Phosphorylation of histone H2AX on serine 139 (gamma-H2AX, γH2AX) occurs at sites flanking DNA double-strand breaks (DSBs) and can provide a measure of the number of DSBs within a cell. Here we describe a rapid and simple flow-cytometry-based method, optimized to measure gamma-H2AX in non-fixed peripheral blood cells. No DSB induced signal was observed in H2AX−/− cells indicating that our FACS method specifically recognized gamma-H2AX accumulation. The gamma-H2AX assay was capable of detecting DNA damage at levels 100-fold below the detection limit of the alkaline comet assay. The gamma-H2AX signal was quantitative with a linear increase of the gamma-H2AX signal over two orders of magnitude. We found that all nucleated blood cell types examined, including the short-lived neutrophils induce gamma-H2AX in response to DSBs. Interindividual difference in the gamma-H2AX signal in response to ionizing radiation and the DSB-inducing drug calicheamicin was almost 2-fold in blood cells from patients, indicating that the amount of gamma-H2AX produced in response to a given dose of radiation varies significantly in the human population. This simple method could be used to monitor response to radiation or DNA-damaging drugs.
The crucial ‘flaw’ in the existing treatment paradigm for Non-small Cell Lung Cancer (NSCLC) is the ‘one size fits all approach’. Consequently, adjuvant chemotherapy is given to all patients to benefit a minority and, in the metastatic setting doublet chemotherapy only provides modest improvements in response rates and survival. A personalized approach of treatment selection is therefore desperately needed.
Genetic information is stored in the chemical structure of DNA. To maintain the structural integrity of DNA, an intricate network of DNA repair systems have evolved. One of these is the nucleotide excision repair (NER), a highly versatile and sophisticated DNA damage removal pathway. We show here that this DNA repair mechanism is instrumental in defining prognosis and response to treatment. ERCC1, one of the proteins in this pathway, is measured to assess its functional status of the NER pathway. In patients with early stage NSCLC, low ERCC1 predicts for relapse and selects for patients who will benefit from adjuvant cisplatin-based chemotherapy. Conversely, ERCC1 positive resected patients have a better intrinsic prognosis and are not likely to benefit from platinum based chemotherapy. In a phase II trial in metastatic disease, we show that by tailoring chemotherapy using ERCC1 and RRM1 we can obtain one-year survival of 60% (versus approximately 36% in historical controls) and response rates of 42% (versus 25% in historical controls). This approach is currently being validated in a prospective phase III trial. In the future, assessment of NER function may play a central role in NSCLC treatment decision making.
Lung Cancer; Nuclear Excision Repair; ERCC1; Chemotherapy; Cisplatin; Adjuvant and Metastatic
Sadia Ismail and Graham Mulley discuss the evolution of rules surrounding visiting patients in hospital
To investigate the role of the major histocompatibility complex in Irish patients with optic neuritis (ON) and determine whether HLA‐DRB1 genotypes are a risk factor for the development of multiple sclerosis (MS) in such patients.
All patients were Caucasian, had Irish ancestry and had MRI of brain and optic nerves within 2–3 weeks of presentation. Patients were referred to a neurologist if MRI findings were consistent with a diagnosis of MS. HLA‐DRB1 allele and phenotype frequencies for 78 patients with a clinical diagnosis of acute ON were compared with those for 250 healthy bone marrow donors.
An ON/MS positive patient was 3.4 times more likely than an ON/MS negative patient to be DRB1*15 positive. No difference in age profile was detected between ON/MS positive and ON/MS negative patients or between the ON male and female subgroups. No gender or HLA‐DRB1 association was identified for ON/MS negative patients. Female gender was significantly increased among ON/MS positive patients with a p value of 0.0053.
DRB1*15 is a significant predisposing factor for ON. This ON patient cohort has also provided an opportunity to evaluate the relationship of HLA genotype with the risk of MS development. The findings of this study indicate that Irish individuals presenting with ON and who are HLA DRB1*15 positive have a higher risk than HLA DRB1*15 negative patients of presenting with MRI findings indicative of MS. This study has also demonstrated that female gender is a risk factor for developing MS in the Irish population.
optic neuritis; multiple sclerosis; HLA typing; demyelination
Various demyelinating disorders have been reported in association with anti‐tumour necrosis factor α (TNFα) agents. The objective of this study was to review the occurrence, clinical features and outcome of optic neuritis (ON) during treatment with anti‐TNFα agents.
A PubMed search was conducted to identify literature addressing the potential association between anti‐TNFα agents and ON, following our experience with a patient having rheumatoid arthritis in whom ON developed while being treated with infliximab.
15 patients including the case presented here with ON in whom the symptoms developed following TNFα antagonist therapy were evaluated. Eight of these patients had received infliximab, five had received etanercept and two patients had received adalimumab. Among them, nine patients experienced complete resolution, and two patients had partial resolution, while four patients continued to have symptoms.
Patients being treated with a TNFα antagonist should be closely monitored for the development of ophthalmological or neurological signs and symptoms. Furthermore, consideration should be given to avoiding such therapies in patients with a history of demyelinating disease. If clinical evaluation leads to the diagnosis of ON, discontinuation of the medication and institution of steroid treatment should be a priority.
To determine the uptake of current antenatal HIV testing, the prevalence of risk factors for HIV in pregnant women and the acceptability of the rapid point‐of‐care HIV test (RPOCT) among pregnant women and their midwives.
A retrospective review of 717 notes to determine current HIV screening practices and a cross‐sectional survey using a self‐completed questionnaire for pregnant women and midwives.
The antenatal clinic (ANC) and postnatal wards (PNW) at a university teaching hospital in the West Midlands.
486 women attending the ANC or admitted to the PNW during a fortnight in May–June 2006. 72 midwives on the delivery ward completed a second questionnaire.
The questionnaire showed that 90.4% of those offered the standard HIV test accepted it, with 7.2% having at least one risk factor for HIV. Over half of the decliners perceived themselves as not at risk. 85.2% would accept the rapid test, including 35.6% of the decliners. 92.8% of midwives agreed/strongly agreed the RPOCT has a role on the delivery ward and 97.2% would be happy to offer the test with appropriate training and guidance.
Midwives deem the RPOCT to be appropriate for a variety of perinatal settings. It is also acceptable to a clinically significant proportion of those who decline the standard test (21 of 59) and therefore has the potential to increase screening and detection rates. Hence, by allowing early diagnosis and the initiation of antenatal interventions, it could reduce the rate of mother‐to‐child transmission (MTCT) in the UK.
HIV in pregnancy; mother‐to‐child transmission; point‐of‐care test; rapid test; antenatal screening
Tyrosine kinase inhibitors (TKIs) have advanced cancer treatment. Sunitinib, a recently-approved, multi-targeted TKI, prolongs survival for patients with metastatic renal cell carcinoma (RCC) and gastrointestinal stromal tumor (GIST), but concerns about cardiac safety have arisen with this agent.
To determine the cardiovascular risk associated with sunitinib, we reviewed all cardiovascular events in patients with imatinib-resistant, metastatic GIST at the Dana-Farber Cancer Institute enrolled in a Phase I/II protocol evaluating the efficacy of the drug (n=75). Sunitinib’s effects on left ventricular ejection fraction (LVEF) and blood pressure (BP) were also examined. Studies in isolated cardiomyocytes and mice investigated potential mechanisms of sunitinib-associated cardiac effects.
Eleven percent (8/75) of subjects suffered a cardiovascular event with congestive heart failure (CHF) occurring in 8% (6/75) of the population. Twenty-eight percent (10/36) of patients treated at the FDA-approved dose had LVEF declines of ≥ 10 EF%, and nineteen percent (7/36) experienced LVEF declines of ≥ 15 EF%. Sunitinib induced significant increases in mean systolic and diastolic BP in patients, and 47% (35/75) of individuals developed hypertension (HTN) (>150/100 mmHg). CHF and LV dysfunction generally responded to withholding drug and instituting medical management. In mice and cultured cardiomyocytes, sunitinib caused mitochondrial injury and cardiomyocyte apoptosis.
Sunitinib treatment can lead to HTN, LVEF decline, and/or CHF. Experimental studies suggest that this is due, at least in part, to direct cardiomyocyte toxicity which may be exacerbated by HTN. Patients treated with sunitinib should receive close monitoring and prompt treatment for HTN and/or LVEF decline.
The purpose of this research was to prepare acyclovir niosomes in a trial to improve its poor and variable oral bioavailability. The nonionic surfactant vesicles were prepared by the conventional thin film hydration method. The lipid mixture consisted of cholesterol, span 60, and dicetyl phosphate in the molar ratio of 65:60:5, respectively. The percentage entrapment was ∼11% of acyclovir used in the hydration process. The vesicles have an average size of 0.95 µm, a most probable size of 0.8 µm, and a size range of 0.4 to 2.2 µm. Most of the niosomes have unilamellar spherical shape. In vitro drug release profile was found to follow Higuchi’s equation for free and niosomal drug. The niosomal formulation exhibited significantly retarded release compared with free drug. The in vivo study revealed that the niosomal dispersion significantly improved the oral bioavailability of acyclovir in rabbits after a single oral dose of 40 mg kg−1. The average relative bioavailability of the drug from the niosomal dispersion in relation to the free solution was 2.55 indicating more than 2-fold increase in drug bioavailability. The niosomal dispersion showed significant increase in the mean residence time (MRT) of acyclovir reflecting sustained release characteristics. In conclusion, the niosomal formulation could be a promising delivery system for acyclovir with improved oral bioavailability and prolonged drug release profiles.
Acyclovir niosomes; oral acyclovir; bioavailability of acyclovir
Early detection of polyps or colorectal carcinoma can reduce colorectal carcinoma-associated deaths. Previous studies have demonstrated raised serum levels of matrix metalloproteinase 9 (sMMP-9) in a range of cancers. The aim of this study was to investigate the role of sMMP-9 levels in identifying colorectal neoplasia. Consenting patients donated a blood sample and were assessed by proforma-led history and physical examination. Samples were analysed for sMMP-9 concentration (enzyme-linked immuno-sorbant assay) and compared to final diagnoses. Logistic regression modelling determined independent factors associated with neoplasia. A total of 365 patients were recruited of whom 300 were analysed, including 46 normal controls. A total of 27 significant adenomas and 63 malignancies were identified. The median sMMP-9 concentration was 443ng ml−1 (IQR: 219–782; mean: 546). Patients with neoplasia had significantly elevated sMMP-9 levels (P<0.001). Logistic regression modelling identified elevated log(sMMP-9) as the most significant predictor of neoplasia (χ2=38.33, P<0.001). Other significant factors were age, sex, smoking history, abdominal pain and weight loss. The model accurately predicted neoplasia in 77.3% of cases. Sensitivity and specificity were 77.9 and 77.1%. sMMP-9 estimation can accurately stratify patient to low- or high-risk cohorts. Serum sampling is a potential means of avoiding unnecessary colonoscopy and reducing patient anxiety, iatrogenic morbidity and mortality, and cost.
colorectal; matrix metalloproteinase; screening; risk
The previously known potent cytotoxic agent silvestrol (1) (0.002% w/w yield) and five new flavagline derivatives (2-6) were isolated from the leaves of Aglaia foveolata collected in Indonesia. The new compound 5 has an unprecedented cyclic amide moiety in its cyclopenta[b]benzopyran skeleton, while compound 6 is a novel benzo[b]oxepine derivative in which the oxepine ring is cleaved. Pyramidatine (7), a biogenetic precursor of the new flavaglines 2-6, was isolated from the leaf extract investigated. Silvestrol was also isolated from the stem bark of A. foveolata (yield of 0.02% w/w) along with a new baccharane-type triterpenoid (8). The structures of the new compounds were elucidated on the basis of their NMR and mass spectrometric data. All new compounds isolated were tested against a panel of cancer cell lines, but only compound 2 was cytotoxic (IC50 range = 1.4-1.8 μM), and is the first member of the cyclopenta[b]benzopyran class found to exhibit this type of activity. Compound 2 also showed significant NF-κB inhibitory activity in an Elisa assay (IC50 = 0.37 μM).
Aglaia foveolata; Meliaceae; Flavaglines; Cyclopenta[b]benzofuran; Cyclopenta[b]benzopyrans; Benzo[b]oxepine; Bisamide; Cytotoxicity; NF-κB inhibitory activity
The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Δ1-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Δ1-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Δ1-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.
Background and Aims
Salinity is a widespread soil problem limiting productivity of cereal crops worldwide. Rice is particularly sensitive to salt stress during the seedling stage, with consequent poor crop establishment, as well as during reproduction where salinity can severely disrupt grain formation and yield. Tolerance at the seedling stage is weakly associated with tolerance during reproduction. Physiological responses to salinity were evaluated for contrasting genotypes, during the seedling and reproductive stages.
Three rice genotypes differing in their tolerance of salinity were evaluated in a set of greenhouse experiments under salt stress during both seedling stage and reproduction.
Photosynthetic CO2 fixation, stomatal conductance (gs) and transpiration decreased substantially because of salt stress, but with greater reduction in the sensitive cultivar IR29. The tolerant lines IR651 and IR632 had more responsive stomata that tended to close faster during the first few hours of stress, followed by partial recovery after a brief period of acclimation. However, in the sensitive line, gs continued to decrease for longer duration and with no recovery afterward. Chlorophyll fluorescence measurements revealed that non-photochemical quenching increased, whereas the electron transport rate decreased under salt stress. Salt-tolerant cultivars exhibited much lower lipid peroxidation, maintained elevated levels of reduced ascorbic acid and showed increased activities of the enzymes involved in the reactive oxygen scavenging system during both developmental stages.
Upregulation of the anti-oxidant system appears to play a role in salt tolerance of rice, with tolerant genotypes also maintaining relatively higher photosynthetic function; during both the vegetative and reproductive stages.
Chlorophyll fluorescence; photosynthesis; reactive oxygen species; rice; Oryza sativa; salinity
The initial enzymes and genes involved in the anoxic metabolism of cholesterol were studied in the denitrifying bacterium Sterolibacterium denitrificans Chol-1ST. The second enzyme of the proposed pathway, cholest-4-en-3-one-Δ1-dehydrogenase (AcmB), was partially purified. Based on amino acid sequence analysis, a gene probe was derived to screen a cosmid library of chromosomal DNA for the acmB gene. A positive clone comprising a 43-kbp DNA insert was sequenced. In addition to the acmB gene, the DNA fragment harbored the acmA gene, which encodes the first enzyme of the pathway, cholesterol dehydrogenase/isomerase. The acmA gene was overexpressed, and the recombinant dehydrogenase/isomerase was purified. This enzyme catalyzes the predicted transformation of cholesterol to cholest-4-en-3-one. S. denitrificans cells grown aerobically with cholesterol exhibited the same pattern of soluble proteins and cell extracts formed the same 14C-labeled products from [14C]cholesterol as cells that were grown under anoxic, denitrifying conditions. This is especially remarkable for the late products that are formed by anaerobic hydroxylation of the cholesterol side chain with water as the oxygen donor. Hence, this facultative anaerobic bacterium may use the anoxic pathway lacking any oxygenase-dependent reaction also under oxic conditions. This confers metabolic flexibility to such facultative anaerobic bacteria.
The aim of this study is to investigate the association between breast cancer and serum selenium level as well as other risk factors for breast cancer.
A matched case-control study was conducted in a hospital in Malaysia from July 2000 to January 2001 and from May 2001 to June 2001. Sixty-two newly diagnosed breast cancer patients were selected as the cases. Each control, selected from the same hospital population was matched to each case according to age, ethnic group, and menopausal status.
The mean selenium concentration among the cases was significantly lower than that among the control. There was a significant association (p<0.05) between breast cancer and low selenium serum level, nulliparity (OR=5.5,95% CI=1.22 to 24.81), exposure to cigarette smoke (OR=2.2, 95% CI=1.04 to 4.65) and use of oral contraceptives (OR=3.0, 95% CI=1.09 to 8.25) as determined by the McNemar test. Multivariate analysis showed that nulliparity (OR=10.08, 95% CI=1.48 to 68.52) and use of oral contraceptives (OR=3.66, 95% CI=1.36 to 9.87) were associated with increased breast cancer risk. An increased selenium concentration contributes to a reduced risk of breast cancer (OR=0.89, 95% CI=0.84 to 0.94).
The results suggest that use of oral contraceptive pills, being nulliparous, and a low serum selenium level are associated with breast cancer.
breast cancer; selenium; glutathione peroxides enzyme; oral contraceptives; nulliparity
To identify noninvasive factors predicting the presence of large varices (LV) in patients hospitalized with gastroesophageal variceal hemorrhage (GEVH).
Case records of patients admitted with GEVH between January 1998 and June 2005 were retrospectively analyzed. Relevant clinical parameters assessed included Child-Pugh class, ascites (clinical and/or on ultrasound), portosystemic encephalopathy (PSE), splenomegaly (clinical and/or on ultrasound), and hemodynamic instability. The laboratory parameters assessed were hemoglobin level, platelet count, prothrombin time, serum bilirubin, and albumin. The ultrasonographic characteristics noted were splenic size, presence of splenic varices, and portal vein diameter.
A total of 420 patients (264 men) presented with GEVH during the study period. The mean age, gender distribution, and presence of cirrhosis were similar in the two groups. Liver cirrhosis with hepatocellular carcinoma (HCC), Child-Pugh class C, presence of clinically detectable ascites, grade 3–4 PSE, detectable splenomegaly, previous history of GEVH, hemodynamic instability and platelet count <91,000 were more common in the LV group. The frequency of radiologically detected ascites, splenomegaly, and portal vein diameter were similar in both groups. On multivariate analysis, the independent predictors for the presence of LV were cirrhosis with HCC, clinically detectable splenomegaly, hemodynamic instability, a previous history of GEVH, platelet count <91,000, and splenic size ≥158 mm.
Cirrhosis with HCC, clinical splenomegaly, hemodynamic instability, a previous history of GEVH, thrombocytopenia (i.e., platelet count <91,000), and splenic size ≥158 mm are independent noninvasive predictors of large varices in patients hospitalized with gastroesophageal variceal hemorrhage.
Gastroesophageal variceal hemorrhage; Predictors; Varices
Objective: Carotid artery (CA) invasion by head and neck tumors is a challenging problem for the cranial base surgeon. Proposed methods for management have the disadvantage of constant or temporary interruption of the arterial blood flow and, as a consequence, cerebral ischemic complications. The objective of the study was to investigate the long-term effects of a novel technique, “extarterectomy,” on the vascular wall and the arterial blood flow in an ovine model. Methods: Wallstents were implanted bilaterally in the common CA of 5 sheep by the Seldinger technique. Six weeks after stent implantation, a segment of the arterial wall of about 2 cm in length was peeled off the external surface of the stent. Six months later, control angiography was performed. The animals were sacrificed after 18 months and the “extarterectomized” arteries were removed for the microscopic and macroscopic evaluation. Results: There were no acquired neurological deficits in the study group. Extarterectomy was performed without any complication in every case. Control angiography confirmed patent CAs. Evaluation by light microscopy showed the “neointimal” layer within the interstices of the stent. Conclusion: Extarterectomy allows aggressive tumor removal together with the maintenance of blood flow through the CA and offers hope for those with tumors invading the CA.
Carotid artery; head and neck cancer; carotid stenting
Hyperthyroidism may present with signs and symptoms related to dysfunction of a variety of organs. Cardiovascular pathology in hyperthyroidism is common. A few case reports describe isolated right heart failure, tricuspid regurgitation, and pulmonary hypertension as the prominent cardiovascular manifestations of hyperthyroidism. Although most textbooks do not mention hyperthyroidism as a cause of pulmonary hypertension and isolated right heart failure, the literature suggests that some hyperthyroid patients may develop reversible pulmonary hypertension and isolated right heart failure. We report a case of hyperthyroidism presenting with signs and symptoms of isolated right heart failure, tricuspid regurgitation, and pulmonary hypertension, which resolved with treatment of hyperthyroidism.
hyperthyroidism; cardiovascular pathology; heart failure; tricuspid regurgitation; pulmonary hypertension
Using PCR-based denaturing gradient gel electrophoresis analyses of oral bacterial samples in 20 mother-child dyads, this study demonstrated a high degree of similarity of bacterial compositions between the mothers and their children; the two may share as much as 94% of their oral bacterial spectra, including cariogenic species.
Infection with gram-negative monocytotropic Ehrlichia strains results in a fatal toxic shock-like syndrome characterized by a decreased number of Ehrlichia-specific CD4+ Th1 cells, the expansion of tumor necrosis factor alpha (TNF-α)-producing CD8+ T cells, and the systemic overproduction of interleukin-10 (IL-10) and TNF-α. Here, we investigated the role of CD4+ and CD8+ T cells in immunity to Ehrlichia and the pathogenesis of fatal ehrlichiosis caused by infection with low- and high-dose (103 and 105 bacterial genomes/mouse, respectively) ehrlichial inocula. The CD4+ T-cell-deficient mice showed exacerbated susceptibility to a lethal high- or low-dose infection and harbored higher bacterial numbers than did wild-type (WT) mice. Interestingly, the CD8+ T-cell-deficient mice were resistant to a low dose but succumbed to a high dose of Ehrlichia. The absence of CD8+ T cells abrogated TNF-α and IL-10 production, reduced tissue injury and bacterial burden, restored splenic CD4+ T-cell numbers, and increased the frequency of Ehrlichia-specific CD4+ Th1 cells in comparison to infected WT mice. Although fatal disease is perforin independent, our data suggested that perforin played a critical role in controlling bacterial burden and mediating liver injury. Similar to WT mice, mortality of infected perforin-deficient mice was associated with CD4+ T-cell apoptosis and a high serum concentration of IL-10. Depletion of IL-10 restored the number of CD4+ and CD8+ T cells in infected WT mice. Our data demonstrate a novel mechanism of immunopathology in which CD8+ T cells mediate Ehrlichia-induced toxic shock, which is associated with IL-10 overproduction and CD4+ T-cell apoptosis.