Carl V. Crawford

Genetic transformation of a Corynebacterial symbiont from the Chagas disease vector Triatoma infestans

Insect-borne diseases have experienced a troubling resurgence in recent years. Emergence of resistance to pesticides greatly hampers control efforts. Paratransgenesis, or the genetic transformation of bacterial symbionts of disease vectors, is an alternative to traditional approaches. Previously, we developed paratransgenic lines of Rhodnius prolixus, a vector of Chagas disease in Central America. Here, we report identification of a Corynebacterial species as a symbiont of Triatoma infestans, a leading vector of Chagas disease in South America. We have modified this bacterium to produce an immunologically active single chain antibody fragment, termed rDB3. This study establishes the basis for generating paratransgenic T. infestans as a strategy for control of Chagas disease.

Use of probiotics in prevention and treatment of patients with Clostridium difficile infection.

Clostridium difficile is an anaerobic, gram positive, sporulating, toxin-producing bacillus which causes a spectrum of clinical disease ranging from an asymptomatic carrier state to toxic megacolon and fulminant disease. Infection with C. difficile is an expensive and pervasive health care burden. The current theory regarding the development of C. difficile infection (CDI) suggests that disruption of the structure and/or function of an individuals normal intestinal microbiota enables colonization by C. difficile, and in the absence of an effective immune response, the bacteria causes illness. In this article we discuss the role of the colonic microbiota in the development of CDI and the potential role of probiotics in preventing and treating CDI. We review the evidence from in vitro laboratory and pre-clinical studies, as well as evidence from clinical studies and discuss the current recommendations for the use of probiotics for CDI in clinical practice.

Expression of a functional single-chain antibody via Corynebacterium pseudodiphtheriticum.

Antibody-based therapeutics are effective against conditions ranging from acute infections to malignancy. They may prove crucial in combating bioterrorism and responding to drug-resistant and emerging pathogens. At present the cost of producing therapeutic monoclonal antibodies is between

Heterotopic Pancreatic Neoplasm Presenting as an Obstructing Mass at the Fourth Portion of the Duodenum

1,000 to

Malakoplakia-induced diarrhea in a kidney transplant recipient.

6,000 per gram. The need to administer antibodies parenterally at frequent intervals further drives the cost of this treatment. Here we present an antibody delivery system, termed paratransgenesis, with the potential to overcome these limitations. The paratransgenic approach involves genetically transforming a commensal or symbiont bacterium to express foreign molecules that target pathogens. We describe transformation of Corynebacterium pseudodiptheriticum, a commensal bacterium found in the human respiratory tract, to express a murine single-chain antibody binding progesterone. The antibody was functional and bound specifically to progesterone in a concentration-dependent manner. This marker antibody system is the precursor to development of expression systems producing recombinant humanized single-chain antibodies. Studies are in progress evaluating fitness, transgene stablility, and pathogenecity of the genetically engineered C. pseudodiptheriticum. We anticipate developing a repertoire of expressed molecules targeting infectious agents and surface epitopes of pulmonary mass lesions. If expression systems for anti-pathogen molecules in C. pseudodiptheriticum and other respiratory commensal bacteria can be optimized, these bacteria have the potential for a range of therapeutic and prophylactic applications.

Single Delivery of High-diversity Fecal Microbiota Preparation by Colonoscopy Is Safe and Effective in Increasing Microbial Diversity in Active Ulcerative Colitis.

CONTEXT Heterotopic pancreatic adenocarcinoma is a rare finding at laparotomy. Herein we present the case of a patient with malignant transformation of a heterotopic pancreas located in the fourth portion of the duodenum. CASE REPORT A 79-year-old woman was admitted to the surgery service with complaints of early satiety and abdominal fullness progressively worsening over the previous two years. A computed tomography scan of the abdomen and an upper endoscopy revealed an obstructing mass in the fourth portion of the duodenum, biopsies were negative for carcinoma. A segmentectomy of the third and fourth portions of the duodenum was performed. Post-operative histology revealed malignant transformation of a heterotopic pancreas. The patient had an unremarkable postoperative recovery and was discharged home. CONCLUSION In evaluation of patients with distal duodenal masses, we report that heterotopic pancreatic neoplasms should be considered in the differential diagnosis.

201 Early Referral for 24-Hour Esophageal pH Monitoring Is More Cost-Effective Than Prolonged Use of Proton Pump Inhibitors in Patients With Suspected Gastroesophageal Reflux Disease

ter cardiac transplantation. N Engl J Med 2010; 362: 1890. 5. Deng MC, Halpern B, Wolters H, et al. Patientspecific longitudinal pattern of AlloMap test scores [abstract]. J Heart Lung Transplant 2009; 28: S229. 6. Proust-Lima C, Taylor JMG. Development and validation of a dynamic prognostic tool for prostate cancer recurrence using repeated measures of posttreatment PSA: A joint modeling approach. Biostatistics 2009; 10: 535.

Cost-Effectiveness Analysis of Probiotic Use to Prevent Clostridium difficile Infection in Hospitalized Adults Receiving Antibiotics

Background: Recent trials suggest fecal microbiota transplantation (FMT) with repeated enemas and high-diversity FMT donors is a promising treatment to induce remission in ulcerative colitis. Methods: We designed a prospective, open-label pilot study to assess the safety, clinical efficacy, and microbial engraftment of single FMT delivery by colonoscopy for active ulcerative colitis using a 2-donor fecal microbiota preparation (FMP). Safety and clinical endpoints of response, remission, and mucosal healing at week 4 were assessed. Fecal DNA and rectal biopsies were used to characterize the microbiome and mucosal CD4+ T cells, respectively, before and after FMT. Results: Of the 20 patients enrolled in this study, 7 patients (35%) achieved a clinical response by week 4. Three patients (15%) were in remission at week 4 and 2 of these patients (10%) achieved mucosal healing. Three patients (15%) required escalation of care. No serious adverse events were observed. Microbiome analysis revealed that restricted diversity of recipients pre-FMT was significantly increased by high-diversity 2-donor FMP. The microbiome of recipients post-transplant was more similar to the donor FMP than the pretransplant recipient sample in both responders and nonresponders. Notably, donor composition correlated with clinical response. Mucosal CD4+ T-cell analysis revealed a reduction in both Th1 and regulatory T-cells post-FMT. Conclusions: High-diversity, 2-donor FMP delivery by colonoscopy seems safe and effective in increasing fecal microbial diversity in patients with active ulcerative colitis. Donor composition correlated with clinical response and further characterization of immunological parameters may provide insight into factors influencing clinical outcome.

To Scope or Not to Scope? The Safety of Upper Endoscopy in the Setting of Pulmonary Embolism

Introduction: Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils and constitutes a major component of the prominent desmoplastic reaction in pancreatic ductal adenocarcinoma (PDA). We have demonstrated recently that a subunit of COL6, COL6A3, is expressed in high levels in PDA tissue. We also showed that COL6A3 gene undergoes tumor-specific alternative splicing to produce 3 isoforms E3, E4 and E6 that are tumor tissue-specific. The aim of this study is to investigate the diagnostic value and clinical significance of circulating COL6A3 isoforms mRNA in PDA. Methods: Serum samples were obtained frompatients that underwent pancreatic resection at a single institution between 2006 and 2009. COL6A3 levels in the sera from patients with pathologically confirmed PDA (n=40), intraductal papillary mucinous neoplasms (IPMN) (n=20), and chronic pancreatitis (n=10) were analyzed by real time PCR using isoform-specific primers for E3, E4 and E6 . In addition, sera from age-matched healthy volunteers were analyzed (n=30). The prediction levels for malignancy were determined by the area under the receiver operating characteristic curve (AUC). In vitro, wound healing, cell proliferation and softagar colony formation assays evaluated the functional impact of each isoform in PDA cells (MIAPACA-2 and ASP-C-1) transfectedwith isoform-specific siRNA. A panel of inflammationand invasion/angiogenesis-related genes was also evaluated. Results: Circulating E6 mRNA levels were significantly (p=0.006) elevated in PDA patients when compared to all benign lesions. E3 and E4 were expressed at extremely low levels in all patients. Compared to IPMN alone, E6 levels were significantly higher in PDA (p=0.0036). There were no significant differences between E6 levels in IPMN and Normal sera (p= 0.59). Using a logistic regression model, we found that for each increasing unit of log E6 COL6A3, patients are 9.5 times more likely to harbor a cancer rather than a benign lesion, 95% CI (2.4, 38.1), p=0.002. The area under the ROC curve, AUC, was 0.72. Knocking down E3 or E4 or E6 with isoform-specific siRNA resulted in reduced PDA cell migration and invasion and concomitant reduction of the expression of several inflammation and angiogenesis-related genes, such as MMP-9, OPN, MCP-1 and VEGF. Interestingly, knocking down any of the 3 isoforms resulted in increased expression of TNF-alpha. Conclusions: Our data show for the first time the potential clinical significance of circulating E6 COL6A3 levels in the diagnosis of pancreatic malignancy. Our in vitro data suggests a role for COL6A3 isoforms in PDA progression and metastatic potential.

PORTAL VEIN THROMBOSIS PREVALENCE AND ASSOCIATED MORTALITY IN CIRRHOSIS IN A NATIONALLY REPRESENTATIVE INPATIENT COHORT: PORTAL VEIN THROMBOSIS PREVALENCE AND ASSOCIATED MORTALITY IN CIRRHOSIS IN A NATIONALLY REPRESENTATIVE INPATIENT COHORT

Abstract Background Systematic reviews with meta-analyses and meta-regression suggest that timely probiotic use can prevent Clostridium difficile infection (CDI) in hospitalized adults receiving antibiotics, but the cost effectiveness is unknown. We sought to evaluate the cost effectiveness of probiotic use for prevention of CDI versus no probiotic use in the United States. Methods We programmed a decision analytic model using published literature and national databases with a 1-year time horizon. The base case was modeled as a hypothetical cohort of hospitalized adults (mean age 68) receiving antibiotics with and without concurrent probiotic administration. Projected outcomes included quality-adjusted life-years (QALYs), costs (2013 US dollars), incremental cost-effectiveness ratios (ICERs;