Low-dose clarithromycin therapy modulates CD4(+) T-cell responses in a mouse model of chronic Pseudomonas aeruginosa lung infection.

BACKGROUND AND OBJECTIVE: Low-dose clarithromycin (CAM) is widely used for the treatment of chronic respiratory infections. However, its anti-inflammatory mechanisms have not been fully explored. As CD4(+) T cells play an important role in the initiation of immune responses to infectious microorganisms, we aimed to investigate the effects of low-dose CAM on CD4(+) T-cell responses. METHODS: Fifty-four BALB/c mice were randomly divided into three groups: a control group (inoculated with sterile agarose beads and treated with saline from day 7), a saline group (inoculated with Pseudomonas aeruginosa-loaded beads and treated with saline from day 7) and a CAM group (identical to the saline group, except that saline was replaced by CAM solution). Bronchoalveolar lavage (BAL) fluid cell counts, bacterial load, lung tissue histology and pulmonary CD3(+) CD4(+) cell numbers were assessed. Levels of T helper (Th)1/Th2/Th17 cytokines and suppressor cytokines (interleukin (IL)-10 and transforming growth factor-ß1) were analysed. Messenger RNA (mRNA) levels for transcription factors for CD4(+) T-cell subsets were determined. RESULTS: The CAM group had lower BAL fluid cell counts, pathological scores and pulmonary CD3(+) CD4(+) cell numbers compared with the saline group, whereas the bacterial load was not significantly different. Levels of Th1/Th17 cytokines and expression of a transcription factor for naturally occurring regulatory T cells (Treg) were significantly decreased in the CAM group compared with the saline group, whereas there was no significant difference in GATA-3 mRNA expression. CONCLUSIONS: This study demonstrated a downregulation of Th1/Th17/naturally occurring Treg responses after treatment with low-dose CAM in mice with chronic P. aeruginosa lung infection.

Effect of Lactobacillus plantarum LP-Onlly on gut flora and colitis in interleukin-10 knockout mice.

BACKGROUND AND AIM: Probiotics are used in the therapy of inflammatory bowel disease. This study aimed to determine the effects of probiotic Lactobacillus plantarum LP-Onlly (LP) on gut flora and colitis in interleukin-10 knockout (IL-10(-/-) ) mice, a model of spontaneous colitis. METHODS: IL-10(-/-) and wild-type mice were used at 8 weeks of age and LP by gavage was administered at a dose of 10(9) cells/day per mice for 4 weeks. Mice were maintained for another one week without LP treatment. The colonic tissues were collected for histological and ultrastructural analysis at death after 4 weeks treatment of LP, and the feces were collected at 1-week intervals throughout the experiment for the analysis of gut flora and LP using selective culture-based techniques. RESULTS: Compared with control mice, IL-10(-/-) mice developed a severe intestinal inflammation and tissue damage, and had an abnormal composition of gut microflora. LP administration attenuated colitis with the decreased inflammatory scoring and histological injury in the colon of IL-10(-/-) mice. In addition, LP administration increased the numbers of beneficial total bifidobacteria and lactobacilli, and decreased the numbers of potential pathogenic enterococci and Clostridium perfringens, although the decrease of coliforms was not significant after LP treatment in IL-10(-/-) mice. CONCLUSIONS: Oral administration of LP was effective in the treatment of colitis, with the direct modification of gut microflora in IL-10(-/-) mice. This probiotic strain could be used as a potential adjuvant in the therapy of inflammatory bowel disease, although further studies are required in human.

Lactobacillus plantarum ameliorates colonic epithelial barrier dysfunction by modulating the apical junctional complex and PepT1 in IL-10 knockout mice.

Probiotics are efficacious in the treatment of inflammatory bowel disease. However, the precise mechanisms remain unknown. To determine whether probiotic Lactobacillus plantarum (LP) ameliorates colonic epithelial barrier dysfunction present in interleukin-10 knockout (IL-10⁻(/)⁻) mice, IL-10⁻(/)⁻ and wild-type mice received LP or the vehicle for 4 wk. Colitis was assessed by histological scores and clinical manifestation, and gut paracellular permeability was measured by Ussing chamber. Oligopeptide transporter 1 (PepT1)-mediated transepithelial transport was evaluated by measuring the plasma cephalexin concentration. The expression and distribution of apical junctional complex (AJC) proteins and PepT1 were determined by Western blotting and immunofluorescence and their mRNA by reverse transcriptase-PCR. Spontaneous colitis was observed in all IL-10⁻(/)⁻ mice in which paracellular permeability was increased, in conjunction with decreased expression and redistribution of zonula occludens-1, occludin, claudin-1, and ß-catenin. PepT1 expression was increased, accompanied with an enhanced cephalexin transport. Colonic epithelial barrier dysfunction was further confirmed by increased bacterial translocation and proinflammatory cytokine production. Treatment with LP decreased colonic paracellular permeability with restoration of expression and distribution of AJC proteins and partially prevented PepT1 expression and cephalexin transport in IL-10⁻(/)⁻ mice. Moreover, treatment with LP also prevented bacterial translocation and proinflammatory cytokine production in IL-10⁻(/)⁻ mice. Results from this study indicated that treatment with LP may ameliorate colonic epithelial barrier dysfunction in IL-10⁻(/)⁻ mice, by modulating the AJC- and PepT1-mediated transepithelial transport.

[Effects of erythromycin on the twitching motility of Pseudomonas aeruginosa].

OBJECTIVE: To observe the effects of low concentration of erythromycin on the twitching motility of Pseudomonas aeruginosa. METHODS: Pseudomonas aeruginosa 1244 (PA1244) was cultured in LB plate or LB broth with added erythromycin at different concentrations 2.5, 0.5, 0.25 mg/L, and in cultures without erythromycin as the control. The changes of PA1244's twitching motility was observed by naked eyes, immunofluorescence, Western blot, dot blot and electron microscope. RESULTS: SubMIC erythromycin inhibited the halation of twitching motility on the culture plates. The average diameters of bacterial halation after culture for 18 h were as below: group of 2.5 mg/L was (0.48 +/- 0.14) cm, group of 0.5 mg/L was (0.64 +/- 0.20) cm, group of 0.25 mg/L was (0.95 +/- 0.18) cm; the control group was (1.40 +/- 0.21) cm (F = 123.15, P < 0.01). After culture for 24 h: group of 2.5 mg/L was (0.67 +/- 0.12) cm, group of 0.5 mg/L was (0.82 +/- 0.23) cm, group of 0.25 mg/L was (1.18 +/- 0.24) cm; the control group was (1.58 +/- 0.28) cm (F = 76.37, P < 0.01). After culture for 36 h: group of 2.5 mg/L was (0.91 +/- 0.17) cm, group of 0.5 mg/L was (1.04 +/- 0.32) cm, group of 0.25 mg/L was (1.49 +/- 0.31) cm; the control group was (2.07 +/- 0.38) cm (F = 54.75, P < 0.01). Immunofluorescence showed that the key component of twitching motility was pilus VI located at the pole of the PA body. Western blot showed that the expression of pilus VI was increased with the decreasing concentration of erythromycin. Dot blot showed that pilus VI was expressed mostly at the outmost of the twitching zone and there was no significant difference between groups. Through transmission electron microscope, PA of the group of 2.5 mg/L had fewer pilus than the control group. CONCLUSION: Diverse concentrations of erythromycin have inhibitory actions on the twitching motility of Pseudomonas aeruginosa.

Influences of enteral nutrition combined with probiotics on gut microflora and barrier function of rats with abdominal infection.

AIM: To investigate the influences of enteral, parenteral nutrition and probiotics delivered by gut on intestinal microecology, epithelial tight junctions, immune and barrier function of rats with abdominal infection. METHODS: Rat abdominal infection models established with cecal ligation and perforation method, were divided into three groups: parenteral nutrition (PN group, n = 7), PN+enteral nutrition (EN group, n = 7) and PN + EN + probiotics (probiotics group, n = 7) via the needle jejunostomy and neck vein for five days. The total nutritional supplement of the three groups was isonitrogenic and isocaloric. Probiotics was delivered by jejunostomy 10 mL/d (1 x 10(8) cfu/mL). The rats were killed on the sixth day. The feces in the cecum were cultured for anaerobic bacterial growth and analyzed with bacterial group DNA fingerprint profile with random amplified polymorphic DNA. The transmembrane binding proteins (occludin) and IgA level in plasma cells of intestine epithelium in colon and terminal ileum were measured by an immunohistochemistry method. The ultrastructure of intestinal epithelial tight junctions in colon and small intestine was observed by electron-microscopy. Vena cava blood and the homogenated tissue of liver, lung and mesenteric lymph nodes were cultured to determine the bacterial translocations, and endotoxin in the blood from portal vein was detected. RESULTS: (1) The amount of bacteria of gut species in EN group and probiotic group was higher than that in PN group. The DNA-profiles in EN group and probiotic group were similar to that of normal rats. The number of DNA-profiles in probiotics group was much more than that in PN group and EN group. Moreover, there were strange stripes in PN group. (2) The expression of occludin and IgA in the small and large intestine in EN group (2.309 +/- 0.336, 15.440 +/- 2.383) and probiotic group (2.938 +/- 0.515, 16.230 +/- 3.183) was improved as compared with PN group (1.207 +/- 0.587, P < 0.05, 11.189 +/- 2.108, P < 0.01). The expression of occludin in probiotic group (intestine: 2.93 +/- 0.515; cecum: 3.40 +/- 0.617) was higher than that in EN group (intestine: 2.309 +/- 0.336; cecum: 2.076 +/- 0.670; P < 0.05). The expression of IgA, especially in EN group (intestine: 15.440 +/- 2.383) and probiotic EN group (large intestine: 12.516 +/- 1.542) significantly increased as compared with PN group (intestine: 11.189 +/- 2.108; cecum: 10.160 +/- 1.643; P<0.01). The intestinal epithelial tight junctions and microvilli of the probiotic group were more intact than those in the PN group. (3) The bacterial translocations in blood, liver, lung and mesenteric lymph nodes, and the levels of endotoxin were significantly reduced in probiotic (0.082 +/- 0.029) and EN (0.125 +/- 0.040) groups as compared with PN group (0.403 +/- 0.181, P < 0.05). CONCLUSION: Application of EN combined with probiotics could improve the expression of transmembrane binding proteins (occludin) and IgA, correct the intestinal flora disturbance, maintain gut barrier functions and tight junctions, and reduce the occurrence of gut bacterial translocation.

[Characterization of macrolide-resistance mediated by transposable elements Tn1545 and Tn917 in clinical Isolates of Streptococcus pneumoniae].

OBJECTIVE: To investigate the characterization of ermB gene expression and dissemination in macrolide-resistant Streptococcus pneumoniae (Sp) in Shanghai. METHODS: Eighty-six erythromycin-resistant isolates of Sp were isolated from 3 hospitals in Shanghai. E-test or K-B disk diffusion test were used to determine the susceptibility to 12 antibiotics according to the National Committee for Clinical Laboratory Standards. Macrolide resistant genes ermB and mefE, and transposable elements Tn1545 and Tn917 were amplified by PCR. The isolates were divided into Tn1545 and Tn917 groups according to the transposable elements thereof. Double disc test with erythromycin and clindamycin discs divided the isolates into 2 macrolide resistant phenotypes: cMLS(B) (inducible) phenotype and M phenotype (resistant to erythromycin and sensitive to clindamycin). BOX-PCR was used to analyze the homology of the S. pneumoniae. RESULTS: (1) Of the 86 erythromycin-resistant isolates, the positive rates of ermB, mefE, Tn1545, and Tn917 were 94%, 46%, 87% and 7% respectively. Tn1545 and Tn917 were not detected in 5 ermB-mdfE + strains. (2) Most strains in the Tn1545 and Tn917 groups were highly resistant to erythromycin with a MIC50 of 256 microg/ml. The Tn917 group had a lower MIC to beta-lactam antibiotics and lower resistance to tetracycline, levofloxacin, and compound sulfonamide in comparison with the Tn1545 group. (3) The most common macrolide resistance phenotype of the Tn1545 group was cMLS(B) phenotype. Three strains in the Tn917 group had a 194 bp deletion in the promoter region of ermB and an insertion of TAAA motif in the N end of leader peptide, resulting in the change of the ermB gene from inducible to constitutive expression. (4) BOX-PCR showed that Tn1545 and Tn917 might spread horizontally. CONCLUSION: In Shanghai ermB-mediated cMLS(B) is the most prevalent phenotype in macrolide-resistant Streptococcus pneumoniae isolates. Primarily, the ermB gene was carried and spread horizontally by Tn1545.

[Influences of enteral nutrition combined with probiotics on the gut microecology and barrier function of the rats with abdominal infection].

OBJECTIVE: To investigate the influences of enteral nutrition (EN), parenteral nutrition (PN) and probiotics supplement on the intestinal microecology, and barrier function of the rats with abdominal infection. METHODS: Twenty-one Sprague-Dawley (SD) rats with abdominal infection were randomly divided into three groups, and received PN (PN group, n=7), PN+ EN (PN+ EN group, n=7) or PN+ EN+ probiotics (probiotics group, n=7) respectively with isonitrogen and isocaloric nutrition. The rats were sacrificed after six days. The feces in cecum were cultured for anaerobic bacterial growth and DNA fingerprint spectrum was analyzed by randomly amplified polymorphic DNA technique. The transmembrane binding protein (occludin) and IgA levels in colon and terminal ileum were detected by immunohistochemistry method. The bacterial translocation rate and endotoxin level were also measured. RESULTS: The germ numbers of different species were both higher in PN+ EN and probiotic group than those in PN group. The bands of DNA fingerprint spectrum were significantly decreased in PN group, but the bands in both PN+ EN group and probiotic group were similar to that in the normal rats. The expression levels of occludin and IgA in the intestine and colorectum were higher in both PN+ EN group and probiotic group compared with those of PN group (P< 0.05, P< 0.01, respectively), the expression level of occludin was higher in probiotic group than that in PN+ EN group (P< 0.05). The overall bacterial translocation rates and endotoxin levels were significantly reduced in both probiotic and PN+ EN group (P< 0.05), but there was no difference between probiotic group and EN group. CONCLUSION: EN combined with probiotics can increase occluding and IgA expressions, improve the intestinal microecology,maintain the gut barrier function, and decrease the incidence of gut bacterial translocation.

Effect of lactobacillus on the gut microflora and barrier function of the rats with abdominal infection.

AIM: To investigate the effect of probiotics supplemented by gut on the tight junctions of epithelial cells, barrier function and the microflora of rats with abdominal infection. METHODS: After the model of cecal ligation and perforation established, SD rats were divided into two groups: parenteral nutrition (PN) group and PN+probiotics (probiotics) group, PN solution was supplemented by neck vein and probiotics was delivered via the jejunostomy tube for five days. Vena cava blood and the homogenated tissue of liver, lung and mesenteric lymph nodes were cultured to determine the bacterial translocation rate (BTR). The ultra-structure of epithelial tight junctions and microvilli of the gut were observed by electron microscopy; occluding expression was measured by indirect-immune fluorescence method; anaerobic bacterial growth by anaerobic culture and DNA fingerprint of bacterial colonies of the feces by PCR. RESULTS: The quantity of lactobacteria and bifydobacteria in probiotics group was higher than that of PN group. The profiles of DNA fingerprint expression in probiotics group were similar to that in the normal group, a new 16S rDNA sequence appeared in the profile in PN group. The occludin expression, the integrality of the gut epithelial tight junction and microvilli in probiotics group were improved as compared with PN group. The BTR and endotoxin in blood were reduced more significantly in probiotics group as compared with PN group. CONCLUSION: The probiotics could improve the gut microflora disturbance, increase occludin expression, maintain the gut epithelial tight junction and decrease the bacterial translocations rate.