Evaluation of the Gastric Microbiome in Patients with Chronic Superficial Gastritis and Intestinal Metaplasia.

Objective To evaluate the gastric microbiome in patients with chronic superficial gastritis (CSG) and intestinal metaplasia (IM) and investigate the influence of Helicobacter pylori (H. pylori) on the gastric microbiome. Methods Gastric mucosa tissue samples were collected from 54 patients with CSG and IM, and the patients were classified into the following four groups based on the state of H. pylori infection and histology: H. pylori-negative CSG (n=24), H. pylori-positive CSG (n=14), H. pylori-negative IM (n=11), and H. pylori-positive IM (n=5). The gastric microbiome was analyzed by 16S rRNA gene sequencing. Results H. pylori strongly influenced the bacterial abundance and diversity regardless of CSG and IM. In H. pylori-positive subjects, the bacterial abundance and diversity were significantly lower than in H. pylori-negative subjects. The H. pylori-negative groups had similar bacterial composition and bacterial abundance. The H. pylori-positive groups also had similar bacterial composition but different bacterial relative abundance. The relative abundance of Neisseria, Streptococcus, Rothia, and Veillonella were richer in the I-HP group than in G-HP group, especially Neisseria (t=175.1, P<0.001). Conclusions The gastric microbial abundance and diversity are lower in H. pylori- infected patients regardless of CSG and IM. Compared to H. pylori-positive CSG group and H. pylori-positive IM, the relative abundance of Neisseria, Streptococcus, Rothia, and Veillonella is higher in H. pylori-positive patients with IM than in H. pylori-positive patients with CSG, especially Neisseria.

Urinary orosomucoid 1 protein to creatinine ratio as a potential biomarker for early screening of kidney impairment in type-2 diabetes patients.

BACKGROUND: Early screening of diabetic kidney disease (DKD) remains a major challenge. Our aim was to evaluate the value of urinary orosomucoid 1 protein (UORM1) in early renal impairment screening in type-2 diabetes patients. METHODS: The concentration of UORM1, the UORM1-to-creatinine ratio (UORM1CR), the urinary albumin-to-creatinine ratio (ACR), the alpha-1-microglobulin-to-creatinine ratio (A1MCR) and estimated glomerular filtration rate (eGFR) were measured in 406 type-2 diabetes patients. Any positive values for ACR, A1MCR and/or eGFR were considered as indicative of renal impairment. RESULTS: On average, the levels of UORM1 and UORM1CR were about seven times higher in subjects with renal injury than in those without. Both UORM1 and UORM1CR, when adjusted via logarithm-transformation, were significantly related to ACR, A1MCR and eGFR levels. The highest correlation was observed between UORM1CR and A1MCR (r = 0.85, P < .001). The cut-off values for UORM1 (2.53 mg/L) and UORM1CR (3.69 mg/g) for the early diagnosis of kidney impairment were obtained from receiver operating characteristic curves. UORM1CR obviously had higher diagnostic efficiency corresponding to 83.26% sensitivity and 90.32% specificity than UORM1. Likewise, its sensitivity was higher than those of ACR, A1MCR and eGFR. Bad glycaemic control had the highest risk of increased UORM1CR (odds ratio [OR] = 2.81, P < .001), while high HDL-C (high-density lipoprotein cholesterol) decreased the risk of increased UORM1CR (OR = 0.38, P = .017). CONCLUSION: The UORM1CR (>3.69 mg/g) has the high diagnostic efficiency for the early screening of renal impairment in type-2 diabetes patients. Furthermore, good glycaemic control and high HDL-C might be protective factors against UORM1CR increase.

Excreted-secreted antigens of Toxoplasma gondii inhibit Foxp3 via IL-2Rγ/JAK3/Stats pathway.

Toxoplasma gondii excreted-secreted antigens (ESA) could lead to the fetal abortion especially in the early stage of pregnancy. Deficit in regulatory T cells is a critical event in the fetal abortion. Transcription factor forkhead box p3 (Foxp3) mediates differentiation and functional roles on regulatory T cells. Previously, we revealed that ESA inhibited Foxp3 through the suppression of transforming growth factor-ß type II receptor, phosphorylation of Smad2, Smad3, and Smad4. Knockdown of Smad2 collaborated with ESA to further inhibit Foxp3. The decrease in Foxp3 caused by ESA reversed via forced expression of Smad2, Smad3, and Smad4, respectively. In this study, we investigate whether other signaling pathways are implicated in ESA-induced Foxp3 downregulation. EL4 cells were cultured and stimulated with ESA. Interleukin-2 receptor γ (IL-2Rγ) chain, Janus kinase 3 (JAK3), signal transducer and activator of transcription 5 (Stat5), Stat3, phosphorylation of Stat5 and Stat3 were assayed by Western blot analysis. Phosphorylation of Stat5 and Stat3 was further measured by cellular immunofluorescence. The expression plasmid of pcDNA3.1-Stat3 and pcDNA3.1-Stat5b was constructed, respectively. The concentration of interleukin-2 (IL-2) in the culture supernatants was detected by enzyme-linked immunosorbent assay. ESA inhibited the level of JAK3, phosphorylation of Stat5 and Stat3, and Foxp3 in EL4 cells. The suppressive effects of ESA on Foxp3 were attenuated by forced expression of Stat5 and Stat3. In addition, ESA suppressed IL-2Rγ in EL4 cells, while IL-2Rγ agonist could markedly reverse the diminished Foxp3 caused by ESA. Furthermore, ESA directly influenced the expression of IL-2Rγ, rather than the availability of IL-2 indirectly. ESA suppressed the level of Foxp3 via inhibiting IL-2Rγ/JAK3/Stats signaling pathway in EL4 cells.

Chinese 1 strain of Toxoplasma gondii excreted-secreted antigens negatively modulate Foxp3 via inhibition of the TGFßRII/Smad2/Smad3/Smad4 pathway.

Toxoplasma gondii is an opportunistic intracellular parasite and is considered an important aetiological factor in the process of abortion, especially as occurs in early gestation. Chinese 1 strain of T. gondii is a dominant genotype prevalent in China. Although it is known that early foetal resorption triggered by RH strain of T. gondii is attributable to immune mechanisms rather than its direct effect in uterus, the underlying mechanism of the abortion caused by Chinese 1 strain remains unclear. This study was designed to investigate the effect of excreted-secreted antigens (ESA) of Chinese 1 strain of T. gondii on the expression of forkhead box transcription factor (Foxp3) as it pertains to early pregnancy and abortion. ESA caused a marked inhibition in the expression of Foxp3 both in vivo and in vitro. In addition, ESA negatively modulated Smad2 and Smad3 at the posttranslational level. Smad2 siRNA cooperated with ESA to further suppress the level of Foxp3. This inhibitory effect on Foxp3 expression was partially abrogated by overexpression of Smad2, Smad3 and Smad4. Additionally, ESA attenuated the expression of TGFßRII, whereas TGFßRII agonist could profoundly reversed the decreased Foxp3 triggered by ESA. Collectively, the findings suggested that ESA restricted Foxp3 expression by inhibiting TGFßRII/Smad2/Smad3/Smad4 signalling, ultimately resulting in abortion.

Toll-like receptor 4 (TLR4) deficiency impedes Toxoplasma gondii excreted-secreted antigens (ESA)-induced abortion.

INTRODUCTION: Toxoplasma gondii is an opportunistic intracellular parasite that is a major pathogenic factor in miscarriage, especially when it occurs early in pregnancy. We have previously demonstrated that the regulation of forkhead box transcription factor (Foxp3) is associated with abortion in early pregnancy caused by excretory-secretory antigen (ESA) of strain China 1. We aimed to reveal the underlying mechanism of miscarriage caused by ESA. METHODS: A TLR4-/- pregnant mouse model was successfully constructed. Pregnant mice at gestational day 5 (G5) were injected with ESA. All animals were sacrificed on G13, pregnancy outcomes were observed, and abortion rates were calculated. Placental status observed by Hematoxylin-eosin staining; gene expression was measured by IHC; flow cytometry analysis was used to determine the number and function of regulatory T cells. In EL4 cells, real-time PCR and Western blot were used to evaluate gene expression and cytokines assay. RESULTS: In vivo studies revealed that ESA injection caused 83% abortion in pregnant mice but only 35% abortion in TLR4-/- pregnant mice. In addition, ESA attenuated the number and function of regulatory T cells, further suppressed Foxp3, FOXO1 levels, and upregulated CD127 expression. TLR4-/- mice partially reversed this inhibitory effect on regulatory T cells. Furthermore, in vitro studies revealed that ESA inhibited TLR4/NF-κB signaling pathway expression and that TLR4 agonists significantly restored the ESA-induced decrease in Foxp3. DISCUSSION: These findings suggest that ESA suppresses Foxp3 expression by blocking TLR4/NF-κB signaling, resulting in miscarriage. More importantly, the results indicated that miscarriage caused by ESA is TLR4 dependent.

Laminin­332 mediates proliferation, apoptosis, invasion, migration and epithelial­to­mesenchymal transition in pancreatic ductal adenocarcinoma.

The poor prognosis of patients with pancreatic ductal adenocarcinoma (PDAC) is primarily due to the invasive and metastatic behaviors of this disease. Laminin­332 (LM­332) is a key component of the basement membrane barrier, and is associated with tumor metastasis. The present study provides evidence towards the potential function of LM­332 in carcinoma, indicating the distinct roles of the three LM­332 subunits (α3, ß3 and γ2) in cell proliferation, migration, invasion, apoptosis and the epithelial­to­mesenchymal transition (EMT) in cancer. The roles of the α3, ß3 and γ2 subunits in the malignant biological behavior of PDAC were investigated in the present study. It was revealed that the α3, ß3 and γ2 subunits were upregulated in PDAC. Inhibition of all LM­332 subunits abrogated the tumorigenic outcomes, which included cell proliferation, apoptosis, invasion, migration and EMT in vitro. However, the three LM­332 subunits had different degrees of effects on biological behavior. It was observed that LAMA3 (α3) had a stronger effect on cell proliferation, migration and invasion. In addition, LAMB3 (ß3) knockdown significantly increased E­cadherin levels and decreased vimentin levels, indicating that LAMB3 was associated with EMT. Likewise, LAMC2 (γ2) mediated proliferation, apoptosis, invasion and migration. However, small interfering (si)­LAMC2 promoted the progression of EMT, which was the opposite effect to that of si­LAMB3. The LM­332 subunits (α3, ß3 and γ2) may be novel therapeutic targets of PDAC in the future.

High ACR level is a strong risk factor for renal tubular impairment in patients with type 2 diabetes: A longitudinal observational study.

BACKGROUND: Several studies have indicated that high albuminuria is associated with renal function decline. However, the relationship between the urinary albumin-to-creatinine ratio (ACR) and risk of developing tubular injury remains unclear. Our aim was to investigate the association of ACR with the risk of developing tubular impairment in patients with type 2 diabetes. METHODS: This longitudinal observational study compared baseline with follow-up data in 183 patients with type 2 diabetes. ACR, urinary alpha-1-microglobulin-to-creatinine ratio (A1MCR) and estimated glomerular filtration rate (eGFR) were used to evaluate albuminuira, tubular injury and glomerular filtration function, respectively. RESULTS: Levels of high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and A1MCR were significantly different at the two-year follow-up compared with baseline levels. Among patients both with baseline ACR above and below 30 mg/g, the percentage with A1MCR > 15 mg/g clearly increased after follow-up (P < 0.05). The risk of A1MCR rising from normal ranges to >15 mg/g over the follow-up increased with increasing baseline ACR values lower baseline eGFR. Among the patients with baseline ACR > 63.10 mg/g, all showed increased A1MCR values at follow-up compared with baseline. In the multivariate regression analysis, the patients with baseline ACR > 63.10 mg/g had a strong risk of A1MCR rising from normal to >15 mg/g (odds ratio (OR) = 11.12, P = 0.001) over the follow-up, while the males had a 2.89-fold risk of A1MCR increasing from normal to >15 mg/g compared with females. CONCLUSION: Baseline ACR level is related to increased risk of developing renal tubular injury; in particular, this association is much stronger in patients with type 2 diabetes and baseline ACR > 63.10 mg/g.

TRPA1 and substance P mediate stress induced duodenal lesions in water immersion restraint stress rat model.

BACKGROUND/AIMS: Transient receptor potential ankyrin 1 (TRPA1) and substance P (SP), both expression in sensory neurons, have important roles in stress-induced duodenal lesions. The possible contribution of TRPA1 and SP to stress-induced duodenal lesions was explored by using the water immersion restraint stress (WIRS) rat model. MATERIALS AND METHODS: Western blotting, Real-time polymerase chain reaction (RT-PCR), and immunohistochemistry assay were used to evaluate the changes of TRPA1and SP expression in the dorsal root ganglia (DRG, T8-11), the corresponding segment of the spinal cord (T8-11), and the duodenum in a duodenal lesions rat model. The SP concentrations of duodenal mucosa were investigated using an enzyme-linked immunosorbent assay (ELISA). Duodenal lesions were assessed according to histopathological changes. TRPA1 specific antagonist HC-030031 was intrathecally or intraperitoneally performed to suppress the expression of both TRPA1 and SP for evaluating the roles of TRPA1 and SP in duodenal lesions. RESULTS: In contrast to the control group, TRPA1 and substance P in the DRG (T8-11) and duodenum were up-regulated, and concentrations of SP in the duodenal mucosa were increased after WIRS (p<0.05), which are closely associated with duodenal lesions. SP concentrations in the duodenal mucosa were decreased and duodenal lesions were alleviated by pretreatment with TRPA1 antagonist HC-030031. We identified a protective role for HC-030031 in WIRS-induced duodenal lesions. Furthermore, we demonstrated that WIRS increased the concentrations of SP in the duodenal mucosa in a TRPA1-dependent manner. However, WIRS caused no significant changes of TRPA1 and SP in the spinal cord (T8-11) compared with the control group (p>0.05). CONCLUSION: Our study indicates that TRPA1 antagonist HC-030031 alleviates duodenal lesions. TRPA1 is activated and sensitized, therefore concomitant neuropeptide SP is released, which exerts a critical role in inducing and maintaining duodenal lesions following WIRS in rats. This provides evidence that neuroimmune interactions may control duodenal injury. TRPA1 may be a potential drug target to inhibit the development of duodenal lesions by stress-induced in patients.

Soluble egg antigens of Schistosoma japonicum induce senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway.

Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. Based on previously observed anti-fibrotic effects of soluble egg antigens from Schistosoma japonicum in vitro, we hypothesized that SEA might play a crucial role in alleviating liver fibrosis through promoting senescence of activated HSCs. We show here that SEA inhibited expression of α-SMA and pro-collagen I and promoted senescence of activated HSCs in vitro. In addition, SEA induced an increased expression of P-p53 and p21. Knockdown of p53 inhibited the expression of p21 and failed to induce senescence of activated-HSCs. Phosphorylated STAT3 was elevated upon SEA stimulation, while loss of STAT3 decreased the level of p53 and senescence of HSCs. Results from immunoprecipitation analysis demonstrated that SOCS3 might be involved in the SEA-induced senescence in HSCs through its interaction with p53. This study demonstrates the potential capacity of SEA in restricting liver fibrosis through promoting senescence in HSCs. Furthermore, a novel STAT3-p53-p21 pathway might participate in the observed SEA-mediated senescence of HSCs. Our results suggest that SEA might carry potential therapeutic effects of restraining liver fibrosis through promoting senescence.

Egg antigen p40 of Schistosoma japonicum promotes senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway.

Liver fibrosis is a serious disease that is characterized by the excess deposition of extracellular matrix (ECM) components. Activated hepatic stellate cells (HSCs) are a major source of ECM and serve as a key regulator in liver fibrogenesis. Inactivation of HSCs is essential for liver fibrotic regression. The present study explores the underlying mechanisms of Schistosoma japonicum egg antigen p40 (Sjp40) promoting senescence in HSCs and antifibrosis. For the first time we report that Sjp40 inhibits the activation and proliferation of an immortalized human HSC line (LX-2 cells) and promotes cellular senescence and cell cycle arrest. Sjp40 through action on the STAT3/p53/p21 pathway triggered cellular senescence, while knockdown of p53 or STAT3 partly restored cell senescence. In addition, Sjp40-induced cellular senescence caused LX-2 cells to be more sensitive to a human NK cell line (YT cells). Together these findings provide novel insights into the mechanism of antifibrosis and may have implications for the development of antifibrosis therapies.

Septin4_i1 regulates apoptosis in hepatic stellate cells through peroxisome proliferator-activated receptor-γ/Akt/B-cell lymphoma 2 pathway.

Apoptosis of activated hepatic stellate cells (HSCs) has been verified as a potential mechanism to aid in hepatic fibrosis remission. Earlier research suggests that Septin4_i1 may sensitize hepatocellular carcinoma cells to serum starvation-induced apoptosis. Here, we aimed to investigate the effect of Septin4_i1 on HSC apoptosis and explore the associated signaling pathways. We found that Septin4_i1 can induce apoptosis in LX-2 cells and that this is accompanied by an up-regulation in cleaved-caspase-3 and peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and a down-regulation in α-SMA expression. Over-expression of Septin4_i1 reduced phosphorylated Akt and B-cell lymphoma 2 (Bcl-2) expression but had no effect on the expression of p53 and death receptor (DR)-5. The decreased expression of Bcl-2 and the increased expression of cleaved-caspase-3 induced by Sept4_i1 could be reversed by GW501516, a PPAR-ß/δ agonist that has been reported by others to enhance Akt signaling. In addition, GW9662, an antagonist of PPAR-γ, could also inhibit apoptosis in LX-2 cells induced by Sept4_i1. In conclusion, our data suggest that Sept4_i1 induces HSC apoptosis by inhibiting Akt and Bcl-2 expression and up-regulating PPAR-γ expression.