Dysbiosis of Gut Microbiota Promotes Hepatocellular Carcinoma Progression by Regulating the Immune Response.

METHOD: This study included 74 Chinese male patients with HCC. They were divided into early (n = 19), intermediate (n = 37), and terminal (n = 18) groups, referred to as Barcelona Clinic Liver Cancer stage 0+A, B, and C+D, respectively. Paired fecal and plasma samples were collected. Microbial composition and profiles were analyzed by 16S rRNA gene sequencing. The levels of gut damage marker (regenerating islet-derived protein 3α (REG3α)) and microbial translocation markers (soluble CD14 (sCD14), lipopolysaccharide-binding protein (LBP), peptidoglycan recognition proteins (PGRPs)) were determined in plasma samples of patients by ELISA. Twenty plasma cytokine and chemokines were determined by Luminex. RESULTS: In early, intermediate, and terminal groups, the abundance of the Bifidobacteriaceae family decreased significantly (3.52%, 1.55%, and 0.56%, respectively, P = 0.003), while the abundance of the Enterococcaceae family increased significantly (1.6%, 2.9%, and 13.4%, respectively, P = 0.022). Levels of REG3α and sCD14 were markedly elevated only in the terminal group compared with the early (P = 0.025 and P = 0.048) and intermediate groups (P = 0.023 and P = 0.046). The level of LBP significantly increased in the intermediate (P = 0.035) and terminal (P = 0.025) groups compared with the early group. The PGRP levels were elevated only in the terminal group compared with the early group (P = 0.018). The ratio of Enterococcaceae to Bifidobacteriaceae was significantly associated with the levels of REG3α, LBP, sCD14, and PGRPs. With HCC progression, increased levels of inflammatory cytokines accompanied by a T cell-immunosuppressive response and microbial translocation were observed. CONCLUSION: Gut microbiota compositional and functional shift, together with elevated gut damage and microbial translocation, may promote HCC development by stimulating inflammatory response and suppressing T cell response.

Microbiota in vitro modulated with polyphenols shows decreased colonization resistance against Clostridioides difficile but can neutralize cytotoxicity.

While the knowledge on gut microbiota - C. difficile interactions has improved over the years, the understanding of the underlying mechanisms providing colonization resistance as well as preventative measures against the infection remain incomplete. In this study the antibiotic clindamycin and polyphenol extracts from pomegranate and blueberries were used individually and in combination to modulate fecal microbial communities in minibioreactor arrays (MBRA). Modulated communities were inoculated with C. difficile (ribotype 027). Subsequent 7-day periodical monitoring included evaluation of C. difficile growth and activity of toxins TcdA and TcdB as well as analysis of MBRA bacterial community structure (V3V4 16 S metagenomics). Polyphenols affected multiple commensal bacterial groups and showed different synergistic and antagonistic effects in combination with clindamycin. Exposure to either clindamycin or polyphenols led to the loss of colonization resistance against C. difficile. The successful growth of C. difficile was most significantly correlated with the decrease in Collinsella and Lachnospiraceae. Additionally, we demonstrated that Clostridium sporogenes decreased the activity of both C. difficile toxins TcdA and TcdB. The feature was shown to be common among distinct C. sporogenes strains and could potentially be applicable as a non-antibiotic agent for the alleviation of C. difficile infection.

Half of 'Micrococcus spp.' cases identified by conventional methods are revealed as other life-threatening bacteria with different drug susceptibility patterns by 16S ribosomal RNA gene sequencing.

Bacterial infection during chemotherapy is a fatal complication, therefore precise identification of the pathogenic microorganism is required for treatment. We report that 2 of 4 pediatric patients with malignancy who were diagnosed with Micrococcus spp. infection by conventional methods were finally revealed to have Kytococcus schroeteri and Kocuria marina infection by 16S ribosomal RNA gene sequence analysis (16S rRNA analysis). Although K. schroeteri is morphologically similar to Micrococcus spp., its drug susceptibility profile is quite different from that of Micrococcus spp. K. schroeteri is resistant to penicillin and cephalosporin, which are effective for Micrococcus spp. In fact, penicillin-resistant lethal pneumonia caused by K. schroeteri has been reported in compromised hosts. Based on our results, Micrococcus spp. determined by conventional methods could contain other life-threatening bacteria with different drug susceptibility patterns from Micrococcus spp. To develop an effective empirical treatment for immunocompromised hosts, accumulation of pathogen data by 16S rRNA analysis is required.

Catheter-related bloodstream infection by Microbacterium paraoxydans in a pediatric patient with B-cell precursor acute lymphocytic leukemia: A case report and review of literature on Microbacterium bacteremia.

Microbacterium species are coryneform gram-positive rods that are widely distributed in the environment and have been recently recognized as rare pathogens in humans. However, information about the epidemiologic and clinical characteristics of Microbacterium species is scarce. We herein reported an 11-year-old girl with acute leukemia who was found to have catheter-related bloodstream infection in her neutropenic phase. Gram-positive bacilli repeatedly grew on the blood cultures and were later confirmed by 16S rRNA analysis as Microbacterium paraoxydans. A literature review found available clinical courses in 21 cases (7 pediatric cases) of Microbacterium spp. bacteremia. Our case and those in literature suggested that Microbacterium spp. bacteremia often occurs in patients with indwelling central venous catheters; the literature review further suggested that removal of central venous catheters is required in most cases and that 16S rRNA sequence was useful in identifying in detail the species of Microbacterium.

Lung inflammation and disease: A perspective on microbial homeostasis and metabolism.

It is now well appreciated that the human microbiome plays a significant role in a number of processes in the body, significantly affecting its metabolic, inflammatory, and immune homeostasis. Recent research has revealed that almost every mucosal surface in the human body is associated with a resident commensal microbiome of its own. While the gut microbiome and its role in regulation of host metabolism along with its alteration in a disease state has been well studied, there is a lacuna in understanding the resident microbiota of other mucosal surfaces. Among these, the scientific information on the role of lung microbiota in pulmonary diseases is currently severely limited. Historically, lungs have been considered to be sterile and lung diseases have only been studied in the context of bacterial pathogenesis. Recently however, studies have revealed a resilient microbiome in the upper and lower respiratory tracts and there is increased evidence on its central role in respiratory diseases. Knowledge of lung microbiome and its metabolic fallout (local and systemic) is still in its nascent stages and attracting immense interest in recent times. In this review, we will provide a perspective on lung-associated metabolic disorders defined for lung diseases (e.g., chronic obstructive pulmonary disease, asthma, and respiratory depression due to infection) and correlate it with lung microbial perturbation. Such perturbations may be due to altered biochemical or metabolic stress as well. Finally, we will draw evidence from microbiome and classical microbiology literature to demonstrate how specific lung morbidities associate with specific metabolic characteristics of the disease, and with the role of microbiome in this context. © 2018 IUBMB Life, 71(1):152-165, 2019.

A semi-synthetic natural product blocks collagen induced arthritis by preferentially suppressing the production of IL-6.

Rheumatoid arthritis (RA), an autoimmune-inflammatory disease is characterized by dysregulation of signal transduction pathways, increased production of pro-inflammatory cytokines, enhanced leukocyte infiltration into synovial microvascular endothelium, extensive formation of hyper proliferative pannus, degradation of cartilage and bone erosion. Several compounds that abrogate cytokine production demonstrate a therapeutic effect in experimental models of arthritis. In this study, we report that a novel semi-synthetic natural product (Compound A) being a preferential IL-6 inhibitor, is efficacious in a murine model of arthritis. In vitro evaluations of pro-inflammatory cytokine production reveal that Compound A preferentially inhibits induced production of IL-6 and not TNF-α from THP-1 cells and isolated human monocytes. Furthermore, Compound A robustly inhibits the spontaneous production of IL-6 from pathologically relevant synovial tissue cells isolated from patients with active RA. In a physiologically relevant assay, Compound A selectively inhibits the activated T cell contact-mediated production of IL-6 from human monocytes. Compound A, at pharmacologically efficacious concentrations, does not significantly curtail the LPS-induced activation of p38 MAPKs. In the collagen-induced arthritis (CIA) mouse model (i) macroscopic observations demonstrate that Compound A, administered subcutaneously in a therapeutic regimen, significantly and dose-dependently inhibits disease associated increases in articular index and paw thickness; (ii) histological analyses of paw tissues reveal that Compound A prominently diminishes joint destruction, hyperproliferative pannus formation and infiltration of inflammatory cells. Collectively, these results provide direct evidence that Compound A, a novel preferential IL-6 inhibitor, suppresses collagen-induced arthritis, and may be a potential therapeutic for treating patients with active RA.

Putative Serine Protease Effectors of Clavibacter michiganensis Induce a Hypersensitive Response in the Apoplast of Nicotiana Species.

Clavibacter michiganensis subspp. michiganensis and sepedonicus cause diseases on solanaceous crops. The genomes of both subspecies encode members of the pat-1 family of putative serine proteases known to function in virulence on host plants and induction of hypersensitive responses (HR) on nonhosts. One gene of this family in C. michiganensis subsp. sepedonicus, chp-7, is required for triggering HR in Nicotiana tabacum. Here, further investigation revealed that mutation of the putative catalytic serine residue at position 232 to threonine abolished the HR induction activity of Chp-7, suggesting that enzymatic activity is required. Purified Chp-7 triggered an HR in N. tabacum leaves in the absence of the pathogen, indicating Chp-7 itself is the HR elicitor from C. michiganensis subsp. sepedonicus. Ectopic expression of chp-7 constructs in N. tabacum leaves revealed that Chp-7 targeted to the apoplast triggered an HR while cytoplasmic Chp-7 did not, indicating that Chp-7 induces the HR in the apoplast of N. tabacum leaves. Chp-7 also induced HR in N. sylvestris, a progenitor of N. tabacum, but not in other Nicotiana species tested. ChpG, a related protein from C. michiganensis subsp. michiganensis, also triggered HR in N. tabacum and N. sylvestris. Unlike Chp-7, ChpG triggered HR in N. clevelandii and N. glutinosa.

Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner.

BACKGROUND: Bacterial vaginosis increases the susceptibility to sexually transmitted infections and negatively affects women's reproductive health. METHODS: To investigate host-vaginal microbiota interactions and the impact on immune barrier function, we colonized 3-dimensional (3-D) human vaginal epithelial cells with 2 predominant species of vaginal microbiota (Lactobacillus iners and Lactobacillus crispatus) or 2 prevalent bacteria associated with bacterial vaginosis (Atopobium vaginae and Prevotella bivia). RESULTS: Colonization of 3-D vaginal epithelial cell aggregates with vaginal microbiota was observed with direct attachment to host cell surface with no cytotoxicity. A. vaginae infection yielded increased expression membrane-associated mucins and evoked a robust proinflammatory, immune response in 3-D vaginal epithelial cells (ie, expression of CCL20, hBD-2, interleukin 1ß, interleukin 6, interleukin 8, and tumor necrosis factor α) that can negatively affect barrier function. However, P. bivia and L. crispatus did not significantly upregulate pattern-recognition receptor-signaling, mucin expression, antimicrobial peptides/defensins, or proinflammatory cytokines in 3-D vaginal epithelial cell aggregates. Notably, L. iners induced pattern-recognition receptor-signaling activity, but no change was observed in mucin expression or secretion of interleukin 6 and interleukin 8. CONCLUSIONS: We identified unique species-specific immune signatures from vaginal epithelial cells elicited by colonization with commensal and bacterial vaginosis-associated bacteria. A. vaginae elicited a signature that is consistent with significant disruption of immune barrier properties, potentially resulting in enhanced susceptibility to sexually transmitted infections during bacterial vaginosis.

Detection of reaginic antibodies against Faenia rectivirgula from the serum of horses affected with Recurrent Airway Obstruction by an in vitro bioassay.

Reaginic antibodies, mainly of the IgE and some IgG subclasses, play an important role in the induction of type I immediate hypersensitivity reactions. These antibodies bind through their Fc fragment to high affinity receptors (FcεRI) present in the membrane of mast cells and basophils. Previously, several studies have investigated the role of reaginic antibodies in the pathogenesis of RAO. However, whereas immunological aspects of RAO have been extensively studied, the precise sequence of events is still not well understood and role of IgE in this disease still remains controversial. Therefore, in this study a bioassay was developed for reaginic antibody determination in serum from RAO-affected horses in order to determine the etiology of disease. The technique involves measuring in vitro calcium mobilization in RBL-2H3 cells following incubation with horse serum from RAO-affected or unaffected horses and one of the RAO antigens (Faenia rectivirgula). Results demonstrated that 15% of samples from the RAO-affected horses reacted positively in this in vitro bioassay, whereas the samples from unaffected horses did not. This bioassay indicates that reaginic antibodies could be involved in the immunological mechanism leading to RAO; and this technique may facilitate future research in other allergic diseases in horses.

Innate inflammatory responses of human decidual cells to periodontopathic bacteria.

OBJECTIVE: The purpose of this study was to test the hypothesis that periodontopathic bacteria exert potent proinflammatory effects in human decidua. STUDY DESIGN: The immunostimulatory effects of Gram-positive and negative periodontopathic bacteria and their lipopolysaccharides were tested in human decidual cell cultures in comparison with Escherichia coli. Cytokine production was measured by enzyme-linked immunosorbent assay; inflammatory gene expression was measured by oligonucleotide arrays and quantitative real time-polymerase chain reaction. RESULTS: All bacteria that were tested elicited an inflammatory response, although concentration-dependence and efficacy varied considerably with organism and culture. Lipopolysaccharides were more potent stimuli than intact bacterial cells, although bacteria exerted greater effects at high concentrations. Of 112 genes on the arrays, 18 genes were stimulated significantly by one or more lipopolysaccharide preparation. CONCLUSION: The ability of periodontopathic bacteria to stimulate a decidual inflammatory response is highly variable and partly dependent on the presence and structure of constituent lipopolysaccharides. This adds to our understanding of the causal association between periodontal disease and preterm birth.

Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide.

Peptidoglycan-derived muramyl dipeptide (MDP) activates innate immunity via the host sensor NOD2. Although MDP is N-acetylated in most bacteria, mycobacteria and related Actinomycetes convert their MDP to an N-glycolylated form through the action of N-acetyl muramic acid hydroxylase (NamH). We used a combination of bacterial genetics and synthetic chemistry to investigate whether N-glycolylation of MDP alters NOD2-mediated immunity. Upon infecting macrophages with 12 bacteria, tumor necrosis factor (TNF) alpha secretion was NOD2 dependent only with mycobacteria and other Actinomycetes (Nocardia and Rhodococcus). Disruption of namH in Mycobacterium smegmatis obrogated NOD2-mediated TNF secretion, which could be restored upon gene complementation. In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor kappaB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion. In mice challenged intraperitoneally with live or killed mycobacteria, NOD2-dependent immune responses depended on the presence of bacterial namH. Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy. Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

Atopobium vaginae triggers an innate immune response in an in vitro model of bacterial vaginosis.

Bacterial vaginosis is the most common vaginal disorder among women of reproductive age. The pathogenesis of bacterial vaginosis is poorly understood, but is defined by a transition in the vaginal flora from the predominant Lactobacillus species to other bacterial species such as Atopobium vaginae and Gardnerella vaginalis. This change is associated with an increase in vaginal cytokine secretion. We hypothesize that vaginal epithelial cells respond to bacterial vaginosis-associated bacteria by triggering an innate immune response. We observed that vaginal epithelial cells secreted interleukin-6 and interleukin-8 in response to Atopobium vaginae and Gardnerella vaginalis, but not to Lactobacillus crispatus. Atopobium vaginae induced increased levels of interleukin-6 and interleukin-8 transcripts, as well as increased transcripts for the antimicrobial peptide beta-defensin 4. This innate immune response required live bacteria capable of protein synthesis in direct contact with vaginal epithelial cells. The response of vaginal epithelial cells was mediated by Toll-like receptor 2, required the adaptor protein MyD88, and involved activation of the NFkappaB signaling pathway. These results suggest that Atopobium vaginae stimulates an innate immune response from vaginal epithelial cells, leading to localized cytokine and defensin production, and possibly contributes to the pathogenesis of bacterial vaginosis.

Whipple's disease: immunospecific and quantitative immunohistochemical study of intestinal biopsy specimens.

Whipple's disease may be diagnosed by periodic acid-Schiff (PAS) staining, electron microscopy, or polymerase chain reaction of intestinal biopsy specimens. The aim of this study was to evaluate the diagnostic value of immunohistochemistry and the quantification of infected cells in intestinal Whipple's disease. A total of 29 duodenal biopsy specimens from 15 patients with untreated and treated Whipple's disease were examined and compared with biopsy specimens from control patients with normal intestinal mucosa or various pathologic processes. Percentages of staining surfaces with PAS stain and antibodies directed against CD68, a macrophage marker, or the Whipple bacillus, Tropheryma whipplei, were studied quantitatively using a computerized system of image analysis. Positive detection of T. whipplei was obtained using immunohistochemistry in all 15 patients with Whipple's disease. No bacteria were detected in any of the negative controls. The use of quantitative image analysis showed a massive intestinal macrophagic infiltration before (20.3%) and after (13.4%) antibiotic therapy completion as compared with controls (2.1%). The 2 detection methods for T. whipplei, PAS stain and immunohistochemistry, were quantitatively similar before therapy (19.9% versus 17.5%), but the immunodetection-based surface area was significantly lower than the PAS staining surface area after therapy (2.8% versus 7.9%). Our findings indicate that immunohistochemistry is highly specific and sensitive and is applicable as a diagnostic method on intestinal tissue specimens to detect T. whipplei during active infection or in retrospective studies.

Monoclonal antibodies to immunodominant epitope of Tropheryma whipplei.

Recent isolation of Tropheryma whipplei (formerly Trophyrema whippelii), the agent of Whipple's disease, from the cardiac valve of a patient with Whipple's disease endocarditis now allows the detection of reactive epitopes that could be used in a serological assay. In order to propose an enzyme-linked immunosorbent assay (ELISA) that uses recombinant T. whipplei antigen, we first determined by Western blotting of human, mouse, and rabbit antisera that the common immunodominant epitope is an 84-kDa protein. We then produced 13 monoclonal antibodies (MAbs) against T. whipplei, 12 of which recognize this immunodominant epitope. These MAbs did not react with phylogenetically closely related bacteria or bacteria previously shown to be cross-reactive with T. whipplei, but they did react with two other strains of T. whipplei isolated, one from an ocular sample and the other from a duodenal biopsy specimen. By confocal microscopy, the MAbs allowed detection of T. whipplei within infected fibroblasts. The identification of the 84-kDa antigen with our MAbs will make it possible to develop a diagnostic antigen for use in a diagnostic ELISA for Whipple's disease.

Culture and immunological detection of Tropheryma whippelii from the duodenum of a patient with Whipple disease.

CONTEXT: Culture of Tropheryma whippelii has been established only once, in human fibroblast cell lines from a heart valve inoculum. Molecular-based diagnostic techniques, although highly sensitive, may be less specific. New diagnostic tools involving isolation of bacteria from contaminated intestinal biopsies and immunohistological detection need to be developed. OBJECTIVE: To describe a novel method for detection and culture of T whippelii strains. DESIGN, SETTING, AND SUBJECTS: Laboratory analysis of duodenal biopsy specimens from a patient with typical relapsing Whipple disease with intestinal involvement, performed Marseille, France, in March 2000. Biopsy specimens were decontaminated with antimicrobial agents and inoculated onto cell cultures. Mouse anti-T whippelii polyclonal antibodies were used to detect T whippelii in fixed specimens taken from the patient before and after relapse, compared with specimens from 10 controls. The genotype of the isolate was determined by amplification and sequencing of 2 DNA fragments (ITS and 23S rRNA). MAIN OUTCOME MEASURE: Isolation and genotyping of a new strain(s) of T whippelii from the case patient's biopsy specimens. RESULTS: A strain was grown from the case patient's intestinal specimen that has a genotype different from the first strain isolated. During 2 episodes of Whipple disease, T whippelii bacteria were detected by immunochemistry in the patient's duodenal biopsy specimens, but not in controls. CONCLUSIONS: A second strain of T whippelii has been isolated and a protocol for isolation from the intestine has been proven to be efficient. Immunodetection of T whippelii in intestinal biopsy specimens may provide a useful tool for the diagnosis and follow-up of patients with Whipple disease. Both techniques need further evaluation and confirmation.