Real-Time Monitoring of \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 Adherence to Beef Carcass Surface Tissues with a Bioluminescent Reporter

A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues

Detection of non-melanoma skin cancer by in vivo fluorescence imaging with fluorocoxib A.

Non-melanoma skin cancer (NMSC) is the most common form of cancer in the US and its incidence is increasing. The current standard of care is visual inspection by physicians and/or dermatologists, followed by skin biopsy and pathologic confirmation. We have investigated the use of in vivo fluorescence imaging using fluorocoxib A as a molecular probe for early detection and assessment of skin tumors in mouse models of NMSC. Fluorocoxib A targets the cyclooxygenase-2 (COX-2) enzyme that is preferentially expressed by inflamed and tumor tissue, and therefore has potential to be an effective broadly active molecular biomarker for cancer detection. We tested the sensitivity of fluorocoxib A in a BCC allograft SCID hairless mouse model using a wide-field fluorescence imaging system. Subcutaneous allografts comprised of 1000 BCC cells were detectable above background. These BCC allograft mice were imaged over time and a linear correlation (R(2) = 0.8) between tumor volume and fluorocoxib A signal levels was observed. We also tested fluorocoxib A in a genetically engineered spontaneous BCC mouse model (Ptch1(+/-) K14-Cre-ER2 p53(fl/fl)), where sequential imaging of the same animals over time demonstrated that early, microscopic lesions (100 μm size) developed into visible macroscopic tumor masses over 11 to 17 days. Overall, for macroscopic tumors, the sensitivity was 88% and the specificity was 100%. For microscopic tumors, the sensitivity was 85% and specificity was 56%. These results demonstrate the potential of fluorocoxib A as an in vivo imaging agent for early detection, margin delineation and guided biopsies of NMSCs

Heme Oxygenase-1 Deletion Affects Stress Erythropoiesis

BACKGROUND:Homeostatic erythropoiesis leads to the formation of mature red blood cells under non-stress conditions, and the production of new erythrocytes occurs as the need arises. In response to environmental stimuli, such as bone marrow transplantation, myelosuppression, or anemia, erythroid progenitors proliferate rapidly in a process referred to as stress erythropoiesis. We have previously demonstrated that heme oxygenase-1 (HO-1) deficiency leads to disrupted stress hematopoiesis. Here, we describe the specific effects of HO-1 deficiency on stress erythropoiesis. METHODOLOGY/PRINCIPAL FINDINGS:We used a transplant model to induce stress conditions. In irradiated recipients that received hmox(+/-) or hmox(+/+) bone marrow cells, we evaluated (i) the erythrocyte parameters in the peripheral blood; (ii) the staining intensity of CD71-, Ter119-, and CD49d-specific surface markers during erythroblast differentiation; (iii) the patterns of histological iron staining; and (iv) the number of Mac-1(+)-cells expressing TNF-α. In the spleens of mice that received hmox(+/-) cells, we show (i) decreases in the proerythroblast, basophilic, and polychromatophilic erythroblast populations; (ii) increases in the insoluble iron levels and decreases in the soluble iron levels; (iii) increased numbers of Mac-1(+)-cells expressing TNF-α; and (iv) decreased levels of CD49d expression in the basophilic and polychromatophilic erythroblast populations. CONCLUSIONS/SIGNIFICANCE:As reflected by effects on secreted and cell surface proteins, HO-1 deletion likely affects stress erythropoiesis through the retention of erythroblasts in the erythroblastic islands of the spleen. Thus, HO-1 may serve as a therapeutic target for controlling erythropoiesis, and the dysregulation of HO-1 may be a predisposing condition for hematologic diseases

Fibered Confocal Microscopy of Bladder Tumors: An ex Vivo Study

Background and Purpose: The inadequacy of white-light cystoscopy to detect flat bladder tumors is well recognized. Great interest exists in developing other imaging technologies to augment or supplant conventional cystoscopy. Fibered confocal microscopy offers the promise of providing in vivo histopathologic information to help distinguish malignant from benign bladder lesions. We report the initial use of this technology to visualize tumors in the human bladder. Materials and Methods: We performed ex vivo fibered confocal imaging of fresh radical cystectomy specimens using the Mauna Kea Technologies Cellvizio system. The findings were compared with results from standard histopathology. Results: The bladders of four patients were imaged using the fibered confocal microscope. Normal and neoplastic urothelium manifested differences in cellular and vascular density. Conclusion: This study demonstrates the feasibility of using fibered confocal microscopy to detect histologic differences between normal and neoplastic urothelium, and establishes a foundation for the use of fiber-based confocal microscopy in clinical studies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78112/1/end.2008.0524.pd

Efficacy of cathelicidin-mimetic antimicrobial peptoids against staphylococcus aureus

Staphylococcus aureus is one of the most common pathogens associated with infection in wounds. The current standard of care uses a combination of disinfection and drainage followed by conventional antibiotics such as methicillin. Methicillin and vancomycin resistance has rendered these treatments ineffective, often causing the reemergence of infection. This study examines the use of antimicrobial peptoids (sequence-specific poly-N-substituted glycines) designed to mimic naturally occurring cationic, amphipathic host defense peptides, as an alternative to conventional antibiotics. These peptoids also show efficient and fast (<30 min) killing of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) at low micromolar concentrations without having apparent cytotoxic side effects in vivo. Additionally, these novel peptoids show excellent efficacy against biofilm formation and detachment for both MSSA and MRSA. In comparison, conventional antibiotics were unable to detach or prevent formation of biofilms. One cationic 12mer, Peptoid 1, shows great promise, as it could prevent formation of and detach biofilms at concentrations as low as 1.6 μM. The use of a bioluminescent S. aureus murine incision wound model demonstrated clearance of infection in peptoid-treated mice within 8 days, conveying another advantage these peptoids have over conventional antibiotics. These results provide clear evidence of the potential for antimicrobial peptoids for the treatment of S. aureus wound infections. IMPORTANCE Staphylococcus aureus resistance is a consistent problem with a large impact on the health care system. Infections with resistant S. aureus can cause serious adverse effects and can result in death. These antimicrobial peptoids show efficient killing of bacteria both as a biofilm and as free bacteria, often doing so in less than 30 min. As such, these antimicrobials have the potential to alleviate the burden that Staphylococcus infections have on the health care system and cause better outcomes for infected patients

Development of B Cells and Erythrocytes Is Specifically Impaired by the Drug Celastrol in Mice

Background: Celastrol, an active compound extracted from the root of the Chinese medicine ‘‘Thunder of God Vine’’ (Tripterygium wilfordii), exhibits anticancer, antioxidant and anti-inflammatory activities, and interest in the therapeutic potential of celastrol is increasing. However, described side effects following treatment are significant and require investigation prior to initiating clinical trials. Here, we investigated the effects of celastrol on the adult murine hematopoietic system. Methodology/Principal Findings: Animals were treated daily with celastrol over a four-day period and peripheral blood, bone marrow, spleen, and peritoneal cavity were harvested for cell phenotyping. Treated mice showed specific impairment of the development of B cells and erythrocytes in all tested organs. In bone marrow, these alterations were accompanied by decreases in populations of common lymphoid progenitors (CLP), common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP). Conclusions/Significance: These results indicate that celastrol acts through regulators of adult hematopoiesis and could be used as a modulator of the hematopoietic system. These observations provide valuable information for further assessmen

Use of Reporter Genes for Optical Measurements of Neoplastic Disease In vivo

Revealing the cellular and molecular changes associated with cancer, as they occur in intact living animal models of human neoplastic disease, holds tremendous potential for understanding disease mechanisms and elucidating effective therapies. Since light is transmitted through mammalian tissues, at a low level, optical signatures conferred on tumor cells by expression of reporter genes encoding bioluminescent and fluorescent proteins can be detected externally using sensitive photon detection systems. Expression of reporter genes, such as the bioluminescent enzyme firefly luciferase (Luc) or variants of green fluorescent protein (GFP) in transformed cells, can effectively be used to reveal molecular and cellular features of neoplasia in vivo. Tumor cell growth and regression in response to various therapies have been evaluated non-invasively in living experimental animals using these reporter genes. Detection of Luc-labeled cells in vivo was extremely sensitive with signals over background from as few as 1000 human tumor cells distributed throughout the peritoneal cavity of a mouse with linear relationships between cell number and signal intensity over five logs. GFP offers the strength of high-resolution ex vivo analyses following in vivo localization of the tumor. The dynamic range of Luc detection allows the full disease course to be monitored since disease progression from small numbers of cells to extensive disease can be assessed. As such, therapies that target minimal disease as well as those designed for late stage disease can be readily evaluated in animal models. Real time spatiotemporal analyses of tumor cell growth can reveal the dynamics of neoplastic disease, and facilitate rapid optimization of effective treatment regimens. Thus, these methods improve the predictability of animal models of human disease as study groups can be followed over time, and can accelerate the development of therapeutic strategies

Advancing Molecular Therapies through In Vivo Bioluminescent Imaging

Effective development of therapeutics that target the molecular basis of disease is dependent on testing new therapeutic moieties and delivery strategies in animal models of human disease. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide data in real time and are sensitive to the subtle changes, are crucial for rapid advancement of these approaches. Modalities based on optics are rapid, sensitive, and accessible methods for in vivo analyses with relatively low instrumentation costs. In vivo bioluminescent imaging (BLI) is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity. BLI is based on the use of light-emitting enzymes as internal biological light sources that can be detected externally as biological indicators. BLI has been used to test spatio-temporal expression patterns of both target and therapeutic genes in living laboratory animals where the contextual influences of whole biological systems are preserved. BLI has also been used to analyze gene delivery, immune cell therapies, and the in vivo efficacy of inhibitory RNAs. New tools for BLI are being developed that will offer greater flexibility in detection and analyses. BLI can be used to accelerate the evaluation of experimental therapeutic strategies and whole body imaging offers the opportunity of revealing the effects of novel approaches on key steps in disease processes