biomarkers for
combination cytotoxic chemotherapy sensitivity, especially in oestrogen
receptor-negative breast cancer.
FUNDING: Nottingham Hospitals Charity and the John and Lucille van Geest
Foundation. |
What are some of the effects of Zika Virus in infected individuals? | While most of the symptoms of zika virus are relatively mild, zika virus in pregnant mothers can cause microcephaly and other congenital defects in the fetus. | Conflict of interest statement: Disclosure: Jason C. Kwong has received
ficial assistance from Pfizer to attend an international conference. Karin
Leder has received funding from GlaxoSmithKline for a study on hepatitis B, and
ficial assistance from GlaxoSmithKline and Sanofi Pasteur to attend
international conferences. Zika virus is a flavivirus transmitted by Aedes (Stegomyia) species of
mosquitoes. In May 2015, the World Health Organization confirmed the first local
transmission of Zika virus in the Americas in Brazil. The virus has spread
rapidly to other countries in the Americas; as of January 29, 2016, local
transmission has been detected in at least 22 countries or territories,
including the Commonwealth of Puerto Rico and the U.S. Virgin Islands. Zika
virus can infect pregt women in all three trimesters. Although pregt women
do not appear to be more susceptible to or more severely affected by Zika virus
infection, maternal-fetal transmission has been documented. Several pieces of
evidence suggest that maternal Zika virus infection is associated with adverse
neonatal outcomes, most notably microcephaly. Because of the number of countries
and territories with local Zika virus transmission, it is likely that obstetric
health care providers will care for pregt women who live in or have traveled
to an area of local Zika virus transmission. We review information on Zika
virus, its clinical presentation, modes of transmission, laboratory testing,
effects during pregcy, and methods of prevention to assist obstetric health
care providers in caring for pregt women considering travel or with a history
of travel to areas with ongoing Zika virus transmission and pregt women
residing in areas with ongoing Zika virus transmission. BACKGROUND: The incidence of microcephaly in Brazil in 2015 was 20 times higher
than in previous years. Congenital microcephaly is associated with genetic
factors and several causative agents. Epidemiological data suggest that
microcephaly cases in Brazil might be associated with the introduction of Zika
virus. We aimed to detect and sequence the Zika virus genome in amniotic fluid
samples of two pregt women in Brazil whose fetuses were diagnosed with
microcephaly.
METHODS: In this case study, amniotic fluid samples from two pregt women from
the state of Paraíba in Brazil whose fetuses had been diagnosed with
microcephaly were obtained, on the recommendation of the Brazilian health
authorities, by ultrasound-guided transabdominal amniocentesis at 28 weeks'
gestation. The women had presented at 18 weeks' and 10 weeks' gestation,
respectively, with clinical manifestations that could have been symptoms of Zika
virus infection, including fever, myalgia, and rash. After the amniotic fluid
samples were centrifuged, DNA and RNA were extracted from the purified virus
particles before the viral genome was identified by quantitative reverse
transcription PCR and viral metagenomic next-generation sequencing. Phylogenetic
reconstruction and investigation of recombination events were done by comparing
the Brazilian Zika virus genome with sequences from other Zika strains and from
flaviviruses that occur in similar regions in Brazil.
FINDINGS: We detected the Zika virus genome in the amniotic fluid of both
pregt women. The virus was not detected in their urine or serum. Tests for
dengue virus, chikungunya virus, Toxoplasma gondii, rubella virus,
cytomegalovirus, herpes simplex virus, HIV, Treponema pallidum, and parvovirus
B19 were all negative. After sequencing of the complete genome of the Brazilian
Zika virus isolated from patient 1, phylogenetic analyses showed that the virus
shares 97-100% of its genomic identity with lineages isolated during an outbreak
in French Polynesia in 2013, and that in both envelope and NS5 genomic regions,
it clustered with sequences from North and South America, southeast Asia, and
the Pacific. After assessing the possibility of recombination events between the
Zika virus and other flaviviruses, we ruled out the hypothesis that the
Brazilian Zika virus genome is a recombit strain with other mosquito-borne
flaviviruses.
INTERPRETATION: These findings strengthen the putative association between Zika
virus and cases of microcephaly in neonates in Brazil. Moreover, our results
suggest that the virus can cross the placental barrier. As a result, Zika virus
should be considered as a potential infectious agent for human fetuses.
Pathogenesis studies that confirm the tropism of Zika virus for neuronal cells
are warranted.
FUNDING: Consellho Nacional de Desenvolvimento e Pesquisa (CNPq), Fundação de
Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). The Zika virus outbreak has captivated the attention of the global audience and
information has spread rapidly and wildly through the internet and other media
channels. This virus was first identified in 1947, when it was isolated from a
sentinel rhesus monkey placed by British scientists working at the Yellow Fever
Research Laboratory located in the Zika forest area of Uganda, hence its name,
and is transmitted primarily by the mosquito vector, Aedes aegypti. The fact
that the rhesus macaque is an Asian species being placed in an African forest
brings to mind the possibility of rapid adaptation of the virus from an African
to Asian species, an issue that has not been considered. Whether such adaptation
has played any role in acquiring pathogenicity due to cross species transmission
remains to be identified. The first human infection was described in Nigeria in
1954, with only scattered reports of about a dozen human infections identified
over a 50-year period. It was not until 2007 that Zika virus raised its ugly
head with infections noted in three-quarters of the population on the tiny
island of Yap located between the Philippines and Papua New Guinea in the
western Pacific Ocean, followed by a major outbreak in French Polynesia in 2013.
The virus remained confined to a narrow equatorial band in Africa and Asia until
2014 when it began to spread eastward, first toward Oceania and then to South
America. Since then, millions of infected individuals have been identified in
Brazil, Colombia, Venezuela, including 25 additional countries in the Americas.
While the symptoms associated with Zika virus infection are generally mild,
consisting of fever, maculopapular rash, arthralgia and conjunctivitis, there
have been reports of more severe reactions that are associated with neurological
complications. In pregt women, fetal neurological complications include brain
damage and microcephaly, while in adults there have been several cases of
virus-associated Guillain-Barre syndrome. The virus was until recently believed
to only be transmitted via mosquitoes. But when the Zika virus was isolated from
the semen specimens from a patient in Texas, this provided the basis for the
recent report of possible sexual transmission of the Zika virus. Due to the
neurological complications, various vectors for infection as well as the rapid
spread throughout the globe, it has prompted the World Health Organization to
issue a global health emergency. Various governmental organizations have
recommended that pregt women do not travel to countries where the virus is
epidemic, and within the countries affected by the virus, recommendations were
provided for women of childbearing age to delay pregcy. The overall public
health impact of these above findings highlights the need for a rapid but
specific diagnostic test for blood banks worldwide to identify those infected
and for the counseling of women who are pregt or contemplating pregcy. As
of this date, there are neither commercially licensed diagnostic tests nor a
vaccine. Because cross-reactivity of the Zika virus with dengue and Chikungunya
virus is common, it may pose difficulty in being able to quickly develop such
tests and vaccines. So far the most effective public health measures include
controlling the mosquito populations via insecticides and preventing humans from
direct exposure to mosquitoes. Zika virus is a mosquito-borne flavivirus discovered in Africa in 1947. Most
persons with Zika virus infection are asymptomatic; symptoms when present are
generally mild and include fever, maculopapular rash, arthralgia, and
conjunctivitis. Since early 2015, Zika virus has spread rapidly through the
Americas, with local transmission identified in 31 countries and territories as
of February 29, 2016, including several US territories. All age groups are
susceptible to Zika virus infection, including children. Maternal-fetal
transmission of Zika virus has been documented; evidence suggests that
congenital Zika virus infection is associated with microcephaly and other
adverse pregcy and infant outcomes. Perinatal transmission has been reported
in 2 cases; 1 was asymptomatic, and the other had thrombocytopenia and a rash.
Based on limited information, Zika virus infection in children is mild, similar
to that in adults. The long-term sequelae of congenital, perinatal, and
pediatric Zika virus infection are largely unknown. No vaccine to prevent Zika
virus infection is available, and treatment is supportive. The primary means of
preventing Zika virus infection is prevention of mosquito bites in areas with
local Zika virus transmission. Given the possibility of limited local
transmission of Zika virus in the continental United States and frequent travel
from affected countries to the United States, US pediatric health care providers
need to be familiar with Zika virus infection. This article reviews the Zika
virus, its epidemiologic characteristics, clinical presentation, laboratory
testing, treatment, and prevention to assist providers in the evaluation and
management of children with possible Zika virus infection. Zika virus transmission was detected in the Region of the Americas (Americas) in
Brazil in May 2015, and as of March 21, 2016, local mosquito-borne transmission
of Zika virus had been reported in 32 countries and territories in the Americas,
including Puerto Rico and the U.S. Virgin Islands.* Most persons infected with
Zika virus have a mild illness or are asymptomatic. However, increasing evidence
supports a link between Zika virus infection during pregcy and adverse
pregcy and birth outcomes (1), and a possible association between recent Zika
virus infection and Guillain-Barré syndrome has been reported (2). Although Zika
virus is primarily transmitted through the bite of Aedes species of mosquitoes,
sexual transmission also has been documented (3). Zika virus RNA has been
detected in a number of body fluids, including blood, urine, saliva, and
amniotic fluid (3-5), and whereas transmission associated with occupational
exposure to these body fluids is theoretically possible, it has not been
documented. Although there are no reports of transmission of Zika virus from
infected patients to health care personnel or other patients, minimizing
exposures to body fluids is important to reduce the possibility of such
transmission. CDC recommends Standard Precautions in all health care settings to
protect both health care personnel and patients from infection with Zika virus
as well as from blood-borne pathogens (e.g., human immunodeficiency virus [HIV]
and hepatitis C virus [HCV]) (6). Because of the potential for exposure to large
volumes of body fluids during the labor and delivery process and the sometimes
unpredictable and fast-paced nature of obstetrical care, the use of Standard
Precautions in these settings is essential to prevent possible transmission of
Zika virus from patients to health care personnel. Zika virus belongs to Aedes mosquito-borne flavivirus. In response to the
current cluster of congenital malformations (microcephaly) and other
neurological complications (Guillain-Barré Syndrome) that could be linked to
Zika virus infection, WHO declares that Zika virus is of global public health
importance. Data sources were from peer review articles and WHO documents. The
sources of Zika virus infection would include patients, people with asymptomatic
infections and primates. The infectious period of Zika virus remains unclear.
However, according to the period that RNA of Zika virus can be positively
detected in blood, saliva, urine or semen, we can presume that the communicable
period may last for 2 months or even longer. Zika virus is primarily transmitted
to humans by infected Aedes spp. mosquitoes. Presumptive vertical, blood or
sexual routes of transmission have been reported. More evidence indicated the
existence of a cause-effect relationship between Zika virus infection and
congenital microcephaly/Guillain-Barre syndrome. Strategies include successful
control the amount of mosquitoes and minimize the contacts between mosquitoes
and human beings could effectively prevent the Zika virus transmission. Other
preventive measures as cutting off vertical, blood or sexual routes of
transmission should also be adopted. The epidemiology of Zika virus remains
uncertain which calls for further research. The marked increase in infants born with microcephaly in Brazil after a 2015
outbreak of Zika virus (Zika virus) disease suggests an association between
maternal Zika virus infection and congenital microcephaly. To project the timing
of delivery of infants born to mothers infected during early pregcy in 1 city
in Bahia State, Brazil, we incorporated data on reported Zika virus disease
cases and microcephaly cases into a graphical schematic of weekly birth cohorts.
We projected that these births would occur through February 2016. Applying
similar projections to a hypothetical location at which Zika virus transmission
started in November, we projected that full-term infants at risk for Zika virus
infection would be born during April-September 2016. We also developed a
modifiable spreadsheet tool that public health officials and researchers can use
for their countries to plan for deliveries of infants to women who were infected
with Zika virus during different pregcy trimesters. Zika virus is a flavivirus transmitted primarily by Aedes species mosquitoes,
and symptoms of infection can include rash, fever, arthralgia, and
conjunctivitis (1).* Zika virus infection during pregcy is a cause of
microcephaly and other severe brain defects (2). Infection has also been
associated with Guillain-Barré syndrome (3). In December 2015, Puerto Rico
became the first U.S. jurisdiction to report local transmission of Zika virus,
with the index patient reporting symptom onset on November 23, 2015 (4). This
report provides an update to the epidemiology of and public health response to
ongoing Zika virus transmission in Puerto Rico. During November 1, 2015-April
14, 2016, a total of 6,157 specimens from suspected Zika virus-infected patients
were evaluated by the Puerto Rico Department of Health (PRDH) and CDC Dengue
Branch (which is located in San Juan, Puerto Rico), and 683 (11%) had laboratory
evidence of current or recent Zika virus infection by one or more tests: reverse
transcription-polymerase chain reaction (RT-PCR) or immunoglobulin M (IgM)
enzyme-linked immunosorbent assay (ELISA). Zika virus-infected patients resided
in 50 (64%) of 78 municipalities in Puerto Rico. Median age was 34 years
(range = 35 days-89 years). The most frequently reported signs and symptoms were
rash (74%), myalgia (68%), headache (63%), fever (63%), and arthralgia (63%).
There were 65 (10%) symptomatic pregt women who tested positive by RT-PCR or
IgM ELISA. A total of 17 (2%) patients required hospitalization, including 5
(1%) patients with suspected Guillain-Barré syndrome. One (<1%) patient died
after developing severe thrombocytopenia. The public health response to the
outbreak has included increased laboratory capacity to test for Zika virus
infection (including blood donor screening), implementation of enhanced
surveillance systems, and prevention activities focused on pregt women.
Vector control activities include indoor and outdoor residual spraying and
reduction of mosquito breeding environments focused around pregt women's
homes. Residents of and travelers to Puerto Rico should continue to employ
mosquito bite avoidance behaviors, take precautions to reduce the risk for
sexual transmission (5), and seek medical care for any acute illness with rash
or fever. First isolated in 1947 from a monkey in the Zika forest in Uganda, and from
mosquitoes in the same forest the following year, Zika virus has gained
international attention due to concerns for infection in pregt women
potentially causing fetal microcephaly. More than one million people have been
infected since the appearance of the virus in Brazil in 2015. Approximately 80%
of infected patients are asymptomatic. An association with microcephaly and
other birth defects as well as Guillain-Barre Syndrome has led to a World Health
Organization declaration of Zika virus as a Public Health Emergency of
International Concern in February 2016. Zika virus is a vector-borne disease
transmitted primarily by the Aedes aegypti mosquito. Male to female sexual
transmission has been reported and there is potential for transmission via blood
transfusions. After an incubation period of 2-7 days, symptomatic patients
develop rapid onset fever, maculopapular rash, arthralgia, and conjunctivitis,
often associated with headache and myalgias. Emergency department (ED) personnel
must be prepared to address concerns from patients presenting with symptoms
consistent with acute Zika virus infection, especially those who are pregt or
planning travel to Zika-endemic regions, as well as those women planning to
become pregt and their partners. The identify-isolate-inform (3I) tool,
originally conceived for initial detection and management of Ebola virus disease
patients in the ED, and later adjusted for measles and Middle East Respiratory
Syndrome, can be adapted for real-time use for any emerging infectious disease.
This paper reports a modification of the 3I tool for initial detection and
management of patients under investigation for Zika virus. Following an
assessment of epidemiologic risk, including travel to countries with mosquitoes
that transmit Zika virus, patients are further investigated if clinically
indicated. If after a rapid evaluation, Zika or other arthropod-borne diseases
are the only concern, isolation (contact, droplet, airborne) is unnecessary.
Zika is a reportable disease and thus appropriate health authorities must be
notified. The modified 3I tool will facilitate rapid analysis and triggering of
appropriate actions for patients presenting to the ED at risk for Zika. Zika virus has rapidly spread through the World Health Organization's Region of
the Americas since being identified in Brazil in early 2015. Transmitted
primarily through the bite of infected Aedes species mosquitoes, Zika virus
infection during pregcy can cause spontaneous abortion and birth defects,
including microcephaly (1,2). New York City (NYC) is home to a large number of
persons who travel frequently to areas with active Zika virus transmission,
including immigrants from these areas. In November 2015, the NYC Department of
Health and Mental Hygiene (DOHMH) began developing and implementing plans for
managing Zika virus and on February 1, 2016, activated its Incident Command
System. During January 1-June 17, 2016, DOHMH coordinated diagnostic laboratory
testing for 3,605 persons with travel-associated exposure, 182 (5.0%) of whom
had confirmed Zika virus infection. Twenty (11.0%) confirmed patients were
pregt at the time of diagnosis. In addition, two cases of Zika
virus-associated Guillain-Barré syndrome were diagnosed. DOHMH's response has
focused on 1) identifying and diagnosing suspected cases; 2) educating the
public and medical providers about Zika virus risks, transmission, and
prevention strategies, particularly in areas with large populations of
immigrants from areas with ongoing Zika virus transmission; 3) monitoring
pregt women with Zika virus infection and their fetuses and infants; 4)
detecting local mosquito-borne transmission through both human and mosquito
surveillance; and 5) modifying existing Culex mosquito control measures by
targeting Aedes species of mosquitoes through the use of larvicides and
adulticides. The emergence of Zika virus in the Americas has followed a pattern that is
familiar from earlier epidemics of other viruses, where a new disease is
introduced into a human population and then spreads rapidly with important
public health consequences. In the case of Zika virus, an accumulating body of
recent evidence implicates the virus in the etiology of serious pathologies of
the human nervous system, that is, the occurrence of microcephaly in neonates
and Guillain-Barré syndrome in adults. Zika virus is an arbovirus
(arthropod-borne virus) and a member of the family Flaviviridae, genus
Flavivirus. Zika virions are enveloped and icosahedral, and contain a
nonsegmented, single-stranded, positive-sense RNA genome, which encodes 3
structural and 7 nonstructural proteins that are expressed as a single
polyprotein that undergoes cleavage. Zika genomic RNA replicates in the
cytoplasm of infected host cells. Zika virus was first detected in 1947 in the
blood of a febrile monkey in Uganda's Zika Forest and in crushed suspensions of
the Aedes mosquito, which is one of the vectors for Zika virus. The virus
remained obscure, with a few human cases confined to Africa and Asia. There are
two lineages of the Zika virus, African and Asian, with the Asian strain causing
outbreaks in Micronesia in 2007 and French Polynesia in 2013-2014. From here,
the virus spread to Brazil with the first report of autochthonous Zika
transmission in the Americas in March 2015. The rapid advance of the virus in
the Americas and its likely association with microcephaly and Guillain-Barré
syndrome make Zika an urgent public health concern. Ann Neurol 2016;80:479-489. CONTEXT: -Pathology studies have been important in concluding that Zika virus
infection occurring in pregt women can result in vertical transmission of the
agent from mother to fetus. Fetal and infant autopsies have provided crucial
direct evidence that Zika virus can infect an unborn child, resulting in
microcephaly, other malformations, and, in some cases, death.
OBJECTIVE: -To better understand the etiologic role and mechanism(s) of Zika
virus in causing birth defects such as microcephaly, this communication analyzes
the spectrum of clinical and autopsy studies reported from fetuses and infants
who developed intrauterine Zika virus infection, and compares these findings
with experimental data related to Zika virus infection.
DESIGN: -Retrospective analysis of reported clinical, autopsy, pathology, and
related postmortem studies from 9 fetuses and infants with intrauterine Zika
virus infection and microcephaly.
RESULTS: -All fetuses and infants examined demonstrated an overlapping spectrum
of gross and microscopic neuropathologic abnormalities. Direct cytopathic
effects of infection by the Zika virus were confined to the brain; in cases
where other organs were evaluated, no direct viral effects were identified.
CONCLUSIONS: -There is concordance of the spectrum of brain damage, reinforcing
previous data indicating that the Zika virus has a strong predilection for cells
of the fetal central nervous system following vertical transmission. The
occurrence of additional congenital abnormalities suggests that intrauterine
brain damage from Zika virus interferes with normal fetal development, resulting
in fetal akinesia. Experimental in vitro and in vivo studies of Zika virus
infection corroborate the human autopsy findings of neural specificity. The identification of Zika virus as a significant teratogen has raised
international concern, causing the World Health Organization to declare a Public
Health Emergency of International Concern. This has allowed a global
mobilization of experts in tropical infectious diseases, obstetrics, pediatrics,
virology, public health policy, reproductive health, bioethics, and germ cell
research to name just a few. This worldwide crisis has also raised awareness of
health care disparities and concerns regarding the ability of families and
societies to shoulder the long-term ficial burden that the follow-up of
affected children will require. There is now strong biologic evidence of
causality between Zika virus and microcephaly and other neurologic abnormalities
identified. Multiple national and international organizations have collaborated
to develop guidelines for the management of pregt women who reside in or who
are exposed to Zika virus, whether from travel to affected areas or via sexual
contact with an infected individual. These guidelines are updated frequently as
data are made available. Testing algorithms are available and though testing is
fraught with interpretation issues, the development of better diagnostic tests
is ongoing. IMPORTANCE: Recent studies have reported an increase in the number of fetuses
and neonates with microcephaly whose mothers were infected with the Zika virus
(ZIKV) during pregcy. To our knowledge, most reports to date have focused on
select aspects of the maternal or fetal infection and fetal effects.
OBJECTIVE: To describe the prenatal evolution and perinatal outcomes of 11
neonates who had developmental abnormalities and neurological damage associated
with ZIKV infection in Brazil.
DESIGN, SETTING, AND PARTICIPANTS: We observed 11 infants with congenital ZIKV
infection from gestation to 6 months in the state of Paraíba, Brazil. Ten of 11
women included in this study presented with symptoms of ZIKV infection during
the first half of pregcy, and all 11 had laboratory evidence of the infection
in several tissues by serology or polymerase chain reaction. Brain damage was
confirmed through intrauterine ultrasonography and was complemented by magnetic
resoce imaging. Histopathological analysis was performed on the placenta and
brain tissue from infants who died. The ZIKV genome was investigated in several
tissues and sequenced for further phylogenetic analysis.
MAIN OUTCOMES AND MEASURES: Description of the major lesions caused by ZIKV
congenital infection.
RESULTS: Of the 11 infants, 7 (63.6%) were female, and the median (SD) maternal
age at delivery was 25 (6) years. Three of 11 neonates died, giving a perinatal
mortality rate of 27.3%. The median (SD) cephalic perimeter at birth was 31 (3)
cm, a value lower than the limit to consider a microcephaly case. In all
patients, neurological impairments were identified, including microcephaly, a
reduction in cerebral volume, ventriculomegaly, cerebellar hypoplasia,
lissencephaly with hydrocephalus, and fetal akinesia deformation sequence (ie,
arthrogryposis). Results of limited testing for other causes of microcephaly,
such as genetic disorders and viral and bacterial infections, were negative, and
the ZIKV genome was found in both maternal and neonatal tissues (eg, amniotic
fluid, cord blood, placenta, and brain). Phylogenetic analyses showed an
intrahost virus variation with some polymorphisms in envelope genes associated
with different tissues.
CONCLUSIONS AND RELEVANCE: Combined findings from clinical, laboratory, imaging,
and pathological examinations provided a more complete picture of the severe
damage and developmental abnormalities caused by ZIKV infection than has been
previously reported. The term congenital Zika syndrome is preferable to refer to
these cases, as microcephaly is just one of the clinical signs of this
congenital malformation disorder. PURPOSE: This article describes what pediatric healthcare professionals should
know about Zika virus (ZIKV).
LITERATURE REVIEW: ZIKV is classified as an arthropod-borne, single-stranded RNA
virus of the Flaviviridae family and genus Flavivirus. ZIKV is not new. The
virus was first discovered almost 70 years ago in Uganda. The first isolate of
the virus was found in rhesus monkeys in the Zika Forrest, hence the
nomenclature. The primary route of ZIKV transmission to humans is through the
bite of an infected Aedes species mosquito-primarily Aedes aegypti. When the
mosquito bites individuals infected with the virus, mosquitos then become the
vector of transmitting the infection to others. Women can also pass ZIKV to
their fetus during pregcy and at the time of delivery. ZIKV can also be
transmitted through sexual activity from an individual who is infected with the
virus to his or her partners. It is estimated that approximately 18% of
individuals infected with ZIKV will go on to develop symptoms. When symptoms
develop, it is usually within 3-12 days, although this may vary. Most often,
symptoms are mild and self-limited. The most common symptoms are fever,
arthralgia, maculopapular rash, and conjunctivitis lasting up to seven days.
Less frequent symptoms include headache, vertigo, myalgia, vomiting, and
diarrhea. At present, there is no vaccine available to prevent ZIKV and no
specific antiviral treatment. Supportive care consisting of rest, hydration,
analgesics, antihistamines, and antipyretics is recommended as needed. Given
that there is no vaccine or treatment for ZIKV, considerable efforts must be
focused on prevention. One of the most effective ways of preventing ZIKV
infection is through avoiding mosquito bites, especially when traveling to or
residing in areas where transmission is present. Precautions should include
wearing appropriate attire with the objective of having as little skin exposed
as possible, use of screens for windows and doors, and use of insect repellent.
PRACTICE IMPLICATIONS: What is known about ZIKV changes continually. An
infectious threat that was relatively obscure just a few months ago has now
become a topic of heightened interest worldwide. Pediatric healthcare
professionals must remain cognizant of evolving developments and emerging new
evidence. |
What is the function of Oseltamivir when administered during flu? | Oseltamivir (has known by its brand name 'Tamiflu') is a prodrug, requiring ester hydrolysis for conversion to the active form, Oseltamivir carboxylate. Oseltamivir is the first orally active neuraminidase inhibitor and it is an antiviral drug for the treatment of Swine Flu. | BACKGROUND: Safe and effective antiviral agents are needed to prevent infection
with influenza A and B virus. Oseltamivir (GS4104), which can be administered
orally, is the prodrug of GS4071, a potent and selective inhibitor of
influenzavirus neuraminidases. We studied the use of oseltamivir for long-term
prophylaxis against influenza in two placebo-controlled, double-blind trials at
different U.S. sites during the winter of 1997-1998.
METHODS: We randomly assigned 1559 healthy, nonimmunized adults 18 to 65 years
old to receive either oral oseltamivir (75 mg given once or twice daily, for a
total daily dose of 75 or 150 mg) or placebo for six weeks during a peak period
of local influenzavirus activity. The primary end point with respect to efficacy
was laboratory-confirmed influenza-like illness (defined as a temperature of at
least 37.2 degrees C accompanied by at least one respiratory and at least one
systemic symptom).
RESULTS: In the two studies combined, the risk of influenza among subjects
assigned to either once-daily or twice-daily oseltamivir (1.2 percent and 1.3
percent, respectively) was lower than that among subjects assigned to placebo
(4.8 percent; P<0.001 and P=0.001 for the comparison with once-daily and
twice-daily oseltamivir, respectively). The protective efficacy of oseltamivir
in the two active-treatment groups combined was 74 percent (95 percent
confidence interval, 53 to 88 percent) at all the sites combined and 82 percent
(95 percent confidence interval, 60 to 93 percent) at sites in Virginia, where
the rate of influenza infection was higher than the overall rate. For
culture-proved influenza, the rate of protective efficacy in the two oseltamivir
groups combined was 87 percent (95 percent confidence interval, 65 to 96
percent). The rate of laboratory-confirmed influenza infection was lower with
oseltamivir than with placebo (5.3 percent vs. 10.6 percent, P<0.001).
Oseltamivir was well tolerated but was associated with a greater frequency of
nausea (12.1 percent and 14.6 percent in the once-daily and twice-daily groups,
respectively) and vomiting (2.5 percent and 2.7 percent, respectively) than was
placebo (nausea, 7.1 percent; vomiting, 0.8 percent). However, the frequency of
premature discontinuation of drug or placebo was similar among the three groups
(3.1 to 4.0 percent).
CONCLUSIONS: Oseltamivir administered daily for six weeks by the oral route is
safe and effective for the prevention of influenza. OBJECTIVE: Influenza outbreaks have revealed that elderly persons are a great
risk of death and serious complications after infection. The administration of
oseltamivir, a neuramidase inhibitor, is effective for prophylaxis of influenza
and to reduce disease duration and severity in healthy adults with naturally
acquired febrile influenza. To clarify the usefulness of oseltamivir in the
elderly we administered oseltamivir to all residents when an influenza A
outbreak occurred in a nursing home.
PATIENTS: Sixty-eight residents in the nursing home were investigated in which
the influenza A outbreak occurred; 32 residents had fever and 28 residents were
positive for influenza A with direct enzyme immunoassay.
METHODS: Oseltamivir was administered at 75 mg twice daily for 5 days to all
residents.
RESULTS: Oseltamivir almost inhibited symptom onset in the influenza A-positive
afebrile group. Initiation at 0 hour (22 cases), 1-12 hours (4 cases), 13-24
hours (5 cases) or 72 hours (1 case) from onset of symptoms was associated with
mean fever durations of 26+/-18 hours, 38+/-21 hours, 54+/-12 hours and 120
hours, respectively, indicating that earlier initiation of therapy was
associated with faster resolution of fever in elderly patients. Oseltamivir may
be effective for household prophylaxis in the elderly persons. Oseltamivir
administration was well tolerated in elderly persons.
CONCLUSION: Oseltamivir is effective for the reduction or prophylaxis of
influenza A infection in elderly persons. BACKGROUND: Among asthmatic children, influenza is associated with increased
hospitalizations. Although vaccination is safe and effective among asthmatic
children, its protective efficacy varies and uptake rates can be low. In
comparison, oseltamivir (Tamiflu) is effective against all influenza strains and
can reduce the severity and duration of influenza among adults and children.
This study determined the effects of oseltamivir among influenza-infected
children with asthma.
METHODS: Asthmatic children (6-12 years of age) were randomized to receive
oseltamivir (2 mg/kg) or placebo twice daily, as a syrup. The primary efficacy
endpoint was the time to freedom from illness. Secondary endpoints included the
area under the symptom score-hour curve, the proportion of patients with asthma
exacerbations and changes in forced expiratory volume at 1 second during the
dosing period. Analysis was performed for both the intent-to-treat infected (n =
179) and per protocol (n = 162) populations.
RESULTS: The primary endpoint for this study was not met. Oseltamivir tended to
reduce the time to freedom from illness in the intent-to-treat infected
population (10.4 hours, 8%; P = 0.5420), the per protocol population (24.3
hours, 17%; P = 0.1607) and patients who started treatment <24 hours after
symptom onset (39.8 hours, 25%; P = 0.0780). However, an improvement in
pulmonary function was observed. The improvement in forced expiratory volume at
1 second was significantly greater among oseltamivir-treated patients (10.8%
versus 4.7%; P = 0.0148). Oseltamivir-treated patients also experienced fewer
asthma exacerbations up to day 7 (68% versus 51%; P = 0.031). Oseltamivir was
safe and well-tolerated.
CONCLUSIONS: Oseltamivir is safe and well-tolerated among asthmatic children,
may reduce symptom duration and helps improve lung function and reduce asthma
exacerbations during influenza infection. BACKGROUND: To compare the effectiveness of oseltamivir for treatment of
influenza A and influenza B, we conducted a prospective, multicenter study of
the 2003-2004 and 2004-2005 influenza seasons. The study included 3351 patients
in whom influenza had been diagnosed by use of an antigen detection test kit.
METHODS: Oseltamivir was administered to 1818 patients with influenza A and 1485
patients with influenza B. No anti-influenza drugs were administered to 21
patients with influenza A or to 27 patients with influenza B. Patients receiving
oseltamivir therapy were divided into 4 groups according to the time between the
onset of fever (temperature, > or = 37.5 degrees C) and administration of the
first dose of oseltamivir (0-12 h, 13-24 h, 25-36 h, and 37-48 h). The patients
were also divided into 4 subgroups on the basis of age (0-6 years, 7-15 years,
16-64 years, and >64 years). Virus isolation was performed after completion of
oseltamivir therapy for 44 patients with influenza A and 31 patients with
influenza B.
RESULTS: The duration of fever was significantly shorter for patients with
influenza A and B who were treated with oseltamivir than for patients who were
not treated with an anti-influenza drug (P<.001 for both). The time until the
patient became afebrile after the initial administration of oseltamivir and the
duration of fever were significantly longer for patients with influenza B than
for patients with influenza A for the 0-12 h, 13-24 h, 25-36 h, and 37-48 h
groups (P<.001) and for all age groups (P<.001). After 4-6 days of oseltamivir
therapy, the influenza B virus reisolation rate (51.6%) was significantly higher
than the influenza A virus reisolation rate (15.9%) (P<.001).
CONCLUSION: Oseltamivir is less effective for influenza B than for influenza A
with regard to duration of fever and virus persistence, irrespective of patient
age or the timing of administration of the first dose. Oseltamivir (Tamiflu), a neuraminidase inhibitor, is widely used for treatment
of influenza. Because abnormal behaviors have been observed in some Japanese
teenagers following oseltamivir use, its safety has been questioned. Oseltamivir
is known to alter neuronal function and behavior in animals, particularly when
administered in combination with ethanol. Based on this, it has been
hypothesized that interactions of oseltamivir with other drugs may result in
altered CNS excitability in this study. It has been found that injection of
ephedrine and caffeine overcame inactivity induced by oseltamivir and ethanol
but did not alter changes in novelty seeking behavior in a Y-maze test. In
ex-vivo hippocampal slices, oseltamivir carboxylate (OTC), an active form of
oseltamivir, alters excitability in the absence of ethanol. In slices pretreated
with OTC, long-term depression (LTD), a form of synaptic plasticity that is
correlated with Y-maze performance was not altered if caffeine or ephedrine was
administered individually. However, LTD could not be induced in slices
pretreated with OTC if caffeine and ephedrine were administered simultaneously.
These observations suggest that combination of oseltamivir with other
neurostimulants may alter synaptic plasticity and this may contribute to
behavioral changes associated with the drug. Influenza is a highly contagious and debilitating disease that imposes an excess
burden of complications and mortality. Antiviral therapy is the primary
intervention for treatment and post-exposure prophylaxis (PEP) of influenza.
Amantadine and rimantadine are members of the M2 class of antiviral agents and
are moderately effective in influenza management. However, their utility is
compromised by high levels of resistance, tolerability concerns and a lack of
efficacy against influenza B. An alternative class of agents, the neuraminidase
inhibitors (NIs), represent the most advanced form of antiviral therapy
available, and act by specifically inhibiting the neuraminidase enzymes that are
present on all influenza subtypes. Two NIs, oseltamivir and zanamivir, are
currently available for clinical use. Oseltamivir, the most widely used NI, is
administered orally as a prodrug (oseltamivir carboxylate) and systemically
distributed to all potential infection sites. Zanamivir, a second NI, is
administered by inhalation via a disk inhaler and deposited primarily in the
respiratory tract. When administered within 48 hours of symptom onset, both
agents significantly reduce illness duration and symptom severity, and decrease
the rate of influenza-associated complications. With oseltamivir, greater
benefits are detected with earlier treatment initiation (<12 hours). In PEP,
both NIs effectively protect the close contacts of index cases from symptomatic
influenza. Oseltamivir and zanamivir are generally well tolerated and associated
with a low level of resistance. Emerging evidence supports the activity of both
NIs against the H5N1avian influenza infection, which is a pandemic candidate.
However, the WHO currently recommends the use of oseltamivir for the management
of suspected cases, given the systemic nature of the H5N1 challenge. Ongoing
studies are exploring the effectiveness of oseltamivir, zanamivir and other NIs
for pandemic management. In this work, we study the consequences of sequence variations of the "2009
H1N1" (swine or Mexican flu) influenza A virus strain neuraminidase for drug
treatment and vaccination. We find that it is phylogenetically more closely
related to European H1N1 swine flu and H5N1 avian flu rather than to the H1N1
counterparts in the Americas. Homology-based 3D structure modeling reveals that
the novel mutations are preferentially located at the protein surface and do not
interfere with the active site. The latter is the binding cavity for 3 currently
used neuraminidase inhibitors: oseltamivir (Tamiflu), zanamivir (Relenza) and
peramivir; thus, the drugs should remain effective for treatment. However, the
antigenic regions of the neuraminidase relevant for vaccine development,
serological typing and passive antibody treatment can differ from those of
previous strains and already vary among patients.
REVIEWERS: This article was reviewed by Sandor Pongor and L. Aravind. Oseltamivir (Tamiflu) is the most important antiviral drug available and a
cornerstone in the defence against a future influenza pandemic. Recent
publications have shown that the active metabolite, oseltamivir carboxylate
(OC), is not degraded in sewage treatment plants and is also persistent in
aquatic environments. This implies that OC will be present in aquatic
environments in areas where oseltamivir is prescribed to patients for
therapeutic use. The country where oseltamivir is used most is Japan, where it
is used to treat seasonal flu. We measured the levels of OC in water samples
from the Yodo River system in the Kyoto and Osaka prefectures, Japan, taken
before and during the flu-season 2007/8. No OC was detected before the
flu-season but 2-58 ng L(-1) was detected in the samples taken during the flu
season. This study shows, for the first time, that low levels of oseltamivir can
be found in the aquatic environment. Therefore the natural reservoir of
influenza virus, dabbling ducks, is exposed to oseltamivir, which could promote
the evolution of viral resistance. BACKGROUND: Oseltamivir phosphate (OP; Tamiflu) is a prodrug of the
anti-influenza neuraminidase inhibitor oseltamivir carboxylate (OC) and has been
developed for the treatment and prevention of both A and B strains of influenza.
The recent increase in OP resistance in influenza A virus (H1N1; commonly called
"swine flu") has raised questions about the widespread use of Tamiflu in
seasonal epidemics and the potential ecotoxicologic risk associated with its use
in the event of a pandemic.
OBJECTIVES: The objectives of this study were to develop an analytical method
for quantitative determination of OC in sewage treatment plant (STP) effluent
and receiving river water, and to investigate the occurrence of OC in STP
effluent and river water in Japan during a seasonal flu outbreak.
METHODS: We developed an analytical method based on solid-phase extraction
followed by liquid chromatography-tandem mass spectrometry. Using this method,
we analyzed samples from three sampling campaigns conducted during the 2008-2009
flu season in Kyoto City, Japan.
RESULTS: The highest concentration of OC detected in STP discharge was 293.3
ng/L from a conventional activated-sludge-based STP; however, we detected only
37.9 ng/L from an advanced STP with ozonation as a tertiary treatment. In the
receiving river water samples, we detected 6.6-190.2 ng/L OC, during the peak of
the flu season.
CONCLUSION: OC is present in STP effluent and river water only during the flu
season. Ozonation as tertiary treatment in STP will substantially reduce the OC
load in STP effluent during an influenza epidemic or pandemic. Although neuraminidase inhibitors are active against most 2009-2010 pandemic
influenza A (H1N1) swine-origin strains, sporadic cases of oseltamivir
resistance have been described. Since April 2009, 54 cases of
oseltamivir-resistant H1N1 swine-origin have been reported in the USA
(http://www.cdc.gov/flu/weekly/; accessed 1 February 2010). Approximately 1.4%
of tested isolates are oseltamivir resistant. We report a patient with an
underlying hematological maligcy who was hospitalized with influenza A (H1N1)
swine-origin and whose strain developed oseltamivir resistance during therapy. The aim of this paper was the evaluation of clinical characteristics,
demographics and therapeutic response for oseltamivir, among patients with swine
flu confirmed, hospitalized in the Hospital For Infectious Diseases in Warsaw,
Poland.
MATERIAL: We have observed infection A/H1N1 occurrence in 109 patients
(Female-64, Male-45, aged 17-71 y), hospitalized between August and December
2009. The influenza specific test PCR (TaqMan A/H1N1) were used to pandemic flu
confirmation.
RESULTS: Out of 109 analyzed patients, 67% were young, before 40 y. old. The
largest infected group were patients between 20 and 29 years. Among multiple
acute symptoms we observed high temperature, cough, myalgia and neurological
manifestations, very frequent. In 42 patients (38%) the interstitial pneumonia
were observed. Eight patients developed severe respiratory insufficiencies -ARDS
(7%) and one died. We observed also 10 infections A/H1N1 influenza during
pregcy, with good oseltamivir tolerance and without recent perinathal
complications.
CONCLUSIONS: Among 109 individuals with swine flu influenza, 67% have not
complicated clinical manifestation and they recovered during 3-4 days. Eight
patients developed ARDS and one of them died. Test PCR for influenza A/H1N1 was
the basis in diagnostics procedures of the new pandemic influenza confirmation.
Oseltamivir safety and tolerability were verified in patients with new variant
infection A/H1N1. BACKGROUND: Anti-viral prophylaxis is used to prevent the transmission of
influenza. We studied serological confirmation of 2009 Influenza A (H1N1)
infections during oseltamivir prophylaxis and after cessation of prophylaxis.
METHODS: Between 22 Jun and 16 Jul 09, we performed a cohort study in 3
outbreaks in the Singapore military where post-exposure oseltamivir ring
chemoprophylaxis (75 mg daily for 10 days) was administered. The entire cohort
was screened by RT-PCR (with HA gene primers) using nasopharyngeal swabs three
times a week. Three blood samples were taken for haemagglutination inhibition
testing--at the start of outbreak, 2 weeks after completion of 10 day
oseltamivir prophylaxis, and 3 weeks after the pandemic's peak in Singapore.
Questionnaires were also administered to collect clinical symptoms.
RESULTS: 237 personnel were included for analysis. The overall infection rate of
2009 Influenza A (H1N1) during the three outbreaks was 11.4% (27/237). This
included 11 index cases and 16 personnel (7.1%) who developed four-fold or
higher rise in antibody titres during oseltamivir prophylaxis. Of these 16
personnel, 8 (3.5%) were symptomatic while the remaining 8 personnel (3.5%) were
asymptomatic and tested negative on PCR. Post-cessation of prophylaxis, an
additional 23 (12.1%) seroconverted. There was no significant difference in mean
fold-rise in GMT between those who seroconverted during and post-prophylaxis
(11.3 vs 11.7, p = 0.888). No allergic, neuropsychiatric or other severe
side-effects were noted.
CONCLUSIONS: Post-exposure oseltamivir prophylaxis reduced the rate of infection
during outbreaks, and did not substantially increase subsequent infection rates
upon cessation. Asymptomatic infections occur during prophylaxis, which may
confer protection against future infection. Post-exposure prophylaxis is
effective as a measure in mitigating pandemic influenza outbreaks. Pregt women with influenza are at increased risk of morbidity, particularly
due to respiratory complications. A high excess mortality rate among pregt
women has been observed in previous influenza pandemics and healthcare agencies
have provided recommendations on the use of oseltamivir to treat pregt women
who are infected with the pandemic (H1N1) 2009 virus. This article reviews
pre-clinical and clinical data to assess the safety of oseltamivir administered
during pregcy, in the context of the effects of influenza on adverse
pregcy outcomes and fetal malformations. The effects of influenza during
pregcy, whether mediated directly by the virus or by fever or other events
secondary to the underlying infection, are not yet well understood, but some
data indicate an increased risk of birth defects in women infected with
influenza during the first trimester. Animal and toxicology studies do not
suggest that clinically effective dosages of oseltamivir have the potential to
produce adverse effects on fetal development. Additionally, transplacental
transfer of the drug and its active metabolite was very limited and not
detectable at normal therapeutic doses in an ex vivo human placenta model. To
investigate the safety of oseltamivir in pregcy, the Roche oseltamivir safety
database was searched for all exposures to oseltamivir during pregcy in the 9
years up to 14 December 2008. In addition, a search of the literature was
carried out. Of 232 maternal exposures to oseltamivir in the Roche database,
pregcy outcomes were known for 115 of these exposures. The incidence of
adverse pregcy outcomes was as follows: spontaneous abortions 6.1% (7/115),
therapeutic abortions 11.3% (13/115) and pre-term deliveries 2.1% (2/94 live
births), values that are not higher than background incidence rates. Fetal
outcomes were known in 100 of the 232 exposures. For the nine cases of birth
defect that were reported, the timing of oseltamivir exposure in relation to the
sensitive period for inducing the birth defect was analysed. Two cases of
ventricular septal defect, a more common birth defect, and one case of
anophthalmos, an uncommon birth defect, were consistent with exposure to
oseltamivir during the sensitive period for these birth defects. For other birth
defects, there was either no exposure to oseltamivir during the sensitive period
for the defect or insufficient information for assessment. These findings were
consistent with other reports in the published literature, including a series of
79 Japanese women exposed to oseltamivir during the first trimester. Together
with the other evidence reviewed herein, review of the company safety database
suggests that oseltamivir is unlikely to cause adverse pregcy or fetal
outcomes, but available data are limited. Clinicians who use oseltamivir in
pregt women should consider the available safety information, the
pathogenicity of the circulating influenza virus strain, the woman's general
health and the guidance provided by health authorities. Roche will continue to
monitor all reports of oseltamivir use during pregcy. Oseltamivir (has known by its brand name 'Tamiflu') is a prodrug, requiring
ester hydrolysis for conversion to the active form, Oseltamivir carboxylate.
Oseltamivir was the first orally active neuraminidase inhibitor commercially
developed by US based Gilead Sciences and is currently marketed by F.
Hoffmann-La Roche (Roche). Oseltamivir is an antiviral drug which works by
blocking the function of the viral neuraminidase protein. US FDA approved
Oseltamivir for prophylaxis of uncomplicated influenza A and B. Currently,
Oseltamivir is the only first line defense drug available for the treatment of
Swine Flu. Orally Oseltamivir is well tolerated and effective in treatment of
influenza in adolescents and adults, including the elderly and patients with
chronic cardiac and/or respiratory disease. Many of the pharmaceutical companies
targeted Oseltamivir as a block buster molecule. In present review, we have
tried to cover chemistry, mode of binding, total synthesis, current patent
status, adverse effect and clinical status of Oseltamivir giving emphasis on
medicinal chemistry aspect. Oseltamivir (Tamiflu) is currently the frontline antiviral drug employed to
fight the flu virus in infected individuals by inhibiting neuraminidase, a flu
protein responsible for the release of newly synthesized virions. However,
oseltamivir resistance has become a critical problem due to rapid mutation of
the flu virus. Unfortunately, how mutations actually confer drug resistance is
not well understood. In this study, we employ molecular dynamics (MD) and
steered molecular dynamics (SMD) simulations, as well as graphics processing
unit (GPU)-accelerated electrostatic mapping, to uncover the mechanism behind
point mutation induced oseltamivir-resistance in both H5N1 "avian" and H1N1pdm
"swine" flu N1-subtype neuraminidases. The simulations reveal an electrostatic
binding funnel that plays a key role in directing oseltamivir into and out of
its binding site on N1 neuraminidase. The binding pathway for oseltamivir
suggests how mutations disrupt drug binding and how new drugs may circumvent the
resistance mechanisms. An independent reanalysis of 11 randomized clinical trials shows that
oseltamivir treatment reduces the risk of lower respiratory tract complications
requiring antibiotic treatment by 28% overall (95% confidence interval [CI],
11%-42%) and by 37% among patients with confirmed influenza infections (95% CI,
18%-52%). During the H1N1 (swine flu) pandemic of 2009, the World Health Organization
(WHO) confirmed more than 14,000 deaths globally; this included a death toll of
147 in Iran. In order to evaluate (a) the appropriateness of the Oseltamivir
dose through calculation of a patient’s creatinine clearance (CrCl) and (b) the
quality of data in the medical charts, we conducted a retrospective study at the
Shariati Hospital in Tehran. All admissions to the hospital between the dates 1
October 2009 and 31 January 2010 were evaluated, amounting to a total of 51
patients’ charts, including 8 outpatient charts. Of these 51 charts, 26 (51%)
contained all the information necessary to evaluate the CrCl. However, there was
at least one piece of information missing (e.g. the patient’s weight; serum
creatinine) from each of the remaining 25 charts (49% of the sample), which made
it impossible for us to evaluate the dose. These results demonstrate how
crucially important it is to ensure that all the necessary patient information
is correctly registered at the time of admission in order to minimise medication
errors. The H1N1 2009 virus is pandemic in many countries. The genome of this virus
contains eight segments. Among the eight segments maximum numbers of mutation
occur at the segment 1 and segment 4 which codes for PB2 subunit and
hemagglutinin (HA) and less number of mutations occur in segment 6 which codes
for neuraminidase (NA) protein. Neuraminidase (NA) inhibitors (Oseltamivir and
Zanamivir) are presently used as an anti-flu drugs. In the present study, the in
silico efficacy of different drugs was tested against the swine flu virus. 3D
structures of neuraminidase (NA) proteins of H1N1 2009 were generated using
Geno3D. The 3D structure of H1N1 1918 was downloaded from PDB. Interaction study
was done using Arguslab 4 and PyMol view. Oseltamivir and Zanamivir have good
number of interactions with H1N1 2009 virus and the scoring function also
support to this result. When compared with the 1918 H1N1 viral protein, 2009
H1N1 NA protein shows more number of interaction and good scoring function. The
RMSD value of before and after docking are found to be same at 0.04A° for both
the drugs. The force field energy of NA protein 2009 was found to be -15603.529
KJ/mol before docking. The force field energy was found to be decreased after
docking at -17620.740 KJ/mol with Tamiflu and -17652.242 KJ/mol with Zanamivir.
The number of interaction and scoring function shows that Oseltamivir and
Zanamivir will be able to effectively control the present pandemic H1N1 virus
2009. STUDY OBJECTIVE: To investigate oseltamivir and oseltamivir carboxylate
pharmacokinetics in critically ill patients who were receiving continuous
venovenous hemodialysis (CVVHD) and/or extracorporeal membrane oxygenation
(ECMO).
DESIGN: Prospective, open-label, pharmacokinetic study.
SETTING: Intensive care units of an academic medical center.
PATIENTS: Thirteen critically ill patients aged 13 years or older with suspected
or confirmed H1N1 influenza who had a prescription for oseltamivir and were
concurrently receiving CVVHD and/or ECMO between October 2009 and January 2010.
INTERVENTION: Oseltamivir 150 mg was administered nasogastrically or
nasoenterically every 12 hours. Blood samples were collected at baseline and at
1, 2, 4, 6, 8, 10, and 12 hours after administration of the fourth oseltamivir
dose or subsequent doses. In patients receiving CVVHD, effluent also was
collected at the same time points. Urine was collected throughout the 12-hour
dosing interval.
MEASUREMENTS AND MAIN RESULTS: Eight patients received CVVHD only, four patients
received both CVVHD and ECMO, and one patient received ECMO only.
Pharmacokinetic parameters for the patient who received only ECMO were not
reported. The median maximum plasma concentration and area under the plasma
concentration-time curve for the 12-hour dosing interval (AUC(0-12) ) for the
remaining 12 patients were 83.4 ng/ml and 216 ng•hour/ml, respectively, for
oseltamivir and 2000 ng/ml and 21,500 ng•hour/ml, respectively, for oseltamivir
carboxylate. Mean clearance due to CVVHD was 33.8 ml/minute for oseltamivir and
50.2 ml/minute for oseltamivir carboxylate. For patients who received ECMO, no
substantial differences between pre- and post-ECMO oxygenator plasma
concentrations were found for oseltamivir or oseltamivir carboxylate.
CONCLUSION: Although the optimal pharmacokinetic-pharmacodynamic targets for
oseltamivir carboxylate remain unclear, in the patients receiving CVVHD with or
without ECMO, a regimen of oseltamivir 150 mg every 12 hours yielded a median
oseltamivir carboxylate AUC(0-12) considerably higher than would be expected in
non-critically ill patients receiving the same dosage regimen. The clinical manifestations associated with H5N1 infection in humans range from
asymptomatic infection to mild upper respiratory illness, severe pneumonia, and
multiple organ failure. The ratio of symptomatic cases to asymptomatic cases is
not known, because it is not possible to precisely define the number of
asymptomatic cases. A total of 97 cases suffering from avian flu were suspected
based on history taking, demographic data, clinical manifestations, laboratory
and radiological investigations. The followings were done for all cases;
complete blood picture (differential leucocytic count), coagulation profile,
renal and liver function tests. H5N1 influenza virus was diagnosed thorough PCR
technique. Changes in arterial blood gases and repeated chest X-rays were
reported frequently. All patients were given specific antiviral therapy
(oseltamivir). The study described the clinical picture and laboratory results
of 81 confirmed avian influenza human cases in an Egyptian hospital (Abassia
chest hospital), and reviewed the avian influenza current situation covering
from March 2006 to June 2009 with very high pick in the first half of 2009. The
significant apparent symptoms were fever as initial and main symptom (93.75%),
followed by shortness of breathing (73%), cough (66.6%), muscle & joint pain
(60%) and sore throat (40%). H5N1 is a subtype of the influenza A virus that can cause disease in humans and
many other animal species. Oseltamivir (Tamiflu) is a potent and selective
antiviral drug employed to fight the flu virus in infected individuals by
inhibiting neuraminidase (NA), a flu protein responsible for the release and
spread of the progeny virions. However, oseltamivir resistance has become a
critical problem. In particular, influenza strains with a R292K NA mutation are
highly resistant to the oseltamivir. Though the biological functions of the
mutations have previously been characterized, the structural basis behind the
reduced catalytic activity and reduced protein level is not clear. In this
study, molecular docking and molecular dynamics (MD) approach were employed to
investigate the structural and dynamical effects throughout the protein
structure and specifically, at the drug-binding pocket. Furthermore, potential
of mean force was analyzed using explicit solvent MD simulations with the
umbrella sampling method to explore the free energy of binding. It is believed
that this study provides valuable guidance for the resistance management of
oseltamivir and designing of more potent antiviral inhibitor. BACKGROUND: Monitoring the emergence of drug-resistant influenza variants is
crucial in influenza surveillance programs.
OBJECTIVES: Influenza A kinetics and the emergence of drug-resistant strains in
hospitalized patients treated with oseltamivir were investigated.
STUDY DESIGN: Sequential samples from oseltamivir-treated and -untreated
hospitalized patients in the period November 2011 through April 2012 were
analyzed. NA gene was sequenced in samples from oseltamivir treated patients.
Clonal analysis of the viral population was performed in patients unresponsive
to treatment. Viral kinetics was determined in 24 (14 immunocompromised and 10
immunocompetent) A(H3N2)-positive patients treated and 24 (10 immunocompromised
and 14 immunocompetent) untreated patients.
RESULTS: Viral shedding was significantly reduced in treated vs untreated
immunocompromised patients (7 vs 22 days, p<0.05, respectively). Viral load
decreased significantly in immunocompromised and immunocompetent treated
patients as compared with immunocompromised and immunocompetent untreated
patients (0.73 and 0.93 vs 0.47 and 0.45 log10/day, p<0.05). In two (8.3%)
treated patients with prolonged virus shedding, the oseltamivir resistance R292K
mutation was revealed. In these patients, clonal analysis of the virus
population showed the presence of additional oseltamivir-resistant mutants
(E119V, N294S and deletion Del247-250).
CONCLUSIONS: Oseltamivir resistance is reported for the first time in A(H3N2)
virus strains during the 2011-2012 influenza season. Different drug-resistant
viruses emerged in hospitalized immunocompromised patients showing prolonged
virus shedding. OBJECTIVES: We evaluated the implementation of three commercially available
neuraminidase inhibition assays in a public health laboratory (PHL) setting. We
also described the drug susceptibility patterns of human influenza A and B
circulating in Maryland during the 2011-2012 influenza season.
METHODS: From January to May 2012, 169 influenza virus isolates were tested for
phenotypic susceptibility to oseltamivir, zanamivir, and peramivir using
NA-Fluor(TM), NA-Star®, and NA-XTD(TM) concurrently. A 50% neuraminidase
inhibitory concentration (IC50) value was calculated to determine drug
susceptibility. We used the standard deviation based on the median absolute
deviation of the median analysis to determine the potential for reduced drug
susceptibility. We evaluated each assay for the use of resources in high- and
low-volume testing scenarios.
RESULTS: One of the 25 2009 influenza A (H1N1) pandemic isolates tested was
resistant to oseltamivir and peramivir, and sensitive to zanamivir, on all three
platforms. Eighty-two influenza A (H3N2) and 62 B isolates were sensitive to all
three drugs in all three assays. For a low-volume scenario, NA-Star and NA-XTD
took 120 minutes to complete, while NA-Fluor required 300 minutes to complete.
The lowest relative cost favored NA-Star. In a high-volume scenario, NA-Fluor
had the highest throughput. Reagent use was most efficient when maximizing
throughput. Cost efficiency from low- to high-volume testing improved the most
for NA-Star.
CONCLUSIONS: Our evaluation showed that both chemiluminescent and fluorescent
neuraminidase inhibition assays can be successfully implemented in a PHL setting
to screen circulating influenza strains for neuraminidase inhibitor resistance.
For improved PHL influenza surveillance, it may be essential to develop
guidelines for phenotypic drug-resistance testing that take into consideration a
PHL's workload and available resources. OBJECTIVE: To report the treatment process of the first case of human pneumonia
resulted from H10N8 avian influenza virus infection in the world for providing
the data for clinical diagnosis and treatment.
METHODS: On November 30, 2013, the first case of human infection with H10N8
avian influenza virus was discovered in Nanchang City, Jiangxi Province. Its
clinical symptoms and epidemiology were analyzed and compared with the
characteristics of human infection with H7N9 avian influenza virus.
RESULTS: A 73-year old female patient complaining of cough and chest tightness
for 3 days and fever for 1 day was admitted to the Department of Respiratory
Diseases of the Third Affiliated Hospital of Nanchang University on November 30,
2013. As the illness became worse, the patient was transferred into Intensive
Care Unit (ICU) of the Department of Critical Care Medicine on December 2. The
patient's condition deteriorated, manifesting multiple organ failure (MOF) on
December 5. At 08:30 on December 6, cardiac arrest occurred, and the patient
died after inefficient resuscitation. (1) Epidemiological investigation: the
patient was an elderly woman, suffering from a variety of chronic diseases
(hypertension, coronary heart disease, myasthenia gravis, etc) and impaired
immune function (undergone thymectomy), all of them were predisposing factors
for deterioration of her health. She had visited the live poultry market one
week before admission, and developed symptoms of influenza. The transmission
route was the respiratory tract, which was similar to H7N9 avian influenza. (2)
CLINICAL MANIFESTATIONS: the patient had flu-like symptoms, such as cough and
fever (39.1 centigrade), but no headache or myalgia. Two days later pneumonia
accompanied with respiratory distress developed and a large amount of bloody
sputum was sucked out through tracheostomy tube (2 000 mL/24 h). Acute kidney
injury, acute respiratory distress syndrome (ARDS), septic shock, and
unconsciousness occurred, all of which was consistent with the diagnosis of H7N9
avian influenza. (3) Auxiliary examination: with the exception of a decrease in
lymphocyte ratio (0.070), aspartate aminotransferase (AST) was slightly
increased (57 U/L), C- reactive protein (CRP) was elevated (>200 mg/L), but the
platelet count, creatine kinase, lactate dehydrogenase, alanine aminotransferase
and myoglobin were not increased, while leucocyte count was increased slightly
(10.34×10(9)/L). The changes in above indexes did not match the characteristics
of H7N9 avian influenza. However, the aggravated consolidation of the lung
conformed to that of H7N9 avian influenza. (4) DIAGNOSIS AND TREATMENT:
according to the clinical manifestations, aggravation of consolidation of the
lung, and epidemiological evidence, the diagnosis of avian influenza was
considered. Though therapeutic dose of oseltamivir was given as antiviral
treatment for the early therapy, and other therapeutic measures such as
energetic respiratory and circulatory support, and immunosuppressant therapy
were given, the patient eventually died from respiratory failure and shock. (5)
The Chinese disease prevention and control center (CDC) confirmed that, the
patient was infected H10N8 avian influenza virus. No person with close contact
with the patient was infected, as screened by Nanchang City and Chinese CDC.
CONCLUSIONS: Human infection with H10N8 avian influenza was not exactly the same
as that of H7N9. It was difficult to get true information from the conventional
laboratory examinations, while the clinical characteristic and epidemiology were
essential for the diagnosis. Referring to the treatment regime for human
infection with H7N9 avian influenza virus, therapeutic dose of neuraminidase
inhibitors could not reverse deterioration of pulmonary pathology. Chinese CDC
found that the risk of human infection and transmission of H10N8 avian influenza
virus through personal contact was low. OBJECTIVES: We assessed the relationship between individual characteristics and
receipt of oseltamivir (Tamiflu) in the United States during the H1N1 pandemic
and other flu seasons.
METHODS: In a cohort of individuals enrolled in pharmacy benefit plans, we used
a multivariate logistic regression model to measure associations between
subscriber characteristics and filling a prescription for oseltamivir during 3
flu seasons (October 2006-May 2007, October 2007-May 2008, and October 2008-May
2010). In 19 states with county-level influenza rates reported, we controlled
for disease burden.
RESULTS: Approximately 56 million subscribers throughout the United States were
included in 1 or more study periods. During pandemic flu, beneficiaries in the
highest income category had 97% greater odds of receiving oseltamivir than those
in the lowest category (P < .001). After we controlled for disease burden,
subscribers in the 2 highest income categories had 2.18 and 1.72 times the odds
of receiving oseltamivir compared with those in the lowest category (P < .001
for both).
CONCLUSIONS: Income was a stronger predictor of oseltamivir receipt than
prevalence of influenza. These findings corroborate concerns about equity of
treatment in pandemics, and they call for improved approaches to distributing
potentially life-saving treatments. BACKGROUND: Influenza is a highly contagious viral infection of the respiratory
system. To attack two processes involved in flu pathogenesis-viral replication
in the infected body and oxidative damages, we studied the combination effect of
neuraminidase inhibitor oseltamivir and antioxidant α-tocopherol in experimental
model of influenza.
METHODS: After inoculation of albino mice with 10 MLD50 (50% mouse lethal dose)
of influenza virus A/Aichi/2/68 (H3N2), oseltamivir was applied orally at three
doses, 2.5 mg/kg, 1.25 mg/kg, and 0.625 mg/kg, for five days post infection.
α-Tocopherol (120 mg/kg, in sunflower oil) was administered intraperitoneally.
Three schemes of α-tocopherol five-day course were tested: onset five or two
days before infection, or on the virus inoculation day.
RESULTS: Strongly dose-dependent augmented antiviral effect of the combination
α-tocopherol and 0.625 mg/kg oseltamivir was demonstrated when α-tocopherol was
administered simultaneously with oseltamivir: a pronounced decrease in mortality
rate (a 78% protection), and a lengthening of mean survival time by 3.2-4 days.
Lung parameters showed a substantial decrease in infectious virus content
(Δ logs = 3.8/4.1) and a marked diminishment of lung index and pathology.
Combination α-tocopherol with 1.25 mg/kg oseltamivir manifested a marked
protective effect, but the effect on lung parameters was less. The combination
effect of α-tocopherol with 2.5 mg/kg oseltamivir did not surpass the
monotherapeutic effect of oseltamivir. When α-tocopherol was applied in courses
starting five or two days before infection, its combination with oseltamivir was
ineffective.
CONCLUSIONS: Evidently, α-tocopherol could be considered as prospective
component of influenza therapy in combination with oseltamivir. |
What is the composition of the gamma-secretase complex? | Gamma-secretase is a multisubunit enzyme complex which is consists of four proteins: presenilin 1 (PS1) or presenilin 2 (PS2), nicastrin, Aph-1 and Pen-2. | Presenilin heterodimers apparently contain the active site of gamma-secretase, a
polytopic aspartyl protease involved in the transmembrane processing of both the
Notch receptor and the amyloid-beta precursor protein. Although critical to
embryonic development and the pathogenesis of Alzheimer's disease, this protease
is difficult to characterize, primarily because it is a multicomponent complex
of integral membrane proteins. Here the functional gamma-secretase complex was
isolated by using an immobilized active site-directed inhibitor of the protease.
Presenilin heterodimers and nicastrin bound specifically to this inhibitor under
conditions tightly correlating with protease activity, whereas several other
presenilin-interacting proteins (beta-catenin, calsenilin, and
presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies
immunoprecipitated gamma-secretase activity from detergent-solubilized
microsomes. Unexpectedly, C83, the major endogenous amyloid-beta precursor
protein substrate of gamma-secretase, was also quantitatively associated with
the complex. These results provide direct biochemical evidence that nicastrin is
a member of the active gamma-secretase complex, indicate that beta-catenin,
calsenilin, and presenilin-associated protein are not required for gamma
activity, and suggest an unprecedented mechanism of substrate-protease
interaction. Gamma-secretase is a protease complex of four integral membrane proteins, with
presenilin (PS) as the apparent catalytic component, and this enzyme processes
the transmembrane domains of a variety of substrates, including the amyloid
beta-protein precursor and the Notch receptor. Here we explore the mechanisms of
structurally diverse gamma-secretase inhibitors by examining their ability to
displace an active site-directed photoprobe from PS heterodimers. Most
gamma-secretase inhibitors, including a potent inhibitor of the PS-like signal
peptide peptidase, blocked the photoprobe from binding to PS1, indicating that
these compounds either bind directly to the active site or alter it through an
allosteric interaction. Conversely, some reported inhibitors failed to displace
this interaction, demonstrating that these compounds do not interfere with the
protease by affecting its active site. Differential effects of the inhibitors
with respect to photoprobe displacement and in cell-based and cell-free assays
suggest that these compounds are important mechanistic tools for deciphering the
workings of this intramembrane-cleaving protease complex and its similarity to
other polytopic aspartyl proteases. Gamma-secretase cleavage is the final enzymatic step generating beta-amyloid via
intramembranous cleavage of the amyloid precursor protein (APP). Presenilin
(PS), initially identified as a gene in which mutations account for the vast
majority of early-onset autosomal domit Alzheimer's disease, is a major
component of gamma-secretase. Enzymatic activity also depends on nicastrin,
Aph-1, and Pen-2. We propose a model in which gamma-secretase components
assemble, interact with substrates initially at a docking site, and then cleave
and release substrates. To test this model, we developed a novel morphological
technique on the basis of advanced fluorescence microscopy methods, fluorescence
lifetime imaging microscopy (FLIM). FLIM allows us to examine protein-protein
"proximity" in intact cells. We show that, although the strongest colocalization
of APP and PS1 is in the perinuclear area, the strongest interactions detected
by FLIM are at or near the cell surface. We also found that APP-PS1 interactions
occur even when gamma-secretase inhibitors or "domit-negative" PS1 mutations
are used to block gamma-secretase activity. Finally, using nicastrin RNA
interference, we demonstrate that nicastrin is critical for APP association with
PS1. We interpret these results to suggest that there is a noncatalytic docking
site closely associated with PS1-gamma-secretase. Two secretases are involved in the generation of amyloid beta-peptide, the
principal component of amyloid plaques in the brains of Alzheimer's disease
patients. While beta-secretase is a classical aspartyl protease, gamma-secretase
activity is associated with a high molecular weight complex. One of the complex
components, which is critically required for gamma-secretase activity is
nicastrin (NCT). Here we investigate the assembly of NCT into the
gamma-secretase complex. NCT mutants either lacking the entire cytoplasmic tail,
the cytoplasmic tail, and the transmembrane domain (TMD), or containing a set of
heterologous TMDs were expressed in cells with strongly reduced levels of
endogenous NCT. Maturation of exogenous NCT, gamma-secretase complex formation
and proteolytic function was then investigated. This revealed that the
cytoplasmic tail of NCT is dispensable for gamma-secretase complex assembly and
function. In contrast, the authentic TMD of NCT is critically required for the
interaction with gamma-secretase complex components and for formation of an
active gamma-secretase complex. Neither soluble NCT lacking any membrane anchor
nor NCT containing a heterologous TMD were inserted into the gamma-secretase
complex. We identified the N-terminal region of the NCT TMD as a functionally
important entity of NCT. These data thus demonstrate that NCT interacts with
other gamma-secretase complex components via its TMD. The gamma-secretase complex catalyzes intramembrane proteolysis of a number of
transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and
E-cadherin. gamma-Secretase is known to contain four major protein constituents:
presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane
proteins. There is increasing evidence that the formation of the complex and the
stability of the individual components are tightly controlled in the cell,
assuring correct composition of functional complexes. In this report, we
investigate the topology, localization, and mechanism for destabilization of
Pen-2 in relation to PS function. We show that PS1 regulates the subcellular
localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the
endoplasmic reticulum (ER) and not transported to post-ER compartments, where
the mature gamma-secretase complexes reside. PS deficiency also leads to
destabilization of Pen-2, which is alleviated by proteasome inhibitors. In
keeping with this, we show that Pen-2, which adopts a hairpin structure with the
N and C termini facing the luminal space, is ubiquitylated prior to degradation
and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our
data suggest that failure to become incorporated into the gamma-secretase
complex leads to degradation of Pen-2 through the ER-associated
degradation-proteasome pathway. Gamma-secretase catalyzes the proteolytic processing of a number of integral
membrane proteins, including amyloid precursor protein (APP) and Notch. The
native gamma-secretase is a heterogeneous population of large membrane protein
complexes containing presenilin 1 (PS1) or presenilin 2 (PS2), aph-1a or aph-1b,
nicastrin, and pen-2. Here we report the reconstitution of a gamma-secretase
complex in Sf9 cells by co-infection with baculoviruses carrying the PS1,
nicastrin, pen-2, and aph-1a genes. The reconstituted enzyme processes C99 and
the Notch-like substrate N160 and displays the characteristic features of
gamma-secretase in terms of sensitivity to a gamma-secretase inhibitor,
upregulation of Abeta42 production by a familial Alzheimer's disease (FAD)
mutation in the APP gene, and downregulation of Notch processing by PS1 FAD
mutations. However, the ratio of Abeta42:Abeta40 production by the reconstituted
gamma-secretase is significantly higher than that of the native enzyme from 293
cells. Unlike in mammalian cells where PS1 FAD mutations cause an increase in
Abeta42 production, PS1 FAD missense mutations in the reconstitution system
alter the cleavage sites in the C99 substrate without changing the
Abeta42:Abeta40 ratio. In addition, PS1DeltaE9 is a loss-of-function mutation in
both C99 and N160 processing. Reconstitution of gamma-secretase provides a
homogeneous system for studying the individual gamma-secretase complexes and
their roles in Abeta production, Notch processing and AD pathogenesis. These
studies may provide important insight into the development of a new generation
of selective gamma-secretase inhibitors with an improved side effect profile. The gamma-secretase complex processes substrate proteins within membranes and
consists of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. PS
harbours the enzymatic activity of the complex, and there are two mammalian PS
homologues: PS1 and PS2. PS undergoes endoproteolysis, generating the N- and
C-terminal fragments, NTF and CTF, which represent the active species of PS. To
characterize the functional similarity between complexes of various PS
composition, we analysed PS1, PS2, and chimeric PS composed of the NTF from PS1
and CTF from PS2, or vice versa, in assembly and function of the gamma-secretase
complex. Chimeric PSs, like PS1 and PS2, undergo normal endoproteolysis when
introduced into cells devoid of endogenous PS. Furthermore, PS2 CTF can, at
least partially, restore processing in a truncated PS1, which cannot undergo
endoproteolysis. All PS forms enable maturation of nicastrin and cleave full
length Notch receptors, indicating that both PS1 and PS2 are present at the cell
surface. Finally, when co-introduced as separate molecules, NTF and CTF of
different PS origin reconstitute gamma-secretase activity. In conclusion, these
data show that endoproteolysis, NTF-CTF interactions, and the assembly and
activity of gamma-secretase complexes are very conserved between PS1 and PS2. gamma-Secretase is a key enzyme involved in the processing of the beta-amyloid
precursor protein into amyloid beta-peptides (Abeta). Abeta accumulates and
forms plaques in Alzheimer's disease (AD) brains. A progressive
neurodegeneration and cognitive decline occurs during the course of the disease,
and Abeta is believed to be central for the molecular pathogenesis of AD.
Apoptosis has been implicated as one of the mechanisms behind the neuronal cell
loss seen in AD. We have studied preservation and activity of the
gamma-secretase complex during apoptosis in neuroblastoma cells (SH-SY5Y)
exposed to staurosporine (STS). We report that the known components (presenilin,
Nicastrin, Aph-1 and Pen-2) interact and form active gamma-secretase complexes
in apoptotic cells. In addition, the fragments corresponding to the PS1
N-terminal fragment and the caspase-cleaved PS1 C-terminal fragment
(PS1-caspCTF) were found to form active gamma-secretase complexes when
co-expressed in presenilin (PS) knockout cells. Interestingly, PS1-caspCTF
replaced the normal PS1 C-terminal fragment and was co-immunoprecipitated with
the gamma-secretase complex in SH-SY5Y cells exposed to STS. In addition, Abeta
was detected in medium from apoptotic HEK APP(swe) cells. Together, the data
show that gamma-secretase complexes containing PS1-caspCTF are active, and
suggest that this proteolytic activity is also important in dying cells and may
affect the progression of AD. gamma-Secretase is a membrane-embedded multi-protein complex that catalyzes the
final cut of the Alzheimer's disease-related amyloid precursor protein (APP) to
amyloid-beta peptides of variable length (37-43 amino acids) via an unusual
intramembrane cleavage. Recent findings propose that some commonly used
non-steroidal anti-inflammatory drugs (NSAIDs) have the ability to modulate
specifically gamma-secretase activity without inhibiting the enzyme as a whole.
These drugs may shift the processing of APP from the longer amyloid-beta 42
peptide towards shorter, less fibrillogenic and less toxic amyloid-beta species.
We hypothesize that gamma-secretase activity, as an enzyme that is strictly
associated with cellular membranes, is sensitive to alterations of the
hydrophobic membrane environment. Here, we show that the gamma-secretase
modulator and amyloid-beta 42-lowering drug sulindac sulfide alters the physical
state of the membrane and strongly decreases fluidity of cellular membranes.
Furthermore, sulindac sulfide changed the protein composition of membrane
microdomains, the so-called lipid rafts. Most significantly, APP C-terminal
fragments (CTFs) were redistributed from rafts towards non-raft membrane
domains. This could be demonstrated also in cell-free assays, where in addition
presenilin-1, the catalytic subunit of the gamma-secretase complex, was shifted
out of lipid rafts. Together, these findings suggest that sulindac sulfide
directly alters the membrane architecture and shifts the
gamma-secretase-mediated cleavage of APP towards a hydrophobic environment where
the enzyme-substrate complex is in a conformation for processing preferentially
shorter amyloid-beta peptides. Gamma-secretase mediates the final proteolytic cleavage, which liberates amyloid
beta-peptide (Abeta), the major component of senile plaques in the brains of
Alzheimer disease patients. Therefore, gamma-secretase is a prime target for
Abeta-lowering therapeutic strategies. gamma-Secretase is a protein complex
composed of four different subunits, presenilin (PS), APH-1, nicastrin, and
PEN-2, which are most likely present in a 1:1:1:1 stoichiometry. PS harbors the
catalytically active site, which is critically required for the aspartyl
protease activity of gamma-secretase. Moreover, numerous familial Alzheimer
disease-associated mutations within the PSs increase the production of the
aggregation-prone and neurotoxic 42-amino acid Abeta. Nicastrin may serve as a
substrate receptor, although this has recently been challenged. PEN-2 is
required to stabilize PS within the gamma-secretase complex. No particular
function has so far been assigned to APH-1. The four components are sufficient
and required for gamma-secretase activity. At least six different
gamma-secretase complexes exist that are composed of different variants of PS
and APH-1. All gamma-secretase complexes can exert pathological Abeta
production. Assembly of the gamma-secretase complex occurs within the
endoplasmic reticulum, and only fully assembled and functional gamma-secretase
complexes are transported to the plasma membrane. Structural analysis by
electron microscopy and chemical cross-linking reveals a water-containing
cavity, which allows intramembrane proteolysis. Specific and highly sensitive
gamma-secretase inhibitors have been developed; however, they interfere with the
physiological function of gamma-secretase in Notch signaling and thus cause
rather significant side effects in human trials. Modulators of gamma-secretase,
which selectively affect the production of the pathological 42-amino acid Abeta,
do not inhibit Notch signaling. Gamma-secretase is a multiprotein, intramembrane-cleaving protease with a
growing list of protein substrates, including the Notch receptors and the
amyloid precursor protein. The four components of gamma-secretase
complex--presenilin (PS), nicastrin (NCT), Pen2, and Aph1--are all thought to be
essential for activity. The catalytic domain resides within PS proteins, NCT has
been suggested to be critical for substrate recognition, and the contributions
of Pen2 and Aph1 remain unclear. The role of NCT has been challenged recently by
the observation that a critical residue (E332) in NCT, which had been thought to
be essential for gamma-secretase activity, is instead involved in complex
maturation. Here, we report that NCT is dispensable for gamma-secretase
activity. NCT-independent gamma-secretase activity can be detected in two
independent NCT-deficient mouse embryonic fibroblast lines and blocked by the
gamma-secretase inhibitors
N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester and
L-685,458. This catalytic activity requires prior ectodomain shedding of the
substrate and can cleave ligand-activated endogenous Notch receptors, indicating
presence of this activity at the plasma membrane. Small interfering RNA
knockdown experiments demonstrated that NCT-independent gamma-secretase activity
requires the presence of PS1, Pen2, and Aph1a but can tolerate knockdown of PS2
or Aph1b. We conclude that a PS1/Pen2/Aph1a trimeric complex is an active
enzyme, displaying biochemical properties similar to those of gamma-secretase
and roughly 50% of its activity when normalized to PS1 N-terminal fragment
levels. This PS1/Pen2/Aph1a complex, however, is highly unstable. Thus, NCT acts
to stabilize gamma-secretase but is not required for substrate recognition. Presenilin (PS1 or PS2) is the catalytic component of the gamma-secretase
complex, which mediates the final proteolytic processing step leading to the
Alzheimer's disease (AD)-characterizing amyloid beta-peptide. PS is cleaved
during complex assembly into its characteristic N- and C-terminal fragments.
Both fragments are integral components of physiologically active gamma-secretase
and harbor the two critical aspartyl residues of the active site domain. While
the minimal subunit composition of gamma-secretase has been defined and numerous
substrates were identified, the cellular mechanism of the endoproteolytic
cleavage of PS is still unclear. We addressed this pivotal question by
investigating whether familial AD (FAD)-associated PS1 mutations affect the
precision of PS endoproteolysis in a manner similar to the way that such
mutations shift the intramembrane cleavage of gamma-secretase substrates. We
demonstrate that all FAD mutations investigated still allow endoproteolysis to
occur. However, the precision of PS1 endoproteolysis is affected by PS1
mutations. Comparing the cleavage products generated by a variety of PS1 mutants
revealed that specifically cleavages at positions 293 and 296 of PS1 are
selectively affected. Systematic mutagenesis around the cleavage sites revealed
a stepwise three amino acid spaced cleavage mechanism of PS endoproteolysis
reminiscent to the epsilon-, zeta-, and gamma-cleavages described for typical
gamma-secretase substrates, such as the beta-amyloid precursor protein. Our
findings therefore suggest that intramembranous cleavage by gamma-secretase and
related intramembrane-cleaving proteases may generally occur via stepwise
endoproteolysis. γ-Secretase complexes are involved in the generation of amyloid-β (Aβ) in the
brain. Therefore, γ-secretase has been proposed as a potential therapeutic
target in Alzheimer disease (AD). Targeting γ-secretase activity in AD requires
the pharmacological dissociation of the processing of physiological relevant
substrates and the generation of "toxic" Aβ. Previous reports suggest the
differential targeting of γ-secretase complexes, based on their subunit
composition, as a valid strategy. However, little is known about the biochemical
properties of the different complexes, and key questions regarding their Aβ
product profiles should be first addressed. Here, we expressed, purified, and
analyzed, under the same conditions, the endopeptidase and carboxypeptidase-like
activities of the four γ-secretase complexes present in humans. We find that the
nature of the catalytic subunit in the complex affects both activities.
Interestingly, PSEN2 complexes discriminate between the Aβ40 and Aβ38 production
lines, indicating that Aβ generation in one or the other pathway can be
dissociated. In contrast, the APH1 subunit mainly affects the
carboxypeptidase-like activity, with APH1B complexes favoring the generation of
longer Aβ peptides. In addition, we determined that expression of a single human
γ-secretase complex in cell lines retains the intrinsic attributes of the
protease while present in the membrane, providing validation for the in vitro
studies. In conclusion, our data show that each γ-secretase complex produces a
characteristic Aβ signature. The qualitative and quantitative differences
between different γ-secretase complexes could be used to advance drug
development in AD and other disorders. γ-Secretase is involved in the regulated intramembrane proteolysis of amyloid-β
protein precursor (AβPP) and of many other important physiological substrates.
γ-secretase is a multiproteic complex made of four main core components, namely
presenilin 1 or 2, APH-1, PEN-2, and Nicastrin. Since APH-1 exists as different
variants, combinations of these proteins can theoretically yield distinct
γ-secretase complexes. Whether γ-secretase complexes trafficking and targeting
to either similar or distinct subcellular compartments depend upon their
molecular composition remains unknown. A differential complex-specific
distribution may drive a narrow specificity for a subset of substrates that
would traffic within the same cellular compartments. Here, we generated bigenic
expression vectors to co-express untagged nicastrin or presenilin 1 together
with either PEN-2 or distinct variants of APH-1 (aL, aS and b) tagged with
complementary fragments of the fluorescent protein Venus. We show that these
constructs allow the formation of functional γ-secretase complexes and their
visualization with bimolecular fluorescence complementation (BiFC). BiFC can be
detected at the plasma membrane as well as in endosomes/lysosomes in addition to
the endoplasmic reticulum (ER) of COS-7 cells transfected with the different
variants of APH-1. However, the majority of cells co-transfected with APH-1b
presented BiFC signal only in the ER, suggesting enhanced retention/retrieval of
APH-1b-containing γ-secretase complexes. Therefore, the new tools described here
should be helpful to decipher the precise subcellular trafficking of γ-secretase
complexes and to delineate the distinct variant-linked pathways in various
cellular systems. Gamma-secretase is a multisubunit complex with intramembrane proteolytic
activity. In humans it was identified in genetic screens of patients suffering
from familial forms of Alzheimer's disease, and since then it was shown to
mediate cleavage of more than 80 substrates, including amyloid precursor protein
or Notch receptor. Moreover, in animals, γ-secretase was shown to be involved in
regulation of a wide range of cellular events, including cell signalling,
regulation of endocytosis of membrane proteins, their trafficking, and
degradation. Here we show that genes coding for γ-secretase homologues are
present in plant genomes. Also, amino acid motifs crucial for γ-secretase
activity are conserved in plants. Moreover, all γ-secretase subunits: PS1/PS2,
APH-1, PEN-2, and NCT colocalize and interact with each other in Arabidopsis
thaliana protoplasts. The intracellular localization of γ-secretase subunits in
Arabidopsis protoplasts revealed a distribution in endomembrane system
compartments that is consistent with data from animal studies. Together, our
data may be considered as a starting point for analysis of γ-secretase in
plants. γ-Secretase complexes achieve the production of amyloid peptides playing a key
role in Alzheimer disease. These proteases have many substrates involved in
important physiological functions. They are composed of two constant subunits,
nicastrin and PEN2, and two variable ones, presenilin (PS1 or PS2) and APH1
(APH1aL, APH1aS, or APH1b). Whether the composition of a given γ-secretase
complex determines a specific cellular targeting remains unsolved. Here we
combined a bidirectional inducible promoter and 2A peptide technology to
generate constructs for the temporary, stoichiometric co-expression of six
different combinations of the four γ-secretase subunits including EGFP-tagged
nicastrin. These plasmids allow for the formation of functional γ-secretase
complexes displaying specific activities and maturations. We show that
PS1-containing γ-secretase complexes were targeted to the plasma membrane,
whereas PS2-containing ones were addressed to the trans-Golgi network, to
recycling endosomes, and, depending on the APH1-variant, to late endocytic
compartments. Overall, these novel constructs unravel a presenilin-dependent
subcellular targeting of γ-secretase complexes. These tools should prove useful
to determine whether the cellular distribution of γ-secretase complexes
contributes to substrate selectivity and to delineate regulations of their
trafficking. |
List kinases that phosphorylates the protein Bora. | During cell division Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, and GSK3β and Cdk1 albeit at distinctive sites. | Cdk1 and Plk1/Plx1 activation leads to their inactivation through negative
feedback loops. Cdk1 deactivates itself by activating the APC/C, consequently
generating embryonic cell cycle oscillations. APC/C inhibition by the mitotic
checkpoint in somatic cells and the cytostatic factor (CSF) in oocytes sustain
the mitotic state. Plk1/Plx1 targets its co-activator Bora for degradation, but
it remains unclear how embryonic oscillations in Plx1 activity are generated,
and how Plk1/Plx1 activity is sustained during mitosis. We show that
Plx1-mediated degradation of Bora in interphase generates oscillations in Plx1
activity and is essential for development. In CSF extracts, phosphorylation of
Bora on the Cdk consensus site T52 blocks Bora degradation. Upon fertilization,
Calcineurin dephosphorylates T52, triggering Plx1 oscillations. Similarly, we
find that GFP-Bora is degraded when Plk1 activity spreads to somatic cell
cytoplasm before mitosis. Interestingly, GFP-Bora degradation stops upon mitotic
entry when Cdk1 activity is high. We hypothesize that Cdk1 controls Bora through
an incoherent feedforward loop synchronizing the activities of mitotic kinases. Mitosis is orchestrated by several protein kinases including Cdks, Plks and
Aurora kinases. Despite considerable progress toward understanding the
individual function of these protein kinases, how their activity is coordinated
in space and time during mitosis is less well understood. In a recent article
published in the Journal of Cell Biology, we show that CDK-1 regulates PLK-1
activity during mitosis in C. elegans embryos through multisite phosphorylation
of the PLK-1 activator SPAT-1 (Aurora Borealis, Bora in human). SPAT-1 variants
mutated on CDK-1 phosphorylation sites results in severe delays in mitotic
entry, mimicking embryos lacking spat-1 or plk-1 function. We further show that
SPAT-1 phosphorylation by CDK-1 promotes its binding to PLK-1 and stimulates
PLK-1 phosphorylation on its activator T-loop by Aurora A kinase in vitro.
Likewise, we find that phosphorylation of Bora by Cdk1 promotes phosphorylation
of human Plk1 by Aurora A suggesting that this mechanism is conserved in humans.
These results indicate that Cdk1 regulates Plk1 by boosting its kinase activity.
Here we discuss these recent findings and open questions regarding the
regulation of Plk1/PLK-1 by Cdk1/CDK-1 and Bora/SPAT-1. Polo-like kinase 1 (Plk1) is an important mitotic kinase that is crucial for
entry into mitosis after recovery from DNA damage-induced cell cycle arrest.
Plk1 activation is promoted by the conserved protein Bora (SPAT-1 in C.
elegans), which stimulates the phosphorylation of a conserved residue in the
activation loop by the Aurora A kinase. In a recent article published in Cell
Reports, we show that the master mitotic kinase Cdk1 contributes to Plk1
activation through SPAT-1/Bora phosphorylation. We identified 3 conserved Sp/Tp
residues that are located in the N-terminal, most conserved part, of
SPAT-1/Bora. Phosphorylation of these sites by Cdk1 is essential for Plk1
function in mitotic entry in C. elegans embryos and during DNA damage checkpoint
recovery in mammalian cells. Here, using an untargeted Förster Resoce Energy
Transfer (FRET) biosensor to monitor Plk1 activation, we provide additional
experimental evidence supporting the importance of these phosphorylation sites
for Plk1 activation and subsequent mitotic entry after DNA damage. We also
briefly discuss the mechanism of Plk1 activation and the potential role of Bora
phosphorylation by Cdk1 in this process. As Plk1 is overexpressed in cancer
cells and this correlates with poor prognosis, understanding how Bora
contributes to Plk1 activation is paramount for the development of innovative
therapeutical approaches. |
Are deletions of chromosomal regulatory boundaries associated with congenital disease? | Yes. Enhancer adoption caused by deletions of regulatory boundaries may contribute to a substantial minority of copy-number variation phenotypes and should thus be taken into account in their medical interpretation. | BACKGROUND: Recent data from genome-wide chromosome conformation capture
analysis indicate that the human genome is divided into conserved megabase-sized
self-interacting regions called topological domains. These topological domains
form the regulatory backbone of the genome and are separated by regulatory
boundary elements or barriers. Copy-number variations can potentially alter the
topological domain architecture by deleting or duplicating the barriers and
thereby allowing enhancers from neighboring domains to ectopically activate
genes causing misexpression and disease, a mutational mechanism that has
recently been termed enhancer adoption.
RESULTS: We use the Human Phenotype Ontology database to relate the phenotypes
of 922 deletion cases recorded in the DECIPHER database to monogenic diseases
associated with genes in or adjacent to the deletions. We identify combinations
of tissue-specific enhancers and genes adjacent to the deletion and associated
with phenotypes in the corresponding tissue, whereby the phenotype matched that
observed in the deletion. We compare this computationally with a gene-dosage
pathomechanism that attempts to explain the deletion phenotype based on
haploinsufficiency of genes located within the deletions. Up to 11.8% of the
deletions could be best explained by enhancer adoption or a combination of
enhancer adoption and gene-dosage effects.
CONCLUSIONS: Our results suggest that enhancer adoption caused by deletions of
regulatory boundaries may contribute to a substantial minority of copy-number
variation phenotypes and should thus be taken into account in their medical
interpretation. |
What is the role of the UBC9 enzyme in the protein sumoylation pathway? | The small ubiquitin-like modifier (SUMO) pathway in eukaryotes is an essential post-translational modification required for a variety of cellular processes, development and organelle biogenesis. SUMO-conjugating enzyme (Ubc9) is the sole conjunction enzyme in the SUMO pathway. | The activity of the p53 tumor suppressor protein and the c-Jun protooncogene is
regulated by posttranslational modifications, such as phosphorylation or
ubiquitination. In addition, covalent attachment of the ubiquitin-like modifier
SUMO appears to modulate their transcriptional activity. Sumoylation proceeds
via an enzymatic pathway that is mechanistically analogous to ubiquitination,
but requires a different E1-activating enzyme and Ubc9, a SUMO-specific
E2-conjugating enzyme. Here, we show that two members of the PIAS family, PIAS1
and PIASxbeta, act as specific E3-like ligases that promote sumoylation of p53
and c-Jun in vitro and in vivo. The PIAS proteins physically interact with both
p53 and c-Jun. In addition, they bind to Ubc9, suggesting that they recruit the
E2 enzyme to their respective substrate. The SUMO ligase activity requires the
conserved zinc-finger domain, which is distantly related to the essential
RING-finger motif, found in a subset of ubiquitin ligases. Furthermore, similar
to RING-type ubiquitin ligases, PIASxbeta can catalyze its own modification.
Hence, these data further extend the analogy between the ubiquitin and SUMO
pathway. Strikingly, PIAS proteins strongly repress the transcriptional activity
of p53, suggesting that the PIAS-SUMO pathway plays a crucial role in the
regulation of p53 and presumably other transcription factors. Ubc9 is an E2-conjugating enzyme required for sumoylation and has been
implicated in regulating several critical cellular pathways. We have shown
previously that Ubc9 is important for sumoylation and nucleolar delocalization
of topoisomerase (topo) I in response to topo I inhibitors such as topotecan.
However, the role for Ubc9 in tumor drug responsiveness is not clear. In this
study, we found that although MCF7 cells expressing a Ubc9 domit-negative
mutant (Ubc9-DN) display decreased activity of topo I, these cells are more
sensitive to the topo I inhibitor topotecan and other anticancer agents such as
VM-26 and cisplatin. In addition, we found that alteration of Ubc9 expression
correlates with drug responsiveness in tumor cell lines. To understand possible
mechanisms of Ubc9-associated drug responsiveness, we examined several proteins
that have been shown to interact with Ubc9 and that may be involved in drug
responsiveness. One such protein is Daxx, which is a Fas-associated protein that
plays a role in Fas-mediated apoptosis by participating in a caspase-independent
pathway through activation of apoptosis signal-regulating kinase 1 and c-Jun
NH(2)-terminal kinase. We found that cells expressing Ubc9-DN accumulate more
cytoplasmic Daxx than the control cells. Because cytoplasmic Daxx is believed to
participate in cellular apoptosis, we suggest that the interaction of Ubc9 with
Daxx and subsequent alteration in the subcellular localization of Daxx may
contribute to the increased sensitivity to anticancer drugs in the cells
expressing Ubc9-DN. Finally, we found that overexpression of Daxx sensitizes
cells to anticancer drugs possibly in part through alterations of the ratio of
cytoplasmic and nuclear Daxx. Together, our results suggest a role for Ubc9 in
tumor drug responsiveness. The SUMO pathway parallels the classical ubiquitinylation pathway with three
discrete steps: activation involving the enzyme E1, conjugation involving the E2
enzyme UBC9, and substrate modification through the cooperative association of
UBC9 and E3 ligases. We report here that the adenoviral protein Gam1 inhibits
the SUMO pathway by interfering with the activity of E1 (SAE1/SAE2). In vivo,
Gam1 expression leads to SAE1/SAE2 inactivation, both SAE1/SAE2 and UBC9
disappearance, and overall inhibition of protein sumoylation. This results in
transcriptional activation of some promoters and is directly linked to
inhibition of sumoylation of the transcriptional activators involved. Our
results identify a mechanism for interfering with the SUMO pathway and with
transcription that could have an impact in the design of novel pharmaceutical
agents. They also point out once again to the extraordinary ability of
eukaryotic viruses to interfere with the biology of host cells by targeting
fundamental biochemical processes. Sumoylation and ubiquitinylation reversibly regulate the activity of
transcription factors through covalent attachment to lysine residues of target
proteins. We examined whether the Ets-1 transcription factor is modified by
sumoylation and/or ubiquitinylation. Among four potential SUMO motifs in Ets-1,
we identified lysines 15 and 227 within the LK(15)YE and IK(227)QE motifs, as
being the sumoylation acceptor sites. Using transfection of Ets-1 wildtype (WT)
or its sumoylation deficient version (Ets-1 K15R/K227R), as well as WT or mutant
proteins of the SUMO pathway, we further demonstrated that the E2
SUMO-conjugating enzyme Ubc9 and a E3 SUMO ligase, PIASy, can enhance Ets-1
sumoylation, while a SUMO protease, SENP1, can desumoylate Ets-1. We also found
that Ets-1 is modified by K48-linked polyubiquitinylation independently of the
sumoylation acceptor sites and is degraded through the 26S proteasome pathway,
while sumoylation of Ets-1 does not affect its stability. Finally, sumoylation
of Ets-1 leads to reduced transactivation and we demonstrated that previously
identified critical lysine residues in Synergistic Control motifs are the
sumoylation acceptor sites of Ets-1. These data show that Ets-1 can be modified
by sumoylation and/or ubiquitinylation, with sumoylation repressing
transcriptional activity of Ets-1 and having no clear antagonistic action on the
ubiquitin-proteasome degradation pathway. RAP80, a nuclear protein with two functional ubiquitin-interaction motifs (UIMs)
at its N-terminus, plays a critical role in the regulation of estrogen receptor
alpha and DNA damage response signaling. A yeast two-hybrid screen identified
the SUMO-conjugating enzyme UBC9 as a protein interacting with RAP80. The
interaction of RAP80 with UBC9 was confirmed by co-immunoprecipitation and GST
pull-down analyses. The region between aa 122-204 was critical for the
interaction of RAP80 with UBC9. In addition, we demonstrate that RAP80 is a
target for SUMO-1 modification in intact cells. Expression of UBC9 enhanced
RAP80 mono-sumoylation and also induced multi-sumoylation of RAP80. In addition
to SUMO-1, RAP80 was efficiently conjugated to SUMO-3 but was only a weak
substrate for SUMO-2 conjugation. These findings suggest that sumoylation plays
a role in the regulation of RAP80 functions. A primary step in activating the alternative nuclear factor-kappaB (NF-kappaB)
pathway requires NF-kappaB2/p100 processing to generate p52. In most cases,
stimuli-induced p100 processing is dependent on NF-kappaB-inducing
kinase/IkappaB kinase alpha-mediated phosphorylation and ubiquitination. Here,
we report that post-translational modification of p100 at specific sites by the
small ubiquitin-like modifier (SUMO) is another determining factor for
stimuli-induced p100 processing. The results show that basal SUMO modification
is required for stimuli-induced p100 phosphorylation and that blocking
SUMOylation of p100, either by site-directed mutation or by short interfering
RNA-targeted diminution of E2 SUMO-conjugating enzyme Ubc9, inhibits various
physiological stimuli-induced p100 processing and ultimate activation of the
alternative NF-kappaB pathway. Together, these findings show the crucial role of
SUMO1 modification in p100 processing and provide mechanistic insights into the
participation of SUMO1 modification in the regulation of signal transduction. BACKGROUND: In astrocytes, the inflammatory induction of Nitric Oxide Synthase
type 2 (NOS2) is inhibited by noradrenaline (NA) at the transcriptional level
however its effects on specific transcription factors are not fully known.
Recent studies show that the activity of several transcription factors including
C/EBPbeta, which is needed for maximal NOS2 expression, is modulated by
conjugation of the small molecular weight protein SUMO. We examined whether the
expression of SUMO Related Genes (SRGs: SUMO-1, the conjugating enzyme Ubc9, and
the protease SENP1) are affected by inflammatory conditions or NA and whether
SUMO-1 regulates NOS2 through interaction with C/EBPbeta.
METHODS: Bacterial endotoxin lipopolysaccharide (LPS) was used to induce
inflammatory responses including NOS2 expression in primary astrocytes. The mRNA
levels of SRGs were determined by QPCR. A functional role for SUMOylation was
evaluated by determining effects of over-expressing SRGs on NOS2 promoter and
NFkappaB binding-element reporter constructs. Interactions of SUMO-1 and
C/EBPbeta with the NOS2 promoter were examined by chromatin immunoprecipitation
assays. Interactions of SUMO-1 with C/EBPbeta were examined by
immunoprecipitation and Western blot analysis and by fluorescence resoce
energy transfer (FRET) assays.
RESULTS: LPS decreased mRNA levels of SUMO-1, Ubc9 and SENP1 in primary
astrocytes and a similar decrease occurred during normal aging in brain. NA
attenuated the LPS-induced reductions and increased SUMO-1 above basal levels.
Over-expression of SUMO-1, Ubc9, or SENP1 reduced the activation of a NOS2
promoter, whereas activation of a 4 x NFkappaB binding-element reporter was only
reduced by SUMO-1. ChIP studies revealed interactions of SUMO-1 and C/EBPbeta
with C/EBP binding sites on the NOS2 promoter that were modulated by LPS and NA.
SUMO-1 co-precipitated with C/EBPbeta and a close proximity was confirmed by
FRET analysis.
CONCLUSION: Our results demonstrate that SUMOylation regulates NOS2 expression
in astrocytes, and point to modification of C/EBPbeta as a possible mechanism of
action. Targeting the SUMOylation pathway may therefore offer a novel means to
regulate inflammatory NOS2 expression in neurological conditions and diseases. Tec1 is a transcription factor in the yeast mitogen-activated protein kinase
(MAPK) pathway that controls invasive growth. Previously we reported that a
fraction of Tec1 protein is sumoylated on residue lysine 54 in normally growing
cells. Here we describe regulation and functional consequences of Tec1
sumoylation. We found that activation of Kss1, the MAPK that directly activates
Tec1, results in a decrease in Tec1 sumoylation and a concurrent increase of
Tec1 transcriptional activity. Consistent with a role of sumoylation in
inhibiting Tec1 activity, specifically increasing sumoylation of Tec1 by fusing
it to the sumoylating enzyme Ubc9 leads to a dramatic decrease of Tec1
transcriptional activity. Invasive growth is also compromised in Tec1-Ubc9. In
contrast, fusing sumoylation-site mutant Tec1, i.e., Tec1(K54R), to Ubc9 did not
significantly alter transcriptional activation and had a less effect on invasive
growth. Taken together, these findings provide evidence for regulated
sumoylation as a mechanism to modulate the activity of Tec1 and validate Ubc9
fusion-directed sumoylation as a useful approach for studying protein
sumoylation. Here we have identified host cell proteins involved with the cellular
SUMOylation pathway, SUMO-1 (small ubiquitin-like modifier) and UBC9, a SUMO-1
conjugating enzyme that interact with classical swine fever virus (CSFV) Core
protein. Five highly conserved lysine residues (K179, K180, K220, K221, and
K246) within the CSFV Core were identified as putative SUMOylation sites.
Analysis of these interactions showed that K179A, K180A, and K221A substitutions
disrupt Core-SUMO-1 binding, while K220A substitution precludes Core-UBC9
binding. In vivo, Core mutant viruses (K179A, K180A, K220A, K221A) and (K220A,
K221A) harboring those substitutions were attenuated in swine. These data shows
a clear correlation between the disruption of Core protein binding to SUMO-1 and
UBC9 and CSFV attenuation. Overall, these data suggest that the interaction of
Core with the cellular SUMOylation pathway plays a significant role in the CSFV
growth cycle in vivo. Sumoylation, the covalent attachment of SUMO (Small Ubiquitin-Like Modifier) to
proteins, differs from other Ubl (Ubiquitin-like) pathways. In sumoylation, E2
ligase Ubc9 can function without E3 enzymes, albeit with lower reaction
efficiency. Here, we study the mechanism through which E3 ligase RanBP2 triggers
target recognition and catalysis by E2 Ubc9. Two mechanisms were proposed for
sumoylation. While in both the first step involves Ubc9 conjugation to SUMO, the
subsequent sequence of events differs: in the first E2-SUMO forms a complex with
the target and E3, followed by SUMO transfer to the target. In the second,
Ubc9-SUMO binds to the target and facilitates SUMO transfer without E3. Using
dynamic correlations obtained from explicit solvent molecular dynamic
simulations we illustrate the key roles played by allostery in both mechanisms.
Pre-existence of conformational states explains the experimental observations
that sumoylation can occur without E3, even though at a reduced rate.
Furthermore, we propose a mechanism for enhancement of sumoylation by E3.
Analysis of the conformational ensembles of the complex of E2 conjugated to SUMO
illustrates that the E2 enzyme is already largely pre-organized for target
binding and catalysis; E3 binding shifts the equilibrium and enhances these
pre-existing populations. We further observe that E3 binding regulates
allosterically the key residues in E2, Ubc9 Asp100/Lys101 E2, for the target
recognition. The Drosophila protein Sex Comb on Midleg (Scm) is a member of the Polycomb
group (PcG), a set of transcriptional repressors that maintain silencing of
homeotic genes during development. Recent findings have identified PcG proteins
both as targets for modification by the small ubiquitin-like modifier (SUMO)
protein and as catalytic components of the SUMO conjugation pathway. We have
found that the SUMO-conjugating enzyme Ubc9 binds to Scm and that this
interaction, which requires the Scm C-terminal sterile α motif (SAM) domain, is
crucial for the efficient sumoylation of Scm. Scm is associated with the major
Polycomb response element (PRE) of the homeotic gene Ultrabithorax (Ubx), and
efficient PRE recruitment requires an intact Scm SAM domain. Global reduction of
sumoylation augments binding of Scm to the PRE. This is likely to be a direct
effect of Scm sumoylation because mutations in the SUMO acceptor sites in Scm
enhance its recruitment to the PRE, whereas translational fusion of SUMO to the
Scm N terminus interferes with this recruitment. In the metathorax, Ubx
expression promotes haltere formation and suppresses wing development. When SUMO
levels are reduced, we observe decreased expression of Ubx and partial
haltere-to-wing transformation phenotypes. These observations suggest that SUMO
negatively regulates Scm function by impeding its recruitment to the Ubx major
PRE. BACKGROUND: One aspect of brain death is cardiovascular deregulation because
asystole invariably occurs shortly after its diagnosis. A suitable neural
substrate for mechanistic delineation of this aspect of brain death resides in
the rostral ventrolateral medulla (RVLM). RVLM is the origin of a life-and-death
signal that our laboratory detected from blood pressure of comatose patients
that disappears before brain death ensues. At the same time, transcriptional
upregulation of heme oxygenase-1 in RVLM by hypoxia-inducible factor-1α (HIF-1α)
plays a pro-life role in experimental brain death, and HIF-1α is subject to
sumoylation activated by transient cerebral ischemia. It follows that
sumoylation of HIF-1α in RVLM in response to hypoxia may play a modulatory role
on brain stem cardiovascular regulation during experimental brain death.
METHODOLOGY/PRINCIPAL FINDINGS: A clinically relevant animal model that employed
mevinphos as the experimental insult in Sprague-Dawley rat was used. Biochemical
changes in RVLM during distinct phenotypes in systemic arterial pressure
spectrum that reflect maintained or defunct brain stem cardiovascular regulation
were studied. Western blot analysis, EMSA, ELISA, confocal microscopy and
immunoprecipitation demonstrated that drastic tissue hypoxia, elevated levels of
proteins conjugated by small ubiquitin-related modifier-1 (SUMO-1), Ubc9 (the
only known conjugating enzyme for the sumoylation pathway) or HIF-1α, augmented
sumoylation of HIF-1α, nucleus-bound translocation and enhanced transcriptional
activity of HIF-1α in RVLM neurons took place preferentially during the pro-life
phase of experimental brain death. Furthermore, loss-of-function manipulations
by immunoneutralization of SUMO-1, Ubc9 or HIF-1α in RVLM blunted the
upregulated nitric oxide synthase I/protein kinase G signaling cascade, which
sustains the brain stem cardiovascular regulatory machinery during the pro-life
phase.
CONCLUSIONS/SIGNIFICANCE: We conclude that sumoylation of HIF-1α in RVLM
ameliorates brain stem cardiovascular regulatory failure during experimental
brain death via upregulation of nitric oxide synthase I/protein kinase G
signaling. This information should offer new therapeutic initiatives against
this fatal eventuality. Global sumoylation, SUMO chain formation, and genome stabilization are all
outputs generated by a limited repertoire of enzymes. Mechanisms driving
selectivity for each of these processes are largely uncharacterized. Here,
through crystallographic analyses we show that the SUMO E2 Ubc9 forms a
noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and
Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that
differential recruitment of Ubc9 by SUMO or Rad60 provides a novel means for
such selectivity. Indeed, deconvoluting Ubc9 function by disrupting either the
Ubc9:SLD2 or Ubc9:SUMO noncovalent complex reveals distinct roles in
facilitating sumoylation. Ubc9:SLD2 acts in the Nse2 SUMO E3 ligase-dependent
pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation
and chain formation, via the Pli1 E3 SUMO ligase. Moreover, this Pli1-dependent
SUMO chain formation causes the genome instability phenotypes of SUMO-targeted
ubiquitin ligase (STUbL) mutants. Overall, we determine that, unexpectedly, Ubc9
noncovalent partner choice dictates the role of sumoylation in distinct cellular
pathways. The human papillomavirus oncogenic protein, E6, interacts with a number of
cellular proteins, and for some targets, E6 directs their degradation through
the ubiquitin-proteasome pathway. Post-translational modification with
ubiquitin-like modifiers, such as SUMO, also influences protein activities,
protein-protein interactions, and protein stability. We report that the high
risk HPVE6 proteins reduce the intracellular quantity of the sole SUMO
conjugation enzyme, Ubc9, concomitant with decreased host sumoylation. E6 did
not significantly influence transcription of Ubc9, indicating that the effects
were likely at the protein level. Consistent with typical E6-mediated
proteasomal degradation, E6 bound to Ubc9 in vitro, and required E6AP for
reduction of Ubc9 levels. Under stable E6 expression conditions in
differentiating keratinocytes there was a decrease in Ubc9 and a loss of
numerous sumoylated targets indicating a significant perturbation of the normal
sumoylation profile. While E6 is known to inhibit PIASy, a SUMO ligase, our
results suggest that HPV E6 also targets the Ubc9 protein to modulate host cell
sumoylation, suggesting that the sumoylation system may be an important target
during viral reproduction and possibly the subsequent development of cervical
cancer. Sumoylation regulates a wide range of cellular processes. However, little is
known about the regulation of the SUMO machinery. In this study, we demonstrate
that two lysine residues (Lys-153 and Lys-157) in the C-terminal region of the
yeast E2-conjugating enzyme Ubc9 are the major and minor autosumoylation sites,
respectively. Surprisingly, mutation of Lys-157 (ubc9(K157R)) significantly
stimulates the level of Ubc9 autosumoylation at Lys-153. The functional role of
Ubc9 autosumoylation is exemplified in our findings that cell cycle-dependent
sumoylation of cytoskeletal septin proteins is inversely correlated with the
Ubc9 autosumoylation level and that mutation of the Ubc9 autosumoylation sites
results in aberrant cell morphology. Our study elucidates a regulatory mechanism
that utilizes automodification of the E2 enzyme of the sumoylation machinery to
control substrate sumoylation. Small ubiquitin-like modifier (SUMO1-3) constitutes a group of proteins that
conjugate to lysine residues of target proteins thereby modifying their
activity, stability, and subcellular localization. A large number of SUMO target
proteins are transcription factors and other nuclear proteins involved in gene
expression. Furthermore, SUMO conjugation plays key roles in genome stability,
quality control of newly synthesized proteins, proteasomal degradation of
proteins, and DNA damage repair. Any marked increase in levels of
SUMO-conjugated proteins is therefore expected to have a major impact on the
fate of cells. We show here that SUMO conjugation is activated in human
astrocytic brain tumors. Levels of both SUMO1- and SUMO2/3-conjugated proteins
were markedly increased in tumor samples. The effect was least pronounced in
low-grade astrocytoma (WHO Grade II) and most pronounced in glioblastoma
multiforme (WHO Grade IV). We also found a marked rise in levels of Ubc9, the
only SUMO conjugation enzyme identified so far. Blocking SUMO1-3 conjugation in
glioblastoma cells by silencing their expression blocked DNA synthesis, cell
growth, and clonogenic survival of cells. It also resulted in DNA-dependent
protein kinase-induced phosphorylation of H2AX, indicative of DNA double-strand
damage, and G(2) /M cell cycle arrest. Collectively, these findings highlight
the pivotal role of SUMO conjugation in DNA damage repair processes and imply
that the SUMO conjugation pathway could be a new target of therapeutic
intervention aimed at increasing the sensitivity of glioblastomas to
radiotherapy and chemotherapy. Successful viruses have evolved superior strategies to escape host defenses or
exploit host biological pathways. Most of the viral immediate-early (ie) genes
are essential for viral infection and depend solely on host proteins; however,
the molecular mechanisms are poorly understood. In this study, we focused on the
modification of viral IE proteins by the crayfish small ubiquitin-related
modifier (SUMO) and investigated the role of SUMOylation during the viral life
cycle. SUMO and SUMO ubiquitin-conjugating enzyme 9 (UBC9) involved in
SUMOylation were identified in red swamp crayfish (Procambarus clarkii). Both
SUMO and UBC9 were upregulated in crayfish challenged with white spot syndrome
virus (WSSV). Replication of WSSV genes increased in crayfish injected with
recombit SUMO or UBC9, but injection of mutant SUMO or UBC9 protein had no
effect. Subsequently, we analyzed the mechanism by which crayfish SUMOylation
facilitates WSSV replication. Crayfish UBC9 bound to all three WSSV IE proteins
tested, and one of these IE proteins (WSV051) was covalently modified by SUMO in
vitro. The expression of viral ie genes was affected and that of late genes was
significantly inhibited in UBC9-silenced or SUMO-silenced crayfish, and the
inhibition effect was rescued by injection of recombit SUMO or UBC9. The
results of this study demonstrate that viral IE proteins can be modified by
crayfish SUMOylation, prompt the expression of viral genes, and ultimately
benefit WSSV replication. Understanding of the mechanisms by which viruses
exploit host components will greatly improve our knowledge of the virus-host
"arms race" and contribute to the development of novel methods against virulent
viruses. Lung cancer is the leading cause of cancer-related mortality worldwide. The
mortality is high mainly due to the lack of known effective screening
procedures; there is a high tendency for early spread and systemic therapies do
not cure metastatic disease. Thus, it is important to investigate the molecular
mechanism(s) of lung cancer development and, specifically, to identify an
effective method by which to inhibit the invasion and metastasis of lung cancer.
Ubiquitin-conjugating enzyme 9 (Ubc9), the sole conjugating enzyme for
sumoylation, regulates protein function and plays a key role in tumorigenesis.
Whether Ubc9 is involved in the invasion and metastasis of lung cancer remains
unknown. Herein, we report that Ubc9 exhibits an important role in lung cancer
invasion and metastasis. We first investigated the biological effect of Ubc9 on
lung cancer by cloning the Ubc9 gene into a eukaryotic expression plasmid and
stably expressing it in the human small cell lung cancer cell line NCI-H446 in
order to observe any biological changes. We further analyzed the effect of Ubc9
in an in vivo experiment, injecting NCI-H446 cells stably overexpressing Ubc9
into nude mice and analyzing their metastatic ability. Our results demonstrated
that Ubc9 is expressed at higher levels in primary lung cancer tissue and
metastatic nodules as compared to premaligt and/or normal tissue.
Furthermore, we demonstrated that upregulation of Ubc9 expression promotes
migration and invasion. Ubc9 likely plays an important role in cancer
progression by promoting invasion and metastasis in lung cancer. INTRODUCTION: Small ubiquitin-like modifiers (SUMO) conjugate to target proteins
in a dynamic, reversible manner to function as post-translational modifiers.
SUMOylation of target proteins can impinge on their localization, in addition to
their activity or stability. Differential expression of deSUMOylating enzymes
(SENP 1 and 2) contributes to altered mammalian placental development and
function in mice. Severe preeclampsia (sPE) is associated with abnormal
placental development and chronic ischemic injury. Extra- and intracellular
stimuli/stressors that include hypoxic-activated pathways are known modulators
of SUMOylation. In this current study we hypothesized that placentas from sPE
patients will display up regulation in the SUMO regulatory pathway.
METHODS: Utilizing qRT-PCR, immuno-blotting and Western techniques, we
determined the expression levels of SUMO pathway genes in healthy and diseased
placentas. We also exposed placental explants to hypoxia to study the effect on
the SUMOylation pathway.
RESULTS: We observed steady-state expression of SUMO1-3, SUMO-conjugated
enzyme-UBC9 and deSUMOylating enzymes - SENPs, throughout normal gestation. An
elevated level of free SUMO1-3 and SUMO-protein conjugates was observed in sPE
placentas. Furthermore, placental UBC9 levels were strikingly increased in the
same sPE patients. Hypoxia-induced SUMOylation in first trimester placental
explants.
DISCUSSION: Our data demonstrate an elevated steady-state of SUMOylation in sPE
placentas compared with gestational aged-matched controls. The observed
hyper-SUMOylation in sPE placentas correlates with elevated expression of UBC9
rather than with reduced expression of SENPs Hypoxia may contribute to
alterations in placental SUMOylation pathway.
CONCLUSION: Increased placental SUMOylation may contribute to the pathogenesis
of serious placental pathology that causes extreme preterm birth. Whether NF-κB promoter transactivation by the human T-cell leukemia virus type 1
(HTLV-1) Tax protein requires Tax SUMOylation is still a matter of debate. In
this study, we revisited the role of Tax SUMOylation using a strategy based on
the targeting of Ubc9, the unique E2 SUMO-conjugating enzyme. We show that
either a catalytically inactive form of Ubc9 (Ubc9-C93S) or Ubc9 small
interfering RNA (siRNA) dramatically reduces Tax conjugation to endogenous
SUMO-1 or SUMO-2/3, demonstrating that as expected, Tax SUMOylation is under the
control of the catalytic activity of Ubc9. We further report that a
non-SUMOylated Tax protein produced in 293T cells is still able to activate
either a transfected or an integrated NF-κB reporter promoter and to induce
expression of an NF-κB-regulated endogenous gene. Importantly, blocking Ubc9
activity in T cells also results in the production of a non-SUMOylated Tax that
is still fully functional for the activation of a NF-κB promoter. These results
provide the definitive evidence that Tax SUMOylation is not required for
NF-κB-driven gene induction.
IMPORTANCE: Human T-cell leukemia virus type 1 is able to transform CD4(+) T
lymphocytes. The viral oncoprotein Tax plays a key role in this process by
promoting cell proliferation and survival, mainly through permanent activation
of the NF-κB pathway. Elucidating the molecular mechanisms involved in NF-κB
pathway activation by Tax is therefore a key issue to understand HTLV-1-mediated
transformation. Tax SUMOylation was initially proposed to be critical for
Tax-induced NF-κB promoter activation, which was challenged by our later
observation that a low-level-SUMOylated Tax mutant was still functional for
activation of NF-κB promoters. To clarify the role of Tax SUMOylation, we set up
a new approach based on the inhibition of the SUMOylation machinery in
Tax-expressing cells. We show that blocking the SUMO-conjugating enzyme Ubc9
abolishes Tax SUMOylation and that a non-SUMOylated Tax still activates NF-κB
promoters in either adherent cells or T cells. RATIONALE: Impairment of proteasomal function is pathogenic in several cardiac
proteinopathies and can eventually lead to heart failure. Loss of proteasomal
activity often results in the accumulation of large protein aggregates. The
ubiquitin proteasome system (UPS) is primarily responsible for cellular protein
degradation, and although the role of ubiquitination in this process is well
studied, the function of an ancillary post-translational modification,
SUMOylation, in protein quality control is not fully understood.
OBJECTIVE: To determine the role of ubiquitin-conjugating enzyme 9 (UBC9), a
small ubiquitin-like modifier-conjugating enzyme, in cardiomyocyte protein
quality control.
METHODS AND RESULTS: Gain- and loss-of-function approaches were used to
determine the importance of UBC9. Overexpression of UBC9 enhanced UPS function
in cardiomyocytes, whereas knockdown of UBC9 by small interfering RNA caused
significant accumulations of aggregated protein. UPS function and relative
activity was analyzed using a UPS reporter protein consisting of a short degron,
CL1, fused to the COOH-terminus of green fluorescent protein (GFPu).
Subsequently, the effects of UBC9 on UPS function were tested in a proteotoxic
model of desmin-related cardiomyopathy, caused by cardiomyocyte-specific
expression of a mutated αB crystallin, CryAB(R120G). CryAB(R120G) expression
leads to aggregate formation and decreased proteasomal function. Coinfection of
UBC9-adenovirus with CryAB(R120G) virus reduced the proteotoxic sequelae,
decreasing overall aggregate concentrations. Conversely, knockdown of UBC9
significantly decreased UPS function in the model and resulted in increased
aggregate levels.
CONCLUSIONS: UBC9 plays a significant role in cardiomyocyte protein quality
control, and its activity can be exploited to reduce toxic levels of misfolded
or aggregated proteins in cardiomyopathy. Sumoylation is a post-translational modification that plays an important role in
a wide range of cellular processes. Among the proteins involved in the
sumoylation pathway, Ubc9 is the sole E2-conjugating enzyme required for
sumoylation and plays a central role by interacting with almost all of the
partners required for sumoylation. Ubc9 has been implicated in a variety of
human maligcies. In order to exploit the therapeutic potential of Ubc9, we
have identified the potential site to target for rational drug design using
molecular modeling approaches. The structural information derived was then used
to prioritize hits from a small-molecule library for biological assay using a
virtual screening protocol that involves shape matching with a known inhibitor
inhibitors and docking of a small-molecule library utilizing computational
approaches that incorporate both ligand and protein flexibility. Nineteen
compounds were acquired from different chemical vendors and were tested for Ubc9
inhibitory activity. Five compounds showed inhibitory activity against Ubc9, out
of which one compound was selected for further optimization. A similarity search
was then carried out to retrieve commercially available derivatives, which were
further acquired and assayed, resulting in two compounds with acceptable
potency. These two compounds can be used as starting points for the development
of more potent inhibitors of Ubc9 targeting the predicted site. Sumoylation plays important roles in the modulation of protein function,
neurotransmission and plasticity, but the mechanisms regulating this
post-translational system in neurons remain largely unknown. Here we demonstrate
that the synaptic diffusion of Ubc9, the sole conjugating enzyme of the
sumoylation pathway, is regulated by synaptic activity. We use restricted
photobleaching/photoconversion of individual hippocampal spines to measure the
diffusion properties of Ubc9 and show that it is regulated through an
mGlu5R-dependent signalling pathway. Increasing synaptic activity with a GABAA
receptor antagonist or directly activating mGlu5R increases the synaptic
residency time of Ubc9 via a Gαq/PLC/Ca(2+)/PKC cascade. This activation
promotes a transient synaptic trapping of Ubc9 through a PKC
phosphorylation-dependent increase of Ubc9 recognition to phosphorylated
substrates and consequently leads to the modulation of synaptic sumoylation. Our
data demonstrate that Ubc9 diffusion is subject to activity-dependent regulatory
processes and provide a mechanism for the dynamic changes in sumoylation
occurring during synaptic transmission. As the sole E2 enzyme for SUMOylation, Ubc9 is predomitly nuclear. However,
the underlying mechanisms of Ubc9 nuclear localization are still not well
understood. Here we show that RNAi-depletion of Imp13, an importin known to
mediate Ubc9 nuclear import, reduces both Ubc9 nuclear accumulation and global
SUMOylation. Furthermore, Ubc9-R13A or Ubc9-H20D mutation previously shown to
interrupt the interaction of Ubc9 with nucleus-enriched SUMOs reduces the
nuclear enrichment of Ubc9, suggesting that the interaction of Ubc9 with the
nuclear SUMOs may enhance Ubc9 nuclear retention. Moreover, Ubc9-R17E mutation,
which is known to disrupt the interaction of Ubc9 with both SUMOs and Imp13,
causes a greater decrease in Ubc9 nuclear accumulation than Ubc9-R13A or
Ubc9-H20D mutation. Lastly, Ubc9-K74A/S89D mutations that perturb the
interaction of Ubc9 with nucleus-enriched SUMOylation-consensus motifs has no
effect on Ubc9 nuclear localization. Altogether, our results have elucidated
that the amino acid residues within the N-terminal region of Ubc9 play a pivotal
role in regulation of Ubc9 nuclear localization. OBJECTIVE: To explore the hypoxic regulation of sumoylation pathways and cell
viability in nucleus pulposus (NP) and annulus fibrosus (AF) cells.
DESIGN: Expression of small ubiquitin-like modifier (SUMO) molecules, SUMO E1
activating enzymes SAE1 and SAE2, SUMO E2 conjugating enzyme UBC9, and
de-sumoylation enzyme sentrin/SUMO-specific proteases (SENP)1 was
immunolocalized in rat intervertebral disc (IVD) cells. NP and AF cells were
cultured in hypoxia and cell viability was evaluated by quantifying cell
proliferation, cellular senescence, apoptosis, and cell cycle distribution.
Hypoxic regulation of sumoylation pathways was studied by analyzing the
transcription and expression of SUMO molecules and sumoylation enzymes. Loss of
function study using SENP1 siRNA was performed to investigate the regulatory
role of sumoylation on the function of hypoxia inducible factor 1α (HIF-1α) and
the hypoxic tolerance of IVD cells.
RESULTS: Sumoylation pathways were expressed in IVD cells and localized
predomitly in nuclei. Both NP and AF cells maintained viability under hypoxia
and upregulated the expression of SENP1. In NP cells hypoxia transiently
increased the expression of SUMO-1, SUMO-2/3, SAE2, and UBC9, whereas SUMO-1 was
elevated while SUMO-2/3, SAE1, SAE2, and UBC9 were reduced by low oxygen
tensions in AF cells. Although downregulation of SENP1 decreased the
transcriptional activity of HIF-1α, the viability of disc cells showed no
significant loss under hypoxia.
CONCLUSIONS: NP and AF cells equally tolerate oxygen deficiency, but differently
regulate the sumoylation pathways under hypoxia. The distinct sumoylation
dynamics may help extend our understanding of the cell-specific regulation of
the molecular basis that promotes cell survival in the hypoxic IVD. Small ubiquitin-like modifier (SUMO) participates in a reversible
posttranslational modification process (SUMOylation) that regulates a wide
variety of cellular processes and plays important roles for numerous viruses
during infection. However, the roles of viral protein SUMOylation in dengue
virus (DENV) infection have not been elucidated. In this study, we found that
the SUMOylation pathway was involved in the DENV life cycle, since DENV
replication was reduced by silencing the cellular gene Ubc9, which encodes the
sole E2-conjugating enzyme required for SUMOylation. By in vivo and in vitro
SUMOylation assays, the DENV NS5 protein was identified as an authentic
SUMO-targeted protein. By expressing various NS5 mutants, we found that the SUMO
acceptor sites are located in the N-terminal domain of NS5 and that a putative
SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation. A
DENV replicon harboring the SUMOylation-defective SIM mutant showed a severe
defect in viral RNA replication, supporting the notion that NS5 SUMOylation is
required for DENV replication. SUMOylation-defective mutants also failed to
suppress the induction of STAT2-mediated host antiviral interferon signaling.
Furthermore, the SUMOylation of NS5 significantly increased the stability of NS5
protein, which could account for most of the biological functions of SUMOylated
NS5. Collectively, these findings suggest that the SUMOylation of DENV NS5 is
one of the mechanisms regulating DENV replication.
IMPORTANCE: SUMOylation is a common posttranslational modification that
regulates cellular protein functions but has not been reported in the proteins
of dengue virus. Here, we found that the replicase of DENV, nonstructural
protein 5 (NS5), can be SUMOylated. It is well known that providing
RNA-dependent RNA polymerase activity and antagonizing host antiviral IFN
signaling are a "double indemnity" of NS5 to support DENV replication. Without
SUMOylation, NS5 fails to maintain its protein stability, which consequently
disrupts its function in viral RNA replication and innate immunity antagonism.
DENV threatens billions of people worldwide, but no licensed vaccine or specific
therapeutics are currently available. Thus, our findings suggest that rather
than specifically targeting NS5 enzyme activity, NS5 protein stability is a
novel drug target on the growing list of anti-DENV strategies. RATIONALE: SUMOylation plays an important role in cardiac function and can be
protective against cardiac stress. Recent studies show that SUMOylation is an
integral part of the ubiquitin proteasome system, and expression of the small
ubiquitin-like modifier (SUMO) E2 enzyme UBC9 improves cardiac protein quality
control. However, the precise role of SUMOylation on other protein degradation
pathways, particularly autophagy, remains undefined in the heart.
OBJECTIVE: To determine whether SUMOylation affects cardiac autophagy and
whether this effect is protective in a mouse model of proteotoxic cardiac
stress.
METHODS AND RESULTS: We modulated expression of UBC9, a SUMO E2 ligase, using
gain- and loss-of-function in neonatal rat ventricular cardiomyocytes. UBC9
expression seemed to directly alter autophagic flux. To confirm this effect in
vivo, we generated transgenic mice overexpressing UBC9 in cardiomyocytes. These
mice have an increased level of SUMOylation at baseline and, in confirmation of
the data obtained from neonatal rat ventricular cardiomyocytes, demonstrated
increased autophagy, suggesting that increased UBC9-mediated SUMOylation is
sufficient to upregulate cardiac autophagy. Finally, we tested the protective
role of SUMOylation-mediated autophagy by expressing UBC9 in a model of cardiac
proteotoxicity, induced by cardiomyocyte-specific expression of a mutant
α-B-crystallin, mutant CryAB (CryAB(R120G)), which shows impaired autophagy.
UBC9 overexpression reduced aggregate formation, decreased fibrosis, reduced
hypertrophy, and improved cardiac function and survival.
CONCLUSIONS: The data showed that increased UBC9-mediated SUMOylation is
sufficient to induce relatively high levels of autophagy and may represent a
novel strategy for increasing autophagic flux and ameliorating morbidity in
proteotoxic cardiac disease. Foxp3-expressing regulatory T (Treg) cells are essential for immune tolerance;
however, the molecular mechanisms underlying Treg cell expansion and function
are still not well understood. SUMOylation is a protein post-translational
modification characterized by covalent attachment of SUMO moieties to lysines.
UBC9 is the only E2 conjugating enzyme involved in this process, and loss of
UBC9 completely abolishes the SUMOylation pathway. Here, we report that
selective deletion of Ubc9 within the Treg lineage results in fatal early-onset
autoimmunity similar to Foxp3 mutant mice. Ubc9-deficient Treg cells exhibit
severe defects in TCR-driven homeostatic proliferation, accompanied by impaired
activation and compromised suppressor function. Importantly, TCR ligation
enhanced SUMOylation of IRF4, a critical regulator of Treg cell function
downstream of TCR signals, which regulates its stability in Treg cells. Our data
thus have demonstrated an essential role of SUMOylation in the expansion and
function of Treg cells. |
Can the Micro-C XL method achieve mononucleosome resolution? | Yes. Micro-C XL is an improved method for analysis of chromosome folding at mononucleosome resolution. | We present Micro-C XL, an improved method for analysis of chromosome folding at
mononucleosome resolution. Using long crosslinkers and isolation of insoluble
chromatin, Micro-C XL increases signal-to-noise ratio. Micro-C XL maps of
budding and fission yeast genomes capture both short-range chromosome fiber
features such as chromosomally interacting domains and higher order features
such as centromere clustering. Micro-C XL provides a single assay to interrogate
chromosome folding at length scales from the nucleosome to the full genome. |
Can Diabetes be caused by a defect in a potassium chanel? | Mutations in the KATP channel can lead to neonatal diabetes. | OBJECTIVE: The increased perinatal morbidity in diabetes may be partly related
to vascular dysfunction. Because potassium channels play an important role in
the regulation of vascular tone, this study explores the impact of diabetes on
potassium channel function in the fetoplacental vascular bed.
STUDY DESIGN: Vascular potassium channel function was investigated by ex vivo
dual perfusion of isolated placental cotyledons (n = 47). Appropriate control
experiments were carried out to exclude nonspecific effects.
RESULTS: Glibenclamide (KATP channel blocker) increased perfusion pressure to a
maximum fetoplacental arterial pressure of 37 +/- 6 mm Hg in controls versus 15
+/- 6 mm Hg in diabetes (P < .05). 4-Aminopyridine (KV channel blocker) equally
increased fetoplacental arterial pressure in controls, and in diabetes (21 +/- 4
mm Hg vs 22 +/- 2 mm Hg). Apamin and charybdotoxin (KCa channel blockers) caused
a negligible rise in fetoplacental arterial pressure.
CONCLUSION: In the fetoplacental circulation, KATP channels and KV channels
significantly contribute to baseline vascular tone. In diabetes, vascular KATP
channel function is impaired. We report a case of a 6-week-old infant with diabetes mellitus based on a
genetic defect in the sulfonylurea receptor 1 (SUR1), an ATP-sensitive potassium
(KATP) channel protein. A spectacular improvement in glucose regulation was
shown by real-time continuous glucose monitoring when switching her from insulin
to oral glibenclamide. Children with neonatal onset of diabetes deserve genetic
testing in order to replace insulin with oral medication. |
Which fimA genotypes are associated with disease? | FimA has been characterized as an important virulence factor for P. gingivalis, and many studies, both animal experiments and clinical investigations, have characterized fimA genotypes II, Ib, and IV to be associated with disease (periodontitis and cardiovascular disease) | OBJECTIVE: Long fimbriae (FimA) are important virulence factors of Porphyromonas
gingivalis. Based on the diversity of the fimA gene, this species is classified
into 6 genotypes. This study surveyed samples from primary endodontic infections
for the presence of these P. gingivalis fimA variants.
STUDY DESIGN: Genomic DNA isolated from samples taken from 25 root canals of
teeth with chronic apical periodontitis and 25 aspirates from acute apical
abscess was used as template in polymerase chain reaction (PCR) assays directed
toward the detection of the different P. gingivalis fimA genotypes.
RESULTS: Porphyromonas gingivalis was detected by a 16S rRNA gene-based PCR in
36% of the total number of cases sampled (44% of chronic apical periodontitis
and 28% of abscess aspirates). In cases of chronic apical periodontitis, P.
gingivalis variant type IV was the most prevalent (24%), followed by types I
(20%), II (16%), and III (8%). In acute abscess samples, variant type II was the
most prevalent (12%), followed by types III and IV (8% of each) and type I (4%).
Combinations of up to 3 different genotypes were detected in a few cases. No
single fimA genotype variant or combination thereof was significantly associated
with symptoms. Overall, fimA types IV (16%), II (14%), and I (12%) were the most
prevalent.
CONCLUSIONS: Findings demonstrated that different P. gingivalis fimA genotypes
can be present in primary endodontic infections. Porphyromonas gingivalis is a primary pathogen involved in the initiation and
progression of adult chronic periodontitis. Its colonization on oral surfaces is
a necessary first step leading to infection. FimA, a subunit protein of major
(long) fimbriae, is a well-known virulence factor. Based on its nucleotide
sequence, FimA is classified into several genotypes. We compared here the
transcriptional levels of the fimA gene in several P. gingivalis strains using
real-time polymerase chain reaction analysis, fimbrial display on the P.
gingivalis surface using transmission electronic microscopy, and the adherence
competencies of P. gingivalis strains carrying different types of FimAs towards
saliva and Streptococcus gordonii surfaces using mutagenesis analysis. We
demonstrated differential expression of each fimA gene in these P. gingivalis
strains. A correlation of the transcription level of fimA and binding activity
of P. gingivalis was revealed. We show that P. gingivalis strains with genotype
I and II of FimA are efficient in interaction with saliva or S. gordonii. This
work highlights the important role of FimA type I and II in P. gingivalis
attachment to oral surfaces. Porphyromonas gingivalis FimA fimbriae have been classified into 6 genotypes
(types I-V and Ib) based on the diversity of the fimA genes encoding the
fimbrial subunits. We investigated the prevalence of fimA genotype in Japanese
children. Dental plaque specimens were obtained from 400 subjects (age; 2 to 15
years), including 134 with healthy gingiva, 239 with gingivitis and 27 with
periodontitis, and then analyzed by polymerase chain reaction. P. gingivalis was
detected in 1.5%, 10.0% and 29.6% of these subjects, respectively. Significant
differences were observed with regard to P. gingivalis infection among the
groups [chi-squared analysis: gingivitis vs. healthy, P < 0.01, odds ratio (OR)
= 7.4; periodontitis vs. healthy, P < 0.001, OR = 27.8]. In P.
gingivalis-positive subjects with periodontitis, the most prevalent fimA types
were type Ib/type II combination (37.5%) and type IV (37.5%), followed by type
II (25.0%), while type IV (33.3%) and type II (29.2%) were most often detected
in those with gingivitis. Our results suggest that the presence of P. gingivalis
is associated with periodontal diseases, and that the type II, IV and Ib/II
combination are the most common among fimA genotypes. OBJECTIVES: The aim of this study was to determine the prevalence of the
different fimA genotypes of Porphyromonas gingivalis in adult Spanish patients
with chronic periodontitis, patients with gingivitis and periodontally healthy
subjects, and the relationship between these genotypes and other
periodontopathogenic bacteria.
STUDY DESIGN: Samples of subgingival plaque were taken from 86 patients (33 with
chronic periodontitis, 16 with gingivitis, and 37 periodontally healthy) in the
course of a full periodontal examination. PCR was employed to determine the
presence of the 6 fimA genotypes of Porphyromonas gingivalis (I-V and Ib) and of
Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema
denticola.
RESULTS: Porphyromonas gingivalis fimA genotypes II and Ib were present in
significantly higher percentages in periodontal patients (39.4% and 12.1%
respectively) than in healthy or gingivitis subjects. The prevalence of
Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis fimA
genotype IV was significantly higher in the group that presented bleeding
greater than 30%. A positive correlation was found between Porphyromonas
gingivalis fimA genotype IV and Treponema denticola.
CONCLUSIONS: A strong association between Porphyromonas gingivalis fimA
genotypes II and Ib and chronic periodontitis exists in the Spanish population.
The most prevalent genotype in periodontal patients is II. Strains of Porphyromonas gingivalis, a periodontopathic bacterium, are
classified into six genotypic variants based on nucleotide sequence differences
in the fimA gene encoding FimA. A PCR assay using primer sets specific for each
genotype has demonstrated that the most predomit fimA genotype in
periodontitis patients is type II, which is now commonly referred to as the
periodontitis-associated fimA genotype of P. gingivalis. However, the potential
for false type II fimA positives caused by cross-hybridization of type II
fimA-specific primers with type Ib fimA has complicated the genotyping. A
previous study developed new primers that specifically amplified only the DNA
fragment of type II fimA. The aim of the present study was to assess the
prevalence of P. gingivalis fimA genotypes in Korean adults and to reconfirm the
relationship between type II fimA and periodontitis using the new primers. Among
412 Korean adults, P. gingivalis was detected in 97.5 % of patients and 57.8 %
of healthy subjects. Type II fimA was the most widely distributed type among
healthy and periodontitis subjects. Organisms with types II, Ib and IV fimA had
a significant frequency of occurrence in periodontitis subjects. Statistical
analysis, however, revealed that a more significant correlation was found
between periodontitis and the occurrence of type Ib fimA. BACKGROUND: The microbiologic feature of aggressive periodontitis (AgP) in
Chinese patients has not yet been determined. This study aims to investigate the
prevalence of eight periodontal microorganisms and the distribution of the
Porphyromonas gingivalis fimA genotype in a cohort of Chinese patients with AgP.
METHODS: Saliva and pooled subgingival plaque samples were collected from 81
patients with AgP (25 with incisor-first molar type and 56 with generalized type
[GAgP]) and 34 periodontally healthy controls. Eight periodontal microorganisms,
including Aggregatibacter actinomycetemcomitans, P. gingivalis, Tannerella
forsythia, Treponema denticola, Campylobacter rectus, Prevotella intermedia,
Prevotella nigrescens, and Fusobacterium nucleatum were detected in these
samples by the polymerase chain reaction (PCR). In addition, the distribution of
fimA genotypes was assessed in P. gingivalis-positive individuals by PCR.
RESULTS: The prevalence of P. gingivalis, T. forsythia, T. denticola, C. rectus,
P. intermedia, F. nucleatum, and A. actinomycetemcomitans in patients with AgP
was significantly higher than that in healthy controls. The prevalence of A.
actinomycetemcomitans in patients with GAgP was relatively low (30.4%) compared
with other pathogens. Results of logistic regression analysis showed that
younger patients were more likely to harbor A. actinomycetemcomitans (odds ratio
= 2.85). Type II was the most prevalent fimA genotype of P. gingivalis in
patients with AgP.
CONCLUSIONS: P. gingivalis, T. forsythia, T. denticola, C. rectus, P.
intermedia, and F. nucleatum were the predomit periodontal pathogens of
patients with GAgP in China. Type II of fimA was the most prevalent genotype of
P. gingivalis in patients with AgP. The prevalence of A. actinomycetemcomitans
in patients with GAgP was relatively low. Increasing evidence has shown periodontal pathogen Porphyromonas gingivalis
(P.gingivalis) infection contributes to atherosclerosis (AS) progression.
P.gingivalis fimbriae act as an important virulence factor in AS. Regulatory T
cells (Tregs) may play a crucial role in autoimmune response during this
process. However, whether P.gingivalis infection is associated with Tregs
dysregulation during AS is still unknown and the prevalence of different
P.gingivalis FimA genotypes during this process is unclear. Here we analyzed the
distribution of Tregs and in P.gingivalis-infected atherosclerotic patients to
reveal the relationship between P.gingivalis infection and Tregs
reduction/dysfunction and to elucidate their role in periodontitis-AS
interaction. FimA genotype was also examined to determine the prevalence of
fimbriae. Our results showed that P.gingivalis infection reduced Tregs in
atherosclerotic patients compared with non-atherosclerotic patients and health
controls. Concentration of TGF-β1, which plays an important role in the
development of Tregs, also decreased in P.gingivalis infected patients.
Furthermore, type II FimA seems to show higher prevalence than the other five
detected types. The population of Tregs further decreased in patients with type
II FimA compared with the other types. P.gingivlias FimA genotype II was the
domit type associated with decreased Treg population. These results indicate
that P.gingivalis infection may be associated with Tregs dysregulation in AS;
type II FimA may be a predomit genotype in this process. BACKGROUND: Porphyromonas gingivalis produces outer membrane-attached proteins
that include the virulence-associated cysteine proteinases RgpA and RgpB
(Arg-gingipains) and Kgp (Lys-gingipain). The gingipains provide P. gingivalis
with a general proteolytic tool for degradation of proteinaceous nutrients for
growth. They are also essential for the processing and maturation of the major
fimbriae (FimA) which are important in facilitating bacterial adhesion to host
tissues. FimA has been characterized as an important virulence factor for P.
gingivalis, and many studies, both animal experiments and clinical
investigations, have characterized fimA genotypes II, Ib, and IV to be
associated with disease (periodontitis and cardiovascular disease) while
genotypes I, III, and V represent avirulent strains. The relationship between
virulence and gene variation of the rgpB gene has not been investigated
extensively. However, nucleotide variations of the rgpB gene result in four
amino acid substitutions in the catalytic domain identifying five different rgpB
genotypes. They are named according to the different amino acid sequences in the
primary protein structure of RGPB and named NYPN, NSSN, NSSK, NYPK, and DYPN
(Beikler et al. 2005).
AIM: The aim of the present study is to elucidate a possible relationship
between fimA virulent and avirulent genotypes to the corresponding rgpB
genotypes.
METHODS: The total length of the rgpB (2212 bp) and fimA genes (1140 bp) from 20
clinical isolates was sequenced. Primers for pcr and sequencing of the genes
were selected according to earlier investigations.
RESULTS: The rgpB genotypes, NSSN and NYPN (n=2 and 6, respectively), were
detected in the fimA II genotypes of P. gingivalis, while NSSK was detected in
one fimA Ib genotype in the population. For the eight fimA IV genotypes, five
NSSK and three NSSN rgpB genotypes were detected. In two fima I genotypes were
detected one DYPN and one NYPN of the rgpB variants, while one fimA III genotype
was associated with NSSK.
CONCLUSION: The rgpB genotypes NSSN, NYPN, NSSK of P. gingivalis may be
associated with the virulent fimA genotypes II, Ib and IV, indicating a possible
connection to their virulent properties. PURPOSE: Porphyromonas gingivalis fimA is a virulence factor associated with
periodontal diseases, but its role in the pathogenesis of peri-implantitis
remains unclear. We aimed to evaluate the relationship between the condition of
peri-implant tissue and the distribution of P. gingivalis fimA genotypes in
Koreans using a new primer.
METHODS: A total of 248 plaque samples were taken from the peri-implant sulci of
184 subjects. The control group consisted of sound implants with a peri-implant
probing depth (PD) of 5 mm or less with no bleeding on probing (BOP). Test group
I consisted of implants with a peri-implant PD of 5 mm or less and BOP, and test
group II consisted of implants with a peri-implant PD of more than 5 mm and BOP.
DNA was extracted from each sample and analyzed a using a polymerase chain
reaction (PCR) with P. gingivalis-specific primers, followed by an additional
PCR assay to differentiate the fimA genotypes in P. gingivalis-positive
subjects.
RESULTS: The Prevalence of P. gingivalis in each group did not significantly
differ (P>0.05). The most predomit fimA genotype in all groups was type II.
The prevalence of type Ib fimA was significantly greater in test group II than
in the control group (P<0.05).
CONCLUSIONS: The fimA type Ib genotype of P. gingivalis was found to play a
critical role in the destruction of peri-implant tissue, suggesting that it may
be a distinct risk factor for peri-implantitis. |
Is rucaparib used for ovarian cancer treatment? | Yes, rucaparib is a PARP inhibitor that is used for ovarian cancer treatment. | Here, we investigate the potential role of the PARP inhibitor rucaparib (CO-338,
formerly known as AG014699 and PF-01367338) for the treatment of sporadic
ovarian cancer. We studied the growth inhibitory effects of rucaparib in a panel
of 39 ovarian cancer cell lines that were each characterized for mutation and
methylation status of BRCA1/2, baseline gene expression signatures, copy number
variations of selected genes, PTEN status, and sensitivity to platinum-based
chemotherapy. To study interactions with chemotherapy, we used multiple drug
effect analyses and assessed apoptosis, DNA fragmentation, and γH2AX formation.
Concentration-dependent antiproliferative effects of rucaparib were seen in 26
of 39 (67%) cell lines and were not restricted to cell lines with BRCA1/2
mutations. Low expression of other genes involved in homologous repair (e.g.,
BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to
platinum-based chemotherapy was associated with sensitivity to rucaparib. Drug
interactions with rucaparib were synergistic for topotecan, synergistic, or
additive for carboplatin, doxorubicin or paclitaxel, and additive for
gemcitabine. Synergy was most pronounced when rucaparib was combined with
topotecan, which resulted in enhanced apoptosis, DNA fragmentation, and γH2AX
formation. Importantly, rucaparib potentiated chemotherapy independent of its
activity as a single agent. PARP inhibition may be a useful therapeutic strategy
for a wider range of ovarian cancers bearing deficiencies in the homologous
recombination pathway other than just BRCA1/2 mutations. These results support
further clinical evaluation of rucaparib either as a single agent or as an
adjunct to chemotherapy for the treatment of sporadic ovarian cancer. BACKGROUND: Rucaparib is a potent, orally available, small-molecule inhibitor of
poly ADP-ribose polymerase (PARP) 1 and 2. Ongoing clinical trials are assessing
the efficacy of rucaparib alone or in combination with other cytotoxic drugs,
mainly in breast and ovarian cancer patients with mutations in the breast cancer
associated (BRCA) genes.
PURPOSE: We aimed to establish whether the multidrug efflux transporters ABCG2
(BCRP) and ABCB1 (P-gp, MDR1) affect the oral availability and brain penetration
of rucaparib in mice.
RESULTS: In vitro, rucaparib was efficiently transported by both human ABCB1 and
ABCG2, and very efficiently by mouse Abcg2. Transport could be inhibited by the
small-molecule ABCB1 and ABCG2 inhibitors zosuquidar and Ko143, respectively. In
vivo, oral availability (plasma AUC0-1 and AUC0-24) and brain levels of
rucaparib at 1 and 24 h were increased by the absence of both Abcg2 and
Abcb1a/1b after oral administration of rucaparib at 10 mg/kg.
CONCLUSIONS: Our data show to our knowledge for the first time that oral
availability and brain accumulation of a PARP inhibitor are markedly and
additively restricted by Abcg2 and Abcb1a/1b. This may have clinical relevance
for improvement of rucaparib therapy in PARP inhibitor-resistant tumors with
ABCB1 and/or ABCG2 expression and in patients with brain (micro)metastases
positioned behind a functional blood-brain barrier. Poly(ADP-ribose) polymerases(PARP) synthesize the ADP-ribose polymers onto
proteins and play a role in DNA repair. PARP inhibitors block the repair of
single-strand breaks, which in turn gives rise to double-strand breaks during
DNA replication. Thus, PARP inhibitors elicit synthetic lethality in cancer with
BRCA1/2 loss-of-function mutations that hamper homologous recombination repair
of double-strand breaks. Olaparib, the first-in-class PARP inhibitor, was
approved for treatment of BRCA-mutated ovarian cancer in Europe and the United
States in 2014. Other PARP inhibitors under clinical trials include rucaparib,
niraparib, veliparib, and the "PARP-trapping" BMN-673. BRCA1/2 sequencing is an
FDA-approved companion diagnostics, which predicts the cancer vulnerability to
PARP inhibition. Together, synthetic lethal PARP inhibition is a novel promising
strategy for cancer intervention even in cases without prominent driver
oncogenes. BACKGROUND: Rucaparib is an orally available potent selective small-molecule
inhibitor of poly(ADP-ribose) polymerase (PARP) 1 and 2. Rucaparib induces
synthetic lethality in cancer cells defective in the homologous recombination
repair pathway including BRCA-1/2. We investigated the efficacy and safety of
single-agent rucaparib in germline (g) BRCA mutation carriers with advanced
breast and ovarian cancers.
METHODS: Phase II, open-label, multicentre trial of rucaparib in proven BRCA-1/2
mutation carriers with advanced breast and or ovarian cancer, WHO PS 0-1 and
normal organ function. Intravenous (i.v.) and subsequently oral rucaparib were
assessed, using a range of dosing schedules, to determine the safety,
tolerability, dose-limiting toxic effects and pharmacodynamic (PD) and
pharmacokinetic (PK) profiles.
RESULTS: Rucaparib was well tolerated in patients up to doses of 480 mg per day
and is a potent inhibitor of PARP, with sustained inhibition ⩾24 h after single
doses. The i.v. rucaparib (intermittent dosing schedule) resulted in an
objective response rate (ORR) of only 2% but with 41% (18 out of 44) patients
achieved stable disease for ⩾12 weeks and 3 patients maintaining disease
stabilisation for >52 weeks. The ORR for oral rucaparib (across all six dose
levels) was 15%. In the oral cohorts, 81% (22 out of 27) of the patients had
ovarian cancer and 12 out of 13, who were dosed continuously, achieved RECIST
complete response/partial response (CR/PR) or stable disease (SD) ⩾12 weeks,
with a median duration of response of 179 days (range 84-567 days).
CONCLUSIONS: Rucaparib is well tolerated and results in high levels of PARP
inhibition in surrogate tissues even at the lowest dose levels. Rucaparib is
active in gBRCA-mutant ovarian cancer and this activity correlates with
platinum-free interval. The key lessons learned from this study is that
continuous rucaparib dosing is required for optimal response, the recommended
phase 2 dose (RP2D) for continuous oral scheduling has not been established and
requires further exploration and, thirdly, the use of a PD biomarker to evaluate
dose-response has its limitations. : High-grade serous ovarian cancer is characterized by genomic instability, with
one half of all tumors displaying defects in the important DNA repair pathway of
homologous recombination. Given the action of poly(ADP-ribose) polymerase (PARP)
inhibitors in targeting tumors with deficiencies in this repair pathway by loss
of BRCA1/2, ovarian tumors could be an attractive population for clinical
application of this therapy. PARP inhibitors have moved into clinical practice
in the past few years, with approval from the Food and Drug Administration (FDA)
and European Medicines Agency (EMA) within the past 2 years. The U.S. FDA
approval of olaparib applies to fourth line treatment in germline BRCA-mutant
ovarian cancer, and European EMA approval to olaparib maintece in both
germline and somatic BRCA-mutant platinum-sensitive ovarian cancer. In order to
widen the ovarian cancer patient population that would benefit from PARP
inhibitors, predictive biomarkers based on a clear understanding of the
mechanism of action are required. Additionally, a better understanding of the
toxicity profile is needed if PARP inhibitors are to be used in the curative,
rather than the palliative, setting. We reviewed the development of PARP
inhibitors in phase I-III clinical trials, including combination trials of PARP
inhibitors and chemotherapy/antiangiogenics, the approval for these agents, the
mechanisms of resistance, and the outstanding issues, including the development
of biomarkers and the rate of long-term hematologic toxicities with these
agents.
IMPLICATIONS FOR PRACTICE: The poly(ADP-ribose) polymerase (PARP) inhibitor
olaparib has recently received approval from the Food and Drug Administration
(FDA) and European Medicines Agency (EMA), with a second agent (rucaparib)
likely to be approved in the near future. However, the patient population with
potential benefit from PARP inhibitors is likely wider than that of germline
BRCA mutation-associated disease, and biomarkers are in development to enable
the selection of patients with the potential for clinical benefit from these
agents. Questions remain regarding the toxicities of PARP inhibitors, limiting
the use of these agents in the prophylactic or adjuvant setting until more
information is available. The indications for olaparib as indicated by the FDA
and EMA are reviewed. Rucaparib camsylate (CO-338;
8-fluoro-2-{4-[(methylamino)methyl]phenyl}-1,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indol-6-one
((1S,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]hept-1-yl)methanesulfonic acid salt) is
a PARP1, 2 and 3 inhibitor. Phase I studies identified a recommended Phase II
dose of 600 mg orally twice daily. ARIEL2 Part 1 established a tumor genomic
profiling test for homologous recombination loss of heterozygosity
quantification using a next-generation sequencing companion diagnostic (CDx).
Rucaparib received US FDA Breakthrough Therapy designation for treatment of
platinum-sensitive BRCA-mutated advanced ovarian cancer patients who received
greater than two lines of platinum-based therapy. Comparable to rucaparib
development, other PARP inhibitors, such as olaparib, niraparib, veliparib and
talazoparib, are developing CDx tests for targeted therapy. PARP inhibitor
clinical trials and CDx assays are discussed in this review, as are potential
PARP inhibitor combination therapies and likely resistance mechanisms. For several years, a major obstacle in the systemic treatment of ovarian cancer
has been the lack of a therapeutic strategy tailored to specific biomarkers
present in the individual patient's tumour. However, considerable progress has
been made recently through the development of drugs targeting cells deficient in
the key mechanism of double-strand DNA repair, known as homologous recombination
(HRD). These drugs, inhibitors of the enzyme poly (ADP) ribose polymerase
(PARP), selectively kill HRD cells through a process known as tumour-selective
synthetic lethality. Olaparib is the first such agent, now approved for the
treatment of ovarian cancer associated with mutations in the BRCA 1/2 genes,
since these are characterised by cells with HRD. Importantly, another group of
patients with tumours bearing a similar repair deficiency but without BRCA
mutations may also be susceptible to PARP inhibition and efforts to develop an
HRD assay are therefore a priority so that these patients can be identified as
PARPi candidates. In addition, combination strategies are an area of intense
research; these include combinations with antiangiogenic agents and with
inhibitors of the P13K/AKT pathway and others are likely to merit assessment
since resistance to PARP inhibitors will certainly emerge as the next challenge.
While olaparib is the first PARP inhibitor to receive approval for ovarian
cancer treatment, others including rucaparib and niraparib are clearly effective
in this disease and, within the next year or two, the results of ongoing
randomised trials will clarify their respective roles. PARP inhibitors are
generally well tolerated; regulatory approval at present supports their use as a
maintece therapy (in Europe) and as treatment for advanced recurrent disease
(in the United States), but it is likely that these indications will extend as
the results of ongoing trials become available. Ten years have elapsed between
the first pre-clinical publications and the regulatory approval of PARP
inhibitors and the next 10 years promise to be even more productive. BACKGROUND: Slow progress in improving the outcome of ovarian cancer with
chemotherapy over the last decade has stimulated research into molecularly
targeted therapy. Poly(ADP-ribose) polymerase (PARP) inhibitors target DNA
repair and are specifically active in cells that have impaired repair of DNA by
the homologous recombination (HR) pathway. Cells with mutated BRCA function have
HR deficiency (HRD), which is also present in a significant proportion of
non-BRCA-mutated ovarian cancer.
DESIGN: In the last decade, olaparib, the first and most-investigated oral PARP
inhibitor, has undergone phase I-III trials as a single agent, in comparison
with and in addition to chemotherapy, and as a maintece therapy following
chemotherapy.
RESULTS: The greatest benefit to-date has been in the maintece setting,
prolonging the progression-free survival of high-grade serous ovarian cancer
with a BRCA1/2 mutation. In this group of patients, olaparib has received
approval as maintece following chemotherapy from the EMA, and accelerated
approval as a single agent in women who have had three or more lines of therapy.
Olaparib can be given for a prolonged period with few significant side-effects
in most patients. Similar trials with other PARP inhibitors (rucaparib,
niraparib and veliparib) are in progress and include non-BRCA-mutated ovarian
cancer. Second-generation studies are exploring the combination of PARP
inhibitors with anti-angiogenic drugs.
CONCLUSIONS: PARP inhibitors represent a step change in the management of
ovarian cancer. BRCA mutations are the first genotypic predictive markers in
ovarian cancer and can be used to select patients who will most likely benefit
from PARP inhibitors. BRCA testing is now becoming a routine part of the
evaluation of women with ovarian cancer, and tests for HRD are being used to
evaluate PARP inhibitors in an extended population of non-BRCA-mutated ovarian
cancer. Purpose: DNA damage defects are common in ovarian cancer and can be used to
stratify treatment. Although most work has focused on homologous recombination
(HR), DNA double-strand breaks are repaired primarily by nonhomologous end
joining (NHEJ). Defects in NHEJ have been shown to contribute to genomic
instability and have been associated with the development of
chemoresistance.Experimental Design: NHEJ was assessed in a panel of ovarian
cancer cell lines and 47 primary ascetic-derived ovarian cancer cultures, by
measuring the ability of cell extracts to end-join linearized plasmid monomers
into multimers. mRNA and protein expression of components of NHEJ was determined
using RT-qPCR and Western blotting. Cytotoxicities of cisplatin and the PARP
inhibitor rucaparib were assessed using sulforhodamine B (SRB) assays. HR
function was assessed using γH2AX/RAD51 foci assay.Results: NHEJ was defective
(D) in four of six cell lines and 20 of 47 primary cultures. NHEJ function was
independent of HR competence (C). NHEJD cultures were resistant to rucaparib (P
= 0.0022). When HR and NHEJ functions were taken into account, only NHEJC/HRD
cultures were sensitive to rucaparib (compared with NHEJC/HRC P = 0.034,
NHEJD/HRC P = 0.0002, and NHEJD/HRD P = 0.0045). The DNA-PK inhibitor, NU7441,
induced resistance to rucaparib (P = 0.014) and HR function recovery in a
BRCA1-defective cell line.Conclusions: This study has shown that NHEJ is
defective in 40% of ovarian cancers, which is independent of HR function and
associated with resistance to PARP inhibitors in ex vivo primary cultures. Clin
Cancer Res; 23(8); 2050-60. ©2016 AACR. Recent advances in our understanding of the molecular biology of epithelial
ovarian cancer have led to the development of a number of targeted therapies,
including poly-ADP-ribose polymerase (PARP) inhibitors. PARP inhibitors are a
novel class of therapeutic agents that target tumors with deficiencies in the
homologous recombination DNA repair pathway. Early studies have shown
significant efficacy for PARP inhibitors in patients with germline BRCA1/2
mutations. It has become evident that BRCA wild-type patients with other defects
in the homologous recombination repair pathway benefit from this therapeutic
approach. Importantly, companion homologous recombination deficiency scores are
being developed to help guide the selection of patients most likely to gain
clinical benefit from PARP inhibition. Olaparib, the first and most extensively
investigated PARP inhibitor, is now licensed in Europe for maintece treatment
of patients with platinum-sensitive relapsed BRCA-mutated (germline or somatic)
high-grade serous ovarian cancer who have responded to platinum-based
chemotherapy. In the United States, olaparib is licensed for treatment of
patients with germline BRCA-mutated ovarian cancer who have received 3 or more
lines of chemotherapy. There are a number of other PARP inhibitors in late phase
clinical development in ovarian cancer including rucaparib, niraparib,
veliparib, and talazoparib. This review will focus on the current evidence for
PARP inhibitors in ovarian cancer and discuss ongoing clinical trials and future
research directions in this rapidly evolving area. 1. |
What is the role of cohesins at the IFNG locus? | Cohesins form cell-type-specific long-range chromosomal cis-interactions at the developmentally regulated IFNG locus. Hence, the ability of cohesin to constrain chromosome topology is used not only for the purpose of sister chromatid cohesion, but also to dynamically define the spatial conformation of specific loci. This new aspect of cohesin function is probably important for normal development and disease. | Cohesin-mediated sister chromatid cohesion is essential for chromosome
segregation and post-replicative DNA repair. In addition, evidence from model
organisms and from human genetics suggests that cohesin is involved in the
control of gene expression. This non-canonical role has recently been
rationalized by the findings that mammalian cohesin complexes are recruited to a
subset of DNase I hypersensitive sites and to conserved noncoding sequences by
the DNA-binding protein CTCF. CTCF functions at insulators (which control
interactions between enhancers and promoters) and at boundary elements (which
demarcate regions of distinct chromatin structure), and cohesin contributes to
its enhancer-blocking activity. The underlying mechanisms remain unknown, and
the full spectrum of cohesin functions remains to be determined. Here we show
that cohesin forms the topological and mechanistic basis for cell-type-specific
long-range chromosomal interactions in cis at the developmentally regulated
cytokine locus IFNG. Hence, the ability of cohesin to constrain chromosome
topology is used not only for the purpose of sister chromatid cohesion, but also
to dynamically define the spatial conformation of specific loci. This new aspect
of cohesin function is probably important for normal development and disease. |
Describe Wellens' Syndrome. | Wellens Syndrome (WS) is a condition characterized by typical changes in ECG, which are biphasic T-wave inversions (less common) or symmetric and deeply inverted T waves (including 75%) in lead V2-V3 chest derivations. | The pattern of clinical findings and electrocardiography (ECG) changes known as
Wellens' syndrome is associated with significant stenosis of the proximal left
anterior descending coronary artery. Cases can be classified according to the
ECG pattern into type 1 (biphasic T waves) or type 2 (deeply inverted T waves,
especially in leads V2 and V3). We present here an unusual case of Wellens'
syndrome in which the ECG pattern changed from type 2 to type 1 during
observation, and in which the coronary lesion was in the middle rather than the
proximal part of the left anterior descending artery. Wellens' syndrome is characterized by symmetrically inverted T-waves in the
precordial leads suggestive of impending myocardial infarction due to a critical
proximal left anterior descending (LAD) stenosis. We describe three unusual
cases of patients with such electrocardiographic abnormality in which coronary
angiography ruled out the presence of critical coronary stenosis and cardiac
magnetic resoce imaging excluded the presence of acute or chronic myocardial
infarction. We describe the case of a 40-year-old woman who presented to the Emergency
Department with resolving chest pain. The initial electrocardiogram (ECG) showed
biphasic T-waves in V2-V4, which was recognized as Wellens' syndrome, or acute
coronary T-wave syndrome. Emergent cardiac catheterization revealed 95% stenosis
of the previously placed stent in the proximal left anterior descending artery
(LAD). Review of her records from 3 years prior revealed similar ECG findings
consistent with Wellens' syndrome, at which time a stent was placed for critical
proximal LAD stenosis. We report this case to increase awareness of the T-wave
abnormalities associated with Wellens' syndrome. There is significant morbidity
and mortality that can occur in the absence of emergent coronary
revascularization. This report of repeated Wellens' syndrome in the same patient
demonstrates both types of precordial T-wave abnormalities that characterize
Wellens' syndrome. Wellens syndrome is a clinical-electrocardiographic entity also referred to as
left anterior descending (LAD) coronary T-wave syndrome or acute coronary T-wave
syndrome. It is a complex of symptoms and signals indicating the existence of an
undesirable condition secondary to critical high-grade proximal stenosis of the
LAD coronary artery characterized by the association of prior history of acute
coronary syndrome with little or no elevation of markers of myocardial damage
(unstable angina) and characteristic electrocardiographic changes consistent
with subepicardial anterior ischemic pattern (persistently symmetrical, deep
negative and broad-based T waves) or plus-minus T waves with inversion of the
terminal portion in the LAD coronary artery territory (V1 through V5 or V6). We
present a case of a variant of Wellens syndrome that reveals association and,
transitorily, the criteria described in literature for left septal fascicular
block. The diagnosis of acute coronary syndrome relies on clinical history,
electrocardiographic (ECG) changes, and cardiac biomarkers; but within the
spectrum of acute coronary syndrome, there exist subtle presentations that
cannot afford to be overlooked. Wellens syndrome is one such example, in which a
patient can present with both ECG changes that are not classic for myocardial
ischemia and negative cardiac biomarkers. The characteristic ECG findings
associated with Wellens syndrome consist of deep, symmetric T-wave inversions in
the anterior precordial leads. However, Wellens syndrome can also present as
biphasic T-wave inversions in those same ECG leads. The associated critical
stenosis of the proximal left anterior descending artery carries an immediately
life-threatening prognosis if not recognized promptly (Am Heart J. 1982;103[4 Pt
2]:730-736). We describe a case of a less common manifestation of Wellens
syndrome (type 1) followed by a discussion of its implications and management. Wellens syndrome is characterised by negative or biphasic T waves in V2-V4 leads
and critical stenosis of proximal part of the left descending coronary artery.
These ECG changes without atherosclerotic changes in coronary angiography, i.e.
coronary artery spasm are called pseudo-Wellens syndrome. We describe a patient
with acute coronary syndrome and pseudo-Wellens syndrome as a cause of
vasospastic angina. These ECG abnormalities need differentiation with acute
pulmonary embolism. BACKGROUND: The Wellens' electrocardiogram (ECG) pattern of dynamic T-wave
inversion in the anterior leads is observed in clinical conditions characterized
by reversible left ventricular (LV) dysfunction (stunned myocardium), either
ischemic or nonischemic. The pathophysiologic basis of this ECG pattern remains
to be elucidated.
OBJECTIVE: The purpose of this study was to report the contrast-enhanced cardiac
magnetic resoce (CE-CMR) findings in 4 cases of Wellens' ECG pattern
associated with transient LV dysfunction from a variety of clinical conditions
such as myocardial bridge, coronary artery dissection, cholecystitis, and
takotsubo syndrome.
METHODS: All patients underwent CE-CMR at the time of acute clinical
manifestations and after 6 to 8 weeks of follow-up to assess the presence and
dynamics of LV myocardial changes.
RESULTS: In all patients, the Wellens' ECG abnormalities were associated with
increased signal intensity of the LV myocardium on T2-weighted sequences
suggesting myocardial edema, in the absence of late enhancement on postcontrast
sequences. Repolarization abnormalities and myocardial edema had a parallel time
course with persistence beyond recovery of mechanical abnormalities. T-wave
inversion was associated with transient prolongation of the QTc interval in all
cases.
CONCLUSION: The study results suggest that myocardial edema rather than systolic
dysfunction underlies the Wellens' ECG pattern, regardless of the causative
mechanism. We describe a case of previously healthy 72-year-old man, who presented with
rest angina. The ECG revealed sinus rhythm, biphasic T waves with preserved R
waves in V1-V4 precordial leads. Subsequent evaluation revealed the normal serum
cardiac marker levels and echocardiography with the coronary angiography showing
a critical lesion in the proximal left anterior descending artery. Thus it was
diagnosed as Wellens' syndrome. In our case, we highlight the subtle though
classical ECG findings of Wellens' syndrome and its specific angiographic
correlation. It can be of vital importance to identify these changes and
intervene in time appropriately so as to avoid the development of myocardial
infarction that carries a substantial morbidity and mortality. Important aspects
of diagnosis and management have also been reviewed. We describe a case of a 42-year-old man, with a previous episode of angina and a
normal ECG and serum cardiac markers, and a two months later finding of biphasic
T wave in leads V2-V3 and deeply inverted T wave in V4-V5 at a asymptomatic
occupational evaluation. This is a typical ECG pattern of Wellens' syndrome. A
subsequent coronary angiography showed a critical stenosis of proximal left
anterior descendent. We underline the careful value of prolonged observation in
chest pain unit and repetitive ECG evaluation also during pain-free period after
an angina episode, to exclude an earlier T wave pseudonormalization. Coronary artery vasospasm is an important cause of chest pain syndromes that can
lead to myocardial infarction, ventricular arrhythmias, and sudden death. In
1959, Prinzmetal et al described a syndrome of nonexertional chest pain with
ST-segment elevation on electrocardiography. Persistent angina is challenging,
and repeated coronary angioplasty may be required in this syndrome. Calcium
antagonists are extremely effective in treating and preventing coronary spasm,
and may provide long-lasting relief for the patient. Whereas the Wellens'
syndrome is characterized by symmetrically inverted T-waves with preserved R
waves in the precordial leads suggestive of impending myocardial infarction due
to a critical proximal left anterior descending stenosis, the pseudo-Wellens'
syndrome caused by coronary artery spasm has also rarely been reported in
literature. We present a pseudo-Wellens syndrome as a cause of vasospastic
angina, and a diffuse ST segment elavation on electrocardiogram resembling the
Greek letter lambda, called also 'action potential-like' ECG in a patient with
vasospastic-type Printzmetal angina. INTRODUCTION: Wellens Syndrome (WS) is a condition characterized by typical
changes in ECG, which are biphasic T-wave inversions (less common) or symmetric
and deeply inverted T waves (including 75%) in lead V2-V3 chest derivations. WS
is considered important because it has not only diagnostic value but also
prognostic value.
CASE REPORT: A 52-year-old male patient without cardiovascular disease or risk
factors was admitted to the emergency department (ED) suffering with chest pain
and syncope, just after having been involved in a discussion at work. Chest pain
was radiating to the left arm and was not precipitated by exertion. Shortness of
breath was not accompanied by angina. The patient underwent cardiac
catheterization at Department of Cardiology. Stents were positioned in both LADA
and a severe lesion in the left main coronary artery. The patient was discharged
two days following catheterization, due to no chest pain and hemodynamic
instability during the hospitalization. The patient has approved the inform
consent for to be used for this case report. BACKGROUND: Reperfusion after coronary occlusion (myocardial infarction, MI), as
in Wellens' syndrome, is often represented on ECG as T-wave inversion in the
leads overlying the affected myocardial wall(s). As an extension of this logic,
reperfusion of the posterior wall should manifest on right precordial leads
(which are opposite the posterior wall) as enlarged T-waves.
OBJECTIVE: We sought to determine whether T-wave amplitude (TWa) in leads V2 and
V3 after reperfusion in posterior MI (PMI) is greater than in patients without
PMI.
METHODS: Review of ECGs from patients with ST elevation MI of the left
circumflex or right coronary artery with post-procedure thrombolysis in MI
(TIMI) flow >0 between 2007 and 2009. Blinded experts reviewed admission ECGs to
determine the presence of PMI and measure TWa before and after reperfusion.
Maximum TWa in V2 and V3 and the difference between maximum and admission V2 and
V3 TWa were compared between those with and without PMI.
RESULTS: Of 72 patients, 48 had PMI. Values expressed are medians and IQRs.
Maximum TWa after reperfusion was greater in PMI than in non-PMI in V2 (5.00 mm
(3.5 to 8.25) vs 3.9 mm (2.75 to 5.5), p=0.04), but not in V3 (4.0 mm (2 to 5.5)
vs 3.0 mm (1.75 to 4), p=0.09). The increase in TWa in V2 and V3 after
reperfusion was greater in PMI compared with non-PMI: (V2, 3.4 mm (2 to 5.25) vs
1.25 mm (-0.25 to 2), p=0.0005; V3, 2 mm (-0.5 to 3.25) vs 0.25 mm (-1 to 1.75),
p=0.03).
CONCLUSIONS: Reperfusion of the posterior wall results in higher right
precordial TWa, and an even greater increase in TWa, as measured in leads V2 and
V3. This observation has important implications for emergency physicians to
accurately identify recent posterior infarction in patients who may be symptom
free on presentation but at risk of reocclusion. Wellens' syndrome is characterized by T-wave changes in electrocardiogram (EKG)
during pain-free period in a patient with intermittent angina chest pain. It
carries significant diagnostic and prognostic value because this syndrome
represents a pre-infarction stage of coronary artery disease involving proximal
left anterior descending (LAD) artery, which can subsequently lead to extensive
anterior myocardial infarctions (MIs) and even death without coronary
angioplasty. Therefore, it is crucial for every physician to recognize EKG
features of Wellens' syndrome in order to take appropriate immediate
intervention to reduce mortality and morbidity for MI. Here, we report a case of
an overweight man with 35 pack-year of smoking history who presented to Easton
Hospital with intermittent pressing chest pain of 5/6 times within 10 day-period
and was found to have type A Wellens' sign, which was biphasic T-waves in
precordial leads V2 and V3 during pain-free period with no cardiac enzymes
elevation. He was given therapeutic lovenox and subsequently underwent coronary
angioplasty and had 95-99% occlusion in proximal LAD artery. The unique feature
of our case was that Wellens' type B EKG changes were seen after reduction of
stenosis with LAD artery stent, which was likely explained by the reperfusion of
the ischemic myocardium. Therefore, it is important for physicians to recognize
EKG features of Wellens' syndrome in order to take appropriate therapy to
reducing mortality and morbidity form impending MI. We report the case of a woman in her 70s presenting to the emergency department
with syncope, troponemia, and an electrocardiogram with deep symmetric T-wave
inversions in V2 and V3 and prolonged QTc. Her presentation was concerning for
acute coronary syndrome, Wellens syndrome in particular, given the elevated
troponin levels, lack of ST segment changes, and characteristic T-wave findings.
The diagnosis was confirmed with angiography that showed a critical left
anterior descending (LAD) artery occlusion. Since myocardial infarction does not
typically present with syncope, we explored the differential diagnoses for
T-wave inversions, which include electrolyte abnormalities, medications,
intracranial hemorrhage, pulmonary embolism, and other cardiac diseases that
were ruled out in our patient. We also explored the pathophysiology leading to
syncope in the setting of acute myocardial infarction including arrhythmias and
exaggerated neurally mediated response. Our patient received two drug-eluting
stents to the LAD artery and was started on dual antiplatelet therapy,
beta-blockers, and an angiotensin-converting enzyme inhibitor. |
Which human disease is associated with mutated UBQLN2 | Ggene mutations in UBQLN2 cause dominant inheritance of amyotrophic lateral sclerosis (ALS). | Amyotrophic Lateral Sclerosis (ALS) is the most frequent motor neuron disease in
adults. Classical ALS is characterized by the death of upper and lower motor
neurons leading to progressive paralysis. Approximately 10 % of ALS patients
have familial form of the disease. Numerous different gene mutations have been
found in familial cases of ALS, such as mutations in superoxide dismutase 1
(SOD1), TAR DNA-binding protein 43 (TDP-43), fused in sarcoma (FUS), C9ORF72,
ubiquilin-2 (UBQLN2), optineurin (OPTN) and others. Multiple animal models were
generated to mimic the disease and to test future treatments. However, no animal
model fully replicates the spectrum of phenotypes in the human disease and it is
difficult to assess how a therapeutic effect in disease models can predict
efficacy in humans. Importantly, the genetic and phenotypic heterogeneity of ALS
leads to a variety of responses to similar treatment regimens. From this has
emerged the concept of personalized medicine (PM), which is a medical scheme
that combines study of genetic, environmental and clinical diagnostic testing,
including biomarkers, to individualized patient care. In this perspective, we
used subgroups of specific ALS-linked gene mutations to go through existing
animal models and to provide a comprehensive profile of the differences and
similarities between animal models of disease and human disease. Finally, we
reviewed application of biomarkers and gene therapies relevant in personalized
medicine approach. For instance, this includes viral delivering of antisense
oligonucleotide and small interfering RNA in SOD1, TDP-43 and C9orf72 mice
models. Promising gene therapies raised possibilities for treating differently
the major mutations in familial ALS cases. Author information:
(1)Institute of Molecular, Cell and Systems Biology, College of Medical,
Veterinary and Life Sciences, Davidson Building, Henry Wellcome Lab of Cell
Biology, University of Glasgow, G12 8QQ Glasgow, UK; The MRC Protein
Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of
Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.
(2)Institute of Molecular, Cell and Systems Biology, College of Medical,
Veterinary and Life Sciences, Davidson Building, Henry Wellcome Lab of Cell
Biology, University of Glasgow, G12 8QQ Glasgow, UK; The MRC Protein
Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of
Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.
Electronic address: [email protected].
(3)The MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black
Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1
5EH, Scotland.
(4)Newcastle University Protein and Proteome Analysis, Devonshire Building,
Devonshire Terrace, Newcastle upon Tyne NE1 7RU, UK.
(5)Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa,
Israel.
(6)Department of Medical and Molecular Genetics, King's College London, 8th
Floor Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, UK.
(7)School of Veterinary Medicine, College of Medical, Veterinary and Life
Sciences, University of Glasgow, 464 Bearsden Road, Glasgow G61 1QH, UK.
(8)Institute of Molecular, Cell and Systems Biology, College of Medical,
Veterinary and Life Sciences, Davidson Building, Henry Wellcome Lab of Cell
Biology, University of Glasgow, G12 8QQ Glasgow, UK; The MRC Protein
Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of
Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.
Electronic address: [email protected]. Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease,
which causes progressive and eventually fatal loss of motor function. Here, we
describe genetic and pathologic characterization of brain tissue banked from 19
ALS patients over nearly 20 years at the Department of Anatomy and the Centre
for Brain Research, University of Auckland, New Zealand. We screened for
mutations in SOD1, TARDBP, FUS, and C9ORF72 genes and for neuropathology caused
by phosphorylated TDP-43, dipeptide repeats (DPRs), and ubiquilin. We identified
2 cases with C9ORF72 repeat expansions. Both harbored phosphorylated TDP-43 and
DPR inclusions. We show that DPR inclusions can incorporate or occur
independently of ubiquilin. We also identified 1 case with a UBQLN2 mutation,
which showed phosphorylated TDP-43 and characteristic ubiquilin protein
inclusions. This is the first study of ALS genetics in New Zealand, adding New
Zealand to the growing list of countries in which C9ORF72 repeat expansion and
UBQLN2 mutations are detected in ALS cases. Missense mutations in ubiquilin 2 (UBQLN2) cause ALS with frontotemporal
dementia (ALS-FTD). Animal models of ALS are useful for understanding the
mechanisms of pathogenesis and for preclinical investigations. However, previous
rodent models carrying UBQLN2 mutations failed to manifest any sign of motor
neuron disease. Here, we show that lines of mice expressing either the
ALS-FTD-linked P497S or P506T UBQLN2 mutations have cognitive deficits,
shortened lifespans, and develop motor neuron disease, mimicking the human
disease. Neuropathologic analysis of the mice with end-stage disease revealed
the accumulation of ubiquitinated inclusions in the brain and spinal cord,
astrocytosis, a reduction in the number of hippocampal neurons, and reduced
staining of TAR-DNA binding protein 43 in the nucleus, with concomitant
formation of ubiquitin+ inclusions in the cytoplasm of spinal motor neurons.
Moreover, both lines displayed denervation muscle atrophy and age-dependent loss
of motor neurons that correlated with a reduction in the number of large-caliber
axons. By contrast, two mouse lines expressing WT UBQLN2 were mostly devoid of
clinical and pathological signs of disease. These UBQLN2 mouse models provide
valuable tools for identifying the mechanisms underlying ALS-FTD pathogenesis
and for investigating therapeutic strategies to halt disease. |
Which ApoE isoform is associated with hyperlipoproteinemia? | Type III hyperlipoproteinemia (HLP) is characterized by the accumulation of remnant lipoproteins in the plasma and it is associated with ApoE2 isoform. ApoE2 binds poorly to low density lipoprotein receptors, resulting in defective remnant lipoprotein clearance. | Type III hyperlipoproteinemia typically is associated with homozygosity for
apolipoprotein (apo) E2(Arg158----Cys). Domit expression of type III
hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by
heterozygosity for a human apoE variant, apoE3(Cys112----Arg, Arg142----Cys).
However, this apoE3 variant was not separable from the normal apoE3 in these
patients' plasma because the two proteins have identical amino acid composition,
charge, and molecular weight. Therefore, to determine the functional
characteristics of this protein, we used recombit DNA techniques to produce
this apoE variant in bacteria. We also produced a non-naturally occurring
variant, apoE(Arg142----Cys), that had only the cysteine substituted at residue
142. These two apoE variants were purified from cell lysates of the transfected
Escherichia coli by ultracentrifugal flotation in the presence of phospholipid,
by gel filtration chromatography, and by heparin-Sepharose chromatography. Both
Cys142 apoE variants bound to lipoprotein receptors on human fibroblasts with
only about 20% of normal binding activity. Therefore, cysteine at residue 142,
not arginine at residue 112, is responsible for the decreased receptor binding
activity of the variants. Cysteamine treatment and removal of the
carboxyl-terminal domain had little effect on the binding activity, whereas both
modulate the receptor binding activity of apoE2(Arg158----Cys). The mutation at
residue 142 decreased the binding activity of apoE to both heparin and the
monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE),
whereas apoE2(Arg158----Cys), which is associated with recessive expression of
type III hyperlipoproteinemia, binds normally to both. The Arg112, Cys142
variant predomites 3:1 over normal apoE3 in the very low density lipoproteins
of plasma from an affected subject, as assessed by differential reactivity with
the antibody 1D7. The unique combination of functional properties of the Arg112,
Cys142 variant provides a possible explanation for its association with domit
expression of type III hyperlipoproteinemia. There is agreement about the influence of the genetic apolipoprotein (apo E)
polymorphism on plasma lipid and apoprotein levels in man. Whereas the
association of the apo E2 isoform with primary dysbetalipoproteinemia and
hyperlipoproteinemia type III is well established, the plasma- and
LDL-cholesterol lowering effects of apo E2 and the phenomenon of apo E4 raising
these parameters on the development of coronary heart disease is still a matter
of controversial discussion. Despite these uncertainties the knowledge of an
individual's apo E phenotype may provide additional information in future to
judge its risk to develop atherosclerosis. From a variety of methods meanwhile
described to analyze the polymorphism and evaluate the apo E phenotype the
author has modified three procedures to apply to different questions concerning
apo E isoform analysis. They concern the visualization of the apo E pattern in
diluted solutions and those containing high salt concentrations, the apo E
phenotyping on a large scale basis from whole plasma avoiding a possible
misinterpretation by a thrombin fragment of apo E and finally the search for new
apo E variants with a high resolution system on immobilized pH gradient gels. Apolipoprotein E (apoE) is important in the modulation of the catabolism of
chylomicron and very low density lipoprotein (VLDL) remts. ApoE has three
major genetically determined isoproteins in plasma, designated apoE-2, apoE-3
and apoE-4, with homozygosity for the allele coding for apoE-2 being associated
with dysbetalipoproteinemia or type III hyperlipoproteinemia (HLP). We describe
a new variant of apoE, apoE-1Harrisburg, which is, in contrast to apoE-2,
domitly associated with type III HLP. Five of twelve members of the affected
kindred are heterozygous for the mutant form of apoE, and four of the five have
type III HLP, while the fifth member has dysbetalipoproteinemia on diet therapy.
Neuraminidase digestion, which removes charged sialic acid residues, did not
alter the electrophoretic position of the apoE-1Harrisburg isoprotein,
indicating that the altered charge of apoE-1Harrisburg was not due to sialic
acid addition to the apolipoprotein. Cysteamine modification, which adds a
positively charged group to cysteine, resulted in a shift of apoE-1Harrisburg
from the E-1 to the E-2 isoform position, indicating that there is one cysteine
in apoE-1Harrisburg as is the case for apoE-3. These results are consistent with
apoE-1Harrisburg originating in the allele for apoE-3 with the mutation leading
to a negative two-unit charge shift. The definitive identification of a kindred
with an apoE variant, apoE-1Harrisburg, domitly associated with
dysbetalipoproteinemia and type III HLP provides a unique opportunity to gain
important insights into the structure-function requirements of the E
apolipoprotein as well as the mechanisms by which apoE modulates lipoprotein
metabolism. Minigel methodology has been utilized for apolipoprotein (apo) E isoform
phenotyping and criteria for distinguishing the apoE4/4 phenotype (mean
apoE4/apoE3 ratio: 5.81) from the apoE4/3 phenotype (mean ratio: 1.01) and the
apoE3/3 phenotype (mean apoE3/apoE2 ratio: 2.67) from the apoE3/2 phenotype
(mean ratio: 0.76) based on gel scanning were developed. ApoE allele frequencies
in 1209 subjects were: apoE3, 0.786; apoE4, 0.135; apoE2, 0.075; apoE5, 0.002;
and apoE1, 0.002. Subjects with the apoE2 allele tended to have higher plasma
very low density lipoprotein (VLDL) cholesterol and lower low density
lipoprotein (LDL) cholesterol concentrations than subjects with the apoE3
allele, while the converse was true for subjects with the apoE4 allele. Subjects
with the rare apoE1 allele had values similar to those with the apoE2 allele,
while subjects with the rare apoE5 allele had values similar to those with the
apoE4 allele. Apolipoprotein E (apoE) is associated with plasma lipoproteins and is important
in modulating the catabolism of remts of triglyceride-rich lipoproteins. ApoE
is a polymorphic protein, and homozygosity for the E2 allele is associated with
type III hyperlipoproteinemia. A variant of apoE, apoEBethesda, was found that
migrates in the E1 position on isoelectric focusing. Evaluation of the proband
and her family showed that both individuals with apoEBethesda had type III
hyperlipoproteinemia, and the analysis was consistent with the concept that
apoEBethesda was coded by a new E allele and that this allele was expressed in a
codomit fashion. The discovery of apoEBethesda and its association with type
III hyperlipoproteinemia provides additional evidence for the genetic
heterogeneity of the E allele and suggests that a number of different structural
mutations of the E apolipoprotein may be associated with the type III phenotype. Type III hyperlipoproteinemia is characterized by delayed chylomicron and VLDL
remt catabolism and is associated with homozygosity for the apoE-2 allele. We
have identified a kindred in which heterozygosity for an apoE mutant, apoE-1
(Lys146-->Glu), is domitly associated with the expression of type III
hyperlipoproteinemia. DNA sequence analysis of the mutant apoE gene revealed a
single-point mutation that resulted in the substitution of glutamic acid (GAG)
for lysine (AAG) at residue 146 in the proposed receptor-binding domain of apoE.
The pathophysiological effect of this mutation was investigated in vivo by
kinetic studies in the patient and six normal subjects, and in vitro by binding
studies of apoE-1 (Lys146-->Glu) to LDL receptors on human fibroblasts and to
heparin. The kinetic studies revealed that apoE-1 (Lys146-->Glu) was catabolized
significantly slower than apoE-3 in normals (P < 0.005). In the proband, the
plasma residence times of both apoEs were substantially longer and the
production rate of total apoE was about two times higher than in the control
subjects. ApoE-1 (Lys146-->Glu) was defective in interacting with LDL receptors,
and its ability to displace LDL in an in vitro assay was reduced to 7.7%
compared with apoE-3. The affinity of apoE-1 (Lys146-->Glu) to heparin was also
markedly reduced compared with both apoE-2 (Arg158-->Cys) and apoE-3. These
abnormal in vitro binding characteristics and the altered in vivo metabolism of
apoE-1 (Lys146-->Glu) are proposed to result in the functional domice of this
mutation in the affected kindred. The initial step in the clearance of apolipoprotein (apo) E-enriched remt
lipoproteins from the plasma appears to be sequestration within the liver
mediated by their binding to heparan sulfate proteoglycans (HSPG). The
surface-bound remts are believed to be internalized by their interaction with
the low density lipoprotein (LDL) receptor-related protein or by the LDL
receptor. Cholesterol-induced rabbit beta-very low density lipoproteins
(beta-VLDL) enriched in human apoE3 display 4-5-fold enhanced binding to
cultured cells. The present study attempts to determine whether recessive versus
domit type III hyperlipoproteinemia might be explained, at least in part, by
a variable interaction of the mutant forms of apoE with the HSPG and impaired
uptake. The beta-VLDL+apoE2(Arg158-->Cys), which is associated with recessive
type III hyperlipoproteinemia, bound more poorly than beta-VLDL+apoE3 but still
possessed significant enhanced binding (approximately 2-2.5-fold compared with
beta-VLDL without added apoE) to HepG2 and McA-RH7777 cells. In comparison,
beta-VLDL+apoE(Arg142-->Cys), beta-VLDL+apoE(Arg145-->Cys), and
beta-VLDL+apoE-Leiden, which are associated with domit type III
hyperlipoproteinemia, bound more poorly. This same hierarchy of binding and
uptake was determined by [14C]oleate incorporation into cholesteryl esters in
LDL receptor-negative cells and by secretion of apoE3 and the variant apoE forms
from McA-RH7777 cells. Furthermore, the enhanced binding of the apoE-enriched
beta-VLDL was almost totally inhibited by heparinase treatment of the cells, and
the basal binding activity was inhibited by 80-90% following addition of an LDL
receptor antibody capable of blocking receptor-ligand interaction. The beta-VLDL
enriched in apoE or apoE-dimyristoylphosphatidylcholine complexes bound to
isolated HSPG from McA-RH7777 cells or the rat liver to a very similar degree.
Likewise, the binding of beta-VLDL plus the various forms of apoE to the LDL
receptor-related protein on ligand blots paralleled the results of other
studies. In conclusion, all of the type III hyperlipoproteinemic apoE variants
are defective in displaying enhanced binding to HSPG and in the cellular uptake
initiated by HSPG. However, apoE2(Arg158-->Cys) displayed more activity than the
variants associated with the domit forms of type III hyperlipoproteinemia.
The hierarchy of binding and uptake was as follows: apoE3 > apoE2(Arg158-->Cys)
> apoE(Arg145-->Cys) > apoE(Arg142-->Cys) approximately apoE-Leiden (the latter
two usually displaying very little, if any, enhanced binding and uptake). Thus,
a correlation exists between the mode of expression of type III
hyperlipoproteinemia and the binding and uptake of the specific apoE mutation. Apolipoprotein E (apo E) plays a role in the regulation of the lipid metabolism
of humans. Apo E, 229 amino acid polypeptide, is classified into three major
isoform (E2, E3, E4) according to the differences of amino acid in position 112
and 158. In the normal population apo E3 isoform is most prevalent and apo E2 or
E4 is frequently associated with hyperlipoproteinemia. To find out the frequency
of apo E isoform distribution in the Korean population, apo E genotyping was
performed. After amplification of apoE gene by polymerase chain reaction (PCR),
restriction isotyping was done by cleavage with restriction enzyme Hha I and
polyacrylamide gel electrophoresis. The apo E allele frequency in 73 normal
subjects was 4.8% for E2, 84.9% for E3 and 10.3% for E4. In diabetic patient
with hyperlipoproteinemia, the frequency of apo E allele was 6.3% for E2, 81.0%
for E3 and 12.7% for E4. There was no significant difference in apo E isoform
distribution between diabetics and normal populations. But in patients with
cardiovascular disease with hyperlipidemia, the apo E4 allele frequency was
significantly higher than normal (20.0% vs 10.3%, p < 0.005). Apo E3 was the
most common isoform in normal and diabetic subjects and apo E2 isoform was
rather low frequency compared to Caucasians. This pattern is similar to the
Japanese population but somewhat different from other populations. From the data
of a high association of apo E4 allele and cardiovascular disease with
hypercholesterolemia, apo E isoform may be one of the determits of
hyperlipoproteinemia. The PCR method may be useful in apo E genotyping. To study isoform-specific effects of apolipoprotein E (apoE) in vivo, we
generated mice with a human APOE*2 allele in place of the mouse Apoe gene via
targeted gene replacement in embryonic stem cells. Mice expressing human apoE2
(2/2) have virtually all the characteristics of type III hyperlipoproteinemia.
Their plasma cholesterol and triglyceride levels are both twice to three times
those in (normolipidemic) mice that are expressing human apoE3 (3/3) made in an
identical manner. The 2/2 mice are markedly defective in clearing beta-migrating
VLDL particles, and spontaneously develop atherosclerotic plaques, even on a
regular diet. An atherogenic diet, high in fat and cholesterol, exacerbates
development of atherosclerosis and xanthomas in the 2/2 mice. Thus, comparisons
between the 2/2 and 3/3 mice unequivocally demonstrate that a single amino acid
difference (Arg158 Cys) in the apoE protein is sufficient to cause type III HLP
and spontaneous atherosclerosis in mice. Apolipoprotein (apo) E plays an important role in lipid metabolism, and the
major isoforms of apoE (apoE2, apoE3, and apoE4) have significantly different
metabolic effects. Apolipoprotein E4 is associated with a higher risk of both
heart disease and Alzheimer's disease (AD). Patients homozygous for
apolipoprotein E2 are predisposed to type III hyperlipoproteinemia, and apoE2
may be protective against AD. Structure/function studies have proved to be a
useful tool in understanding how the different apoE isoforms result in different
pathological consequences. As these studies continue, it is essential to have a
reliable method to produce large quantities of apoE and mutants of apoE. We
describe here a method of apoE production in Escherichia coli strain BL21(DE3).
The cDNA from apoE isoforms was inserted into a pET32a vector with a T7 promoter
and a fusion partner (thioredoxin). The T7 promoter results in high expression
of an easily purified His-tagged fusion protein. A thrombin recognition site was
positioned in the expression vector so that only two novel amino acids (Gly-Ser)
are added to the amino terminus of apoE following the removal of thioredoxin.
Approximately 20 mg of apoE is obtained from a 1-liter culture. The major
isoforms of apoE produced with this system were extensively characterized for
their ability to bind the low-density lipoprotein (LDL) receptor, for their
characteristic lipid association preferences, and for their stability as
measured by guanidine denaturation. The recombit proteins behaved identically
to plasma-derived apoE isoforms. The primary receptor mediating clearance of apolipoprotein (apo)E- and
apoB100-containing lipoproteins from the circulation is the low density
lipoprotein (LDL) receptor. Reduced expression of the LDLR is believed to be a
precipitating factor in the pathogenesis of type III hyperlipoproteinemia (HLP)
in some humans homozygous for the apoE2 allele (APOE*2). To test the effect of
genetic changes in LDL receptor expression on the pathogenesis of type III HLP,
we have generated a variant allele at the endogenous mouse Ldlr locus that
expresses the human LDL receptor transcript. Transcription of the human LDLR
minigene is regulated by the endogenous mouse promoter sequence, but a
truncation of 3'-untranslated region results in increased mRNA stability.
Consequently, in liver of heterozygotes, steady state levels of mouse and human
LDLR transcripts are 50 and 180% the levels of total transcript in wild type
mice, respectively. Overall, the 2.3-fold normal level of LDLR message in
heterozygotes completely ameliorates type III HLP caused by the homozygosity for
the human APOE*2 allele, normalizing their plasma lipoprotein profile. We
conclude that a modest increase in expression of the LDLR through message
stabilization is sufficient to prevent precipitation of type III HLP in mice. Apolipoprotein (apo) E mediates the removal of chylomicron and very low density
lipoprotein remts from plasma. It is polymorphic in sequence and the products
of the three common alleles (epsilon 2, epsilon 3, epsilon 4) differ from one
another in their binding to lipoprotein receptors. ApoE2 is defective in binding
and homozygosity for apoE2 is associated with type III hyperlipoproteinemia
(HLP). Other rare isoforms of apoE have been found to be associated either with
domit type III HLP or with the development of hypertriglyceridemia. We
identified a 42 year-old hypertriglyceridemic woman with an apoE phenotype 3/1.
Restriction isotyping using AflIII/HaeII resulted in an apparent apoE genotype
3/2, suggesting that the mutation occurred in an epsilon 2 allele. DNA sequence
analysis revealed a C-->T point mutation at the first position of the codon for
amino acid residue 180 of the mature apoE. This predicted a change
Arg(180)-->Cys. The mutation altered a recognition site for the endonuclease
HaeII, which allowed us rapidly to screen for this mutation. In relatives of the
proband, apoE1 Baden was consistently associated with hypertriglyceridemia.
Similar to other apoE variants linked to hypertriglyceridemia, the
Arg(180)-->Cys mutation is located within the lipid binding domain of apoE. We
therefore suggest that apoE1 Baden may cause hypertrigylceridemia, possibly by
inhibiting the hydrolysis of triglycerides associated with very low density
lipoproteins. First recognized as a major determit in lipoprotein metabolism and
cardiovascular disease, apolipoprotein (apo) E has emerged as an important
molecule in several biological processes not directly related to its lipid
transport function, including Alzheimer's disease and cognitive function,
immunoregulation, and possibly even infectious diseases. ApoE is a polymorphic
protein arising from three alleles at a single gene locus. The three major
isoforms, apoE4, apoE3, and apoE2, differ from one another only by single amino
acid substitutions, yet these changes have profound functional consequences at
both the cellular and molecular levels. ApoE3 seems to be the normal isoform in
all known functions, while apoE4 and apoE2 can each be dysfunctional. Isoform
(allele)-specific effects include the association of apoE2 with the genetic
disorder type III hyperlipoproteinemia and with both increased and decreased
risk for atherosclerosis and the association of apoE4 with increased risk for
both atherosclerosis and Alzheimer's disease, impaired cognitive function, and
reduced neurite outgrowth; isoform-specific differences in cellular signaling
events may also exist. Functional differences in the apoE isoforms that affect
(or did affect) survival before the reproductive years probably account, at
least in part, for the allele frequencies of the present day. Apolipoprotein E (apoE) is a major constituent of lipoproteins in the plasma and
in the brain. There are three common apoE isoforms, termed E2, E3, and E4. By
virtue of its ability to bind to lipoprotein receptors, apoE plays a key role in
the metabolism of triglyceride-rich lipoproteins in the plasma. Homozygous
carriers of apoE2 have an increased risk to develop type III
hyperlipoproteinemia, whereas apoE4 is associated with elevated levels of
low-density lipoprotein cholesterol. In the brain, apoE is associated with
cholesterol-rich lipoproteins and is involved in the transport of cholesterol to
neurons. The genetic polymorphism of apoE is among the strongest determits of
the risk and mean age of onset of Alzheimer's disease. The mechanism by which
apoE isoforms differentially contribute to disease expression is not known. BACKGROUND: Apolipoprotein E (ApoE) is a molecular scavenger in the blood and
brain. Aberrant function of the molecule causes formation of protein and lipid
deposits or "plaques" that characterize Alzheimer's disease (AD) and
atherosclerosis. There are three human isoforms of ApoE designated epsilon2,
epsilon3, and epsilon4. Each isoform differentially affects the structure and
function of the protein and thus the development of disease. Homozygosity for
ApoE epsilon4 is associated with atherosclerosis and Alzheimer's disease whereas
ApoE epsilon2 and epsilon3 tend to be protective. Furthermore, the epsilon2 form
may cause forms of hyperlipoproteinemia. Therefore, introduction of ApoE
epsilon3 may be beneficial to patients that are susceptible to or suffering from
these diseases. Mesenchymal stem cells or multipotent mesenchymal stromal cells
(MSCs) are adult progenitor cells found in numerous tissues. They are easily
expanded in culture and engraft into host tissues when administered
appropriately. Furthermore, MSCs are immunosuppressive and have been reported to
engraft as allogeneic transplants. In our previous study, mouse MSCs (mMSCs)
were implanted into the brains of ApoE null mice, resulting in production of
small amounts of ApoE in the brain and attenuation of cognitive deficits.
Therefore human MSCs (hMSCs) are a promising vector for the administration of
ApoE epsilon3 in humans.
RESULTS: Unlike mMSCs, hMSCs were found not to express ApoE in culture;
therefore a molecular screen was performed for compounds that induce expression.
PPARgamma agonists, neural stem cell conditioned medium, osteo-inductive media,
dexamethasone, and adipo-inductive media (AIM) were tested. Of the conditions
tested, only AIM or dexamethasone induced sustained secretion of ApoE in MSCs
and the duration of secretion was only limited by the length of time MSCs could
be sustained in culture. Upon withdrawal of the inductive stimuli, the ApoE
secretion persisted for a further 14 days.
CONCLUSION: The data demonstrated that pre-treatment and perhaps
co-administration of MSCs homozygous for ApoE epsilon3 and dexamethasone may
represent a novel therapy for severe instances of AD, atherosclerosis and other
ApoE-related diseases. BACKGROUND: Apolipoprotein E (ApoE) plays a role in the regulation of lipid
metabolism in humans. ApoE, a 229-amino-acid polypeptide, is classified into
three major isoforms (E2, E3, and E4) according to the differences in amino
acids at positions 112 and 158. In the normal population, ApoE3 isoform is the
most prevalent, and ApoE2 or E4 is frequently associated with
hyperlipoproteinemia. The objective of this work was to investigate the
relationship between ApoE gene polymorphism and coronary heart disease (CHD) in
Gaza Strip and investigate the association between serum lipid levels and CHD.
MATERIAL AND METHODS: The study population consisted of 137 subjects including
69 CHD cases (45 male, 24 female) and 68 healthy subjects (33 male and 35
female).
RESULTS: The ApoE3/E3 genotype was the most common in the control and the CHD
groups. ApoE2/E3 and ApoE3/E4 were the next most common genotypes. The
frequencies of ApoE alleles in the CHD subjects were 0.826 for E3, 0.137 for E4,
and 0.0362 for E2. These frequencies are comparable to those found in the
control group which were 0.875 for the E3, 0.073 for E4, and 0.0515 for E2. No
statistically significant differences in ApoE genotypes were found between the
patients and the control groups. Moreover, there was no significant difference
between the mean of triglyceride (TG) and HDL levels among different ApoE
genotypes. However, there was a significant difference in the mean of LDL and
ApoE genotypes where the mean of LDL was 218.17 mg/dl in ApoE4, 149.67 mg/dl in
ApoE2, and 184.52 mg/dl in ApoE3. A significant difference was also evident
between the mean of LDL levels in the CHD and the control group where the mean
of LDL was 126 mg/dl in CHD and 111.47 mg/dl in the control group. Our study
indicated that there was no significant difference between the mean of
cholesterol and TG levels of the CHD and the control groups.
CONCLUSIONS: To our knowledge, this is the first study in Gaza Strip
investigating the relation between ApoE genotypes and CHD. Further
investigations are needed to link other genetic factors to CHD. BACKGROUND: Type III hyperlipoproteinemia (HLP), a disorder associated with a
high incidence of premature cardiovascular diseases, is characterized by the
accumulation of remt lipoproteins in the plasma. The primary genetic defect
in patients with type III HLP is the presence of apolipoprotein E2 (apoE2), an
isoform of apoE, and accumulation of remt lipoproteins in the plasma has been
thought to be attributable to the presence of apoE2, which bind poorly to low
density lipoprotein receptors, resulting in defective remt lipoprotein
clearance. On the other hand, the activity of hepatic lipase (HL), the enzyme
that plays a pivotal role in the removal of remt lipoproteins, in type III
HLP has not been investigated.
METHODS: We examined post-heparin plasma lipoprotein lipase (LPL) and HL
activities in 7 patients with type III HLP. The activities of HL and LPL in
post-heparin plasma were measured separately by an immunochemical method using
antiserum specifically directed against HL.
RESULTS: The post-heparin plasma HL activity was significantly reduced, while
the LPL activity was normal.
CONCLUSIONS: Reduced HL activity may account, at least in part, for the
accumulation of remt lipoproteins in the plasma, a characteristic feature of
type III HLP. Apolipoprotein (apo) E is a 299-residue protein which functions as a key
regulator of plasma lipid levels. Human apoE exists as three common isoforms and
the parent form, apoE3, operates optimally in promoting clearance of
triglyceride (TG)-rich lipoproteins and is associated with normal plasma lipid
levels. This result occurs because apoE3 possesses both the requisite
lipid-binding ability and affinity for the low density lipoprotein receptor
(LDLR) to mediate appropriate lipolytic processing and endocytosis of TG-rich
lipoprotein remt particles. ApoE2 which differs from apoE3 by the single
amino acid substitution Arg158Cys located near the LDLR recognition site
exhibits impaired binding to the receptor and an inability to promote clearance
of TG-rich lipoprotein remt particles; this isoform is associated with
Type-III hyperlipoproteinemia. ApoE4 which differs from apoE3 by the single
amino acid substitution Cys112Arg is also associated with dyslipidemia although
binding of this isoform to the LDLR is unaffected. The amino acid substitution
affects the organization and stability of both the N-terminal helix bundle
domain and separately folded C-terminal domain so that apoE4 has enhanced lipid
binding ability. As a consequence, apoE4 binds better than apoE3 to the surface
of very low density lipoprotein (VLDL) particles and impairs their lipolytic
processing in the circulation so that apoE4 is associated with a more
pro-atherogenic lipoprotein-cholesterol distribution (higher
VLDL-cholesterol/high density lipoprotein-cholesterol ratio). This review
summarizes current understanding of the structural differences between apoE2,
apoE3, and apoE4, and the molecular mechanisms responsible for the alterations
in lipoprotein metabolism resulting from this polymorphism of apoE. Detailed
knowledge of how expression of structurally distinct apoE variants modifies
lipoprotein metabolism provides a basis for developing ways to manipulate the
functionality of apoE in vivo. |
Could hypophosphatemic rickets cause craniosynostosis? | Yes, hypophosphatemic rickets could cause craniosynostosis. | A report of three cases of craniosynostosis in X-linked hypophosphataemic
rickets (XLH) is presented. The literature is reviewed, suggesting that
craniosynostosis is relatively common in XLH and that boys may be more at risk
than girls. It is recommended that radiological screening be offered to all
patients with XLH. PURPOSE: INTRODUCTION TO THE OPHTHALMIC LITERATURE OF AN UNUSUAL CAUSE OF
PAPILLEDEMA AND SUBSEQUENT OPTIC ATROPHY: X-linked hypophosphatemic rickets
(XLH).
METHODS: Case report of a 3-year-old female presenting with papilledema
resulting from craniosynostosis secondary to XLH.
RESULTS: Early intervention with craniofacial surgery prevented the development
of optic atrophy.
CONCLUSION: Children with XLH should be screened for ophthalmic evidence of
elevated intracranial pressure to aid early intervention and prevention of
permanent loss of vision. BACKGROUND: The severity and dysmorphology that results from the premature
fusion of one or more cranial sutures is not uniform. Less striking phenotypes
may be more easily missed on routine screening, possibly leading to delayed
diagnosis and treatment. The purpose of this study was to compare the age at
initial presentation for the different forms of craniosynostosis.
METHODS: The authors reviewed the records of all patients who underwent open
craniofacial repair of craniosynostosis at a single institution from 1996 to
2009. Relationships between type of suture fusion and age at initial
consultation were compared.
RESULTS: Two hundred eleven patients (136 males, 75 females) were identified.
Indications included sagittal (n = 96), metopic (n = 39), unicoronal (n = 33),
bicoronal (n = 24), multisutural (n = 15), bilambdoidal (n = 3), and
unilambdoidal (n = 1) synostoses. Seventeen patients (8.1%) had a
craniosynostosis syndrome and 5 (2.4%) had a syndrome or disorder not typically
associated with craniosynostosis [X-linked hypophosphatemic rickets (n = 3),
achondroplasia (n = 1), and Beckwith Wiedemann (n = 1)]. Median age at initial
consultation was 4.1 months; there was no gender difference. Patients with
X-linked hypophosphatemic rickets presented at a significantly older age than
nonsyndromic patients or those with a known craniosynostosis syndrome. Those
with multisutural synostosis presented at a significantly older age than
patients with sagittal or bicoronal synostosis.
CONCLUSIONS: Patients with multisutural involvement or X-linked hypophosphatemic
rickets had a significant delay in presentation for craniosynostosis. The latter
group of patients may especially benefit from routine surveillance for
craniosynostosis given their advanced age at diagnosis. Craniosynostosis can be gene-linked, or caused by metabolic diseases, such as
rickets, which results from a deficiency or impaired metabolism of vitamin D,
magnesium, phosphorus or calcium leading to hypomineralization of the bone.
X-linked domit hypophosphatemic rickets (XLHR) is the most prevalent genetic
type of hypophosphatemic rickets and is caused by germ line mutations in the
PHEX-gene. In XLHR, only few case reports of craniosynostosis were described.
Here, we present a clinical report of an 18 months old child with XLHR and
bilateral coronal and sagittal synostosis who was treated by subtotal cranial
vault remodelling with fronto-orbital advancement and right-angled
Z-osteotomies. As a consequence of the child's diminished bone regeneration
capacity, surgery that is performed after the age of 1 year requires more
extensive craniectomy, multiple osteotomies and rigid fixation for calvarial
vault remodelling to prevent extensive bone defects. PURPOSE: A defect in a phosphate-regulating gene leads to the most common form
of rickets: X-linked hypophosphatemic rickets (XLH) or vitamin D-resistant
rickets (VDDR). XLH has been associated with craniosynostosis, the sagittal
suture being the most commonly involved.
METHODS: We present three patients with rickets and symptomatic sagittal suture
craniosynostosis all of whom presented late (>2 years of age). Two had a severe
phenotype and papilledema, while the third presented with an osseous bulging
near the anterior fontanel and experienced chronic headaches.
RESULTS: All underwent successful cranial vault expansion.
CONCLUSIONS: Rachitic patients with scaphocephaly should be screened for
craniosynostosis. OBJECTIVE This study examines a series of patients with hypophosphatemic rickets
and craniosynostosis to characterize the clinical course and associated
craniofacial anomalies. METHODS A 20-year retrospective review identified
patients with hypophosphatemic rickets and secondary craniosynostosis at 3 major
craniofacial centers. Parameters examined included sex, age at diagnosis of head
shape anomaly, affected sutures, etiology of rickets, presenting symptoms,
number and type of surgical interventions, and associated diagnoses. A review of
the literature was performed to optimize treatment recommendations. RESULTS Ten
patients were identified (8 males, 2 females). Age at presentation ranged from 1
to 9 years. The most commonly affected suture was the sagittal (6/10 patients).
Etiologies included antacid-induced rickets, autosomal domit hypophosphatemic
rickets, and X-linked hypophosphatemic (XLH) rickets. Nine patients had
undergone at least 1 cranial vault remodeling (CVR) surgery. Three patients
underwent subsequent surgeries in later years. Four patients underwent formal
intracranial pressure (ICP) monitoring, 3 of which revealed elevated ICP. Three
patients were diagnosed with a Chiari Type I malformation. CONCLUSIONS Secondary
craniosynostosis develops postnatally due to metabolic or mechanical factors.
The most common metabolic cause is hypophosphatemic rickets, which has a variety
of etiologies. Head shape changes occur later and with a more heterogeneous
presentation compared with that of primary craniosynostosis. CVR may be required
to prevent or relieve elevated ICP and abnormalities of the cranial vault.
Children with hypophosphatemic rickets who develop head shape abnormalities
should be promptly referred to a craniofacial specialist. We present the case of a preterm 6-month-old African American infant who
developed craniosynostosis secondary to rickets. This child developed rickets
and macrocephaly by the age of 6 months. His head continued to enlarge, and a 3D
CT obtained when the child was 2 years old revealed metopic and bilateral
coronal craniosynostosis. This CT suggested increased intracranial pressure, and
therefore, corrective cranial vault reconstruction was performed.
Craniosynostosis secondary to rickets is rarely reported, but since neither
rickets nor craniosynostosis is a reportable disease, the exact incidence of
both diseases is unknown. Craniosynostosis should be suspected in any rachitic
child with an abnormal head circumference or shape and craniofacial CT
evaluation should be performed, so that a corrective surgery can be performed at
an appropriate age. |
Is vortioxetine effective for treatment of depression? | Yes. Vortioxetine is the most recently approved medication for the treatment of major depressive disorder. | Lu-AA21004, an oral, multimodal serotonergic agent, is currently under
development by H Lundbeck and Takeda Pharmaceutical, for the potential treatment
of depression and anxiety. Lu-AA21004 belongs to a novel chemical class of
antidepressant agents, the bisarylsulfanyl amines, and possesses a novel
pharmacological profile, with activity at serotonergic receptors 5-HT3, 5-HT7
and 5-HT1A, and also at the 5-HT transporter. Acute administration of Lu-AA21004
in rats inhibited the firing activity of serotonergic neurons of the dorsal
raphe nucleus through 5-HT3 receptor blockade, with rapid recovery of firing
activity upon cessation of treatment compared with an antidepressant of the SSRI
class. Results from phase II clinical trials have reported improvement in
depression and anxiety symptoms after 6 weeks of treatment. Lu-AA21004 was
generally well tolerated, with adverse events related to sexual dysfunction
occurring in a lower number of patients receiving Lu-AA21004 compared with
venlafaxine. Phase III clinical trials with Lu-AA21004 in patients with major
depressive disorder are underway and phase III trials in patients with
generalized anxiety disorder have been completed. If initial outcomes from these
clinical trials prove positive, Lu-AA21004 may pave the way for new multimodal
therapies for the treatment of depression and anxiety. The efficacy, safety, and tolerability of Lu AA21004 vs. placebo using
venlafaxine XR as active reference in patients with DSM-IV-TR major depressive
disorder (MDD) were evaluated. Lu AA21004 is a novel antidepressant that is a
5-HT3 and 5-HT7 receptor antagonist, 5-HT1A receptor agonist, 5-HT1B receptor
partial agonist and inhibitor of the 5-HT transporter in recombit cell lines.
In this 6-wk, multi-site study, 429 patients were randomly assigned (1:1:1:1) to
5 or 10 mg Lu AA21004, placebo or 225 mg venlafaxine XR. All patients had a
baseline Montgomery-Åsberg Depression Rating Scale (MADRS) total score ≥ 30. The
primary efficacy analysis was based on the MADRS total score adjusting for
multiplicity using a hierarchical testing procedure starting with the highest
dose vs. placebo. Lu AA21004 was statistically significantly superior to placebo
(n=105) in mean change from baseline in MADRS total score at week 6 (p<0.0001,
last observation carried forward), with a mean treatment difference vs. placebo
of 5.9 (5 mg, n=108), and 5.7 (10 mg, n=100) points. Venlafaxine XR (n=112) was
also significantly superior to placebo at week 6 (p<0.0001). In total, 30
patients withdrew due to adverse events (AEs)--placebo: four (4%); 5 mg Lu
AA21004: three (3%); 10 mg Lu AA21004: seven (7%); and venlafaxine: 16 (14%).
The most common AEs were nausea, headache, hyperhidrosis, and dry mouth. No
clinically relevant changes over time were seen in the clinical laboratory
results, vital signs, weight, or ECG parameters. In this study, treatment with 5
mg and 10 mg Lu AA21004 for 6 wk was efficacious and well tolerated in patients
with MDD. 1-[2-(2,4-Dimethylphenyl-sulfanyl)-phenyl]-piperazine (Lu AA21004) is a human
(h) serotonin (5-HT)(3A) receptor antagonist (K(i) = 3.7 nM), h5-HT(7) receptor
antagonist (K(i) = 19 nM), h5-HT(1B) receptor partial agonist (K(i) = 33 nM),
h5-HT(1A) receptor agonist (K(i) = 15 nM), and a human 5-HT transporter (SERT)
inhibitor (K(i) = 1.6 nM) (J Med Chem 54:3206-3221, 2011). Here, we confirm that
Lu AA21004 is a partial h5-HT(1B) receptor agonist [EC(50) = 460 nM, intrinsic
activity = 22%] using a whole-cell cAMP-based assay and demonstrate that Lu
AA21004 is a rat (r) 5-HT(7) receptor antagonist (K(i) = 200 nM and IC(50) =
2080 nM). In vivo, Lu AA21004 occupies the r5-HT(1B) receptor and rSERT (ED(50)
= 3.2 and 0.4 mg/kg, respectively) after subcutaneous administration and is a
5-HT(3) receptor antagonist in the Bezold-Jarisch reflex assay (ED(50) = 0.11
mg/kg s.c.). In rat microdialysis experiments, Lu AA21004 (2.5-10.0 mg/kg s.c.)
increased extracellular 5-HT, dopamine, and noradrenaline in the medial
prefrontal cortex and ventral hippocampus. Lu AA21004 (5 mg/kg per day for 3
days; minipump subcutaneously), corresponding to 41% rSERT occupancy,
significantly increased extracellular 5-HT in the ventral hippocampus.
Furthermore, the 5-HT(3) receptor antagonist, ondansetron, potentiated the
increase in extracellular levels of 5-HT induced by citalopram. Lu AA21004 has
antidepressant- and anxiolytic-like effects in the rat forced swim (Flinders
Sensitive Line) and social interaction and conditioned fear tests (minimal
effective doses: 7.8, 2.0, and 3.9 mg/kg). In conclusion, Lu AA21004 mediates
its pharmacological effects via two pharmacological modalities: SERT inhibition
and 5-HT receptor modulation. In vivo, this results in enhanced release of
several neurotransmitters and antidepressant- and anxiolytic-like profiles at
doses for which targets in addition to the SERT are occupied. The multimodal
activity profile of Lu AA21004 is distinct from that of current antidepressants. The efficacy, safety, and tolerability of Lu AA21004 versus placebo, using
duloxetine as active reference, in patients with DSM-IV-TR diagnosed major
depressive disorder (MDD) were evaluated in this 8-week, multi-site study.
Patients (n=766) had a baseline Montgomery-Åsberg Depression Rating Scale
(MADRS) total score ≥26 and were randomly assigned (1:1:1:1:1) to 2.5, 5 or 10
mg Lu AA21004, placebo, or 60 mg duloxetine. The 5mg and 10mg doses of Lu
AA21004 were tested separately versus placebo at p≤0.025 in a pre-specified
order. In the pre-defined primary efficacy analysis [mean change from baseline
in MADRS total score at Week 8, full analysis set, ANCOVA, last observation
carried forward (LOCF)], the differences to placebo (n=145) of -1.7 (Lu AA21004
5 mg, n=155) and -1.5 points (Lu AA21004 10 mg, n=151) were not statistically
significant; nor were those for Lu AA21004 2.5 mg (-1.4 points, n=155) or
duloxetine (-2.0 points, n=149). Using mixed model, repeated measures (MMRM)
analyses of the primary endpoint and most secondary endpoints were supportive of
likely efficacy for Lu AA21004 5 mg and 10 mg and duloxetine. Treatment-emergent
adverse events led to the withdrawal of 72 patients: 8% (placebo), 12%
(duloxetine), and 6%, 11% and 9% in the Lu AA21004 groups (2.5 mg, 5 mg and 10
mg, respectively). The most common adverse events were nausea, headache,
dizziness, and dry mouth. No clinically relevant changes were seen in vital
signs, weight, ECG, or laboratory results. In summary, none of the active
treatment groups, including duloxetine, separated from placebo in the primary
analysis in this 'failed' study. Findings on secondary outcome measures, using
MMRM instead of LOCF, were supportive of likely efficacy for Lu AA21004 5mg and
10mg and duloxetine. Lu AA21004 (2.5, 5 and 10 mg) was well tolerated. The efficacy and tolerability of Lu AA21004 in the prevention of relapse of
major depressive disorder (MDD) in patients in remission after acute treatment
was evaluated. Patients (n=639) aged 18-75 years with a primary diagnosis of MDD
with a current major depressive episode (MDE) ≥4 weeks' duration, at least one
prior MDE and a MADRS total score ≥26 received 12-week, open-label Lu AA21004 at
5 or 10mg/day. Patients in remission (MADRS ≤10) at both weeks 10 and 12 were
assigned to double-blind treatment with either placebo or Lu AA21004 (fixed dose
from Week 8).Patients (n=396) were treated, after random assignment to placebo
(n=192) or Lu AA21004 (n=204). The primary analysis of time to relapse
(full-analysis set, Cox proportional hazard model) showed a statistically
significant difference in favour of Lu AA21004 versus placebo with a hazard
ratio of 2.01 (95% confidence interval: 1.26-3.21; p=0.0035). The proportion of
patients who relapsed was 13% in the Lu AA21004 group (n=27) and 26% in the
placebo group (n=50). The withdrawal rates due to adverse events were 8%
(open-label), and 3% (placebo) and 8% (Lu AA21004) (double-blind). Thus, Lu
AA21004 was effective in preventing relapse of MDD and was well tolerated as
maintece treatment. The efficacy and tolerability of Lu AA21004 at 5 mg/day, a novel multimodal
antidepressant, were assessed in elderly patients with recurrent major
depressive disorder. Patients were randomly assigned (1:1:1) to Lu AA21004 5
mg/day, duloxetine 60 mg/day (reference) or to placebo in an 8-week double-blind
study. The primary efficacy measure was the 24-item Hamilton Depression Scale
(HAM-D(24)) total score (analysis of covariance, last observation carried
forward). Patients (mean age 70.6 years) had a mean baseline HAM-D(24) score of
29.0. Lu AA21004 showed significantly (P = 0.0011) greater improvement on the
primary efficacy endpoint compared with placebo at week 8 (3.3 points).
Duloxetine also showed superiority to placebo at week 8, thereby validating the
study. HAM-D(24) response (53.2 vs. 35.2%) and HAM-D(17) remission (29.2 vs.
19.3%) rates at endpoint were higher for Lu AA21004 than for placebo. Lu AA21004
showed superiority to placebo in cognition tests of speed of processing, verbal
learning and memory. The withdrawal rate due to adverse events was 5.8% (Lu
AA21004), 9.9% (duloxetine) and 2.8% (placebo). Whereas nausea was the only
adverse event with a significantly higher incidence on treatment with Lu AA21004
(21.8%) compared with placebo (8.3%), the incidence of nausea, constipation, dry
mouth, hyperhidrosis and somnolence was higher for duloxetine. In conclusion, Lu
AA21004 was efficacious and well tolerated in the treatment of elderly patients
with recurrent major depressive disorder. Vortioxetine (Lu AA21004) is a multi-modal antidepressant in clinical
development for the treatment of major depressive disorder (MDD). The current
study evaluated the efficacy and tolerability of 5 mg vortioxetine compared to
placebo after 6 wk of treatment in adults with MDD in an out-patient setting.
Adults aged 18-75 yr, with a diagnosis of MDD and a baseline Montgomery-Asberg
Depression Rating Scale (MADRS) total score ≥30, were randomized to receive
either 5 mg vortioxetine or placebo over 6 wk, followed by a 2-wk
medication-free discontinuation period. The primary efficacy measure was change
from baseline in Hamilton Rating Scale for Depression (HAMD)-24 total score at
week 6 compared to placebo. Additional measures included response and remission
rates, Clinical Global Impression Scale - Improvement scores, HAMD-24 total
score in subjects with baseline Hamilton Anxiety Scale (HAMA) >19 and MADRS-S
total score. Adverse events (AEs) were assessed throughout the study. A total of
600 adults were randomized. There were no significant differences in efficacy
measures between subjects in the 5 mg vortioxetine and placebo groups at week 6.
HAMD-24 total score in subjects with baseline HAMA >19 in the 5 mg vortioxetine
group was improved at weeks 3-6 compared to the placebo group (nominal p value
<0.05). The most common AEs for the vortioxetine and placebo groups were nausea
(19.1 and 9.4%), headache (17.1 and 15.1%) and diarrhoea (11.4 and 7.0%),
respectively. In this study of adults with MDD, 5 mg vortioxetine did not differ
significantly from placebo in reducing depression symptoms after 6 wk of
treatment. OBJECTIVE: The primary objective of this study was to evaluate the safety and
tolerability of the investigational drug vortioxetine (Lu AA21004) in the
long-term treatment of patients with major depressive disorder.
METHODS: Patients entered this 52-week, open-label extension study after
completing an 8-week lead-in study. Safety and tolerability were evaluated at
regular intervals on the basis of spontaneously reported adverse events (AEs),
clinical safety laboratory tests, vital signs, ECG and physical examination.
Effectiveness of treatment was assessed using the Montgomery-Åsberg Depression
Rating Scale (MADRS) total score.
RESULTS: A total of 535 patients were treated and 61.3% (n = 328) completed the
study, resulting in 393 patient years of exposure to vortioxetine. AEs reported
by ≥10% of patients were nausea, headache, and nasopharyngitis. Taken together,
six patients had eight AEs related to sexual dysfunction. There were no
clinically significant safety findings with respect to mean changes of vital
signs, weight, ECG parameters, or clinical laboratory values. Patients entered
the extension study with a mean MADRS total score of 13.5 ± 8.7. The mean MADRS
total score decreased (improved) by approximately 8 points to 5.5 ± 6.0 at Week
52 (OC). By the end of the study, the proportion of responders had increased
from 63% to 94% (OC), as had the proportion in remission (MADRS ≤10), increasing
from 42% to 83% (OC). Patients in remission (n = 226) at the start of this study
had a relapse rate (MADRS ≥22) of 9.7%.
CONCLUSIONS: As with all open-label studies, the conclusions that can be drawn
are limited by the lack of a placebo control, making it difficult to assess
causality of any changes in outcome measures. However, on the basis of these
findings, vortioxetine (2.5, 5, 10 mg/day) demonstrated a favourable safety and
tolerability profile and maintained effectiveness over 12 months of treatment.
TRIAL REGISTRATION: This study has the ClinicalTrials.gov identifier:
NCT00694304. OBJECTIVE: Vortioxetine (Lu AA21004) is an investigational antidepressant. In
vitro studies indicate that vortioxetine is a 5-HT(3), 5-HT(7), and 5-HT(1D)
receptor antagonist, 5-HT(1B) receptor partial agonist, 5-HT(1A) receptor
agonist and inhibitor of the 5-HT transporter. This trial assessed the efficacy
and tolerability of 2.5 and 5 mg vortioxetine for the treatment of MDD.
RESEARCH DESIGN AND METHODS: Adults (N = 611) with MDD were randomized to 8
weeks of double-blind treatment with placebo, vortioxetine (2.5 or 5 mg) or
active reference (duloxetine 60 mg). The primary measure was change from
baseline in the 24-item Hamilton Depression Scale (HAM-D24). Secondary endpoints
included responder rate, Clinical Global Impression Scale-Global Improvement
scale (CGI-I), and remission rate. Participants were monitored for adverse
events (AEs), and treatment-emergent sexual dysfunction using the Arizona Sexual
Experiences (ASEX) scale.
RESULTS: Both doses of vortioxetine were associated with declines in HAM-D24
total scores compared to placebo but were not statistically significant. At 8
weeks, changes from baseline were [mean (SE)]: -10.50 (0.76) placebo, -12.04
(0.74) 2.5 mg vortioxetine, and -11.08 (0.74) 5 mg vortioxetine. Secondary
outcome measures in the vortioxetine groups, including responder rate, CGI-I,
and remission rate, were also not significantly different from placebo.
Duloxetine treatment was associated with declines in HAM-D24 total score
[-13.47(0.75); p = 0.005] as well as significant improvements in secondary
outcome measures versus placebo (p ≤ 0.05). The most common AEs for vortioxetine
were nausea, dry mouth, and headache. Rates of sexual dysfunction (ASEX) were
51.0%, 37.5%, 46.9%, and 33.3% in the vortioxetine 2.5 mg, vortioxetine 5 mg,
duloxetine, and placebo groups, respectively.
CONCLUSIONS: In this study of adults with MDD treated for 8 weeks with
vortioxetine 2.5 mg or 5 mg per day, reductions in depression symptoms were not
statistically significant compared with placebo. Study limitations are
discussed, including patient characteristics, MDD severity, drug dosing, and
aspects of trial design. Both doses of vortioxetine were well tolerated. This
trial has been registered at clinicaltrials.gov #NCT00672620. The current generation of antidepressant drugs acts predomitly by targeting
the serotonin transporter (SERT). The original trend to do this selectively
(e.g., with SSRIs or selective serotonin reuptake inhibitors) has given way to
combining various additional pharmacologic mechanisms with SERT inhibition,
including dual actions by single drugs (e.g., SNRIs or serotonin norepinephrine
reuptake inhibitors), or by augmenting SSRIs with a second drug of a different
mechanism (e.g., bupropion with dopamine and norepinephrine reuptake inhibition;
trazodone with 5HT2A antagonism; mirtazapine with 5HT2A/5HT2C/5HT3/alpha2
antagonism; buspirone or some atypical antipsychotics with 5HT1A partial
agonism; other atypical antipsychotics with 5HT2C/5HT7 antagonism and other
mechanisms). Novel drugs in development include those that combine multiple
simultaneous pharmacologic mechanisms in addition to SERT inhibition within the
same molecule, such as vilazodone (combining 5HT1A partial agonism with SERT
inhibition), triple reuptake inhibitors (combining norepinephrine and dopamine
reuptake inhibition with SERT inhibition), and vortioxetine, a multimodal
antidepressant combining actions at the G protein receptor mode (5HT1A and 5HT1B
partial agonism and 5HT7 antagonism), at the ion channel mode (5HT3 antagonism)
as well as the neurotransmitter transporter mode (SERT inhibition). These
various strategies that build upon SERT inhibition provide promise for novel
therapeutic approaches to depression, including the possibility of targeting
residual symptoms not well treated by SERT inhibition alone, and reducing side
effects, such as sexual dysfunction. Psychiatric disorders represent a large economic burden in modern societies.
However, pharmacological treatments are still far from optimal. Drugs used in
the treatment of major depressive disorder (MDD) and anxiety disorders
(selective serotonin [5-HT] reuptake inhibitors [SSRIs] and
serotonin-noradrenaline reuptake inhibitors [SNRIs]) are pharmacological
refinements of first-generation tricyclic drugs, discovered by serendipity, and
show low efficacy and slowness of onset. Moreover, antipsychotic drugs are
partly effective in positive symptoms of schizophrenia, yet they poorly treat
negative symptoms and cognitive deficits. The present article reviews the
neurobiological basis of 5-HT1A receptor (5-HT1A-R) function and the role of
pre- and postsynaptic 5-HT1A-Rs in the treatment of MDD, anxiety and psychotic
disorders. The activation of postsynaptic 5-HT1A-Rs in corticolimbic areas
appears beneficial for the therapeutic action of antidepressant drugs. However,
presynaptic 5-HT1A-Rs play a detrimental role in MDD, since individuals with
high density or function of presynaptic 5-HT1A-Rs are more susceptible to mood
disorders and suicide, and respond poorly to antidepressant drugs. Moreover, the
indirect activation of presynaptic 5-HT1A-Rs by SSRIs/SNRIs reduces 5-HT neuron
activity and terminal 5-HT release, thus opposing the elevation of extracellular
5-HT produced by blockade of the serotonin transporter (SERT) in the forebrain.
Chronic antidepressant treatment desensitizes presynaptic 5-HT1A-Rs, thus
reducing the effectiveness of the 5-HT1A autoreceptor-mediated negative
feedback. The prevention of this process by the non-selective partial agonist
pindolol accelerates clinical antidepressant effects. Two new antidepressant
drugs, vilazodone (marketed in the USA) and vortioxetine (in development)
incorporate partial 5-HT1A-R agonist properties with SERT blockade. Several
studies with transgenic mice have also established the respective role of pre-
and postsynaptic 5-HT1A-Rs in MDD and anxiety. In agreement with pharmacological
studies, presynaptic and postsynaptic 5-HT1A-R activation appears necessary for
anxiolytic and antidepressant effects, respectively, yet, neurodevelopmental
roles for 5-HT1A-Rs are also involved. Likewise, the use of small interference
RNA has enabled the showing of robust antidepressant-like effects in mice after
selective knock-down of 5-HT1A autoreceptors. Postsynaptic 5-HT1A-Rs in the
prefrontal cortex (PFC) also appear important for the superior clinical effects
of clozapine and other second-generation (atypical) antipsychotic drugs in the
treatment of schizophrenia and related psychotic disorders. Despite showing a
moderate in vitro affinity for 5-HT1A-Rs in binding assays, clozapine displays
functional agonist properties at this receptor type in vivo. The stimulation of
5-HT1A-Rs in the PFC leads to the distal activation of the mesocortical pathway
and to an increased dopamine release in PFC, an effect likely involved in the
clinical actions of clozapine in negative symptoms and cognitive deficits in
schizophrenia. The anxiolytic/antidepressant properties of 5-HT1A-R agonists in
preclinical tests raised expectations enormously. However, these agents have
achieved little clinical success, possibly due to their partial agonist
character at postsynaptic 5-HT1A-Rs, together with full agonist properties at
presynaptic 5-HT1A autoreceptors, as well as their gastrointestinal side
effects. The partial 5-HT1A-R agonists buspirone, gepirone, and tandospirone are
marketed as anxiolytic drugs, and buspirone is also used as an augmentation
strategy in MDD. The development of new 5-HT1A-R agonists with selectivity for
postsynaptic 5-HT1A-Rs may open new perspectives in the field. Monoamine-based treatments for depression have evolved greatly over the past
several years, but shortcomings such as suboptimal efficacy, treatment lag, and
residual cognitive dysfunction are still significant. Preclinical and clinical
studies using compounds directly targeting glutamatergic neurotransmission
present new opportunities for antidepressant treatment, with ketamine having a
surprisingly rapid and sustained antidepressant effect that is presumably
mediated through glutamate-dependent mechanisms. While direct modulation of
glutamate transmission for antidepressant and cognition-enhancing actions may be
hampered by nonspecific effects, indirect modulation through the serotonin
(5-HT) system may be a viable alternative approach. Based on localization and
function, 5-HT can modulate glutamate neurotransmission at least through the
5-HT1A, 5-HT1B, 5-HT3, and 5-HT7 receptors, which presents a rational
pharmacological opportunity for modulating glutamatergic transmission without
the direct use of glutamatergic compounds. Combining one or more of these
glutamate-modulating 5-HT targets with 5-HT transporter inhibition may offer new
therapeutic opportunities. The multimodal compounds vortioxetine and vilazodone
are examples of this approach with diverse mechanisms, and their different
clinical effects will provide valuable insights into serotonergic modulation of
glutamate transmission for the potential treatment of depression and associated
cognitive dysfunction. OBJECTIVE: To describe the efficacy and safety of vortioxetine for the treatment
of major depressive disorder (MDD).
DATA SOURCES: The pivotal registration trials were accessed by querying
http://www.ncbi.nlm.nih.gov/pubmed/, http://www.clinicaltrialsregister.eu and
http://www.clinicaltrials.gov for the search terms 'vortioxetine' and 'Lu
AA21004', and by obtaining posters presented at congresses. Product labelling
provided additional information.
STUDY SELECTION: All available clinical reports of studies were identified.
DATA EXTRACTION: Descriptions of the principal results and calculation of number
needed to treat (NNT) and number needed to harm (NNH) for relevant dichotomous
outcomes were extracted from the available study reports and other sources of
information.
DATA SYNTHESIS: Vortioxetine is a multi-modal antidepressant that functions as a
human 5-HT3A and 5-HT7 receptor antagonist, 5-HT1B receptor partial agonist,
5-HT1A receptor agonist, and inhibitor of the serotonin transporter. The
recommended dose range is 5-20 mg/day. Approval for the treatment of MDD was
based on a clinical development programme that included six positive 6-8 week
studies, including one study in elderly people, and one positive maintece
study in adults. In the informative short-term studies in non-elderly patients,
NNT for response with vortioxetine vs. placebo was 7 (95% CI 6-9), and NNT for
remission vs. placebo was 11 (95% CI 8-17). NNH for discontinuation because of
an adverse event (AE) was 36 (95% CI 24-70). The most commonly encountered AEs
(incidence ≥ 5% and at least twice the rate of placebo) as identified in product
labelling were nausea, constipation and vomiting, with NNH values vs. placebo of
6 (95% CI 6-7), 64 (95% CI 37-240), and 28 (95% CI 23-38), respectively. Changes
in weight were not clinically relevant.
CONCLUSIONS: Vortioxetine represents another option for the treatment of MDD.
Vortioxetine appears to have a favourable weight-gain profile. Additional
information regarding the time course of response/remission and for the commonly
occurring AE of nausea would be helpful to better characterise this agent.
Pending clinical trials include those examining cognitive dysfunction that can
accompany MDD. Patients with major depressive disorder often experience relapse after
responding to treatment; therefore, maintece therapy with antidepressants is
recommended for maintaining response or remission. This multicenter, open-label,
flexible-dose, 52-week extension study evaluated the long-term safety,
tolerability, and maintece of efficacy in study participants who had
completed one of two randomized, double-blind, placebo-controlled, 8-week
dose-ranging vortioxetine trials in study participants with major depressive
disorder. At the open-label baseline, all study participants were switched to
vortioxetine 5 mg/day for the first week, with subsequent dose adjustments from
2.5 to 10 mg/day on the basis of response and tolerability. Treatment with
vortioxetine for 52 weeks was well tolerated, with no new safety signals
identified. Among the 834 evaluable study participants, treatment-emergent
adverse events were reported in 70.6%, with the most common in the combined (all
doses) population of nausea (15.2%), headache (12.4%), nasopharyngitis (9.8%),
diarrhea (7.2%), and dizziness (6.8%). The rate of adverse events related to
sexual dysfunction was low and weight gain was minimal. Laboratory values, vital
signs, ECGs, physical examinations, and Columbia-Suicide Severity Rating Scale
results showed no trends of clinical concern. The change in the severity of
depressive and anxiety symptoms was maintained throughout the study as reflected
by a 24-item Hamilton Depression Scale total score of 8.2 at week 52 (from 17.6
at open-label baseline) in the observed case data set. This study assessed the efficacy, tolerability and safety of vortioxetine versus
placebo in adults with recurrent major depressive disorder. This double-blind,
randomized, placebo-controlled study included 608 patients [Montgomery-Åsberg
Depression Rating Scale (MADRS) total score ≥ 26 and Clinical Global Impression
- Severity score ≥ 4]. Patients were randomly assigned (1 : 1 : 1 : 1) to
vortioxetine 15 mg/day, vortioxetine 20 mg/day, duloxetine 60 mg/day or placebo.
The primary efficacy endpoint was change from baseline in MADRS total score at
week 8 (mixed model for repeated measurements). Key secondary endpoints were:
MADRS responders; Clinical Global Impression - Improvement scale score; MADRS
total score in patients with baseline Hamilton Anxiety Rating Scale ≥ 20;
remission (MADRS ≤ 10); and Sheehan Disability Scale total score at week 8. On
the primary efficacy endpoint, both vortioxetine doses were statistically
significantly superior to placebo, with a mean difference to placebo (n = 158)
of -5.5 (vortioxetine 15 mg, P < 0.0001, n = 149) and -7.1 MADRS points
(vortioxetine 20 mg, P < 0.0001, n = 151). Duloxetine (n = 146) separated from
placebo, thus validating the study. In all key secondary analyses, both
vortioxetine doses were statistically significantly superior to placebo.
Vortioxetine treatment was well tolerated; common adverse events (incidence ≥
5%) were nausea, headache, diarrhea, dry mouth and dizziness. No clinically
relevant changes were seen in clinical safety laboratory values, weight, ECG or
vital signs parameters. Vortioxetine was efficacious and well tolerated in the
treatment of patients with major depressive disorder. Depressed patients suffer from cognitive dysfunction, including memory deficits.
Acute serotonin (5-HT) depletion impairs memory and mood in vulnerable patients.
The investigational multimodal acting antidepressant vortioxetine is a 5-HT3,
5-HT7 and 5-HT1D receptor antagonist, 5-HT1B receptor partial agonist, 5-HT1A
receptor agonist and 5-HT transporter (SERT) inhibitor that enhances memory in
normal rats in novel object recognition (NOR) and conditioned fear (Mørk et al.,
2013). We hypothesized that vortioxetine's 5-HT receptor mechanisms are involved
in its memory effects, and therefore investigated these effects in 5-HT depleted
rats. Four injections of the irreversible tryptophan hydroxylase inhibitor
4-chloro-dl-phenylalanine methyl ester hydrochloride (PCPA, 86mg/kg, s.c.)
induced 5-HT depletion, as measured in hippocampal homogenate and
microdialysate. The effects of acute challenge with vortioxetine or the 5-HT
releaser fenfluramine on extracellular 5-HT were measured in PCPA-treated and
control rats. PCPA's effects on NOR and spontaneous alternation (SA) performance
were assessed along with the effects of acute treatment with
5-hydroxy-l-tryptophan (5-HTP), vortioxetine, the selective 5-HT reuptake
inhibitor escitalopram, or the 5-HT norepinephrine reuptake inhibitor
duloxetine. SERT occupancies were estimated by ex vivo autoradiography. PCPA
depleted central 5-HT by >90% in tissue and microdialysate, and impaired NOR and
SA performance. Restoring central 5-HT with 5-HTP reversed these deficits. At
similar SERT occupancies (>90%) vortioxetine, but not escitalopram or
duloxetine, restored memory performance. Acute fenfluramine significantly
increased extracellular 5-HT in control and PCPA-treated rats, while
vortioxetine did so only in control rats. Thus, vortioxetine restores 5-HT
depletion impaired memory performance in rats through one or more of its
receptor activities. Vortioxetine is an orally administered small molecule developed by Lundbeck A/S
for the once-daily treatment of major depressive disorder (MDD) and generalized
anxiety disorder (GAD). Vortioxetine received its first global approval for MDD
in the USA in September 2013 and regulatory approval for its use in this
indication in the EU (where it has received a positive opinion) and Canada is
awaited. The drug is a bis-aryl-sulphanyl amine compound that combines serotonin
(5-HT) reuptake inhibition with other characteristics, including receptor
activity modulation. In vitro studies indicate that vortioxetine is an inhibitor
of the 5-HT transporter and is a 5-HT(1D), 5-HT₃ and 5-HT₇ receptor antagonist,
a 5-HT(1A) receptor agonist and a 5-HT(1B) receptor partial agonist. Animal and
in vitro studies indicate that several neurotransmitter systems may be impacted
by vortioxetine, with the drug enhancing levels of 5-HT, noradrenaline,
dopamine, acetylcholine and histamine in certain areas of the brain, as well as
modulating γ-aminobutyric acid and glutamate neurotransmission. Phase III trials
of vortioxetine in both MDD and GAD have been conducted worldwide. This article
summarizes the milestones in the development of vortioxetine leading to this
first approval for MDD. BACKGROUND: Vortioxetine is a recently approved multimodal antidepressant with
anxiolytic properties in preclinical studies.
OBJECTIVE: This double-blind, placebo-controlled study assessed the efficacy and
tolerability of vortioxetine in subjects with a primary diagnosis of generalized
anxiety disorder.
METHODS: Subjects (n = 457) were randomized 1:1:1 to treatment with placebo or
vortioxetine 2.5 or 10 mg once daily. The primary efficacy endpoint was
reduction in Hamilton Anxiety Scale (HAM-A) total scores from baseline after
8 weeks of treatment. Key secondary outcomes were changes from baseline in HAM-A
total scores for the 2.5 and 10 mg dose, Hospital Anxiety and Depression anxiety
subscore, 36-Item Short-Form Health Survey, Sheehan Disability Scale, and
Clinical Global Impression-Improvement Scale score, as well as HAM-A response
rate at week 8.
RESULTS: Neither vortioxetine dose achieved a statistically significant
improvement over placebo on the primary endpoint (least-squares mean
difference ± standard error from placebo: -0.87 ± 0.803 [p = 0.279] for 2.5 mg
and -0.81 ± 0.791 [p = 0.306] for 10 mg vortioxetine) or on any secondary
efficacy endpoints. Common adverse events (≥5% in either vortioxetine group)
were nausea, dry mouth, headache, diarrhea, constipation, and vomiting.
CONCLUSIONS: Vortioxetine 2.5 and 10 mg treatment did not significantly improve
generalized anxiety disorder symptoms versus placebo. Vortioxetine was safe and
well tolerated in this patient population. Vortioxetine (Lu-AA-21004; 1-[2-(2,4-dimethylphenylsulfanyl)phenyl]piperazine
hydrobromide) is a novel orally active molecule that is being investigated by
Lundbeck and Takeda for the treatment of major depression and generalized
anxiety disorders. Vortioxetine has a unique "multi-modal" mechanism of action.
It inhibits the activity of serotonin transporters and is an agonist of
serotonin 5-HT1A receptor, partial agonist of 5-HT1B and antagonist of 5-HT3A,
5-HT7 and 5-HT1D receptors. Vortioxetine has been effective in various animal
models of depression and anxiety and clinical studies have shown the
antidepressant and antianxiety properties of vortioxetine in a dose range of
5-20 mg/day. Vortioxetine reverses cognitive decline in patients with depression
making it a unique molecule. The molecule lacks any serious side effects and
drug-drug interactions. However, dose adjustments are required if vortioxetine
is co-administered with rifampicin or bupropion. The molecule is under review by
various regulatory agencies around the world for the treatment of major
depression. Vortioxetine is a novel antidepressant with effects on multiple 5-HT receptors
and on the serotonin transporter. This paper reviews preclinical and clinical
evidence regarding its mechanism of action, its tolerability, and its efficacy
in treating major depression. Clinical studies indicate that vortioxetine is
effective in the treatment of major depression, though there is no suggestion of
superiority over active comparators. There may be a clinically meaningful
advantage in terms of tolerability. OBJECTIVE: To evaluate the clinical literature and potential clinical role of
vortioxetine (Brintellix) for the treatment of major depressive disorder (MDD).
DATA SOURCES: A MEDLINE search (1966-February 2014) was conducted using the
search terms vortioxetine, Lu AA21004, and depression. Bibliographies of all
articles retrieved were also reviewed. All references included were published
between 1999 and 2014.
STUDY SELECTION/DATA EXTRACTION: All studies that included humans and were
published in English, with data describing vortioxetine for the treatment of
MDD, were reviewed.
DATA SYNTHESIS: Vortioxetine is a novel multimodal antidepressant agent, which
inhibits the 5-HT transporter protein, acts as a 5-HT3 antagonist, 5-HT1A
receptor agonist, 5-HT7 receptor antagonist, and a partial agonist of the 5-HT1B
receptor. It has been studied in 10 short-term (6-8 weeks), 1
relapse-prevention, and 3 long-term extension trials. Vortioxetine demonstrated
efficacy in reducing Montgomery-Asberg Depression Rating Scale or Hamilton
Rating Scale for Depression scores in 6 of the short-term trials. The proportion
of individuals who responded to treatment and achieved remission increased over
time in all 3 long-term trials. The most common adverse effects, consistently
reported by >10% of individuals in the clinical trials include nausea and
headache.
CONCLUSIONS: Vortioxetine is an effective agent for the treatment of MDD, but it
does not have any clear advantages over other available treatment options. INTRODUCTION: Major depressive disorder (MDD), one of the most common disorders
in medical practice and one of the leading causes of disability worldwide, is
frequently comorbid with anxiety disorders. Vortioxetine (Lu AA21004) is a new
antidepressant that combines a number of neurotransmitter reuptake and receptor
effects that have been thought to predict efficacy as a treatment for depressive
and anxiety disorders.
AREAS COVERED: This review summarizes the pharmacology and neurobiology of
vortioxetine. Studies of its efficacy and tolerability in major depression and
generalized anxiety disorder are critically reviewed.
EXPERT OPINION: Despite the fact that industry-sponsored studies are more likely
than other clinical trials to support efficacy of the experimental drug, results
have been mixed. Some studies supported that vortioxetine is superior to placebo
in the treatment of MDD and some do not. Two studies supported the efficacy of
vortioxetine in the treatment of generalized anxiety disorder and two do not.
The incidence of sexual dysfunction has varied considerably in different
studies, but cardiac effects and psychomotor impairment seem to be minimal.
Advantages of vortioxetine over existing antidepressants are not yet clear. The efficacy of vortioxetine 10 and 20 mg/d vs. placebo on cognitive function
and depression in adults with recurrent moderate-to-severe major depressive
disorder (MDD) was evaluated. Patients (18-65 yr, N = 602) were randomized
(1:1:1) to vortioxetine 10 or 20 mg/d or placebo for 8 wk in a double-blind
multi-national study. Cognitive function was assessed with objective
neuropsychological tests of executive function, processing speed, attention and
learning and memory, and a subjective cognitive measure. The primary outcome
measure was change from baseline to week 8 in a composite z-score comprising the
Digit Symbol Substitution Test (DSST) and Rey Auditory Verbal Learning Test
(RAVLT) scores. Depressive symptoms were assessed using the Montgomery-Åsberg
Depression Rating Scale (MADRS). In the pre-defined primary efficacy analysis,
both doses of vortioxetine were significantly better than placebo, with mean
treatment differences vs. placebo of 0.36 (vortioxetine 10 mg, p < 0.0001) and
0.33 (vortioxetine 20 mg, p < 0.0001) on the composite cognition score.
Significant improvement vs. placebo was observed for vortioxetine on most of the
secondary objectives and subjective patient-reported cognitive measures. The
differences to placebo in the MADRS total score at week 8 were -4.7 (10 mg:
p < 0.0001) and -6.7 (20 mg: p < 0.0001). Path and subgroup analyses indicate
that the beneficial effect of vortioxetine on cognition is largely a direct
treatment effect. No safety concern emerged with vortioxetine. Vortioxetine
significantly improved objective and subjective measures of cognitive function
in adults with recurrent MDD and these effects were largely independent of its
effect on improving depressive symptoms. OBJECTIVE: This randomised, double-blind, 12-week study compared efficacy and
tolerability of flexible-dose treatment with vortioxetine(10-20 mg/day) versus
agomelatine (25-50 mg/day) in major depressive disorder patients with inadequate
response to selective serotonin reuptake inhibitor
(SSRI)/serotonin-noradrenaline reuptake inhibitor (SNRI) monotherapy.
METHODS: Patients were switched directly from SSRI/SNRI to vortioxetine or
agomelatine. Primary endpoint was change from baseline to week 8 in the
Montgomery-Åsberg Depression Rating Scale (MADRS) total score analysed by mixed
model for repeated measurements, using a noninferiority test followed by a
superiority test. Secondary endpoints included response and remission rates,
anxiety symptoms(Hamilton Anxiety Rating Scale), Clinical Global Impression,
overall functioning (Sheehan Disability Scale), health-related quality of
life(EuroQol 5 Dimensions), productivity (work limitation questionnaire) and
family functioning (Depression and Family Functioning Scale).
RESULTS: Primary endpoint noninferiority was established and vortioxetine (n =
252) was superior to agomelatine (n = 241) by 2.2 MADRS points (p<0.01).
Vortioxetine was also significantly superior in response and remission rates at
weeks 8 and 12; MADRS, Hamilton Anxiety Rating Scale, Clinical Global
Impression, Sheehan Disability Scale and EuroQol 5 Dimensions scores at week 4
onwards; work limitation questionnaire at week 8 and Depression and Family
Functioning Scale at weeks 8 and 12. Fewer patients withdrew because of adverse
events with vortioxetine (5.9% vs 9.5%). Adverse events (incidence ≥5%) were
nausea, headache, dizziness and somnolence.
CONCLUSIONS: Vortioxetine was noninferior and significantly superior to
agomelatine in major depressive disorder patients with previous inadequate
response to a single course of SSRI/SNRI monotherapy. Vortioxetine was safe and
well tolerated. INTRODUCTION: Vortioxetine is an antidepressant with multimodal activity which
has shown efficacy in major depressive disorder (MDD) patients in six of ten
short-term, randomized, placebo-controlled trials (completed end 2012).
METHODS: We performed meta-regression analyses to indirectly compare
vortioxetine to seven marketed antidepressants with different mechanisms of
action. To ensure study comparability, only experimental drug and placebo arms
from placebo-controlled registration studies were included in primary analyses.
The main outcomes were efficacy (standardized mean difference in change from
baseline to 2 months on primary endpoint [MADRS/HAM-D]), and tolerability
(withdrawal rate due to adverse events).
RESULTS: For efficacy, estimates of treatment effect (negative estimates favor
vortioxetine) for vortioxetine versus comparators were: agomelatine, -0.16 (p =
0.11); desvenlafaxine, 0.03 (p = 0.80); duloxetine, 0.09 (p = 0.42);
escitalopram, -0.05 (p = 0.70); sertraline, -0.04 (p = 0.83); venlafaxine IR/XR,
0.12 (p = 0.33); and vilazodone, -0.25 (p = 0.11). For tolerability, all but one
combination was numerically in favor of vortioxetine (odds ratio < 1), although
not all differences were statistically significant: agomelatine, 1.77 (p =
0.03); desvenlafaxine, 0.58 (p = 0.04); duloxetine, 0.75 (p = 0.26);
escitalopram, 0.67 (p = 0.28); sertraline, 0.30 (p = 0.01); venlafaxine, 0.47 (p
= 0.01); and vilazodone, 0.64 (p = 0.18). Sensitivity analyses did not
significantly alter antidepressant effect estimates or relative ranking.
CONCLUSION: These meta-regression data show that vortioxetine offers a
comparable or favorable combination of efficacy (measured by MADRS/HAM-D) and
tolerability (measured by withdrawal rate due to adverse events) versus other
antidepressants in registration studies in MDD. Alternative methods like
mixed-treatment comparison and inclusion of all randomized studies and active
reference arms may provide complementary information to this analysis (more
evidence but also more heterogeneity). Key messages: Indirect comparisons based
on registration studies allow a useful comparison between a recently approved
antidepressant and an approved drug. Vortioxetine offers a comparable or
favorable combination of efficacy (measured by MADRS/HAM-D assessments) and
tolerability (measured by withdrawal rate due to adverse events) versus other
antidepressants in registration studies in MDD. BACKGROUND: Vortioxetine was approved by the U.S. Food and Drug Administration
(FDA) in September 2013 for treating major depressive disorder (MDD). Thus far,
a number of randomized, double-blind, placebo-controlled clinical trials (RCTs)
of vortioxetine have been conducted in patients with MDD. We performed a
meta-analysis to increase the statistical power of these studies and enhance our
current understanding of the role of vortioxetine in the treatment of MDD.
METHODS: We performed an extensive search of databases and the clinical trial
registry. The mean change in total scores on the 24-item Hamilton Rating Scale
for Depression (HAM-D) and the Montgomery- Åsberg Depression Rating Scale
(MADRS) from the baseline were the primary outcome measures. The secondary
efficacy measures were the response and remission rates, as defined by a 50% or
greater reduction in HAM-D/MADRS total scores and as a score of 10 or less in
the MADRS and 7 or less in the HAM-D total scores at the end of treatment.
RESULTS: We included 7 published and 5 unpublished short-term (6-12 wk) RCTs in
our meta-analysis. Vortioxetine was significantly more effective than placebo,
with an effect size (standardized mean difference [SMD]) of -0.217 (95%
confidence interval [CI] -0.313 to -0.122) and with odds ratios (ORs) for
response and remission of 1.652 (95% CI 1.321 to 2.067) and 1.399 (95% CI 1.104
to 1.773), respectively. Those treated with vortioxetine did not differ
significantly from those treated with selective norepinephrine reuptake
inhibitors/agomelatine with regard to the SMD of the primary outcome measure
(0.081, -0.062 to 0.223) or for response (OR 0.815, 95% CI 0.585 to 1.135) and
remission (OR 0.843, 95% CI 0.575 to 1.238) rates. Discontinuation owing to lack
of efficacy (OR 0.541, 95% CI 0.308 to 0.950) was significantly less common
among those treated with vortioxetine than among those who received placebo,
whereas discontinuation owing to adverse events (AEs; OR 1.530, 95% CI 1.144 to
2.047) was significantly more common among those treated with vortioxetine than
among those receiving placebo. There was no significant difference in
discontinuation rates between vortioxetine and comparators owing to inefficacy
(OR 0.983, 95% CI 0.585 to 1.650), whereas discontinuation owing to AEs was
significantly less common in the vortioxetine than in the comparator group (OR
0.728, 95% CI 0.554 to 0.957).
LIMITATIONS: Studies examining the role of vortioxetine in the treatment of MDD
are limited.
CONCLUSION: Although our results suggest that vortioxetine may be an effective
treatment option for MDD, they should be interpreted and translated into
clinical practice with caution, as the meta-analysis was based on a limited
number of heterogeneous RCTs. OBJECTIVE: This article summarizes the US Food and Drug Administration's (FDA's)
review of the New Drug Application for vortioxetine, especially the clinical
efficacy and safety data. It emphasizes the issues that were important to the
FDA's approval decision, particularly the difference in the effective dose in
domestic and foreign studies, and notes several new labeling features,
specifically, description of time course of treatment response and detailed
sexual dysfunction evaluation.
DATA SOURCES: The data sources were the original raw data sets for all clinical
trials included in the development program for vortioxetine, as well as the
sponsor's original analyses of these data. Data were available from 51 human
trials involving vortioxetine, and included a total of 7,666 healthy volunteers
and patients with a diagnosis of major depressive disorder (MDD) or generalized
anxiety disorder who were exposed to at least 1 dose of vortioxetine for a total
of 2,743 patient-years.
RESULTS: Vortioxetine was effective in treating MDD in the United States at a
dose of 20 mg/d. The recommended starting dose is 10 mg once daily without
regard to food, with increase to 20 mg/d if the 10 mg/d dose is tolerated. For
patients who do not tolerate 20 mg/d, 10 mg/d can be used and 5-mg/d dose can be
considered. Vortioxetine can be discontinued abruptly, but it is recommended
that doses of 15 mg/d or 20 mg/d be reduced to 10 mg/d for 1 week prior to full
discontinuation to avoid potential withdrawal symptoms. Although the non-US
maintece study showed that maintece doses of 5 to 10 mg/d were effective,
a clinical judgment needs to be made to decide the maintece dose in the
United States. The applicant has agreed to conduct a US maintece
dose-response study covering the US-approved dose range. Vortioxetine's adverse
event profile is similar to that of other selective serotonin reuptake
inhibitors (SSRIs). Nausea is the most common adverse event and is dose
dependent. No dose adjustment is needed based on age, gender, or the presence of
renal or mild to moderate hepatic impairment. The maximum recommended dose is 10
mg/d in known cytochrome P450 2D6 poor metabolizers.
CONCLUSIONS: Vortioxetine is a new treatment for MDD, and its adverse event
profile is similar to that of other SSRIs. OBJECTIVE: This randomized, double-blind 8 week study compared the efficacy and
tolerability of fixed-dose treatment with vortioxetine (10 mg/day) and
venlafaxine extended release (XR) (150 mg/day) in major depressive disorder
(MDD) patients.
RESEARCH DESIGN AND METHODS: Patients aged 18-65 years with a primary diagnosis
of recurrent MDD, a Montgomery-Åsberg Depression Rating Scale (MADRS) total
score ≥26 and a Clinical Global Impression-Severity (CGI-S) score ≥4 were
randomized (1:1) to treatment with either vortioxetine or venlafaxine XR. The
primary endpoint was change from baseline to Week 8 in MADRS total score
(analysis of covariance [ANCOVA], full-analysis set [FAS], last observation
carried forward [LOCF]), using a non-inferiority margin of +2.5 points.
Pre-specified secondary endpoints included MADRS response and remission rates,
anxiety symptoms (HAM-A), CGI, overall functioning (SDS), and health-related
quality of life (Q-LES-Q).
CLINICAL TRIAL REGISTRATION: This study (SOLUTION) has the
www.ClinicalTrials.gov identifier: NCT01571453.
RESULTS: On the primary efficacy endpoint at Week 8, non-inferiority was
established with a difference of -1.2 MADRS points in favor of vortioxetine (95%
CI: -3.0 to 0.6). The MADRS total score decreased (improved) from 32.3 ± 4.6 at
baseline to 13.6 ± 9.6 (vortioxetine: n = 209) and from 32.3 ± 4.5 to
14.8 ± 10.4 (venlafaxine XR: n = 215) (FAS, LOCF). At Week 8, the HAM-A and SDS
total scores, CGI and Q-LES-Q scores, and response and remission rates
demonstrated similar improvement for vortioxetine and venlafaxine XR, with
remission rates (MADRS ≤10) of 43.1% (vortioxetine) versus 41.4% (venlafaxine
XR) (LOCF). Fewer vortioxetine than venlafaxine XR patients withdrew for any
reason (18.0% versus 27.4%) or for adverse events (6.6% versus 13.7%). The most
frequent adverse events (≥5%) for both treatments were nausea, dizziness,
headache, and dry mouth. In addition, accidental overdose, decreased appetite,
constipation and insomnia were reported by (≥5%) of patients treated with
venlafaxine XR.
LIMITATIONS: The inclusion and exclusion criteria may limit the generalizability
of the study. Since patients with a history of lack of response to venlafaxine
XR were excluded from this study, there is a selection bias in favor of
venlafaxine XR.
CONCLUSION: Vortioxetine was at least as efficacious as venlafaxine XR and was
safe and better tolerated than venlafaxine XR. Vortioxetine has a beneficial pharmacological profile for reducing anxiety and
depression. Recently, a number of randomized, double-blind, placebo-controlled
clinical trials (RCTs) of vortioxetine have been conducted in patients with
generalized anxiety disorder (GAD); however, the results from GAD RCTs are
inconsistent. With an extensive search of databases and clinical trial
registries, four published short-term RCTs were identified and included in the
present meta-analysis. The mean change in total scores on the Hamilton Anxiety
Rating Scale (HAMA) from baseline was the primary endpoint. The secondary
endpoints included the response and remission rates, as defined by a ≥50%
reduction in HAMA total scores and a ≤7 change in the HAMA total score at the
end of treatment. In addition, the mean change in the HAMA total score from
baseline in the subgroup with a HAMA total score ≥25 at baseline was included.
Vortioxetine was significantly more effective than was placebo, with a
standardized mean difference (SMD) of -0.118 (95% CIs, -0.203 to -0.033,
P = 0.007). In particular, those with severe GAD (HAMA total score ≥25 at
baseline) had a significantly greater benefit from vortioxetine than those
without (SMD = -0.338, 95% CIs = -0.552 to -0.124, p = 0.002). The odds ratios
(ORs) for vortioxetine for response and remission were 1.221 (95% CIs, 1.027 to
1.452, P = 0.024) and 1.052 (95% CIs, 0.853 to 1.296, P = 0.637), respectively.
Discontinuation due to adverse events (AEs) (OR = 1.560, 1.006 to 2.419,
p = 0.047) was marginally higher in vortioxetine than placebo treatment, whereas
discontinuation due to any reason (OR = 0.971, 0.794 to 1.187, p = 0.771) and
inefficacy (OR = 0.687, 0.380 to 1.243, p = 0.215) were not significantly
different among treatment groups. Although our results suggest that vortioxetine
may have a potential as an another treatment option for GAD (especially for
severe GAD), they should be interpreted and translated into clinical practice
with caution, as the meta-analysis was based on a limited number of RCTs. BACKGROUND: Vortioxetine is the first mixed serotonin agonist and antagonist
antidepressant approved in the US. We sought to evaluate all published and
unpublished data available to determine the efficacy and harms of vortioxetine
in adults with major depressive disorder.
METHODS: We used a predefined search strategy of MEDLINE, the Cochrane Central
Register of Controlled Trials, ClinicalTrials.gov, and Drugs@FDA to identify
studies evaluating vortioxetine in the acute treatment of major depressive
disorder. Only randomized controlled trials (RCTs) that provided results on
relevant clinical efficacy and safety outcomes were included. Study quality was
assessed and results were pooled using mixed effect meta-analyses where
applicable.
RESULTS: We identified 11 RCTs with 6,145 participants meeting inclusion
criteria (eight were published and three were unpublished). The trials did not
exceed 8 weeks in duration. The response rate with vortioxetine was
significantly higher for 1-mg (relative risk (RR) = 1.91; 95% confidence
interval (CI) 1.36 to 2.69), 5-mg (RR = 1.33; 95% CI 1.10 to 1.61), 10-mg
(RR = 1.42; 95% CI 1.21 to 1.67), and 20-mg doses (RR = 1.58; 95% CI 1.19 to
2.08) compared to placebo. Remission rates were significantly higher for the
10-mg group (RR = 1.45; 95% CI 1.18 to 1.77) and the 20-mg group (RR = 1.68; 95%
CI 1.19 to 2.37) compared to placebo. Meta-regression of dose on the log odds
ratio of response was not statistically significant (β = 0.01; P = 0.46).
Vortioxetine response rates were lower than active serotonin and norepinephrine
reuptake inhibitor (SNRI) comparators for the 5-mg (RR = 0.88; 95% CI 0.80 to
0.98), 15-mg (RR = 0.78; 95% CI 0.68 to 0.90), and 20-mg (RR = 0.82; 95% CI 0.72
to 0.94) doses. The most common adverse events were nausea and vomiting which
increased in frequency with higher doses.
CONCLUSIONS: Vortioxetine was significantly more effective than placebo for
acute treatment of major depressive disorder (MDD). Although treatment effect
estimates varied substantially between studies, a dose effect was not observed.
Vortioxetine does not appear to be more effective, and is potentially less
effective, than an SNRI.
SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42013006198 . Depression and cerebrovascular atherosclerosis often occur in comorbidity
showing neuropsychological impairment and poor response to antidepressant
treatment. Objective is to evaluate if new antidepressant vortioxetine may be a
potential treatment option. Mechanism of Action : Vortioxetine has 5-HT3, 5-HT7
and 5-HT1D antagonists, 5-HT1B partial agonist and a 5-HT1A agonist and
serotonin transporter inhibitor property. Efficacy and safety in Major
Depressive Disorders and in cognitive impairment : The majority of trials (one
of them in older people) showed efficacy for vortioxetine against placebo and no
differences against other active treatments. The Adverse Effects ranged from
15.8% more to 10.8% less than placebo. In the elderly, only nausea was found
higher than placebo. Effects on arterial blood pressure and cardiac parameters
including the ECG-QT segment were similar to placebo. Elderly depressive
patients on vortioxetine showed improvement versus placebo and other active
comparators in Auditory Verbal Learning Test and Digit Symbol Substitution Test
scores. The inclusion criteria admitted cases with middle cerebrovascular
disease. Conclusion : The mechanism of action, the efficacy on depression and
safety profile and early data on cognitive impairment make Vortioxetine a strong
candidate for use in depression associated with cerebrovascular disease. This
information must be supported by future randomized controlled trials. OBJECTIVE: The treatment of depressive disorders remains unsatisfactory for many
patients with regard to efficacy and tolerability. Vortioxetine has been
registered by regulatory authorities for the treatment of major depressive
disorder. This paper aims to provide clinicians with a brief overview of
vortioxetine and its place in treatment.
CONCLUSIONS: Vortioxetine is a serotonin reuptake inhibitor with additional
serotonergic receptor effects of uncertain significance; hence, its
classification as 'multimodal'. The half-life is about 2.75 days and steady
state requires about 14 days. Metabolism is hepatic and involves cytochromes 2D6
and 3A4/5. Antidepressant efficacy in major depressive disorder has been
established in registration studies, but the effectiveness of vortioxetine in
'real world' patients and in comparison to other antidepressants needs further
investigation. The recommended dose range is 5-20 mg. Nausea, constipation and
vomiting are the most common side effects. Sexual dysfunction may occur at
higher doses but there appears to be low risk of weight gain and sedation. There
is still much to learn about this drug, particularly whether it has unique
characteristics in comparison to existing antidepressants. At present,
vortioxetine can be considered as an antidepressant option in patients with
established major depressive disorder who have not responded adequately to other
antidepressants. Vortioxetine is approved for the treatment of adults with major depressive
disorder. This open-label extension (OLE) study evaluated the safety and
tolerability of vortioxetine in the long-term treatment of major depressive
disorder patients, as well as evaluated its effectiveness using measures of
depression, anxiety, and overall functioning. This was a 52-week, flexible-dose,
OLE study in patients who completed one of three randomized, double-blind,
placebo-controlled, 8-week vortioxetine trials. All patients were switched to
10 mg/day vortioxetine for week 1, then adjusted between 15 and 20 mg for the
remainder of the study, but not downtitrated below 15 mg. Safety and
tolerability were assessed on the basis of treatment-emergent adverse events
(TEAEs), vital signs, laboratory values, physical examination, and the
Columbia-Suicide Severity Rating Scale. Efficacy measures included the
Montgomery-Åsberg Depression Rating Scale, the Hamilton Anxiety Scale, the
Clinical Global Impression Scale-Severity of Illness, and the Sheehan Disability
Scale. Of the 1075 patients enrolled, 1073 received at least one dose of
vortioxetine and 538 (50.0%) completed the study. A total of 537 patients
withdrew early, with 115 (10.7% of the original study population) withdrawing
because of TEAEs. Long-term treatment with vortioxetine was well tolerated; the
most common TEAEs (≥10%) were nausea and headache. Laboratory values, vital
signs, and physical examinations revealed no trends of clinical concern. The
mean Montgomery-Åsberg Depression Rating Scale total score was 19.9 at the start
of the extension study and 9.0 after 52 weeks of treatment (observed cases).
Similar improvements were observed with the Hamilton Anxiety Scale (Δ-4.2), the
Clinical Global Impression Scale-Severity of Illness (Δ-1.2), and the Sheehan
Disability Scale (Δ-4.7) total scores after 52 weeks of treatment (observed
case). In this 52-week, flexible-dose OLE study, 15 and 20 mg vortioxetine were
safe and well tolerated. After entry into this study, patients continued to show
improvement in depression and anxiety symptoms, as well as overall functioning,
throughout the treatment period. CONTEXT: Vortioxetine (Lu AA21004) is an antidepressant with a mechanism of
action thought to be related to a combination of 2 pharmacologic actions: direct
modulation of several receptors and inhibition of the serotonin transporter.
OBJECTIVE: To evaluate the efficacy of vortioxetine 10 and 20 mg once daily in
outpatients with major depressive disorder.
DESIGN, SETTING, AND PARTICIPANTS: This 8-week, multicenter, randomized,
double-blind, placebo-controlled, parallel-group study was conducted from July
2010 to January 2012 among adults with a primary diagnosis of recurrent major
depressive disorder (DSM-IV-TR).
INTERVENTION: Eligible subjects were randomized in 1:1:1 ratio to 1 of 3
treatment arms: vortioxetine 10 mg, vortioxetine 20 mg, or placebo once daily
for 8 weeks. Subjects who completed the 8-week trial entered a 2-week blinded
discontinuation period to assess potential discontinuation symptoms.
MAIN OUTCOME MEASURE: The primary endpoint was the least squares mean change in
Montgomery-Asberg Depression Rating Scale (MADRS) total score from baseline. Key
secondary outcomes were analyzed in the following prespecified sequential order:
MADRS response (≥ 50% decrease from baseline in total score), Clinical Global
Impressions-Improvement score, change from baseline in MADRS total score in
subjects with baseline Hamilton Anxiety Rating Scale score ≥ 20, MADRS remission
(total score ≤ 10), and change from baseline in Sheehan Disability Scale total
score (all at week 8).
RESULTS: A total of 462 subjects were randomized to placebo (n = 157),
vortioxetine 10 mg (n = 155), and vortioxetine 20 mg (n = 150). Mean (SE)
reductions from baseline in MADRS total score (week 8) were -10.77 (± 0.807),
-12.96 (± 0.832), and -14.41 (± 0.845) for the placebo, vortioxetine 10 mg (P =
.058 vs placebo), and vortioxetine 20 mg (P = .002 vs placebo) groups. MADRS
response/remission was achieved in 28.4%/14.2%, 33.8%/21.4%, and 39.2%/22.3% of
subjects, respectively, in the 3 groups. Only MADRS response for vortioxetine 20
mg significantly separated from placebo (P = .044). Treatment was well
tolerated, with the most frequently reported adverse events consisting of
nausea, headache, diarrhea, and dizziness.
CONCLUSIONS: Vortioxetine 20 mg significantly reduced MADRS total score at 8
weeks in this study population. Overall, vortioxetine was well tolerated in this
study.
TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01163266. BACKGROUND: This 8-week, randomized, double-blind, placebo-controlled study,
conducted August 2010-May 2012 in the United States, evaluated the safety and
efficacy of vortioxetine 10 mg and 15 mg in patients with major depressive
disorder (MDD). The mechanism of action of vortioxetine is thought to be related
to direct modulation of serotonin (5-HT) receptor activity and inhibition of the
serotonin transporter.
METHOD: Adults aged 18-75 years with MDD (DSM-IV-TR) and Montgomery-Asberg
Depression Rating Scale (MADRS) total score ≥ 26 were randomized (1:1:1) to
receive vortioxetine 10 mg or 15 mg or placebo once daily, with the primary
efficacy end point being change from baseline at week 8 in MADRS analyzed by
mixed model for repeated measures. Adverse events were recorded during the
study, suicidal ideation and behavior were assessed using the Columbia-Suicide
Severity Rating Scale (C-SSRS), and sexual dysfunction was assessed using the
Arizona Sexual Experience (ASEX) scale.
RESULTS: Of the 1,111 subjects screened, 469 subjects were randomized: 160 to
placebo, 157 to vortioxetine 10 mg, and 152 to vortioxetine 15 mg. Differences
from placebo in the primary efficacy end point were not statistically
significant for vortioxetine 10 mg or vortioxetine 15 mg. Nausea, headache, dry
mouth, constipation, diarrhea, vomiting, dizziness, and flatulence were reported
in ≥ 5% of subjects receiving vortioxetine. Discontinuation due to adverse
events occurred in 7 subjects (4.4%) in the placebo group, 8 (5.2%) in the
vortioxetine 10 mg group, and 12 (7.9%) in the vortioxetine 15 mg group. ASEX
total scores were similar across groups. There were no clinically significant
trends within or between treatment groups on the C-SSRS, laboratory values,
electrocardiogram, or vital sign parameters.
CONCLUSIONS: In this study, vortioxetine did not differ significantly from
placebo on MADRS total score after 8 weeks of treatment in MDD subjects.
TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01179516. PURPOSE: Major depressive disorder (MDD) has detrimental effects on
health-related quality of life (HRQoL). We describe the effect of vortioxetine
on HRQoL in MDD patients by using patient-reported outcome instruments.
METHODS: HRQoL was evaluated in 5 short-term (6-8 weeks), randomized studies of
vortioxetine (5-20 mg/d; n = 2155) versus placebo (n = 1316) in adults with MDD
by using the 36-item Short-Form Health Survey (SF-36), the Quality of Life
Enjoyment and Satisfaction Questionnaire-Short Form, the EuroQol 5-Dimension
Questionnaire (EQ-5D), and the 12-item Health Status Questionnaire in 1 study in
elderly patients. Only patients receiving the approved doses of vortioxetine 5,
10, 15, or 20 mg/d were included in the analysis. A random effects meta-analysis
was performed on the 4 adult MDD studies that used the SF-36. A within-studies
mixed model for repeated measures analysis based on the full analysis set (FAS)
was used unless otherwise specified. Standardized effect size (SES) was
calculated to reflect clinical relevance, based on a Cohen's d of 0.2.
FINDINGS: Vortioxetine produced significantly better results compared with
placebo in the SF-36 mental component summary score (5 mg: 2.6, P = 0.001, SES
of 0.22, n = 604; 10 mg: 4.8, P < 0.001, SES of 0.42, n = 328) and 4 domain
scores (vitality, social functioning, role emotional, and mental health).
Vortioxetine was also significantly better in the EuroQoL-5 Dimension
Questionnaire Health State score (10 mg: 7.5, P < 0.05, SES of 0.33, n = 86) and
Quality of Life Enjoyment and Satisfaction Questionnaire-Short Form total score
(15 mg: 3.3, P < 0.01, SES of 0.38, n = 127; 20 mg: 4.5, P < 0.0001, SES of
0.52, n = 134) (FAS, last-observation-carried-forward). In the study of elderly
patients, vortioxetine 5 mg (n = 136) improved 12-item Health Status
Questionnaire scores significantly more than placebo (n = 148) for the domains
of health perception (10.4, P < 0.0001, SES of 0.54), mental health (7.9, P <
0.001, SES of 0.44), and energy (6.4, P < 0.05, SES of 0.28) (FAS, mixed model
for repeated measures).
IMPLICATIONS: Vortioxetine yielded significant, meaningful HRQoL improvements in
6 MDD studies of 6 to 8 weeks' duration. Vortioxetine is a new antidepressant, which mechanism of action is multimodal,
targeting the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT3, 5-HT7 receptors and the serotonin
transporter (5-HTT). Its efficacy and safety were assessed in fourteen studies
including more than 3700 patients with a major depressive episode and treated
with vortioxetine. In short-term studies (8 weeks), vortioxetine is more
efficacious than placebo in decreasing depressive symptoms as measured by the
MADRS total score, response rate (vortioxetine: 53.2% vs placebo: 35.2%) and
remission rate (vortioxetine: 29.2% vs placebo: 19.3%). In a long-term study
(52 weeks), vortioxetine is also superior to placebo in preventing relapses and
recurrences. Moreover, in second line treatment, after failure of a first line
selective serotonin reuptake inhibitor (SSRI) or serotonin norepinephrin
reuptake inhibitor (SNRI), vortioxetine is superior to agomelatine in improving
depressive symptoms and achieving response and remission. Furthermore, the
positive effects of vortioxetine on improvement of cognitive symptoms of major
depressive episodes are particularly well established in several clinical
trials. The tolerability profile of vortioxetine is favourable. The recommended
daily posology of vortioxetine is 10mg/d. Vortioxetine is a new antidepressant
drug with a multimodal mechanism of action, well-documented efficacy and safety
profiles. Vortioxetine is approved for the treatment of major depressive disorder (MDD).
This analysis aimed to develop pharmacokinetic (PK) and PK/Efficacy models to
evaluate the exposure-response relationship for vortioxetine in patients with
MDD. PK data from 10 MDD and two generalized anxiety disorder studies of
vortioxetine (3160 patients), and efficacy data [Montgomery-Åsberg Depression
Rating Scale (MADRS)] from seven MDD studies (2537 patients), were used for the
development of PK and PK/Efficacy models. One- and two-compartment models were
evaluated as structural PK models, and linear and nonlinear (Emax) models were
used to describe the relationship between average vortioxetine concentration at
steady-state (Cav) and change in MADRS score from baseline (ΔMADRS). The impact
of selected covariates on the PK and efficacy parameters of vortioxetine was
also investigated. PK of vortioxetine was best characterized by a
two-compartment model with first-order absorption and elimination. Mean
estimates for oral clearance (CL/F) and volume of distribution for the central
compartment of vortioxetine were 42 L/hr and 2920 L. Creatinine clearance,
height and geographic region had statistically significant effects on
vortioxetine CL/F, but the effect of each of these covariates was not considered
clinically relevant, as they lead to ±26% change in area under the curve or Cmax
of vortioxetine. An Emax model best described the relationship between ΔMADRS
and Cav. Half-maximal effective concentration (EC50) and Emax estimates were
24.9 ng/mL and 7.0. No identified covariates, except region, had clinically
meaningful effects on vortioxetine efficacy. These PK/Efficacy models adequately
characterized the vortioxetine exposure-response relationship. BACKGROUND: We recently conducted a comprehensive review of the psychiatric
inclusion/exclusion criteria used in 170 placebo-controlled antidepressant
efficacy trials (AETs) published during the past 20 years and found that the
criteria of more recent studies were significantly more restrictive than prior
studies. Vortioxetine is the most recently approved medication for the treatment
of major depressive disorder (MDD). We compared the inclusion/exclusion criteria
of the vortioxetine studies to the criteria used in other AETs, and discuss the
broader issue of the generalizability of AETs and the implications this might
have for the labeling of antidepressants receiving FDA approval.
METHODS: We conducted a comprehensive literature review of placebo-controlled
AETs published from January, 1995 through December, 2014. We identified 170 AETs
published during this 20 year period and compared the inclusion/exclusion
criteria used in the 12 studies of vortioxetine to those used in the
nonvortioxetine studies. A second analysis compared vortioxetine to the 3
antidepressants most recently approved prior to vortioxetine (desvenlafaxine,
levomilnacipran extended release, vilazodone).
RESULTS: Compared to the nonvortioxetine AETs, the vortioxetine studies
significantly more often excluded patients with any comorbid Axis I disorder
(p<.001) and more often required the current depressive episode to be longer
than the DSM minimum symptom duration requirement of 2 weeks (p<.01). The cutoff
on the Montgomery Asberg Depression Rating Scale required for inclusion in the
vortioxetine studies was higher than the cutoff used in the other AETs (p<.01).
LIMITATIONS: A limitation of the present analysis is that it was based on
published placebo-controlled studies of antidepressants.
CONCLUSION: The inclusion/exclusion criteria in the studies of vortioxetine were
more restrictive than the criteria used in other AETs. Inconsistent with FDA
guidelines on the labeling of medications, the label of vortioxetine does not
include a description of the limits to the group of patients with MDD for whom
the medication has been shown to be effective. OBJECTIVE: To assess the cost-effectiveness of vortioxetine versus venlafaxine
XR (extended-release) in major depressive disorder (MDD) patients in South
Korea.
METHODS: A 1-year cost-effectiveness analysis from a limited societal
perspective was performed using a combined model consisting of a decision-tree
and a Markov model. Patients entered the model when initiating or switching
antidepressant treatment following inadequate response to previous treatment.
Remission, relapse and recovery were the main health states.
RESULTS: Vortioxetine dominated venlafaxine XR, with quality-adjusted life year
(QALY) gains of 0.0131 and cost savings of KRW 623,229/year [US$530/year] from a
limited societal perspective. Safety contributed more than efficacy to the
incremental QALY gains. More patients were in recovery after initial treatment
and after 1 year with vortioxetine (31%, 40%) compared to venlafaxine XR (23%,
36%). Vortioxetine remained domit in 98% of probabilistic simulations.
CONCLUSION: Vortioxetine dominated venlafaxine XR in South Korea and is a
relevant treatment option for MDD patients initiating or switching therapy. INTRODUCTION: Vortioxetine is a structurally novel medication that has recently
been approved for treatment of major depressive disorder (MDD). This medication
is a serotonin reuptake inhibitor that also has a number of other potentially
relevant effects on serotoninergic receptors, which may differentiate the drug's
effects from those of current first-line antidepressants, such as selective
serotonin reuptake inhibitors (SSRIs) and serotonin norepinephrine reuptake
inhibitors (SNRIs).
AREAS COVERED: This article will review the basic clinical pharmacology of
vortioxetine, summarize the major clinical trials that were performed prior to
approval by the US Food and Drug Administration (FDA), discuss relevant
post-marketing studies of this drug, and offer expert commentary on the
significance of this new agent in clinical practice. Pre-approval studies were
identified as all randomized, placebo-controlled studies of vortioxetine listed
on clinicaltrials.gov. Other referenced studies were identified via a MEDLINE
database literature search in August 2015 using the key search terms,
vortioxetine and Lu AA21004, combined with additional terms that included
pharmacological profile, pharmacokinetics, drug interactions, adverse effects,
side effects, safety, major depression, and major depressive disorder. We
identified relevant systematic reviews, meta-analyses, randomized trials and
preclinical studies of importance.
EXPERT OPINION: Results of placebo-controlled trials suggest efficacy and an
overall safety profile comparable to existing first-line antidepressants. The
most common side effects are nausea, vomiting and constipation. Results of
several studies indicate that vortioxetine may have therapeutic effects on
cognition (e.g., memory and executive functioning) that exceed that of standard
antidepressants. Disadvantages include cost and the current paucity of long-term
efficacy data from large clinical trials. The authors suggest that vortioxetine
is currently a good second-line antidepressant option and shows promise, pending
additional long-term data, to become a first-line antidepressant option. BACKGROUND AND OBJECTIVE: Vortioxetine and duloxetine are two new antidepressant
drugs that have been used clinically in the treatment of major depressive
disorder (MDD). The objectives of this meta-analysis were to evaluate the
efficacy and tolerability of vortioxetine compared with duloxetine in MDD.
METHODS: Randomized controlled trials (RCTs) published in PubMed, EMBASE, Web of
Science, and ClinicalTrials.gov were systematically reviewed to compare
vortioxetine with duloxetine in terms of efficacy and tolerability in patients
with MDD. Results were expressed as the risk ratio (RR) with 95 % confidence
intervals (CIs), and weighted mean difference (WMD). Pooled estimates were
calculated by using a fixed-effects model or a randomized-effects model,
depending on the heterogeneity among studies.
RESULTS: A total of five RCTs involving 2287 patients met the inclusion criteria
and were included in this meta-analysis. Pooled results showed that duloxetine
was associated with a higher response rate than vortioxetine, as well as showing
a similar remission rate with vortioxetine. The changes from baseline in the
Montgomery-Åsberg Depression Rating Scale (MADRS), 24-item Hamilton Rating Scale
for Depression (HAM-D24), Clinical Global Impression-Improvement scale (CGI-I),
CGI-Severity scale (CGI-S), Sheehan Disability Scale (SDS), and Hamilton Anxiety
Rating Scale (HAM-A) scores were significantly greater in the duloxetine group
than in the vortioxetine group. The incidence of treatment-emergent adverse
events was significantly higher in the duloxetine group than in the vortioxetine
group.
CONCLUSION: Duloxetine was more effective but less well-tolerated than
vortioxetine in MDD. Considering the potential limitations of this
meta-analysis, more large-scale RCTs are needed to confirm these findings. The efficacy and safety of vortioxetine, an antidepressant approved for the
treatment of adults with major depressive disorder (MDD), was studied in 11
randomized, double-blind, placebo-controlled trials of 6/8 weeks׳ treatment
duration. An aggregated study-level meta-analysis was conducted to estimate the
magnitude and dose-relationship of the clinical effect of approved doses of
vortioxetine (5-20mg/day). The primary outcome measure was change from baseline
to endpoint in Montgomery-Åsberg Depression Rating Scale (MADRS) total score.
Differences from placebo were analyzed using mixed model for repeated
measurements (MMRM) analysis, with a sensitivity analysis also conducted using
last observation carried forward. Secondary outcomes included MADRS single-item
scores, response rate (≥50% reduction in baseline MADRS), remission rate (MADRS
≤10), and Clinical Global Impressions scores. Across the 11 studies, 1824
patients were treated with placebo and 3304 with vortioxetine (5mg/day: n=1001;
10mg/day: n=1042; 15mg/day: n=449; 20mg/day: n=812). The MMRM meta-analysis
demonstrated that vortioxetine 5, 10, and 20mg/day were associated with
significant reductions in MADRS total score (Δ-2.27, Δ-3.57, and Δ-4.57,
respectively; p<0.01) versus placebo. The effects of 15mg/day (Δ-2.60; p=0.105)
were not significantly different from placebo. Vortioxetine 10 and 20mg/day were
associated with significant reductions in 9 of 10 MADRS single-item scores.
Vortioxetine treatment was also associated with significantly higher rates of
response and remission and with significant improvements in other
depression-related scores versus placebo. This meta-analysis of vortioxetine
(5-20mg/day) in adults with MDD supports the efficacy demonstrated in the
individual studies, with treatment effect increasing with dose. |
Which server is used for simulation of macromolecular diffusional association? | Macromolecular interactions play a crucial role in biological systems. Simulation of diffusional association (SDA) is a software for carrying out Brownian dynamics simulations that can be used to study the interactions between two or more biological macromolecules. webSDA allows users to run Brownian dynamics simulations with SDA to study bimolecular association and encounter complex formation, to compute association rate constants, and to investigate macromolecular crowding using atomically detailed macromolecular structures. webSDA facilitates and automates the use of the SDA software, and offers user-friendly visualization of results. | Macromolecular interactions play a crucial role in biological systems.
Simulation of diffusional association (SDA) is a software for carrying out
Brownian dynamics simulations that can be used to study the interactions between
two or more biological macromolecules. webSDA allows users to run Brownian
dynamics simulations with SDA to study bimolecular association and encounter
complex formation, to compute association rate constants, and to investigate
macromolecular crowding using atomically detailed macromolecular structures.
webSDA facilitates and automates the use of the SDA software, and offers
user-friendly visualization of results. webSDA currently has three modules: 'SDA
docking' to generate structures of the diffusional encounter complexes of two
macromolecules, 'SDA association' to calculate bimolecular diffusional
association rate constants, and 'SDA multiple molecules' to simulate the
diffusive motion of hundreds of macromolecules. webSDA is freely available to
all users and there is no login requirement. webSDA is available at
http://mcm.h-its.org/webSDA/. |
Can beans induce apoptosis? | White kidney bean lectin has been shown to exert anti-proliferative and apoptotic effects on cancer cells | Treatment of human stomach cancer KATO III cells with hot-water extracts from
adzuki beans led to their growth inhibition as well as apoptosis induction.
There are morphological changes in the cultured cells treated with the extracts,
by which DNA fragmentation characteristic of apoptosis was actualized both
concentration- and time-dependently. In contrast, N-acetyl-L-cysteine suppressed
such DNA fragmentation, implying that the extracts from adzuki beans might exert
antitumorigenicity via active oxygen-induced apoptosis. In order to verify this
hypothesis in animal experiment, the 40% ethanol fraction of hot-water extracts
was examined for its preventive effect against benzo(a)pyrene-induced
tumorigenesis in the forestomach of A/J mice, given as drinking water containing
the above fraction at 0.5-2.0% levels. Consequently, forestomach cancer has
turned out to be reduced by 36-62% in tumor weight relative to the control.
These results suggest that the fraction of hot-water adzuki extracts may serve
as a nutrapharmaceutical or functional food available for cancer prevention. In this study, we investigated the molecular basis of Korean kidney bean husk
extract, with emphasis on its ability to control intracellular signaling
cascades of AMP-activated protein kinase (AMPK) responsible for inducing
antitumor activities in colon cancer cells. Recently, the evolutionarily
conserved serine/threonine kinase, AMPK, has emerged as a possible target
molecule of tumor control. We investigated the effects of Korean kidney bean
husk extract on apoptosis regulation and the activation of AMPK. Korean kidney
bean husk extract exhibited a series of antitumor effects such as cell death and
apoptotic body appearance. These antitumor potentials were accompanied by the
increase in p-AMPK and p-Acc as well as antitumor proteins p53 and p21. The
stimulation of AMPK by this extract was blocked with the synthetic AMPK
inhibitor Compound C at 10 micromol/L, and the combined treatment of Compound C
and the AMPK activator AICAR
(5-aminoimiazole-4-carboxamide-1-beta-D-ribofuranoside) showed that Compound C
could inhibit the activation of AMPK at the concentration of 20 micromol/L. In
conclusion, the ability of carcinogenesis control by Korean kidney bean husk
extract with high potency suggests its value as an antitumor agent in colon
cancer therapy. Previous studies have shown that soybean fermentation products can act as cancer
chemoprevention or therapeutic agents. In this study, the anticancer activities
of a fermentation product of soybean, black bean, and green bean mixture (BN999)
were investigated. We found that BN999 inhibited the growth of human breast
cancer AU565 cells and prostate adenocarcinoma PC-3 cells but not that of normal
human cells. BN999 induced apoptosis in various human cancer cells but not in
normal human cells. BN999 treatment of AU565 cancer cells resulted in activation
of calpain and caspase-8, -9, and -3, suggesting that BN999 induces apoptosis
via receptor-, mitochondria-, and endoplasmic reticulum-mediated pathways.
Finally, we showed that BN999 inhibited the growth of mouse CT-26 colon cancer
xenografts in syngenic BALB/c mice without causing obvious side effects.
Together, these data suggest that BN999 has potential to be used as a cancer
chemoprevention or therapeutic agent. PHA-E is a natural product extracted from red kidney beans, and it has been
reported to induce cell apoptosis by blocking EGFR in lung cancer cells. Because
EGF is the major in vivo competitor to PHA-E in clinical application, PHA-E must
be proved that has better affinity to EGFR than EGF. This study would focus on
how PHA-E tightly bind to EGFR and the results would compare with EGF. The
adhesion force, measured by AFM, between EGFR and PHA-E was 207.14±74.42 pN that
was higher than EGF (183.65±86.93 pN). The equilibrium dissociation constant of
PHA-E and EGF to EGFR was 2.4 10(-9)±1.4 10(-9) and 7.3 10(-8)±2.7 10(-8),
respectively, that could evaluate binding affinity. The result showed that
binding affinity of PHA-E to EGFR was one order higher than EGF to EGFR. In the
results of flow cytometer and confocal microscope, we found binding efficiency
of EGF to EGFR was decrease as the concentration of PHA-E increased. In the
analysis of Western blot, treatment of A-549 cells with PHA-E resulted in a
dose-dependent decrease in EGFR phosphorylation. In conclusion, we found that
PHA-E had better adhesion force and binding affinity to EGFR than that of the
EGF. The interaction between PHA-E and EGFR could block EGF binding and then
inhibit EGFR phosphorylation. PHA-E could be developed into a new target
molecule for lung cancer treatment that could be immobilized on the drug carrier
to guide therapeutic particles to the tumor site. We have previously demonstrated that the non-digestible fraction (NDF) from
common cooked beans (P. vulgaris L., cv Negro 8025) inhibits azoxymethane
(AOM)-induced colon cancer and influences the expression of genes involved in
the induction of apoptosis and cell cycle arrest through the action of butyrate.
The objective of this study was to identify cell cycle alterations and
morphological changes induced by treatment with AOM and to examine the formation
of colonic aberrant crypt foci (ACF) in male Sprague Dawley rats fed with these
beans. Rats were fed control diets upon arrival and were randomly placed into
four groups after one week of acclimatization: control, NDF (intragastric
administration), NDF + AOM and AOM. Rats treated with NDF + AOM exhibited a
significantly lower number of total colonic ACF with a notable increase in the
number of cells present in the G1 phase (83.14%); a decreased proliferation
index was observed in the NDF + AOM group when compared to AOM group. NDF + AOM
also displayed a higher number of apoptotic cells compared to AOM group. NDF of
cooked common beans inhibited colon carcinogenesis at an early stage by inducing
cell cycle arrest of colon cells and morphological changes linked to apoptosis,
thus confirming previous results obtained with gene expression studies. The anticancer activity of δ-tocotrienol, a bioactive vitamin E present in whole
grain cereals, annatto beans and palm fruit, is strongly dependent on its effect
on the induction of apoptosis. δ-Tocotrienol-induced apoptosis is associated
with consistent induction in the expression of the proapoptotic protein
Bcl-2-associated X protein (Bax). The molecular mechanism by which δ-tocotrienol
regulates Bax expression is unknown. We carried out a DNA microarray study that
identified δ-tocotrienol induction of the zinc finger transcription factor EGR-1
in pancreatic cancer cells. Here, we provide evidence linking
δ-tocotrienol-induced apoptosis in pancreatic cancer cells to EGR-1 regulation
of Bax expression. Forced expression of EGR-1 induces Bax expression and
apoptosis in pancreatic cancer cells. In contrast, knockdown of
δ-tocotrienol-induced EGR-1 by small interfering RNA attenuated
δ-tocotrienol-induced Bax expression and reduced δ-tocotrienol-induced
apoptosis. Further analyses showed that de novo protein synthesis was not
required for δ-tocotrienol-induced EGR-1 expression, suggesting a direct effect
of δ-tocotrienol on EGR-1 expression. Furthermore, a chromatin
immunoprecipitation assay demonstrated that EGR-1 binds to the Bax gene
promoter. Finally, δ-tocotrienol treatment induced Bax expression and activated
EGR-1 in the pancreatic neoplastic cells of the PDX-Cre Kras genetically
engineered model of pancreatic cancer. Our study provides the first evidence for
EGR-1 as a direct target of vitamin E δ-tocotrienol, suggesting that EGR-1 may
act as a proapoptotic factor in pancreatic cancer cells via induction of Bax. A lectin exhibiting antiproliferative activity on tumor cell lines but devoid of
antifungal activity has been purified from Phaseolus vulgaris cv. Green Dragon
no. 8 seeds. The lectin was a 60 kDa dimeric protein with two 30 kDa subunits.
It was a glucosamine-specific lectin as implied from the inhibitory effect of
glucosamine on hemagglutinating activity of the lectin. The steps for isolation
of the lectin involved Affi-gel blue gel (affinity gel), Mono Q (anion
exchanger), and Superdex 75 column (size exclusion). The lectin was purified
20.8-fold from the crude extract of the beans. The purified lectin showed
antiproliferative activity on breast cancer MCF7 cell line and nasopharyngeal
cancer HONE1 and CNE2 cell lines, but a low activity on normal skin fibroblast
HSF98 cell line. The lectin was shown to induce apoptosis on HONE1 cells, as
indicated by increased phosphatidylserine externalization and mitochondrial
depolarization. It also blocked HONE1 cell division and kept the cells at the
G2/M phase of the cell cycle. A 60-kDa glucosamine binding lectin, white kidney bean lectin (WKBL), was
purified from Phaseolus vulgaris cv. white kidney beans, by application of anion
exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue
gel, and FPLC-size exclusion on Superdex 75. The anti-proliferative activity of
WKBL on HONE1 cells and HepG2 cells was stronger than the activity on MCF7 cells
and WRL68 cells (IC50 values for a 48-h treatment with WKBL on HONE1 cells: 18.8
μM; HepG2 cells: 19.7 μM; MCF7 cells: 26.9 μM; and WRL68 cells: >80 μM). The
activity could be reduced by addition of glucosamine, which occupies the binding
sites of WKBL, indicating that carbohydrate binding is crucial for the activity.
Annexin V-FITC and PI staining, JC-1 staining and Hoechst 33342 staining
revealed that apoptosis was induced on WKBL-treated HONE1 cells and HepG2 cells,
but not as obviously on MCF7 cells. Cell cycle analysis also showed a slight
cell cycle arrest on HONE1 cells after WKBL treatment. Western blotting
suggested that WKBL induced apoptosis of HONE1 cells occurred through the
extrinsic apoptosis pathway, with detection of increased level of active caspase
3, 8 and 9. |
What is the function of transthyretin in cerebrospinal fluid? | Transthyretin (TTR) is a protein synthesized in the choroid plexus, which forms the blood-cerebrospinal fluid barrier. TTR is the physiological carrier of thyroxine (T4) and retinol from the blood into the cerebrospinal fluid and their distribution in the brain. The transport of T4 is important for normal development of the brain. | This paper reviews studies on the synthesis and secretion of the thyroid
hormone-binding protein, transthyretin by the choroid plexus. The secretion of
transthyretin by the choroid plexus into the cerebrospinal fluid may have an
important function in the transport of thyroxine from the blood to the brain.
The transthyretin gene is expressed in the choroid plexus of most vertebrates
and synthesis of this protein may have evolved in the brain before the liver. The transport of thyroxine from the bloodstream to the brain and the synthesis
and secretion of transthyretin (formerly called prealbumin) were studied in rats
and in sheep choroid plexus perfused in vitro. Rat choroid plexus contained 4.4
micrograms and rat liver 0.39 micrograms transthyretin mRNA per gram wet tissue.
The specific radioactivity of transthyretin isolated from cerebrospinal fluid of
rats 60 min after intravenous injection of [14C]leucine was greater than 50
times that of transthyretin from serum. After adding [14C]leucine to the
perfusion medium of an in vitro perfused sheep choroid plexus, highly
radioactive transthyretin was isolated from freshly secreted cerebrospinal fluid
collected from the exposed choroid plexus surface. Secretion of newly
synthesized transthyretin into the perfusion medium could not be demonstrated.
After intravenous injection of [125I]-thyroxine into rats, a maximum in the
curve of radioactivity in tissue plotted against time after injection was
observed first for choroid plexus, thereafter for cerebrospinal fluid, and still
later for cortex and striatum. Based on the obtained data, a hypothesis is
derived for the mechanism of the transport of thyroid hormones from the
bloodstream to the brain involving transthyretin synthesized in choroid plexus
and secreted into the cerebrospinal fluid. Transthyretin is an important choroid plexus-specific transport protein that has
been reported to be both elevated and decreased in the CSF of multiple sclerosis
patients. We report that CSF transthyretin levels are not altered in MS,
indicating that choroid plexus function with respect to this protein is
unaffected in this disease. This paper reviews knowledge on the structure and function and evolution of the
thyroid hormone binding protein transthyretin (TTR), with particular reference
to factors affecting thyroid hormone distribution and delivery to the brain. The
pool of thyroid hormones critical for the biological actions of the hormones is
the pool of free thyroid hormone. The size of this pool is determined for short
time periods by uptake/release of thyroid hormones into/from cell and
binding/release of thyroid hormones by thyroid hormone-binding proteins. Both
proportions and absolute concentrations of these proteins differ in blood plasma
and cerebrospinal fluid (CSF). The most pronounced difference is found for TTR
which is the only thyroid hormone-binding plasma protein synthesized in the
brain. TTR is also distinct from the other two thyroid hormone-binding plasma
proteins in humans by the absence of genetic deficiencies. TTR gene expression
was initiated during evolution much earlier in the brain than in the liver. The
structure of the domains of TTR involved in thyroxine (TR) T4 binding has been
completely conserved for 350 million years. These observations point to a
special functional significance of TTR in the brain. It is proposed that this is
the determination of the level of free T4 in the extracellular compartment of
the brain. T4 can then be converted in the brain to triiodothyronine T3 by
specific deiodinases. This T3 can interact with receptors in the cell nuclei,
regulating gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS) The presence and synthesis of transthyretin, a major carrier protein of
thyroxine in rat cerebrospinal fluid, was investigated in choroid plexus
epithelial cells and ependymal cells by immunocytochemistry, in situ
hybridization, and analysis by Northern and Western blot using a specific
oligonucleotide probe and a specific polyclonal antibody to transthyretin.
Choroid plexus epithelial cells expressed transthyretin at high levels in
developing rat cerebral hemispheres and in cultured cells. These cells secreted
transthyretin into the cerebrospinal fluid. In the developing rat brain
transthyretin was present in the cytoplasm of ependymal cells, in vesicles in
contact with the apical membrane and in cilia. In ependymal cell cultures this
protein was particularly abundant in the cilia of these cells. In contrast,
ependymal cells did not synthesize transthyretin. It is postulated that
transthyretin is transported to ependymal cells from the cerebrospinal fluid by
endocytosis. Transthyretin (TTR), synthesized by the choroid plexus, is proposed to have a
role in transport of thyroid hormones in the brain. Our previous studies in
animals suggest that sequestration of lead (Pb) in the choroid plexus may lead
to a marked decrease in TTR levels in the cerebrospinal fluid (CSF). The
objectives of this study were to establish in humans whether TTR and thyroxine
(T(4)) are correlated in the CSF, and whether CSF levels of Pb are associated
with those of TTR, T(4), and/or retinol-binding protein (RBP). Eighty-two paired
CSF and blood/serum samples were collected from patients undergoing clinical
diagnosis of CSF chemistry. Results showed that the mean value of CSF
concentrations for TTR was 3.33 +/- 1.60 microg/mg of CSF proteins (mean +/- SD,
n = 82), for total T(4) (TT(4)) was 1.56 +/- 1.68 ng/mg (n = 82), for RBP was
0.34 +/- 0.19 microg/mg (n = 82), and for Pb was 0.53 +/- 0.69 microg/dl (n = 61
for those above the detection limit). Linear regression analyses revealed that
CSF TTR levels were positively associated with those of CSF TT(4) (r = 0.33, p <
0.005). CSF TTR concentrations, however, were inversely associated with CSF Pb
concentrations (r = -0.29, p < 0.05). There was an inverse, albeit weak,
correlation between CSF TT(4) and CSF Pb concentrations (r = -0.22, p = 0.09).
The concentrations of TTR, TT(4), and Pb in the CSF did not vary as the function
of their levels in blood or serum, but RBP concentrations in the CSF did
correlate to those of serum (r = 0.39, p < 0.0005). Unlike TTR, CSF RBP
concentrations were not influenced by PB: These human data are consistent with
our earlier observations in animals, which suggest that TTR is required for
thyroxine transport in the CSF and that Pb exposure is likely associated with
diminished TTR levels in the CSF. Transthyretin (TTR), synthesized by the choroid plexuses (CP) has an important
role in transporting thyroxine from blood to cerebrospinal fluid (CSF). However,
the role of TTR on thyroxine transport from CSF to either blood or brain is not
clear. By using the incubated isolated ovine brain tissues technique, we found
the CP accumulated most 125I-T4 compared to ventricular ependymal, frontal
cortex or cerebellum. The accumulation was higher in the young CP than the old.
There was dose-dependent inhibition by TTR on 125I-T4 accumulation in the brain
tissues, and kinetics of T4 accumulation in presence of TTR was obtained by
plotting a double reciprocal of B (bound) versus TTR concentration curve. The KD
of 125I-T4 binding to TTR was higher in the CP compared to other tissues,
suggesting that CP competes with TTR for T4 binding to a greater extent than the
other tissues. Using the isolated perfused CP preparation, TTR significantly
inhibited 125I-T4 efflux across CP from the CSF to blood side. Bovine serum
albumin (BSA) was also able to inhibit 125I-T4 accumulation in the incubated
tissues, but required higher concentrations to reach the level of inhibition
seen with TTR. In conclusion, this study found a significant role for CSF TTR in
preventing T4 loss to blood across the CP, and TTR inhibited both CP and
selected brain tissue uptake in a dose-dependent manner. The physiological
relevance of TTR may relate to preventing T4 loss from CSF and encouraging
redistribution of hormone around the brain in CSF. Transthyretin (TTR) is a homotetrameric serum and cerebrospinal fluid protein
that transports both thyroxine (T(4)) and the retinol-retinol binding protein
complex (holoRBP). Rate-limiting tetramer dissociation and rapid monomer
misfolding and misassembly of variant TTR results in familial amyloid
polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), or familial central
nervous system amyloidosis. Analogous misfolding of wild-type TTR results in
senile systemic amyloidosis (SSA) characterized by sporadic amyloidosis in
elderly populations. With the availability of genetic and immunohistochemical
diagnostic tests, patients with TTR amyloidosis have been found in many nations
worldwide. Recent studies indicate that TTR amyloidosis is not a rare endemic
disease as previously thought. The only effective treatment for the familial TTR
amyloidoses is liver transplantation; however, this strategy has a number of
limitations, including a shortage of donors, a requirement for surgery for both
the recipient and living donor, and the high cost. Furthermore, a large number
of patients are not good transplant candidates. Recent studies focused on the
TTR gene and protein have provided insight into the pathogenesis of TTR
amyloidosis and suggested new strategies for therapeutic intervention. TTR
tetramer (native state) kinetic stabilization by small molecule binding, immune
therapy, and gene therapy with small interfering RNAs, antisense
oligonucleotides, and single-stranded oligonucleotides are promising strategies
based on our understanding of the pathogenesis of TTR amyloidosis. Among these,
native state kinetic stabilization by diflunisal and Fx-1006A, a novel
therapeutic strategy against protein misfolding diseases, are currently in Phase
II/III clinical trials. Transthyretin (TTR), a plasma and cerebrospinal fluid protein secreted by the
liver and choroid plexus, is mainly known as the physiological carrier of
thyroxine (T(4)) and retinol. Under pathological conditions, various TTR
mutations are related to familial amyloid polyneuropathy (FAP), a
neurodegenerative disorder characterized by deposition of TTR amyloid fibrils,
particularly in the peripheral nervous system (PNS), leading to axonal loss and
neuronal death. Recently, a number of TTR functions in neurobiology have been
described; these may explain the preferential TTR deposition, when mutated, in
the PNS of FAP patients. In this respect, and with a particular relevance in the
PNS, TTR has been shown to have the ability to enhance neurite outgrowth in
vitro and nerve regeneration following injury, in vivo. In the following pages,
this novel TTR function, as well as its importance in nerve biology and repair
will be discussed. The Inuit population of Nunavik (Northern Quebec, Canada) is highly exposed to
persistent organic pollutants (POPs) through their traditional diet. Some POPs,
i.e., hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs),
pentachlorophenol (PCP), and perfluorooctane sulfonate (PFOS), compete with
thyroxin (T4) for binding sites on transthyretin (TTR), a T4 transport protein
found in plasma and cerebrospinal fluid. We tested the hypothesis that these
TTR-binding compounds decrease circulating concentrations of T4 bound to TTR
(T4-TTR) in Inuit women of reproductive age. We measured the concentration of
T4-TTR in plasma samples obtained from 120 Inuit women (18-39 years old) by
combining native-polyacrylamide gel electrophoresis and liquid
chromatography-tandem mass spectrometry (LC-MS/MS) techniques. Total T4, TTR,
and thyroxin-binding globulin (TBG) concentrations were also determined, while
POPs levels had been previously measured. The mean T4-TTR concentration was 8.4
nmol/L (SD = 2.4) with values ranging from 2.9 to 14.4 nmol/L. Linear regression
analysis revealed that TTR, TBG, and total T4 concentrations were significant
predictors (p < 0.002) of T4-TTR levels (total adjusted R-squared = 0.26, p <
0.0001) but not levels of OH-PCBs, chlorophenols, or PFOS. Our results suggest
that circulating levels of these TTR-binding compounds in Inuit women of
childbearing age are not high enough to affect TTR-mediated thyroid hormone
transport. The possibility of increased delivery of these compounds to the
developing brain requires further investigation. Transthyretin (TTR) is a visceral protein, which facilitates the transport of
thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure
of TTR enables the simultaneous binding of two thyroid hormones per molecule.
Each TTR subunit provides a single cysteine residue (Cys10 ), which is
frequently affected by oxidative post-translational modifications. As Cys10 is
part of the thyroid hormone-binding channel within the TTR molecule, PTM of
Cys10 may influence the binding of thyroid hormones. Therefore, we analysed the
effects of Cys10 modification with sulphonic acid, cysteine, cysteinylglycine
and glutathione on binding of triiodothyronine (T3) by molecular modelling.
Furthermore, we determined the PTM pattern of TTR in serum of patients with
thyroid disease by immunoprecipitation and mass spectrometry to evaluate this
association in vivo. The in silico assays demonstrated that oxidative PTM of TTR
resulted in substantial reorganization of the intramolecular interactions and
also affected the binding of T3 in a chemotype- and site-specific manner with
S-glutathionylation as the most potent modulator of T3 binding. These findings
were supported by the in vivo results, which indicated thyroid function-specific
patterns of TTR with a substantial decrease in S-sulphonated,
S-cysteinylglycinated and S-glutathionylated TTR in hypothyroid patients. In
conclusion, this study provides evidence that oxidative modifications of Cys10
seem to affect binding of T3 to TTR probably because of the introduction of a
sterical hindrance and induction of conformational changes. As oxidative
modifications can be dynamically regulated, this may represent a sensitive
mechanism to adjust thyroid hormone availability. Transthyretin (TTR) is a protein that binds and distributes thyroid hormones
(THs). TTR synthesised in the liver is secreted into the bloodstream and
distributes THs around the body, whereas TTR synthesised in the choroid plexus
is involved in movement of thyroxine from the blood into the cerebrospinal fluid
and the distribution of THs in the brain. This is important because an adequate
amount of TH is required for normal development of the brain. Nevertheless,
there has been heated debate on the role of TTR synthesised by the choroid
plexus during the past 20 years. We present both sides of the debate and how
they can be reconciled by the discovery of TH transporters. New roles for TTR
have been suggested, including the promotion of neuroregeneration, protection
against neurodegeneration, and involvement in schizophrenia, behaviour, memory
and learning. Recently, TTR synthesis was revealed in neurones and peripheral
Schwann cells. Thus, the synthesis of TTR in the central nervous system (CNS) is
more extensive than previously considered and bolsters the hypothesis that TTR
may play wide roles in neurobiological function. Given the high conservation of
TTR structure, function and tissue specificity and timing of gene expression,
this implies that TTR has a fundamental role, during development and in the
adult, across vertebrates. An alarming number of 'unnatural' chemicals can bind
to TTR, thus potentially interfering with its functions in the brain. One role
of TTR is delivery of THs throughout the CNS. Reduced TH availability during
brain development results in a reduced IQ. The combination of the newly
discovered sites of TTR synthesis in the CNS, the increasing number of
neurological diseases being associated with TTR, the newly discovered functions
of TTR and the awareness of the chemicals that can interfere with TTR biology
render this a timely review on TTR in neurobiology. |
Can you define iatrogenic disease? | An iatrogenic disease is one that arises from treatment of another illness, such as an arrythmia that results from surgery or and hospital aquired infection in an immunocompromised patient. | A report of a patient with granulomatous peritonitis following general surgery
is presented. Laparoscopy was performed as the final diagnostic procedure.
Prevention is the remedy for this iatrogenic problem. A 3-year-old child diagnosed as having acute lymphatic leukemia (ALL), developed
meningeal leukemia 36 months after the onset of the disease. He was twice
subjected to cranial irradiation plus intrathecal methotrexate (i.t. MTX). Skull
radiology showed bilateral gyriform calcification of both cerebral hemispheres.
Hematological relapse was first detected 5 years after diagnosis and the child
died 5 months later. The most striking findings of a right frontal lobe biopsy
and the postmortem examination were wide calcium deposits located in the cortex
and in the adjacent white matter. Intense demyelination as well as areas of
neuron poverty were apparent in the necropsy but in the biopsy specimen. The
possible interrelationship between such deposition and cranial irradiation
and/or i.t. MTX suggests a new iatrogenic disorder. Nearly 30 years have elapsed since Rowe and Weller and their colleagues
discovered human cytomegalovirus (CMV). Because of its complex structure, long
replicative cycle, low yield in vitro, and highly species-specific
cell-substrate requirement, the cellular and molecular biologic analyses of
human CMV have been slow, but recombit DNA and monoclonal antibody
technologies are bringing about rapid changes. Because of the long period of
latency and wide range of disease presentations, epidemiologic and medical
insights have also come slowly. However, the clinical events that occur during
iatrogenic immunosuppression (transplantation and cancer therapy) and as a
result of immunocompromise due to human immunodeficiency virus infection are
currently promoting our understanding of the epidemiology of CMV disease and the
definition of its clinical spectrum. Rapid diagnostic methods, antiviral drugs,
and vaccines for CMV are becoming available. We may not yet understand
completely the impact of this agent on the nonimmunosuppressed or aspects of its
pathogenesis: e.g., the immune functions controlling recrudescence and the
possibility of increased disease severity in those with no detectable immune
defect. With the availability of new approaches, other issues should be
clarified, such as the functions of host and virus involved in the mechanism of
persistence. With the increased use of prophylactic and broad-spectrum antibiotics,
pseudomembranous colitis has emerged as a significant clinical problem.
Management with specific anti-Clostridium difficile therapy (vancomycin or
metronidazole) has reduced mortality to less than 2%. Nevertheless, the disease
may progress to a fulmit toxic colitis or colonic perforation. Additionally,
another subset of patients will present with a dramatic clinical picture,
suggesting acute peritonitis, eventuating in unnecessary laparotomy. This report
reviews both the medical and surgical literature during the past 15 years of
patients treated for pseudomembranous colitis. Analysis of this clinical data
has provided us with the opportunity to both define the role of surgery in this
disorder and illustrate the necessity for a combined medical and surgical
cooperative approach in the early management of this iatrogenic disease. We report a case of acne secondary to amineptine (Survector) in a 54-year-old
woman. Clinical picture and relationship with excessive drug intake of this drug
define this new iatrogenic acne. To our knowledge, this is the first case
described in Spain. Iatrogenic diseases are defined as illnesses due to diagnostic or therapeutic
procedures. The authors explore the possibility of recording iatrogenic diseases
by a physician self-monitoring system. The responsible physician himself records
his/her patients' iatrogenic complications on hospital discharge, after which
the authors examine retrospectively all charts of the patients involved and a
random sample of patients not included. The results show that in one year 208 of
1618 patients were recorded as having an iatrogenic disease which was severe in
88. 7.1% of these cases were not confirmed retrospectively (false positives). On
the other hand, about one third of iatrogenic illnesses were not recorded (false
negatives), though these were less severe cases only. We calculated that 17% of
our patients had iatrogenic diseases, 22% of these were predictable. About a
third of the patients had iatrogenic disease before hospital admission, and
these patients had more severe, and other, iatrogenic diseases than those who
acquired them during hospitalization. Iatrogenic complications affect other
organ systems than non-iatrogenic acquired diseases. On average, patients with
iatrogenic diseases were 6 years older and hospitalized 10 days longer than
other patients. Although not all cases may be recorded, we believe that
physician self-monitoring is preferable to other systems for recording
iatrogenic diseases, since it may have a direct teaching effect on those who
cause the illness. The authors observed one case of an iatrogenic subarachnoid-pleural fistula
secondary to the resection of an upper lobe carcinoma of the lung. The clinical
presentation was characterized by a sudden deterioration of mental status and
level of consciousness immediately after the removal of the thoracotomy chest
tube. The diagnosis was substantiated by the demonstration of pneumocephalus by
a computed tomographic scan of the head and by the identification of a left T5
nerve root fistula by a postmyelographic computed tomographic scan. The
excellent anatomical definition achieved by this modality emphasizes its
usefulness in the identification and therapeutic management of these lesions.
Operative treatment consisted of the suture ligature of the nerve root and a
chest drain. The postoperative course was uneventful, and the outcome was
excellent, with the only finding of sensory loss in the T5 nerve root territory.
A review of the literature disclosed 11 similar cases, with some differences in
the choice of the most appropriate diagnostic procedure and significant
differences in the therapeutic options, which were related to the various
mechanisms of injury. OBJECTIVES: a) To evaluate the frequency, types, severity, and morbidity of
iatrogenic complications; b) determine associated factors that favor iatrogenic
complications; and c) suggest new or more efficient protective measures that may
be taken to improve patient safety.
DESIGN: Prospective, observational study.
SETTING: Two ICUs in France.
PATIENTS AND METHODS: The study included 382 patients (age > or = 15 yrs; 400
consecutive admissions). Patients were monitored by two physicians in each ICU
to assess all iatrogenic complications occurring during their ICU stay, with the
exception of adverse effects of drugs. An iatrogenic complication was defined as
an adverse event that was independent of the patient's underlying disease.
RESULTS: We observed 316 iatrogenic complications in 124 (31%) of the 400
admissions. Of these iatrogenic complications, 107 (in 53 [13%] of the 400
admissions) complications were major, three leading to death. Severe
hypotension, respiratory distress, pneumothorax, and cardiac arrest represented
78% of the major iatrogenic complications. Fifty-nine percent of the major
iatrogenic complications had clearly identified associated factors. Human errors
accounted for 67% of these factors. Patients > 65 yrs (adjusted odds ratio =
2.6, 95% confidence interval: 1.4 to 4.9) and those patients admitted with two
or more organ failures (adjusted odds ratio = 4.8, 95% confidence interval: 2.5
to 9.2) were more likely to develop major iatrogenic complications. High or
excessive nursing workload also led to an increased risk of major iatrogenic
complications. Persistent morbidity, secondary to iatrogenic complications at
the time of discharge, was present in five survivors. The risk of ICU death was
about two-fold higher for the patients with major iatrogenic complications than
in the remaining patients after adjusting for the Organ System Failure Score and
the prognosis of the disease (relative risk = 1.92, 95% confidence interval:
1.28 to 2.56).
CONCLUSIONS: Major iatrogenic complications were frequent, associated with
increased morbidity and mortality rates, related to high or excessive nursing
workload, and were often secondary to human errors. To improve patient safety in
our ICUs, preventive measures should be targeted primarily on the elderly and
the most severely ill patients. Special attention should be given to improving
the organization of workload and training, and promoting wider use of
noninvasive monitoring. Restenosis following angioplasty is an iatrogenic disease of increasing
frequency. Restenosis may be defined in terms of either angiographic or clinical
criteria. Definitions of angiographic restenosis have varied in different
studies, accounting in part for the differences in reported restenosis rates.
Most studies now define angiographic restenosis as either a > 50% loss of
initial gain or an absolute lesion stenosis of > or = 50% at follow-up
angiogram. Common clinical end points used in defining restenosis include
recurrent angina, need for repeat revascularization, or myocardial infarction.
Despite technical advances and multiple pharmacologic interventions, most
studies have found that the incidence of angiographic restenosis remains in the
range of 40%; in none of these studies, however, was complete angiographic
follow-up obtained, and thus actual restenosis rates may be somewhat higher. In
several studies, clinical restenosis has been found to occur in approximately
36-40% of patients. Thus, a minority of patients with angiographic restenosis
have no clinical manifestations. Most patients who develop symptoms of
restenosis develop these symptoms within the first 3 months after angioplasty.
The presenting symptom in the majority of these patients is progressive
exertional angina. Patients occasionally will present with unstable angina and
only rarely with acute myocardial infarction. In patients who present with
recurrent chest pain, several features have been found to be helpful in
predicting whether they will have angiographic restenosis at follow-up
angiography. Patients who present 1-6 months after angioplasty with typical
anginal symptoms have a high likelihood of having angiographic restenosis. By
contrast, patients who present more than 6 months after percutaneous
transluminal coronary angioplasty with recurrent chest pain are more likely to
have new, significant coronary lesions to account for their symptoms.
Noninvasive testing in patients with clinical presentations suggestive of
restenosis can, in general, add only modest information in predicting whether
restenosis is indeed present. A negative exercise thallium test appears to have
a high specificity in ruling out restenosis and may be helpful in patients who
present with more atypical symptoms. Repeat angioplasty is the therapy most
frequently utilized to treat restenosis, although coronary artery bypass surgery
or medical therapy may be reasonable alternative therapies. Clinical success
rates with repeat angioplasty are > 90%, and major complications are rare;
however, restenosis will recur in a significant percentage of these patients.
Some patients who develop such recurrent restenoses will ultimately benefit from
a strategy of repeat angioplasties, although many will require surgical
revascularization. BACKGROUND: Epilepsy is a common chronic neurological disorder, usually
requiring long-term treatment with anti-epileptic drugs (AED). Many studies have
reported that AED therapy is associated with metabolic bone disease and is a
major iatrogenic risk factor for fractures. There remains uncertainty about the
type(s) of bone disease due to AED treatment, and the pathogenesis of
AED-associated fractures.
RATIONALE: Deficits in bone mineral density (BMD) are widely reported in
AED-treated patient populations. However, much of the research conducted to date
has been limited by factors such as small sample size, potentially biased
subject selection, a lack of selection of appropriate control data, and failure
to take account of important confounding influences. The pathogenesis of
AED-associated fractures is likely to be multifactorial, due to factors
including reduced BMD, impaired bone quality (due to osteoporosis and/or
osteomalacia), increased propensity to fall, and fractures associated with
seizures or loss of consciousness.
RECOMMENDATIONS: Patients receiving long-term AED should be monitored for
indices of bone health, including BMD and vitamin D status. Lifestyle factors
should be optimized, vitamin D status maintained, and fall prevention strategies
introduced as appropriate. Good seizure control is important. The use of
additional, specific osteoporosis therapy is not evidence-based in this setting,
but would appear reasonable in patients with clinically significant decreases in
BMD, applying current treatment guidelines for osteoporosis.
CONCLUSION: There is a pressing need for improved understanding of the
pathogenesis of AED-associated bone disease, for better definition of the risk
associated with specific AED regimens, and for the development of evidence-based
preventive and treatment approaches in this common but neglected disorder. BACKGROUND: Failed back surgery syndrome (FBSS) is well known and physicians
often fear this outcome. Its definition is difficult to understand because it is
multifactorial. However, we analyze its etiology in order to determine if it is
from iatrogenic causes. This syndrome can be categorized as follows: mistaken
diagnoses, transoperative error, technique error, poor application, poor
indication.
METHODS: We undertook a prospective, observational and lineal study in 20
patients, 313 surgeries and 4,500 consultations. Age and gender variables were
analyzed, number of prior surgeries, and diagnosis prior to first surgery, as
well as predomit symptoms for the last surgery, surgical time, and involved
segment. Patients were evaluated with Oswestry preoperative scale and followed
up for 2 years.
RESULTS: There were 16 females and 4 males with an average age of 53.2 years.
Eight patients had 1 prior surgery, 8 patients had 2 prior surgeries, 3 patients
had 3 prior surgeries, and 1 patient had 4 prior surgeries. According to the
Oswestry preoperative scale, 12 patients had scores higher than 60% and at
2-year follow-up, 11 patients had scores lower than 20%. Despite the persistent
symptomatology and complications, in almost all patients the satisfaction index
was 100%. According to the evaluation, the main cause was poor indication in
three patients, poor indication + technique error in 10, and technique error in
7 patients.
CONCLUSIONS: The most reported initial etiology was lumbar disc hernia with
minimally invasive treatment with questionable surgical indication. Radiological procedures require the intravascular administration of iodinated
contrast media, which are becoming a great source of an iatrogenic disease known
as contrast-induced nephropathy. The development of contrast-induced nephropathy
is associated with prolonged hospitalization, the potential need for renal
replacement therapy, and increased mortality. Despite numerous clinical and
experimental studies, several important issues regarding contrast-induced
nephropathy remain controversial. One of the controversial points is its very
definition: a universally accepted definition of contrast-induced nephropathy
does not exist. This can be a major problem. Differing definitions of
contrast-induced nephropathy and the clinical importance of these definitions
were discussed in this letter. PURPOSE: This study was designed to determine whether incidental splenectomy for
iatrogenic injury affects long-term cancer-specific survival in patients having
resection of an adenocarcinoma of the sigmoid or rectum.
METHODS: A retrospective case-matched review of patients undergoing surgery for
colorectal cancer with incidental splenectomy between January 1, 1990 and
December 31, 1999 was undertaken. Data were analysed for age, American Society
of Anesthesiologists physical status, gender, disease stage, operation type, and
outcome. These cases were matched with patients from the same center, of the
same age and gender, with the same stage of disease and operation, who did not
require a splenectomy at the time of their surgery.
RESULTS: Fifty-five patients were identified who had an iatrogenic splenectomy.
Matched gender, stage, and American Society of Anesthesiologists-matched
controls were identified. Follow-up from time of surgery to death or last
follow-up ranged from 2 to 205 (median, 43) months. A Kaplan-Meier survival
analysis using the Cox proportional hazards model to define the statistical
significance found a significant difference between the groups favoring those
without splenectomy (hazard ratio, 1.8; 95 percent confidence interval (CI),
1-3.3; P=0.0399). Cancer-specific survival at five years was 70 vs. 47 percent
and at ten years was 55 vs. 38 percent.
DISCUSSION: Patients with colorectal cancer who had splenectomy as a result of
iatrogenic damage of the spleen while undergoing resection of the sigmoid or
rectum for adenocarcinoma had a significantly worse prognosis. BACKGROUND: Iatrogenic events are increasingly recognised as an important
problem in all people admitted to hospital. However, few epidemiological data
are available for iatrogenic events in neonatal high-risk units. We aimed to
assess the incidence, nature, preventability, and severity of iatrogenic events
in a neonatal centre and to establish the association of patient characteristics
with the occurrence of iatrogenic events in neonates.
METHODS: We undertook an observational, prospective study from Jan 1, 2005, to
Sept 1, 2005, including all neonates admitted in the Division of Neonatology of
an academic, tertiary neonatal centre in southern France. Iatrogenic events were
defined as any event that compromised the safety margin for the patient, in the
presence or absence of harm. The report of an iatrogenic event was voluntary,
anonymous, and non-punitive. The primary outcome was the rate of iatrogenic
events per 1000 patient days.
FINDINGS: A total of 388 patients were studied during 10 436 patient days. We
recorded 267 iatrogenic events in 116 patients. The incidence of iatrogenic
events was 25.6 per 1000 patient days. 92 (34%) were preventable and 78 (29%)
were severe. Two iatrogenic events (1%) were fatal, but neither was preventable.
The most severe iatrogenic events were nosocomial infections (49/62 [79%]) and
respiratory events (nine of 26 [35%]). Cutaneous injuries were frequent (n=94)
but generally minor (89 [95%]), as were medication errors (15/19 [76%]). Most
medication errors occurred during administration stage (12/19 [63%]) and were
ten-fold errors (nine of 19 [47%]). The major risk factors were low birthweight
and gestational age (both p<0.0001), length of stay (p<0.0001), a central venous
line (p<0.0001), mechanical ventilation (p=0.0021), and support with continuous
positive airwary pressure (p=0.0076).
INTERPRETATION: Iatrogenic events occur frequently and are often serious in
neonates, especially in infants of low birthweight. Improved knowledge of the
incidence and characteristics of iatrogenic events, and continuous monitoring
could help to improve quality of health care for this vulnerable population. A detailed hierarchal nomenclature of arrhythmias is offered with definition of
its applications to diagnosis and complications. The conceptual and
organizational approach to discussion of arrhythmias employs the following
sequence: location--mechanism--aetiology--duration. The classification of
arrhythmias is heuristically divided into an anatomical hierarchy: atrial,
junctional, ventricular, or atrioventricular. Mechanisms are most simplistically
classified as either reentrant, such as macro-reentrant atrial tachycardia,
previously described as atrial flutter, or focal, such as automatic or
micro-reentrant tachycardia, for example, junctional ectopic tachycardia. The
aetiology of arrhythmias can be either iatrogenic, such as postsurgical, or
non-iatrogenic, such as genetic or congenital, and in many cases is
multi-factorial. Assigning an aetiology to an arrhythmia is distinct from
understanding the mechanism of the arrhythmia, yet assignment of a possible
aetiology of an arrhythmia may have important therapeutic implications in
certain clinical settings. For example, postoperative atrial arrhythmias in
patients after cardiac transplantation may be harbingers of rejection or
consequent to remediable imbalances of electrolytes. The duration, frequency of,
and time to occurrence of arrhythmia are temporal measures that further refine
arrhythmia definition, and may offer insight into ascription of aetiology.
Finally, arrhythmias do not occur in a void, but interact with other organ
systems. Arrhythmias not only can result from perturbations of other organ
systems, such as renal failure, but can produce dysfunction in other organ
systems due to haemodynamic compromise or embolic phenomena. A recent meta-analysis in this journal showed incidences between 3.4 and 33.9%.
Studies performed by pharmacists and epidemiologists produced lower incidences
than internists' studies. We reassessed the prevalence of iatrogenic admissions
in a study of internists. Iatrogenic disease was defined as adverse drug
reactions according to the World Health Organization Definition and
complications induced by non-drug medical interventions. Subsequent admissions
at the Departments of Medicine/Cardiology/Pulmonology in a 1,250 bed general
hospital in the Netherlands from May 2007 to August 2007 were studied. 2,000
consecutive admissions were studied: 576 (29%, 26-32%) were classified as
possibly iatrogenic; out of these 380 (19%, 17-22%) as definitely iatrogenic,
out of whom 229 (12%, 10-14%) had already been classified as iatrogenic by the
admitting physicians. Patients with cardiac disease, hypertension,
gastrointestinal conditions, anticoagulant treatment and use of NSAIDs were,
particularly, at risk of iatrogenic admission with percentages of 22 (16-24), 13
(11-18), 12 (9-15), and 7 (5-11) %. An independent predictor of iatrogenic
admissions was age with an odds ratio of 1.27 per 10 years (p = 0.0001). 1. At
least 19% of admissions to the Departments of Internal
Medicine/Cardiology/Pulmonology, and, maybe, even percentages up to 29% were due
to adverse drug effects. 2. A large difference between the numbers of iatrogenic
admission according to the physicians in charge of admission and the
investigators, 229 versus 380 patients, was observed. 3. Most often iatrogenic
admissions were observed with cardiac disease, hypertension, gastrointestinal
conditions, anticoagulant treatment, and use of NSAIDs. INTRODUCTION: Iatrogenic injury to the spleen is not an uncommon complication.
Left nephrectomy has been reported as the second commonest cause of iatrogenic
splenectomy with a reported incidence between 1.3 and 24%. Iatrogenic
splenectomy is associated with significant morbidity and mortality.
AIMS: We reviewed the occurrence of iatrogenic splenectomy during left
nephrectomy at our centre. Our aims were to determine the incidence of
iatrogenic splenectomy within the Mid Yorkshire Hospitals NHS Trust in order to
understand the nature of the splenic injury and the morbidity and mortality
associated with it.
METHODS: All splenectomy and nephrectomy histology reports from January 2000 to
December 2007 were reviewed retrospectively. Indications for splenectomy and
nephrectomy were identified. Patients' demographic data, tumour characteristics,
operative details, length of hospital stay and any reported morbidity or
mortality were collected.
RESULTS: A total of 447 nephrectomies were identified which included 234 left
nephrectomies. Within the same period 136 cases of splenectomy were performed.
Thirty-four cases were iatrogenic splenectomies and 12 were caused by left
nephrectomy. The incidence was 5.13%. The male to female ratio was 1:1 with an
average age of 66 years. Grade 2 and stage pT2 renal cancer were the commonest
tumour characteristics. All iatrogenic injuries occurred during mobilisation of
the colon or division of adhesion. The average operative time was 4.7 h. Average
length of hospital stay was 14 days. Five patients had postoperative
complications and 1 died of respiratory failure and sepsis.
CONCLUSION: Splenic injury during left nephrectomy is a morbid complication. A
good understanding of anatomy and surgical approach may reduce the incidence,
morbidity and mortality of iatrogenic splenectomy during left nephrectomy. Hallux valgus is a common forefoot pathology often requiring surgical
intervention for symptomatic relief. One complication of hallux valgus
correction is flexible hallux varus. Iatrogenic flexible hallux varus often
requires surgical repair; however, the most advantageous surgical procedure for
repair of iatrogenic flexible hallux varus and their sustainability remains
unclear. Therefore, we performed a systematic review to determine the
sustainability of soft-tissue release with tendon transfer for the correction of
iatrogenic flexible hallux varus. Studies were eligible for inclusion only if
they involved failure of soft-tissue release with tendon transfer for flexible
iatrogenic hallux varus. Eight studies met our inclusion criteria, seven of
which were evidence-based medicine level IV studies and one was level V. A total
of 52 patients, all female, involving 68 feet, were included. All studies
included soft-tissue release of the first metatarsal-phalangeal joint capsule
and 1 of the following procedures: Johnson transfer of the extensor hallucis
longus tendon with arthrodesis of the hallux interphalangeal joint (41 feet);
Hawkins transfer of the abductor hallucis tendon (9 feet); reverse Hawkins
transfer (7 feet); Valtin transfer of the first dorsal interosseous tendon (7
feet); and Myerson transfer of the extensor hallucis brevis tendon (4 feet). The
weighted mean age of the patients was 50.4 years, and the weighted mean
follow-up was 30.2 months. A total of 11 complications (16.2%) occurred. Of
note, only 3 cases (4.4%) of recurrent hallux varus deformity developed, all of
which occurred after Johnson transfer of the extensor hallucis longus tendon,
with arthrodesis of the hallux interphalangeal joint. Our results support that
sustainable correction of iatrogenic flexible hallux varus can be achieved with
soft-tissue release of the first metatarsal-phalangeal joint combined with a
variety of tendon transfer procedures. However, given the limited data
available, potential areas for additional prospective investigation remain. BACKGROUND: An increase in the incidence of intraoperative aortic dissection has
been reported recently, attributed to the increasingly elderly patient
population undergoing cardiac surgery and more off-pump coronary artery bypass.
We performed this study to examine current trends, identify risk factors for
iatrogenic dissection, and compare iatrogenic intraoperative aortic dissection
with spontaneous aortic dissection.
METHODS: The 15,144 consecutive patients who underwent cardiac surgery from
April 1999 to April 2011 were studied retrospectively on data collected
prospectively.
RESULTS: Iatrogenic type A aortic dissection following cardiac surgery was
diagnosed intraoperatively in 7 (0.04%) patients. Of the 4784 patients who had
off-pump coronary artery bypass, only 2 (0.04%) developed iatrogenic
intraoperative aortic dissection. Patients in the iatrogenic aortic dissection
group were older by a decade (median age 72 vs. 62 years, p = 0.01). The
cannulation site in conventional coronary artery bypass grafting and injury by
the side-biting clamp in off-pump coronary artery bypass were the most common
causes of dissection. Atheromatous disease was identified at the site of
cannulation in 5 (71.4%) of the 7 cases.
CONCLUSIONS: Intraoperative aortic dissection remains a rare and unpredictable
complication of cardiac surgery, with worse outcomes than spontaneous aortic
dissection. Increased age and atheromatous disease at the site of cannulation
are significant risk factors for iatrogenic dissection. In this series, off-pump
coronary artery bypass did not appear to be a risk factor for iatrogenic aortic
dissection. BACKGROUND: Advances in neonatology have markedly improved prognosis for
premature babies in recent years. However, they have also entailed the need for
recourse to considerable intensive care involving potentially iatrogenic
diagnostic and therapeutic acts. Among the resulting iatrogenic events,
cutaneous lesions are the most frequent but have been the subject of very few
studies. Our own study thus aimed to assess the rate of iatrogenic cutaneous
events in premature infants born at less than 33 weeks of amenorrhea and
hospitalised at Besançon university hospital and to identify the factors
associated with the occurrence of these events.
PATIENTS AND METHODS: This was a prospective study carried out in the department
of paediatric intensive care and neonatology at Besançon university hospital
between May 2011 and April 2012. All babies born before 33 weeks of amenorrhea
hospitalised over this period were included. An iatrogenic event was defined as
"an adverse event related to a medical procedure". Iatrogenic cutaneous events
were reported to the dermatologist by medical and paramedical staff.
RESULTS: One hundred and thirthteen newborn babies were included during the
study period. Twenty-six iatrogenic cutaneous events were recorded in 19
infants, representing 16.8% of the population involved: nine were associated
with ventilation techniques, six with the use of intravenous catheters, five
with electrodes, two involved pressure sores, two were linked to the birth, one
to disinfectants and one to dressings. The main risk factor was low birth weight
(P=0.016). High prematurity and the duration of ventilation increased the risk,
although not significantly. The death rate was higher in children with
iatrogenic events but the difference was not significant. The duration of
hospitalisation was unaffected by the presence or absence of an iatrogenic
event.
CONCLUSION: The frequency of iatrogenic cutaneous events is high in hospital
departments in charge of very premature infants. Awareness by the medical and
paramedical staff of the frequency of such iatrogenic events should improve the
quality of care. Iatrogenic neurologic deficits after lumbar spine surgery are rare
complications, but important to recognize and manage. Complications such as
radiculopathy, spinal cord compression, motor deficits (i.e. foot drop with L5
radiculopathy), and new onset radiculitis, while uncommon do occur. Attempts at
mitigating these complications with the use of neuromonitoring have been
successful. Guidance in the literature as to the true rate of iatrogenic
neurologic deficit is limited to several case studies and retrospective designed
studies describing the management, prevention and treatment of these deficits.
The authors review the lumbar spinal surgery literature to examine the incidence
of iatrogenic neurologic deficit in the lumbar spinal surgery literature. An
advanced MEDLINE search conducted on May 14th, 2015 from January 1, 2004 through
May 14, 2015, using the following MeSH search terms "postoperative
complications," then subterms "lumbar vertebrae," treatment outcome," "spinal
fusion," and "radiculopathy" were included together with "postoperative
complications" in a single search. Postoperative complications including
radiculopathy, weakness, and spinal cord compression were included. The
definition of iatrogenic neurologic complication was limited to post-operative
radiculopathy, motor weakness or new onset pain/radiculitis. An advanced MEDLINE
search conducted on May 14th, 2015 using all of the above terms together yielded
21 results. After careful evaluation, 11 manuscripts were excluded and 10 were
carefully reviewed. The most common indications for surgery were degenerative
spondylolisthesis, spondylosis, scoliosis, and lumbar stenosis. In 2783 patients
in 12 total studies, there were 56 patients who had reported a postoperative
neurologic deficit for a rate of 5.7. The rates of deficits ranged from 0.46% to
17% in the studies used. The average rate of reported neurologic complications
within these papers was 9% (range 0.46-24%). Thirty patients of a total of 731
(4.1%) had a new onset neurologic injury after anterior lumber interbody fusion
or lateral lumber interbody fusion. Thirty-seven out of 2052 (1.9%) patients had
a neurologic injury after posterior decompression and fusion. Screw malposition
was responsible for 11 deficits. Spinal surgery for lumbar degenerative disease
carries a low but definite rate of neurologic deficits. Despite the introduction
of neuromonitoring, these complications still occur. Interpretation of
neurologic injury rates for lumbar surgery is limited by the few prospective and
cohort-matched controlled studies. Likewise, most injuries were associated with
the placement of instrumentation despite the type of approach. BACKGROUND: Baastrup's Syndrome is a condition that occurs when there is
abnormal contact between two adjacent spinous processes resulting in back pain.
An alteration in lumbar spinal alignment and/or adjacent segment compensatory
motion is thought to be potential causative factors. The objective of this study
was to present a case series of what appears to be iatrogenic Baastrup's
Syndrome as a mid-to-late term complication following anterior lumbar interbody
surgery.
METHODS: A retrospective chart review was performed of all patients undergoing
anterior lumbar surgery for either fusion or disc replacement to determine the
prevalence of Baastrup's Syndrome.
RESULTS: Over a 12-year period, 855 patients who had undergone an anterior
approach for lumbar spine surgery were identified. Of them 8 patients with
evidence of Baastrup's Syndrome were found; this demonstrated a prevalence of
0.9%. Diagnostic injection was a helpful clinical tool in confirming the
diagnosis of iatrogenic Baastrup's Syndrome. The partial removal of the
impinging spinous processes resulted in excellent clinical relief.
CONCLUSIONS: Iatrogenic Baastrup's Syndrome may be an iatrogenic result of
anterior lumbar surgery in small group of patients. Spinous process excision is
a suggested treatment option. Further studies are necessary to explore the above
phenomenon. This study is a Level 3 retrospective case series. |
Where do the Schwann cells and melanocytes originate from? | Schwann cells and melanocytes originate from the multipotent population of neural crest cells. | The presence of melanin granules in Schwann cells of unmyelinated nerve fibres
in the normal skin of a black woman is demonstrated by electron microscopy.
Pathological conditions associated with the differentiation ability of Schwann
cells for melanogenic are reviewed. This capacity may be due to the common
origin of Schwann cells and melanocytes in the neural crest. In the vertebrate embryo, Schwann cells lining the peripheral nerves originate
from the neural crest (NC), a structure that also gives rise to ganglion
satellite cells, most of the neurons of the peripheral nervous system,
melanocytes, and part of the cranial mesenchyme. We have studied the emergence
of the Schwann cell lineage in vitro in clonal cultures of quail mesencephalic
NC cells by using the Schwann cell myelin protein antigen as an early and
specific marker for myelinating and nonmyelinating cells. After 13-16 days in
culture, numerous clones contained Schwann cell myelin protein-positive cells,
sometimes isolated and sometimes associated with other NC-derived cell types.
Detailed phenotypic analysis of the clones allowed us to infer the presence of
differently committed Schwann-cell ancestors in the NC during the migration
stage. In particular, we found evidence for the existence of a bipotent
precursor of Schwann cells and nonneuronal satellite cells; a common precursor
of neurons, satellite cells, and Schwann cells; and a pluripotent precursor of
Schwann cells, satellite cells, neurons, and melanocytes. These founder cell
types coexist in the NC with a committed Schwann cell progenitor of
high-proliferative potential that differentiates in vitro in the absence of
other peripheral cells and axons. Immunohistochemical analyses were performed on 64 nevocellular nevi (12 compound
nevi and 52 intradermal nevi). S-100 protein and its alpha- and beta-subunits
were almost always demonstrated in type A, B and C cells, and the staining
intensity tended to increase in the type C cells. Neuron-specific enolase was
detected in each type of cell; however, the population of positive cells was
smaller among type C cells. Beta 2-microglobulin was occasionally demonstrated,
but only in type A cells. Vimentin was frequently revealed in every type of
cell. Neither myelin basic protein nor glial fibrillary acidic protein was
observed in any type of cell. In contrast, normal epidermal melanocytes were
positive for vimentin, but negative for S-100 protein and its subunits and
neuron-specific enolase. Schwann cells were positive for S-100 protein and its
beta-subunit, but negative for the alpha-subunit. Thus, the nevus cells shared a
common nature with epidermal melanocytes and Schwann cells which originate from
the neural crest; however, the former cells were somewhat different from the
latter two kinds and from benign and maligt tumors derived from these cells
in the expression of these antigenic substances. Such differences in the
expression of antigenic substances may be due to dysontogenic manifestations in
nevus cells. After heterotopically grafting premigratory neural crest cells from quail
embryos to the wing buds of chicken embryos, Schwann cells, non-epidermal
melanocytes and epidermal melanocytes develop in the wings of the host embryos.
This indicates that these neural crest derived cell populations are determined
before the onset of emigration of neural crest cells from the crest. Epidermal
melanocytes of donor origin are found essentially distally from the grafts,
whereas non-epidermal melanocytes and Schwann cells are found proximally and
distally therefrom. If the apical ectodermal ridge is removed from wing buds
that have received a graft of neural crest cells, the directionality of the
migration of epidermal melanocytes is lost. This indicates that the apical
ectodermal ridge might account for the directional movement of epidermal
melanoblasts. This situation is compared to myoblasts that behave in a similar
way within the avian embryonic wing bud. Human hand anlagen of different developmental stages are studied light and
scanning electron microscopically. The findings are compared with experimental
and ultrastructural results obtained from avian limb anlagen. Shaping, cell
differentiation and the spatial arrangement of different cells are found to be
the basic processes of hand development. The shaping of the arm and hand seems
to anticipate future grasping movements. Factors controlling this developmental
process are on the one hand the apical ectodermal ridge (AER) that maintains in
the underlying mesoderm a high level of mitotic activity, and on the other hand
a species-specific pattern of cell death in different zones of arm and hand.
Interdigital cell death, microfilament bundles included in the basal compartment
of AER cells, and local anchorings of the AER ectoderm by collagen fibrils are
involved in finger separation. The flexion creases are genetically fixed and
their development cannot be explained by mechanical factors. It is found that
the early hand anlage is already composed of relatively autonomous founder cells
committed to different lineages. This is true for the muscle precursor cells
which originate from the brachial somites. These migrating somite cells are
determined to belong to the myogenic lineage. However, their distribution,
mitotic activity and later arrangement in single muscles are controlled by
factors localized within the hand itself. Tendons develop autonomously from
somatopleural cells. Other already committed cells are the angioblasts forming
the endothelial lining of the blood vessels, the neural crest cells
differentiating into melanocytes and Schwann cells, and the blood-derived cells
like chrondro- or osteoclasts. The differentiation of somatopleural cells into
cartilage, connective tissue or smooth muscle depends on their position within
the hand anlage. Possible mechanisms leading to the specific pattern are
discussed. Analysis of lineage segregation during mammalian neural crest development has
not been sufficiently performed due to technical difficulties. In the present
study, therefore, we established a clonal culture system of mouse neural crest
cells in order to analyze developmental potentials of individual neural crest
cells and their patterns of lineage segregation.
12-O-Tetradecanoylphorbol-13-acetate (TPA) and cholera toxin (CT) were applied
to culture medium to trigger melanogenic differentiation of mouse neural crest
cells. Three morphologically distinct types of clones were observed. (1)
"Pigmented clones" consisted of melanocytes only, suggesting that the
clone-forming cells were committed to the melanogenic lineage. These clones were
observed only in the presence of TPA and CT. The proportion of this type of
clone (8%) was much lower than that of the equivalent type of clone in quail
trunk neural crest (40-60%; Sieber-Blum and Cohen, 1980, Dev. Biol. 80, 96-106).
It therefore appears that the segregation pattern to the melanogenic lineage
during mouse neural crest development in vitro differs quantitatively from that
in the quail. (2) "Mixed clones" consisted of pigmented and unpigmented cells.
Like pigmented clones, they were observed only in the presence of TPA and CT.
The clones contained up to four types of cells: melanocytes, S100-positive cells
(Schwann cells or melanogenic precursor cells), serotonin (5-HT)-positive
autonomic neuron-like cells, and substance P (SP)-immunoreactive sensory
neuron-like cells. Thus, at least some mixed clone-forming cells are
pluripotent. (3) Two classes of "unpigmented clones" were observed that
consisted of unpigmented cells only. These clones developed in the presence and
absence of TPA and CT. Unpigmented clones in one class contained up to three
types of cells as well as other, as yet unidentified cells: S100-, 5-HT-, and
SP-positive cells. This observation suggests that at least some of these clones
originate from cells with a partially restricted developmental potential. Clones
in another class consisted of S100- or SP-positive cells only. These clones
might be derived from cells restricted to the SP-positive neuronal cell or
melanocyte/Schwann cell lineage. The present data indicate that at initiation of
migration, the mouse neural crest of the trunk region is a heterogeneous
population of cells containing pluripotent cells, cells with a restricted
developmental potential, and cells apparently committed to the melanogenic cell
lineage. We previously found that cultured neural crest-derived cells from embryonic
quail peripheral nerves, which consist mostly of Schwann cell precursors, gave
rise to melanocytes following treatment with basic fibroblast growth factor
(bFGF) or 12-O-tetradecanoyl phorbol-13-acetate (TPA). Here, we show that
antisense deoxyoligonucleotides targeted against two regions of the bFGF mRNA
transcript blocked this TPA-induced transdifferentiation of Schwann cell
precursors. Neither sense nor scrambled antisense control oligonucleotides had
any effect in this regard. TPA increased bFGF protein expression in cell lysates
but not in conditioned media from these cultures, and this expression was
localized to the nucleus and cytoplasm. Furthermore, bFGF-neutralizing
antibodies and inositol-hexakisphosphate (InsP6) both inhibited pigmentation
caused by exogenous bFGF, but had no affect on TPA-induced melanogenesis,
suggesting that bFGF is not released by these cells. These data indicate that
bFGF is necessary for the TPA-induced transdifferentiation of Schwann cell
precursors into melanocytes and that bFGF acts via an intracrine mechanism. Melanocytic tumors as well as multiple neurofibromas were induced in 35 of 88
Syrian golden hamsters by a single s.c. administration of 100 mg/kg of
N-nitroso-N-ethylurea applied 48 h after birth. The lesions were all observed
proliferating in the dermis and demonstrated melanosomes and premelanosomes.
High cellularity, nuclear atypia and transplantability of the tumors in outbred
hamsters suggested a maligt nature. Some of the melanomas were
morphologically heterogenous and contained Schwann-like cells as minor
components. In addition, transplantation of the melanomas resulted in increased
schwannian differentiation even for primary tumors which did not contain any
Schwann-like cell foci. One of the transplanted melanomas mimicked maligt
peripheral nervous tumor. Schwannian differentiation was also proved by the fact
that glial fibrillary acidic protein was positive in 22.2% of the cases. The
present results suggest that the induced hamster melanomas originate from neural
crest-derived cells which are able to differentiate into both melanocytes and
Schwann cells. The urodele amphibians are nearly the only adult vertebrates able to regenerate
their missing or amputated tail. The most striking feature of this model lies in
the ability of the spinal cord (SC) to differentiate, within the regenerating
tail, a new ependymal tube from which the SC and the peripheral nervous system
originate. A fundamental question is whether, in response to tail excision, the
ependymoglia of the old SC stump behaves as an embryonic neuroepithelium. To
evaluate this possibility, cell lines from primary cell cultures of adult SC
were established for the first time in newts, and two cell clones,
immunochemically characterized as ependymoglial cell populations, could be
obtained. To analyze the potentialities of these clonal cells, after
transplantation in tail regenerates, cell-marking experiments, using either in
vitro transfection with lacZ gene or the lineage tracer lysinated rhodamine
dextran (LRD), were performed. One to 2 weeks postimplantation, most of labeled
derivatives were identified as melanocytes. Interestingly, labeled cells were
also seen integrated in the ependymoglia of the regenerating SC. Two to 6 weeks
after implantation in young regenerates, we also observed LRD-labeled elongated
cells close to nerves or myofibers which were unambiguously identified as
Schwann cells by galactocerebroside staining. Taken together, these findings
showed that clonal cells derived from adult newt SC cultures could largely find,
in regenerate mesenchyme, suitable environmental conditions to differentiate
into melanocytes or Schwann cells. Because these two cells types arise from
neural crest cells during embryo-genesis, this supports the interesting view
that multipotent cells are still present in the SC of adult urodeles. The peptide endothelin 3 (EDN3) is essential for normal neural crest development
in vivo, and is a potent mitogen for quail truncal crest cells in vitro. It is
not known which subpopulations of crest cells are targets for this response,
although it has been suggested that EDN3 is selective for melanoblasts. In the
absence of cell markers for different precursor types in the quail crest, we
have characterised EDN3-responsive cell types using in vitro colony assay and
clonal analysis. Colonies were analysed for the presence of Schwann cells,
melanocytes, adrenergic cells or sensory-like cells. We provide for the first
time a description of the temporal pattern of lineage segregation in neural
crest cultures. In the absence of exogenous EDN3, crest cells proliferate and
then differentiate. Colony assay indicates that in these differentiated cultures
few undifferentiated precursors remain and there is a low replating efficiency.
By contrast, in the presence of 100 ng/ml EDN3 differentiation is inhibited and
most of the cells maintain the ability to give rise to mixed colonies and clones
containing neural crest derivatives. A high replating efficiency is maintained.
In secondary culture there was a progressive decline in the number of cell types
per colony in control medium. This loss of developmental potential was not seen
when exogenous EDN3 was present. Cell type analysis suggests two novel cellular
targets for EDN3 under these conditions. Contrary to expectations, one is a
multipotent precursor whose descendants include melanocytes, adrenergic cells
and sensory-like cells; the other can give rise to melanocytes and Schwann
cells. Our data do not support previous claims that the action of EDN3 in neural
crest culture is selective for cells in the melanocyte lineage. In this study, we have analyzed the melanogenic potential of Schwann cells using
in vitro cell cultures of embryonic quail peripheral nerves. It is shown that in
Schwann cells, two factors, 12-O-tetradecanoylphorbol-13 acetate (TPA) and
endothelin 3, trigger a differentiation pathway toward melanocytes, and that
Steel factor has no effect on these cells unless treated simultaneously with
TPA. In these cultures, TPA induces the expression of c-kit, whereas Steel
factor enhances the development of melanocytes. In the assay system we employed,
neither neuronal nor catecholaminergic phenotypes were obtained, regardless of
various combinations of related factors added to the culture medium. These data
support our previous observations indicating the existence of bipotent
progenitors that are capable of differentiating into Schwann cells or into
melanocytes, and the regulatory role of endothelin 3 on those precursors, as
revealed by the clonal culture of neural crest cells. The avian spinal cord is characterized by an absence of motor nerves and sensory
nerves and ganglia at its caudalmost part. Since peripheral sensory neurons
derive from neural crest cells, three basic mechanisms could account for this
feature: (i) the caudalmost neural tube does not generate any neural crest
cells; (ii) neural crest cells originating from the caudal part of the neural
tube cannot give rise to dorsal root ganglia or (iii) the caudal environment is
not permissive for the formation of dorsal root ganglia. To solve this problem,
we have first studied the pattern of expression of ventral (HNF3beta) and dorsal
(slug) marker genes in the caudal region of the neural tube; in a second
approach, we have recorded the emergence of neural crest cells using the HNK1
monoclonal antibody; and finally, we have analyzed the developmental potentials
of neural crest cells arising from the caudalmost part of the neural tube in
avian embryo in in vitro culture and by means of heterotopic transplantations in
vivo. We show here that neural crest cells arising from the neural tube located
at the level of somites 47-53 can differentiate both in vitro and in vivo into
melanocytes and Schwann cells but not into neurons. Furthermore, the neural tube
located caudally to the last pair of somites (i.e. the 53rd pair) does not give
rise to neural crest cells in any of the situations tested. The specific
anatomical aspect of the avian spinal cord can thus be accounted for by limited
developmental potentials of neural crest cells arising from the most caudal part
of the neural tube. Blue nevi (BN) and neurocristic hamartomas (NCH) are pigmented lesions that
originate from the aberrant development of cells derived from the neural crest.
BN are composed of dermal melanocytes, whereas NCH have a broader histology and
show Schwann cell differentiation as well. In addition, BN and NCH might differ
in their potential to develop into a maligcy. We describe a patient with a
lesion showing features of both a plaque-type BN and NCH. In the vertebrate embryo, multiple cell types originate from a common structure,
the neural crest (NC), which forms at the dorsal tips of the neural epithelium.
The NC gives rise to migratory cells that colonise a wide range of embryonic
tissues and later differentiate into neurones and glial cells of the peripheral
nervous system (PNS), pigment cells (melanocytes) in the skin and endocrine
cells in the adrenal and thyroid glands. In the head and the neck, the NC also
yields mesenchymal cells that form craniofacial cartilages, bones, dermis,
adipose tissue, and vascular smooth muscle cells. The NC is therefore a model
system to study cell diversification during embryogenesis and phenotype
maintece in the adult. By analysing the developmental potentials of quail NC
cells in clonal cultures, we have shown that the migratory NC is a collection of
heterogeneous progenitors, including various types of intermediate precursors
and highly multipotent cells, some of which being endowed of self-renewal
capacity. We also have identified common progenitors for mesenchymal derivatives
and neural/melanocytic cells in the cephalic NC. These results are consistent
with a hierarchical model of lineage segregation wherein environmental cytokines
control the fate of progenitors and stem cells. One of these cytokines, the
endothelin3 peptide, promotes the survival, proliferation, and self-renewal
capacity of common progenitors for glial cells and melanocytes. At
post-migratory stages, when they have already differentiated, NC-derived cells
exhibit phenotypic plasticity. Epidermal pigment cells and Schwann cells from
peripheral nerves in single-cell culture are able to reverse into multipotent
NC-like progenitors endowed with self-renewal. Therefore, stem cell properties
are expressed by a variety of NC progenitors and can be re-acquired by
differentiated cells of NC origin, suggesting potential function for repair. Mature cystic teratomas have been widely studied relative to their tissue
components derived from all 3 embryonic layers, and immunohistochemical methods
have demonstrated a variety of neurohormonal polypeptides. To our knowledge,
Langerhans cells (LCs), which are a peculiar component of epidermis, have not
been reported in ovarian teratomas. The origin of these cells is still a matter
of debate, ranging from bone marrow stem cells to neural elements. Thirty mature
teratomas of the ovary were studied by immunohistochemistry using CD1 (specific
against dendritic LCs), S100 protein (against LCs and melanocytes), and melan-A
and HMB45 (against melanocytes). Furthermore, antibodies for identifying subsets
of lymphocytes and monocytes (CD3, CD4, CD8, CD20, and CD68) were used.
Histologic examination showed teratomas with the presence of all 3 embryonic
layers in variable proportions in 23 cases, while 7 teratomas were composed only
of epidermis without appendages or other tissues. Immunohistochemistry
identified LCs among the suprabasal layers of epidermis in the same sites at
which melanocytes were seen in the basal layer. CD1-positive LCs sometimes
appeared to cross the basal membrane and penetrate the subepidermal tissue
(related to their known migratory ability), and they were associated there with
T-cell line lymphocytes (CD3 positive). These findings were commonly observed in
teratomas that included all 3 embryonic layers and neural tissues. Notably, LCs
and melanocytes were undetectable in the 7 teratomas composed of epidermis only.
Our observations of LCs in ovarian teratomas led us to consider these cells to
be derived from neural cells, possibly related to Schwann cells, in accord with
the original description by Langerhans. In fact, LCs are always associated with
melanocytes, which are universally considered to be derived from the neural
crest, as are Schwann cells and peripheral nerves. Finally, we propose that LCs
may be part of a cytoimmunologic system related to the T-cell compartment, with
a stem cell derived from the neural crest. S100 protein is a sensitive marker for melanomas and peripheral nerve sheath
tumors. It is, however, expressed by other mesenchymal and epithelial tumors.
Despite its low specificity, S100 protein is valuable for the diagnosis of
desmoplastic melanomas and peripheral nerve sheath tumors, for which no specific
marker is available. Sox10 is a neural crest transcription factor crucial for
specification, maturation, and maintece of Schwann cells and melanocytes.
Anti-Sox10 antibody was applied to a variety of neural crest-derived tumors,
mesenchymal and epithelial neoplasms, and normal tissues. Sox10 nuclear
expression was found in 76 of 78 melanomas (97%) and 38 of 77 maligt
peripheral nerve sheath tumors (49%) whereas S100 protein was expressed in 71
melanomas (91%) and 23 maligt peripheral nerve sheath tumors (30%). Sox10 was
diffusely expressed in schwannomas and neurofibromas. Sox10 reaction was seen
only in sustentacular cells of pheochromocytomas/paragangliomas, and
occasionally carcinoid tumors from various organs, but it was not seen in the
tumor cells. In normal tissues, Sox10 was expressed in Schwann cells,
melanocytes, and myoepithelial cells of salivary, bronchial, and mammary glands.
Sox10 reaction was not identified in any other mesenchymal and epithelial tumors
except for myoepitheliomas and diffuse astrocytomas. Sox10 was expressed by
metastatic melanomas and nodal capsular nevus in sentinel lymph nodes, but not
by other lymph node components such as dendritic cells. Our results indicate
that Sox10 will serve as a more sensitive and specific marker for the diagnosis
of melanocytic and schwannian tumors than S100 protein. Neural crest cells are the primary innovation that led to evolution of the
vertebrates, and transcription factors of the SoxE family (Sox8, Sox9 and Sox10)
are among the central players regulating the development of these cells. In all
vertebrates examined to date, one or more SoxE proteins are required for the
formation of neural crest cells, the maintece of their multipotency, and
their survival. Later, SoxE proteins drive the formation of multiple neural
crest derivatives including chondrocytes, melanocytes, and cells of the
peripheral nervous system, particularly Schwann cells/peripheral glia. Given
their multiple diverse roles in the development of the neural crest, it is
important to understand how the activity of SoxE factors is controlled such that
they direct the correct developmental outcome. While combinatorial control with
other regulatory factors is clearly one mechanism for generating such functional
versatility, modulation of SoxE activity, both by SoxD family factors and by
post-translational modification, also appears to be important. Elucidating the
mechanisms that control SoxE function is essential to understand the
evolutionary origin of the vertebrates, as well as a host of SoxE-linked
syndromes and diseases, and may prove crucial for developing stem cell based
therapies that target SoxE-regulated cell types. Melanocytes and Schwann cells are derived from the multipotent population of
neural crest cells. Although both cell types were thought to be generated
through completely distinct pathways and molecular processes, a recent study has
revealed that these different cell types are intimately interconnected far
beyond previously postulated limits in that they share a common post-neural
crest progenitor, i.e. the Schwann cell precursor. This finding raises
interesting questions about the lineage relationships of hitherto unrelated cell
types such as melanocytes and Schwann cells, and may provide clinical insights
into mechanisms of pigmentation disorders and for cancer involving Schwann cells
and melanocytes. Melanocytes, the pigmented cells of the skin, and the glial Schwann cells lining
peripheral nerves are developmentally derived from an early and transient
ectodermal structure of the vertebrate embryo, the neural crest, which is also
at the origin of multiple neural and non-neural cell types. Besides melanocytes
and neural cells of the peripheral nervous system, the neural crest cells give
rise to mesenchymal cell types in the head, which form most of the craniofacial
skeleton, dermis, fat tissue and vascular musculo-connective components. How
such a wide diversity of differentiation fates is established during
embryogenesis and is later maintained in adult tissues are among key questions
in developmental and stem cell biology. The analysis of the developmental
potentials of single neural crest cells cultured in vitro led to characterizing
multipotent stem/progenitor cells as well as more restricted precursors in the
early neural crest of avian and mammalian embryos. Data support a hierarchical
model of the diversification of neural crest lineages through progressive
restrictions of multipotent stem cell potentials driven by local environmental
factors. In particular, melanocytes and glial Schwann cells were shown to arise
from a common bipotent progenitor, which depends upon the peptide endothelin-3
for proliferation and self-renewal ability. In vivo, signaling by endothelin-3
and its receptor is also required for the early development of melanocytes and
proper pigmentation of the vertebrate body. It is generally assumed that, after
lineage specification and terminal differentiation, specialized cell types, like
the melanocytes and Schwann cells, do not change their identity. However, this
classic notion that somatic cell differentiation is a stable and irreversible
process has been challenged by emerging evidence that dedifferentiation can
occur in different biological systems through nuclear transfer, cell fusion,
epigenetic modifications and ectopic gene expression. This review considers the
issue of whether neural crest-derived lineages are endowed with some phenotypic
plasticity. Emphasis is put on the ability of pigment cells and Schwann cells to
dedifferentiate and reprogram their fate in vitro. To address this question, we
have studied the clonal progeny of differentiated Schwann cells and melanocytes
after their isolation from the sciatic nerve and the back skin of quail embryos,
respectively. When stimulated to proliferate in vitro in the presence of
endothelin-3, both cell types were able to dedifferentiate and produce
alternative neural crest-derived cell lineages. Individual Schwann cells
isolated by FACS, using a glial-specific surface marker, gave rise in culture to
pigment cells and myofibroblasts/smooth muscle cells. Treatment of the cultures
with endothelin-3 was required for Schwann cell conversion into melanocytes,
which involved acquisition of multipotency. Moreover, Schwann cell plasticity
could also be induced in vivo: following transplantation into the branchial arch
of a young chick host embryo, dedifferentiating Schwann cells were able to
integrate the forming head structures of the host and, specifically, to
contribute smooth muscle cells to the wall of cranial blood vessels. We also
analyzed the in vitro behavior of individual pigment cells obtained by
microdissection and enzymatic treatment of quail epidermis at embryonic and
hatching stages. In single cell cultures treated with endothelin-3, pigment
cells strongly proliferated while rapidly dedifferentiating into unpigmented
cells, leading to the formation of large colonies that comprised glial cells and
myofibroblasts in addition to melanocytes. By serially subcloning these primary
colonies, we could efficiently propagate a bipotent glial-melanocytic precursor
that is generated in the progeny of the melanocytic founder. These data
therefore suggest that pigment cells have the ability to revert back to the
state of self-renewing neural crest-like progenitors. Altogether, these studies
have shown that Schwann cells and pigment cells display an unstable status of
differentiation, which can be disclosed if these differentiated cells are
displaced out of their native tissue. When challenged with new environmental
conditions in vitro, differentiated Schwann cells and pigment cells can
reacquire stem cell properties of their neural crest ancestors. Notably, such
reprogramming was achieved through the effect of a single exogenous factor and
without the need of any induced genetic modification. Deciphering the cellular
and molecular mechanisms that regulate the plasticity and maintece of neural
crest-derived differentiated cells is likely to be an important step towards the
understanding of the neurocristopathies and cancers that target neural crest
derivatives in humans. During the process of development, neural crest cells migrate out from their
niche between the newly formed ectoderm and the neural tube. Thereafter, they
give rise not only to ectodermal cell types, but also to mesodermal cell types.
Cell types with neural crest ancestry consequently comprise a number of
specialized varieties, such as ectodermal neurons, melanocytes and Schwann
cells, as well as mesodermal osteoblasts, adipocytes and smooth muscle cells.
Numerous recent studies suggest that stem cells with a neural crest origin
persist into adulthood, especially within the mammalian craniofacial
compartment. This review discusses the sources of adult neural crest-derived
stem cells (NCSCs) derived from the cranium, as well as their differentiation
potential and expression of key stem cell markers. Furthermore, the expression
of marker genes associated with embryonic stem cells and the issue of multi-
versus pluripotency of adult NCSCs is reviewed. Stringent tests are proposed,
which, if performed, are anticipated to clarify the issue of adult NCSC potency.
Finally, current pre-clinical and clinical data are discussed in light of the
clinical impact of adult NCSCs. Melanocytes are pigment-producing cells generated from neural crest cells (NCCs)
that delaminate from the dorsal neural tube. The widely accepted premise that
NCCs migrating along the dorsolateral pathway are the main source of melanocytes
in the skin was recently challenged by the finding that Schwann cell precursors
are the major cellular source of melanocytes in the skin. Still, in a wide
variety of vertebrate embryos, melanocytes are exclusively derived from NCCs. In
this study, we show that a NCC population that is not derived from Sox1(+)
dorsal neuroepithelial cells but are derived from Sox1(-) cells differentiate
into a significant population of melanocytes in the skin of mice. Later, these
Sox1(-) cells clearly segregate from cells that originated from Sox1(+) dorsal
neuroepithelial cell-derived NCCs. The possible derivation of Sox1(-) cells from
epidermal cells also strengthens their non-neuroepithelial origin. Melanocytes are pigment-producing cells that reside in the skin, eyes, ears,
heart, and central nervous system meninges of mammals. Schwann cells are glial
cells, which closely associate with peripheral nerves, myelinating, and
sheathing them. Melanocytes and Schwann cells both arise from the neural crest
during development, and some melanocytes arise directly from Schwann cell
precursors lining developing spinal nerves. In this review, we explore the
connections between melanocytes and Schwann cells in development and
transformation. Skin melanocytes arise from two sources: either directly from neural crest
progenitors or indirectly from neural crest-derived Schwann cell precursors
after colonization of peripheral nerves. The relationship between these two
melanocyte populations and the factors controlling their specification remains
poorly understood. Direct lineage tracing reveals that neural crest and Schwann
cell progenitor-derived melanocytes are differentially restricted to the epaxial
and hypaxial body domains, respectively. Furthermore, although both populations
are initially part of the Foxd3 lineage, hypaxial melanocytes lose Foxd3 at late
stages upon separation from the nerve, whereas we recently found that epaxial
melanocytes segregate earlier from Foxd3-positive neural progenitors while still
residing in the dorsal neural tube. Gain- and loss-of-function experiments in
avians and mice, respectively, reveal that Foxd3 is both sufficient and
necessary for regulating the balance between melanocyte and Schwann cell
development. In addition, Foxd3 is also sufficient to regulate the switch
between neuronal and glial fates in sensory ganglia. Together, we propose that
differential fate acquisition of neural crest-derived cells depends on their
progressive segregation from the Foxd3-positive lineage. The neural crest is a transient migratory multipotent cell population that
originates from the neural plate border and is formed at the end of gastrulation
and during neurulation in vertebrate embryos. These cells give rise to many
different cell types of the body such as chondrocytes, smooth muscle cells,
endocrine cells, melanocytes, and cells of the peripheral nervous system
including different subtypes of neurons and peripheral glia. Acquisition of
lineage-specific markers occurs before or during migration and/or at final
destination. What are the mechanisms that direct specification of neural crest
cells into a specific lineage and how do neural crest cells decide on a specific
migration route? Those are fascinating and complex questions that have existed
for decades and are still in the research focus of developmental biologists.
This review discusses transcriptional events and regulations occurring in neural
crest cells and derived lineages, which control specification of peripheral
glia, namely Schwann cell precursors that interact with peripheral axons and
further differentiate into myelinating or nonmyelinating Schwann cells,
satellite cells that remain tightly associated with neuronal cell bodies in
sensory and autonomous ganglia, and olfactory ensheathing cells that wrap
olfactory axons, both at the periphery in the olfactory mucosa and in the
central nervous system in the olfactory bulb. Markers of the different
peripheral glia lineages including intermediate multipotent cells such as
boundary cap cells, as well as the functions of these specific markers, are also
reviewed. Enteric ganglia, another type of peripheral glia, will not be
discussed in this review. GLIA 2015;63:1883-1896. |
Please list 3 diseases associated with the PIEZO2 gene. | Recently, Gordon syndrome has been associated to heterozygous mutations in the piezo-type mechanosensitive ion channel component 2 gene (PIEZO2). Different mutations of this gene also cause distal arthrogryposis type 5 and Marden-Walker syndrome. | Mechanotransduction, the pathway by which mechanical forces are translated to
biological signals, plays important but poorly characterized roles in
physiology. PIEZOs are recently identified, widely expressed, mechanically
activated ion channels that are hypothesized to play a role in
mechanotransduction in mammals. Here, we describe two distinct PIEZO2 mutations
in patients with a subtype of Distal Arthrogryposis Type 5 characterized by
generalized autosomal domit contractures with limited eye movements,
restrictive lung disease, and variable absence of cruciate knee ligaments.
Electrophysiological studies reveal that the two PIEZO2 mutations affect
biophysical properties related to channel inactivation: both E2727del and I802F
mutations cause the PIEZO2-dependent, mechanically activated currents to recover
faster from inactivation, while E2727del also causes a slowing of inactivation.
Both types of changes in kinetics result in increased channel activity in
response to a given mechanical stimulus, suggesting that Distal Arthrogryposis
Type 5 can be caused by gain-of-function mutations in PIEZO2. We further show
that overexpression of mutated PIEZO2 cDNAs does not cause constitutive activity
or toxicity to cells, indicating that the observed phenotype is likely due to a
mechanotransduction defect. Our studies identify a type of channelopathy and
link the dysfunction of mechanically activated ion channels to developmental
malformations and joint contractures. Author information:
(1)Department of Pediatrics, University of Washington, Seattle, WA 98195, USA.
(2)Department of Pediatrics, University of Washington, Seattle, WA 98195, USA;
Division of Genetic Medicine, Seattle Children's Hospital, Seattle, WA 98105,
USA.
(3)Genetic Unit, Hospital Dr. Luis Calvo Mackenna, Santiago 7500539, Chile;
Division of Pediatrics, Pontificia Universidad Católica de Chile, Santiago
8330074, Chile.
(4)Departments of Pediatrics and Genetics, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599, USA.
(5)Service de Pédiatrie, Hôpital Jean Verdier, Assistance Publique - Hôpitaux de
Paris, Bondy 93143, France.
(6)Department of Pediatrics, University of Utah, Salt Lake City, UT 84108, USA.
(7)Department of Pediatrics, University of New Mexico, Albuquerque, NM 87131,
USA.
(8)Manchester Academic Health Science Centre and University of Manchester,
Manchester M13 9NT, UK.
(9)Genetic Medicine Central California, University of California, San Francisco,
Fresno, CA 93701, USA.
(10)Centre for Human Genetics, University Hospitals KU Leuven, 3000 Leuven,
Belgium.
(11)Greenwood Genetic Center, Greenwood, SC 29646, USA.
(12)Department of Clinical Genetics, Alder Hey Children's Hospital, Liverpool
L12 2AP, UK.
(13)Genetic Health Service New Zealand, Christchurch Hospital, Christchurch
8140, New Zealand.
(14)Genetics and Molecular Medicine, Dipartimento di Scieze della Salute,
University of Florence, Florence 50132, Italy.
(15)Division of Clinical Genetics and Dysmorphology, Cedars-Sinai Medical
Center, Los Angeles, CA 90048, USA.
(16)Departments of Medical Genetics and Pediatrics, University of British
Columbia and BC Children's Hospital, Vancouver, BC V6H 3N1, Canada.
(17)Department of Pediatrics, University of Texas Medical School, Houston, TX
77030, USA.
(18)North East Thames Regional Genetic Service, Great Ormond Street Hospital,
London WC1N 3BH, UK.
(19)Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital,
University of Oxford, Oxford OX3 9DU, UK.
(20)Department of Clinical Genetics, School for Oncology and Developmental
Biology, Maastricht UMC+, Maastricht 6229 GR, the Netherlands.
(21)Princess Elisabeth Children's Hospital, Ghent University Hospital, 9000
Ghent, Belgium.
(22)Department of Medical Genetics, Sydney Children's Hospital, Sydney, NSW
2031, Australia; School of Women's and Children's Health, UNSW Medicine,
University of New South Wales, Sydney, NSW 2052, Australia.
(23)National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N
3BG, UK.
(24)Department of Women's and Children's Health, University of Otago, Dunedin
9054, New Zealand.
(25)Division of Human Genetics, Cincinnati Children's Hospital Medical Center,
Cincinnati, OH 45229, USA.
(26)Department of Pediatrics, University of Hawai'i John A. Burns School of
Medicine, Honolulu, HI 96826, USA.
(27)Department of Neurology, University of California, San Francisco, San
Francisco, CA 94143, USA.
(28)Northern Genetics Service, Institute of Genetic Medicine, Newcastle upon
Tyne NE1 3BZ, UK.
(29)Department of Clinical Genetics, Churchill Hospital, Oxford University
Hospitals NHS Trust, Oxford OX3 7LJ, UK.
(30)Department of Medical Genetics, Faculty of Medicine, Uludag University,
Bursa 16059, Turkey; Department of Histology & Embryology, Faculty of Medicine,
Uludag University, Bursa 16059, Turkey; Department of Histology & Embryology,
Faculty of Medicine, Near East University, TRNC Mersin 10, Turkey.
(31)Department of Medical and Molecular Genetics, Indiana University School of
Medicine, Indianapolis, IN 46202, USA.
(32)Department of Clinical Genetics, Southern General Hospital, Glasgow G51 4TF,
UK.
(33)Genomic Medicine Institute, Geisinger Health System, Danville, PA 17822,
USA.
(34)Division of Genetic Medicine, Seattle Children's Hospital, Seattle, WA
98105, USA; Treuman Katz Center for Pediatric Bioethics, Seattle Children's
Research Institute, Seattle, WA 98101, USA.
(35)Department of Genome Sciences, University of Washington, Seattle, WA 98195,
USA.
(36)Department of Pediatrics, University of Washington, Seattle, WA 98195, USA;
Division of Genetic Medicine, Seattle Children's Hospital, Seattle, WA 98105,
USA; Department of Genome Sciences, University of Washington, Seattle, WA 98195,
USA. Electronic address: [email protected]. Domit mutations in PIEZO2, which codes for the principal mechanotransduction
channel for proprioception and touch sensation, have been found to cause
different forms of distal arthrogryposis. Some observations suggest that these
domit mutations induce a gain-of-function effect on the channel. Here, we
report a consanguineous family with three siblings who showed short stature,
scoliosis, gross motor impairment, and a progressive form of contractures
involving the distal joints that is distinct from that found in patients with
domit mutations in PIEZO2. These siblings also displayed deficits in
proprioception and touch sensation. Whole-exome sequencing performed in the
three affected siblings revealed the presence of a rare homozygous variant
(c.2708C>G; p.S903*) in PIEZO2. This variant is predicted to disrupt PIEZO2
function by abolishing the pore domain. Sanger sequencing confirmed that all
three siblings are homozygous whereas their parents and an unaffected sibling
are heterozygous for this variant. Recessive mutations in PIEZO2 thus appear to
cause a progressive phenotype that overlaps with, while being mostly distinct
from that associated with domit mutations in the same gene. Gordon syndrome or distal arthrogryposis type 3 is a rare autosomal domit
disorder characterized by contractures of upper and lower limbs. It is
distinguishable from other forms of distal arthrogryposis by cleft palate and
short stature. Recently, Gordon syndrome has been associated to heterozygous
mutations in the piezo-type mechanosensitive ion channel component 2 gene
(PIEZO2). Different mutations of this gene also cause distal arthrogryposis type
5 and Marden-Walker syndrome. Dysfunction of this ion channel provides
pleiotropic effects on joints, ocular muscles, and bone development. Here, we
present a family with three affected individuals exhibiting multiple
contractures (metacarpo-phalangeal and interphalangeal joints as well as elbow,
shoulder, knee, and ankle joints), clubfeet, short stature, bifid uvula/cleft
palate, and a distinct facial phenotype including ptosis. In addition, mild
intellectual disability and delay in psychomotor development are obvious. The
multigenerational phenotypic spectrum of Gordon syndrome is present in the
37-year-old father, his 4-year-old son and a male neonate showing typical signs
of arthrogryposis in the prenatal ultrasound examination already seen at 13 week
of gestation. In all affected family members, we identified the PIEZO2 mutation
c.8057G>A (p.Arg2686His) by Sanger sequencing. Our analysis indicated that mild
delay in psychomotor development and intellectual disability could be part of
the phenotypic spectrum of Gordon syndrome. © 2016 Wiley Periodicals, Inc. We report ten individuals of four independent consanguineous families from
Turkey, India, Libya, and Pakistan with a variable clinical phenotype that
comprises arthrogryposis, spontaneously resolving respiratory insufficiency at
birth, muscular atrophy predomitly of the distal lower limbs, scoliosis, and
mild distal sensory involvement. Using whole-exome sequencing, SNPchip-based
linkage analysis, DNA microarray, and Sanger sequencing, we identified three
independent homozygous frameshift mutations and a homozygous deletion of two
exons in PIEZO2 that segregated in all affected individuals of the respective
family. The mutations are localized in the N-terminal and central region of the
gene, leading to nonsense-mediated transcript decay and consequently to lack of
PIEZO2 protein. In contrast, heterozygous gain-of-function missense mutations,
mainly localized at the C terminus, cause domit distal arthrogryposis 3
(DA3), distal arthrogryposis 5 (DA5), or Marden-Walker syndrome (MWKS), which
encompass contractures of hands and feet, scoliosis, ophthalmoplegia, and
ptosis. PIEZO2 encodes a mechanosensitive ion channel that plays a major role in
light-touch mechanosensation and has recently been identified as the principal
mechanotransduction channel for proprioception. Mice ubiquitously depleted of
PIEZO2 are postnatally lethal. However, individuals lacking PIEZO2 develop a not
life-threatening, slowly progressive disorder, which is likely due to loss of
PIEZO2 protein in afferent neurons leading to disturbed proprioception causing
aberrant muscle development and function. Here we report a recessively inherited
PIEZO2-related disease and demonstrate that depending on the type of mutation
and the mode of inheritance, PIEZO2 causes clinically distinguishable
phenotypes. |
Is treatment resistant depression related to vitamin B9? | Treatment with SAMe, as well as with various formulations of folates and analogues, appears to be efficacious and well tolerated in reducing depressive symptoms in treatment resistant depression. | BACKGROUND: Although major depressive disorder (MDD) is a treatable disease, the
remission rates associated with antidepressant monotherapy are still far from
optimal. Folate is an inexpensive, easily tolerated natural augmenting agent,
which has been reported to improve medication treatment outcomes in patients
with MDD.
OBJECTIVE: The aim of this study was to review the literature on the clinical
utility of folate augmentation for patients with MDD. FOLATE AND DEPRESSION:
Patients with depression have consistently been found to have lower levels of
serum and red blood cell folate than normal or nondepressed psychiatric
patients. Decreased folate levels have been associated with lowered response
rates to standard antidepressant pharmacotherapy. Recent studies have shown that
augmentation with a folate supplement increases medication response in both
treatment-naïve and treatment-resistant depressed patients irrespective of
whether there is folate deficiency.
CONCLUSIONS: Depressed patients with both low and normal folate levels may
benefit from augmenting a primary antidepressant medication either initially, at
the onset of treatment, or later after some degree of treatment resistance has
been recognized. Interest in nonpharmaceutical supplements for treating major depressive disorder
(MDD) has increased significantly, both among patients and among clinicians
during the past decades. Despite the large array of antidepressants (ADs)
available, many patients continue to experience relatively modest response and
remission rates, in addition to a burden of side effects that can hinder
treatment compliance and acceptability. In this article, we review the
literature on folates and S-adenosylmethionine (SAMe), 2 natural compounds
linked in the 1-carbon cycle metabolic pathway, for which substantial evidence
supports their involvement in mood disorders. Background information, efficacy
data, proposed mechanisms of action, and side effects are reviewed. Based on
existing data, supplementation with SAMe, as well as with various formulations
of folates, appears to be efficacious and well tolerated in reducing depressive
symptoms. Compared with other forms of folates, 5-methyltetrahydrofolate
(L-methylfolate or 5-MTHF) may represent a preferable treatment option for MDD
given its greater bioavailability in patients with a genetic polymorphism, and
the lower risk of specific side effects associated with folic acid. Although
further randomized controlled trials in this area appear warranted, SAMe and
L-methylfolate may represent a useful addition to the AD armamentarium. Folate (vitamin B9) and cobalamin (vitamin B12) are essential for the normal
development and function of the central nervous system. The metabolism of these
vitamins is intimately linked and supports the synthesis of
S-adenosylmethionine (SAMe), the major methyl group donor in methylation
reactions. This article reviews the metabolic and clinical importance of folate,
vitamin B12, and SAMe, as well as clinical trials in relation to depression and
dementia. Depression in students is a major public health problem. Although several risk
factors associated with depression have been identified, the cause of depression
is still not clear. Several studies have demonstrated that physical activity and
nutrient intake, such as increased levels of B vitamins in serum, decrease
symptoms of depression. The aim of this study was to investigate the association
between physical activity and dietary intake of vitamins B₆, B₉, and B₁₂ and
symptoms of depression among postgraduate students. The results of this study
suggest that intake of vitamin B9 may modulate the total score of Center for
Epidemiological Studies Depression Scale (CES-D) and two subscales of the CES-D
including depressive affect and interpersonal difficulties. This study also
showed that moderate/high levels of physical activity were inversely and
significantly associated with symptoms of depression (total scores) and three
subscales of the CES-D including depressive affect, positive affect, and somatic
complaints. Depression is an important and often recurrent illness. An initial
antidepressant trial is effective at achieving remission for about 30 % of
patients when prescribed as monotherapy, with the majority of patients returning
as partial or non-responders. Suboptimal serum and red blood cell folate levels
have been associated with a poorer response to antidepressant therapy, a greater
severity of symptoms, later onset of clinical improvement, and overall treatment
resistance. This article reviews the evidence for L-methylfolate and folic acid
as antidepressive agents in depression and discusses their clinical use. Depression is a severe and heterogeneous disorder resulting from interacting
genetic, environmental, and epigenetic factors. Nutrient deficiency resulting
from bariatric bypass surgery has been involved in the pathophysiological
mechanisms of depression and treatment response.We report the case of a patient
who developed, after a bariatric bypass surgery, a severe depressive episode,
refractory to both pharmacological treatment and electroconvulsivotherapy (ECT).
Folate deficiency was evidenced. A dramatic response to ECT was observed after
folate supplementation. We focused on the involvement of folate in the
pathophysiological mechanisms of depression and response to both pharmacological
treatment and ECT. We emphasize on the need for close monitoring of patients
experiencing psychiatric disorder (in particular, depressive unipolar disorder)
after bariatric surgery. |
Where is the protein slitrk1 localized? | Slitrk1 is enriched in postsynaptic fractions and is localized to excitatory synapses. | Slitrks are a family of structurally related transmembrane proteins belonging to
the leucine-rich repeat (LRR) superfamily. Six family members exist (Slitrk1-6)
and all are highly expressed in the central nervous system (CNS). Slitrks have
been implicated in mediating basic neuronal processes, ranging from neurite
outgrowth and dendritic elaboration to neuronal survival. Recent studies in
humans and genetic mouse models have led to the identification of Slitrks as
candidate genes that might be involved in the development of neuropsychiatric
conditions, such as obsessive compulsive spectrum disorders and schizophrenia.
Although these system-level approaches have suggested that Slitrks play
prominent roles in CNS development, key questions remain regarding the molecular
mechanisms through which they mediate neuronal signaling and connectivity. BACKGROUND: Dynamic changes to the epigenome play a critical role in
establishing and maintaining cellular phenotype during differentiation, but
little is known about the normal methylomic differences that occur between
functionally distinct areas of the brain. We characterized intra- and
inter-individual methylomic variation across whole blood and multiple regions of
the brain from multiple donors.
RESULTS: Distinct tissue-specific patterns of DNA methylation were identified,
with a highly significant over-representation of tissue-specific differentially
methylated regions (TS-DMRs) observed at intragenic CpG islands and low CG
density promoters. A large proportion of TS-DMRs were located near genes that
are differentially expressed across brain regions. TS-DMRs were significantly
enriched near genes involved in functional pathways related to neurodevelopment
and neuronal differentiation, including BDNF, BMP4, CACNA1A, CACA1AF, EOMES,
NGFR, NUMBL, PCDH9, SLIT1, SLITRK1 and SHANK3. Although between-tissue variation
in DNA methylation was found to greatly exceed between-individual differences
within any one tissue, we found that some inter-individual variation was
reflected across brain and blood, indicating that peripheral tissues may have
some utility in epidemiological studies of complex neurobiological phenotypes.
CONCLUSIONS: This study reinforces the importance of DNA methylation in
regulating cellular phenotype across tissues, and highlights genomic patterns of
epigenetic variation across functionally distinct regions of the brain,
providing a resource for the epigenetics and neuroscience research communities. The membrane protein SLITRK1 functions as a developmentally regulated stimulator
of neurite outgrowth and variants in this gene have been implicated in Tourette
syndrome. In the current study we have cloned and characterized the porcine
SLITRK1 gene. The genomic organization of SLITRK1 lacks introns, as does its
human and mouse counterparts. RT-PCR cloning revealed two SLITRK1 transcripts: a
full-length mRNA and a transcript variant that results in a truncated protein.
The encoded SLITRK1 protein, consisting of 695 amino acids, displays a very high
homology to human SLITRK1 (99%). The porcine SLITRK1 gene is expressed
exclusively in brain tissues. Following the migration of the axonal growth cone to its target area, the
initial axo-dendritic contact needs to be transformed into a functional synapse.
This multi-step process relies on overlapping but distinct combinations of
molecules that confer synaptic identity. Slitrk molecules are transmembrane
proteins that are highly expressed in the central nervous system. We found that
two members of the Slitrk family, Slitrk1 and Slitrk2, can regulate synapse
formation between hippocampal neurons. Slitrk1 is enriched in postsynaptic
fractions and is localized to excitatory synapses. Overexpression of Slitrk1 and
Slitrk2 in hippocampal neurons increased the number of synaptic contacts on
these neurons. Furthermore, decreased expression of Slitrk1 in hippocampal
neurons led to a reduction in the number of excitatory, but not inhibitory,
synapses formed in hippocampal neuron cultures. In addition, we demonstrate that
different leucine rich repeat domains of the extracellular region of Slitrk1 are
necessary to mediate interactions with Slitrk binding partners of the LAR
receptor protein tyrosine phosphatase family, and to promote dimerization of
Slitrk1. Altogether, our results demonstrate that Slitrk family proteins
regulate synapse formation. |
Describe clinical manifestation of the Harlequin syndrome. | Harlequin syndrome is characterized by the sudden onset of unilateral facial flushing and sweating, often preceded by exercise, excessive heat, or, rarely, regional anesthesia. It is a rare autonomic disorder due to a hemifacial cutaneous sympathetic denervation. | A 45-year-old woman presented with a 10 year history of asymmetrical facial
flushing and sweating after exertion or in hot weather. During these episodes
the right side of her face remained dry and white, while the left side normally
flushed. Sweating was impaired on the left side in the limbs and trunk. She also
had areflexia in the lower limbs and slow pupillary reactions to light and
darkness, as seen in Adie's syndrome. The topography of the sweating disorder
suggested that the lesion involved the sympathetic pathways at the level of
spinal cord. The relationship with the harlequin syndrome and related disorders
is discussed. When trying to establish the likely anatomical site (preganglionic or
postganglionic) of a lesion causing congenital Horner's syndrome, the
distribution of facial flushing (the "harlequin" sign), may be seen. In babies
and young children, facial flushing is a relatively simple clinical sign to
demonstrate, compared with facial sweating. In unilateral facial flushing the
areas that do not flush are almost always identical to the anhidrotic areas.
However, neither facial flushing nor testing the pupil reactions with pholedrine
or hydroxyamphetamine can be relied on to predict the probable site of any
lesion causing congenital Horner's syndrome. Two patients with congenital
Horner's syndrome are presented which demonstrated the "harlequin" sign and in
whom clinical examination and pharmacological testing gave conflicting evidence
for localisation of the site of the causative lesion. The presentation of
congenital Horner's syndrome should be investigated and include MRI or CT to
exclude a serious underlying cause. We report a Japanese infant with Horner syndrome whose clinical examination and
testing suggested the location of the causative lesion. A 4-year-old Japanese
girl had an acquired right ocular ptosis and unequal pupils presenting shortly
after birth. She also exhibited left hemifacial flushing and loss of sweating on
the contralateral side (harlequin sign). Physical examination demonstrated 2.0
mm of ptosis of the right upper lid with normal elevator function. The diameters
of the pupils were 4 mm on the left and 2.5 mm on the right. No sweating was
induced in the right frontal region at 40 degrees C for 15 minutes of sweat
challenge test. Otherwise, no abnormalities were found by the neurophysiologic
examinations or magnetic resoce imaging of the brain. Based on the clinical
examination, we speculated that the responsible lesion might be in the
preganglionic areas. Harlequin sign was informative for making the diagnosis of
Horner syndrome. Harlequin sign and harlequin syndrome, which are used interchangeably in the
literature, are characterized by sudden onset of hemifacial sweating and
flushing, induced by exercise and heat. Hemifacial sweating and flushing with
normal ocular sympathetic innervation, known as harlequin syndrome, is rarely
associated with tonic pupils, parasympathetic oculomotor lesion and pre- or
postganglionic sudomotor sympathetic deficit. In the literature, hemifacial
sweating and flushing in patients with apparently abnormal ocular sympathetic
innervation has been defined as harlequin sign. To date, a few reports of
excessive hemifacial sweating and flushing in structural lesion have been
documented. Herein, we report five patients with excessive hemifacial sweating
and flushing, two of whom had a syrinx. In presenting the patients, we have
attempted to distinguish harlequin syndrome from harlequin sign. With this in
mind, Case 1 can be described as harlequin syndrome resembling Ross syndrome,
Case 2 as harlequin syndrome with normal ocular sympathetic innervation, Case 3
as harlequin sign with congenital Horner syndrome, Case 4 as harlequin sign with
sympathetic and parasympathetic denervation sensitivity, and Case 5 as harlequin
syndrome associated with occult sympathetic denervation sensitivity. These cases
are discussed together with a review of the literature. BACKGROUND: Harlequin syndrome is a curious phenomenon in which one half of the
face fails to flush during thermal or emotional stress as a result of damage to
vasodilator sympathetic fibers. Anecdotal reports suggest that some of these
patients have abnormal pupils. In this study we set out to systematically
investigate autonomic pupil disturbances in an unselected cohort of patients
with harlequin syndrome.
METHODS: A consecutive series of 39 patients with harlequin syndrome who were
referred to a tertiary autonomic function laboratory underwent slit-lamp
examinations, testing of deep tendon reflexes, infrared video pupillography and,
where needed, additional pharmacologic pupillary testing. Results were compared
with a meta-analysis of all previously reported cases of harlequin syndrome (n =
39) identified from a literature search.
RESULTS: In 65% of patients, no underlying causative medical disturbance could
be identified. In 64% of patients, there were abnormal pupils, most commonly
Horner syndrome, which was always present ipsilateral to the side of the face
with impaired facial sweating and flushing. The lesion was postganglionic in 9
of 10 patients tested pharmacologically. Five (13%) patients had tonic pupils,
most of whom also had tendon areflexia but no other neurologic findings, a
pattern consistent with Holmes-Adie syndrome. In 2 of these patients, tonic and
Horner pupils coexisted. Normal pupils were present in 36% of patients. These
results are similar to those for the 39 previously reported patients with
harlequin syndrome.
CONCLUSIONS: The frequent coexistence of harlequin and Horner syndromes without
other neurologic deficits suggests pathologic changes affecting the superior
cervical ganglion. Because either syndrome may occur alone, damage is apparently
selective. Among the patients with harlequin syndrome who also have tonic pupils
and tendon areflexia (Holmes-Adie syndrome), we postulate a ganglionopathy
affecting not merely the (sympathetic) superior cervical ganglion, but also the
(parasympathetic) ciliary and dorsal root ganglia. Because we found that more
than 10% of patients had an undisclosed mass lesion in the chest or neck or a
generalized autonomic neuropathy, we recommend a targeted evaluation in selected
patients with harlequin syndrome. BACKGROUND AND IMPORTANCE: Harlequin syndrome is a rare neurological condition
involving various degrees of unilateral hyperhidrosis and erythema of the head
and neck. We present a clinical presentation and description of curative therapy
in a patient with a sudden onset of Harlequin syndrome following a thoracotomy.
CLINICAL PRESENTATION: A 42-year-old female with a history of mastectomy for
right-sided breast cancer subsequently had a left partial pneumonectomy for a
metastasis. Postoperatively, she had onset of contralateral neck and facial
flushing and sweating. Flushing was triggered by emotion and exercise, but also
occurred spontaneously at random intervals. Magnetic resoce imaging of the
brain, cervical spine, and thoracic spine were negative for pathology. Because
of the patient's surgical history and negative workup, she was given a diagnosis
of Harlequin syndrome. Surgical intervention consisted of a partial right T3
costotransversectomy with T2 sympathectomy. Postoperatively, the patient's
symptoms of Harlequin syndrome resolved. The procedure was complicated by T1
radicular pain, which responded well to Gabapentin.
CONCLUSION: The diagnosis of Harlequin syndrome is relatively new, and the
majority of the scientific literature is concerned with descriptive case
presentations. We present a surgical technique for the treatment of Harlequin
syndrome. INTRODUCTION: Harlequin phenomenon is characterized by a strictly unilateral
erythrosis of the face with flushing and hyperhydrosis, and controlaterally a
pale anhydrotic aspect. This syndrome can occur alone or associated to other
dysautonomic phenomena such as Horner syndrome, Adie syndrome or Ross syndrome.
PATIENTS AND METHODS: We report three cases: two patients presented a Harlequin
sign, associated with Horner syndrome for one and Ross syndrome for the second.
The etiologic investigation was normal, allowing recognizing the idiopathic
nature of the disorder. For the third patient, Harlequin syndrome was observed
in a neoplastic context due to breast cancer, metastatic dissemination, and bone
metastases involving the right side of the T2 body.
DISCUSSION: We reviewed the literature: 108 cases have been described. This
syndrome occurred alone in 48 patients and was associated with other
dysautonomic syndromes such as Horner syndrome in 38 patients, Holmes Adie
syndrome in six, and Ross syndrome in six; both Ross and Holmes Adie syndrome
were associated five cases and associations were not reported in five patients.
The pathophysiological mechanisms of this autonomic cranial neuropathy, the
possible etiologies, and therapeutic management were discussed.
CONCLUSION: Harlequin phenomenon with flushing and unilateral hyperhydrosis is
rare, occurring alone or in combination with other autonomic syndromes of the
face. Idiopathic in two-thirds of cases, Harlequin phenomenon does not require
specific treatment; sympathectomy may be discussed in the severe cases with a
significant social impact. Harlequin syndrome is characterized by the sudden onset of unilateral facial
flushing and sweating, often preceded by exercise, excessive heat, or, rarely,
regional anesthesia. Although the exact mechanism remains unclear, it is often
referred to as transient or permanent interruption of the sympathetic nervous
system. We present a case of Harlequin syndrome without Horner syndrome in a
patient with unilateral right-sided facial flushing that started shortly after a
left-sided thoracic paravertebral nerve block for a mastectomy. We discuss the
interruption of the sympathetic and parasympathetic nervous system and the
levels of spinal nerve block associated with a thoracic paravertebral nerve
block. Author information:
(1)Department of Pediatrics, Hospital Universitario de Fuenlabrada, Madrid,
Spain. Electronic address: [email protected].
(2)Department of Pediatrics, Hospital Universitario de Fuenlabrada, Madrid,
Spain. Electronic address: [email protected].
(3)Department of Dermatology, Hospital Universitario de Fuenlabrada, Madrid,
Spain. Electronic address: [email protected].
(4)Department of Ophtalmology, Hospital Universitario de Fuenlabrada, Madrid,
Spain. Electronic address: [email protected].
(5)Department of Pediatrics, Hospital Universitario de Fuenlabrada, Madrid,
Spain. Electronic address: [email protected].
(6)Department of Pediatrics, Hospital Universitario de Fuenlabrada, Madrid,
Spain. Electronic address: [email protected].
(7)Department of Pediatrics, Hospital Clínico San Carlos, Madrid, Spain.
Electronic address: [email protected]. Harlequin syndrome is a rare, clinically striking syndrome characterized by
distinctly demarcated asymmetric facial flushing and sweating. It may be of
idiopathic aetiology or caused by demonstrable ipsilateral damage to the
sympathetic nervous system. A case is described where a patient presented to her
general dental practitioner complaining of distinctly demarcated unilateral
facial flushing and sweating. Onward referral resulted in a diagnosis of
Harlequin syndrome. CPD/CLINICAL RELEVANCE: This article highlights the
neurological signs and symptoms of Harlequin syndrome, making it easier to
recognize if it presents in general dental practice. |
What is the role of the constitutive photomorphogenesis 9 signalosome (CSN)? | The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a protein complex involved in the ubiquitin proteasome system and plays a role in regulating the degradation of polyubiquitinated proteins. The mammalian CSN is also involved in embryonic development, in cancer and the DNA damage response, whereas in plants CSN plays a role in innate immunity. | SCF ubiquitin ligases control various processes by marking regulatory proteins
for ubiquitin-dependent proteolysis. To illuminate how SCF complexes are
regulated, we sought proteins that interact with the human SCF component CUL1.
The COP9 signalosome (CSN), a suppressor of plant photomorphogenesis, associated
with multiple cullins and promoted cleavage of the ubiquitin-like protein NEDD8
from Schizosaccharomyces pombe CUL1 in vivo and in vitro. Multiple
NEDD8-modified proteins uniquely accumulated in CSN-deficient S. pombe cells. We
propose that the broad spectrum of activities previously attributed to CSN
subunits--including repression of photomorphogenesis, activation of JUN, and
activation of p27 nuclear export--underscores the importance of dynamic cycles
of NEDD8 attachment and removal in biological regulation. Angiogenesis is a prerequisite for solid tumor growth and metastasis.
Elucidation of the signaling pathways that control tumor angiogenesis
constitutes the basis for a rational antiangiogenic tumor therapy. Here we show
that the production of vascular endothelial growth factor (VEGF) in HeLa and
HL-60 cells is directed by the constitutive photomorphogenesis 9 signalosome
(CSN). The CSN is a kinase complex that cooperates with the ubiquitin/26S
proteasome system in regulating the stability of proteins involved in signal
transduction. VEGF expression is controlled by the transcription factors
activator protein (AP)-1, AP-2, SP-1, and hypoxia-inducible factor 1. Inhibition
of CSN kinase activity by 50 microM curcumin for 2 h decreases the cellular
c-Jun concentration, resulting in a reduction of the VEGF production by
approximately 75%. The removal of the inhibitor from the cells led to a
time-dependent recovery of endogenous c-Jun that is paralleled by increasing
VEGF production. Elevated cellular CSN activity induced by CSN subunit 2
overexpression causes increased VEGF production in HeLa cells. A competitor of
CSN-dependent c-Jun phosphorylation, the NH(2)-terminal c-Jun fragment
Deltac-Jun(1-226), inhibits VEGF production in HeLa cells. The transcription
factors AP-2 and SP-1 act independently of the CSN. They contribute less than a
quarter to basal VEGF production. Under our experimental conditions,
hypoxia-inducible factor 1alpha protein was not detected. Overexpression of the
tumor suppressor p53 reduces VEGF production in HeLa cells. p53 competes with
c-Jun for CSN-specific phosphorylation with the consequence of c-Jun
destabilization. We conclude that CSN-directed c-Jun signaling mediates high
VEGF production in HeLa and HL-60 cells. The data provide an explanation for the
known antiangiogenic and antitumorigenic activities of curcumin. Because the CSN
regulates the major part of VEGF production in the tested tumor cells, it
constitutes a potentially important target for tumor therapy. The COP9 signalosome (CSN) is a complex of eight proteins first identified as a
repressor of plant photomorphogenesis. A protein kinase activity associated with
the COP9 signalosome has been reported but not identified; we present evidence
for inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) as a protein kinase
associated with the COP9 signalosome. We have shown that 5/6-kinase exists in a
complex with the eight-component COP9 signalosome both when purified from bovine
brain and when transfected into HEK 293 cells. 5/6-kinase phosphorylates the
same substrates as those of the COP9 signalosome, including IkappaBalpha, p53,
and c-Jun but fails to phosphorylate several other substrates, including c-Jun
1-79, which are not substrates for the COP9-associated kinase. Both the COP9
signalosome- associated kinase and 5/6-kinase are inhibited by curcumin. The
association of 5/6-kinase with the COP9 signalosome is through an interaction
with CSN1, which immunoprecipitates with 5/6-kinase. In addition, the inositol
kinase activity of 5/6-kinase is inhibited when in a complex with CSN1. We
propose that 5/6-kinase is the previously described COP9 signalosome-associated
kinase. The COP9 signalosome (CSN) is an evolutionarily conserved multiprotein complex
that mediates the repression of photomorphogenesis in the dark in Arabidopsis
through the degradation of transcription factors such as HY5 and HYH.
CSN-mediated HY5 and HYH degradation also requires the activity of the putative
E3 ubiquitin ligase (E3) component COP1 and the E2-conjugating enzyme variant
COP10. Recently, it was shown that CSN also is required for auxin responses
mediated by the SCF-type E3 SCF(TIR1). To determine whether Arabidopsis CSN is
required for E3-mediated processes in a more general manner, we generated plants
with reduced E3 function by suppressing AtRBX1, an essential core subunit of
SCF-type E3s. We observed that AtRBX1 transgenic plants share multiple
phenotypes with CSN reduced-function plants, such as morphological defects and
reduced responses to auxin, jasmonic acid, and cold stress, suggesting that CSN
is required for multiple AtRBX1-mediated processes. Furthermore, we observed
that mutants with defects in AXR1, a protein that had been described only as a
regulator of SCF(TIR1) function, also is required for other E3-mediated
processes and for the COP1/COP10/CSN-mediated repression of photomorphogenesis
in the dark. We conclude that CSN and AXR1 are of general importance for
different pathways that are controlled by E3-mediated protein degradation. The COP9 signalosome (CSN) is a multiprotein complex that was initially
identified in plants as a repressor of photomorphogenesis. It is now known to
play major roles in several other developmental pathways, from auxin response to
flower development. Furthermore, the COP9 signalosome shares homologies with the
lid sibcomplex of the proteasome and is evolutionarily conserved from fission
yeast to humans. It is important for the proper development of virtually all
higher eukaryotes. In recent years, significant progress has been made in
unraveling the molecular, cellular, and physiological mode of action of the COP9
signalosome. This review discusses our current understanding of the COP9
signalosome function with particular emphasis on its recently defined role in
modulating a wide variety of cellular processes by regulating specific protein
degradation events. COP10 is a ubiquitin-conjugating enzyme variant (UEV), which is thought to act
together with COP1, DET1, and the COP9 signalosome (CSN) in Arabidopsis to
repress photomorphogenesis. Here, we demonstrate that COP10 interacts with
ubiquitin-conjugating enzymes (E2s) in vivo, and can enhance their activity in
vitro, an activity distinct from previous characterized UEVs such as MMS2 and
UEV1. Furthermore, we show that COP10 forms a complex with UV-damaged
DNA-binding protein 1a (DDB1a) and de-etiolated 1 (DET1), and physically
interacts with COP1 and the CSN. Purified CDD (COP10, DDB1, DET1) complex also
shows enhancement of E2 activity (UEA) similar to that observed with COP10
itself. Our data suggests that COP10, along with COP1 and the CSN, promotes the
degradation of positive regulators of photomorphogenesis, such as the
transcription factor HY5, via the ubiquitin/26S proteasome system. Thus, the CDD
complex may act as a ubiquitylation-promoting factor to regulate
photomorphogenesis. PURPOSE: The Int6 gene was originally identified as a common insertion site for
the mouse mammary tumor virus in virally induced mouse mammary tumors. Recent
studies indicate that Int6 is a multifaceted protein involved in the regulation
of protein translation and degradation through binding with three complexes: the
eukaryotic translation initiation factor 3, the proteasome regulatory lid, and
the constitutive photomorphogenesis 9 signalosome. This study aimed to
investigate the prognostic role of Int6 in a large series of stage I non-small
cell lung cancers (NSCLC) patients with long-term follow-up.
EXPERIMENTAL DESIGN: We determined the methylation status of Int6 DNA by
methylation-specific PCR and the steady-state levels of Int6 RNA by quantitative
real-time reverse transcription-PCR in 101 NSCLCs and matched normal lung
tissues.
RESULTS: In 27% of the tumors, Int6 RNA levels were reduced relative to normal
tissue. In 85% of the tumors with reduced Int6 expression, the transcription
promoter and first exon were hypermethylated, whereas only 4% of the tumors with
elevated Int6 RNA levels were hypermethylated (P <0.000001). Low levels of Int6
RNA were found a significant predictor of overall and disease-free survival
(P=0.0004 and P=0.0020, respectively). A multivariate analysis confirmed that
low Int6 expression was the only independent factor to predict poor prognosis,
for both overall (P=0.0006) and disease-free (P=0.024) survival.
CONCLUSIONS: Our results suggest that Int6 expression, evaluated by quantitative
real-time PCR, may represent a new prognostic factor in patients with stage I
NSCLC. The COP9 signalosome (CSN) is a multimeric protein complex that occurs in all
eukaryotic cells. Originally described in plants as a regulator of
photomorphogenesis, its purification and characterization from mammalian cells
revealed significant sequence homologies to subunits of the 26S proteasome lid
complex, as well as of the eukaryotic translation initiation factor 3. Recent
studies disclosed its participation in processes such as DNA repair, cell cycle
regulation, development, and angiogenesis. At the moment, the pleiotropic
effects of the CSN point to a regulatory role in the ubiquitin/26S proteasome
system, but its exact function still remains to be clarified. This chapter
describes the method to purify human CSN from red blood cells. Two outdated
erythrocyte concentrates are sufficient to prepare approximately 0.5 mg of CSN.
Washed cells are first lysed and then proteins are separated by a DEAE
anion-exchange column. The CSN-containing fractions are pooled and subjected to
an ammonium sulfate precipitation followed by dialysis. The concentrated
proteins are then loaded onto a glycerol density gradient and
ultracentrifugation is performed. The purification procedure is continued using
two succeeding anion-exchange columns, resulting in a sufficiently pure CSN
complex. Optionally, an additional density gradient centrifugation can be
attached. The purified CSN complex possesses kinase, deneddylase, and
deubiquitinase activities and can be stored for at least 2 months on ice at 4
degrees . The COP9 signalosome (CSN) plays important roles in multifaceted cellular
processes. Study has shown that inositol 1,3,4-trisphosphate 5/6 kinase (5/6
kinase) interacts with CSN in mammalian cells. However, the biological function
of the interaction still remains unknown. Here, we report that the Arabidopsis
inositol 1,3,4-trisphosphate 5/6 kinase (AtItpk-1) is also associated with CSN
and involved in photomorphogenesis under red light (RL) conditions, as
demonstrated by co-immunoprecipitation of AtItpk-1 with CSN and characterization
of the atitpk-1 mutants. Expression analysis showed that AtItpk-1 had the same
sub-cellular localization and organ expression pattern as CSN. Furthermore,
autophosphorylation analysis showed that AtItpk-1 has protein kinase activity.
Under RL, the atitpk-1 mutants exhibited phenotype slightly similar with that of
the csn mutants, indicating that 5/6 kinase might be involved in the same
developmental pathway as CSN. This study suggests that AtItpk-1 may function as
a protein kinase that is involved in photomorphogenesis possibly via interaction
with COP9 signalosome under red light. Repression of photomorphogenesis in Arabidopsis thaliana requires activity of
the COP9 signalosome (CSN), CDD, and COP1 complexes, but how these three
complexes work in concert to accomplish this important developmental switch has
remained unknown. Here, we demonstrate that Arabidopsis CULLIN4 (CUL4)
associates with the CDD complex and a common catalytic subunit to form an active
E3 ubiquitin ligase both in vivo and in vitro. The partial loss of function of
CUL4 resulted in a constitutive photomorphogenic phenotype with respect to
morphogenesis and light-regulated gene expression. Furthermore, CUL4 exhibits a
synergistic genetic interaction with COP10 and DET1. Therefore, this CUL4-based
E3 ligase is essential for the repression of photomorphogenesis. This CUL4-based
E3 ligase appears to associate physically with COP1 E3 ligase and positively
regulates the COP1-dependent degradation of photomorphogenesis-promoting
transcription factors, whereas the CSN controls the biochemical modification of
CUL4 essential for E3 activity. Thus, this study suggests a biochemical activity
connection between CSN and CDD complexes in their cooperation with COP1 in
orchestrating the repression of photomorphogenesis. Jab1 is a co-activator of activating protein-1 (AP-1) transcription factor and
the fifth subunit of the constitutive photomorphogenesis 9 (COP9) signalosome,
which has been shown to mediate nuclear exportation and ubiquitin-dependent
degradation of the tumor suppressor p27(Kip1). Jab1 is overexpressed in several
types of human cancer. However, de-regulation of Jab1 gene expression in cancer
cells is largely unclear. In this study, we reported that expression of Jab1 was
stimulated by HER-2/neu oncogene via transcriptional activation. Promoter
deletion and mutation analysis indicated that HER-2/neu stimulated Jab1 via the
T cell factor (TCF) binding site located at the -380/-368 region of the human
Jab1 promoter. DNA affinity precipitation assay and chromatin
immunoprecipitation assay verified that binding of beta-catenin and TCF-4 to
this consensus site was increased by HER-2/neu. In addition, domit-negative
mutant of TCF significantly attenuated the stimulatory effect of HER-2/neu. We
also demonstrated that HER-2/neu increased beta-catenin/TCF-mediated Jab1
expression via the AKT signaling pathway because chemical inhibitor or
domit-negative mutant of AKT effectively attenuated the stimulatory action of
HER-2/neu. IGF-I, which is a well-known AKT activator, also up-regulated the
expression of Jab1 in NIH/3T3 and MCF-7 cells. Knockdown of Jab1 by small
interfering RNA (siRNA) preferentially inhibited proliferation of
HER-2/neu-overexpressing breast cancer cells. Taken together, our results
suggest that HER-2/neu transcriptionally activates Jab1 expression to promote
proliferation of breast cancer cells. RATIONALE: Ubiquitin-proteasome system (UPS) dysfunction has been implicated in
cardiac pathogenesis. Understanding how cardiac UPS function is regulated will
facilitate delineating the pathophysiological significance of UPS dysfunction
and developing new therapeutic strategies. The COP9 (constitutive
photomorphogenesis mutant 9) signalosome (CSN) may regulate the UPS, but this
has not been tested in a critical vertebrate organ. Moreover, the role of CSN in
a postmitotic organ and the impact of cardiomyocyte-restricted UPS dysfunction
on the heart have not been reported.
OBJECTIVE: We sought to determine the role of CSN-mediated deneddylation in UPS
function and postnatal cardiac development and function.
METHODS AND RESULTS: Cardiomyocyte-restricted Csn8 gene knockout (CR-Csn8KO) in
mice was achieved using a Cre-LoxP system. CR-Csn8KO impaired CSN holocomplex
formation and cullin deneddylation and resulted in decreases in F-box proteins.
Probing with a surrogate misfolded protein revealed severe impairment of UPS
function in CR-Csn8KO hearts. Consequently, CR-Csn8KO mice developed cardiac
hypertrophy, which rapidly progressed to heart failure and premature death.
Massive cardiomyocyte necrosis rather than apoptosis appears to be the primary
cause of the heart failure. This is because (1) massive necrotic cell death and
increased infiltration of leukocytes were observed before increased apoptosis;
(2) increased apoptosis was not detectable until overt heart failure was
observed; and (3) cardiac overexpression of Bcl2 failed to ameliorate CR-Csn8KO
mouse premature death.
CONCLUSIONS: Csn8/CSN plays an essential role in cullin deneddylation,
UPS-mediated degradation of a subset of proteins, and the survival of
cardiomyocytes and, therefore, is indispensable in postnatal development and
function of the heart. Cardiomyocyte-restricted UPS malfunction can cause heart
failure. The mammalian constitutive photomorphogenesis 9 (COP9) signalosome (CSN), a
protein complex involved in embryonic development, is implicated in cell cycle
regulation and the DNA damage response. Its role in tumor development, however,
remains unclear. Here, we have shown that the COP9 subunit 6 (CSN6) gene is
amplified in human breast cancer specimens, and the CSN6 protein is upregulated
in human breast and thyroid tumors. CSN6 expression positively correlated with
expression of murine double minute 2 (MDM2), a potent negative regulator of the
p53 tumor suppressor. Expression of CSN6 appeared to prevent MDM2
autoubiquitination at lysine 364, resulting in stabilization of MDM2 and
degradation of p53. Mice in which Csn6 was deleted died early in embryogenesis
(E7.5). Embryos lacking both Csn6 and p53 survived to later in embryonic
development (E10.5), which suggests that loss of p53 could partially rescue the
effect of loss of Csn6. Mice heterozygous for Csn6 were sensitized to
γ-irradiation-induced, p53-dependent apoptosis in both the thymus and the
developing CNS. These mice were also less susceptible than wild-type mice to
γ-irradiation-induced tumorigenesis. These results suggest that loss of CSN6
enhances p53-mediated tumor suppression in vivo and that CSN6 plays an important
role in regulating DNA damage-associated apoptosis and tumorigenesis through
control of the MDM2-p53 signaling pathway. COP9 plays a role in plant innate immunity. The role of COP9 in mammalian innate
immune responses is unknown. Here, we show that the COP9 signalosome subunit 5
(CSN5) is required for activation of proinflammatory kinases p38 and Erk and for
down-regulation of the expression of genes regulated by nuclear factor
E2-related factor 2. Mice with myeloid-specific CSN5 deficiency have lower
mortality in polymicrobial sepsis. CSN5 is required for both Toll-like receptor
(TLR) and reactive oxygen species-mediated deneddylation of Cul3, which is
essential for Cul3/Keap1-mediated degradation of nuclear factor E2-related
factor 2. On the basis of our results COP9 subunit CSN5 is considered to be an
essential component of mammalian innate immunity. The COP9 signalosome (CSN) is a eukaryotic protein complex, which regulates a
wide range of biological processes mainly through modulating the cullin
ubiquitin E3 ligases in the ubiquitin-proteasome pathway. The CSN possesses a
highly conserved deneddylase activity that centers at the JAMM motif of the Csn5
subunit but requires other subunits in a complex assembly. The classic CSN is
composed of 8 subunits (Csn1-8), yet in several Ascomycota, the complex is
smaller and lacks orthologs for a few CSN subunits, but nevertheless contains a
conserved Csn5. This feature makes yeast a powerful model to determine the
minimal assemblage required for deneddylation activity. Here we report, that
Csi1, a diverged S. cerevisiae CSN subunit, displays significant homology with
the carboxyl terminal domain of the canonical Csn6, but lacks the amino terminal
MPN(-) domain. Through the comparative and experimental analyses of the budding
yeast and the mammalian CSNs, we demonstrate that the MPN(-) domain of the
canonical mouse Csn6 is not part of the CSN deneddylase core. We also show that
the carboxyl domain of Csn6 has an indispensable role in maintaining the
integrity of the CSN complex. The CSN complex assembled with the carboxyl
fragment of Csn6, despite its lack of an MPN(-) domain, is fully active in
deneddylation of cullins. We propose that the budding yeast Csi1 is a functional
equivalent of the canonical Csn6, and thus the composition of the CSN across
phyla is more conserved than hitherto appreciated. The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large
multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a
central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs).
The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1),
resides in the deneddylation of the CRLs that is the hydrolysis of the
cullin-neural precursor cell expressed developmentally downregulated gene 8
(Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase
activity, it is intrinsically inactive in other physiologically relevant forms.
Here we analyze the crystal structure of CSN5 in its catalytically inactive form
to illuminate the molecular basis for its activation state. We show that CSN5
presents a catalytic domain that brings essential elements to understand its
activity control. Although the CSN5 active site is catalytically competent and
compatible with di-isopeptide binding, the Ins-1 segment obstructs access to its
substrate-binding site, and structural rearrangements are necessary for the
Nedd8-binding pocket formation. Detailed study of CSN5 by molecular dynamics
unveils signs of flexibility and plasticity of the Ins-1 segment. These analyses
led to the identification of a molecular trigger implicated in the
active/inactive switch that is sufficient to impose on CSN5 an active
isopeptidase state. We show that a single mutation in the Ins-1 segment restores
biologically relevant deneddylase activity. This study presents detailed
insights into CSN5 regulation. Additionally, a dynamic monomer-dimer equilibrium
exists both in vitro and in vivo and may be functionally relevant. CSN6 is one subunit of the constitutive photomorphogenesis 9 (COP9) signalosome
(CSN), which is an evolutionarily conserved multiprotein complex found in plants
and animals and originally described as a repressor of light-dependent growth
and transcription in Arabidopsis. CSN is homologous to the 19S lid subcomplex of
the 26S proteasome, thus it has been postulated to be a regulator of the
ubiquitin-proteasome pathway. In mammalian cells, it consists of eight subunits
(CSN1-CSN8). Among the CSN subunits, CSN5 and CSN6 are the only two that each
contains an MPN (Mpr1p and Pad1p N-terminal) domain. The deneddylating activity
of an MPN domain toward cullin-RING ubiquitin ligases (CRL) may coordinate
CRL-mediated ubiquitination activity. More and more studies about CSN6 are
emerging, and its overexpression is found in many types of cancers. Evidence has
shown that CSN6 is a molecule platform between protein degradation and signal
transduction. Here, we provide a summary of human CSN6, especially its roles in
cancer, hoping that it can lay the groundwork for cancer prevention or therapy. The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome
system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases
conferring substrate specificity. The CSN controls a large family of Ub ligases
called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators,
transcription factors and DNA damage response proteins. The CSN possesses
structural similarities with the 26S proteasome Lid complex and the translation
initiation complex 3 (eIF3) indicating similar ancestry and function. Initial
structures were obtained 14years ago by 2D electron microscopy (EM). Recently,
first 3D molecular models of the CSN were created on the basis of negative-stain
EM and single-particle analysis, mostly with recombit complexes. Here, we
compare deneddylating activity and structural features of CSN complexes purified
in an elaborate procedure from human erythrocytes and efficiently pulled down
from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the
human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates.
3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN
were generated by negative stain EM and by cryo-EM. Both complexes show a
central U-shaped segment from which several arms emanate. This structure, called
the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away
from the horseshoe. Compared to 3D models of negatively stained CSN complexes,
densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because
biochemical and structural results obtained with CSN complexes isolated from
human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts
are very similar, Flag-CSN pulldowns are a proper alternative to CSN preparation
from erythrocytes. C-Jun activation domain-binding protein-1 (Jab1) not only is full but also a
subunit (CSN5) of the constitutive photomorphogenesis 9 signalosome (CSN), which
is an evolutionarily conserved and multifunctional protein that involves in
controlling cellular proliferation and apoptosis, affecting a series of
pathways, as well as regulating genomic instability and DNA damage and repair.
The CSN is a highly conservative protein from yeast to human and interacts with
the cullin-RING family of ubiquitin ligases so that it could be execute a
process of removing NEDD8, a ubiquitin-like polypeptide (deneddylase activity).
The role of Jab1/CSN5's multi-function has been proved as being oncogenic in
nature, what is more, Jab1/CSN5 has been confirmed by much evidence that it
participates in the carcinogenesis progression and is tightly associated with
poor prognosis. However, the biologic implication of Jab1/CSN5 activity during
the cancer's development is unclear. We performed a systematic literature review
and assessment from PubMed and Medline databases in this article. Jab1/CSN5 is
participate in a lot of biologic responses, including cell proliferation,
apoptosis, cell cycle regulation, DNA metabolism, invasion, DNA damage and
repair, and recurrence. It also promotes cell transformation and tumorigenesis.
In this review, we mainly expound the progress in the function and research
advances of Jab1/CSN5 and in untangling the Jab1/CSN5 signaling pathway. Based
on these bases, its potential as a therapeutic target for cancer can play a
greater role in future cancer treatment. CSN6, a critical subunit of the constitutive photomorphogenesis 9 (COP9)
signalosome (CSN), has received attention as a regulator of the degradation of
cancer-related proteins such as p53, c-myc and c-Jun, through the
ubiquitin-proteasome system, suggesting its importance in cancerogenesis.
However, the biological functions and molecular mechanisms of CSN6 in
glioblastoma (GBM) remain poorly understood. Here, we report that GBM tumors
overexpressed CSN6 compared with normal brain tissues and that CSN6 promoted GBM
cell proliferation, migration, invasion and tumorigenesis. Erlotinib, a
small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase
inhibitor, was used to reveal that the proliferative and metastatic effects of
CSN6 on GBM cells were EGFR dependent. We also found that CSN6 positively
regulated EGFR stability via reduced levels of EGFR ubiquitination, thereby
elevating steady expression of EGFR. In addition, this study is the first
description of a novel role for the CSN6-interacting E3 ligase, CHIP (carboxyl
terminus of heat-shock protein 70-interacting protein), regulating EGFR
ubiquitination in cancer cells. We showed that CSN6 associated with CHIP and led
to CHIP destabilization by increasing CHIP self-ubiquitination. Moreover, CSN6
decreased CHIP expression and increased EGFR expression in the tumor samples.
Deregulation of this axis promoted GBM cell's proliferation and metastasis.
Thus, our study provides insights into the applicability of using the
CSN6-CHIP-EGFR axis as a potential therapeutic target in cancer. The COP9 (constitutive photomorphogenesis 9) signalosome (CSN) is a protein
complex involved in the ubiquitin proteasome system and a common host target of
diverse pathogens in Arabidopsis. The known derubylation function of the COP9
complex is carried out by subunit 5 encoded by AtCSN5A or AtCSN5B in
Arabidopsis. A single CSN5-like gene (designated as TaCSN5) with three
homeologues was identified on the long arms of wheat (Triticum aestivum L.)
group 2 chromosomes. In this study, we identified and characterized the function
of TaCSN5 in response to infection by the leaf rust pathogen. Down-regulation of
all three TaCSN5 homeologues or mutations in the homeologues on chromosomes 2A
or 2D resulted in significantly enhanced resistance to leaf rust. Enhanced leaf
rust resistance corresponded to a seven-fold increase in PR1
(pathogenesis-related gene 1) expression. Collectively, the data indicate that
the wheat COP9 subunit 5-like gene acts as a negative regulator of wheat leaf
rust resistance. The COP9 signalosome (CSN) is an essential regulator of cullin-RING-ubiquitin
(Ub) ligases (CRLs), which ubiquitinate important cellular regulators and target
them for degradation by the Ub proteasome system (UPS). The CSN exhibits
deneddylating activity localized on subunit CSN5, which removes the
ubiquitin-like protein Nedd8 from the cullins of CRLs. CSN-mediated
deneddylation is an important step in the process of CRL remodeling, in which
new substrate recognition units are incorporated into Ub ligases to meet changed
requirements for proteolysis in cells. For instance, extensive CRL remodeling
occurs during adipogenic differentiation when new CRL3s are formed.
Diversification of CSN complexes during evolution is most likely another
adaptation to meet different cellular requirements. Best known CSN variants are
formed by different CSN subunit isoforms. For instance, in plant cells, isoforms
have been identified for the MPN-domain subunits CSN5 (CSN5A and CSN5B) and CSN6
(CSN6A and CSN6B) which form four distinct CSN variants. In mammalian cells
CSNCSN7A and CSNCSN7B variants are generated by CSN7 isoforms. We demonstrate
that the two variants coexist in human LiSa-2 cells and in mouse embryonic
fibroblasts. During adipogenic differentiation of LiSa-2 cells CSN7B increases
in parallel with an elevation of the total CSN complex. Permanent overexpression
of Flag-CSN7B but not of Flag-CSN7A accelerates adipogenesis in LiSa-2 cells
indicating a specific function of the CSNCSN7B variant in stimulating
adipogenesis. Silencing of CSN7A as well as of CSN7B in LiSa-2 cells and in
mouse embryonic fibroblasts (MEFs) reduces adipogenic differentiation
demonstrating that both CSNCSN7A and CSNCSN7B variants are involved in the
process. |
Is adalimumab effective for hidradenitis suppurativa? | Yes, Adalimumab, a recombinant, fully humanized, anti-tumor necrosis factor alpha (anti-TNF-α) monoclonal antibody, is the only officially approved treatment for the management of moderate-to-severe hidradenitis suppurativa. | Adalimumab is a biological agent, one of the tumour necrosis factor-alpha
inhibitors. Pivotal studies evaluating its efficacy in plaque psoriasis
(CHAMPION, REVEAL) and psoriatic arthritis (PsA) (ADEPT) were carried out in
recent years. Adalimumab proved highly effective in psoriasis patients and in
PsA patients previously unresponsive to nonsteroidal anti-inflammatory drugs.
Results of smaller studies suggest therapy with the drug may be successful in
psoriasis resistant to other biologics and PsA unresponsive to disease-modifying
antirheumatic drugs. Adalimumab has also been shown to improve patients' quality
of life significantly. Although they should be further extended as far as
dermatological conditions are concerned, available data indicate adalimumab is
safe and well tolerated. Numerous case reports featuring its off-label use
suggest the drug could be helpful in treating hidradenitis suppurativa, pyoderma
gangrenosum, Sweet's syndrome, cutaneous sarcoidosis, pemphigus, systemic
vasculitides, multicentric reticulohistiocytosis and stomatitis. Hidradenitis suppurativa (HS), a chronic, inflammatory, relapsing disease of the
apocrine glands in the skin, commonly occurs in women aged 20 to 40 years.
Patients typically present with discomfort and/or itching associated with
papules or nodules that may recur and lead to abscess formation and sinus
tracts. Recent reports have demonstrated that adalimumab, a tumor necrosis
factor (TNF) antagonist, may be effective in the treatment of patients with HS
who have failed conventional therapy. The authors describe 3 patients who
experienced the resolution of skin lesions associated with HS after treatment
with adalimumab. The patients found this treatment to be convenient, as they
could administer the therapy at home, and 1 patient was able to avoid surgical
intervention through use of TNF-antagonist therapy. Adalimumab may resolve
symptoms of HS when conventional therapy fails. More studies are necessary to
investigate the efficacy and safety of adalimumab for the treatment of HS. BACKGROUND: Several studies report the use of tumor necrosis factor alpha
(TNF-alpha) inhibitors in refractory hidradenitis suppurativa (HS), particularly
infliximab and etanercept. However, very limited data have been reported for
adalimumab, the newest fully human anti-TNF-alpha monoclonal antibody. We
evaluated the long-term efficacy and safety of adalimumab therapy in 6 patients
with refractory HS. In the case of positive culture findings from any draining
lesion, antibiotic therapy was administered for at least 2 weeks before
initiating adalimumab therapy. Adalimumab (in 40-mg subcutaneous injections) was
prescribed every other week. If the disease was inadequately controlled, the
dosage was increased to 40 mg/wk. If HS was in persistent clinical remission,
adalimumab therapy was gradually decreased to 40 mg every 3 weeks. Quality of
life was assessed using the Dermatology Life Quality Index.
OBSERVATIONS: Six patients (mean [SD] age, 39.3 [12.9] years) with severe HS
(mean [SD] duration, 22.5 [11.7] years) were treated with adalimumab.
Significant improvements after 1 month of treatment were seen in the Dermatology
Life Quality Index; in the number of affected regions, nodules, and fistulas;
and in the basic laboratory findings. Improvements were maintained for a mean
(SD) follow-up of 21.5 (7.1) (range, 13-29) months. Adalimumab was well
tolerated. Conclusion Adalimumab appears to be an effective and safe treatment
for refractory HS. Hidradenitis suppurativa (HS) is a common inflammatory skin disease. Medical
treatment is often disappointing and in severe disease surgery remains the
therapy of choice. Extensive surgery may be effective but also mutilating.
Patients experience a significant reduction in quality of life and the need for
new treatment modalities are urgent. In recent years patients with HS have been
treated off-label with tumour necrosis factor-alpha (TNF-alpha) inhibitors with
a varying degrees of effect. We performed a systematic review of papers
retrieved from two databases (PubMed and Web of Science) using the follow-ing
keywords: hidradenitis suppurativa, acne inversa, infliximab, etanercept, and
adalimumab. A total of 34 publications were retrieved, describing treatment of
105 patients. Most cases report treatment with infliximab (52/105). A positive
treatment outcome was reported in 90/105 cases, with only 7/105 non-responders
and 8/105 patients experiencing side-effects. The side-effects were comparable
to those seen in other TNF-alpha inhibitor studies. In the majority of cases the
treatment was effective when given as a suppressive therapy, but 15/105 cases
were described with long-term remission (>or= 3 months) after the end of
therapy. In most publications follow-up was, however, insufficient to allow a
systematic exploration of this. TNF-alpha inhibitors seem to be effective in the
treatment of HS. However, several questions remain to be answered through
specific studies. This review has also identified a need for more standardized
reporting of the outcomes as well as randomized controlled trials in this
disease. PURPOSE: To report a patient who presented with bilateral interstitial keratitis
in association with severe hidradenitis suppurativa.
METHODS: Case report.
RESULTS: An 18-year-old African American woman with severe active hidradenitis
suppurativa of the axillae and groin presented with a 2-week history of
bilateral blurry vision. On examination, best-corrected visual acuity was
counting fingers in the right eye and 20/70 in the left eye. Slit-lamp
examination revealed diffuse vascularization of the corneal stroma with
surrounding infiltrates bilaterally. In the left eye, corneal thinning and an
epithelial defect were present in an area of infiltrate. Our clinical impression
at that time was bilateral interstitial keratitis with secondary bacterial
keratitis in the left eye. Topical therapy, prednisolone acetate 1% in the right
eye, and ofloxacin in the left eye, was instituted. A systemic workup, including
antinuclear antibody, rheumatoid factor, Lyme titer, cytoplasmatic staining
antineutrophil cytoplasmic antibodies, perinuclear staining antineutrophil
cytoplasmic antibodies, erythrocyte sedimentation rate, Venereal Disease
Research Laboratory, rapid plasma reagin, basic metabolic panel,
angiotensin-converting enzyme level, and a chest x-ray was negative. Topical
steroids were used in the left eye after resolution of the bacterial keratitis.
The interstitial keratitis responded to topical steroids and remained in
remission after steroid taper. However, bilateral interstitial keratitis
recurred coincident with a severe flare of hidradenitis suppurativa within 1
month of discontinuing the topical steroids. A course of subcutaneous adalimumab
injections (40 mg/mL every 2 weeks) for hidradenitis suppurativa was
implemented. Both her dermatological and ocular conditions responded to this
therapy and have remained in remission through 7 months of follow-up.
CONCLUSIONS: Hidradenitis suppurativa is a rare cause of bilateral interstitial
keratitis. Patients may experience simultaneous exacerbations of both
dermatological and ocular manifestations. Systemic treatment with adalimumab can
improve both dermatological and ocular conditions. Hidradenitis suppurativa is a chronic disease that affects the apocrine
gland-bearing regions of the body. The etiology of this disorder is poorly
understood, but most likely is a complex process involving follicular apocrine
occlusion with subsequent perifolliculitis. Many treatment options have been
reported with varying degrees of success, including topical and oral therapy and
surgical procedures. Recently, TNF-alpha antagonists have been reported as
effective therapy in a few patients. We report here a patient who initially
responded to infliximab but developed an infusion reaction to this medication.
After subsequent treatment with adalimumab, the patient's disease improved
dramatically and has been maintained under excellent control for over 15 months.
We propose that TNF-alpha inhibitors, particularly monoclonal antibody based
agents, are a viable treatment option in patients with severe, recalcitrant HS
and that a patient may be safely and successfully treated with the fully human
monoclonal antibody adalimumab in cases in which the chimeric monoclonal
antibody infliximab therapy is not tolerated. OBJECTIVE: To evaluate the safety and efficacy of adalimumab for the management
of hidradenitis suppurativa (HS).
METHODS: In a prospective open-label phase II study, adalimumab was administered
subcutaneously in a dose of 160 mg induction regimen at week 0, followed by 80
mg at week 1, and 40 mg at alternate weeks for 12 weeks in 10 patients. The
patients were followed up to 13 weeks and their disease activity was assessed
using the HS Severity Index (HSSI) as well as with the numbers of daily dressing
changes, the Visual Analogue Scale (VAS), Dermatologic Life Quality Index
(DLQI), and Physician's Global Assessment of disease severity (PGA).
RESULTS: Ten patients were enrolled in this study. Of these, six patients
completed the 12-week treatment period. A ≥ 50% decrease of HSSI score was not
found in any of the patients at week 2, 4, 8, and 12. None of the 10 patients
was classified as a responder at week 12 compared with baseline. Statistically
significant difference in HSSI score was found between baseline and week 8 (P <
0.05) only but no significant differences were found between baseline and week
2, 4, and 12. Comparison of baseline with week 12 VAS and DLQI scores failed to
show statistically significant improvement. Adalimumab was well tolerated and
there were no serious adverse events reported.
CONCLUSIONS: Our study demonstrated statistically clinical improvement is not
observed in the treatment of HS with adalimumab. Future studies using higher
doses of adalimumab are warranted. Hidradenitis suppurativa (HS) is a difficult disease to treat. Although the
pathogenesis of this inflammatory skin disease is largely unknown, the important
role of the immune system has been demonstrated in both experimental and
clinical studies. Clinicians are therefore increasingly prescribing systemic
treatments with immunosuppressive agents, but the more traditionally used
systemic retinoids, especially isotretinoin, also remain relatively common
therapies. In order to provide an overview of all currently available systemic
immunosuppressive agents and retinoids for the treatment of HS, a systematic
search was performed using the Medline and Embase databases. All published
papers concerning systemic retinoids or immunosuppressive treatments for HS in
adults were included. The primary endpoints were the percentages of significant
responders, moderate responders and nonresponders. Other endpoints were the
relapse rate and adverse events. In total 87 papers were included, comprising
518 patients with HS who were treated with systemic retinoids, biological agents
or another immunosuppressive agents, including colchicine, ciclosporin, dapsone
or methotrexate. The highest response rates were observed with infliximab,
adalimumab and acitretin. Overall, the quality of evidence was low and differed
between the agents, making direct comparisons difficult. However, based on the
amount of evidence, infliximab and adalimumab were the most effective agents.
Acitretin was also effective in HS, although the quality of the evidence was
low. The therapeutic effect of isotretinoin is questionable. Randomized
controlled trials are needed to confirm the effectiveness of acitretin, and to
identify the most effective immunosuppressive agents in HS. BACKGROUND: Hidradenitis suppurativa (HS) is a chronic, painful skin disease
characterized by abscesses, nodules, and draining fistulas in the axilla and
groin of young adults.
OBJECTIVE: To evaluate the efficacy and safety of adalimumab, an anti-tumor
necrosis factor-α antibody, in patients with moderate to severe HS.
DESIGN: Phase 2, parallel, randomized, placebo-controlled trial consisting of a
blinded 16-week period (period 1) and an open-label 36-week period (period 2).
All study personnel, investigators, and patients remained blinded to treatment
group throughout the study. (ClinicalTrials.gov: NCT00918255)
SETTING: 26 academic and private practice medical centers in the United States
and Europe.
PATIENTS: 154 adult patients with moderate to severe HS who were unresponsive or
intolerant to oral antibiotics.
INTERVENTION: Patients were assigned in a 1:1:1 ratio to adalimumab, 40 mg/wk;
adalimumab, 40 mg every other week (EOW); or placebo. All patients received
adalimumab, 40 mg EOW, at the beginning of period 2 but switched to weekly
dosing if the response was suboptimal (HS Physician's Global Assessment [PGA]
score of moderate or worse) at weeks 28 or 31.
MEASUREMENTS: The primary outcome measure (clinical response) was the proportion
of patients achieving an HS-PGA score of clear, minimal, or mild with at least a
2-grade improvement relative to baseline at week 16.
RESULTS: At week 16, 3.9% of placebo patients (2 of 51), 9.6% of EOW patients (5
of 52), and 17.6% of weekly patients (9 of 51) achieved clinical response (EOW
vs. placebo strata-adjusted difference, 5.6% [95% CI, -4.0% to 15.3%]; P = 0.25;
weekly vs. placebo strata-adjusted difference, 13.7% [CI, 1.7% to 25.7%]; P =
0.025). Serious adverse event rates were 3.9%, 5.8%, and 7.8% for placebo, EOW,
and weekly patients, respectively (EOW vs. placebo difference, 1.8% [CI, -6.4%
to 10.1%]; weekly vs. placebo difference, 3.9% [CI, -5.2% to 13.0%]).
Significantly greater improvements in patient-reported outcomes and pain were
seen in the weekly dosing group than in the placebo group. A decrease in
response was seen after the switch from weekly to EOW dosing in period 2.
LIMITATIONS: Weeks 16 to 52 of the study were open-label. The study was not
powered to assess the risk for known serious adverse effects of adalimumab, such
as tuberculosis, other serious infections, and demyelinating disorders.
CONCLUSION: Adalimumab dosed once per week alleviates moderate to severe HS.
PRIMARY FUNDING SOURCE: Abbott Laboratories. Aim of the study was to investigate the impact of TNF-α antagonists on
health-related quality of life (HRQoL) in selected skin diseases, i.e. chronic
plaque psoriasis, Behçet's disease (BD), hidradenitis suppurativa (HS) and
pyoderma gangrenosum (PG). We have carried out a systematic literature search of
Medline (2000 to April 2013) using the Cochrane highly sensitive and specific
search strategy. Citations were screened for randomized, controlled trials of
TNF-α antagonists (adalimumab, etanercept and infliximab) versus placebo in
adults with psoriasis, BD, HS or PG. From the literature it is evident that skin
diseases can affect physical, psychological, social and occupational aspects of
everyday life. TNF-α antagonists induced consistent benefits across health
outcomes in psoriasis, but only monoclonal antibodies, infliximab and adalimumab
were effective in improving QoL in patients with BD, HS and PG. Dermatology Life
Quality Index was the most common used tool for investigating HRQoL. For the
majority of patients with skin diseases, the most important negative impacts on
QoL were appearance related. Generally, the burden on QoL was correlated to the
severity of skin disease and the improvement in QoL achieved by TNF-α blockers
was proportional to the degree of disease remission. HRQoL issues are becoming
even more important in evaluating medical care, including treatment of skin
diseases. In general, achieving the highest clearing of skin disease with
anti-TNF-α agents is required for optimal improvement in QoL. BACKGROUND: Hidradenitis suppurativa (HS) is characterized by chronic,
suppurative abscesses, sinus tracts, and fistulas affecting the axilla, groin,
and perianal region resulting from hyperkeratosis and occlusion of the terminal
hair follicle.
OBJECTIVE: This report highlights the use of biologic agents for the treatment
of recalcitrant HS.
METHOD: We report on a 48-year-old male with a 15-year history of refractory
perianal-inguinal-buttock HS who, despite receiving numerous surgical drainages
and traditional medical treatment for HS, still had severe pain. After trialing
etanercept and infliximab with methotrexate, the patient had marked improvement
with adalimumab. A literature review of biologics therapy was also performed.
RESULTS: After trialing many traditional therapies, we found that adalimumab
appears to be the most effective treatment modality for our patient. A
literature search revealed 53 articles on biologics therapy in HS. These
articles are summarized.
DISCUSSION: Biologic agents have been shown to have variable results in the
treatment of refractory HS. Enough low-grade evidence has been accumulated to
make the use of these agents suitable in HS. Until more clinical trials are
performed on this topic, physicians should use clinical judgment when treating
HS with biologic agents and be cautious by watching for significant adverse
effects. BACKGROUND: Quantification of disease severity supports the development of
evidence-based treatments. Assessments to capture clinical improvement in
hidradenitis suppurativa (HS) can be improved.
OBJECTIVES: This study aimed to validate the Hidradenitis Suppurativa Clinical
Response (HiSCR), which is defined as a ≥ 50% reduction in inflammatory lesion
count (sum of abscesses and inflammatory nodules, AN), and no increase in
abscesses or draining fistulas in HS when compared with baseline as a meaningful
clinical endpoint for HS treatment.
METHODS: Patients with ≥ 3 ANs at baseline in a Phase II adalimumab trial for HS
were included for analysis. HiSCR achievers vs. nonachievers were assessed at
week 16 and week 52. Criteria measures included physician-rated assessments
[Hurley stage, modified Sartorius score (MSS), and HS Physician's Global
Assessment] and patient-reported outcomes (PROs: visual analogue pain scale,
Dermatology Life Quality Index, and Work Productivity and Activity Impairment
questionnaire). Test-retest reliability, convergent validity, responsiveness and
predictive validity of HiSCR, and its meaningfulness to patients were assessed.
RESULTS: Among 138 eligible study participants, the majority were female (69·6%)
with a mean age of 36·7 years. The mean (median) MSS was 125·2 (85·5) at
baseline. Test-retest reliability of the AN count was 0·91. HiSCR was
significantly correlated with improvements in all physician-rated and PRO
measures (Spearman's rho between -0·61 and -0·27, all P < 0·001). Improvements
of all PROs in HiSCR achievers exceeded the respective meaningful improvement
thresholds.
CONCLUSIONS: In patients with HS with ≥ 3 ANs, HiSCR achievers had significant
improvements in physician-rated and patient-reported HS disease severity and
impact. HiSCR is a valid and meaningful endpoint for assessing HS treatment
effectiveness in controlling inflammatory manifestations in this population. Hidradenitis suppurativa (HS) is a chronic, inflammatory disease characterized
by recurring abscesses, nodules, and fistulas predomitly in the area of the
groin and axillae. The association between HS and Crohn's disease (CD) has
already been documented. We report on a case of a patient with CD associated HS,
refractory to multiple local and systemic agents.A complete resolution of both
diseases was finally achieved after treatment with adalimumab. Our case report
supports the co-occurrence of both diseases and suggests that adalimumab
approved for CD might also be a safe and effective therapeutic option in the
treatment of HS. More than 50 interventions have been used to treat hidradenitis suppurativa
(HS), and so therapy decisions can be challenging. Our objective was to
summarize and appraise randomized controlled trial (RCT) evidence for HS
interventions in adults. Searches were conducted in Medline, Embase, CENTRAL,
LILACS, five trials registers and abstracts from eight dermatology conferences
until 13 August 2015. Two review authors independently assessed study
eligibility, extracted data and assessed methodological quality. Primary
outcomes were quality of life and adverse effects of the interventions. Twelve
trials, from 1983 to 2015, investigating 15 different interventions met our
inclusion criteria. The median trial duration was 16 weeks and the median number
of participants was 27. Adalimumab 40 mg weekly improved the Dermatology Life
Quality Index (DLQI) by 4·0 points, which equates to the minimal clinically
important difference for the scale, compared with placebo (95% confidence
interval -6·5 to -1·5 points). Evidence quality was reduced to 'moderate'
because the results are based on only a single study. Adalimumab 40 mg every
other week was ineffective in a meta-analysis of two studies comprising 124
participants. Infliximab 5 mg kg(-1) improved the DLQI score by 8·4 points after
8 weeks in a moderate-quality study completed by 33 of 38 participants.
Etanercept 50 mg twice weekly was ineffective. Inclusion of a gentamicin sponge
prior to primary closure did not improve outcomes. Other interventions,
including topical and oral antibiotics, were investigated by relatively small
studies, preventing treatment recommendations due to imprecision. More, larger
RCTs are required to investigate most HS interventions, particularly oral
treatments and surgical therapy. Moderate-quality evidence suggests that
adalimumab given weekly and infliximab are effective, whereas adalimumab every
other week is ineffective. BACKGROUND: Hidradenitis suppurativa (HS) is a chronic, recurrent, inflammatory
skin disease with frequent comorbidities of paid depression. Adalimumab
treatment for 16 weeks improved HS lesions significantly versus
placebo (NCT00918255).
OBJECTIVE: The relationship between pain and depressive symptoms and the effects
of adalimumab on each was examined in this post hoc analysis.
METHODS: Patients with moderate to severe HS (N=154) were randomized 1:1:1 to
adalimumab 40 mg weekly (ew), adalimumab 40 mg every other week (eow), or
placebo. Skin pain was assessed using a visual analog scale (VAS; 0-100 mm).
Depressive symptoms were assessed using the 9-item Patient Health Questionnaire
(PHQ-9; score ≥10 indicative of depression).
RESULTS: At baseline, overall mean±SD pain VAS was 54.3±26.5 mm and 41.8% of
patients had PHQ-9 scores ≥10. At baseline, VAS pain scores (mean±SD) were
significantly higher (P<0.001) for patients with PHQ-9 scores ≥10 (63.9±23.3)
versus <10 (47.4±26.7). At Week 16, clinically relevant pain reduction was
observed for ew-treated patients with baseline PHQ-9 score ≥10 (ew, 45.8%;
eow, 29.4%; placebo, 23.8%) and <10 (ew, 50.0%; eow, 37.9%; placebo, 29.6%),
but did not reach statistical significance. In patients with high baseline pain
(≥median VAS score), adalimumab ew significantly decreased depressivesymptoms
versus placebo (PHQ-9 scores, -34.03% vs +2.26%; P<0.01).
CONCLUSION: Patients with moderate to severe HS had a high degree of pain and
depressive symptoms at baseline. Adalimumabtherapy was associated with decreased
pain and depressive symptoms compared to baseline. Subcutaneous adalimumab (Humira®) is a tumour necrosis factor-α blocker that is
the only approved agent for the treatment of moderate to severe hidradenitis
suppurativa (HS) in several countries worldwide. This article reviews the
clinical efficacy and safety of subcutaneous adalimumab in patients with
moderate to severe HS. In clinical trials (PIONEER I and II), a greater
proportion of adalimumab than placebo recipients reached HS clinical response
(HiSCR) at week 12. The main secondary endpoints, such as the proportion of
patients with an abscess and inflammatory nodule count of ≤2 at week 12, were
significantly greater with adalimumab than with placebo in PIONEER II, but not
in PIONEER I. In addition, adalimumab showed the potential to reduce the high
health-related quality of life burden of HS and increase patient satisfaction.
HiSCR rates were generally maintained in the longer term, and the safety profile
of adalimumab in patients with moderate to severe HS was consistent with the
known safety profile of the drug for other indications, with no new emerging
safety signals. Adalimumab is an effective and generally well tolerated
treatment for patients with moderate to severe HS, and is the first agent
approved for this difficult-to-treat disease. Hidradenitis suppurativa (HS) is a chronic, recurrent, inflammatory skin disease
that affects the hair follicles of the aprocrine gland-bearing anatomical areas
of the body. It is characterized by deep painful nodules and abscesses that
rupture and contribute to the formation of sinus tracks and scarring. The
management of HS is based on the assessment of disease severity and a
combination of medical and surgical treatment according to the European
Guidelines. Adalimumab, a recombit, fully humanized, anti-tumor necrosis
factor alpha (anti-TNF-α) monoclonal antibody, is the only officially approved
treatment for the management of moderate-to-severe HS. Case reports, concerning
42 patients who received adalimumab for severe HS (with the standard dose
regimen for psoriasis), reported a cumulative response rate of 58% (≥50% in 23
patients) with a relapse rate of 71% (10 out of 14 patients). The most recent
and most well-powered phase III, randomized placebo-controlled trials for the
evaluation of the efficacy and safety of adalimumab in treatment of
moderate-to-severe HS (PIONEER studies I and II) showed that the Hidradenitis
Suppurativa Clinical Response (HiSCR) rate at week 12 was significantly higher
for patients randomized to adalimumab compared to placebo. Adverse events were
comparable to placebo. In conclusion, adalimumab, to date, holds the most robust
data regarding treatment efficacy in HS. Larger, registry-based studies are
needed to further establish the efficacy and safety profile of this anti-TNF-α
agent in HS. |
What is Legg-Calvé-Perthes Disease | Legg-Calve-Perthes disease is idiopathic avascular necrosis of the hip, usually occurring in pediatric populations. | BACKGROUND: Legg-Perthes disease is associated with ischemia of the capital
femoral epiphysis in children. Thrombophilia has been implicated as a potential
cause of the condition, and screening of patients with Legg-Perthes disease for
thrombophilia has been recommended. We analyzed the value of screening for
inherited thrombophilia in patients with Legg-Perthes disease by examining the
association between Legg-Perthes disease and abnormalities in the thrombotic
pathway.
METHODS: A random series of consecutive patients with Legg-Perthes disease were
prospectively enrolled in this study. Assays for the detection of factor-V
Leiden mutation and the plasma concentrations of protein C, protein S,
antithrombin III, and lipoprotein (a) were performed on plasma samples from
children with Legg-Perthes disease, and the results were compared with those for
pooled plasma from normal controls. Plasma concentrations below the 95% midrange
of the control values were classified as protein deficiencies. The estimated
population frequency of each coagulation abnormality was compared with the
proportion of the study group with the corresponding abnormality.
RESULTS: The proportion of abnormalities observed in the study group did not
differ from the estimated population frequency for protein C, protein S,
antithrombin III, or factor-V Leiden mutation. A lipoprotein (a) level of >30
mg/dL (>1.07 micro mol/L) was found in 16% of the study group.
CONCLUSIONS: Our data do not suggest that thrombotic diatheses due to deficiency
of protein C, protein S, or antithrombin III or due to factor-V Leiden mutation
are major causes of Legg-Perthes disease. The elevated levels of lipoprotein (a)
in children with Legg-Perthes disease suggest that they may be at risk for
atherosclerosis as adults. BACKGROUND: Legg-Calvé-Perthes disease is a childhood hip disorder that may
result in a deformed and poorly functioning hip. The purpose of this study was
to evaluate the correlation between hip deformity at skeletal maturity and
degenerative osteoarthritis and to present the long-term results of proximal
femoral varus derotational osteotomy in Legg-Calvé-Perthes disease.
METHODS: We analyzed the results of 40 patients (43 hips), who underwent
proximal femoral varus derotational osteotomy for Legg-Calvé-Perthes disease in
our institution between 1959 and 1983. All available patients underwent a single
long-term follow-up examination. Hips were classified with the classification
system of Stulberg. Osteoarthritis was evaluated using the Tönnis
classification. The long-term outcomes were evaluated after a mean follow-up
period of 33 years.
RESULTS: When examining the outcome using the Stulberg classification system,
there were 8 Stulberg class I hips (19.5%), 15 Stulberg class II hips (36.6%), 8
Stulberg class III hips (19.5%), 9 Stulberg class IV hips (22%), and 1 Stulberg
class V hip (2.4%). One patient, who had a bilateral Legg-Calvé-Perthes disease,
underwent total hip replacement for osteoarthritis. Seven patients had poor
clinical results.
CONCLUSIONS: Proximal femoral varus derotational osteotomy provides good
long-term results for Legg-Calvé-Perthes disease. The Stulberg classification is
a good predictor for patient outcome.
LEVEL OF EVIDENCE: Level IV, therapeutic study. Legg-Calvé-Perthes' (Perthes') disease is a developmental disease of the hip
joint that may result in numerous short and long term problems. The etiology of
the disease remains largely unknown, but the mechanism is believed to be
vascular and/or biomechanical in nature. There are several anatomical
characteristics that tend to be prevalent in children with Perthes' disease,
namely: skeletal immaturity, reduced height, and rostral sparing. We present an
overview of the literature, summarizing the current understanding of the
pathogenesis, particularly related to how the formation of the vasculature to
the femoral epiphysis places children aged 5-8 at a higher risk for Perthes'
disease, how skeletal immaturity and rostral sparing could increase the
probability of developing Perthes' disease, and how animal models have aided our
understanding of the disease. In doing so, we also explore why Perthes' disease
is correlated to latitude, with populations at higher latitudes having higher
incidence rates than populations closer to the Equator. Finally, we present five
hypotheses detailing how Perthes' disease could have a biomechanical cause.
Clin. Anat. 29:759-772, 2016. © 2016 Wiley Periodicals, Inc. Legg-Calve-Perthes disease (LCPD) is the insidious onset of idiopathic avascular
necrosis of the hip in the pediatric population. The disease encompasses a wide
spectrum of pathology, from mild with no long-term sequelae to severe with
permanent degenerative change of the hip joint. A pediatric patient with hip
pathology may initially only present with knee or thigh pain, thus obligating
the clinician to maintain awareness about the hip during examination. Common
physical examination findings include deficits in hip abduction and internal
rotation, along with Trendelenburg gait in late stages. Plain radiographs
demonstrate changes in the anterolateral aspect of the femoral head, sometimes
only seen on the frog lateral X-ray in early stages. Treatment options aim to
restore range of motion and maintain adequate coverage of the femoral head. When
appropriate, surgery is used to reorient the femoral head or pelvis to maintain
coverage. |
What is promoted by ERAP1-ERAP2 dimerization? | ERAP1-ERAP2 dimerization increases peptide-trimming efficiency. | The endoplasmic reticulum aminopeptidases (ERAP)1 and ERAP2 play a critical role
in the production of final epitopes presented by MHC class I molecules.
Formation of heterodimers by ERAP1 and ERAP2 has been proposed to facilitate
trimming of epitope precursor peptides, but the effects of dimerization on ERAP
function remain unknown. In this study, we produced stabilized ERAP1-ERAP2
heterodimers and found that they produced several mature epitopes more
efficiently than a mix of the two enzymes unable to dimerize. Physical
interaction with ERAP2 changes basic enzymatic parameters of ERAP1 and improves
its substrate-binding affinity. Thus, by bringing the two enzymes in proximity
and by producing allosteric effects on ERAP1, dimerization of ERAP1/2 creates
complexes with superior peptide-trimming efficacy. Such complexes are likely to
enhance Ag presentation by cells displaying coordinated expression of the two
enzymes. |
What is plantar fasciitis | Plantar fascia (PF) disorders like plantar fasciitis commonly cause heel pain and disability and are thought to be degenerative rather than inflammatory in nature | The authors review histologic findings from 50 cases of heel spur surgery for
chronic plantar fasciitis. Findings include myxoid degeneration with
fragmentation and degeneration of the plantar fascia and bone marrow vascular
ectasia. Histologic findings are presented to support the thesis that "plantar
fasciitis" is a degenerative fasciosis without inflammation, not a fasciitis.
These findings suggest that treatment regimens such as serial corticosteroid
injections into the plantar fascia should be reevaluated in the absence of
inflammation and in light of their potential to induce plantar fascial rupture. BACKGROUND: Plantar fasciitis is a common foot disorder that impacts many
functional activities. Research that quantifies the impact that plantar
fasciitis has on function is lacking. In addition, little is known about which
variables are associated with disability in patients with plantar fasciitis. The
first purpose of this study was to determine if age, gender, body mass index,
pain intensity, chronicity of symptoms, or ankle dorsiflexion range of motion
was associated with disability in patients with plantar fasciitis. The second
purpose was to describe the impact that plantar fasciitis has on functional
status in the context of five functional domains: household activities of daily
living, usual work and hobbies, nonweightbearing activities, walking-related
activities, and running-related activities.
METHODS: Fifty consecutive patients diagnosed with unilateral plantar fasciitis
were recruited. Demographic and impairment data were collected and all patients
completed the Lower Extremity Functional Scale (LEFS), a validated self-report
measure of disability. Multiple regression analysis was used to describe the
association between the variables and disability. Graphs depicting five domains
of function derived from the LEFS were generated to describe the extent of
disability.
RESULTS: Body mass index (BMI) was the only variable that was significantly
associated with disability (F = 9.87, p =.003). Measures of pain intensity,
ankle dorsiflexion, age, gender, chronicity, and time spent weightbearing were
not related to disability. Plantar fasciitis showed distinct patterns of
disability depending on the functional domain that was assessed.
CONCLUSIONS: With the exception of BMI, impairment and demographic variables do
not predict the extent of functional loss in patients with plantar fasciitis.
The most likely domains of function to be at least moderately affected in
patients with plantar fasciitis are running-related activities and usual work or
hobbies. BACKGROUND: Although plantar fasciitis is the most common cause of heel pain,
little has been reported on the incidence rates of this disorder. We sought to
determine the incidence rate and demographic risk factors of plantar fasciitis
in an ethnically diverse and physically active population of United States
military service members.
METHODS: A query was performed with use of the Defense Medical Epidemiology
Database for the International Classification of Diseases, Ninth Revision,
Clinical Modification, code for plantar fasciitis (728.71). Multivariate Poisson
regression analysis was used to estimate the rate of plantar fasciitis per 1000
person-years, while controlling for sex, race, rank, service, and age.
RESULTS: The overall unadjusted incidence rate of plantar fasciitis was 10.5 per
1000 person-years. Compared with men, women had a significantly increased
adjusted incidence rate ratio for plantar fasciitis of 1.96 (95% confidence
interval, 1.94 to 1.99). The adjusted incidence rate ratio for black service
members compared with white service members was 1.12 (95% confidence interval,
1.09 to 1.12). With junior officers as the referent category, junior enlisted,
senior enlisted, and senior officer rank groups had a significantly increased
adjusted incidence rate ratio for plantar fasciitis: 1.20 (95% confidence
interval, 1.18 to 1.23), 1.19 (95% confidence interval, 1.17 to 1.22), and 1.56
(95% confidence interval, 1.52 to 1.61), respectively. Compared with service
members in the Air Force, those in the Army and Marines had a significantly
increased adjusted incidence rate ratio for plantar fasciitis of 1.85 (95%
confidence interval, 1.82 to 1.87) and 1.28 (95% confidence interval, 1.25 to
1.30), respectively. The adjusted incidence rate ratio for the age group of
forty years old or more compared with the twenty to twenty-four-year-old group
was 3.42 (95% confidence interval, 3.34 to 3.51).
CONCLUSIONS: Female sex; black race; junior enlisted, senior enlisted, and
senior officer rank groups; service in the Army or Marines; and increasing age
are all risk factors for plantar fasciitis. Plantar fasciitis is a common cause of heel pain and is the result of a
degenerative process of the plantar fascia at its calcaneal attachment. A case
study of a preliminary experience with local injections of botulinum toxin A
(BTX-A) for the treatment of chronic plantar fasciitis in a 43-year-old woman is
presented. We injected the patient with 70 units of BTX-A (0.7mL) in 2 divided
doses: 40 units (0.4mL) in the tender region of the heel, and 30 units (0.3mL)
in the most tender point of the foot arch. Visual analog scale (VAS) and
pressure pain threshold (PPT) were measured to evaluate the efficacy of BTX-A
injections. Real-time, high-resolution ultrasonographic findings of the plantar
fascia after BTX-A injections were also used for serial follow-ups. After BTX-A
injection, decreased VAS values were reported and increased PPT was observed. In
ultrasonographic studies, the thickness of the plantar fascia and the
hypoechogenicity of the fascia were reduced. Decreased plantar fascia thickness
was observed on the first and third week after BTX-A injections. The findings
were compatible with the changes in pain assessed by VAS and PPT.
Ultrasonographic findings also indicated a progressive decrease in the thickness
of the underlying muscle belly. Ultrasonography seems to be a valuable,
noninvasive diagnostic tool for the evaluation of plantar fasciitis treated with
BTX-A injections. It can offer objective measurements of therapeutic effects and
is feasible for serial follow-ups. Plantar fasciitis, a self-limiting condition, is a common cause of heel pain in
adults. It affects more than 1 million persons per year, and two-thirds of
patients with plantar fasciitis will seek care from their family physician.
Plantar fasciitis affects sedentary and athletic populations. Obesity, excessive
foot pronation, excessive running, and prolonged standing are risk factors for
developing plantar fasciitis. Diagnosis is primarily based on history and
physical examination. Patients may present with heel pain with their first steps
in the morning or after prolonged sitting, and sharp pain with palpation of the
medial plantar calcaneal region. Discomfort in the proximal plantar fascia can
be elicited by passive ankle/first toe dorsiflexion. Diagnostic imaging is
rarely needed for the initial diagnosis of plantar fasciitis. Use of
ultrasonography and magnetic resoce imaging is reserved for recalcitrant
cases or to rule out other heel pathology; findings of increased plantar fascia
thickness and abnormal tissue signal the diagnosis of plantar fasciitis.
Conservative treatments help with the disabling pain. Initially,
patient-directed treatments consisting of rest, activity modification, ice
massage, oral analgesics, and stretching techniques can be tried for several
weeks. If heel pain persists, then physician-prescribed treatments such as
physical therapy modalities, foot orthotics, night splinting, and corticosteroid
injections should be considered. Ninety percent of patients will improve with
these conservative techniques. Patients with chronic recalcitrant plantar
fasciitis lasting six months or longer can consider extracorporeal shock wave
therapy or plantar fasciotomy. BACKGROUND: Due to complexity of the plantar intrinsic foot muscles, little is
known about their muscle architecture in vivo. Chronic plantar fasciitis may be
accompanied by muscle atrophy of plantar intrinsic foot muscles and tibialis
posterior compromising the dynamic support of the foot prolonging the injury.
Magnetic resoce images of the foot may be digitized to quantify muscle
architecture. The first purpose of this study was to estimate in vivo the volume
and distribution of healthy plantar intrinsic foot muscles. The second purpose
was to determine whether chronic plantar fasciitis is accompanied by atrophy of
plantar intrinsic foot muscles and tibialis posterior.
METHODS: Magnetic resoce images were taken bilaterally in eight subjects with
unilateral plantar fasciitis. Muscle perimeters were digitally outlined and
muscle signal intensity thresholds were determined for each image for volume
computation.
FINDINGS: The mean volume of contractile tissue in healthy plantar intrinsic
foot muscles was 113.3 cm(3). Forefoot volumes of plantar fasciitis plantar
intrinsic foot muscles were 5.2% smaller than healthy feet (P=0.03, ES=0.26),
but rearfoot (P=0.26, ES=0.08) and total foot volumes (P=0.07) were similar. No
differences were observed in tibialis posterior size.
INTERPRETATIONS: While the total volume of plantar intrinsic foot muscles was
similar in healthy and plantar fasciitis feet, atrophy of the forefoot plantar
intrinsic foot muscles may contribute to plantar fasciitis by destabilizing the
medial longitudinal arch. These results suggest that magnetic resoce imaging
measures may be useful in understanding the etiology and rehabilitation of
chronic plantar fasciitis. Author information:
(1)From the Department of Orthopaedic Surgery at Memorial Hospital in York,
Pennsylvania (Dr Thompson); the Rowan University School of Osteopathic Medicine
(RowanSOM) in Stratford, New Jersey (Student Doctor Saini); the Department of
Orthopedics at RowanSOM in Stratford, New Jersey (Dr Reb); and the Department of
Surgery at Jefferson Medical College in Philadelphia, Pennsylvania (Dr Daniel).
Dr Thompson is in his second year of residency training, and Dr Reb is in his
fifth year of residency training.
(2)From the Department of Orthopaedic Surgery at Memorial Hospital in York,
Pennsylvania (Dr Thompson); the Rowan University School of Osteopathic Medicine
(RowanSOM) in Stratford, New Jersey (Student Doctor Saini); the Department of
Orthopedics at RowanSOM in Stratford, New Jersey (Dr Reb); and the Department of
Surgery at Jefferson Medical College in Philadelphia, Pennsylvania (Dr Daniel).
Dr Thompson is in his second year of residency training, and Dr Reb is in his
fifth year of residency training [email protected]. OBJECTIVES: Plantar fasciitis, a common injury in runners, has been speculated
to be associated with weakness of the intrinsic foot muscles. A recent study
reported that atrophy of the intrinsic forefoot muscles might contribute to
plantar fasciitis by destabilizing the medial longitudinal arch. However,
intrinsic foot muscle volume difference between individuals with plantar
fasciitis and healthy counterparts remains unknown. This study examined the
relationship of intrinsic foot muscle volume and incidence of plantar fasciitis.
DESIGN: Case-control study.
METHODS: 20 experienced (≥5 years) runners were recruited. Ten of them had
bilateral chronic (≥2 years) plantar fasciitis while the others were healthy
characteristics-matched runners. Intrinsic muscle volumes of the participants'
right foot were scanned with a 1.5T magnetic resoce system and segmented
using established methods. Body-mass normalized intrinsic foot muscle volumes
were compared between runners with and without chronic plantar fasciitis.
RESULTS: There was significant greater rearfoot intrinsic muscle volume in
healthy runners than runners with chronic plantar fasciitis (Cohen's d=1.13;
p=0.023). A similar trend was also observed in the total intrinsic foot muscle
volume but it did not reach a statistical significance (Cohen's d=0.92;
p=0.056). Forefoot volume was similar between runners with and without plantar
fasciitis.
CONCLUSIONS: These results suggest that atrophy of intrinsic foot muscles may be
associated with symptoms of plantar fasciitis in runners. These findings may
provide useful information in rehabilitation strategies of chronic plantar
fasciitis. Plantar fasciitis (PF) is present in 10% of the population and is the most
common cause of plantar heel pain. PF is painful, can alter daily activities and
presents as a sharp pain localized to the plantar foot and medial heel. The
underlying etiology involves microtrauma to the plantar fascia, specifically at
its insertion point on the calcaneus. Successful management of plantar fasciitis
is typically achieved with the conservative therapy approaches discussed. Plantar fascia (PF) disorders commonly cause heel pain and disability in the
general population. Imaging is often required to confirm diagnosis. This review
article aims to provide simple and systematic guidelines for imaging assessment
of PF disease, focussing on key findings detectable on plain radiography,
ultrasound and magnetic resoce imaging (MRI). Sonographic characteristics of
plantar fasciitis include PF thickening, loss of fibrillar structure,
perifascial collections, calcifications and hyperaemia on Doppler imaging.
Thickening and signal changes in the PF as well as oedema of adjacent soft
tissues and bone marrow can be assessed on MRI. Radiographic findings of plantar
fasciitis include PF thickening, cortical irregularities and abnormalities in
the fat pad located deep below the PF. Plantar fibromatosis appears as
well-demarcated, nodular thickenings that are iso-hypoechoic on ultrasound and
show low-signal intensity on MRI. PF tears present with partial or complete
fibre interruption on both ultrasound and MRI. Imaging description of further PF
disorders, including xanthoma, diabetic fascial disease, foreign-body reactions
and plantar infections, is detailed in the main text. Ultrasound and MRI should
be considered as first- and second-line modalities for assessment of PF
disorders, respectively. Indirect findings of PF disease can be ruled out on
plain radiography. Teaching Points • PF disorders commonly cause heel pain and
disability in the general population.• Imaging is often required to confirm
diagnosis or reveal concomitant injuries.• Ultrasound and MRI respectively
represent the first- and second-line modalities for diagnosis.• Indirect
findings of PF disease can be ruled out on plain radiography. |
Which is the enzymatic activity of OTULIN? | OTULIN is a deubiquitinase, that specifically cleaves Met1-linked polyUb. | The linear ubiquitin (Ub) chain assembly complex (LUBAC) is an E3 ligase that
specifically assembles Met1-linked (also known as linear) Ub chains that
regulate nuclear factor κB (NF-κB) signaling. Deubiquitinases (DUBs) are key
regulators of Ub signaling, but a dedicated DUB for Met1 linkages has not been
identified. Here, we reveal a previously unotated human DUB, OTULIN (also
known as FAM105B), which is exquisitely specific for Met1 linkages. Crystal
structures of the OTULIN catalytic domain in complex with diubiquitin reveal
Met1-specific Ub-binding sites and a mechanism of substrate-assisted catalysis
in which the proximal Ub activates the catalytic triad of the protease. Mutation
of Ub Glu16 inhibits OTULIN activity by reducing kcat 240-fold. OTULIN
overexpression or knockdown affects NF-κB responses to LUBAC, TNFα, and
poly(I:C) and sensitizes cells to TNFα-induced cell death. We show that OTULIN
binds LUBAC and that overexpression of OTULIN prevents TNFα-induced NEMO
association with ubiquitinated RIPK1. Our data suggest that OTULIN regulates
Met1-polyUb signaling. Conjugation of Met1-linked polyubiquitin (Met1-Ub) by the linear ubiquitin chain
assembly complex (LUBAC) is an important regulatory modification in innate
immune signaling. So far, only few Met1-Ub substrates have been described, and
the regulatory mechanisms have remained elusive. We recently identified that the
ovarian tumor (OTU) family deubiquitinase OTULIN specifically disassembles
Met1-Ub. Here, we report that OTULIN is critical for limiting Met1-Ub
accumulation after nucleotide-oligomerization domain-containing protein 2 (NOD2)
stimulation, and that OTULIN depletion augments signaling downstream of NOD2.
Affinity purification of Met1-Ub followed by quantitative proteomics uncovered
RIPK2 as the predomit NOD2-regulated substrate. Accordingly, Met1-Ub on RIPK2
was largely inhibited by overexpressing OTULIN and was increased by OTULIN
depletion. Intriguingly, OTULIN-depleted cells spontaneously accumulated Met1-Ub
on LUBAC components, and NOD2 or TNFR1 stimulation led to extensive Met1-Ub
accumulation on receptor complex components. We propose that OTULIN restricts
Met1-Ub formation after immune receptor stimulation to prevent unwarranted
proinflammatory signaling. Linear ubiquitin chains generated by the linear ubiquitin chain assembly complex
(LUBAC) play an important role in NF-κB activation. However, the regulation of
linear ubiquitin chain generation by LUBAC is not well characterized. Here, we
identified two deubiquitinating enzymes (DUBs), ovarian tumor DUB with linear
linkage specificity (OTULIN/Gumby/FAM105B) and cylindromatosis (CYLD) that can
cleave linear polyubiquitin chains and interact with LUBAC via the N-terminal
PNGase/UBA or UBX (PUB) domain of HOIP, a catalytic subunit of LUBAC. HOIP
interacts with both CYLD and OTULIN even in unstimulated cells. The interaction
of CYLD and OTULIN with HOIP synergistically suppresses LUBAC-mediated linear
polyubiquitination and NF-κB activation. Moreover, introduction of a HOIP mutant
unable to bind either deubiquitinase into HOIP-null cells augments the
activation of NF-κB by TNF-α stimulation. Thus, the interactions between these
two deubiquitinases and the LUBAC ubiquitin ligase are involved in controlling
the extent of TNF-α-induced NF-κB activation in cells by fine-tuning the
generation of linear ubiquitin chains by LUBAC. The interaction of HOIP with
OTULIN is also involved in OTULIN suppressing the canonical Wnt signaling
pathway activation by LUBAC. Our observations provide molecular insights into
the roles of ligase-deubiquitinase interactions in regulating molecular events
resulting from linear ubiquitin conjugation. The linear ubiquitin (Ub) chain assembly complex (LUBAC) generates Met1-linked
"linear" Ub chains that regulate the activation of the nuclear factor κB (NFκB)
transcription factor and other processes. We recently discovered OTULIN as a
deubiquitinase that specifically cleaves Met1-linked polyUb. Now, we show that
OTULIN binds via a conserved PUB-interacting motif (PIM) to the PUB domain of
the LUBAC component HOIP. Crystal structures and nuclear magnetic resoce
experiments reveal the molecular basis for the high-affinity interaction and
explain why OTULIN binds the HOIP PUB domain specifically. Analysis of
LUBAC-induced NFκB signaling suggests that OTULIN needs to be present on LUBAC
in order to restrict Met1-polyUb signaling. Moreover, LUBAC-OTULIN complex
formation is regulated by OTULIN phosphorylation in the PIM. Phosphorylation of
OTULIN prevents HOIP binding, whereas unphosphorylated OTULIN is part of the
endogenous LUBAC complex. Our work exemplifies how coordination of ubiquitin
assembly and disassembly activities in protein complexes regulates individual Ub
linkage types. Linear ubiquitin chains are implicated in the regulation of the NF-κB pathway,
immunity, and inflammation. They are synthesized by the LUBAC complex containing
the catalytic subunit HOIL-1-interacting protein (HOIP) and are disassembled by
the linear ubiquitin-specific deubiquitinase OTULIN. Little is known about the
regulation of these opposing activities. Here we demonstrate that HOIP and
OTULIN interact and act as a bimolecular editing pair for linear ubiquitin
signals in vivo. The HOIP PUB domain binds to the PUB interacting motif (PIM) of
OTULIN and the chaperone VCP/p97. Structural studies revealed the basis of
high-affinity interaction with the OTULIN PIM. The conserved Tyr56 of OTULIN
makes critical contacts with the HOIP PUB domain, and its phosphorylation
negatively regulates this interaction. Functionally, HOIP binding to OTULIN is
required for the recruitment of OTULIN to the TNF receptor complex and to
counteract HOIP-dependent activation of the NF-κB pathway. Linear ubiquitination is a post-translational protein modification recently
discovered to be crucial for innate and adaptive immune signaling. The function
of linear ubiquitin chains is regulated at multiple levels: generation,
recognition, and removal. These chains are generated by the linear ubiquitin
chain assembly complex (LUBAC), the only known ubiquitin E3 capable of forming
the linear ubiquitin linkage de novo. LUBAC is not only relevant for activation
of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) in
various signaling pathways, but importantly, it also regulates cell death
downstream of immune receptors capable of inducing this response. Recognition of
the linear ubiquitin linkage is specifically mediated by certain ubiquitin
receptors, which is crucial for translation into the intended signaling outputs.
LUBAC deficiency results in attenuated gene activation and increased cell death,
causing pathologic conditions in both, mice, and humans. Removal of ubiquitin
chains is mediated by deubiquitinases (DUBs). Two of them, OTULIN and CYLD, are
constitutively associated with LUBAC. Here, we review the current knowledge on
linear ubiquitination in immune signaling pathways and the biochemical
mechanisms as to how linear polyubiquitin exerts its functions distinctly from
those of other ubiquitin linkage types. Protein ubiquitination, a major post-translational modification in eukaryotes,
requires an adequate pool of free ubiquitin. Cells maintain this pool by two
pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from
ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo.
Although many advances have been made in recent years regarding ubiquitin
recycling, our knowledge on ubiquitin precursor processing is still limited, and
questions such as when are these precursors processed and which DUBs are
involved remain largely uswered. Here we provide data suggesting that two of
the four mammalian ubiquitin precursors, UBA52 and UBA80, are processed mostly
post-translationally whereas the other two, UBB and UBC, probably undergo a
combination of co- and post-translational processing. Using an unbiased
biochemical approach we found that UCHL3, USP9X, USP7, USP5 and
Otulin/Gumby/FAM105b are by far the most active DUBs acting on these precursors.
The identification of these DUBs together with their properties suggests that
each ubiquitin precursor can be processed in at least two different manners,
explaining the robustness of the ubiquitin de novo synthesis pathway. Modification of proteins with Met1-linked 'linear' ubiquitin chains has emerged
as a key regulatory signal to control inflammatory signalling via the master
regulator, the transcription factor nuclear factor κB (NF-κB). While the
assembly machinery, the linear ubiquitin chain assembly complex (LUBAC), and
receptors for this ubiquitin chain type have been known for years, it was less
clear which deubiquitinating enzymes (DUBs) hydrolyse Met1 linkages
specifically. In 2013, two labs reported the previously unotated protein
FAM105B/OTULIN to be this missing Met1 linkage-specific DUB. Structural studies
have revealed how OTULIN achieves its remarkable specificity, employing a
mechanism of ubiquitin-assisted catalysis in which a glutamate residue on the
substrate complements the active site of the enzyme. The specificity of OTULIN
enables it to regulate global levels of Met1-linked polyubiquitin in cells. This
ability led to investigations of NF-κB activation from new angles, and also
revealed involvement of Met1-polyubiquitin in Wnt signalling. Interestingly,
OTULIN directly interacts with LUBAC, and this interaction is dynamic and can be
regulated by OTULIN phosphorylation. This provides a new paradigm for how
individual linkage types can be regulated by dedicated enzyme complexes
mediating assembly and removal. Here we review what has been learned about
OTULIN's mechanism, regulation and function, discuss the open questions in the
field, and discuss how DUBs regulate the NF-κB response. Author information:
(1)Inflammatory Disease Section, National Human Genome Research Institute,
Bethesda, MD 20892;
(2)Genetics and Pathogenesis of Allergy Section, Laboratory of Allergic
Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD
20892;
(3)Familial Mediterranean Fever Arthritis Vasculitis and Orphan Disease Research
Center, Institute of Health Sciences, R&D Center, Gulhane Military Medical
Academy, Ankara 06018, Turkey;
(4)Translational Immunology Section, National Institute of Arthritis and
Musculoskeletal and Skin Diseases, Bethesda, MD 20892;
(5)Department of Laboratory Medicine, National Institutes of Health Clinical
Center, Bethesda, MD 20892;
(6)Laboratory of Cardiovascular Regenerative Medicine, National Heart, Lung, and
Blood Institute, Bethesda, MD 20892;
(7)National Institutes of Health Intramural Sequencing Center, National Human
Genome Research Institute, Rockville, MD 20852;
(8)Heart of England National Health Service Foundation Trust, Birmingham B9 5ST,
United Kingdom;
(9)Department of Pediatric Nephrology and Rheumatology, Faculty of Medicine,
Hacettepe University, Ankara 06100, Turkey;
(10)Institute of Cellular Medicine, Newcastle University, Newcastle NE2 4HH,
United Kingdom.
(11)Inflammatory Disease Section, National Human Genome Research Institute,
Bethesda, MD 20892; [email protected] [email protected]. |
List diseases that could be targeted by disaggregases? | UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. | The structural basis by which Hsp104 dissolves disordered aggregates and prions
is unknown. A single subunit within the Hsp104 hexamer can solubilize disordered
aggregates, whereas prion dissolution requires collaboration by multiple Hsp104
subunits. Here, we establish that the poorly understood Hsp104 N-terminal domain
(NTD) enables this operational plasticity. Hsp104 lacking the NTD (Hsp104(ΔN))
dissolves disordered aggregates but cannot dissolve prions or be potentiated by
activating mutations. We define how Hsp104(ΔN) invariably stimulates Sup35
prionogenesis by fragmenting prions without solubilizing Sup35, whereas Hsp104
couples Sup35 prion fragmentation and dissolution. Volumetric reconstruction of
Hsp104 hexamers in ATPγS, ADP-AlFx (hydrolysis transition state mimic), and ADP
via small-angle X-ray scattering revealed a peristaltic pumping motion upon ATP
hydrolysis, which drives directional substrate translocation through the central
Hsp104 channel and is profoundly altered in Hsp104(ΔN). We establish that the
Hsp104 NTD enables cooperative substrate translocation, which is critical for
prion dissolution and potentiated disaggregase activity. Intracellular amyloid fibrils linked to neurodegenerative disease typically
accumulate in an age-related manner, suggesting inherent cellular capacity for
counteracting amyloid formation in early life. Metazoan molecular chaperones
assist native folding and block polymerization of amyloidogenic proteins,
preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly,
however, is unclear. Here, we show that a specific combination of human Hsp70
disaggregase-associated chaperone components efficiently disassembles
α-synuclein amyloid fibrils characteristic of Parkinson's disease in vitro.
Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110
family nucleotide exchange factor (NEF) provide ATP-dependent activity that
disassembles amyloids within minutes via combined fibril fragmentation and
depolymerization. This ultimately generates non-toxic α-synuclein monomers.
Concerted, rapid interaction cycles of all three chaperone components with
fibrils generate the power stroke required for disassembly. This identifies a
powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid
fibrils and points to crucial yet undefined biology underlying amyloid-based
diseases. |
Can NEECHAM Confusion Scale be used for evaluation of postoperative delirium? | Yes, NEECHAM Confusion Scale can be used for evaluation of postoperative delirium. | PURPOSE: To determine if elderly patients undergoing hip surgery became
delirious postoperatively and, if so, whether age and/or time of day were
related to delirium.
DESIGN: Repeated measures.
SAMPLE: A convenience sample of 70 hip surgery patients 60 years of age and
older at a large Midwestern teaching hospital were studied. Patients were
excluded who were unconscious, unable to hear, see, and/or verbally communicate
in English. Patients were also excluded who had a known history of dementia,
Alzheimer's dementia, addiction to alcohol and/or sedative hypnotics, functional
psychosis, or any other psychiatric diagnosis. Of the 70 patients, 37 were
female and 33 were male. Mean age of the patients was 72.9 years (S.D. = 8.13).
Patients were placed into one of three groups: Group 1--age 60 to 69 years (n =
25); Group 2--age 70 to 79 years (n = 25); or Group 3--80 years and older (n =
20). The most common procedure for all groups was total hip replacement (n =
48).
METHOD: Data were collected primarily by both objective and subjective
assessment of the patients. Both the Folstein's Mini Mental Status Exam (MMSE)
and the NEECHAM Confusion Tool were used to collect data. Chart reviews provided
additional data. Patients were assessed preoperatively to obtain baseline
assessment and screen out patients with preexisting confusion. Assessments were
then done once in the morning and once in the evening for 5 days following
surgery.
MAIN RESEARCH CLASSIFICATIONS: Delirium, sundowning, sundown syndrome.
FINDINGS: The MMSE and NEECHAM were found to be highly correlated: Morning
NEECHAM vs morning MMSE (Correlation Coefficient = .6515; p = .000), evening
NEECHAM vs evening MMSE (Correlation Coefficient = .8301; p = .000). A test of
repeated measures was used to examine the data. The Within factor was time, the
Between factor was age, and the interaction effect was age by time of day.
Dependent variables were total NEECHAM scores and total MMSE scores, in addition
to total scores of these tests' subsections. An alpha level of .05 was used for
all statistical tests. Age had a significant main effect on the NEECHAM (F =
7.44; p = .001) and MMSE (F = 6.04; p = .004). Time did not have a significant
effect on either the MMSE (F = .00; p = .953) or the NEECHAM (F = .43; p =
.513). There was no statistically significant interaction between age and time
of day (NEECHAM, F = .97, p = .384 and MMSE (F = .19, p = .826). No subjects
were assessed as demented per the MMSE. Only 12 episodes of acute confusion were
noted using the NEECHAM.
CONCLUSION: Patients in this population rarely, if ever, became confused. The
older the individual, the more likely he/she was to have confusion. Time of day
was neither significant in development of delirium nor on mental status
assessment scores in this population.
IMPLICATIONS FOR NURSING RESEARCH: Current research literature notes that 10% to
50% of elderly postoperative patients experience delirium. Patients who have had
femoral neck fractures can experience delirium three times more than patients
having nonorthopaedic surgery. This study found that delirium in hip surgery
patients is rare. A study of the more subtle components of delirium such as
attention and memory might reveal less obvious changes in mental status
following surgery. Type of orthopaedic surgery also might impact incidence of
delirium. A comparative study between elderly total knee and total hip
replacement patients would be interesting. Studying patients who have emergency
hip surgery related to fracture in comparison to patients having elective hip
surgery for degenerative conditions might identify other etiologies for delirium
following hip surgery. Fatigue rather than time of day might be studied to
observe whether it has a significant impact on mental status or delirium scores. Acute confusion (AC), also referred to as delirium (AC/delirium), is a common
problem seen by health professionals who work in a variety of care settings.
This is an evaluative report on the clinical usability of instruments to assess
AC/delirium as a part of nursing practice. Specifically, five instruments [the
Confusion Assessment Method (CAM), Delirium Rating Scale (DRS), Delirium Symptom
Inventory (DSI), Mini-Mental State Examination (MMSE), and Neelon/Champagne
(NEECHAM) Confusion Scale] are discussed. The work demonstrates how the
cooperation of nurses in practice, education, and research can improve both
patient and staff outcomes. Delirium is a severe psychiatric syndrome that is highly prevalent in elderly
general hospital patients. However, the diagnosis of delirium is often missed.
The use of rating scales can be helpful in detecting and measuring delirium
symptom severity. This article reviews recent developments with regard to
psychometric qualities, measurement goal, content and rating procedures of some
of the available rating scales in clinical practise. Studies that used delirium
rating scales were searched for using the MEDLINE and subsequent examination of
reference lists. Ten rating scales were selected for further evaluation. The
Confusional Assessment Method (CAM), NEECHAM Confusion Scale (NEECHAM) and
Delirium Observation Scale (DOS) appear to be most suitable as a screening
instrument, depending on the type of rater (physician or nurse). The (revised)
Delirium Rating Scale (DRS(-R-98)) seems to be particularly useful for measuring
delirium severity or monitoring change. BACKGROUND: Determination of a patient's cognitive status by use of a valid and
reliable screening instrument is of major importance as early recognition and
accurate diagnosis of delirium is necessary for effective management. This study
determined the reliability, validity and diagnostic value of the Flemish
translation of the NEECHAM Confusion Scale.
METHODS: A sample of 54 elderly hip fracture patients with a mean age of 80.9
years (SD = 7.85) were included. To test the psychometric properties of the
NEECHAM Confusion Scale, performance on the NEECHAM was compared to the
Confusion Assessment Method (CAM) and the Mini-Mental State Examination (MMSE),
by using aggregated data based on 5 data collection measurement points (repeated
measures). The CAM and MMSE served as gold standards.
RESULTS: The alpha coefficient for the total NEECHAM score was high (0.88).
Principal components analysis yielded a two-component solution accounting for
70.8% of the total variance. High correlations were found between the total
NEECHAM scores and total MMSE (0.75) and total CAM severity scores (-0.73),
respectively. Diagnostic values using the CAM algorithm as gold standard showed
76.9% sensitivity, 64.6% specificity, 13.5% positive and 97.5% negative
predictive values, respectively.
CONCLUSION: This validation of the Flemish version of the NEECHAM Confusion
Scale adds to previous evidence suggesting that this scale holds promise as a
valuable screening instrument for delirium in clinical practice. Further
validation studies in diverse clinical populations; however, are needed. The aim of the present study was to identify clinical signs detective of the
postoperative delirium at the early stage for nursing management. A total of 66
inpatients undergoing cardiac surgery were interviewed using the Delirium Rating
Scale (DRS) and NEECHAM Confusion Scale (NCS) preoperatively and on days 1 and 3
postoperatively. The mean onset of delirium occurred on postoperative day 1.3.
Development of delirium was detected early by cognitive impairments in the DRS
subscales of perceptual disturbance, hallucination, and cognitive status, and
the NCS subscales of attention, command, orientation, and verbal skill. These
results suggest that assessment of cognitive status on postoperative day is an
important strategy in the early detection of postoperative delirium. BACKGROUND: Several reports indicate a high incidence of intensive care
delirium. To develop strategies to prevent this complication, validated
instruments are needed. The Confusion Assessment Method for the Intensive Care
Unit (CAM-ICU) is widely used. A binary result diagnoses delirium. The Neelon
and Champagne (NEECHAM) Confusion Scale recently has been validated for use in
the ICU and has a numeric assessment. This scale allows the patients to be
classified in four categories: non-delirious, at risk, confused, and delirious.
In this study, we investigated the results of the NEECHAM scale in comparison
with the CAM-ICU.
METHODS: A consecutive sample of 172 non-intubated patients in a mixed ICU was
assessed after a stay in the ICU for at least 24 hours. All adult patients with
a Glasgow Coma Scale score of greater than 9 were included. A nurse researcher
simultaneously assessed both scales once daily in the morning. A total of 599
paired observations were made.
RESULTS: The CAM-ICU showed a 19.8% incidence of delirium. The NEECHAM scale
detected incidence rates of 20.3% for delirious, 24.4% for confused, 29.7% for
at risk, and 25.6% for normal patients. The majority of the positive CAM-ICU
patients were detected by the NEECHAM scale. The sensitivity of the NEECHAM
scale was 87% and the specificity was 95%. The positive predictive value and the
negative predictive value were 79% and 97%, respectively. The diagnostic
capability in cardiac surgery patients proved to be lower than in other
patients.
CONCLUSION: In non-intubated patients, the NEECHAM scale identified most cases
of delirium which were detected by the CAM-ICU. Additional confused patients
were identified in the categorical approach of the scale. The NEECHAM scale
proved to be a valuable screening tool compared with the CAM-ICU in the early
detection of intensive care delirium by nurses. BACKGROUND: The incidences of surgery-field disorders such as femur neck
fracture and colorectal cancer in elderly persons have increased with the rapid
aging of society. In such patients, postoperative delirium is also frequent.
Patients should be generally assessed from the aspect of both physical and
mental conditions in order to predict a high-delirium risk group. If so,
delirium may be prevented more efficiently. In this study, we investigated
whether the early detection of postoperative delirium in elderly patients is
possible using a simple, useful behavior-assessing scale, the NEECHAM Confusion
Scale, and a method for comprehensively evaluating elderly persons' stress
related to surgery, E-PASS.
METHODS: The subjects were 160 patients aged more than 75 years who underwent
surgery. Among them, three patients had vascular surgery-field disorders, 67 had
orthopedic-field disorders, and 90 had digestive surgery-field disorders. To
comprehensively evaluate surgery-related stress, E-PASS was employed. In
addition, we assessed recognition, activities of daily living (ADL), and the
quality of life (QOL). For delirium diagnosis and severity assessment, we used
the NEECHAM Confusion Scale. The cut-off value of the NEECHAM score was
established as 20 points, and patients showing values less than this after
surgery were regarded as having postoperative delirium. Evaluation was performed
until 10 days after surgery.
RESULTS: Postoperative delirium was noted in 54.7% of the subjects. There was a
decrease in the NEECHAM score between the first and fourth postoperative days,
but it gradually increased thereafter. Both uni- and multivariate analyses
showed that postoperative delirium was associated with an advanced age (more
than 80 years), low preoperative NEECHAM and MMSE scores, the preoperative QOL,
and E-PASS. In groups showing an MMSE score of less than 25 or a preoperative
NEECHAM score of less than 27, the incidence of postoperative delirium was 76%.
CONCLUSION: The results suggest that E-PASS and the NEECHAM score facilitate
assessment of the risk of postoperative delirium in elderly patients,
contributing to early prevention/treatment. PURPOSE: To determine if there is an association between perioperative
administration of beta-blockers and postoperative delirium in patients
undergoing vascular surgery.
METHODS: After Institutional Review Board approval, data were retrospectively
collected on patients who underwent vascular surgery in an academic hospital
during the period January 2006 to January 2007. Patients with preoperative
altered level of consciousness, carotid endarterectomy, or discharge within 24 h
of surgery were excluded from the study. Identification of delirium was based on
evaluation of the level of consciousness with the NEECHAM Confusion Scale and/or
a chart-based instrument for delirium. Multivariable logistic regression
analysis was used to identify independent perioperative predictors of
postoperative delirium. Beta-blockers were tested for a potential effect.
RESULTS: The incidence of postoperative delirium was 128/582 (22%). Independent
predictors included age (OR 1.04, 95% CI [1.02-1.07]), history of
cerebrovascular accident/transient ischemic attack (OR 2.64, 95% CI
[1.57-4.55]), and depression (OR 3.56, 95% CI [1.53-8.28]). Open aortic
reconstruction was associated with an OR of 5.34, 95% CI (2.54-11.2) and
amputation with an OR of 4.66, 95% CI (1.96-11.09). Preoperative beta-blocker
administration increased the odds of postoperative delirium 2.06 times (95% CI
[1.18-3.6]). Statin administration reduced the odds of delirium by 44% (95% CI
[0.37-0.88]). The model was reliable (Hosmer-Lemeshow test, P = 0.72) and
discriminative (area under the receiver operating characteristic [ROC] curve =
0.729).
CONCLUSIONS: Preoperative administration of beta-blockers is associated with an
increased risk of postoperative delirium after vascular surgery. Conversely,
preoperative statin administration is associated with a lower risk of
postoperative delirium. A randomized prospective controlled trial is required to
validate these findings. OBJECTIVES: The prevention of delirium is an important issue in the field of
perioperative nursing. The objective of this study was to verify the usefulness
of acute-stage bright light exposure on patients following oesophagectomy.
METHODS: The participants were oesophagectomy patients that were removed from
their ventilators the day after surgery. After extubation, we assigned the
participants to either the exposure group or control group. At Day 2 after
surgery, the exposure group underwent two hours of bright light exposure for
four days. In both groups, we monitored physical activity and autonomic
activity. In addition, we scored the participants on the NEECHAM Scale and
evaluated their postoperative delirium and postoperative arrhythmia.
RESULTS: On the nights of Days 4 and 5, the amount of activity of the exposure
group was significantly lower and The sympathetic nervous index was
significantly lower on the night of Day 5. The level of arrhythmia was lower in
the exposure group and we observed a significant difference on the night of Day
4 and the daytime of Day 5 after surgery. The occurrence rate of postoperative
delirium tended to be lower in the exposure group, but there was no significant
difference. None of the participants in the exposure group had NEECHAM Scale
scores below the cut-off value from the night of Day 4 onwards.
CONCLUSION: We conclude that postoperative bright light exposure adjusted the
sleep-wakefulness cycle and improved the bed rest of patients. It was also
indicated that bright light therapy is useful for reducing postoperative
delirium. BACKGROUND: Delirium is common among older people in hospital and various
instruments have been developed for detecting delirium. One of these, the
NEECHAM Confusion Scale, is easy for nurses to administrate but needs to be
tested further.
AIMS AND OBJECTIVES: The aim of the present study was to assess the validity and
predictive value of the NEECHAM Confusion Scale.
METHODS: The study was conducted among 149 patients aged ≥ 65, who had undergone
surgery for a hip fracture. The patients were observed daily using DSM-IV
criteria for delirium. The NEECHAM Confusion Scale was performed upon admission
and prior to discharge.
RESULTS: The incidence of DSM-IV related delirium was 24%. Patients who scored
below 25 points on the NEECHAM scale had a 12 times higher risk of developing
DSM-IV related delirium. During admission, the sensitivity of NEECHAM was zero
because all patients with DSM-IV delirium were excluded, the specificity was
75%. On discharge, it was 100% and 91% respectively.
CONCLUSIONS: This study adds to the body of knowledge that NEECHAM discriminates
for delirium. It is a valid and reliable screening instrument for predicting
delirium. The instrument can be used for clinical practice to identify patients
who are at risk of contracting delirium and when considering prevention
measures. PURPOSE: Our objective was to compare open and endovascular aortic aneurysm
repair with respect to postoperative delirium.
METHODS: After Institutional Ethics Review Board approval, we conducted a
retrospective review of all patients who underwent abdominal and
thoraco-abdominal aortic aneurysm repair surgery at Toronto General Hospital
during June 2006 to December 2007. Patients were classed into either the OPEN or
the endovascular (EVAR) group based on the type of surgery and were assessed for
the presence of delirium after surgery. The NEECHAM Confusion Scale and the
validated chart review instrument were used for diagnosis of delirium. Patients
with dementia and/or abnormal levels of consciousness preoperatively were
excluded.
RESULTS: There were 256 patients included in the study, 149 (58%) in the OPEN
group and 107 (42%) in the EVAR group. Patients in the EVAR group were
considerably older, 74 (10) yr vs 68 (9) yr, and they had shorter duration of
surgery, 150 [119, 180] min vs 200 [165, 260] min, respectively, P < 0.0001.
Postoperative delirium was present in 43 (29%) patients in the OPEN group and 14
(13%) patients in the EVAR group (95% confidence interval [CI], 22 to 36 vs 95%
CI, 7 to 19, respectively; P = 0.003). Hospital length of stay was 8.3 [6.6,
13.4] days in the OPEN group and 4.5 [3.1, 6.4] days in the EVAR group, P <
0.0001.
CONCLUSIONS: Perioperative management of patients undergoing endovascular aortic
aneurysm repair was associated with lower rates of delirium after surgery than
that of patients undergoing open aortic aneurysm repair. AIMS AND OBJECTIVES: To estimate the diagnostic value and determine the
feasibility of the NEECHAM Confusion Scale on critically ill older patients.
BACKGROUND: Delirium is a common syndrome in hospitalised older patients,
especially in surgical intensive care units, and the consequences of
under-detection can be very serious for older people. Therefore, assessment of
the cognitive status of older patients using a valid instrument is important in
intensive care units.
DESIGN: A descriptive prospective design was used.
METHODS: Consecutive nonintubated patients aged 65 and older, admitted to a
surgical intensive care unit of an Italian hospital during a seven months
period, were assessed for delirium using the NEECHAM scale and the Confusion
Assessment Method for intensive care unit, once per shift, for 48 hours after
admission. Cohen's kappa coefficient, ROC curve, sensitivity and specificity
were estimated. An open ended questionnaire was used to assess user-friendliness
of the scale.
RESULTS: A sample of 41 older patients with a mean age of 78·3 years was
studied. The kappa coefficient was 0·95. The sensitivity was 99·19%, specificity
95% at cut-off of 25, and the area under the curve was 0·99 (CI 0·99-1·00).
Nurses evaluated positively the scale as they were able to collect data during
care process in maximum 10 minutes, but experienced problems in rating the
appearance behaviour and physiological control items of the scale.
CONCLUSIONS: Findings from this study confirm the good diagnostic value and ease
of application of the NEECHAM scale with nonventilated intensive care patients.
RELEVANCE TO CLINICAL PRACTICE: The NEECHAM scale can be used to detect delirium
during the routine nursing assessment of nonintubated older patients as it
requires minimal demand and stress on the patient as well as on the bedside
nurse. Over the years many scales have been designed for screening, diagnosis and
assessing the severity of delirium. In this paper we review the various
instruments available to screen the patients for delirium, instruments available
to diagnose delirium, assess the severity, cognitive functions, motoric
subtypes, etiology and associated distress. Among the various screening
instruments, NEECHAM confusion scale and delirium observation scale appear to be
most suitable screening instrument for patients' in general medical and surgical
wards, depending on the type of rater (physician or nurse). In general, the
instruments which are used for diagnosis [i.e., confusion assessment method
(CAM), CAM for intensive care unit (CAM-ICU), Delirium Rating Scale-revised
version (DRS-R-98), memorial selirium assessment scale, etc.] are based on
various Diagnostic and Statistical Manual criteria and have good to excellent
reliability and fair to good validity. Among the various diagnostic instruments,
CAM is considered to be most useful instrument because of its accuracy, brevity,
and ease of use by clinicians and lay interviewers. In contrast, DRS-R-98
appears to be a comprehensive instrument useful for diagnosis, severity rating
and is sensitive to change and hence can be used for monitoring patients over a
period. In the ICU setting, evidence suggests that CAM-ICU and Nursing Delirium
Screening Scale had comparable sensitivities, but CAM-ICU has higher
specificity. With regard to assessment of delirium in pediatric age group,
certain instruments like Pediatric Anesthesia Emergence Delirium scale and
pediatric CAM-ICU has been designed and have been found to be useful. PURPOSE: The purpose of this study was to develop and test a scale for
predicting POD in patients undergoing cerebrovascular surgery.
METHODS: The predictive scale for POD was composed of 32 items reflecting the
strongest risk factors as determined by a literature review. The NEECHAM
Confusion Scale determined POD onset and severity.
RESULTS: Delirium developed in 38 (31.1%) of the 122 patients in our sample.
Logistic regression revealed the following risk factors: dehydration, age,
disturbance of consciousness, underlying illness, and anxiety or depression. The
final scale was weighted by referring to odds ratios. The area under the curve
was 0.844 (95% CI=0.766-0.921). The possible total score on this scale was 20
points. A cutoff score of 11 was set for risk of POD (patients scoring over 12
were considered at higher risk). The median score was 8 (range: 4-9) in the
non-delirium group and 13 (range: 9-16) in the delirium group (U=499.0; df=120;
p<0.001). Scale scores were moderately correlated with delirium duration
(ρ=0.532; p<0.001).
CONCLUSION: The present scale is a promising a tool for predicting POD but needs
to be studied further. OBJECTIVE: The objective of the present study is to determine the incidence of
delirium and the associated factors in patients undergoing open heart surgery.
METHOD: This is an Analytic-descriptive study conducted on 404 patients
undergoing elective open heart surgery in Fatemeh Zahra Heart Center, Sari, over
the period of 6 months from July to December 2011. Sampling was achieved in a
nonrandomized targeted manner and delirium was assessed using NeeCham
questionnaire. A trained nurse evaluated the patients for delirium and completed
the risk factor checklist on days 1 to 5 after surgery. Data analyses were
accomplished using survival analysis (Kaplan-Meier and Cox regression) on SPSS
software version 15.
RESULTS: We found that variables, including ventilation time, increased drainage
during the first 24 hours, the need for re-operation in the first 24 hours,
dysrhythmias, use of inotropic agents, increased use of analgesics, increased
arterial carbon dioxide, lack of visitors, and use of physical restrainers were
associated with the development of delirium. In addition, we found a delirium
incidence of 29%.
CONCLUSION: Diagnosis of cognitive disorders is of utmost value; therefore,
further studies are required to clarify the risk factors because controlling
them will help prevent delirium. BACKGROUND/AIMS: Despite the presence of several diagnosis scales for delirium,
no prediction scale that is specific for postoperative delirium after abdominal
surgery is available. We sought to create a novel delirium prediction system
that is specific for abdominal surgery.
METHODS: This study included 213 consecutive patients who required management in
the surgical ICU following abdominal surgery. The Neelon and Champagne (NEECHAM)
Confusion score was monitored throughout the postoperative course and patients
with low NEECHAM score (≤26) were diagnosed as having delirium.
RESULTS: Seventy-three patients (34%) were categorized in the delirium group.
Multivariate analyses indicated that an age >70 years, hypertension, those
undergoing hepatopancreatobiliary or upper gastrointestinal surgeries, a serum
albumin level <2.5 g/dl on postoperative day (POD) 3 or 5 and a ≥6 mEq/l gap in
the serum sodium level between the preoperative value and that on POD 3 were
independently associated with a low NEECHAM score (≤26). When the presence of
each risk was counted as 1 point, 21 patients had ≥4 points and 15 of them (71%)
had low NEECHAM score.
CONCLUSION: The scoring system combining multiple risk factors may be useful for
predicting patients with an elevated risk for postoperative delirium after
abdominal surgery. |
What is adhesive capsulitis | Adhesive capsulitis also known as "frozen shoulder" is characterized by painful, gradual loss of active and passive shoulder motion resulting from fibrosis and contracture of the joint capsule. | Adhesive capsulitis of the shoulder is an idiopathic condition characterized
clinically by pain and limitation of movement. The characteristic arthrographic
features are those of limited capacity of the joint, pain during injection, and
retracted recesses. Progressive distension of the capsule with 1% lidocaine and
40 mg of triamcinolone acetonide (Kenalog) is a recommended form of treatment
for this condition. Twenty-five patients treated by this method were reviewed
retrospectively. Eleven (44%) were completely relieved of their symptoms, 6
(24%) had a 50% improvement, and 8 (32%) had no relief. Patients with idiopathic
adhesive capsulitis responded more favorably to the treatment than those with a
traumatic etiology. The duration of symptoms had no correlation with the results
of therapy. There were no complications from the treatment. Although adhesive capsulitis of the shoulder is a common clinical syndrome,
adhesive capsulitis in other joints has rarely been reported. Four patients who
developed adhesive capsulitis of the hip joint after trauma and two patients
with adhesive capsulitis of the ankle, also precipitated by trauma, are
described. The diagnosis of adhesive capsulitis is easily made during
arthrography when a joint of low volume and high intracapsular pressure is
encountered. OBJECTIVE: Adhesive capsulitis is a clinical syndrome of pain and severely
decreased joint motion ("frozen shoulder") caused by thickening and contraction
of the joint capsule and synovium. Although arthrographic criteria for the
diagnosis have been described, to our knowledge, the MR characteristics have not
been reported. Accordingly, we studied the MR findings in 10 patients with this
syndrome.
MATERIALS AND METHODS: MR images of 25 subjects were included in the study. Nine
had adhesive capsulitis documented by arthrography, and one had adhesive
capsulitis proved at surgery. The MR findings in these patients were compared
with those of 15 asymptomatic volunteers. Images were assessed for thickness of
the joint capsule and synovium, for thickness of the coracohumeral ligament, and
for volume of articular fluid. Capsule and synovium thickness was measured
adjacent to the axillary recess. The volume of intraarticular fluid was
calculated from direct measurements of the axillary recess and biceps tendon
sheath. The rotator cuff interval was qualitatively evaluated for the presence
of abnormal tissue.
RESULTS: Thickening of capsule and synovium on MR images was characteristic of
adhesive capsulitis, with a significant difference between mean thickness in
patients with adhesive capsulitis (5.2 mm) and in asymptomatic volunteers (2.9
mm) (p < .01). Capsule and synovium thickness greater than 4 mm was a specific
(95%) and sensitive (70%) criterion for the diagnosis of adhesive capsulitis.
There was no significant difference in volume of articular fluid or thickness of
the coracohumeral ligament between patients with adhesive capsulitis and
asymptomatic volunteers (p > .5). The rotator cuff interval was not useful for
assessing changes of adhesive capsulitis.
CONCLUSION: Joint capsule and synovium thickness greater than 4 mm is a useful
MR criterion for the diagnosis of adhesive capsulitis. The volume of articular
fluid seen on MR images is not significantly diminished in patients with
adhesive capsulitis. Adhesive capsulitis of the hip is a not a common clinical presentation. We
report a case of adhesive capsulitis of the hip in a patient with hypothyroidism
and previous adhesive capsulitis of the shoulder who was receiving
thyroid-hormone replacement. The adhesive capsulitis of both hip and shoulder
were treated successfully with physical therapy. Orthopedic surgeons should be
aware of this diagnosis and its association with shoulder adhesive capsulitis
and thyroid dysfunction, to allow them to recognize it and intervene early. Painful stiffening of the shoulder, 'frozen shoulder' is a common cause of
shoulder pain and disability. Continuous passive motion (CPM) is an established
method of preventing joint stiffness and of overcoming it. A randomized,
comparative prospective clinical trial was planned to compare the early response
with different rehabilitation methods [CPM vs. conventional physiotherapy
treatment (CPT) protocol] for adhesive capsulitis taking into consideration the
clinical efficacy. A total of 57 patients with frozen shoulder were included in
this study. Patients were assigned randomly to receive daily CPM treatments or
CPT protocol. Parameters were measured at baseline, and at weeks 4 and 12. All
patients were evaluated with respect to pain (visual anologue scale) at rest,
pain at movement, pain at night, measurement of range of motion (shoulder
flexion, abduction, internal-external rotation were assessed), constant
functional shoulder score and the shoulder pain and disability index. The first
group (n=29) (CPM group) received CPM treatments for 1 h once a day for 20 days
during a period of 4 weeks. The second group (n=28) (CPT group) had a daily
physiotherapy treatment protocol including active stretching and pendulum
exercises for 1 h once a day for 20 days during a period of 4 weeks. All
patients in both groups were also instructed in a standardized home exercise
programme consisting of passive range of motion and pendulum exercises to be
performed every day. In both groups, statistically significant improvements were
detected in all outcome measures compared with baseline. Pain reduction,
however, evaluated with respect to pain at rest, at movement and at night was
better in CPM group. In addition the CPM group showed better shoulder pain index
scores than the CPT group. CPM treatment provides better response in pain
reduction than the conventional physiotherapy treatment protocol in the early
phase of treatment in adhesive capsulitis. BACKGROUND: Adhesive capsulitis often is difficult to diagnose in its early
stage and to differentiate from other commonly seen shoulder disorders with the
potential to cause pain and limited range of movement.
OBJECTIVES: The purpose of this study was to establish consensus among a group
of experts regarding the clinical identifiers for the first or early stage of
primary (idiopathic) adhesive capsulitis.
DESIGN: A correspondence-based Delphi technique was used in this study.
METHODS: Three sequential questionnaires, each building on the results of the
previous round, were used to establish consensus.
RESULTS: A total of 70 experts from Australia and New Zealand involved in the
diagnosis and treatment of adhesive capsulitis completed the 3 rounds of
questionnaires. Following round 3, descriptive statistics were used to screen
the data into a meaningful subset. Cronbach alpha and factor analysis then were
used to determine agreement among the experts. Consensus was achieved on 8
clinical identifiers. These identifiers clustered into 2 discrete domains of
pain and movement. For pain, the clinical identifiers were a strong component of
night pain, pain with rapid or unguarded movement, discomfort lying on the
affected shoulder, and pain easily aggravated by movement. For movement, the
clinical identifiers included a global loss of active and passive range of
movement, with pain at the end-range in all directions. Onset of the disorder
was at greater than 35 years of age.
CONCLUSIONS: This is the first study to use the Delphi technique to establish
clinical identifiers indicative of the early stage of primary (idiopathic)
adhesive capsulitis. Although limited in differential diagnostic ability, these
identifiers may assist the clinician in recognizing early-stage adhesive
capsulitis and may inform management, as well as facilitate future research. Adhesive capsulitis is one of the most common conditions affecting the shoulder;
however, early clinical diagnosis can be challenging. Treatment is most
effective when commenced prior to the onset of capsular thickening and
contracture; consequently, the role of imaging is increasing. The aim of this
review is to demonstrate the typical imaging appearances of adhesive capsulitis
and to examine some of the evidence regarding each of these imaging modalities.
An evaluation of the various management options available to the clinician is
also presented. BACKGROUND: Adhesive capsulitis is often difficult to diagnose in its early
stage and to differentiate from other common shoulder disorders.
OBJECTIVE: The aim of this study was to validate any or all of the 8 clinical
identifiers of early-stage primary/idiopathic adhesive capsulitis established in
an earlier Delphi study.
DESIGN: This was a cross-sectional study.
METHODS: Sixty-four patients diagnosed with early-stage adhesive capsulitis by a
physical therapist or medical practitioner were included in the study. Eight
active and 8 passive shoulder movements and visual analog scale pain scores for
each movement were recorded prior to and immediately following an
intra-articular injection of corticosteroid and local anesthetic. Using the
local anesthetic as the reference standard, pain relief of ≥70% for passive
external rotation was deemed a positive anesthetic response (PAR).
RESULTS: Sixteen participants (25%) demonstrated a PAR. Univariate logistic
regression identified that of the proposed identifiers, global loss of passive
range of movement (odds ratio [OR]=0.26, P=.03), pain at the end of range of all
measured active movements (OR=0.06, P=.02), and global loss of passive
glenohumeral movements (OR=0.23, P=.02) were associated with a PAR. Following
stepwise removal of the variables, pain at the end of range of all measured
active movements remained the only identifier but was associated with reduced
odds of a PAR.
LIMITATIONS: The lack of a recognized reference standard for diagnosing
early-stage adhesive capsulitis remains problematic in all related research.
CONCLUSIONS: None of the clinical identifiers for early-stage adhesive
capsulitis previously proposed by expert consensus have been validated in this
study. Clinicians should be aware that commonly used clinical identifiers may
not be applicable to this stage. PURPOSE: The present study investigated the prevalence and risk factors of
adhesive capsulitis of the shoulder in breast cancer patients between 13 and
18 months after surgery.
METHODS: This study included 271 women who underwent surgery for breast cancer
with a postoperative period of 13-18 months. Current adhesive capsulitis was
defined as restriction of external rotation and one or more additional
directional restrictions with history of shoulder pain. Cumulative adhesive
capsulitis was defined as current adhesive capsulitis or a previous history of
adhesive capsulitis after breast cancer surgery. Multivariate logistic
regression analysis was performed to examine associations between current or
cumulative adhesive capsulitis and potential risk factors.
RESULTS: Among the 271 study patients, 28 (10.3%) and 21 (7.7%) had cumulative
or current adhesive capsulitis, respectively. The incidences of cumulative and
current adhesive capsulitis were higher in those aged 50-59 years (odds ratio
[OR], 9.912; 95% confidence interval [CI], 1.790-54.880; and OR, 12.395; 95% CI,
1.187-129.444, respectively) and those who underwent mastectomy (OR, 6.805; 95%
CI, 1.800-25.733; and OR, 9.645; 95% CI, 2.075-44.829, respectively) or
mastectomy with reconstruction (OR, 13.122; 95% CI, 2.488-69.218; and OR,
20.075; 95% CI, 2.873-140.261, respectively).
CONCLUSIONS: Adhesive capsulitis of the shoulder is a common problem after
breast cancer treatment. An age of 50-59 years and mastectomy are major risk
factors for adhesive capsulitis, and breast reconstruction additionally
increases the risk. Patients with these risk factors require greater attention
for early diagnosis and proper treatment. |
Describe the applicability of Semantic MediaWiki in the case of FANTOM5 | To make the heterogeneous data set accessible and useful for investigators, a web-based database called Semantic catalog of Samples, Transcription initiation And Regulators (SSTAR) has been developed. SSTAR utilizes the open source wiki software MediaWiki along with the Semantic MediaWiki (SMW) extension, which provides flexibility to model, store, and display a series of data sets produced during the course of the FANTOM5 project. The use of SMW demonstrates the utility of the framework for dissemination of large-scale analysis results. SSTAR is a case study in handling biological data generated from a large-scale research project in terms of maintenance and growth alongside research activities | Author information:
(1)Division of Genomic Technologies (DGT), RIKEN Center for Life Science
Technologies (CLST), Kanagawa 230-0045, Japan.
(2)RIKEN Omics Science Center (OSC), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama
230-0045, Japan.
(3)Division of Genomic Technologies (DGT), RIKEN Center for Life Science
Technologies (CLST), Kanagawa 230-0045, Japan RIKEN Omics Science Center (OSC),
1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
(4)RIKEN Omics Science Center (OSC), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama
230-0045, Japan RIKEN Preventive Medicine and Diagnosis Innovation Program,
Wako, Saitama 351-0198, Japan.
(5)Division of Genomic Technologies (DGT), RIKEN Center for Life Science
Technologies (CLST), Kanagawa 230-0045, Japan RIKEN Omics Science Center (OSC),
1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan Harry Perkins Institute
of Medical Research, QEII Medical Centre and Centre for Medical Research, the
University of Western Australia, Nedlands, Western Australia, Australia.
(6)Division of Genomic Technologies (DGT), RIKEN Center for Life Science
Technologies (CLST), Kanagawa 230-0045, Japan [email protected].
(7)Division of Genomic Technologies (DGT), RIKEN Center for Life Science
Technologies (CLST), Kanagawa 230-0045, Japan RIKEN Omics Science Center (OSC),
1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan RIKEN Preventive
Medicine and Diagnosis Innovation Program, Wako, Saitama 351-0198, Japan
Preventive Medicine and Applied Genomics Unit, RIKEN Advanced Center for
Computing and Communication, Kanagawa 230-0045, Japan [email protected]. |
Is there an association between Muenke Syndrome and FGFR3 gene mutation? | Yes, Muenke syndrome is caused by point mutation (C749G) in the Fibroblast Growth Factor Receptor3 (FGFR3) gene. It affects 1 in 30,000 newborns and accounts for 25% to 30% of genetic causes of craniosynostosis. | Achondroplasia, the most common form of short-limbed dwarfism in humans, occurs
between 1 in 15,000 and 40,000 live births. More than 90% of cases are sporadic
and there is, on average, an increased paternal age at the time of conception of
affected individuals. More then 97% of persons with achondroplasia have a
Gly380Arg mutation in the transmembrane domain of the fibroblast growth factor
receptor (FGFR) 3 gene. Mutations in the FGFR3 gene also result in
hypochondroplasia, the lethal thanatophoric dysplasias, the recently described
SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans)
dysplasia, and two craniosynostosis disorders: Muenke coronal craniosynostosis
and Crouzon syndrome with acanthosis nigricans. Recent evidence suggests that
the phenotypic differences may be due to specific alleles with varying degrees
of ligand-independent activation, allowing the receptor to be constitutively
active. Since the Gly380Arg achondroplasia mutation was recognized, similar
observations regarding the conserved nature of FGFR mutations and resulting
phenotype have been made regarding other skeletal phenotypes, including
hypochondroplasia, thanatophoric dysplasia, and Muenke coronal craniosynostosis.
These specific genotype-phenotype correlations in the FGFR disorders seem to be
unprecedented in the study of human disease. The explanation for this high
degree of mutability at specific bases remains an intriguing question. Fibroblast growth factors are structurally related proteins associated with cell
growth, differentiation, migration, wound healing, angiogenesis, and
oncogenesis. At the cellular level, their function is mediated by transmembrane
tyrosinekinase receptors, fibroblast growth factor receptors. Four genes
encoding fibroblast growth factor receptors have been identified, and mutations
in three of these, FGFR1, FGFR2, and FGFR3, can cause different congenital,
autosomal domit disorders affecting the craniofacial and skeletal
development: craniosynostosis and chondrodysplasias. The craniosynostosis
syndromes: Apert syndrome, Beare-Stevenson syndrome, Crouzon syndrome,
Jackson-Weiss syndrome, Muenke syndrome, Pfeiffer syndrome and Saethre-Chotzen
syndrome can be caused by mutation in either FGFR1, FGFR2, or FGFR3.
Saethre-Chotzen syndrome can also be caused by mutation in a functionally
related gene, ACS. The same mutation can cause different syndromes, and the same
syndrome can be caused by mutations in different genes. The chondrodysplasias:
achondroplasia, hypochondroplasia, and thanatophoric dysplasia are all caused by
mutations in FGFR3. Identical proline-->arginine gain-of-function mutations in fibroblast growth
factor receptor (FGFR) 1 (Pro252Arg), FGFR2 (Pro253Arg) and FGFR3 (Pro250Arg),
result in type I Pfeiffer, Apert and Muenke craniosynostosis syndromes,
respectively. Here, we characterize the effects of proline-->arginine mutations
in FGFR1c and FGFR3c on ligand binding using surface plasmon resoce and X-ray
crystallography. Both Pro252Arg FGFR1c and Pro250Arg FGFR3c exhibit an
enhancement in ligand binding in comparison to their respective wild-type
receptors. Interestingly, binding of both mutant receptors to FGF9 was notably
enhanced and implicates FGF9 as a potential pathophysiological ligand for mutant
FGFRs in mediating craniosynostosis. The crystal structure, of Pro252Arg FGFR1c
in complex with FGF2, demonstrates that the enhanced ligand binding is due to an
additional set of receptor-ligand hydrogen bonds, similar to those
gain-of-function interactions that occur in the Apert syndrome Pro253Arg
FGFR2c-FGF2 crystal structure. However, unlike the Apert syndrome Pro253Arg
FGFR2c mutant, neither the Pfeiffer syndrome Pro250Arg FGFR1c mutant nor the
Muenke syndrome Pro250Arg FGFR3c mutant bound appreciably to FGF7 or FGF10. This
observation provides a potential explanation for why the limb phenotypes,
observed in type I Pfeiffer and Muenke syndromes, are less severe than the limb
abnormalities observed in Apert syndrome. Hence, although analogous
proline-->arginine mutations in FGFR1-3 act through a common structural
mechanism to result in gain-of-function, differences in the primary sequence
among FGFRs result in varying effects on ligand binding specificity. BACKGROUND: Muenke syndrome is a genetically determined craniosynostosis that
involves one or both coronal sutures. In some patients it is associated with
skeletal abnormalities such as thimble-like middle phalanges, coned epiphysis,
and/or neurological impairment, namely sensorineural hearing loss or mental
retardation. In spite of a variable phenotype, Muenke syndrome has been related
to a unique mutation on the FGFR3 gene, Pro 250 to Arg, which is characteristic
of this disease. Because of the incomplete penetrance of this anomaly, the
suspicion of Muenke syndrome must be raised in any child with uni- or bilateral
coronal craniosynostosis, and the genetic analysis propounded even in the
absence of extracranial features.
ILLUSTRATIVE CASES: We report the cases of two sisters who presented with Muenke
syndrome and whose affected mother did not have any form of craniosynostosis. Muenke syndrome, also known as FGFR3-associated coronal synostosis, is defined
molecularly by the presence of a heterozygous nucleotide transversion, c.749C>G,
encoding the amino acid substitution Pro250Arg, in the fibroblast growth factor
receptor type 3 gene (FGFR3). This frequently occurs as a new mutation,
manifesting one of the highest documented rates for any transversion in the
human genome. To understand the biology of this mutation, we have investigated
its parental origin, and the ages of the parents, in 19 families with de novo
c.749C>G mutations. All ten informative cases originated from the paternal
allele (95% confidence interval 74-100% paternal); the average paternal age at
birth overall was 34.7 years. An exclusive paternal origin of mutations, and
increased paternal age, were previously described for a different mutation
(c.1138G>A) of the FGFR3 gene causing achondroplasia, as well as for mutations
of the related FGFR2 gene causing Apert, Crouzon and Pfeiffer syndromes. We
conclude that similar biological processes are likely to shape the occurrence of
this c.749C>G mutation as for other mutations of FGFR3 as well as FGFR2. BACKGROUND/PURPOSE: Despite the similar clinical phenotype of the
Saethre-Chotzen and Muenke craniosynostoses, the 2 syndromes are now
genotypically distinct. Patients with Saethre-Chotzen and Muenke syndromes carry
mutations in the TWIST and fibroblast growth factor receptor (FGFR) 3 genes,
respectively. We sought to assess possible ocular phenotypic differences in
patients with mutations of either gene previously grouped according to phenotype
only.
METHODS: A retrospective chart review was performed for 21 children with known
mutations of the TWIST (n=10) or the FGFR3 (n=11) genes. Data gathered included
patient sex, age, family craniofacial history, craniofacial and ophthalmic
surgeries, type of strabismus, ptosis, cycloplegic refraction, visual acuity,
the presence of amblyopia, nasolacrimal duct obstruction (NLDO), nystagmus,
hypertelorism, epicanthal fold anomalies, and any ocular structural
abnormalities.
RESULTS: In the TWIST group, ptosis was present in 90%, amblyopia in 70%,
horizontal strabismus in 70%, vertical strabismus in 60%, NLDO in 60%,
astigmatism in 50%, inferior oblique overaction (IOOA) in 40%, hyperopia in 40%,
myopia in 30%, nystagmus in 30%, and optic nerve findings in 30%. In the FGFR3
group, ptosis was present in 36%, amblyopia in 18%, horizontal strabismus in
55%, vertical strabismus in 36%, NLDO in 0%, astigmatism in 9%, IOOA in 45%,
hyperopia in 27%, myopia in 18%, nystagmus in 18%, and optic nerve findings in
27%.
CONCLUSIONS: Patients with TWIST gene mutations may have more ophthalmic
abnormalities, including more strabismus, ptosis, NLDO, astigmatism, vertical
deviations, and amblyopia compared with patients with FGFR3 gene mutations. P250R mutation in the FGFR3 gene also known as Muenke syndrome is associated
with coronal craniosynostosis, sensorineural deafness, craniofacial, and digital
abnormalities. We report a family with this mutation associated with sudden
death in an affected newborn, most probably due to upper airway obstruction. Muenke syndrome is an autosomal domit disorder characterized by coronal
suture craniosynostosis, hearing loss, developmental delay, carpal and tarsal
fusions, and the presence of the Pro250Arg mutation in the FGFR3 gene. Reduced
penetrance and variable expressivity contribute to the wide spectrum of clinical
findings in Muenke syndrome. To better define the clinical features of this
syndrome, we initiated a study of the natural history of Muenke syndrome. To
date, we have conducted a standardized evaluation of nine patients with a
confirmed Pro250Arg mutation in FGFR3. We reviewed audiograms from an additional
13 patients with Muenke syndrome. A majority of the patients (95%) demonstrated
a mild-to-moderate, low frequency sensorineural hearing loss. This pattern of
hearing loss was not previously recognized as characteristic of Muenke syndrome.
We also report on feeding and swallowing difficulties in children with Muenke
syndrome. Combining 312 reported cases of Muenke syndrome with data from the
nine NIH patients, we found that females with the Pro250Arg mutation were
significantly more likely to be reported with craniosynostosis than males (P <
0.01). Based on our findings, we propose that the clinical management should
include audiometric and developmental assessment in addition to standard
clinical care and appropriate genetic counseling. The heterozygous Pro250Arg substitution mutation in fibroblast growth factor
receptor 3 (FGFR3), which increases ligand-dependent signalling, is the most
common genetic cause of craniosynostosis in humans and defines Muenke syndrome.
Since FGF signalling plays dosage-sensitive roles in the differentiation of the
auditory sensory epithelium, we evaluated hearing in a large group of Muenke
syndrome subjects, as well as in the corresponding mouse model (Fgfr3(P244R)).
The Muenke syndrome cohort showed significant, but incompletely penetrant,
predomitly low-frequency sensorineural hearing loss, and the Fgfr3(P244R)
mice showed domit, fully penetrant hearing loss that was more severe than
that in Muenke syndrome individuals, but had the same pattern of relative
high-frequency sparing. The mouse hearing loss correlated with an alteration in
the fate of supporting cells (Deiters'-to-pillar cells) along the entire length
of the cochlear duct, with the most extreme abnormalities found at the apical or
low-frequency end. In addition, there was excess outer hair cell development in
the apical region. We conclude that low-frequency sensorineural hearing loss is
a characteristic feature of Muenke syndrome and that the genetically equivalent
mouse provides an excellent model that could be useful in testing hearing loss
therapies aimed at manipulating the levels of FGF signalling in the inner ear. Muenke syndrome, defined by heterozygosity for a Pro250Arg substitution in
fibroblast growth factor receptor 3 (FGFR3), is the most common genetic cause of
craniosynostosis in humans. We have used gene targeting to introduce the Muenke
syndrome mutation (equivalent to P244R) into the murine Fgfr3 gene. A rounded
skull and shortened snout (often skewed) with dental malocclusion was observed
in a minority of heterozygotes and many homozygotes. Development of this
incompletely penetrant skull phenotype was dependent on genetic background and
sex, with males more often affected. However, these cranial abnormalities were
rarely attributable to craniosynostosis, which was only present in 2/364
mutants; more commonly, we found fusion of the premaxillary and/or zygomatic
sutures. We also found decreased cortical thickness and bone mineral densities
in long bones. We conclude that although both cranial and long bone development
is variably affected by the murine Fgfr3(P244R) mutation, coronal
craniosynostosis is not reliably reproduced. Mutations in the gene that encodes Fibroblast Growth Factor Receptor 3 (FGFR3)
are associated with Achondroplasia (MIM 100800), Hypochondroplasia (MIM 146000),
Muenke Syndrome (MIM 602849), Thanatophoric Dysplasia (MIM 187600, MIM 187601)
and Lacrimo-Auriculo-Dento-Digital Syndrome (MIM 149730).Here we report a
clinical and molecular study in a large cohort of 125 Portuguese patients with
these skeletal disorders. The identification of the P250R mutation allowed the
confirmation of the Muenke Syndrome in 9 out of the 52 cases referred. Two known
mutations were found in the Thanatophoric Dysplasia referred cases. No mutations
were identified in the LADD syndrome patient. In Achondroplasia and
Hypochondroplasia, genetic heterogeneity was present amongst the 70 clinically
diagnosed patients with 5 different mutations identified. As in other studies,
complex phenotypic heterogeneity amongst patients carrying the same gene defect
was observed. In several cases, the new amino acids encoded, as a consequence of
mutations, were related to the severity of patients' phenotype. The presence of
10 misdiagnosed cases emphasizes the importance of performing mutation analysis
of the hotspot regions responsible for both dysplasias (Ach and Hch). For
patients with an unquestionable clinical diagnosis, lacking the most common
mutations, a complete screening of FGFR3 is necessary. Muenke syndrome (MS), also known as Muenke nonsyndromic coronal
craniosynostosis, is an autosomal domit condition which can be distinguished
from the more common forms of acrocephalosyndactyly but presents a significant
variable phenotype. We report on a set of identical twins with a de novo C749G
mutation in the FGFR3 gene codon 250 after a pregcy complicated by prenatal
exposure to Nortriptyline. These patients illustrate the variable expressivity
of MS in association with an identical gene mutation. The Muenke syndrome (MS) is characterized by unicoronal or bicoronal
craniosynostosis, midfacial hypoplasia, ocular hypertelorism, and a variety of
minor abnormalities associated with a mutation in the fibroblast growth factor
receptor 3 (FGFR3) gene. The birth prevalence is approximately one in 10,000
live births, accounting for 8-10% of patients with coronal synostosis. Although
MS is a relatively common diagnosis in patients with craniosynostosis syndromes,
with autosomal domit inheritance, there has been no report of MS, in an
affected Korean family with typical cephalo-facial morphology that has been
confirmed by molecular studies. Here, we report a familial case of MS in a
female patient with a Pro250Arg mutation in exon 7 (IgII-IGIII linker domain) of
the FGFR3 gene. This patient had mild midfacial hypoplasia, hypertelorism,
downslanting palpebral fissures, a beak shaped nose, plagio-brachycephaly, and
mild neurodevelopmental delay. The same mutation was confirmed in the patient's
mother, two of the mother's sisters and the maternal grandfather. The severity
of the cephalo-facial anomalies was variable among these family members. BACKGROUND: Muenke syndrome is a fibroblast growth factor receptor 3
(FGFR-3)-associated coronal craniosynostosis syndrome, which was first described
in 1997.
CASE: We report an infant girl who was born to a 29-year-old primapara at 38
weeks' gestation. When evaluated at 3 days old, physical examination revealed a
high forehead with frontal bossing, upturned nose, arched palate, shallow
midface structures, and heavily ridged coronal sutures bilaterally. Clinically,
the infant seemed to be neurologically normal. Skull radiographs and computed
tomography confirmed the presence of bilateral coronal synostosis, with patency
of all other sutures. Family history was remarkable, in that the infant's
father, paternal grandmother, and a paternal cousin demonstrated subtle
craniofacial features, which had not been previously identified. Mutation
analysis of FGFR-3 revealed a missense mutation in exon 6, c.749 C>G, with a
resultant amino acid change from proline to arginine at codon 250 (P250R), in
keeping with Muenke syndrome (Am J Hum Genet 1997;60:555-564). The mutation was
subsequently identified in her father, suggesting variable expression in this
family, as he had only mild midfacial flattening. At 9 months of age, our
patient underwent anterior cranial expansion, correction of orbital
hypertelorism, intracranial orbital osteotomies, and advancement of the frontal
bandeau. She tolerated the procedure well and has done well postoperatively.
CONCLUSIONS: We report the case of an infant with Muenke syndrome, with evidence
of variable expressivity within the paternal family. The pertinent literature,
in which only 2 prior Canadian cases were identified, is reviewed. In about 30% of the patients with syndromal craniosynostosis, a genetic mutation
can be traced. For the purpose of adequate genetic counseling and treatment of
these patients, the full spectrum of clinical findings for each specific
mutation needs to be appreciated. The Pro250Arg mutation in the FGFR3 gene is
found in patients with Muenke syndrome and is one of the most frequently
encountered mutations in craniosynostosis syndromes. A number of studies on the
relationship between genotype and phenotype concerning this specific mutation
have been published. Two Dutch families with Muenke syndrome were screened for
the reported characteristics of this syndrome and for additional features. New
phenotypical findings were hypoplasia of the frontal sinus, ptosis of the upper
eyelids, dysplastic elbow joints with restricted elbow motion, and mild
cutaneous syndactyly. Incidentally, polydactyly, severe ankylosis of the elbow,
fusion of cervical vertebrae, and epilepsy were found. Upper eyelid ptosis is
thought to be pathognomonic for Saethre-Chotzen syndrome but was also observed
in our series of patients with Muenke syndrome. Because Muenke and
Saethre-Chotzen syndrome can have similar phenotypes, DNA analysis is needed to
distinguish between these syndromes, even when a syndrome diagnosis is already
made in a family member. Muenke syndrome caused by the FGFR3 Pro250Arg mutation is associated with
craniosynostosis, hearing loss, and various bony anomalies. Although this
mutation is involved in bone growth and development, bony tumors are rare in
this condition. We describe a patient with a molecular diagnosis of Muenke
syndrome who also presented with multiple osteochondromas of the upper and lower
extremities. This association has only been described once before in a patient
with an isolated osteochondroma of the proximal tibia. Altered expression of
FGFR3, an important mediator of chondrocyte proliferation and differentiation
during in the growth plates of long bones, may help to explain the development
of osteochondromatous lesions in this patient. PURPOSE: The Muenke syndrome mutation (FGFR3 (P250R)), which was discovered 15
years ago, represents the single most common craniosynostosis mutation. Muenke
syndrome is characterized by coronal suture synostosis, midface hypoplasia,
subtle limb anomalies, and hearing loss. However, the spectrum of clinical
presentation continues to expand. To better understand the pathophysiology of
the Muenke syndrome, we present collective findings from several recent studies
that have characterized a genetically equivalent mouse model for Muenke syndrome
(FgfR3 (P244R)) and compare them with human phenotypes.
CONCLUSIONS: FgfR3 (P244R) mutant mice show premature fusion of facial sutures,
premaxillary and/or zygomatic sutures, but rarely the coronal suture. The mice
also lack the typical limb phenotype. On the other hand, the mutant mice display
maxillary retrusion in association with a shortening of the anterior cranial
base and a premature closure of intersphenoidal and spheno-occipital
synchondroses, resembling human midface hypoplasia. In addition, sensorineural
hearing loss is detected in all FgfR3 (P244R) mutant mice as in the majority of
Muenke syndrome patients. It is caused by a defect in the mechanism of cell fate
determination in the organ of Corti. The mice also express phenotypes that have
not been previously described in humans, such as reduced cortical bone
thickness, hypoplastic trabecular bone, and defective temporomandibular joint
structure. Therefore, the FgfR3 (P244R) mouse provides an excellent opportunity
to study disease mechanisms of some classical phenotypes of Muenke syndrome and
to test novel therapeutic strategies. The mouse model can also be further
explored to discover previously unreported yet potentially significant
phenotypes of Muenke syndrome. Muenke syndrome is an autosomal domit craniosynostosis syndrome resulting
from a defining point mutation in the Fibroblast Growth Factor Receptor3 (FGFR3)
gene. Muenke syndrome is characterized by coronal craniosynostosis (bilateral
more often than unilateral), hearing loss, developmental delay, and carpal
and/or tarsal bone coalition. Tarsal coalition is a distinct feature of Muenke
syndrome and has been reported since the initial description of the disorder in
the 1990s. Although talocalcaneal coalition is the most common tarsal coalition
in the general population, it has never previously been reported in a patient
with Muenke syndrome. We present a 7-year-old female patient with Muenke
syndrome and symptomatic talocalcaneal coalition. She presented at the age of 7
with limping, tenderness and pain in her right foot following a fall and strain
of her right foot. She was treated with ibuprofen, shoe inserts, a CAM walker
boot, and stretching exercises without much improvement in symptoms. A computed
tomography (CT) scan revealed bilateral talocalcaneal coalitions involving the
middle facet. She underwent resection of the talocalcaneal coalitions, remaining
pain-free post-operatively with an improvement in her range of motion, gait, and
mobility. This report expands the phenotype of tarsal coalition in Muenke
syndrome to include talocalcaneal coalition. A literature review revealed a high
incidence of tarsal coalition in all FGFR related craniosynostosis syndromes
when compared to the general population, a difference that is statistically
significant. The most common articulation involved in all syndromic
craniosynostoses associated with FGFR mutations is the calcaneocuboid
articulation. Muenke is a fibroblast growth factor receptor 3 (FGFR-3)-associated syndrome,
which was first described in late 1990 s. Muenke syndrome is an autosomal
domit disorder characterized mainly by coronal suture craniosynostosis,
hearing impairment and intellectual disability. The syndrome is defined
molecularly by a unique point mutation c.749C > G in exon 7 of the FGFR3 gene
which results to an amino acid substitution p.Pro250Arg of the protein product.
Despite the fact that the mutation rate at this nucleotide is one of the most
frequently described in human genome, few Muenke familial case reports are
published in current literature. We describe individuals among three generations
of a Greek family who are carriers of the same mutation. Medical record and
physical examination of family members present a wide spectrum of clinical
manifestations. In particular, a 38-year-old woman and her father appear milder
clinical findings regarding craniofacial characteristics compared to her uncle
and newborn female child. This familial case illustrates the variable
expressivity of Muenke syndrome in association with an identical gene mutation. Muenke syndrome caused by point mutation (C749G) in the FGFR3 gene affects 1 in
30,000 newborns and accounts for 25% to 30% of genetic causes of
craniosynostosis. Anomalies in patients with Muenke syndrome include
craniosynostosis, hypertelorism, sensorineural hearing loss, and developmental
delay, among others. Most craniosynostoses in patients with Muenke syndrome
involve bicoronal suture fusion. This article reports, for the first time, the
existence of squamosal craniosynostosis in patients with Muenke syndrome. PURPOSE: There are a number of craniosynostosis syndromes with hearing
loss-including Muenke, Apert, Pfeiffer, Crouzon, Beare-Stevenson, Crouzon with
acanthosis nigricans, and Jackson-Weiss syndromes-that result from mutations in
the fibroblast growth factor receptor (FGFR) genes. Studies of FGFRs and their
ligands, fibroblast growth factors (FGFs), have revealed clues to the precise
contribution of aberrant FGFR signaling to inner ear morphogenesis and the
hearing loss encountered in craniosynostoses. The purpose of this article is to
review basic studies of FGFRs with emphasis on their function and expression in
the inner ear and surrounding structures.
METHOD: A Medline search was performed to find basic science articles regarding
FGFR, their ligands, and their expression and relevant mouse models. Additional
items searched included clinical descriptions and studies of individuals with
FGFR-related craniosynostosis syndromes.
RESULTS: The FGF signaling pathway is essential for the morphogensis and proper
function of the inner ear and auditory sensory epithelium.
CONCLUSION: The variable auditory phenotypes seen in individuals with Muenke
syndrome may have a genetic basis, likely due to multiple interacting factors in
the genetic environment or modifying factors. Further analysis and studies of
mouse models of Muenke syndrome, in particular, may provide clues to the
specific effects of the defining mutation in FGFR3 in the inner ear not only at
birth but also into adulthood. In particular, investigations into these models
may give insight into the variable expression and incomplete penetrance of this
phenotype. Muenke syndrome is a nonsyndromic coronal craniosynostosis, characterised by
clinical and radiological variability, with occurrence of both familial and
sporadic cases. Pro250Arg (P250R) is a pathogenic mutation, causing this highly
clinically heterogeneous syndrome reported worldwide irrespective of race and
ethnicity. The authors describe three Indian cases in two different families
showing phenotypic spectrum of the disease, which was later confirmed by genetic
testing. OBJECTIVE: To investigate executive function and adaptive behavior in
individuals with Muenke syndrome using validated instruments with a normative
population and unaffected siblings as controls.
STUDY DESIGN: Participants in this cross-sectional study included individuals
with Muenke syndrome (P250R mutation in FGFR3) and their mutation-negative
siblings. Participants completed validated assessments of executive functioning
(Behavior Rating Inventory of Executive Function [BRIEF]) and adaptive behavior
skills (Adaptive Behavior Assessment System, Second Edition [ABAS-II]).
RESULTS: Forty-four with a positive FGFR3 mutation, median age 9 years, range 7
months to 52 years were enrolled. In addition, 10 unaffected siblings served as
controls (5 males, 5 females; median age, 13 years; range, 3-18 years). For the
General Executive Composite scale of the BRIEF, 32.1% of the cohort had scores
greater than +1.5 SD, signifying potential clinical significance. For the
General Adaptive Composite of the ABAS-II, 28.2% of affected individuals scored
in the 3rd-8th percentile of the normative population, and 56.4% were below the
average category (<25th percentile). Multiple regression analysis did not
identify craniosynostosis as a predictor of BRIEF (P = .70) or ABAS-II scores (P
= .70). In the sibling pair analysis, affected siblings performed significantly
poorer on the BRIEF General Executive Composite and the ABAS-II General Adaptive
Composite.
CONCLUSION: Individuals with Muenke syndrome are at an increased risk for
developing adaptive and executive function behavioral changes compared with a
normative population and unaffected siblings. Muenke syndrome is an autosomal domit disorder characterized by coronal
suture craniosynostosis, hearing loss, developmental delay, carpal, and
calcaneal fusions, and behavioral differences. Reduced penetrance and variable
expressivity contribute to the wide spectrum of clinical findings. Muenke
syndrome constitutes the most common syndromic form of craniosynostosis, with an
incidence of 1 in 30,000 births and is defined by the presence of the
p.Pro250Arg mutation in FGFR3. Participants were recruited from international
craniofacial surgery and genetic clinics. Affected individuals, parents, and
their siblings, if available, were enrolled in the study if they had a
p.Pro250Arg mutation in FGFR3. One hundred and six patients from 71 families
participated in this study. In 51 informative probands, 33 cases (64.7%) were
inherited. Eighty-five percent of the participants had craniosynostosis (16 of
103 did not have craniosynostosis), with 47.5% having bilateral and 28.2% with
unilateral synostosis. Females and males were similarly affected with bicoronal
craniosynostosis, 50% versus 44.4% (P = 0.84), respectively. Clefting was rare
(1.1%). Hearing loss was identified in 70.8%, developmental delay in 66.3%,
intellectual disability in 35.6%, attention deficit/hyperactivity disorder in
23.7%, and seizures in 20.2%. In patients with complete skeletal surveys (upper
and lower extremity x-rays), 75% of individuals were found to have at least a
single abnormal radiographical finding in addition to skull findings. This is
the largest study of the natural history of Muenke syndrome, adding valuable
clinical information to the care of these individuals including behavioral and
cognitive impairment data, vision changes, and hearing loss. |
Which R package is used for visualization of linear and circular karyotypes? | The chromDraw graphical tool was developed as a user-friendly graphical tool for visualizing both linear and circular karyotypes based on the same input data matrix. The output graphics, saved in two different formats (EPS and SVG), can be easily imported to and modified in presentation and image-editing computer programs. The tool is freely distributed under GNU General Public License (GPL) and can be installed from Bioconductor or from the chromDraw home page. | Species-specific sets of chromosomes-karyotypes-are traditionally depicted as
linear ideograms with individual chromosomes represented by vertical bars.
However, linear visualization has its limitations when the shared collinearity
and/or chromosomal rearrangements differentiating two or more karyotypes need to
be demonstrated. In these instances, circular visualization might provide easier
comprehension and interpretation of inter-species chromosomal collinearity. The
chromDraw graphical tool was developed as a user-friendly graphical tool for
visualizing both linear and circular karyotypes based on the same input data
matrix. The output graphics, saved in two different formats (EPS and SVG), can
be easily imported to and modified in presentation and image-editing computer
programs. The tool is freely distributed under GNU General Public License (GPL)
and can be installed from Bioconductor or from the chromDraw home page. |
Has small pox been eradicated from the world? | smallpox is now eradicated. | At the beginning of this century the compulsory vaccination and revaccination
which was legally founded after the introduction of the vaccination by Jenner
(1796) led to the removal of the smallpox in Europe and Northern America.
However, up to the sixties in the developing countries of Asia, Africa as well
as of Southern America and Middle America still fell ill and died of small-pox
millions of people. Between 1953 and 1973 importations into countries of Europe
and Northern America took place in 51 cases. In 1959 on the motion of the USSR
the WHO decided performance of a world-wide eradication programme of small-pox
which could be led to success with comprehensive personal, material and
ficial support of many countries. Flanking scientific, technological and
methodical measures were of essential importance. In May 1980 the World Health
Assembly in Geneva announced in solemn form the world-wide eradication of the
small-pox and gave recommendations to the member countries for concluding
measures concerning the small-pox vaccination, the foundation of vaccine
reserves and the control of the epidemiological situation in the world. Also in
the GDR the small-pox vaccination in childhood could be abolished. In December 1801, the first vaccination against smallpox in Norway took place.
Vaccine material came from Denmark, England, Ireland, and other countries; it
was also obtained from a few local cowpox cases. What mattered was the effect,
not the origin. Several reports indicate that variola virus itself, the cause of
smallpox, was also used for human vaccination after passages through cows and
horses. A vaccine institute for production of vaccine in calves was established
in Kristiania in 1891. Cowpox was once a rare disease in cattle, but a total of
70,985 bovine cases were reported between 1889 and 1928. The source of infection
was thought to be humans vaccinated against smallpox. Pox-like diseases were
also registered in horses, pigs, sheep, goats and dogs at that time. Compulsory
vaccination continued in Norway until 1976; smallpox is now eradicated. During
the last decades, however, cowpox virus infections have re-emerged among zoo
animals, domestic cats and humans in Western Europe, with small wild rodents and
shrews as wildlife reservoirs. Vaccinia virus is also met with new interest as a
vector in recombit vaccines. Given the fact that the human population no
longer has immunity against orthopoxviruses and the new possible exposure
through pets and wildlife, it may be appropriate to reflect on poxvirus history
in Norway in the light of the present situation. In Italy until sec. XIX, the beginning of vaccination duty allows to obtain some
weighty aims in Public Health, like small pox eradication, polio and diphtheria
elimination, or like the great reduction of tetanus and hepatitis B incidence.
At the some, however, the vaccination duty doesn't allow to develop a
"vaccination conscience" to do to accept the vaccines like the most important
instrument, for effectiveness and utility, in the infectious diseases
prevention. So, if from a side it's necessary to leave the vaccination duty from
the another side it's necessary to make e leaded way of health education in the
people and it's necessary to make a share of resources and aims in the sanitary
world. Technological advancements, including landmark innovations in vaccinology
through molecular virology, and significant transformation and changes in the
society have taken place since the eradication of smallpox thirty years ago. The
success with eradicating smallpox gave confidence for initiating the eradication
of other diseases, such as malaria and polio. However, these efforts have not
been as effective, as recorded for small pox, for a variety of reasons. There is
now a debate within the global health community as to whether eradication
campaigns should be abandoned in favor of less costly and perhaps more effective
primary health and containment or control programmes. Significant changes that
have taken place in the last thirty years, since the eradication of smallpox
include, among others, (i) post-colonial political changes, with varying
commitment to disease eradication initiatives, especially in the parts of the
world most burdened by infectious and vaccine preventable diseases, (ii)
innovations leading to the development of new and highly effective vaccines,
targeted to specific diseases, (iii) the transformation brought about by
improvement in education and the new global access to information (cell phones,
internet, etc.), leading to an unlimited access to different types of
information, subject to either positive or negative use. At the onset of
eradication of smallpox, global health was confined in its operation. Today,
global health is at the intersection of medical and social science
disciplines-including demography, economics, epidemiology, political economy and
sociology. Therefore, in considering the issue of disease eradication, medical
and social perspectives must be brought into play, if future eradication
programmes must succeed. The paper discusses the roles of these disciplines in
disease control and eradication, especially as it affects sub Saharan Africa,
the melting pot and verdant pasture of infectious diseases. Edward Jenner, who discovered that it is possible to vaccinate against Small Pox
using material from Cow Pox, is rightly the man who started the science of
immunology. However, over the passage of time many of the details surrounding
his astounding discovery have been lost or forgotten. Also, the environment
within which Jenner worked as a physician in the countryside, and the state of
the art of medicine and society are difficult to appreciate today. It is
important to recall that people were still being bled at the time, to relieve
the presence of evil humors. Accordingly, this review details Jenner's discovery
and attempts to place it in historical context. Also, the vaccine that Jenner
used, which decreased the prevalence of Small Pox worldwide in his own time, and
later was used to eradicate Small Pox altogether, is discussed in light of
recent data. |
Where is Akkermansia muciniphila found? | Akkermansia muciniphila is a Gram-negative mucin-degrading bacterium that resides in the gastrointestinal tracts of humans and animals. | OBJECTIVE: Individuals with obesity and type 2 diabetes differ from lean and
healthy individuals in their abundance of certain gut microbial species and
microbial gene richness. Abundance of Akkermansia muciniphila, a mucin-degrading
bacterium, has been inversely associated with body fat mass and glucose
intolerance in mice, but more evidence is needed in humans. The impact of diet
and weight loss on this bacterial species is unknown. Our objective was to
evaluate the association between faecal A. muciniphila abundance, faecal
microbiome gene richness, diet, host characteristics, and their changes after
calorie restriction (CR).
DESIGN: The intervention consisted of a 6-week CR period followed by a 6-week
weight stabilisation diet in overweight and obese adults (N=49, including 41
women). Faecal A. muciniphila abundance, faecal microbial gene richness, diet
and bioclinical parameters were measured at baseline and after CR and weight
stabilisation.
RESULTS: At baseline A. muciniphila was inversely related to fasting glucose,
waist-to-hip ratio and subcutaneous adipocyte diameter. Subjects with higher
gene richness and A. muciniphila abundance exhibited the healthiest metabolic
status, particularly in fasting plasma glucose, plasma triglycerides and body
fat distribution. Individuals with higher baseline A. muciniphila displayed
greater improvement in insulin sensitivity markers and other clinical parameters
after CR. These participants also experienced a reduction in A. muciniphila
abundance, but it remained significantly higher than in individuals with lower
baseline abundance. A. muciniphila was associated with microbial species known
to be related to health.
CONCLUSIONS: A. muciniphila is associated with a healthier metabolic status and
better clinical outcomes after CR in overweight/obese adults. The interaction
between gut microbiota ecology and A. muciniphila warrants further
investigation.
TRIAL REGISTRATION NUMBER: NCT01314690. |
Is there alternative polyadenylation during zebrafish development? | Yes. There is extensive alternative polyadenylation during zebrafish development. | The post-transcriptional fate of messenger RNAs (mRNAs) is largely dictated by
their 3' untranslated regions (3' UTRs), which are defined by cleavage and
polyadenylation (CPA) of pre-mRNAs. We used poly(A)-position profiling by
sequencing (3P-seq) to map poly(A) sites at eight developmental stages and
tissues in the zebrafish. Analysis of over 60 million 3P-seq reads substantially
increased and improved existing 3' UTR annotations, resulting in confidently
identified 3' UTRs for >79% of the annotated protein-coding genes in zebrafish.
mRNAs from most zebrafish genes undergo alternative CPA, with those from more
than a thousand genes using different domit 3' UTRs at different stages.
These included one of the poly(A) polymerase genes, for which alternative CPA
reinforces its repression in the ovary. 3' UTRs tend to be shortest in the
ovaries and longest in the brain. Isoforms with some of the shortest 3' UTRs are
highly expressed in the ovary, yet absent in the maternally contributed RNAs of
the embryo, perhaps because their 3' UTRs are too short to accommodate a
uridine-rich motif required for stability of the maternal mRNA. At 2 h
post-fertilization, thousands of unique poly(A) sites appear at locations
lacking a typical polyadenylation signal, which suggests a wave of widespread
cytoplasmic polyadenylation of mRNA degradation intermediates. Our insights into
the identities, formation, and evolution of zebrafish 3' UTRs provide a resource
for studying gene regulation during vertebrate development. |
Which NADPH oxidase family member requires interaction with NOXO1 for function? | NADPH oxidase 1 (NOX1) requires interaction with NOXO1 for function. | The members of the NOX family of enzymes are expressed in a variety of tissues
and serve a number of functions. There is a high level of conservation of
primary protein sequence, as well as functional features, although specialized
responses are beginning to emerge. In this context, our data demonstrate that
the NOX1 cytoplasmic domains interact efficiently with the cytoplasmic subunits
of the phagocyte NADPH oxidase and identify the second cytoplasmic loop of NOX
electron transporters as a crucial domain for enzyme function. Studies of
cytosolic co-factors showed that the C-terminal cytoplasmic domain of NOX1 was
absolutely required for activation with NOXO1 and NOXA1 and that this activity
required interaction of the putative NADPH-binding region of this domain with
NOXA1. Finally, we have provided the first example of how alternative splicing
of a NOX co-factor may be involved in the regulation of NADPH oxidase function. The phagocyte NADPH oxidase is dormant in resting cells but becomes activated
during phagocytosis to produce superoxide, a precursor of microbicidal oxidants,
thereby playing a crucial role in host defence. The catalytic core of this
enzyme comprises the two membranous subunits gp91phox/Nox2 and p22phox. The
oxidase activation requires the small GTPase Rac and the SH3 domain-containing
proteins p47phox and p67phox; they normally exist in the cytoplasm and
translocate upon cell stimulation to the membrane. The translocation depends on
a stimulus-induced conformational change of p47phox, which leads to the SH3
domain-mediated interaction with p22phox, a binding required for the
gp91phox/Nox2-dependent superoxide production. Activation of Nox1, an oxidase
that is likely involved in host defence at the colon, requires novel proteins
homologous to p47phox and p67phox, designated Noxo1 and Noxa1, respectively.
Noxo1 and Noxa1, both expressed abundantly in the colon, are capable of
constitutively activating Nox1. The constitutive activation may be due to the
property of Noxo1: in contrast with p47phox, Noxo1 seems to normally associate
with p22phox, which is required for the Nox1 activation. We will also describe
the mechanism underlying regulation of the third oxidase Nox3, which exits in
fetal kidney and inner ears. It is well established that growth-factor-induced reactive oxygen species (ROS)
act as second messengers in cell signaling. We have previously reported that
betaPix, a guanine nucleotide exchange factor for Rac, interacts with NADPH
oxidase 1 (Nox1) leading to EGF-induced ROS generation. Here, we report the
identification of the domains of Nox1 and betaPix responsible for the
interaction between the two proteins. GST pull-down assays show that the PH
domain of betaPix binds to the FAD-binding region of Nox1. We also show that
overexpression of the PH domain of betaPix results in inhibition of superoxide
anion generation in response to EGF. Additionally, NADPH oxidase Organizer 1
(NoxO1) is shown to interact with the NADPH-binding region of Nox1. These
results suggest that the formation of the complex consisting of Nox1, betaPix,
and NoxO1 is likely to be a critical step in EGF-induced ROS generation. Activation of the superoxide-producing NADPH oxidase Nox1 requires both the
organizer protein Noxo1 and the activator protein Noxa1. Here we describe an
alternative splicing form of Noxo1, Noxo1gamma, which is expressed in the testis
and fetal brain. The Noxo1gamma protein contains an additional five amino acids
in the N-terminal PX domain, a phosphoinositide-binding module; the domain plays
an essential role in supporting superoxide production by NADPH oxidase (Nox)
family oxidases including Nox1, gp91(phox)/Nox2, and Nox3, as shown in this
study. The PX domain isolated from Noxo1gamma shows a lower affinity for
phosphoinositides than that from the classical splicing form Noxo1beta.
Consistent with this, in resting cells, Noxo1gamma is poorly localized to the
membrane, and thus less effective in activating Nox1 than Noxo1beta, which is
constitutively present at the membrane. On the other hand, cell stimulation with
phorbol 12-myristate 13-acetate (PMA), an activator of Nox1-3, facilitates
membrane translocation of Noxo1gamma; as a result, Noxo1gamma is equivalent to
Noxo1beta in Nox1 activation in PMA-stimulated cells. The effect of the
five-amino-acid insertion in the Noxo1 PX domain appears to depend on the type
of Nox; in activation of gp91(phox)/Nox2, Noxo1gamma is less active than
Noxo1beta even in the presence of PMA, whereas Noxo1gamma and Noxo1beta support
the superoxide-producing activity of Nox3 to the same extent in a manner
independent of cell stimulation. The superoxide-producing phagocyte NADPH oxidase gp91(phox)/Nox2 and the
non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with
p22(phox), which provides both stabilization and a docking site for organizer
proteins. The p22(phox)-complexed Nox2 and Nox1 are dormant on their own, and
their activation requires soluble supportive proteins such as a Nox organizer
(p47(phox) or Noxo1) and a Nox activator (p67(phox) or Noxa1). The small GTPase
Rac directly binds to the activators, and thus plays an essential role in the
Nox2-based oxidase containing p47(phox) and p67(phox) or a positive role in Nox1
activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22(phox)
constitutively produce superoxide, the production can be enhanced by supportive
proteins. Here we compare the roles of Rac in these p22(phox)-dependent oxidases
using the organizer and activator in different combinations. Expression of
constitutively active Rac1(Q61L) is essential for activation of the Nox2- or
Nox1-based oxidase containing the organizer p47(phox) and either p67(phox) or
Noxa1. When these oxidases use Noxo1 as an organizer instead of p47(phox), they
produce a small but significant amount of superoxide without expression of
Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47(phox) is
likely related to strict dependence on Rac. The Nox3-based oxidase has a similar
tendency in the change of the dependence: Rac plays a positive role in Nox3
activation in the presence of p47(phox) and either p67(phox) or Noxa1, whereas
Rac fails to upregulate Nox3 activity when p47(phox) is replaced with Noxo1. We
also demonstrate that, in the Nox3-based oxidase containing solely p67(phox) as
supportive protein, expression of Rac1(Q61L) enhances not only superoxide
production but also membrane translocation of p67(phox). Since the enhancements
are not observed with a mutant p67(phox) defective in binding to Rac, this
GTPase appear to directly recruit p67(phox) to the membrane. BACKGROUND: The reactive oxygen-generating NADPH oxidases (Noxes) function in a
variety of biological roles, and can be broadly classified into those that are
regulated by subunit interactions and those that are regulated by calcium. The
prototypical subunit-regulated Nox, Nox2, is the membrane-associated catalytic
subunit of the phagocyte NADPH-oxidase. Nox2 forms a heterodimer with the
integral membrane protein, p22phox, and this heterodimer binds to the regulatory
subunits p47phox, p67phox, p40phox and the small GTPase Rac, triggering
superoxide generation. Nox-organizer protein 1 (NOXO1) and Nox-activator 1
(NOXA1), respective homologs of p47phox and p67phox, together with p22phox and
Rac, activate Nox1, a non-phagocytic homolog of Nox2. NOXO1 and p22phox also
regulate Nox3, whereas Nox4 requires only p22phox. In this study, we have
assembled and analyzed amino acid sequences of Nox regulatory subunit orthologs
from vertebrates, a urochordate, an echinoderm, a mollusc, a cnidarian, a
choanoflagellate, fungi and a slime mold amoeba to investigate the evolutionary
history of these subunits.
RESULTS: Ancestral p47phox, p67phox, and p22phox genes are broadly seen in the
metazoa, except for the ecdysozoans. The choanoflagellate Monosiga brevicollis,
the unicellular organism that is the closest relatives of multicellular animals,
encodes early prototypes of p22phox, p47phox as well as the earliest known
Nox2-like ancestor of the Nox1-3 subfamily. p67phox- and p47phox-like genes are
seen in the sea urchin Strongylocentrotus purpuratus and the limpet Lottia
gigantea that also possess Nox2-like co-orthologs of vertebrate Nox1-3.
Duplication of primordial p47phox and p67phox genes occurred in vertebrates,
with the duplicated branches evolving into NOXO1 and NOXA1. Analysis of
characteristic domains of regulatory subunits suggests a novel view of the
evolution of Nox: in fish, p40phox participated in regulating both Nox1 and
Nox2, but after the appearance of mammals, Nox1 (but not Nox2) became
independent of p40phox. In the fish Oryzias latipes, a NOXO1 ortholog retains an
autoinhibitory region that is characteristic of mammalian p47phox, and this was
subsequently lost from NOXO1 in later vertebrates. Detailed amino acid sequence
comparisons identified both putative key residues conserved in characteristic
domains and previously unidentified conserved regions. Also, candidate
organizer/activator proteins in fungi and amoeba are identified and hypothetical
activation models are suggested.
CONCLUSION: This is the first report to provide the comprehensive view of the
molecular evolution of regulatory subunits for Nox enzymes. This approach
provides clues for understanding the evolution of biochemical and physiological
functions for regulatory-subunit-dependent Nox enzymes. Nox activator 1 (NoxA1) is a homologue of p67(phox) that acts in conjunction
with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS)
production by the NADPH oxidase Nox1. The phosphorylation of cytosolic
regulatory components by multiple kinases plays important roles in assembly and
activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about
regulation by phosphorylation in the Nox1 system. Here we identify Ser(172) and
Ser(461) of NoxA1 as phosphorylation sites for protein kinase A (PKA). A
consequence of this phosphorylation was the enhancement of NoxA1 complex
formation with 14-3-3 proteins. Using both a transfected human embryonic kidney
293 cell Nox1 model system and endogenous Nox1 in colon cell lines, we showed
that the elevation of cAMP inhibits, whereas the inhibition of PKA enhances,
Nox1-dependent ROS production through effects on NoxA1. Inhibition of Nox1
activity was intensified by the availability of 14-3-3zeta protein, and this
regulatory interaction was dependent on PKA-phosphorylatable sites at Ser(172)
and Ser(461) in NoxA1. We showed that phosphorylation and 14-3-3 binding induce
the dissociation of NoxA1 from the Nox1 complex at the plasma membrane,
suggesting a mechanism for the inhibitory effect on Nox1 activity. Our data
establish that PKA-phosphorylated NoxA1 is a new binding partner of 14-3-3
protein(s) and that this forms the basis of a novel mechanism regulating the
formation of ROS by Nox1 and, potentially, other NoxA1-regulated Nox family
members. NADPH oxidase 1 (Nox1) is a multicomponent enzyme consisting of p22(phox), Nox
organizer 1 (NOXO1), Nox1 activator 1, and Rac1. Interleukin-1beta, flagellin,
interferon-gamma, and tumor necrosis factor alpha (TNF-alpha) similarly induced
Nox1 in a colon cancer cell line (T84), whereas only TNF-alpha fully induced
NOXO1 and upregulated superoxide-producing activity by ninefold. This
upregulation was canceled by knockdown of NOXO1 with small interfering RNAs.
TNF-alpha rapidly phosphorylated p38 mitogen-activated protein kinase and c-Jun
N-terminal kinase 1/2, followed by phosphorylation of c-Jun and c-Fos and
appearance of an AP-1 binding activity within 30 min. We cloned the 5' flank of
the human NOXO1 gene (-3888 to +263 bp), and found that the region between -585
and -452 bp, which contains consensus elements of YY-1, AP-1, and Ets, and the
GC-rich region encoding three putative binding sites for SP-1, was crucial for
TNF-alpha-dependent promoter activity. Serial mutation analysis of the elements
identified an AP-1 binding site (from -561 to -551 bp, agtAAGtcatg) as a crucial
element for TNF-alpha-stimulated transcription of the human NOXO1 gene, which
was also confirmed by the AP-1 decoy experiments. Thus, TNF-alpha acts as a
potent activator of Nox1-based oxidase in colon epithelial cells, suggesting a
potential role of this oxidase in inflammation of the colon. Reactive oxygen species (ROS) have been known for a long time to play important
roles in host defense against microbial infections. In addition, it has become
apparent that they also perform regulatory roles in signal transduction and cell
proliferation. The source of these chemicals are members of the NOX family of
NADPH oxidases that are found in a variety of tissues. NOX1, an NADPH oxidase
homologue that is most abundantly expressed in colon epithelial cells, requires
the regulatory subunits NOXO1 (NOX organizing protein 1) and NOXA1 (NOX
activating protein 1), as well as the flavocytochrome component p22(phox) for
maximal activity. Unlike NOX2, the phagocytic NADPH oxidase whose activity is
tightly repressed in the resting state, NOX1 produces superoxide constitutively
at low levels. These levels can be further increased in a stimulus-dependent
manner, yet the molecular details regulating this activity are not fully
understood. Here we present the first quantitative characterization of the
interactions made between the cytosolic regulators NOXO1 and NOXA1 and
membrane-bound p22(phox). Using isothermal titration calorimetry we show that
the isolated tandem SH3 domains of NOXO1 bind to p22(phox) with high affinity,
most likely adopting a superSH3 domain conformation. In contrast, complex
formation is severely inhibited in the presence of the C-terminal tail of NOXO1,
suggesting that this region competes for binding to p22(phox) and thereby
contributes to the regulation of superoxide production. Furthermore, we provide
data indicating that the molecular details of the interaction between NOXO1 and
NOXA1 is significantly different from that between the homologous proteins of
the phagocytic oxidase, suggesting that there are important functional
differences between the two systems. Taken together, this study provides clear
evidence that the assembly of the NOX1 oxidase complex can be regulated through
reversible protein-protein interactions. NADPH oxidase (Nox) family enzymes are one of the main sources of cellular
reactive oxygen species (ROS), which have been implicated in several
physiological and pathophysiological processes. To date seven members of this
family have been reported, including Nox1-5 and Duox1 and 2. With the exception
of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is
highly expressed in the colon, and requires two cytosolic regulators, the
organizer subunit NoxO1 and the activator subunit NoxA1, as well as the binding
of Rac1 GTPase, for its activity. Recently, we identified the c-Src substrate
proteins Tks4 and Tks5 as functional members of a p47(phox)-related organizer
superfamily. As a functional consequence of this interaction, Nox1 localizes to
invadopodia, actin-rich membrane protrusions of cancer cells which facilitate
pericellular proteolysis and invasive behavior. Here, we report that Tks4 and
Tks5 directly bind to NoxA1. Moreover, the integrity of the N-terminal PRR of
NoxA1 is essential for this direct interaction with the Tks proteins. When the
PRR in NoxA1 is disrupted, Tks proteins cannot bind NoxA1 and lose their ability
to support Nox1-dependent ROS generation. Consistent with this, Tks4 and Tks5
are unable to act as organizers for Nox2 because of their inability to interact
with p67(phox), which lacks the N-terminal PRR, thus conferring a unique
specificity to Tks4 and 5. Taken together, these results clarify the molecular
basis for the interaction between NoxA1 and the Tks proteins and may provide new
insights into the pharmacological design of a more effective anti-metastatic
strategy. AIMS/HYPOTHESIS: We have previously shown that NADPH oxidase (NOX) lies upstream
of uncoupled endothelial nitric oxide synthase (eNOS), which is known to occur
in diabetic endothelium. However, it remains unclear which specific NOX
isoform(s) is responsible for eNOS uncoupling and endothelial dysfunction in
diabetic mouse models. The aim of the present study was to test the hypothesis
that one or more NOX isoform(s) mediate(s) diabetic uncoupling of eNOS, which
has been shown to occur in patients with diabetes to contribute to endothelial
dysfunction.
METHODS: Diabetes was induced by streptozotocin administration. The N
(ω)-nitro-L-arginine methyl ester (L-NAME)-sensitive superoxide production of
aortic segments, reflective of eNOS uncoupling activity, was determined by
electron spin resoce.
RESULTS: The L-NAME-sensitive superoxide production was more than doubled in
wild-type diabetic mice, implicating uncoupling of eNOS. This was abolished in
diabetic p47 ( phox-/-) (also known as Ncf1 (-/-)) mice, but preserved in Nox2
(-/y) (also known as Cybb (-/-)) mice made diabetic. The eNOS uncoupling
activity was markedly attenuated in diabetic mice transfected with Nox1 or Nox1
organiser 1 (Noxo1) short interfering RNA (siRNA), and abolished in Nox1 (-/y)
diabetic mice. Diabetes-induced impairment in endothelium-dependent
vasorelaxation was also significantly attenuated in the Nox1 (-/y) mice made
diabetic. By contrast, Nox4 siRNA, or inhibition of mitochondrial complex I or
III with rotenone or siRNA, respectively, had no effect on diabetic uncoupling
of eNOS. Overexpression of Dhfr, or oral administration of folic acid to improve
dihydrofolate reductase (DHFR) function, recoupled eNOS in diabetes to improve
endothelial function.
CONCLUSIONS/INTERPRETATION: Our data demonstrate for the first time that the
p47(phox) and NOXO1-dependent activation of NOX1, but not that of NOX2, NOX4 or
mitochondrion, mediates diabetic uncoupling of eNOS. NOX1-null mice are
protected from diabetic endothelial dysfunction. Novel approaches to inhibit
NOX1 and/or improve DHFR function, may prove to have therapeutic potential for
diabetic endothelial dysfunction. The generation of reactive oxygen species (ROS) is required for proper cell
signaling, but must be tightly regulated to minimize deleterious oxidizing
effects. Activation of the NADPH oxidases (Nox) triggers ROS production and,
thus, regulatory mechanisms exist to properly control Nox activity. In this
study, we report a novel mechanism in which Nox1 activity is regulated through
the proteasomal degradation of Nox organizer 1 (NoxO1). We found that through
the interaction between NoxO1 and growth receptor-bound protein 2 (Grb2), the
Casitas B-lineage lymphoma (Cbl) E3 ligase was recruited, leading to decreased
NoxO1 stability and a subsequent reduction in ROS generation upon epidermal
growth factor (EGF) stimulation. Additionally, we show that EGF-mediated
phosphorylation of NoxO1 induced its release from Grb2 and facilitated its
association with Nox activator 1 (NoxA1) to stimulate ROS production.
Consistently, overexpression of Grb2 resulted in decreased Nox1 activity,
whereas knockdown of Grb2 led to increased Nox1 activity in response to EGF.
CRISPR/Cas9-mediated NoxO1 knockout in human colon cancer cells abrogated
anchorage-independent growth on soft agar and tumor-forming ability in athymic
nude mice. Moreover, the expression and stability of NoxO1 were significantly
increased in human colon cancer tissues compared with normal colon. Taken
together, these results support a model whereby Nox1 activity and ROS generation
are regulated by Grb2/Cbl-mediated proteolysis of NoxO1 in response to EGF,
providing new insight into the processes by which excessive ROS production may
promote oncogenic signaling to drive colorectal tumorigenesis. Despite numerous reports implicating NADPH oxidases (Nox) in the pathogenesis of
many diseases, precise regulation of this family of professional reactive oxygen
species (ROS) producers remains unclear. A unique member of this family, Nox1
oxidase, functions as either a canonical or hybrid system using Nox organizing
subunit 1 (NoxO1) or p47(phox), respectively, the latter of which is functional
in vascular smooth muscle cells (VSMC). In this manuscript, we identify critical
requirement of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50; aka
NHERF1) for Nox1 activation and downstream responses. Superoxide (O2 (•-))
production induced by angiotensin II (AngII) was absent in mouse EBP50 KO VSMC
vs. WT. Moreover, ex vivo incubation of aortas with AngII showed a significant
increase in O2 (•-) in WT but not EBP50 or Nox1 nulls. Similarly,
lipopolysaccharide (LPS)-induced oxidative stress was attenuated in femoral
arteries from EBP50 KO vs. WT. In silico analyses confirmed by confocal
microscopy, immunoprecipitation, proximity ligation assay, FRET, and
gain-/loss-of-function mutagenesis revealed binding of EBP50, via its PDZ
domains, to a specific motif in p47(phox) Functional studies revealed
AngII-induced hypertrophy was absent in EBP50 KOs, and in VSMC overexpressing
EBP50, Nox1 gene silencing abolished VSMC hypertrophy. Finally, ex vivo
measurement of lumen diameter in mouse resistance arteries exhibited attenuated
AngII-induced vasoconstriction in EBP50 KO vs. WT. Taken together, our data
identify EBP50 as a previously unidentified regulator of Nox1 and support that
it promotes Nox1 activity by binding p47(phox) This interaction is pivotal for
agonist-induced smooth muscle ROS, hypertrophy, and vasoconstriction and has
implications for ROS-mediated physiological and pathophysiological processes. |
Are alterations in ultraconserved elements implicated in breast cancer? | Yes. SNPs in ultraconserved elements (UCEs) might be associated with cancer risk. | Ultraconserved elements (UCEs) are segments of >200 bp length showing absolute
sequence identity between orthologous regions of human, rat and mouse genomes.
The selection factors acting on these UCEs are still unknown. Recent studies
have shown that UCEs function as long-range enhancers of flanking genes or are
involved in splicing when overlapping with exons. The depletion of UCEs among
copy number variation as well as the significant under-representation of
single-nucleotide polymorphisms (SNPs) within UCEs have also revealed their
evolutional and functional importance indicating their potential impact on
disease, such as cancer. In the present study, we investigated the influence of
six SNPs within UCEs on familial breast cancer risk. Two out of six SNPs showed
an association with familial breast cancer risk. Whereas rs9572903 showed only a
borderline significant association, the frequency of the rare [G] allele of
rs2056116 was higher in cases than in controls indicating an increased familial
breast cancer risk ([G] versus [A]: odds ratio (OR) = 1.18, 95% confidence
interval (CI) 1.06-1.30, P = 0.0020; [GG] versus [AA]: OR = 1.41, 95% CI
1.15-1.74, P = 0.0011). Interestingly, comparing with the older age group, the
ORs were increased in woman younger than 50 years of age ([G] versus [A]: OR =
1.27, 95% CI 1.11-1.45, P = 0.0005; [GG] versus [AA]: OR = 1.60, 95% CI
1.22-2.10, P = 0.0007) pointing to an age- or hormone-related effect. This is
the first study indicating that SNPs in UCEs might be associated with cancer
risk. |
What is the mechanism of action of Osimertinib? | Osimertinib is a third-generation irreversible tyrosine kinase inhibitor of both activating EGFR mutations and resistance-associated T790M point mutation. | EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell
lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase
inhibitors (EGFR-TKIs) have been developed to target mutated EGFR. However,
there is little information regarding mutation specific potency of EGFR-TKIs
against various types of EGFR mutations. The purpose of this study is to
establish an in vitro model to determine the "therapeutic window" of EGFR-TKIs
against various types of EGFR mutations, including EGFR exon 20 insertion
mutations. The potency of 1st (erlotinib), 2nd (afatinib) and 3rd (osimertinib
and rociletinib) generation EGFR-TKIs was compared in vitro for human lung
cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild
type EGFR. An in vitro model of mutation specificity was created by calculating
the ratio of IC50 values between mutated and wild type EGFR. The in vitro model
identified a wide therapeutic window of afatinib for exon 19 deletions and L858R
and of osimertinib and rociletinib for T790M positive mutations. The results
obtained with our models matched well with previously reported preclinical and
clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of
which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib
was potent and presented a wide therapeutic window. To our knowledge, this is
the first report that has identified the therapeutic window of osimertinib for
EGFR exon 20 insertion mutations. In conclusion, this model will provide a
preclinical rationale for proper selection of EGFR-TKIs against
clinically-relevant EGFR mutations. Precision oncology is now the evidence-based standard of care for the management
of many advanced non-small cell lung cancers (NSCLCs). Expert consensus has
defined minimum requirements for routine testing and identification of epidermal
growth factor (EGFR) mutations (15% of tumors harbor EGFR exon 19 deletions or
exon 21 L858R substitutions) and anaplastic lymphoma kinase (ALK) rearrangements
(5% of tumors) in advanced lung adenocarcinomas (ACs). Application of palliative
targeted therapies with oral tyrosine kinase inhibitors (TKIs) in
advanced/metastatic lung ACs harboring abnormalities in EGFR (gefitinib,
erlotinib, afatinib) and ALK/ROS1/MET (crizotinib) has consistently led to more
favorable outcomes compared with traditional cytotoxic agents. In addition,
mutations leading to resistance to first-line EGFR and ALK TKIs can now be
successfully inhibited by soon to be approved third-generation EGFR TKIs
(osimertinib, rociletinib) and second-generation ALK TKIs (ceritinib,
alectinib). Notably, increasing feasibility, accessibility, and application of
molecular profiling technologies has permitted dynamic growth in the
identification of actionable driver oncogenes. Emerging genomic aberrations for
which TKIs have shown impressive results in clinical trials and expansion of
drug labels for approved agents are awaited include ROS1 rearrangements (1-2% of
tumors, drug: crizotinib) and BRAF-V600E mutations (1-3% of tumors, drugs:
vemurafenib, dafrafenib + trametinib). Evolving genomic events in which TKI
responses have been reported in smaller series include MET exon 14 skipping
mutations (2-4% of tumors, drug: crizotinib); high-level MET amplification (1-2%
of tumors, drug: crizotinib); RET rearrangements (1% of tumors, drug:
cabozantinib); and ERBB2 mutations (2-3% of tumors, drug: afatinib), among
others. Unfortunately, the most common genomic event in NSCLC, KRAS mutations
(25-30% of tumors), is not targetable with approved or in development small
molecule inhibitors. Here, we review currently approved, emerging, and evolving
systemic precision therapies matched with their driver oncogenes for the
management of advanced NSCLC. Since the discovery of sensitizing EGFR mutations as a predictive marker of
sensitivity to EGFR tyrosine kinase inhibitors (TKIs), the field of targeted
therapy in non-small cell lung cancer (NSCLC) has been revolutionized. Patients
harbouring these sensitizing mutations treated with EGFR TKI have derived
significant clinical outcome when compared with standard platinum based
chemotherapy doublets. However disease progression invariably occurs at a median
of about 9-13 months from initiation treatment, if acquired resistance commonly
due to the development of EGFR T790M mutation. A novel class of "third
generation" EGFR TKIs have been developed that is sensitising and T790M
mutant-specific whilst sparing WT EGFR, representing a significant breakthrough
in the treatment in NSCLC patients with acquired resistance harboring these
genotypes. Early phase clinical data suggest the third generation EGFR TKIs such
as osimertinib, rociletinib, and HM61713 are highly efficacious and well
tolerated. Another promising class of EGFR TKI such as AZD3759 has been designed
to penetrate blood brain barrier to treat brain metastases and leptomeningeal
disease and has showed promising responses in patients with brain metastases.
Acquired resistance to third generation EGFR TKIs has been reported including
EGFR C797S. Given its non-invasive nature, plasma ctDNA is being explored as a
possible approach to detect T790M mutation and to also inform on novel molecular
mechansims of tertiary resistance to third generation EGFR TKIs. An
understanding of the mechanisms of acquired resistance to the third-generation
EGFR TKIs will greatly aid in the development of the next generation of EGFR
TKIs. PURPOSE: Osimertinib (AZD9291) 80 mg once daily is approved by the US FDA for
the treatment of patients with metastatic EGFR T790M-positive NSCLC whose
disease has previously progressed on EGFR-TKI therapy. Osimertinib PK was
evaluated to define the dose and dosing interval, whether a fixed-dosing
approach can be used globally, and the impact of formulation and food on
exposure.
METHODS: AURA (NCT01802632): single- and multiple-dose PK of osimertinib
(20-240 mg daily) was determined in patients with advanced NSCLC.
Bioavailability study (NCT01951599): single-dose PK of osimertinib (20 mg) was
determined in healthy volunteers with administration of capsule, solution, or
tablet formulations fasted, and as a tablet in the fed and fasted state.
RESULTS: Osimertinib was slowly absorbed and displayed dose-proportional
increases in exposure from 20 to 240 mg. Distribution was extensive and
clearance low to moderate, resulting in a mean half-life of 48.3 h. Steady state
was achieved by 15 days of dosing, consistent with single-dose PK, with a
peak-to-trough ratio of 1.6. Two active metabolites circulated at ~10 % of
osimertinib exposure. Ethnicity did not appear to affect exposure. Osimertinib
PK profiles in healthy volunteers were similar to those in patients and were
unaffected by formulation. Food caused a clinically insignificant increase in
exposure.
CONCLUSIONS: Osimertinib PK supports once-daily dosing; the same dose for Asian
and non-Asian populations; a fixed-dosing approach; a minimal effect of food on
exposure; and a switch to tablet formulation without alteration to dose or
schedule. Osimertinib plasma concentrations are sustained throughout the dosing
period, which is considered optimal for efficacy. First- and second-generation epidermal growth factor receptor (EGFR) tyrosine
kinase inhibitors (TKIs) are the evidence-based first-line treatment for
metastatic non-small-cell lung cancers (NSCLCs) that harbor sensitizing EGFR
mutations (i.e. exon 19 deletions or L858R). However, acquired resistance to
EGFR TKI monotherapy occurs invariably within a median time frame of one year.
The most common form of biological resistance is through the selection of tumor
clones harboring the EGFR T790M mutation, present in >50% of repeat biopsies.
The presence of the EGFR T790M mutation negates the inhibitory activity of
gefitinib, erlotinib, and afatinib. A novel class of third-generation EGFR TKIs
has been identified by probing a series of covalent pyrimidine EGFR inhibitors
that bind to amino-acid residue C797 of EGFR and preferentially inhibit mutant
forms of EGFR versus the wild-type receptor. We review the rapid clinical
development and approval of the third-generation EGFR TKI osimertinib for
treatment of NSCLCs with EGFR-T790M. The tyrosine kinase inhibitors (TKI) against epidermal growth factor receptor
(EGFR) are widely used in patients with non-small cell lung cancer (NSCLC).
However, EGFR T790M mutation leads to resistance to most clinically available
EGFR TKIs. Third-generation EGFR TKIs against the T790M mutation have been in
active clinical development. These agents include osimertinib, rociletinib,
HM61713, ASP8273, EGF816, and PF-06747775. Osimertinib and rociletinib have
shown clinical efficacy in phase I/II trials in patients who had acquired
resistance to first- or second-generation TKIs. Osimertinib (AZD9291, TAGRISSO)
was recently approved by FDA for metastatic EGFR T790M mutation-positive NSCLC.
HM61713, ASP8237, EGF816, and PF-06747775 are still in early clinical
development. This article reviews the emerging data regarding third-generation
agents against EGFR T790M mutation in the treatment of patients with advanced
NSCLC. The effectiveness of cancer chemotherapy is often circumvented by multidrug
resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) drug
transporter ABCB1 (MDR1, P-glycoprotein). Several epidermal growth factor
receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been shown previously
capable of modulating the function of ABCB1 and reversing ABCB1-mediated MDR in
human cancer cells. Furthermore, some TKIs are transported by ABCB1, which
results in low oral bioavailability, reduced distribution, and the development
of acquired resistance to these TKIs. In this study, we investigated the
interaction between ABCB1 and osimertinib, a novel selective, irreversible
third-generation EGFR TKI that has recently been approved by the U.S. Food and
Drug Administration. We also evaluated the potential impact of ABCB1 on the
efficacy of osimertinib in cancer cells, which can present a therapeutic
challenge to clinicians in the future. We revealed that although osimertinib
stimulates the ATPase activity of ABCB1, overexpression of ABCB1 does not confer
resistance to osimertinib. Our results suggest that it is unlikely that the
overexpression of ABCB1 can be a major contributor to the development of
osimertinib resistance in cancer patients. More significantly, we revealed an
additional action of osimertinib that directly inhibits the function of ABCB1
without affecting the expression level of ABCB1, enhances drug-induced
apoptosis, and reverses the MDR phenotype in ABCB1-overexpressing cancer cells.
Considering that osimertinib is a clinically approved third-generation EGFR TKI,
our findings suggest that a combination therapy with osimertinib and
conventional anticancer drugs may be beneficial to patients with MDR tumors. The overexpression of ATP-binding cassette (ABC) transporters has been proved to
be a major trigger for multidrug resistance (MDR) in certain types of cancer. In
our study, we investigated whether osimertinib (AZD9291), a third-generation
irreversible tyrosine kinase inhibitor of both activating EGFR mutations and
resistance-associated T790M point mutation, could reverse MDR induced by ABCB1
and ABCG2 in vitro, in vivo, and ex vivo Our results showed that osimertinib
significantly increased the sensitivity of ABCB1- and ABCG2-overexpressing cells
to their substrate chemotherapeutic agents in vitro and in the model of
ABCB1-overexpressing KBv200 cell xenograft in nude mice. Mechanistically,
osimertinib increased the intracellular accumulations of doxorubicin (DOX) and
Rhodamine 123 (Rho 123) by inhibiting the efflux function of the transporters in
ABCB1- or ABCG2-overexpressing cells but not in their parental sensitive cells.
Furthermore, osimertinib stimulated the ATPase activity of both ABCB1 and ABCG2
and competed with the [(125)I] iodoarylazidoprazosin photolabeling bound to
ABCB1 or ABCG2, but did not alter the localization and expression of ABCB1 or
ABCG2 in mRNA and protein levels nor the phosphorylations of EGFR, AKT, and ERK.
Importantly, osimertinib also enhanced the cytotoxicity of DOX and intracellular
accumulation of Rho 123 in ABCB1-overexpressing primary leukemia cells. Overall,
these findings suggest osimertinib reverses ABCB1- and ABCG2-mediated MDR via
inhibiting ABCB1 and ABCG2 from pumping out chemotherapeutic agents and provide
possibility for cancer combinational therapy with osimertinib in the clinic. Mol
Cancer Ther; 15(8); 1845-58. ©2016 AACR. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are
the evidence-based first-line treatment for advanced non-small-cell lung cancer
that harbors sensitizing EGFR mutations (EGFRm(+)) such as exon 19 deletions and
L858R substitutions in exon 21. However, acquired resistance to EGFR TKIs is
mostly driven by a second-site EGFR T790M mutation, which negates their
inhibitory activity. Osimertinib (AZD9291, Tagrisso™), an oral, third-generation
EGFR TKI, has been designed to target the EGFR T790M mutation, while sparing
wild-type EGFR. In this up-to-date review, focus is not only on the structure,
mechanisms, and pharmacokinetics of osimertinib but also on summarizing clinical
trials and making recommendations of osimertinib for patients with
non-small-cell lung cancer. Osimertinib (OSI, also known as AZD9291) is the newest FDA-approved epidermal
growth factor receptor (EGFR) tyrosine kinase inhibitor for non-small cell lung
cancer (NSCLC) patients with EGFR T790M mutation. However, resistance to OSI is
likely to progress and the study of potential OSI-resistant mechanisms in
advanced is necessary. Here, the OSI-resistant NCI-H1975/OSIR cells were
established. After cells developed resistance to OSI, cell proliferation was
decreased while cell migration and invasion were increased. The NCI-H1975/OSIR
cells exhibited more resistance to gefitinib, erlotinib, afatinib, rociletinib,
doxorubicin, and fluorouracil, meanwhile showing higher sensitivity to
paclitaxel, when compared with NCI-H1975 cells. In addition, the NCI-H1975/OSIR
cells did not display multidrug resistance phenotype. The activation and
expression of EGFR were decreased after cells exhibited resistance. Compared
with NCI-H1975 cells, the activation of ERK and AKT in NCI-H1975/OSIR cells
could not be significantly inhibited by OSI treatment. Navitoclax
(ABT-263)-induced cell viability inhibition and apoptosis were more significant
in NCI-H1975/OSIR cells than that in NCI-H1975 cells. Moreover, these effects of
navitoclax in NCI-H1975/OSIR cells could be reversed by pretreatment of
Z-VAD-FMK. Collectively, loss of EGFR could pose as one of the OSI-resistant
mechanisms and navitoclax might be the candidate drug for OSI-resistant NSCLC
patients. Metabolic plasticity is an emerging hallmark of cancer, and increased glycolysis
is often observed in transformed cells. Small molecule inhibitors that target
driver oncogenes can potentially inhibit the glycolytic pathway. Osimertinib
(AZD9291) is a novel EGFR tyrosine kinase inhibitor (TKI) that is potent and
selective for sensitising (EGFRm) and T790M resistance mutations. Clinical
studies have shown osimertinib to be efficacious in patients with EGFRm/ T790M
advanced NSCLC who have progressed after EGFR-TKI treatment. However experience
with targeted therapies suggests that acquired resistance may emerge. Thus there
is a need to characterize resistance mechanisms and to devise ways to prevent,
delay or overcome osimertinib resistance. We show here that osimertinib
suppresses glycolysis in parental EGFR-mutant lung adenocarcinoma lines, but has
not in osimertinib-resistant cell lines. Critically, we show osimertinib
treatment induces a strict dependence on mitochondrial oxidative phosphorylation
(OxPhos), as OxPhos inhibitors significantly delay the long-term development of
osimertinib resistance in osimertinib-sensitive lines. Accordingly, growth
conditions which promote a less glycolytic phenotype confer a degree of
osimertinib resistance. Our data support a model in which the combination of
osimertinib and OxPhos inhibitors can delay or prevent resistance in
osimertinib-naïve tumour cells, and represents a novel strategy that warrants
further pre-clinical investigation. INTRODUCTION: AZD9291 (osimertinib) is designed for acquired T790M mutation
after first- and second-generation EGFR) tyrosine kinase inhibitors have been
used. Some of the resistance mechanisms that present after osimertinib
treatment, including a newly acquired EGFR C797S mutation, have been identified.
It is unclear, however, whether the bypass pathway is also a mechanism of
resistance in patients after osimertinib treatment.
METHODS: Cells from maligt pleural effusion were collected and cultured at
the time of progression in a patient being treated with osimertinib. Tumor
genotyping was done by matrix-assisted laser desorption ionization-time of
flight mass spectrometry. EGFR, AKT, MEK, and ERK phosphorylation were
determined. An anchorage-dependent colony formation assay was used for drug
sensitivity.
RESULTS: An acquired mutation, BRAF V600E, was found in the patient at the time
of progression while being treated with osimertinib. Cells grown from maligt
pleural effusion were sensitive to BRAF V600E inhibitor and were more vulnerable
to a combination treatment with osimertinib.
CONCLUSIONS: A potential mechanism of acquired resistance to osimertinib in
patients with T790M is through the BRAF pathway. Simultaneous blockade of the
BRAF and EGFR had a significant inhibitory effect. |
What is Bartter syndrome? | All forms of Bartter syndrome are characterized by hypokalemia, metabolic alkalosis, and secondary hyperaldosteronism | Five patients with pseudo-Bartter's syndrome from surreptitious diuretic abuse
were compared with six patients with true Bartter's syndrome, diagnosed as a
normotensive, hyperreninaemic, hypokalaemic metabolic alkalosis with normal
urine chloride excretion, low CH2O/(CH2O+CCl) ratio during maximal water
diuresis and negative urine screen for diuretics. The latter was positive for
frusemide in four and for hydrochlorothiazide in the remaining pseudo-Bartter's
patients. The two groups of patients did not differ as for plasma Na+, Cl-, K+,
HCO3-, renin, and aldosterone, while uric acid and Mg2+ were greater in
pseudo-Bartter's patients. Daily and fasting urine Na+, Cl- and K+ excretion
were less in pseudo-Bartter's patients; however, there was substantial overlap
of values between the two groups. Fractional distal solute reabsorption during
maximal water diuresis was low in the six patients with Bartter's syndrome and
in two pseudo-Bartter's patients; thus, this parameter could not be taken as a
specific diagnostic marker of Bartter's syndrome. Frusemide administration, 40
mg i.v., induced a brisk increase of urine flow (11.7-21.8 ml/min), UOsm
(148-186 mOsm/kg H2O) and FENa (14.6-24%) in Bartter's syndrome, but not
pseudo-Bartter's patients; in all pseudo-Bartter's patients frusemide-induced
changes of UOsm (13-97) and FENa (-0.5 to 10.2) were markedly less than in
Bartter's syndrome patients. Frusemide resistance in pseudo-Bartter's patients
was most probably related to diuretic-induced ECF volume contraction and
increased proximal tubule solute reabsorption; in fact fractional lithium
clearance (FELi, a marker of post-proximal solute delivery) was low in
pseudo-Bartter's, but not in Bartter's syndrome patients.(ABSTRACT TRUNCATED AT
250 WORDS) BACKGROUND: Hypokalemia due to renal potassium wasting in the absence of
hypertension, moderate metabolic alkalosis, hyperreninism and hyperaldosteronism
suggest the presence of Bartter's syndrome. The underlying cause is an inherited
defect of sodium chloride reabsorption in the thick ascending limb of Henle. A
differential diagnosis of Bartter's syndrome is Gitelman's syndrome, another
hypokalemia-hypomagnesemia syndrome, which is thought to be caused by a
transport defect in the distal tube.
PATIENTS AND METHODS: We report 3 patients presenting with signs primarily
suggestive of Bartter's syndrome, who turned out to have Gitelman's syndrome
after determining the excretion of calcium in the urine.
RESULTS: Two women, 36- and 55-year old, suffered from paresthesias in the hands
and feet and from tetanic convulsions. The brother of the 36-year old woman
presented in our hospital because of an accidentally discovered hypokalemia
without any clinical symptoms. In all patients the outstanding biochemical
features were hypokalemia, hypomagnesemia and moderate metabolic alcalosis. The
renin and aldosterone values were inappropriately high. The most characteristic
finding in the urine, besides the presence of hyperkaliuria was the diminution
of calcium excretion, despite normocalcemia.
CONCLUSION: The association between sodium and calcium reabsorption in the loop
of Henle predicts hypercalciuria in patients with a defect in salt reabsorption
in this segment, as in Bartter's syndrome. In Gitelman's syndrome the laboratory
features resemble the findings in Bartter's syndrome, except for the presence of
hypocalciuria. Since hypocalciuria follows also the administration of thiazide
diuretics, which act in the early part of distal tube, a transport defect in
this part of the tube is thought to be responsible for the electrolyte
disturbances in Gitelman's syndrome. The measurement of the urinary calcium
excretion in patients with an unclear hypokalemia-hypomagnesemia-syndrome allows
easily the differentiation between Bartter's and Gitelman's syndrome. Bartter syndrome involves an overlapping set of closely related renal tubular
disorders which can be subdivided into at least three clinical phenotypes: (1)
classic Bartter syndrome (2) Gitelman syndrome, and (3) a neonatal variant of
Bartter syndrome. In contrast to classic Bartter syndrome and Gitelman syndrome,
the neonatal variant of Bartter syndrome has both the features of renal tubular
hypokalemic alkalosis as well as profound systemic manifestations. Specifically,
neonatal Bartter syndrome is characterized by intrauterine polyhydramnios,
premature delivery, and life-threatening episodes of fever and dehydration. Most
of these infants also have severe hypercalciuria with associated
nephrocalcinosis and osteopenia. Over a 22-year period, 20 Costa Rican patients
with a congenital syndrome that resembles neonatal Bartter syndrome have been
identified and characterized. While these patients exhibit some of the clinical
characteristics previously described for neonatal Bartter syndrome, this cohort
also has a set of distinct features. They are predomitly female, have a later
age of diagnosis, manifest a relatively unique set of physical traits, and
appear to have milder clinical disease. Given these differences, it will be
important to apply the emerging molecular tools to determine whether the
phenotypic variability indicates genetic heterogeneity in neonatal-onset Bartter
syndrome. The molecular defects responsible for the two most-common forms of inherited
normotensive hypokalemic metabolic alkalosis have recently been defined. Most
patients with Bartter syndrome have defects in transporters in the thick
ascending limb of the loop of Henle, such as the Na-K-2Cl cotransporter, NKCC2,
or the ATP-sensitive potassium channel, ROMK. Patients with Gitelman syndrome
usually have mutations in the thiazide-sensitive Na-Cl cotransporter in the
distal convoluted tubule. The location of the affected transporters correlates
well with the typical presentation of these syndromes. Patients with Bartter
syndrome typically present with normal or increased calcium excretion.
Hypomagnesemia is present in only one-third of affected individuals. In
contrast, hypomagnesemia and hypocalciuria are considered hallmarks of Gitelman
syndrome. This report describes siblings presenting as young adults with mild
symptoms associated with normotensive hypokalemic metabolic alkalosis. One
sibling has hypocalciuria and hypomagnesemia, consistent with Gitelman syndrome.
Surprisingly, the other sibling has normal serum magnesium and urinary calcium
excretion. These siblings demonstrate the biochemical heterogeneity that can
exist in patients with normotensive hypokalemic metabolic alkalosis. This report
indicates that hypocalciuria does not always distinguish Gitelman and Bartter
syndromes. ABSTRACT.: Inherited hypokalemic renal tubulopathies are differentiated into at
least three clinical subtypes: (1) the Gitelman variant of Bartter syndrome
(GS); (2) hyperprostaglandin E syndrome, the antenatal variant of Bartter
syndrome (HPS/aBS); and (3) the classic Bartter syndrome (cBS). Hypokalemic
metabolic alkalosis and renal salt wasting are the common characteristics of all
three subtypes. Hypocalciuria and hypomagnesemia are specific clinical features
of Gitelman syndrome, while HPS/aBS is a life-threatening disorder of the
newborn with polyhydramnios, premature delivery, hyposthenuria, and
nephrocalcinosis. The Gitelman variant is uniformly caused by mutations in the
gene for the thiazide-sensitive NaCl-cotransporter NCCT (SLC12A3) of the distal
tubule, while HPS/aBS is caused by mutations in the gene for either the
furosemide-sensitive NaK-2Cl-cotransporter NKCC2 (SLC12A1) or the inwardly
rectifying potassium channel ROMK (KCNJ1). Recently, mutations in a basolateral
chloride channel CLC-Kb (CLCNKB) have been described in a subset of patients
with a Bartter-like phenotype typically lacking nephrocalcinosis. In this study,
the screening for CLCNKB mutations showed 20 different mutations in the affected
children from 30 families. The clinical characterization revealed a highly
variable phenotype ranging from episodes of severe volume depletion and
hypokalemia during the neonatal period to almost asymptomatic patients diagnosed
during adolescence. This study adds 16 novel mutations to the nine already
described, providing further evidence that mutations in the gene for the
basolateral chloride channel CLC-Kb are the molecular basis of classic Bartter
syndrome. Interestingly, the phenotype elicited by CLCNKB mutations occasionally
includes HPS/aBS, as well as a Gitelman-like phenotype. PURPOSE OF REVIEW: This review describes recent advances in our understanding of
the genetic heterogeneity, pathophysiology and treatment of Bartter syndrome, a
group of autosomal recessive disorders that are characterized by markedly
reduced or absent salt transport by the thick ascending limb of Henle.
Consequently, individuals with Bartter syndrome exhibit renal salt wasting and
lowered blood pressure, hypokalemic metabolic alkalosis and hypercalciuria with
a variable risk of renal stones.
RECENT FINDINGS: Previously, three genes (SLC12A2, the sodium-potassium-chloride
co-transporter; KCNJ1, the ROMK potassium ion channel; ClC-Kb, the basolateral
chloride ion channel) had been identified as causing antenatal and 'classic'
Bartter syndrome. Two additional genes have now been identified. Barttin is a
beta-subunit that is required for the trafficking of CLC-K (both ClC-Ka and
ClC-Kb) channels to the plasma membrane in both the thick ascending limb and the
marginal cells in the scala media of the inner ear that secrete potassium
ion-rich endolymph. Loss-of-function mutations in barttin thus cause Bartter
syndrome with sensorineural deafness. In addition, severe gain-of-function
mutations in the extracellular calcium ion-sensing receptor can result in a
Bartter phenotype because activation of this G protein-coupled receptor inhibits
salt transport in the thick ascending limb (a furosemide-like effect).
SUMMARY: Five genes have been identified as causing Bartter syndrome (types
I-V), with the unifying pathophysiology being the loss of salt transport by the
thick ascending limb. Phenotypic differences in Bartter types I-V relate to the
specific physiological roles of the individual genes in the kidney and other
organ systems. The term "Bartter syndrome" encompasses a group of closely related inherited
tubulopathies characterized by markedly reduced NaCl transport by the distal
nephron. At present, five different genetic variants have been demonstrated. The
majority of patients with so-called classic Bartter syndrome carry inactivating
mutations of the CLCNKB gene encoding the basolateral ClC-Kb chloride channel
(Bartter syndrome type III). The purpose of this study was to investigate the
underlying mutation in cases of classic Bartter syndrome followed at our center.
Ten patients, including two sisters, with clinical and biochemical features of
classic Bartter syndrome were included in the mutational analysis. They
originated from different regions of Spain with either Basque or Spanish
ancestry. There was no history of consanguineous marriage in any of the
kindreds. The parents and siblings of each patient, as well as a population of
300 healthy control adult subjects, were also analyzed. All ten patients were
found to be homozygous for an identical missense mutation in the CLCNKB gene,
substituting a threonine for an alanine at codon 204 (A204T) in the putative
fifth transmembrane domain of the protein. None of the 300 control subjects were
homozygous for the A204T allele. Overall, the A204T mutation was detected on
2/600 control chromosomes. Despite sharing a common mutation, the clinical
manifestations of the syndrome in the patients varied from lack of symptoms to
severe growth retardation. Demonstration of a point mutation within the CLCNKB
gene as the apparently unique cause of Bartter syndrome type III in Spain is
highly suggestive of a founder effect. Our results also support the lack of
correlation between genotype and phenotype in this disease. Bartter's syndrome belongs to a group of hypokalemic renal channel diseases.
These channels are located in the lipid layer of cell membranes where they exist
as water channels through which ion transport is performed. Based on the type of
genetic disorder and clinical presentation, Bartter's syndrome is classified as
neonatal, classical and Gitelman's syndrome. Most of the cases have been noted
in pediatric age groups and adult-onset cases are very rare. Moreover, an
association between Bartter's syndrome and empty sella has recently been
reported in 3 children. We report here the second case of an adult patient
affected by Bartter's syndrome with partial empty sella. The patient showed some
clinical and histological characteristics of both classic Bartter's syndrome and
Gitelman's syndrome, suggesting that genotype and phenotype of Bartter's
syndrome are not so clear-cut and that phenotypic overlap may occur, according
to a recent hypothesis. Magnetic resoce imaging disclosed a partial empty
sella. A thorough endocrinological investigation showed normal hypophyseal,
thyroidal, adrenal and gonadal function. Good therapeutic effects were achieved
using spironolactone, ACE-inhibitor and potassium supplementation, with
normalization of the kalemia. At present, the value of the association of
Bartter's syndrome and empty sella remains unclear and future studies are needed
to clarify the importance of this association, both in children and in adult
patients affected by Bartter's syndrome. Bartter syndrome, a group of disorders that encompasses multiple genetic defects
with similar clinical presentation, has been divided into six different
genotypes, according to different genetic defects, and into three main clinical
variants (or phenotypes). Classic laboratory findings in all variants include
hypochloremia, hypokalemia, and metabolic alkalosis with excessive excretion of
chloride and potassium. Classic Bartter syndrome, neonatal Bartter syndrome, and
Gitelman syndrome are the three main clinical variants. Classic Bartter syndrome
and neonatal Bartter syndrome have defects in genes that affect transport
channels in the ascending loop of Henle, where as in Gitleman syndrome the
defect occurs in the transport channels of the distal convoluted tubule. Classic
Bartter syndrome and neonatal Bartter syndrome have similar presenting symptoms,
potential outcomes, and treatment, but different ages at presentation. Gitelman
syndrome, a more benign condition than the other clinical variants, has the
classic hallmark finding of hypomagnesemia and low to normal excretion of
calcium. This differentiates it from the classic and neonatal variants of the
disease. With early diagnosis and proper treatment, Bartter syndrome has a good
prognosis. But failure to identify it can lead to tubulointerstitial nephritis
and renal failure. We present a case of a 6-month-old boy with Bartter syndrome
who presented with poor weight gain and an abdominal mass. BACKGROUND: antenatal variant of Bartter syndrome is characterized by a history
of polyhydramnios, premature birth, metabolic alkalosis, hypokalemia, polyuria
and renal salt wasting. In this report we present a premature female baby with
antenatal Barter syndrome who had three episodes of urinary tract infection
(UTI), without evidence for congenital anomaly of the kidneys or urinary tract.
METHODS: antenatal Bartter syndrome was diagnosed according to the standard
criteria. Ultrasound scan and voiding cystourethrography were performed to
exclude congenital anomaly of the kidneys and urinary tract.
RESULTS: the baby presented with early hyperkalemia and acidosis. The typical
biochemical features of the Bartter syndrome were observed in the second month.
Despite appropriate treatment she had persistent hypercalciuria. The clinical
course was complicated with recurrent episodes of febrile UTIs. Urinary tract
system imaging did not demonstrate congenital anomalies. She finally died of
severe dehydration, acidosis and renal failure.
CONCLUSION: since no congenital anomaly of the kidneys or urinary tract was
demonstrated in our patient, we believe that severe, persistent hypercalciuria
is the most important risk factor for development of recurrent UTIs. Bartter syndrome (BS) is a rare renal tubular disorder presenting with
hypokalemic metabolic alkalosis, which is classified into five types. KCNJ1
mutations usually cause the neonatal form of BS, type II BS (OMIM 241200).
However, this report concerns a female patient with a novel, compound
heterozygous KCNJ1 mutation that causes late-onset BS. The unique clinical
findings of this case include persistently elevated 1,25(OH)(2) vitamin D
levels, possibly due to increase prostaglandin E(2) levels, and medullary
nephrocalcinosis. Treatment with COX-2 inhibitors resolved her hypercalciuria
and improved her height and weight; renal function remains stable and there is
no progression of nephrocalcinosis. BACKGROUND: Hypokalaemic nephropathy has been described in patients with chronic
potassium depletion; it is a condition in which proximal tubular vacuolization
and interstitial fibrosis occur, resulting in a decline in glomerular filtration
rate (GFR) and, in some cases, renal failure. It has been described in patients
with chronic diarrhoea, eating disorders, laxative abuse and primary
hyperaldosteronism; also occasionally in Bartter syndrome (BS), in which severe
hypokalaemia accompanies significant renal sodium and water losses, though
rarely in Gitelman syndrome (GS), in which there is equally severe hypokalaemia,
but only modest sodium losses.
AIM: We hypothesized that hypokalaemic nephropathy may not be due to potassium
depletion per se, but persistently elevated circulating levels of aldosterone,
possibly with superimposed episodes of renal hypoperfusion.
DESIGN AND METHODS: We searched UK and European data sets to retrospectively
compare serum and urinary parameters in patients with GS and BS.
RESULTS: The patients with GS often had lower serum potassium concentrations
than patients with BS, but the BS patients had significantly higher serum
creatinine concentrations and lower estimated GFRs (eGFR). BS patients had
significantly higher fractional excretions of sodium compared with GS patients,
as well as higher plasma renin activities and serum aldosterone levels.
CONCLUSION: These findings show that in genetically confirmed cases of BS and
GS, the degree of hypokalaemia (as an index of chronic potassium depletion) does
not correlate with GFR, and that on-going sodium and water losses, and
consequent secondary hyperaldosteronism, may play a more important role in the
aetiology of hypokalaemic nephropathy. BACKGROUND: We aim to review the clinical features of two renal tubular
disorders characterized by sodium and potassium wasting: Bartter syndrome and
Gitelman syndrome.
DATA SOURCES: Selected key references concerning these syndromes were analyzed,
together with a PubMed search of the literature from 2000 to 2011.
RESULTS: The clinical features common to both conditions and those which are
distinct to each syndrome were presented. The new findings on the genetics of
the five types of Bartter syndrome and the discrete mutations in Gitelman
syndrome were reviewed, together with the diagnostic workup and treatment for
each condition.
CONCLUSIONS: Patients with Bartter syndrome types 1, 2 and 4 present at a
younger age than classic Bartter syndrome type 3. They present with symptoms,
often quite severe in the neonatal period. Patients with classic Bartter
syndrome type 3 present later in life and may be sporadically asymptomatic or
mildly symptomatic. The severe, steady-state hypokalemia in Bartter syndrome and
Gitelman syndrome may abruptly become life-threatening under certain aggravating
conditions. Clinicians need to be cognizant of such renal tubular disorders, and
promptly treat at-risk patients. Acquired Bartter-like syndrome (BLS), characterized by hypokalemic metabolic
alkalosis, hypomagnesemia, hypocalcemia, and normal kidney function, can be
induced by diuretics or antibiotics. It is a very rare condition and only
anecdotal cases mostly in adults were reported. Although tubulopathy associated
with colistin was reported in adults, to the best of our knowledge,
colistin-associated BLS neither in adults nor in children has been reported in
the literature. We here report a-28-week, 740 g female preterm infant who
developed BLS just after colistin treatment for Acinetobacter baumannii
infection and recovered few days after the drug cessation, and discuss the
possible association of colistin and tubulopathy. More research on colistin
pharmacokinetics and pharmacodynamics in critically ill patients and preterm
infants is needed to guide adequate colistin dosing at the least toxicity. Bartter syndrome is a group of inherited, salt-losing tubulopathies presenting
as hypokalemic metabolic alkalosis with normotensive hyperreninemia and
hyperaldosteronism. Around 150 cases have been reported in literature till now.
Mutations leading to salt losing tubulopathies are not routinely tested in
Indian population. The authors have done the genetic analysis for the first time
in the Bartter syndrome on two cases from India. First case was antenatal
Bartter syndrome presenting with massive polyuria and hyperkalemia. Mutational
analysis revealed compound heterozygous mutations in KCNJ1(ROMK) gene
[p(Leu220Phe), p(Thr191Pro)]. Second case had a phenotypic presentation of
classical Bartter syndrome however, genetic analysis revealed only heterozygous
novel mutation in SLC12A gene p(Ala232Thr). Bartter syndrome is a clinical
diagnosis and genetic analysis is recommended for prognostication and genetic
counseling. Bartter syndrome (BS) is a hereditary disease, with an autosomal recessive or
autosomal domit mode of transmission. It is characterized by salt wasting
hypochloraemic, hypokalaemic metabolic alkalosis and hyperreninaemia with normal
blood pressure. The primary defect is in the thick ascending limb of loop of
Henle (TAL). Herein, we report a case that had typical features of BS like
severe dehydration, severe hypokalaemia, metabolic alkalosis and failure to
thrive but had normal aldosterone level which is very uncommon. |
Is there any tool that facilitates the functional analysis of cis-regulatory regions in zebrafish? | Yes. The zebrafish enhancer detection (ZED) vector is a tool that facilitates transgenesis and the functional analysis of cis-regulatory regions in zebrafish. | |
List RNA modifications databases | RMBase and MODOMICS | MODOMICS is a database of RNA modifications that provides comprehensive
information concerning the chemical structures of modified ribonucleosides,
their biosynthetic pathways, RNA-modifying enzymes and location of modified
residues in RNA sequences. In the current database version, accessible at
http://modomics.genesilico.pl, we included new features: a census of human and
yeast snoRNAs involved in RNA-guided RNA modification, a new section covering
the 5'-end capping process, and a catalogue of 'building blocks' for chemical
synthesis of a large variety of modified nucleosides. The MODOMICS collections
of RNA modifications, RNA-modifying enzymes and modified RNAs have been also
updated. A number of newly identified modified ribonucleosides and more than one
hundred functionally and structurally characterized proteins from various
organisms have been added. In the RNA sequences section, snRNAs and snoRNAs with
experimentally mapped modified nucleosides have been added and the current
collection of rRNA and tRNA sequences has been substantially enlarged. To
facilitate literature searches, each record in MODOMICS has been
cross-referenced to other databases and to selected key publications. New
options for database searching and querying have been implemented, including a
BLAST search of protein sequences and a PARALIGN search of the collected nucleic
acid sequences. tRNA molecules contain numerous chemically altered nucleosides, which are formed
by enzymatic modification of the primary transcripts during the complex tRNA
maturation process. Some of the modifications are introduced by single
reactions, while other require complex series of reactions carried out by
several different enzymes. The location and distribution of various types of
modifications vary greatly between different tRNA molecules, organisms and
organelles. We have developed a computational method tRNAmodpred, for predicting
modifications in tRNA sequences. Briefly, our method takes as an input one or
more unmodified tRNA sequences and a set of protein sequences corresponding to a
proteome of a cell. Subsequently it identifies homologs of known tRNA
modification enzymes in the proteome, predicts tRNA modification activities and
maps them onto known pathways of RNA modification from the MODOMICS database.
Thereby, theoretically possible modification pathways are identified, and
products of these modification reactions are proposed for query tRNAs. This
method allows for predicting modification patterns for newly sequenced genomes
as well as for checking tentative modification status of tRNAs from one species
treated with enzymes from another source, e.g. to predict the possible
modifications of eukaryotic tRNAs expressed in bacteria. tRNAmodpred is freely
available as a web server at http://genesilico.pl/trnamodpred/. |
Please list the 3 findings in HELLP syndrome. | hemolysis, elevated liver enzymes and low platelets constitute the hellp syndrome. | OBJECTIVE: Our objective was to categorize the histologic findings in the liver
in patients with HELLP syndrome (hemolysis, elevated liver enzymes, and low
platelet count) and to correlate these findings with the severity of clinical
laboratory abnormalities.
STUDY DESIGN: Eleven patients with laboratory criteria for HELLP syndrome who
required cesarean delivery underwent needle biopsy of the liver under direct
visualization.
RESULTS: Eight patients had periportal hemorrhage, and six had fibrin
deposition. Fatty infiltration was seen in four, one with large-droplet fat,
three with microvesicular fat. There was no statistically significant
correlation between the severity of the histologic findings of periportal
hemorrhage and fibrin deposition and the clinical laboratory findings. Fatty
infiltration did not correlate with the severity of the HELLP syndrome's
histologic condition, but, in contrast, did correlate with thrombocytopenia and
aminotransferase elevations.
CONCLUSIONS: Laboratory abnormalities do not accurately reflect the severity of
the underlying histopathologic condition in HELLP syndrome. We propose that all
patients with HELLP syndrome, regardless of the degree of their laboratory
abnormalities, be treated aggressively, primarily with delivery. OBJECTIVE: Our purpose was to test the hypothesis that the HELLP (hemolysis,
elevated liver enzymes, and low platelets) syndrome is the result of excessive
vasoconstriction of the hepatic arterial circulation.
STUDY DESIGN: Doppler ultrasonography was used to measure the pulsatility index
of the common hepatic artery in 14 women with preeclampsia, 15 with preeclampsia
complicated by HELLP syndrome, and 8 with HELLP syndrome but without
proteinuria. Gestational age ranged from 24 to 38 weeks. The study group was
compared with a reference group (n = 42).
RESULTS: Both in preeclampsia and in the HELLP syndrome the hepatic artery
pulsatility index values were significantly increased compared with the
reference group. However, no significant differences were found between the
preeclamptic group, the HELLP group with proteinuria, and those with HELLP
without proteinuria.
CONCLUSIONS: These findings indicate that hepatic artery resistance to blood
flow is increased in preeclampsia in the presence or absence of the HELLP
syndrome. The results also demonstrate that vasoconstriction of the hepatic
arteries is not more pronounced in the HELLP syndrome than in other
manifestations of preeclampsia. Therefore factors other than vasoconstriction
are likely to be responsible for the development of the HELLP syndrome. OBJECTIVES: Our objective was to describe the hepatic imaging findings in
selected patients with HELLP syndrome (hemolysis, elevated liver enzymes, and
low platelet count) and to correlate these findings with the severity of
concurrent clinical and laboratory abnormalities.
STUDY DESIGN: Patients with laboratory criteria for HELLP syndrome with
complaints of severe right upper quadrant abdominal pain in association with
either shoulder pain, neck pain, or relapsing hypotension underwent imaging of
the liver. Clinical and laboratory parameters were then correlated with the
hepatic imaging findings.
RESULTS: Thirty-four patients were evaluated in this study. Computed tomographic
scanning of the liver was used for 33 patients. Additional imaging evaluations
included magnetic resoce imaging for 4 patients and ultrasonographic
evaluation of the liver for 5 patients. In 15 cases (45%) the computed
tomographic results were abnormal. The most frequent abnormal hepatic imaging
findings were subcapsular hematoma (n = 13) and intraparenchymal hemorrhage (n =
6). There was no statistically significant correlation between the presence of
an abnormal hepatic imaging finding and the severity of liver function test
abnormalities. However, the severity of thrombocytopenia did correlate with
hepatic imaging findings (p = 0.04). In particular, an abnormal hepatic imaging
finding was noted for 10 of 13 patients (77%) with a platelet count of < or = 20
x 10(9)/L (p = 0.012).
CONCLUSIONS: Abnormalities in liver function test results do not accurately
reflect the presence of abnormal hepatic imaging findings in HELLP syndrome.
Patients with HELLP syndrome having complaints of right upper quadrant pain and
neck pain, shoulder pain, or relapsing hypotension should undergo imaging of the
liver. OBJECTIVE: Our purpose was to compare neonatal outcome after preterm delivery of
infants whose gestation was complicated by the HELLP (hemolysis, elevated liver
enzymes, and low platelet count) syndrome, partial HELLP syndrome, or severe
preeclampsia.
STUDY DESIGN: We reviewed the maternal and neonatal charts from 269 consecutive
pregcies complicated by the HELLP syndrome or severe preeclampsia managed at
our perinatal center. The HELLP syndrome was defined by previously published
laboratory criteria. Viable pregcies were divided into 3 groups: HELLP
syndrome, partial HELLP syndrome (at least 1, but not all 3, features of the
HELLP syndrome), and severe preeclampsia (no features of the HELLP syndrome).
Results were compared by means of chi2 analysis and Student t test where
appropriate. Logistic regression was used to evaluate outcome variables at
different gestational ages.
RESULTS: There were no significant differences in complications among the 3
groups at each gestational age. There was, as expected, a significant decrease
in morbidity and mortality rates with advanced gestational age.
CONCLUSIONS: In severe preeclampsia, neonatal morbidity and death are related to
gestational age rather than to the presence or absence of the HELLP syndrome.
Whether expectant management is safe for women with the HELLP syndrome requires
further study. The coincidence of hemolysis, elevated liver enzymes, and low platelet count
(HELLP) syndrome and cortical blindness is an uncommon but very dramatic event.
We describe a case of HELLP syndrome complicating with acute cortical blindness
before delivery. A 27 year-old woman, gravida 1, para 0, with normal medical
history, was referred to our emergency department at the 33th week of gestation
due to headache, vomiting, and blurred vision. The ophthalmologic examination
showed intact pupillary light reflexes and normal ophthalmoscopic findings, but
no light perception in either eye. Brain computed tomography showed normal
findings. HELLP syndrome and preeclampsia was diagnosed based on the findings of
hypertension and proteinuria as well as laboratory data. Prompt delivery was
performed in order to achieve good maternal and neonatal outcomes. OBJECTIVE: A novel human endogenous retroviral element, designated as syncytin,
has been suggested as a contributor to normal placental architecture, especially
in the fusion processes of cytotrophoblasts to syncytiotrophoblasts. We tested
the hypothesis of whether the gene expression of syncytin may be altered in
cases with placental dysfunction such as preeclampsia or HELLP (hemolysis,
elevated liver enzymes, low platelets) syndrome.
STUDY DESIGN: We included 30 women with normal pregcies, 16 with
preeclampsia, and 6 with HELLP syndrome. After delivery, messenger ribonucleic
acids (mRNA) of syncytin, glyceraldehyde-3-phosphate dehydrogenase and
beta-actin were analyzed in placental villi with use of quantitative real-time
polymerase chain reaction.
RESULTS: In placental villi, syncytin mRNA/beta-actin mRNA and syncytin
mRNA/glyceraldehyde-3-phosphate dehydrogenase mRNA ratios were lower in patients
with preeclampsia (P <.05) or HELLP syndrome than in healthy control subjects.
CONCLUSION: A reduced placental expression of syncytin may contribute to altered
cell fusion processes in placentogenesis and disturbed placental function in
hypertensive disorders of pregcy. We describe the imaging findings of the HELLP (haemolysis, elevated liver
enzymes and low platelets) syndrome in a gravid 36-year-old-woman who presented
at 19.5 weeks gestation with severe right upper quadrant pain. Ultrasound
findings suggested a focal hyperechoic liver lesion suggesting pregcy-related
hepatopathy prior to development of biochemical signs of HELLP syndrome. Within
3 days, the patient developed characteristic clinical and laboratory evidence of
the HELLP syndrome. MR imaging at this time demonstrated localized hepatic
ischaemia, further strengthening the diagnosis of HELLP syndrome. In preeclampsia and hemolysis, elevated liver enzymes and low platelet (HELLP)
syndrome, impaired trophoblast invasion and excessive fibrin deposition in the
placental intervillous space is associated with fetal compromise. However,
little information is available whether modulation of placental protease
expression--potentially causing impaired trophoblast invasion--is associated
with the HELLP syndrome. Total RNA and protein were extracted from placental
tissue from 11 females with HELLP syndrome and 8 controls matched for
gestational age. mRNA expression of matrix metalloprotease (MMP) -2 and -9,
tissue inhibitors of metalloprotease (TIMP) -1, -2, and -3, and urokinase-type
plasminogen activator receptor (uPAR) was determined by Northern blotting.
Protein expression of MMP-2 and -9, and TIMP-1 and -2 was detected by Western
blotting and that of uPA, uPAR, and plasminogen activator inhibitor (PAI) -1 by
ELISA. In patients with HELLP syndrome, mRNA expression of MMP-2 and TIMP-2 was
decreased, whereas TIMP-1 and -3 levels were unchanged. MMP-9 and uPAR mRNA was
undetectable in both groups. Protein expression of all investigated proteolytic
factors remained unchanged. Our findings at the mRNA level suggest a decrease in
matrix remodeling in placentae from patients with HELLP syndrome compared with
control pregcies, although this is not supported at the protein level. PROBLEM: Hemolysis, elevated liver enzymes, and low platelet count syndrome
(HELLP syndrome) is a life-threatening variant of severe pre-eclampsia in
pregt women. The complement system may play a role in the pathogenesis of
this condition. We sought to determine serum complement 3 (C3) levels and its
regulatory protein complement factor H (FH) in the HELLP syndrome.
METHOD OF STUDY: Twenty-two pre-eclamptic patients with HELLP syndrome (mean
age: 27.8 +/- 6.2 years), 21 pre-eclamptic patients without HELLP syndrome (mean
age: 27.5 +/- 6.8 years) and 24 normotensive, healthy pregt women (mean age:
26.1 +/- 4.4 years) were included in this study. Serum concentrations of C3 and
FH were measured in all participants.
RESULTS: Concentrations of C3 and FH did not differ significantly between the
study groups. In patients with the HELLP syndrome, FH levels were positively
associated with platelet count.
CONCLUSION: These findings did not support a major role of complement activation
in the HELLP syndrome. In patients with HELLP, lower levels of FH are correlated
with a reduced platelet count. OBJECTIVE: To investigate apoptosis, proliferation and Fas ligand expression of
placental trophoblast in the hemolysis, elevated liver enzymes, low platelets
(HELLP) syndrome and in pre-eclampsia (PE), and to compare this with normal
pregcies.
DESIGN: Prospective study.
SETTING: University hospital in Croatia.
SAMPLE: Placentae from women with HELLP syndrome (n=10), PE (n=10) and normal
pregcies (n=10).
METHODS: The HELLP syndrome was diagnosed with platelets <100×10(9) /L,
aspartate aminotransferase (AST) and alanine transaminase (ALT) >70 U/L and
lactic acid dehydrogenase (LDH) > 600 U/L. Pre-eclampsia was diagnosed at blood
pressure >140/90 mmHg, with proteinuria >300 mg/L/24 hours. For detection of
apoptosis and proliferation in villous trophoblast, antibodies M30 and Ki-67
were used. Expression of Fas ligand was assessed using immunohistochemistry and
the semiquantitative HSCORE method.
MAIN OUTCOME MEASURES: Apoptosis, proliferation and Fas ligand expression in
villous trophoblast.
RESULTS: Apoptosis, proliferation and Fas ligand expression were higher in
villous trophoblast in HELLP syndrome than in the PE group (p=0.015, p=0.018 and
p=0.002, respectively) and the control group (p=0.000, p=0.012 and p=0.049,
respectively). Placentae from the PE group had higher levels of apoptosis
(p=0.019), lower Fas ligand expression (p=0.029) and no difference in
proliferation (p=0.887) compared with the control group.
CONCLUSIONS: There is an increase in apoptosis, proliferation and Fas ligand
expression in placentae from women with HELLP syndrome compared with placentae
from PE and normal pregcies. Our findings indicate the possibility of
differential mechanisms behind HELLP syndrome and PE. BACKGROUND: Hepatic hemorrhage occurs in less than 5% of patients with
hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome but it is a
profound cause of maternal/perinatal morbidity and mortality.
OBJECTIVES: To determine when liver bleeding occurs during the development of
HELLP syndrome.
SEARCH STRATEGY: The English literature was searched for all reports of HELLP
syndrome associated with liver bleeding.
SELECTION CRITERIA: Eighty-seven case summaries of liver bleeding in the setting
of HELLP syndrome were included. The standard definition of HELLP syndrome was
used with expansion into the Mississippi classification system, supplemented by
patients with partial HELLP syndrome.
DATA COLLECTION AND ANALYSIS: Demographic and clinical data were collected and
recorded in an Excel database.
MAIN RESULTS: Liver bleeding was detected in 18 (20.7%) patients with class 1
HELLP syndrome, 24 (27.6%) with class 2 HELLP syndrome, and 12 (13.8%) with
class 3 or partial HELLP syndrome. In 33 (37.9%) patients, the exact class of
HELLP syndrome at the time liver bleeding was detected could not be determined
from the published descriptions.
CONCLUSIONS: Liver bleeding can occur early during HELLP syndrome development,
not only in patients with advanced, class 1 illness. Hypertensive disorders of pregcy are one of the most common complications of
pregcy, but one of the most serious expressions of this pathology is HELLP
syndrome. The HELLP syndrome is characterized by the presence of hypertension
disorder more a triad: microangiopathic hemolysis, elevated liver enzymes and
low platelet count. Patient with HELLP syndrome is associated with increased
maternal risk complications such as: cerebral hemorrhage, retinal detachment,
hematoma/ hepatic rupture, acute renal failure, disseminated intravascular
coagulation, placental abruption and therefore a maternal death. For all these
reasons it is recommended to search for findings of HELLP syndrome in patients
with hypertensive disorder of pregcy. The main clinical confusion of HELLP
syndrome is acute fatty liver of pregcy, however there are parameters that
help correct identification. The presence of HELLP syndrome involves a rapid
termination of pregcy and the administration of corticosteroids does not
improve maternal morbidity and mortality but may help raise the platelet count,
thus decreasing the need for transfusion and shorten hospital stay. Much of the
decline in maternal morbidity and mortality associated with hypertensive
disorders of pregcy is in proper diagnosis and effective management of HELLP
syndrome. Neurological disorders caused by pregcy and puerperium include the posterior
reversible encephalopathy syndrome, the amniotic fluid embolism syndrome (AFES),
the postpartum angiopathy due to reversible vasoconstriction syndrome, and the
Sheehan syndrome. Hypertension and proteinuria are the hallmarks of
preeclampsia, seizures define eclampsia. Hemolysis, elevated liver enzymes and
low platelets constitute the HELLP syndrome. Vision disturbances including
cortical blindness occur in the posterior reversible encephalopathy syndrome
(PRES). The Sheehan syndrome presents with panhypopituitarism post partum due to
apoplexia of the pituitary gland in severe peripartal blood loss leading to
longstanding hypotension. Some neurological disorders occur during pregcy and
puerperium with an increased frequency. These include stroke, sinus thrombosis,
the restless legs syndrome and peripheral nerve syndromes, especially the carpal
tunnel syndrome. Chronic neurologic diseases need an interdisciplinary approach
during pregcy. Some anticonvulsants double the risk of birth defects. The
highest risk exists for valproic acid, the lowest for lamotrigine and
levetiracetam. For MS interval treatment, glatiramer acetate and interferones
seem to be safe during pregcy. All other drugs should be avoided. |
Which mutated gene is associated with Waardenburg and Tietz syndromes? | Mutations in microphthalmia-associated transcription factor (MITF) gene cause Waardenburg and Tietz syndromes. | On some occasions, mutations of a gene cause different syndromes that may have
similar phenotypes. For example, mutations of the MITF gene cause Waardenburg
syndrome type 2 (Tassabehji et al, 1994; Nobukuni et al, 1996) as well as Tietz
syndrome (Smith et al, 1997). On other occasions, mutations of different genes
cause an identical syndrome. Molecular analyses of these genes may provide a
good opportunity to not only understand such syndromes themselves but also the
biologic aspects of cells relevant to these syndromes. By analyzing the genes
for Waardenburg syndrome, we showed that PAX3, the gene responsible for
Waardenburg syndrome type 1, regulates MITF, the gene responsible for
Waardenburg syndrome type 2. Such epistatic relationships have been shown
between other genes related to Waardenburg syndrome, and likely to construct a
cascade. This paper proposes such a cascade, one that involves genes for PAX3,
MITF, human MyoD, MYF5, c-MET, c-KIT, tyrosinase, TRP-1, human QNR-71, SOX10,
EDNRB, and EDN3. Mouse microphthalmia transcription factor (Mitf) mutations affect the
development of four cell types: melanocytes, mast cells, osteoclasts, and
pigmented epithelial cells of the eye. The mutations are phenotypically diverse
and can be arranged in an allelic series. In humans, MITF mutations cause
Waardenburg syndrome type 2A (WS2A) and Tietz syndrome, autosomal domit
disorders resulting in deafness and hypopigmentation. Mitf mice thus represent
an important model system for the study of human disease. Here we report the
complete exon/intron structure of the mouse Mitf gene and show it to be similar
to the human gene. We also found that the mouse gene is transcriptionally
complex and is capable of generating at least 13 different Mitf isoforms. Some
of these isoforms are missing important functional domains of the protein,
suggesting that they might play an inhibitory role in Mitf function and signal
transduction. In addition, we determined the molecular basis for six
microphthalmia mutations. Two of the mutations are reported for the first time
here (Mitf(mi-enu198) and Mitf(mi-x39)), while the others (Mitf(mi-ws),
Mitf(mi-bws), Mitf(mi-ew), and Mitf(mi-di)) have been described but the
molecular basis for the mutation not determined. When analyzed in terms of the
genomic and transcriptional data presented here, it is apparent that these
mutations result from RNA processing or transcriptional defects. Interestingly,
three of the mutations (Mitf(mi-x39), Mitf(mi-bws), and Mitf(mi-ws)) produce
proteins that are missing important functional domains of the protein identified
in in vitro studies, further confirming a biological role for these domains in
the whole animal. Microphthalmia-associated transcription factor (MITF) is a transcription factor
with a basic-helix-loop-helix-leucine zipper (bHLHZip) structure. Mutations of
the MITF gene cause a variety of phenotypes, most notably in pigmented cells, in
several species. In humans, haploinsufficiency of MITF causes Waardenburg
syndrome type 2, while a domit-negative mutation causes Tietz syndrome. Four
isoforms have been cloned so far: MITF-M is the most abundant and is expressed
in neural-crest-derived melanocytes; MITF-A is expressed in various cultured
cells including retinal pigment epithelium (RPE) and enriched in RPE of
embryonal and developing eyes; MITF-H are expressed in many types of cultured
cells and in the heart tissue; MITF-C is expressed in many types of cultured
cells, but not in melanocytes. Many growth factor signaling pathways have been
implicated for regulation of MITF at both protein and promoter levels. Most
notably, Steel factor/c-Kit signaling pathway was linked to phosphorylation of
MITF at Ser73 and Ser409 through activation of MAP kinase and RSK-1,
respectively. Phosphorylation of MITF is also conducted at Ser298 through
GSK3beta, although the signaling pathway for this event still remains to be
elucidated. IGF-1 and HGF/SF pathways may merge with the c-Kit signaling
pathway. WNT and MSH signaling pathways regulate MITF positively at the promoter
level. Endothelins may regulate MITF at the protein and promoter levels. MITF is
involved in the differentiation, growth and survival of pigment cells, employing
a number of signaling pathways. Waardenburg syndrome (WS) is a series of auditory-pigmentary disorders inherited
in an autosomal domit manner. In most patients, WS2 results from mutations in
the MITF gene. MITF encodes a basic helix-loop-helix transcription factor that
activates transcription of tyrosinase and other melanocyte proteins. The
clinical presentation of WS is highly variable, and we believe that Tietz
syndrome and WS2 with ocular albinism (OA) are likely two variations of WS2 due
to the presence of modifiers. One family with a molecular diagnosis of WS2
co-segregating with OA has previously been reported. A digenic mutation
mechanism including both a MITF mutation and the TYR(R402Q) hypomorphic allele
was proposed to be the cause of OA in this family. Here, we present a second WS2
family with OA and provide evidence suggesting the TYR(R402Q) allele does not
cause OA in this family. We hypothesize the presence of a novel OCA3 mutation
together with the MITF del p.R217 mutation account for the OA phenotype in this
family. Since MITF is a transcription factor for pigmentation genes, a mutation
in MITF plus a heterozygous mutation in OCA3 together provide an adverse effect
crossing a quantitative threshold; therefore, WS2 with OA occurs. We have
hypothesized previously that the clinical spectrum and mutation mechanism of OCA
depend on the pigmentation threshold of an affected individual. This unique
family has provided further evidence supporting this hypothesis. We suggest that
by studying OCA patients alongside WS patients with various pigmentation
profiles we can facilitate further understanding of the pigmentation pathway. Mutations in microphthalmia-associated transcription factor (MITF) lead to
Waardenburg syndrome type 2 (WS2), a domitly inherited disorder involving
hearing loss and pigment disturbances caused by a lack of melanocytes. On rare
occasions, mutations in MITF lead to Tietz syndrome (TS), which is characterized
by a severe WS2 phenotype. The MITF gene is the human homolog of the mouse
microphthalmia (mi) gene in some families. Mi/mi mice show decreased numbers and
an abnormal phenotype of mast cells (MC). In contrast, the number and phenotype
of MC in WS2/TS patients who also have an alteration in their MITF gene are
unclear. In this study, we identified a mutation in the MITF gene, delR217,
which was equivalent to that found in mi/mi mice, in a case of TS. None of the
MITF isoforms with the mutation were able to transactivate the tyrosinase gene
promoter. In addition, mutant MITF-M showed domit negative activity toward
wild-type MITF-M, inhibiting its transactivation of the tyrosinase gene
promoter. The patient's peripheral blood CD34 cells showed no differences with
respect to total cell number or their expression levels of tryptase mRNA in a
serum-deprived liquid culture system for 6 weeks when compared with normal
control cells. These findings suggest that MITF does not play a critical role in
MC development in humans. Waardenburg syndrome type II (WS2) is associated with syndromic deafness. A
subset of WS2, WS2A, accounting for approximately 15% of patients, is attributed
to mutations in the microphthalmia-associated transcription factor (MITF) gene.
We examined the genetic basis of WS2 in a large Chinese family. All 9 exons of
the MITF gene, the single coding exon (exon 2) of the most common hereditary
deafness gene GJB2 and the mitochondrial DNA (mtDNA) 12S rRNA were sequenced. A
novel heterozygous mutation c.[742_743delAAinsT;746_747delCA] in exon 8 of the
MITF gene co-segregates with WS2 in the family. The MITF mutation results in a
premature termination codon and a truncated MITF protein with only 247 of the
419 wild type amino acids. The deaf proband had this MITF gene heterozygous
mutation as well as a c.[109G>A]+[235delC] compound heterozygous pathogenic
mutation in the GJB2 gene. No pathogenic mutation was found in mtDNA 12S rRNA in
this family. Thus, a novel compound heterozygous mutation,
c.[742_743delAAinsT;746_747delCA] in MITF exon 8 was the key genetic reason for
WS2 in this family, and a digenic effect of MITF and GJB2 genes may contribute
to deafness of the proband. The article presents the melanogenesis pathway and the role of cyclic adenosine
monophosphate (cAMP) and microphthalmia transcription factor (MITF) in
regulation of this process. Products of melanogenesis are eu- and/or
pheomelanins synthesized in a multistage process of tyrosine oxidation and
polymerization. The conversions require the presence of tyrosinase (TYR, key
enzyme), tyrosine hydroxylase isoform I (THI) and tyrosinase related proteins
(TRP1 and TRP2). Many types of signal molecules and transcription factors
participate in regulation of melanin synthesis, but the most important are cAMP
and MITF. cAMP is the second messenger in the intracellular signal cascade,
which is synthesized from adenosine triphosphate (ATP) by adenylyl cyclase,
activated among others by the melanocortin receptor and the αS subunit of G
protein. The signal molecule cAMP regulates MITF, TYR, THI, GTP-cyclohydroxylase
I (GTP-CHI) transcription and phenylalanine hydroxylase (PAH) phosphorylation at
Ser16 by protein kinase A (PKA). Mutations of genes encoding proteins belonging
to the cAMP signal cascade may lead to McCune-Albright and Carney syndromes.
MITF is one of the most important nuclear transcription factors regulating
melanogenesis. Currently 10 isoforms of human MITF are known, but in melanocytes
only MITF-M, MITF-Mdel, MITF-A and MITF-H occur. MITF transcription factor
regulates melanogenesis by activation of tyrosinase, TRP1 and TRP2
transcription. It also affects expression of other factors regulating melanosome
maturation, biogenesis and transport. Moreover, it regulates melanocyte
proliferation and protection against apoptosis. Mutations of the MITF gene may
lead to hereditary diseases: Waardenburg type II and Tietz syndromes. The human deafness-pigmentation syndromes, Waardenburg syndrome (WS) type 2a,
and Tietz syndrome are characterized by profound deafness but only partial
cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in
MITF. To illuminate differences between cutaneous and otic melanocytes in these
syndromes, their development and survival in heterozygous Microphthalmia-White
(Mitf(Mi-wh) /+) mice were studied and hearing function of these mice
characterized. Mitf(Mi-wh) /+ mice have a profound hearing deficit,
characterized by elevated auditory brainstem response thresholds, reduced
distortion product otoacoustic emissions, absent endocochlear potential, loss of
outer hair cells, and stria vascularis abnormalities. Mitf(Mi-wh) /+ embryos
have fewer melanoblasts during embryonic development than their wild-type
littermates. Although cochlear melanocytes are present at birth, they disappear
from the Mitf(Mi-wh) /+ cochlea between P1 and P7. These findings may provide
insight into the mechanism of melanocyte and hearing loss in human
deafness-pigmentation syndromes such as WS and Tietz syndrome and illustrate
differences between otic and follicular melanocytes. MITF mutations results in an abnormal melanocyte development and lead to
Waardenburg syndrome type 2 (WS2). Here, we analyzed the in vitro activities of
two recently identified WS2-associated MITF mutations (p.R217I and p.T192fsX18).
The R217I MITF retained partial activity, normal DNA-binding ability and nuclear
distribution, whereas the T192fsX18 MITF failed to activate TYR promoter and
showed aberrant subcellular localization which may be caused by deletion of
nuclear localization signal (NLS) at aa 213-218 (ERRRRF). These results suggest
that haploinsufficiency may be the underlying mechanism for the mild phenotypes
of WS2 caused by these two mutations. OBJECTIVES: Till date, mutations in the genes PAX3 and MITF have been described
in Waardenburg syndrome (WS), which is clinically characterised by congenital
hearing loss and pigmentation anomalies. Our study intended to determine the
frequency of mutations and deletions in these genes, to assess the clinical
phenotype in detail and to identify rational priorities for molecular genetic
diagnostics procedures.
DESIGN: Prospective analysis.
PATIENTS: 19 Caucasian patients with typical features of WS underwent stepwise
investigation of PAX3 and MITF. When point mutations and small
insertions/deletions were excluded by direct sequencing, copy number analysis by
multiplex ligation-dependent probe amplification was performed to detect larger
deletions and duplications. Clinical data and photographs were collected to
facilitate genotype-phenotype analyses.
SETTING: All analyses were performed in a large German laboratory specialised in
genetic diagnostics.
RESULTS: 15 novel and 4 previously published heterozygous mutations in PAX3 and
MITF were identified. Of these, six were large deletions or duplications that
were only detectable by copy number analysis. All patients with PAX3 mutations
had typical phenotype of WS with dystopia canthorum (WS1), whereas patients with
MITF gene mutations presented without dystopia canthorum (WS2). In addition, one
patient with bilateral hearing loss and blue eyes with iris stroma dysplasia had
a de novo missense mutation (p.Arg217Ile) in MITF. MITF 3-bp deletions at amino
acid position 217 have previously been described in patients with Tietz syndrome
(TS), a clinical entity with hearing loss and generalised hypopigmentation.
CONCLUSIONS: On the basis of these findings, we conclude that sequencing and
copy number analysis of both PAX3 and MITF have to be recommended in the routine
molecular diagnostic setting for patients, WS1 and WS2. Furthermore, our
genotype-phenotype analyses indicate that WS2 and TS correspond to a clinical
spectrum that is influenced by MITF mutation type and position. The basic-helix-loop-helix-leucine zipper (bHLHZip) protein MITF
(microphthalmia-associated transcription factor) is a master regulator of
melanocyte development. Mutations in the MITF have been found in patients with
the domitly inherited hypopigmentation and deafness syndromes Waardenburg
syndrome type 2A (WS2A) and Tietz syndrome (TS). Additionally, both somatic and
germline mutations have been found in MITF in melanoma patients. Here, we
characterize the DNA-binding and transcription activation properties of 24 MITF
mutations found in WS2A, TS and melanoma patients. We show that most of the WS2A
and TS mutations fail to bind DNA and activate expression from
melanocyte-specific promoters. Some of the mutations, especially R203K and
S298P, exhibit normal activity and may represent neutral variants. Mutations
found in melanomas showed normal DNA-binding and minor variations in
transcription activation properties; some showed increased potential to form
colonies. Our results provide molecular insights into how mutations in a single
gene can lead to such different phenotypes. OBJECTIVE: To determine the genetic cause for a patient featuring decreased
pigmentation of the skin and iris, hearing loss and multiple congenital
anomalies.
METHODS: Routine chromosomal banding was performed to analyze the karyotype of
the patient and his parents. Single nucleotide polymorphism array (SNP array)
was employed to identify cryptic chromosome aberrations, and quantitative
real-time PCR was used to confirm the results.
RESULTS: Karyotype analysis has revealed no obvious anomaly for the patient and
his parents. SNP array analysis of the patient has demonstrated a 3.9 Mb
deletion encompassing 3p13p14.1, which caused loss of entire MITF gene. The
deletion was confirmed by quantitative real-time PCR. Clinical features of the
patient have included severe bilateral hearing loss, decreased pigmentation of
the skin and iris and multiple congenital anomalies.
CONCLUSION: The patient, carrying a 3p13p14.1 deletion, has features of Tietz
syndrome/Waardenburg syndrome type IIa. This case may provide additional data
for the study of genotype-phenotype correlation of this disease. Tietz syndrome and Waardenburg syndrome type 2A are allelic conditions caused by
MITF mutations. Tietz syndrome is inherited in an autosomal domit pattern and
is characterized by congenital deafness and generalized skin, hair, and eye
hypopigmentation, while Waardenburg syndrome type 2A typically includes variable
degrees of sensorineural hearing loss and patches of de-pigmented skin, hair,
and irides. In this paper, we report two unrelated families with MITF mutations.
The first family showed an autosomal domit pattern and variable expressivity.
The second patient was isolated. MITF gene analysis in the first family
demonstrated a c.648A>C heterozygous mutation in exon 8 c.648A>C; p. (R216S),
while in the isolated patient, an apparently de novo heterozygous
c.1183_1184insG truncating mutation was demonstrated in exon 10. All patients
except one had bilateral reduced ocular anteroposterior axial length and a high
hyperopic refractive error corresponding to posterior microphthalmos, features
that have not been described as part of the disease. Our results suggest that
posterior microphthalmos might be part of the clinical characteristics of
Tietz/Waardenburg syndrome type 2A and expand both the clinical and molecular
spectrum of the disease. © 2016 Wiley Periodicals, Inc. |
List the three most abundant bacterial phyla present in mouse feces. | Firmicutes
Proteobacteria
Bacteroidetes | An extraction method was developed to recover high-quality RNA from rumen
digesta and mouse feces for phylogenetic analysis of metabolically active
members of the gut microbial community. Four extraction methods were tested on
different amounts of the same samples and compared for efficiency of recovery
and purity of RNA. Trizol extraction after bead beating produced a higher
quantity and quality of RNA than a similar method using phenol/chloroform.
Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery
compared with phosphate-buffered saline during the dissociation of
microorganisms from rumen digesta or fecal particles. The identity of
metabolically active bacteria in the samples was analyzed by sequencing 87
amplicons produced using bacteria-specific 16S rDNA primers, with cDNA
synthesized from the extracted RNA as the template. Amplicons representing the
major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%;
Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered,
showing that development of the RNA extraction method enables RNA-based analysis
of metabolically active bacterial groups from the rumen and other environments.
Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA
amplicons were related to the Proteobacteria, providing the first evidence that
this group may have greater importance in rumen metabolism than previously
attributed by DNA-based analysis. BACKGROUND AND AIMS: Increased efficiency of energy harvest, due to alterations
in the gut microbiota (increased Firmicutes and decreased Bacteroidetes), has
been implicated in obesity in mice and humans. However, a causal relationship is
unproven and contributory variables include diet, genetics and age. Therefore,
we explored the effect of a high-fat (HF) diet and genetically determined
obesity (ob/ob) for changes in microbiota and energy harvesting capacity over
time.
METHODS: Seven-week-old male ob/ob mice were fed a low-fat diet and wild-type
mice were fed either a low-fat diet or a HF-diet for 8 weeks (n=8/group). They
were assessed at 7, 11 and 15 weeks of age for: fat and lean body mass (by NMR);
faecal and caecal short-chain fatty acids (SCFA, by gas chromatography); faecal
energy content (by bomb calorimetry) and microbial composition (by metagenomic
pyrosequencing).
RESULTS: A progressive increase in Firmicutes was confirmed in both HF-fed and
ob/ob mice reaching statistical significance in the former, but this phylum was
unchanged over time in the lean controls. Reductions in Bacteroidetes were also
found in ob/ob mice. However, changes in the microbiota were dissociated from
markers of energy harvest. Thus, although the faecal energy in the ob/ob mice
was significantly decreased at 7 weeks, and caecal SCFA increased, these did not
persist and faecal acetate diminished over time in both ob/ob and HF-fed mice,
but not in lean controls. Furthermore, the proportion of the major phyla did not
correlate with energy harvest markers.
CONCLUSION: The relationship between the microbial composition and energy
harvesting capacity is more complex than previously considered. While
compositional changes in the faecal microbiota were confirmed, this was
primarily a feature of high-fat feeding rather than genetically induced obesity.
In addition, changes in the proportions of the major phyla were unrelated to
markers of energy harvest which changed over time. The possibility of microbial
adaptation to diet and time should be considered in future studies. |
What gene is mutated in Sickle Cell Anemia? | Sickle cell anemia (SCA) is an autosomal recessive disease caused by by the HBB:c.20A>T mutation that leads to hemoglobin S synthesis. | Sickle-cell anemia results from an A leads to T transversion in the second
nucleotide of codon 6 of the beta-globin gene. We now report an uncommon
beta-thalassemia gene that contains a deletion of this nucleotide. Thus, one
mutation (GAG leads to GTG) produces sickle-cell anemia, while the other (GAG
leads to GG) eliminates beta-globin production. These data establish that
different alterations affecting one specific nucleotide can produce either an
abnormal hemoglobin or beta-thalassemia. Moreover, the nucleotide sequence
comprising codons 6-8 of the beta-globin gene appears to be particularly
susceptible to mutations affecting nucleotide number. Retrovirus-mediated gene transfer into hematopoietic cells may provide a means
of treating both inherited and acquired diseases involving hematopoietic cells.
Implementation of this approach for disorders resulting from mutations affecting
the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however,
has been hampered by the inability to generate recombit viruses able to
efficiently and faithfully transmit the necessary sequences for appropriate gene
expression. We have addressed this problem by carefully examining the
interactions between retroviral and beta-globin gene sequences which affect
vector transmission, stability, and expression. First, we examined the
transmission properties of a large number of different recombit proviral
genomes which vary both in the precise nature of vector, beta-globin structural
gene, and locus control region (LCR) core sequences incorporated and in the
placement and orientation of those sequences. Through this analysis, we
identified one specific vector, termed M beta 6L, which carries both the human
beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully
transmits recombit proviral sequences to cells with titers greater than 10(6)
per ml. Populations of murine erythroleukemia (MEL) cells transduced by this
virus expressed levels of human beta-globin transcript which, on a per gene copy
basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which
carries human chromosome 11, the site of the beta-globin locus. Analysis of
individual transduced MEL cell clones, however, indicated that, while expression
was detected in every clone tested (n = 17), the levels of human beta-globin
treatment varied between 4% and 146% of the levels in Hu11. This clonal
variation in expression levels suggests that small beta-globin LCR sequences may
not provide for as strict chromosomal position-independent expression of
beta-globin as previously suspected, at least in the context of
retrovirus-mediated gene transfer. Sickle cell anemia is a genetic blood disorder arising from a point mutation in
the beta-globin gene that leads to the replacement of glutamic acid residue by
valine at the sixth position of the beta--chain of hemoglobin. At low oxygen
tension, the mutant hemoglobin, sickle hemoglobin, polymerizes inside the red
blood cells into a gel or further into fibers leading to a drastic decrease in
the red cell deformability. As a result, micro-vascular occlusion arises which
may lead to serious, sometimes fatal, crises. The present article reviews the
historical, genetic, molecular, cellular, and clinical aspects of the disease. A
review for the development and design of drugs to treat sickle cell anemia is
presented. Anti-sickling agents are classified, based on the target to be
modified, into three classes: the gene, the sickle hemoglobin molecule, and the
red cell membrane modifiers. In spite of the full understanding of the
pathology, physiology, and the molecular nature of the disease, and the
development of large number of antisickling agents, a cure for sickle cell
anemia still is unavailable. Strategies to treat sickle cell anemia since the
early times of the disease state discovery in 1910, has focussed mainly on
prophylactic measures to alleviate the painful crises. The article addresses
clinical approaches used then and now to treat the disease, and the rationale of
their use. Currently in clinical practice, hydroxyurea is the most commonly used
agent to treat the disease, and it has been recently approved by the united
states Food and Drug Administration as a drug for that purpose. BACKGROUND: Sickle cell anemia is caused by a single type of mutation, a
homozygous A→T substitution in the ß globin gene. Clinical severity is diverse,
partially due to additional, disease-modifying genetic factors. We are studying
one such modifier locus, HMIP (HBS1L-MYB intergenic polymorphism, chromosome
6q23.3). Working with a genetically admixed patient population, we have
encountered the necessity to generate haplotype signatures of genetic markers to
label genomic fragments with distinct genealogical origin at this locus. With
the goal to generate haplotype signatures from patients experimentally, we have
investigated the suitability of an existing ofluidic assay platform to
perform phase alignment with single-nucleotide polymorphism alleles.
METHODOLOGY/PRINCIPAL FINDINGS: Patient DNA samples were loaded onto Fluidigm
Digital Arrays and individual DNA molecules were assayed with allele-specific
probes for SNP markers. Here we present data showing the utility of the
ofluidic approach, yielding haplotype data identical to those obtained with a
family-based method. We then determined haplotype composition in a group of
patients with sickle cell disease, including in those where a mathematical
inference approach gave ambiguous or misleading results. Experimental phasing of
genotypes across 3.8 kb for rs9399137, rs9402685, and rs11759553 created
unequivocal haplotype signatures for each of the patients. In 68 patients, we
found 8 copies of a haplotype signature ('C-C-T'), which is known to be
prevalent in Europeans but to be absent in West African populations. We have
confirmed the identity of our phased allele pairs by single-molecule sequencing
and have demonstrated, in principle, that three-allele phasing (using three
colors) is a potential extension to this method.
CONCLUSIONS/SIGNIFICANCE: Phased haplotypes yield more information than the
individual marker genotypes. Procedures such as the one described here would
therefore benefit genetic mapping and functional studies as well as diagnostic
procedures where the identity or parental origin of short genetic fragments is
of importance. BACKGROUND: Sickle cell anemia (SCA) is an inherited blood disorder. SCA
patients present clinical and hematologic variability that cannot be only
explained by the single mutation in the beta-globin gene. Others genetic
modifiers and environmental effects are important for the clinical phenotype.
SCA patients present arginine deficiency that contributes to a lower nitric
oxide (NO) bioactivity.
OBJECTIVE: The aim of this work is to determine the association between
hematological and biochemical parameters and genetic variants from eNOS gene, in
pediatric SCA patients.
METHODS: 26 pediatric SCA patients were genotyped using polymerase chain
reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques in
three important eNOS gene polymorphisms - rs2070744, rs1799983 and intron 4
VNTR.
RESULTS: Results from this study show a significant statistical association
between some parameters and genetic variants: an increased reticulocyte count
and high serum lactate dehydrogenase levels were associated with both the
rs2070744_TT and the rs1799983_GG genotypes at eNOS gene and high levels of
neutrophils were associated with the eNOS4a allele at intron 4 VNTR.
CONCLUSIONS: Our results reinforce the importance of NO bioactivity in SCA. We
presume that NO, and its precursors might be used as therapy to improve the
quality of life of SCA patients. Sickle cell anemia (SCA) is an autosomal recessive disease caused by the
HBB:c.20A>T mutation that leads to hemoglobin S synthesis. The disease presents
with high clinical heterogeneity characterized by chronic hemolysis, recurrent
episodes of vaso-oclusion and infection. This work aimed to characterize by in
silico studies some genetic modulators of severe hemolysis and stroke risk in
children with SCA, and understand their consequences at the hemorheological
level.Association studies were performed between hemolysis biomarkers as well as
the degree of cerebral vasculopathy and the inheritance of several polymorphic
regions in genes related with vascular cell adhesion and vascular tonus in
pediatric SCA patients. In silico tools (e.g. MatInspector) were applied to
investigate the main variant consequences.Variants in vascular adhesion
molecule-1 (VCAM1) gene promoter and endothelial nitric oxide synthase (NOS3)
gene were significantly associated with higher degree of hemolysis and stroke
events. They potentially modify transcription factor binding sites (e.g. VCAM1
rs1409419_T allele may lead to an EVI1 gain) or disturb the corresponding
protein structure/function. Our findings emphasize the relevance of genetic
variation in modulating the disease severity due to their effect on gene
expression or modification of protein biological activities related with sickled
erythrocyte/endothelial interactions and consequent hemorheological
abnormalities. |
What is the function of the Indian hedgehog protein in chondrocytes? | Indian hedgehog is a protein that regulates endochondral differentiation and ossification in both a parathyroid hormone-related protein (PTHrP)-dependent and -independent manner by activating transcriptional mediator Gli2 and is an essential factor for proper skeletal development. | Proper regulation of chondrocyte differentiation is necessary for the
morphogenesis of skeletal elements, yet little is known about the molecular
regulation of this process. A chicken homolog of Indian hedgehog (Ihh), a member
of the conserved Hedgehog family of secreted proteins that is expressed during
bone formation, has now been isolated. Ihh has biological properties similar to
those of Sonic hedgehog (Shh), including the ability to regulate the conserved
targets Patched (Ptc) and Gli. Ihh is expressed in the prehypertrophic
chondrocytes of cartilage elements, where it regulates the rate of hypertrophic
differentiation. Misexpression of Ihh prevents proliferating chondrocytes from
initiating the hypertrophic differentiation process. The direct target of Ihh
signaling is the perichondrium, where Gli and Ptc flank the expression domain of
Ihh. Ihh induces the expression of a second signal, parathyroid hormone-related
protein (PTHrP), in the periarticular perichondrium. Analysis of PTHrP (-/-)
mutant mice indicated that the PTHrP protein signals to its receptor in the
prehypertrophic chondrocytes, thereby blocking hypertrophic differentiation. In
vitro application of Hedgehog or PTHrP protein to normal or PTHrP (-/-) limb
explants demonstrated that PTHrP mediates the effects of Ihh through the
formation of a negative feedback loop that modulates the rate of chondrocyte
differentiation. A complex signaling pathway involving members of the Hedgehog, Bone
morphogenetic protein (Bmp) and Gli families regulates early patterning events
in fetal skeletogenesis (Hui and Joyner, 1993. A mouse model of Greig
cephalopolysyndactyly syndrome: the extra-toes mutation contains an intragenic
deletion of the Gli3 gene. Nat. Genet. 3, 241-246; Bitgood and McMahon, 1995.
Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell
interaction in the mouse embryo. Dev. Biol. 172, 126-138; Lanske et al., 1996.
PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone
growth. Science 273, 663-666; Vortkamp et al., 1996. Regulation of rate of
cartilage differentiation by Indian hedgehog and PTH-related protein. Science
273, 613-622). Hedgehog genes encode secreted proteins that mediate patterning
and growth through the induction of secondary signals (reviewed in Hammerschmidt
et al., 1997. The world according to hedgehog. Trends Genet. 13, 14-21). Two
potential targets of Ihh are bmp6 and gli (Johnson et al., 1995. Patched
overexpression alters wing disc size and pattern: transcriptional and
post-transcriptional effects on hedgehog targets. Development 121, 4161-4170;
Dominguez et al., 1996. Sending and receiving the hedgehog signal: control by
the Drosophila Gli protein Cubitus interruptus. Science 272, 1621-1625; Marigo
et al., 1996. Sonic hedgehog differentially regulates expression of GLI and GLI3
during limb development. Dev. Biol. 180, 273-283). We investigated the molecular
similarities and differences between fetal and postnatal skeletal development by
analyzing the coincident and complimentary expression domains of indian hedgehog
(ihh), bmp6 and gli in adjacent sections throughout the process of
skeletogenesis. In almost all of the skeletal tissues examined, the expression
domains of ihh and bmp6 were adjacent to one another and this region was
surrounded by gli-expressing cells. These observations are in keeping with the
proposed function of gli as a negative regulator of Ihh signaling and the
induction of Bmps by Hedgehog proteins (Roberts et al., 1995. Sonic hedgehog is
an endodermal signal inducing Bmp-4 and Hox genes during induction and
regionalization of the chick hindgut. Development 121, 3163-3174; Kawakami et
al., 1996. BMP signaling during bone pattern determination in the developing
limb. Development 122, 3557-3566). By puberty, ihh, bmp6 and gli transcripts
were no longer detected in the growth plate, despite the fact that physeal
chondrocytes continued to hypertrophy and differentiate. Although bmp6 was
expressed, ihh transcripts were not found in primordia of intramembranous bones,
nor in cells lining the future articular surfaces. Collectively our findings
suggest that ihh participates in, but is not required for chondrocyte
hypertrophy. We investigated the regulation of Sox9, a transcription factor known to play a
role in chondrogenesis, by bone morphogenetic protein-2 (BMP-2) and hedgehog
proteins in order to better understand their signaling function in endochondral
bone formation. The mesenchymal progenitor cell line C3H10T1/2 was stimulated
with BMP-2. Sox9 expression levels were measured by quantitative reverse
transcriptase-polymerase chain reaction and Northern analysis. We found that
Sox9 was up-regulated by BMP-2 in a dose-dependent manner. The expression of
Col2a1, a downstream response gene of Sox9, was also significantly increased
upon BMP-2 addition. We also monitored Sox9 expression after the addition of
BMP-2 to osteosarcoma cell lines; BMP-2 treatment increased Sox9 mRNA levels in
MG63, considered to be early osteoblast-like, but not in human osteogenic
sarcoma (HOS) cells, which are thought to be more advanced in the osteoblastic
lineage. This response seems to be influenced by differences in BMP receptor
expression; MG63 cells express BMP receptor IA (BMPR-IA), whereas HOS cells
express BMPR-IA and BMPR-IB. We also saw an increase in Sox9 mRNA levels in
BMP-2-treated primary human bone cells (HBCs) derived from femoral heads. We
found that in addition to BMP-2, Sonic and Indian hedgehog can increase Sox9
expression in C3H10T1/2 and primary HBCs. Time course studies with C3H10T1/2
cells after BMP-2 stimulation showed increasing expression of cartilage markers,
decrease of collagen I mRNA, and a late induction of osteocalcin expression.
Moreover, the treatment of C3H10T1/2 cells with Sox9 antisense oligonucleotides
revealed that Sox9 is a downstream mediator of BMP-2 affecting the expression of
chondrocyte and osteoblast marker genes. Our data show that Sox9 is an important
downstream mediator of the BMP-2 and hedgehog signaling pathways in osteogenic
cells. Indian hedgehog (Ihh) and Parathyroid Hormone-related Protein (PTHrP) play a
critical role in the morphogenesis of the vertebrate skeleton. Targeted deletion
of Ihh results in short-limbed dwarfism, with decreased chondrocyte
proliferation and extensive hypertrophy, features shared by mutants in PTHrP and
its receptor. Activation of Ihh signaling upregulates PTHrP at the articular
surface and prevents chondrocyte hypertrophy in wild-type but not PTHrP null
explants, suggesting that Ihh acts through PTHrP. To investigate the
relationship between these factors during development of the appendicular
skeleton, mice were produced with various combinations of an Ihh null mutation
(Ihh(-/-)), a PTHrP null mutation (PTHrP(-/-)), and a constitutively active
PTHrP/Parathyroid hormone Receptor expressed under the control of the Collagen
II promoter (PTHrPR*). PTHrPR* rescues PTHrP(-/-) embryos, demonstrating this
construct can completely compensate for PTHrP signalling. At 18.5 dpc, limb
skeletons of Ihh, PTHrP compound mutants were identical to Ihh single mutants
suggesting Ihh is necessary for PTHrP function. Expression of PTHrPR* in
chondrocytes of Ihh(-/-) mice prevented premature chondrocyte hypertrophy but
did not rescue either the short-limbed dwarfism or decreased chondrocyte
proliferation. These experiments demonstrate that the molecular mechanism that
prevents chondrocyte hypertrophy is distinct from that which drives
proliferation. Ihh positively regulates PTHrP, which is sufficient to prevent
chondrocyte hypertrophy and maintain a normal domain of cells competent to
undergo proliferation. In contrast, Ihh is necessary for normal chondrocyte
proliferation in a pathway that can not be rescued by PTHrP signaling. This
identifies Ihh as a coordinator of skeletal growth and morphogenesis, and
refines the role of PTHrP in mediating a subset of Ihh's actions. Mutant BMP receptors were transfected into cultured embryonic upper sternal
chrondrocytes using retroviral vectors to determine if BMP signaling is required
for chondrocyte maturation and the expression of a key regulatory molecule,
Indian hedgehog (Ihh). Chondrocytes infected with replication competent avian
retroviruses (RCAS) viruses carrying constitutive active (CA) BMPR-IA and
BMPR-IB had enhanced expression of type X collagen and Ihh mRNA. Addition of
PTHrP, a known inhibitor of chondrocyte maturation, abolished the expression of
type X collagen, BMP-6, and Ihh mRNAs in control cells. In contrast, PTHrP
treated cultures infected with of CA BMPR-IA or CA BMPR-IB had low levels of
BMP-6 and type X collagen, but high levels of Ihh expression. Although domit
negative (DN) BMPR-IA had no effect, DN BMPR-IB inhibited the expression of type
X collagen and BMP-6, and decreased alkaline phosphatase activity, even in the
presence of exogenously added BMP-2 and BMP-6. DN BMPR-IB also completely
blocked Ihh expression. Overall, the effect of DN BMPR-IB mimicked the effects
of PTHrP. To determine if there is an autocrine role for the BMPs in chondrocyte
maturation, the cultures were treated with noggin and follistatin, molecules
that bind BMP-2/-4 and BMP-6/-7, respectively. While noggin and follistatin
inhibited the effects of recombit BMP-2 and BMP-6, respectively, they had
only minimal effects on the spontaneous maturation of chondrocytes in culture,
suggesting that more than one subgroup of BMPs regulates chondrocyte maturation.
The results demonstrate that: (i) BMP signaling stimulates chondrocyte
maturation; (ii) BMP signaling increases Ihh expression independent of
maturational effects; and (iii) BMP signaling can partially overcome the
inhibitory effects of PTHrP on maturation. Indian hedgehog (Ihh), a member of the vertebrate hedgehog morphogen family, is
a key signaling molecule that controls chondrocyte proliferation and
differentiation. In this study, we show a novel function of Ihh. Namely, it acts
as an essential mediator of mechanotransduction in cartilage. Cyclic mechanical
stress greatly induces the expression of Ihh by chondrocytes. This induction is
abolished by gadolinium, an inhibitor of stretch-activated channels. This
suggests that the IHH gene is mechanoresponsive. The mechano-induction of Ihh is
essential for stimulating chondrocyte proliferation by mechanical loading. The
presence of an Ihh functional blocking antibody during loading completely
abolishes the stimulatory effect of mechanical load on proliferation.
Furthermore, Ihh mediates the mechanotransduction process in a bone morphogenic
protein (BMP)-dependent and parathyroid hormone-related peptide-independent
manner. BMP 2/4 are up-regulated by mechanical stress through the induction of
Ihh, and BMP antagonist noggin inhibits mechanical stimulation of chondrocyte
proliferation. This suggests BMP lies downstream of Ihh in mechanotransduction
pathway. Our data suggest that Ihh may transduce mechanical signals during
cartilage growth and repair processes. During endochondral ossification, two secreted signals, Indian hedgehog (Ihh)
and parathyroid hormone-related protein (PTHrP), have been shown to form a
negative feedback loop regulating the onset of hypertrophic differentiation of
chondrocytes. Bone morphogenetic proteins (BMPs), another family of secreted
factors regulating bone formation, have been implicated as potential interactors
of the Ihh/PTHrP feedback loop. To analyze the relationship between the two
signaling pathways, we used an organ culture system for limb explants of mouse
and chick embryos. We manipulated chondrocyte differentiation by supplementing
these cultures either with BMP2, PTHrP and Sonic hedgehog as activators or with
Noggin and cyclopamine as inhibitors of the BMP and Ihh/PTHrP signaling systems.
Overexpression of Ihh in the cartilage elements of transgenic mice results in an
upregulation of PTHrP expression and a delayed onset of hypertrophic
differentiation. Noggin treatment of limbs from these mice did not antagonize
the effects of Ihh overexpression. Conversely, the promotion of chondrocyte
maturation induced by cyclopamine, which blocks Ihh signaling, could not be
rescued with BMP2. Thus BMP signaling does not act as a secondary signal of Ihh
to induce PTHrP expression or to delay the onset of hypertrophic
differentiation. Similar results were obtained using cultures of chick limbs. We
further investigated the role of BMP signaling in regulating proliferation and
hypertrophic differentiation of chondrocytes and identified three functions of
BMP signaling in this process. First we found that maintaining a normal
proliferation rate requires BMP and Ihh signaling acting in parallel. We further
identified a role for BMP signaling in modulating the expression of IHH:
Finally, the application of Noggin to mouse limb explants resulted in advanced
differentiation of terminally hypertrophic cells, implicating BMP signaling in
delaying the process of hypertrophic differentiation itself. This role of BMP
signaling is independent of the Ihh/PTHrP pathway. Indian hedgehog (Ihh), one of the three mammalian hedgehog (Hh) proteins,
coordinates proliferation and differentiation of chondrocytes during
endochondral bone development. Smoothened (Smo) is a transmembrane protein that
transduces all Hh signals. In order to discern the direct versus indirect roles
of Ihh in cartilage development, we have used the Cre-loxP approach to remove
Smo activity specifically in chondrocytes. Animals generated by this means
develop shorter long bones when compared to wild-type littermates. In contrast
to Ihh mutants (Ihh(n)/Ihh(n)), chondrocyte differentiation proceeds normally.
However, like Ihh(n)/Ihh(n) mice, proliferation of chondrocytes is reduced by
about 50%, supporting a direct role for Ihh in the regulation of chondrocyte
proliferation. Moreover, by overexpressing either Ihh or a constitutively active
Smo allele (Smo*) specifically in the cartilage using the bigenic UAS-Gal4
system, we demonstrate that activation of the Ihh signaling pathway is
sufficient to promote chondrocyte proliferation. Finally, expression of cyclin
D1 is markedly downregulated when either Ihh or Smo activity is removed from
chondrocytes, indicating that Ihh regulates chondrocyte proliferation at least
in part by modulating the transcription of cyclin D1. Taken together, the
present study establishes Ihh as a key mitogen in the endochondral skeleton. Enchondromas are common benign cartilage tumors of bone. They can occur as
solitary lesions or as multiple lesions in enchondromatosis (Ollier and Maffucci
diseases). Clinical problems caused by enchondromas include skeletal deformity
and the potential for maligt change to chondrosarcoma. The extent of skeletal
involvement is variable in enchondromatosis and may include dysplasia that is
not directly attributable to enchondromas. Enchondromatosis is rare, obvious
inheritance of the condition is unusual and no candidate loci have been
identified. Enchondromas are usually in close proximity to, or in continuity
with, growth-plate cartilage. Consequently, they may result from abnormal
regulation of proliferation and terminal differentiation of chondrocytes in the
adjoining growth plate. In normal growth plates, differentiation of
proliferative chondrocytes to post-mitotic hypertrophic chondrocytes is
regulated in part by a tightly coupled signaling relay involving parathyroid
hormone related protein (PTHrP) and Indian hedgehog (IHH). PTHrP delays the
hypertrophic differentiation of proliferating chondrocytes, whereas IHH promotes
chondrocyte proliferation. We identified a mutant PTH/PTHrP type I receptor
(PTHR1) in human enchondromatosis that signals abnormally in vitro and causes
enchondroma-like lesions in transgenic mice. The mutant receptor constitutively
activates Hedgehog signaling, and excessive Hedgehog signaling is sufficient to
cause formation of enchondroma-like lesions. Differentiation and growth of chondrocytes in fetal growth plates of vertebrate
long bones and ribs appear to occur in a gradual, continuous manner between the
resting zone through the proliferation zone, maturation zone, and upper and
lower hypertrophic zones, with a continuous increase in cell size up to 10-fold
of the volume of a resting chondrocyte. Here we provide evidence, however, that
after centrifugation through a continuous Percoll gradient growth plate
chondrocytes separate into four distinct cell populations (B1 to B4) which
differ markedly in density, size, and gene expression. These populations collect
in the absence of any phase borders in the gradient which might serve as
concentration barriers. Fractions B1 and B2 contained the largest cells with the
lowest buoyant density and showed the highest expression levels for type X
collagen (Col X), but only the B1 population expressed high levels of matrix
metalloproteinase-13 (collagenase 3). Cells in fraction B3 were significantly
smaller and expressed little Col X, while cells in fraction B4 were of similar
size to cells in the resting zone without significant Col X expression. The
highest levels of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor
(PTHR-1), and Indian hedgehog (Ihh) expression were also found in the
hypertrophic fractions B1 and B2 and not in the prehypertrophic fraction B3, as
expected from in situ hybridization data on PTHR-1 expression in fetal rodent or
chicken growth plates. Incubation of fractions B1 to B3 with the amino-terminal
fragments PTH (1-34) or PTHrP (1-40) suppressed the expression of Col X and
PTHR-1 by more than 50% and the expression of Ihh nearly completely. In
contrast, the mid-regional PTH fragment PTH (28-48) and PTH (52-84) consistently
stimulated the expression of PTHR-1 by 10-20% in fractions B1 to B3. These
findings confirm the existence of distinct differentiation stages within
chondrocytes of the growth plate and support the hypothesis proposed by Vortkamp
et al. (Science 273(1996)613) of a regulatory feedback loop of Ihh and PTH/PTHrP
fragments controlling the differentiation of proliferating to prehypertrophic
chondrocytes, but extend the ability to respond to PTH/PTHrP hypertrophic
chondrocytes. Tibial dyschondroplasia (TD) is a form of aberrant endochondral ossification in
chickens, in that a plug of avascular cartilage (TD lesion) is formed within the
growth plate. Histologically, the lesion is filled with apparently transitional
chondrocytes that have been unable to differentiate to hypertrophic
chondrocytes. We have examined the spatial expression of mRNAs for type X
collagen, Indian hedgehog (Ihh) and Parathyroid Hormone-related protein (PTHrP)
in the TD growth plate by in situ hybridization in order to ascertain at which
stage chondrocyte differentiation is arrested in TD. In the normal growth plate,
type X collagen mRNA was expressed by both prehypertrophic and hypertrophic
chondrocytes. Indian Hedgehog mRNA was detected in a band of prehypertrophic
chondrocytes and PTHrP expression was localized to a narrow band of
prehypertrophic chondrocytes and in osteoblasts within the diaphysis. In TD
sections, collagen X expression was seen within differentiating cells, within a
small number of lesion cells, and within hypertrophic chondrocytes on the
diaphyseal side of the lesion. Ihh expression was also seen within the
differentiating cells and throughout the lesion. These data indicate that
chondrocyte differentiation is arrested at the transitional stage just prior to
hypertrophy. Contrary to the previously reported PTHrP expression patterns in TD
chicks by immunohistochemistry, PTHrP mRNA was not detected in the TD lesion.
This observation probably reflects the cessation of PTHrP gene expression by
chondrocytes in the more severe TD lesions. The results from the present study
also imply that the arrest of cell differentiation in TD is independent of PTHrP
and that endochondral ossification in the post-hatch avian growth plate may
involve additional regulatory pathways. Hedgehog proteins exert critical roles in embryogenesis and require heparan
sulfate proteoglycans (HS-PGs) for action. Indian hedgehog (Ihh) is produced by
prehypertrophic chondrocytes in developing long bones and regulates chondrocyte
proliferation and other events, but it is not known whether it requires HS-PGs
for function. Because the HS-PG syndecan-3 is preferentially expressed by
proliferating chondrocytes, we tested whether it mediates Ihh action. Primary
chick chondrocyte cultures were treated with recombit Ihh (rIhh-N) in absence
or presence of heparinase I or syndecan-3 neutralizing antibodies. While rIhh-N
stimulated proliferation in control cultures, it failed to do so in heparinase-
or antibody-treated cultures. In reciprocal gain-of-function studies,
chondrocytes were made to overexpress syndecan-3 by an RCAS viral vector. Cells
became more responsive to rIhh-N, but even this response was counteracted by
heparinase or antibody treatment. To complement the in vitro data, RCAS viral
particles were microinjected in day 4-5 chick wing buds and effects of
syndecan-3 misexpression were monitored over time. Syndecan-3 misexpression led
to widespread chondrocyte proliferation and, interestingly, broader expression
and distribution of Ihh. In addition, the syndecan-3 misexpressing skeletal
elements were short, remained cartilaginous, lacked osteogenesis, and exhibited
a markedly reduced expression of collagen X and osteopontin, products
characteristic of hypertrophic chondrocytes and bone cells. The data are the
first to indicate that Ihh action in chondrocyte proliferation involves
syndecan-3 and to identify a specific member of the syndecan family as mediator
of hedgehog function. The role of Hedgehogs (Hh) in murine skeletal development was studied by
overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice
using the collagen II promoter/enhancer. Overexpression caused a lethal
craniorachischisis with major alterations in long bones because of defects in
chondrocyte differentiation.
INTRODUCTION: Hedgehogs (Hhs) are a family of secreted polypeptides that play
important roles in vertebrate development, controlling many critical steps of
cell differentiation and patterning. Skeletal development is affected in many
different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways
cause severe abnormalities in the appendicular, axial, and cranial skeleton in
man and other vertebrates.
MATERIALS AND METHODS: Genetic manipulation of mouse embryos was used to study
in vivo the function of SHH in skeletal development. By DNA microinjection into
pronuclei of fertilized oocytes, we have generated transgenic mice that express
SHH specifically in chondrocytes using the cartilage-specific collagen II
promoter/enhancer. Transgenic skeletal development was studied at different
embryonic stages by histology. The expression pattern of specific chondrocyte
molecules was studied by immunohistochemistry and in situ hybridization.
RESULTS: Transgenic mice died at birth with severe craniorachischisis and other
skeletal defects in ribs, sternum, and long bones. Detailed analysis of long
bones showed that chondrocyte differentiation was blocked at prehypertrophic
stages, hindering endochondral ossification and trabecular bone formation, with
specific defects in different limb segments. The growth plate was highly
disorganized in the tibia and was completely absent in the femur and humerus,
leading to skeletal elements entirely made of cartilage surrounded by a thin
layer of bone. In this cartilage, chondrocytes maintained a columnar
organization that was perpendicular to the bone longitudinal axis and directed
toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was
greatly increased in all cartilage, as well as the expression of parathyroid
hormone-related protein (PTHrP) at the articular surface; while the expression
of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of
chondrocyte maturation, was greatly reduced and restricted to the displaced
chondrocyte columns. Transgenic mice also revealed the ability of SHH to
upregulate the expression of Sox9, a major transcription factor implicated in
chondrocyte-specific gene expression, in vivo and in vitro, acting through the
proximal 6.8-kb-long Sox9 promoter.
CONCLUSION: Transgenic mice show that continuous expression of SHH in
chondrocytes interferes with cell differentiation and growth plate organization
and induces high levels and diffuse expression of Sox9 in cartilaginous bones. Growth plate chondrocytes undergo a tightly regulated process of
differentiation, allowing for the longitudinal growth of bones. Although it is
known that parathyroid hormone related protein (PTHrP) and Indian hedgehog
regulate the differentiation of growth plate chondrocytes, how these pathways
interact to regulate chondrocyte development is not fully elucidated. We
examined how the interaction between PTHrP and the hedgehog activated
transcription factors, Gli2 and Gli3, regulates growth plate chondrocyte
differentiation and proliferation. Analysis of fetal limbs showed that Gli2 is a
negative regulator and Gli3 a positive regulator of type X collagen expression.
Limb explant cultures showed that PTHrP treatment inhibited type X collagen
expression and increased chondrocyte proliferation. This effect was
substantially enhanced in Gli2-/- limbs, was blocked in Gli3-/- limbs, and was
only partially inhibited by hedgehog ligand blockade. PTHrP negatively regulated
Gli mediated transcription in cell cultures, and regulated the level of the
repressor form of Gli3 in a PKA dependent manner. These results show that PTHrP
regulates growth plate chondrocyte proliferation and differentiation in part
through the activity of Gli3, suggesting a crucial role for Gli3 in growth plate
chondrocyte development. Indian Hedgehog (Ihh)--Parathyroid related protein (PTHrP) and Fibroblast Growth
Factor 3 (FGFR3) signaling pathways are important in regulating endochondral
bone formation. In the growth plate, Ihh and PTHrP are involved in a feedback
loop to increase proliferation and delay differentiation of chondrocytes.
Fibroblast Growth Factor Receptor 3 (FGFR3) conversely decreases proliferation
and hastens differentiation with an agonist. Since proliferation is the hallmark
of chondrosarcoma cells, we hypothesized that Ihh/PTHrP and FGF3R pathways may
be dysfunctional on these cells. Therefore, we sought to investigate the role of
these signaling pathways in the Swarm rat chondrosarcoma cells utilizing
expression and functional studies. Semiquantitative RT-PCR analysis demonstrated
difference in expression between normal growth plate chondrocytes and
chondrosarcoma cells (JWS). JWS had an increased mRNA expression of FGF2 and
FGFR3 suggesting a mechanism to reverse the proliferative rate of the cells.
Immunohistochemical analysis showed increased staining for FGFR3 and patched-1
(Ihh receptor) in JWS compared to the rat tibia growth plate (p = 0.O004 and
0.02 respectively). In vitro functional experiments demonstrated that the use of
FGF2, a FGFR3 receptor agonist, dramatically decreased the proliferative rate of
Swarm chondrosarcoma cells (LTC). Cyclopamine, a hedgehog inhibitor, did not
have a significant effect on their proliferative rate. However, when cyclopamine
was used on normal chondrocytes, it effectively decreased the proliferative rate
of these cells, suggesting abnormalities in this pathway in the chondrosarcoma
cells. In conclusion, our investigation describes dissimilarity in the Indian
Hedgehog and FGFR3 signaling pathways between the rat chondrosarcoma cells and
native rat chondrocytes. Understanding the underlying mechanisms may provide a
target for future therapy for chondrosarcoma. Chondrocyte hypertrophy is an essential process required for endochondral bone
formation. Proper regulation of chondrocyte hypertrophy is also required in
postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play
crucial roles in regulating the onset of chondrocyte hypertrophy by forming a
negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy
by controlling PTHrP expression. To understand whether there is a
PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy,
we have both activated and inactivated Ihh signaling in the absence of PTHrP
during endochondral skeletal development. We found that upregulating Ihh
signaling in the developing cartilage by treating PTHrP(-/-) limb explants with
sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of
PTHrP(-/-) embryos or inactivating patched 1 (Ptch1), a negative regulator of
hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP(-/-)
embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing
Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy
was delayed in the PTHrP(-/-) embryo. Furthermore, we show that upregulated Hh
signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy
during secondary ossification, which in turn caused reduction of joint
cartilage. Our results revealed a novel role of Ihh signaling in promoting
chondrocyte hypertrophy independently of PTHrP, which is particularly important
in postnatal cartilage development and homeostasis. In addition, we found that
bone morphogenetic protein (Bmp) and Wnt/beta-catenin signaling in the cartilage
may both mediate the effect of upregulated Ihh signaling in promoting
chondrocyte hypertrophy. OBJECTIVE: Chondrocytes of the epiphyseal growth zone are regulated by the
Indian hedgehog (IHH)-parathyroid hormone-related protein (PTHrP) axis. In
weight-bearing joints, this growth zone comes to be subdivided by the secondary
ossification center into distinct articular and growth cartilage structures. The
purpose of this study was to explore the cells of origin, localization,
regulation of expression, and putative functions of IHH and PTHrP in articular
cartilage in the mouse.
METHODS: We assessed IHH and PTHrP expression in an allelic PTHrP-LacZ-knockin
mouse and several versions of PTHrP-null mice. Selected joints were unloaded
surgically to examine load-induction of PTHrP and IHH.
RESULTS: The embryonic growth zone appears to serve as the source of
PTHrP-expressing proliferative chondrocytes that populate both the forming
articular cartilage and growth plate structures. In articular cartilage, these
cells take the form of articular chondrocytes in the midzone. In PTHrP-knockout
mice, mineralizing chondrocytes encroach upon developing articular cartilage but
appear to be prevented from mineralizing the joint space by IHH-driven surface
chondrocyte proliferation. In growing and adult mice, PTHrP expression in
articular chondrocytes is load-induced, and unloading is associated with rapid
changes in PTHrP expression and articular chondrocyte differentiation.
CONCLUSION: We conclude that the IHH-PTHrP axis participates in the maintece
of articular cartilage. Dysregulation of this system might contribute to the
pathogenesis of arthritis. The advent of molecular targeted therapies offers the hope of therapeutic
advance in the fight against cancer. However, this hope is tempered by recent
findings that certain targeted therapies may have unique side effects. The
Hedgehog (HH) pathway is a potential target for treatment of several cancers,
including basal cell carcinoma and a subset of medulloblastoma. Recent clinical
trials in adults have shown responses to HH pathway inhibition in both basal
cell carcinoma and medulloblastoma. However, concerns have been raised about the
use of HH pathway inhibitors in children because of the role the HH pathway
plays in development. Indeed, young mice treated with the HH pathway inhibitor
HhAntag developed severe bone defects, including premature differentiation of
chondrocytes, thinning of cortical bone, and fusion of the growth plate. In an
effort to lessen the severity of bone defects caused by HhAntag, we treated
young mice simultaneously with HhAntag and parathyroid hormone-related protein
(PTHrP), which functions downstream of Indian Hedgehog to maintain chondrocytes
in a proliferative state. The results show that whereas treatment with PTHrP
causes a significant increase in trabecular bone, it does not prevent fusion of
the growth plate induced by HhAntag. Primary cilia regulate limb and axial skeletal formation and hedgehog signaling,
but their roles in temporomandibular joint (TMJ) development are unknown. Thus,
we created conditional mouse mutants deficient in ciliary transport protein
Kif3a in cartilage. In post-natal wild-type mice, primary cilia were
occasionally observed on the superior, inferior, or lateral side of condylar
cells. Cilia were barely detectable in mutant chondrocytes but were evident in
surrounding tissues, attesting to the specificity of chondrocyte Kif3a ablation.
Mutant condyles from 3-month-old mice were narrow and flat along their
antero-posterior and medio-lateral axes, were often fused with the articular
disc, and displayed an irregular bony surface. The polymorphic layer in P15
mutants contained fewer Sox9-expressing chondroprogenitor cells because of
reduced mitotic activity, and newly differentiated chondrocytes underwent
precocious hypertrophic enlargement accompanied by early activation of Indian
hedgehog (Ihh). Interestingly, there was excessive intramembranous ossification
along the perichondrium, accompanied by local expression of the hedgehog
receptor Patched-1 and up-regulation of Osterix and Collagen I. In summary,
Kif3a and primary cilia are required for coordination of chondrocyte maturation,
intramembranous bone formation, and chondrogenic condylar growth. Defects in
these processes in Kif3a condylar cartilage are likely to reflect abnormal
hedgehog signaling topography and dysfunction. Proper regulation of Indian hedgehog (Ihh) signaling is vital for chondrocyte
proliferation and differentiation in the growth plate. Its dysregulation causes
skeletal dysplasia, osteoarthritis or cartilaginous neoplasia. Here, we show
that Suppressor of fused (Sufu) and Kif7 are essential regulators of Ihh
signaling. While Sufu acts as a negative regulator of Gli transcription factors,
Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role
in the turnover of Sufu and the exclusion of Sufu-Gli complexes from the primary
cilium. Importantly, halving the dose of Sufu restores normal hedgehog pathway
activity and chondrocyte development in Kif7-null mice, demonstrating that the
positive role of Kif7 is to restrict the inhibitory activity of Sufu.
Furthermore, Kif7 also inhibits Gli transcriptional activity in the chondrocytes
when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli
transcription factors through both Sufu-dependent and -independent mechanisms. Atf4 is a leucine zipper-containing transcription factor that activates
osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The
relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation
of skeletal development and bone formation is poorly understood. Investigations
of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively
overexpressed in chondrocytes in an Atf4-null background, demonstrate that
chondrocyte-derived Atf4 regulates osteogenesis during development and bone
remodeling postnatally. Atf4 overexpression in chondrocytes of the
Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb
in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling,
proliferation and accelerated hypertrophy that characterize the Atf4(-/-)
developing growth plate cartilages. Unexpectedly, this genetic manipulation also
restores the expression of osteoblastic marker genes, namely Ocn and bone
sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult
mice, all the defective bone parameters found in Atf4(-/-) mice, including bone
volume, trabecular number and thickness, and bone formation rate, are rescued.
In addition, the conditioned media of ex vivo cultures from wild-type or
Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation
defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody
eliminates this effect. Together, these data indicate that Atf4 in chondrocytes
is required for normal Ihh expression and for its paracrine effect on osteoblast
differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes
dominates the role of Atf4 in osteoblasts during development for the control of
early osteogenesis and skeletal growth. The Indian hedgehog (Ihh) signal plays a vital role in regulating proliferation
and hypertrophy of chondrocytes. To investigate its function in postnatal
chicken (Gallus gallus) chondrocytes, cyclopamine was used to inhibit Ihh
signaling. The MTT and ALP assays revealed the downgrade-proliferation and
upgrade-differentiation of chondrocytes. To further elucidate the mechanism, the
mRNA expression levels of Ihh, parathyroid hormone related protein (PTHrP),
Gli-2, Bcl-2, Bone Morphogenetic Protein 6 (BMP-6), type X collagen (Col X) and
type II collagen (Col II) were detected by quantitative real-time RT-PCR
analysis, and the protein expressions of Ihh, Col X, and Col II were determined
using Western blot analysis. After the Ihh signal was blocked, chondrocytes
demonstrated high expression levels of PTHrP and Col X and low levels of Gli-2,
BMP-6, Bcl-2 and Col II although Ihh expression was increased. Based on these
results, the Ihh signal is essential for balancing chicken chondrocyte
proliferation and hypertrophy, and the regulatory function of PTHrP acts in an
Ihh-dependent manner. Furthermore, BMP-6 and Bcl-2 played roles in maintaining
the development of chondrocytes and may be downstream regulatory factors of Ihh
signaling. Core binding factor beta (Cbfβ) is essential for embryonic bone morphogenesis.
Yet the mechanisms by which Cbfβ regulates chondrocyte proliferation and
differentiation as well as postnatal cartilage and bone formation remain
unclear. Hence, using paired-related homeobox transcription factor 1-Cre
(Prx1-Cre) mice, mesenchymal stem cell-specific Cbfβ-deficient (Cbfβ(f/f)
Prx1-Cre) mice were generated to study the role of Cbfβ in postnatal cartilage
and bone development. These mutant mice survived to adulthood but exhibited
severe sternum and limb malformations. Sternum ossification was largely delayed
in the Cbfβ(f/f) Prx1-Cre mice and the xiphoid process was noncalcified and
enlarged. In newborn and 7-day-old Cbfβ(f/f) Prx1-Cre mice, the resting zone was
dramatically elongated, the proliferation zone and hypertrophic zone of the
growth plates were drastically shortened and disorganized, and trabecular bone
formation was reduced. Moreover, in 1-month-old Cbfβ(f/f) Prx1-Cre mice, the
growth plates were severely deformed and trabecular bone was almost absent. In
addition, Cbfβ deficiency impaired intramembranous bone formation both in vivo
and in vitro. Interestingly, although the expression of Indian hedgehog (Ihh)
was largely reduced, the expression of parathyroid hormone-related protein
(PTHrP) receptor (PPR) was dramatically increased in the Cbfβ(f/f) Prx1-Cre
growth plate, indicating that that Cbfβ deficiency disrupted the Ihh-PTHrP
negative regulatory loop. Chromatin immunoprecipitation (ChIP) analysis and
promoter luciferase assay demonstrated that the Runx/Cbfβ complex binds putative
Runx-binding sites of the Ihh promoter regions, and also the Runx/Cbfβ complex
directly upregulates Ihh expression at the transcriptional level. Consistently,
the expressions of Ihh target genes, including CyclinD1, Ptc, and Pthlh, were
downregulated in Cbfβ-deficient chondrocytes. Taken together, our study reveals
not only that Cbfβ is essential for chondrocyte proliferation and
differentiation for the growth and maintece of the skeleton in postnatal
mice, but also that it functions in upregulating Ihh expression to promoter
chondrocyte proliferation and osteoblast differentiation, and inhibiting PPR
expression to enhance chondrocyte differentiation. The Hedgehog (Hh) family of proteins consists of Indian hedgehog (Ihh), sonic
hedgehog (Shh), and desert hedgehog (Dhh). These proteins serve as essential
regulators in a variety of developmental events. Ihh is mainly produced and
secreted by prehypertrophic chondrocytes and regulates chondrocyte hypertrophy
and endochondral bone formation during growth plate development. Tissue-specific
deletion of the Ihh gene (targeted by Col2a1-Cre) causes early lethality in
mice. Transgenic mice with induced Ihh expression exhibit increased chondrocyte
hypertrophy and cartilage damage resembling human osteoarthritis (OA). During OA
development, chondrocytes recapitulate the differentiation process that happens
during the fetal status and which does not occur to an appreciable degree in
adult articular cartilage. Ihh expression is up-regulated in human OA cartilage,
and this upregulation correlates with OA progression and changes in chondrocyte
morphology. A genetic study in mice further showed that conditional deletion of
Ihh in chondrocytes attenuates OA progression, suggesting the possibility that
blocking Ihh signaling can be used as a therapeutic approach to prevent or delay
cartilage degeneration. However, Ihh gene deletion is currently not a
therapeutic option as it is lethal in animals. RNA interference (RNAi) provides
a means to knockdown Ihh without the severe side effects caused by chemical
inhibitors. The currently available delivery methods for RNAi are oparticles
and liposomes. Both have problems that need to be addressed. In the future, it
will be necessary to develop a safe and effective RNAi delivery system to target
Ihh signaling for preventing and treating OA. Wnt/β-catenin, Indian hedgehog (Ihh)/Parathyroid-related peptide (PTHrP) and
retinoid signaling pathways regulate cartilage differentiation, growth, and
function during development and play a key role in endochondral ossification.
The objective of this study was to elucidate the gene and protein expression of
signaling molecules of these regulatory pathways in chondrocytes surrounding
cartilage canals and the osteochondral junction during neonatal and
pre-adolescent development. This study revealed cell-specific and age-related
differences in gene and protein expression of signaling molecules of these
regulatory pathways. A trend for higher gene expression of PTHrP along the
cartilage canals and Ihh along the osteochondral junction suggests the presence
of paracrine feedback in articular-epiphyseal cartilage. Differential expression
of canonical (β-catenin, Wnt-4, Lrp4, Lrp6) and noncanonical Wnt signaling
(Wnt-5b, Wnt-11) and their inhibitors (Dkk1, Axin1, sFRP3, sFRP5, Wif-1)
surrounding the cartilage canals and osteochondral junction provides evidence of
the complex interactions occurring during endochondral ossification. |
What is the function of R-spondin 1 and noggin in non-damaged gallbladders? | R-spondin 1 and noggin facilitate expansion of resident stem cells from non-damaged gallbladders. | |
List the classical triad of symptoms of the Melkersson–Rosenthal syndrome. | The Melkersson-Rosenthal syndrome consists of the classical triad of symptoms:
1) orofacial oedema
2) fissured tongue (lingua plicata) and
3) facial paralysis. | Melkersson-Rosenthal syndrome (MRS) is characterized by the triad of facial
paralysis, facial oedema, and lingua plicata in association with other symptoms,
such as headache and mental changes. Because the origin of this syndrome is
still unknown, only symptomatic treatment is possible. In 18 patients suffering
from MRS, whether with a complete or with an incomplete picture, we used the
antileprosy drug clofazimine for therapy. A decrease in the frequency and
intensity of oedema was achieved by this therapy in 94% of patients. However,
improvement persisting throughout a follow-up period of up to 3 years was seen
in only 62% of patients. We demonstrate that clofazimine is an alternative to
glucocorticosteroids for the treatment of MRS. Peripheral facial nerve palsy, recurrent or persistent oral or facial swelling,
and fissured tongue constitute a triad of symptoms known as Melkersson-Rosenthal
syndrome. Granulomatous labial enlargement, known as cheilitis granulomatosa, is
considered the single most important diagnostic feature of this syndrome. This
lesion has been difficult to treat. This article describes a case of 8 months'
duration of cheilitis granulomatosa of the lower lip, which was successfully
managed with intralesional steroid injections. The Melkersson-Rosenthal Syndrome consists of recurrent edema of the lips,
intermittent facial edema and a furrowed tongue. This is the classic triad which
defines the syndrome, although it is accepted that the presence of two
manifestations is sufficient to make the diagnosis. Distribution is universal
although the majority of the cases are described in the European literature. The
case of a 37 year-old female is presented. She came to consult for persistent
edema of the upper lip, of a three-year duration, which started abruptly without
any clear etiologic correlation with acute episodes that disappear spontaneously
or with corticoid treatment. Associated symptoms included migraine headaches
which started years earlier. The complimentary examinations were normal except
for a mild elevation of the sedimentary rate, and the biopsy was compatible with
granulomatous cheilitis. In conclusion, MRS has to be considered as a diagnostic
possibility in a patient who consulted for recurrent edema associated with other
dermatologic and neurologic manifestations, although not necessarily having the
complete triad. Melkersson-Rosenthal syndrome is a triad of recurrent orofacial swelling,
relapsing facial paralysis, and fissured tongue. However, the classic triad is
not frequently seen in its complete form. Monosymptomatic and oligosymptomatic
forms are more common. The histological findings of sarcoid-like granuloma in
skin or mucosal biopsy specimens support the diagnosis. The course is chronic
but benign. Treatment is difficult, but intralesional or systemic
corticosteroids may be helpful. The Melkersson-Rosenthal syndrome is a rare disease of unknown pathogenesis.
Classical signs include recurrent facial palsy, lingua plicata and orofacial
edema. The diagnosis is often difficult when all features are not present at the
same time: in the literature complete triads occurred in 25-30% of the patients.
We report a case of Melkersson-Rosenthal syndrome with classical triad of signs
in a 13 year old boy. The pathology, clinical features and management of this
disease are discussed: the possible role of food allergy or additives
intolerance is also examined. The Melkersson-Rosenthal syndrome is a rare disorder of unknown etiology
characterized by a triad of recurrent orofacial swelling, relapsing facial
paralysis, and fissured tongue. Exacerbations and recurrences are common. The
orofacial swelling is characterized by fissured, reddish-brown, swollen,
nonpruritic lips or firm edema of the face. The facial palsy is
indistinguishable from Bell's palsy. The fissured tongue is seen in one third to
one half of patients and, although the least common manifestation, its presence
assists in diagnosis. The classic triad is not seen frequently in its complete
form; therefore, diagnosis is difficult. This is particularly true because
monosymptomatic and oligosymptomatic variants are seen more commonly. Cheilitis
granulomatosa of Miescher is an example of a monosymptomatic variant of the
Melkersson-Rosenthal syndrome. The histologic findings of noncaseating,
sarcoidal granulomas support the diagnosis. These granulomas are not invariably
present, and their absence does not exclude the diagnosis of the
Melkersson-Rosenthal syndrome. Thus, the Melkersson-Rosenthal syndrome is a
disease with elements of orofacial granulomatosis. Orofacial granulomatosis is a
clinicopathologic entity describing oral lesions with noncaseating granulomas.
The spectrum of this entity includes patients with oral Crohn's disease,
patients with oral lesions who will develop typical bowel symptoms of Crohn's
disease in the ensuing months to years, patients with tooth-associated
infections, patients with sarcoidosis, and patients with food or contact
allergies. The value of the clinicopathologic construct of orofacial
granulomatosis is to provoke the careful search for provocative causes for the
reactive symptom complex of the Melkersson-Rosenthal syndrome. OBJECTIVE: The objective of our study was to describe the ophthalmic features
and histologic eyelid findings of Melkersson-Rosenthal syndrome.
DESIGN: Three patients with eyelid edema underwent eyelid biopsy to establish
the diagnosis of Melkersson-Rosenthal syndrome.
RESULTS: Of the three patients, only one patient manifest the classic triad of
facial edema, facial paralysis, and furrowed tongue. Histoloppgically, the
eyelid skin in all patients showed characteristic perilymphatic granulomas with
marked dermal edema.
CONCLUSIONS: The histopathologic features assist in establishing the diagnosis
of Melkersson-Rosenthal syndrome in oligo- and monosymptomatic patients.
Melkersson-Rosenthal syndrome should be considered in patients presenting with
eyelid edema of unknown etiology and biopsy performed. The Melkersson-Rosenthal syndrome consists of a triad of recurrent lip and/or
face swelling, fissured tongue, and intermittent facial palsy. Onset of the
symptoms may occur during childhood, and treatment of the condition is
difficult. We describe two children with Melkersson-Rosenthal syndrome in whom
combination treatment with prednisone and minocycline proved effective and well
tolerated. Melkersson-Rosenthal syndrome is a rare disorder consisting of the triad of
persistent or recurrent orofacial edema, relapsing facial paralysis and fissured
tongue. It is far more uncommon to find the complete triad since it generally
presents in oligosymptomatic forms. We present three cases of the
Melkersson-Rosenthal syndrome with the classic triad of symptoms and discuss the
etiology and the clinical and electromyography findings of this syndrome. BACKGROUND: Melkersson-Rosenthal syndrome (MRS) is a rare disorder of unknown
etiology characterized by a triad of symptoms: recurrent orofacial swelling,
relapsing facial palsy. and a fissured tongue. A differential diagnosis must be
made with other granulomatous diseases, such as sarcoidosis and oral Crohn
disease; however, the histologic findings of noncaseating, sarcoidal granulomas
support the diagnosis of MRS.
RESULTS: Many therapeutic modalities have been described for this disease. In
this case report, we present a patient with MRS that was treated with
thalidomide because of the identification of tumor necrosis factor a in the
lesion by immunohistochemical analysis. This is the first reported detection of
tumor necrosis factor a in an MRS lesion, as well the first reported use of
thalidomide to treat this clinical condition. Melkersson-Rosenthal syndrome (MRS) is a rare idiopathic non-caseating
granulomatous condition. It is rarely described in otorhinolaryngology-related
journals, although facial palsy, lip-swelling, and lingua plicata, are its most
common presenting features. This classical triad however is not always present
in MRS. Other forms of orofacial swelling exist. This paper describes a patient
with a variant of MRS, treated by us with intra-lesional steroid injection. We
also discuss the other differential diagnoses that may mimic MRS. A triad of facial palsy, facial edema, and furrowed tongue characterizes
Melkersson-Rosenthal syndrome, a rare, noncaseating granulomatous disease of
unknown cause. Although most reported cases of Melkersson-Rosenthal syndrome
involve swelling of the perioral area, the authors present a case of
Melkersson-Rosenthal syndrome involving the periocular area. Because of its
rarity, the syndrome is usually ignored and misdiagnosed; however, the syndrome
should not only be considered in the classic perioral presentation but also in
the rare periocular form, which may be confused with orbital tumors and orbital
pseudotumors. Biopsies should be performed routinely in all patients who present
with eyelid edema of unknown etiology. The physician and surgeon who see
patients with head and neck pathology should be familiar with
Melkersson-Rosenthal syndrome, and with the possibility of its presentation in
the orbit and periocular region. BACKGROUND: Melkersson-Rosenthal syndrome may manifest as the classical triad
(orofacial edema, facial nerve palsy and stable lingua plicata) but
monosymptomatic manifestations or combinations of typical symptoms are not
infrequent. The available therapeutic options provide only limited success or
temporary benefit.
CASE REPORT: A 20-year-old man presented with a 7-month history of recurrent
episodes of swelling of the upper lip without pain, burning or local pruritus.
No causative factors, such as food, drugs or latex, or physical, chemical or
emotional conditions could be identified. The patient had been treated with oral
antihistamines and corticosteroids with no clinical improvement. Physical
examination showed firm edema without fovea, limited to the central area of the
upper lip without epidermal changes or symptoms on palpation. The patient had a
previous history of facial palsy 6 years previously and recurrent episodes of
herpes simplex labialis. Skin prick tests with inhalant aeroallergens, food,
latex and Anisakis allergens were negative. Laboratory investigation revealed
normal complete blood count, erythrocyte sedimentation rate, thyroid hormones,
biochemistry, complement components (C3, C4 and C1-esterase inhibitor) and CH50,
rheumatoid factor, antinuclear antibodies, immune complexes, protein
electrophoresis and immunoglobulins. Thorax and paranasal sinus radiographs were
clear. Biopsy of the involved area of the lip showed edema with lymphocytic and
plasma cell infiltration and mononuclear perivascular infiltrates without
granulomas, suggesting initial granulomatous cheilitis. Because the patient
showed lack of response and/or poor tolerance to prior treatments (deflazacort,
clofazimine and metronidazole), intralesional triamcinolone injections were
administered with satisfactory response from the first session.
CONCLUSIONS: Response to available treatments for Melkersson-Rosenthal syndrome
is highly variable. In the present case, intralesional triamcinolone injections
were effective. The Melkersson-Rosenthal syndrome consists of triad of symptoms: recurrent
oedema of lips, recurrent facial nerve paralysis and lingua plicata. Treatment
is usually symptomatic and required cooperation of different specialists as:
dermatologists, neurologists, dentists, laryngologists, surgeons. A rare case of
Melkersson-Rosenthal syndrome in 49-year-old man was observed in the Clinic of
Dermatology Silesian Medical Academy in Katowice. Melkersson-Rosenthal Syndrome (MRS) is a systemic neuro-mucocutaneous
granulomatous disease, characterized in its classical form by a triad of
recurrent facial nerve paralysis, swelling of the lips and lingua plicata.
However, this classical triad is rarely present, while the monosymptomatic or
oligosymptomatic forms are more frequent. The presence of two or one of the
manifestations mentioned above, with granulomatous cheilitis in the biopsy, is
sufficient to make the diagnosis of monosymptomatic or oligosymptomatic form of
MRS. This syndrome is very rare in childhood, instead, it is more frequent in
young adults between the second and third decades of life. We present the case
of an 8 years old boy who was brought to us because of a non painful swelling of
the upper lip, associated with gingival hypertrophy, that had persisted for more
than two months. Given the negative results of the hemato-chemical and
instrumental assessments, we performed an upper lip biopsy whose histological
study showed granulomatous cheilitis. We diagnosed this case as a
monosymptomatic MRS and administered an intralesional steroid therapy using
triamcinolone, with complete recovery. Melkersson-Rosenthal syndrome (MRS) is an uncommon granulomatous condition
characterized by persistent or recurrent orofacial oedema, relapsing facial
paralysis and fissured tongue. We report here a case of MRS with the classical
triad of signs accompanied by unilateral anterior uveitis. A 35-year-old man
with a fissured tongue, recurrent facial palsy and orofacial oedema presented
with a 3-month history of conjunctival redness and decreased vision in the right
eye. On evaluation of visual acuity, the patient was only able to count fingers.
Slit lamp examination revealed severe conjunctival injection, cells and flare,
posterior synechiae and keratic precipitates. Examination of the left eye
revealed no evidence of inflammation. Examination of the other systems was
normal. The patient was treated with topical corticosteroids and cycloplegics
and the visual acuity improved to 7/10. To our knowledge, there are no previous
reports of an association between uveitis and MRS. We present a rare case of bilateral crocodile tears syndrome (CTS) in the course
of Melkersson-Rosenthal syndrome. Melkersson-Rosenthal syndrome is characterised
by a triad of recurrent orofacial swelling, relapsing facial paralysis, and
fissured tongue. The classic triad is infrequent and oligosymptomatic variants
are seen more frequently. CTS is a rare complication of facial nerve paralysis
characterised by inappropriate lacrimation on the side of the palsy in response
to salivary stimuli. It results from aberrant reinnervation of the lacrimal
gland by salivary parasympathetic fibres. The therapeutic approach for an acute
bout of Melkersson-Rosenthal syndrome consists mainly of steroid administration.
CTS management is composed of anticholinergic drugs and surgical procedures.
Botulin toxin injection into the lacrimal gland is the most modern therapeutic
option. In the case presented CTS developed in a 50-year-old man after 5
incidents of facial palsy due to Melkersson-Rosenthal syndrome. The case
deserves attention due to the rarity of the observed symptoms and signs. Melkersson-Rosenthal syndrome (MRS) is a rare neuromucocutaneous syndrome marked
by the triad of recurrent nonpitting orofacial edema, fissured dorsal tongue
(lingua plicata), and lower motoneuron facial paralysis. Large case series
including treatment are limited. A retrospective records review was performed
for the diagnoses Melkersson-Rosenthal syndrome, granulomatous cheilitis, and
orofacial granulomatosis, confirmed by noncaseating granulomas on biopsy, at the
Mayo Clinic in Rochester, Minnesota (1979-2009). There were 72 patients [51
women (71 %), mean age at presentation 39 years (range 8-79)] identified with
facial edema with noncaseating granulomas on skin biopsy. Lingua plicata
occurred in 34 cases (47 %, 95 % confidence interval 35.3-59.3 %). Unilateral or
partial facial nerve palsy occurred in 14 cases (19.4, 95 % confidence interval
11.4-30.8 %). Comorbidities among those with facial edema included periodontal
disease (n = 10, 14 %), history of allergic disease (n = 10, 14 %), Crohn's
Disease (n = 6, 8 %), migraine headaches (n = 5, 7 %), and systemic lupus
erythematosus (n = 2, 3 %). There were no patients who had low C1q or C4 levels
among those who were tested. Overall, the full triad canonical of
Melkersson-Rosenthal syndrome was observed in nine patients (seven female,
median age at symptomatic presentation 35 years (range 10-74 years), 13 %, (95 %
confidence interval 6.2-22.9 %) with a median time from first symptoms to
diagnosis of 4 years (range 1-35). The medication treatments attempted in the
nine patients with the full triad of symptoms included non-steroidal
anti-inflammatory drugs, oral and intra-lesional steroids, metronidazole,
dapsone, acyclovir, methotrexate, and thalidomide with no consistent treatment
responses. The Melkersson-Rosenthal syndrome may present over the course of most
of the lifespan and may require several years of observation to be diagnosed.
Neurologists who observe a combination of facial edema and facial palsy in a
patient should consider the diagnosis of MRS and proceed to a diagnostic skin
biopsy and a trial of steroid treatment for their patient. BACKGROUND: Melkersson-Rosenthal syndrome (MRS) is a rare disorder of unknown
cause. The classical triad of MRS is orofacial edema, recurrent facial
paralysis, and a fissured tongue.
PATIENT: We present a 9-year-old girl with a recurrent peripheral facial
paralysis. She experienced the first episode of a peripheral facial paralysis on
the same side without orofacial swelling and lingua plicata 1 year ago. She was
diagnosed with Hashimoto thyroiditis 9 months earlier, as confirmed by an
endocrinologic investigation.
RESULTS: While the patient was hospitalized with recurrent facial paralysis, we
found that serum levels of free thyroxine (1.3 ng/dL) and thyrotropin (0.4
uIU/mL) were within normal range, but the level of antithyroperoxidase
antibodies (772.0 IU/mL) was very increased. She had been taking an oral
prednisolone orally for 2 weeks. At the 1-month follow-up, the patient's
symptoms had completely disappeared.
DISCUSSION: The possible correlation between MRS and autoimmune disorders has
been documented in only one report, which described an adult with autoimmune
thyroiditis (Hashimoto thyroiditis) and MRS. We suggest that the co-occurrence
of MRS and Hashimoto thyroiditis is not coincidental but linked to autoimmunity. Melkersson-Rosenthal syndrome (MRS) is a rare granulomatous inflammatory disease
characterised by the triad of orofacial oedema, facial nerve palsy and furrowed
tongue. We describe the case of a 29-year-old patient suffering from an
oligosymptomatic form of the disease with orofacial oedema, cobblestone pattern
on the buccal mucosa and swelling of the tongue, accompanied by intermittent
fatigue, influenza-like symptoms, intermittent tinnitus and acute hearing loss.
An increase of several autoimmune-associated antibodies was also detected.
Treatment with prednisolone, azathioprine or methotrexate failed to adequately
control all symptoms in the long term. In the absence of a specific and
well-established therapy for MRS, treatment with adalimumab was administered.
Under adalimumab, total remission of all symptoms was achieved, indicating that
tumour necrosis factor-α blockers are a promising therapeutic option for
patients with Melkersson-Rosenthal syndrome. Melkersson-Rosenthal Syndrome (MRS) is a rare otoneurologic condition, which is
poorly understood and often underdiagnosed. Etiology and incidence are unclear,
although infectious, inflammatory, and genetic causes have been implicated.
Recurrent facial nerve palsy, facial swelling, and fissured tongue are the
symptoms and signs of this condition. However, this triad is not typical in all
patients as patients may present with one or more of the symptoms, which makes
management of this condition difficult. Steroids may prove to be useful
especially in patients who have facial nerve palsy. In this case report, we have
described a 46 year-old Caucasian male who presented to the clinic for the
evaluation of orofacial swelling and left facial deviation with a history of
multiple treatments for recurrent lower motor neuron type facial nerve palsy. PURPOSE OF REVIEW: We aim to illustrate the potential viability of MCTD as an
underlying aetiology of Melkersson-Rosenthal syndrome. The case is probably the
first description available in the literature of the Melkersson-Rosenthal as an
early manifestation of mixed connective tissue disease.
RECENT FINDINGS: The Melkersson-Rosenthal syndrome consists of a triad of
recurrent lip and/or face swelling, fissured tongue, and intermittent facial
palsy. Mixed connective tissue disease is a multisystemic disorder with
overlapping features of systemic lupus erythematosus, scleroderma, and
polymyositis, and is differentiated from them by a high titer of antibodies to
ribonucleoprotein. The paper presents a case report of Melkersson-Rosenthal
syndrome with an onset in childhood that derived from vasculitis that turned out
to be an early manifestation of mixed connective tissue disease. We used MRI to
evaluate patient's brain structure and Immunoblot Ena Profil 1 test to test
serum autoantibodies level. The patient has a typical for Melkersson-Rosenthal
syndrome triad of symptoms: bilateral facial nerve palsy, lingua plicata and
facial oedema. Both TC and MRI of the head show no changes as well as laboratory
tests except Anti-SS-A (Anti-Ro) and Anti-RNP autoantibody serum level that was
highly positive. Neurological involvement of the MCTD usually includes,
according to the frequency of the occurrence, trigeminal neuralgia, headaches,
sensorineural hearing, cerebral haemorrhage, transverse myelitis, cauda equina
syndrome, retinal vasculitis, progressive multifocal encephalopathy, and
demyelinating neuropathy. For clinical practice it is important to remember that
Melkersson-Rosenthal syndrome can also be the neurological manifestation of
MCTD, especially when accompanied by other systemic symptoms. |
Which disease is treated with taliglucerase alfa? | Taliglucerase alfa, the first available plant cell-expressed recombinant therapeutic protein, is an enzyme replacement therapy approved for Gaucher disease. | Gaucher disease is inherited as an autosomal recessive disorder. The absence of
β-glucocerebrosidase whose purpose is to cleave the glucose from ceramide
results in accumulation of glucocerebroside; storage of this glycolipid results
in Gaucher disease. There is tremendous clinical heterogeneity: prediction of
onset of symptoms (if at all), which organs will be affected, and the degree of
severity of the signs and symptoms are areas of current research. Lysosomal
storage diseases may be treatable by enzyme replacement therapy. Enzyme
replacement for Gaucher disease has been attempted intermittently since the
middle 1970s but was not successful until removal of sugars to expose the inner
mannose residues allowed the targeting of the enzyme to macrophages via mannose
receptors. The use of the recombit imiglucerase (Cerezyme™) as intravenous
therapy has been safe and effective for the visceral symptoms and signs of
Gaucher disease in more than 5000 patients world-wide for more than 18 years.
Nonetheless, beyond enzymes not being able to traverse the blood-brain barrer,
dependence on a single modality is problematic since not all patients are
responders, some develop adverse events, and supply may not be forthcoming for
non-medical reasons. Thus, the availability of new enzymatic preparations,
velaglucerase alfa (VPRIV™) and taliglucerase alfa (UPLYSO™), as well as
alternative modalities such as substrate reduction and pharmacological
chaperones, are important additions to the management portfolio of this disease. Taliglucerase alfa (Protalix Biotherapeutics, Carmiel, Israel) is a novel plant
cell-derived recombit human β-glucocerebrosidase for Gaucher disease. A phase
3, double-blind, randomized, parallel-group, comparison-dose (30 vs 60 U/kg body
weight/infusion) multinational clinical trial was undertaken. Institutional
review board approvals were received. A 9-month, 20-infusion trial used
inclusion/exclusion criteria in treatment-naive adult patients with splenomegaly
and thrombocytopenia. Safety end points were drug-related adverse events: Ab
formation and hypersensitivity reactions. Primary efficacy end point was
reduction in splenic volume measured by magnetic resoce imaging. Secondary
end points were: changes in hemoglobin, hepatic volume, and platelet counts.
Exploratory parameters included biomarkers and bone imaging. Twenty-nine
patients (11 centers) completed the protocol. There were no serious adverse
events; drug-related adverse events were mild/moderate and transient. Two
patients (6%) developed non-neutralizing IgG Abs; 2 other patients (6%)
developed hypersensitivity reactions. Statistically significant spleen reduction
was achieved at 9 months: 26.9% (95% confidence interval [CI]: -31.9, -21.8) in
the 30-unit dose group and 38.0% (95% CI: -43.4, -32.8) in the 60-unit dose
group (both P < .0001); and in all secondary efficacy end point measures, except
platelet counts at the lower dose. These results support safety and efficacy of
taliglucerase alfa for Gaucher disease. Author information:
(1)New York University School of Medicine, Neurogenetics Unit, 403 E 34th St,
Suite 2, New York, NY 10016 USA. Electronic address: [email protected].
(2)Belgrade University Medical School, Dr Subotica 13, Belgrade 11000, Serbia;
Clinic for Endocrinology, Clinical Center of Serbia, Institute of Endocrinology,
Diabetes and Metabolic Diseases, Belgrade, Serbia. Electronic address:
[email protected].
(3)Hospital Universitario Miguel Servet, CIBERER, Paseo de Isabel La Católica
1-3, Zaragoza 50009, Spain. Electronic address: [email protected].
(4)Hematology Department, Rambam Health Care Campus, 8 Haaliya Street, Haifa
31096, Israel. Electronic address: [email protected].
(5)Royal Melbourne Hospital, 300 Grattan Street, Parkville, Victoria 3050,
Australia. Electronic address: [email protected].
(6)Lysosomal Diseases Unit, Addenbrooke's Hospital, Department of Medicine,
University of Cambridge, Level 5, (Box 157) Hills Road, Cambridge,
Cambridgeshire CB2 2QQ, UK. Electronic address:
[email protected].
(7)Mount Sinai Hospital, Joseph and Wolf Lebovic Health Complex, 600 University
Avenue, Toronto, Ontario M5G 1X5, Canada. Electronic address:
[email protected].
(8)Gutenberg-University Mainz, Saarstrasse 21, Mainz D 55099, Germany.
Electronic address: [email protected].
(9)KK Women's and Children's Hospital, Department of Paediatric Medicine, 100
Bukit Timah Road, 229899, Singapore. Electronic address:
[email protected].
(10)Protalix BioTherapeutics, 2 Snunit St., Science Park, POB 455 Carmiel,
Israel. Electronic address: [email protected].
(11)Protalix BioTherapeutics, 2 Snunit St., Science Park, POB 455 Carmiel,
Israel. Electronic address: [email protected].
(12)Gaucher Clinic, Shaare Zedek Medical Center, 12 Bayit Street, Jerusalem
91031, Israel. Electronic address: [email protected]. Taliglucerase alfa is a plant cell-expressed beta-glucocerebrosidase approved in
the United States, Israel, Australia, Canada, and other countries for enzyme
replacement therapy in adults with Type 1 Gaucher disease (GD), for treatment of
pediatric patients in the United States, Australia, and Canada, and for the
hematologic manifestations of Type 3 GD in pediatric patients in Canada. This
multicenter, randomized, double-blind, parallel-dose, 12-month study assessed
efficacy and safety of taliglucerase alfa in pediatric patients with GD. Eleven
children were randomized to taliglucerase alfa 30U/kg (n=6) or 60U/kg (n=5) per
infusion every other week. From baseline to month 12, the following changes were
noted in the taliglucerase alfa 30-U/kg and 60-U/kg dose groups, respectively:
median hemoglobin concentrations increased by 12.2% and 14.2%; the interquartile
ranges of median percent change in hemoglobin levels from baseline were 20.6 and
10.4, respectively; mean spleen volume decreased from 22.2 to 14.0 multiples of
normal (MN) and from 29.4 to 12.9 MN; mean liver volume decreased from 1.8 to
1.5 MN and from 2.2 to 1.7 MN; platelet counts increased by 30.9% and 73.7%; and
chitotriosidase activity was reduced by 58.5% and 66.1%. Nearly all adverse
events were mild/moderate, unrelated to treatment, and transient. One patient
presented with treatment-related gastroenteritis reported as a serious adverse
event due to the need for hospitalization for rehydration. No patient
discontinued. These data suggest that taliglucerase alfa has the potential to be
a therapeutic treatment option for children with GD. This study was registered
at www.clinicaltrials.gov as NCT01132690. Taliglucerase alfa is an intravenous enzyme replacement therapy approved for
treatment of type 1 Gaucher disease (GD), and is the first available plant
cell-expressed recombit therapeutic protein. Herein, we report long-term
safety and efficacy results of taliglucerase alfa in treatment-naïve adult
patients with GD. Patients were randomized to receive taliglucerase alfa 30 or
60 U/kg every other week, and 23 patients completed 36 months of treatment.
Taliglucerase alfa (30 U/kg; 60 U/kg, respectively) resulted in mean decreases
in spleen volume (50.1%; 64.6%) and liver volume (25.6%; 24.4%) with mean
increases in hemoglobin concentration (16.0%; 35.8%) and platelet count (45.7%;
114.0%), and mean decreases in chitotriosidase activity (71.5%; 82.2%). All
treatment-related adverse events were mild to moderate in intensity and
transient. The most common adverse events were nasopharyngitis, arthralgia,
upper respiratory tract infection, headache, pain in extremity, and
hypertension. These 36-month results of taliglucerase alfa in treatment-naïve
adult patients with GD demonstrate continued improvement in disease parameters
with no new safety concerns. These findings extend the taliglucerase alfa
clinical safety and efficacy dataset. www.clinicaltrials.gov identifier
NCT00705939. Am. J. Hematol. 91:656-660, 2016. © 2016 Wiley Periodicals, Inc. BACKGROUND: Gaucher disease is a rare lysosomal storage disease resulting from a
deficiency or reduced activity in the acid β-glucocosidase enzyme. Only 1
treatment option was available for 15 years, but several new treatment options
have come to market since 2003.
OBJECTIVE: The article will detail the pathophysiology and review current
therapies in the literature for all 3 major clinical types of Gaucher disease,
with a focus on considerations for selecting therapy in type 1 disease.
METHODS: Extracted and summarized applicable studies and reviews from Cochrane
Review, ClinicalTrials.gov, CINAHL, IPA, and PubMed.
RESULTS: Enzyme replacement therapy is preferred for the management of Gaucher
disease. Current literature does not favor any enzyme replacement product over
another. However, velaglucerase alfa and taliglucerase alfa theoretically have a
lower risk of immunogenicity reactions compared with imiglucerase. Alternative
treatments for type 1 disease include substrate reduction therapy; however,
these treatments require evaluation of patient-specific variables (eg, genotype
evaluation, renal function) and consideration of adverse effect and dosing
profiles. Evaluation of current literature found no substrate reduction therapy
is preferred over another. There are no approved therapies for type 2 and type 3
disease, but enzyme replacement therapy may be used with limited efficacy for
symptom management.
CONCLUSION: Enzyme replacement therapy is preferred for treating type 1 Gaucher
disease and substrate replacement therapy may be considered in patients who do
not tolerate or cannot receive enzyme replacement therapy. Taliglucerase alfa is an enzyme replacement therapy approved for treatment of
Gaucher disease (GD) in children and adults in several countries. This
multicenter extension study assessed the efficacy and safety of taliglucerase
alfa in pediatric patients with GD who were treatment-naïve (n=10) or switched
from imiglucerase (n=5). Patients received taliglucerase alfa 30 or 60U/kg
(treatment-naïve) or the same dose as previously treated with imiglucerase every
other week. In treatment-naïve patients, taliglucerase alfa 30 and 60U/kg,
respectively, reduced mean spleen volume (-18.6 multiples of normal [MN] and
-26.0MN), liver volume (-0.8MN and -0.9MN), and chitotriosidase activity (-72.7%
and -84.4%), and increased mean Hb concentration (+2.0g/dL and +2.3g/dL) and
mean platelet count (+38,200/mm3 and +138,250/mm3) from baseline through 36
total months of treatment. In patients previously treated with imiglucerase,
these disease parameters remained stable through 33 total months of treatment
with taliglucerase alfa. Most adverse events were mild/moderate; treatment was
well tolerated. These findings extend the taliglucerase alfa safety and efficacy
profile and demonstrate long-term clinical improvement in treatment-naïve
children receiving taliglucerase alfa and maintece of disease stability in
children switched to taliglucerase alfa. Treatment was well-tolerated, with no
new safety signals. This study is registered at www.clinicaltrials.gov as
NCT01411228. |
What is formin associated with in the snail? | Formin is associated with Left-Right asymmetry in the pond snail and the frog. | While components of the pathway that establishes left-right asymmetry have been
identified in diverse animals, from vertebrates to flies, it is striking that
the genes involved in the first symmetry-breaking step remain wholly unknown in
the most obviously chiral animals, the gastropod snails. Previously, research on
snails was used to show that left-right signaling of Nodal, downstream of
symmetry breaking, may be an ancestral feature of the Bilateria [1 and 2]. Here,
we report that a disabling mutation in one copy of a tandemly duplicated,
diaphanous-related formin is perfectly associated with symmetry breaking in the
pond snail. This is supported by the observation that an anti-formin drug
treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs,
drug inhibition or overexpression by microinjection of formin has a
chirality-randomizing effect in early (pre-cilia) embryos. Contrary to
expectations based on existing models [3, 4 and 5], we discovered asymmetric
gene expression in 2- and 4-cell snail embryos, preceding morphological
asymmetry. As the formin-actin filament has been shown to be part of an
asymmetry-breaking switch in vitro [6 and 7], together these results are
consistent with the view that animals with diverse body plans may derive their
asymmetries from the same intracellular chiral elements [8]. |
What is the major difference between eucaryotes and procaryotes? | Eucaryotes have a nucleolus, Procaryotes do not. Eucrayotes have a nuclear membrane, organelles and differ in transcription and translation. Procaryotes have polycistronic RNA and the initiation of protein synthesis proceeds with an initiator tRNA which is found to be modified (formylated) in procaryotes and not in eucaryotes. | In eucaryotes the 5'-terminal guanylate moiety of mature tRNAHis is added
posttranscriptionally. To determine whether the same mechanism occurs in
procaryotes, we processed in vitro-derived Escherichia coli tRNAHis precursors
to mature tRNA, either in E. coli extracts or by using pure M1-RNA, the
catalytic component of RNase P. The results show that the extra guanylate at the
5' end of mature E. coli tRNAHis is encoded in the gene and is found in tRNA as
the result of an unusual cleavage by RNase P. Comparisons of toxicities elicited by nonpolar and polar narcotics, weak acid
uncouplers of oxidative phosphorylation, and bioreactive chemicals between the
eucaryotic systems Pimephales promelas and Tetrahymena pyriformis and the
procaryotic systems Escherichia coli and Photobacterium phosphoreum were
performed. Each chemical had been a priori assigned a mechanism/mode of action
based on the results from previous studies with eucaryotic systems.
Hydrophobicity-dependent QSARs for nonpolar narcosis for both the E. coli and
the P. phosphoreum endpoints was developed. However, due to the lack of a
significant relationship between P. phosphoreum toxicity and log Kow, such a
QSAR for polar narcosis was developed only for the E. coli endpoint. Except for
4-nitroaniline (the only chemical in the examined group that required activation
to become the Michael receptor), all chemicals containing reactive substructures
revealed excess toxicity over polar narcosis QSAR for E. coli endpoints.
Moreover, chloroacidic acid and ethyl chloroacetate in this system also appear
to be bioreactive. The only mechanism that seemed to not exist in the
procaryotic system was uncoupling of oxidative phosphorylation. Chemicals from
this group, except 2,4-dinitroaniline, did not exhibit excess toxicity over
polar narcosis QSAR. This was thought to be explained by the lack of
mitochondria in procaryotes, the target site of uncoupling agents in eucaryotes.
In addition, evaluation of toxicities of halogen-substituted short-chain
carboxylic alcohols indicated that their mechanisms vary, depending upon the
type of substitution and the system. Arachaebacteria have been recently placed in evolution as a separate kingdom of
organisms between procaryotes and eucaryotes. Although these organisms contain
both glycolipids and glycoproteins, they possess no Golgi. No biosynthetic work
has been published on the complex carbohydrates of these newly reassigned
organisms. This report describes preliminary results from one member of this
kingdom, Haloferax volcanii, which suggest that all glycosylation proceeds
through lipid intermediates. Evidence for novel glycolipid structure was also
found during this study. H. volcanii plasma membranes contain all of the enzyme
activities for synthesis of N-linked glycoproteins and archaeol-based
glycolipids. For glucose transfer, all reactions apparently proceed through
glucose-phosphopolyisoprenol using UDP-glucose as primary donor. Incorporation
of D-[3H]glucose from UDP-D-[3H]glucose into glycoproteins and glycolipids of H.
volcanii was stimulated by addition of C55-polyisoprenol phosphate, but not by
C85-105 dolichol phosphate, and was inhibited by amphomycin and two recently
described sugar nucleotide analogs, PP36
(5'-[N-(2-decanoylamino-3-hydroxy-3-phenylpropyloxy
carbonyl)glycyl]amino]-5'-deoxyuridine) and PP55
(5'-O-[[(2-decanoylamino-3-phenylpropyloxycarbonyl) amino]sulfonyl]uridine). All
three inhibitors are reported to block transfer of sugar from UDP-sugars to
phosphopolyisoprenols in eucaryotes. However, in H. volcanii these inhibitors
apparently block transfer of glucose from polyprenyl intermediates to final
glycoproteins and glycolipid products. The sulfodihexosyl archaeol glycolipid
fraction was partially characterized by mass spectrometry and was found to
contain a previously unreported structure with sulfate on the reducing-end
sugar. Four major glycoproteins 190, 105, 56, and 52 kDa and an archaeol-based
glycolipid fraction were labeled by amphomycin-sensitive pathways. Photoaffinity
labeling of H. volcanii homogenate with 5-azido-[32P]UDP-Glc tagged only one
45-kDa polypeptide which is a probable glucosyl-phosphorylpolyisoprenol
synthase. The fact that only one polypeptide band was photoaffinity-labeled
indicated that no other transferase utilized UDP-glucose directly in H.
volcanii. The salt requirement of the UDP-glucose-dependent pathways suggests
that cytoplasmic enzymes function in a high salt environment in H. volcanii. The
archaebacterial plasma membrane thus expresses many functions for glycosylation
of both glycoproteins and glycolipids, normally found in the endoplasmic
reticulum and Golgi of eucaryotes. |
What is the HSP70-HSP110 disaggregase machinery? | Clearance of misfolded and aggregated proteins is central to cell survival. UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. | Author information:
(1)Institute of Molecular, Cell and Systems Biology, College of Medical,
Veterinary and Life Sciences, Davidson Building, Henry Wellcome Lab of Cell
Biology, University of Glasgow, G12 8QQ Glasgow, UK; The MRC Protein
Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of
Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.
(2)Institute of Molecular, Cell and Systems Biology, College of Medical,
Veterinary and Life Sciences, Davidson Building, Henry Wellcome Lab of Cell
Biology, University of Glasgow, G12 8QQ Glasgow, UK; The MRC Protein
Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of
Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.
Electronic address: [email protected].
(3)The MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black
Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1
5EH, Scotland.
(4)Newcastle University Protein and Proteome Analysis, Devonshire Building,
Devonshire Terrace, Newcastle upon Tyne NE1 7RU, UK.
(5)Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa,
Israel.
(6)Department of Medical and Molecular Genetics, King's College London, 8th
Floor Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, UK.
(7)School of Veterinary Medicine, College of Medical, Veterinary and Life
Sciences, University of Glasgow, 464 Bearsden Road, Glasgow G61 1QH, UK.
(8)Institute of Molecular, Cell and Systems Biology, College of Medical,
Veterinary and Life Sciences, Davidson Building, Henry Wellcome Lab of Cell
Biology, University of Glasgow, G12 8QQ Glasgow, UK; The MRC Protein
Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of
Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.
Electronic address: [email protected]. |
Has whole exome sequencing been performed in Alzheimer patients? | Yes, numerous whole exome sequencing studies of ALzheimer patients have been conducted. | Alzheimer's disease (AD) is a genetically complex disorder for which the
definite diagnosis is only accomplished postmortem. Mutations in 3 genes (APP,
PSEN1, and PSEN2) are known to cause AD, but a large number of familial cases do
not harbor mutations in these genes and several unidentified genes that contain
disease-causing mutations are thought to exist. We performed whole exome
sequencing in a Turkish patient clinically diagnosed with Alzheimer's disease
from a consanguineous family with a complex history of neurological and
immunological disorders and identified a mutation in NOTCH3 (p.R1231C),
previously described as causing cerebral autosomal domit arteriopathy with
subcortical infarcts and leukoencephalopathy (CADASIL). Complete screening of
NOTCH3 in a cohort of 95 early onset AD cases and 95 controls did not reveal any
additional pathogenic mutations. Although the complex history of disease in this
family precluded us to establish segregation of the mutation found with disease,
our results show that exome sequencing is a rapid, cost-effective and
comprehensive tool to detect genetic mutations, allowing for the identification
of unexpected genetic causes of clinical phenotypes. As etiological based
therapeutics become more common, this method will be key in diagnosing and
treating disease. Performing exome sequencing in 14 autosomal domit early-onset Alzheimer
disease (ADEOAD) index cases without mutation on known genes (amyloid precursor
protein (APP), presenilin1 (PSEN1) and presenilin2 (PSEN2)), we found that in
five patients, the SORL1 gene harbored unknown nonsense (n=1) or missense (n=4)
mutations. These mutations were not retrieved in 1500 controls of same ethnic
origin. In a replication sample, including 15 ADEOAD cases, 2 unknown
non-synonymous mutations (1 missense, 1 nonsense) were retrieved, thus yielding
to a total of 7/29 unknown mutations in the combined sample. Using in silico
predictions, we conclude that these seven private mutations are likely to have a
pathogenic effect. SORL1 encodes the Sortilin-related receptor LR11/SorLA, a
protein involved in the control of amyloid beta peptide production. Our results
suggest that besides the involvement of the APP and PSEN genes, further genetic
heterogeneity, involving another gene of the same pathway is present in ADEOAD. In the search for new genes in Alzheimer's disease, classic linkage-based and
candidate-gene-based association studies have been supplanted by exome
sequencing, genome-wide sequencing (for mendelian forms of Alzheimer's disease),
and genome-wide association studies (for non-mendelian forms). The
identification of new susceptibility genes has opened new avenues for
exploration of the underlying disease mechanisms. In addition to detecting novel
risk factors in large samples, next-generation sequencing approaches can deliver
novel insights with even small numbers of patients. The shift in focus towards
translational studies and sequencing of individual patients places each
patient's biomaterials as the central unit of genetic studies. The notional
shift needed to make the patient central to genetic studies will necessitate
strong collaboration and input from clinical neurologists. Here, we describe a nonsense haplotype in PRNP associated with clinical
Alzheimer's disease. The patient presented an early-onset of cognitive decline
with memory loss as the primary cognitive problem. Whole-exome sequencing
revealed a nonsense mutation in PRNP (NM_000311, c.C478T; p.Q160*; rs80356711)
associated with homozygosity for the V allele at position 129 of the protein,
further highlighting how very similar genotypes in PRNP result in strikingly
different phenotypes. Early-onset Alzheimer's disease (EOAD) accounts for 1%-2% of all Alzheimer's
disease (AD) subjects, with large variation in the reported genetic contribution
of known dementia genes. In this pilot study, we genetically characterized a
German EOAD cohort (23 subjects) by whole-exome sequencing, capturing variants
in all recognized AD and frontotemporal dementia genes. After variant filtering,
we identified 7 events of altogether 6 different rare variants in 6 subjects,
including 4 novel variants. Four of the 6 variants, observed in 5 different
index subjects (5/23 = 22%), were considered to be possibly pathogenic. These
included 2 presenilin 2 (PSEN2) variants (p.N141I-previously denoted as a Volga
German variant, observed in 2 index subjects; and p.L238P), 1 amyloid precursor
protein (p.I716M), and 1 presenilin 1 (ΔE9). Using a control exome data set of
96 ethnically matched neurodegenerative disease controls (Parkinson's disease),
we identified only 1 variant (PSEN2 p.T18M) (1%), demonstrating a significantly
higher mutational burden in the EOAD group (p > 0.0001). Our findings
demonstrate a substantial frequency of variants in dementia genes in EOAD,
including several seemingly "sporadic" subjects. This indicates that
heritability in EOAD might be higher than assumed. The finding of 3 subjects
carrying potential pathogenic PSEN2 variants suggests that, in specific
populations PSEN2 variants might be as frequent as (or more frequent than)
presenilin 1, for example, in German populations which are influenced by Volga
German heritage. Variants in AD genes were also associated with rare phenotypes
such as frontal AD or primary progressive aphasia, demonstrating the need to
screen AD genes in frontotemporal dementia-like phenotypes. OBJECTIVE: To characterize the clinical and molecular effect of mutations in the
sortilin-related receptor (SORL1) gene.
METHODS: We performed whole-exome sequencing in early-onset Alzheimer disease
(EOAD) and late-onset Alzheimer disease (LOAD) families followed by functional
studies of select variants. The phenotypic consequences associated with SORL1
mutations were characterized based on clinical reviews of medical records.
Functional studies were completed to evaluate β-amyloid (Aβ) production and
amyloid precursor protein (APP) trafficking associated with SORL1 mutations.
RESULTS: SORL1 alterations were present in 2 EOAD families. In one, a SORL1
T588I change was identified in 4 individuals with AD, 2 of whom had parkinsonian
features. In the second, an SORL1 T2134 alteration was found in 3 of 4 AD cases,
one of whom had postmortem Lewy bodies. Among LOAD cases, 4 individuals with
either SORL1 A528T or T947M alterations had parkinsonian features. Functionally,
the variants weaken the interaction of the SORL1 protein with full-length APP,
altering levels of Aβ and interfering with APP trafficking.
CONCLUSIONS: The findings from this study support an important role for SORL1
mutations in AD pathogenesis by way of altering Aβ levels and interfering with
APP trafficking. In addition, the presence of parkinsonian features among select
individuals with AD and SORL1 mutations merits further investigation. |
Which is the genome browser database for DNA shape annotations? | GBshape provides minor groove width, propeller twist, roll, helix twist and hydroxyl radical cleavage predictions for the entire genomes of 94 organisms. Additional genomes can easily be added using the GBshape framework. GBshape can be used to visualize DNA shape annotations qualitatively in a genome browser track format, and to download quantitative values of DNA shape features as a function of genomic position at nucleotide resolution. | Many regulatory mechanisms require a high degree of specificity in protein-DNA
binding. Nucleotide sequence does not provide an answer to the question of why a
protein binds only to a small subset of the many putative binding sites in the
genome that share the same core motif. Whereas higher-order effects, such as
chromatin accessibility, cooperativity and cofactors, have been described, DNA
shape recently gained attention as another feature that fine-tunes the DNA
binding specificities of some transcription factor families. Our Genome Browser
for DNA shape annotations (GBshape; freely available at
http://rohslab.cmb.usc.edu/GBshape/) provides minor groove width, propeller
twist, roll, helix twist and hydroxyl radical cleavage predictions for the
entire genomes of 94 organisms. Additional genomes can easily be added using the
GBshape framework. GBshape can be used to visualize DNA shape annotations
qualitatively in a genome browser track format, and to download quantitative
values of DNA shape features as a function of genomic position at nucleotide
resolution. As biological applications, we illustrate the periodicity of DNA
shape features that are present in nucleosome-occupied sequences from human, fly
and worm, and we demonstrate structural similarities between transcription start
sites in the genomes of four Drosophila species. |
Is Stat4 a transcription factor? | Yes, Stat4 is a transcription factor.
Stat4 is a member of the signal transducer and activator of transcription (STAT) family of molecules that localizes to the cytoplasm. STAT4 regulates various genes expression as a transcription factor after it is phosphorylated, dimerizes and translocates to the nucleus. | STAT4 is a latent cytosolic factor that encodes a transcription factor
transmitting signals stimulated by cytokines. Previous studies with different
study designs in diverse ethnic populations have assessed the influence of STAT4
rs7574865 polymorphism on HBV-induced HCC risk. The aim of the current study was
to investigate the effects in a larger sample. The individual reports published
up to Dec. 30, 2013 were systematically identified by searching the PubMed and
Embase databases. To combine the OR and corresponding 95% CI, we used the fixed
effects model during meta-analysis. Based on eight independent populations with
a total of 5,719 cases and 6,525 controls, we found a slightly reduced risk of
HBV-induced HCC in individuals with the minor T allele compared with individuals
with the common G allele (T versus G: OR = 0.87, 95% CI = 0.82-0.91, P(Het) =
0.974). Similar reductions were also indicated in all subgroups. The combined
data indicate that STAT4 rs7574865 polymorphism may be associated with
significantly reduced risk of HBV-induced HCC in Asian. During infection, the release of damage-associated molecular patterns, so-called
"alarmins," orchestrates the immune response. The alarmin IL-33 plays a role in
a wide range of pathologies. Upon release, IL-33 signals through its receptor
ST2, which reportedly is expressed only on CD4(+) T cells of the Th2 and
regulatory subsets. Here we show that Th1 effector cells also express ST2 upon
differentiation in vitro and in vivo during lymphocytic choriomeningitis virus
(LCMV) infection. The expression of ST2 on Th1 cells was transient, in contrast
to constitutive ST2 expression on Th2 cells, and marked highly activated
effector cells. ST2 expression on virus-specific Th1 cells depended on the
Th1-associated transcription factors T-bet and STAT4. ST2 deficiency resulted in
a T-cell-intrinsic impairment of LCMV-specific Th1 effector responses in both
mixed bone marrow-chimeric mice and adoptive cell transfer experiments.
ST2-deficient virus-specific CD4(+) T cells showed impaired expansion, Th1
effector differentiation, and antiviral cytokine production. Consequently, these
cells mediated little virus-induced immunopathology. Thus, IL-33 acts as a
critical and direct cofactor to drive antiviral Th1 effector cell activation,
with implications for vaccination strategies and immunotherapeutic approaches. AIM: To investigate the role of signal transduction and activation of
transcription 4 (STAT4) in the development and progression of human
hepatocellular carcinoma (HCC).
METHODS: Recent genetic investigations have identified that a genetic variant of
STAT4 is associated with hepatitis B virus (HBV)-related HCC. The level of STAT4
in 90 HCC patients was examined via Western blot and immunohistochemical
analyses. The correlation between STAT4 expression and the clinicopathological
characteristics of the patients was analyzed. The level of STAT4 expression in
the HCC liver tissues was significantly lower than that in the non-HCC liver
tissues and correlated with tumor size, histological grade of HCC and serum
hepatitis B surface antigen level in HCC patients. The data were statistically
analyzed using SPSS. Furthermore, siRNA oligos targeting STAT4 were employed to
investigate the influence of STAT4 RNA interference on HCC cell physiology.
Based on Cell Counting Kit-8 and flow cytometric assays, we found that depletion
of STAT4 expression significantly enhanced the proliferation of L02 cells.
RESULTS: STAT4 protein expression was significantly lower in HCC tissues than in
normal liver tissues. Immunohistochemistry followed by statistical analysis
revealed that the expression of STAT4 negatively correlated with Ki67 expression
(r = 0.851; P < 0.05) and positively correlated with maximal tumor size (P <
0.05), HBV (P = 0.012) and histological grade (P < 0.05). Kaplan-Meier analysis
revealed significant differences in the survival curves between HCC patients
expressing low and high levels of STAT4 and Ki67 (P < 0.05). Based on a
multivariate Cox proportional hazard model, STAT4 expression was an independent
prognostic indicator for HCC patients who underwent curative resection. In
vitro, following the release of L02 cell lines from serum starvation, the
expression of STAT4 was downregulated, and transfection of L02 cells with siRNA
targeting STAT4 inhibited cell proliferation.
CONCLUSION: Our data indicate that STAT4 may inhibit HCC development by
modulating HCC cell proliferation. |
Have the promoter regions of the genes implicated in Rett Syndrome been characterized with CAGE? | Yes. Promoter regions of the genes implicated in Rett Syndrome have been characterized using CAGE. |
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