Identification and engraftment of new bacterial strains by shotgun metagenomic sequence analysis in patients with recurrent Clostridioides difficile infection before and after fecal microbiota transplantation and in healthy human subjects.

BACKGROUND: Recurrent Clostridioides diffícile infection (RCDI) is associated with major bacterial dysbiosis and colitis. Fecal microbiota transplantation (FMT) is a highly effective therapeutic modality for RCDI. While several studies have identified bacterial species associated with resolution of symptoms in patients, characterization of the fecal microbiome at the bacterial strain level in RCDI patients before and after FMT and healthy donors, has been lacking. The aim of this study was to examine the ability of bacterial strains from healthy donors to engraft in the gastrointestinal tract of patients with RCDI following FMT. METHODS: Fecal samples were collected from 22 patients with RCDI before and after FMT and their corresponding healthy donors. Total DNA was extracted from each sample and analyzed by shotgun metagenomic sequencing. The Cosmos-ID analysis platform was used for taxonomic assignment of sequences and calculation of the relative abundance (RA) of bacterial species and strains. From these data, the total number of bacterial strains (BSI), Shannon diversity index, dysbiosis index (DI), and bacterial engraftment factor, were calculated for each strain. FINDINGS: A marked reduction (p<0·0001) in the RA of total and specific bacterial strains, especially from phylum Firmicutes, was observed in RCDI patients prior to FMT. This change was associated with an increase in the DI (p<0·0001) and in pathobiont bacterial strains from phylum Proteobacteria, such as Escherichia coli O157:H7 and Klebsiella pneumoniae UCI 34. BSI was significantly lower in this group of patients as compared to healthy donors and correlated with the Shannon Index. (p<0·0001). Identification and engraftment of bacterial strains from healthy donors revealed a greater diversity and higher relative abundance of short-chain fatty acid (SCFA)-producing bacterial strains, including Lachnospiraceae bacterium 5_1_63FAA_u_t, Dorea formicigenerans ATCC 27755, Anaerostipes hadrusand others, in RCDI patients after FMT. INTERPRETATION: These observations identify a group of SCFA-producing bacterial strains from healthy donors that engraft well in patients with RCDI following FMT and are associated with complete resolution of clinical symptoms and bacterial dysbiosis.

Superiority of Higher-Volume Fresh Feces Compared to Lower-Volume Frozen Feces in Fecal Microbiota Transplantation for Recurrent Clostridioides Difficile Colitis.

GOALS: To compare the clinical outcomes of different protocols for fecal microbiota transplantation (FMT) in two community hospitals with similar patient demographics. BACKGROUND: FMT is commonly performed for recurrent or refractory Clostridioides difficile infection (rCDI). The clinical efficacy of FMT for this indication has been well established. However, there has been no standardization or optimization of the amount of fecal material, method of feces preparation, or route of delivery for FMT. STUDY: In this retrospective study, patients with rCDI received FMT using commercially available frozen fecal preparation (22.7 g) at Center A and locally prepared fresh fecal filtrate (30-50 g) at Center B. The primary outcome was defined as complete resolution of clinical symptoms related to rCDI after at least 8 weeks of follow-up. RESULTS: Fifty patients from each center were included in the study. Clinical success after initial FMT with lower-volume frozen fecal preparation at Center A was 32/50 (64.0%) compared to 49/50 (98.0%) with higher-volume fresh fecal filtrate at Center B (p < 0.0001). Seventeen patients in Center A and 1 patient in Center B underwent at least one repeat FMT. Overall clinical success was achieved in 43/50 (86%) of patients in Center A and 50/50 (100%) in Center B (p = 0.012). CONCLUSIONS: Our results suggest superior clinical efficacy of a larger amount of fresh fecal filtrate over a smaller amount of commercially available frozen fecal preparation. Further studies are needed to examine the effect of varying amounts of feces and the optimal protocol for FMT in patients with rCDI.

Parkinson's Disease: The Emerging Role of Gut Dysbiosis, Antibiotics, Probiotics, and Fecal Microbiota Transplantation.

The role of the microbiome in health and human disease has emerged at the forefront of medicine in the 21st century. Over the last 2 decades evidence has emerged to suggest that inflammation-derived oxidative damage and cytokine induced toxicity may play a significant role in the neuronal damage associated with Parkinson's disease (PD). Presence of pro-inflammatory cytokines and T cell infiltration has been observed in the brain parenchyma of patients with PD. Furthermore, evidence for inflammatory changes has been reported in the enteric nervous system, the vagus nerve branches and glial cells. The presence of α-synuclein deposits in the post-mortem brain biopsy in patients with PD has further substantiated the role of inflammation in PD. It has been suggested that the α-synuclein misfolding might begin in the gut and spread "prion like" via the vagus nerve into lower brainstem and ultimately to the midbrain; this is known as the Braak hypothesis. It is noteworthy that the presence of gastrointestinal symptoms (constipation, dysphagia, and hypersalivation), altered gut microbiota and leaky gut have been observed in PD patients several years prior to the clinical onset of the disease. These clinical observations have been supported by in vitro studies in mice as well, demonstrating the role of genetic (α-synuclein overexpression) and environmental (gut dysbiosis) factors in the pathogenesis of PD. The restoration of the gut microbiome in patients with PD may alter the clinical progression of PD and this alteration can be accomplished by carefully designed studies using customized probiotics and fecal microbiota transplantation.

Evidence of multimeric forms of HSP70 with phosphorylation on serine and tyrosine residues--implications for roles of HSP70 in detection of GI cancers.

BACKGROUND: Heat-shock protein70 (HSP70) are intracellular protein chaperones, with emerging evidence of their association with various diseases. We have previously reported significantly elevated plasma-HSP70 (pHSP70) in pancreatic cancer. Current methods of pHSP70 isolation are ELISA-based which lack specificity due to cross-reactivity by similarities in the amino-acid sequence in regions of the protein backbone resulting in overestimated HSP70 value. MATERIALS AND METHODS: This study was undertaken to develop a methodology to capture all isoforms of pHSP70, while further defining their tyrosine and serine phosphorylation status. RESULTS: The methodology included gel electrophoresis on centrifuged supernatant obtained from plasma incubated with HSP70 antibody-coupled beads. After blocking non-specific binding sites, blots were immunostained with monoclonal-antibody specific for human-HSP70, phosphoserine and phosphotyrosine. CONCLUSIONS: Our novel immunocapture approach has distinct advantages over the commercially available methods of pHSP70 quantification by allowing isolation of molecular aggregates of HSP70 with additional ability to precisely distinguish phosphorylation state of HSP70 molecules at serine and tyrosine residues.

Barrett's esophagus and ß-carotene therapy: symptomatic improvement in GERD and enhanced HSP70 expression in esophageal mucosa.

INTRODUCTION: Epidemiological studies suggest a protective role for ß-carotene with several malignancies. Esophageal adenocarcinoma frequently arises from Barrett's esophagus (BE). We postulated that ß-carotene therapy maybe protective in BE. MATERIALS AND METHOD: We conducted a prospective study in which 25 mg of ß-carotene was administered daily for six-months to six patients. Each patient underwent upper endoscopy before and after therapy and multiple mucosal biopsies were obtained. Additionally, patients completed a gastroesophageal reflux disease (GERD) symptoms questionnaire before and after therapy and severity score was calculated. To study the effect of ß-carotene at molecular level, tissue extracts of the esophageal mucosal biopsy were subjected to assessment of heat-shock protein 70 (HSP70). RESULTS: A significant (p<0.05) reduction in mean GERD symptoms severity score from 7.0±2.4 to 2.7±1.7 following ß-carotene therapy was noted. Measurement of Barrett's segment also revealed a significant reduction in mean length after therapy. In fact, two patients had complete disappearance of intestinal metaplasia. Furthermore, marked enhancement of HSP70 expression was demonstrated in biopsy specimens from Barrett's epithelium in four cases that were tested. CONCLUSIONS: Long- term ß-carotene therapy realizes amelioration of GERD symptoms along with restitution of the histological and molecular changes in esophageal mucosa of patients with BE, associated with concurrent increase in mucosal HSP70 expression.

Serum HSP70: a novel biomarker for early detection of pancreatic cancer.

OBJECTIVES: Heat shock protein 70 (HSP70) is overexpressed in human pancreatic cancer cell lines. To determine if serum HSP70 levels are elevated in patients with pancreatic cancer and can function as a biomarker for early detection of pancreatic cancer. METHODS: Study subjects were divided into 3 groups: histologically proven pancreatic cancer (PC; n = 23), chronic pancreatitis (CP; n = 12), and matched normal control subjects (C; n = 10). Serum HSP70 levels were determined using a novel immunoelectrophoresis method developed and validated by the authors. Significance of difference between the groups was analyzed with analysis of variance (ANOVA). Receiver operating characteristic (ROC) curve analysis was performed to discriminate patients with pancreatic cancer from normal controls. RESULTS: The mean ± SE serum HSP70 levels in the PC, CP, and C groups were 1.68 ± 0.083 ng/mL, 0.40 ± 0.057 ng/mL, and 0.04 ng/mL, respectively. Serum HSP70 levels in the PC group were significantly higher compared with either the CP or C groups (P < 0.01). The sensitivity and specificity of elevated serum HSP70 in the PC group was 74% and 90%, respectively. CONCLUSIONS: Serum HSP70 levels are significantly increased in patients with pancreatic cancer and may be useful as an additional biomarker for the detection of pancreatic cancer.

Markers of Inflammation and Lineage on Exfoliated Colonic Cells In Pediatric Inflammatory Bowel Disease.

OBJECTIVES: The diagnosis (endoscopy, and biopsy) and continued clinical management of Inflammatory Bowel Disease (IBD), remain highly invasive, expensive, and inconvenient for the pediatric patient. The objective of this study was to see if colonocytes obtained from stools of subjects with IBD and normal controls would demonstrate higher levels of inflammatory markers (Cox 2 in CD45+ and CD45- cells) and if the inflammatory process and treatment effects would be reflected in an altered cytokine expression in the subjects compared to controls. SETTING: Outpatient hospital based pediatric gastroenterology clinic. METHODS AND MAIN OUTCOME MEASURES: Stool samples (~ 1 gm), were obtained from 18 children between the ages of 4 and 18 diagnosed with IBD, and from a normal first degree relative. Colonocytes were isolated using the Somatic Cell Sampling Recovery (SCSR) system and assessed for the expression of COX-2, CD-45, IgA, IgG, IL6, IL18, TGF ß, TNF, and IL16ß using flow cytometry. In addition, levels of COX-2 and cytokeratin 19 transcripts were measured by microwell plate hybridization assay. RESULTS: Expression of COX-2 and co-expression of IgA and IgG were significantly higher in the IBD cases compared to the controls. In ulcerative colitis, the expression of COX-2 and co-expression of COX-2 and CD45 were greater than that in patients with Crohn's disease. In contrast, cells expressing IgA and IgG were higher in Crohn's. Subjects on immunosuppressants and/or anti-inflammatory medications, expressed significantly lower levels of COX-2 and IL-18 compared to those who were not on treatment. CONCLUSIONS: This study indicates that the use of disease markers on exfoliated colonic cells can be used for non-invasive assessment of disease status, for follow-up of response to treatment and for forecasting flare-up of disease before its symptomatic manifestations.

Non-invasive detection of c-myc p64, c-myc p67 and c-erbb-2 in colorectal cancer.

OBJECTIVE: Colorectal cancer is a major cause of cancer mortality in the industrialized nations in the West. Because mortality is closely related to the stage of the disease at the time of diagnosis, detection at an early stage is likely to result in improved recovery rates. Since current diagnostic procedures such as colonoscopy are invasive and the fecal occult blood test (FOBT) lacks sensitivity and specificity for the detection of early lesions, the development of non-invasive methods based on molecular markers of neoplasia can lead to earlier diagnosis and more favorable outcomes for patients with colorectal cancer. Recent advances in the technology for isolating colonocytes from stool (SCSR (somatic cell sampling and recovery)) provide a non-invasive tool for the study of biomarkers expressed in colorectal cancer. The aim of this study was to detect mRNA expression of three biomarkers: (c-erbb-2 and two forms of c-myc: p64 and p67) in fecal colonocytes and to evaluate its use in diagnosing colorectal cancer. MATERIAL AND METHODS: Colonocytes (SCSR cells) were isolated from stools from 30 subjects: 15 colorectal cancer patients and 15 normal controls. One cancer patient was excluded from the final data analysis because the tumor was a gastrointestinal lymphoma. Each sample yielded two fractions: a pellet and an interphase. Expression of c-myc p64, c-myc p67 and c-erbb-2 mRNA was evaluated in each of the fractions by reverse transcriptase-polymerase chain reaction (RT-PCR). A marker was considered positive upon detecting an amplicon of the expected size in agarose gel electrophoresis. RESULTS: c-myc p64 mRNA expression was observed in both fractions in 78.5% of colorectal cancer patients, compared with 13.3% in the control group (p=0.009). For c-myc p67, 78.6% of the colorectal cancer patients showed mRNA expression in both fractions in comparison with only 13.3% of the controls (p=0.003). C-erbb-2 showed no significant difference in mRNA expression between colorectal cancer and controls. When the data were analyzed for co-expression of c-myc p64 and c-myc p67, in both pellet and interphase, sensitivity was 64% and specificity was 100%. CONCLUSIONS: Fecal colonocytes isolated by somatic cell sampling and recovery (SCSR) technology could be used for the non-invasive assessment of the expression of biomarkers of colon cancer such as c-myc p64, c-myc p67 and c-erbb-2. The expression of c-myc p64 and c-myc p67 in colonocytes showed a significant association with colorectal cancer and may be helpful as a biomarker for the non-invasive detection of colorectal cancer.

Exfoliated colonic epithelial cells: surrogate targets for evaluation of bioactive food components in cancer prevention.

There is significant evidence supporting the hypotheses that lifestyle, diet, and bioactive components in foods are important modifiers of cancer risk. However, our ability to assess host response noninvasively is limited. To overcome this, we have developed a technology to isolate several million viable exfoliated somatic colonic cells from a small sample of stool (0.5-1.0 g) by a procedure known as somatic cell sampling and recovery (SCSR). Orally administered carotenoids appear in these cells several days after consuming the supplement, usually showing a peak concentration between 5-7 d after their ingestion. The time lag observed for the appearance of orally administered carotenoids in SCSR cells corresponds to the turnover rate of the colonic mucosa. This is an example of how changes in cell turnover rates can be carefully assessed using the SCSR system. The specific mechanisms by which individual constituents of diet affect the cancer process are not fully understood. However, host response to dietary constituents may be investigated, noninvasively, by following the modulation of tumor-associated molecular markers in these exfoliated SCSR cells. We have demonstrated the feasibility of using SCSR cells to detect the expression of carcinoembryonic antigen, CD44, and its splice variants, c-myc, c-erbb2, and mutations in the p53 gene. In this regard, SCSR cells are a readily available surrogate cellular target that may serve to monitor changes in cell turnover, differentiation, and expression of cancer-associated biomarkers that are likely to be modulated by bioactive food components.

Coprocytobiology: on the nature of cellular elements from stools in the pathophysiology of colonic disease.

The gastrointestinal epithelium is known to undergo constant and rapid renewal resulting in millions of cells being shed into the fecal stream every day. The conventional wisdom was that these cells disintegrate upon exfoliation and will not survive the transit through the intestinal tract. In 1990, we (P.N.) made the discovery that a significant number of these cells remain intact and viable and that they can be isolated. The implications of this important discovery became apparent when we demonstrated that these cells are exclusively of colonic origin, are anatomically representative of the entire colon, and can be used for clinical investigations of disease processes. The term coprocytobiology (CCB) was coined to encompass the broad range of applications of this new technology. The somatic cell sampling and recovery (SCSR) process involves the isolation of exfoliated colonocytes from a small sample of stool ( approximately 1 g) collected and transported in a unique medium at ambient temperature, providing cells for the detection of a number of biomarkers of disease propensity. These exfoliated colonocytes express cytokeratins indicating epithelial lineage as well as colon-specific antigen. Over the years, the study of exfoliated colonocytes has provided striking new insights into the biology of colon cancer and inflammatory bowel disease, including detection of p53 gene mutations, reverse transcriptase polymerase chain reaction amplification, and identification of CD44 splice variants, neoplasia-associated specific binding of plant lectins, and expression of COX-2, the inducible form of cyclooxygenase. The functional diversity of cells isolated by SCSR is revealed by the demonstration of cell surface markers such as secretory component, IgA, and IgG on the one hand and the amplification and cloning of the human insulin receptor and the expression of the multidrug resistance gene mdr-1 on the other hand. This review portrays the immense potential of CCB as a powerful tool for investigating the pathophysiology of disease, identifying genetic variants in pharmacogenetics, assessment of mucosal immunity, and several other applications that use somatic cells.