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In this study, we adapt a Cox-based model for recurrent events; the Prentice, Williams and Peterson Total -Time (PWP-TT) that has largely, been used under the assumption of non-informative censoring and evaluate it under an informative censoring setting. Empirical evaluation was undertaken with the aid of the semi-parametric framework for recurrent events suggested by Huang [1] and implemented in R Studio software. For validation we used data from a typical HIV care setting in Kenya. Of the three models under consideration; the standard Cox Model had gender hazard ratio (HR) of 0.66 (p-value=0.165), Andersen-Gill had HR 0.46 (with borderline p-value=0.054) and extended PWP TT had HR 0.22 (p-value=0.006). The PWP-TT model performed better as compared to other models under informative setting. In terms of risk factors under informative setting, LTFU due to stigma; gender [base=Male] had HR 0.544 (p-value =0.002), age [base is < 37] had HR 0.772 (p-value=0.008), ART regimen [base= First line] had HR 0.518 (p-value= 0.233) and differentiated care model (Base=not on DCM) had HR 0.77(p-value=0.036). In conclusion, in spite of the multiple interventions designed to address incidences of LTFU among HIV patients, within-person cases of LTFU are usually common and recurrent in nature, with the present likelihood of a person getting LTFU influenced by previous occurrences and therefore informative censoring should be checked.

Summary Background In giant cell arteritis, temporal artery biopsies often show vasculitis with giant cell formation, but optimal biopsy length for diagnosis is debated. We reviewed temporal artery biopsies from a 10-year period in the province of Alberta, Canada, to identify an ideal biopsy length in the diagnostic process for giant cell arteritis. Methods We retrospectively reviewed electronic medical records of patients who had undergone a temporal artery biopsy procedure in Alberta between Jan 1, 2008, and Jan 1, 2018, as reported in the Data Integration and Management Repository of Alberta Health Services. We extracted data on baseline demographic characteristics (sex and age), inflammatory markers (erythrocyte sedimentation rate [ESR] and C-reactive protein [CRP]), temporal artery biopsy characteristics (side of biopsy and postfixation length), and final pathological diagnoses. All positive biopsies were reviewed by a single pathologist to ensure uniformity of pathological interpretation, with subsequent discordant results removed from analysis. Predictors of positive pathological diagnosis of giant cell arteritis were modeled by logistic regression, and the Akaike information criterion was used to compare logistic regression models with varying biopsy length cutoffs (0·5, 1·0, 1·5, 2·0, and 2·5 cm) to determine a change point for diagnostic sensitivity in postfixation length. Findings We extracted data on 1203 temporal artery biopsies; after removal of 13 discordant biopsies, 1190 biopsies from 1163 patients were reviewed. The mean age of patients was 72·0 years (SD 10·3) and 799 (68·7%) patients were women. 222 (18·7%) temporal artery biopsies were positive for giant cell arteritis. In univariable analysis, increases in age (71·3 years [SD 10·6] in negative biopsies vs 75·3 years [8·3] in positive biopsies; odds ratio [OR] 1·04 [95% CI 1·02–1·06]; p Interpretation Accounting for postfixation shrinkage, our findings suggest a 1·5–2·0 cm prefixation length as the optimal biopsy length to diagnose patients with giant cell arteritis, with greater lengths unlikely to provide significant additional diagnostic yield to justify risks associated with surgery. Funding None.

Purpose:Circulating tumor DNA (ctDNA) has been suggested as a major prognostic factor in resected stage-III colon cancer. We analyzed ctDNA of patients randomized in the phase III IDEA-France trial.Experimental Design:ctDNA was tested for WIF1 and NPY by droplet digital PCR with method developed and validated for colorectal cancer. Disease-free survival (DFS) and overall survival (OS) were analyzed via multivariable analysis in patients with ctDNA samples and in sub-groups according to treatment duration (3/6 months) and disease stage (high/low-risk stage III).Results:Of 2,010 randomized patients, 1,345 had available ctDNA samples (1,017 collected both post-surgery and pre-chemotherapy). More Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 (78% versus 69%) and T4 and/or N2 (40% versus 36%) were observed in patients studied (n = 1017) versus not analyzed (n = 993). There were 877 ctDNA-negative (86.2%) and 140 ctDNA-positive (13.8%) patients; their baseline characteristics were similar. With a median follow-up of 6.6 years, the 3-year DFS rate was 66.39% for ctDNA-positive patients and 76.71% for ctDNA-negative patients (P = 0.015). ctDNA was confirmed as an independent prognostic marker for DFS (adjusted HR = 1.55, 95% CI 1.13–2.12, P = 0.006) and OS (HR = 1.65, 95% CI 1.12–2.43, P = 0.011). ctDNA was prognostic in patients treated for 3 months and with T4 and/or N2 tumors, but not in those treated for 6 months and with T1–3/N1 tumors.Conclusions:In this first ctDNA assessment of a large series of patients with stage III colon cancer enrolled in phase III trial, post-surgery ctDNA was found in 13.8% of them and was confirmed as an independent prognostic marker.See related commentary by Bent and Kopetz, p. 5449

In this study, we investigate the effects of missing data when estimating HIV/TB co-infection. We revisit the concept of missing data and examine three available approaches for dealing with missingness. The main objective is to identify the best method for correcting missing data in TB/HIV Co-infection setting. We employ both empirical data analysis and extensive simulation study to examine the effects of missing data, the accuracy, sensitivity, specificity and train and test error for different approaches. The novelty of this work hinges on the use of modern statistical learning algorithm when treating missingness. In the empirical analysis, both HIV data and TB-HIV co-infection data imputations were performed, and the missing values were imputed using different approaches. In the simulation study, sets of 0% (Complete case), 10%, 30%, 50% and 80% of the data were drawn randomly and replaced with missing values. Results show complete cases only had a co-infection rate (95% Confidence Interval band) of 29% (25%, 33%), weighted method 27% (23%, 31%), likelihood-based approach 26% (24%, 28%) and multiple imputation approach 21% (20%, 22%). In conclusion, MI remains the best approach for dealing with missing data and failure to apply it, results to overestimation of HIV/TB co-infection rate by 8%.

Background The drive for muscularity behaviors are very common in male athletes, especially in male bodybuilders. Studies have related drive for muscularity behaviors to body dissatisfaction, eating disorders and muscle dysmorphia. Methods This study applied the trans-contextual model of motivation to the drive for muscularity behaviors of male bodybuilders at risk of developing muscle dysmorphia. The relationships between self-determination theory constructs and drive for muscularity behaviors, via the theory of planned behavior variables (i.e., attitude, subjective norm, perceived behavioral control, and intention) were examined. A total of 175 Swiss male bodybuilders (Mage = 27.34; SDage = 7.53) completed measures on motivation for sport, theory of planned behavior variables, and drive for muscularity behaviors. They practiced bodybuilding from three to 24 h per week (Mhours per week = 6.59; SDhours per week = 3.45) and had done so for 7.19 years on average (SDnumber of years = 6.91). Using bootstrapped maximum likelihood estimation with the AMOS 7.0 program, a series of confirmatory factor analyses was performed on each subscale and a series of path analyses was performed to determine the final model. Results The fit indices of the final model were satisfactory: χ2 (11) = 13.81; p = .244; TLI = .98; CFI = .99; RMSEA = .04. The model explained 29% of the variance of drive for muscularity behaviors. The final path analysis supported the motivational sequence, with autonomous motivation for sport showing a positive, significant and indirect association with the drive for muscularity behaviors via perceived behavioral control and intention to gain muscle mass, and controlled motivation for sport showing a positive association with the drive for muscularity behaviors both directly and via attitude and intention to gain muscle mass. Conclusions It was concluded that the trans-contextual model of motivation applies only partially to the drive for muscularity behaviors in male bodybuilders. Perspective The motivational mechanisms explaining the development of drive for muscularity behaviors might be better understood through complementary analyses of motivational profiles. Such investigations would guide the design of programs to lower the risks associated with these behaviors.

Anti-PD-1/PD-L1 blockade can restore tumour-specific T-cell immunity and is an emerging therapy in non-small-cell lung cancer (NSCLC). We investigated the correlation between 18F-FDG PET/CT-based markers and tumour tissue expression of PD-L1, necrosis and clinical outcome in patients receiving checkpoint inhibitor treatment. PD-Li expression in biopsy or resection specimens from 49 patients with confirmed NSCLC was investigated by immunohistochemistry. Maximum standardized uptake value (SUVmax), mean SUV (SUVmean), metabolic tumour volume (MTV) and total lesion glycolysis (TLG) were obtained from 18F-FDG PET/CT images. The ratio of metabolic to morphological lesion volumes (MMVR) and its association with PD-L1 expression in each lesion were calculated. The associations between histologically reported necrosis and 18F-FDG PET imaging patterns and radiological outcome (evaluated by iRECIST) following anti-PD-1/PD-L1 therapy were also analysed. In 14 patients, the association between necrosis and MMVR and tumour immune contexture were analysed by multiple immunofluorescent (IF) staining for CD8, PD-1, granzyme B (GrzB) and NFATC2. In total, 25 adenocarcinomas and 24 squamous cell carcinomas were analysed. All tumours showed metabolic 18F-FDG PET uptake. MMVR was correlated inversely with PD-L1 expression in tumour cells. Furthermore, PD-L1 expression and low MMVR were significantly correlated with clinical benefit. Necrosis was correlated negatively with MMVR. Multiplex IF staining showed a greater frequency of activated CD8+ cells in necrotic tumours than in nonnecrotic tumours in both stromal and epithelial tumour compartments. This study introduces MMVR as a new imaging biomarker and its ability to noninvasively capture increased PD-L1 tumour expression and predict clinical benefit from checkpoint blockade in NSCLC should be further evaluated.

El Sindrome de Takotsubo es una disfuncion ventricular aguda reversible en ausencia de obstruccion coronaria. Una mujer de 85 anos de edad con antecedentes de reemplazo valvular aortico transcateter, ingreso por dos semanas de dolor severo por una cadera desplazada por osteosintesis fallida. Mientras se programaba para cirugia, se documento hiponatremia severa secundaria a secrecion inapropiada de hormona antidiuretica. Subitamente desarrollo edema agudo pulmonar. El ecocardiograma confirmo una valvula protesica funcional y aquinesia medial y apical de las paredes del ventriculo izquierdo. Recibio tratamiento con ventilacion mecanica no invasiva, restriccion de liquidos y diureticos. La hiponatremia y la cardiomiopatia resolvieron. Takotsubo syndrome is a form of acute reversible left ventricular dysfunction in the absence of coronary obstruction. An 85-year-old lady with a medical history of transcatheter aortic valve replacement was readmitted complaining of 2 weeks of severe pain by a displaced hip and failed osteosynthesis. While she was scheduled for hip surgery, severe hyponatremia secondary to inappropriate antidiuretic hormone secretion was documented, and sudden-onset pulmonary edema ensued. Echocardiography confirmed normally functioning aortic prosthetic valve and classical features of Takotsubo. She was treated with non-invasive mechanical ventilation, water restriction, and diuretics. Hyponatremia and the cardiomyopathy resolved and the patient recovered completely.

Long-chain fatty acids (LCFAs) are a tremendous source of metabolic energy, an essential component of membranes, and important effector molecules that regulate a myriad of cellular processes. As an energy-rich nutrient source, the role of LCFAs in promoting bacterial survival and infectivity is well appreciated. LCFA degradation generates a large number of reduced cofactors that may confer redox stress; therefore, it is imperative to understand how bacteria deal with this paradoxical situation. Although the LCFA utilization pathway has been studied in great detail, especially in Escherichia coli, where the earliest studies date back to the 1960s, the interconnection of LCFA degradation with bacterial stress responses remained largely unexplored. Recent work in E. coli shows that LCFA degradation induces oxidative stress and also impedes oxidative protein folding. Importantly, both issues arise due to the insufficiency of ubiquinone, a lipid-soluble electron carrier in the electron transport chain. However, to maintain redox homeostasis, bacteria induce sophisticated cellular responses. Here, we review these findings in light of our current knowledge of the LCFA metabolic pathway, metabolism-induced oxidative stress, the process of oxidative protein folding, and stress combat mechanisms. We discuss probable mechanisms for the activation of defense players during LCFA metabolism and the likely feedback imparted by them. We suggest that besides defending against intrinsic stresses, LCFA-mediated upregulation of stress response pathways primes bacteria to adapt to harsh external environments. Collectively, the interplay between LCFA metabolism and stress responses is likely an important factor that underlies the success of LCFA-utilizing bacteria in the host.

Kidney transplantation has become the preferred treatment option in end-stage chronic renal failure as it provides significant improvements over dialysis in terms of both quality and duration of life. Even after several randomized studies conducted in the last 20 years, the combination of CNI, MMF, and steroids continues to be considered the gold standard for kidney transplantation. However, novel molecules aimed at minimizing renal and cardiovascular toxicity, particularly with CNI sparing, are being identified. The present review assesses various such molecules available currently and briefly discusses the existing combination strategies and novel perspectives for the redesigning of protocols based on our novel therapeutic arsenal.

Several GntR/FadR transcriptional regulators govern sugar acid metabolism in bacteria. Although effectors have been identified for a few sugar acid regulators, the mode of effector binding is unknown. Even in the overall FadR subfamily, there are limited details on effector-regulator interactions. Here, we identified the effector-binding cavity in Escherichia coli DgoR, a FadR subfamily transcriptional repressor of D-galactonate metabolism that employs D-galactonate as its effector. Using a genetic screen, we isolated several dgoR superrepressor alleles. Blind docking suggested eight amino acids corresponding to these alleles to form a part of the effector-binding cavity. In vivo and in vitro assays showed that these mutations compromise the inducibility of DgoR without affecting its oligomeric status or affinity for target DNA. Taking Bacillus subtilis GntR as a representative, we demonstrated that the effector-binding cavity is similar among FadR subfamily sugar acid regulators. Finally, a comparison of sugar acid regulators with other FadR members suggested conserved features of effector-regulator recognition within the FadR subfamily. Sugar acid metabolism is widely implicated in bacterial colonization and virulence. The present study sets the basis to investigate the influence of natural genetic variations in FadR subfamily regulators on their sensitivity to sugar acids and ultimately on host-bacterial interactions.

Background and Aims IgG4 disease is a systemic fibroinflammatory disorder that may affect virtually any organ. IgG4-related kidney disease (IgG4-RKD) is a major manifestation, occurring in almost one third of patient. The present study addresses the diagnosis, management and outcome of French patients with IgG4-RKD. Method We conducted a retrospective observational study including patients with renal impairment due to IgG4-RKD from 32 centers. Clinical, biological, and imaging data, as well as management modalities and outcome, were retrieved from medical records. Results 74 patients, 64 males and 10 females, were diagnosed with IgG4-RKD between 1997 and 2019 and were included in the present study. The mean age at diagnosis was 65.2 ± 15-year-old. Renal involvement was present at diagnosis in 63% of cases. Nine (12%) patients had isolated renal involvement, whereas extra-renal involvement included retroperitoneal fibrosis (20%), auto-immune pancreatitis (49%), cholangitis (27%), and lung disease (22%). Renal imaging lesions were observed in 48 (64%) patients, and included renal hypertrophy, patchy lesions and pseudo-tumor solitary renal lesion. PET-CT scan was performed in 47 (65%) patients and showed hypermetabolic renal lesion(s) in 36% of cases. The presence of extra-renal hypermetabolic lesions was observed in 35 cases (74%). 43 %, 25% and 14% of patients presented with AKI, AKI-on-CKD, and isolated CKD, respectively. The mean serum creatinine level at diagnosis was 244 ± 140 µmol/L, corresponding to an eGFR of 69 ± 22ml/min/1.73m. Urinary sediment was most often bland. Mean urine protein-to-creatinine ratio was 1.27 g/g. of note, 26% of patients had more than 1 g/g. 82% of patients underwent a kidney biopsy showing tubulointerstitial involvement in all cases, 12 (16%) had additional glomerular involvement, mainly membranous nephropathy. The major additional laboratory abnormalities included polyclonal hypergammaglobulinemia, and increased serum IgG4 increase in 83% and 86%, respectively. Complement levels were decreased in 27 (37%) patients. Two patients were lost for follow-up and 69 patients were treated with corticosteroid therapy in 66 (89%) patients and 10 received Rituximab as first line therapy. The mean duration of follow-up was 28.8 ± 30 months. During follow-up, 22 (30%) patients relapsed, while 45 (62%) patients had persistent CKD at last follow-up, with a mean eGFR of 47±27 ml/min/1.73m. Seven (9%) patients progressed to ESRD and 7 died. A significant response was achieved in 46 (62%) patients. Patients who received steroids higher than 0.5mg/kg/day had lower serum IgG4 levels (1.8 g/l versus 5.7 g/l, p=0.007) at last follow up but there was no significant difference observed in terms of renal function between the two groups (45.5 vs 48.8 ml/min/1.73m, p= 0.72). By univariate analysis, predictors of relapse were low C3 or C4 serum level (p=0.01), ANCA positivity (p= 0.02), renal radiological lesions (p=0.05). At last follow-up the eGFR was worse in patients with a serum creatinine diagnosis greater than 300 µmol/L (30.25 vs 57 ml/min/1.73m p=0.0001) and more progressed to ESRD (p=0.03). Conclusion IgG4-RKD has been recently described and may affect middle-aged males. The disease presents as tubulointerstitial nephritis with glomerular involvement in 16% of cases. Disease response to corticosteroid therapy is favorable but is characterized by a high rate of relapses and a significant risk of persistent CKD in the majority of patient. Risk of ESRD and death in almost 20% of patients.

The envelope of gram-negative bacteria serves as the first line of defense against environmental insults. Therefore, its integrity is continuously monitored and maintained by several envelope stress response (ESR) systems. Due to its oxidizing environment, the envelope represents an important site for disulfide bond formation. In Escherichia coli, the periplasmic oxidoreductase, DsbA introduces disulfide bonds in substrate proteins and transfers electrons to the inner membrane oxidoreductase, DsbB. Under aerobic conditions, the reduced form of DsbB is re-oxidized by ubiquinone, an electron carrier in the electron transport chain (ETC). Given the critical role of ubiquinone in transferring electrons derived from the oxidation of reduced cofactors, we were intrigued whether metabolic conditions that generate a large number of reduced cofactors render ubiquinone unavailable for disulfide bond formation. To test this, here we investigated the influence of metabolism of long-chain fatty acid (LCFA), an energy-rich carbon source, on the redox state of the envelope. We show that LCFA degradation increases electron flow in the ETC. Further, whereas cells metabolizing LCFAs exhibit characteristics of insufficient disulfide bond formation, these hallmarks are averted in cells exogenously provided with ubiquinone. Importantly, the ESR pathways, Cpx and σE, are activated by envelope signals generated during LCFA metabolism. Our results argue that Cpx is the primary ESR that senses and maintains envelope redox homeostasis. Amongst the two ESRs, Cpx is induced to a greater extent by LCFAs and senses redox-dependent signal. Further, ubiquinone accumulation during LCFA metabolism is prevented in cells lacking Cpx response, suggesting that Cpx activation helps maintain redox homeostasis by increasing the oxidizing power for disulfide bond formation. Taken together, our results demonstrate an intricate relationship between cellular metabolism and disulfide bond formation dictated by ETC and ESR, and provide the basis for examining whether similar mechanisms control envelope redox status in other gram-negative bacteria., Author summary Disulfide bonds contribute to the folding and stability of many extracytoplasmic proteins in all domains of life. In gram-negative bacteria, including Escherichia coli, disulfide bond formation occurs in the oxidizing environment of the periplasmic space enclosed within the outer and inner membrane layers of the envelope. Because disulfide-bonded proteins are involved in diverse biological processes, bacteria must monitor the envelope redox status and elicit an appropriate response when perturbations occur; however, these mechanisms are not well elucidated. Here, we demonstrated that the metabolism of an energy-rich carbon source, long-chain fatty acid (LCFA) hampers disulfide bond formation in E. coli. An envelope stress response (ESR) system, Cpx, senses this redox imbalance and increases the oxidizing power for disulfide bond formation. LCFA metabolism, disulfide bond formation, and ESR systems have independently been implicated in the pathogenesis of several gram-negative bacteria. The present study sets the basis to explore whether LCFA metabolism impacts the virulence of these bacteria by influencing the redox status of their envelope and activation of ESR pathways.

The removal of ciprofloxacin (CIPRO) and cinoxacin (CINO) antibiotics from wastewater was investigated using V2O5-ZnO coated carbon nanofibers (V2O5-ZnO@CNF) as an adsorbent. The V2O5-ZnO@CNF nanocomposite was characterized using characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transforms infrared spectroscopy (FTIR), scanning electron microscopy/energy dispersion x-ray spectroscopy (SEM/EDX), and UV–vis spectrophotometry. The characterization results revealed that the V2O5-ZnO nanoparticles were incorporated on the surface of carbon nanofibers. The effects of different experimental parameters such as adsorbent amounts, contact time, initial pH, that affect the removal process were optimized using response surface methodology based on Box-Behnken design. The optimum conditions were found to be 018 g, 6.5 and 20 min for the adsorbent amount, sample pH and contact time, respectively. Under optimized conditions, the maximum adsorption capacity was found to be 71.4 and 87.7 mg g−1 for cinoxacin and ciprofloxacin, respectively. In addition, the adsorption isotherms and kinetics were used to investigate the adsorption mechanism. The data followed the order: Langmuir (R2 = 0.09942–0.9996) > Dubinin-Radushkevich (R2 = 0.9257–0.9557 > Temkin (R2 = 0.79991–0.9132),> Flory-Huggins (R2 = 0.7432–0.8813) > Freundlich (R2 = 0.7057–0.7808). Furthermore, the data was better described by pseudo- second-order kinetics, this implied that the adsorption process was dominated by chemisorption process as the rate-limiting mechanism through sharing or exchange of electrons between adsorbent and CIPRO or CINO.

Paper presented at the 4th Strathmore International Mathematics Conference (SIMC 2017), 19 - 23 June 2017, Strathmore University, Nairobi, Kenya. Melanoma of the skin is the 5th and seventh most commonly diagnosed carcinoma in men and women, respectively, in the USA. So far, gene signatures prognostic for overall and distant metastasis-free survival, for example, have been promising in the identification of therapeutic targets for primary and metastatic melanoma. But most of these gene signatures have been selected using statistics that depend entirely on the parametric distributions of the data (e.g. t -statistics). In this study, we assessed the impact of relaxing the parametric assumptions on the power of the models used for gene selection. We developed a semi-parametric model for feature selection that does not depend on the distributions of the covariates. This copula based model only assumed that the marginal distributions of the covariates are continuous. Simulations indicated that the copula-based model had reasonable power at various levels of the false discovery rate (FDR). These results were validated in a publicly available melanoma dataset. Relaxing parametric assumptions on microarray data may yield procedures that have good power for differential gene expression analysis.

A cross-sectional study was conducted with 227 adults, 162 using antiretroviral therapy (ART), both sexes, in Secondary Immunodeficiency Outpatient Clinic of the Department of Dermatology of the Hospital das Clínicas of the Faculty of Medicine of University of São Paulo. The patients were grouped into 92 under ART and self-reported lipodystrophy (G1); 70 under ART and without self-reported lipodystrophy (G2); 65 without ART (G3). We evaluated: (1) self-reported lipodystrophy, self-perception and feeling about body image; (2) Anthropometric and lipemic profile. We included 67% (

Background The dual challenge of low diagnostic sensitivity of microscopy test and technical challenge of performing a TB culture test poses a problem for case detection and initiation of Tuberculosis (TB) second-line treatment. There is thus need for a rapid, reliable and easily accessible assay. This comparative analysis was performed to assess diagnostic performance characteristics of GeneXpert MTB/RIF and Line Probe Assay (LPA). Methods Three hundred twenty nine sputum samples of patients across the 47 counties in Kenya suspected to have drug resistant TB were picked and subjected to GeneXpert, LPA and Culture MGIT at the National TB Reference Laboratory. Sensitivity, specificity and predictive values were then determined to assess the performance characteristics of the various assays. Results Against culture MGIT as the gold standard for TB diagnosis, GeneXpert had a sensitivity, specificity, positive predictive value, and negative predictive value of 78.5, 64.9, 59.4 and 82.2% respectively while LPA had 98.4, 66.0, 65.4 and 98.4%. For diagnosis of rifampicin mono-resistance GeneXpert had a moderate agreement (Kappa 0.59, P p Conclusion LPA has a better performance characteristic to GeneXpert and an alternative to culture with regards to detection of RIF’s mono-resistance.

d-Galactonate, an aldonic sugar acid, is used as a carbon source by Escherichia coli, and the structural dgo genes involved in its metabolism have previously been investigated. Here, using genetic, biochemical and bioinformatics approaches, we present the first detailed molecular and functional insights into the regulation of d-galactonate metabolism in E. coli K-12 by the transcriptional regulator DgoR. We found that dgoR deletion accelerates the growth of E. coli in d-galactonate concomitant with the strong constitutive expression of dgo genes. In the dgo locus, sequence upstream of dgoR alone harbors the d-galactonate-inducible promoter that likely drives the expression of all dgo genes. DgoR exerts repression on the dgo operon by binding two inverted repeats overlapping the dgo promoter. Binding of d-galactonate induces a conformational change in DgoR to derepress the dgo operon. The findings from our work firmly place DgoR in the GntR family of transcriptional regulators: DgoR binds an operator sequence [5′-TTGTA(G/C)TACA(A/T)-3′] matching the signature of GntR family members that recognize inverted repeats [5′-(N)yGT(N)xAC(N)y-3′, where x and y indicate the number of nucleotides, which varies], and it shares critical protein-DNA contacts. We also identified features in DgoR that are otherwise less conserved in the GntR family. Recently, missense mutations in dgoR were recovered in a natural E. coli isolate adapted to the mammalian gut. Our results show these mutants to be DNA binding defective, emphasizing that mutations in the dgo-regulatory elements are selected in the host to allow simultaneous induction of dgo genes. The present study sets the basis to explore the regulation of dgo genes in additional enterobacterial strains where they have been implicated in host-bacterium interactions. IMPORTANCEd-Galactonate is a widely prevalent aldonic sugar acid. Despite the proposed significance of the d-galactonate metabolic pathway in the interaction of enteric bacteria with their hosts, there are no details on its regulation even in Escherichia coli, which has been known to utilize d-galactonate since the 1970s. Here, using multiple methodologies, we identified the promoter, operator, and effector of DgoR, the transcriptional repressor of d-galactonate metabolism in E. coli. We establish DgoR as a GntR family transcriptional regulator. Recently, a human urinary tract isolate of E. coli introduced in the mouse gut was found to accumulate missense mutations in dgoR. Our results show these mutants to be DNA binding defective, hence emphasizing the role of the d-galactonate metabolic pathway in bacterial colonization of the mammalian gut.

In spite of adjuvant chemotherapy, a significant fraction of patients with localized breast cancer (BC) relapse after optimal treatment. We determined the occurrence of cytoplasmic MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3B)-positive puncta, as well as the presence of nuclear HMGB1 (high mobility group box 1) in cancer cells within surgical BC specimens by immunohistochemistry, first in a test cohort (152 patients) and then in a validation cohort of localized BC patients who all received adjuvant anthracycline-based chemotherapy (1646 patients). Cytoplasmic LC3B(+) puncta inversely correlated with the intensity of SQSTM1 staining, suggesting that a high percentage cells of LC3B(+) puncta reflects increased autophagic flux. After setting optimal thresholds in the test cohort, cytoplasmic LC3B(+) puncta and nuclear HMGB1 were scored as positive in 27.2% and 28.6% of the tumors, respectively, in the validation cohort, while 8.7% were considered as double positive. LC3B(+) puncta or HMGB1 expression alone did not constitute independent prognostic factors for metastasis-free survival (MFS) in multivariate analyses. However, the combined positivity for LC3B(+) puncta and nuclear HMGB1 constituted an independent prognostic factor significantly associated with prolonged MFS (hazard ratio: 0.49 95% confidence interval [0.26-0.89]; P = 0.02), and improved breast cancer specific survival (hazard ratio: 0.21 95% confidence interval [0.05-0.85]; P = 0.029). Subgroup analyses revealed that within patients with poor-prognosis BC, HMGB1(+) LC3B(+) double-positive tumors had a better prognosis than BC that lacked one or both of these markers. Altogether, these results suggest that the combined positivity for LC3B(+) puncta and nuclear HMGB1 is a positive predictor for longer BC survival.

Ever accumulating evidence indicates that the long-term effects of radiotherapy and chemotherapy largely depend on the induction (or restoration) of an anticancer immune response. Here, we investigated this paradigm in the context of esophageal carcinomas treated by neo-adjuvant radiochemotherapy, in a cohort encompassing 196 patients. We found that the density of the FOXP3+ regulatory T cell (Treg) infiltrate present in the residual tumor (or its scar) correlated with the pathological response (the less Tregs the more pronounced was the histological response) and predicted cancer-specific survival. In contrast, there was no significant clinical impact of the frequency of CD8+ cytotoxic T cells. At difference with breast or colorectal cancer, a loss-of-function allele of toll like receptor 4 (TLR4) improved cancer-specific survival of patients with esophageal cancer. While a loss-of-function allele of purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7) failed to affect cancer-specific survival, its presence did correlate with an increase in Treg infiltration. Altogether, these results corroborate the notion that the immunosurveillance seals the fate of patients with esophageal carcinomas treated with conventional radiochemotherapy.

BACKGROUND & AIMS: Phosphatidylethanolamine N-methyltransferase (PEMT), a liver enriched enzyme, is responsible for approximately one third of hepatic phosphatidylcholine biosynthesis. When fed a high-fat diet (HFD), Pemt(-/-) mice are protected from HF-induced obesity; however, they develop steatohepatitis. The vagus nerve relays signals between liver and brain that regulate peripheral adiposity and pancreas function. Here we explore a possible role of the hepatic branch of the vagus nerve in the development of diet induced obesity and steatohepatitis in Pemt(-/-) mice.METHODS: 8-week old Pemt(-/-) and Pemt(+/+) mice were subjected to hepatic vagotomy (HV) or capsaicin treatment, which selectively disrupts afferent nerves, and were compared to sham-operated or vehicle-treatment, respectively. After surgery, mice were fed a HFD for 10weeks.RESULTS: HV abolished the protection against the HFD-induced obesity and glucose intolerance in Pemt(-/-) mice. HV normalized phospholipid content and prevented steatohepatitis in Pemt(-/-) mice. Moreover, HV increased the hepatic anti-inflammatory cytokine interleukin-10, reduced chemokine monocyte chemotactic protein-1 and the ER stress marker C/EBP homologous protein. Furthermore, HV normalized the expression of mitochondrial electron transport chain proteins and of proteins involved in fatty acid synthesis, acetyl-CoA carboxylase and fatty acid synthase in Pemt(-/-) mice. However, disruption of the hepatic afferent vagus nerve by capsaicin failed to reverse either the protection against the HFD-induced obesity or the development of HF-induced steatohepatitis in Pemt(-/-) mice.CONCLUSIONS: Neuronal signals via the hepatic vagus nerve contribute to the development of steatohepatitis and protection against obesity in HFD fed Pemt(-/-) mice.

The immune system avoids oncogenesis and slows down tumor progression through a mechanism called immunosurveillance. Nevertheless, some malignant cells manage to escape from immune control and form clinically detectable tumors. Tetraploidy, which consists in the intrinsically unstable duplication of the genome, is considered as a (pre)-cancerous event that can result in aneuploidy and contribute to oncogenesis. We previously described the fact that tetraploid cells can be eliminated by the immune system. Here, we investigate the role of different innate and acquired immune effectors by inoculating hyperploid cancer cells into wild type or mice bearing different immunodeficient genotypes (Cd1d(−/−), FcRn(−/−), Flt3l(−/−), Foxn1(nu/nu), MyD88(−/−), Nlrp3(−)(/)(−), Ighm(tm1Cgn), Rag2(−/−)), followed by the monitoring of tumor incidence, growth and final ploidy status. Our results suggest that multiple different immune effectors including B, NK, NKT and T cells, as well as innate immune responses involving the interleukine-1 receptor and the Toll-like receptor systems participate to the immunoselection against hyperploid cells. Hence, optimal anticancer immunosurveillance likely involves the contribution of multiple arms of the immune system.

Decrease in bone mineral density (BMD) has been a complication among people living with HIV/AIDS. To investigate the prevalence of osteopenia/osteoporosis among HIV-infected people living in São Paulo city, we studied 108 HIV-infected patients (79 men and 29 women). We extracted data from patients’ medical records and BMD was measured by dual-energy X-ray absorptiometry (DXA). Median age of participants was 42 years (interquartile range [IQR] 36-48 years), and the median time since HIV diagnosis was 4.01 years (IQR 2-11 years). Patients had acquired HIV primarily by the sexual route (men who have sex with men 44%, heterosexual 49%). Median age, duration of HIV infection, duration of ART and CD4 nadir were similar for men and women. Plasma viral load was undetectable for 53 patients (49%). Median CD4 T cell count was 399 cells/µL (IQR 247 - 568). Twenty five patients (23%) had LBMD, and there was no statistically significant difference between men and women (

cis-Diamminedichloroplatinum(II) (CDDP), which is mostly referred to as cisplatin, is a widely used antineoplastic. The efficacy of cisplatin can be improved by combining it with the vitamin B6 precursor pyridoxine. Here, we evaluated the putative synergistic interaction of CDDP with pyridoxine in the treatment of an orthotopic mouse model of non-small-cell lung cancer (NSCLC). CDDP and pyridoxine exhibited hyperadditive therapeutic effects. However, this synergy was only observed in the context of an intact immune system and disappeared when the otherwise successful drug combination was applied to the same NSCLC cancer implanted in the lungs of athymic mice (which lack T lymphocytes). Immunocompetent mice that had been cured from NSCLC by the combined regimen of CDDP plus pyridoxine became resistant against subcutaneous rechallenge with the same (but not with an unrelated) cancer cell line. In vitro, CDDP and pyridoxine did not only cause synergistic killing of NSCLC cells but also elicited signs of immunogenic cell death including an endoplasmic reticulum stress response and exposure of calreticulin at the surface of the NSCLC cells. NSCLC cells treated with CDDP plus pyridoxine in vitro elicited a protective anticancer immune response upon their injection into immunocompetent mice. Altogether, these results suggest that the combined regimen of cisplatin plus pyridoxine mediates immune-dependent antineoplastic effects against NSCLC.

International audience; Although chemically non-reactive, inert noble gases may influence multiple physiological and pathological processes via hitherto uncharacterized physical effects. Here we report a cell-based detection system for assessing the effects of pre-defined gas mixtures on the induction of apoptotic cell death. In this setting, the conventional atmosphere for cell culture was substituted with gas combinations, including the same amount of oxygen (20%) and carbon dioxide (5%) but 75% helium, neon, argon, krypton, or xenon instead of nitrogen. The replacement of nitrogen with noble gases per se had no effects on the viability of cultured human osteosarcoma cells in vitro. Conversely, argon and xenon (but not helium, neon, and krypton) significantly limited cell loss induced by the broad-spectrum tyrosine kinase inhibitor staurosporine, the DNA-damaging agent mitoxantrone and several mitochondrial toxins. Such cytoprotective effects were coupled to the maintenance of mitochondrial integrity, as demonstrated by means of a mitochondrial transmembrane potential-sensitive dye and by assessing the release of cytochrome c into the cytosol. In line with this notion, argon and xenon inhibited the apoptotic activation of caspase-3, as determined by immunofluorescence microscopy coupled to automated image analysis. The antiapoptotic activity of argon and xenon may explain their clinically relevant cytoprotective effects.

International audience; Despite effective targeted therapy acting on KIT and PDGFRA tyrosine kinases, gastrointestinal stromal tumors (GIST) escape treatment by acquiring mutations conveying resistance to imatinib mesylate (IM). Following the identification of NKp30-based immunosurveillance of GIST and the off-target effects of IM on NK cell functions, we investigated the predictive value of NKp30 isoforms and NKp30 soluble ligands in blood for the clinical response to IM. The relative expression and the proportions of NKp30 isoforms markedly impacted both event-free and overall survival, in two independent cohorts of metastatic GIST. Phenotypes based on disbalanced NKp30B/NKp30C ratio (Delta BClow) and low expression levels of NKp30A were identified in one third of patients with dismal prognosis across molecular subtypes. This Delta BClow blood phenotype was associated with a pro-inflammatory and immunosuppressive tumor microenvironment. In addition, detectable levels of the NKp30 ligand sB7-H6 predicted a worse prognosis in metastatic GIST. Soluble BAG6, an alternate ligand for NKp30 was associated with low NKp30 transcription and had additional predictive value in GIST patients with high NKp30 expression. Such GIST microenvironments could be rescued by therapy based on rIFN-alpha and anti-TRAIL mAb which reinstated innate immunity.

Identifying sexually transmitted infections (STI) in HIV-infected individuals has potential to benefit individual and public health. There are few guidelines regarding routine STI screening in sub-Saharan African HIV programs. We determined sexual risk behavior and prevalence and correlates of STI in a national survey of large HIV treatment programs in Kenya.A mobile screening team visited 39 (95%) of the 42 largest HIV care programs in Kenya and enrolled participants using population-proportionate systematic sampling. Participants provided behavioral and clinical data. Genital and blood specimens were tested for trichomoniasis, gonorrhea, chlamydia, syphilis, and CD4 T-lymphocyte counts.Among 1661 adults, 41% reported no sexual partners in the past 3 months. Among those who reported sex in the past 3 months, 63% of women reported condom use during this encounter compared with 77% of men (P0.001). Trichomoniasis was the most common STI in women (10.9%) and men (2.8%); prevalences of gonorrhea, chlamydia, and syphilis were low (1%-2%). Among women, younger age (adjusted odds ratio [OR], 0.96 per year; 95% confidence interval [CI], 0.94-0.98) and primary school education or lower level (adjusted OR, 2.16; 95% CI, 1.37-3.40) were independently associated with trichomoniasis, whereas CD4 count, cotrimoxazole use, and reported condom use were not. Reported condom use at last sex was associated with reporting that the clinic provided condoms among both women (OR, 1.7; 95% CI, 1.17-2.35) and men (OR, 2.4; 95% CI, 1.18-4.82).Women attending Kenyan HIV care programs had a 10.9% prevalence of trichomoniasis, suggesting that screening for this infection may be useful. Condom provision at clinics may enhance secondary HIV prevention efforts.

Cancer immunosurveillance relies on effector/memory tumor-infiltrating CD8+ T cells with a T-helper cell 1 (TH1) profile. Evidence for a natural killer (NK) cell-based control of human malignancies is still largely missing. The KIT tyrosine kinase inhibitor imatinib mesylate markedly prolongs the survival of patients with gastrointestinal stromal tumors (GIST) by direct effects on tumor cells as well as by indirect immunostimulatory effects on T and NK cells. Here, we investigated the prognostic value of tumor-infiltrating lymphocytes (TIL) expressing CD3, Foxp3, or NKp46 (NCR1) in a cohort of patients with localized GIST. We found that CD3+ TIL were highly activated in GIST and were especially enriched in areas of the tumor that conserve class I MHC expression despite imatinib mesylate treatment. High densities of CD3+ TIL predicted progression-free survival (PFS) in multivariate analyses. Moreover, GIST were infiltrated by a homogeneous subset of cytokine-secreting CD56bright (NCAM1) NK cells that accumulated in tumor foci after imatinib mesylate treatment. The density of the NK infiltrate independently predicted PFS and added prognostic information to the Miettinen score, as well as to the KIT mutational status. NK and T lymphocytes preferentially distributed to distinct areas of tumor sections and probably contributed independently to GIST immunosurveillance. These findings encourage the prospective validation of immune biomarkers for optimal risk stratification of patients with GIST. Cancer Res; 73(12); 3499–510. ©2013 AACR.

International audience; The immune system is routinely confronted with cell death resulting from the physiological turnover of renewable tissues, as well as from pathological insults of several types. We hypothesize the existence of a mechanism that allows the immune system to discriminate between physiological and pathological instances of cell death, but the factors that determine whether cellular demise is perceived as a neutral, tolerogenic or immunogenic event remain unclear 1. Infectious insults are accompanied by so-called microbe-associated molecular patterns (MAMPs), i.e., viral or bacterial products that activate immune cells through a panel of pattern-recognition receptors (PRRs) 2. Moreover, intracellular pathogens generally trigger adaptive mechanisms aimed toward the re-establishment of homeosta-sis, including the unfolded protein response (UPR) and autophagy 3,4. In mammals, MAMPs coupled to the activation of stress responses Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell-mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN- and IFN- receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN-related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy. npg

Autophagy is an evolutionarily conserved catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Alterations in autophagic signaling are found in several human diseases including cancer. Here, we describe a validated immunohistochemical protocol for the detection of LC3 puncta in human formalin-fixed, paraffin-embedded cancer specimens that can also be applied to mouse tissues. In response to systemic chemotherapy, autophagy-competent mouse tumors exhibited LC3 puncta, which did not appear in mouse cancers that had been rendered autophagy-deficient by the knockdown of Atg5 or Atg7. As compared with normal tissues, LC3 staining was moderately to highly elevated in the large majority of human cancers studied, albeit tumors of the same histological type tended to be highly heterogeneous in the number and intensity of LC3 puncta per cell. Moreover, tumor-infiltrating immune cells often were highly positive for LC3. Altogether, this protocol for LC3 staining appears suitable for the specific detection of LC3 puncta in human specimens, including tissue microarrays. We surmise that this technique can be employed for retrospective or prospective studies involving large series of human tumor samples.

A major predictor of failed liver resection and transplantation is nonalcoholic fatty liver disease (NAFLD). NAFLD is linked to a wide spectrum of diseases including obesity and diabetes that are increasingly prevalent in Western populations. Thus, it is important to develop therapies aimed at improving posthepatectomy outcomes in patients with NAFLD, as well as to improve the evaluation of patients slated for hepatic surgery. Decreased hepatic phosphatidylcholine (PC) content and decreased ratio of hepatic PC to phosphatidylethanolamine (PE) have previously been linked to NAFLD. To determine if decreased hepatic PC/PE could predict survival after hepatectomy, we used mouse models lacking key enzymes in PC biosynthesis, namely, phosphatidylethanolamine N-methyltransferase and hepatic-specific CTP:phosphocholine cytidylyltransferase α. These mice were fed a high-fat diet to induce NAFLD. We then performed a 70% partial hepatectomy and monitored postoperative survival. We identified hepatic PC/PE to be inversely correlated with the development of steatosis and inflammation in the progression of NAFLD. Decreased hepatic PC/PE before surgery was also strongly associated with decreased rates of survival after partial hepatectomy. Choline supplementation to the diet increased hepatic PC/PE in Pemt−/− mice with NAFLD, decreased inflammation, and increased the survival rate after partial hepatectomy. Conclusion: Decreased hepatic PC/PE is a predictor of NAFLD and survival following partial hepatectomy. Choline supplementation may serve as a potential therapy to prevent the progression of NAFLD and to improve postoperative outcome after liver surgery. (HEPATOLOGY 2012)

Renal injury is one of the severe and common complications that occurs early in neonates with asphyxia, and reactive oxygen species have been implicated to play an important role on its pathogenesis. Improved renal recovery has been shown previously with N-acetyl-l-cysteine (NAC) in various acute kidney injuries. Using a subacute swine model of neonatal hypoxia-reoxygenation (H/R), we examined whether NAC can sustain its beneficial effect on renal recovery for 48 h. Newborn piglets were randomly assigned into a sham-operated group (without H/R, n = 6) and two H/R experimental groups (n = 8 each) with 2 h normocapnic alveolar hypoxia and 1 h 100% oxygen of reoxygenation followed by 21% oxygen for 47 h. Five minutes after reoxygenation, piglets received either normal saline (H/R control) or NAC (150-mg/kg bolus and 20 mg/kg per hour i.v. for 24 h) in a blinded, randomized fashion. All piglets were acidotic and in cardiogenic shock after hypoxia. Treating the piglets with NAC significantly increased both renal blood flow and oxygen delivery throughout the reoxygenation period. N-acetyl-l-cysteine treatment also improved the renal function with the attenuation of elevated urinary N-acetyl-β-d-glucosaminidase activity and plasma creatinine concentration observed in H/R controls (both P < 0.05). The tissue levels of lipid hydroperoxides and caspase 3 in the kidney of NAC-treated animals were significantly lower than those of H/R controls. Conclusively, postresuscitation administration of NAC elicits a prolonged beneficial effect in improving renal functional recovery and reducing oxidative stress in newborn piglets with H/R insults for 48 h.

Protracted diarrhea is used to describe infants with loose and frequent stools of sufficient severity to require nutritional support, most commonly parenteral nutrition. Despite similar clinical presentations, the causes of protracted diarrhea in infants are varied and diverse in management and prognosis. The following cases represent the two more common causes of protracted diarrhea in young infants in the developed world - allergic and autoimmune enteropathy. Both patients demonstrate diagnostic challenges related to clinical and/or laboratory features. These cases illustrate the important role histological assessment plays in determining the correct diagnosis, treatment course and prognosis in infants with protracted diarrhea.

International audience; Melanoma is known to be under latent immunosurveillance. Here, we studied four biomarkers of immunogenic cell stress and death (microtubule-associated proteins 1A/1B light chain 3B (MAP-LC3B, best known as LC3B)-positive puncta in the cytoplasm as a sign of autophagy; presence of nuclear HMGB1; phosphorylation of eIF2 alpha; increase in ploidy) in melanoma cells, in tissue microarrays (TMA) from metastases from 147 melanoma patients. These biomarkers of immunogenicity were correlated with the density of immune cells infiltrating the metastases and expressing CD3, CD4(+), CD8(+), CD20, CD45, CD56, CD138, CD163, DC-LAMP or FOXP3. LC3B puncta positively correlated with the infiltration of metastases by CD163(+) macrophages, while expression of HMGB1 correlated with infiltration by FOXP3(+) regulatory T cells and CD56(+) lymphocytes. eIF2 alpha phosphorylation was associated with an augmentation of nuclear diameters, reflecting an increase in ploidy. Interestingly, therapeutic vaccination led to a reduction of eIF2 alpha phosphorylation suggestive of immunoselection against cells bearing this sign of endoplasmic reticulum (ER) stress. None of the stress/death-related biomarkers had a significant prognostic impact, contrasting with the major prognostic effect of the ratio of cytotoxic T lymphocytes (CTL) over immunosuppressive FOXP3(+) and CD163(+) cells. Altogether, these results support the idea of a mutual dialog between, on one hand, melanoma cells with their cell-intrinsic stress pathways and, on the other hand, immune effectors. Future work is required to understand the detailed mechanisms of this interaction.Keywords

International audience; Several cell-intrinsic alterations have poor prognostic features in human breast cancer, as exemplified by the absence of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 )-positive puncta in the cytoplasm (which indicates reduced autophagic flux) or the loss of nuclear HMGB1 expression by malignant cells. It is well established that breast cancer is under strong immunosurveillance, as reflected by the fact that scarce infiltration of the malignant lesion by CD8(+) cytotoxic T lymphocytes or comparatively dense infiltration by immunosuppressive cell types (such as FOXP3(+) regulatory T cells or CD68(+) tumor-associated macrophages), resulting in low CD8(+):FOXP3(+) or CD8(+):CD68(+) ratios, has a negative prognostic impact. Here, we reveal the surprising finding that cell-intrinsic features may influence the composition of the immune infiltrate in human breast cancer. Thus, the absence of LC3B puncta is correlated with intratumoral (but not peritumoral) infiltration by fewer CD8(+) cells and more FOXP3(+) or CD68(+) cells, resulting in a major drop in the CD8(+):FOXP3(+) or CD8(+):CD68(+) ratios. Moreover, absence of HMGB1 expression in nuclei correlated with a general drop in all immune effectors, in particular FOXP3(+) and CD68(+) cells, both within the tumor and close to it. Combined analysis of LC3B puncta and HMGB1 expression allowed for improved stratification of patients with respect to the characteristics of their immune infiltrate as well as overall and metastasis-free survival. It can be speculated that blocked autophagy in, or HMGB1 loss from, cancer cells may favor tumor progression due to their negative impact on anticancer immunosurveillance.

How dying tumor cells get noticed Besides killing tumor cells directly, some chemotherapies, such as anthracyclines, also activate the immune system to kill tumors. Vacchelli et al. discovered that in mice, anthracycline-induced antitumor immunity requires immune cells to express the protein formyl peptide receptor 1 (FPR1). Dendritic cells (DCs) near tumors expressed especially high amounts of FPR1. DCs normally capture fragments of dying tumor cells and use them to activate nearby T cells to kill tumors, but DCs lacking FPR1 failed to do this effectively. Individuals with breast or colon cancer expressing a variant of FPR1 and treated with anthracyclines showed poor metastasis-free and overall survival. Thus, FPR1 may affect anti-tumor immunity in people, too. Science , this issue p. 972

Proteolytic cascades are widely implicated in signaling between cellular compartments. In Escherichia coli, accumulation of unassembled outer membrane porins (OMPs) in the envelope leads to expression of σE-dependent genes in the cytoplasmic cellular compartment. A proteolytic cascade conveys the OMP signal by regulated proteolysis of RseA, a membrane-spanning anti-sigma factor whose cytoplasmic domain inhibits σE-dependent transcription. Upon activation by OMP C termini, the membrane localized DegS protease cleaves RseA in its periplasmic domain, the membrane-embedded protease RseP (YaeL) cleaves RseA near the inner membrane, and the released cytoplasmic RseA fragment is further degraded. Initiation of RseA degradation by activated DegS makes the system sensitive to a wide range of OMP concentrations and unresponsive to variations in the levels of DegS and RseP proteases. These features rely on the inability of RseP to cleave intact RseA. In the present report, we demonstrate that RseB, which binds to the periplasmic face of RseA, and DegS each independently inhibits RseP cleavage of intact RseA. Thus, the function of RseB, widely conserved among bacteria using the σE pathway, and the second role of DegS (in addition to RseA proteolysis initiation) is to improve the performance characteristics of this signal transduction system.

Among human immunodeficiency virus (HIV) -infected adults and children in Africa, diarrheal disease remains a major cause of morbidity and mortality. We evaluated the effectiveness of provision and home-based reinforcement of a point-of-use water filtration device to reduce diarrhea among 361 HIV-infected adults in western Kenya by comparing prevalence of self-reported diarrhea before and after these interventions. After provision of the filter, 8.7% of participants reported diarrhea compared with 17.2% in the 3 months before filter provision (odds ratio [OR] = 0.39, 95% confidence interval [95% CI] = 0.23–0.66, P < 0.001). The association was similar among 231 participants who were already taking daily cotrimoxazole prophylaxis before being given a filter (OR = 0.47, 95% CI = 0.25–0.88, P = 0.019). Educational reinforcement was also associated with a modest reduction in self-reported diarrhea (OR = 0.50, 95% CI = 0.20–0.99, P = 0.047). Provision and reinforcement of water filters may confer significant benefit in reducing diarrhea among HIV-infected persons, even when cotrimoxazole prophylaxis is already being used.

Beyond cancer-cell intrinsic factors, the immune status of the host has a prognostic impact on patients with cancer and influences the effects of conventional chemotherapies. Metastatic melanoma is intrinsically immunogenic, thereby facilitating the search for immune biomarkers of clinical responses to cytotoxic agents. Here, we show that a multi-tyrosine kinase inhibitor, sorafenib, upregulates interleukin (IL)-15Rα in vitro and in vivo in patients with melanoma, and in conjunction with natural killer (NK) group 2D (NKG2D) ligands, contributes to the Th1 polarization and accumulation of peripheral CD4+NKG2D+ T cells. Hence, the increase of blood CD4+NKG2D+ T cells after two cycles of sorafenib (combined with temozolomide) was associated with prolonged survival in a prospective phase I/II trial enrolling 63 patients with metastatic melanoma who did not receive vemurafenib nor immune checkpoint-blocking antibodies. In contrast, in metastatic melanoma patients treated with classical treatment modalities, this CD4+NKG2D+ subset failed to correlate with prognosis. These findings indicate that sorafenib may be used as an "adjuvant" molecule capable of inducing or restoring IL-15Rα/IL-15 in tumors expressing MHCclass I-related chain A/B (MICA/B) and on circulating monocytes of responding patients, hereby contributing to the bioactivity of NKG2D+ Th1 cells., peer-reviewed

In a series of 248 tumor samples obtained from image-guided biopsies from patients diagnosed with ductal carcinoma in situ of the breast, we attempted to identify biomarkers that predict microinfiltration at definitive surgery or relapse during follow-up. For this, we used immunohistochemical methods, followed by automated image analyses, to measure the mean diameter of nuclei (which correlates with ploidy), the phosphorylation of eukaryotic initiation factor 2α (eIF2α, which reflects endoplasmic reticulum stress) as well as the density and ratio of CD8+ cytotoxic T lymphocytes and FOXP3+ regulatory T cells. The median nuclear diameter of malignant cells correlated with eIF2α phosphorylation (in cancerous tissue), which in turn correlated with the density of the CD8+ infiltrate and the CD8+/FOXP3 ratio (both in cancerous and the adjacent non-cancerous parenchyma). Neither microinfiltration nor lymph node involvement was associated with the probability of relapse. Both correlated positively with the CD8+/FOXP3 ratio in the malignant area. In contrast, relapse was associated with a paucity of the CD8+ infiltrate as well as an unfavorable CD8+/FOXP3 ratio, both in malignant and non-malignant parenchyma. The combined analysis of the CD8+/FOXP3 ratio in cancerous and non-cancerous tissues revealed a significant impact of their interaction on the probability of relapse, but not on the presence of microinfiltration or lymph node metastasis. Altogether, these results support the idea of an immunosurveillance system that determines the risk of relapse in ductal carcinoma in situ of the breast.

It is clear from epidemiological studies that excess iron is associated with increased risk of colorectal cancer; however, questions regarding the mechanism of how iron increases cancer risk, the source of the excess iron (circulating or luminal), and whether iron reduction represents a potential therapeutic option remain unanswered. In this study, we show that after Apc deletion, the cellular iron acquisition proteins TfR1 and DMT1 are rapidly induced. Conversely, restoration of APC reduces cellular iron due to repression of these proteins. To test the functional importance of these findings, we performed in vivo investigations of the impact of iron levels on intestinal tumorigenesis. Strikingly, depletion of luminal (but not systemic) iron strongly suppressed murine intestinal tumorigenesis, whereas increased luminal iron strongly promoted tumorigenesis. Taken together, our data definitively delineate iron as a potent modifier of intestinal tumorigenesis and have important implications for dietary iron supplementation in patients at high risk of colorectal cancer.

Several studies suggest that low levels of hepatic phosphatidylcholine (PC) play a role in the pathogenesis of non-alcoholic steatohepatitis (NASH). CTP: phosphocholine cytidylyltransferase (CT) is the key regulatory enzyme in the CDP-choline pathway for PC biosynthesis. Liver-specific elimination of CTα (LCTα(-/-)) in mice fed a chow diet decreases very-low-density lipoprotein secretion, reduces lipid efflux from liver, and causes mild steatosis. We fed LCTα(-/-) mice a high fat diet to determine if impaired PC biosynthesis played a role in development of NASH. LCTα(-/-) mice developed NASH within one week of high fat feeding. Hepatic CTα deficiency caused hepatic steatosis, a 2-fold increase in ceramide mass, and a 20% reduction in PC content. In an attempt to prevent NASH, LCTα(-/-) mice were either injected daily with CDP-choline or fed the high fat diet supplemented with betaine. In addition, LCTα(-/-) mice were injected with adenoviruses expressing CTα. CDP-choline injections and adenoviral expression of CTα increased hepatic PC, while dietary betaine supplementation normalized hepatic triacylglycerol but did not alter hepatic PC mass in LCTα(-/-) mice. Interestingly, none of the treatments normalized hepatic ceramide mass or fully prevented the development of NASH in LCTα(-/-) mice. These results show that normalizing the amount of hepatic PC is not sufficient to prevent NASH in LCTα(-/-) mice.

In Escherichia coli , the σ E transcription factor monitors and maintains outer membrane (OM) integrity by activating genes required for assembly of its two key components, outer membrane proteins (OMPs) and lipopolysaccharide (LPS) and by transcribing small RNAs to down-regulate excess unassembled OMPs. σ E activity is governed by the rate of degradation of its membrane-spanning anti-σ factor, RseA. Importantly, the DegS protease can initiate RseA cleavage only when activated by binding to unassembled OMPs. The prevalent paradigm has been that the σ E response is controlled by the amount of activated DegS. Here we demonstrate that inactivation of a second negative regulator, the periplasmic protein RseB, is also required for σ E induction in vivo. Moreover, OMPs, previously known only to activate DegS, also generate a signal to antagonize RseB inhibition. This signal may be lipid related, as RseB is structurally similar to proteins that bind lipids. We propose that the use of an AND gate enables σ E to sense and integrate multivariate signals from the envelope.

International audience; The natural killer (NK) cell receptor NKp30 is involved in the recognition of tumor and dendritic cells (DCs). Here we describe the influence of three NKp30 splice variants on the prognosis of gastrointestinal sarcoma (GIST), a malignancy that expresses NKp30 ligands and that is treated with NK-stimulatory KIT tyrosine kinase inhibitors. Healthy individuals and those with GIST show distinct patterns of transcription of functionally different NKp30 isoforms. In a retrospective analysis of 80 individuals with GIST, predominant expression of the immunosuppressive NKp30c isoform (over the immunostimulatory NKp30a and NKp30b isoforms) was associated with reduced survival of subjects, decreased NKp30-dependent tumor necrosis factor-α (TNF-α) and CD107a release, and defective interferon-γ (IFN-γ) and interleukin-12 (IL-12) secretion in the NK-DC cross-talk that could be restored by blocking of IL-10. Preferential NKp30c expression resulted partly from a single-nucleotide polymorphism at position 3790 in the 3' untranslated region of the gene encoding NKp30. The genetically determined NKp30 status predicts the clinical outcomes of individuals with GIST independently from KIT mutation.

Proteolytic cascades often transduce signals between cellular compartments, but the features of these cascades that permit efficient conversion of a biological signal into a transcriptional output are not well elucidated. σE mediates an envelope stress response in Escherichia coli, and its activity is controlled by regulated degradation of RseA, a membrane-spanning anti-σ factor. Examination of the individual steps in this protease cascade reveals that the initial, signal-sensing cleavage step is rate-limiting; that multiple ATP-dependent proteases degrade the cytoplasmic fragment of RseA and that dissociation of σE from RseA is so slow that most free σE must be generated by the active degradation of RseA. As a consequence, the degradation rate of RseA is set by the amount of inducing signal, and insulated from the “load” on and activity of the cytoplasmic proteases. Additionally, changes in RseA degradation rate are rapidly reflected in altered σE activity. These design features are attractive as general components of signal transduction pathways governed by unstable negative regulators.

A 71-year-old previously healthy man, vacationing in Arizona, was admitted for a 1-month history of cough and progressive fatigue limiting his ambulation. With paroxysms of coughing, he described bilateral vision loss for 5 to 10 seconds. His vision grayed and “tunneled down” (i.e., from periphery to center) before blindness. Sometimes, he had transient bilateral blurring with “stars” but no diplopia. He denied presyncope. There was no hemoptysis, dyspnea, or pleurisy. He had no headache, scalp tenderness, jaw claudication, or morning stiffness, but had diffuse bone and muscle pains. He had constitutional symptoms including anorexia, night sweats, and 20-lb weight loss. Other than his Arizona holiday, his infectious contact and medical history was unremarkable. He had low risk of atherosclerosis: he was fit and a nonsmoker, had no family history, and had systolic pressures

Summary: Prilocaine–lidocaine emulsion (EMLA cream) is a topical anaesthetic commonly used prior to diagnostic and therapeutic procedures. While undergoing clinical investigation for the suspicion of a metabolic disorder, a series of children underwent skin biopsy with EMLA cream pretreatment. In each case, the pathologist identified ultrastructural features consistent with a lysosomal storage disorder, yet the clinical features were not consistent with the pathological findings. Ultrastructural artefact was suspected, resulting from the use of the EMLA cream. All patients underwent repeat skin biopsy without EMLA cream. Biopsies were reviewed by two pathologists blinded to the previous biopsy findings. Electron microscopy repeated without the use of EMLA cream was normal. It is concluded that the use of EMLA cream causes ultrastructural artefact and should be avoided prior to skin biopsy for electron microscopy.