Gadolinium chloride pre-treatment reduces the inflammatory response and preserves intestinal barrier function in a rat model of sepsis.

The inflammatory response is closely associated with sepsis occurrence and progression. Damage to the function of the intestinal mucosal barrier is considered to be the ῾initiation factor᾿ for the development of multiple organ dysfunction syndrome, which is the most severe progression of sepsis. The aim of the present study was to investigate whether gadolinium chloride (GdCl3) could alleviate the systemic inflammatory response and protect the function of the intestinal mucosal barrier in a rat model of sepsis. The mechanism underlying this protective effect was also explored. Sprague-Dawley rats were divided into four groups: Sham, sham + GdCl3, cecal ligation and puncture (CLP; a model of sepsis) and CLP + GdCl3. In each group, blood was collected from the abdominal aorta, and intestinal tissue was collected after 6, 12 and 24 h of successful modeling. Levels of tumor necrosis factor-α, interleukin (IL)-6 and IL-1ß were determined using ELISA. Western blot analysis was used to determine levels of occludin, tight junction protein ZO-1 (ZO-1), myosin light chain kinase 3 (MLCK), NF-κB and caspase-3 in intestinal tissues. Hematoxylin-eosin staining was used to observe the degree of damage to intestinal tissue. The results indicated that in CLP sepsis model rats treated with GdCl3, the release of systemic and intestinal pro-inflammatory factors was reduced and tissue damage was alleviated when compared with untreated CLP rats. Additionally, the expression of occludin and ZO-1 was increased, while that of NF-κB, MLCK, and caspase-3 was reduced in the CLP + GdCl3 rats compared with the CLP rats. GdCl3 may alleviate systemic and intestinal inflammatory responses and reduce the expression of MLCK through inhibition of the activation of NF-kB. The results of the present study also indicated that GdCl3 promoted the expression of occludin and ZO-1. GdCl3 was also demonstrated to reduce cell apoptosis through the inhibition of caspase-3 expression.

Effects of imipenem combined with low-dose cyclophosphamide on the intestinal barrier in septic rats.

Anti-infection therapy combined with immunotherapy is one of the important research approaches for treating sepsis. However, the combination of anti-infection and immunotherapy therapeutic agents may have an adverse effect on intestinal barrier function. In the present study, it was hypothesized that imipenem combined with low-dose cyclophosphamide (CTX) could improve the sepsis survival rate compared with imipenem treatment alone. In addition, the alterations in the intestinal barrier were investigated and the possible mechanisms of altering intestinal barrier function in septic rats treated with imipenem combined with low-dose CTX or imipenem alone were explored. To investigate the effect of imipenem combined with low-dose CTX on the intestinal barrier, the markers of histopathology, intestinal permeability, intestinal epithelial apoptosis, cytokines interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-α, and tight junction proteins zonula occludens (ZO)-1, occludin and claudin-2, were quantitatively and qualitatively evaluated. The results indicated that imipenem combined with low-dose CTX significantly improved the survival rate of rats compared with imipenem alone (P<0.05). However, no significantly difference between the treatment with imipenem combined with low-dose CTX and imipenem treatment alone was indicated with regard to histopathology, intestinal permeability, intestinal epithelial apoptosis and the expression of claudin-2, ZO-1 and TNF-α. However, imipenem combined with low-dose CTX significantly reduced IL-6 and IL-10 expression and significantly increased occludin expression compared with imipenem alone (P<0.05). It was concluded that imipenem combined with low-dose CTX could improve the survival rate of rats with sepsis compared with rats treated with imipenem alone. The present findings suggest that imipenem combined with low-dose CTX may cause damage to the intestinal barrier function and the mechanism may be associated with a reduction in IL-10 expression.

Effect of early fluid resuscitation combined with low dose cyclophosphamide on intestinal barrier function in severe sepsis rats.

To investigate the effect of early fluid resuscitation on intestinal microecology in rats with severe sepsis. The severe sepsis model used was mainly cecal ligation perforation (CLP) model. Male SD rats were randomly divided into five groups: sham, CLP, CLP + normal saline (NS), CLP + cyclophosphamide (CTX), and CLP + NS + CTX. (1) The levels of IL-6, IL-10, and TNF-α in peripheral blood were measured by ELISA. (2) The expression of occludin/ß-action in colonic tissue of mice was examined by Western Blot. (3) The intestinal permeability was measured by FD70 detection. (4) The length of the chorionic membrane was measured by colon histopathological staining. (5) The intestinal epithelial cell apoptosis was measured with the apoptosis index. (1) The rat model of severe sepsis was successfully replicated, and the 7-day survival rate of sepsis mice in each group was analyzed. (2) The expression level of splenic junction protein and the pathological damage in colonic tissue of the severe sepsis mice was significantly different between sham, CLP, CTX, NS, and NS + CTX (P < 0.05). The expression of tight junction protein in the NS + CTX mice was the highest, and the pathological damage was the smallest. (3) The colonic tissue apoptosis and intestinal permeability in the severe sepsis mice were compared with those of the colon tissues (P < 0.05). (4) The expression levels of IL-6, IL-10, and TNF-α in peripheral blood were significantly increased after severe sepsis (P < 0.01). The expression of IL-6 and TNF-alpha in each treatment group decreased (P < 0.05), while the expression of IL-10 in NS + CTX group increased significantly (P < 0.01). (1) We successfully replicated the rat model of severe sepsis. (2) Early fluid intervention and cyclophosphamide treatment can significantly improve the 7-day survival rate of the sepsis mice. (3) The fluid resuscitation and cyclophosphamide can delay intestinal damage to the intestinal tract barrier function and play a protective role.

Comparison of mono- and combination antibiotic therapy for the treatment of Pseudomonas aeruginosa bacteraemia: A cumulative meta-analysis of cohort studies.

It is currently unknown whether antibiotic monotherapy or combination therapy is a more effective treatment for patients with Pseudomonas aeruginosa bacteraemia. The present study consists of a systematic review and meta-analysis of cohort studies in associated studies. The treatment options of monotherapy and combination therapy have been compared, to determine which is more effective against P. aeruginosa bacteraemia. Several electronic bibliographic databases were systematically searched and clinical studies that compared combination therapy with monotherapy for P. aeruginosa bacteraemia were identified. Dersimonian and Laird's random-effects models were used to generate summary estimates of the effects and to assess their association according to different patient characteristics and research quality standards. A total of 17 studies were selected, 3 of which were prospective while the remaining 14 were retrospective. The studies involved a total of 2,504 patients. Significant differences between combination therapy and monotherapy treatment were not found when the data were combined (odds ratio (OR)=0.81, 95% confidence interval (CI)=0.61-1.08; P=0.035). The results demonstrated strength in a number of stratification and sensitivity analyses. The variables used included study type, treatment quality score and survival rate of subgroup analysis. To conduct cumulative meta-analysis, the number of years and samples were calculated. The OR value and 95% CI were stable and demonstrated good change trend. According to the size of the sample order following accumulation, OR values and 95% CI (0.89, 0.76-1.04) exhibited a narrow range. Neither combination therapy or monotherapy exhibited significant effects on the mortality of patients with P. aeruginosa bacteraemia. Future research is required and should include large, well-designed prospective cohorts, and grouped clinical studies.

Effects of peritoneal macrophage autophagy on the immune function of sepsis mice.

OBJECTIVE: To investigate the effect of peritoneal macrophage autophagy on immune function in sepsis mice. METHODS: Seventy-two male BALB/C mice were intraperitoneally injected with LPS to induce sepsis. The mice were randomly divided into six groups: LPS+2 h, LPS+6 h, LPS+12 h, LPS+24 h and LPS+36 h. LPS with a dose of 10 mg/kg was intraperitoneally injected into the abdominal cavity of the sepsis mice, and the control group was injected with the same dose of saline. ELISA was used to detect the concentrations of inflammatory factors IL-2, IL-10 and TNF-α in the peripheral blood, and the CD4+T/CD8+T ratio in the peripheral blood was detected by flow cytometry. The expression levels of LC3II and Beclin-1/beta-action in the mouse macrophages were measured using Western blot to determine the level of autophagy. RESULTS: The expression levels of LC3II and Beclin-1 were significantly higher in the peritoneal macrophages of the mice from the LPS+2 h group than in those of the mice from the normal group (P<0.05). Meanwhile, these levels continuously declined in the LPS+6 h, LPS+12 h, LPS+24 h and LPS+36 h groups (P<0.05). The peripheral blood CD4+T/CD8+T cell ratio was significantly higher in the LPS+2 h and LPS+6 h groups than in the normal group (P<0.05). The ratio peaked at 6 h and then continuously declined (P<0.05). Furthermore, the concentrations of IL-2 and Tnf-α were significantly higher in the peripheral blood serum of the LPS+2 h, LPS+6 h and LPS+12 h groups than in those of the normal group (P<0.05). The peak was observed at 12 h followed by a continuous decline in the LPS+24 h and 3 LPS+6 h groups (P<0.05). The peripheral serum IL-10 concentration was significantly higher in the LPS+2 h, LPS+6 h, LPS+12 h, LPS+24 h and LPS+36 h groups than in the normal group (P<0.05). In the LPS+6 h, LPS+12 h, LPS+24 h and LPS+36 h groups, the peritoneal macrophages LC3II, Beclin-1 and peripheral serum CD+4T/CD+8T ratio correlation index R2=0.716 (P=0.043), R2=0.954 (P=0.023). CONCLUSION: Autophagy in peritoneal macrophages plays an important role in the immune function of sepsis mice. In addition, the autophagy of peritoneal macrophages and the immune function of sepsis mice are strongly correlated. Furthermore, macrophage autophagy plays an important role in the immune function changes in sepsis mice, and the underlying mechanism may be involved in inflammation and macrophage antigen presentation by regulating the secretion of inflammatory cytokines and lymphocyte apoptosis antagonism.

A small hairpin RNA targeting myeloid cell leukemia-1 enhances apoptosis in host macrophages infected with Mycobacterium tuberculosis.

Myeloid cell leukemia-1 (Mcl-1) plays an important role in various cell survival pathways. Some studies indicated that the expression of Mcl-1 was upregulated in host cells during infection with the virulent Mycobacterium tuberculosis strain, H37Rv. The present study was designed to investigate the effect of inhibiting Mcl-1 expression both in vivo and in vitro on apoptosis of host macrophages infected with M. tuberculosis using a small hairpin (sh)RNA. Mcl-1 expression was detected by the real time-polymerase chain reaction, western blotting, and immunohistochemistry. Flow cytometry and transmission electron microscopy were used to measure host macrophage apoptosis. We found elevated Mcl-1 levels in host macrophages infected with M. tuberculosis H37Rv. The expression of Mcl-1 was downregulated efficiently in H37Rv-infected host macrophages using shRNA. Knockdown of Mcl-1 enhanced the extent of apoptosis in H37Rv-infected host macrophages significantly. The increased apoptosis correlated with a decrease in M. tuberculosis colony forming units recovered from H37Rv-infected cells that were treated with Mcl-1-shRNA. Reducing Mcl-1 accumulation by shRNA also reduced accumulation of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax, in H37Rv-infected host macrophages. Our results showed that specific knockdown of Mcl-1 expression increased apoptosis of host macrophages significantly and decreased the intracellular survival of a virulent strain of M. tuberculosis. These data indicate that interference with Mcl-1 expression may provide a new avenue for tuberculosis therapy.

Suppression of Mcl-1 induces apoptosis in mouse peritoneal macrophages infected with Mycobacterium tuberculosis.

The effect of myeloid cell leukemia-1 (Mcl-1) inhibition on apoptosis of peritoneal macrophages in mice infected with Mycobacterium tuberculosis was investigated and the primary signaling pathway associated with the transcriptional regulation of Mcl-1 was identified. Real-time PCR and western blotting indicated that Mcl-1 transcript and protein expression are upregulated during infection with virulent M. tuberculosis H37Rv and Xinjiang strains but not with attenuated M. tuberculosis strain H37Ra or Bacillus Calmette-Guérin. Mcl-1 transcript and protein expression were downregulated by specific inhibitors of the Janus kinase/signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways (AG490, PD98059 and LY294002, respectively). The strongest inhibitor of Mcl-1 expression was PD98059, the MAPK inhibitor. Flow cytometry demonstrated that the rate of apoptosis in peritoneal macrophages is significantly higher in mice infected with M. tuberculosis and the rate of apoptosis is correlated with the virulence of the strain of M. tuberculosis. Apoptosis was found to be upregulated by AG490, PD98059 and LY294002, whereas inhibition of the MAPK pathway sensitized the infected macrophages to apoptosis. Taken together, these results suggest that specific downregulation of Mcl-1 significantly increases apoptosis of peritoneal macrophages and that the MAPK signaling pathway is the primary mediator of Mcl-1 expression.

[Identification of A Novel HLA Allele DRB1 * 16:36 by Sequencing-Based Typing].

OBJECTIVE: To identify a novel HLA allele DRB1 * 16:36 from a Uygur woman. METHODS: PCR-SBT technology was applied to the extracted DNA for genotyping, and a possible new gene was sequenced by using sequence specific primers and single stranded SBT. This novel allele was compared with known most homologous gene sequences and their difference was analyzed. RESULTS: This novel allele was different from HLA alle DRB1 * 16:23, and had highest similarity in 2 nucleotides at position 227 A→T and 236 T→C in exon 2, resulting in 3 amino acid changes from Tyr to Phe at codon 47 and Val to Ala at codon 50. The sequence of this novel allele had been submitted to GenBank. CONCLUSION: This HLA allele DRB1 * 16:23 has been confirmed to be a novel allele, and has been officially named DRB1 * 16:36 by the World Health Organization (WHO) Nomenclature Committee in May 2015.