Zika virus triggers autophagy to exploit host lipid metabolism and drive viral replication.

BACKGROUND: Zika virus (ZIKV), an arbovirus of global concern, has been associated with neurological complications including microcephaly in newborns and Guillain-Barré syndrome in adults. Like other flaviviruses, ZIKV depends on cholesterol to facilitate its replication; thus, cholesterol has been proposed as a therapeutic target to treat the infection using FDA-approved statins. Cholesterol is stored in intracellular lipid droplets (LD) in the form of cholesterol esters and can be regulated by autophagy. We hypothesize that the virus hijacks autophagy machinery as an early step to increase the formation of LD and viral replication, and that interference with this pathway will limit reproduction of virus. METHODS: We pretreated MDCK cells with atorvastatin or other inhibitors of autophagy prior to infection with ZIKV. We measured viral expression by qPCR for NS1 RNA and immunofluorescence for Zika E protein. RESULTS: Autophagy increases in virus-infected cells as early as 6 h post infection (hpi). In the presence of atorvastatin, LD are decreased, and cholesterol is reduced, targeting key steps in viral replication, resulting in suppression of replication of ZIKV is suppressed. Other both early- and late-acting autophagy inhibitors decrease both the number of LD and viral replication. Bafilomycin renders cholesterol is inaccessible to ZIKV. We also confirm previous reports of a bystander effect, in which neighboring uninfected cells have higher LD counts compared to infected cells. CONCLUSIONS: We conclude that atorvastatin and inhibitors of autophagy lead to lower availability of LD, decreasing viral replication. We conclude that bafilomycin A1 inhibits viral expression by blocking cholesterol esterification to form LD. Video Abstract.

Ceramide from sphingomyelin hydrolysis induces neuronal differentiation, whereas de novo ceramide synthesis and sphingomyelin hydrolysis initiate apoptosis after NGF withdrawal in PC12 Cells.

BACKGROUND: Ceramide, important for both neuronal differentiation and dedifferentiation, resides in several membranes, is synthesized in the endoplasmic reticulum, mitochondrial, and nuclear membranes, and can be further processed into glycosphingolipids or sphingomyelin. Ceramide may also be generated by hydrolysis of sphingomyelin by neutral or acidic sphingomyelinases in lysosomes and other membranes. Here we asked whether the differing functions of ceramide derived from different origins. METHODS: We added NGF to PC12 cells and to TrkA cells. These latter overexpress NGF receptors and are partially activated to differentiate, whereas NGF is required for PC12 cells to differentiate. We differentiated synthesis from hydrolysis by the use of appropriate inhibitors. Ceramide and sphingomyelin were measured by radiolabeling. RESULTS: When NGF is added, the kinetics and amounts of ceramide and sphingomyelin indicate that the ceramide comes primarily from hydrolysis but, when hydrolysis is inhibited, can also come from neosynthesis. When NGF is removed, the ceramide comes from both neosynthesis and hydrolysis. CONCLUSION: We conclude that the function of ceramide depends heavily on its intracellular location, and that further understanding of its function will depend on resolving its location during changes of cell status. Video Abstract.

Ceramide and sphingomyelin reportedly are important both for differentiation of nerve cells and for their death. We studied PC12 cells, which can differentiate into neuron-like cells in the presence of nerve growth factor and cells that overexpress receptors for nerve growth factor. By combining various inhibitors, we conclude that in the presence of nerve growth factor ceramide comes from hydrolysis of sphingomyelin, but when nerve growth factor is removed and the cells atrophy and die, sphingomyelin comes from both neosynthesis and hydrolysis.

Atorvastatin restricts the ability of influenza virus to generate lipid droplets and severely suppresses the replication of the virus.

Influenza virus causes infected cells to generate large numbers of lipid droplets. Because the virus envelope contains substantial cholesterol, we applied atorvastatin (ATV) to Madin-Darby Canine Kidney cells before infecting them. Five micromolars ATV, within physiologic range, strongly (>95%) inhibits reproduction of influenza A as measured by PCR of viral RNA, plaque assay for viable virus, and production of virus nucleoprotein (NP). Inhibition of any of the following can suppress formation of lipid droplets (>-50%) but does not interfere with the production of NP: endoplasmic reticulum stress, autophagy, or production of reactive oxygen substances (ROS). We conclude that, regardless of whether this widely used statin, which is generally considered to be safe, can prevent infection or minimize its severity, inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway to protect against infection by influenza virus or to mitigate its severity warrants further exploration.-Episcopio, D., Aminov, S., Benjamin, S., Germain, G., Datan, E., Landazuri, J., Lockshin, R. A., Zakeri, Z. Atorvastatin restricts the ability of influenza virus to generate lipid droplets and severely suppresses the replication of the virus.

Higher sensitivity of female cells to ethanol: methylation of DNA lowers Cyp2e1, generating more ROS.

BACKGROUND: Cells taken from mouse embryos before sex differentiation respond to insults according to their chromosomal sex, a difference traceable to differential methylation. We evaluated the mechanism for this difference in the controlled situation of their response to ethanol. METHODS: We evaluated the expression of mRNA for alcohol dehydrogenase (ADH), aldehyde dehyrogenases (ALDH), and a cytochrome P450 isoenzyme (Cyp2e1) in male and female mice, comparing the expressions to toxicity under several experimental conditions evaluating redox and other states. RESULTS: Females are more sensitive to ethanol. Disulfiram, which inhibits alcohol dehydrogenase (ADH), increases cell death in males, eliminating the sex dimorphism. The expressions ADH Class 1 to 4 and ALDH Class 1 and 2 do not differ by sex. However, females express approximately 8X more message for Cyp2e1, an enzyme in the non-canonical pathway. Female cells produce approximately 15% more ROS (reactive oxygen species) than male cells, but male cells contain approximately double the concentration of GSH, a ROS scavenger. Scavenging ROS with N-acetyl cysteine reduces cell death and eliminates sex dimorphism. Finally, since many of the differences in gene expression derive from methylation of DNA, we exposed cells to the methyltransferase inhibitor 5-aza- 2-deoxycytidine; blocking methylation eliminates both the difference in expression of Cyp2e1 and cell death. CONCLUSION: We conclude that the sex-differential cell death caused by ethanol derives from sex dimorphic methylation of Cyp2e1 gene, resulting in generation of more ROS.

Microglial-induced apoptosis is potentially responsible for hyperalgesia variations during CFA-induced inflammation.

AIMS: Activated microglia is known as a main mediator of inflammatory pain, but the possible mechanisms of its operation are poorly understood. Microglial cells have considered as one of the main sources of pro-inflammatory cytokines in the CNS. PTEN is one of the important targets of pro-inflammatory cytokines and the main mediator of apoptotic cell death. In this study, we investigated the possible effect of microglial activation on PTEN/PI3K/Akt signaling pathway and apoptosis in an inflammatory rat model of Complete Freund's adjuvant (CFA). METHODS: Persistent peripheral inflammation was induced by a subcutaneous injection of CFA into the rats' right hind paw on day 0. Minocycline (a potent selective inhibitor of microglial) was administered intraperitoneally during days 1-21 after CFA injection. Hyperalgesia was assessed on days 0, 7, and 21 using plantar test, then lumbar spinal cord segments were isolated, and the amount of spinal Iba1 (microglial marker), PTEN, P.Akt, and cleaved caspase-3 (a marker of apoptosis activation) were analyzed using Western blot. The spinal TNF-α levels were assayed by ELISA and the microglia numbers were determined using immunohistochemical technique. RESULTS: Results revealed that increased hyperalgesia was concurrent with an increment of Iba1 (P < 0.001), TNF-α (P < 0.001), PTEN (P < 0.01), cleaved caspase-3 (P < 0.001), and a decrement of P.Akt (P < 0.01) during the acute phase of CFA-induced inflammation, while, at the same time as decreasing hyperalgesia during the chronic phase of study, Iba1 and TNF-α expression significantly decreased and PTEN, cleaved caspase-3, and P.Akt restored to baseline on day 0. Minocycline administration reduced the elevation of spinal Iba1 (P < 0.001), TNF-α (0.001), PTEN (P < 0.01), and cleaved caspase-3 (P < 0.001) expression induced by CFA injection, and also restored Akt activity to the baseline on day 0 (P < 0.001). CONCLUSIONS: These results suggest that microglial-mediated pain following CFA injection might be related in part to increased spinal cell apoptosis which probably is mediated by PTEN/PI3K/Akt deregulation.

MicroRNA and exosome: Key players in rheumatoid arthritis.

Rheumatoid arthritis (RA) is known as one of important autoimmune disorders which can lead to joint pain and damage throughout body. Given that internal (ie, genetic and epigenetic alterations) and external factors (ie, lifestyle changes, age, hormones, smoking, stress, and obesity) involved in RA pathogenesis. Increasing evidence indicated that cellular and molecular alterations play critical roles in the initiation and progression of RA. Among various targets and molecular signaling pathways, microRNAs (miRNAs) and their regulatory networks have key roles in the RA pathogenesis. It has been showed that deregulation of many miRNAs involved in different stages of RA. Hence, identification of miRNAs and their signaling pathways in RA, could contribute to new knowledge which help to better treatment of patients with RA. Besides miRNAs, exosomes have been emerged as key messengers in RA pathogenesis. Exsosomes are nanocarriers which could be released from various cells and lead to changing of behaviors recipient cells via targeting their cargos (eg, proteins, messenger RNAs, miRNAs, long noncoding RNAs, DNAs). Here, we summarized several miRNAs involved in RA pathogenesis. Moreover, we highlighted the roles of exosomes in RA pathogenesis.

Comparison of main molecular markers involved in autophagy and apoptosis pathways between spermatozoa of infertile men with varicocele and fertile individuals.

Abnormal dilatation and tortuosity of the pampiniform plexus within the spermatic cord are termed varicocele which leads to impaired spermatogenesis due to heat-related oxidative stress and cell death. Previously, it was shown that both apoptosis and autophagy pathways were activated by heat in germ cells of mouse in vivo and in vitro. But, status of these pathways is not clear in chronic state of heat stress such as varicocele. Therefore, we aimed to access sperm apoptotic markers (active caspases 3/7 and DNA fragmentation), and autophagic markers (Atg7 and LC3 proteins) as primary outcomes, and also sperm parameters and protamine deficiency as secondary outcomes between 23 infertile men with varicocele and 16 fertile individuals. Sperm parameters were assessed according to World Health Organization 2010 protocol. Apoptotic markers (active caspases 3/7 and DNA fragmentation), autophagic markers (Atg7 and LC3 proteins), and protamine deficiency were evaluated by flow cytometry, fluorescence microscope, and western blotting techniques. Mean of autophagy and apoptosis markers, and also protamine deficiency have significantly increased in infertile men with varicocele compared to fertile individuals, but autophagy and apoptosis markers did not significantly correlate with each other. In conclusion, it seems that both apoptosis and autophagy pathways are independently active in spermatozoa of infertile men with varicocele.

Interleukin-1ß (IL-1ß) & IL-4 gene polymorphisms in patients with systemic lupus erythematosus (SLE) & their association with susceptibility to SLE.

BACKGROUND & OBJECTIVES: Interleukin-1 (IL-1) is one of the pro-inflammatory cytokines that plays a main role in the regulation of immune and inflammatory responses. Interleukin 4 (IL-4) as an anti-inflammatory cytokine regulates balance between Th1 and Th2 immune responses. this study was undertaken to investigate the IL-1ß and IL-4 genes polymorphisms in patients with systemic lupus erythematosus (SLE) and also association between the polymorphisms and susceptibility to SLE. METHODS: One hundred and sixty three SLE patients and 180 healthy controls were genotyped for the IL-4 VNTR (variable number tandem repeat), IL-1ß C-511T and IL-1ß T-31C polymorphisms by polymerase chain reaction (PCR) or PCR-RFLP (restriction fragment length polymorphism) method. RESULTS: The frequencies of CC genotype and C allele of the IL-1ß T-31C polymorphism were significantly (P<0.01) lower in SLE patients than controls. Moreover, the frequencies of RP1/RP2 genotype and RP2 allele of IL-4 VNTR polymorphism were significantly (P<0.05) higher in the SLE patients. No association was observed between IL-1ß C-511T polymorphism and increased risk of SLE. We observed increased frequency of CT and TT genotypes of IL-1ß C-511T polymorphism in SLE patients with malar rash compared to SLE patients without this manifestation. INTERPRETATION & CONCLUSIONS: The present findings suggest that IL-1ß T-31C and IL-4 VNTR polymorphisms but not IL-1ß C-511T polymorphism may contribute in SLE pathogenesis. In addition, CT and TT genotypes of IL-1ß C-511T polymorphism were associated with SLE.

Sex-dependent regulation of cytochrome P450 family members Cyp1a1, Cyp2e1, and Cyp7b1 by methylation of DNA.

Sexual differences are only partially attributable to hormones. Cultured male or female cells, even from embryos before sexual differentiation, differ in gene expression and sensitivity to toxins, and these differences persist in isolated primary cells. Male and female cells from Swiss Webster CWF mice manifest sex-distinct patterns of DNA methylation for X-ist and for cytochrome P450 (CYP; family members 1a1, 2e1m, and 7b1. Dnmt3l is differentially expressed but not differentially methylated, and Gapdh is neither differentially methylated nor expressed. CYP family genes differ in expression in whole tissue homogenates and cell cultures, with female Cyp expression 2- to 355-fold higher and Dnmt3l 12- to 32-fold higher in males. DNA methylation in the promoters of these genes is sex dimorphic; reducing methylation differences reduces to 1- to 6-fold differences in the expression of these genes. Stress or estradiol alters both methylation and gene expression. We conclude that different methylation patterns partially explain the sex-based differences in expression of CYP family members and X-ist, which potentially leads to inborn differences between males and females and their different responses to chronic and acute changes. Sex-differential methylation may have medical effects.

Association of FAS and FAS ligand genes polymorphism and risk of systemic lupus erythematosus.

UNLABELLED: FAS/FASL pathway plays a critical role in maintaining peripheral immune tolerance; therefore, the apoptosis genes, Fas and Fas ligand (FasL), could be suitable candidate genes in human SLE susceptibility. MATERIALS AND METHODS: In this case-control study, 106 SLE patients and 149 sex, age, and ethnicity matched healthy controls were genotyped for the Fas A-670G and FasLC-844T polymorphisms by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). RESULTS: The frequency of -670AA genotype was significantly higher in SLE patients than control group and the risk of SLE was 2.1-fold greater in subjects with AA genotype (P=0.03). The frequency of -670A allele was significantly higher in SLE patients than in controls too (58% versus 49%, P=0.03). The -844CC genotype frequency was significantly higher in SLE patients than in healthy controls and the risk of SLE was 2.8-fold greater in these subjects (P=0.01). The C allele frequency was significantly higher in patients than in controls (69% versus 49%, P=0.001). Increased SLE risk was observed in individuals with combined effect of Fas-670AA and FasL-844CC genotypes (P=0.001). CONCLUSION: Fas-670AA and FasL-844CC genotypes were associated with SLE risk, and combined effect of -670AA and -844CC genotypes might increase SLE susceptibility.

Flavivirus NS4A-induced autophagy protects cells against death and enhances virus replication.

Flaviviruses include the most prevalent and medically challenging viruses. Persistent infection with flaviviruses of epithelial cells and hepatocytes that do not undergo cell death is common. Here, we report that, in epithelial cells, up-regulation of autophagy following flavivirus infection markedly enhances virus replication and that one flavivirus gene, NS4A, uniquely determines the up-regulation of autophagy. Dengue-2 and Modoc (a murine flavivirus) kill primary murine macrophages but protect epithelial cells and fibroblasts against death provoked by several insults. The flavivirus-induced protection derives from the up-regulation of autophagy, as up-regulation of autophagy by starvation or inactivation of mammalian target of rapamycin also protects the cells against insult, whereas inhibition of autophagy via inactivation of PI3K nullifies the protection conferred by flavivirus. Inhibition of autophagy also limits replication of both Dengue-2 and Modoc virus in epithelial cells. Expression of flavivirus NS4A is sufficient to induce PI3K-dependent autophagy and to protect cells against death; expression of other viral genes, including NS2A and NS4B, fails to protect cells against several stressors. Flavivirus NS4A protein induces autophagy in epithelial cells and thus protects them from death during infection. As autophagy is vital to flavivirus replication in these cells, NS4A is therefore also identified as a critical determinant of flavivirus replication.

Prevalence of depression and depressive symptoms in patients with systemic lupus erythematosus: Iranian experience.

Psychiatric disorders including depression represent clinical manifestation of systemic lupus erythematosus (SLE). Recognition of depression in SLE patients is of utmost importance since it is treatable and can be of fatal consequences if unrecognized. This study was conducted to determine the prevalence of depression and depressive symptoms in SLE patients in terms of age, gender, disease duration and severity, and duration of steroid treatment in SLE patients. Eighty-five SLE patients (77 women, 8 men) with verified SLE diagnosis completed Beck's depression inventory, a self-reported measure of depression. Clinical data on disease and treatment were obtained from patient files. In total, 60% of patients achieved scores indicating depression. The most common depressive symptoms in participants were fatigue and weakness (88.2%), irritability (82.3%), sadness (77.6%), and somatic preoccupation (76.4%), while the least common symptoms were weight loss (34.1%), low level of energy (28.2%), and suicide ideation (10.5%). There was a significant difference between the disease activity and the severity of depression (P = 0.0001). Our findings show higher prevalence of depression in our sample in comparison with previous studies, suggesting that the prevalence of depression varies across different populations. Severity of depression increases with more severe disease course.

Caspase-independent cell deaths.

A very common and the best understood of the mechanisms of physiological cell death is apoptosis, resulting from the activation, through either of two primary pathways, of site-specific proteases called caspases. There are, however, many other routes to cell death, prominently including autophagy and proteasomal degradation of critical constituents of cells. These routes are frequently seen in experimental situations in which initiator or effector caspases are inhibited or blocked through genetic means, but they are also encountered during normal physiological and pathological processes. Most frequently, autophagic or proteasomal degradation is used to eliminate massive cytoplasm of very large cells, especially post-mitotic cells, and these pathways are prominent even though caspase genes, messages, and pro-enzymes are found in the cells. These forms of cell death are fully physiological and not simply a default pathway for a defective cell; and they are distinct from necrosis. We do not yet understand the extent to which the pathways are linked, what mechanisms trigger the caspase-independent deaths, and how the choices are made.

Evaluation of bone mineral density in premenopausal women with type-2 diabetes mellitus in Zahedan, southeast Iran.

OBJECTIVE: To determine the BMD in premenopausal women with type-2 diabetes mellitus. METHODS: This case-control study was performed on 60 premenopausal women with type-2 diabetes mellitus and 60 normal premenopausal subjects. The groups were not completely matched regarding BMI; but they were in the range of obesity. Bone mineral density was determined using dual energy X-ray absorptiometry (DXA) to define bone mineral density (BMD) in second to fourth lumbar vertebrae and the neck of the femur (g/cm2). RESULTS: The results showed that BMD, T- and Z-score of femoral neck, total femur, L2, and Ward's were not significantly different between type 2 diabetic and normal premenopausal women (p > 0.5). A significant increase of L3 BMD and L2-L4 Z-score was observed in diabetic group (p < 0.05). In addition BMD, T- and Z-score of L4 were significantly higher in type 2 diabetic women than normal premenopausal women (P < 0.05). CONCLUSION: Higher BMD was noted over the spine in diabetic group which may be due to higher BMI in this group.

Association of P2X7 receptor genetic polymorphisms and expression with rheumatoid arthritis susceptibility in a sample of the Iranian population: a case-control study.

OBJECTIVE: Rheumatoid arthritis (RA) is a complex inflammatory autoimmune disease with joint eruption, systemic manifestation, and numerous predisposing genetic factors. The P2X7 receptor is an essential ligand-gated channel that contributes to many physiological processes, especially inflammation. However, genetic variations can alter the P2X7 receptor function. Therefore, the present study aimed to explore the impact of P2X7 genetic polymorphisms and expression on susceptibility to RA in a sample of the Iranian population. METHODS: We enrolled 160 (145 female, 15 male) RA patients and 160 (142 female, 18 male) healthy individuals in this study. Genotyping was performed using tetra amplification refractory mutation system-polymerase chain reaction (TARMS-PCR) for rs1718119, rs2230912, rs2393799, rs28360457, rs35933842, and allele-specific PCR for rs1653624 and rs3751143. Furthermore, 44 new cases of RA and 48 healthy controls were recruited to investigate whether P2X7 mRNA expression is associated with RA susceptibility. RESULTS: The results revealed that the rs2393799 significantly increased the risk of RA in all genetic models (p<0.05), while rs3751143 in codominant (CC vs. AA, OR=0.49, 95% CI=0.26-0.92), dominant (AC+CC, OR=0.59, 95% CI=0.37-0.94), C allele (OR=0.63, 95% CI=0.46-0.88), and rs2230912 in codominant (AG vs. AA, OR=0.56, 95% CI=0.34-0.94), dominant (AG+GG vs. AA, OR=0.59, 95% CI=0.35-0.99), and overdominant (AG vs. AA+GG, OR=0.57, 95% CI=0.33-0.98) significantly decreased the RA risk (p<0.05). Furthermore, the rs1718119 and rs1653624 were not associated with susceptibility of RA (p>0.05), and rs28360457 and rs35933842 were not polymorphic in our study. The mRNA expression level of P2X7 in both groups revealed that the P2X7 gene was significantly upregulated in RA (3.18±0.43) compared to healthy subjects (1.47±0.15, p<0.001). CONCLUSION: Our results suggest that rs2393799, rs3751143, and rs2230912 variants of the P2X7 gene are associated with RA's susceptibility in a sample of the Iranian population. Also, P2X7 mRNA expression was higher in our new RA patients. The P2X7 receptor has been considered as a potential pharmacologic target in RA. Key Points • P2X7 variants (rs2393799, rs2230912, rs3751143) were associated with RA susceptibility in a sample of the Iranian population. • rs2393799 increases the risk of RA, while rs2230912 and rs3751143 decrease the risk of RA. • P2X7 expression was significantly upregulated in new RA patients compared to controls.

Assessment of Atg7 and LC3II/LC3, as The Markers of Autophagy, in Sperm of Infertile Men with Globozoospermia: A Case-Control Study.

OBJECTIVE: Assessment of relationship between LC3II/LC3 and Autophagy-related 7 (Atg7) proteins, as markers of autophagy, as well as evaluating the sperm parameters and DNA fragmentation in spermatozoa of infertile men with globozoospermia. MATERIALS AND METHODS: In this case-control study, 10 semen samples from infertile men with globozoospermia and 10 fertile individuals were collected, and the sperm parameters, sperm DNA fragmentation, and main autophagy markers (Atg7 and LC3II/LC3) were assessed according to World Health Organization (WHO) criteria, TUNEL assay, and western blot technique, respectively. RESULTS: The mean of sperm concentration and motility were significantly lower, while the percentage of abnormal spermatozoa and DNA fragmentation were significantly higher in infertile men with globozoospermia compared to fertile individuals (P<0.01). Unlike the relative expression of LC3II/LC3 that did not significantly differ between the two groups, the relative expression of ATG7 was significantly higher in infertile men with globozoospermia compared to fertile individuals (P <0.05). There was a significantly negative correlation between the sperm concentration (r=-0.679; P=0.005) and motility (r=-0.64; P=0.01) with the expression of ATG7, while a significantly positive association was founf between the percentage of DNA fragmentation and expression of ATG7 (0.841; P =0.018). CONCLUSION: The increased expression of ATG7 and unaltered expression of LC3II/LC3 may indicate that the autophagy pathway is initiated but not completely executed in spermatozoa of individuals with globozoospermia. A significant correlation of ATG7 expression with increased sperm DNA fragmentation, reduced sperm concentration, and sperm motility may associate with the activation of a compensatory mechanism for promoting deficient spermatozoa to undergo cell death by the autophagy pathway. Therfore, this pathway could act as a double-edged sword that, at the physiological level, is involved in acrosome biogenesis, while, at the pathological level, such as globozoospermia, could act as a compensatory mechanism.

Prevalence of celiac disease in patients with atypical presentations.

BACKGROUND AND STUDY AIMS: Unawareness about atypical forms of celiac disease (CD) leads to the underdiagnoses of CD. This study has investigated the prevalence of CD in patients with atypical presentations, such as idiopathic low bone mineral density (ILBMD) and dyspepsia, in the Iranian population. PATIENTS AND METHODS: Two separate groups of patients who have been diagnosed with dyspepsia and ILBMD (including either osteopenia or osteoporosis of unknown cause) were screened for CD during 2016-2019. Patients were serologically screened by means of IgA anti-tissue transglutaminase (IgA anti-tTG); in case of positive results, the patients underwent endoscopic intestinal biopsy to confirm the diagnosis of CD. RESULTS: Of 200 patients with ILBMD, six (3%) had a positive result for IgA anti-tTG; in five cases (2.5%), duodenal histology confirmed the CD diagnosis. Of 290 patients with dyspepsia, 25 (8.6%) had a positive result for anti-tTG IgA; nine cases (3.7%) were histologically compatible with CD. No significant differences were found between the two groups of patients. CONCLUSIONS: The prevalence of CD in patients with atypical presentations, such as ILBMD and dyspepsia, is consistent (pvalue = 0.788) and higher than that in the general population (p value = 0.001); therefore, screening program for CD in these patients is highly recommended.

Lack of Bax prevents influenza A virus-induced apoptosis and causes diminished viral replication.

The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.

Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells.

Sexual dimorphisms are typically attributed to the hormonal differences arising once sex differentiation has occurred. However, in some sexually dimorphic diseases that differ in frequency but not severity, the differences cannot be logically connected to the sex hormones. Therefore, we asked whether any aspect of sexual dimorphism could be attributed to chromosomal rather than hormonal differences. Cells taken from mice at d 10.5 postconception (PC) before sexual differentiation, at d 17.5 PC after the first embryonic assertion of sexual hormones, and at postnatal day 17 (puberty) were cultured and exposed to 400 microM ethanol or 20 microM camptothecin or to infection with influenza A virus (multiplicity of infection of 5). The results showed that untreated male and female cells of the same age grew at similar rates and manifested similar morphology. However, they responded differently to the applied stressors, even before the production of fetal sex hormones. Furthermore, microarray and qPCR analyses of the whole 10.5 PC embryos also revealed differences in gene expression between male and female tissues. Likewise, the exposure of cells isolated from fetuses and adolescent mice to the stressors and/or sex hormones yielded expression patterns that reflected chromosomal sex, with ethanol feminizing male cells and masculinizing female cells. We conclude that cells differ innately according to sex irrespective of their history of exposure to sex hormones. These differences may have consequences in the course of sexually dimorphic diseases and their therapy.