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The impact of Pseudomonas aeruginosa (PA) was explored in a mouse model with non-diarrheal gut permeability defect using 1.5% dextran sulfate solution (DSS) plus antibiotics (ATB) with or without orally administered PA. As such, ATB+DSS+PA mice induced more severe intestinal injury as indicated by stool consistency and leaky gut (FITC-dextran assay, bacteremia, and endotoxemia) with an increase in serum cytokines, liver enzyme, and hepatocyte apoptosis when compared with ATB+DSS mice. There was no abnormality by these parameters in the non-DSS group, including water alone (control), antibiotics alone (ATB+water), and antibiotics with PA (ATB+water+PA). Despite a similarly fecal microbiome patterns between ATB+DSS and ATB+DSS+PA groups, a higher abundance of Pseudomonas, Enterococci, and Escherichia-Shigella was detected in ATB+DSS+PA mice. Additionally, the additive pro-inflammation between pathogen molecules, using heat-killed P. aeruginosa preparations, and LPS against enterocytes (Caco2) and hepatocytes (HegG2), as indicated by supernatant IL-8 and expression of several genes (IL-8, NF-kB, and NOS2) are demonstrated. In conclusion, presence of P. aeruginosa in the gut exacerbated DSS-induced intestinal injury with spontaneous translocation of LPS and bacteria from the gut into the blood circulation (leaky gut) that induced more severe systemic inflammation. The presence of pathogenic bacteria, especially PA in stool of the healthy individuals might have some adverse effect. More studies are in needed.

BackgroundMost of the current bacteriophages (phages) are mostly isolated from environments. However, phages isolated from feces might be more specific to the bacteria that are harmful to the host. Meanwhile, some phages from the environment might affect non-pathogenic bacteria for the host.MethodsHere, bacteriophages isolated from mouse feces were intratracheally (IT) or intravenously (IV) administered in pneumonia mice caused by Pseudomonas aeruginosa at 2 hours post-intratracheal bacterial administration. As such, the mice with phage treatment, using either IT or IV administration, demonstrated less severe pneumonia as indicated by mortality, serum cytokines, bacteremia, bacterial abundance in bronchoalveolar lavage fluid (BALF), and neutrophil extracellular traps (NETs) in lung tissue (immunofluorescence of neutrophil elastase and myeloperoxidase).ResultsInterestingly, the abundance of phages in BALF from the IT and IV injections was similar, supporting a flexible route of phage administration. With the incubation of bacteria with neutrophils, the presence of bacteriophages significantly improved bactericidal activity, but not NETs formation, with the elevated supernatant IL-6 and TNF-α, but not IL-1β. In conclusion, our findings suggest that bacteriophages against Pseudomonas aeruginosa can be discovered from feces of the host.ConclusionsThe phages attenuate pneumonia partly through an enhanced neutrophil bactericidal activity, but not via inducing NETs formation. The isolation of phages from the infected hosts themselves might be practically useful for future treatment. More studies are warranted.

Although patients with either β-thalassemia or chronic kidney disease (CKD) clinically correlate with severe osteoporosis, the mechanism by which CKD exposed to high phosphate affects bone turnover has not been characterized in β-thalassemia. We aimed to determine the effects of renal insufficiency on high phosphate intake induced changes in bone metabolism after 5/6th nephrectomy in hemizygous β-globin knockout (BKO) mice. Male BKO mice manifested severe anemia and osteopenia. Nephrectomy induced renal fibrosis and reduced renal function as assessed by increased serum urea nitrogen levels. Moreover, nephrectomy increased bone turnover leading to bone loss in wild type (WT) but not BKO mice. In nephrectomized BKO, PBS in drinking water induced hyperphosphatemia, and hypercalcemia along with osteopenia in both cancellous and cortical bone. Histomorphometric analysis confirmed reduced cancellous bone volume due to decreased bone formation rate, osteoblast number and osteoclast number. The mRNA levels for Alpl, Sp7, Kl, Tnfsf11, and Tnfsf11/Tnfrsf11b were decreased in nephrectomized BKO mice drinking PBS. Interestingly, Fgf23, a bone-derived hormone produced by osteocytes and osteoblasts in response to hyperphosphatemia, were remarkably increased in nephrectomized BKO mice following PBS intake. Serum FGF23 and erythropoietin levels were markedly elevated in BKO mice. Nephrectomy decreased serum erythropoietin but not FGF23 levels. Hyperphosphatemia in BKO mice increased serum erythropoietin, FGF23, and PTH levels, nominating these factors as candidate mediators of bone loss in thalassemic mice with CKD during phosphate retention.

Patients with systemic lupus erythematosus are at increased risk for alveolar bone loss due to periodontitis possibly as a result of a pathogenic immune response to oral bacteria and inflammation. The aim of the present study was to investigate whether an anti-TNF-α antagonist could prevent mandibular bone loss in the FcγRIIb-/- mouse model of lupus. Mice lacking FcγRIIb had decreased cancellous and cortical bone volume at 6 months of age. Etanercept increased cancellous but not cortical bone volume in WT and increased both cancellous bone volume and cortical thickness in FcγRIIb-deficient mice. FcγRIIb deficiency decreased mRNA levels for osteoblast marker genes, Osx, Col1a1 and Alp without any change in osteoclast marker genes. Etanercept increased Osx, Alp, and Ocn in both WT and FcγRIIb-/- mice. Osteoclast marker genes including TNF-α, Trap and RANKL/OPG ratio was decreased in WT. Serum markers of proinflammatory cytokines, TNF-α, IFNγ, IL-6, and IL-17A, were increased in FcγRIIb-/- mice and etanercept antagonized these effects in FcγRIIb-/- mice. Etanercept increased serum PTH levels in the FcγRIIb-/- mouse model of lupus. Our results suggest that deletion of FcγRIIb induces osteopenia by increasing the level of proinflammatory cytokines. Etanercept is effective in preventing mandibular bone loss in FcγRIIb-/- mice, suggesting that anti-TNF-α therapy may be able to ameliorate mandibular bone loss in SLE patients with periodontitis.

Because of a possible impact of capsaicin in the high concentrations on enterocyte injury (cytotoxicity) and bactericidal activity on probiotics, Lactobacillus rhamnosus L34 (L34) and Lactobacillus rhamnosus GG (LGG), the probiotics derived from Thai and Caucasian population, respectively, were tested in the chili-extract administered C57BL/6 mice and in vitro experiments. In comparison with placebo, 2 weeks administration of the extract from Thai chili in mice caused loose feces and induced intestinal permeability defect as indicated by FITC-dextran assay and the reduction in tight junction molecules (occludin and zona occludens-1) using fluorescent staining and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the chili extracts also induced the translocation of gut pathogen molecules; lipopolysaccharide (LPS) and (1→3)-β-d-glucan (BG) and fecal dysbiosis (microbiome analysis), including reduced Firmicutes, increased Bacteroides, and enhanced total Gram-negative bacteria in feces. Both L34 and LGG attenuated gut barrier defect (FITC-dextran, the fluorescent staining and gene expression of tight junction molecules) but not improved fecal consistency. Additionally, high concentrations of capsaicin (0.02-2 mM) damage enterocytes (Caco-2 and HT-29) as indicated by cell viability test, supernatant cytokine (IL-8), transepithelial electrical resistance (TEER) and transepithelial FITC-dextran (4.4 kDa) but were attenuated by Lactobacillus condition media (LCM) from both probiotic-strains. The 24 h incubation with 2 mM capsaicin (but not the lower concentrations) reduced the abundance of LGG (but not L34) implying a higher capsaicin tolerance of L34. However, Lactobacillus rhamnosus fecal abundance, using qRT-PCR, of L34 or LGG after 3, 7, and 20 days of the administration in the Thai healthy volunteers demonstrated the similarity between both strains. In conclusion, high dose chili extracts impaired gut permeability and induced gut dysbiosis but were attenuated by probiotics. Despite a better capsaicin tolerance of L34 compared with LGG in vitro, L34 abundance in feces was not different to LGG in the healthy volunteers. More studies on probiotics with a higher intake of chili in human are interesting.

The attenuation of hyper-inflammation in sepsis with the administration of anti-inflammatory macrophages is an interesting adjuvant therapy for sepsis. Because the induction of anti-inflammatory macrophages by microRNA (miR), a regulator of mRNA, has been mentioned, the exploration on miR-induced anti-inflammatory macrophages was performed. The over-expression of miR-223 and miR-146a in RAW264.7 induced M2 macrophage-polarization (anti-inflammatory macrophages) as evaluated by the enhanced expression of Arginase-1 and Fizz. However, miR-223 over-expressed cells demonstrated the more potent anti-inflammatory property against LPS stimulation as lesser iNOS expression, lower supernatant IL-6 and higher supernatant IL-10 compared with miR-146a over-expressed cells. Interestingly, LPS stimulation in miR-223 over-expressed cells, compared with LPS-stimulated control cells, demonstrated lower activity of glycolysis pathway and higher mitochondrial respiration, as evaluated by the extracellular flux analysis, and also down-regulated HIF-1α, an important enzyme of glycolysis pathway. In addition, the administration of miR-223 over-expressed macrophages with IL-4 pre-conditioning, but not IL-4 stimulated control cells, attenuated sepsis severity in LPS injected mice as evaluated by serum creatinine, liver enzymes, lung histology and serum cytokines. In conclusion, miR-223 interfered with the glycolysis pathway through the down-regulation of HIF-1α, resulting in the anti-inflammatory status. The over-expression of miR-223 in macrophages prevented the conversion into M1 macrophage polarization after LPS stimulation. The administration of miR-223 over-expressed macrophages, with IL-4 preconditioning, attenuated sepsis severity in LPS model. Hence, a proof of concept in the induction of anti-inflammatory macrophages through the cell-energy interference for sepsis treatment was proposed as a basis of cell-based therapy in sepsis.

Gut fungi may influence the course of Clostridium difficile infection either positively or negatively for the host. Fungi are not prominent in the mouse gut, and C. albicans, the major human gastrointestinal commensal yeast, is in low abundance or absent in mice. Bifidobacterium is one of the probiotics that may attenuate the severity of C. difficile infection. Inflammatory synergy between C. albicans and C. difficile, in gut, may provide a state that more closely resembles human infection and be more suitable for testing probiotic effects. We performed fecal mycobiota analysis and administered C. albicans at 1 day prior to C. difficile dosing. Fecal eukaryotic 18S rDNA analysis demonstrated the presence of Ascomycota, specifically, Candida spp., after oral antibiotics, despite negative fecal fungal culture. C. albicans administration enhanced the severity of the C. difficile infection model as determined by mortality rate, weight loss, gut leakage (FITC-dextran assay), and serum and intestinal tissue cytokines. This occurred without increased fecal C. difficile or bacteremia, in comparison with C. difficile gavage alone. Candida lysate with C. difficile increased IL-8 production from HT-29 and Caco-2 human intestinal epithelial cell-lines. Bifidobacterium attenuated the disease severity of the C. difficile plus Candida model. The reduced severity was associated with decreased Candida burdens in feces. In conclusion, gut C. albicans worsened C. difficile infection, possibly through exacerbation of inflammation. Hence, a mouse model of Clostridium difficile infection with C. albicans present in the gut may better model the human patient condition. Gut fungal mycobiome investigation in patients with C. difficile is warranted and may suggest therapeutic targets.

Despite tacrolimus (TAC) drug-level monitoring, TAC-induced chronic renal allograft fibrosis remains an important problem. This study investigated the potential of urinary neutrophil gelatinase-associated lipocalin (uNGAL) as a chronic renal allograft fibrosis biomarker in a two-phase study (proof of concept and cohort). In the proof of concept stage of the study, increased TAC-doses at 3 days after dose adjustment compared with the baseline were associated with elevated uNGAL (+ΔuNGAL) and urinary interleukin 18 (IL-18), but normal serum creatinine (SCr), despite the therapeutic trough levels of TAC. In the cohort study, the patients with elevated uNGAL post-recruitment in comparison with the baseline (+ΔuNGAL) was associated with the more severe renal allograft fibrosis from renal pathology of the protocol biopsy at 12 months post kidney transplantation (post-KT). A cut-off value of uNGAL ≥ 125.2 ng/mL during a 3, 6, 9 and 12 months post-KT was associated with a higher fibrosis score, with an area under the receiver operating characteristics curve of 0.80 (95% confidence interval [CI] 0.72 to 0.88, p < 0.0001) and a hazard ratio (HR) of 2.54 (95% CI 1.45 to 9.33; p < 0.001). We conclude that uNGAL is a sensitive biomarker of TAC induced subtle renal injury and TAC-induced chronic renal allograft fibrosis. We propose that uNGAL measurements, in addition to trough levels of TAC, should be used to predict TAC-induced chronic renal allograft fibrosis in the recipients of KT.

Intimal hyperplasia (IH) is a common cause of vasculopathy due to direct endothelial damage (such as post-coronary revascularization) or indirect injury (such as chronic kidney disease, or CKD). Although the attenuation of coronary revascularization-induced IH (direct-vascular-injury-induced IH) by cilostazol, a phosphodiesterase III inhibitor, has been demonstrated, our understanding of the effect on CKD-induced IH (indirect-vascular-injury-induced IH) is limited. Herein, we tested if cilostazol attenuated CKD-induced IH in a mouse model of ischemic-reperfusion injury with unilateral nephrectomy (Chr I/R), a normotensive non-proteinuria CKD model. Cilostazol (50 mg/kg/day) or placebo was orally administered once daily from 1-week post-nephrectomy. At 20 weeks, cilostazol significantly attenuated aortic IH as demonstrated by a 34% reduction in the total intima area with 50% and 47% decreases in the ratios of tunica intima area/tunica media area and tunica intima area/(tunica intima + tunica media area), respectively. The diameters of aorta and renal function were unchanged by cilostazol. Interestingly, cilostazol decreased miR-221, but enhanced miR-143 and miR-145 in either in vitro or aortic tissue, as well as attenuated several pro-inflammatory mediators, including asymmetrical dimethylarginine, high-sensitivity C-reactive protein, vascular endothelial growth factor in aorta and serum pro-inflammatory cytokines (IL-6 and TNF-α). We demonstrated a proof of concept of the effectiveness of cilostazol in attenuating IH in a Chr I/R mouse model, a CKD model with predominantly indirect-vascular-injury-induced IH. These considerations warrant further investigation to develop a new primary prevention strategy for CKD-related IH.

Candida albicans is the most common fungus in the human intestinal microbiota but not in mice. To make a murine sepsis model more closely resemble human sepsis and to explore the role of intestinal C. albicans, in the absence of candidemia, in bacterial sepsis, live- or heat-killed C. albicans was orally administered to mice at 3h prior to cecal ligation and puncture (CLP). A higher mortality rate of CLP was demonstrated with Candida-administration (live- or heat-killed) prior to CLP. Fecal Candida presented only in experiments with live-Candida administration. Despite the absence of candidemia, serum (1→3)-β-D-glucan (BG) was higher in CLP with Candida-administration than CLP-controls (normal saline administration) at 6h and/or 18h post-CLP. Interestingly, fluconazole attenuated the fecal Candida burden and improved survival in mice with live-Candida administration, but not CLP-control. Microbiota analysis revealed increased Bacteroides spp. and reduced Lactobacillus spp. in feces after Candida administration. Additionally, synergy in the elicitation of cytokine production from bone marrow-derived macrophages, in vitro, was demonstrated by co-exposure to heat-killed E. coli and BG. In conclusion, intestinal abundance of fungi and/or fungal-molecules was associated with increased bacterial sepsis-severity, perhaps through enhanced cytokine elicitation induced by synergistic responses to molecules from gut-derived bacteria and fungi. Conversely, reducing intestinal fungal burdens decreased serum BG and attenuated sepsis in our model.

Critically ill patients with acute kidney injury (AKI) who receive renal replacement therapy (RRT) have very high mortality rate. During RRT, there are markedly loss of macro- and micronutrients which may cause malnutrition and result in impaired renal recovery and patient survival. We aimed to examine the predictive role of macro- and micronutrients on survival and renal outcomes in critically ill patients undergoing continuous RRT (CRRT). This prospective observational study enrolled critically ill patients requiring CRRT at Intensive Care Unit of King Chulalongkorn Memorial Hospital from November 2012 until November 2013. The serum, urine, and effluent fluid were serially collected on the first three days to calculate protein metabolism including dietary protein intake (DPI), nitrogen balance, and normalized protein catabolic rate (nPCR). Serum zinc, selenium, and copper were measured for micronutrients analysis on the first three days of CRRT. Survivor was defined as being alive on day 28 after initiation of CRRT.Dialysis status on day 28 was also determined. Of the 70 critically ill patients requiring CRRT, 27 patients (37.5%) survived on day 28. The DPI and serum albumin of survivors were significantly higher than non-survivors (0.8± 0.2 vs 0.5 ±0.3g/kg/day, p = 0.001, and 3.2±0.5 vs 2.9±0.5 g/dL, p = 0.03, respectively) while other markers were comparable. The DPI alone predicted patient survival with area under the curve (AUC) of 0.69. A combined clinical model predicted survival with AUC of 0.78. When adjusted for differences in albumin level, clinical severity score (APACHEII and SOFA score), and serum creatinine at initiation of CRRT, DPI still independently predicted survival (odds ratio 4.62, p = 0.009). The serum levels of micronutrients in both groups were comparable and unaltered following CRRT. Regarding renal outcome, patients in the dialysis independent group had higher serum albumin levels than the dialysis dependent group, p = 0.01. In conclusion, in critically ill patients requiring CRRT, DPI is a good predictor of patient survival while serum albumin is a good prognosticator of renal outcome.