query: Healthy volunteers exhibit rapid and transient increase of cellular ATP after being bolus-injected with fructose.

title: Alterations in liver ATP homeostasis in human nonalcoholic steatohepatitis: a pilot study.

text:
CONTEXT The mechanisms that drive progression from fatty liver to steatohepatitis and cirrhosis are unknown. In animal models, obese mice with fatty livers are vulnerable to liver adenosine triphosphate (ATP) depletion and necrosis, suggesting that altered hepatic energy homeostasis may be involved.
 OBJECTIVE To determine if patients with fatty liver disease exhibit impaired recovery from hepatic ATP depletion.
 DESIGN Laboratory analysis of liver ATP stores monitored by nuclear magnetic resonance spectroscopy before and after transient hepatic ATP depletion was induced by fructose injection. The study was conducted between July 15 and August 30, 1998.
 SETTING University hospital.
 PATIENTS Eight consecutive adults with biopsy-proven nonalcoholic steatohepatitis and 7 healthy age- and sex-matched controls.
 MAIN OUTCOME MEASURE Level of ATP 1 hour after fructose infusion in patients vs controls.
 RESULTS In patients, serum aminotransferase levels were increased (P = .02 vs controls); albumin and bilirubin values were normal and clinical evidence of portal hypertension was absent in both groups. However, 2 patients had moderate fibrosis and 1 had cirrhosis on liver biopsy. Mean serum glucose, cholesterol, and triglyceride levels were similar between groups but patients weighed significantly more than controls (P = .02). Liver ATP levels were similar in the 2 groups before fructose infusion and decreased similarly in both after fructose infusion (P = .01 vs initial ATP levels). However, controls replenished their hepatic ATP stores during the 1-hour follow-up period (P<.02 vs minimum ATP) but patients did not. Hence, patients' hepatic ATP levels were lower than those of controls at the end of the study (P = .04). Body mass index (BMI) correlated inversely with ATP recovery, even in controls (R = -0.768; P = .07). Although BMI was greater in patients than controls (P = .02) and correlated strongly with fatty liver and serum aminotransferase elevations, neither of the latter 2 parameters nor the histologic severity of fibrosis strongly predicted hepatic ATP recovery.
 CONCLUSIONS These data suggest that recovery from hepatic ATP depletion becomes progressively less efficient as body mass increases in healthy controls and is severely impaired in patients with obesity-related nonalcoholic steatohepatitis.

title: The cause of hepatic accumulation of fructose 1-phosphate on fructose loading.

text: 1. The changes in the metabolite content in freeze-clamped livers of fed rats occurring on perfusion with 10mm-d-fructose have been examined. 2. The most striking effects of fructose were an accumulation of fructose 1-phosphate, as already known, up to 8.7mumol/g of liver within 10min, a loss of total adenine nucleotides (up to 35% after 40min) with a decrease in the ATP content to 23% within 10min, a sevenfold rise in the concentration of IMP to 1.1mumol/g and an eightfold rise of alpha-glycerophosphate to 1.1mumol/g. 3. There was a transient decrease in P(i) from 4.2 to 1.7mumol/g. Within 40min the P(i) content recovered to the normal value, probably because of an uptake of P(i) from the perfusion medium. 4. The degradation of the adenine nucleotides beyond the stage of AMP can be accounted for by the decrease of ATP and P(i). As ATP inhibits 5-nucleotidase, and as P(i) inhibits AMP deaminase any AMP arising in the tissue is liable to undergo dephosphorylation or deamination under the conditions occurring after fructose loading. 5. The content of lactate increased to 4.3mumol/g at 80min; pyruvate also increased and the [lactate]/[pyruvate] ratio remained within physiological limits. 6. The concentration of free fructose within the liver remained much below that in the perfusion medium, indicating that the rate of penetration of fructose into the tissue was lower than the rate of utilization. 7. The fission of fructose 1-phosphate by liver aldolase is inhibited by several phosphorylated intermediates, especially by IMP. This inhibition is competitive with a K(i) of 0.1mm. 8. The maximal rates of the enzymes synthesizing and splitting fructose 1-phosphate are about equal. The accumulation of fructose 1-phosphate on fructose loading is due to the inhibition of the fission of fructose 1-phosphate by the IMP arising from the degradation of the adenine nucleotides.

title: T1 mapping for myocardial extracellular volume measurement by CMR: bolus only versus primed infusion technique.

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OBJECTIVES The aim of this study was to determine the accuracy of the contrast "bolus only" T1 mapping cardiac magnetic resonance (CMR) technique for measuring myocardial extracellular volume fraction (ECV).
 BACKGROUND Myocardial ECV can be measured with T1 mapping before and after contrast agent if the contrast agent distribution between blood/myocardium is at equilibrium. Equilibrium distribution can be achieved with a primed contrast infusion (equilibrium contrast-CMR [EQ-CMR]) or might be approximated by the dynamic equilibration achieved by delayed post-bolus measurement. This bolus only approach is highly attractive, but currently limited data support its use. We compared the bolus only technique with 2 independent standards: collagen volume fraction (CVF) from myocardial biopsy in aortic stenosis (AS); and the infusion technique in 5 representative conditions.
 METHODS One hundred forty-seven subjects were studied: healthy volunteers (n = 50); hypertrophic cardiomyopathy (n = 25); severe AS (n = 22); amyloid (n = 20); and chronic myocardial infarction (n = 30). Bolus only (at 15 min) and infusion ECV measurements were performed and compared. In 18 subjects with severe AS the results were compared with histological CVF.
 RESULTS The ECV by both techniques correlated with histological CVF (n = 18, r² = 0.69, p < 0.01 vs. r² = 0.71, p < 0.01, p = 0.42 for comparison). Across health and disease, there was strong correlation between the techniques (r² = 0.97). However, in diseases of high ECV (amyloid, hypertrophic cardiomyopathy late gadolinium enhancement, and infarction), Bland-Altman analysis indicates the bolus only technique has a consistent and increasing offset, giving a higher value for ECVs above 0.4 (mean difference ± limit of agreement for ECV <0.4 = -0.004 ± 0.037 vs. ECV >0.4 = 0.040 ± 0.075, p < 0.001).
 CONCLUSIONS Bolus only, T1 mapping-derived ECV measurement is sufficient for ECV measurement across a range of cardiac diseases, and this approach is histologically validated in AS. However, when ECV is >0.4, the bolus only technique consistently measures ECV higher compared with infusion.

title: Exosomal miR-99a-5p is elevated in sera of ovarian cancer patients and promotes cancer cell invasion by increasing fibronectin and vitronectin expression in neighboring peritoneal mesothelial cells

text:
BACKGROUND microRNAs (miRNAs) stably exist in circulating blood and are encapsulated in extracellular vesicles such as exosomes. The aims of this study were to identify which exosomal miRNAs are highly produced from epithelial ovarian cancer (EOC) cells, to analyze whether serum miRNA can be used to discriminate patients with EOC from healthy volunteers, and to investigate the functional role of exosomal miRNAs in ovarian cancer progression.
 METHODS Exosomes were collected from the culture media of serous ovarian cancer cell lines, namely TYK-nu and HeyA8 cells. An exosomal miRNA microarray revealed that several miRNAs including miR-99a-5p were specifically elevated in EOC-derived exosomes. Expression levels of serum miR-99a-5p in 62 patients with EOC, 26 patients with benign ovarian tumors, and 20 healthy volunteers were determined by miRNA quantitative reverse transcription-polymerase chain reaction. To investigate the role of exosomal miR-99a-5p in peritoneal dissemination, neighboring human peritoneal mesothelial cells (HPMCs) were treated with EOC-derived exosomes and then expression levels of miR-99a-5p were examined. Furthermore, mimics of miR-99a-5p were transfected into HPMCs and the effect of miR-99a-5p on cancer invasion was analyzed using a 3D culture model. Proteomic analysis with the tandem mass tag method was performed on HPMCs transfected with miR-99a-5p and then potential target genes of miR-99a-5p were examined.
 RESULTS The serum miR-99a-5p levels were significantly increased in patients with EOC, compared with those in benign tumor patients and healthy volunteers (1.7-fold and 2.8-fold, respectively). A receiver operating characteristic curve analysis showed with a cut-off of 1.41 showed sensitivity and specificity of 0.85 and 0.75, respectively, for detecting EOC (area under the curve = 0.88). Serum miR-99a-5p expression levels were significantly decreased after EOC surgeries (1.8 to 1.3, p = 0.002), indicating that miR-99a-5p reflects tumor burden. Treatment with EOC-derived exosomes significantly increased miR-99a-5p expression in HPMCs. HPMCs transfected with miR-99a-5p promoted ovarian cancer invasion and exhibited increased expression levels of fibronectin and vitronectin.
 CONCLUSIONS Serum miR-99a-5p is significantly elevated in ovarian cancer patients. Exosomal miR-99a-5p from EOC cells promotes cell invasion by affecting HPMCs through fibronectin and vitronectin upregulation and may serve as a target for inhibiting ovarian cancer progression.

title: Respiratory and cardiovascular responses to walking down a traffic-polluted road compared with walking in a traffic-free area in participants aged 60 years and older with chronic lung or heart disease and age-matched healthy controls: a randomised, crossover study

text:
BACKGROUND Long-term exposure to pollution can lead to an increase in the rate of decline of lung function, especially in older individuals and in those with chronic obstructive pulmonary disease (COPD), whereas shorter-term exposure at higher pollution levels has been implicated in causing excess deaths from ischaemic heart disease and exacerbations of COPD. We aimed to assess the effects on respiratory and cardiovascular responses of walking down a busy street with high levels of pollution compared with walking in a traffic-free area with lower pollution levels in older adults.
 METHODS In this randomised, crossover study, we recruited men and women aged 60 years and older with angiographically proven stable ischaemic heart disease or stage 2 Global initiative for Obstructive Lung Disease (GOLD) COPD who had been clinically stable for 6 months, and age-matched healthy volunteers. Individuals with ischaemic heart disease or COPD were recruited from existing databases or outpatient respiratory and cardiology clinics at the Royal Brompton & Harefield NHS Foundation Trust and age-matched healthy volunteers using advertising and existing databases. All participants had abstained from smoking for at least 12 months and medications were taken as recommended by participants' doctors during the study. Participants were randomly assigned by drawing numbered disks at random from a bag to do a 2 h walk either along a commercial street in London (Oxford Street) or in an urban park (Hyde Park). Baseline measurements of participants were taken before the walk in the hospital laboratory. During each walk session, black carbon, particulate matter (PM) concentrations, ultrafine particles, and nitrogen dioxide (NO2) concentrations were measured.
 FINDINGS Between October, 2012, and June, 2014, we screened 135 participants, of whom 40 healthy volunteers, 40 individuals with COPD, and 39 with ischaemic heart disease were recruited. Concentrations of black carbon, NO2, PM10, PM2.5, and ultrafine particles were higher on Oxford Street than in Hyde Park. Participants with COPD reported more cough (odds ratio [OR] 1·95, 95% CI 0·96-3·95; p<0·1), sputum (3·15, 1·39-7·13; p<0·05), shortness of breath (1·86, 0·97-3·57; p<0·1), and wheeze (4·00, 1·52-10·50; p<0·05) after walking down Oxford Street compared with Hyde Park. In all participants, irrespective of their disease status, walking in Hyde Park led to an increase in lung function (forced expiratory volume in the first second [FEV1] and forced vital capacity [FVC]) and a decrease in pulse wave velocity (PWV) and augmentation index up to 26 h after the walk. By contrast, these beneficial responses were attenuated after walking on Oxford Street. In participants with COPD, a reduction in FEV1 and FVC, and an increase in R5-20 were associated with an increase in during-walk exposure to NO2, ultrafine particles and PM2.5, and an increase in PWV and augmentation index with NO2 and ultrafine particles. In healthy volunteers, PWV and augmentation index were associated both with black carbon and ultrafine particles.
 INTERPRETATION Short-term exposure to traffic pollution prevents the beneficial cardiopulmonary effects of walking in people with COPD, ischaemic heart disease, and those free from chronic cardiopulmonary diseases. Medication use might reduce the adverse effects of air pollution in individuals with ischaemic heart disease. Policies should aim to control ambient levels of air pollution along busy streets in view of these negative health effects.
 FUNDING British Heart Foundation.

title: Pharmacodynamics and pharmacokinetics of eptifibatide in patients with acute coronary syndromes: prospective analysis from PURSUIT.

text:
BACKGROUND Platelet deposition and aggregation are central to the pathogenesis of ischemic complications of acute coronary syndromes (ACS). Pharmacodynamic effects of the platelet glycoprotein IIb/IIIa antagonist eptifibatide have been delineated in healthy subjects but not in patients with ACS. We assessed effects of eptifibatide on ex vivo platelet aggregation in patients enrolled in the Platelet glycoprotein IIb/IIIa in Unstable angina: Receptor Suppression Using Integrilin (eptifibatide) Therapy (PURSUIT) trial of ACS.
 METHODS AND RESULTS Patients were randomly assigned to an intravenous bolus (180 microgram/kg) and 72-hour infusion of eptifibatide (2.0 microgram/kg per minute, n=48) or placebo (n=50). We assessed correlations of plasma eptifibatide levels with receptor occupancy and inhibition of ex vivo platelet aggregation at 5 minutes and 1, 4, 24, 48, and 72 hours during treatment and 4 and 8 hours after termination of infusion. Blood was collected in buffered citrate and D-phenylalanyl-L-prolyl-L-arginine chloromethylketone anticoagulants. Although eptifibatide produced profound, prolonged inhibition of platelet aggregation during therapy, aggregation appeared to recover partially by 4 hours after the bolus. The aggregation response was greater with thrombin receptor agonist peptide versus ADP stimulation; inhibition of platelet aggregation was greater in blood samples anticoagulated with citrate versus D-phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK). Plasma eptifibatide levels correlated significantly with receptor occupancy but not with inhibition of platelet aggregation.
 CONCLUSIONS A bolus and infusion of eptifibatide inhibits platelet aggregation profoundly in patients with ACS and is followed by brief, partial recovery. These results enhance our understanding of the relation between pharmacodynamic and clinical effects of eptifibatide in such patients and may have important implications for its use in percutaneous interventions.

title: High-dose recombinant tumor necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion of the limbs for melanoma and sarcoma.

text:
PURPOSE To determine the toxicity and the therapeutic efficacy of the combination of the recombinant tumor necrosis factor alpha (rTNF alpha), recombinant interferon gamma (rIFN-gamma), and melphalan, we designed a protocol using isolation limb perfusion (ILP) with hyperthermia for in-transit metastases of melanoma and recurrent sarcoma. The triple combination was chosen because of the reported synergistic antitumor effect of rTNF alpha with IFN-gamma and of rTNF alpha with alkylating agents.
 PATIENTS AND METHODS Twenty-three patients received a total of 25 ILPs with the triple combination. There were 19 females and four males with either multiple progressive in-transit melanoma metastases of the extremities (stage IIIa or IIIab; 19 patients) or recurrent soft tissue sarcoma (five). The rTNF alpha was injected as a bolus in the arterial line, and total dose ranged between 2 and 4 mg, under hyperthermic conditions (40 degrees C to 40.5 degrees C) for 90 minutes. The rIFN-gamma was given subcutaneously (SC) on days -2 and -1 and in the perfusate, with rTNF alpha at the dose of 0.2 mg. Melphalan (Alkeran; Burroughs Wellcome Co, London, England) was administered in the perfusate at 40 micrograms/mL.   RESULTS Toxicity observed during three ILPs in a pilot study with rTNF alpha included only two severe toxicities: one severe hypotension with tachycardia and transient oliguria and one moderate hypotension for 4 hours followed by severe kidney failure with complete recovery on day 29. In all 18 ILPs performed in the triple combination protocol, the patients received continuous infusion dopamine at 3 micrograms/kg/min from the start of ILP and for 72 hours and showed only mild hypotension and transient chills and temperature. Regional toxicity attributable to rTNF alpha was minimal. There have been 11 cases with hematologic toxicity consisting of neutropenia (one grade 4 and one grade 3) and neutropenia with thrombocytopenia (one grade 4 and three grade 2). Twelve patients had been previously treated with melphalan in ILP (11) or with cisplatin (one). The 23 patients are assessable: there have been 21 complete responses (CRs; range, 4 to 29 months; 89%), two partial responses (PRs; range, 2 to 3 months), and no failures. Overall disease-free survival and survival have been 70% and 76%, respectively, at 12 months. In all cases, softening of the nodules was obvious within 3 days after ILP and time to definite response ranged between day 5 and 30.
 CONCLUSION This preliminary analysis of a phase II study suggests that high-dose rTNF alpha can be administered with acceptable toxicity by ILP with dopamine and hyperhydration. Tumor responses can be evidenced in melanoma and sarcoma. Furthermore, combination of rTNF alpha, rIFN-gamma, and melphalan seems to achieve high efficacy with minimal toxicity, even after failure of prior therapy with melphalan alone.

title: Translocated LPS Might Cause Endotoxin Tolerance in Circulating Monocytes of Cystic Fibrosis Patients

text: Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene codes of a chloride channel. The lungs of CF patients are chronically infected by several pathogens but bacteraemia have rarely been reported in this pathology. Besides that, circulating monocytes in CF patients exhibit a patent Endotoxin Tolerance (ET) state since they show a significant reduction of the inflammatory response to bacterial stimulus. Despite a previous description of this phenomenon, the direct cause of ET in CF patients remains unknown. In this study we have researched the possible role of microbial/endotoxin translocation from a localized infection to the bloodstream as a potential cause of ET induction in CF patients. Plasma analysis of fourteen CF patients revealed high levels of LPS compared to healthy volunteers and patients who suffer from Chronic Obstructive Pulmonary Disease. Experiments in vitro showed that endotoxin concentrations found in plasma of CF patients were enough to induce an ET phenotype in monocytes from healthy controls. In agreement with clinical data, we failed to detect bacterial DNA in CF plasma. Our results suggest that soluble endotoxin present in bloodstream of CF patients causes endotoxin tolerance in their circulating monocytes.

title: Evaluation of oxidative status and depression-like responses in Brown Norway rats with acute myeloid leukemia

text: It has been proved that oxidative stress increases when leukemia is accompanied by depression. This fact may indicate the role of oxidative stress in the development of depression in cancer patients. The aim of this study was to determine whether the acute myeloid leukemia of Brown Norway rats, which is accompanied by oxidative stress, evoked behavioral and receptor changes resembling alterations characteristic of rat models of depression. The rats were divided into two groups: leukemic rats and healthy control. Leukemia was induced through intraperitoneal injection of 10(7) promyelocytic leukemia cells to the Brown Norway rats. Depression-like behavior was evaluated in the forced swim test at 30 or 34 days after leukemic cells injection. The rats were killed after the evaluation and the spleen, brain cortex and hippocampus were excised. The red-ox state was assessed in homogenates of tissues by measuring total glutathione (GSH) content, the ferric ion reducing ability of plasma (FRAP) level, expression of heme oxygenase-1 (HO-1), biliverdin reductase (BvR) and ferritin mRNA, superoxide dismutase (SOD) activity, as well as malondialdehyde (MDA) concentration. Radioligand binding assay was used to assess of the effect of leukemia on cortical receptors. Leukemic cells were identified using RM-124 antibody by FACS Calibur flow cytometry. Leukemia influenced locomotory activity as well as forced swim test behavior in a 34-day series of experiments. Signs of oxidative stress in leukemic rats were observed in each examined stage of leukemia development. The FRAP values and glutathione contents, were significantly lowered whereas HO-1 mRNA expression, and malonodialdehyde concentrations were significantly increased in the spleen and brain structures of leukemic rats in comparison with the healthy controls. A significant increase in the potency of glycine to displace [(3)H]L-689,560 from the strychnine-insensitive glycine site of the N-methyl-D-aspartic (NMDA) receptors receptor complex in cortical homogenates of the leukemic rats in 30- and 34-day experimental series was observed in comparison with the control. Upregulation of 5-HT(2A) receptors was observed in rat cortex after 30 days of leukemia development but not in 34-days series compared with the control. It is concluded that disturbances in antioxidant system in brain cortex were accompanied by an activation of glycine sites of the NMDA receptor complex, regardless of stage of leukemia development, which are characteristic of model of depression. Findings of our study demonstrate the link between glutamatergic activity, oxidative stress and leukemia.

title: Chronic stress, glucocorticoid receptor resistance, inflammation, and disease risk.

text: We propose a model wherein chronic stress results in glucocorticoid receptor resistance (GCR) that, in turn, results in failure to down-regulate inflammatory response. Here we test the model in two viral-challenge studies. In study 1, we assessed stressful life events, GCR, and control variables including baseline antibody to the challenge virus, age, body mass index (BMI), season, race, sex, education, and virus type in 276 healthy adult volunteers. The volunteers were subsequently quarantined, exposed to one of two rhinoviruses, and followed for 5 d with nasal washes for viral isolation and assessment of signs/symptoms of a common cold. In study 2, we assessed the same control variables and GCR in 79 subjects who were subsequently exposed to a rhinovirus and monitored at baseline and for 5 d after viral challenge for the production of local (in nasal secretions) proinflammatory cytokines (IL-1β, TNF-α, and IL-6). Study 1: After covarying the control variables, those with recent exposure to a long-term threatening stressful experience demonstrated GCR; and those with GCR were at higher risk of subsequently developing a cold. Study 2: With the same controls used in study 1, greater GCR predicted the production of more local proinflammatory cytokines among infected subjects. These data provide support for a model suggesting that prolonged stressors result in GCR, which, in turn, interferes with appropriate regulation of inflammation. Because inflammation plays an important role in the onset and progression of a wide range of diseases, this model may have broad implications for understanding the role of stress in health.

title: Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells.

text: Telomere extension has been proposed as a means to improve cell culture and tissue engineering and to treat disease. However, telomere extension by nonviral, nonintegrating methods remains inefficient. Here we report that delivery of modified mRNA encoding TERT to human fibroblasts and myoblasts increases telomerase activity transiently (24-48 h) and rapidly extends telomeres, after which telomeres resume shortening. Three successive transfections over a 4 d period extended telomeres up to 0.9 kb in a cell type-specific manner in fibroblasts and myoblasts and conferred an additional 28 ± 1.5 and 3.4 ± 0.4 population doublings (PDs), respectively. Proliferative capacity increased in a dose-dependent manner. The second and third transfections had less effect on proliferative capacity than the first, revealing a refractory period. However, the refractory period was transient as a later fourth transfection increased fibroblast proliferative capacity by an additional 15.2 ± 1.1 PDs, similar to the first transfection. Overall, these treatments led to an increase in absolute cell number of more than 10(12)-fold. Notably, unlike immortalized cells, all treated cell populations eventually stopped increasing in number and expressed senescence markers to the same extent as untreated cells. This rapid method of extending telomeres and increasing cell proliferative capacity without risk of insertional mutagenesis should have broad utility in disease modeling, drug screening, and regenerative medicine.

title: Chronophin mediates an ATP-sensing mechanism for cofilin dephosphorylation and neuronal cofilin-actin rod formation.

text: Actin and its key regulatory component, cofilin, are found together in large rod-shaped assemblies in neurons subjected to energy stress. Such inclusions are also enriched in Alzheimer's disease brain, and appear in transgenic models of neurodegeneration. Neuronal insults, such as energy loss and/or oxidative stress, result in rapid dephosphorylation of the cellular cofilin pool prior to its assembly into rod-shaped inclusions. Although these events implicate a role for phosphatases in cofilin rod formation, a mechanism linking energy stress, phosphocofilin turnover, and subsequent rod assembly has been elusive. We demonstrate the ATP-sensitive interaction of the cofilin phosphatase chronophin (CIN) with the chaperone hsp90 to form a biosensor that mediates cofilin/actin rod formation. Our results suggest a model whereby attenuated interactions between CIN and hsp90 during ATP depletion enhance CIN-dependent cofilin dephosphorylation and consequent rod assembly, thereby providing a mechanism for the formation of pathological actin/cofilin aggregates during neurodegenerative energy flux.

title: Cellular and histopathological changes in the infrapatellar fat pad in the monoiodoacetate model of osteoarthritis pain.

text:
OBJECTIVE The infrapatellar fat pad (IPFP) has been identified as a source of anterior knee pain. Fibrosis and marked inflammatory infiltrate in the IPFP of patients with arthritis of the knee and reduction in pain post knee replacement in patients following resection of the IPFP have been observed. We have investigated changes in the IPFP of rats undergoing the monoiodoacetate (MIA) model of degenerative joint disease, a model that exhibits some histopathological similarities to osteoarthritis (OA).
 METHODS Rats were injected intra-articularly with MIA and the development of weight bearing asymmetry was followed for 21 days as compared to vehicle-injected animals. In addition, IPFPs were removed from both ipsilateral and contralateral joints. Both inflammatory infiltrate and histopathological changes were analysed.
 RESULTS MIA injection caused marked weight bearing asymmetry. Ipsilateral IPFP wet weights were significantly increased on days 1 and 3 in MIA-treated animals. MIA treatment also resulted in significant increases in IPFP total white blood cells and monocytes on days 1, 3, and 7 and neutrophils on days 1 and 3. This was supported by histopathological findings at early time points which progressed to adipocyte necrosis, IPFP fibrosis, patellar cartilage and subchondral bone necrosis with synovial hyperplasia at later timepoints.
 CONCLUSIONS The current study clearly demonstrated that marked inflammatory changes in the IPFP occur during the early stage of the MIA model of OA which may contribute to the pain observed at this early stage. The role of the IPFP in later stages of the model needs to be further explored.

title: Postnatal development of adipocyte cellularity in the normal rat.

text: It has been shown by several investigators that adipocyte number is stable in mature human beings and several species of rodents. Although the number of new cells appearing in the adipose depot can be measured histometrically and by Coulter counting of osmium-fixed cells, such methods do not distinguish between "lipid filling" of preexistent adipocytes and synthesis of new adipocytes. The experiments reported here using in vivo injection of [(3)H]thymidine show that synthesis of new adipocytes in the Sprague-Dawley rat continues after birth and ceases before sexual maturity. Furthermore, during the second and third postnatal weeks, a "bed" of preadipocytes is synthesized. Preadipocytes may take as long as 30 days to appear as mature adipocytes.

title: Modification of the Constant-Murley shoulder score-introduction of the individual relative Constant score Individual shoulder assessment.

text: The Constant-Murley shoulder assessment score has proven to be a valuable diagnostic instrument. Thus, in the literature it has been mentioned that the clinical accuracy of this score varies especially when comparing patients in larger, inhomogeneous patient groups. The "relative Constant score" (CS(rel)) tries to minimize these problems by using reference parameters out of healthy age and gender related control groups. The authors of this study tried to show that it is even more accurate to use the functional performance of the uninjured collateral shoulder of the same individual as reference, introducing the "individual relative Constant score" (CS(indiv)). The CS(indiv) and the CS(rel) were compared for 125 consecutive patients with shoulder disorders, and a group of 125 healthy volunteers as a control group. In a non-parametric comparison of the reciever operating characteristics the CS(indiv) shows the higher ability to discriminate between patients and healthy volunteers (p=0.004). This indicates that the individual relative Constant score gives a more accurate view about the functional result for shoulder disorders. It is expected to be more reliable for larger and incoherent patient populations, because specific interindividual differences, regarding the patient's age, gender and constitution are eliminated as well as other individual physiological parameters.

title: A diffusion tensor magnetic resonance imaging study of brain tissue from patients with migraine.

text:
OBJECTIVE To measure in vivo, using diffusion tensor magnetic resonance imaging (DT-MRI) the extent of pathological damage of normal appearing brain tissue (NABT) from patients with migraine.
 METHODS Dual echo and DT-MRI scans of the brain were acquired from 34 patients with migraine and 17 sex and age matched healthy volunteers. Mean diffusivity (MD) and fractional anisotropy (FA) histograms of the NABT were obtained from all subjects and the histograms' peak heights and average NABT MD and FA measured. When present, average MD and FA values of T2 visible lesions were also measured.
 RESULTS In comparison with healthy volunteers, patients with migraine had lower MD histogram peak height (p=0.02) of the NABT. No differences were found in FA histogram derived metrics between migraine patients and healthy subjects. No difference was found for any MD and FA histogram derived metrics between migraine patients with and without brain MRI lesions, and between patients with and without aura.
 CONCLUSIONS This study shows that, although brain damage may extend beyond T2 weighted abnormalities in patients with migraine, the severity of these "occult" changes is mild compared with that found in other diseases associated with white matter abnormality.

title: Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos.

text: Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17+ endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17+ endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.

title: Cytokine production patterns and lymphoproliferative responses in volunteers orally immunized with attenuated vaccine strains of Salmonella typhi.

text: New recombinant strains of attenuated Salmonella typhi used as live oral vaccines elicit potent immune responses. This study examined the patterns of cytokine production and proliferation to specific S. typhi antigens in subjects orally immunized with attenuated S. typhi vaccines CVD 906, CVD 908, and CVD 908 expressing the circumsporozoite protein of Plasmodium falciparum. After immunization, sensitized lymphocytes were found in subjects' blood that exhibited significantly increased proliferative responses and interferon-gamma production to purified S. typhi flagella when compared with preimmunization levels. Significant negative correlations were observed between interleukin-4 production and both interferon-gamma production and proliferation to S. typhi flagella. These results demonstrate that oral immunization with attenuated S. typhi strains alone or with those carrying a foreign gene elicits strong systemic cell-mediated immunity to purified S. typhi antigens, including the production of cytokines compatible with T1-type responses.

title: Human ‘brite / beige’ adipocytes develop from capillary networks and their implantation improves metabolic homeostasis in mice

text: Uncoupling protein 1 (UCP1) is highly expressed in brown adipose tissue, where it generates heat by uncoupling electron transport from ATP production. UCP1 is also found outside classical brown adipose tissue depots, in adipocytes that are termed 'brite' (brown-in-white) or 'beige'. In humans, the presence of brite or beige (brite/beige) adipocytes is correlated with a lean, metabolically healthy phenotype, but whether a causal relationship exists is not clear. Here we report that human brite/beige adipocyte progenitors proliferate in response to pro-angiogenic factors, in association with expanding capillary networks. Adipocytes formed from these progenitors transform in response to adenylate cyclase activation from being UCP1 negative to being UCP1 positive, which is a defining feature of the beige/brite phenotype, while displaying uncoupled respiration. When implanted into normal chow-fed, or into high-fat diet (HFD)-fed, glucose-intolerant NOD-scid IL2rg(null) (NSG) mice, brite/beige adipocytes activated in vitro enhance systemic glucose tolerance. These adipocytes express neuroendocrine and secreted factors, including the pro-protein convertase PCSK1, which is strongly associated with human obesity. Pro-angiogenic conditions therefore drive the proliferation of human beige/brite adipocyte progenitors, and activated beige/brite adipocytes can affect systemic glucose homeostasis, potentially through a neuroendocrine mechanism.

title: Increased lean body mass as an early indicator of olanzapine-induced weight gain in healthy men.

text: One of the primary limitations of many psychiatric medications is weight gain, the mechanism of which remains to be fully elucidated. We conducted a 2-week double-blind placebo-controlled study on weight gain with olanzapine, which is frequently but unpredictably associated with this side effect, to address the possible mechanisms of weight gain independent of changes in the psychiatric condition for which it is prescribed. Healthy male volunteers were randomly assigned to olanzapine (5 mg/day for 7 days, then 10 mg/day for 7 days) or a matching placebo. Of the 24 participants, 19 completed the study (olanzapine, n=13; placebo, n=6). Body weight, glucose, triglyceride, total cholesterol, lipid, leptin, insulin, and aldosterone levels, resting metabolic rate, body composition, physical activity, and 24-h dietary intake were assessed. A significant increase in weight as well as triglyceride, insulin, and leptin levels were found in the olanzapine group as a whole. In participants receiving olanzapine who actually gained weight (n=8), lean but not fat mass increased, as did insulin, fasting glucose, total cholesterol, low-density lipoprotein, and non-high-density lipoprotein levels, whereas aldosterone levels decreased. There were no significant metabolic or endocrine changes in participants receiving placebo or in those receiving olanzapine who did not gain weight. Early metabolic changes appear to be independent of accumulation of fat.


query: Helicobacter pylori-induced aberrant NF-kB-dependent expression of activation-induced cytidine deaminase contributes to the mutagenesis of host DNA.

title: Helicobacter pylori infection triggers aberrant expression of activation-induced cytidine deaminase in gastric epithelium

text: Infection with Helicobacter pylori (H. pylori) is a risk factor for the development of gastric cancer. Here we show that infection of gastric epithelial cells with 'cag' pathogenicity island (cagPAI)-positive H. pylori induced aberrant expression of activation-induced cytidine deaminase (AID), a member of the cytidine-deaminase family that acts as a DNA- and RNA-editing enzyme, via the IκB kinase–dependent nuclear factor-κB activation pathway. H. pylori–mediated upregulation of AID resulted in the accumulation of nucleotide alterations in the TP53 tumor suppressor gene in gastric cells in vitro. Our findings provide evidence that aberrant AID expression caused by H. pylori infection might be a mechanism of mutation accumulation in the gastric mucosa during H. pylori–associated gastric carcinogenesis.

title: The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA

text: High mutation frequency during reverse transcription has a principal role in the genetic variation of primate lentiviral populations. It is the main driving force for the generation of drug resistance and the escape from immune surveillance. G to A hypermutation is one of the characteristics of primate lentiviruses, as well as other retroviruses, during replication in vivo and in cell culture. The molecular mechanisms of this process, however, remain to be clarified. Here, we demonstrate that CEM15 (also known as apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G; APOBEC3G), an endogenous inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is a cytidine deaminase and is able to induce G to A hypermutation in newly synthesized viral DNA. This effect can be counteracted by the HIV-1 virion infectivity factor (Vif). It seems that this viral DNA mutator is a viral defence mechanism in host cells that may induce either lethal hypermutation or instability of the incoming nascent viral reverse transcripts, which could account for the Vif-defective phenotype. Importantly, the accumulation of CEM15-mediated non-lethal hypermutation in the replicating viral genome could potently contribute to the genetic variation of primate lentiviral populations.

title: Chronic Helicobacter pylori infections induce gastric mutations in mice.

text:
BACKGROUND & AIMS Helicobacter pylori is an important etiologic factor in the development of gastric cancer. The aim of this study was to analyze the role of H. pylori infections in the induction of mutagenic events in gastric epithelial cells. The effect of a high-salt diet as a genotoxic risk factor was also investigated.
 METHODS Big Blue transgenic male mice (C57Bl/6) were inoculated with H. pylori (strain SS1) or Helicobacter felis (strain CS1) for 6 and 12 months. The frequency and spectrum of mutations at the stomach level were assessed. Inflammatory host response and inducible nitric oxide synthase (iNOS) expression by reverse-transcription polymerase chain reaction and immunohistochemistry analysis were also performed.
 RESULTS After 6 months, the gastric mutant frequency was 4-fold and 1.7-fold higher in mice infected with H. pylori and H. felis, respectively, than in uninfected mice. It was associated with a high frequency of transversions (AT --> CG and GC --> TA) known to result from oxidative damages. The Helicobacter-infected mice exhibited severe gastritis and a high level of iNOS messenger RNA expression. Hyperplasia developed 12 months after inoculation, and both the mutagenic effects and iNOS expression decreased in H. pylori- and H. felis-infected mice. No synergistic effects of a high-salt diet and Helicobacter infection were observed regarding the frequency of gastric mutation.
 CONCLUSIONS A direct gastric mutagenic effect due to H. pylori infection in the Big Blue transgenic mouse model has been shown 6 months after inoculation. This genotoxicity can be attributable to oxidative DNA damage involving the inflammatory host response.

title: Acetylation of apurinic/apyrimidinic endonuclease-1 regulates Helicobacter pylori-mediated gastric epithelial cell apoptosis.

text:
BACKGROUND & AIMS Helicobacter pylori-induced gastric epithelial cell (GEC) apoptosis is a complex process that includes activation of the tumor suppressor p53. p53-mediated apoptosis involves p53 activation, bax transcription, and cytochrome c release from mitochondria. Apurinic/apyrimidinic endonuclease-1 (APE-1) regulates transcriptional activity of p53, and H pylori induce APE-1 expression in human GECs. H pylori infection increases intracellular calcium ion concentration [Ca2+]i of GECs, which induces APE-1 acetylation. We investigated the effects of H pylori infection and APE-1 acetylation on GEC apoptosis.
 METHODS AGS cells (wild-type or with suppressed APE-1), KATO III cells, and cells isolated from gastric biopsy specimens were infected with H pylori. Effects were examined by immunoblotting, real-time reverse-transcription polymerase chain reaction, immunoprecipitation, immunofluorescence microscopy, chromatin immunoprecipitation, mobility shift, DNA binding, and luciferase assays.
 RESULTS H pylori infection increased [Ca2+]i and acetylation of APE-1 in GECs, but the acetylation status of APE-1 did not affect the transcriptional activity of p53. In GECs, expression of a form of APE-1 that could not be acetylated increased total and mitochondrial levels of Bax and induced release of cytochrome c and fragmentation of DNA; expression of wild-type APE-1 reduced these apoptotic events. We identified a negative calcium response element in the human bax promoter and found that poly (adenosine diphosphate-ribose) polymerase 1 recruited the acetylated APE-1/histone deacetylase-1 repressor complex to bax nCaRE.
 CONCLUSIONS H pylori-mediated acetylation of APE-1 suppresses Bax expression; this prevents p53-mediated apoptosis when H pylori infect GECs.

title: Tolerance rather than immunity protects from Helicobacter pylori-induced gastric preneoplasia.

text:
BACKGROUND & AIMS Chronic infection with the bacterial pathogen Helicobacter pylori causes gastric disorders, ranging from chronic gastritis to gastric adenocarcinoma. Only a subset of infected persons will develop overt disease; most remains asymptomatic despite lifelong colonization. This study aims to elucidate the differential susceptibility to H pylori that is found both across and within populations.
 METHODS We have established a C57BL/6 mouse model of H pylori infection with a strain that is capable of delivering the virulence factor cytotoxin-associated gene A (CagA) into host cells through the activity of a Cag-pathogenicity island-encoded type IV secretion system.
 RESULTS Mice infected at 5-6 weeks of age with CagA(+)H pylori rapidly develop gastritis, gastric atrophy, epithelial hyperplasia, and metaplasia in a type IV secretion system-dependent manner. In contrast, mice infected during the neonatal period with the same strain are protected from preneoplastic lesions. Their protection results from the development of H pylori-specific peripheral immunologic tolerance, which requires transforming growth factor-β signaling and is mediated by long-lived, inducible regulatory T cells, and which controls the local CD4(+) T-cell responses that trigger premalignant transformation. Tolerance to H pylori develops in the neonatal period because of a biased ratio of T-regulatory to T-effector cells and is favored by prolonged low-dose exposure to antigen.
 CONCLUSIONS Using a novel CagA(+)H pylori infection model, we report here that the development of tolerance to H pylori protects from gastric cancer precursor lesions. The age at initial infection may thus account for the differential susceptibility of infected persons to H pylori-associated disease manifestations.

title: Helicobacter pylori AlpA and AlpB bind host laminin and influence gastric inflammation in gerbils.

text: Helicobacter pylori persistently colonizes humans, causing gastritis, ulcers, and gastric cancer. Adherence to the gastric epithelium has been shown to enhance inflammation, yet only a few H. pylori adhesins have been paired with targets in host tissue. The alpAB locus has been reported to encode adhesins involved in adherence to human gastric tissue. We report that abrogation of H. pylori AlpA and AlpB reduces binding of H. pylori to laminin while expression of plasmid-borne alpA or alpB confers laminin-binding ability to Escherichia coli. An H. pylori strain lacking only AlpB is also deficient in laminin binding. Thus, we conclude that both AlpA and AlpB contribute to H. pylori laminin binding. Contrary to expectations, the H. pylori SS1 mutant deficient in AlpA and AlpB causes more severe inflammation than the isogenic wild-type strain in gerbils. Identification of laminin as the target of AlpA and AlpB will facilitate future investigations of host-pathogen interactions occurring during H. pylori infection.

title: Cholesterol glucosylation promotes immune evasion by Helicobacter pylori

text: Helicobacter pylori infection causes gastric pathology such as ulcer and carcinoma. Because H. pylori is auxotrophic for cholesterol, we have explored the assimilation of cholesterol by H. pylori in infection. Here we show that H. pylori follows a cholesterol gradient and extracts the lipid from plasma membranes of epithelial cells for subsequent glucosylation. Excessive cholesterol promotes phagocytosis of H. pylori by antigen-presenting cells, such as macrophages and dendritic cells, and enhances antigen-specific T cell responses. A cholesterol-rich diet during bacterial challenge leads to T cell–dependent reduction of the H. pylori burden in the stomach. Intrinsic α-glucosylation of cholesterol abrogates phagocytosis of H. pylori and subsequent T cell activation. We identify the gene hp0421 as encoding the enzyme cholesterol-α-glucosyltransferase responsible for cholesterol glucosylation. Generation of knockout mutants lacking hp0421 corroborates the importance of cholesteryl glucosides for escaping phagocytosis, T cell activation and bacterial clearance in vivo. Thus, we propose a mechanism regulating the host–pathogen interaction whereby glucosylation of a lipid tips the scales towards immune evasion or response.

title: Bax translocation and mitochondrial fragmentation induced by Helicobacter pylori.

text:
BACKGROUND AND AIMS Previous in vitro and in vivo studies have revealed an association between Helicobacter pylori infection and apoptosis in gastric epithelial cells. Although involvement of the Bcl-2 family of proteins as well as cytochrome c release has been demonstrated in H pylori induced cell death, the exact role of the mitochondria during this type of programmed cell death has not been fully elucidated. Therefore, we sought to determine whether or not Bax translocation and mitochondrial fragmentation occur on exposure of gastric epithelial cells to H pylori, resulting in cell death.
 METHODS Experiments were performed with human gastric adenocarcinoma (AGS) cells, AGS cells transfected with the HPV-E6 gene (which inactivates p53 function), AGS-neo cells (transfected with the backbone construct), mouse embryonic fibroblasts (MEFs), and p19(ARF) null (ARF(-/-)) MEFs. Cells were incubated with a cag positive H pylori strain for up to 24 hours, lysed, and cytoplasmic and mitochondrial membrane fractions were analysed by western blot for Bax translocation.
 RESULTS Bax translocation was detected in AGS, AGS-neo, and normal MEF cells after exposure to H pylori for three hours, but not in ARF(-/-) MEFs cells. Translocation of Bax after H pylori incubation was also detected in AGS-E6 cells (inactive p53 gene) but to a lesser degree than in AGS-neo cells. In parallel studies, the mitochondrial morphology of living cells infected with H pylori was assessed by confocal microscopy. Mitochondrial fragmentation was detectable after 10 hours of H pylori incubation with AGS cells and after seven hours with MEF cells. In wild-type MEFs, mitochondrial fragmentation was significantly increased in comparison with ARF null MEFs (43% v 10.4%, respectively). Furthermore, mitochondrial depolarisation and caspase-3 activity were initiated within four hours in cells incubated with H pylori, and these events were inhibited by forced expression of Bcl-2.
 CONCLUSIONS These data suggest that during H pylori induced apoptosis, Bax translocates to the mitochondria which subsequently undergo depolarisation and profound fragmentation. Functional ARF and p53 proteins may play an important role in H pylori induced mitochondrial modification.

title: Helicobacter pylori induces AGS cell motility and elongation via independent signaling pathways.

text: Helicobacter pylori induces motogenic and cytoskeletal responses in gastric epithelial cells. We demonstrate that these responses can be induced via independent signaling pathways that often occur in parallel. The cag pathogenicity island appears to be nonessential for induction of motility, whereas the elongation phenotype depends on translocation and phosphorylation of CagA.

title: A study of high cell density cultivation process of recombinant Helicobacter pylori multi-epitope vaccine engineering bacteria.

text:
OBJECTIVE To establish high cell density cultivation process of recombinant Helicobacter pylori multi-epitope vaccine engineering bacteria BIB.
 METHODS Based on the results of shake flask fermentation, the process was magnified into volume of a 50 L fermenter to optimize and verify the factors affecting the yield of the target protein, such as the fermentation medium, working seed inoculation amount, inducer concentration, induction starting time, induction duration, inducer adding mode and feeding strategy.
 RESULTS After activated in modified TB medium at 37°C for 8 h, the BIB working seed was inoculated at 5% (v/v) and was induced for expression for another 11 h by the final concentration of 5 mmol/L lactose. In growth phase, glucose at rate of 80 ml/h was used as carbon source, and in induction phase, glycerol at rate of 40 ml/h was used as carbon source; ammonia water was added dropwise to control pH at about 7.0, and revolution speed is adjusted to control the dissolved oxygen at above 30%; ultimately the output of bacterial body was 70 g/L and protein expression amount was about 32%.
 CONCLUSION After high cell density cultivation of the recombinant engineering bacteria, expression and yield of the target protein rBIB significantly increased.

title: Interaction of Helicobacter pylori with gastric epithelial cells is mediated by the p53 protein family.

text:
BACKGROUND & AIMS Although the p53 tumor suppressor has been extensively studied, many critical questions remain unanswered about the biological functions of p53 homologs, p73 and p63. Accumulating evidence suggests that both p73 and p63 play important roles in regulation of apoptosis, cell differentiation, and therapeutic drug sensitivity.
 METHODS Gastric epithelial cells were cocultured with Helicobacter pylori, and the roles of p63 and p73 proteins were assessed by luciferase reporter, real-time polymerase chain reaction, immunoblotting, and cell survival assays. Short hairpin RNA and dominant-negative mutants were used to inhibit activity of p73 and p63 isoforms. Human and murine gastric tissues were analyzed by immunohistochemistry with p73 and p63 antibodies and modified Steiner's silver method.
 RESULTS Interaction of H pylori with gastric epithelial cells leads to robust up-regulation of p73 protein in vitro and in vivo in human gastritis specimens and H pylori-infected mice. The p73 increase resulted in up-regulation of pro-apoptotic genes, NOXA, PUMA, and FAS receptor in gastric epithelial cells. Down-regulation of p73 activity suppressed cell death and Fas receptor induced by H pylori. Bacterial virulence factors within the cag pathogenicity island, c-Abl tyrosine kinase, and interaction with p63 isoforms control the activity of p73.
 CONCLUSION Our findings implicate p73 in H pylori-induced apoptosis and more generally suggest that the p53 family may play a role in the epithelial cell response to H pylori infection.

title: Prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA against Helicobacter pylori infection in a BALB/c mice model

text: Epitope vaccine based on the enzyme urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In our previous study, the epitope vaccine CTB-UA, which was composed of the mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA183–203) from the H. pylori urease A subunit (UreA) was constructed. This particular vaccine was shown to have good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies, which exhibited effectively inhibitory effects on the enzymatic activity of H. pylori urease. In this study, the prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA was evaluated in a BALB/c mice model. The experimental results indicated that oral prophylactic or therapeutic immunization with CTB-UA significantly decreased H. pylori colonization compared with oral immunization with PBS. The results also revealed that the protection was correlated with antigen-specific IgG, IgA, and mucosal secretory IgA antibody responses. CTB-UA may be a promising vaccine candidate for the control of H. pylori infection.

title: A Role for the Vacuolating Cytotoxin, VacA, in Colonization and Helicobacter pylori–Induced Metaplasia in the Stomach

text: Carriage of Helicobacter pylori strains producing more active (s1/i1) forms of VacA is strongly associated with gastric adenocarcinoma. To our knowledge, we are the first to determine effects of different polymorphic forms of VacA on inflammation and metaplasia in the mouse stomach. Bacteria producing the less active s2/i2 form of VacA colonized mice more efficiently than mutants null for VacA or producing more active forms of it, providing the first evidence of a positive role for the minimally active s2/i2 toxin. Strains producing more active toxin forms induced more severe and extensive metaplasia and inflammation in the mouse stomach than strains producing weakly active (s2/i2) toxin. We also examined the association in humans, controlling for cagPAI status. In human gastric biopsy specimens, the vacA i1 allele was strongly associated with precancerous intestinal metaplasia, with almost complete absence of intestinal metaplasia in subjects infected with i2-type strains, even in a vacA s1, cagA(+) background.

title: Increased stress-induced inflammatory responses in male patients with major depression and increased early life stress.

text:
OBJECTIVE The authors sought to determine innate immune system activation following psychosocial stress in patients with major depression and increased early life stress.
 METHOD Plasma interleukin (IL)-6, lymphocyte subsets, and DNA binding of nuclear factor (NF)-kB in peripheral blood mononuclear cells were compared in medically healthy male subjects with current major depression and increased early life stress (N=14) versus nondepressed male comparison subjects (N=14) before and after completion of the Trier Social Stress Test.
 RESULTS Trier Social Stress Test-induced increases in IL-6 and NF-kappaB DNA-binding were greater in major depression patients with increased early life stress and independently correlated with depression severity, but not early life stress. Natural killer (NK) cell percentages also increased following stress. However, there were no differences between groups and no correlation between NK cell percentage and stress-induced NF-kappaB DNA-binding or IL-6.
 CONCLUSIONS Male major depression patients with increased early life stress exhibit enhanced inflammatory responsiveness to psychosocial stress, providing preliminary indication of a link between major depression, early life stress and adverse health outcomes in diseases associated with inflammation.

title: Therapeutic efficacy of a multi-epitope vaccine against Helicobacter pylori infection in BALB/c mice model.

text: Epitope vaccine is a promising option for therapeutic vaccination against Helicobacter pylori (H. pylori) infection. In this study, we constructed a multi-epitope vaccine with five epitopes and mucosal adjuvant E. coli heat-labile enterotoxin B subunit (LTB) named HUepi-LTB and evaluated its therapeutic effect against H. pylori infection in BALB/c mice model. HUepi-LTB containing three Th epitopes from UreB and two B cell epitopes from UreB and HpaA was constructed and expressed in E. coli. Oral therapeutic immunization with HUepi-LTB significantly decreased H. pylori colonization compared with oral immunization with PBS, and the protection was correlated with antigen-specific CD4+ T cells and IgG and mucosal IgA antibody responses. This multi-epitope vaccine may be a promising vaccine candidate that may help to control H. pylori infection.

title: The myeloid transcription factor KLF2 regulates the host response to polymicrobial infection and endotoxic shock.

text: Precise control of myeloid cell activation is required for optimal host defense. However, this activation process must be under exquisite control to prevent uncontrolled inflammation. Herein, we identify the Kruppel-like transcription factor 2 (KLF2) as a potent regulator of myeloid cell activation in vivo. Exposure of myeloid cells to hypoxia and/or bacterial products reduced KLF2 expression while inducing hypoxia inducible factor-1α (HIF-1α), findings that were recapitulated in human septic patients. Myeloid KLF2 was found to be a potent inhibitor of nuclear factor-kappaB (NF-κB)-dependent HIF-1α transcription and, consequently, a critical determinant of outcome in models of polymicrobial infection and endotoxemia. Collectively, these observations identify KLF2 as a tonic repressor of myeloid cell activation in vivo and an essential regulator of the innate immune system.

title: Inhibition of mammalian target of rapamycin potentiates thrombin-induced intercellular adhesion molecule-1 expression by accelerating and stabilizing NF-kappa B activation in endothelial cells.

text: We addressed the regulatory function of mammalian target of rapamycin (mTOR) in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. Pretreatment of HUVECs with rapamycin, an inhibitor of mTOR, augmented thrombin-induced ICAM-1 expression. Inhibition of mTOR by this approach promoted whereas over-expression of mTOR inhibited thrombin-induced transcriptional activity of NF-kappaB, an essential regulator of ICAM-1 transcription. Analysis of the NF-kappaB signaling pathway revealed that inhibition of mTOR potentiated IkappaB kinase activation resulting in a rapid and persistent phosphorylation of IkappaBalpha on Ser32 and Ser36, a requirement for IkappaBalpha degradation. Consistent with these data, we observed a more efficient and stable nuclear localization of RelA/p65 and, subsequently, the DNA binding activity of NF-kappaB by thrombin following mTOR inhibition. These data define a novel role of mTOR in down-regulating thrombin-induced ICAM-1 expression in endothelial cells by controlling a delayed and transient activation of NF-kappaB.

title: Activator protein-1 (AP-1) DNA binding activity is induced by hydroxyurea in organogenesis stage mouse embryos

text: Hydroxyurea is a potent teratogen; free radical scavengers or antioxidants reduce its teratogenicity. Activator Protein-1 (AP-1) and NF-kappaB are redox-sensitive transcription factors with important roles in normal development and the stress response. This study was designed to determine if exposure to teratogenic doses of hydroxyurea induces oxidative stress and alters gene expression by activating these transcription factors. Pregnant mice were treated with saline or hydroxyurea (400, 500, or 600 mg/kg) on gestation day 9 (GD 9) and killed either on GD 9, 0.5, 3, or 6 h after treatment, to assess oxidative stress and transcription factor activities, or on GD 18, to assess fetal development. Exposure to 400 mg/kg hydroxyurea did not affect the progeny, whereas exposure to 500 or 600 mg/kg resulted in dose-dependent increases in fetal resorptions and malformations, including curly tails, abnormal limbs (oligodactyly, hemimelia, and amelia), and short ribs. Hydroxyurea did not induce oxidative stress, as assessed by the ratio of oxidized to reduced glutathione, nor did it alter NF-kappaB DNA binding activity in the GD 9 conceptus. In contrast, exposure to hydroxyurea at any dose increased AP-1 DNA binding activity in embryos and yolk sacs 0.5 or 3 h after treatment. Hydroxyurea-induced c-Fos heterodimer activity in the embryo peaked 3-4-fold above control at 3 h and remained elevated by 6 h; in contrast, the activity of c-Jun dimers was not altered by drug exposure. A dramatic and region-specific increase in c-Fos immunoreactivity was found in hydroxyurea-treated embryos. The induction of AP-1 DNA binding activity by hydroxyurea represents an early, sensitive marker of the embryonic response to insult.

title: Association between infection with Helicobacter pylori and risk of gastric cancer: evidence from a prospective investigation.

text:
OBJECTIVE To investigate the association between gastric cancer and prior infection with Helicobacter pylori.
 DESIGN Case-control comparison of prevalence of IgG antibodies to H pylori in blood samples collected prospectively, before diagnosis of gastric cancer in the cases. Presence of H pylori antibody (greater than 10 micrograms IgG/ml) determined by enzyme linked immunosorbent assay (ELISA).    SUBJECTS 29 men with a subsequent diagnosis of gastric cancer and 116 aged matched controls selected from over 22,000 middle aged men participating in two ongoing cohort studies (the British United Provident Association study and the Caerphilly collaborative heart disease study), who had provided blood samples during 1975-1982.
 RESULTS 20 of the 29 cases (69%) and 54 of the 116 controls (47%) were positive for H pylori specific antibody. The median specific IgG concentration was significantly higher in the cases than controls (90 micrograms/ml v 3.6 micrograms/ml, p less than 0.01). The estimated odds ratio for the risk of gastric cancer in those with a history of infection with H pylori was 2.77 (95% confidence interval 1.04 to 7.97, 2p = 0.039).
 CONCLUSIONS H pylori infection may be an important cause of gastric cancer; between 35% and 55% of all cases may be associated with such an infection.

title: Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori

text: Half the world's population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease produces NH(3) and CO(2), neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.


query: Helminths interfere with immune system control of coinfections by microbial pathogens 16.

title: Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by engaging the IL-4 receptor pathway

text: Tuberculosis and helminthic infections coexist in many parts of the world, yet the impact of helminth-elicited Th2 responses on the ability of the host to control Mycobacterium tuberculosis (Mtb) infection has not been fully explored. We show that mice infected with the intestinal helminth Nippostrongylus brasiliensis (Nb) exhibit a transitory impairment of resistance to airborne Mtb infection. Furthermore, a second dose of Nb infection substantially increases the bacterial burden in the lungs of co-infected mice. Interestingly, the Th2 response in the co-infected animals did not impair the onset and development of the protective Mtb-specific Th1 cellular immune responses. However, the helminth-induced Th2 environment resulted in the accumulation of alternatively activated macrophages (AAMs) in the lung. Co-infected mice lacking interleukin (IL) 4Rα exhibited improved ability to control Mtb infection, which was accompanied by significantly reduced accumulation of AAMs. Moreover, IL-4Rα(-/-) mice adoptively transferred with wild-type macrophages had a significantly higher Mtb load in their lungs compared with those that received IL-4Rα(-/-) macrophages, suggesting a direct contribution for the IL-4R pathway to the heightened susceptibility of co-infected animals. The Th2 response can thus enhance the intracellular persistence of Mtb, in part by mediating the alternative activation of macrophages via the IL-4Rα signaling pathway.

title: Candida albicans-Staphylococcus aureus polymicrobial peritonitis modulates host innate immunity.

text: Despite advances in medical device fabrication and antimicrobial treatment therapies, fungal-bacterial polymicrobial peritonitis remains a serious complication for surgery patients, those on peritoneal dialysis, and the critically ill. Using a murine model of peritonitis, we have demonstrated that monomicrobial infection with Candida albicans or Staphylococcus aureus is nonlethal. However, coinfection with these same doses leads to a 40% mortality rate and increased microbial burden in the spleen and kidney by day 1 postinfection. Using a multiplex enzyme-linked immunosorbent assay, we have also identified a unique subset of innate proinflammatory cytokines (interleukin-6, granulocyte colony-stimulating factor, keratinocyte chemoattractant, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α) that are significantly increased during polymicrobial versus monomicrobial peritonitis, leading to increased inflammatory infiltrate into the peritoneum and target organs. Treatment of coinfected mice with the cyclooxygenase (COX) inhibitor indomethacin reduces the infectious burden, proinflammatory cytokine production, and inflammatory infiltrate while simultaneously preventing any mortality. Further experiments demonstrated that the immunomodulatory eicosanoid prostaglandin E2 (PGE2) is synergistically increased during coinfection compared to monomicrobial infection; indomethacin treatment also decreased elevated PGE2 levels. Furthermore, addition of exogenous PGE2 into the peritoneal cavity during infection overrode the protection provided by indomethacin and restored the increased mortality and microbial burden. Importantly, these studies highlight the ability of fungal-bacterial coinfection to modulate innate inflammatory events with devastating consequences to the host.

title: Neutrophil extracellular chromatin traps connect innate immune response to autoimmunity

text: Autoantibodies to DNA and histones (chromatin) are the defining antigen specificity in systemic lupus erythematosus (SLE) and related musculoskeletal disorders but the mechanisms responsible for their induction remain mysterious. That situation rapidly changed once neutrophil extracellular chromatin traps (NETs) were discovered and observed to play a conserved role in innate immune responses to a broad variety of microbial pathogens. At the center of an infectious process, neutrophils exert various antimicrobial defenses, including the release of nuclear chromatin into the extracellular space. The externalized NETs, a complex meshwork of nuclear chromatin and antimicrobial proteins, serve to immobilize and degrade microbial pathogens. Here, we critically evaluate the evidence supporting NETs versus apoptotic bodies as a source for nuclear antigens in autoimmunity. We also discuss the possibility that NET chromatin forms an essential component of immune deposits in the pathogenesis of glomerulonephritis in SLE and other autoimmune immune complex diseases.

title: Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus.

text: Infection of Sulfolobus islandicus REY15A with mixtures of different Sulfolobus viruses, including STSV2, did not induce spacer acquisition by the host CRISPR immune system. However, coinfection with the tailed fusiform viruses SMV1 and STSV2 generated hyperactive spacer acquisition in both CRISPR loci, exclusively from STSV2, with the resultant loss of STSV2 but not SMV1. SMV1 was shown to activate adaptation while itself being resistant to CRISPR-mediated adaptation and DNA interference. Exceptionally, a single clone S-1 isolated from an SMV1 + STSV2-infected culture, that carried STSV2-specific spacers and had lost STSV2 but not SMV1, acquired spacers from SMV1. This effect was also reproducible on reinfecting wild-type host cells with a variant SMV1 isolated from the S-1 culture. The SMV1 variant lacked a virion protein ORF114 that was shown to bind DNA. This study also provided evidence for: (i) limits on the maximum sizes of CRISPR loci; (ii) spacer uptake strongly retarding growth of infected cultures; (iii) protospacer selection being essentially random and non-directional, and (iv) the reversible uptake of spacers from STSV2 and SMV1. A hypothesis is presented to explain the interactive conflicts between SMV1 and the host CRISPR immune system.

title: Th2-mediated host protective immunity to intestinal nematode infections.

text: Despite many years of study, relatively little is known about the effector mechanisms that operate against intestine-dwelling nematodes. Most of the current understanding comes from studies of laboratory model systems in rodents. It is clear that when an intestinal helminth infection takes place the immune system generates a strong Th2-mediated response, which regulates a variety of responses characteristic of helminth infections such as eosinophilia, intestinal mastocytosis and elevated IgE production. The ability to modulate the host's immune response in vivo with cytokine-specific monoclonal antibodies and recombinant cytokines, together with the use of animals with disruption of key genes involved in the immune response, have provided powerful tools with which to dissect the potential effector mechanisms operating. In the absence of a T-cell compartment the host is unable to expel the parasite. If a Th1-dominated response is generated, protective immunity is almost universally compromised. Thus, it it would appear that some aspect of a Th2-mediated response controls effector mechanisms. Although it is clear that for some infections the mast cell appears to be involved in protection, probably through the generation of a non-specific inflammatory response, how these cells become activated remains unclear. Data from infections in transgenic animals suggest that activation is not through the high-affinity receptor for IgE. Such studies also call into doubt the importance of conventional interactions between effector leucocytes and antibody. There is little evidence to support a protective role for eosinophilia in any system. New data also imply that, although interleukin 4 (IL-4) is generally important (and can exert effects independent of an adaptive immune response), it is not always sufficient to mediate protection; other Th2 cytokines (e.g. IL-13) may warrant closer investigation. It is apparent that a number of potential Th2-controlled effector mechanisms (some of which may be particularly important at mucosal surfaces) remain to be explored. Overall, it is likely that worm expulsion is the result of a combination of multiple mechanisms, some of which are more critical to some species of parasite than to others.

title: Infection and apoptosis as a combined inflammatory trigger.

text: While inflammatory phagocytosis of microbial pathogens and non-inflammatory phagocytosis of apoptotic cells have each been studied extensively, the consequences of innate immune recognition of host cells undergoing apoptosis as a direct result of infection are unclear. In this situation, the innate immune system is confronted with mixed signals, those from apoptotic cells and those from the infecting pathogen. Nuclear receptor activation has been implicated downstream of apoptotic cell recognition while Toll-like receptors are the prototypical inflammatory receptors engaged during infection. When the two signals combine, a new set of events takes place beginning with transrepression of a subset of inflammatory-response genes and ending with the induction of a T helper-17 adaptive immune response. This response is best suited for clearing the infecting pathogen and repairing the damage that occurred to the host tissue during infection.

title: Current knowledge and future directions of TLR and NOD signaling in sepsis

text: The incidence of sepsis is increasing over time, along with an increased risk of dying from the condition. Sepsis care costs billions annually in the United States. Death from sepsis is understood to be a complex process, driven by a lack of normal immune homeostatic functions and excessive production of proinflammatory cytokines, which leads to multi-organ failure. The Toll-like receptor (TLR) family, one of whose members was initially discovered in Drosophila, performs an important role in the recognition of microbial pathogens. These pattern recognition receptors (PRRs), upon sensing invading microorganisms, activate intracellular signal transduction pathways. NOD signaling is also involved in the recognition of bacteria and acts synergistically with the TLR family in initiating an efficient immune response for the eradication of invading microbial pathogens. TLRs and NOD1/NOD2 respond to different pathogen-associated molecular patterns (PAMPs). Modulation of both TLR and NOD signaling is an area of research that has prompted much excitement and debate as a therapeutic strategy in the management of sepsis. Molecules targeting TLR and NOD signaling pathways exist but regrettably thus far none have proven efficacy from clinical trials.

title: Beyond pattern recognition: five immune checkpoints for scaling the microbial threat

text: Pattern recognition by the innate immune system enables the detection of microorganisms, but how the level of microbial threat is evaluated — a process that is crucial for eliciting measured antimicrobial responses with minimal inflammatory tissue damage — is less well understood. New evidence has shown that features of microbial viability can be detected by the immune system and thereby induce robust responses that are not warranted for dead microorganisms. Here, we propose five immune checkpoints that, as defined here, collectively determine the gravity of microbial encounters.

title: Critical role of CD28 in protective immunity against Salmonella typhimurium.

text: Efficient T cell activation requires both TCR signals and costimulatory signals. CD28 is one of the molecules that provide costimulatory signals for T cells. We used mice deficient in CD28 expression (CD28-/- mice) to analyze the role of CD28 in the immune response against the intracellular bacterium Salmonella typhimurium, the causative agent of murine typhoid fever. CD28-/- mice were highly susceptible to infection with wild-type S. typhimurium and even failed to control infection with attenuated aroA- S. typhimurium. More detailed analysis revealed that CD28-/- animals did not mount a T-dependent Ab response and were highly impaired in the production of IFN-gamma. Thus, CD28 cosignaling is crucial for immunity against S. typhimurium. To our knowledge, this is the first report describing an essential role for CD28 in protective immunity against an intracellular microbial pathogen.

title: Effects of treatment for intestinal helminth infection on growth and cognitive performance in children: systematic review of randomised trials.

text:
OBJECTIVE To summarise the effects of anthelmintic drug treatment on growth and cognitive performance in children.
 DATA SOURCES Electronic databases: Cochrane Infectious Diseases Group controlled trial register, Cochrane controlled trials register, Embase, and Medline. Citations of all identified trials. Contact with the World Health Organization and field researchers.    REVIEW METHODS Systematic review of randomised controlled trials in children aged 1-16 that compared anthelmintic treatment with placebo or no treatment. Assessment of validity and data abstraction conducted independently by two reviewers.
 MAIN OUTCOME MEASURES Growth and cognitive performance.
 RESULTS Thirty randomised controlled trials in more than 15 000 children were identified. Effects on mean weight were unremarkable, and heterogeneity was evident in the results. There were some positive effects on mean weight change in the trials reporting this outcome: after a single dose (any anthelmintic) the pooled estimates were 0.24 kg (95% confidence interval 0.15 kg to 0. 32 kg; fixed effects model assumed) and 0.38 kg (0.01 kg to 0.77 kg; random effects model assumed). Results from trials of multiple doses showed mean weight change in up to one year of follow up of 0.10 kg (0.04 kg to 0.17 kg; fixed effects) or 0.15 kg (0.00 to 0.30; random effects). At more than one year of follow up, mean weight change was 0.12 kg (-0.02 kg to 0.26 kg; fixed effects) and 0.43 (-0.61 to 1. 47; random effects). Results from studies of cognitive performance were inconclusive.
 CONCLUSIONS There is some limited evidence that routine treatment of children in areas where helminths are common has effects on weight gain, but this is not consistent between trials. There is insufficient evidence as to whether this intervention improves cognitive performance.

title: The N-end rule pathway regulates pathogen responses in plants.

text: To efficiently counteract pathogens, plants rely on a complex set of immune responses that are tightly regulated to allow the timely activation, appropriate duration and adequate amplitude of defense programs. The coordination of the plant immune response is known to require the activity of the ubiquitin/proteasome system, which controls the stability of proteins in eukaryotes. Here, we demonstrate that the N-end rule pathway, a subset of the ubiquitin/proteasome system, regulates the defense against a wide range of bacterial and fungal pathogens in the model plant Arabidopsis thaliana. We show that this pathway positively regulates the biosynthesis of plant-defense metabolites such as glucosinolates, as well as the biosynthesis and response to the phytohormone jasmonic acid, which plays a key role in plant immunity. Our results also suggest that the arginylation branch of the N-end rule pathway regulates the timing and amplitude of the defense program against the model pathogen Pseudomonas syringae AvrRpm1.

title: Hygiene hypothesis in inflammatory bowel disease: a critical review of the literature.

text: The hygiene hypothesis is thought to be a significant contributor to the growing incidence of inflammatory bowel disease (IBD) around the world, although the evidence for specific factors that underlie the hygiene hypothesis in IBD is unclear. We aimed to systematically review the literature to determine which hygiene-related factors are associated with the development of IBD. Publications identified from a broad based MEDLINE and Current Contents search between 1966 and 2007 on key terms relevant to the 'hygiene hypothesis' and IBD including H pylori exposure, helminths, cold chain hypothesis, measles infection and vaccination, antibiotic use, breastfeeding, family size, sibship, urban upbringing, day care attendance and domestic hygiene were reviewed. The literature suggests that the hygiene hypothesis and its association with decreased microbial exposure in childhood probably plays an important role in the development of IBD, although the strength of the supporting data for each of the factors varies considerably. The most promising factors that may potentially be associated with development of IBD include H pylori exposure, helminths, breastfeeding and sibship. However, the vast majority of studies in this area are plagued by serious methodological shortcomings, particularly the reliance on retrospective recall of information making it difficult to truly ascertain the importance of a 'hygiene hypothesis' in IBD. The 'hygiene hypothesis' in IBD is an important area of research that may give clues to the aetiology of this disease. Directions for future research are recommended.

title: Role of the Parasite-Derived Prostaglandin D2 in the Inhibition of Epidermal Langerhans Cell Migration during Schistosomiasis Infection

text: Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-α, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-α–triggered migration of LCs through the adenylate cyclase–coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.

title: Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease.

text: Crohn's disease and ulcerative colitis, the two main types of chronic inflammatory bowel disease, are multifactorial conditions of unknown aetiology. A susceptibility locus for Crohn's disease has been mapped to chromosome 16. Here we have used a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping, to identify three independent associations for Crohn's disease: a frameshift variant and two missense variants of NOD2, encoding a member of the Apaf-1/Ced-4 superfamily of apoptosis regulators that is expressed in monocytes. These NOD2 variants alter the structure of either the leucine-rich repeat domain of the protein or the adjacent region. NOD2 activates nuclear factor NF-kB; this activating function is regulated by the carboxy-terminal leucine-rich repeat domain, which has an inhibitory role and also acts as an intracellular receptor for components of microbial pathogens. These observations suggest that the NOD2 gene product confers susceptibility to Crohn's disease by altering the recognition of these components and/or by over-activating NF-kB in monocytes, thus documenting a molecular model for the pathogenic mechanism of Crohn's disease that can now be further investigated.

title: The composition of the gut microbiota throughout life, with an emphasis on early life

text: The intestinal microbiota has become a relevant aspect of human health. Microbial colonization runs in parallel with immune system maturation and plays a role in intestinal physiology and regulation. Increasing evidence on early microbial contact suggest that human intestinal microbiota is seeded before birth. Maternal microbiota forms the first microbial inoculum, and from birth, the microbial diversity increases and converges toward an adult-like microbiota by the end of the first 3-5 years of life. Perinatal factors such as mode of delivery, diet, genetics, and intestinal mucin glycosylation all contribute to influence microbial colonization. Once established, the composition of the gut microbiota is relatively stable throughout adult life, but can be altered as a result of bacterial infections, antibiotic treatment, lifestyle, surgical, and a long-term change in diet. Shifts in this complex microbial system have been reported to increase the risk of disease. Therefore, an adequate establishment of microbiota and its maintenance throughout life would reduce the risk of disease in early and late life. This review discusses recent studies on the early colonization and factors influencing this process which impact on health.

title: Foxp3(+) T cells regulate immunoglobulin a selection and facilitate diversification of bacterial species responsible for immune homeostasis.

text: Foxp3(+) T cells play a critical role for the maintenance of immune tolerance. Here we show that in mice, Foxp3(+) T cells contributed to diversification of gut microbiota, particularly of species belonging to Firmicutes. The control of indigenous bacteria by Foxp3(+) T cells involved regulatory functions both outside and inside germinal centers (GCs), consisting of suppression of inflammation and regulation of immunoglobulin A (IgA) selection in Peyer's patches, respectively. Diversified and selected IgAs contributed to maintenance of diversified and balanced microbiota, which in turn facilitated the expansion of Foxp3(+) T cells, induction of GCs, and IgA responses in the gut through a symbiotic regulatory loop. Thus, the adaptive immune system, through cellular and molecular components that are required for immune tolerance and through the diversification as well as selection of antibody repertoire, mediates host-microbial symbiosis by controlling the richness and balance of bacterial communities required for homeostasis.

title: Viruses and microRNAs

text: The discovery of RNA interference and cellular microRNAs (miRNAs) has not only affected how biological research is conducted but also revealed an entirely new level of post-transcriptional gene regulation. Here, I discuss the potential functions of the virally encoded miRNAs recently identified in several pathogenic human viruses and propose that cellular miRNAs may have had a substantial effect on viral evolution and may continue to influence the in vivo tissue tropism of viruses. Our increasing knowledge of the role and importance of virally encoded miRNAs will probably offer new insights into how viruses that establish latent infections, such as herpesviruses, avoid elimination by the host innate or adaptive immune system. Research into viral miRNA function might also suggest new approaches for treating some virally induced diseases.

title: Memory CD4+ T cells induce innate responses independently of pathogen

text: Inflammation induced by recognition of pathogen-associated molecular patterns markedly affects subsequent adaptive responses. We asked whether the adaptive immune system can also affect the character and magnitude of innate inflammatory responses. We found that the response of memory, but not naive, CD4+ T cells enhances production of multiple innate inflammatory cytokines and chemokines (IICs) in the lung and that, during influenza infection, this leads to early control of virus. Memory CD4+ T cell–induced IICs and viral control require cognate antigen recognition and are optimal when memory cells are either T helper type 1 (TH1) or TH17 polarized but are independent of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production and do not require activation of conserved pathogen recognition pathways. This represents a previously undescribed mechanism by which memory CD4+ T cells induce an early innate response that enhances immune protection against pathogens.

title: CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA

text: Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.

title: Loss of allergen 1 confers a hypervirulent phenotype that resembles mucoid switch variants of Cryptococcus neoformans.

text: Microbial survival in a host is usually dependent on the ability of a pathogen to undergo changes that promote escape from host defense mechanisms. The human-pathogenic fungus Cryptococcus neoformans undergoes phenotypic switching in vivo that promotes persistence in tissue. By microarray and real-time PCR analyses, the allergen 1 gene (ALL1) was found to be downregulated in the hypervirulent mucoid switch variant, both during logarithmic growth and during intracellular growth in macrophages. The ALL1 gene encodes a small cytoplasmic protein that is involved in capsule formation. Growth of an all1Delta gene deletion mutant was normal. Similar to cells of the mucoid switch variant, all1Delta cells produced a larger polysaccharide capsule than cells of the smooth parent and the complemented strain produced, and the enlarged capsule inhibited macrophage phagocytosis. The mutant exhibited a modest defect in capsule induction compared to all of the other variants. In animal models the phenotype of the all1Delta mutant mimicked the hypervirulent phenotype of the mucoid switch variant, which is characterized by decreased host survival and elevated intracranial pressure. Decreased survival is likely the result of both an ineffective cell-mediated immune response and impaired phagocytosis by macrophages. Consequently, we concluded that, unlike loss of most virulence-associated genes, where loss of gene function results in attenuated virulence, loss of the ALL1 gene enhances virulence by altering the host-pathogen interaction and thereby impairing clearance. Our data identified the first cryptococcal gene associated with elevated intracranial pressure and support the hypothesis that an environmental opportunistic pathogen has modified its virulence in vivo by epigenetic downregulation of gene function.